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Phylogenetic inference with q2-phylogeny

Note

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

Inferring phylogenies

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

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

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

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

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

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

  1. generate a sequence alignment within QIIME 2

  2. mask the alignment if needed

  3. construct a phylogenetic tree

  4. root the phylogenetic tree

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

Sequence Alignment

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

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

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

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

Next, download the data:

Please select a download option that is most appropriate for your environment:
wget \
  -O "rep-seqs.qza" \
  "https://data.qiime2.org/2022.8/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2022.8/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 several tools that attempt to reduce the amount of ambiguously aligned regions by using curated reference alignments. Traditional, de novo alignment methods mututally align a set of unaligned sequences to create a multiple sequence alignment (MSA) from scratch. Re-running these methods with additional sequences will create MSAs with varying numbers of columns and assignments of bases to each column. These alignments is therefore incompatible with one another and may not be joined through concatenation.

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

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

Note

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

Construct a phylogeny

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

  1. Phylogeny for the faint of heart - a tutorial

  2. Molecular phylogenetics - principles and practice

  3. Phylogenetics - An Introduction

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

  1. FastTree

  2. RAxML

  3. IQ-TREE

Methods

fasttree

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

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

Output artifacts:

Tip

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

raxml

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

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

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid hill-climbing mode

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


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

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 8571 -N 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp3npx9lx -n q2 


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

Inference[0]: Time 0.359312 CAT-based likelihood -1249.682690, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1394.064223 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp3npx9lx/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1394.064223 .... 

Final GAMMA-based Score of best tree -1388.585712

Program execution info written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp3npx9lx/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp3npx9lx/RAxML_bestTree.q2

Overall execution time: 0.813990 secs or 0.000226 hours or 0.000009 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 8571 -N 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp3npx9lx -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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r -n q2 


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

Inference[0]: Time 0.421948 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.349659 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.351034 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.445239 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.319800 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r/RAxML_bestTree.q2

Overall execution time: 2.398480 secs or 0.000666 hours or 0.000028 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpox292f2r -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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec -n q2bootstrap 



Time for BS model parameter optimization 0.039563
Bootstrap[0]: Time 0.123594 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.079859 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.074715 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.066403 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.074875 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.079225 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.079191 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.070687 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.077070 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.084300 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.058013 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.084380 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.095610 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.074554 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.084201 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.079886 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.080463 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.070237 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.066962 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.069577 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.062797 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.059261 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.067997 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.079803 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.070505 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.070065 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.070330 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.087988 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.085280 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.056698 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.071459 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.075376 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.078311 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.068704 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.065264 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.076532 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.063127 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.079562 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.063310 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.058920 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.070405 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.070591 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.066066 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.082446 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.063777 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.071864 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.057860 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.084590 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.055784 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.078005 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.088726 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.063722 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.074346 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.063973 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.074404 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.085111 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.070036 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.061965 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.088467 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.069820 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.060392 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.069991 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.082334 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.064137 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.061787 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.062648 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.077924 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.078683 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.062319 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.063159 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.066523 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.064870 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.054981 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.065406 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.057686 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.061171 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.071426 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.086812 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.069915 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.071863 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.064986 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.070165 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.083228 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.062561 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.068217 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.071421 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.061543 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.060356 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.073302 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.066829 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.079408 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.071707 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.028819 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.075709 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.063221 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.060873 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.072994 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.075910 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.027583 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.036025 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 7.066032 seconds
Average Time per Rapid Bootstrap 0.070660 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 2.808639 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: 1.419676 seconds
Thorough ML search Time: 0.402453 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 4.634939 secs or 0.001287 hours

Combined Bootstrap and ML search took 11.701428 secs or 0.003250 hours

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



Found 1 tree in File /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 11.709542 secs or 0.003253 hours or 0.000136 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpk4s7tqec -n q2bootstrap

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

Output artifacts:

Tip

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

iqtree

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

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree -nt 1
Seed:    912530 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:00:44 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000151873 secs using 73.75% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000230074 secs using 74.76% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.28746e-05 secs using 85.44% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 3.88622e-05 secs using 72.05% CPU


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Optimizing NNI: done in 0.013303 secs using 99.26% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.281
2. Current log-likelihood: -1389.723
Optimal log-likelihood: -1388.882
Rate parameters:  A-C: 0.33811  A-G: 2.30746  A-T: 2.15809  C-G: 1.19223  C-T: 3.30165  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: 1.423
Parameters optimization took 2 rounds (0.008 sec)
Time for fast ML tree search: 0.036 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.278     46  2866.557     2892.449     3021.392
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.422
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 14  GTR+F+I+I+R2  1380.757     48  2857.513     2886.023     3019.080
 15  GTR+F+I+I+R3  1380.696     50  2861.392     2892.681     3029.691
 25  SYM+G4        1387.155     43  2860.310     2882.568     3005.047
 27  SYM+R2        1382.244     44  2852.489     2875.921     3000.592
 36  SYM+I+I+R2    1382.405     45  2854.809     2879.452     3006.278
 47  TVM+F+G4      1388.425     45  2866.850     2891.493     3018.319
 49  TVM+F+R2      1382.482     46  2856.965     2882.857     3011.799
 58  TVM+F+I+I+R2  1382.560     47  2859.119     2886.300     3017.320
 69  TVMe+G4       1387.122     42  2858.244     2879.367     2999.615
 71  TVMe+R2       1382.298     43  2850.596     2872.855     2995.333
 80  TVMe+I+I+R2   1382.453     44  2852.907     2876.339     3001.010
 91  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 93  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
102  TIM3+F+I+I+R2 1384.573     46  2861.146     2887.039     3015.981
113  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
115  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
124  TIM3e+I+I+R2  1385.391     43  2856.782     2879.040     3001.519
135  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
137  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
146  TIM2+F+I+I+R2 1386.377     46  2864.755     2890.647     3019.590
157  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
159  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
168  TIM2e+I+I+R2  1391.272     43  2868.544     2890.802     3013.281
179  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
181  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
190  TIM+F+I+I+R2  1383.340     46  2858.680     2884.572     3013.515
201  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
203  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
212  TIMe+I+I+R2   1388.456     43  2862.912     2885.171     3007.649
223  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
225  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
234  TPM3u+F+I+I+R2 1386.501     45  2863.002     2887.645     3014.471
245  TPM3+G4       1390.540     40  2861.080     2880.039     2995.719
247  TPM3+R2       1385.307     41  2852.614     2872.637     2990.619
256  TPM3+I+I+R2   1385.459     42  2854.917     2876.040     2996.288
267  TPM2u+F+G4    1395.283     43  2876.566     2898.825     3021.303
269  TPM2u+F+R2    1388.114     44  2864.229     2887.661     3012.332
278  TPM2u+F+I+I+R2 1388.249     45  2866.497     2891.140     3017.966
289  TPM2+G4       1397.749     40  2875.499     2894.458     3010.138
291  TPM2+R2       1391.198     41  2864.396     2884.419     3002.401
300  TPM2+I+I+R2   1391.346     42  2866.692     2887.815     3008.063
311  K3Pu+F+G4     1392.050     43  2870.100     2892.359     3014.837
313  K3Pu+F+R2     1385.122     44  2858.245     2881.677     3006.348
322  K3Pu+F+I+I+R2 1385.245     45  2860.491     2885.134     3011.960
333  K3P+G4        1394.786     40  2869.573     2888.532     3004.212
335  K3P+R2        1388.359     41  2858.717     2878.740     2996.722
344  K3P+I+I+R2    1388.531     42  2861.061     2882.184     3002.432
355  TN+F+G4       1394.628     43  2875.256     2897.515     3019.993
357  TN+F+R2       1386.824     44  2861.647     2885.079     3009.750
366  TN+F+I+I+R2   1386.967     45  2863.933     2888.576     3015.402
377  TNe+G4        1397.748     40  2875.497     2894.456     3010.136
379  TNe+R2        1391.134     41  2864.269     2884.292     3002.274
388  TNe+I+I+R2    1391.289     42  2866.578     2887.701     3007.949
399  HKY+F+G4      1395.752     42  2875.504     2896.627     3016.875
401  HKY+F+R2      1388.691     43  2863.382     2885.640     3008.119
410  HKY+F+I+I+R2  1388.820     44  2865.639     2889.071     3013.742
421  K2P+G4        1397.753     39  2873.507     2891.438     3004.780
423  K2P+R2        1391.216     40  2862.433     2881.392     2997.072
432  K2P+I+I+R2    1391.363     41  2864.726     2884.750     3002.731
443  F81+F+G4      1406.478     41  2894.955     2914.979     3032.960
445  F81+F+R2      1400.602     42  2885.203     2906.326     3026.574
454  F81+F+I+I+R2  1400.799     43  2887.598     2909.857     3032.335
465  JC+G4         1408.432     38  2892.865     2909.802     3020.772
467  JC+R2         1403.021     39  2884.041     2901.972     3015.314
476  JC+I+I+R2     1403.194     40  2886.388     2905.348     3021.028
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree.model.gz
CPU time for ModelFinder: 0.957 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.995 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.307
Optimal log-likelihood: -1385.304
Rate parameters:  A-C: 0.39693  A-G: 1.57495  A-T: 1.00000  C-G: 0.39693  C-T: 1.57495  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.403) (0.277,2.560)
Parameters optimization took 1 rounds (0.003 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000741959 secs using 94.34% CPU
Computing ML distances took 0.000810 sec (of wall-clock time) 0.000747 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.69413e-05 secs using 81.66% CPU
Constructing RapidNJ tree: done in 0.000109911 secs using 141.9% CPU
Computing RapidNJ tree took 0.000191 sec (of wall-clock time) 0.000217 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.836
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.083 second
Computing log-likelihood of 98 initial trees ... 0.057 seconds
Current best score: -1385.304

