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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 Exact Sequence Variants (ESVs) 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/2020.6/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2020.6/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 7092 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-c1f18dg8/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp845ry07c -n q2 


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

Inference[0]: Time 0.486469 CAT-based likelihood -1260.366573, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1387.515474 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp845ry07c/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.515474 .... 

Final GAMMA-based Score of best tree -1387.322305

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp845ry07c/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp845ry07c/RAxML_bestTree.q2

Overall execution time: 1.102060 secs or 0.000306 hours or 0.000013 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 7092 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-c1f18dg8/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp845ry07c -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-19hriesj/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2 -n q2 


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

Inference[0]: Time 0.629416 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.515001 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.519138 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.668655 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.503173 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/tmpwsu38uv2/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2/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/tmpwsu38uv2/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2/RAxML_bestTree.q2

Overall execution time: 3.583277 secs or 0.000995 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-19hriesj/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpwsu38uv2 -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-oexl3o75/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a -n q2bootstrap 



Time for BS model parameter optimization 0.056078
Bootstrap[0]: Time 0.185074 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.121611 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.107505 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.089611 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.112485 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.113935 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.111682 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.101907 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.110658 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.115804 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.082405 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.123558 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.133073 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.107332 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.114167 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.114152 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.111000 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.101283 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.108047 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.107751 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.096659 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.093042 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.105184 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.125952 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.105803 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.105963 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.109685 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.136913 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.139343 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.088225 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.112265 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.112991 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.119235 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.105518 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.100942 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.121336 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.101479 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.121082 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.097355 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.088922 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.110313 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.111957 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.104273 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.129481 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.100915 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.110257 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.089835 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.134818 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.087453 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.117611 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.134486 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.098659 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.114902 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.099655 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.114255 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.118403 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.099021 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.093381 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.138287 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.103345 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.093473 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.108319 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.125102 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.098949 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.095430 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.101183 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.119905 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.122746 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.093321 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.099022 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.102629 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.101329 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.087596 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.104316 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.090045 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.103983 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.109994 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.133881 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.114246 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.113244 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.096659 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.110906 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.123701 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.097868 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.107620 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.113322 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.095103 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.106982 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.109915 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.105585 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.114366 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.111256 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.046022 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.119051 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.097659 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.095031 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.114128 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.114778 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.058552 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.056111 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.799327 seconds
Average Time per Rapid Bootstrap 0.107993 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.352431 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: 2.223355 seconds
Thorough ML search Time: 0.584558 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 7.164111 secs or 0.001990 hours

Combined Bootstrap and ML search took 17.963752 secs or 0.004990 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.973583 secs or 0.004993 hours or 0.000208 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-oexl3o75/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpiqqiko1a -n q2bootstrap

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

Output artifacts:

