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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/2022.2/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2022.2/tutorials/phylogeny/rep-seqs.qza" > \
  "rep-seqs.qza"

Run MAFFT

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

Output artifacts:

Reducing alignment ambiguity: masking and reference alignments

Why mask an alignment?

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

Note

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

How to mask alignment.

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

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

Output artifacts:

Reference based alignments

There are a variety of tools such as PyNAST) (using NAST), Infernal, and SINA, etc., that attempt to reduce the amount of ambiguously aligned regions by using curated reference alignments (e.g. SILVA. Reference alignments are particularly powerful for rRNA gene sequence data, as knowledge of secondary structure is incorporated into the curation process, thus increasing alignment quality. For a more in-depth and eloquent overview of reference-based alignment approaches, check out the great SINA community tutorial).

Note

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

Construct a phylogeny

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

  1. Phylogeny for the faint of heart - a tutorial

  2. Molecular phylogenetics - principles and practice

  3. Phylogenetics - An Introduction

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

  1. FastTree

  2. RAxML

  3. IQ-TREE

Methods

fasttree

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

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

Output artifacts:

Tip

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

raxml

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

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

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid hill-climbing mode

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


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

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 2484 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-nawidg6k/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpc7yohcap -n q2 


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

Inference[0]: Time 0.592248 CAT-based likelihood -1241.820506, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1388.324170 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpc7yohcap/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1388.324170 .... 

Final GAMMA-based Score of best tree -1387.275825

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpc7yohcap/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpc7yohcap/RAxML_bestTree.q2

Overall execution time: 1.216451 secs or 0.000338 hours or 0.000014 days

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

Command: raxmlHPC -m GTRCAT -p 2484 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-nawidg6k/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpc7yohcap -n q2

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

Output artifacts:

Perform multiple searches using raxml

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

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

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid hill-climbing mode

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


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

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-4cki8dpr/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9 -n q2 


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

Inference[0]: Time 0.591545 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.512294 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.514611 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.655594 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.495589 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/tmpxaucj8f9/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9/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/tmpxaucj8f9/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9/RAxML_bestTree.q2

Overall execution time: 3.525764 secs or 0.000979 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-archive-4cki8dpr/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxaucj8f9 -n q2

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

Output artifacts:

raxml-rapid-bootstrap

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

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

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

  2. Find best scoring ML tree through multiple independent searches using the original input alignment. The number of independent searches is determined by the number of bootstrap replicates set in the 1st step. That is, your search becomes more thorough with increasing bootstrap replicates. The ML optimization of RAxML uses every 5th bootstrap tree as the starting tree for an ML search on the original alignment.

  3. Map the bipartitions (bootstrap supports, 1st step) onto the best scoring ML tree (2nd step).

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

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid bootstrapping and subsequent ML search

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



Executing 100 rapid bootstrap inferences and thereafter a thorough ML search 

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-w58qsbgd/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp -n q2bootstrap 



Time for BS model parameter optimization 0.053214
Bootstrap[0]: Time 0.176107 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.116814 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.107875 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.095814 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.111717 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.114141 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.111630 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.102155 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.110671 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.117030 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.085221 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.119856 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.132877 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.112091 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.117228 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.114450 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.108611 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.098844 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.103680 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.106273 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.097301 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.096399 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.105059 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.123990 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.104976 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.106219 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.109160 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.133407 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.133844 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.088228 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.110866 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.112616 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.119959 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.105513 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.101130 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.120824 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.097998 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.120680 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.094161 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.096817 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.110331 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.109308 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.104607 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.132777 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.094301 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.111346 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.090018 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.129902 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.094719 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.117032 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.139497 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.098654 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.111662 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.096127 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.115316 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.122265 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.099054 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.093640 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.138473 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.106545 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.093879 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.108406 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.124893 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.100164 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.095990 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.097657 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.119499 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.118478 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.092889 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.099699 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.102796 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.102971 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.087155 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.101048 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.090339 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.097645 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.110693 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.133455 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.113731 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.113266 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.100970 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.108406 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.127233 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.097603 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.112748 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.114030 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.096299 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.098558 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.106532 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.098010 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.114252 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.109930 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.045898 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.119239 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.097775 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.094776 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.108806 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.117463 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.045511 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.059061 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.747096 seconds
Average Time per Rapid Bootstrap 0.107471 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.271557 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: 2.206338 seconds
Thorough ML search Time: 0.580224 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 7.064088 secs or 0.001962 hours

Combined Bootstrap and ML search took 17.811479 secs or 0.004948 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.824805 secs or 0.004951 hours or 0.000206 days

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

Command: raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-w58qsbgd/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpdq267zdp -n q2bootstrap

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

Output artifacts:

Tip

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

iqtree

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

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-6hbw_zdd/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree -nt 1
Seed:    129212 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:27:29 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-archive-6hbw_zdd/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000218868 secs using 81.33% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000463009 secs using 78.62% 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 75.77% CPU


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Optimizing NNI: done in 0.083215 secs using 99.6% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1407.502
2. Current log-likelihood: -1400.070
3. Current log-likelihood: -1396.375
4. Current log-likelihood: -1394.887
Optimal log-likelihood: -1393.863
Rate parameters:  A-C: 0.18680  A-G: 2.16102  A-T: 2.20604  C-G: 1.11230  C-T: 2.82171  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.022
Gamma shape alpha: 1.074
Parameters optimization took 4 rounds (0.037 sec)
Time for fast ML tree search: 0.161 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 299 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1413.964     45  2917.928     2942.571     3069.397
  2  GTR+F+I       1411.832     46  2915.664     2941.556     3070.499
  3  GTR+F+G4      1392.724     46  2877.447     2903.339     3032.282
  4  GTR+F+I+G4    1393.053     47  2880.106     2907.287     3038.307
  5  GTR+F+R2      1386.427     47  2866.854     2894.035     3025.055
  6  GTR+F+R3      1386.566     49  2871.131     2901.009     3036.064
 16  SYM+G4        1393.581     43  2873.162     2895.421     3017.899
 18  SYM+R2        1388.979     44  2865.958     2889.390     3014.061
 29  TVM+F+G4      1393.240     45  2876.480     2901.123     3027.949
 31  TVM+F+R2      1387.337     46  2866.674     2892.566     3021.508
 42  TVMe+G4       1393.700     42  2871.399     2892.522     3012.770
 44  TVMe+R2       1388.983     43  2863.967     2886.226     3008.704
 55  TIM3+F+G4     1397.789     44  2883.579     2907.011     3031.682
 57  TIM3+F+R2     1391.101     45  2872.202     2896.845     3023.671
 68  TIM3e+G4      1398.114     41  2878.228     2898.251     3016.233
 70  TIM3e+R2      1393.075     42  2870.150     2891.272     3011.521
 81  TIM2+F+G4     1401.888     44  2891.776     2915.208     3039.879
 83  TIM2+F+R2     1394.794     45  2879.587     2904.230     3031.056
 94  TIM2e+G4      1407.166     41  2896.331     2916.354     3034.336
 96  TIM2e+R2      1401.544     42  2887.088     2908.211     3028.459
107  TIM+F+G4      1397.999     44  2883.999     2907.431     3032.102
109  TIM+F+R2      1391.093     45  2872.186     2896.829     3023.655
120  TIMe+G4       1404.065     41  2890.130     2910.153     3028.135
122  TIMe+R2       1398.405     42  2880.810     2901.933     3022.181
133  TPM3u+F+G4    1398.298     43  2882.597     2904.855     3027.334
135  TPM3u+F+R2    1392.056     44  2872.112     2895.544     3020.215
146  TPM3+G4       1398.232     40  2876.464     2895.423     3011.103
148  TPM3+R2       1393.083     41  2868.167     2888.190     3006.172
159  TPM2u+F+G4    1402.414     43  2890.828     2913.086     3035.565
161  TPM2u+F+R2    1395.759     44  2879.518     2902.949     3027.620
172  TPM2+G4       1407.298     40  2894.595     2913.555     3029.234
174  TPM2+R2       1401.478     41  2884.955     2904.979     3022.960
185  K3Pu+F+G4     1398.695     43  2883.391     2905.650     3028.128
187  K3Pu+F+R2     1392.170     44  2872.341     2895.773     3020.444
198  K3P+G4        1404.158     40  2888.315     2907.275     3022.954
200  K3P+R2        1398.390     41  2878.779     2898.803     3016.784
211  TN+F+G4       1402.054     43  2890.109     2912.368     3034.846
213  TN+F+R2       1395.027     44  2878.054     2901.486     3026.157
224  TNe+G4        1407.200     40  2894.399     2913.359     3029.038
226  TNe+R2        1401.479     41  2884.959     2904.982     3022.964
237  HKY+F+G4      1402.597     42  2889.194     2910.317     3030.565
239  HKY+F+R2      1396.061     43  2878.122     2900.380     3022.859
250  K2P+G4        1407.335     39  2892.671     2910.602     3023.944
252  K2P+R2        1401.478     40  2882.957     2901.916     3017.596
263  F81+F+G4      1410.825     41  2903.650     2923.673     3041.655
265  F81+F+R2      1404.980     42  2893.960     2915.083     3035.331
276  JC+G4         1415.573     38  2907.146     2924.084     3035.054
278  JC+R2         1410.586     39  2899.172     2917.103     3030.445
289  UNREST+FO+G4  1388.830     50  2877.659     2908.948     3045.958
291  UNREST+FO+R2  1384.547     51  2871.093     2903.834     3042.758
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree.model.gz
CPU time for ModelFinder: 1.094 seconds (0h:0m:1s)
Wall-clock time for ModelFinder: 1.098 seconds (0h:0m:1s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1393.083
Optimal log-likelihood: -1393.065
Rate parameters:  A-C: 0.33096  A-G: 1.35570  A-T: 1.00000  C-G: 0.33096  C-T: 1.35570  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.693,0.329) (0.307,2.511)
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 0.000867844 secs using 98.06% CPU
Computing ML distances took 0.000939 sec (of wall-clock time) 0.000905 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 5.48363e-05 secs using 89.36% CPU
Constructing RapidNJ tree: done in 9.39369e-05 secs using 146.9% CPU
Computing RapidNJ tree took 0.000215 sec (of wall-clock time) 0.000257 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1402.161
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.034 second
Computing log-likelihood of 98 initial trees ... 0.102 seconds
Current best score: -1393.065

