Fork me on GitHub

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/2020.8/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2020.8/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 3292 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-br1glp4y/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsmfd533g -n q2 


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

Inference[0]: Time 0.644953 CAT-based likelihood -1243.188861, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1387.901970 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsmfd533g/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.901970 .... 

Final GAMMA-based Score of best tree -1387.230194

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsmfd533g/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsmfd533g/RAxML_bestTree.q2

Overall execution time: 1.234595 secs or 0.000343 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 3292 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-br1glp4y/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpsmfd533g -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-mrli9ecg/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p -n q2 


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

Inference[0]: Time 0.599204 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.494217 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.509194 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.645469 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.492101 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/tmp3koilo6p/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p/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/tmp3koilo6p/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p/RAxML_bestTree.q2

Overall execution time: 3.591913 secs or 0.000998 hours or 0.000042 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-mrli9ecg/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3koilo6p -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-qtggddv3/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo -n q2bootstrap 



Time for BS model parameter optimization 0.052233
Bootstrap[0]: Time 0.169091 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.115116 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.106058 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.096876 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.107694 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.112493 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.110869 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.097126 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.109027 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.115948 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.084349 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.120544 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.127477 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.106124 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.113790 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.117109 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.107067 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.099392 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.102568 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.102311 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.095706 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.096347 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.104302 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.122358 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.104090 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.104919 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.108205 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.131106 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.129628 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.087631 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.109806 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.112362 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.118484 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.104628 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.099987 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.115580 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.096331 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.120668 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.097271 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.092041 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.110409 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.107807 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.103152 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.125607 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.097342 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.110027 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.089245 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.131602 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.087224 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.118262 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.137284 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.098121 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.114505 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.098484 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.114281 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.115462 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.098438 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.093054 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.134695 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.105265 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.093015 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.107282 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.123827 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.100263 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.094881 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.096011 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.119897 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.117892 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.094982 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.102031 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.101948 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.100246 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.094176 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.100459 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.089151 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.097082 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.113965 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.136899 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.108751 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.112620 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.099987 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.108504 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.129963 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.096898 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.107509 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.109257 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.099415 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.094538 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.110779 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.101714 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.113858 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.110134 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.045855 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.120769 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.097283 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.098526 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.108158 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.117069 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.045418 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.062055 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.658823 seconds
Average Time per Rapid Bootstrap 0.106588 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.190279 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: 2.175820 seconds
Thorough ML search Time: 0.572394 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 6.945187 secs or 0.001929 hours

Combined Bootstrap and ML search took 17.604288 secs or 0.004890 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.617315 secs or 0.004894 hours or 0.000204 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-qtggddv3/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8v4c1tlo -n q2bootstrap

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

Output artifacts:

Tip

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

iqtree

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

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-qtneq_oy/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpvyaxcv_5/q2iqtree -nt 1
Seed:    51331 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:35:15 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-qtneq_oy/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1396.575
2. Current log-likelihood: -1395.213
Optimal log-likelihood: -1394.464
Rate parameters:  A-C: 0.21819  A-G: 2.03593  A-T: 1.93394  C-G: 1.05109  C-T: 2.56337  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: 1.322
Parameters optimization took 2 rounds (0.013 sec)
Time for fast ML tree search: 0.056 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         1411.054     45  2912.108     2936.751     3063.577
  2  GTR+F+I       1409.135     46  2910.270     2936.162     3065.105
  3  GTR+F+G4      1392.992     46  2877.983     2903.876     3032.818
  4  GTR+F+I+G4    1393.280     47  2880.561     2907.741     3038.762
  5  GTR+F+R2      1387.712     47  2869.423     2896.604     3027.624
  6  GTR+F+R3      1387.747     49  2873.494     2903.372     3038.427
 16  SYM+G4        1393.506     43  2873.012     2895.271     3017.749
 18  SYM+R2        1389.901     44  2867.802     2891.234     3015.905
 29  TVM+F+G4      1393.474     45  2876.947     2901.590     3028.416
 31  TVM+F+R2      1388.475     46  2868.950     2894.842     3023.785
 42  TVMe+G4       1393.632     42  2871.264     2892.387     3012.635
 44  TVMe+R2       1389.912     43  2865.824     2888.083     3010.561
 55  TIM3+F+G4     1396.957     44  2881.914     2905.346     3030.017
 57  TIM3+F+R2     1391.441     45  2872.881     2897.524     3024.350
 68  TIM3e+G4      1397.005     41  2876.010     2896.033     3014.015
 70  TIM3e+R2      1393.195     42  2870.390     2891.513     3011.761
 81  TIM2+F+G4     1401.480     44  2890.961     2914.393     3039.064
 83  TIM2+F+R2     1395.762     45  2881.524     2906.167     3032.993
 94  TIM2e+G4      1406.407     41  2894.815     2914.838     3032.820
 96  TIM2e+R2      1402.269     42  2888.539     2909.662     3029.910
107  TIM+F+G4      1397.972     44  2883.943     2907.375     3032.046
109  TIM+F+R2      1392.182     45  2874.365     2899.008     3025.834
120  TIMe+G4       1403.787     41  2889.575     2909.598     3027.580
122  TIMe+R2       1399.417     42  2882.835     2903.958     3024.206
133  TPM3u+F+G4    1397.423     43  2880.846     2903.105     3025.583
135  TPM3u+F+R2    1392.257     44  2872.514     2895.946     3020.617
146  TPM3+F+G4     1397.423     43  2880.846     2903.105     3025.583
148  TPM3+F+R2     1392.257     44  2872.514     2895.946     3020.617
159  TPM2u+F+G4    1401.944     43  2889.889     2912.147     3034.626
161  TPM2u+F+R2    1396.513     44  2881.027     2904.458     3029.129
172  TPM2+F+G4     1401.944     43  2889.889     2912.147     3034.626
174  TPM2+F+R2     1396.513     44  2881.027     2904.458     3029.129
185  K3Pu+F+G4     1398.573     43  2883.146     2905.405     3027.883
187  K3Pu+F+R2     1393.066     44  2874.132     2897.564     3022.235
198  K3P+G4        1403.919     40  2887.838     2906.798     3022.477
200  K3P+R2        1399.438     41  2880.876     2900.899     3018.881
211  TN+F+G4       1401.600     43  2889.200     2911.459     3033.937
213  TN+F+R2       1395.970     44  2879.940     2903.372     3028.043
224  TNe+G4        1406.476     40  2892.951     2911.911     3027.590
226  TNe+R2        1402.298     41  2886.596     2906.620     3024.601
237  HKY+F+G4      1402.073     42  2888.146     2909.268     3029.517
239  HKY+F+R2      1396.722     43  2879.444     2901.703     3024.181
250  K2P+G4        1406.650     39  2891.299     2909.230     3022.572
252  K2P+R2        1402.334     40  2884.668     2903.627     3019.307
263  F81+F+G4      1410.301     41  2902.603     2922.626     3040.608
265  F81+F+R2      1405.908     42  2895.816     2916.939     3037.187
276  JC+G4         1414.958     38  2905.915     2922.852     3033.822
278  JC+R2         1411.527     39  2901.054     2918.985     3032.327
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/tmpvyaxcv_5/q2iqtree.model.gz
CPU time for ModelFinder: 0.634 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.638 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1389.912
Optimal log-likelihood: -1389.906
Rate parameters:  A-C: 0.10868  A-G: 1.62939  A-T: 1.39318  C-G: 0.63110  C-T: 1.62939  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.687,0.344) (0.313,2.442)
Parameters optimization took 1 rounds (0.004 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.952
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.085 second
Computing log-likelihood of 98 initial trees ... 0.080 seconds
Current best score: -1389.906