Tip

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

iqtree

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

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-3rh2tljy/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree -nt 1
Seed:    376420 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:54:17 2020
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-3rh2tljy/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.605
Optimal log-likelihood: -1388.793
Rate parameters:  A-C: 0.37543  A-G: 2.37167  A-T: 2.15334  C-G: 1.24272  C-T: 3.32366  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.400
Parameters optimization took 1 rounds (0.006 sec)
Time for fast ML tree search: 0.068 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.600     45  2895.200     2919.843     3046.669
  2  GTR+F+I       1401.121     46  2894.242     2920.135     3049.077
  3  GTR+F+G4      1387.358     46  2866.716     2892.609     3021.551
  4  GTR+F+I+G4    1387.726     47  2869.452     2896.633     3027.653
  5  GTR+F+R2      1382.364     47  2858.729     2885.910     3016.930
  6  GTR+F+R3      1382.420     49  2862.840     2892.718     3027.773
 16  SYM+G4        1387.134     43  2860.269     2882.528     3005.006
 18  SYM+R2        1383.095     44  2854.189     2877.621     3002.292
 29  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 31  TVM+F+R2      1383.789     46  2859.578     2885.470     3014.413
 42  TVMe+G4       1387.122     42  2858.245     2879.368     2999.616
 44  TVMe+R2       1383.079     43  2852.159     2874.418     2996.896
 55  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 57  TIM3+F+R2     1385.912     45  2861.825     2886.468     3013.294
 68  TIM3e+G4      1390.358     41  2862.715     2882.738     3000.720
 70  TIM3e+R2      1385.918     42  2855.836     2876.959     2997.207
 81  TIM2+F+G4     1393.635     44  2875.270     2898.702     3023.373
 83  TIM2+F+R2     1387.681     45  2865.362     2890.005     3016.831
 94  TIM2e+G4      1396.795     41  2875.589     2895.613     3013.594
 96  TIM2e+R2      1391.574     42  2867.148     2888.270     3008.519
107  TIM+F+G4      1390.363     44  2868.726     2892.158     3016.829
109  TIM+F+R2      1384.933     45  2859.866     2884.509     3011.335
120  TIMe+G4       1394.002     41  2870.005     2890.028     3008.010
122  TIMe+R2       1389.000     42  2862.000     2883.123     3003.371
133  TPM3u+F+G4    1392.306     43  2870.611     2892.870     3015.348
135  TPM3u+F+R2    1387.329     44  2862.659     2886.091     3010.762
146  TPM3+F+G4     1392.306     43  2870.611     2892.870     3015.348
148  TPM3+F+R2     1387.329     44  2862.659     2886.091     3010.762
159  TPM2u+F+G4    1394.536     43  2875.071     2897.330     3019.808
161  TPM2u+F+R2    1389.054     44  2866.107     2889.539     3014.210
172  TPM2+F+G4     1394.536     43  2875.071     2897.330     3019.808
174  TPM2+F+R2     1389.054     44  2866.107     2889.539     3014.210
185  K3Pu+F+G4     1391.405     43  2868.811     2891.070     3013.548
187  K3Pu+F+R2     1386.385     44  2860.769     2884.201     3008.872
198  K3P+G4        1394.023     40  2868.046     2887.005     3002.685
200  K3P+R2        1389.010     41  2860.020     2880.043     2998.025
211  TN+F+G4       1394.039     43  2874.077     2896.336     3018.814
213  TN+F+R2       1388.239     44  2864.479     2887.911     3012.582
224  TNe+G4        1396.796     40  2873.593     2892.552     3008.232
226  TNe+R2        1391.590     41  2865.180     2885.204     3003.185
237  HKY+F+G4      1394.954     42  2873.909     2895.032     3015.280
239  HKY+F+R2      1389.609     43  2865.217     2887.476     3009.954
250  K2P+G4        1396.829     39  2871.658     2889.589     3002.931
252  K2P+R2        1391.598     40  2863.197     2882.156     2997.836
263  F81+F+G4      1405.747     41  2893.495     2913.518     3031.500
265  F81+F+R2      1400.802     42  2885.604     2906.727     3026.975
276  JC+G4         1407.653     38  2891.306     2908.243     3019.213
278  JC+R2         1402.856     39  2883.713     2901.644     3014.986
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree.model.gz
CPU time for ModelFinder: 0.602 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.614 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1383.079
Optimal log-likelihood: -1383.077
Rate parameters:  A-C: 0.22953  A-G: 1.98475  A-T: 1.54898  C-G: 0.78167  C-T: 1.98475  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.713,0.411) (0.287,2.461)
Parameters optimization took 1 rounds (0.004 sec)
Computing ML distances based on estimated model parameters... 0.007 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.274
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.092 second
Computing log-likelihood of 98 initial trees ... 0.078 seconds
Current best score: -1383.077

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.824
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1382.305
Iteration 10 / LogL: -1382.851 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.316 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.305 / CPU time: 0.440
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1382.339 / Time: 0h:0m:0s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.297
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1382.089
UPDATE BEST LOG-LIKELIHOOD: -1382.087
Iteration 40 / LogL: -1383.689 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 50 / LogL: -1382.422 / Time: 0h:0m:0s (0h:0m:1s left)
BETTER TREE FOUND at iteration 51: -1382.087
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 54: -1382.010
Iteration 60 / LogL: -1382.205 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1383.091 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.009
UPDATE BEST LOG-LIKELIHOOD: -1382.007
Iteration 80 / LogL: -1383.086 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1406.184 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 100 / LogL: -1382.424 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 110 / LogL: -1382.090 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.007
UPDATE BEST LOG-LIKELIHOOD: -1382.007
Iteration 120 / LogL: -1382.427 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1382.008 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 140 / LogL: -1382.097 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 150 / LogL: -1390.587 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.007
TREE SEARCH COMPLETED AFTER 155 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.007
Optimal log-likelihood: -1382.003
Rate parameters:  A-C: 0.19019  A-G: 1.83594  A-T: 1.52388  C-G: 0.77358  C-T: 1.83594  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.409) (0.277,2.545)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.003
Total tree length: 7.110

Total number of iterations: 155
CPU time used for tree search: 2.678 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.683 sec (0h:0m:2s)
Total CPU time used: 2.706 sec (0h:0m:2s)
Total wall-clock time used: 2.713 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree.log

Date and Time: Wed Jun 24 17:54:20 2020
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-3rh2tljy/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9y1wa90q/q2iqtree -nt 1

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

Output artifacts:

Specifying a substitution model

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-s5niso85/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpknowh5ex/q2iqtree -nt 1
Seed:    110458 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:54:25 2020
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-s5niso85/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.287 / LogL: -1395.194
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.376 / LogL: -1395.464
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.390 / LogL: -1395.530
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.384 / LogL: -1395.523
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.375 / LogL: -1395.492
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.374 / LogL: -1395.529
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.369 / LogL: -1395.476
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.365 / LogL: -1395.493
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.373 / LogL: -1395.500
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.374 / LogL: -1395.511
Optimal pinv,alpha: 0.000, 1.287 / LogL: -1395.194