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00153899 secs using 190.1% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.303
Optimizing NNI: done in 0.00788307 secs using 192.8% CPU
Optimizing NNI: done in 0.00860786 secs using 189.4% CPU
Optimizing NNI: done in 0.00729799 secs using 190.8% CPU
Optimizing NNI: done in 0.00740814 secs using 194.8% CPU
Optimizing NNI: done in 0.00749588 secs using 189.7% CPU
Optimizing NNI: done in 0.00709486 secs using 192.1% CPU
Optimizing NNI: done in 0.00784421 secs using 118.5% CPU
Optimizing NNI: done in 0.0071609 secs using 99.78% CPU
Optimizing NNI: done in 0.00801706 secs using 97.89% CPU
Iteration 10 / LogL: -1385.338 / Time: 0h:0m:0s
Optimizing NNI: done in 0.00707412 secs using 98.63% CPU
Optimizing NNI: done in 0.0075202 secs using 96.66% CPU
Optimizing NNI: done in 0.00700283 secs using 99.23% CPU
Optimizing NNI: done in 0.00662208 secs using 98.91% CPU
Optimizing NNI: done in 0.00734806 secs using 95.92% CPU
Optimizing NNI: done in 0.00656796 secs using 99.36% CPU
Optimizing NNI: done in 0.0066061 secs using 97.88% CPU
Optimizing NNI: done in 0.00642586 secs using 99.35% CPU
Optimizing NNI: done in 0.00652909 secs using 99.14% CPU
Optimizing NNI: done in 0.00658989 secs using 99.68% CPU
Iteration 20 / LogL: -1385.339 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1385.303 / CPU time: 0.288
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.00786901 secs using 98.54% CPU
Optimizing NNI: done in 0.00799179 secs using 99.63% CPU
Optimizing NNI: done in 0.00870585 secs using 99.61% CPU
Optimizing NNI: done in 0.00611806 secs using 99.9% CPU
Optimizing NNI: done in 0.00938797 secs using 99.39% CPU
Optimizing NNI: done in 0.0116849 secs using 99.74% CPU
Optimizing NNI: done in 0.0107291 secs using 98.85% CPU
Optimizing NNI: done in 0.010026 secs using 98.87% CPU
Optimizing NNI: done in 0.00877118 secs using 96.36% CPU
Optimizing NNI: done in 0.016408 secs using 98.94% CPU
Iteration 30 / LogL: -1386.017 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.0103171 secs using 98.27% CPU
Optimizing NNI: done in 0.00536919 secs using 99.9% CPU
Optimizing NNI: done in 0.00623393 secs using 98.17% CPU
Optimizing NNI: done in 0.0105181 secs using 99.04% CPU
Optimizing NNI: done in 0.0117881 secs using 99.86% CPU
Optimizing NNI: done in 0.010097 secs using 98.62% CPU
Optimizing NNI: done in 0.0122759 secs using 98.92% CPU
Optimizing NNI: done in 0.0106502 secs using 99.85% CPU
Optimizing NNI: done in 0.011656 secs using 99.82% CPU
Optimizing NNI: done in 0.00808001 secs using 99.06% CPU
Iteration 40 / LogL: -1386.361 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.00804186 secs using 99.63% CPU
Optimizing NNI: done in 0.00822997 secs using 99.15% CPU
Optimizing NNI: done in 0.00939178 secs using 99.29% CPU
Optimizing NNI: done in 0.011996 secs using 99.02% CPU
Optimizing NNI: done in 0.0102441 secs using 99.73% CPU
Optimizing NNI: done in 0.013972 secs using 99.36% CPU
Optimizing NNI: done in 0.00928712 secs using 98.42% CPU
Optimizing NNI: done in 0.00755787 secs using 99.21% CPU
Optimizing NNI: done in 0.00638413 secs using 99.86% CPU
Optimizing NNI: done in 0.00929594 secs using 99.52% CPU
Iteration 50 / LogL: -1386.362 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.0115681 secs using 98.99% CPU
Optimizing NNI: done in 0.0059092 secs using 99.66% CPU
Optimizing NNI: done in 0.00925303 secs using 99.47% CPU
Optimizing NNI: done in 0.00826287 secs using 99.78% CPU
Optimizing NNI: done in 0.00792789 secs using 98.83% CPU
Optimizing NNI: done in 0.00979805 secs using 99.32% CPU
Optimizing NNI: done in 0.0133359 secs using 98.85% CPU
Optimizing NNI: done in 0.00393581 secs using 99.98% CPU
Optimizing NNI: done in 0.012254 secs using 99.57% CPU
Optimizing NNI: done in 0.012743 secs using 98.79% CPU
Iteration 60 / LogL: -1394.215 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.00951505 secs using 99.07% CPU
Optimizing NNI: done in 0.009408 secs using 99.78% CPU
Optimizing NNI: done in 0.00804687 secs using 99.59% CPU
Optimizing NNI: done in 0.0082469 secs using 99.37% CPU
Optimizing NNI: done in 0.00900817 secs using 99.17% CPU
Optimizing NNI: done in 0.00692797 secs using 99.94% CPU
Optimizing NNI: done in 0.00751996 secs using 99% CPU
Optimizing NNI: done in 0.00435519 secs using 98.5% CPU
Optimizing NNI: done in 0.00494909 secs using 99.94% CPU
Optimizing NNI: done in 0.00731206 secs using 99.94% CPU
Iteration 70 / LogL: -1385.310 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.0055058 secs using 99.95% CPU
Optimizing NNI: done in 0.00714803 secs using 98.88% CPU
Optimizing NNI: done in 0.00615692 secs using 99.94% CPU
Optimizing NNI: done in 0.00520515 secs using 99.13% CPU
Optimizing NNI: done in 0.00779986 secs using 99.55% CPU
Optimizing NNI: done in 0.00995588 secs using 99.15% CPU
Optimizing NNI: done in 0.00927091 secs using 99.87% CPU
Optimizing NNI: done in 0.00766182 secs using 99.58% CPU
Optimizing NNI: done in 0.013288 secs using 99.75% CPU
Optimizing NNI: done in 0.00961113 secs using 99.83% CPU
Iteration 80 / LogL: -1385.617 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.00857091 secs using 99.83% CPU
Optimizing NNI: done in 0.0133781 secs using 99.33% CPU
Optimizing NNI: done in 0.00977707 secs using 98.68% CPU
Optimizing NNI: done in 0.00773311 secs using 99.73% CPU
Optimizing NNI: done in 0.012713 secs using 99.94% CPU
Optimizing NNI: done in 0.0069499 secs using 99.54% CPU
Optimizing NNI: done in 0.00682497 secs using 99.14% CPU
Optimizing NNI: done in 0.00697994 secs using 99.56% CPU
Optimizing NNI: done in 0.00730705 secs using 99.42% CPU
Optimizing NNI: done in 0.00585008 secs using 98.58% CPU
Iteration 90 / LogL: -1385.564 / Time: 0h:0m:0s (0h:0m:0s left)
Optimizing NNI: done in 0.00891519 secs using 99.48% CPU
Optimizing NNI: done in 0.008605 secs using 99.94% CPU
Optimizing NNI: done in 0.0109408 secs using 99.78% CPU
Optimizing NNI: done in 0.0103612 secs using 99.94% CPU
Optimizing NNI: done in 0.00822306 secs using 99.43% CPU
Optimizing NNI: done in 0.012568 secs using 99.73% CPU
Optimizing NNI: done in 0.00906801 secs using 99.11% CPU
Optimizing NNI: done in 0.00874186 secs using 99.66% CPU
Optimizing NNI: done in 0.00550508 secs using 98.98% CPU
Optimizing NNI: done in 0.00942993 secs using 99.11% CPU
Iteration 100 / LogL: -1385.942 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.011765 secs using 99.7% CPU
Optimizing NNI: done in 0.0126839 secs using 99.17% CPU
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.303
Optimal log-likelihood: -1385.303
Rate parameters:  A-C: 0.39653  A-G: 1.57284  A-T: 1.00000  C-G: 0.39653  C-T: 1.57284  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.402) (0.276,2.564)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.303
Total tree length: 6.855

Total number of iterations: 102
CPU time used for tree search: 1.239 sec (0h:0m:1s)
Wall-clock time used for tree search: 1.071 sec (0h:0m:1s)
Total CPU time used: 1.257 sec (0h:0m:1s)
Total wall-clock time used: 1.088 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree.treefile -m "TPM3{0.396529,1.57284}+FQ+R2{0.723515,0.402303,0.276485,2.56407}" --length 214

Date and Time: Tue Sep 20 15:00:47 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpx78q7mf5/q2iqtree -nt 1

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

Output artifacts:

Specifying a substitution model

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree -nt 1
Seed:    510673 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:00:54 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000128031 secs using 83.57% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000228167 secs using 74.51% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 3.31402e-05 secs using 57.33% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 4.79221e-05 secs using 75.12% CPU

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.245 / LogL: -1394.426
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.308 / LogL: -1394.715
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.319 / LogL: -1394.789
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.316 / LogL: -1394.785
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.311 / LogL: -1394.749
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.314 / LogL: -1394.777
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.309 / LogL: -1394.722
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.310 / LogL: -1394.735
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.312 / LogL: -1394.747
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.313 / LogL: -1394.756
Optimal pinv,alpha: 0.000, 1.245 / LogL: -1394.426

