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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:

  1. generate a sequence alignment within QIIME 2

  2. mask the alignment if needed

  3. construct a phylogenetic tree

  4. 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:
wget \
  -O "rep-seqs.qza" \
  "https://data.qiime2.org/2021.11/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2021.11/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:

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:

Reference based alignments

There are a variety of tools such as PyNAST) (using NAST), Infernal, and SINA, etc., that attempt to reduce the amount of ambiguously aligned regions by using curated reference alignments (e.g. 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. For a more in-depth and eloquent overview of reference-based alignment approaches, check out the great SINA community tutorial).

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:

  1. Phylogeny for the faint of heart - a tutorial

  2. Molecular phylogenetics - principles and practice

  3. Phylogenetics - An Introduction

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

  1. FastTree

  2. RAxML

  3. IQ-TREE

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:

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 9627 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-jwjpdhq8/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps4rst7jw -n q2 


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

Inference[0]: Time 0.629874 CAT-based likelihood -1242.732284, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1387.757761 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps4rst7jw/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.757761 .... 

Final GAMMA-based Score of best tree -1387.351596

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps4rst7jw/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps4rst7jw/RAxML_bestTree.q2

Overall execution time: 1.230376 secs or 0.000342 hours or 0.000014 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 9627 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-jwjpdhq8/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps4rst7jw -n q2

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

Output artifacts:

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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-g6tduit1/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po -n q2 


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

Inference[0]: Time 0.587736 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.482767 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.496469 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.636286 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.478449 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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po/RAxML_bestTree.q2

Overall execution time: 3.468199 secs or 0.000963 hours or 0.000040 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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-g6tduit1/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpy04pz0po -n q2

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

Output artifacts:

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:

  1. Bootstrap the input alignment 100 times and perform a Maximum Likelihood (ML) search on each.

  2. 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.

  3. 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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-fzl4h_gh/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o -n q2bootstrap 



Time for BS model parameter optimization 0.048698
Bootstrap[0]: Time 0.164074 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.109902 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.103879 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.091332 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.105927 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.107430 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.111256 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.099820 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.107501 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.115591 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.079048 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.116969 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.121491 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.104648 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.111953 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.109227 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.105969 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.093611 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.101721 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.101600 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.094543 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.087232 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.103115 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.120925 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.109031 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.108022 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.107155 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.129447 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.127295 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.085918 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.107896 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.112838 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.114167 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.103613 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.094929 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.118013 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.095094 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.118240 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.091463 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.086950 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.108154 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.112306 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.107222 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.120238 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.096154 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.108516 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.084264 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.131374 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.085591 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.117099 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.134679 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.096570 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.105733 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.101284 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.107876 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.116101 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.093054 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.091278 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.130824 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.106465 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.092266 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.102652 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.122047 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.093146 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.093539 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.091601 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.117315 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.115933 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.091150 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.093157 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.100998 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.099004 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.086882 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.099235 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.088444 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.095473 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.103640 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.133046 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.107543 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.110927 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.099113 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.105983 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.124326 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.095567 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.102498 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.107821 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.090391 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.095776 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.107576 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.099830 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.112539 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.107814 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.045192 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.124739 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.092357 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.092847 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.103141 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.118619 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.040919 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.058114 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.410240 seconds
Average Time per Rapid Bootstrap 0.104102 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.158886 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: 2.134417 seconds
Thorough ML search Time: 0.558732 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 6.858234 secs or 0.001905 hours

Combined Bootstrap and ML search took 17.268669 secs or 0.004797 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.278257 secs or 0.004800 hours or 0.000200 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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-fzl4h_gh/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmph613ox_o -n q2bootstrap

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

Output artifacts:

