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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/2022.11/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2022.11/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 several tools that attempt to reduce the amount of ambiguously aligned regions by using curated reference alignments. Traditional, de novo alignment methods mututally align a set of unaligned sequences to create a multiple sequence alignment (MSA) from scratch. Re-running these methods with additional sequences will create MSAs with varying numbers of columns and assignments of bases to each column. These alignments is therefore incompatible with one another and may not be joined through concatenation.

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

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

Note

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

Construct a phylogeny

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

  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 4787 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptxgxppt6 -n q2 


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

Inference[0]: Time 0.682235 CAT-based likelihood -1242.537192, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1387.781776 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptxgxppt6/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.781776 .... 

Final GAMMA-based Score of best tree -1387.637559

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptxgxppt6/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptxgxppt6/RAxML_bestTree.q2

Overall execution time: 1.305106 secs or 0.000363 hours or 0.000015 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 4787 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptxgxppt6 -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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak -n q2 


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

Inference[0]: Time 0.624630 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.517125 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.525780 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.679197 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.507800 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/tmpt97cqfak/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak/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/tmpt97cqfak/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak/RAxML_bestTree.q2

Overall execution time: 3.576888 secs or 0.000994 hours or 0.000041 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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpt97cqfak -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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb -n q2bootstrap 



Time for BS model parameter optimization 0.052752
Bootstrap[0]: Time 0.177800 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.122693 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.113021 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.096906 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.113858 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.119477 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.116693 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.106583 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.116023 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.123995 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.087550 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.126606 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.135856 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.112439 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.119418 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.119437 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.112390 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.104104 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.108394 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.113624 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.141889 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.101156 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.110022 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.128886 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.111348 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.111008 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.113976 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.138939 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.125867 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.086524 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.108149 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.110555 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.126362 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.103655 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.099606 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.118424 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.096077 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.123330 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.095574 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.091579 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.108637 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.107543 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.102190 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.124105 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.100324 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.108733 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.088126 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.132367 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.085572 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.118823 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.136255 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.092691 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.109999 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.097658 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.112010 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.120369 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.098136 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.091508 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.131501 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.104305 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.092823 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.107526 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.122189 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.097147 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.093802 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.095252 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.117957 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.112160 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.091240 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.101430 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.101048 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.099191 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.085972 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.099237 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.088268 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.100185 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.108331 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.130837 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.108573 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.111270 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.098770 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.110637 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.126605 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.096003 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.102703 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.109006 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.094438 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.092781 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.107713 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.099967 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.107884 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.108053 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.045073 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.116150 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.096184 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.095134 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.110306 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.115628 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.044557 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.057857 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.776281 seconds
Average Time per Rapid Bootstrap 0.107763 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.173317 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: 2.175799 seconds
Thorough ML search Time: 0.550966 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 6.906880 secs or 0.001919 hours

Combined Bootstrap and ML search took 17.683474 secs or 0.004912 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.697314 secs or 0.004916 hours or 0.000205 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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_kwuz1yb -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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree -nt 1
Seed:    261007 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:10:05 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000224113 secs using 83.44% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000552893 secs using 73.07% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 2.00272e-05 secs using 84.88% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 7.51019e-05 secs using 77.23% CPU


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...
Optimizing NNI: done in 0.0267479 secs using 98.9% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.311
2. Current log-likelihood: -1389.733
Optimal log-likelihood: -1388.881
Rate parameters:  A-C: 0.33789  A-G: 2.29237  A-T: 2.14761  C-G: 1.19040  C-T: 3.28358  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: 1.424
Parameters optimization took 2 rounds (0.014 sec)
Time for fast ML tree search: 0.069 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 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      1387.279     46  2866.558     2892.450     3021.393
  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     3013.423
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 14  GTR+F+I+I+R2  1380.757     48  2857.513     2886.023     3019.080
 15  GTR+F+I+I+R3  1380.696     50  2861.392     2892.681     3029.691
 25  SYM+G4        1387.157     43  2860.314     2882.573     3005.051
 27  SYM+R2        1382.244     44  2852.489     2875.920     3000.591
 36  SYM+I+I+R2    1382.405     45  2854.809     2879.452     3006.278
 47  TVM+F+G4      1388.424     45  2866.848     2891.491     3018.317
 49  TVM+F+R2      1382.481     46  2856.963     2882.855     3011.798
 58  TVM+F+I+I+R2  1382.560     47  2859.119     2886.300     3017.320
 69  TVMe+G4       1387.122     42  2858.245     2879.367     2999.616
 71  TVMe+R2       1382.298     43  2850.596     2872.854     2995.333
 80  TVMe+I+I+R2   1382.453     44  2852.907     2876.339     3001.010
 91  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 93  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
102  TIM3+F+I+I+R2 1384.573     46  2861.146     2887.039     3015.981
113  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
115  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
124  TIM3e+I+I+R2  1385.391     43  2856.782     2879.040     3001.519
135  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
137  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
146  TIM2+F+I+I+R2 1386.377     46  2864.755     2890.647     3019.590
157  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
159  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
168  TIM2e+I+I+R2  1391.272     43  2868.544     2890.802     3013.281
179  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
181  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
190  TIM+F+I+I+R2  1383.340     46  2858.680     2884.572     3013.515
201  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
203  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
212  TIMe+I+I+R2   1388.456     43  2862.912     2885.171     3007.649
223  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
225  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
234  TPM3u+F+I+I+R2 1386.501     45  2863.002     2887.645     3014.471
245  TPM3+G4       1390.540     40  2861.080     2880.039     2995.719
247  TPM3+R2       1385.307     41  2852.614     2872.637     2990.619
256  TPM3+I+I+R2   1385.459     42  2854.917     2876.040     2996.288
267  TPM2u+F+G4    1395.283     43  2876.566     2898.825     3021.303
269  TPM2u+F+R2    1388.114     44  2864.229     2887.661     3012.332
278  TPM2u+F+I+I+R2 1388.249     45  2866.497     2891.140     3017.966
289  TPM2+G4       1397.749     40  2875.499     2894.458     3010.138
291  TPM2+R2       1391.198     41  2864.396     2884.419     3002.401
300  TPM2+I+I+R2   1391.346     42  2866.692     2887.815     3008.063
311  K3Pu+F+G4     1392.050     43  2870.100     2892.359     3014.837
313  K3Pu+F+R2     1385.122     44  2858.245     2881.677     3006.348
322  K3Pu+F+I+I+R2 1385.245     45  2860.491     2885.134     3011.960
333  K3P+G4        1394.786     40  2869.573     2888.532     3004.212
335  K3P+R2        1388.359     41  2858.717     2878.740     2996.722
344  K3P+I+I+R2    1388.531     42  2861.061     2882.184     3002.432
355  TN+F+G4       1394.628     43  2875.256     2897.515     3019.993
357  TN+F+R2       1386.824     44  2861.647     2885.079     3009.750
366  TN+F+I+I+R2   1386.967     45  2863.933     2888.576     3015.402
377  TNe+G4        1397.748     40  2875.497     2894.456     3010.136
379  TNe+R2        1391.134     41  2864.269     2884.292     3002.274
388  TNe+I+I+R2    1391.289     42  2866.578     2887.701     3007.949
399  HKY+F+G4      1395.752     42  2875.504     2896.627     3016.875
401  HKY+F+R2      1388.691     43  2863.382     2885.640     3008.119
410  HKY+F+I+I+R2  1388.820     44  2865.639     2889.071     3013.742
421  K2P+G4        1397.753     39  2873.507     2891.438     3004.780
423  K2P+R2        1391.216     40  2862.433     2881.392     2997.072
432  K2P+I+I+R2    1391.363     41  2864.726     2884.750     3002.731
443  F81+F+G4      1406.478     41  2894.955     2914.979     3032.960
445  F81+F+R2      1400.602     42  2885.203     2906.326     3026.574
454  F81+F+I+I+R2  1400.799     43  2887.598     2909.857     3032.335
465  JC+G4         1408.432     38  2892.865     2909.802     3020.772
467  JC+R2         1403.021     39  2884.041     2901.972     3015.314
476  JC+I+I+R2     1403.194     40  2886.388     2905.348     3021.028
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree.model.gz
CPU time for ModelFinder: 1.529 seconds (0h:0m:1s)
Wall-clock time for ModelFinder: 1.573 seconds (0h:0m:1s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.307
Optimal log-likelihood: -1385.304
Rate parameters:  A-C: 0.39693  A-G: 1.57493  A-T: 1.00000  C-G: 0.39693  C-T: 1.57493  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.403) (0.277,2.560)
Parameters optimization took 1 rounds (0.004 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000849009 secs using 96.82% CPU
Computing ML distances took 0.000980 sec (of wall-clock time) 0.000891 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.4121e-05 secs using 88.69% CPU
Constructing RapidNJ tree: done in 0.00013113 secs using 134.2% CPU
Computing RapidNJ tree took 0.000278 sec (of wall-clock time) 0.000308 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.836
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.111 second
Computing log-likelihood of 98 initial trees ... 0.102 seconds
Current best score: -1385.304