Parameters optimization took 0.303 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000965834 secs using 98.77% CPU
Computing ML distances took 0.001075 sec (of wall-clock time) 0.001049 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.90871e-05 secs using 82.51% CPU
Constructing RapidNJ tree: done in 5.48363e-05 secs using 158.7% CPU
Computing RapidNJ tree took 0.000126 sec (of wall-clock time) 0.000139 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.974
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.078 second
Computing log-likelihood of 98 initial trees ... 0.079 seconds
Current best score: -1392.974

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.0106921 secs using 192.2% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.274
Optimizing NNI: done in 0.013521 secs using 192.7% CPU
Optimizing NNI: done in 0.00760913 secs using 134.9% CPU
Optimizing NNI: done in 0.00729418 secs using 99.41% CPU
Optimizing NNI: done in 0.00728202 secs using 99.09% CPU
Optimizing NNI: done in 0.008044 secs using 96.81% CPU
Optimizing NNI: done in 0.00711584 secs using 99.12% CPU
Optimizing NNI: done in 0.0071671 secs using 99.44% CPU
Optimizing NNI: done in 0.007581 secs using 97.24% CPU
Optimizing NNI: done in 0.00745106 secs using 97.73% CPU
Iteration 10 / LogL: -1387.731 / Time: 0h:0m:0s
Optimizing NNI: done in 0.0135429 secs using 97.96% CPU
Optimizing NNI: done in 0.0139449 secs using 98.62% CPU
Optimizing NNI: done in 0.0138628 secs using 97.8% CPU
Optimizing NNI: done in 0.013814 secs using 99.22% CPU
Optimizing NNI: done in 0.0135491 secs using 98.1% CPU
Optimizing NNI: done in 0.0145941 secs using 92.76% CPU
Optimizing NNI: done in 0.0148611 secs using 97.13% CPU
Optimizing NNI: done in 0.0139711 secs using 98.93% CPU
Optimizing NNI: done in 0.0145991 secs using 97.85% CPU
Optimizing NNI: done in 0.0134411 secs using 97.87% CPU
Iteration 20 / LogL: -1387.285 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1387.274 / CPU time: 0.404
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.0224371 secs using 98.03% CPU
Optimizing NNI: done in 0.016897 secs using 98.05% CPU
Optimizing NNI: done in 0.016011 secs using 97.81% CPU
Optimizing NNI: done in 0.0183609 secs using 96.12% CPU
Optimizing NNI: done in 0.0140259 secs using 98.3% CPU
Optimizing NNI: done in 0.0135341 secs using 99.11% CPU
Optimizing NNI: done in 0.0148971 secs using 98.64% CPU
Optimizing NNI: done in 0.0171251 secs using 98.63% CPU
Optimizing NNI: done in 0.0164199 secs using 96.96% CPU
Optimizing NNI: done in 0.012445 secs using 98.56% CPU
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 30 / LogL: -1387.255 / Time: 0h:0m:0s (0h:0m:2s left)
Optimizing NNI: done in 0.0138841 secs using 97.77% CPU
Optimizing NNI: done in 0.023351 secs using 98.59% CPU
Optimizing NNI: done in 0.00868487 secs using 98.06% CPU
Optimizing NNI: done in 0.020818 secs using 98.32% CPU
Optimizing NNI: done in 0.0103838 secs using 98.78% CPU
Optimizing NNI: done in 0.012279 secs using 97.97% CPU
Optimizing NNI: done in 0.007792 secs using 98.4% CPU
Optimizing NNI: done in 0.015028 secs using 98.97% CPU
Optimizing NNI: done in 0.0150349 secs using 98.21% CPU
Optimizing NNI: done in 0.0183761 secs using 98.2% CPU
Iteration 40 / LogL: -1387.433 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.015687 secs using 98.76% CPU
Optimizing NNI: done in 0.016283 secs using 98.12% CPU
Optimizing NNI: done in 0.013967 secs using 98.6% CPU
Optimizing NNI: done in 0.0151312 secs using 97.86% CPU
Optimizing NNI: done in 0.0137219 secs using 98.51% CPU
Optimizing NNI: done in 0.0226059 secs using 97.97% CPU
Optimizing NNI: done in 0.027061 secs using 98.74% CPU
Optimizing NNI: done in 0.036957 secs using 98.71% CPU
Optimizing NNI: done in 0.019634 secs using 97.83% CPU
Optimizing NNI: done in 0.0177209 secs using 97.24% CPU
Iteration 50 / LogL: -1410.920 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.017838 secs using 97.15% CPU
Optimizing NNI: done in 0.0322042 secs using 96.93% CPU
Optimizing NNI: done in 0.0212181 secs using 98.38% CPU
Optimizing NNI: done in 0.0160191 secs using 98.28% CPU
Optimizing NNI: done in 0.010046 secs using 98.65% CPU
Optimizing NNI: done in 0.016207 secs using 98.11% CPU
Optimizing NNI: done in 0.0209961 secs using 97.45% CPU
Optimizing NNI: done in 0.018362 secs using 96.79% CPU
Optimizing NNI: done in 0.020623 secs using 97.08% CPU
Optimizing NNI: done in 0.0171411 secs using 98.95% CPU
Iteration 60 / LogL: -1387.358 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.0145481 secs using 99.68% CPU
Optimizing NNI: done in 0.0081439 secs using 99.78% CPU
Optimizing NNI: done in 0.0203688 secs using 99.4% CPU
Optimizing NNI: done in 0.0089848 secs using 98.94% CPU
Optimizing NNI: done in 0.0125539 secs using 98.59% CPU
Optimizing NNI: done in 0.00941014 secs using 98.36% CPU
Optimizing NNI: done in 0.0240691 secs using 98.88% CPU
Optimizing NNI: done in 0.017211 secs using 99.53% CPU
Optimizing NNI: done in 0.0180831 secs using 99.56% CPU
Optimizing NNI: done in 0.006603 secs using 99.32% CPU
Iteration 70 / LogL: -1387.358 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0115261 secs using 99.95% CPU
Optimizing NNI: done in 0.017066 secs using 99.71% CPU
Optimizing NNI: done in 0.0111449 secs using 99.23% CPU
Optimizing NNI: done in 0.01721 secs using 99.58% CPU
Optimizing NNI: done in 0.0250039 secs using 99.65% CPU
Optimizing NNI: done in 0.015486 secs using 99.69% CPU
Optimizing NNI: done in 0.0126278 secs using 99.97% CPU
Optimizing NNI: done in 0.00899792 secs using 99.66% CPU
Optimizing NNI: done in 0.0114219 secs using 99.05% CPU
Optimizing NNI: done in 0.0133252 secs using 99.56% CPU
Iteration 80 / LogL: -1387.505 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0157139 secs using 99.57% CPU
Optimizing NNI: done in 0.0125089 secs using 98.99% CPU
Optimizing NNI: done in 0.02091 secs using 99.55% CPU
Optimizing NNI: done in 0.0199251 secs using 99.48% CPU
Optimizing NNI: done in 0.0182719 secs using 99.73% CPU
Optimizing NNI: done in 0.0154071 secs using 99.23% CPU
Optimizing NNI: done in 0.022336 secs using 99.49% CPU
Optimizing NNI: done in 0.0232549 secs using 99.49% CPU
Optimizing NNI: done in 0.0177929 secs using 99.17% CPU
Optimizing NNI: done in 0.012218 secs using 99.06% CPU
Iteration 90 / LogL: -1387.359 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0165019 secs using 99.56% CPU
Optimizing NNI: done in 0.0148492 secs using 98.67% CPU
Optimizing NNI: done in 0.013597 secs using 99.82% CPU
Optimizing NNI: done in 0.0139179 secs using 99.94% CPU
Optimizing NNI: done in 0.014719 secs using 99.71% CPU
Optimizing NNI: done in 0.00753713 secs using 99.79% CPU
Optimizing NNI: done in 0.0116 secs using 98.91% CPU
Optimizing NNI: done in 0.0146852 secs using 99.69% CPU
Optimizing NNI: done in 0.0143552 secs using 98.89% CPU
Optimizing NNI: done in 0.0240672 secs using 99.59% CPU
Iteration 100 / LogL: -1387.360 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.019855 secs using 99.56% CPU
Optimizing NNI: done in 0.0147569 secs using 98.8% CPU
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.255
Optimal log-likelihood: -1387.254
Rate parameters:  A-C: 0.33252  A-G: 2.22621  A-T: 2.10604  C-G: 1.15614  C-T: 3.23097  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: 1.329
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1387.254
Total tree length: 6.708

Total number of iterations: 102
CPU time used for tree search: 1.941 sec (0h:0m:1s)
Wall-clock time used for tree search: 1.809 sec (0h:0m:1s)
Total CPU time used: 2.248 sec (0h:0m:2s)
Total wall-clock time used: 2.127 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree.treefile -m "GTR{0.332516,2.22621,2.10604,1.15614,3.23097}+F{0.243216,0.181967,0.319196,0.255621}+I{2.66502e-07}+G4{1.32867}" --length 214

Date and Time: Tue Sep 20 15:00:56 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmptmtancma/q2iqtree -nt 1

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

Output artifacts:

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree -nt 1 -fast
Seed:    122540 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:01:04 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000123978 secs using 84.69% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000326872 secs using 74.34% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Analyzing sequences: done in 1.5974e-05 secs using 87.64% CPU
Checking for duplicate sequences: done in 4.88758e-05 secs using 71.61% CPU