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:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-5nliengb/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree -nt 1
Seed:    685591 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:35:22 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-5nliengb/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)... 0.003 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: 0.33813  A-G: 2.30739  A-T: 2.15805  C-G: 1.19220  C-T: 3.30147  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 (0.013 sec)
Time for fast ML tree search: 0.069 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 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  2866.557     2892.449     3021.392
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.422
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 16  SYM+G4        1387.155     43  2860.310     2882.568     3005.047
 18  SYM+R2        1382.244     44  2852.489     2875.921     3000.592
 29  TVM+F+G4      1388.425     45  2866.850     2891.493     3018.319
 31  TVM+F+R2      1382.482     46  2856.965     2882.857     3011.799
 42  TVMe+G4       1387.122     42  2858.244     2879.367     2999.615
 44  TVMe+R2       1382.298     43  2850.596     2872.855     2995.333
 55  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 57  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
 68  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
 70  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
 81  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
 83  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
 94  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
 96  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
107  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
109  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
120  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
122  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
133  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
135  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
146  TPM3+F+G4     1392.567     43  2871.134     2893.393     3015.871
148  TPM3+F+R2     1386.368     44  2860.736     2884.168     3008.839
159  TPM2u+F+G4    1395.282     43  2876.564     2898.823     3021.301
161  TPM2u+F+R2    1388.115     44  2864.231     2887.663     3012.334
172  TPM2+F+G4     1395.282     43  2876.564     2898.823     3021.301
174  TPM2+F+R2     1388.115     44  2864.231     2887.663     3012.334
185  K3Pu+F+G4     1392.067     43  2870.133     2892.392     3014.870
187  K3Pu+F+R2     1385.124     44  2858.247     2881.679     3006.350
198  K3P+G4        1394.798     40  2869.597     2888.556     3004.236
200  K3P+R2        1388.380     41  2858.761     2878.784     2996.766
211  TN+F+G4       1394.627     43  2875.254     2897.513     3019.991
213  TN+F+R2       1386.824     44  2861.648     2885.079     3009.750
224  TNe+G4        1397.746     40  2875.492     2894.452     3010.131
226  TNe+R2        1391.135     41  2864.270     2884.293     3002.275
237  HKY+F+G4      1395.753     42  2875.505     2896.628     3016.876
239  HKY+F+R2      1388.692     43  2863.383     2885.642     3008.120
250  K2P+G4        1397.751     39  2873.502     2891.433     3004.775
252  K2P+R2        1391.217     40  2862.434     2881.394     2997.073
263  F81+F+G4      1406.484     41  2894.968     2914.991     3032.973
265  F81+F+R2      1400.605     42  2885.210     2906.333     3026.581
276  JC+G4         1408.433     38  2892.866     2909.803     3020.773
278  JC+R2         1403.022     39  2884.045     2901.976     3015.318
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree.model.gz
CPU time for ModelFinder: 0.666 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.699 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1382.298
Optimal log-likelihood: -1382.296
Rate parameters:  A-C: 0.21620  A-G: 2.01102  A-T: 1.56832  C-G: 0.77577  C-T: 2.01102  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.406) (0.278,2.543)
Parameters optimization took 1 rounds (0.005 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.003816 sec (of wall-clock time) 0.003790 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000086 sec (of wall-clock time) 0.000108 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1396.935
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 98 initial trees ... 0.083 seconds
Current best score: -1382.296

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.296
Iteration 10 / LogL: -1382.305 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.311 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.296 / CPU time: 0.437
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 21: -1382.087
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 24: -1382.003
Iteration 30 / LogL: -1382.921 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 40 / LogL: -1383.097 / Time: 0h:0m:0s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 50 / LogL: -1382.003 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.002
Iteration 60 / LogL: -1413.280 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1382.483 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1382.087 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 90 / LogL: -1382.006 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1382.477 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 110 / LogL: -1382.109 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 120 / LogL: -1382.003 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 125 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.002
Optimal log-likelihood: -1382.001
Rate parameters:  A-C: 0.19151  A-G: 1.84202  A-T: 1.53312  C-G: 0.77297  C-T: 1.84202  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.409) (0.276,2.554)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.001
Total tree length: 7.110

Total number of iterations: 125
CPU time used for tree search: 2.430 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.248 sec (0h:0m:2s)
Total CPU time used: 2.464 sec (0h:0m:2s)
Total wall-clock time used: 2.278 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree.log

Date and Time: Mon Nov 22 20:35:25 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-5nliengb/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp00jhrf79/q2iqtree -nt 1

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

Output artifacts:

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:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-lannsjhr/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzk2x2ijk/q2iqtree -nt 1
Seed:    550184 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:35:35 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-lannsjhr/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)... 0.001 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: -1394.544
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: -1394.544

Parameters optimization took 0.466 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007676 sec (of wall-clock time) 0.007641 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000085 sec (of wall-clock time) 0.000080 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.665
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.100 second
Computing log-likelihood of 98 initial trees ... 0.113 seconds
Current best score: -1394.544

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.260
Iteration 10 / LogL: -1387.281 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.267 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.260 / CPU time: 0.612
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.277 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1388.284 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1388.151 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 60 / LogL: -1387.570 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 70 / LogL: -1387.348 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1387.335 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 90 / LogL: -1387.348 / Time: 0h:0m:2s (0h:0m:0s left)
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 100 / LogL: -1387.618 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    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: 0.34646  A-G: 2.32383  A-T: 2.14454  C-G: 1.23513  C-T: 3.22073  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 (0.006 sec)
BEST SCORE FOUND : -1387.167
Total tree length: 7.608

Total number of iterations: 102
CPU time used for tree search: 2.966 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.785 sec (0h:0m:2s)
Total CPU time used: 3.469 sec (0h:0m:3s)
Total wall-clock time used: 3.283 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzk2x2ijk/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzk2x2ijk/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzk2x2ijk/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzk2x2ijk/q2iqtree.log

Date and Time: Mon Nov 22 20:35:38 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-lannsjhr/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzk2x2ijk/q2iqtree -nt 1

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

Output artifacts:

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:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-mnne0g2g/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree -nt 1 -fast
Seed:    746350 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:35:48 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-mnne0g2g/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)