Parameters optimization took 0.502 sec
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.836
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.094 second
Computing log-likelihood of 98 initial trees ... 0.110 seconds
Current best score: -1392.836

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.260
Iteration 10 / LogL: -1387.280 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.280 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.260 / CPU time: 0.631
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.590 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.636 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1396.702 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 60 / LogL: -1387.321 / Time: 0h:0m:2s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 63: -1387.169
Iteration 70 / LogL: -1387.341 / Time: 0h:0m:2s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 80 / LogL: -1388.631 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 90 / LogL: -1387.341 / Time: 0h:0m:3s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 100 / LogL: -1387.169 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 110 / LogL: -1387.383 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 120 / LogL: -1387.412 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 130 / LogL: -1396.677 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 140 / LogL: -1407.631 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 150 / LogL: -1387.190 / Time: 0h:0m:5s (0h:0m:0s left)
Iteration 160 / LogL: -1391.993 / Time: 0h:0m:5s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 164 ITERATIONS / Time: 0h:0m:5s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.169
Optimal log-likelihood: -1387.168
Rate parameters:  A-C: 0.34427  A-G: 2.30324  A-T: 2.12300  C-G: 1.22342  C-T: 3.18566  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.289
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.168
Total tree length: 7.583

Total number of iterations: 164
CPU time used for tree search: 4.963 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.968 sec (0h:0m:4s)
Total CPU time used: 5.493 sec (0h:0m:5s)
Total wall-clock time used: 5.509 sec (0h:0m:5s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpknowh5ex/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpknowh5ex/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpknowh5ex/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpknowh5ex/q2iqtree.log

Date and Time: Wed Jun 24 17:54:30 2020
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-s5niso85/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpknowh5ex/q2iqtree -nt 1

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

Output artifacts:

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-so2eco19/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree -nt 1 -fast
Seed:    993001 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:54:34 2020
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-so2eco19/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

---> START RUN NUMBER 1 (seed: 993001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.290 / LogL: -1395.208
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.019, 1.361 / LogL: -1395.971
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.023, 1.415 / LogL: -1396.188
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.023, 1.430 / LogL: -1396.157
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.026, 1.433 / LogL: -1396.340
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.028, 1.436 / LogL: -1396.497
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.023, 1.426 / LogL: -1396.261
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.024, 1.430 / LogL: -1396.321
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.025, 1.430 / LogL: -1396.366
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.026, 1.434 / LogL: -1396.414
Optimal pinv,alpha: 0.000, 1.290 / LogL: -1395.208

Parameters optimization took 0.350 sec
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.833
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.271
UPDATE BEST LOG-LIKELIHOOD: -1387.265
Finish initializing candidate tree set (3)
Current best tree score: -1387.265 / CPU time: 0.050
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.265
Optimal log-likelihood: -1387.255
Rate parameters:  A-C: 0.33350  A-G: 2.25974  A-T: 2.13775  C-G: 1.16813  C-T: 3.30009  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.319
Parameters optimization took 1 rounds (0.006 sec)
BEST SCORE FOUND : -1387.255
Total tree length: 6.745

Total number of iterations: 2
CPU time used for tree search: 0.050 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.051 sec (0h:0m:0s)
Total CPU time used: 0.421 sec (0h:0m:0s)
Total wall-clock time used: 0.433 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 994001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters... 0.014 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1394.002
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.046
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.950
2. Current log-likelihood: -1387.791
3. Current log-likelihood: -1387.671
4. Current log-likelihood: -1387.582
5. Current log-likelihood: -1387.514
6. Current log-likelihood: -1387.461
Optimal log-likelihood: -1387.419
Rate parameters:  A-C: 0.33533  A-G: 2.25857  A-T: 2.13278  C-G: 1.17254  C-T: 3.26710  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 6 rounds (0.028 sec)
BEST SCORE FOUND : -1387.419
Total tree length: 6.707