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00284696 secs using 99.26% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.303
Optimizing NNI: done in 0.013592 secs using 98.98% CPU
Optimizing NNI: done in 0.014385 secs using 98.56% CPU
Optimizing NNI: done in 0.0141699 secs using 99.32% CPU
Optimizing NNI: done in 0.013526 secs using 98.6% CPU
Optimizing NNI: done in 0.0195889 secs using 98.74% CPU
Optimizing NNI: done in 0.013073 secs using 99.02% CPU
Optimizing NNI: done in 0.01352 secs using 98.31% CPU
Optimizing NNI: done in 0.0148649 secs using 98.36% CPU
Optimizing NNI: done in 0.014097 secs using 98.63% CPU
Iteration 10 / LogL: -1385.338 / Time: 0h:0m:0s
Optimizing NNI: done in 0.014029 secs using 98.81% CPU
Optimizing NNI: done in 0.013206 secs using 99.09% CPU
Optimizing NNI: done in 0.012912 secs using 99.24% CPU
Optimizing NNI: done in 0.0122399 secs using 98.33% CPU
Optimizing NNI: done in 0.0130279 secs using 98.9% CPU
Optimizing NNI: done in 0.0127461 secs using 99.19% CPU
Optimizing NNI: done in 0.0132172 secs using 99.28% CPU
Optimizing NNI: done in 0.01316 secs using 99.03% CPU
Optimizing NNI: done in 0.0113862 secs using 98.71% CPU
Optimizing NNI: done in 0.0135431 secs using 98.76% CPU
Iteration 20 / LogL: -1385.339 / Time: 0h:0m:0s
Finish initializing candidate tree set (3)
Current best tree score: -1385.303 / CPU time: 0.489
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.014503 secs using 98.66% CPU
Optimizing NNI: done in 0.0297918 secs using 98.55% CPU
Optimizing NNI: done in 0.0200369 secs using 99% CPU
Optimizing NNI: done in 0.020638 secs using 98.64% CPU
Optimizing NNI: done in 0.0127709 secs using 99.06% CPU
Optimizing NNI: done in 0.0121748 secs using 98.88% CPU
Optimizing NNI: done in 0.00877595 secs using 98.78% CPU
Optimizing NNI: done in 0.0115311 secs using 99.26% CPU
Optimizing NNI: done in 0.0256569 secs using 98.79% CPU
Optimizing NNI: done in 0.0196528 secs using 99.52% CPU
Iteration 30 / LogL: -1385.707 / Time: 0h:0m:0s (0h:0m:1s left)
Optimizing NNI: done in 0.012574 secs using 99.17% CPU
Optimizing NNI: done in 0.0176771 secs using 98.9% CPU
Optimizing NNI: done in 0.0153069 secs using 98.98% CPU
Optimizing NNI: done in 0.021528 secs using 98.71% CPU
Optimizing NNI: done in 0.016993 secs using 99.23% CPU
Optimizing NNI: done in 0.0242 secs using 99% CPU
Optimizing NNI: done in 0.012531 secs using 99.03% CPU
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Optimizing NNI: done in 0.0265892 secs using 99.05% CPU
Optimizing NNI: done in 0.0125179 secs using 99.34% CPU
Iteration 40 / LogL: -1385.881 / Time: 0h:0m:0s (0h:0m:1s left)
Optimizing NNI: done in 0.0134759 secs using 98.81% CPU
Optimizing NNI: done in 0.0208821 secs using 98.85% CPU
Optimizing NNI: done in 0.018235 secs using 98.74% CPU
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Optimizing NNI: done in 0.0215828 secs using 99.18% CPU
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Optimizing NNI: done in 0.019454 secs using 99.03% CPU
Optimizing NNI: done in 0.017009 secs using 98.79% CPU
Optimizing NNI: done in 0.0153179 secs using 98.82% CPU
Iteration 50 / LogL: -1385.975 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.01582 secs using 98.74% CPU
Optimizing NNI: done in 0.018393 secs using 98.74% CPU
Optimizing NNI: done in 0.013016 secs using 98.53% CPU
Optimizing NNI: done in 0.0196841 secs using 98.91% CPU
Optimizing NNI: done in 0.016021 secs using 98.99% CPU
Optimizing NNI: done in 0.0246501 secs using 99.27% CPU
Optimizing NNI: done in 0.0179009 secs using 99.02% CPU
Optimizing NNI: done in 0.0213251 secs using 98.92% CPU
Optimizing NNI: done in 0.0145299 secs using 99.17% CPU
Optimizing NNI: done in 0.0148978 secs using 99.3% CPU
Iteration 60 / LogL: -1385.891 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.013443 secs using 99% CPU
Optimizing NNI: done in 0.0231791 secs using 98.95% CPU
Optimizing NNI: done in 0.017554 secs using 99.24% CPU
Optimizing NNI: done in 0.0294929 secs using 98.43% CPU
Optimizing NNI: done in 0.0218899 secs using 98.86% CPU
Optimizing NNI: done in 0.0142782 secs using 98.79% CPU
Optimizing NNI: done in 0.022656 secs using 98.84% CPU
Optimizing NNI: done in 0.00497293 secs using 99.08% CPU
Optimizing NNI: done in 0.016506 secs using 98.97% CPU
Optimizing NNI: done in 0.011802 secs using 99.19% CPU
Iteration 70 / LogL: -1385.304 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0205591 secs using 98.85% CPU
Optimizing NNI: done in 0.0143371 secs using 99.27% CPU
Optimizing NNI: done in 0.0216689 secs using 97.79% CPU
Optimizing NNI: done in 0.0105031 secs using 98.95% CPU
Optimizing NNI: done in 0.0170259 secs using 98.42% CPU
Optimizing NNI: done in 0.0164559 secs using 98.7% CPU
Optimizing NNI: done in 0.015245 secs using 98.83% CPU
Optimizing NNI: done in 0.0215039 secs using 98.36% CPU
Optimizing NNI: done in 0.022331 secs using 98.98% CPU
Optimizing NNI: done in 0.015111 secs using 99.94% CPU
Iteration 80 / LogL: -1394.218 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0123539 secs using 99.95% CPU
Optimizing NNI: done in 0.0138621 secs using 99.93% CPU
Optimizing NNI: done in 0.0141599 secs using 99.94% CPU
Optimizing NNI: done in 0.0150442 secs using 99.95% CPU
Optimizing NNI: done in 0.0109682 secs using 99.94% CPU
Optimizing NNI: done in 0.0143139 secs using 99.94% CPU
Optimizing NNI: done in 0.0152819 secs using 99.95% CPU
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Optimizing NNI: done in 0.0124531 secs using 99.94% CPU
Optimizing NNI: done in 0.0210421 secs using 99.95% CPU
Iteration 90 / LogL: -1385.859 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.011955 secs using 99.94% CPU
Optimizing NNI: done in 0.0209742 secs using 99.94% CPU
Optimizing NNI: done in 0.015192 secs using 99.95% CPU
Optimizing NNI: done in 0.017307 secs using 99.95% CPU
Optimizing NNI: done in 0.0111351 secs using 99.95% CPU
Optimizing NNI: done in 0.0173252 secs using 99.95% CPU
Optimizing NNI: done in 0.0124321 secs using 99.95% CPU
Optimizing NNI: done in 0.016021 secs using 99.94% CPU
Optimizing NNI: done in 0.0207522 secs using 99.95% CPU
Optimizing NNI: done in 0.013715 secs using 100% CPU
Iteration 100 / LogL: -1385.306 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0106652 secs using 99.93% CPU
Optimizing NNI: done in 0.0122459 secs using 99.95% CPU
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.303
Optimal log-likelihood: -1385.303
Rate parameters:  A-C: 0.39652  A-G: 1.57285  A-T: 1.00000  C-G: 0.39652  C-T: 1.57285  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.402) (0.276,2.564)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1385.303
Total tree length: 6.855

Total number of iterations: 102
CPU time used for tree search: 2.087 sec (0h:0m:2s)
Wall-clock time used for tree search: 1.926 sec (0h:0m:1s)
Total CPU time used: 2.116 sec (0h:0m:2s)
Total wall-clock time used: 1.950 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/q2iqtree.treefile -m "TPM3{0.396517,1.57285}+FQ+R2{0.723516,0.402304,0.276484,2.56408}" --length 214

Date and Time: Wed Dec 21 19:10:08 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpi7orr6zj/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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/q2iqtree -nt 1
Seed:    573285 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:10:18 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000216961 secs using 84.81% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000457048 secs using 78.33% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.88351e-05 secs using 90.26% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 7.39098e-05 secs using 75.77% CPU

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, 1.287 / LogL: -1395.194
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.376 / LogL: -1395.464
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.390 / LogL: -1395.530
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.384 / LogL: -1395.523
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.375 / LogL: -1395.492
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.374 / LogL: -1395.529
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.369 / LogL: -1395.476
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.365 / LogL: -1395.493
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.373 / LogL: -1395.500
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.374 / LogL: -1395.511
Optimal pinv,alpha: 0.000, 1.287 / LogL: -1395.194

Parameters optimization took 0.512 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00124383 secs using 97.04% CPU
Computing ML distances took 0.001347 sec (of wall-clock time) 0.001276 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.38826e-05 secs using 90.94% CPU
Constructing RapidNJ tree: done in 9.39369e-05 secs using 150.1% CPU
Computing RapidNJ tree took 0.000217 sec (of wall-clock time) 0.000254 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.836
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.108 second
Computing log-likelihood of 98 initial trees ... 0.131 seconds
Current best score: -1392.836

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.0193992 secs using 98.9% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.260
Optimizing NNI: done in 0.0269868 secs using 98.99% CPU
Optimizing NNI: done in 0.0143731 secs using 99.07% CPU
Optimizing NNI: done in 0.0148611 secs using 98.77% CPU
Optimizing NNI: done in 0.014185 secs using 99.29% CPU
Optimizing NNI: done in 0.015137 secs using 98.67% CPU
Optimizing NNI: done in 0.0144601 secs using 98.96% CPU
Optimizing NNI: done in 0.0140481 secs using 99.69% CPU
Optimizing NNI: done in 0.0144138 secs using 98.74% CPU
Optimizing NNI: done in 0.014266 secs using 99.12% CPU
Iteration 10 / LogL: -1387.730 / Time: 0h:0m:0s
Optimizing NNI: done in 0.0264869 secs using 98.61% CPU
Optimizing NNI: done in 0.0255589 secs using 99.63% CPU
Optimizing NNI: done in 0.026654 secs using 98.78% CPU
Optimizing NNI: done in 0.0260201 secs using 99.22% CPU
Optimizing NNI: done in 0.0259199 secs using 99.16% CPU
Optimizing NNI: done in 0.0264139 secs using 98.92% CPU
Optimizing NNI: done in 0.0264399 secs using 98.84% CPU
Optimizing NNI: done in 0.0264721 secs using 99.27% CPU
Optimizing NNI: done in 0.0260601 secs using 99.28% CPU
Optimizing NNI: done in 0.0264618 secs using 98.82% CPU
Iteration 20 / LogL: -1387.280 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.260 / CPU time: 0.685
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.020699 secs using 99.08% CPU
Optimizing NNI: done in 0.0190799 secs using 98.93% CPU
Optimizing NNI: done in 0.0688949 secs using 98.92% CPU
Optimizing NNI: done in 0.0332582 secs using 98.97% CPU
Optimizing NNI: done in 0.0331481 secs using 98.69% CPU
Optimizing NNI: done in 0.0274601 secs using 99.26% CPU
Optimizing NNI: done in 0.0310991 secs using 98.98% CPU
Optimizing NNI: done in 0.025357 secs using 99% CPU
Optimizing NNI: done in 0.0246849 secs using 98.97% CPU
Optimizing NNI: done in 0.038347 secs using 98.63% CPU
Iteration 30 / LogL: -1387.401 / Time: 0h:0m:1s (0h:0m:3s left)
Optimizing NNI: done in 0.0365891 secs using 99.06% CPU
Optimizing NNI: done in 0.0378742 secs using 98.37% CPU
Optimizing NNI: done in 0.0454102 secs using 98.55% CPU
Optimizing NNI: done in 0.033551 secs using 98.97% CPU
Optimizing NNI: done in 0.023309 secs using 99.05% CPU
Optimizing NNI: done in 0.020159 secs using 98.44% CPU
Optimizing NNI: done in 0.019711 secs using 99.47% CPU
Optimizing NNI: done in 0.0362921 secs using 98.99% CPU
Optimizing NNI: done in 0.026089 secs using 98.95% CPU
Optimizing NNI: done in 0.0155919 secs using 98.78% CPU
Iteration 40 / LogL: -1387.285 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.0342271 secs using 98.69% CPU
Optimizing NNI: done in 0.0346389 secs using 98.3% CPU
Optimizing NNI: done in 0.025681 secs using 97.6% CPU
Optimizing NNI: done in 0.051019 secs using 98.45% CPU
Optimizing NNI: done in 0.021888 secs using 99.12% CPU
Optimizing NNI: done in 0.0222442 secs using 98.92% CPU
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Optimizing NNI: done in 0.02038 secs using 98.12% CPU
Optimizing NNI: done in 0.01741 secs using 98.9% CPU
Optimizing NNI: done in 0.0267589 secs using 99.13% CPU
Iteration 50 / LogL: -1387.381 / Time: 0h:0m:2s (0h:0m:2s left)
Optimizing NNI: done in 0.0188189 secs using 98.88% CPU
Optimizing NNI: done in 0.0217819 secs using 98.3% CPU
Optimizing NNI: done in 0.0364749 secs using 94.74% CPU
Optimizing NNI: done in 0.0254118 secs using 98.93% CPU
Optimizing NNI: done in 0.028791 secs using 99.1% CPU
Optimizing NNI: done in 0.025671 secs using 98.89% CPU
Optimizing NNI: done in 0.0294602 secs using 99.1% CPU
Optimizing NNI: done in 0.0203559 secs using 99.22% CPU
Optimizing NNI: done in 0.0450871 secs using 98.74% CPU
Optimizing NNI: done in 0.012131 secs using 99.18% CPU
Iteration 60 / LogL: -1387.379 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0192721 secs using 99.51% CPU
Optimizing NNI: done in 0.0299778 secs using 98.88% CPU
Optimizing NNI: done in 0.0222878 secs using 99.13% CPU
Optimizing NNI: done in 0.0153949 secs using 98.94% CPU
Optimizing NNI: done in 0.0285208 secs using 98.88% CPU
Optimizing NNI: done in 0.037766 secs using 98.82% CPU
Optimizing NNI: done in 0.0294759 secs using 98.95% CPU
Optimizing NNI: done in 0.0146322 secs using 98.37% CPU
Optimizing NNI: done in 0.0246329 secs using 99% CPU
Optimizing NNI: done in 0.0341299 secs using 98.96% CPU
Iteration 70 / LogL: -1387.382 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0341921 secs using 99.06% CPU
Optimizing NNI: done in 0.0244648 secs using 99.05% CPU
Optimizing NNI: done in 0.0224729 secs using 98.79% CPU
Optimizing NNI: done in 0.0493729 secs using 98.98% CPU
Optimizing NNI: done in 0.03473 secs using 98.76% CPU
Optimizing NNI: done in 0.0285029 secs using 98.95% CPU
Optimizing NNI: done in 0.031065 secs using 99.2% CPU
Optimizing NNI: done in 0.0512631 secs using 98.89% CPU
Optimizing NNI: done in 0.0308499 secs using 99.02% CPU
Optimizing NNI: done in 0.018234 secs using 98.95% CPU
Iteration 80 / LogL: -1387.500 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0230439 secs using 99.01% CPU
Optimizing NNI: done in 0.0366771 secs using 99.29% CPU
Optimizing NNI: done in 0.030189 secs using 98.71% CPU
Optimizing NNI: done in 0.0343542 secs using 98.82% CPU
Optimizing NNI: done in 0.0111558 secs using 98.96% CPU
Optimizing NNI: done in 0.0328109 secs using 98.81% CPU
Optimizing NNI: done in 0.030463 secs using 99.07% CPU
Optimizing NNI: done in 0.0199931 secs using 99.96% CPU
Optimizing NNI: done in 0.0128419 secs using 99.96% CPU
Optimizing NNI: done in 0.0298619 secs using 99.97% CPU
Iteration 90 / LogL: -1387.368 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0439639 secs using 99.95% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.039299 secs using 99.95% CPU
Optimizing NNI: done in 0.0388188 secs using 99.96% CPU
Optimizing NNI: done in 0.0148909 secs using 99.95% CPU
Optimizing NNI: done in 0.017261 secs using 99.96% CPU
Optimizing NNI: done in 0.0425701 secs using 99.96% CPU
Optimizing NNI: done in 0.0347259 secs using 99.95% CPU
Optimizing NNI: done in 0.0462651 secs using 99.82% CPU
Optimizing NNI: done in 0.0417581 secs using 99.97% CPU
Optimizing NNI: done in 0.0309339 secs using 99.96% CPU
Iteration 100 / LogL: -1387.354 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.027797 secs using 99.96% CPU
Optimizing NNI: done in 0.0373781 secs using 99.79% CPU
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: -1387.260
Optimal log-likelihood: -1387.255
Rate parameters:  A-C: 0.32423  A-G: 2.24117  A-T: 2.12015  C-G: 1.16411  C-T: 3.24849  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: 1.327
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.255
Total tree length: 6.712