---> START RUN NUMBER 1 (seed: 122540)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.50)
1. Initial log-likelihood: -1493.26
2. Current log-likelihood: -1403.08
3. Current log-likelihood: -1398.35
4. Current log-likelihood: -1396.98
5. Current log-likelihood: -1396.26
Optimal log-likelihood: -1395.75
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98595  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.024 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00096488 secs using 98.87% CPU
Computing ML distances took 0.001053 sec (of wall-clock time) 0.000996 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 82.45% CPU
Constructing RapidNJ tree: done in 5.50747e-05 secs using 161.6% CPU
Computing RapidNJ tree took 0.000141 sec (of wall-clock time) 0.000162 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.173
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0103199 secs using 193.5% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Optimizing NNI: done in 0.028353 secs using 190.2% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / CPU time: 0.050
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.961
2. Current log-likelihood: -1387.803
3. Current log-likelihood: -1387.684
4. Current log-likelihood: -1387.593
5. Current log-likelihood: -1387.523
6. Current log-likelihood: -1387.468
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.33414  A-G: 2.26635  A-T: 2.14117  C-G: 1.17550  C-T: 3.28158  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.353
Parameters optimization took 6 rounds (0.013 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.743

Total number of iterations: 2
CPU time used for tree search: 0.096 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.050 sec (0h:0m:0s)
Total CPU time used: 0.159 sec (0h:0m:0s)
Total wall-clock time used: 0.101 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 123540)

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 (0.023 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00138116 secs using 193% CPU
Computing ML distances took 0.001454 sec (of wall-clock time) 0.002790 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 5.19753e-05 secs using 86.58% CPU
Constructing RapidNJ tree: done in 8.2016e-05 secs using 108.5% CPU
Computing RapidNJ tree took 0.000223 sec (of wall-clock time) 0.000174 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0112002 secs using 191.7% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.017195 secs using 189.9% CPU
BETTER TREE FOUND at iteration 2: -1388.189
Finish initializing candidate tree set (4)
Current best tree score: -1388.189 / CPU time: 0.034
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: 2.31018  A-T: 2.11746  C-G: 1.22267  C-T: 3.27882  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 (0.016 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.066 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.035 sec (0h:0m:0s)
Total CPU time used: 0.160 sec (0h:0m:0s)
Total wall-clock time used: 0.085 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 124540)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20301  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.025 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.0013001 secs using 164.7% CPU
Computing ML distances took 0.001384 sec (of wall-clock time) 0.002269 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.38554e-05 secs using 70.89% CPU
Constructing RapidNJ tree: done in 6.10352e-05 secs using 116.3% CPU
Computing RapidNJ tree took 0.000144 sec (of wall-clock time) 0.000127 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0112441 secs using 177.4% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.016567 secs using 189.8% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.045
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.016 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.074 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.045 sec (0h:0m:0s)
Total CPU time used: 0.185 sec (0h:0m:0s)
Total wall-clock time used: 0.110 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 125540)

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: 2.06956  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 (0.024 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00117517 secs using 186.2% CPU
Computing ML distances took 0.001243 sec (of wall-clock time) 0.002292 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.59876e-05 secs using 80.81% CPU
Constructing RapidNJ tree: done in 5.50747e-05 secs using 92.6% CPU
Computing RapidNJ tree took 0.000164 sec (of wall-clock time) 0.000118 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.184
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0116911 secs using 191.8% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.955
Optimizing NNI: done in 0.00601006 secs using 192.3% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.955 / CPU time: 0.028
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.955
2. Current log-likelihood: -1387.798
3. Current log-likelihood: -1387.680
4. Current log-likelihood: -1387.590
5. Current log-likelihood: -1387.521
6. Current log-likelihood: -1387.467
Optimal log-likelihood: -1387.423
Rate parameters:  A-C: 0.33566  A-G: 2.27095  A-T: 2.14605  C-G: 1.17829  C-T: 3.29012  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.352
Parameters optimization took 6 rounds (0.014 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.744

Total number of iterations: 2
CPU time used for tree search: 0.054 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.028 sec (0h:0m:0s)
Total CPU time used: 0.149 sec (0h:0m:0s)
Total wall-clock time used: 0.079 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 126540)

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: 2.05006  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 (0.025 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000958204 secs using 195.9% CPU
Computing ML distances took 0.001023 sec (of wall-clock time) 0.001959 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.60012e-05 secs using 80.55% CPU
Constructing RapidNJ tree: done in 9.10759e-05 secs using 131.8% CPU
Computing RapidNJ tree took 0.000205 sec (of wall-clock time) 0.000204 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.724
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.010963 secs using 190.7% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.981
Optimizing NNI: done in 0.0147252 secs using 190.2% CPU
Finish initializing candidate tree set (4)
Current best tree score: -1387.981 / CPU time: 0.033
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: -1387.686
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: 2.25269  A-T: 2.12563  C-G: 1.16855  C-T: 3.25524  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 (0.014 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.701

Total number of iterations: 2
CPU time used for tree search: 0.062 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.033 sec (0h:0m:0s)
Total CPU time used: 0.157 sec (0h:0m:0s)
Total wall-clock time used: 0.084 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 127540)

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 (0.023 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000940084 secs using 180.9% CPU
Computing ML distances took 0.001000 sec (of wall-clock time) 0.001792 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.69413e-05 secs using 81.66% CPU
Constructing RapidNJ tree: done in 5.60284e-05 secs using 99.95% CPU
Computing RapidNJ tree took 0.000121 sec (of wall-clock time) 0.000103 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0115809 secs using 186.3% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.018086 secs using 188.4% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.036
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.016 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.069 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.037 sec (0h:0m:0s)
Total CPU time used: 0.163 sec (0h:0m:0s)
Total wall-clock time used: 0.087 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 128540)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.199
2. Current log-likelihood: -1404.591
3. Current log-likelihood: -1399.228
4. Current log-likelihood: -1397.831
5. Current log-likelihood: -1397.074
Optimal log-likelihood: -1396.495
Rate parameters:  A-C: 0.24620  A-G: 2.08306  A-T: 1.99580  C-G: 1.06240  C-T: 2.85598  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.432
Parameters optimization took 5 rounds (0.025 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000983953 secs using 189.4% CPU
Computing ML distances took 0.001050 sec (of wall-clock time) 0.001968 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 82.45% CPU
Constructing RapidNJ tree: done in 8.70228e-05 secs using 98.82% CPU
Computing RapidNJ tree took 0.000178 sec (of wall-clock time) 0.000147 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.972
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.010504 secs using 194.1% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.188
Optimizing NNI: done in 0.0158269 secs using 191.8% CPU
UPDATE BEST LOG-LIKELIHOOD: -1388.187
Finish initializing candidate tree set (3)
Current best tree score: -1388.187 / CPU time: 0.033
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: 0.33228  A-G: 2.23741  A-T: 2.11202  C-G: 1.16006  C-T: 3.23503  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 (0.016 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.064 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.033 sec (0h:0m:0s)
Total CPU time used: 0.164 sec (0h:0m:0s)
Total wall-clock time used: 0.087 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 129540)

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 (0.042 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00116801 secs using 189.3% CPU
Computing ML distances took 0.001235 sec (of wall-clock time) 0.002319 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 4.3869e-05 secs using 79.78% CPU
Constructing RapidNJ tree: done in 6.48499e-05 secs using 120.3% CPU
Computing RapidNJ tree took 0.000173 sec (of wall-clock time) 0.000156 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.0105679 secs using 182.5% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.017303 secs using 191.9% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.037
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.017 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.068 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.038 sec (0h:0m:0s)
Total CPU time used: 0.185 sec (0h:0m:0s)
Total wall-clock time used: 0.109 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 130540)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
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: 2.10646  C-G: 1.20301  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.024 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00100303 secs using 194.1% CPU
Computing ML distances took 0.001063 sec (of wall-clock time) 0.002042 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.40939e-05 secs using 76.26% CPU
Constructing RapidNJ tree: done in 5.48363e-05 secs using 129.5% CPU
Computing RapidNJ tree took 0.000130 sec (of wall-clock time) 0.000121 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.011039 secs using 191.1% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.016839 secs using 191.6% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.035
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.016 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.066 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.035 sec (0h:0m:0s)
Total CPU time used: 0.165 sec (0h:0m:0s)
Total wall-clock time used: 0.087 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 131540)

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: 2.10646  C-G: 1.20301  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.024 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00105309 secs using 187.4% CPU
Computing ML distances took 0.001110 sec (of wall-clock time) 0.002061 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 82.45% CPU
Constructing RapidNJ tree: done in 0.000113964 secs using 70.2% CPU
Computing RapidNJ tree took 0.000196 sec (of wall-clock time) 0.000149 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Optimizing NNI: done in 0.01053 secs using 190.5% CPU
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
Optimizing NNI: done in 0.016515 secs using 190.8% CPU
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.034
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.016 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.065 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.034 sec (0h:0m:0s)
Total CPU time used: 0.161 sec (0h:0m:0s)
Total wall-clock time used: 0.086 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 5 gave best log-likelihood: -1387.423
Total CPU time for 10 runs: 1.675 seconds.
Total wall-clock time for 10 runs: 0.932 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree.treefile
  Trees from independent runs:   /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree.runtrees
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree.treefile -m "GTR{0.323517,2.25837,2.12825,1.16566,3.23836}+F{0.243216,0.181967,0.319196,0.255621}+I{0.0163393}+G4{1.37314}" --length 214

Date and Time: Tue Sep 20 15:01:05 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 10 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpzrfrqctf/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

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

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

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

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    612396 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:01:12 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000130892 secs using 79.45% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000235081 secs using 74.44% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.69277e-05 secs using 82.7% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 3.91006e-05 secs using 71.61% CPU

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.239 / LogL: -1394.543
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.340 / LogL: -1394.887
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.353 / LogL: -1394.887
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.352 / LogL: -1394.871
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.836
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.862
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.884
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.346 / LogL: -1394.826
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.838
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.841
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.543