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00694299 secs using 196.8% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1392.032
Optimizing NNI: done in 0.010772 secs using 196.3% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.326
Optimizing NNI: done in 0.0373728 secs using 147.5% CPU
Optimizing NNI: done in 0.0406878 secs using 99.95% CPU
Optimizing NNI: done in 0.0288322 secs using 99.95% CPU
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Optimizing NNI: done in 0.0441151 secs using 99.95% CPU
Iteration 10 / LogL: -1385.915 / Time: 0h:0m:0s
Optimizing NNI: done in 0.0481391 secs using 99.94% CPU
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Optimizing NNI: done in 0.0499659 secs using 99.95% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.0321341 secs using 99.95% CPU
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Optimizing NNI: done in 0.0498161 secs using 99.95% CPU
Iteration 20 / LogL: -1516.381 / Time: 0h:0m:1s
Finish initializing candidate tree set (19)
Current best tree score: -1385.326 / CPU time: 0.996
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.0171871 secs using 99.46% CPU
Optimizing NNI: done in 0.017936 secs using 99.88% CPU
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Optimizing NNI: done in 0.0178671 secs using 99.9% CPU
Optimizing NNI: done in 0.0147061 secs using 99.93% CPU
BETTER TREE FOUND at iteration 25: -1385.323
Optimizing NNI: done in 0.011766 secs using 99.75% CPU
Optimizing NNI: done in 0.0155859 secs using 99.87% CPU
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Iteration 30 / LogL: -1385.326 / Time: 0h:0m:1s (0h:0m:3s left)
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Iteration 40 / LogL: -1385.719 / Time: 0h:0m:1s (0h:0m:2s left)
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Iteration 50 / LogL: -1385.720 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.012754 secs using 99.95% CPU
Optimizing NNI: done in 0.00704193 secs using 99.93% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.322
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Iteration 60 / LogL: -1385.920 / Time: 0h:0m:1s (0h:0m:1s left)
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Iteration 70 / LogL: -1385.930 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.0228751 secs using 99.93% CPU
Optimizing NNI: done in 0.019407 secs using 99.89% CPU
Optimizing NNI: done in 0.013742 secs using 99.95% CPU
Optimizing NNI: done in 0.0128019 secs using 99.81% CPU
Optimizing NNI: done in 0.0197771 secs using 99.95% CPU
Optimizing NNI: done in 0.00595999 secs using 99.95% CPU
Optimizing NNI: done in 0.0191498 secs using 99.81% CPU
Optimizing NNI: done in 0.0182941 secs using 99.44% CPU
Optimizing NNI: done in 0.0210791 secs using 99.78% CPU
Optimizing NNI: done in 0.0147901 secs using 99.2% CPU
Iteration 80 / LogL: -1385.720 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.011024 secs using 99.95% CPU
Optimizing NNI: done in 0.0136602 secs using 99.95% CPU
Optimizing NNI: done in 0.029969 secs using 99.88% CPU
Optimizing NNI: done in 0.012584 secs using 99.87% CPU
Optimizing NNI: done in 0.0133619 secs using 99.95% CPU
Optimizing NNI: done in 0.0149641 secs using 99.93% CPU
Optimizing NNI: done in 0.019809 secs using 99.95% CPU
Optimizing NNI: done in 0.017031 secs using 99.96% CPU
Optimizing NNI: done in 0.0162499 secs using 99.96% CPU
Optimizing NNI: done in 0.0218918 secs using 99.95% CPU
Iteration 90 / LogL: -1385.323 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.010711 secs using 99.94% CPU
Optimizing NNI: done in 0.016731 secs using 99.95% CPU
Optimizing NNI: done in 0.0140181 secs using 99.94% CPU
Optimizing NNI: done in 0.0178919 secs using 99.95% CPU
Optimizing NNI: done in 0.00527692 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.322
Optimizing NNI: done in 0.020215 secs using 99.81% CPU
Optimizing NNI: done in 0.016995 secs using 99.89% CPU
Optimizing NNI: done in 0.015146 secs using 99.95% CPU
Optimizing NNI: done in 0.0159302 secs using 99.95% CPU
Optimizing NNI: done in 0.018604 secs using 99.95% CPU
Iteration 100 / LogL: -1394.070 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.0194921 secs using 99.63% CPU
Optimizing NNI: done in 0.0156829 secs using 99.96% CPU
Optimizing NNI: done in 0.0148799 secs using 99.93% CPU
Optimizing NNI: done in 0.013876 secs using 99.95% CPU
Optimizing NNI: done in 0.0189519 secs using 99.94% CPU
Optimizing NNI: done in 0.016773 secs using 99.94% CPU
Optimizing NNI: done in 0.0163388 secs using 99.95% CPU
Optimizing NNI: done in 0.0117059 secs using 99.94% CPU
Optimizing NNI: done in 0.0272141 secs using 99.77% CPU
Optimizing NNI: done in 0.0124669 secs using 99.95% CPU
Iteration 110 / LogL: -1385.719 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.015934 secs using 99.94% CPU
Optimizing NNI: done in 0.0188909 secs using 99.94% CPU
Optimizing NNI: done in 0.0114579 secs using 99.95% CPU
Optimizing NNI: done in 0.01649 secs using 99.95% CPU
Optimizing NNI: done in 0.024936 secs using 99.84% CPU
Optimizing NNI: done in 0.0149262 secs using 99.84% CPU
Optimizing NNI: done in 0.0164611 secs using 99.93% CPU
Optimizing NNI: done in 0.0224798 secs using 99.96% CPU
Optimizing NNI: done in 0.0155289 secs using 99.94% CPU
Optimizing NNI: done in 0.0137899 secs using 99.94% CPU
Iteration 120 / LogL: -1469.954 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.0174961 secs using 99.77% CPU
Optimizing NNI: done in 0.0112441 secs using 99.78% CPU
Optimizing NNI: done in 0.0139301 secs using 99.93% CPU
Optimizing NNI: done in 0.0102959 secs using 99.94% CPU
Optimizing NNI: done in 0.0131249 secs using 99.95% CPU
Optimizing NNI: done in 0.012594 secs using 99.94% CPU
TREE SEARCH COMPLETED AFTER 126 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.322
Optimal log-likelihood: -1385.311
Rate parameters:  A-C: 0.39450  A-G: 1.57508  A-T: 1.00000  C-G: 0.39450  C-T: 1.57508  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.717,0.394) (0.283,2.537)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1385.311
Total tree length: 6.965

Total number of iterations: 126
CPU time used for tree search: 2.912 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.734 sec (0h:0m:2s)
Total CPU time used: 2.940 sec (0h:0m:2s)
Total wall-clock time used: 2.758 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree.log

Date and Time: Mon Feb 21 12:27:32 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-archive-6hbw_zdd/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8fqc3j5z/q2iqtree -nt 1

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

Output artifacts:

Specifying a substitution model

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-eofzjwbk/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9wt85luo/q2iqtree -nt 1
Seed:    744872 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:27:42 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-archive-eofzjwbk/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000217915 secs using 85.35% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000464916 secs using 78.94% 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 84.95% 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)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 0.397 / LogL: -1823.354
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.019, 0.465 / LogL: -1823.521
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.030, 0.492 / LogL: -1823.178
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.035, 0.500 / LogL: -1822.673
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.038, 0.499 / LogL: -1822.073
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.037, 0.491 / LogL: -1821.357
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.038, 0.488 / LogL: -1820.740
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.039, 0.482 / LogL: -1820.015
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.040, 0.477 / LogL: -1819.309
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.041, 0.471 / LogL: -1818.588
Optimal pinv,alpha: 0.041, 0.471 / LogL: -1818.588

Parameters optimization took 0.629 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00232697 secs using 99.14% CPU
Computing ML distances took 0.002411 sec (of wall-clock time) 0.002363 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 5.57899e-05 secs using 89.62% CPU
Constructing RapidNJ tree: done in 0.000108004 secs using 141.7% CPU
Computing RapidNJ tree took 0.000232 sec (of wall-clock time) 0.000274 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1413.667
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.034 second
Computing log-likelihood of 98 initial trees ... 0.145 seconds
Current best score: -1413.667

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.012588 secs using 194.9% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.651
Optimizing NNI: done in 0.0629721 secs using 99.96% CPU
Optimizing NNI: done in 0.083672 secs using 99.95% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 3: -1387.488
Optimizing NNI: done in 0.071444 secs using 99.97% CPU
Optimizing NNI: done in 0.0655179 secs using 99.97% CPU
Optimizing NNI: done in 0.0531249 secs using 99.97% CPU
Optimizing NNI: done in 0.0723941 secs using 99.96% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.06691 secs using 99.97% CPU
Optimizing NNI: done in 0.036448 secs using 99.96% CPU
Optimizing NNI: done in 0.0721941 secs using 99.97% CPU
Iteration 10 / LogL: -1520.689 / Time: 0h:0m:1s
Optimizing NNI: done in 0.0863349 secs using 99.97% CPU
Optimizing NNI: done in 0.066752 secs using 99.91% CPU
Optimizing NNI: done in 0.0803308 secs using 99.96% CPU
Optimizing NNI: done in 0.0891919 secs using 99.92% CPU
Optimizing NNI: done in 0.079345 secs using 99.96% CPU
Optimizing NNI: done in 0.0805988 secs using 99.97% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.087435 secs using 99.68% CPU
Optimizing NNI: done in 0.033854 secs using 99.96% CPU
Optimizing NNI: done in 0.05759 secs using 99.97% CPU
Optimizing NNI: done in 0.0561099 secs using 99.97% CPU
Iteration 20 / LogL: -1406.637 / Time: 0h:0m:2s
Finish initializing candidate tree set (18)
Current best tree score: -1387.488 / CPU time: 1.583
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.035645 secs using 99.86% CPU
Optimizing NNI: done in 0.0300579 secs using 99.93% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 22: -1387.351
Optimizing NNI: done in 0.028111 secs using 99.8% CPU
Optimizing NNI: done in 0.023325 secs using 99.82% CPU
Optimizing NNI: done in 0.0288851 secs using 99.89% CPU
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1387.280
Optimizing NNI: done in 0.016644 secs using 99.76% CPU
Optimizing NNI: done in 0.0260649 secs using 99.96% CPU
Optimizing NNI: done in 0.0418391 secs using 99.86% CPU
Optimizing NNI: done in 0.0690269 secs using 99.95% CPU
Optimizing NNI: done in 0.0420132 secs using 99.89% CPU
Iteration 30 / LogL: -1388.256 / Time: 0h:0m:2s (0h:0m:8s left)
Optimizing NNI: done in 0.0316679 secs using 99.96% CPU
Optimizing NNI: done in 0.0232692 secs using 99.94% CPU
BETTER TREE FOUND at iteration 32: -1387.280
Optimizing NNI: done in 0.0393538 secs using 99.75% CPU
Optimizing NNI: done in 0.0490012 secs using 99.96% CPU
Optimizing NNI: done in 0.033406 secs using 99.96% CPU
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Optimizing NNI: done in 0.019891 secs using 99.96% CPU
Iteration 40 / LogL: -1387.559 / Time: 0h:0m:2s (0h:0m:6s left)
Optimizing NNI: done in 0.0206971 secs using 99.96% CPU
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Optimizing NNI: done in 0.050602 secs using 99.96% CPU
Optimizing NNI: done in 0.0216331 secs using 99.96% CPU
Iteration 50 / LogL: -1396.567 / Time: 0h:0m:3s (0h:0m:5s left)
Optimizing NNI: done in 0.026777 secs using 99.96% CPU
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Optimizing NNI: done in 0.0162742 secs using 99.92% CPU
Optimizing NNI: done in 0.0200601 secs using 99.84% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.280
Optimizing NNI: done in 0.043077 secs using 99.86% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.279
Optimizing NNI: done in 0.0270021 secs using 99.84% CPU
Optimizing NNI: done in 0.0692711 secs using 99.96% CPU
Iteration 60 / LogL: -1387.448 / Time: 0h:0m:3s (0h:0m:4s left)
Optimizing NNI: done in 0.0345271 secs using 99.96% CPU
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Optimizing NNI: done in 0.034019 secs using 99.96% CPU
Optimizing NNI: done in 0.0409889 secs using 99.97% CPU
Iteration 70 / LogL: -1387.320 / Time: 0h:0m:3s (0h:0m:3s left)
Optimizing NNI: done in 0.0189939 secs using 99.95% CPU
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Optimizing NNI: done in 0.0268569 secs using 99.96% CPU
Optimizing NNI: done in 0.0320351 secs using 99.95% CPU
Iteration 80 / LogL: -1387.509 / Time: 0h:0m:4s (0h:0m:2s left)
Optimizing NNI: done in 0.02055 secs using 99.97% CPU
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Optimizing NNI: done in 0.028795 secs using 99.79% CPU
Optimizing NNI: done in 0.019119 secs using 99.86% CPU
Iteration 90 / LogL: -1387.280 / Time: 0h:0m:4s (0h:0m:2s left)
Optimizing NNI: done in 0.022809 secs using 99.84% CPU
Optimizing NNI: done in 0.0243859 secs using 99.96% CPU
Optimizing NNI: done in 0.0474122 secs using 99.96% CPU
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Optimizing NNI: done in 0.0314069 secs using 99.96% CPU
Optimizing NNI: done in 0.031337 secs using 99.96% CPU
Iteration 100 / LogL: -1387.283 / Time: 0h:0m:4s (0h:0m:1s left)
Optimizing NNI: done in 0.024379 secs using 99.96% CPU
Optimizing NNI: done in 0.0291388 secs using 99.96% CPU
Optimizing NNI: done in 0.047951 secs using 99.96% CPU
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Optimizing NNI: done in 0.039984 secs using 99.96% CPU
Optimizing NNI: done in 0.0258222 secs using 99.96% CPU
Optimizing NNI: done in 0.030107 secs using 99.97% CPU
Iteration 110 / LogL: -1387.280 / Time: 0h:0m:5s (0h:0m:1s left)
Optimizing NNI: done in 0.029587 secs using 99.96% CPU
Optimizing NNI: done in 0.0211229 secs using 99.81% CPU
Optimizing NNI: done in 0.040602 secs using 99.96% CPU
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Optimizing NNI: done in 0.0296869 secs using 99.96% CPU
Optimizing NNI: done in 0.020494 secs using 99.96% CPU
Optimizing NNI: done in 0.030174 secs using 99.82% CPU
Optimizing NNI: done in 0.0353281 secs using 99.96% CPU
Iteration 120 / LogL: -1387.305 / Time: 0h:0m:5s (0h:0m:0s left)
Optimizing NNI: done in 0.0112879 secs using 99.95% CPU
Optimizing NNI: done in 0.0254529 secs using 99.97% CPU
Optimizing NNI: done in 0.026248 secs using 99.85% CPU
Optimizing NNI: done in 0.0428178 secs using 99.86% CPU
Optimizing NNI: done in 0.0295799 secs using 99.96% CPU
Optimizing NNI: done in 0.038702 secs using 99.96% CPU
Optimizing NNI: done in 0.040941 secs using 99.96% CPU
Optimizing NNI: done in 0.029923 secs using 99.96% CPU
Optimizing NNI: done in 0.0334852 secs using 99.97% CPU
Optimizing NNI: done in 0.0299928 secs using 99.96% CPU
Iteration 130 / LogL: -1387.323 / Time: 0h:0m:5s (0h:0m:0s left)
Optimizing NNI: done in 0.0317531 secs using 99.97% CPU
Optimizing NNI: done in 0.02036 secs using 99.96% CPU
Optimizing NNI: done in 0.043314 secs using 99.96% CPU
TREE SEARCH COMPLETED AFTER 133 ITERATIONS / Time: 0h:0m:5s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.279
2. Current log-likelihood: -1387.261
3. Current log-likelihood: -1387.245
4. Current log-likelihood: -1387.233
5. Current log-likelihood: -1387.222
Optimal log-likelihood: -1387.213
Rate parameters:  A-C: 0.34958  A-G: 2.33258  A-T: 2.15590  C-G: 1.24042  C-T: 3.23753  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.001
Gamma shape alpha: 1.287
Parameters optimization took 5 rounds (0.017 sec)
BEST SCORE FOUND : -1387.213
Total tree length: 7.621

