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Phylogenetic inference with q2-phylogeny

Note

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

Inferring phylogenies

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

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

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

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

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

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

  1. generate a sequence alignment within QIIME 2

  2. mask the alignment if needed

  3. construct a phylogenetic tree

  4. root the phylogenetic tree

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

Sequence Alignment

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

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

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

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

Next, download the data:

Please select a download option that is most appropriate for your environment:
wget \
  -O "rep-seqs.qza" \
  "https://data.qiime2.org/2020.11/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2020.11/tutorials/phylogeny/rep-seqs.qza" > \
  "rep-seqs.qza"

Run MAFFT

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

Output artifacts:

Reducing alignment ambiguity: masking and reference alignments

Why mask an alignment?

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

Note

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

How to mask alignment.

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

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

Output artifacts:

Reference based alignments

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

Note

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

Construct a phylogeny

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

  1. Phylogeny for the faint of heart - a tutorial

  2. Molecular phylogenetics - principles and practice

  3. Phylogenetics - An Introduction

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

  1. FastTree

  2. RAxML

  3. IQ-TREE

Methods

fasttree

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

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

Output artifacts:

Tip

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

raxml

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

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

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid hill-climbing mode

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


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

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 6029 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-4xa4_9f9/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9l5vne7 -n q2 


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

Inference[0]: Time 0.592496 CAT-based likelihood -1242.800535, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1387.542672 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9l5vne7/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.542672 .... 

Final GAMMA-based Score of best tree -1387.231349

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9l5vne7/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9l5vne7/RAxML_bestTree.q2

Overall execution time: 1.213617 secs or 0.000337 hours or 0.000014 days

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

Command: raxmlHPC -m GTRCAT -p 6029 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-4xa4_9f9/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg9l5vne7 -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-83gf6_8_/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9 -n q2 


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

Inference[0]: Time 0.622635 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.494979 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.501749 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.647470 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.487555 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/tmpeu2u6wj9/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9/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/tmpeu2u6wj9/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9/RAxML_bestTree.q2

Overall execution time: 3.500996 secs or 0.000972 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-83gf6_8_/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpeu2u6wj9 -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-50xfe34m/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0 -n q2bootstrap 



Time for BS model parameter optimization 0.051174
Bootstrap[0]: Time 0.172528 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.113470 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.105118 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.094437 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.106514 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.112570 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.109708 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.100803 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.108574 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.116927 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.084227 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.118650 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.126655 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.106150 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.112832 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.115825 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.107068 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.098862 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.102751 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.106741 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.095408 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.092270 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.103083 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.125781 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.103894 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.105215 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.107515 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.131299 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.128682 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.087077 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.110945 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.110546 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.121055 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.104294 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.100986 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.123980 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.099252 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.121541 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.096741 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.091542 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.109774 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.107773 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.102929 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.127988 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.097848 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.109597 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.088548 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.129318 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.086704 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.118459 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.134089 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.098051 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.110445 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.098283 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.114188 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.117310 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.094265 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.093729 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.133200 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.105201 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.092922 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.108327 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.123546 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.097677 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.090485 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.093805 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.114375 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.112810 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.087646 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.096061 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.102518 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.100853 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.086235 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.101517 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.088877 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.093051 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.110345 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.132068 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.110082 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.114067 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.100089 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.107106 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.127940 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.100983 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.103703 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.111023 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.095615 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.094808 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.109233 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.101623 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.113758 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.111353 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.045664 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.117640 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.097002 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.093885 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.108553 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.116876 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.044840 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.059058 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.589718 seconds
Average Time per Rapid Bootstrap 0.105897 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.193859 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: 2.147984 seconds
Thorough ML search Time: 0.562621 seconds

Final ML Optimization Likelihood: -1387.204993

Model Information:

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

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


ML search took 6.909638 secs or 0.001919 hours

Combined Bootstrap and ML search took 17.499828 secs or 0.004861 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.509964 secs or 0.004864 hours or 0.000203 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-50xfe34m/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4b5jq6d0 -n q2bootstrap