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1388.680
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1382.315
Iteration 10 / LogL: -1382.343 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.354 / Time: 0h:0m:0s
Finish initializing candidate tree set (4)
Current best tree score: -1382.315 / CPU time: 0.411
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1382.313
UPDATE BEST LOG-LIKELIHOOD: -1382.313
UPDATE BEST LOG-LIKELIHOOD: -1382.312
Iteration 30 / LogL: -1383.052 / Time: 0h:0m:0s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.311
Iteration 40 / LogL: -1391.011 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 50 / LogL: -1382.891 / Time: 0h:0m:0s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 54: -1382.090
Iteration 60 / LogL: -1382.437 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1383.104 / Time: 0h:0m:1s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 75: -1382.013
UPDATE BEST LOG-LIKELIHOOD: -1382.013
Iteration 80 / LogL: -1382.477 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1382.417 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.009
Iteration 100 / LogL: -1382.556 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 110 / LogL: -1382.901 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 120 / LogL: -1382.896 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1383.090 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 140 / LogL: -1382.419 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.008
Iteration 150 / LogL: -1382.431 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 160 / LogL: -1384.334 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1383.867 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 176 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.008
Optimal log-likelihood: -1382.004
Rate parameters:  A-C: 0.19334  A-G: 1.85162  A-T: 1.54270  C-G: 0.77640  C-T: 1.85162  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.408) (0.277,2.545)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.004
Total tree length: 7.125

Total number of iterations: 176
CPU time used for tree search: 2.975 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.979 sec (0h:0m:2s)
Total CPU time used: 3.005 sec (0h:0m:3s)
Total wall-clock time used: 3.012 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpvyaxcv_5/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpvyaxcv_5/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpvyaxcv_5/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpvyaxcv_5/q2iqtree.log

Date and Time: Fri Aug 21 12:35:18 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-qtneq_oy/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpvyaxcv_5/q2iqtree -nt 1

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

Output artifacts:

Specifying a substitution model

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-ipzmjqip/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4py7hstn/q2iqtree -nt 1
Seed:    74904 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:35:23 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-ipzmjqip/9d9e927a-ab0b-4a36-9073-34185507633e/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, 1.261 / LogL: -1392.813
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.354 / LogL: -1393.088
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.366 / LogL: -1393.160
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.363 / LogL: -1393.150
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.359 / LogL: -1393.113
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.361 / LogL: -1393.140
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.007, 1.356 / LogL: -1393.088
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.358 / LogL: -1393.104
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.360 / LogL: -1393.109
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.361 / LogL: -1393.119
Optimal pinv,alpha: 0.000, 1.261 / LogL: -1392.813

Parameters optimization took 0.483 sec
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.727
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.086 second
Computing log-likelihood of 98 initial trees ... 0.115 seconds
Current best score: -1392.727