---> START RUN NUMBER 1 (seed: 746350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.266 / LogL: -1392.814
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.018, 1.339 / LogL: -1393.581
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.023, 1.392 / LogL: -1393.793
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.022, 1.407 / LogL: -1393.766
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.025, 1.408 / LogL: -1393.929
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.027, 1.405 / LogL: -1394.069
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.023, 1.401 / LogL: -1393.833
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.024, 1.400 / LogL: -1393.905
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.025, 1.401 / LogL: -1393.956
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.026, 1.402 / LogL: -1394.004
Optimal pinv,alpha: 0.000, 1.266 / LogL: -1392.814

Parameters optimization took 0.331 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007216 sec (of wall-clock time) 0.007162 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000080 sec (of wall-clock time) 0.000075 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.769
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.270
Finish initializing candidate tree set (3)
Current best tree score: -1387.270 / CPU time: 0.048
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: -1387.270
Optimal log-likelihood: -1387.256
Rate parameters:  A-C: 0.33192  A-G: 2.27065  A-T: 2.14835  C-G: 1.17849  C-T: 3.29080  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: 1.312
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.256
Total tree length: 6.762

Total number of iterations: 2
CPU time used for tree search: 0.094 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.048 sec (0h:0m:0s)
Total CPU time used: 0.462 sec (0h:0m:0s)
Total wall-clock time used: 0.408 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 747350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 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: 1.98596  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 (0.039 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.008152 sec (of wall-clock time) 0.016006 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000096 sec (of wall-clock time) 0.000082 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.687
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.087
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: -1387.950
2. Current log-likelihood: -1387.817
3. Current log-likelihood: -1387.717
4. Current log-likelihood: -1387.641
5. Current log-likelihood: -1387.582
Optimal log-likelihood: -1387.535
Rate parameters:  A-C: 0.36767  A-G: 2.30158  A-T: 2.10668  C-G: 1.21806  C-T: 3.25866  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: 1.325
Parameters optimization took 5 rounds (0.018 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.544

Total number of iterations: 2
CPU time used for tree search: 0.172 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.088 sec (0h:0m:0s)
Total CPU time used: 0.332 sec (0h:0m:0s)
Total wall-clock time used: 0.170 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 748350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 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 (0.039 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007731 sec (of wall-clock time) 0.015184 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000063 sec (of wall-clock time) 0.000066 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1400.020
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.190
Finish initializing candidate tree set (3)
Current best tree score: -1388.190 / CPU time: 0.058
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: -1388.190
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.686
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.470
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33256  A-G: 2.23866  A-T: 2.11314  C-G: 1.16082  C-T: 3.23616  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 (0.029 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.733

Total number of iterations: 2
CPU time used for tree search: 0.115 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.059 sec (0h:0m:0s)
Total CPU time used: 0.291 sec (0h:0m:0s)
Total wall-clock time used: 0.150 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 749350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 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 (0.040 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007495 sec (of wall-clock time) 0.014721 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000069 sec (of wall-clock time) 0.000085 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1400.020
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.190
Finish initializing candidate tree set (3)
Current best tree score: -1388.190 / CPU time: 0.060
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: -1388.190
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.686
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.470
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33256  A-G: 2.23866  A-T: 2.11314  C-G: 1.16082  C-T: 3.23616  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 (0.026 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.733

Total number of iterations: 2
CPU time used for tree search: 0.119 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.060 sec (0h:0m:0s)
Total CPU time used: 0.291 sec (0h:0m:0s)
Total wall-clock time used: 0.150 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 750350)

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: 1.99581  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 (0.039 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007773 sec (of wall-clock time) 0.015259 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000069 sec (of wall-clock time) 0.000067 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1400.020
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.190
Finish initializing candidate tree set (3)
Current best tree score: -1388.190 / CPU time: 0.063
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: -1388.190
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.686
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.470
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33256  A-G: 2.23866  A-T: 2.11314  C-G: 1.16082  C-T: 3.23616  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 (0.028 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.733

Total number of iterations: 2
CPU time used for tree search: 0.124 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.063 sec (0h:0m:0s)
Total CPU time used: 0.299 sec (0h:0m:0s)
Total wall-clock time used: 0.154 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 751350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 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 (0.039 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007726 sec (of wall-clock time) 0.015168 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000059 sec (of wall-clock time) 0.000064 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1400.020
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.190
Finish initializing candidate tree set (3)
Current best tree score: -1388.190 / CPU time: 0.059
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: -1388.190
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.686
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.470
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33256  A-G: 2.23866  A-T: 2.11314  C-G: 1.16082  C-T: 3.23616  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 (0.026 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.733