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

Output artifacts:

Tip

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

iqtree

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

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-7jytd2sk/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree -nt 1
Seed:    847309 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:56:27 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-7jytd2sk/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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: -1391.311
2. Current log-likelihood: -1389.733
Optimal log-likelihood: -1388.881
Rate parameters:  A-C: 0.33789  A-G: 2.29237  A-T: 2.14761  C-G: 1.19040  C-T: 3.28358  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.033
Gamma shape alpha: 1.424
Parameters optimization took 2 rounds (0.013 sec)
Time for fast ML tree search: 0.063 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.279     46  2866.558     2892.450     3021.393
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.423
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 16  SYM+G4        1387.157     43  2860.314     2882.573     3005.051
 18  SYM+R2        1382.244     44  2852.489     2875.920     3000.591
 29  TVM+F+G4      1388.424     45  2866.848     2891.491     3018.317
 31  TVM+F+R2      1382.481     46  2856.963     2882.855     3011.798
 42  TVMe+G4       1387.122     42  2858.245     2879.367     2999.616
 44  TVMe+R2       1382.298     43  2850.596     2872.854     2995.333
 55  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 57  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
 68  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
 70  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
 81  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
 83  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
 94  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
 96  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
107  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
109  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
120  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
122  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
133  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
135  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
146  TPM3+F+G4     1392.567     43  2871.134     2893.393     3015.871
148  TPM3+F+R2     1386.368     44  2860.736     2884.168     3008.839
159  TPM2u+F+G4    1395.282     43  2876.564     2898.823     3021.301
161  TPM2u+F+R2    1388.115     44  2864.231     2887.663     3012.334
172  TPM2+F+G4     1395.282     43  2876.564     2898.823     3021.301
174  TPM2+F+R2     1388.115     44  2864.231     2887.663     3012.334
185  K3Pu+F+G4     1392.067     43  2870.133     2892.392     3014.870
187  K3Pu+F+R2     1385.124     44  2858.247     2881.679     3006.350
198  K3P+G4        1394.798     40  2869.597     2888.556     3004.236
200  K3P+R2        1388.380     41  2858.761     2878.784     2996.766
211  TN+F+G4       1394.627     43  2875.254     2897.513     3019.991
213  TN+F+R2       1386.824     44  2861.647     2885.079     3009.750
224  TNe+G4        1397.746     40  2875.492     2894.452     3010.131
226  TNe+R2        1391.135     41  2864.270     2884.293     3002.275
237  HKY+F+G4      1395.753     42  2875.505     2896.628     3016.876
239  HKY+F+R2      1388.692     43  2863.383     2885.642     3008.120
250  K2P+G4        1397.751     39  2873.502     2891.433     3004.775
252  K2P+R2        1391.217     40  2862.434     2881.394     2997.073
263  F81+F+G4      1406.484     41  2894.968     2914.991     3032.973
265  F81+F+R2      1400.605     42  2885.210     2906.333     3026.581
276  JC+G4         1408.433     38  2892.866     2909.803     3020.773
278  JC+R2         1403.022     39  2884.045     2901.976     3015.318
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree.model.gz
CPU time for ModelFinder: 0.669 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.692 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1382.298
Optimal log-likelihood: -1382.296
Rate parameters:  A-C: 0.21625  A-G: 2.01118  A-T: 1.56840  C-G: 0.77587  C-T: 2.01118  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.406) (0.278,2.543)
Parameters optimization took 1 rounds (0.004 sec)
Computing ML distances based on estimated model parameters... 0.006 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.305
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 98 initial trees ... 0.079 seconds
Current best score: -1382.296