Parameters optimization took 0.300 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00101709 secs using 98.81% CPU
Computing ML distances took 0.001104 sec (of wall-clock time) 0.001068 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 4.69685e-05 secs using 68.13% CPU
Constructing RapidNJ tree: done in 5.98431e-05 secs using 165.4% CPU
Computing RapidNJ tree took 0.000158 sec (of wall-clock time) 0.000183 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.870
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.080 second
Computing log-likelihood of 98 initial trees ... 0.078 seconds
Current best score: -1392.870

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.010453 secs using 190% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
Optimizing NNI: done in 0.011672 secs using 170.9% CPU
Optimizing NNI: done in 0.00931716 secs using 175.5% CPU
Optimizing NNI: done in 0.00786591 secs using 96.59% CPU
Optimizing NNI: done in 0.00817609 secs using 98.34% CPU
Optimizing NNI: done in 0.00815105 secs using 99.75% CPU
Optimizing NNI: done in 0.00813293 secs using 95.53% CPU
Optimizing NNI: done in 0.0151291 secs using 97.98% CPU
Optimizing NNI: done in 0.0142889 secs using 98.39% CPU
Optimizing NNI: done in 0.013917 secs using 98.35% CPU
Iteration 10 / LogL: -1387.282 / Time: 0h:0m:0s
Optimizing NNI: done in 0.013725 secs using 97.92% CPU
Optimizing NNI: done in 0.014487 secs using 97.74% CPU
Optimizing NNI: done in 0.0137241 secs using 97.94% CPU
Optimizing NNI: done in 0.0145459 secs using 97.99% CPU
Optimizing NNI: done in 0.013391 secs using 98.02% CPU
Optimizing NNI: done in 0.01406 secs using 98.34% CPU
Optimizing NNI: done in 0.0140572 secs using 98.97% CPU
Optimizing NNI: done in 0.0144589 secs using 96.96% CPU
Optimizing NNI: done in 0.0150242 secs using 97.72% CPU
Optimizing NNI: done in 0.0110559 secs using 96.07% CPU
Iteration 20 / LogL: -1387.267 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1387.265 / CPU time: 0.422
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.028811 secs using 97.98% CPU
Optimizing NNI: done in 0.012409 secs using 98.59% CPU
Optimizing NNI: done in 0.0157802 secs using 99.17% CPU
Optimizing NNI: done in 0.017242 secs using 98.16% CPU
Optimizing NNI: done in 0.00925303 secs using 99.42% CPU
Optimizing NNI: done in 0.0160651 secs using 98.73% CPU
Optimizing NNI: done in 0.018651 secs using 97.39% CPU
Optimizing NNI: done in 0.010989 secs using 98.64% CPU
Optimizing NNI: done in 0.0142388 secs using 98.13% CPU
Optimizing NNI: done in 0.023138 secs using 97.86% CPU
Iteration 30 / LogL: -1387.351 / Time: 0h:0m:0s (0h:0m:2s left)
Optimizing NNI: done in 0.017132 secs using 97.84% CPU
Optimizing NNI: done in 0.012224 secs using 98.73% CPU
Optimizing NNI: done in 0.0172091 secs using 98.49% CPU
Optimizing NNI: done in 0.00676203 secs using 98.8% CPU
Optimizing NNI: done in 0.013047 secs using 97.42% CPU
Optimizing NNI: done in 0.0220239 secs using 97.9% CPU
Optimizing NNI: done in 0.018672 secs using 98.73% CPU
Optimizing NNI: done in 0.0212748 secs using 98.09% CPU
Optimizing NNI: done in 0.0104759 secs using 97.74% CPU
Optimizing NNI: done in 0.0225959 secs using 98.58% CPU
Iteration 40 / LogL: -1387.606 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.00713682 secs using 97.94% CPU
Optimizing NNI: done in 0.0176389 secs using 97.91% CPU
Optimizing NNI: done in 0.0236299 secs using 98.32% CPU
Optimizing NNI: done in 0.01775 secs using 98.67% CPU
Optimizing NNI: done in 0.00692916 secs using 98.09% CPU
Optimizing NNI: done in 0.0120461 secs using 96.94% CPU
Optimizing NNI: done in 0.0163889 secs using 96.95% CPU
Optimizing NNI: done in 0.0237498 secs using 98.87% CPU
Optimizing NNI: done in 0.0213048 secs using 98.67% CPU
Optimizing NNI: done in 0.015497 secs using 98.75% CPU
Iteration 50 / LogL: -1387.436 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.0167389 secs using 98.23% CPU
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Optimizing NNI: done in 0.0131688 secs using 99.39% CPU
Iteration 60 / LogL: -1387.347 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.017844 secs using 98.68% CPU
Optimizing NNI: done in 0.0207999 secs using 99.59% CPU
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Optimizing NNI: done in 0.0128739 secs using 98.08% CPU
Iteration 70 / LogL: -1387.304 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.0201049 secs using 99.52% CPU
Optimizing NNI: done in 0.015744 secs using 99.94% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 72: -1387.171
Optimizing NNI: done in 0.00572801 secs using 99.83% CPU
Optimizing NNI: done in 0.0140021 secs using 99.4% CPU
Optimizing NNI: done in 0.013392 secs using 99.96% CPU
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Iteration 80 / LogL: -1396.664 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.013865 secs using 99.21% CPU
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Iteration 90 / LogL: -1387.327 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.0207779 secs using 98.85% CPU
Optimizing NNI: done in 0.0149381 secs using 99.95% CPU
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Iteration 100 / LogL: -1387.408 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0179808 secs using 99.52% CPU
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Iteration 110 / LogL: -1414.815 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0117409 secs using 99.01% CPU
Optimizing NNI: done in 0.0140851 secs using 99.36% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.171
Optimizing NNI: done in 0.01402 secs using 98.76% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Optimizing NNI: done in 0.0127008 secs using 98.66% CPU
Optimizing NNI: done in 0.017107 secs using 99.44% CPU
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Iteration 120 / LogL: -1387.320 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.00848603 secs using 98.44% CPU
BETTER TREE FOUND at iteration 121: -1387.167
Optimizing NNI: done in 0.01952 secs using 99.44% CPU
Optimizing NNI: done in 0.00698495 secs using 98.83% CPU
Optimizing NNI: done in 0.00720096 secs using 99.93% CPU
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Optimizing NNI: done in 0.00915289 secs using 98.96% CPU
Iteration 130 / LogL: -1387.344 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.012207 secs using 98.79% CPU
Optimizing NNI: done in 0.0171402 secs using 99.61% CPU
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Iteration 140 / LogL: -1387.418 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0164621 secs using 99.26% CPU
Optimizing NNI: done in 0.00941586 secs using 99.94% CPU
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Iteration 150 / LogL: -1394.875 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0150208 secs using 99.71% CPU
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Optimizing NNI: done in 0.0118718 secs using 99.86% CPU
Iteration 160 / LogL: -1387.402 / Time: 0h:0m:2s (0h:0m:1s left)
Optimizing NNI: done in 0.0255511 secs using 99.02% CPU
Optimizing NNI: done in 0.0216951 secs using 99.65% CPU
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Optimizing NNI: done in 0.0136321 secs using 99.7% CPU
Optimizing NNI: done in 0.0100729 secs using 98.89% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.167
Optimizing NNI: done in 0.0101001 secs using 99.5% CPU
Optimizing NNI: done in 0.0130711 secs using 99.51% CPU
Optimizing NNI: done in 0.0117791 secs using 99.75% CPU
Optimizing NNI: done in 0.0164661 secs using 99.56% CPU
Iteration 170 / LogL: -1387.316 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0117478 secs using 98.7% CPU
Optimizing NNI: done in 0.0135839 secs using 99.6% CPU
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Optimizing NNI: done in 0.022526 secs using 99.44% CPU
Iteration 180 / LogL: -1387.194 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.00840807 secs using 99.93% CPU
Optimizing NNI: done in 0.0132999 secs using 99.46% CPU
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Iteration 190 / LogL: -1387.409 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0153189 secs using 99.07% CPU
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Optimizing NNI: done in 0.0105891 secs using 98.88% CPU
Iteration 200 / LogL: -1387.186 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0168381 secs using 98.95% CPU
Optimizing NNI: done in 0.016583 secs using 99.6% CPU
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Iteration 210 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.017628 secs using 99.63% CPU
Optimizing NNI: done in 0.018507 secs using 99.14% CPU
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Optimizing NNI: done in 0.013504 secs using 99.1% CPU
Iteration 220 / LogL: -1387.338 / Time: 0h:0m:3s (0h:0m:0s left)
Optimizing NNI: done in 0.0142448 secs using 99.29% CPU
Optimizing NNI: done in 0.00586891 secs using 98.25% CPU
TREE SEARCH COMPLETED AFTER 222 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.167
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34767  A-G: 2.31953  A-T: 2.14096  C-G: 1.23342  C-T: 3.21606  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 (0.002 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...
0.046 sec.
Total tree length: 7.604

Total number of iterations: 222
CPU time used for tree search: 3.622 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.498 sec (0h:0m:3s)
Total CPU time used: 3.976 sec (0h:0m:3s)
Total wall-clock time used: 3.859 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree.treefile -m "GTR{0.347668,2.31953,2.14096,1.23342,3.21606}+F{0.243216,0.181967,0.319196,0.255621}+I{2.96528e-07}+G4{1.28436}" --length 214

Date and Time: Tue Sep 20 15:01:16 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_agvu5a2/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

Tip

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    946692 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:01:23 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000127077 secs using 83.41% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000324011 secs using 70.37% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 3.09944e-05 secs using 48.4% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 4.98295e-05 secs using 70.24% CPU