Total number of iterations: 2
CPU time used for tree search: 0.117 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.060 sec (0h:0m:0s)
Total CPU time used: 0.289 sec (0h:0m:0s)
Total wall-clock time used: 0.148 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 752350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.491
2. Current log-likelihood: -1404.606
3. Current log-likelihood: -1399.220
4. Current log-likelihood: -1397.821
5. Current log-likelihood: -1397.063
Optimal log-likelihood: -1396.484
Rate parameters:  A-C: 0.24153  A-G: 2.03298  A-T: 1.94373  C-G: 1.02159  C-T: 2.79340  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: 1.438
Parameters optimization took 5 rounds (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007912 sec (of wall-clock time) 0.015543 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000069 sec (of wall-clock time) 0.000073 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1400.067
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.199
Finish initializing candidate tree set (3)
Current best tree score: -1388.199 / CPU time: 0.059
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: -1388.199
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.809
4. Current log-likelihood: -1387.688
5. Current log-likelihood: -1387.597
6. Current log-likelihood: -1387.527
7. Current log-likelihood: -1387.472
Optimal log-likelihood: -1387.427
Rate parameters:  A-C: 0.33146  A-G: 2.22541  A-T: 2.09978  C-G: 1.15373  C-T: 3.21465  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: 1.358
Parameters optimization took 7 rounds (0.026 sec)
BEST SCORE FOUND : -1387.427
Total tree length: 6.730

Total number of iterations: 2
CPU time used for tree search: 0.116 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.059 sec (0h:0m:0s)
Total CPU time used: 0.285 sec (0h:0m:0s)
Total wall-clock time used: 0.146 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 753350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1496.015
2. Current log-likelihood: -1403.630
3. Current log-likelihood: -1398.533
4. Current log-likelihood: -1397.077
5. Current log-likelihood: -1396.256
6. Current log-likelihood: -1395.746
Optimal log-likelihood: -1395.367
Rate parameters:  A-C: 0.23678  A-G: 2.05007  A-T: 1.94889  C-G: 1.06765  C-T: 2.81222  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.021
Gamma shape alpha: 1.337
Parameters optimization took 6 rounds (0.040 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007747 sec (of wall-clock time) 0.015212 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000061 sec (of wall-clock time) 0.000059 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.460
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.984
Finish initializing candidate tree set (3)
Current best tree score: -1387.984 / CPU time: 0.055
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: -1387.984
2. Current log-likelihood: -1387.819
3. Current log-likelihood: -1387.696
4. Current log-likelihood: -1387.602
5. Current log-likelihood: -1387.530
6. Current log-likelihood: -1387.474
Optimal log-likelihood: -1387.428
Rate parameters:  A-C: 0.33235  A-G: 2.26206  A-T: 2.13756  C-G: 1.17331  C-T: 3.27687  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 (0.023 sec)
BEST SCORE FOUND : -1387.428
Total tree length: 6.753

Total number of iterations: 2
CPU time used for tree search: 0.108 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.055 sec (0h:0m:0s)
Total CPU time used: 0.274 sec (0h:0m:0s)
Total wall-clock time used: 0.141 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 754350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 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 (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007741 sec (of wall-clock time) 0.015234 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000061 sec (of wall-clock time) 0.000065 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.687
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.087
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: -1387.950
2. Current log-likelihood: -1387.817
3. Current log-likelihood: -1387.717
4. Current log-likelihood: -1387.641
5. Current log-likelihood: -1387.582
Optimal log-likelihood: -1387.535
Rate parameters:  A-C: 0.36767  A-G: 2.30158  A-T: 2.10668  C-G: 1.21806  C-T: 3.25866  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: 1.325
Parameters optimization took 5 rounds (0.018 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.544

Total number of iterations: 2
CPU time used for tree search: 0.171 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.087 sec (0h:0m:0s)
Total CPU time used: 0.332 sec (0h:0m:0s)
Total wall-clock time used: 0.170 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 755350)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 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 (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007577 sec (of wall-clock time) 0.014918 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000074 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.687
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.087
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: -1387.950
2. Current log-likelihood: -1387.817
3. Current log-likelihood: -1387.717
4. Current log-likelihood: -1387.641
5. Current log-likelihood: -1387.582
Optimal log-likelihood: -1387.535
Rate parameters:  A-C: 0.36767  A-G: 2.30158  A-T: 2.10668  C-G: 1.21806  C-T: 3.25866  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: 1.325
Parameters optimization took 5 rounds (0.018 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.544

Total number of iterations: 2
CPU time used for tree search: 0.172 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.088 sec (0h:0m:0s)
Total CPU time used: 0.326 sec (0h:0m:0s)
Total wall-clock time used: 0.168 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 1 gave best log-likelihood: -1387.256
Total CPU time for 10 runs: 3.211 seconds.
Total wall-clock time for 10 runs: 1.823 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree.log

Date and Time: Mon Nov 22 20:35:50 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-mnne0g2g/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpod6ewh2x/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

  1. SH-aLRT via --p-alrt [INT >= 1000]

  2. aBayes via --p-abayes [TRUE | FALSE]