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.296
Iteration 10 / LogL: -1382.305 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.308 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.296 / CPU time: 0.418
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1382.297 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 40 / LogL: -1382.868 / Time: 0h:0m:0s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 41: -1382.001
Iteration 50 / LogL: -1382.887 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 60 / LogL: -1382.094 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1382.094 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1382.099 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1383.123 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1382.242 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 110 / LogL: -1382.094 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 120 / LogL: -1382.097 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1382.211 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.001
Iteration 140 / LogL: -1382.098 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 142 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.001
Optimal log-likelihood: -1382.001
Rate parameters:  A-C: 0.19132  A-G: 1.83777  A-T: 1.52755  C-G: 0.77037  C-T: 1.83777  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.725,0.409) (0.275,2.561)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.001
Total tree length: 7.109

Total number of iterations: 142
CPU time used for tree search: 2.417 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.419 sec (0h:0m:2s)
Total CPU time used: 2.446 sec (0h:0m:2s)
Total wall-clock time used: 2.450 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree.log

Date and Time: Sat Dec  5 17:56:30 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-7jytd2sk/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmps7tqfmwp/q2iqtree -nt 1

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

Output artifacts:

Specifying a substitution model

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-4m8h5sho/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpj8re7soa/q2iqtree -nt 1
Seed:    466955 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:56:35 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-4m8h5sho/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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, 1.242 / LogL: -1394.542
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.345 / LogL: -1394.882
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.351 / LogL: -1394.883
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.355 / LogL: -1394.867
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.832
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.354 / LogL: -1394.859
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.355 / LogL: -1394.881
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.349 / LogL: -1394.822
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.833
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.843
Optimal pinv,alpha: 0.000, 1.242 / LogL: -1394.542

Parameters optimization took 0.454 sec
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.914
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.089 second
Computing log-likelihood of 98 initial trees ... 0.109 seconds
Current best score: -1392.914

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.266
Iteration 10 / LogL: -1393.565 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.266 / CPU time: 0.619
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 22: -1387.167
UPDATE BEST LOG-LIKELIHOOD: -1387.167
Iteration 30 / LogL: -1387.167 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 40 / LogL: -1387.392 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 50 / LogL: -1387.168 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 60 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 70 / LogL: -1387.210 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1396.400 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 90 / LogL: -1387.182 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 100 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 110 / LogL: -1387.177 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 120 / LogL: -1387.167 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 123 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.167
Optimal log-likelihood: -1387.166
Rate parameters:  A-C: 0.34484  A-G: 2.31013  A-T: 2.13040  C-G: 1.22723  C-T: 3.19829  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: 1.285
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.166
Total tree length: 7.599

Total number of iterations: 123
CPU time used for tree search: 3.391 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.394 sec (0h:0m:3s)
Total CPU time used: 3.873 sec (0h:0m:3s)
Total wall-clock time used: 3.878 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpj8re7soa/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpj8re7soa/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpj8re7soa/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpj8re7soa/q2iqtree.log

Date and Time: Sat Dec  5 17:56:39 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-4m8h5sho/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpj8re7soa/q2iqtree -nt 1

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

Output artifacts:

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-8fnq9x92/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree -nt 1 -fast
Seed:    809908 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:56:43 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-8fnq9x92/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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: 809908)

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 0.342 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: 0.054
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: 2.13776  C-G: 1.16813  C-T: 3.30010  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: 0.054 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.055 sec (0h:0m:0s)
Total CPU time used: 0.424 sec (0h:0m:0s)
Total wall-clock time used: 0.428 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 810908)

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 (0.039 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: 0.080
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: 0.080 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.080 sec (0h:0m:0s)
Total CPU time used: 0.171 sec (0h:0m:0s)
Total wall-clock time used: 0.173 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 811908)