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.357
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1387.260
Iteration 10 / LogL: -1387.734 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.280 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.260 / CPU time: 0.603
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1396.734 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1388.161 / Time: 0h:0m:1s (0h:0m:2s left)
BETTER TREE FOUND at iteration 41: -1387.256
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 50: -1387.168
Iteration 50 / LogL: -1387.168 / Time: 0h:0m:1s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 60 / LogL: -1392.686 / Time: 0h:0m:2s (0h:0m:3s left)
WARNING: NNI search needs unusual large number of steps (20) to converge!
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 70 / LogL: -1387.322 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 80 / LogL: -1406.262 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 90 / LogL: -1387.170 / Time: 0h:0m:3s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 100 / LogL: -1389.291 / Time: 0h:0m:3s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 110 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 120 / LogL: -1387.600 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 130 / LogL: -1387.190 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 140 / LogL: -1396.395 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 150 / LogL: -1387.168 / Time: 0h:0m:4s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 151 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34620  A-G: 2.32996  A-T: 2.15014  C-G: 1.23835  C-T: 3.22811  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.284
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.167
Total tree length: 7.605

Total number of iterations: 151
CPU time used for tree search: 4.159 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.163 sec (0h:0m:4s)
Total CPU time used: 4.674 sec (0h:0m:4s)
Total wall-clock time used: 4.682 sec (0h:0m:4s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4py7hstn/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4py7hstn/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4py7hstn/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4py7hstn/q2iqtree.log

Date and Time: Fri Aug 21 12:35:27 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-ipzmjqip/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp4py7hstn/q2iqtree -nt 1

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

Output artifacts:

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-7p7xs66n/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree -nt 1 -fast
Seed:    125777 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:35:32 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-7p7xs66n/9d9e927a-ab0b-4a36-9073-34185507633e/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: 125777)

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, 1.295 / LogL: -1395.202
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.019, 1.365 / LogL: -1395.967
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.023, 1.420 / LogL: -1396.181
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.023, 1.432 / LogL: -1396.153
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.026, 1.438 / LogL: -1396.335
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.028, 1.440 / LogL: -1396.491
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.023, 1.432 / LogL: -1396.254
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.024, 1.435 / LogL: -1396.310
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.025, 1.436 / LogL: -1396.361
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.026, 1.437 / LogL: -1396.404
Optimal pinv,alpha: 0.000, 1.295 / LogL: -1395.202

Parameters optimization took 0.325 sec
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.873
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.278
UPDATE BEST LOG-LIKELIHOOD: -1387.267
Finish initializing candidate tree set (3)
Current best tree score: -1387.267 / CPU time: 0.052
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: -1387.267
Optimal log-likelihood: -1387.255
Rate parameters:  A-C: 0.33186  A-G: 2.26184  A-T: 2.14950  C-G: 1.17498  C-T: 3.30626  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: 1.320
Parameters optimization took 1 rounds (0.007 sec)
BEST SCORE FOUND : -1387.255
Total tree length: 6.743

Total number of iterations: 2
CPU time used for tree search: 0.052 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.052 sec (0h:0m:0s)
Total CPU time used: 0.410 sec (0h:0m:0s)
Total wall-clock time used: 0.413 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 126777)

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: -1491.633
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 (0.038 sec)
Computing ML distances based on estimated model parameters... 0.011 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: 0.059
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

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

Total number of iterations: 2
CPU time used for tree search: 0.059 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.059 sec (0h:0m:0s)
Total CPU time used: 0.155 sec (0h:0m:0s)
Total wall-clock time used: 0.157 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 127777)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84175  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.040 sec)
Computing ML distances based on estimated model parameters... 0.013 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1394.002
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.043
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.950
2. Current log-likelihood: -1387.791
3. Current log-likelihood: -1387.671
4. Current log-likelihood: -1387.582
5. Current log-likelihood: -1387.514
6. Current log-likelihood: -1387.461
Optimal log-likelihood: -1387.419
Rate parameters:  A-C: 0.33533  A-G: 2.25857  A-T: 2.13278  C-G: 1.17254  C-T: 3.26710  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 6 rounds (0.024 sec)
BEST SCORE FOUND : -1387.419
Total tree length: 6.707

Total number of iterations: 2
CPU time used for tree search: 0.042 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.043 sec (0h:0m:0s)
Total CPU time used: 0.134 sec (0h:0m:0s)
Total wall-clock time used: 0.137 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 128777)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.199
2. Current log-likelihood: -1404.591
3. Current log-likelihood: -1399.228
4. Current log-likelihood: -1397.831
5. Current log-likelihood: -1397.074
Optimal log-likelihood: -1396.495
Rate parameters:  A-C: 0.24620  A-G: 2.08306  A-T: 1.99581  C-G: 1.06240  C-T: 2.85598  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.432
Parameters optimization took 5 rounds (0.042 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.972
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

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

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

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

---> START RUN NUMBER 5 (seed: 129777)

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: -1492.389
2. Current log-likelihood: -1401.890
3. Current log-likelihood: -1396.534
4. Current log-likelihood: -1395.117
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.814
Rate parameters:  A-C: 0.27026  A-G: 2.39526  A-T: 2.16931  C-G: 1.24752  C-T: 3.29290  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.415
Parameters optimization took 5 rounds (0.036 sec)
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.793
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.213
Finish initializing candidate tree set (3)
Current best tree score: -1388.213 / CPU time: 0.047
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.213
2. Current log-likelihood: -1388.014
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.759
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.518
Rate parameters:  A-C: 0.35532  A-G: 2.35215  A-T: 2.13939  C-G: 1.20296  C-T: 3.37021  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.361
Parameters optimization took 7 rounds (0.028 sec)
BEST SCORE FOUND : -1387.518
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.047 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.047 sec (0h:0m:0s)
Total CPU time used: 0.137 sec (0h:0m:0s)
Total wall-clock time used: 0.140 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 130777)