Total number of iterations: 102
CPU time used for tree search: 3.318 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.298 sec (0h:0m:3s)
Total CPU time used: 3.848 sec (0h:0m:3s)
Total wall-clock time used: 3.833 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/q2iqtree.treefile -m "GTR{0.324234,2.24117,2.12015,1.16411,3.24849}+F{0.243216,0.181967,0.319196,0.255621}+I{3.33554e-07}+G4{1.32696}" --length 214

Date and Time: Wed Dec 21 19:10:22 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp51h1cwmz/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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree -nt 1 -fast
Seed:    156709 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:10:32 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000256062 secs using 79.67% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000467062 secs using 76.86% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Analyzing sequences: done in 1.88351e-05 secs using 90.26% CPU
Checking for duplicate sequences: done in 7.39098e-05 secs using 75.77% CPU

---> START RUN NUMBER 1 (seed: 156709)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.50)
1. Initial log-likelihood: -1492.39
2. Current log-likelihood: -1401.89
3. Current log-likelihood: -1396.53
4. Current log-likelihood: -1395.12
5. Current log-likelihood: -1394.39
Optimal log-likelihood: -1393.81
Rate parameters:  A-C: 0.27026  A-G: 2.39526  A-T: 2.16931  C-G: 1.24752  C-T: 3.29290  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: 1.415
Parameters optimization took 5 rounds (0.039 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00121808 secs using 98.76% CPU
Computing ML distances took 0.001313 sec (of wall-clock time) 0.001265 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.48363e-05 secs using 114.9% CPU
Constructing RapidNJ tree: done in 9.29832e-05 secs using 151.6% CPU
Computing RapidNJ tree took 0.000218 sec (of wall-clock time) 0.000266 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.793
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0212669 secs using 191% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.213
Optimizing NNI: done in 0.02057 secs using 192.2% CPU
Finish initializing candidate tree set (3)
Current best tree score: -1388.213 / CPU time: 0.051
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: -1388.213
2. Current log-likelihood: -1388.014
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.759
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35532  A-G: 2.35213  A-T: 2.13937  C-G: 1.20295  C-T: 3.37020  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: 1.361
Parameters optimization took 7 rounds (0.026 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.816

Total number of iterations: 2
CPU time used for tree search: 0.097 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.051 sec (0h:0m:0s)
Total CPU time used: 0.207 sec (0h:0m:0s)
Total wall-clock time used: 0.134 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 157709)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.097
2. Current log-likelihood: -1401.816
3. Current log-likelihood: -1396.523
4. Current log-likelihood: -1395.122
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.818
Rate parameters:  A-C: 0.27163  A-G: 2.41073  A-T: 2.17144  C-G: 1.24911  C-T: 3.27679  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.416
Parameters optimization took 5 rounds (0.041 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00129604 secs using 192.3% CPU
Computing ML distances took 0.001415 sec (of wall-clock time) 0.002682 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 4.60148e-05 secs using 82.58% CPU
Constructing RapidNJ tree: done in 9.20296e-05 secs using 131.5% CPU
Computing RapidNJ tree took 0.000213 sec (of wall-clock time) 0.000210 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.794
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.020205 secs using 191.8% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.214
Optimizing NNI: done in 0.020211 secs using 192.4% CPU
Finish initializing candidate tree set (3)
Current best tree score: -1388.214 / CPU time: 0.049
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.214
2. Current log-likelihood: -1388.015
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.760
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35522  A-G: 2.35151  A-T: 2.13874  C-G: 1.20261  C-T: 3.36909  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.362
Parameters optimization took 7 rounds (0.026 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.094 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.049 sec (0h:0m:0s)
Total CPU time used: 0.252 sec (0h:0m:0s)
Total wall-clock time used: 0.133 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 158709)

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 (0.041 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00141191 secs using 185.8% CPU
Computing ML distances took 0.001493 sec (of wall-clock time) 0.002762 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 6.79493e-05 secs using 88.3% CPU
Constructing RapidNJ tree: done in 0.000130892 secs using 131.4% CPU
Computing RapidNJ tree took 0.000296 sec (of wall-clock time) 0.000308 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.173
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.020499 secs using 191.5% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Optimizing NNI: done in 0.0487859 secs using 191.7% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / CPU time: 0.086
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.961
2. Current log-likelihood: -1387.803
3. Current log-likelihood: -1387.684
4. Current log-likelihood: -1387.593
5. Current log-likelihood: -1387.523
6. Current log-likelihood: -1387.468
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.33414  A-G: 2.26635  A-T: 2.14117  C-G: 1.17550  C-T: 3.28158  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.353
Parameters optimization took 6 rounds (0.022 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.743

Total number of iterations: 2
CPU time used for tree search: 0.166 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.087 sec (0h:0m:0s)
Total CPU time used: 0.318 sec (0h:0m:0s)
Total wall-clock time used: 0.167 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 159709)

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: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28732  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.040 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00127888 secs using 194.5% CPU
Computing ML distances took 0.001369 sec (of wall-clock time) 0.002625 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 6.29425e-05 secs using 73.08% CPU
Constructing RapidNJ tree: done in 0.000100136 secs using 141.8% CPU
Computing RapidNJ tree took 0.000231 sec (of wall-clock time) 0.000247 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0214698 secs using 191.5% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.033468 secs using 192% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.066
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.026 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.126 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.066 sec (0h:0m:0s)
Total CPU time used: 0.283 sec (0h:0m:0s)
Total wall-clock time used: 0.149 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 160709)

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: -1493.550
2. Current log-likelihood: -1403.090
3. Current log-likelihood: -1398.358
4. Current log-likelihood: -1396.975
5. Current log-likelihood: -1396.257
Optimal log-likelihood: -1395.748
Rate parameters:  A-C: 0.23785  A-G: 2.06889  A-T: 1.95307  C-G: 1.06292  C-T: 2.77329  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: 1.358
Parameters optimization took 5 rounds (0.041 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00160384 secs using 193.1% CPU
Computing ML distances took 0.001708 sec (of wall-clock time) 0.003275 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 4.79221e-05 secs using 81.38% CPU
Constructing RapidNJ tree: done in 9.17912e-05 secs using 136.2% CPU
Computing RapidNJ tree took 0.000222 sec (of wall-clock time) 0.000215 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.197
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0201702 secs using 192.4% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Optimizing NNI: done in 0.050328 secs using 191.9% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / 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: -1387.961
2. Current log-likelihood: -1387.803
3. Current log-likelihood: -1387.684
4. Current log-likelihood: -1387.593
5. Current log-likelihood: -1387.523
6. Current log-likelihood: -1387.469
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.33442  A-G: 2.26003  A-T: 2.13495  C-G: 1.17227  C-T: 3.27212  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.353
Parameters optimization took 6 rounds (0.022 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.742

Total number of iterations: 2
CPU time used for tree search: 0.168 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.088 sec (0h:0m:0s)
Total CPU time used: 0.323 sec (0h:0m:0s)
Total wall-clock time used: 0.170 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 161709)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.039 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00130701 secs using 189.6% CPU
Computing ML distances took 0.001392 sec (of wall-clock time) 0.002620 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 5.10216e-05 secs using 82.32% CPU
Constructing RapidNJ tree: done in 8.98838e-05 secs using 146.9% CPU
Computing RapidNJ tree took 0.000222 sec (of wall-clock time) 0.000226 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.184
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.023751 secs using 192.1% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.955
Optimizing NNI: done in 0.0125101 secs using 191.7% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.955 / CPU time: 0.053
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.955
2. Current log-likelihood: -1387.798
3. Current log-likelihood: -1387.680
4. Current log-likelihood: -1387.590
5. Current log-likelihood: -1387.521
6. Current log-likelihood: -1387.467
Optimal log-likelihood: -1387.423
Rate parameters:  A-C: 0.33566  A-G: 2.27095  A-T: 2.14605  C-G: 1.17829  C-T: 3.29012  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.352
Parameters optimization took 6 rounds (0.022 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.744

Total number of iterations: 2
CPU time used for tree search: 0.101 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.053 sec (0h:0m:0s)
Total CPU time used: 0.250 sec (0h:0m:0s)
Total wall-clock time used: 0.131 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 162709)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1491.633
2. Current log-likelihood: -1402.007
3. Current log-likelihood: -1396.792
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.654
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.28077  A-G: 2.37447  A-T: 2.10134  C-G: 1.20130  C-T: 3.28121  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.039 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00129986 secs using 193.1% CPU
Computing ML distances took 0.001382 sec (of wall-clock time) 0.002640 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 4.69685e-05 secs using 80.91% CPU
Constructing RapidNJ tree: done in 9.17912e-05 secs using 126.4% CPU
Computing RapidNJ tree took 0.000200 sec (of wall-clock time) 0.000214 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.810
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.020407 secs using 192.1% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.033215 secs using 191.5% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.064
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36985  A-G: 2.31003  A-T: 2.11728  C-G: 1.22260  C-T: 3.27851  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.026 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.123 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.064 sec (0h:0m:0s)
Total CPU time used: 0.278 sec (0h:0m:0s)
Total wall-clock time used: 0.146 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 163709)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.097
2. Current log-likelihood: -1401.816
3. Current log-likelihood: -1396.523
4. Current log-likelihood: -1395.122
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.818
Rate parameters:  A-C: 0.27163  A-G: 2.41073  A-T: 2.17144  C-G: 1.24911  C-T: 3.27679  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.416
Parameters optimization took 5 rounds (0.043 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00132704 secs using 188.9% CPU
Computing ML distances took 0.001410 sec (of wall-clock time) 0.002637 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 4.79221e-05 secs using 81.38% CPU
Constructing RapidNJ tree: done in 0.000126123 secs using 121.3% CPU
Computing RapidNJ tree took 0.000255 sec (of wall-clock time) 0.000276 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.794
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0207231 secs using 191.7% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.214
Optimizing NNI: done in 0.020632 secs using 191.7% CPU
Finish initializing candidate tree set (3)
Current best tree score: -1388.214 / CPU time: 0.050
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.214
2. Current log-likelihood: -1388.015
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.760
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35522  A-G: 2.35151  A-T: 2.13874  C-G: 1.20261  C-T: 3.36909  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.362
Parameters optimization took 7 rounds (0.026 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.096 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.050 sec (0h:0m:0s)
Total CPU time used: 0.259 sec (0h:0m:0s)
Total wall-clock time used: 0.136 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 164709)