Total number of iterations: 133
CPU time used for tree search: 5.295 sec (0h:0m:5s)
Wall-clock time used for tree search: 5.118 sec (0h:0m:5s)
Total CPU time used: 5.964 sec (0h:0m:5s)
Total wall-clock time used: 5.784 sec (0h:0m:5s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9wt85luo/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9wt85luo/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9wt85luo/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9wt85luo/q2iqtree.log

Date and Time: Mon Feb 21 12:27:48 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-archive-eofzjwbk/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9wt85luo/q2iqtree -nt 1

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

Output artifacts:

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-831ch4_r/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree -nt 1 -fast
Seed:    370492 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:27:58 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-archive-831ch4_r/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000235081 secs using 84.23% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000504971 secs using 78.62% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Analyzing sequences: done in 2.00272e-05 secs using 84.88% CPU
Checking for duplicate sequences: done in 7.89165e-05 secs using 74.76% CPU

---> START RUN NUMBER 1 (seed: 370492)

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: -2058.24
2. Current log-likelihood: -1852.37
3. Current log-likelihood: -1841.32
4. Current log-likelihood: -1839.47
Optimal log-likelihood: -1838.94
Rate parameters:  A-C: 0.00010  A-G: 1.78167  A-T: 2.22392  C-G: 1.90153  C-T: 4.54949  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.055
Gamma shape alpha: 0.549
Parameters optimization took 4 rounds (0.049 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00205183 secs using 99.13% CPU
Computing ML distances took 0.002139 sec (of wall-clock time) 0.002090 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 5.50747e-05 secs using 88.97% CPU
Constructing RapidNJ tree: done in 9.58443e-05 secs using 153.4% CPU
Computing RapidNJ tree took 0.000218 sec (of wall-clock time) 0.000249 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1409.513
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0124321 secs using 196.1% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1389.026
Optimizing NNI: done in 0.087559 secs using 196.8% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 2: -1387.969
Finish initializing candidate tree set (4)
Current best tree score: -1387.969 / CPU time: 0.149
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: -1387.969
2. Current log-likelihood: -1387.834
3. Current log-likelihood: -1387.726
4. Current log-likelihood: -1387.647
5. Current log-likelihood: -1387.587
Optimal log-likelihood: -1387.539
Rate parameters:  A-C: 0.37899  A-G: 2.35435  A-T: 2.15803  C-G: 1.24720  C-T: 3.34425  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: 1.311
Parameters optimization took 5 rounds (0.018 sec)
BEST SCORE FOUND : -1387.539
Total tree length: 7.606

Total number of iterations: 2
CPU time used for tree search: 0.294 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.149 sec (0h:0m:0s)
Total CPU time used: 0.402 sec (0h:0m:0s)
Total wall-clock time used: 0.237 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 371492)

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: -2171.038
2. Current log-likelihood: -1980.051
3. Current log-likelihood: -1923.533
4. Current log-likelihood: -1921.371
5. Current log-likelihood: -1920.591
Optimal log-likelihood: -1920.191
Rate parameters:  A-C: 0.46540  A-G: 4.42811  A-T: 5.73340  C-G: 5.22017  C-T: 12.34775  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.047
Gamma shape alpha: 0.513
Parameters optimization took 5 rounds (0.057 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00223708 secs using 99.59% CPU
Computing ML distances took 0.002306 sec (of wall-clock time) 0.002280 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 4.91142e-05 secs using 93.66% CPU
Constructing RapidNJ tree: done in 9.29832e-05 secs using 154.9% CPU
Computing RapidNJ tree took 0.000212 sec (of wall-clock time) 0.000257 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1409.567
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0124841 secs using 196.3% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.647
Optimizing NNI: done in 0.0856218 secs using 196.7% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1388.647 / CPU time: 0.151
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: -1388.647
2. Current log-likelihood: -1388.179
3. Current log-likelihood: -1387.902
4. Current log-likelihood: -1387.788
5. Current log-likelihood: -1387.693
6. Current log-likelihood: -1387.615
7. Current log-likelihood: -1387.549
Optimal log-likelihood: -1387.509
Rate parameters:  A-C: 0.33876  A-G: 2.32970  A-T: 2.21066  C-G: 1.20503  C-T: 3.38894  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.003
Gamma shape alpha: 1.331
Parameters optimization took 7 rounds (0.031 sec)
BEST SCORE FOUND : -1387.509
Total tree length: 6.865

Total number of iterations: 2
CPU time used for tree search: 0.296 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.151 sec (0h:0m:0s)
Total CPU time used: 0.464 sec (0h:0m:0s)
Total wall-clock time used: 0.257 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 372492)

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: -2203.030
2. Current log-likelihood: -1920.171
3. Current log-likelihood: -1891.219
4. Current log-likelihood: -1888.241
5. Current log-likelihood: -1886.789
6. Current log-likelihood: -1886.096
Optimal log-likelihood: -1885.699
Rate parameters:  A-C: 2.27921  A-G: 13.94801  A-T: 16.57646  C-G: 25.00127  C-T: 56.30754  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: 0.448
Parameters optimization took 6 rounds (0.076 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00232387 secs using 99.62% CPU
Computing ML distances took 0.002401 sec (of wall-clock time) 0.002368 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 4.91142e-05 secs using 93.66% CPU
Constructing RapidNJ tree: done in 9.01222e-05 secs using 157.6% CPU
Computing RapidNJ tree took 0.000206 sec (of wall-clock time) 0.000255 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1417.785
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.024498 secs using 196.5% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.937
Optimizing NNI: done in 0.0829699 secs using 196.6% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.937 / CPU time: 0.177
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: -1387.937
2. Current log-likelihood: -1387.806
3. Current log-likelihood: -1387.711
4. Current log-likelihood: -1387.646
Optimal log-likelihood: -1387.595
Rate parameters:  A-C: 0.35123  A-G: 2.25319  A-T: 2.13485  C-G: 1.20366  C-T: 3.24072  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: 1.288
Parameters optimization took 4 rounds (0.018 sec)
BEST SCORE FOUND : -1387.595
Total tree length: 7.711

Total number of iterations: 2
CPU time used for tree search: 0.347 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.177 sec (0h:0m:0s)
Total CPU time used: 0.493 sec (0h:0m:0s)
Total wall-clock time used: 0.288 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 373492)

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: -2172.151
2. Current log-likelihood: -1968.935
3. Current log-likelihood: -1955.145
4. Current log-likelihood: -1953.495
5. Current log-likelihood: -1952.567
6. Current log-likelihood: -1952.022
Optimal log-likelihood: -1951.639
Rate parameters:  A-C: 0.37558  A-G: 3.11091  A-T: 3.56840  C-G: 4.00162  C-T: 10.74161  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.041
Gamma shape alpha: 0.492
Parameters optimization took 6 rounds (0.065 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00247717 secs using 99.59% CPU
Computing ML distances took 0.002555 sec (of wall-clock time) 0.002525 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 4.91142e-05 secs using 93.66% CPU
Constructing RapidNJ tree: done in 8.91685e-05 secs using 158.1% CPU
Computing RapidNJ tree took 0.000208 sec (of wall-clock time) 0.000252 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1411.154
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.012377 secs using 196.6% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.631
Optimizing NNI: done in 0.0792961 secs using 196.8% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1388.631 / CPU time: 0.139
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: -1388.631
2. Current log-likelihood: -1388.163
3. Current log-likelihood: -1387.885
4. Current log-likelihood: -1387.774
5. Current log-likelihood: -1387.684
6. Current log-likelihood: -1387.608
7. Current log-likelihood: -1387.544
Optimal log-likelihood: -1387.503
Rate parameters:  A-C: 0.34579  A-G: 2.34534  A-T: 2.22721  C-G: 1.21402  C-T: 3.41641  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.003
Gamma shape alpha: 1.325
Parameters optimization took 7 rounds (0.030 sec)
BEST SCORE FOUND : -1387.503
Total tree length: 6.881

Total number of iterations: 2
CPU time used for tree search: 0.274 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.140 sec (0h:0m:0s)
Total CPU time used: 0.419 sec (0h:0m:0s)
Total wall-clock time used: 0.251 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 374492)

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: -2187.914
2. Current log-likelihood: -1945.256
3. Current log-likelihood: -1911.107
4. Current log-likelihood: -1909.125
5. Current log-likelihood: -1908.529
Optimal log-likelihood: -1907.968
Rate parameters:  A-C: 0.00010  A-G: 3.20756  A-T: 4.13615  C-G: 3.02646  C-T: 8.43244  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.050
Gamma shape alpha: 0.524
Parameters optimization took 5 rounds (0.059 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00328207 secs using 99.48% CPU
Computing ML distances took 0.003382 sec (of wall-clock time) 0.003340 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 6.10352e-05 secs using 95.03% CPU
Constructing RapidNJ tree: done in 8.89301e-05 secs using 159.7% CPU
Computing RapidNJ tree took 0.000223 sec (of wall-clock time) 0.000253 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1409.387
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0127108 secs using 196.9% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.656
Optimizing NNI: done in 0.079556 secs using 196.7% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 2: -1387.820
Finish initializing candidate tree set (4)
Current best tree score: -1387.820 / CPU time: 0.145
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: -1387.820
2. Current log-likelihood: -1387.691
3. Current log-likelihood: -1387.591
4. Current log-likelihood: -1387.518
5. Current log-likelihood: -1387.463
Optimal log-likelihood: -1387.420
Rate parameters:  A-C: 0.33318  A-G: 2.27456  A-T: 2.14928  C-G: 1.18018  C-T: 3.29473  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 5 rounds (0.021 sec)
BEST SCORE FOUND : -1387.420
Total tree length: 6.716

Total number of iterations: 2
CPU time used for tree search: 0.286 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.145 sec (0h:0m:0s)
Total CPU time used: 0.435 sec (0h:0m:0s)
Total wall-clock time used: 0.245 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 375492)