  3. 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:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-zlyjd_jv/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpa_oq4k35/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    438935 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:36:00 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-zlyjd_jv/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)... 0.001 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: -1394.430
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.306 / LogL: -1394.720
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.315 / LogL: -1394.793
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.313 / LogL: -1394.791
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.755
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.309 / LogL: -1394.783
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.305 / LogL: -1394.729
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.742
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.308 / LogL: -1394.753
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.312 / LogL: -1394.757
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.430

Parameters optimization took 0.460 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007424 sec (of wall-clock time) 0.007374 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000086 sec (of wall-clock time) 0.000107 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.715
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.095 second
Computing log-likelihood of 98 initial trees ... 0.112 seconds
Current best score: -1394.430

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.264
Iteration 10 / LogL: -1387.281 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.371 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.264 / CPU time: 0.642
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 23: -1387.168
BETTER TREE FOUND at iteration 29: -1387.168
Iteration 30 / LogL: -1410.779 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 40 / LogL: -1387.310 / Time: 0h:0m:1s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 50 / LogL: -1387.168 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 60 / LogL: -1387.188 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 70 / LogL: -1430.139 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 80 / LogL: -1387.175 / Time: 0h:0m:2s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 90 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 100 / LogL: -1387.175 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 110 / LogL: -1396.255 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 120 / LogL: -1387.269 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 130 / LogL: -1387.168 / Time: 0h:0m:4s (0h:0m:-1s left)
TREE SEARCH COMPLETED AFTER 130 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    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: 0.34765  A-G: 2.33581  A-T: 2.15598  C-G: 1.24168  C-T: 3.23772  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: 1.283
Parameters optimization took 1 rounds (0.004 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...
0.055 sec.
Total tree length: 7.613

Total number of iterations: 130
CPU time used for tree search: 3.954 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.772 sec (0h:0m:3s)
Total CPU time used: 4.499 sec (0h:0m:4s)
Total wall-clock time used: 4.314 sec (0h:0m:4s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpa_oq4k35/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpa_oq4k35/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpa_oq4k35/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpa_oq4k35/q2iqtree.log

Date and Time: Mon Nov 22 20:36:04 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-zlyjd_jv/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpa_oq4k35/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

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:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-3uko6xt3/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    662154 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:36:14 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-3uko6xt3/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)... 0.001 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: -1396.594
2. Current log-likelihood: -1395.225
Optimal log-likelihood: -1394.470
Rate parameters:  A-C: 0.22094  A-G: 2.05280  A-T: 1.94947  C-G: 1.06436  C-T: 2.58632  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: 1.320
Parameters optimization took 2 rounds (0.013 sec)
Time for fast ML tree search: 0.059 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1410.735     45  2911.470     2936.113     3062.939
  2  GTR+F+I       1408.913     46  2909.825     2935.717     3064.660
  3  GTR+F+G4      1392.994     46  2877.988     2903.880     3032.823
  4  GTR+F+I+G4    1393.284     47  2880.568     2907.749     3038.769
  5  GTR+F+R2      1387.695     47  2869.389     2896.570     3027.590
  6  GTR+F+R3      1387.734     49  2873.467     2903.346     3038.400
 16  SYM+G4        1393.513     43  2873.027     2895.285     3017.764
 18  SYM+R2        1389.896     44  2867.792     2891.224     3015.895
 29  TVM+F+G4      1393.482     45  2876.964     2901.607     3028.433
 31  TVM+F+R2      1388.482     46  2868.965     2894.857     3023.800
 42  TVMe+G4       1393.649     42  2871.298     2892.421     3012.669
 44  TVMe+R2       1389.915     43  2865.830     2888.089     3010.567
 55  TIM3+F+G4     1396.896     44  2881.792     2905.224     3029.895
 57  TIM3+F+R2     1391.444     45  2872.887     2897.530     3024.356
 68  TIM3e+G4      1396.973     41  2875.945     2895.968     3013.950
 70  TIM3e+R2      1393.201     42  2870.402     2891.525     3011.773
 81  TIM2+F+G4     1401.394     44  2890.789     2914.221     3038.892
 83  TIM2+F+R2     1395.779     45  2881.558     2906.201     3033.027
 94  TIM2e+G4      1406.338     41  2894.676     2914.699     3032.681
 96  TIM2e+R2      1402.241     42  2888.482     2909.605     3029.853
107  TIM+F+G4      1397.923     44  2883.846     2907.278     3031.949
109  TIM+F+R2      1392.152     45  2874.304     2898.946     3025.772
120  TIMe+G4       1403.735     41  2889.469     2909.492     3027.474
122  TIMe+R2       1399.368     42  2882.736     2903.858     3024.107
133  TPM3u+F+G4    1397.362     43  2880.723     2902.982     3025.460
135  TPM3u+F+R2    1392.261     44  2872.521     2895.953     3020.624
146  TPM3+F+G4     1397.362     43  2880.723     2902.982     3025.460
148  TPM3+F+R2     1392.261     44  2872.521     2895.953     3020.624
159  TPM2u+F+G4    1401.856     43  2889.713     2911.972     3034.450
161  TPM2u+F+R2    1396.532     44  2881.064     2904.496     3029.167
172  TPM2+F+G4     1401.856     43  2889.713     2911.972     3034.450
174  TPM2+F+R2     1396.532     44  2881.064     2904.496     3029.167
185  K3Pu+F+G4     1398.521     43  2883.042     2905.300     3027.779
187  K3Pu+F+R2     1393.048     44  2874.096     2897.528     3022.199
198  K3P+G4        1403.868     40  2887.736     2906.696     3022.375
200  K3P+R2        1399.386     41  2880.772     2900.796     3018.777
211  TN+F+G4       1401.516     43  2889.032     2911.291     3033.769
213  TN+F+R2       1395.987     44  2879.974     2903.406     3028.077
224  TNe+G4        1406.405     40  2892.810     2911.770     3027.449
226  TNe+R2        1402.270     41  2886.540     2906.563     3024.545
237  HKY+F+G4      1401.987     42  2887.975     2909.098     3029.346
239  HKY+F+R2      1396.739     43  2879.478     2901.737     3024.215
250  K2P+G4        1406.580     39  2891.160     2909.091     3022.433
252  K2P+R2        1402.302     40  2884.605     2903.564     3019.244
263  F81+F+G4      1410.186     41  2902.372     2922.395     3040.377
265  F81+F+R2      1405.821     42  2895.643     2916.766     3037.014
276  JC+G4         1414.853     38  2905.706     2922.643     3033.613
278  JC+R2         1411.419     39  2900.838     2918.769     3032.111
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree.model.gz
CPU time for ModelFinder: 0.628 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.633 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1389.915
Optimal log-likelihood: -1389.911
Rate parameters:  A-C: 0.11031  A-G: 1.62357  A-T: 1.38384  C-G: 0.62934  C-T: 1.62357  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.689,0.347) (0.311,2.445)
Parameters optimization took 1 rounds (0.004 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.004431 sec (of wall-clock time) 0.004313 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000086 sec (of wall-clock time) 0.000101 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1397.761
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.090 second
Computing log-likelihood of 98 initial trees ... 0.081 seconds
Current best score: -1389.911