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: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20301  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.039 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00129104 secs using 191.1% CPU
Computing ML distances took 0.001377 sec (of wall-clock time) 0.002598 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 7.00951e-05 secs using 72.76% CPU
Constructing RapidNJ tree: done in 0.000118017 secs using 127.1% CPU
Computing RapidNJ tree took 0.000267 sec (of wall-clock time) 0.000261 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.020077 secs using 191.6% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.031868 secs using 192.5% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.062
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.026 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.120 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.063 sec (0h:0m:0s)
Total CPU time used: 0.276 sec (0h:0m:0s)
Total wall-clock time used: 0.145 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 165709)

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: -1492.097
2. Current log-likelihood: -1401.816
3. Current log-likelihood: -1396.523
4. Current log-likelihood: -1395.122
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.818
Rate parameters:  A-C: 0.27163  A-G: 2.41073  A-T: 2.17144  C-G: 1.24911  C-T: 3.27679  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.416
Parameters optimization took 5 rounds (0.042 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00128388 secs using 192.9% CPU
Computing ML distances took 0.001364 sec (of wall-clock time) 0.002596 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 4.81606e-05 secs using 80.98% CPU
Constructing RapidNJ tree: done in 9.10759e-05 secs using 119.7% CPU
Computing RapidNJ tree took 0.000213 sec (of wall-clock time) 0.000195 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.794
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0204771 secs using 191.9% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.214
Optimizing NNI: done in 0.020587 secs using 191.6% CPU
Finish initializing candidate tree set (3)
Current best tree score: -1388.214 / CPU time: 0.050
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.214
2. Current log-likelihood: -1388.015
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.760
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35522  A-G: 2.35151  A-T: 2.13874  C-G: 1.20261  C-T: 3.36909  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.362
Parameters optimization took 7 rounds (0.026 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.095 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.050 sec (0h:0m:0s)
Total CPU time used: 0.255 sec (0h:0m:0s)
Total wall-clock time used: 0.135 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 6 gave best log-likelihood: -1387.423
Total CPU time for 10 runs: 2.736 seconds.
Total wall-clock time for 10 runs: 1.469 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/q2iqtree.treefile -m "GTR{0.337156,2.28043,2.14506,1.18328,3.29428}+F{0.243216,0.181967,0.319196,0.255621}+I{0.0160099}+G4{1.38246}" --length 214

Date and Time: Wed Dec 21 19:10:33 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiv_8ye03/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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    387324 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:10:43 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000231981 secs using 83.2% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000461102 secs using 78.29% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 2.59876e-05 secs using 88.5% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 7.10487e-05 secs using 76% CPU

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, 1.285 / LogL: -1392.552
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.377 / LogL: -1392.829
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.392 / LogL: -1392.898
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.386 / LogL: -1392.889
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.380 / LogL: -1392.852
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.385 / LogL: -1392.879
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.007, 1.381 / LogL: -1392.829
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.382 / LogL: -1392.844
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.385 / LogL: -1392.849
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.384 / LogL: -1392.858
Optimal pinv,alpha: 0.000, 1.285 / LogL: -1392.552

Parameters optimization took 0.493 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00130296 secs using 97.78% CPU
Computing ML distances took 0.001432 sec (of wall-clock time) 0.001361 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.48363e-05 secs using 87.53% CPU
Constructing RapidNJ tree: done in 9.20296e-05 secs using 152.1% CPU
Computing RapidNJ tree took 0.000223 sec (of wall-clock time) 0.000243 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.708
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.109 second
Computing log-likelihood of 98 initial trees ... 0.133 seconds
Current best score: -1392.552

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.012928 secs using 98.62% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.258
Optimizing NNI: done in 0.0216098 secs using 99.44% CPU
Optimizing NNI: done in 0.0142038 secs using 99.04% CPU
Optimizing NNI: done in 0.0142848 secs using 99.09% CPU
Optimizing NNI: done in 0.0145421 secs using 98.78% CPU
Optimizing NNI: done in 0.0144131 secs using 98.97% CPU
Optimizing NNI: done in 0.0144498 secs using 99.04% CPU
Optimizing NNI: done in 0.0263011 secs using 98.96% CPU
Optimizing NNI: done in 0.0278041 secs using 98.25% CPU
Optimizing NNI: done in 0.026782 secs using 98.85% CPU
Iteration 10 / LogL: -1387.279 / Time: 0h:0m:0s
Optimizing NNI: done in 0.026495 secs using 99.07% CPU
Optimizing NNI: done in 0.026603 secs using 99.14% CPU
Optimizing NNI: done in 0.0264981 secs using 99.17% CPU
Optimizing NNI: done in 0.026814 secs using 98.53% CPU
Optimizing NNI: done in 0.0262771 secs using 99.17% CPU
Optimizing NNI: done in 0.0259631 secs using 99.15% CPU
Optimizing NNI: done in 0.0264471 secs using 98.75% CPU
Optimizing NNI: done in 0.0277469 secs using 98.06% CPU
Optimizing NNI: done in 0.026958 secs using 98.82% CPU
Optimizing NNI: done in 0.026134 secs using 99.15% CPU
Iteration 20 / LogL: -1387.279 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.258 / CPU time: 0.717
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.0468829 secs using 98.78% CPU
Optimizing NNI: done in 0.020052 secs using 98.62% CPU
Optimizing NNI: done in 0.014879 secs using 98.77% CPU
Optimizing NNI: done in 0.0323348 secs using 98.74% CPU
Optimizing NNI: done in 0.0242541 secs using 98.7% CPU
Optimizing NNI: done in 0.037302 secs using 98.55% CPU
Optimizing NNI: done in 0.0278091 secs using 99.09% CPU
Optimizing NNI: done in 0.045341 secs using 98.98% CPU
Optimizing NNI: done in 0.0348492 secs using 99.01% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Optimizing NNI: done in 0.028878 secs using 99.02% CPU
Iteration 30 / LogL: -1387.648 / Time: 0h:0m:1s (0h:0m:3s left)
Optimizing NNI: done in 0.0258901 secs using 98.97% CPU
Optimizing NNI: done in 0.0269511 secs using 98.75% CPU
Optimizing NNI: done in 0.0304101 secs using 98.4% CPU
Optimizing NNI: done in 0.03754 secs using 99.08% CPU
Optimizing NNI: done in 0.0410469 secs using 99.04% CPU
Optimizing NNI: done in 0.0276439 secs using 99.01% CPU
Optimizing NNI: done in 0.0322771 secs using 98.74% CPU
Optimizing NNI: done in 0.0225291 secs using 99.04% CPU
Optimizing NNI: done in 0.029856 secs using 99.09% CPU
Optimizing NNI: done in 0.0378909 secs using 99.12% CPU
Iteration 40 / LogL: -1387.352 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.0177629 secs using 98.21% CPU
Optimizing NNI: done in 0.04755 secs using 99.07% CPU
Optimizing NNI: done in 0.020016 secs using 98.8% CPU
Optimizing NNI: done in 0.0391841 secs using 98.92% CPU
Optimizing NNI: done in 0.027298 secs using 98.97% CPU
Optimizing NNI: done in 0.0277131 secs using 99.18% CPU
Optimizing NNI: done in 0.0277779 secs using 99.18% CPU
Optimizing NNI: done in 0.0332351 secs using 98.58% CPU
Optimizing NNI: done in 0.0274451 secs using 98.79% CPU
Optimizing NNI: done in 0.0695369 secs using 98.89% CPU
Iteration 50 / LogL: -1387.431 / Time: 0h:0m:2s (0h:0m:2s left)
Optimizing NNI: done in 0.0318041 secs using 98.69% CPU
Optimizing NNI: done in 0.0278411 secs using 99.08% CPU
Optimizing NNI: done in 0.033685 secs using 98.97% CPU
Optimizing NNI: done in 0.0325611 secs using 98.86% CPU
Optimizing NNI: done in 0.0228798 secs using 96.86% CPU
Optimizing NNI: done in 0.021359 secs using 99.23% CPU
Optimizing NNI: done in 0.026082 secs using 98.95% CPU
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Iteration 60 / LogL: -1387.350 / Time: 0h:0m:2s (0h:0m:1s left)
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Optimizing NNI: done in 0.0479739 secs using 98.87% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 68: -1387.169
Optimizing NNI: done in 0.0551591 secs using 98.82% CPU
Optimizing NNI: done in 0.0199511 secs using 98.43% CPU
Iteration 70 / LogL: -1387.446 / Time: 0h:0m:2s (0h:0m:4s left)
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Iteration 80 / LogL: -1387.337 / Time: 0h:0m:3s (0h:0m:3s left)
Optimizing NNI: done in 0.032444 secs using 99.74% CPU
Optimizing NNI: done in 0.040951 secs using 99.97% CPU
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Iteration 90 / LogL: -1387.337 / Time: 0h:0m:3s (0h:0m:3s left)
Optimizing NNI: done in 0.0496681 secs using 99.84% CPU
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Optimizing NNI: done in 0.027688 secs using 99.93% CPU
Optimizing NNI: done in 0.025466 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Optimizing NNI: done in 0.03438 secs using 99.72% CPU
Optimizing NNI: done in 0.027375 secs using 99.96% CPU
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Iteration 100 / LogL: -1397.858 / Time: 0h:0m:3s (0h:0m:2s left)
Optimizing NNI: done in 0.0313139 secs using 99.73% CPU
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UPDATE BEST LOG-LIKELIHOOD: -1387.169
Optimizing NNI: done in 0.03231 secs using 99.85% CPU
Optimizing NNI: done in 0.040277 secs using 99.96% CPU
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Iteration 110 / LogL: -1387.397 / Time: 0h:0m:4s (0h:0m:2s left)
Optimizing NNI: done in 0.0440562 secs using 99.96% CPU
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Iteration 120 / LogL: -1387.337 / Time: 0h:0m:4s (0h:0m:1s left)
Optimizing NNI: done in 0.0238981 secs using 99.97% CPU
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Iteration 130 / LogL: -1387.170 / Time: 0h:0m:4s (0h:0m:1s left)
Optimizing NNI: done in 0.0198209 secs using 99.97% CPU
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Optimizing NNI: done in 0.0161369 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Optimizing NNI: done in 0.0408149 secs using 99.82% CPU
Iteration 140 / LogL: -1387.359 / Time: 0h:0m:5s (0h:0m:1s left)
Optimizing NNI: done in 0.030967 secs using 99.96% CPU
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Optimizing NNI: done in 0.0357411 secs using 99.95% CPU
Optimizing NNI: done in 0.014786 secs using 99.97% CPU
BETTER TREE FOUND at iteration 144: -1387.169
Optimizing NNI: done in 0.015275 secs using 99.7% CPU
Optimizing NNI: done in 0.0177472 secs using 99.95% CPU
Optimizing NNI: done in 0.047121 secs using 99.86% CPU
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Iteration 150 / LogL: -1387.169 / Time: 0h:0m:5s (0h:0m:3s left)
Optimizing NNI: done in 0.026103 secs using 99.97% CPU
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Iteration 160 / LogL: -1387.191 / Time: 0h:0m:5s (0h:0m:2s left)
Optimizing NNI: done in 0.016701 secs using 99.96% CPU
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Optimizing NNI: done in 0.028116 secs using 99.96% CPU
Optimizing NNI: done in 0.0205009 secs using 99.93% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Optimizing NNI: done in 0.026087 secs using 99.64% CPU
Optimizing NNI: done in 0.028451 secs using 99.88% CPU
Optimizing NNI: done in 0.0250258 secs using 99.97% CPU
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Iteration 170 / LogL: -1387.204 / Time: 0h:0m:5s (0h:0m:2s left)
Optimizing NNI: done in 0.0246289 secs using 99.97% CPU
Optimizing NNI: done in 0.0216072 secs using 99.8% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Optimizing NNI: done in 0.031595 secs using 99.84% CPU
Optimizing NNI: done in 0.0263269 secs using 99.97% CPU
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Iteration 180 / LogL: -1387.169 / Time: 0h:0m:6s (0h:0m:2s left)
Optimizing NNI: done in 0.016537 secs using 99.96% CPU
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Iteration 190 / LogL: -1387.169 / Time: 0h:0m:6s (0h:0m:1s left)
Optimizing NNI: done in 0.0251031 secs using 99.74% CPU
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Iteration 200 / LogL: -1387.175 / Time: 0h:0m:6s (0h:0m:1s left)
Optimizing NNI: done in 0.0228369 secs using 99.95% CPU
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Iteration 210 / LogL: -1387.314 / Time: 0h:0m:6s (0h:0m:1s left)
Optimizing NNI: done in 0.021395 secs using 99.96% CPU
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Iteration 220 / LogL: -1387.169 / Time: 0h:0m:7s (0h:0m:0s left)
Optimizing NNI: done in 0.018419 secs using 99.96% CPU
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Iteration 230 / LogL: -1387.169 / Time: 0h:0m:7s (0h:0m:0s left)
Optimizing NNI: done in 0.0362599 secs using 99.96% CPU
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Iteration 240 / LogL: -1387.169 / Time: 0h:0m:7s (0h:0m:0s left)
Optimizing NNI: done in 0.027554 secs using 99.97% CPU
Optimizing NNI: done in 0.0194011 secs using 99.96% CPU
Optimizing NNI: done in 0.032702 secs using 99.96% CPU
Optimizing NNI: done in 0.0193961 secs using 99.97% CPU
Optimizing NNI: done in 0.020411 secs using 99.97% CPU
TREE SEARCH COMPLETED AFTER 245 ITERATIONS / Time: 0h:0m:7s