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 (0.037 sec)
Computing ML distances based on estimated model parameters... 0.009 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: 0.056
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: 0.36985  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 (0.026 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: 0.144 sec (0h:0m:0s)
Total wall-clock time used: 0.147 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 812908)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.491
2. Current log-likelihood: -1404.606
3. Current log-likelihood: -1399.220
4. Current log-likelihood: -1397.821
5. Current log-likelihood: -1397.063
Optimal log-likelihood: -1396.484
Rate parameters:  A-C: 0.24153  A-G: 2.03298  A-T: 1.94373  C-G: 1.02159  C-T: 2.79340  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.438
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1394.014
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.197
UPDATE BEST LOG-LIKELIHOOD: -1388.190
Finish initializing candidate tree set (3)
Current best tree score: -1388.190 / CPU time: 0.055
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.190
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: -1387.526
7. Current log-likelihood: -1387.471
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33230  A-G: 2.23569  A-T: 2.11033  C-G: 1.15918  C-T: 3.23266  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: 0.055 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.056 sec (0h:0m:0s)
Total CPU time used: 0.147 sec (0h:0m:0s)
Total wall-clock time used: 0.150 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 813908)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.863
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 (0.039 sec)
Computing ML distances based on estimated model parameters... 0.009 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: 0.055
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -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: 0.055 sec (0h:0m:0s)
Total CPU time used: 0.144 sec (0h:0m:0s)
Total wall-clock time used: 0.147 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 814908)

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: -1496.306
2. Current log-likelihood: -1403.641
3. Current log-likelihood: -1398.531
4. Current log-likelihood: -1397.067
5. Current log-likelihood: -1396.244
6. Current log-likelihood: -1395.736
Optimal log-likelihood: -1395.357
Rate parameters:  A-C: 0.22740  A-G: 2.00038  A-T: 1.90797  C-G: 1.02878  C-T: 2.75984  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.021
Gamma shape alpha: 1.340
Parameters optimization took 6 rounds (0.044 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.769
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.988
Finish initializing candidate tree set (4)
Current best tree score: -1387.988 / CPU time: 0.053
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.988
2. Current log-likelihood: -1387.817
3. Current log-likelihood: -1387.688
4. Current log-likelihood: -1387.594
5. Current log-likelihood: -1387.523
6. Current log-likelihood: -1387.468
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.32477  A-G: 2.23464  A-T: 2.10729  C-G: 1.15865  C-T: 3.22565  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: 1.360
Parameters optimization took 6 rounds (0.021 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.697

Total number of iterations: 2
CPU time used for tree search: 0.053 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.053 sec (0h:0m:0s)
Total CPU time used: 0.143 sec (0h:0m:0s)
Total wall-clock time used: 0.145 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 815908)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1496.015
2. Current log-likelihood: -1403.630
3. Current log-likelihood: -1398.533
4. Current log-likelihood: -1397.077
5. Current log-likelihood: -1396.256
6. Current log-likelihood: -1395.746
Optimal log-likelihood: -1395.367
Rate parameters:  A-C: 0.23677  A-G: 2.05007  A-T: 1.94889  C-G: 1.06764  C-T: 2.81222  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.021
Gamma shape alpha: 1.337
Parameters optimization took 6 rounds (0.042 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.724
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.981
Finish initializing candidate tree set (4)
Current best tree score: -1387.981 / CPU time: 0.051
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.981
2. Current log-likelihood: -1387.812
3. Current log-likelihood: -1387.685
4. Current log-likelihood: -1387.592
5. Current log-likelihood: -1387.521
6. Current log-likelihood: -1387.466
Optimal log-likelihood: -1387.423
Rate parameters:  A-C: 0.32763  A-G: 2.25273  A-T: 2.12567  C-G: 1.16858  C-T: 3.25529  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 (0.026 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.701

Total number of iterations: 2
CPU time used for tree search: 0.051 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.052 sec (0h:0m:0s)
Total CPU time used: 0.143 sec (0h:0m:0s)
Total wall-clock time used: 0.145 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 816908)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.259
2. Current log-likelihood: -1403.078
3. Current log-likelihood: -1398.354
4. Current log-likelihood: -1396.979
5. Current log-likelihood: -1396.262
Optimal log-likelihood: -1395.753
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98596  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.040 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: 0.076
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: 0.076 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.076 sec (0h:0m:0s)
Total CPU time used: 0.167 sec (0h:0m:0s)
Total wall-clock time used: 0.170 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 817908)