Total number of iterations: 2
CPU time used for tree search: 0.046 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.047 sec (0h:0m:0s)
Total CPU time used: 0.140 sec (0h:0m:0s)
Total wall-clock time used: 0.144 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 995001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.863
2. Current log-likelihood: -1402.072
3. Current log-likelihood: -1396.809
4. Current log-likelihood: -1395.391
5. Current log-likelihood: -1394.657
Optimal log-likelihood: -1394.080
Rate parameters:  A-C: 0.27275  A-G: 2.35291  A-T: 2.09125  C-G: 1.19606  C-T: 3.26639  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.387
Parameters optimization took 5 rounds (0.035 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.808
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.189
Finish initializing candidate tree set (4)
Current best tree score: -1388.189 / CPU time: 0.056
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: -1388.189
2. Current log-likelihood: -1387.974
3. Current log-likelihood: -1387.831
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36986  A-G: 2.31017  A-T: 2.11745  C-G: 1.22267  C-T: 3.27881  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.028 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.055 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.056 sec (0h:0m:0s)
Total CPU time used: 0.145 sec (0h:0m:0s)
Total wall-clock time used: 0.147 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 996001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28732  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.040 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.058
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31019  A-T: 2.11745  C-G: 1.22269  C-T: 3.27879  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.028 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.057 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.058 sec (0h:0m:0s)
Total CPU time used: 0.152 sec (0h:0m:0s)
Total wall-clock time used: 0.155 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 997001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.199
2. Current log-likelihood: -1404.591
3. Current log-likelihood: -1399.228
4. Current log-likelihood: -1397.831
5. Current log-likelihood: -1397.074
Optimal log-likelihood: -1396.495
Rate parameters:  A-C: 0.24620  A-G: 2.08306  A-T: 1.99580  C-G: 1.06240  C-T: 2.85598  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.432
Parameters optimization took 5 rounds (0.042 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.972
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.188
UPDATE BEST LOG-LIKELIHOOD: -1388.187
Finish initializing candidate tree set (3)
Current best tree score: -1388.187 / CPU time: 0.053
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.187
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.687
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.471
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33227  A-G: 2.23742  A-T: 2.11203  C-G: 1.16007  C-T: 3.23505  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 7 rounds (0.029 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.053 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.054 sec (0h:0m:0s)
Total CPU time used: 0.150 sec (0h:0m:0s)
Total wall-clock time used: 0.152 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 998001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.259
2. Current log-likelihood: -1403.078
3. Current log-likelihood: -1398.354
4. Current log-likelihood: -1396.979
5. Current log-likelihood: -1396.262
Optimal log-likelihood: -1395.753
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98595  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.038 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.985
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.952
Finish initializing candidate tree set (4)
Current best tree score: -1387.952 / CPU time: 0.074
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: -1387.952
2. Current log-likelihood: -1387.793
3. Current log-likelihood: -1387.673
4. Current log-likelihood: -1387.584
5. Current log-likelihood: -1387.515
6. Current log-likelihood: -1387.462
Optimal log-likelihood: -1387.420
Rate parameters:  A-C: 0.33384  A-G: 2.25230  A-T: 2.12617  C-G: 1.16896  C-T: 3.25564  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.024 sec)
BEST SCORE FOUND : -1387.420
Total tree length: 6.702

Total number of iterations: 2
CPU time used for tree search: 0.074 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.075 sec (0h:0m:0s)
Total CPU time used: 0.160 sec (0h:0m:0s)
Total wall-clock time used: 0.163 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 999001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.040 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.056
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31019  A-T: 2.11745  C-G: 1.22269  C-T: 3.27879  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.028 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.056 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.056 sec (0h:0m:0s)
Total CPU time used: 0.151 sec (0h:0m:0s)
Total wall-clock time used: 0.154 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 1000001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1491.633
2. Current log-likelihood: -1402.007
3. Current log-likelihood: -1396.792
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.654
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.28077  A-G: 2.37447  A-T: 2.10134  C-G: 1.20130  C-T: 3.28121  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.041 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.810
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.058
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36985  A-G: 2.31002  A-T: 2.11728  C-G: 1.22260  C-T: 3.27850  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.028 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.058 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.058 sec (0h:0m:0s)
Total CPU time used: 0.151 sec (0h:0m:0s)
Total wall-clock time used: 0.153 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 1001001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.459
2. Current log-likelihood: -1403.057
3. Current log-likelihood: -1398.315
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.224
Optimal log-likelihood: -1395.705
Rate parameters:  A-C: 0.23667  A-G: 2.08551  A-T: 1.97696  C-G: 1.07651  C-T: 2.86649  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.987
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.178
Finish initializing candidate tree set (4)
Current best tree score: -1388.178 / CPU time: 0.038
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: -1388.178
2. Current log-likelihood: -1387.956
3. Current log-likelihood: -1387.791
4. Current log-likelihood: -1387.672
5. Current log-likelihood: -1387.582
6. Current log-likelihood: -1387.514
7. Current log-likelihood: -1387.461
Optimal log-likelihood: -1387.419
Rate parameters:  A-C: 0.33608  A-G: 2.25923  A-T: 2.13353  C-G: 1.17293  C-T: 3.26780  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 7 rounds (0.029 sec)
BEST SCORE FOUND : -1387.419
Total tree length: 6.708

Total number of iterations: 2
CPU time used for tree search: 0.038 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.039 sec (0h:0m:0s)
Total CPU time used: 0.127 sec (0h:0m:0s)
Total wall-clock time used: 0.130 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 1002001)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.459
2. Current log-likelihood: -1403.057
3. Current log-likelihood: -1398.315
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.224
Optimal log-likelihood: -1395.705
Rate parameters:  A-C: 0.23667  A-G: 2.08551  A-T: 1.97696  C-G: 1.07651  C-T: 2.86649  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.036 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.987
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.178
Finish initializing candidate tree set (4)
Current best tree score: -1388.178 / CPU time: 0.042
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.178
2. Current log-likelihood: -1387.956
3. Current log-likelihood: -1387.791
4. Current log-likelihood: -1387.672
5. Current log-likelihood: -1387.582
6. Current log-likelihood: -1387.514
7. Current log-likelihood: -1387.461
Optimal log-likelihood: -1387.419
Rate parameters:  A-C: 0.33608  A-G: 2.25923  A-T: 2.13353  C-G: 1.17293  C-T: 3.26780  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 7 rounds (0.029 sec)
BEST SCORE FOUND : -1387.419
Total tree length: 6.708