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Optimizing NNI: done in 0.0139058 secs using 98.63% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.281
2. Current log-likelihood: -1389.723
Optimal log-likelihood: -1388.882
Rate parameters:  A-C: 0.33813  A-G: 2.30739  A-T: 2.15806  C-G: 1.19220  C-T: 3.30147  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: 1.423
Parameters optimization took 2 rounds (0.008 sec)
Time for fast ML tree search: 0.038 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.278     46  2866.557     2892.449     3021.392
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.422
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 14  GTR+F+I+I+R2  1380.757     48  2857.513     2886.023     3019.080
 15  GTR+F+I+I+R3  1380.696     50  2861.392     2892.681     3029.691
 25  SYM+G4        1387.155     43  2860.310     2882.568     3005.047
 27  SYM+R2        1382.244     44  2852.489     2875.921     3000.592
 36  SYM+I+I+R2    1382.405     45  2854.809     2879.452     3006.278
 47  TVM+F+G4      1388.425     45  2866.850     2891.493     3018.319
 49  TVM+F+R2      1382.482     46  2856.965     2882.857     3011.799
 58  TVM+F+I+I+R2  1382.560     47  2859.119     2886.300     3017.320
 69  TVMe+G4       1387.122     42  2858.244     2879.367     2999.615
 71  TVMe+R2       1382.298     43  2850.596     2872.855     2995.333
 80  TVMe+I+I+R2   1382.453     44  2852.907     2876.339     3001.010
 91  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 93  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
102  TIM3+F+I+I+R2 1384.573     46  2861.146     2887.039     3015.981
113  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
115  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
124  TIM3e+I+I+R2  1385.391     43  2856.782     2879.040     3001.519
135  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
137  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
146  TIM2+F+I+I+R2 1386.377     46  2864.755     2890.647     3019.590
157  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
159  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
168  TIM2e+I+I+R2  1391.272     43  2868.544     2890.802     3013.281
179  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
181  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
190  TIM+F+I+I+R2  1383.340     46  2858.680     2884.572     3013.515
201  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
203  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
212  TIMe+I+I+R2   1388.456     43  2862.912     2885.171     3007.649
223  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
225  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
234  TPM3u+F+I+I+R2 1386.501     45  2863.002     2887.645     3014.471
245  TPM3+G4       1390.540     40  2861.080     2880.039     2995.719
247  TPM3+R2       1385.307     41  2852.614     2872.637     2990.619
256  TPM3+I+I+R2   1385.459     42  2854.917     2876.040     2996.288
267  TPM2u+F+G4    1395.283     43  2876.566     2898.825     3021.303
269  TPM2u+F+R2    1388.114     44  2864.229     2887.661     3012.332
278  TPM2u+F+I+I+R2 1388.249     45  2866.497     2891.140     3017.966
289  TPM2+G4       1397.749     40  2875.499     2894.458     3010.138
291  TPM2+R2       1391.198     41  2864.396     2884.419     3002.401
300  TPM2+I+I+R2   1391.346     42  2866.692     2887.815     3008.063
311  K3Pu+F+G4     1392.050     43  2870.100     2892.359     3014.837
313  K3Pu+F+R2     1385.122     44  2858.245     2881.677     3006.348
322  K3Pu+F+I+I+R2 1385.245     45  2860.491     2885.134     3011.960
333  K3P+G4        1394.786     40  2869.573     2888.532     3004.212
335  K3P+R2        1388.359     41  2858.717     2878.740     2996.722
344  K3P+I+I+R2    1388.531     42  2861.061     2882.184     3002.432
355  TN+F+G4       1394.628     43  2875.256     2897.515     3019.993
357  TN+F+R2       1386.824     44  2861.647     2885.079     3009.750
366  TN+F+I+I+R2   1386.967     45  2863.933     2888.576     3015.402
377  TNe+G4        1397.748     40  2875.497     2894.456     3010.136
379  TNe+R2        1391.134     41  2864.269     2884.292     3002.274
388  TNe+I+I+R2    1391.289     42  2866.578     2887.701     3007.949
399  HKY+F+G4      1395.752     42  2875.504     2896.627     3016.875
401  HKY+F+R2      1388.691     43  2863.382     2885.640     3008.119
410  HKY+F+I+I+R2  1388.820     44  2865.639     2889.071     3013.742
421  K2P+G4        1397.753     39  2873.507     2891.438     3004.780
423  K2P+R2        1391.216     40  2862.433     2881.392     2997.072
432  K2P+I+I+R2    1391.363     41  2864.726     2884.750     3002.731
443  F81+F+G4      1406.478     41  2894.955     2914.979     3032.960
445  F81+F+R2      1400.602     42  2885.203     2906.326     3026.574
454  F81+F+I+I+R2  1400.799     43  2887.598     2909.857     3032.335
465  JC+G4         1408.432     38  2892.865     2909.802     3020.772
467  JC+R2         1403.021     39  2884.041     2901.972     3015.314
476  JC+I+I+R2     1403.194     40  2886.388     2905.348     3021.028
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree.model.gz
CPU time for ModelFinder: 0.948 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.984 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.307
Optimal log-likelihood: -1385.304
Rate parameters:  A-C: 0.39693  A-G: 1.57495  A-T: 1.00000  C-G: 0.39693  C-T: 1.57495  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.403) (0.277,2.560)
Parameters optimization took 1 rounds (0.003 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00067091 secs using 97.33% CPU
Computing ML distances took 0.000734 sec (of wall-clock time) 0.000694 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 82.45% CPU
Constructing RapidNJ tree: done in 7.39098e-05 secs using 121.8% CPU
Computing RapidNJ tree took 0.000157 sec (of wall-clock time) 0.000155 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.836
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.080 second
Computing log-likelihood of 98 initial trees ... 0.058 seconds
Current best score: -1385.304

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00155497 secs using 192.5% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.303
Optimizing NNI: done in 0.00767994 secs using 193.6% CPU
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Optimizing NNI: done in 0.0102298 secs using 97.53% CPU
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Iteration 10 / LogL: -1385.304 / Time: 0h:0m:0s
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Optimizing NNI: done in 0.00668001 secs using 99.03% CPU
Iteration 20 / LogL: -1385.330 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1385.303 / CPU time: 0.324
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.00442791 secs using 97.88% CPU
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Iteration 30 / LogL: -1385.940 / Time: 0h:0m:0s (0h:0m:2s left)
Optimizing NNI: done in 0.00414395 secs using 96.41% CPU
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Iteration 40 / LogL: -1392.791 / Time: 0h:0m:0s (0h:0m:1s left)
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Iteration 50 / LogL: -1385.304 / Time: 0h:0m:0s (0h:0m:1s left)
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Iteration 60 / LogL: -1388.364 / Time: 0h:0m:0s (0h:0m:1s left)
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Optimizing NNI: done in 0.00585699 secs using 99.73% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.303
Optimizing NNI: done in 0.00604606 secs using 99.07% CPU
Iteration 70 / LogL: -1385.304 / Time: 0h:0m:0s (0h:0m:1s left)
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Iteration 80 / LogL: -1385.303 / Time: 0h:0m:0s (0h:0m:1s left)
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Iteration 90 / LogL: -1385.644 / Time: 0h:0m:0s (0h:0m:0s left)
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Iteration 100 / LogL: -1385.304 / Time: 0h:0m:0s (0h:0m:0s left)
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Iteration 110 / LogL: -1385.305 / Time: 0h:0m:0s (0h:0m:0s left)
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Iteration 120 / LogL: -1385.507 / Time: 0h:0m:0s (0h:0m:0s left)
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Iteration 130 / LogL: -1385.767 / Time: 0h:0m:1s (0h:0m:0s left)
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Iteration 140 / LogL: -1385.305 / Time: 0h:0m:1s (0h:0m:0s left)
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Iteration 150 / LogL: -1385.507 / Time: 0h:0m:1s (0h:0m:0s left)
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Iteration 160 / LogL: -1385.305 / Time: 0h:0m:1s (0h:0m:0s left)
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Optimizing NNI: done in 0.00292206 secs using 99.52% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.303
Optimizing NNI: done in 0.00490308 secs using 99.96% CPU
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Iteration 170 / LogL: -1385.312 / Time: 0h:0m:1s (0h:0m:0s left)
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UPDATE BEST LOG-LIKELIHOOD: -1385.303
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Iteration 180 / LogL: -1385.304 / Time: 0h:0m:1s (0h:0m:0s left)
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Optimizing NNI: done in 0.00763488 secs using 99.65% CPU
Iteration 190 / LogL: -1385.304 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.00534606 secs using 98.54% CPU
Optimizing NNI: done in 0.00971484 secs using 98.43% CPU
Optimizing NNI: done in 0.00732899 secs using 99.18% CPU
Optimizing NNI: done in 0.00756216 secs using 99.4% CPU
Optimizing NNI: done in 0.00685692 secs using 99.94% CPU
Optimizing NNI: done in 0.00673485 secs using 99.63% CPU
Optimizing NNI: done in 0.00615311 secs using 99.35% CPU
Optimizing NNI: done in 0.00421906 secs using 99.81% CPU
Optimizing NNI: done in 0.00567102 secs using 99.86% CPU
Optimizing NNI: done in 0.00601506 secs using 99.65% CPU
Iteration 200 / LogL: -1385.304 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.00443411 secs using 98.78% CPU
Optimizing NNI: done in 0.0090549 secs using 98.92% CPU
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.303
Optimal log-likelihood: -1385.303
Rate parameters:  A-C: 0.39653  A-G: 1.57282  A-T: 1.00000  C-G: 0.39653  C-T: 1.57282  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.402) (0.277,2.564)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.303
Total tree length: 6.855