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: -2249.937
2. Current log-likelihood: -1972.913
3. Current log-likelihood: -1926.884
4. Current log-likelihood: -1924.378
5. Current log-likelihood: -1923.567
Optimal log-likelihood: -1923.172
Rate parameters:  A-C: 0.07579  A-G: 4.99960  A-T: 6.87531  C-G: 6.70939  C-T: 15.72924  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.043
Gamma shape alpha: 0.491
Parameters optimization took 5 rounds (0.054 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00222087 secs using 99.65% CPU
Computing ML distances took 0.002298 sec (of wall-clock time) 0.002264 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 93.9% CPU
Constructing RapidNJ tree: done in 8.91685e-05 secs using 160.4% CPU
Computing RapidNJ tree took 0.000205 sec (of wall-clock time) 0.000229 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1412.673
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0142901 secs using 196.1% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.598
Optimizing NNI: done in 0.0776949 secs using 196.7% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1388.598 / CPU time: 0.145
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: -1388.598
2. Current log-likelihood: -1388.147
3. Current log-likelihood: -1387.880
4. Current log-likelihood: -1387.770
5. Current log-likelihood: -1387.679
6. Current log-likelihood: -1387.604
7. Current log-likelihood: -1387.540
Optimal log-likelihood: -1387.504
Rate parameters:  A-C: 0.33979  A-G: 2.34210  A-T: 2.22344  C-G: 1.21181  C-T: 3.41009  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.003
Gamma shape alpha: 1.329
Parameters optimization took 7 rounds (0.029 sec)
BEST SCORE FOUND : -1387.504
Total tree length: 6.870

Total number of iterations: 2
CPU time used for tree search: 0.284 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.145 sec (0h:0m:0s)
Total CPU time used: 0.444 sec (0h:0m:0s)
Total wall-clock time used: 0.244 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 376492)

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: -2131.292
2. Current log-likelihood: -1886.623
3. Current log-likelihood: -1865.156
4. Current log-likelihood: -1863.099
5. Current log-likelihood: -1862.535
Optimal log-likelihood: -1862.269
Rate parameters:  A-C: 0.00010  A-G: 11.57685  A-T: 13.13277  C-G: 13.16951  C-T: 33.38440  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.046
Gamma shape alpha: 0.507
Parameters optimization took 5 rounds (0.066 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00230598 secs using 99.61% CPU
Computing ML distances took 0.002374 sec (of wall-clock time) 0.002349 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 4.81606e-05 secs using 118.4% CPU
Constructing RapidNJ tree: done in 8.70228e-05 secs using 148.2% CPU
Computing RapidNJ tree took 0.000204 sec (of wall-clock time) 0.000259 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1413.332
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.012778 secs using 196.3% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.666
Optimizing NNI: done in 0.0874641 secs using 196.7% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1388.666 / CPU time: 0.167
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: -1388.666
2. Current log-likelihood: -1388.191
3. Current log-likelihood: -1387.908
4. Current log-likelihood: -1387.792
5. Current log-likelihood: -1387.694
6. Current log-likelihood: -1387.614
7. Current log-likelihood: -1387.548
Optimal log-likelihood: -1387.508
Rate parameters:  A-C: 0.33790  A-G: 2.30927  A-T: 2.19120  C-G: 1.19469  C-T: 3.35969  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.003
Gamma shape alpha: 1.331
Parameters optimization took 7 rounds (0.030 sec)
BEST SCORE FOUND : -1387.508
Total tree length: 6.868

Total number of iterations: 2
CPU time used for tree search: 0.328 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.167 sec (0h:0m:0s)
Total CPU time used: 0.497 sec (0h:0m:0s)
Total wall-clock time used: 0.281 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 377492)

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: -2233.377
2. Current log-likelihood: -2020.208
3. Current log-likelihood: -1962.287
4. Current log-likelihood: -1960.737
5. Current log-likelihood: -1960.047
Optimal log-likelihood: -1959.712
Rate parameters:  A-C: 0.31227  A-G: 3.44049  A-T: 5.31178  C-G: 4.20904  C-T: 9.70999  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.050
Gamma shape alpha: 0.507
Parameters optimization took 5 rounds (0.064 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.002177 secs using 99.59% CPU
Computing ML distances took 0.002254 sec (of wall-clock time) 0.002219 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 6.07967e-05 secs using 87.18% CPU
Constructing RapidNJ tree: done in 0.000108957 secs using 140.4% CPU
Computing RapidNJ tree took 0.000251 sec (of wall-clock time) 0.000284 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1410.338
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0127189 secs using 196.8% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.673
Optimizing NNI: done in 0.086112 secs using 196.9% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1388.673 / CPU time: 0.143
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: -1388.673
2. Current log-likelihood: -1388.193
3. Current log-likelihood: -1387.914
4. Current log-likelihood: -1387.796
5. Current log-likelihood: -1387.700
6. Current log-likelihood: -1387.620
7. Current log-likelihood: -1387.553
Optimal log-likelihood: -1387.512
Rate parameters:  A-C: 0.33851  A-G: 2.32579  A-T: 2.20683  C-G: 1.20313  C-T: 3.38371  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: 1.332
Parameters optimization took 7 rounds (0.030 sec)
BEST SCORE FOUND : -1387.512
Total tree length: 6.860

Total number of iterations: 2
CPU time used for tree search: 0.281 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.143 sec (0h:0m:0s)
Total CPU time used: 0.436 sec (0h:0m:0s)
Total wall-clock time used: 0.259 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 378492)

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: -2175.200
2. Current log-likelihood: -1916.392
3. Current log-likelihood: -1903.171
4. Current log-likelihood: -1900.276
5. Current log-likelihood: -1899.281
6. Current log-likelihood: -1898.755
Optimal log-likelihood: -1898.367
Rate parameters:  A-C: 0.78587  A-G: 6.84248  A-T: 6.68372  C-G: 10.82602  C-T: 22.74524  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: 0.441
Parameters optimization took 6 rounds (0.073 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.0023241 secs using 99.61% CPU
Computing ML distances took 0.002400 sec (of wall-clock time) 0.002366 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 5.29289e-05 secs using 92.58% CPU
Constructing RapidNJ tree: done in 8.91685e-05 secs using 160.4% CPU
Computing RapidNJ tree took 0.000216 sec (of wall-clock time) 0.000268 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1416.063
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0246129 secs using 196.6% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.822
Optimizing NNI: done in 0.0805068 secs using 195.9% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
BETTER TREE FOUND at iteration 2: -1387.799
Finish initializing candidate tree set (4)
Current best tree score: -1387.799 / CPU time: 0.162
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: -1387.799
2. Current log-likelihood: -1387.694
3. Current log-likelihood: -1387.616
4. Current log-likelihood: -1387.559
Optimal log-likelihood: -1387.515
Rate parameters:  A-C: 0.37633  A-G: 2.32817  A-T: 2.13387  C-G: 1.23254  C-T: 3.30404  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: 1.305
Parameters optimization took 4 rounds (0.017 sec)
BEST SCORE FOUND : -1387.515
Total tree length: 7.624

Total number of iterations: 2
CPU time used for tree search: 0.316 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.162 sec (0h:0m:0s)
Total CPU time used: 0.462 sec (0h:0m:0s)
Total wall-clock time used: 0.269 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 379492)

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: -2225.628
2. Current log-likelihood: -1974.142
3. Current log-likelihood: -1940.784
4. Current log-likelihood: -1939.115
5. Current log-likelihood: -1938.501
Optimal log-likelihood: -1938.231
Rate parameters:  A-C: 0.91680  A-G: 8.16332  A-T: 9.32969  C-G: 10.41035  C-T: 21.78572  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.049
Gamma shape alpha: 0.493
Parameters optimization took 5 rounds (0.054 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00220919 secs using 99.63% CPU
Computing ML distances took 0.002287 sec (of wall-clock time) 0.002253 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 4.91142e-05 secs using 95.7% CPU
Constructing RapidNJ tree: done in 9.01222e-05 secs using 145.4% CPU
Computing RapidNJ tree took 0.000212 sec (of wall-clock time) 0.000253 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1412.123
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0128419 secs using 196.4% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.719
Optimizing NNI: done in 0.06568 secs using 196.7% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1388.719 / CPU time: 0.133
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: -1388.719
2. Current log-likelihood: -1388.216
3. Current log-likelihood: -1387.923
4. Current log-likelihood: -1387.804
5. Current log-likelihood: -1387.708
6. Current log-likelihood: -1387.627
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.513
Rate parameters:  A-C: 0.34611  A-G: 2.34591  A-T: 2.22722  C-G: 1.21436  C-T: 3.41584  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: 1.328
Parameters optimization took 7 rounds (0.029 sec)
BEST SCORE FOUND : -1387.513
Total tree length: 6.872

Total number of iterations: 2
CPU time used for tree search: 0.262 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.133 sec (0h:0m:0s)
Total CPU time used: 0.409 sec (0h:0m:0s)
Total wall-clock time used: 0.233 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 5 gave best log-likelihood: -1387.420
Total CPU time for 10 runs: 4.492 seconds.
Total wall-clock time for 10 runs: 2.585 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree.log

Date and Time: Mon Feb 21 12:28:00 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-archive-831ch4_r/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpic_js0rc/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

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

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

  3. local bootstrap test via --p-lbp [INT >= 1000]

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-kqe1pbtc/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9s9mgz9/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    716339 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:28:10 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-archive-kqe1pbtc/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000234127 secs using 85% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000509024 secs using 78.78% 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 8.10623e-05 secs using 75.25% CPU

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 0.469 / LogL: -1762.982
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.028, 0.522 / LogL: -1762.832
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.040, 0.538 / LogL: -1762.553
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.042, 0.534 / LogL: -1762.119
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.041, 0.527 / LogL: -1761.641
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.043, 0.523 / LogL: -1761.188
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.045, 0.517 / LogL: -1760.729
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.046, 0.512 / LogL: -1760.260
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.047, 0.505 / LogL: -1759.772
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.048, 0.498 / LogL: -1759.279
Optimal pinv,alpha: 0.048, 0.498 / LogL: -1759.279

Parameters optimization took 0.492 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00219703 secs using 99.13% CPU
Computing ML distances took 0.002282 sec (of wall-clock time) 0.002235 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 5.98431e-05 secs using 86.89% CPU
Constructing RapidNJ tree: done in 0.000101089 secs using 150.4% CPU
Computing RapidNJ tree took 0.000228 sec (of wall-clock time) 0.000256 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1407.925
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.037 second
Computing log-likelihood of 98 initial trees ... 0.148 seconds
Current best score: -1407.925