Total number of iterations: 2
CPU time used for tree search: 0.042 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.042 sec (0h:0m:0s)
Total CPU time used: 0.132 sec (0h:0m:0s)
Total wall-clock time used: 0.134 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 1 gave best log-likelihood: -1387.255
Total CPU time for 10 runs: 1.741 seconds.
Total wall-clock time for 10 runs: 1.783 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree.log

Date and Time: Wed Jun 24 17:54:36 2020
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-so2eco19/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpd6rrf6bc/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

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

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

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

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-alug3308/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxmspqu9k/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    158183 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:54:41 2020
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-alug3308/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.239 / LogL: -1394.544
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.340 / LogL: -1394.887
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.353 / LogL: -1394.887
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.352 / LogL: -1394.871
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.836
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.862
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.884
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.346 / LogL: -1394.826
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.838
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.841
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.544

Parameters optimization took 0.481 sec
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.869
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.085 second
Computing log-likelihood of 98 initial trees ... 0.109 seconds
Current best score: -1392.869

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
Iteration 10 / LogL: -1387.732 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.265 / CPU time: 0.596
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1388.149 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1388.149 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1388.149 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 60 / LogL: -1388.149 / Time: 0h:0m:2s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 65: -1387.174
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 70 / LogL: -1388.134 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 80 / LogL: -1388.345 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 90 / LogL: -1388.134 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 100 / LogL: -1387.338 / Time: 0h:0m:3s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 110 / LogL: -1387.213 / Time: 0h:0m:3s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 120 / LogL: -1387.175 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 130 / LogL: -1387.404 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 140 / LogL: -1387.628 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 150 / LogL: -1387.202 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 160 / LogL: -1387.177 / Time: 0h:0m:4s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 166 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34780  A-G: 2.31928  A-T: 2.14094  C-G: 1.23338  C-T: 3.21631  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.284
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.167

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.056 sec.
Total tree length: 7.604

Total number of iterations: 166
CPU time used for tree search: 4.455 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.460 sec (0h:0m:4s)
Total CPU time used: 5.022 sec (0h:0m:5s)
Total wall-clock time used: 5.040 sec (0h:0m:5s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxmspqu9k/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxmspqu9k/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxmspqu9k/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxmspqu9k/q2iqtree.log

Date and Time: Wed Jun 24 17:54:46 2020
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-alug3308/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpxmspqu9k/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