--------------------------------------------------------------------
|                    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: 0.34679  A-G: 2.32660  A-T: 2.14696  C-G: 1.23684  C-T: 3.22330  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: 1.286
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...
0.067 sec.
Total tree length: 7.597

Total number of iterations: 245
CPU time used for tree search: 7.457 sec (0h:0m:7s)
Wall-clock time used for tree search: 7.323 sec (0h:0m:7s)
Total CPU time used: 8.035 sec (0h:0m:8s)
Total wall-clock time used: 7.905 sec (0h:0m:7s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/q2iqtree.treefile -m "GTR{0.346793,2.3266,2.14696,1.23684,3.2233}+F{0.243216,0.181967,0.319196,0.255621}+I{2.86927e-07}+G4{1.2863}" --length 214

Date and Time: Wed Dec 21 19:10:51 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpx3_xjbco/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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    107350 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:11:01 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000250101 secs using 85.97% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000509024 secs using 74.26% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 2.19345e-05 secs using 82.06% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 7.08103e-05 secs using 74.85% CPU


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...
Optimizing NNI: done in 0.0190251 secs using 98.86% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.711
Optimal log-likelihood: -1388.843
Rate parameters:  A-C: 0.36734  A-G: 2.36723  A-T: 2.14911  C-G: 1.23876  C-T: 3.32308  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.420
Parameters optimization took 1 rounds (0.009 sec)
Time for fast ML tree search: 0.057 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.573     45  2895.147     2919.790     3046.616
  2  GTR+F+I       1401.119     46  2894.238     2920.130     3049.072
  3  GTR+F+G4      1387.362     46  2866.724     2892.616     3021.559
  4  GTR+F+I+G4    1387.729     47  2869.458     2896.638     3027.658
  5  GTR+F+R2      1382.368     47  2858.736     2885.917     3016.937
  6  GTR+F+R3      1382.422     49  2862.845     2892.723     3027.778
 14  GTR+F+I+I+R2  1382.420     48  2860.840     2889.349     3022.406
 15  GTR+F+I+I+R3  1382.451     50  2864.902     2896.191     3033.201
 25  SYM+G4        1387.133     43  2860.266     2882.525     3005.003
 27  SYM+R2        1383.095     44  2854.190     2877.621     3002.292
 36  SYM+I+I+R2    1383.227     45  2856.454     2881.097     3007.923
 47  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 49  TVM+F+R2      1383.790     46  2859.580     2885.472     3014.415
 58  TVM+F+I+I+R2  1383.813     47  2861.626     2888.806     3019.826
 69  TVMe+G4       1387.121     42  2858.243     2879.366     2999.614
 71  TVMe+R2       1383.080     43  2852.159     2874.418     2996.896
 80  TVMe+I+I+R2   1383.224     44  2854.449     2877.881     3002.552
 91  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 93  TIM3+F+R2     1385.913     45  2861.826     2886.469     3013.295
102  TIM3+F+I+I+R2 1386.042     46  2864.083     2889.976     3018.918
113  TIM3e+G4      1390.357     41  2862.713     2882.736     3000.718
115  TIM3e+R2      1385.918     42  2855.835     2876.958     2997.206
124  TIM3e+I+I+R2  1386.072     43  2858.145     2880.404     3002.882
135  TIM2+F+G4     1393.635     44  2875.271     2898.703     3023.374
137  TIM2+F+R2     1387.682     45  2865.363     2890.006     3016.832
146  TIM2+F+I+I+R2 1387.782     46  2867.565     2893.457     3022.400
157  TIM2e+G4      1396.794     41  2875.588     2895.611     3013.593
159  TIM2e+R2      1391.573     42  2867.147     2888.270     3008.518
168  TIM2e+I+I+R2  1391.651     43  2869.302     2891.561     3014.039
179  TIM+F+G4      1390.364     44  2868.727     2892.159     3016.830
181  TIM+F+R2      1384.935     45  2859.870     2884.512     3011.339
190  TIM+F+I+I+R2  1385.017     46  2862.035     2887.927     3016.870
201  TIMe+G4       1394.002     41  2870.004     2890.027     3008.009
203  TIMe+R2       1389.000     42  2862.001     2883.123     3003.372
212  TIMe+I+I+R2   1389.095     43  2864.190     2886.449     3008.927
223  TPM3u+F+G4    1392.305     43  2870.611     2892.870     3015.348
225  TPM3u+F+R2    1387.330     44  2862.659     2886.091     3010.762
234  TPM3u+F+I+I+R2 1387.462     45  2864.924     2889.567     3016.393
245  TPM3+G4       1390.373     40  2860.746     2879.706     2995.385
247  TPM3+R2       1385.925     41  2853.850     2873.873     2991.855
256  TPM3+I+I+R2   1386.070     42  2856.140     2877.263     2997.511
267  TPM2u+F+G4    1394.533     43  2875.067     2897.326     3019.804
269  TPM2u+F+R2    1389.057     44  2866.114     2889.546     3014.217
278  TPM2u+F+I+I+R2 1389.101     45  2868.202     2892.845     3019.671
289  TPM2+G4       1396.823     40  2873.645     2892.605     3008.284
291  TPM2+R2       1391.577     41  2865.155     2885.178     3003.160
300  TPM2+I+I+R2   1391.649     42  2867.297     2888.420     3008.668
311  K3Pu+F+G4     1391.381     43  2868.762     2891.021     3013.499
313  K3Pu+F+R2     1386.372     44  2860.743     2884.175     3008.846
322  K3Pu+F+I+I+R2 1386.425     45  2862.851     2887.494     3014.320
333  K3P+G4        1394.015     40  2868.030     2886.989     3002.669
335  K3P+R2        1389.002     41  2860.005     2880.028     2998.010
344  K3P+I+I+R2    1389.099     42  2862.198     2883.320     3003.569
355  TN+F+G4       1394.039     43  2874.077     2896.336     3018.814
357  TN+F+R2       1388.242     44  2864.484     2887.916     3012.587
366  TN+F+I+I+R2   1388.289     45  2866.579     2891.221     3018.048
377  TNe+G4        1396.791     40  2873.581     2892.541     3008.220
379  TNe+R2        1391.586     41  2865.171     2885.195     3003.176
388  TNe+I+I+R2    1391.666     42  2867.332     2888.454     3008.703
399  HKY+F+G4      1394.951     42  2873.902     2895.024     3015.273
401  HKY+F+R2      1389.609     43  2865.218     2887.477     3009.955
410  HKY+F+I+I+R2  1389.664     44  2867.327     2890.759     3015.430
421  K2P+G4        1396.824     39  2871.648     2889.579     3002.921
423  K2P+R2        1391.594     40  2863.189     2882.148     2997.828
432  K2P+I+I+R2    1391.664     41  2865.327     2885.351     3003.332
443  F81+F+G4      1405.743     41  2893.487     2913.510     3031.492
445  F81+F+R2      1400.806     42  2885.613     2906.736     3026.984
454  F81+F+I+I+R2  1400.909     43  2887.818     2910.077     3032.555
465  JC+G4         1407.650     38  2891.300     2908.237     3019.207
467  JC+R2         1402.858     39  2883.716     2901.648     3014.990
476  JC+I+I+R2     1402.926     40  2885.852     2904.812     3020.491
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree.model.gz
CPU time for ModelFinder: 1.402 seconds (0h:0m:1s)
Wall-clock time for ModelFinder: 1.424 seconds (0h:0m:1s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.925
Optimal log-likelihood: -1385.924
Rate parameters:  A-C: 0.40868  A-G: 1.56207  A-T: 1.00000  C-G: 0.40868  C-T: 1.56207  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.716,0.409) (0.284,2.491)
Parameters optimization took 1 rounds (0.004 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000856876 secs using 96.28% CPU
Computing ML distances took 0.000937 sec (of wall-clock time) 0.000883 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.4121e-05 secs using 77.6% CPU
Constructing RapidNJ tree: done in 9.17912e-05 secs using 114.4% CPU
Computing RapidNJ tree took 0.000220 sec (of wall-clock time) 0.000191 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.820
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|             INITIALIZING CANDIDATE TREE SET                      |
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Generating 98 parsimony trees... 0.103 second
Computing log-likelihood of 98 initial trees ... 0.100 seconds
Current best score: -1385.924