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.012892 secs using 146.4% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.668
Optimizing NNI: done in 0.0815451 secs using 99.71% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1387.498
Optimizing NNI: done in 0.0810869 secs using 99.91% CPU
Optimizing NNI: done in 0.074126 secs using 99.96% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 4: -1387.310
Optimizing NNI: done in 0.0760131 secs using 99.86% CPU
Optimizing NNI: done in 0.062387 secs using 99.96% CPU
Optimizing NNI: done in 0.0753469 secs using 99.97% CPU
Optimizing NNI: done in 0.0787661 secs using 99.96% CPU
Optimizing NNI: done in 0.0756319 secs using 99.96% CPU
Optimizing NNI: done in 0.0810931 secs using 99.97% CPU
Iteration 10 / LogL: -1507.358 / Time: 0h:0m:1s
Optimizing NNI: done in 0.0763841 secs using 99.96% CPU
Optimizing NNI: done in 0.0473402 secs using 99.9% CPU
Optimizing NNI: done in 0.09003 secs using 99.92% CPU
Optimizing NNI: done in 0.078732 secs using 99.93% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.0513439 secs using 99.95% CPU
Optimizing NNI: done in 0.0851171 secs using 99.9% CPU
Optimizing NNI: done in 0.0783741 secs using 99.96% CPU
Optimizing NNI: done in 0.0659561 secs using 99.97% CPU
Optimizing NNI: done in 0.0805922 secs using 99.96% CPU
Optimizing NNI: done in 0.0702441 secs using 99.86% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Iteration 20 / LogL: -1393.172 / Time: 0h:0m:2s
Finish initializing candidate tree set (19)
Current best tree score: -1387.310 / CPU time: 1.690
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.0517349 secs using 99.71% CPU
Optimizing NNI: done in 0.0195539 secs using 99.95% CPU
Optimizing NNI: done in 0.0162179 secs using 99.97% CPU
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Optimizing NNI: done in 0.0267608 secs using 99.96% CPU
Iteration 30 / LogL: -1387.507 / Time: 0h:0m:2s (0h:0m:6s left)
Optimizing NNI: done in 0.030647 secs using 99.96% CPU
Optimizing NNI: done in 0.023025 secs using 99.96% CPU
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Optimizing NNI: done in 0.0284681 secs using 99.96% CPU
Optimizing NNI: done in 0.0346348 secs using 99.95% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.309
Optimizing NNI: done in 0.0305219 secs using 99.84% CPU
Optimizing NNI: done in 0.0432711 secs using 99.96% CPU
Optimizing NNI: done in 0.063673 secs using 99.92% CPU
Optimizing NNI: done in 0.0211139 secs using 99.97% CPU
Iteration 40 / LogL: -1387.479 / Time: 0h:0m:2s (0h:0m:4s left)
Optimizing NNI: done in 0.016181 secs using 99.97% CPU
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Iteration 50 / LogL: -1396.690 / Time: 0h:0m:3s (0h:0m:3s left)
Optimizing NNI: done in 0.0316188 secs using 99.96% CPU
Optimizing NNI: done in 0.0221789 secs using 99.95% CPU
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Optimizing NNI: done in 0.0148151 secs using 99.96% CPU
Iteration 60 / LogL: -1387.309 / Time: 0h:0m:3s (0h:0m:2s left)
Optimizing NNI: done in 0.039804 secs using 99.96% CPU
Optimizing NNI: done in 0.0364778 secs using 99.96% CPU
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Optimizing NNI: done in 0.020098 secs using 99.96% CPU
Optimizing NNI: done in 0.0260601 secs using 99.97% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.309
Optimizing NNI: done in 0.0428321 secs using 99.87% CPU
Optimizing NNI: done in 0.031939 secs using 99.97% CPU
Iteration 70 / LogL: -1387.480 / Time: 0h:0m:3s (0h:0m:1s left)
Optimizing NNI: done in 0.02953 secs using 99.96% CPU
Optimizing NNI: done in 0.0507631 secs using 99.96% CPU
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Optimizing NNI: done in 0.0479362 secs using 99.96% CPU
Iteration 80 / LogL: -1387.335 / Time: 0h:0m:4s (0h:0m:1s left)
Optimizing NNI: done in 0.0221879 secs using 99.97% CPU
Optimizing NNI: done in 0.0438969 secs using 99.8% CPU
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Optimizing NNI: done in 0.0331299 secs using 99.96% CPU
Iteration 90 / LogL: -1387.342 / Time: 0h:0m:4s (0h:0m:0s left)
Optimizing NNI: done in 0.0203331 secs using 99.96% CPU
Optimizing NNI: done in 0.00887489 secs using 99.94% CPU
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Optimizing NNI: done in 0.0284669 secs using 99.96% CPU
Optimizing NNI: done in 0.0241051 secs using 99.87% CPU
Iteration 100 / LogL: -1387.722 / Time: 0h:0m:4s (0h:0m:0s left)
Optimizing NNI: done in 0.0293961 secs using 99.86% CPU
Optimizing NNI: done in 0.0153511 secs using 99.95% CPU
Optimizing NNI: done in 0.0292881 secs using 99.96% CPU
Optimizing NNI: done in 0.0328581 secs using 99.96% CPU
Optimizing NNI: done in 0.0261972 secs using 99.96% CPU
TREE SEARCH COMPLETED AFTER 105 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.309
2. Current log-likelihood: -1387.285
3. Current log-likelihood: -1387.265
4. Current log-likelihood: -1387.249
5. Current log-likelihood: -1387.236
6. Current log-likelihood: -1387.225
Optimal log-likelihood: -1387.216
Rate parameters:  A-C: 0.35235  A-G: 2.33149  A-T: 2.15517  C-G: 1.24020  C-T: 3.23605  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.001
Gamma shape alpha: 1.288
Parameters optimization took 6 rounds (0.020 sec)
BEST SCORE FOUND : -1387.216

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...
0.056 sec.
Total tree length: 7.618

Total number of iterations: 105
CPU time used for tree search: 4.429 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.251 sec (0h:0m:4s)
Total CPU time used: 5.021 sec (0h:0m:5s)
Total wall-clock time used: 4.839 sec (0h:0m:4s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9s9mgz9/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9s9mgz9/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9s9mgz9/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9s9mgz9/q2iqtree.log

Date and Time: Mon Feb 21 12:28:15 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-archive-kqe1pbtc/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9s9mgz9/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

Tip

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-ledkmtyg/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    359207 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:28:25 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-archive-ledkmtyg/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000234127 secs using 85.85% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000464916 secs using 78.08% 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 1.78814e-05 secs using 89.48% 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 73.23% CPU


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Optimizing NNI: done in 0.0886381 secs using 99.95% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1524.136
2. Current log-likelihood: -1517.825
3. Current log-likelihood: -1515.203
Optimal log-likelihood: -1513.848
Rate parameters:  A-C: 0.21227  A-G: 2.66119  A-T: 2.92875  C-G: 1.46743  C-T: 4.51073  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.041
Gamma shape alpha: 0.813
Parameters optimization took 3 rounds (0.030 sec)
Time for fast ML tree search: 0.158 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 299 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1542.305     45  3174.611     3199.253     3326.079
  2  GTR+F+I       1533.642     46  3159.285     3185.177     3314.120
  3  GTR+F+G4      1512.431     46  3116.862     3142.754     3271.697
  4  GTR+F+I+G4    1512.774     47  3119.548     3146.729     3277.749
  5  GTR+F+R2      1509.949     47  3113.897     3141.078     3272.098
  6  GTR+F+R3      1508.490     49  3114.980     3144.858     3279.913
 16  SYM+G4        1513.646     43  3113.291     3135.550     3258.028
 17  SYM+I+G4      1513.917     44  3115.835     3139.267     3263.938
 18  SYM+R2        1511.175     44  3110.351     3133.782     3258.453
 19  SYM+R3        1510.381     46  3112.762     3138.654     3267.597
 29  TVM+F+G4      1514.066     45  3118.132     3142.774     3269.601
 30  TVM+F+I+G4    1514.265     46  3120.530     3146.422     3275.365
 31  TVM+F+R2      1511.878     46  3115.757     3141.649     3270.592
 32  TVM+F+R3      1510.317     48  3116.635     3145.144     3278.201
 42  TVMe+G4       1513.648     42  3111.296     3132.419     3252.667
 43  TVMe+I+G4     1513.871     43  3113.741     3136.000     3258.478
 44  TVMe+R2       1511.238     43  3108.475     3130.734     3253.212
 45  TVMe+R3       1510.422     45  3110.845     3135.488     3262.314
 55  TIM3+F+G4     1518.225     44  3124.451     3147.883     3272.554
 56  TIM3+F+I+G4   1518.605     45  3127.210     3151.853     3278.679
 57  TIM3+F+R2     1515.166     45  3120.333     3144.975     3271.802
 58  TIM3+F+R3     1514.111     47  3122.223     3149.403     3280.423
 68  TIM3e+G4      1518.501     41  3119.002     3139.025     3257.007
 69  TIM3e+I+G4    1518.773     42  3121.546     3142.669     3262.917
 70  TIM3e+R2      1515.624     42  3115.247     3136.370     3256.618
 71  TIM3e+R3      1514.901     44  3117.801     3141.233     3265.904
 81  TIM2+F+G4     1520.982     44  3129.964     3153.396     3278.067
 82  TIM2+F+I+G4   1521.300     45  3132.600     3157.243     3284.069
 83  TIM2+F+R2     1518.617     45  3127.235     3151.878     3278.704
 84  TIM2+F+R3     1516.942     47  3127.885     3155.066     3286.086
 94  TIM2e+G4      1526.233     41  3134.466     3154.490     3272.471
 95  TIM2e+I+G4    1526.417     42  3136.835     3157.957     3278.206
 96  TIM2e+R2      1524.217     42  3132.435     3153.558     3273.806
 97  TIM2e+R3      1522.909     44  3133.818     3157.250     3281.921
107  TIM+F+G4      1516.284     44  3120.567     3143.999     3268.670
108  TIM+F+I+G4    1516.585     45  3123.170     3147.813     3274.639
109  TIM+F+R2      1514.514     45  3119.028     3143.670     3270.497
110  TIM+F+R3      1512.216     47  3118.432     3145.613     3276.633
120  TIMe+G4       1522.561     41  3127.121     3147.144     3265.126
121  TIMe+I+G4     1522.763     42  3129.526     3150.648     3270.897
122  TIMe+R2       1520.529     42  3125.058     3146.181     3266.429
123  TIMe+R3       1518.921     44  3125.843     3149.275     3273.946
133  TPM3u+F+G4    1519.736     43  3125.471     3147.730     3270.208
134  TPM3u+F+I+G4  1519.965     44  3127.931     3151.363     3276.034
135  TPM3u+F+R2    1516.896     44  3121.793     3145.225     3269.896
136  TPM3u+F+R3    1515.886     46  3123.773     3149.665     3278.608
146  TPM3+G4       1518.503     40  3117.007     3135.966     3251.646
147  TPM3+I+G4     1518.778     41  3119.556     3139.579     3257.561
148  TPM3+R2       1515.686     41  3113.372     3133.395     3251.377
149  TPM3+R3       1514.937     43  3115.874     3138.132     3260.611
159  TPM2u+F+G4    1522.774     43  3131.548     3153.807     3276.285
160  TPM2u+F+I+G4  1522.935     44  3133.869     3157.301     3281.972
161  TPM2u+F+R2    1520.655     44  3129.310     3152.742     3277.413
162  TPM2u+F+R3    1519.227     46  3130.453     3156.345     3285.288
172  TPM2+G4       1526.248     40  3132.495     3151.455     3267.134
173  TPM2+I+G4     1526.397     41  3134.794     3154.817     3272.799
174  TPM2+R2       1524.286     41  3130.572     3150.595     3268.577
175  TPM2+R3       1523.068     43  3132.137     3154.395     3276.874
185  K3Pu+F+G4     1518.530     43  3123.060     3145.319     3267.797
186  K3Pu+F+I+G4   1518.700     44  3125.399     3148.831     3273.502
187  K3Pu+F+R2     1516.927     44  3121.854     3145.286     3269.957
188  K3Pu+F+R3     1514.831     46  3121.663     3147.555     3276.498
198  K3P+G4        1522.597     40  3125.194     3144.154     3259.833
199  K3P+I+G4      1522.756     41  3127.512     3147.535     3265.517
200  K3P+R2        1520.703     41  3123.406     3143.430     3261.411
201  K3P+R3        1519.161     43  3124.323     3146.581     3269.060
211  TN+F+G4       1521.251     43  3128.501     3150.760     3273.238
212  TN+F+I+G4     1521.544     44  3131.087     3154.519     3279.190
213  TN+F+R2       1518.893     44  3125.786     3149.218     3273.889
214  TN+F+R3       1517.379     46  3126.758     3152.650     3281.593
224  TNe+G4        1526.252     40  3132.504     3151.464     3267.143
225  TNe+I+G4      1526.418     41  3134.835     3154.859     3272.840
226  TNe+R2        1524.087     41  3130.173     3150.196     3268.178
227  TNe+R3        1522.903     43  3131.807     3154.066     3276.544
237  HKY+F+G4      1523.019     42  3130.037     3151.160     3271.408
238  HKY+F+I+G4    1523.218     43  3132.435     3154.694     3277.172
239  HKY+F+R2      1521.075     43  3128.150     3150.409     3272.887
240  HKY+F+R3      1519.620     45  3129.239     3153.882     3280.708
250  K2P+G4        1526.267     39  3130.534     3148.465     3261.807
251  K2P+I+G4      1526.420     40  3132.840     3151.799     3267.479
252  K2P+R2        1524.288     40  3128.577     3147.536     3263.216
253  K2P+R3        1523.064     42  3130.128     3151.250     3271.499
263  F81+F+G4      1532.526     41  3147.051     3167.075     3285.056
264  F81+F+I+G4    1532.701     42  3149.403     3170.526     3290.774
265  F81+F+R2      1531.031     42  3146.062     3167.185     3287.433
266  F81+F+R3      1529.558     44  3147.116     3170.548     3295.219
276  JC+G4         1535.871     38  3147.741     3164.679     3275.648
277  JC+I+G4       1535.946     39  3149.892     3167.823     3281.165
278  JC+R2         1534.598     39  3147.196     3165.127     3278.469
279  JC+R3         1533.192     41  3148.385     3168.408     3286.390
289  UNREST+FO+G4  1509.112     50  3118.223     3149.512     3286.522
290  UNREST+FO+I+G4 1509.177     51  3120.354     3153.094     3292.018
291  UNREST+FO+R2  1506.908     51  3115.816     3148.557     3287.481
292  UNREST+FO+R3  1505.875     53  3117.750     3153.525     3296.147
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree.model.gz
CPU time for ModelFinder: 2.583 seconds (0h:0m:2s)
Wall-clock time for ModelFinder: 2.599 seconds (0h:0m:2s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1515.686
Optimal log-likelihood: -1515.671
Rate parameters:  A-C: 0.31536  A-G: 1.39782  A-T: 1.00000  C-G: 0.31536  C-T: 1.39782  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.670,0.272) (0.330,2.481)
Parameters optimization took 1 rounds (0.006 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00116205 secs using 98.19% CPU
Computing ML distances took 0.001256 sec (of wall-clock time) 0.001210 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 7.20024e-05 secs using 87.5% CPU
Constructing RapidNJ tree: done in 0.000121117 secs using 147% CPU
Computing RapidNJ tree took 0.000276 sec (of wall-clock time) 0.000334 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1403.144
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.034 second
Computing log-likelihood of 98 initial trees ... 0.106 seconds
Current best score: -1403.144