Tip

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-zdmhliso/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    561205 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:54:50 2020
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-zdmhliso/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1390.939
2. Current log-likelihood: -1389.577
Optimal log-likelihood: -1388.784
Rate parameters:  A-C: 0.37208  A-G: 2.39696  A-T: 2.17455  C-G: 1.26016  C-T: 3.36375  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.405
Parameters optimization took 2 rounds (0.011 sec)
Time for fast ML tree search: 0.063 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.602     45  2895.204     2919.847     3046.673
  2  GTR+F+I       1401.121     46  2894.242     2920.134     3049.077
  3  GTR+F+G4      1387.358     46  2866.716     2892.608     3021.551
  4  GTR+F+I+G4    1387.724     47  2869.448     2896.628     3027.648
  5  GTR+F+R2      1382.364     47  2858.729     2885.910     3016.930
  6  GTR+F+R3      1382.420     49  2862.840     2892.718     3027.773
 16  SYM+G4        1387.134     43  2860.269     2882.528     3005.006
 18  SYM+R2        1383.095     44  2854.189     2877.621     3002.292
 29  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 31  TVM+F+R2      1383.789     46  2859.578     2885.470     3014.413
 42  TVMe+G4       1387.122     42  2858.245     2879.368     2999.616
 44  TVMe+R2       1383.080     43  2852.159     2874.418     2996.896
 55  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 57  TIM3+F+R2     1385.912     45  2861.825     2886.468     3013.294
 68  TIM3e+G4      1390.358     41  2862.715     2882.738     3000.720
 70  TIM3e+R2      1385.918     42  2855.836     2876.959     2997.207
 81  TIM2+F+G4     1393.635     44  2875.270     2898.702     3023.373
 83  TIM2+F+R2     1387.681     45  2865.362     2890.005     3016.831
 94  TIM2e+G4      1396.795     41  2875.589     2895.613     3013.594
 96  TIM2e+R2      1391.574     42  2867.148     2888.270     3008.519
107  TIM+F+G4      1390.363     44  2868.726     2892.158     3016.829
109  TIM+F+R2      1384.933     45  2859.866     2884.509     3011.335
120  TIMe+G4       1394.002     41  2870.005     2890.028     3008.010
122  TIMe+R2       1389.000     42  2862.000     2883.123     3003.371
133  TPM3u+F+G4    1392.306     43  2870.611     2892.870     3015.348
135  TPM3u+F+R2    1387.329     44  2862.659     2886.091     3010.762
146  TPM3+F+G4     1392.306     43  2870.611     2892.870     3015.348
148  TPM3+F+R2     1387.329     44  2862.659     2886.091     3010.762
159  TPM2u+F+G4    1394.536     43  2875.071     2897.330     3019.808
161  TPM2u+F+R2    1389.054     44  2866.107     2889.539     3014.210
172  TPM2+F+G4     1394.536     43  2875.071     2897.330     3019.808
174  TPM2+F+R2     1389.054     44  2866.107     2889.539     3014.210
185  K3Pu+F+G4     1391.405     43  2868.811     2891.070     3013.548
187  K3Pu+F+R2     1386.385     44  2860.769     2884.201     3008.872
198  K3P+G4        1394.023     40  2868.046     2887.005     3002.685
200  K3P+R2        1389.010     41  2860.020     2880.043     2998.025
211  TN+F+G4       1394.039     43  2874.077     2896.336     3018.814
213  TN+F+R2       1388.239     44  2864.479     2887.911     3012.582
224  TNe+G4        1396.796     40  2873.593     2892.552     3008.232
226  TNe+R2        1391.590     41  2865.180     2885.204     3003.185
237  HKY+F+G4      1394.954     42  2873.909     2895.032     3015.280
239  HKY+F+R2      1389.609     43  2865.217     2887.476     3009.954
250  K2P+G4        1396.829     39  2871.658     2889.589     3002.931
252  K2P+R2        1391.598     40  2863.197     2882.156     2997.836
263  F81+F+G4      1405.747     41  2893.495     2913.518     3031.500
265  F81+F+R2      1400.802     42  2885.604     2906.727     3026.975
276  JC+G4         1407.653     38  2891.306     2908.243     3019.213
278  JC+R2         1402.856     39  2883.713     2901.644     3014.986
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree.model.gz
CPU time for ModelFinder: 0.613 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.625 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1383.080
Optimal log-likelihood: -1383.077
Rate parameters:  A-C: 0.22953  A-G: 1.98475  A-T: 1.54898  C-G: 0.78167  C-T: 1.98475  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.713,0.411) (0.287,2.461)
Parameters optimization took 1 rounds (0.007 sec)
Computing ML distances based on estimated model parameters... 0.008 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.274
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.097 second
Computing log-likelihood of 96 initial trees ... 0.077 seconds
Current best score: -1383.077

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.824
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1382.305
Iteration 10 / LogL: -1382.851 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.317 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.305 / CPU time: 0.441
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 25: -1382.003
BETTER TREE FOUND at iteration 27: -1382.003
Iteration 30 / LogL: -1382.422 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 40 / LogL: -1382.090 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 50 / LogL: -1382.092 / Time: 0h:0m:0s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 60 / LogL: -1383.113 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 70 / LogL: -1382.021 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1382.025 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 90 / LogL: -1382.113 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 100 / LogL: -1382.100 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 110 / LogL: -1382.184 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 120 / LogL: -1382.003 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 130 / LogL: -1382.074 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 140 / LogL: -1382.075 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 150 / LogL: -1382.097 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 160 / LogL: -1389.451 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 170 / LogL: -1382.003 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 180 / LogL: -1382.091 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 190 / LogL: -1382.421 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1382.008 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 210 / LogL: -1382.003 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 220 / LogL: -1382.008 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 228 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.003
Optimal log-likelihood: -1382.001
Rate parameters:  A-C: 0.19189  A-G: 1.83715  A-T: 1.52947  C-G: 0.76940  C-T: 1.83715  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.408) (0.276,2.550)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.001
Total tree length: 7.114

Total number of iterations: 228
CPU time used for tree search: 2.644 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.676 sec (0h:0m:2s)
Total CPU time used: 2.677 sec (0h:0m:2s)
Total wall-clock time used: 2.711 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree.log