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: -1496.015
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: 0.23668  A-G: 2.05005  A-T: 1.94886  C-G: 1.06762  C-T: 2.81217  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 (0.038 sec)
Computing ML distances based on estimated model parameters... 0.010 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: 0.055
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.981
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: 0.32762  A-G: 2.25273  A-T: 2.12566  C-G: 1.16857  C-T: 3.25528  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 (0.025 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.701

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: 0.143 sec (0h:0m:0s)
Total wall-clock time used: 0.145 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 131777)

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: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.040 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: -1394.002
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.044
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.950
2. Current log-likelihood: -1387.791
3. Current log-likelihood: -1387.671
4. Current log-likelihood: -1387.582
5. Current log-likelihood: -1387.514
6. Current log-likelihood: -1387.461
Optimal log-likelihood: -1387.419
Rate parameters:  A-C: 0.33533  A-G: 2.25857  A-T: 2.13278  C-G: 1.17254  C-T: 3.26710  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 6 rounds (0.024 sec)
BEST SCORE FOUND : -1387.419
Total tree length: 6.707

Total number of iterations: 2
CPU time used for tree search: 0.043 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.044 sec (0h:0m:0s)
Total CPU time used: 0.134 sec (0h:0m:0s)
Total wall-clock time used: 0.136 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 132777)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.863
2. Current log-likelihood: -1402.072
3. Current log-likelihood: -1396.809
4. Current log-likelihood: -1395.391
5. Current log-likelihood: -1394.657
Optimal log-likelihood: -1394.080
Rate parameters:  A-C: 0.27275  A-G: 2.35291  A-T: 2.09125  C-G: 1.19606  C-T: 3.26638  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.387
Parameters optimization took 5 rounds (0.035 sec)
Computing ML distances based on estimated model parameters... 0.010 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1393.808
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.189
Finish initializing candidate tree set (4)
Current best tree score: -1388.189 / CPU time: 0.059
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.189
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: 0.059 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.060 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)

---> START RUN NUMBER 9 (seed: 133777)

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

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

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.059
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

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

Total number of iterations: 2
CPU time used for tree search: 0.059 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.059 sec (0h:0m:0s)
Total CPU time used: 0.154 sec (0h:0m:0s)
Total wall-clock time used: 0.156 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 134777)

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 (0.039 sec)
Computing ML distances based on estimated model parameters... 0.010 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: 0.056
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: -1388.187
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.687
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.471
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33227  A-G: 2.23742  A-T: 2.11203  C-G: 1.16007  C-T: 3.23505  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 7 rounds (0.029 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.056 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.056 sec (0h:0m:0s)
Total CPU time used: 0.149 sec (0h:0m:0s)
Total wall-clock time used: 0.152 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 1 gave best log-likelihood: -1387.255
Total CPU time for 10 runs: 1.723 seconds.
Total wall-clock time for 10 runs: 1.756 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree.log

Date and Time: Fri Aug 21 12:35:33 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-7p7xs66n/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp52cewpid/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

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

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

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

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-u6d7pvg4/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpo5_z4hbq/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    237532 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:35:38 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-u6d7pvg4/9d9e927a-ab0b-4a36-9073-34185507633e/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, 1.261 / LogL: -1392.812
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.354 / LogL: -1393.088
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.366 / LogL: -1393.159
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.362 / LogL: -1393.150
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.357 / LogL: -1393.112
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.361 / LogL: -1393.138
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.009, 1.364 / LogL: -1393.160
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.358 / LogL: -1393.102
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.359 / LogL: -1393.108
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.361 / LogL: -1393.119
Optimal pinv,alpha: 0.000, 1.261 / LogL: -1392.812

Parameters optimization took 0.477 sec
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.727
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.088 second
Computing log-likelihood of 98 initial trees ... 0.110 seconds
Current best score: -1392.727

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.357
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1387.260
Iteration 10 / LogL: -1387.280 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.280 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.260 / CPU time: 0.603
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1398.869 / Time: 0h:0m:1s (0h:0m:3s left)
BETTER TREE FOUND at iteration 37: -1387.257
Iteration 40 / LogL: -1387.379 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 50 / LogL: -1387.266 / Time: 0h:0m:2s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 55: -1387.168
Iteration 60 / LogL: -1387.335 / Time: 0h:0m:2s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 70 / LogL: -1387.340 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 80 / LogL: -1406.834 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 90 / LogL: -1387.336 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 100 / LogL: -1387.338 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 110 / LogL: -1387.320 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 120 / LogL: -1396.395 / Time: 0h:0m:4s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 130 / LogL: -1387.169 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 140 / LogL: -1387.209 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 150 / LogL: -1387.216 / Time: 0h:0m:4s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
TREE SEARCH COMPLETED AFTER 156 ITERATIONS / Time: 0h:0m:5s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34757  A-G: 2.33789  A-T: 2.15832  C-G: 1.24304  C-T: 3.24083  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.284
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.167

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.059 sec.
Total tree length: 7.609