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1389.527
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1384.255
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 3: -1382.458
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 4: -1382.299
Iteration 10 / LogL: -1382.878 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.348 / Time: 0h:0m:0s
Finish initializing candidate tree set (5)
Current best tree score: -1382.299 / CPU time: 0.487
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 21: -1382.087
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 30: -1382.008
Iteration 30 / LogL: -1382.008 / Time: 0h:0m:0s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.007
Iteration 40 / LogL: -1382.155 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 50 / LogL: -1382.476 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 60 / LogL: -1382.043 / Time: 0h:0m:0s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.006
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 70 / LogL: -1382.417 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1382.887 / Time: 0h:0m:1s (0h:0m:2s left)
BETTER TREE FOUND at iteration 86: -1382.005
Iteration 90 / LogL: -1382.170 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 100 / LogL: -1382.467 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 110 / LogL: -1382.912 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 120 / LogL: -1382.022 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 130 / LogL: -1382.007 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 140 / LogL: -1384.447 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 150 / LogL: -1382.353 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 160 / LogL: -1382.090 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 170 / LogL: -1382.205 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 180 / LogL: -1382.010 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 190 / LogL: -1382.101 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 200 / LogL: -1382.008 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 210 / LogL: -1382.510 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 220 / LogL: -1382.039 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 230 / LogL: -1382.010 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 240 / LogL: -1382.031 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 250 / LogL: -1382.026 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 260 / LogL: -1382.017 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 270 / LogL: -1382.005 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 280 / LogL: -1382.048 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 287 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.005
Optimal log-likelihood: -1382.003
Rate parameters:  A-C: 0.19440  A-G: 1.85866  A-T: 1.54960  C-G: 0.77961  C-T: 1.85866  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.408) (0.276,2.552)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.003
Total tree length: 7.119

Total number of iterations: 287
CPU time used for tree search: 3.515 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.362 sec (0h:0m:3s)
Total CPU time used: 3.546 sec (0h:0m:3s)
Total wall-clock time used: 3.391 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree.log

Date and Time: Mon Nov 22 20:36:18 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-3uko6xt3/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphygkxiug/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