Date and Time: Wed Jun 24 17:54:53 2020
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-zdmhliso/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpzcq3ljt9/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

iqtree-ultrafast-bootstrap

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-fg7n3vmc/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    256942 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:54:58 2020
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-fg7n3vmc/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.885
2. Current log-likelihood: -1390.523
Optimal log-likelihood: -1389.769
Rate parameters:  A-C: 0.33286  A-G: 2.23822  A-T: 2.09604  C-G: 1.18766  C-T: 3.18094  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.359
Parameters optimization took 2 rounds (0.012 sec)
Time for fast ML tree search: 0.064 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1405.418     45  2900.835     2925.478     3052.304
  2  GTR+F+I       1403.836     46  2899.673     2925.565     3054.508
  3  GTR+F+G4      1388.331     46  2868.662     2894.554     3023.497
  4  GTR+F+I+G4    1388.704     47  2871.408     2898.589     3029.609
  5  GTR+F+R2      1382.562     47  2859.124     2886.305     3017.325
  6  GTR+F+R3      1382.602     49  2863.203     2893.081     3028.136
 16  SYM+G4        1388.448     43  2862.897     2885.155     3007.634
 18  SYM+R2        1384.041     44  2856.081     2879.513     3004.184
 29  TVM+F+G4      1389.408     45  2868.815     2893.458     3020.284
 31  TVM+F+R2      1384.278     46  2860.555     2886.448     3015.390
 42  TVMe+G4       1388.431     42  2860.861     2881.984     3002.232
 44  TVMe+R2       1384.070     43  2854.141     2876.399     2998.878
 55  TIM3+F+G4     1392.277     44  2872.555     2895.987     3020.658
 57  TIM3+F+R2     1385.911     45  2861.822     2886.465     3013.291
 68  TIM3e+G4      1391.664     41  2865.328     2885.351     3003.333
 70  TIM3e+R2      1386.836     42  2857.673     2878.795     2999.044
 81  TIM2+F+G4     1395.130     44  2878.260     2901.692     3026.363
 83  TIM2+F+R2     1388.182     45  2866.364     2891.007     3017.833
 94  TIM2e+G4      1398.824     41  2879.647     2899.670     3017.652
 96  TIM2e+R2      1393.016     42  2870.033     2891.156     3011.404
107  TIM+F+G4      1391.782     44  2871.563     2894.995     3019.666
109  TIM+F+R2      1385.369     45  2860.738     2885.381     3012.207
120  TIMe+G4       1396.021     41  2874.043     2894.066     3012.048
122  TIMe+R2       1390.461     42  2864.921     2886.044     3006.292
133  TPM3u+F+G4    1393.267     43  2872.534     2894.792     3017.271
135  TPM3u+F+R2    1387.637     44  2863.274     2886.706     3011.377
146  TPM3+F+G4     1393.267     43  2872.534     2894.792     3017.271
148  TPM3+F+R2     1387.637     44  2863.274     2886.706     3011.377
159  TPM2u+F+G4    1396.097     43  2878.193     2900.452     3022.930
161  TPM2u+F+R2    1389.794     44  2867.587     2891.019     3015.690
172  TPM2+F+G4     1396.097     43  2878.193     2900.452     3022.930
174  TPM2+F+R2     1389.794     44  2867.587     2891.019     3015.690
185  K3Pu+F+G4     1392.948     43  2871.895     2894.154     3016.632
187  K3Pu+F+R2     1387.034     44  2862.069     2885.501     3010.172
198  K3P+G4        1396.027     40  2872.053     2891.013     3006.692
200  K3P+R2        1390.510     41  2863.021     2883.044     3001.026
211  TN+F+G4       1395.483     43  2876.967     2899.225     3021.704
213  TN+F+R2       1388.576     44  2865.152     2888.584     3013.255
224  TNe+G4        1398.824     40  2877.647     2896.607     3012.286
226  TNe+R2        1393.016     41  2868.032     2888.056     3006.037
237  HKY+F+G4      1396.474     42  2876.947     2898.070     3018.318
239  HKY+F+R2      1390.188     43  2866.377     2888.636     3011.114
250  K2P+G4        1398.839     39  2875.678     2893.609     3006.951
252  K2P+R2        1393.063     40  2866.127     2885.086     3000.766
263  F81+F+G4      1406.476     41  2894.952     2914.975     3032.957
265  F81+F+R2      1401.149     42  2886.297     2907.420     3027.668
276  JC+G4         1408.763     38  2893.526     2910.464     3021.434
278  JC+R2         1403.898     39  2885.796     2903.727     3017.069
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.634 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.646 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 256942)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1384.070
Optimal log-likelihood: -1384.067
Rate parameters:  A-C: 0.20495  A-G: 1.86949  A-T: 1.46520  C-G: 0.73846  C-T: 1.86949  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.716,0.399) (0.284,2.516)
Parameters optimization took 1 rounds (0.005 sec)
Computing ML distances based on estimated model parameters... 0.007 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.354
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 98 initial trees ... 0.078 seconds
Current best score: -1384.067