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: -1491.925
2. Current log-likelihood: -1402.064
3. Current log-likelihood: -1396.813
4. Current log-likelihood: -1395.392
5. Current log-likelihood: -1394.652
Optimal log-likelihood: -1394.078
Rate parameters:  A-C: 0.27467  A-G: 2.39505  A-T: 2.12238  C-G: 1.21030  C-T: 3.30514  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: 1.388
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.807
--------------------------------------------------------------------
|             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: -1388.181
Finish initializing candidate tree set (4)
Current best tree score: -1388.181 / CPU time: 0.057
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: -1388.181
2. Current log-likelihood: -1387.974
3. Current log-likelihood: -1387.831
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.646
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36872  A-G: 2.32249  A-T: 2.12948  C-G: 1.22911  C-T: 3.29732  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: 1.331
Parameters optimization took 6 rounds (0.026 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.508

Total number of iterations: 2
CPU time used for tree search: 0.057 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.057 sec (0h:0m:0s)
Total CPU time used: 0.147 sec (0h:0m:0s)
Total wall-clock time used: 0.149 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 818908)

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: 1.99581  C-G: 1.06240  C-T: 2.85598  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.432
Parameters optimization took 5 rounds (0.042 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 (0.025 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: 0.053 sec (0h:0m:0s)
Total CPU time used: 0.146 sec (0h:0m:0s)
Total wall-clock time used: 0.148 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 1 gave best log-likelihood: -1387.255
Total CPU time for 10 runs: 1.788 seconds.
Total wall-clock time for 10 runs: 1.821 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree.log

Date and Time: Sat Dec  5 17:56:45 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-8fnq9x92/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp7jkxv2bh/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

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

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

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

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-_2gdfois/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg5l05a7_/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    96193 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:56:50 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-_2gdfois/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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, 1.282 / LogL: -1392.553
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.377 / LogL: -1392.829
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.391 / LogL: -1392.898
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.388 / LogL: -1392.889
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.853
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.384 / LogL: -1392.879
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.007, 1.379 / LogL: -1392.828
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.382 / LogL: -1392.844
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.849
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.384 / LogL: -1392.858
Optimal pinv,alpha: 0.000, 1.282 / LogL: -1392.553

Parameters optimization took 0.469 sec
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.710
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.090 second
Computing log-likelihood of 98 initial trees ... 0.110 seconds
Current best score: -1392.553

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.258
Iteration 10 / LogL: -1387.279 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.279 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.258 / CPU time: 0.609
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1396.564 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.370 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1387.350 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 60 / LogL: -1387.378 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 70 / LogL: -1387.351 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 80 / LogL: -1387.267 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 90 / LogL: -1387.350 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 100 / LogL: -1387.907 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.258
Optimal log-likelihood: -1387.252
Rate parameters:  A-C: 0.33095  A-G: 2.27170  A-T: 2.15058  C-G: 1.18098  C-T: 3.30169  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: 1.323
Parameters optimization took 1 rounds (0.006 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...
0.057 sec.
Total tree length: 6.716

Total number of iterations: 102
CPU time used for tree search: 3.092 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.094 sec (0h:0m:3s)
Total CPU time used: 3.651 sec (0h:0m:3s)
Total wall-clock time used: 3.657 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg5l05a7_/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg5l05a7_/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg5l05a7_/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg5l05a7_/q2iqtree.log

Date and Time: Sat Dec  5 17:56:53 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-_2gdfois/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpg5l05a7_/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

Tip

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-5b7g8smv/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1x4214ja/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    250130 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:56:58 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-5b7g8smv/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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: 0.33283  A-G: 2.23806  A-T: 2.09600  C-G: 1.18747  C-T: 3.18113  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: 0.065 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/tmp1x4214ja/q2iqtree.model.gz
CPU time for ModelFinder: 0.627 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.631 seconds (0h:0m:0s)