Total number of iterations: 202
CPU time used for tree search: 1.616 sec (0h:0m:1s)
Wall-clock time used for tree search: 1.494 sec (0h:0m:1s)
Total CPU time used: 1.633 sec (0h:0m:1s)
Total wall-clock time used: 1.510 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree.treefile -m "TPM3{0.396533,1.57282}+FQ+R2{0.723444,0.402242,0.276556,2.56368}" --length 214

Date and Time: Tue Sep 20 15:01:26 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp40l8ohvr/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

iqtree-ultrafast-bootstrap

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    190448 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:01:34 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000210047 secs using 79.98% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000334978 secs using 72.84% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 2.00272e-05 secs using 89.88% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 4.88758e-05 secs using 71.61% CPU


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Optimizing NNI: done in 0.0145979 secs using 97.66% CPU
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.311
2. Current log-likelihood: -1389.733
Optimal log-likelihood: -1388.881
Rate parameters:  A-C: 0.33789  A-G: 2.29237  A-T: 2.14761  C-G: 1.19040  C-T: 3.28358  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.033
Gamma shape alpha: 1.424
Parameters optimization took 2 rounds (0.009 sec)
Time for fast ML tree search: 0.039 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.279     46  2866.558     2892.450     3021.393
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.423
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 14  GTR+F+I+I+R2  1380.757     48  2857.513     2886.023     3019.080
 15  GTR+F+I+I+R3  1380.696     50  2861.392     2892.681     3029.691
 25  SYM+G4        1387.157     43  2860.314     2882.573     3005.051
 27  SYM+R2        1382.244     44  2852.489     2875.920     3000.591
 36  SYM+I+I+R2    1382.405     45  2854.809     2879.452     3006.278
 47  TVM+F+G4      1388.424     45  2866.848     2891.491     3018.317
 49  TVM+F+R2      1382.481     46  2856.963     2882.855     3011.798
 58  TVM+F+I+I+R2  1382.560     47  2859.119     2886.300     3017.320
 69  TVMe+G4       1387.122     42  2858.245     2879.367     2999.616
 71  TVMe+R2       1382.298     43  2850.596     2872.854     2995.333
 80  TVMe+I+I+R2   1382.453     44  2852.907     2876.339     3001.010
 91  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 93  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
102  TIM3+F+I+I+R2 1384.573     46  2861.146     2887.039     3015.981
113  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
115  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
124  TIM3e+I+I+R2  1385.391     43  2856.782     2879.040     3001.519
135  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
137  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
146  TIM2+F+I+I+R2 1386.377     46  2864.755     2890.647     3019.590
157  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
159  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
168  TIM2e+I+I+R2  1391.272     43  2868.544     2890.802     3013.281
179  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
181  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
190  TIM+F+I+I+R2  1383.340     46  2858.680     2884.572     3013.515
201  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
203  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
212  TIMe+I+I+R2   1388.456     43  2862.912     2885.171     3007.649
223  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
225  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
234  TPM3u+F+I+I+R2 1386.501     45  2863.002     2887.645     3014.471
245  TPM3+G4       1390.540     40  2861.080     2880.039     2995.719
247  TPM3+R2       1385.307     41  2852.614     2872.637     2990.619
256  TPM3+I+I+R2   1385.459     42  2854.917     2876.040     2996.288
267  TPM2u+F+G4    1395.283     43  2876.566     2898.825     3021.303
269  TPM2u+F+R2    1388.114     44  2864.229     2887.661     3012.332
278  TPM2u+F+I+I+R2 1388.249     45  2866.497     2891.140     3017.966
289  TPM2+G4       1397.749     40  2875.499     2894.458     3010.138
291  TPM2+R2       1391.198     41  2864.396     2884.419     3002.401
300  TPM2+I+I+R2   1391.346     42  2866.692     2887.815     3008.063
311  K3Pu+F+G4     1392.050     43  2870.100     2892.359     3014.837
313  K3Pu+F+R2     1385.122     44  2858.245     2881.677     3006.348
322  K3Pu+F+I+I+R2 1385.245     45  2860.491     2885.134     3011.960
333  K3P+G4        1394.786     40  2869.573     2888.532     3004.212
335  K3P+R2        1388.359     41  2858.717     2878.740     2996.722
344  K3P+I+I+R2    1388.531     42  2861.061     2882.184     3002.432
355  TN+F+G4       1394.628     43  2875.256     2897.515     3019.993
357  TN+F+R2       1386.824     44  2861.647     2885.079     3009.750
366  TN+F+I+I+R2   1386.967     45  2863.933     2888.576     3015.402
377  TNe+G4        1397.748     40  2875.497     2894.456     3010.136
379  TNe+R2        1391.134     41  2864.269     2884.292     3002.274
388  TNe+I+I+R2    1391.289     42  2866.578     2887.701     3007.949
399  HKY+F+G4      1395.752     42  2875.504     2896.627     3016.875
401  HKY+F+R2      1388.691     43  2863.382     2885.640     3008.119
410  HKY+F+I+I+R2  1388.820     44  2865.639     2889.071     3013.742
421  K2P+G4        1397.753     39  2873.507     2891.438     3004.780
423  K2P+R2        1391.216     40  2862.433     2881.392     2997.072
432  K2P+I+I+R2    1391.363     41  2864.726     2884.750     3002.731
443  F81+F+G4      1406.478     41  2894.955     2914.979     3032.960
445  F81+F+R2      1400.602     42  2885.203     2906.326     3026.574
454  F81+F+I+I+R2  1400.799     43  2887.598     2909.857     3032.335
465  JC+G4         1408.432     38  2892.865     2909.802     3020.772
467  JC+R2         1403.021     39  2884.041     2901.972     3015.314
476  JC+I+I+R2     1403.194     40  2886.388     2905.348     3021.028
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.952 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.987 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 190448)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.307
Optimal log-likelihood: -1385.304
Rate parameters:  A-C: 0.39693  A-G: 1.57493  A-T: 1.00000  C-G: 0.39693  C-T: 1.57493  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.403) (0.277,2.560)
Parameters optimization took 1 rounds (0.003 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00068903 secs using 92.88% CPU
Computing ML distances took 0.000742 sec (of wall-clock time) 0.000680 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 78.87% CPU
Constructing RapidNJ tree: done in 8.39233e-05 secs using 141.8% CPU
Computing RapidNJ tree took 0.000176 sec (of wall-clock time) 0.000200 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.836
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.081 second
Computing log-likelihood of 96 initial trees ... 0.059 seconds
Current best score: -1385.304