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00479102 secs using 99% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.880
Optimizing NNI: done in 0.0134859 secs using 98.71% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.311
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Iteration 10 / LogL: -1385.344 / Time: 0h:0m:0s
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Iteration 20 / LogL: -1385.343 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1385.311 / CPU time: 0.475
Number of iterations: 20
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|               OPTIMIZING CANDIDATE TREE SET                      |
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Optimizing NNI: done in 0.010483 secs using 98.62% CPU
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UPDATE BEST LOG-LIKELIHOOD: -1385.310
Iteration 30 / LogL: -1385.310 / Time: 0h:0m:0s (0h:0m:3s left)
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Iteration 40 / LogL: -1386.153 / Time: 0h:0m:0s (0h:0m:2s left)
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Iteration 50 / LogL: -1386.267 / Time: 0h:0m:0s (0h:0m:2s left)
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UPDATE BEST LOG-LIKELIHOOD: -1385.310
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Iteration 60 / LogL: -1385.310 / Time: 0h:0m:0s (0h:0m:2s left)
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Iteration 100 / LogL: -1385.507 / Time: 0h:0m:1s (0h:0m:1s left)
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BETTER TREE FOUND at iteration 107: -1385.310
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UPDATE BEST LOG-LIKELIHOOD: -1385.310
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Iteration 180 / LogL: -1385.311 / Time: 0h:0m:2s (0h:0m:1s left)
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Iteration 190 / LogL: -1385.311 / Time: 0h:0m:2s (0h:0m:1s left)
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Iteration 200 / LogL: -1385.311 / Time: 0h:0m:2s (0h:0m:1s left)
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Iteration 210 / LogL: -1385.328 / Time: 0h:0m:2s (0h:0m:1s left)
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Iteration 220 / LogL: -1385.823 / Time: 0h:0m:2s (0h:0m:1s left)
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Iteration 230 / LogL: -1385.311 / Time: 0h:0m:2s (0h:0m:0s left)
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UPDATE BEST LOG-LIKELIHOOD: -1385.310
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Iteration 240 / LogL: -1385.310 / Time: 0h:0m:2s (0h:0m:0s left)
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Iteration 250 / LogL: -1385.310 / Time: 0h:0m:3s (0h:0m:0s left)
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Iteration 260 / LogL: -1385.311 / Time: 0h:0m:3s (0h:0m:0s left)
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Iteration 270 / LogL: -1385.311 / Time: 0h:0m:3s (0h:0m:0s left)
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Iteration 290 / LogL: -1385.311 / Time: 0h:0m:3s (0h:0m:0s left)
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Iteration 300 / LogL: -1385.310 / Time: 0h:0m:3s (0h:0m:0s left)
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UPDATE BEST LOG-LIKELIHOOD: -1385.310
Optimizing NNI: done in 0.00970006 secs using 99.93% CPU
TREE SEARCH COMPLETED AFTER 308 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.310
Optimal log-likelihood: -1385.306
Rate parameters:  A-C: 0.39450  A-G: 1.56675  A-T: 1.00000  C-G: 0.39450  C-T: 1.56675  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.721,0.402) (0.279,2.542)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1385.306
Total tree length: 6.847

Total number of iterations: 308
CPU time used for tree search: 3.859 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.710 sec (0h:0m:3s)
Total CPU time used: 3.890 sec (0h:0m:3s)
Total wall-clock time used: 3.736 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/q2iqtree.treefile -m "TPM3{0.394497,1.56675}+FQ+R2{0.720585,0.401955,0.279415,2.5423}" --length 214

Date and Time: Wed Dec 21 19:11:06 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpckcd7c3g/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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    956160 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:11:15 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000264883 secs using 79.66% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000491858 secs using 75.43% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 2.90871e-05 secs using 75.63% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 7.41482e-05 secs using 76.87% CPU


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...
Optimizing NNI: done in 0.0348301 secs using 98.72% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.605
Optimal log-likelihood: -1388.793
Rate parameters:  A-C: 0.37543  A-G: 2.37167  A-T: 2.15335  C-G: 1.24272  C-T: 3.32366  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.400
Parameters optimization took 1 rounds (0.006 sec)
Time for fast ML tree search: 0.070 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.600     45  2895.200     2919.843     3046.669
  2  GTR+F+I       1401.121     46  2894.242     2920.135     3049.077
  3  GTR+F+G4      1387.358     46  2866.716     2892.609     3021.551
  4  GTR+F+I+G4    1387.726     47  2869.452     2896.633     3027.653
  5  GTR+F+R2      1382.364     47  2858.729     2885.910     3016.930
  6  GTR+F+R3      1382.420     49  2862.841     2892.719     3027.773
 14  GTR+F+I+I+R2  1382.418     48  2860.837     2889.346     3022.403
 15  GTR+F+I+I+R3  1382.449     50  2864.898     2896.187     3033.197
 25  SYM+G4        1387.134     43  2860.269     2882.528     3005.006
 27  SYM+R2        1383.095     44  2854.189     2877.621     3002.292
 36  SYM+I+I+R2    1383.227     45  2856.454     2881.097     3007.923
 47  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 49  TVM+F+R2      1383.789     46  2859.578     2885.470     3014.413
 58  TVM+F+I+I+R2  1383.812     47  2861.625     2888.805     3019.826
 69  TVMe+G4       1387.122     42  2858.245     2879.368     2999.616
 71  TVMe+R2       1383.079     43  2852.159     2874.418     2996.896
 80  TVMe+I+I+R2   1383.224     44  2854.449     2877.881     3002.552
 91  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 93  TIM3+F+R2     1385.912     45  2861.825     2886.468     3013.294
102  TIM3+F+I+I+R2 1386.041     46  2864.082     2889.975     3018.917
113  TIM3e+G4      1390.358     41  2862.715     2882.738     3000.720
115  TIM3e+R2      1385.918     42  2855.836     2876.959     2997.207
124  TIM3e+I+I+R2  1386.073     43  2858.145     2880.404     3002.882
135  TIM2+F+G4     1393.635     44  2875.270     2898.702     3023.373
137  TIM2+F+R2     1387.681     45  2865.362     2890.005     3016.831
146  TIM2+F+I+I+R2 1387.782     46  2867.564     2893.456     3022.399
157  TIM2e+G4      1396.795     41  2875.589     2895.613     3013.594
159  TIM2e+R2      1391.574     42  2867.148     2888.270     3008.519
168  TIM2e+I+I+R2  1391.651     43  2869.302     2891.561     3014.039
179  TIM+F+G4      1390.363     44  2868.726     2892.158     3016.829
181  TIM+F+R2      1384.933     45  2859.866     2884.509     3011.335
190  TIM+F+I+I+R2  1385.016     46  2862.032     2887.925     3016.867
201  TIMe+G4       1394.002     41  2870.005     2890.028     3008.010
203  TIMe+R2       1389.000     42  2862.000     2883.123     3003.371
212  TIMe+I+I+R2   1389.095     43  2864.190     2886.449     3008.927
223  TPM3u+F+G4    1392.306     43  2870.611     2892.870     3015.348
225  TPM3u+F+R2    1387.329     44  2862.659     2886.091     3010.762
234  TPM3u+F+I+I+R2 1387.462     45  2864.923     2889.566     3016.392
245  TPM3+G4       1390.374     40  2860.748     2879.708     2995.387
247  TPM3+R2       1385.925     41  2853.851     2873.874     2991.856
256  TPM3+I+I+R2   1386.070     42  2856.140     2877.263     2997.511
267  TPM2u+F+G4    1394.533     43  2875.067     2897.325     3019.804
269  TPM2u+F+R2    1389.057     44  2866.113     2889.545     3014.216
278  TPM2u+F+I+I+R2 1389.101     45  2868.201     2892.844     3019.670
289  TPM2+G4       1396.823     40  2873.646     2892.605     3008.285
291  TPM2+R2       1391.578     41  2865.155     2885.178     3003.160
300  TPM2+I+I+R2   1391.649     42  2867.297     2888.420     3008.668
311  K3Pu+F+G4     1391.381     43  2868.762     2891.021     3013.499
313  K3Pu+F+R2     1386.371     44  2860.742     2884.174     3008.845
322  K3Pu+F+I+I+R2 1386.425     45  2862.850     2887.493     3014.319
333  K3P+G4        1394.015     40  2868.030     2886.989     3002.669
335  K3P+R2        1389.002     41  2860.004     2880.028     2998.009
344  K3P+I+I+R2    1389.099     42  2862.197     2883.320     3003.568
355  TN+F+G4       1394.038     43  2874.077     2896.336     3018.814
357  TN+F+R2       1388.241     44  2864.483     2887.915     3012.586
366  TN+F+I+I+R2   1388.289     45  2866.578     2891.221     3018.047
377  TNe+G4        1396.791     40  2873.582     2892.542     3008.221
379  TNe+R2        1391.586     41  2865.172     2885.195     3003.177
388  TNe+I+I+R2    1391.666     42  2867.332     2888.454     3008.703
399  HKY+F+G4      1394.951     42  2873.902     2895.024     3015.273
401  HKY+F+R2      1389.609     43  2865.217     2887.476     3009.954
410  HKY+F+I+I+R2  1389.663     44  2867.327     2890.759     3015.430
421  K2P+G4        1396.825     39  2871.649     2889.580     3002.922
423  K2P+R2        1391.594     40  2863.189     2882.148     2997.828
432  K2P+I+I+R2    1391.664     41  2865.327     2885.351     3003.332
443  F81+F+G4      1405.743     41  2893.486     2913.509     3031.491
445  F81+F+R2      1400.805     42  2885.611     2906.733     3026.982
454  F81+F+I+I+R2  1400.908     43  2887.816     2910.075     3032.553
465  JC+G4         1407.650     38  2891.299     2908.236     3019.206
467  JC+R2         1402.858     39  2883.715     2901.646     3014.988
476  JC+I+I+R2     1402.926     40  2885.851     2904.811     3020.490
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 1.420 seconds (0h:0m:1s)
Wall-clock time for ModelFinder: 1.441 seconds (0h:0m:1s)
Generating 1000 samples for ultrafast bootstrap (seed: 956160)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.925
Optimal log-likelihood: -1385.924
Rate parameters:  A-C: 0.40868  A-G: 1.56206  A-T: 1.00000  C-G: 0.40868  C-T: 1.56206  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.716,0.409) (0.284,2.490)
Parameters optimization took 1 rounds (0.005 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000873089 secs using 96.1% CPU
Computing ML distances took 0.000968 sec (of wall-clock time) 0.000905 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.4121e-05 secs using 88.69% CPU
Constructing RapidNJ tree: done in 9.20296e-05 secs using 150% CPU
Computing RapidNJ tree took 0.000214 sec (of wall-clock time) 0.000243 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.820
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.106 second
Computing log-likelihood of 98 initial trees ... 0.100 seconds
Current best score: -1385.924