Total number of iterations: 156
CPU time used for tree search: 4.551 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.563 sec (0h:0m:4s)
Total CPU time used: 5.117 sec (0h:0m:5s)
Total wall-clock time used: 5.131 sec (0h:0m:5s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpo5_z4hbq/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpo5_z4hbq/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpo5_z4hbq/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpo5_z4hbq/q2iqtree.log

Date and Time: Fri Aug 21 12:35:43 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-u6d7pvg4/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpo5_z4hbq/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

Tip

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-fwp02jx9/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpll4v0igo/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    747202 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:35:47 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-fwp02jx9/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.703
Optimal log-likelihood: -1388.843
Rate parameters:  A-C: 0.35840  A-G: 2.36329  A-T: 2.12617  C-G: 1.22968  C-T: 3.29817  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: 1.424
Parameters optimization took 1 rounds (0.007 sec)
Time for fast ML tree search: 0.057 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         1402.574     45  2895.147     2919.790     3046.616
  2  GTR+F+I       1401.119     46  2894.238     2920.130     3049.072
  3  GTR+F+G4      1387.362     46  2866.724     2892.616     3021.558
  4  GTR+F+I+G4    1387.729     47  2869.457     2896.638     3027.658
  5  GTR+F+R2      1382.368     47  2858.736     2885.917     3016.937
  6  GTR+F+R3      1382.422     49  2862.845     2892.723     3027.778
 16  SYM+G4        1387.133     43  2860.266     2882.525     3005.003
 18  SYM+R2        1383.095     44  2854.190     2877.621     3002.292
 29  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 31  TVM+F+R2      1383.790     46  2859.580     2885.472     3014.415
 42  TVMe+G4       1387.121     42  2858.243     2879.366     2999.614
 44  TVMe+R2       1383.080     43  2852.159     2874.418     2996.896
 55  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 57  TIM3+F+R2     1385.913     45  2861.826     2886.469     3013.295
 68  TIM3e+G4      1390.357     41  2862.713     2882.736     3000.718
 70  TIM3e+R2      1385.918     42  2855.835     2876.958     2997.206
 81  TIM2+F+G4     1393.635     44  2875.271     2898.703     3023.374
 83  TIM2+F+R2     1387.682     45  2865.363     2890.006     3016.832
 94  TIM2e+G4      1396.794     41  2875.588     2895.611     3013.593
 96  TIM2e+R2      1391.573     42  2867.147     2888.270     3008.518
107  TIM+F+G4      1390.364     44  2868.727     2892.159     3016.830
109  TIM+F+R2      1384.935     45  2859.870     2884.512     3011.338
120  TIMe+G4       1394.002     41  2870.004     2890.027     3008.009
122  TIMe+R2       1389.000     42  2862.001     2883.123     3003.372
133  TPM3u+F+G4    1392.305     43  2870.611     2892.870     3015.348
135  TPM3u+F+R2    1387.330     44  2862.659     2886.091     3010.762
146  TPM3+F+G4     1392.305     43  2870.611     2892.870     3015.348
148  TPM3+F+R2     1387.330     44  2862.659     2886.091     3010.762
159  TPM2u+F+G4    1394.536     43  2875.071     2897.330     3019.808
161  TPM2u+F+R2    1389.054     44  2866.108     2889.540     3014.211
172  TPM2+F+G4     1394.536     43  2875.071     2897.330     3019.808
174  TPM2+F+R2     1389.054     44  2866.108     2889.540     3014.211
185  K3Pu+F+G4     1391.406     43  2868.812     2891.070     3013.549
187  K3Pu+F+R2     1386.386     44  2860.771     2884.203     3008.874
198  K3P+G4        1394.023     40  2868.045     2887.005     3002.684
200  K3P+R2        1389.010     41  2860.020     2880.044     2998.025
211  TN+F+G4       1394.039     43  2874.077     2896.336     3018.814
213  TN+F+R2       1388.240     44  2864.480     2887.912     3012.583
224  TNe+G4        1396.796     40  2873.591     2892.551     3008.230
226  TNe+R2        1391.590     41  2865.180     2885.203     3003.185
237  HKY+F+G4      1394.954     42  2873.909     2895.031     3015.280
239  HKY+F+R2      1389.609     43  2865.218     2887.477     3009.955
250  K2P+G4        1396.828     39  2871.657     2889.588     3002.930
252  K2P+R2        1391.598     40  2863.196     2882.156     2997.835
263  F81+F+G4      1405.748     41  2893.496     2913.520     3031.501
265  F81+F+R2      1400.803     42  2885.607     2906.729     3026.978
276  JC+G4         1407.653     38  2891.306     2908.244     3019.214
278  JC+R2         1402.857     39  2883.715     2901.646     3014.988
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/tmpll4v0igo/q2iqtree.model.gz
CPU time for ModelFinder: 0.593 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.597 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1383.080
Optimal log-likelihood: -1383.077
Rate parameters:  A-C: 0.22956  A-G: 1.98471  A-T: 1.54888  C-G: 0.78156  C-T: 1.98471  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.713,0.411) (0.287,2.462)
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: -1389.274
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.087 second
Computing log-likelihood of 97 initial trees ... 0.082 seconds
Current best score: -1383.077