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:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9oa8epds/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    583374 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:36:28 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9oa8epds/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)... 0.001 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 (0.013 sec)
Time for fast ML tree search: 0.063 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 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  2866.557     2892.449     3021.392
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.422
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 16  SYM+G4        1387.155     43  2860.310     2882.568     3005.047
 18  SYM+R2        1382.244     44  2852.489     2875.921     3000.592
 29  TVM+F+G4      1388.425     45  2866.850     2891.493     3018.319
 31  TVM+F+R2      1382.482     46  2856.965     2882.857     3011.799
 42  TVMe+G4       1387.122     42  2858.244     2879.367     2999.615
 44  TVMe+R2       1382.298     43  2850.596     2872.855     2995.333
 55  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 57  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
 68  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
 70  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
 81  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
 83  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
 94  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
 96  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
107  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
109  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
120  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
122  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
133  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
135  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
146  TPM3+F+G4     1392.567     43  2871.134     2893.393     3015.871
148  TPM3+F+R2     1386.368     44  2860.736     2884.168     3008.839
159  TPM2u+F+G4    1395.282     43  2876.564     2898.823     3021.301
161  TPM2u+F+R2    1388.115     44  2864.231     2887.663     3012.334
172  TPM2+F+G4     1395.282     43  2876.564     2898.823     3021.301
174  TPM2+F+R2     1388.115     44  2864.231     2887.663     3012.334
185  K3Pu+F+G4     1392.067     43  2870.133     2892.392     3014.870
187  K3Pu+F+R2     1385.124     44  2858.247     2881.679     3006.350
198  K3P+G4        1394.798     40  2869.597     2888.556     3004.236
200  K3P+R2        1388.380     41  2858.761     2878.784     2996.766
211  TN+F+G4       1394.627     43  2875.254     2897.513     3019.991
213  TN+F+R2       1386.824     44  2861.648     2885.079     3009.750
224  TNe+G4        1397.746     40  2875.492     2894.452     3010.131
226  TNe+R2        1391.135     41  2864.270     2884.293     3002.275
237  HKY+F+G4      1395.753     42  2875.505     2896.628     3016.876
239  HKY+F+R2      1388.692     43  2863.383     2885.642     3008.120
250  K2P+G4        1397.751     39  2873.502     2891.433     3004.775
252  K2P+R2        1391.217     40  2862.434     2881.394     2997.073
263  F81+F+G4      1406.484     41  2894.968     2914.991     3032.973
265  F81+F+R2      1400.605     42  2885.210     2906.333     3026.581
276  JC+G4         1408.433     38  2892.866     2909.803     3020.773
278  JC+R2         1403.022     39  2884.045     2901.976     3015.318
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.662 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.666 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 583374)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1382.298
Optimal log-likelihood: -1382.296
Rate parameters:  A-C: 0.21620  A-G: 2.01102  A-T: 1.56832  C-G: 0.77577  C-T: 2.01102  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.406) (0.278,2.543)
Parameters optimization took 1 rounds (0.006 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.005408 sec (of wall-clock time) 0.005355 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000091 sec (of wall-clock time) 0.000097 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1396.935
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.090 second
Computing log-likelihood of 98 initial trees ... 0.081 seconds
Current best score: -1382.296

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.296
Iteration 10 / LogL: -1382.305 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.311 / Time: 0h:0m:0s
Finish initializing candidate tree set (3)
Current best tree score: -1382.296 / CPU time: 0.578
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1382.296
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 25: -1382.001
Iteration 30 / LogL: -1382.040 / Time: 0h:0m:0s (0h:0m:5s left)
Iteration 40 / LogL: -1383.096 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 50 / LogL: -1382.418 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1405.077
Iteration 60 / LogL: -1382.002 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 70 / LogL: -1382.006 / Time: 0h:0m:1s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.001
Iteration 80 / LogL: -1382.001 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 90 / LogL: -1382.006 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 100 / LogL: -1382.013 / Time: 0h:0m:2s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1404.225
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.995
UPDATE BEST LOG-LIKELIHOOD: -1382.001
Iteration 110 / LogL: -1382.003 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.001
Iteration 120 / LogL: -1382.099 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 130 / LogL: -1382.073 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 140 / LogL: -1382.005 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 150 / LogL: -1382.235 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1403.454
Iteration 160 / LogL: -1382.017 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 170 / LogL: -1382.039 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 180 / LogL: -1382.009 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 190 / LogL: -1382.441 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 200 / LogL: -1382.038 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1403.454
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.999
Iteration 210 / LogL: -1382.002 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.020 / Time: 0h:0m:4s (0h:0m:1s left)
TREE SEARCH COMPLETED AFTER 226 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.001
Optimal log-likelihood: -1382.001
Rate parameters:  A-C: 0.19241  A-G: 1.84128  A-T: 1.53156  C-G: 0.77160  C-T: 1.84128  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.725,0.410) (0.275,2.560)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.001
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.splits.nex
Total tree length: 7.105

Total number of iterations: 226
CPU time used for tree search: 4.937 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.769 sec (0h:0m:4s)
Total CPU time used: 5.049 sec (0h:0m:5s)
Total wall-clock time used: 4.880 sec (0h:0m:4s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.splits.nex...
20 taxa and 145 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.002

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot.log

Date and Time: Mon Nov 22 20:36:34 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9oa8epds/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmphg36yq01/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

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:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-gtmxv0n2/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    27576 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Nov 22 20:36:44 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-gtmxv0n2/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta ... Fasta format detected
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
   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)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 27576)...