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.0108979 secs using 196.9% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.328
Optimizing NNI: done in 0.0171239 secs using 196.8% CPU
Optimizing NNI: done in 0.051857 secs using 109% CPU
Optimizing NNI: done in 0.032558 secs using 99.94% CPU
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Optimizing NNI: done in 0.0499208 secs using 99.93% CPU
Optimizing NNI: done in 0.0438759 secs using 99.88% CPU
BETTER TREE FOUND at iteration 7: -1385.327
Optimizing NNI: done in 0.0481269 secs using 99.95% CPU
Optimizing NNI: done in 0.0381591 secs using 99.93% CPU
Optimizing NNI: done in 0.042057 secs using 99.95% CPU
Iteration 10 / LogL: -1409.351 / Time: 0h:0m:0s
Optimizing NNI: done in 0.0355461 secs using 99.95% CPU
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Optimizing NNI: done in 0.0551362 secs using 99.96% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.0380161 secs using 99.96% CPU
Iteration 20 / LogL: -1386.004 / Time: 0h:0m:0s
Finish initializing candidate tree set (20)
Current best tree score: -1385.327 / CPU time: 0.950
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.00901413 secs using 99.92% CPU
Optimizing NNI: done in 0.00455809 secs using 98.88% CPU
Optimizing NNI: done in 0.0139551 secs using 99.95% CPU
Optimizing NNI: done in 0.0125482 secs using 99.79% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.326
Optimizing NNI: done in 0.019134 secs using 99.78% CPU
Optimizing NNI: done in 0.01263 secs using 99.79% CPU
Optimizing NNI: done in 0.00788689 secs using 99.96% CPU
Optimizing NNI: done in 0.011157 secs using 99.94% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.326
Optimizing NNI: done in 0.016407 secs using 99.75% CPU
Optimizing NNI: done in 0.00925994 secs using 99.85% CPU
Iteration 30 / LogL: -1385.884 / Time: 0h:0m:1s (0h:0m:6s left)
Optimizing NNI: done in 0.0103621 secs using 99.93% CPU
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Optimizing NNI: done in 0.011219 secs using 99.93% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.326
Optimizing NNI: done in 0.0105472 secs using 99.94% CPU
Optimizing NNI: done in 0.0090692 secs using 99.88% CPU
Optimizing NNI: done in 0.0107009 secs using 99.95% CPU
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Iteration 40 / LogL: -1385.720 / Time: 0h:0m:1s (0h:0m:5s left)
Optimizing NNI: done in 0.00580311 secs using 99.93% CPU
Optimizing NNI: done in 0.013799 secs using 99.94% CPU
Optimizing NNI: done in 0.012291 secs using 99.92% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.326
Optimizing NNI: done in 0.0140688 secs using 99.65% CPU
Optimizing NNI: done in 0.0127261 secs using 99.83% CPU
Optimizing NNI: done in 0.00957084 secs using 99.93% CPU
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Iteration 50 / LogL: -1385.720 / Time: 0h:0m:1s (0h:0m:4s left)
Optimizing NNI: done in 0.0127289 secs using 99.95% CPU
Optimizing NNI: done in 0.012085 secs using 99.95% CPU
Optimizing NNI: done in 0.00462604 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.325
Optimizing NNI: done in 0.00550508 secs using 99.93% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.325
Optimizing NNI: done in 0.00711799 secs using 99.13% CPU
Optimizing NNI: done in 0.00678706 secs using 99.85% CPU
Optimizing NNI: done in 0.0161829 secs using 99.9% CPU
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Iteration 60 / LogL: -1385.325 / Time: 0h:0m:1s (0h:0m:3s left)
Optimizing NNI: done in 0.011529 secs using 99.94% CPU
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Iteration 70 / LogL: -1385.723 / Time: 0h:0m:1s (0h:0m:3s left)
Optimizing NNI: done in 0.0125551 secs using 99.95% CPU
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Iteration 80 / LogL: -1385.328 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.012655 secs using 99.94% CPU
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Optimizing NNI: done in 0.0088551 secs using 99.91% CPU
Iteration 90 / LogL: -1385.326 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.014478 secs using 99.95% CPU
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Optimizing NNI: done in 0.01074 secs using 99.93% CPU
Iteration 100 / LogL: -1385.326 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.0113602 secs using 99.95% CPU
Optimizing NNI: done in 0.00903487 secs using 99.95% CPU
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Iteration 110 / LogL: -1385.422 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0085659 secs using 99.94% CPU
Optimizing NNI: done in 0.00490904 secs using 99.96% CPU
Optimizing NNI: done in 0.016294 secs using 99.93% CPU
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Iteration 120 / LogL: -1385.329 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0128119 secs using 99.95% CPU
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Iteration 130 / LogL: -1385.402 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0128062 secs using 99.92% CPU
Optimizing NNI: done in 0.0147841 secs using 99.95% CPU
Optimizing NNI: done in 0.0159888 secs using 99.94% CPU
Optimizing NNI: done in 0.0100651 secs using 99.92% CPU
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Optimizing NNI: done in 0.00879192 secs using 99.94% CPU
Iteration 140 / LogL: -1385.336 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0152502 secs using 99.93% CPU
Optimizing NNI: done in 0.0108879 secs using 99.95% CPU
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Iteration 150 / LogL: -1385.327 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.0126009 secs using 99.92% CPU
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Iteration 160 / LogL: -1385.329 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.00757813 secs using 99.92% CPU
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Iteration 170 / LogL: -1385.325 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.013927 secs using 99.93% CPU
Optimizing NNI: done in 0.0151989 secs using 99.96% CPU
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Iteration 180 / LogL: -1385.497 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.011868 secs using 99.92% CPU
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Iteration 190 / LogL: -1385.329 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0139549 secs using 99.94% CPU
Optimizing NNI: done in 0.00569296 secs using 99.95% CPU
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Iteration 200 / LogL: -1385.327 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0126691 secs using 99.93% CPU
Optimizing NNI: done in 0.0127339 secs using 99.95% CPU
Optimizing NNI: done in 0.0132451 secs using 99.94% CPU
Optimizing NNI: done in 0.0106249 secs using 99.94% CPU
Optimizing NNI: done in 0.0111001 secs using 99.93% CPU
Optimizing NNI: done in 0.010658 secs using 99.92% CPU
Optimizing NNI: done in 0.00711107 secs using 99.97% CPU
Optimizing NNI: done in 0.0105538 secs using 99.93% CPU
TREE SEARCH COMPLETED AFTER 208 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.325
2. Current log-likelihood: -1385.314
Optimal log-likelihood: -1385.307
Rate parameters:  A-C: 0.39478  A-G: 1.57080  A-T: 1.00000  C-G: 0.39478  C-T: 1.57080  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.719,0.397) (0.281,2.542)
Parameters optimization took 2 rounds (0.007 sec)
BEST SCORE FOUND : -1385.307
Total tree length: 6.922

Total number of iterations: 208
CPU time used for tree search: 3.366 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.190 sec (0h:0m:3s)
Total CPU time used: 3.400 sec (0h:0m:3s)
Total wall-clock time used: 3.220 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree.log

Date and Time: Mon Feb 21 12:28:31 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-archive-ledkmtyg/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfphg4jyl/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