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.824
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1382.305
Iteration 10 / LogL: -1382.313 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.315 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.305 / CPU time: 0.454
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 30 / LogL: -1382.078 / Time: 0h:0m:0s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 40 / LogL: -1382.443 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 50 / LogL: -1382.162 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 60 / LogL: -1382.421 / Time: 0h:0m:0s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 70 / LogL: -1382.005 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1382.004 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1382.459 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 100 / LogL: -1384.503 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 110 / LogL: -1382.007 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 120 / LogL: -1382.091 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 130 / LogL: -1382.102 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 140 / LogL: -1382.018 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 150 / LogL: -1382.097 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 160 / LogL: -1382.004 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1382.006 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 180 / LogL: -1382.006 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 190 / LogL: -1382.090 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1382.211 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 203 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.004
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19277  A-G: 1.84601  A-T: 1.53745  C-G: 0.77078  C-T: 1.84601  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.408) (0.276,2.550)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.002
Total tree length: 7.119

Total number of iterations: 203
CPU time used for tree search: 2.550 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.553 sec (0h:0m:2s)
Total CPU time used: 2.579 sec (0h:0m:2s)
Total wall-clock time used: 2.584 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpll4v0igo/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpll4v0igo/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpll4v0igo/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpll4v0igo/q2iqtree.log

Date and Time: Fri Aug 21 12:35:50 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-fwp02jx9/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpll4v0igo/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

iqtree-ultrafast-bootstrap

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-10tfbvom/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    293297 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:35:55 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-10tfbvom/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1396.575
2. Current log-likelihood: -1395.213
Optimal log-likelihood: -1394.464
Rate parameters:  A-C: 0.21819  A-G: 2.03593  A-T: 1.93394  C-G: 1.05109  C-T: 2.56337  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: 1.322
Parameters optimization took 2 rounds (0.013 sec)
Time for fast ML tree search: 0.060 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         1411.054     45  2912.108     2936.751     3063.577
  2  GTR+F+I       1409.135     46  2910.270     2936.162     3065.105
  3  GTR+F+G4      1392.992     46  2877.983     2903.876     3032.818
  4  GTR+F+I+G4    1393.280     47  2880.561     2907.741     3038.762
  5  GTR+F+R2      1387.712     47  2869.423     2896.604     3027.624
  6  GTR+F+R3      1387.747     49  2873.494     2903.372     3038.427
 16  SYM+G4        1393.506     43  2873.012     2895.271     3017.749
 18  SYM+R2        1389.901     44  2867.802     2891.234     3015.905
 29  TVM+F+G4      1393.474     45  2876.947     2901.590     3028.416
 31  TVM+F+R2      1388.475     46  2868.950     2894.842     3023.785
 42  TVMe+G4       1393.632     42  2871.264     2892.387     3012.635
 44  TVMe+R2       1389.912     43  2865.824     2888.083     3010.561
 55  TIM3+F+G4     1396.957     44  2881.914     2905.346     3030.017
 57  TIM3+F+R2     1391.441     45  2872.881     2897.524     3024.350
 68  TIM3e+G4      1397.005     41  2876.010     2896.033     3014.015
 70  TIM3e+R2      1393.195     42  2870.390     2891.513     3011.761
 81  TIM2+F+G4     1401.480     44  2890.961     2914.393     3039.064
 83  TIM2+F+R2     1395.762     45  2881.524     2906.167     3032.993
 94  TIM2e+G4      1406.407     41  2894.815     2914.838     3032.820
 96  TIM2e+R2      1402.269     42  2888.539     2909.662     3029.910
107  TIM+F+G4      1397.972     44  2883.943     2907.375     3032.046
109  TIM+F+R2      1392.182     45  2874.365     2899.008     3025.834
120  TIMe+G4       1403.787     41  2889.575     2909.598     3027.580
122  TIMe+R2       1399.417     42  2882.835     2903.958     3024.206
133  TPM3u+F+G4    1397.423     43  2880.846     2903.105     3025.583
135  TPM3u+F+R2    1392.257     44  2872.514     2895.946     3020.617
146  TPM3+F+G4     1397.423     43  2880.846     2903.105     3025.583
148  TPM3+F+R2     1392.257     44  2872.514     2895.946     3020.617
159  TPM2u+F+G4    1401.944     43  2889.889     2912.147     3034.626
161  TPM2u+F+R2    1396.513     44  2881.027     2904.458     3029.129
172  TPM2+F+G4     1401.944     43  2889.889     2912.147     3034.626
174  TPM2+F+R2     1396.513     44  2881.027     2904.458     3029.129
185  K3Pu+F+G4     1398.573     43  2883.146     2905.405     3027.883
187  K3Pu+F+R2     1393.066     44  2874.132     2897.564     3022.235
198  K3P+G4        1403.919     40  2887.838     2906.798     3022.477
200  K3P+R2        1399.438     41  2880.876     2900.899     3018.881
211  TN+F+G4       1401.600     43  2889.200     2911.459     3033.937
213  TN+F+R2       1395.970     44  2879.940     2903.372     3028.043
224  TNe+G4        1406.476     40  2892.951     2911.911     3027.590
226  TNe+R2        1402.298     41  2886.596     2906.620     3024.601
237  HKY+F+G4      1402.073     42  2888.146     2909.268     3029.517
239  HKY+F+R2      1396.722     43  2879.444     2901.703     3024.181
250  K2P+G4        1406.650     39  2891.299     2909.230     3022.572
252  K2P+R2        1402.334     40  2884.668     2903.627     3019.307
263  F81+F+G4      1410.301     41  2902.603     2922.626     3040.608
265  F81+F+R2      1405.908     42  2895.816     2916.939     3037.187
276  JC+G4         1414.958     38  2905.915     2922.852     3033.822
278  JC+R2         1411.527     39  2901.054     2918.985     3032.327
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/tmp3_250oea/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.628 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.632 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 293297)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1389.912
Optimal log-likelihood: -1389.906
Rate parameters:  A-C: 0.10868  A-G: 1.62939  A-T: 1.39318  C-G: 0.63110  C-T: 1.62939  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.687,0.344) (0.313,2.442)
Parameters optimization took 1 rounds (0.007 sec)
Computing ML distances based on estimated model parameters... 0.007 sec
WARNING: Some pairwise ML distances are too long (saturated)
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1389.952
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.082 second
Computing log-likelihood of 97 initial trees ... 0.078 seconds
Current best score: -1389.906