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.304
UPDATE BEST LOG-LIKELIHOOD: -1382.302
UPDATE BEST LOG-LIKELIHOOD: -1382.301
Iteration 10 / LogL: -1382.307 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.310 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.301 / CPU time: 0.604
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1382.298
UPDATE BEST LOG-LIKELIHOOD: -1382.298
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 28: -1382.087
Iteration 30 / LogL: -1382.087 / Time: 0h:0m:0s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.087
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 36: -1382.005
Iteration 40 / LogL: -1382.413 / Time: 0h:0m:1s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 50 / LogL: -1382.005 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1418.171
Iteration 60 / LogL: -1382.429 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 70 / LogL: -1382.008 / Time: 0h:0m:1s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 80 / LogL: -1382.016 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 90 / LogL: -1382.025 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 100 / LogL: -1382.120 / Time: 0h:0m:2s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1418.171
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 110 / LogL: -1382.004 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 120 / LogL: -1382.017 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 130 / LogL: -1382.430 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 140 / LogL: -1382.090 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 150 / LogL: -1382.021 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1418.885
Iteration 160 / LogL: -1382.180 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 170 / LogL: -1386.479 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 180 / LogL: -1382.515 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 190 / LogL: -1382.033 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 200 / LogL: -1382.240 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1418.885
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 210 / LogL: -1382.590 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.096 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 230 / LogL: -1382.010 / Time: 0h:0m:4s (0h:0m:1s left)
TREE SEARCH COMPLETED AFTER 237 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.004
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19363  A-G: 1.85284  A-T: 1.54522  C-G: 0.77654  C-T: 1.85284  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.408) (0.276,2.551)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.002
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.splits.nex
Total tree length: 7.118

Total number of iterations: 237
CPU time used for tree search: 4.904 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.920 sec (0h:0m:4s)
Total CPU time used: 5.008 sec (0h:0m:5s)
Total wall-clock time used: 5.028 sec (0h:0m:5s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.splits.nex...
20 taxa and 149 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.003

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot.log

Date and Time: Wed Jun 24 17:55:03 2020
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-fg7n3vmc/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9kndsh9d/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

Perform single branch tests alongside ufboot

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

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

stdout:

IQ-TREE multicore version 2.0.3 for Mac OS X 64-bit built Apr 26 2020
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-7qrn4wb7/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    362416 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Wed Jun 24 17:55:08 2020
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-7qrn4wb7/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 362416)...

NOTE: 1 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.218 / LogL: -1394.499
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.293 / LogL: -1394.744
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.008, 1.296 / LogL: -1394.739
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.008, 1.294 / LogL: -1394.737
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.766
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.007, 1.297 / LogL: -1394.718
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.299 / LogL: -1394.735
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.747
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.304 / LogL: -1394.750
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.761
Optimal pinv,alpha: 0.000, 1.218 / LogL: -1394.499

Parameters optimization took 0.500 sec
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.880
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.125 second
Computing log-likelihood of 98 initial trees ... 0.121 seconds
Current best score: -1392.880

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.268
Iteration 10 / LogL: -1387.284 / Time: 0h:0m:1s
UPDATE BEST LOG-LIKELIHOOD: -1387.262
Iteration 20 / LogL: -1387.284 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.262 / CPU time: 0.854
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.346 / Time: 0h:0m:1s (0h:0m:9s left)
Iteration 40 / LogL: -1387.346 / Time: 0h:0m:1s (0h:0m:8s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.258
Iteration 50 / LogL: -1387.258 / Time: 0h:0m:2s (0h:0m:6s left)
Log-likelihood cutoff on original alignment: -1414.208
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 60 / LogL: -1387.346 / Time: 0h:0m:2s (0h:0m:6s left)
Iteration 70 / LogL: -1387.345 / Time: 0h:0m:2s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 80 / LogL: -1387.623 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 90 / LogL: -1387.345 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 100 / LogL: -1387.344 / Time: 0h:0m:3s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1413.503
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
Iteration 110 / LogL: -1387.347 / Time: 0h:0m:4s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.257
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 120 / LogL: -1387.702 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 130 / LogL: -1387.349 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 140 / LogL: -1387.346 / Time: 0h:0m:5s (0h:0m:2s left)
Iteration 150 / LogL: -1387.405 / Time: 0h:0m:5s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1413.503
Iteration 160 / LogL: -1387.306 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 170 / LogL: -1387.288 / Time: 0h:0m:6s (0h:0m:1s left)
Iteration 180 / LogL: -1387.346 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 190 / LogL: -1387.282 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 200 / LogL: -1387.347 / Time: 0h:0m:7s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1413.503
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.993
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:7s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.257
Optimal log-likelihood: -1387.252
Rate parameters:  A-C: 0.33008  A-G: 2.27243  A-T: 2.15023  C-G: 1.17885  C-T: 3.30767  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.314
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.252

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.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.splits.nex
Total tree length: 6.759

Total number of iterations: 202
CPU time used for tree search: 6.775 sec (0h:0m:6s)
Wall-clock time used for tree search: 6.807 sec (0h:0m:6s)
Total CPU time used: 7.448 sec (0h:0m:7s)
Total wall-clock time used: 7.487 sec (0h:0m:7s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.splits.nex...
20 taxa and 174 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.254

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/q2iqtreeufboot.log

Date and Time: Wed Jun 24 17:55:15 2020
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-7qrn4wb7/67af3d05-7ac9-4fdc-b378-b2ededa8b56d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1vlquqjk/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!