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00939703 secs using 97.96% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.880
Optimizing NNI: done in 0.023685 secs using 98.75% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.311
Optimizing NNI: done in 0.025053 secs using 98.99% CPU
BETTER TREE FOUND at iteration 3: -1385.309
Optimizing NNI: done in 0.0241978 secs using 98.94% CPU
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Iteration 10 / LogL: -1385.344 / Time: 0h:0m:0s
Optimizing NNI: done in 0.0241599 secs using 98.8% CPU
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Optimizing NNI: done in 0.021867 secs using 99.05% CPU
Iteration 20 / LogL: -1385.343 / Time: 0h:0m:0s
Finish initializing candidate tree set (3)
Current best tree score: -1385.309 / CPU time: 0.674
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.010803 secs using 99.01% CPU
Optimizing NNI: done in 0.0155399 secs using 98.98% CPU
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Optimizing NNI: done in 0.021096 secs using 99.22% CPU
Iteration 30 / LogL: -1385.895 / Time: 0h:0m:1s (0h:0m:6s left)
Optimizing NNI: done in 0.0193939 secs using 98.77% CPU
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Iteration 40 / LogL: -1390.247 / Time: 0h:0m:1s (0h:0m:5s left)
Optimizing NNI: done in 0.0234308 secs using 99% CPU
Optimizing NNI: done in 0.0154369 secs using 99.02% CPU
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Optimizing NNI: done in 0.0182879 secs using 98.39% CPU
Optimizing NNI: done in 0.018625 secs using 99.04% CPU
Optimizing NNI: done in 0.00952101 secs using 98.72% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Optimizing NNI: done in 0.0238979 secs using 99.15% CPU
Optimizing NNI: done in 0.0189331 secs using 98.77% CPU
Optimizing NNI: done in 0.0156071 secs using 98.62% CPU
Iteration 50 / LogL: -1385.641 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1418.996
Optimizing NNI: done in 0.0164821 secs using 98.24% CPU
Optimizing NNI: done in 0.00567389 secs using 98.27% CPU
Optimizing NNI: done in 0.01703 secs using 98.94% CPU
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Iteration 60 / LogL: -1385.549 / Time: 0h:0m:1s (0h:0m:4s left)
Optimizing NNI: done in 0.0196841 secs using 99.01% CPU
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Iteration 70 / LogL: -1385.310 / Time: 0h:0m:2s (0h:0m:3s left)
Optimizing NNI: done in 0.0155289 secs using 99.96% CPU
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Iteration 80 / LogL: -1385.312 / Time: 0h:0m:2s (0h:0m:3s left)
Optimizing NNI: done in 0.0181401 secs using 99.95% CPU
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Optimizing NNI: done in 0.0175171 secs using 99.95% CPU
Optimizing NNI: done in 0.00836301 secs using 99.96% CPU
Iteration 90 / LogL: -1385.556 / Time: 0h:0m:2s (0h:0m:3s left)
Optimizing NNI: done in 0.0134668 secs using 99.95% CPU
Optimizing NNI: done in 0.017596 secs using 99.95% CPU
Optimizing NNI: done in 0.012526 secs using 99.96% CPU
Optimizing NNI: done in 0.015414 secs using 99.89% CPU
Optimizing NNI: done in 0.0197351 secs using 99.96% CPU
Optimizing NNI: done in 0.0196569 secs using 99.96% CPU
Optimizing NNI: done in 0.010849 secs using 99.96% CPU
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Optimizing NNI: done in 0.0117481 secs using 99.94% CPU
Optimizing NNI: done in 0.0159981 secs using 99.96% CPU
Iteration 100 / LogL: -1385.311 / Time: 0h:0m:2s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1417.213
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
Optimizing NNI: done in 0.0127139 secs using 99.94% CPU
Optimizing NNI: done in 0.0192831 secs using 99.95% CPU
Optimizing NNI: done in 0.0180292 secs using 99.95% CPU
Optimizing NNI: done in 0.0153799 secs using 99.96% CPU
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Iteration 110 / LogL: -1385.530 / Time: 0h:0m:2s (0h:0m:2s left)
Optimizing NNI: done in 0.0117009 secs using 99.97% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Optimizing NNI: done in 0.0122411 secs using 99.63% CPU
Optimizing NNI: done in 0.00881696 secs using 99.86% CPU
Optimizing NNI: done in 0.0210202 secs using 99.95% CPU
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Iteration 120 / LogL: -1385.636 / Time: 0h:0m:3s (0h:0m:2s left)
Optimizing NNI: done in 0.018116 secs using 99.68% CPU
Optimizing NNI: done in 0.00951004 secs using 99.27% CPU
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Optimizing NNI: done in 0.00768399 secs using 99.99% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Optimizing NNI: done in 0.0179009 secs using 99.32% CPU
Iteration 130 / LogL: -1385.570 / Time: 0h:0m:3s (0h:0m:1s left)
Optimizing NNI: done in 0.00906897 secs using 99.32% CPU
Optimizing NNI: done in 0.00855994 secs using 99.45% CPU
Optimizing NNI: done in 0.0177021 secs using 99.08% CPU
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Optimizing NNI: done in 0.010071 secs using 99.96% CPU
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Optimizing NNI: done in 0.021261 secs using 99.95% CPU
Iteration 140 / LogL: -1385.314 / Time: 0h:0m:3s (0h:0m:1s left)
Optimizing NNI: done in 0.00621414 secs using 99.98% CPU
Optimizing NNI: done in 0.021708 secs using 99.95% CPU
Optimizing NNI: done in 0.0178909 secs using 99.95% CPU
Optimizing NNI: done in 0.00494814 secs using 100% CPU
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Optimizing NNI: done in 0.008744 secs using 99.97% CPU
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Optimizing NNI: done in 0.0196569 secs using 99.95% CPU
Optimizing NNI: done in 0.00899196 secs using 99.94% CPU
Iteration 150 / LogL: -1385.309 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1417.213
Optimizing NNI: done in 0.00981784 secs using 99.93% CPU
Optimizing NNI: done in 0.0102921 secs using 99.96% CPU
Optimizing NNI: done in 0.01367 secs using 99.96% CPU
Optimizing NNI: done in 0.007617 secs using 99.96% CPU
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Optimizing NNI: done in 0.00676322 secs using 99.94% CPU
Optimizing NNI: done in 0.00977111 secs using 99.95% CPU
Iteration 160 / LogL: -1385.322 / Time: 0h:0m:3s (0h:0m:1s left)
Optimizing NNI: done in 0.0145228 secs using 99.96% CPU
Optimizing NNI: done in 0.019722 secs using 99.96% CPU
Optimizing NNI: done in 0.00504017 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Optimizing NNI: done in 0.016556 secs using 99.67% CPU
Optimizing NNI: done in 0.018594 secs using 99.94% CPU
Optimizing NNI: done in 0.00803494 secs using 99.96% CPU
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Optimizing NNI: done in 0.0186188 secs using 99.95% CPU
Iteration 170 / LogL: -1385.310 / Time: 0h:0m:4s (0h:0m:0s left)
Optimizing NNI: done in 0.0149601 secs using 99.95% CPU
Optimizing NNI: done in 0.0211992 secs using 99.96% CPU
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Optimizing NNI: done in 0.00778294 secs using 99.96% CPU
Iteration 180 / LogL: -1385.507 / Time: 0h:0m:4s (0h:0m:0s left)
Optimizing NNI: done in 0.0180171 secs using 99.95% CPU
Optimizing NNI: done in 0.017714 secs using 99.96% CPU
Optimizing NNI: done in 0.036788 secs using 99.8% CPU
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Optimizing NNI: done in 0.01108 secs using 99.96% CPU
Iteration 190 / LogL: -1385.309 / Time: 0h:0m:4s (0h:0m:0s left)
Optimizing NNI: done in 0.00551605 secs using 99.94% CPU
Optimizing NNI: done in 0.0126901 secs using 99.95% CPU
Optimizing NNI: done in 0.0155599 secs using 99.95% CPU
Optimizing NNI: done in 0.012121 secs using 99.94% CPU
Optimizing NNI: done in 0.00482678 secs using 99.92% CPU
Optimizing NNI: done in 0.0149879 secs using 99.97% CPU
Optimizing NNI: done in 0.011245 secs using 99.96% CPU
Optimizing NNI: done in 0.025794 secs using 99.96% CPU
Optimizing NNI: done in 0.0193112 secs using 99.95% CPU
Optimizing NNI: done in 0.0125089 secs using 99.96% CPU
Iteration 200 / LogL: -1385.310 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1417.625
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.994
Optimizing NNI: done in 0.012383 secs using 99.94% CPU
Optimizing NNI: done in 0.0240059 secs using 99.96% CPU
Optimizing NNI: done in 0.018254 secs using 99.79% CPU
Optimizing NNI: done in 0.0157881 secs using 99.93% CPU
TREE SEARCH COMPLETED AFTER 204 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.309
Optimal log-likelihood: -1385.305
Rate parameters:  A-C: 0.39447  A-G: 1.56669  A-T: 1.00000  C-G: 0.39447  C-T: 1.56669  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.721,0.402) (0.279,2.542)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1385.305
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.splits.nex
Total tree length: 6.847

Total number of iterations: 204
CPU time used for tree search: 4.896 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.886 sec (0h:0m:4s)
Total CPU time used: 5.010 sec (0h:0m:5s)
Total wall-clock time used: 4.999 sec (0h:0m:4s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.splits.nex...
20 taxa and 153 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1385.306

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/q2iqtreeufboot.treefile -m "TPM3{0.394473,1.56669}+FQ+R2{0.720555,0.401953,0.279445,2.54207}" --length 214

Date and Time: Wed Dec 21 19:11:22 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxcp702f0/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:

IQ-TREE multicore version 2.2.0.3 COVID-edition for Mac OS X 64-bit built Sep  5 2022
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    146896 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Dec 21 19:11:32 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000217199 secs using 84.25% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000458956 secs using 78.44% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.90735e-05 secs using 83.89% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 7.39098e-05 secs using 77.12% CPU

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 146896)...

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, 1.245 / LogL: -1394.426
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.308 / LogL: -1394.715
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.319 / LogL: -1394.789
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.316 / LogL: -1394.785
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.311 / LogL: -1394.749
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.314 / LogL: -1394.777
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.309 / LogL: -1394.722
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.310 / LogL: -1394.735
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.312 / LogL: -1394.747
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.313 / LogL: -1394.756
Optimal pinv,alpha: 0.000, 1.245 / LogL: -1394.426

Parameters optimization took 0.490 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00126481 secs using 97.88% CPU
Computing ML distances took 0.001366 sec (of wall-clock time) 0.001308 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.48363e-05 secs using 89.36% CPU
Constructing RapidNJ tree: done in 9.01222e-05 secs using 154.2% CPU
Computing RapidNJ tree took 0.000241 sec (of wall-clock time) 0.000267 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.974
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.105 second
Computing log-likelihood of 98 initial trees ... 0.132 seconds
Current best score: -1392.974