iqtree-ultrafast-bootstrap

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

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

stdout:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-swijfhdn/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    42654 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:28:41 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-archive-swijfhdn/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000217915 secs using 81.22% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000469923 secs using 78.52% 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 84.95% 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)... 0.000 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 0.0738082 secs using 99.93% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1415.023
2. Current log-likelihood: -1409.585
3. Current log-likelihood: -1408.297
Optimal log-likelihood: -1407.632
Rate parameters:  A-C: 0.31403  A-G: 2.69368  A-T: 2.73260  C-G: 1.45424  C-T: 3.88931  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.036
Gamma shape alpha: 0.821
Parameters optimization took 3 rounds (0.023 sec)
Time for fast ML tree search: 0.133 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 299 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1434.388     45  2958.775     2983.418     3110.244
  2  GTR+F+I       1428.598     46  2949.196     2975.088     3104.031
  3  GTR+F+G4      1406.624     46  2905.248     2931.140     3060.083
  4  GTR+F+I+G4    1406.960     47  2907.919     2935.100     3066.120
  5  GTR+F+R2      1406.162     47  2906.324     2933.505     3064.525
  6  GTR+F+R3      1405.397     49  2908.794     2938.672     3073.727
 16  SYM+G4        1406.038     43  2898.076     2920.335     3042.813
 17  SYM+I+G4      1406.329     44  2900.658     2924.090     3048.761
 18  SYM+R2        1405.917     44  2899.834     2923.266     3047.937
 29  TVM+F+G4      1407.601     45  2905.202     2929.845     3056.671
 30  TVM+F+I+G4    1407.818     46  2907.636     2933.528     3062.471
 31  TVM+F+R2      1407.037     46  2906.074     2931.966     3060.909
 42  TVMe+G4       1406.052     42  2896.105     2917.228     3037.476
 43  TVMe+I+G4     1406.378     43  2898.756     2921.015     3043.493
 44  TVMe+R2       1405.939     43  2897.878     2920.137     3042.615
 55  TIM3+F+G4     1411.680     44  2911.359     2934.791     3059.462
 56  TIM3+F+I+G4   1412.041     45  2914.083     2938.725     3065.552
 57  TIM3+F+R2     1410.654     45  2911.308     2935.951     3062.777
 68  TIM3e+G4      1409.891     41  2901.783     2921.806     3039.788
 69  TIM3e+I+G4    1410.235     42  2904.470     2925.592     3045.841
 70  TIM3e+R2      1409.455     42  2902.909     2924.032     3044.280
 81  TIM2+F+G4     1413.631     44  2915.263     2938.695     3063.366
 82  TIM2+F+I+G4   1413.935     45  2917.870     2942.513     3069.339
 83  TIM2+F+R2     1412.668     45  2915.336     2939.979     3066.805
 94  TIM2e+G4      1416.338     41  2914.677     2934.700     3052.682
 95  TIM2e+I+G4    1416.529     42  2917.059     2938.182     3058.430
 96  TIM2e+R2      1415.338     42  2914.676     2935.798     3056.047
107  TIM+F+G4      1409.708     44  2907.416     2930.848     3055.519
108  TIM+F+I+G4    1410.020     45  2910.039     2934.682     3061.508
109  TIM+F+R2      1408.839     45  2907.678     2932.321     3059.147
120  TIMe+G4       1412.985     41  2907.970     2927.993     3045.975
121  TIMe+I+G4     1413.204     42  2910.408     2931.531     3051.779
122  TIMe+R2       1412.180     42  2908.360     2929.483     3049.731
133  TPM3u+F+G4    1412.579     43  2911.159     2933.417     3055.896
134  TPM3u+F+I+G4  1412.898     44  2913.796     2937.228     3061.899
135  TPM3u+F+R2    1411.509     44  2911.018     2934.450     3059.121
146  TPM3+G4       1409.920     40  2899.840     2918.800     3034.479
147  TPM3+I+G4     1410.222     41  2902.444     2922.468     3040.449
148  TPM3+R2       1409.475     41  2900.949     2920.973     3038.954
159  TPM2u+F+G4    1414.513     43  2915.027     2937.285     3059.764
160  TPM2u+F+I+G4  1414.706     44  2917.412     2940.844     3065.515
161  TPM2u+F+R2    1413.432     44  2914.865     2938.297     3062.968
172  TPM2+G4       1416.372     40  2912.744     2931.703     3047.383
173  TPM2+I+G4     1416.576     41  2915.151     2935.175     3053.156
174  TPM2+R2       1415.392     41  2912.785     2932.808     3050.790
185  K3Pu+F+G4     1410.801     43  2907.602     2929.861     3052.339
186  K3Pu+F+I+G4   1410.996     44  2909.992     2933.423     3058.094
187  K3Pu+F+R2     1409.746     44  2907.492     2930.924     3055.595
198  K3P+G4        1412.990     40  2905.980     2924.939     3040.619
199  K3P+I+G4      1413.209     41  2908.419     2928.442     3046.424
200  K3P+R2        1412.217     41  2906.434     2926.457     3044.439
211  TN+F+G4       1414.297     43  2914.594     2936.852     3059.330
212  TN+F+I+G4     1414.624     44  2917.247     2940.679     3065.350
213  TN+F+R2       1413.063     44  2914.127     2937.559     3062.230
224  TNe+G4        1416.377     40  2912.754     2931.713     3047.393
225  TNe+I+G4      1416.521     41  2915.042     2935.066     3053.047
226  TNe+R2        1415.337     41  2912.673     2932.697     3050.679
237  HKY+F+G4      1415.198     42  2914.396     2935.519     3055.767
238  HKY+F+I+G4    1415.411     43  2916.822     2939.080     3061.559
239  HKY+F+R2      1413.820     43  2913.641     2935.900     3058.378
250  K2P+G4        1416.427     39  2910.854     2928.785     3042.127
251  K2P+I+G4      1416.628     40  2913.255     2932.215     3047.894
252  K2P+R2        1415.391     40  2910.782     2929.741     3045.421
263  F81+F+G4      1424.177     41  2930.354     2950.377     3068.359
264  F81+F+I+G4    1424.331     42  2932.663     2953.785     3074.034
265  F81+F+R2      1423.107     42  2930.213     2951.336     3071.584
276  JC+G4         1425.391     38  2926.783     2943.720     3054.690
277  JC+I+G4       1425.387     39  2928.774     2946.705     3060.048
278  JC+R2         1424.585     39  2927.170     2945.101     3058.443
289  UNREST+FO+G4  1403.073     50  2906.146     2937.434     3074.444
290  UNREST+FO+I+G4 1403.350     51  2908.700     2941.441     3080.365
291  UNREST+FO+R2  1403.013     51  2908.026     2940.767     3079.691
Akaike Information Criterion:           TVMe+G4
Corrected Akaike Information Criterion: TVMe+G4
Bayesian Information Criterion:         TPM3+G4
Best-fit model: TPM3+G4 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 1.622 seconds (0h:0m:1s)
Wall-clock time for ModelFinder: 1.628 seconds (0h:0m:1s)
Generating 1000 samples for ultrafast bootstrap (seed: 42654)...

NOTE: 1 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1409.920
Optimal log-likelihood: -1409.906
Rate parameters:  A-C: 0.34522  A-G: 1.45352  A-T: 1.00000  C-G: 0.34522  C-T: 1.45352  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Gamma shape alpha: 0.891
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 0.00172615 secs using 98.77% CPU
Computing ML distances took 0.001822 sec (of wall-clock time) 0.001778 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 7.20024e-05 secs using 109.7% CPU
Constructing RapidNJ tree: done in 0.00012207 secs using 154.8% CPU
Computing RapidNJ tree took 0.000287 sec (of wall-clock time) 0.000351 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1406.537
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|             INITIALIZING CANDIDATE TREE SET                      |
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Generating 98 parsimony trees... 0.034 second
Computing log-likelihood of 98 initial trees ... 0.141 seconds
Current best score: -1406.537

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.0421259 secs using 134.7% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1390.540
Optimizing NNI: done in 0.028003 secs using 99.87% CPU
Optimizing NNI: done in 0.10122 secs using 99.91% CPU
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Iteration 10 / LogL: -1392.710 / Time: 0h:0m:1s
Optimizing NNI: done in 0.10942 secs using 99.97% CPU
Optimizing NNI: done in 0.074899 secs using 99.97% CPU
Optimizing NNI: done in 0.114695 secs using 99.97% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 13: -1390.373
Optimizing NNI: done in 0.111654 secs using 99.97% CPU
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Iteration 20 / LogL: -1569.674 / Time: 0h:0m:2s
Finish initializing candidate tree set (20)
Current best tree score: -1390.373 / CPU time: 2.035
Number of iterations: 20
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|               OPTIMIZING CANDIDATE TREE SET                      |
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Optimizing NNI: done in 0.0391622 secs using 99.83% CPU
Optimizing NNI: done in 0.0407619 secs using 99.96% CPU
Optimizing NNI: done in 0.00699711 secs using 99.91% CPU
UPDATE BEST LOG-LIKELIHOOD: -1390.372
Optimizing NNI: done in 0.041069 secs using 99.83% CPU
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UPDATE BEST LOG-LIKELIHOOD: -1390.372
Optimizing NNI: done in 0.034302 secs using 99.86% CPU
Iteration 30 / LogL: -1390.686 / Time: 0h:0m:2s (0h:0m:15s left)
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UPDATE BEST LOG-LIKELIHOOD: -1390.371
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Iteration 40 / LogL: -1390.376 / Time: 0h:0m:2s (0h:0m:11s left)
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Iteration 50 / LogL: -1390.373 / Time: 0h:0m:2s (0h:0m:9s left)
Log-likelihood cutoff on original alignment: -1413.674
Optimizing NNI: done in 0.023 secs using 99.94% CPU
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Iteration 60 / LogL: -1390.404 / Time: 0h:0m:3s (0h:0m:8s left)
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Iteration 70 / LogL: -1390.711 / Time: 0h:0m:3s (0h:0m:7s left)
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UPDATE BEST LOG-LIKELIHOOD: -1390.371
Optimizing NNI: done in 0.0323591 secs using 99.83% CPU
Iteration 80 / LogL: -1390.373 / Time: 0h:0m:3s (0h:0m:6s left)
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UPDATE BEST LOG-LIKELIHOOD: -1390.370
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Iteration 90 / LogL: -1390.407 / Time: 0h:0m:4s (0h:0m:5s left)
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Iteration 100 / LogL: -1390.585 / Time: 0h:0m:4s (0h:0m:5s left)
Log-likelihood cutoff on original alignment: -1414.492
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
Optimizing NNI: done in 0.034554 secs using 99.97% CPU
UPDATE BEST LOG-LIKELIHOOD: -1390.370
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Iteration 110 / LogL: -1390.602 / Time: 0h:0m:4s (0h:0m:4s left)
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Iteration 120 / LogL: -1390.373 / Time: 0h:0m:5s (0h:0m:4s left)
Optimizing NNI: done in 0.020443 secs using 99.95% CPU
Optimizing NNI: done in 0.028913 secs using 99.96% CPU
Optimizing NNI: done in 0.021136 secs using 99.96% CPU
Optimizing NNI: done in 0.0265591 secs using 99.96% CPU
Optimizing NNI: done in 0.0187609 secs using 99.96% CPU
UPDATE BEST LOG-LIKELIHOOD: -1390.369
Optimizing NNI: done in 0.0346291 secs using 99.84% CPU
Optimizing NNI: done in 0.0228648 secs using 99.96% CPU
Optimizing NNI: done in 0.0254972 secs using 99.96% CPU
Optimizing NNI: done in 0.0316679 secs using 99.97% CPU
Optimizing NNI: done in 0.0125818 secs using 99.98% CPU
UPDATE BEST LOG-LIKELIHOOD: -1390.369
Iteration 130 / LogL: -1390.369 / Time: 0h:0m:5s (0h:0m:3s left)
Optimizing NNI: done in 0.0289621 secs using 99.8% CPU
Optimizing NNI: done in 0.020637 secs using 99.97% CPU
Optimizing NNI: done in 0.0185909 secs using 99.96% CPU
Optimizing NNI: done in 0.0200849 secs using 99.97% CPU
Optimizing NNI: done in 0.00688601 secs using 99.94% CPU
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Optimizing NNI: done in 0.0224481 secs using 99.96% CPU
Optimizing NNI: done in 0.017555 secs using 99.97% CPU
Iteration 140 / LogL: -1390.369 / Time: 0h:0m:5s (0h:0m:3s left)
Optimizing NNI: done in 0.0140781 secs using 99.96% CPU
Optimizing NNI: done in 0.024869 secs using 99.96% CPU
Optimizing NNI: done in 0.0214739 secs using 99.97% CPU
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Optimizing NNI: done in 0.027703 secs using 99.96% CPU
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Optimizing NNI: done in 0.0295041 secs using 99.96% CPU
Optimizing NNI: done in 0.0234108 secs using 99.97% CPU
Optimizing NNI: done in 0.0242951 secs using 99.95% CPU
Optimizing NNI: done in 0.0169389 secs using 99.94% CPU
Iteration 150 / LogL: -1390.428 / Time: 0h:0m:6s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1414.492
Optimizing NNI: done in 0.0196149 secs using 99.97% CPU
Optimizing NNI: done in 0.023665 secs using 99.96% CPU
Optimizing NNI: done in 0.0168262 secs using 99.96% CPU
Optimizing NNI: done in 0.0147591 secs using 99.97% CPU
Optimizing NNI: done in 0.0251441 secs using 99.97% CPU
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Optimizing NNI: done in 0.028671 secs using 99.97% CPU
Optimizing NNI: done in 0.0506558 secs using 99.96% CPU
Optimizing NNI: done in 0.0272329 secs using 99.97% CPU
Optimizing NNI: done in 0.00648284 secs using 99.96% CPU
Iteration 160 / LogL: -1390.373 / Time: 0h:0m:6s (0h:0m:2s left)
Optimizing NNI: done in 0.013726 secs using 99.97% CPU
Optimizing NNI: done in 0.026212 secs using 99.96% CPU
Optimizing NNI: done in 0.0230341 secs using 99.96% CPU
Optimizing NNI: done in 0.00880504 secs using 99.95% CPU
Optimizing NNI: done in 0.0347118 secs using 99.41% CPU
Optimizing NNI: done in 0.0338259 secs using 99.97% CPU
Optimizing NNI: done in 0.0247409 secs using 99.97% CPU
Optimizing NNI: done in 0.0233731 secs using 99.97% CPU
Optimizing NNI: done in 0.00753903 secs using 99.96% CPU
Optimizing NNI: done in 0.020977 secs using 99.96% CPU
Iteration 170 / LogL: -1390.420 / Time: 0h:0m:6s (0h:0m:1s left)
Optimizing NNI: done in 0.0088861 secs using 99.95% CPU
UPDATE BEST LOG-LIKELIHOOD: -1390.369
Optimizing NNI: done in 0.027283 secs using 99.81% CPU
Optimizing NNI: done in 0.0349438 secs using 99.97% CPU
Optimizing NNI: done in 0.0324211 secs using 99.97% CPU
Optimizing NNI: done in 0.027003 secs using 99.96% CPU
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Optimizing NNI: done in 0.0137591 secs using 99.97% CPU
Optimizing NNI: done in 0.0291162 secs using 99.96% CPU
Optimizing NNI: done in 0.0250809 secs using 99.96% CPU
Iteration 180 / LogL: -1390.375 / Time: 0h:0m:7s (0h:0m:1s left)
Optimizing NNI: done in 0.0242782 secs using 99.97% CPU
Optimizing NNI: done in 0.0241289 secs using 99.47% CPU
Optimizing NNI: done in 0.019181 secs using 99.81% CPU
Optimizing NNI: done in 0.0124369 secs using 99.95% CPU
Optimizing NNI: done in 0.0329752 secs using 99.97% CPU
Optimizing NNI: done in 0.0228639 secs using 99.96% CPU
Optimizing NNI: done in 0.0320032 secs using 99.96% CPU
Optimizing NNI: done in 0.0130472 secs using 99.97% CPU
Optimizing NNI: done in 0.0327229 secs using 99.97% CPU
Optimizing NNI: done in 0.019959 secs using 99.97% CPU
Iteration 190 / LogL: -1390.372 / Time: 0h:0m:7s (0h:0m:0s left)
Optimizing NNI: done in 0.0150089 secs using 99.96% CPU
Optimizing NNI: done in 0.0226889 secs using 99.97% CPU
Optimizing NNI: done in 0.0223219 secs using 99.97% CPU
Optimizing NNI: done in 0.0320361 secs using 99.76% CPU
Optimizing NNI: done in 0.0305662 secs using 99.96% CPU
Optimizing NNI: done in 0.0224411 secs using 99.95% CPU
Optimizing NNI: done in 0.00651288 secs using 99.94% CPU
Optimizing NNI: done in 0.0220721 secs using 99.96% CPU
Optimizing NNI: done in 0.0278552 secs using 99.96% CPU
Optimizing NNI: done in 0.0277259 secs using 99.97% CPU
Iteration 200 / LogL: -1390.402 / Time: 0h:0m:7s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1413.443
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.995
Optimizing NNI: done in 0.0120659 secs using 99.97% CPU
Optimizing NNI: done in 0.019732 secs using 99.96% CPU
Optimizing NNI: done in 0.021955 secs using 99.97% CPU
Optimizing NNI: done in 0.02281 secs using 99.97% CPU
Optimizing NNI: done in 0.0330112 secs using 99.95% CPU
Optimizing NNI: done in 0.0152311 secs using 99.95% CPU
Optimizing NNI: done in 0.0204 secs using 99.97% CPU
Optimizing NNI: done in 0.0238101 secs using 99.96% CPU
Optimizing NNI: done in 0.0240021 secs using 99.97% CPU
Optimizing NNI: done in 0.0335569 secs using 99.97% CPU
Iteration 210 / LogL: -1390.426 / Time: 0h:0m:7s (0h:0m:3s left)
Optimizing NNI: done in 0.0260289 secs using 99.97% CPU
Optimizing NNI: done in 0.00982094 secs using 99.97% CPU
Optimizing NNI: done in 0.0170381 secs using 99.96% CPU
Optimizing NNI: done in 0.018856 secs using 99.97% CPU
TREE SEARCH COMPLETED AFTER 214 ITERATIONS / Time: 0h:0m:8s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1390.369
Optimal log-likelihood: -1390.359
Rate parameters:  A-C: 0.38543  A-G: 1.52207  A-T: 1.00000  C-G: 0.38543  C-T: 1.52207  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Gamma shape alpha: 1.408
Parameters optimization took 1 rounds (0.003 sec)
BEST SCORE FOUND : -1390.359
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.splits.nex
Total tree length: 7.081