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 (0.004 sec)
Computing ML distances based on estimated model parameters... 0.006 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.354
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.087 second
Computing log-likelihood of 98 initial trees ... 0.079 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
Iteration 10 / LogL: -1382.307 / Time: 0h:0m:0s
UPDATE BEST LOG-LIKELIHOOD: -1382.300
Iteration 20 / LogL: -1382.310 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.300 / CPU time: 0.428
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 22: -1382.087
UPDATE BEST LOG-LIKELIHOOD: -1382.087
Iteration 30 / LogL: -1382.863 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 40 / LogL: -1383.091 / Time: 0h:0m:0s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 44: -1382.007
Iteration 50 / LogL: -1390.345 / Time: 0h:0m:0s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.006
UPDATE BEST LOG-LIKELIHOOD: -1382.006
Iteration 60 / LogL: -1382.482 / Time: 0h:0m:0s (0h:0m:2s left)
BETTER TREE FOUND at iteration 68: -1382.005
Iteration 70 / LogL: -1382.038 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1382.021 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1382.021 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 100 / LogL: -1382.090 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 110 / LogL: -1392.448 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 120 / LogL: -1382.017 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 130 / LogL: -1382.099 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 140 / LogL: -1382.006 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 150 / LogL: -1382.017 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 160 / LogL: -1382.005 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 170 / LogL: -1382.093 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 180 / LogL: -1382.013 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 190 / LogL: -1382.016 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1382.063 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 210 / LogL: -1382.017 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 220 / LogL: -1382.079 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 230 / LogL: -1388.681 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 240 / LogL: -1382.018 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 250 / LogL: -1382.017 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 260 / LogL: -1382.005 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 269 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.005
Optimal log-likelihood: -1382.003
Rate parameters:  A-C: 0.19380  A-G: 1.85586  A-T: 1.54657  C-G: 0.77853  C-T: 1.85586  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 : -1382.003
Total tree length: 7.118

Total number of iterations: 269
CPU time used for tree search: 3.065 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.071 sec (0h:0m:3s)
Total CPU time used: 3.095 sec (0h:0m:3s)
Total wall-clock time used: 3.103 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1x4214ja/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1x4214ja/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1x4214ja/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1x4214ja/q2iqtree.log

Date and Time: Sat Dec  5 17:57:01 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-5b7g8smv/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp1x4214ja/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

iqtree-ultrafast-bootstrap

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

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

stdout:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-4lxozo3j/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    459268 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:57:06 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-4lxozo3j/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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: 0.33284  A-G: 2.23808  A-T: 2.09601  C-G: 1.18749  C-T: 3.18112  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: 0.063 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         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/tmptx_hwp5n/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.626 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.630 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 459268)...

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 (0.004 sec)
Computing ML distances based on estimated model parameters... 0.006 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.354
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.086 second
Computing log-likelihood of 95 initial trees ... 0.076 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: -1382.298
Iteration 10 / LogL: -1382.310 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.313 / Time: 0h:0m:0s
Finish initializing candidate tree set (3)
Current best tree score: -1382.298 / CPU time: 0.601
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1384.533 / Time: 0h:0m:0s (0h:0m:4s left)
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1382.088
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 33: -1382.004
Iteration 40 / LogL: -1382.031 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 50 / LogL: -1382.042 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1404.354
Iteration 60 / LogL: -1382.904 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 70 / LogL: -1382.901 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 80 / LogL: -1382.004 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 90 / LogL: -1382.417 / Time: 0h:0m:2s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 100 / LogL: -1382.049 / Time: 0h:0m:2s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1404.537
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.999
Iteration 110 / LogL: -1382.017 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 120 / LogL: -1382.053 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 130 / LogL: -1382.015 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 140 / LogL: -1382.117 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 150 / LogL: -1382.024 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1404.354
Iteration 160 / LogL: -1382.007 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 170 / LogL: -1382.146 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 180 / LogL: -1382.879 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 190 / LogL: -1382.486 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 200 / LogL: -1382.174 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1404.354
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
Iteration 210 / LogL: -1382.091 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.006 / Time: 0h:0m:4s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 230 / LogL: -1382.091 / Time: 0h:0m:4s (0h:0m:1s left)
TREE SEARCH COMPLETED AFTER 234 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.003
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19145  A-G: 1.84354  A-T: 1.53395  C-G: 0.77258  C-T: 1.84354  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.409) (0.276,2.551)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.002
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.splits.nex
Total tree length: 7.115