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.0291929 secs using 98.77% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.274
Optimizing NNI: done in 0.0362139 secs using 99.05% CPU
Optimizing NNI: done in 0.021455 secs using 99.07% CPU
Optimizing NNI: done in 0.021086 secs using 99.26% CPU
Optimizing NNI: done in 0.021687 secs using 98.81% CPU
Optimizing NNI: done in 0.0223598 secs using 98.09% CPU
Optimizing NNI: done in 0.0214801 secs using 99.03% CPU
Optimizing NNI: done in 0.0217459 secs using 98.94% CPU
Optimizing NNI: done in 0.021672 secs using 98.74% CPU
Optimizing NNI: done in 0.0214441 secs using 99.1% CPU
Iteration 10 / LogL: -1387.731 / Time: 0h:0m:1s
Optimizing NNI: done in 0.038969 secs using 98.95% CPU
Optimizing NNI: done in 0.039216 secs using 99.01% CPU
Optimizing NNI: done in 0.0390861 secs using 99% CPU
Optimizing NNI: done in 0.0400729 secs using 98.8% CPU
Optimizing NNI: done in 0.039892 secs using 98.81% CPU
Optimizing NNI: done in 0.0389631 secs using 98.81% CPU
Optimizing NNI: done in 0.039017 secs using 98.87% CPU
Optimizing NNI: done in 0.0393009 secs using 98.96% CPU
Optimizing NNI: done in 0.0392239 secs using 98.88% CPU
Optimizing NNI: done in 0.0393441 secs using 98.8% CPU
Iteration 20 / LogL: -1387.285 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.274 / CPU time: 0.897
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.024416 secs using 98.82% CPU
Optimizing NNI: done in 0.063843 secs using 98.76% CPU
Optimizing NNI: done in 0.0182312 secs using 99.01% CPU
Optimizing NNI: done in 0.00763798 secs using 98.94% CPU
Optimizing NNI: done in 0.0241981 secs using 98.96% CPU
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Optimizing NNI: done in 0.0168231 secs using 98.72% CPU
Optimizing NNI: done in 0.064147 secs using 98.92% CPU
Iteration 30 / LogL: -1387.358 / Time: 0h:0m:1s (0h:0m:10s left)
Optimizing NNI: done in 0.01407 secs using 99% CPU
Optimizing NNI: done in 0.0143819 secs using 98.81% CPU
Optimizing NNI: done in 0.032593 secs using 98.7% CPU
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Optimizing NNI: done in 0.017349 secs using 99% CPU
Optimizing NNI: done in 0.00806689 secs using 98.49% CPU
BETTER TREE FOUND at iteration 38: -1387.266
Optimizing NNI: done in 0.0106299 secs using 98.81% CPU
Optimizing NNI: done in 0.018173 secs using 98.82% CPU
Iteration 40 / LogL: -1387.300 / Time: 0h:0m:2s (0h:0m:10s left)
Optimizing NNI: done in 0.018172 secs using 98.9% CPU
Optimizing NNI: done in 0.02512 secs using 98.8% CPU
Optimizing NNI: done in 0.00855303 secs using 98.69% CPU
Optimizing NNI: done in 0.017714 secs using 98.88% CPU
Optimizing NNI: done in 0.0108168 secs using 98.66% CPU
Optimizing NNI: done in 0.0422521 secs using 98.44% CPU
Optimizing NNI: done in 0.048038 secs using 98.84% CPU
Optimizing NNI: done in 0.016839 secs using 99.08% CPU
Optimizing NNI: done in 0.00711322 secs using 98.27% CPU
Optimizing NNI: done in 0.027323 secs using 99.04% CPU
Iteration 50 / LogL: -1387.356 / Time: 0h:0m:2s (0h:0m:8s left)
Log-likelihood cutoff on original alignment: -1414.443
Optimizing NNI: done in 0.0575581 secs using 98.92% CPU
Optimizing NNI: done in 0.034179 secs using 99.01% CPU
Optimizing NNI: done in 0.0160131 secs using 98.86% CPU
Optimizing NNI: done in 0.026258 secs using 99.09% CPU
Optimizing NNI: done in 0.060756 secs using 99.05% CPU
Optimizing NNI: done in 0.020184 secs using 98.94% CPU
Optimizing NNI: done in 0.014415 secs using 98.49% CPU
Optimizing NNI: done in 0.00705194 secs using 98.64% CPU
Optimizing NNI: done in 0.0287309 secs using 98.84% CPU
Optimizing NNI: done in 0.0213358 secs using 99.26% CPU
Iteration 60 / LogL: -1387.337 / Time: 0h:0m:2s (0h:0m:8s left)
Optimizing NNI: done in 0.0659008 secs using 99.1% CPU
Optimizing NNI: done in 0.0615339 secs using 98.69% CPU
Optimizing NNI: done in 0.0233948 secs using 98.68% CPU
Optimizing NNI: done in 0.0366809 secs using 98.88% CPU
Optimizing NNI: done in 0.0337791 secs using 98.79% CPU
Optimizing NNI: done in 0.0357351 secs using 98.81% CPU
Optimizing NNI: done in 0.0185289 secs using 98.92% CPU
Optimizing NNI: done in 0.0412552 secs using 98.89% CPU
Optimizing NNI: done in 0.00759292 secs using 98.96% CPU
Optimizing NNI: done in 0.0347471 secs using 99.07% CPU
Iteration 70 / LogL: -1387.357 / Time: 0h:0m:3s (0h:0m:7s left)
Optimizing NNI: done in 0.020421 secs using 98.31% CPU
Optimizing NNI: done in 0.0286338 secs using 98.58% CPU
Optimizing NNI: done in 0.024281 secs using 98.81% CPU
Optimizing NNI: done in 0.0425889 secs using 99.24% CPU
Optimizing NNI: done in 0.00622201 secs using 99.94% CPU
Optimizing NNI: done in 0.036649 secs using 99.96% CPU
Optimizing NNI: done in 0.0146339 secs using 99.97% CPU
Optimizing NNI: done in 0.0282331 secs using 99.97% CPU
Optimizing NNI: done in 0.0220439 secs using 99.97% CPU
Optimizing NNI: done in 0.01706 secs using 99.96% CPU
Iteration 80 / LogL: -1387.478 / Time: 0h:0m:3s (0h:0m:6s left)
Optimizing NNI: done in 0.0298321 secs using 99.96% CPU
Optimizing NNI: done in 0.0152869 secs using 99.97% CPU
Optimizing NNI: done in 0.073175 secs using 99.85% CPU
Optimizing NNI: done in 0.0403018 secs using 99.96% CPU
Optimizing NNI: done in 0.0302122 secs using 99.96% CPU
Optimizing NNI: done in 0.012502 secs using 99.94% CPU
Optimizing NNI: done in 0.0936611 secs using 99.96% CPU
Optimizing NNI: done in 0.0307829 secs using 99.97% CPU
Optimizing NNI: done in 0.027163 secs using 99.97% CPU
Optimizing NNI: done in 0.0328019 secs using 99.85% CPU
Iteration 90 / LogL: -1387.357 / Time: 0h:0m:3s (0h:0m:6s left)
Optimizing NNI: done in 0.0226531 secs using 99.96% CPU
Optimizing NNI: done in 0.0121861 secs using 99.52% CPU
Optimizing NNI: done in 0.00665379 secs using 99.91% CPU
Optimizing NNI: done in 0.0110281 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Optimizing NNI: done in 0.038697 secs using 99.83% CPU
Optimizing NNI: done in 0.01507 secs using 99.97% CPU
Optimizing NNI: done in 0.0182409 secs using 99.97% CPU
Optimizing NNI: done in 0.0305202 secs using 99.97% CPU
Optimizing NNI: done in 0.0595739 secs using 99.97% CPU
Optimizing NNI: done in 0.0399561 secs using 99.9% CPU
Iteration 100 / LogL: -1387.358 / Time: 0h:0m:4s (0h:0m:5s left)
Log-likelihood cutoff on original alignment: -1414.928
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.994
Optimizing NNI: done in 0.0211349 secs using 99.96% CPU
Optimizing NNI: done in 0.00614595 secs using 99.98% CPU
Optimizing NNI: done in 0.018924 secs using 99.96% CPU
Optimizing NNI: done in 0.00890422 secs using 99.95% CPU
Optimizing NNI: done in 0.0152991 secs using 99.95% CPU
Optimizing NNI: done in 0.036782 secs using 99.97% CPU
Optimizing NNI: done in 0.011934 secs using 99.97% CPU
Optimizing NNI: done in 0.0335281 secs using 99.97% CPU
Optimizing NNI: done in 0.0260601 secs using 99.96% CPU
Optimizing NNI: done in 0.00845098 secs using 99.96% CPU
Iteration 110 / LogL: -1387.357 / Time: 0h:0m:4s (0h:0m:5s left)
Optimizing NNI: done in 0.031729 secs using 99.97% CPU
Optimizing NNI: done in 0.010432 secs using 99.97% CPU
BETTER TREE FOUND at iteration 112: -1387.259
Optimizing NNI: done in 0.0102398 secs using 99.97% CPU
Optimizing NNI: done in 0.0100169 secs using 99.98% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Optimizing NNI: done in 0.0183859 secs using 99.59% CPU
Optimizing NNI: done in 0.0262909 secs using 99.97% CPU
Optimizing NNI: done in 0.0189071 secs using 99.96% CPU
Optimizing NNI: done in 0.0262198 secs using 99.91% CPU
Optimizing NNI: done in 0.00998211 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Optimizing NNI: done in 0.013741 secs using 99.78% CPU
Iteration 120 / LogL: -1387.259 / Time: 0h:0m:4s (0h:0m:7s left)
Optimizing NNI: done in 0.0222981 secs using 99.91% CPU
Optimizing NNI: done in 0.00688481 secs using 99.93% CPU
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Optimizing NNI: done in 0.026989 secs using 99.97% CPU
Optimizing NNI: done in 0.0132179 secs using 99.97% CPU
Iteration 130 / LogL: -1387.320 / Time: 0h:0m:4s (0h:0m:6s left)
Optimizing NNI: done in 0.0453789 secs using 99.97% CPU
Optimizing NNI: done in 0.014833 secs using 99.97% CPU
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Optimizing NNI: done in 0.0138412 secs using 99.97% CPU
Optimizing NNI: done in 0.0642381 secs using 99.84% CPU
Iteration 140 / LogL: -1387.369 / Time: 0h:0m:5s (0h:0m:6s left)
Optimizing NNI: done in 0.0447679 secs using 99.86% CPU
Optimizing NNI: done in 0.0588241 secs using 99.68% CPU
Optimizing NNI: done in 0.0333002 secs using 99.97% CPU
Optimizing NNI: done in 0.0201561 secs using 99.96% CPU
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Optimizing NNI: done in 0.0144839 secs using 99.96% CPU
Optimizing NNI: done in 0.018265 secs using 99.96% CPU
Iteration 150 / LogL: -1387.276 / Time: 0h:0m:5s (0h:0m:6s left)
Log-likelihood cutoff on original alignment: -1415.310
Optimizing NNI: done in 0.00971889 secs using 99.95% CPU
Optimizing NNI: done in 0.018605 secs using 99.97% CPU
Optimizing NNI: done in 0.0271618 secs using 99.97% CPU
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Optimizing NNI: done in 0.0148668 secs using 99.97% CPU
Iteration 160 / LogL: -1387.311 / Time: 0h:0m:5s (0h:0m:5s left)
Optimizing NNI: done in 0.0227921 secs using 99.97% CPU
Optimizing NNI: done in 0.0184531 secs using 99.96% CPU
Optimizing NNI: done in 0.00928783 secs using 99.96% CPU
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Optimizing NNI: done in 0.0145261 secs using 99.96% CPU
Optimizing NNI: done in 0.0200648 secs using 99.96% CPU
Iteration 170 / LogL: -1387.265 / Time: 0h:0m:6s (0h:0m:5s left)
Optimizing NNI: done in 0.0164962 secs using 99.96% CPU
Optimizing NNI: done in 0.0246611 secs using 99.97% CPU
Optimizing NNI: done in 0.00648713 secs using 99.98% CPU
Optimizing NNI: done in 0.0222261 secs using 99.96% CPU
Optimizing NNI: done in 0.01038 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Optimizing NNI: done in 0.00897598 secs using 99.89% CPU
Optimizing NNI: done in 0.0241332 secs using 99.91% CPU
Optimizing NNI: done in 0.0600319 secs using 99.97% CPU
Optimizing NNI: done in 0.0119681 secs using 99.97% CPU
Optimizing NNI: done in 0.022253 secs using 99.95% CPU
Iteration 180 / LogL: -1387.387 / Time: 0h:0m:6s (0h:0m:4s left)
Optimizing NNI: done in 0.024544 secs using 99.97% CPU
Optimizing NNI: done in 0.017535 secs using 99.98% CPU
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Optimizing NNI: done in 0.015121 secs using 99.97% CPU
Optimizing NNI: done in 0.029073 secs using 99.97% CPU
Iteration 190 / LogL: -1387.275 / Time: 0h:0m:6s (0h:0m:4s left)
Optimizing NNI: done in 0.00801086 secs using 99.95% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Optimizing NNI: done in 0.0109899 secs using 99.95% CPU
Optimizing NNI: done in 0.014308 secs using 99.89% CPU
Optimizing NNI: done in 0.06235 secs using 99.97% CPU
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Iteration 200 / LogL: -1387.357 / Time: 0h:0m:7s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1415.310
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
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UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 210 / LogL: -1387.259 / Time: 0h:0m:7s (0h:0m:3s left)
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Iteration 220 / LogL: -1387.358 / Time: 0h:0m:7s (0h:0m:3s left)
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Iteration 240 / LogL: -1387.359 / Time: 0h:0m:8s (0h:0m:2s left)
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Iteration 250 / LogL: -1387.261 / Time: 0h:0m:8s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1415.310
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UPDATE BEST LOG-LIKELIHOOD: -1387.259
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Iteration 260 / LogL: -1387.309 / Time: 0h:0m:8s (0h:0m:1s left)
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Iteration 270 / LogL: -1387.311 / Time: 0h:0m:9s (0h:0m:1s left)
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UPDATE BEST LOG-LIKELIHOOD: -1387.259
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Iteration 300 / LogL: -1387.314 / Time: 0h:0m:10s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1415.310
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
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Iteration 310 / LogL: -1387.550 / Time: 0h:0m:10s (0h:0m:3s left)
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TREE SEARCH COMPLETED AFTER 313 ITERATIONS / Time: 0h:0m:10s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.259
Optimal log-likelihood: -1387.255
Rate parameters:  A-C: 0.32476  A-G: 2.22309  A-T: 2.10421  C-G: 1.15455  C-T: 3.22830  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: 1.322
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.255

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...
0.059 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.splits.nex
Total tree length: 6.730

Total number of iterations: 313
CPU time used for tree search: 10.056 sec (0h:0m:10s)
Wall-clock time used for tree search: 9.939 sec (0h:0m:9s)
Total CPU time used: 10.705 sec (0h:0m:10s)
Total wall-clock time used: 10.595 sec (0h:0m:10s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.splits.nex...
20 taxa and 184 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.256

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/q2iqtreeufboot.treefile -m "GTR{0.324755,2.22309,2.10421,1.15455,3.2283}+F{0.243216,0.181967,0.319196,0.255621}+I{3.25587e-07}+G4{1.32202}" --length 214

Date and Time: Wed Dec 21 19:11:42 2022
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/caporasolab/data/edb27ebe-b918-4abb-9236-f52b22b3e93b/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptoah5gdk/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!