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, 1.218 / LogL: -1394.499
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.293 / LogL: -1394.744
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.008, 1.296 / LogL: -1394.739
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.008, 1.294 / LogL: -1394.737
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.766
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.007, 1.297 / LogL: -1394.718
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.299 / LogL: -1394.735
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.747
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.304 / LogL: -1394.750
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.761
Optimal pinv,alpha: 0.000, 1.218 / LogL: -1394.499

Parameters optimization took 0.488 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007659 sec (of wall-clock time) 0.007631 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000091 sec (of wall-clock time) 0.000103 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.692
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.090 second
Computing log-likelihood of 97 initial trees ... 0.111 seconds
Current best score: -1394.499

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1394.449
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1387.268
Iteration 10 / LogL: -1387.283 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.283 / Time: 0h:0m:1s
Finish initializing candidate tree set (3)
Current best tree score: -1387.268 / CPU time: 0.803
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1388.162 / Time: 0h:0m:1s (0h:0m:9s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 40: -1387.170
Iteration 40 / LogL: -1387.170 / Time: 0h:0m:1s (0h:0m:9s left)
Iteration 50 / LogL: -1388.142 / Time: 0h:0m:2s (0h:0m:8s left)
Log-likelihood cutoff on original alignment: -1413.535
UPDATE BEST LOG-LIKELIHOOD: -1387.168
UPDATE BEST LOG-LIKELIHOOD: -1387.167
Iteration 60 / LogL: -1387.167 / Time: 0h:0m:2s (0h:0m:7s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.167
Iteration 70 / LogL: -1387.220 / Time: 0h:0m:2s (0h:0m:7s left)
Iteration 80 / LogL: -1387.335 / Time: 0h:0m:3s (0h:0m:6s left)
Iteration 90 / LogL: -1387.176 / Time: 0h:0m:3s (0h:0m:5s left)
Iteration 100 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:5s left)
Log-likelihood cutoff on original alignment: -1413.535
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.982
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 110 / LogL: -1387.168 / Time: 0h:0m:4s (0h:0m:4s left)
Iteration 120 / LogL: -1387.167 / Time: 0h:0m:4s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.167
Iteration 130 / LogL: -1387.168 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 140 / LogL: -1387.167 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 150 / LogL: -1387.216 / Time: 0h:0m:5s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1414.434
Iteration 160 / LogL: -1387.167 / Time: 0h:0m:5s (0h:0m:2s left)
Iteration 170 / LogL: -1387.210 / Time: 0h:0m:5s (0h:0m:2s left)
Iteration 180 / LogL: -1387.207 / Time: 0h:0m:6s (0h:0m:2s left)
Iteration 190 / LogL: -1396.390 / Time: 0h:0m:6s (0h:0m:1s left)
Iteration 200 / LogL: -1387.168 / Time: 0h:0m:6s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1414.886
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.990
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 210 / LogL: -1387.177 / Time: 0h:0m:7s (0h:0m:3s left)
Iteration 220 / LogL: -1389.344 / Time: 0h:0m:7s (0h:0m:2s left)
Iteration 230 / LogL: -1387.168 / Time: 0h:0m:7s (0h:0m:2s left)
Iteration 240 / LogL: -1387.167 / Time: 0h:0m:7s (0h:0m:1s left)
Iteration 250 / LogL: -1387.177 / Time: 0h:0m:8s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1414.434
Iteration 260 / LogL: -1387.167 / Time: 0h:0m:8s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.167
Iteration 270 / LogL: -1387.179 / Time: 0h:0m:8s (0h:0m:0s left)
Iteration 280 / LogL: -1387.167 / Time: 0h:0m:9s (0h:0m:0s left)
Iteration 290 / LogL: -1387.203 / Time: 0h:0m:9s (0h:0m:0s left)
Iteration 300 / LogL: -1387.167 / Time: 0h:0m:9s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1414.434
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
TREE SEARCH COMPLETED AFTER 300 ITERATIONS / Time: 0h:0m:9s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.167
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34930  A-G: 2.33762  A-T: 2.15861  C-G: 1.24330  C-T: 3.24351  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: 1.281
Parameters optimization took 1 rounds (0.004 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...
0.061 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.splits.nex
Total tree length: 7.619

Total number of iterations: 300
CPU time used for tree search: 9.529 sec (0h:0m:9s)
Wall-clock time used for tree search: 9.389 sec (0h:0m:9s)
Total CPU time used: 10.187 sec (0h:0m:10s)
Total wall-clock time used: 10.046 sec (0h:0m:10s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.splits.nex...
20 taxa and 177 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.213

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/q2iqtreeufboot.log

Date and Time: Mon Nov 22 20:36:54 2021
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/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-gtmxv0n2/99d5b257-d26a-4d31-95e1-92a391479844/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8u8tb2n8/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:

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:

  1. qiime alignment mafft ...

  2. qiime alignment mask ...

  3. qiime phylogeny fasttree ...

  4. 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:

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