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1388.680
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1382.315
BETTER TREE FOUND at iteration 3: -1382.311
Iteration 10 / LogL: -1382.352 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.362 / Time: 0h:0m:0s
Finish initializing candidate tree set (6)
Current best tree score: -1382.311 / CPU time: 0.596
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1384.554 / Time: 0h:0m:0s (0h:0m:4s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 31: -1382.088
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
BETTER TREE FOUND at iteration 34: -1382.005
Iteration 40 / LogL: -1382.423 / Time: 0h:0m:0s (0h:0m:4s left)
BETTER TREE FOUND at iteration 42: -1382.005
Iteration 50 / LogL: -1391.036 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1410.054
Iteration 60 / LogL: -1382.089 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 70 / LogL: -1382.090 / Time: 0h:0m:1s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 80 / LogL: -1382.090 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 90 / LogL: -1382.097 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 100 / LogL: -1382.016 / Time: 0h:0m:2s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1410.054
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
Iteration 110 / LogL: -1382.016 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 120 / LogL: -1384.674 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 130 / LogL: -1382.898 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 140 / LogL: -1382.090 / Time: 0h:0m:2s (0h:0m:2s left)
Iteration 150 / LogL: -1382.016 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1410.054
Iteration 160 / LogL: -1382.015 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 170 / LogL: -1382.006 / Time: 0h:0m:3s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 180 / LogL: -1382.060 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 190 / LogL: -1382.090 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 200 / LogL: -1382.037 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1410.054
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
Iteration 210 / LogL: -1382.005 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.009 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 230 / LogL: -1382.007 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 240 / LogL: -1382.007 / Time: 0h:0m:5s (0h:0m:1s left)
TREE SEARCH COMPLETED AFTER 243 ITERATIONS / Time: 0h:0m:5s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.005
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19145  A-G: 1.83967  A-T: 1.53182  C-G: 0.77023  C-T: 1.83967  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.408) (0.277,2.544)
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/tmp3_250oea/q2iqtreeufboot.splits.nex
Total tree length: 7.120

Total number of iterations: 243
CPU time used for tree search: 5.062 sec (0h:0m:5s)
Wall-clock time used for tree search: 5.077 sec (0h:0m:5s)
Total CPU time used: 5.167 sec (0h:0m:5s)
Total wall-clock time used: 5.186 sec (0h:0m:5s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.splits.nex...
20 taxa and 154 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.003

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot.log

Date and Time: Fri Aug 21 12:36:01 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 -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-10tfbvom/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp3_250oea/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

Perform single branch tests alongside ufboot

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

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

stdout:

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

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-_2sjmjvy/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    511844 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Fri Aug 21 12:36:05 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-_2sjmjvy/9d9e927a-ab0b-4a36-9073-34185507633e/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: 511844)...

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, 1.261 / LogL: -1392.812
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.354 / LogL: -1393.088
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.366 / LogL: -1393.159
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.362 / LogL: -1393.150
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.357 / LogL: -1393.112
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.361 / LogL: -1393.138
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.009, 1.364 / LogL: -1393.160
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.358 / LogL: -1393.102
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.359 / LogL: -1393.108
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.361 / LogL: -1393.119
Optimal pinv,alpha: 0.000, 1.261 / LogL: -1392.812

Parameters optimization took 0.479 sec
Computing ML distances based on estimated model parameters... 0.009 sec
Computing BIONJ tree...
0.001 seconds
Log-likelihood of BIONJ tree: -1392.727
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.091 second
Computing log-likelihood of 98 initial trees ... 0.108 seconds
Current best score: -1392.727