Total number of iterations: 234
CPU time used for tree search: 4.803 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.817 sec (0h:0m:4s)
Total CPU time used: 4.908 sec (0h:0m:4s)
Total wall-clock time used: 4.928 sec (0h:0m:4s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.splits.nex...
20 taxa and 159 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.002

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot.log

Date and Time: Sat Dec  5 17:57:12 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-4lxozo3j/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmptx_hwp5n/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

Perform single branch tests alongside ufboot

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

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

stdout:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.0.3 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-mh1mg7rh/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    498303 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Sat Dec  5 17:57:16 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-mh1mg7rh/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/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: 498303)...

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, 1.239 / LogL: -1394.430
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.306 / LogL: -1394.720
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.315 / LogL: -1394.793
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.313 / LogL: -1394.791
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.755
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.309 / LogL: -1394.783
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.305 / LogL: -1394.729
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.742
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.308 / LogL: -1394.753
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.312 / LogL: -1394.757
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.430

Parameters optimization took 0.461 sec
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.898
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.087 second
Computing log-likelihood of 96 initial trees ... 0.106 seconds
Current best score: -1392.898

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.266
Iteration 10 / LogL: -1387.731 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.266 / CPU time: 0.739
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.308 / Time: 0h:0m:1s (0h:0m:8s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.262
Iteration 40 / LogL: -1387.372 / Time: 0h:0m:1s (0h:0m:6s left)
Iteration 50 / LogL: -1387.307 / Time: 0h:0m:1s (0h:0m:5s left)
Log-likelihood cutoff on original alignment: -1409.040
Iteration 60 / LogL: -1387.370 / Time: 0h:0m:2s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 70 / LogL: -1387.361 / Time: 0h:0m:2s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 80 / LogL: -1387.348 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 90 / LogL: -1387.368 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 100 / LogL: -1387.439 / Time: 0h:0m:3s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1409.046
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.988
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 110 / LogL: -1387.258 / Time: 0h:0m:4s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 120 / LogL: -1387.354 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 130 / LogL: -1387.373 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 140 / LogL: -1387.598 / 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: -1408.710
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 160 / LogL: -1387.347 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 170 / LogL: -1390.575 / Time: 0h:0m:6s (0h:0m:1s left)
Iteration 180 / LogL: -1387.268 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 190 / LogL: -1387.346 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 200 / LogL: -1387.346 / Time: 0h:0m:6s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1408.710
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:6s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.255
Optimal log-likelihood: -1387.252
Rate parameters:  A-C: 0.32799  A-G: 2.25616  A-T: 2.13404  C-G: 1.17055  C-T: 3.28011  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.318
Parameters optimization took 1 rounds (0.004 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...
0.055 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.splits.nex
Total tree length: 6.745

Total number of iterations: 202
CPU time used for tree search: 6.430 sec (0h:0m:6s)
Wall-clock time used for tree search: 6.444 sec (0h:0m:6s)
Total CPU time used: 7.055 sec (0h:0m:7s)
Total wall-clock time used: 7.076 sec (0h:0m:7s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.splits.nex...
20 taxa and 161 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.778

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/q2iqtreeufboot.log

Date and Time: Sat Dec  5 17:57:23 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-mh1mg7rh/03a1849a-4bc2-4343-9a49-b17db5bf6f3d/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpmxnxr66z/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!