Total number of iterations: 214
CPU time used for tree search: 8.147 sec (0h:0m:8s)
Wall-clock time used for tree search: 7.985 sec (0h:0m:7s)
Total CPU time used: 8.256 sec (0h:0m:8s)
Total wall-clock time used: 8.091 sec (0h:0m:8s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.splits.nex...
20 taxa and 163 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1390.371

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot.log

Date and Time: Mon Feb 21 12:28:50 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-archive-swijfhdn/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfdh4xk3s/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

Perform single branch tests alongside ufboot

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

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

stdout:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.2.0-beta COVID-edition for Mac OS X 64-bit built Dec 21 2021 built Dec 21 2021
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-archive-lnml3hrr/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    645505 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Mon Feb 21 12:29:00 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-archive-lnml3hrr/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.00022912 secs using 85.11% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000463009 secs using 78.62% 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 75.77% CPU

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 645505)...

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, 0.411 / LogL: -1956.473
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.020, 0.466 / LogL: -1956.320
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.028, 0.479 / LogL: -1955.766
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.030, 0.476 / LogL: -1954.993
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.029, 0.469 / LogL: -1954.124
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.034, 0.474 / LogL: -1953.466
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.036, 0.470 / LogL: -1952.629
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.037, 0.466 / LogL: -1951.816
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.038, 0.462 / LogL: -1950.914
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.039, 0.457 / LogL: -1950.061
Optimal pinv,alpha: 0.039, 0.457 / LogL: -1950.061

Parameters optimization took 0.849 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00243115 secs using 99.25% CPU
Computing ML distances took 0.002507 sec (of wall-clock time) 0.002469 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 6.00815e-05 secs using 88.21% CPU
Constructing RapidNJ tree: done in 0.000101089 secs using 146.4% CPU
Computing RapidNJ tree took 0.000233 sec (of wall-clock time) 0.000275 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1416.742
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.034 second
Computing log-likelihood of 98 initial trees ... 0.145 seconds
Current best score: -1416.742

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.01845 secs using 158.6% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.665
Optimizing NNI: done in 0.0964019 secs using 99.97% CPU
Optimizing NNI: done in 0.0871301 secs using 99.97% CPU
Optimizing NNI: done in 0.106392 secs using 99.97% CPU
Optimizing NNI: done in 0.119042 secs using 99.97% CPU
Optimizing NNI: done in 0.0896769 secs using 99.97% CPU
Optimizing NNI: done in 0.0903761 secs using 99.9% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 7: -1387.353
Optimizing NNI: done in 0.109731 secs using 99.97% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.107748 secs using 99.97% CPU
Optimizing NNI: done in 0.0728669 secs using 99.81% CPU
Iteration 10 / LogL: -1689.748 / Time: 0h:0m:2s
Optimizing NNI: done in 0.122629 secs using 99.9% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.131087 secs using 99.93% CPU
Optimizing NNI: done in 0.127225 secs using 99.91% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Optimizing NNI: done in 0.132074 secs using 99.81% CPU
Optimizing NNI: done in 0.123091 secs using 99.97% CPU
Optimizing NNI: done in 0.121484 secs using 99.97% CPU
Optimizing NNI: done in 0.085392 secs using 99.97% CPU
Optimizing NNI: done in 0.114125 secs using 99.94% CPU
Optimizing NNI: done in 0.128086 secs using 99.94% CPU
Optimizing NNI: done in 0.111441 secs using 99.96% CPU
WARNING: NNI search needs unusual large number of steps (20) to converge!
Iteration 20 / LogL: -1393.064 / Time: 0h:0m:3s
Finish initializing candidate tree set (19)
Current best tree score: -1387.353 / CPU time: 2.373
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.0509739 secs using 99.89% CPU
Optimizing NNI: done in 0.0585711 secs using 99.95% CPU
Optimizing NNI: done in 0.02564 secs using 99.97% CPU
BETTER TREE FOUND at iteration 23: -1387.353
Optimizing NNI: done in 0.05546 secs using 99.87% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.351
Optimizing NNI: done in 0.013247 secs using 99.62% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 25: -1387.298
Optimizing NNI: done in 0.0374629 secs using 99.87% CPU
Optimizing NNI: done in 0.0104868 secs using 99.92% CPU
Optimizing NNI: done in 0.0177429 secs using 99.97% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.298
Optimizing NNI: done in 0.084764 secs using 99.92% CPU
Optimizing NNI: done in 0.019603 secs using 99.97% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.298
Iteration 30 / LogL: -1387.298 / Time: 0h:0m:3s (0h:0m:24s left)
Optimizing NNI: done in 0.053633 secs using 99.89% CPU
Optimizing NNI: done in 0.0357702 secs using 99.96% CPU
Optimizing NNI: done in 0.0475202 secs using 99.96% CPU
Optimizing NNI: done in 0.02685 secs using 99.97% CPU
Optimizing NNI: done in 0.011807 secs using 99.97% CPU
Optimizing NNI: done in 0.0162642 secs using 99.62% CPU
Optimizing NNI: done in 0.062778 secs using 99.96% CPU
Optimizing NNI: done in 0.0135241 secs using 99.96% CPU
Optimizing NNI: done in 0.0187249 secs using 99.97% CPU
Optimizing NNI: done in 0.019259 secs using 99.97% CPU
Iteration 40 / LogL: -1387.298 / Time: 0h:0m:4s (0h:0m:19s left)
Optimizing NNI: done in 0.017334 secs using 99.97% CPU
Optimizing NNI: done in 0.0258701 secs using 99.96% CPU
Optimizing NNI: done in 0.019922 secs using 99.97% CPU
Optimizing NNI: done in 0.0093329 secs using 99.97% CPU
Optimizing NNI: done in 0.029635 secs using 99.97% CPU
Optimizing NNI: done in 0.00702214 secs using 99.96% CPU
Optimizing NNI: done in 0.00777888 secs using 99.98% CPU
Optimizing NNI: done in 0.033087 secs using 99.75% CPU
Optimizing NNI: done in 0.0266712 secs using 99.97% CPU
Optimizing NNI: done in 0.00771785 secs using 99.95% CPU
Iteration 50 / LogL: -1389.649 / Time: 0h:0m:4s (0h:0m:15s left)
Log-likelihood cutoff on original alignment: -1415.849
Optimizing NNI: done in 0.0121369 secs using 99.95% CPU
Optimizing NNI: done in 0.0601451 secs using 99.62% CPU
Optimizing NNI: done in 0.017432 secs using 99.95% CPU
Optimizing NNI: done in 0.033242 secs using 99.97% CPU
Optimizing NNI: done in 0.0194631 secs using 99.96% CPU
Optimizing NNI: done in 0.0133519 secs using 99.96% CPU
Optimizing NNI: done in 0.0395179 secs using 99.96% CPU
Optimizing NNI: done in 0.00930405 secs using 99.96% CPU
Optimizing NNI: done in 0.0189772 secs using 99.97% CPU
Optimizing NNI: done in 0.0225868 secs using 99.97% CPU
Iteration 60 / LogL: -1387.509 / Time: 0h:0m:4s (0h:0m:12s left)
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UPDATE BEST LOG-LIKELIHOOD: -1387.297
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Iteration 100 / LogL: -1387.309 / Time: 0h:0m:5s (0h:0m:7s left)
Log-likelihood cutoff on original alignment: -1415.849
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.987
NOTE: UFBoot does not converge, continue at least 100 more iterations
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TREE SEARCH COMPLETED AFTER 226 ITERATIONS / Time: 0h:0m:9s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.297
2. Current log-likelihood: -1387.275
3. Current log-likelihood: -1387.258
4. Current log-likelihood: -1387.243
5. Current log-likelihood: -1387.231
6. Current log-likelihood: -1387.221
Optimal log-likelihood: -1387.212
Rate parameters:  A-C: 0.34657  A-G: 2.30026  A-T: 2.12337  C-G: 1.22217  C-T: 3.18610  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.001
Gamma shape alpha: 1.290
Parameters optimization took 6 rounds (0.020 sec)
BEST SCORE FOUND : -1387.212

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.055 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.splits.nex
Total tree length: 7.604

Total number of iterations: 226
CPU time used for tree search: 8.889 sec (0h:0m:8s)
Wall-clock time used for tree search: 8.728 sec (0h:0m:8s)
Total CPU time used: 9.916 sec (0h:0m:9s)
Total wall-clock time used: 9.756 sec (0h:0m:9s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.splits.nex...
20 taxa and 173 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.213

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/q2iqtreeufboot.log

Date and Time: Mon Feb 21 12:29:10 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-archive-lnml3hrr/d8ca197e-74a6-4812-bb37-1ce969a79906/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpfcpmvo7j/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!