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.357
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1387.260
Iteration 10 / LogL: -1387.736 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.280 / Time: 0h:0m:1s
Finish initializing candidate tree set (3)
Current best tree score: -1387.260 / CPU time: 0.767
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.438 / Time: 0h:0m:1s (0h:0m:9s left)
Iteration 40 / LogL: -1387.350 / Time: 0h:0m:1s (0h:0m:7s left)
Iteration 50 / LogL: -1387.350 / Time: 0h:0m:2s (0h:0m:6s left)
Log-likelihood cutoff on original alignment: -1407.515
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 55: -1387.169
Iteration 60 / LogL: -1387.912 / Time: 0h:0m:2s (0h:0m:8s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 70 / LogL: -1387.401 / Time: 0h:0m:2s (0h:0m:7s left)
Iteration 80 / LogL: -1387.339 / Time: 0h:0m:3s (0h:0m:7s left)
Iteration 90 / LogL: -1387.636 / Time: 0h:0m:3s (0h:0m:6s left)
Iteration 100 / LogL: -1387.349 / Time: 0h:0m:3s (0h:0m:5s left)
Log-likelihood cutoff on original alignment: -1408.844
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.987
NOTE: UFBoot does not converge, continue at least 100 more iterations
UPDATE BEST LOG-LIKELIHOOD: -1387.169
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 110 / LogL: -1387.212 / Time: 0h:0m:3s (0h:0m:5s left)
Iteration 120 / LogL: -1387.195 / Time: 0h:0m:4s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 130 / LogL: -1387.503 / Time: 0h:0m:4s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 140 / LogL: -1387.169 / Time: 0h:0m:4s (0h:0m:3s left)
Iteration 150 / LogL: -1401.202 / Time: 0h:0m:4s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1409.353
UPDATE BEST LOG-LIKELIHOOD: -1387.169
UPDATE BEST LOG-LIKELIHOOD: -1387.169
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 160 / LogL: -1387.169 / Time: 0h:0m:5s (0h:0m:3s left)
Iteration 170 / LogL: -1387.176 / Time: 0h:0m:5s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 180 / LogL: -1387.169 / Time: 0h:0m:5s (0h:0m:2s left)
Iteration 190 / LogL: -1387.169 / Time: 0h:0m:6s (0h:0m:2s left)
Iteration 200 / LogL: -1387.216 / Time: 0h:0m:6s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1409.353
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.986
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 210 / LogL: -1387.170 / Time: 0h:0m:6s (0h:0m:2s left)
Iteration 220 / LogL: -1387.177 / Time: 0h:0m:7s (0h:0m:2s left)
Iteration 230 / LogL: -1387.169 / Time: 0h:0m:7s (0h:0m:2s left)
Iteration 240 / LogL: -1387.169 / Time: 0h:0m:7s (0h:0m:1s left)
Iteration 250 / LogL: -1387.169 / Time: 0h:0m:7s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1410.030
Iteration 260 / LogL: -1387.169 / Time: 0h:0m:8s (0h:0m:1s left)
Iteration 270 / LogL: -1387.169 / Time: 0h:0m:8s (0h:0m:0s left)
Iteration 280 / LogL: -1387.169 / Time: 0h:0m:8s (0h:0m:0s left)
Iteration 290 / LogL: -1387.169 / Time: 0h:0m:9s (0h:0m:0s left)
Iteration 300 / LogL: -1387.396 / Time: 0h:0m:9s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1410.929
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.986
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 310 / LogL: -1387.192 / Time: 0h:0m:9s (0h:0m:2s left)
Iteration 320 / LogL: -1387.186 / Time: 0h:0m:10s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 330 / LogL: -1387.170 / Time: 0h:0m:10s (0h:0m:2s left)
Iteration 340 / LogL: -1387.169 / Time: 0h:0m:10s (0h:0m:1s left)
Iteration 350 / LogL: -1387.169 / Time: 0h:0m:10s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1411.810
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 360 / LogL: -1390.383 / Time: 0h:0m:11s (0h:0m:1s left)
Iteration 370 / LogL: -1390.383 / Time: 0h:0m:11s (0h:0m:0s left)
Iteration 380 / LogL: -1387.169 / Time: 0h:0m:11s (0h:0m:0s left)
Iteration 390 / LogL: -1387.170 / Time: 0h:0m:12s (0h:0m:0s left)
Iteration 400 / LogL: -1387.169 / Time: 0h:0m:12s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1411.810
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.989
NOTE: UFBoot does not converge, continue at least 100 more iterations
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 410 / LogL: -1387.169 / Time: 0h:0m:12s (0h:0m:2s left)
Iteration 420 / LogL: -1387.379 / Time: 0h:0m:13s (0h:0m:2s left)
Iteration 430 / LogL: -1387.174 / Time: 0h:0m:13s (0h:0m:2s left)
Iteration 440 / LogL: -1387.169 / Time: 0h:0m:13s (0h:0m:1s left)
Iteration 450 / LogL: -1387.169 / Time: 0h:0m:13s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1411.810
Iteration 460 / LogL: -1387.169 / Time: 0h:0m:14s (0h:0m:1s left)
Iteration 470 / LogL: -1387.169 / Time: 0h:0m:14s (0h:0m:0s left)
Iteration 480 / LogL: -1387.169 / Time: 0h:0m:14s (0h:0m:0s left)
Iteration 490 / LogL: -1387.169 / Time: 0h:0m:15s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.169
Iteration 500 / LogL: -1387.176 / Time: 0h:0m:15s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1411.810
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.995
TREE SEARCH COMPLETED AFTER 500 ITERATIONS / Time: 0h:0m:15s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.169
Optimal log-likelihood: -1387.168
Rate parameters:  A-C: 0.34749  A-G: 2.33793  A-T: 2.15825  C-G: 1.24305  C-T: 3.24077  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.284
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.168

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...
0.060 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.splits.nex
Total tree length: 7.608

Total number of iterations: 500
CPU time used for tree search: 15.059 sec (0h:0m:15s)
Wall-clock time used for tree search: 15.093 sec (0h:0m:15s)
Total CPU time used: 15.706 sec (0h:0m:15s)
Total wall-clock time used: 15.746 sec (0h:0m:15s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.splits.nex...
20 taxa and 157 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.213

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/q2iqtreeufboot.log

Date and Time: Fri Aug 21 12:36:21 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-_2sjmjvy/9d9e927a-ab0b-4a36-9073-34185507633e/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp232vfg9f/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!