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.00326419 secs using 190.1% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.303
Optimizing NNI: done in 0.014292 secs using 190.8% CPU
Optimizing NNI: done in 0.0145302 secs using 191.6% CPU
Optimizing NNI: done in 0.014812 secs using 190.1% CPU
Optimizing NNI: done in 0.013581 secs using 147.3% CPU
Optimizing NNI: done in 0.012296 secs using 98.59% CPU
Optimizing NNI: done in 0.0133891 secs using 98.36% CPU
Optimizing NNI: done in 0.013649 secs using 97.96% CPU
Optimizing NNI: done in 0.0130551 secs using 98.95% CPU
Optimizing NNI: done in 0.0141752 secs using 97.73% CPU
Iteration 10 / LogL: -1385.338 / Time: 0h:0m:0s
Optimizing NNI: done in 0.014075 secs using 97.66% CPU
Optimizing NNI: done in 0.013381 secs using 98.08% CPU
Optimizing NNI: done in 0.0135 secs using 98.21% CPU
Optimizing NNI: done in 0.0132051 secs using 97.28% CPU
Optimizing NNI: done in 0.012897 secs using 99.02% CPU
Optimizing NNI: done in 0.0127871 secs using 98.01% CPU
Optimizing NNI: done in 0.0131261 secs using 98.66% CPU
Optimizing NNI: done in 0.0133631 secs using 96.95% CPU
Optimizing NNI: done in 0.0124559 secs using 98.64% CPU
Optimizing NNI: done in 0.0118349 secs using 98.59% CPU
Iteration 20 / LogL: -1385.338 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1385.303 / CPU time: 0.423
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Optimizing NNI: done in 0.0110362 secs using 98.5% CPU
Optimizing NNI: done in 0.0055058 secs using 99.89% CPU
Optimizing NNI: done in 0.00663114 secs using 99.3% CPU
Optimizing NNI: done in 0.00795197 secs using 99.95% CPU
Optimizing NNI: done in 0.010658 secs using 99.06% CPU
Optimizing NNI: done in 0.00914288 secs using 99.45% CPU
Optimizing NNI: done in 0.0112991 secs using 99.41% CPU
Optimizing NNI: done in 0.0086031 secs using 99.08% CPU
Optimizing NNI: done in 0.0124562 secs using 99.73% CPU
Optimizing NNI: done in 0.0104361 secs using 98.82% CPU
Iteration 30 / LogL: -1385.304 / Time: 0h:0m:0s (0h:0m:3s left)
Optimizing NNI: done in 0.0101709 secs using 99.85% CPU
Optimizing NNI: done in 0.00599003 secs using 98.63% CPU
Optimizing NNI: done in 0.00964999 secs using 99.71% CPU
Optimizing NNI: done in 0.0049181 secs using 99.31% CPU
Optimizing NNI: done in 0.0105591 secs using 98.72% CPU
Optimizing NNI: done in 0.0126648 secs using 99.6% CPU
Optimizing NNI: done in 0.008641 secs using 99.71% CPU
Optimizing NNI: done in 0.00998712 secs using 99.81% CPU
Optimizing NNI: done in 0.00932002 secs using 99.66% CPU
Optimizing NNI: done in 0.00838208 secs using 98.97% CPU
Iteration 40 / LogL: -1385.304 / Time: 0h:0m:0s (0h:0m:2s left)
Optimizing NNI: done in 0.00751996 secs using 99.6% CPU
Optimizing NNI: done in 0.0162849 secs using 98.96% CPU
Optimizing NNI: done in 0.00503087 secs using 99.98% CPU
Optimizing NNI: done in 0.00981688 secs using 99.73% CPU
Optimizing NNI: done in 0.0070858 secs using 99.73% CPU
Optimizing NNI: done in 0.0142231 secs using 99.39% CPU
Optimizing NNI: done in 0.010263 secs using 98.97% CPU
Optimizing NNI: done in 0.0118642 secs using 99.21% CPU
Optimizing NNI: done in 0.015825 secs using 99.33% CPU
Optimizing NNI: done in 0.012727 secs using 99.28% CPU
Iteration 50 / LogL: -1385.943 / Time: 0h:0m:0s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1405.890
Optimizing NNI: done in 0.00950408 secs using 99.94% CPU
Optimizing NNI: done in 0.011143 secs using 99.31% CPU
Optimizing NNI: done in 0.00912595 secs using 99.34% CPU
Optimizing NNI: done in 0.00873995 secs using 99.15% CPU
Optimizing NNI: done in 0.00708485 secs using 99.76% CPU
Optimizing NNI: done in 0.006706 secs using 99.25% CPU
Optimizing NNI: done in 0.00722814 secs using 99.64% CPU
Optimizing NNI: done in 0.00942397 secs using 98.59% CPU
Optimizing NNI: done in 0.0130091 secs using 99.27% CPU
Optimizing NNI: done in 0.00479794 secs using 99.92% CPU
Iteration 60 / LogL: -1385.306 / Time: 0h:0m:0s (0h:0m:2s left)
Optimizing NNI: done in 0.0130479 secs using 99.15% CPU
Optimizing NNI: done in 0.0082159 secs using 99.02% CPU
Optimizing NNI: done in 0.013773 secs using 99.53% CPU
Optimizing NNI: done in 0.00282502 secs using 99.4% CPU
Optimizing NNI: done in 0.00809503 secs using 99.62% CPU
Optimizing NNI: done in 0.0116451 secs using 99.35% CPU
Optimizing NNI: done in 0.00256395 secs using 99.88% CPU
Optimizing NNI: done in 0.00882506 secs using 99.76% CPU
Optimizing NNI: done in 0.00294495 secs using 98% CPU
Optimizing NNI: done in 0.00724292 secs using 99.9% CPU
Iteration 70 / LogL: -1385.304 / Time: 0h:0m:1s (0h:0m:2s left)
Optimizing NNI: done in 0.00869513 secs using 99.24% CPU
Optimizing NNI: done in 0.00577688 secs using 99.71% CPU
Optimizing NNI: done in 0.00900221 secs using 99.94% CPU
Optimizing NNI: done in 0.0108631 secs using 99.34% CPU
Optimizing NNI: done in 0.00786901 secs using 98.98% CPU
Optimizing NNI: done in 0.0100169 secs using 99.84% CPU
Optimizing NNI: done in 0.00970507 secs using 99.05% CPU
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Optimizing NNI: done in 0.00542998 secs using 99.39% CPU
Optimizing NNI: done in 0.011749 secs using 99.95% CPU
Iteration 80 / LogL: -1385.941 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.00389481 secs using 99.9% CPU
Optimizing NNI: done in 0.0102482 secs using 99.88% CPU
Optimizing NNI: done in 0.00557089 secs using 99.89% CPU
Optimizing NNI: done in 0.0113571 secs using 99.4% CPU
Optimizing NNI: done in 0.00775409 secs using 99.95% CPU
Optimizing NNI: done in 0.0060091 secs using 99.67% CPU
Optimizing NNI: done in 0.00953412 secs using 99.26% CPU
Optimizing NNI: done in 0.00575399 secs using 98.58% CPU
Optimizing NNI: done in 0.00729203 secs using 99.6% CPU
Optimizing NNI: done in 0.011153 secs using 99.61% CPU
Iteration 90 / LogL: -1385.842 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.00451517 secs using 99.69% CPU
Optimizing NNI: done in 0.00599408 secs using 98.88% CPU
Optimizing NNI: done in 0.00888705 secs using 99.41% CPU
Optimizing NNI: done in 0.00789022 secs using 99.58% CPU
Optimizing NNI: done in 0.00738597 secs using 99.68% CPU
Optimizing NNI: done in 0.012516 secs using 99.56% CPU
Optimizing NNI: done in 0.00576687 secs using 99.92% CPU
Optimizing NNI: done in 0.00760603 secs using 99.74% CPU
Optimizing NNI: done in 0.00467682 secs using 99.88% CPU
Optimizing NNI: done in 0.00825191 secs using 99.59% CPU
Iteration 100 / LogL: -1385.305 / Time: 0h:0m:1s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1405.890
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.993
Optimizing NNI: done in 0.00935817 secs using 99.52% CPU
Optimizing NNI: done in 0.00560904 secs using 99.93% CPU
Optimizing NNI: done in 0.0129371 secs using 99.57% CPU
Optimizing NNI: done in 0.00904322 secs using 99.15% CPU
Optimizing NNI: done in 0.008636 secs using 99.21% CPU
Optimizing NNI: done in 0.00638795 secs using 99.83% CPU
Optimizing NNI: done in 0.00872016 secs using 98.71% CPU
Optimizing NNI: done in 0.00955009 secs using 99.39% CPU
Optimizing NNI: done in 0.0116251 secs using 99.18% CPU
Optimizing NNI: done in 0.00931215 secs using 99.43% CPU
Iteration 110 / LogL: -1385.306 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.01085 secs using 99.2% CPU
Optimizing NNI: done in 0.00890708 secs using 99.89% CPU
Optimizing NNI: done in 0.00897503 secs using 98.95% CPU
Optimizing NNI: done in 0.00559115 secs using 99.91% CPU
Optimizing NNI: done in 0.010767 secs using 99.45% CPU
Optimizing NNI: done in 0.00993514 secs using 99.43% CPU
Optimizing NNI: done in 0.00450802 secs using 98.51% CPU
Optimizing NNI: done in 0.00450206 secs using 99.89% CPU
Optimizing NNI: done in 0.00928307 secs using 99.42% CPU
Optimizing NNI: done in 0.0120971 secs using 98.98% CPU
Iteration 120 / LogL: -1385.992 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.00346303 secs using 99.88% CPU
Optimizing NNI: done in 0.011497 secs using 99.85% CPU
Optimizing NNI: done in 0.011807 secs using 99.04% CPU
Optimizing NNI: done in 0.00690913 secs using 99.74% CPU
Optimizing NNI: done in 0.00469494 secs using 99.75% CPU
Optimizing NNI: done in 0.00844193 secs using 99.11% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.303
Optimizing NNI: done in 0.00807095 secs using 99.93% CPU
Optimizing NNI: done in 0.00882506 secs using 99.74% CPU
Optimizing NNI: done in 0.00644994 secs using 98.09% CPU
Optimizing NNI: done in 0.00847888 secs using 99.77% CPU
Iteration 130 / LogL: -1385.309 / Time: 0h:0m:1s (0h:0m:1s left)
Optimizing NNI: done in 0.00889492 secs using 98.91% CPU
Optimizing NNI: done in 0.00635505 secs using 99.35% CPU
Optimizing NNI: done in 0.00994802 secs using 99.82% CPU
Optimizing NNI: done in 0.00307488 secs using 95.58% CPU
UPDATE BEST LOG-LIKELIHOOD: -1385.303
Optimizing NNI: done in 0.00968003 secs using 99.94% CPU
Optimizing NNI: done in 0.00946093 secs using 99.95% CPU
Optimizing NNI: done in 0.00841999 secs using 99.72% CPU
Optimizing NNI: done in 0.0127101 secs using 99.77% CPU
Optimizing NNI: done in 0.00398493 secs using 99.53% CPU
Optimizing NNI: done in 0.0096159 secs using 99.28% CPU
Iteration 140 / LogL: -1385.330 / Time: 0h:0m:1s (0h:0m:0s left)
Optimizing NNI: done in 0.00589108 secs using 99.64% CPU
Optimizing NNI: done in 0.00665808 secs using 99.8% CPU
Optimizing NNI: done in 0.00509596 secs using 95.39% CPU
Optimizing NNI: done in 0.00257492 secs using 100% CPU
Optimizing NNI: done in 0.00807691 secs using 99.94% CPU
Optimizing NNI: done in 0.00854397 secs using 99.2% CPU
Optimizing NNI: done in 0.012078 secs using 99.51% CPU
Optimizing NNI: done in 0.00856805 secs using 99.77% CPU
Optimizing NNI: done in 0.00673795 secs using 99.84% CPU
Optimizing NNI: done in 0.00906301 secs using 99.94% CPU
Iteration 150 / LogL: -1385.563 / Time: 0h:0m:2s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1406.884
Optimizing NNI: done in 0.0110571 secs using 98.44% CPU
Optimizing NNI: done in 0.00605798 secs using 99.62% CPU
Optimizing NNI: done in 0.01105 secs using 99.24% CPU
Optimizing NNI: done in 0.00736189 secs using 99.95% CPU
Optimizing NNI: done in 0.00835514 secs using 99.41% CPU
Optimizing NNI: done in 0.00396895 secs using 99.93% CPU
Optimizing NNI: done in 0.012579 secs using 99.46% CPU
Optimizing NNI: done in 0.0106752 secs using 99.59% CPU
Optimizing NNI: done in 0.00383401 secs using 99.92% CPU
Optimizing NNI: done in 0.00865793 secs using 99.94% CPU
Iteration 160 / LogL: -1385.307 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.0123041 secs using 99.82% CPU
Optimizing NNI: done in 0.00394702 secs using 99.8% CPU
Optimizing NNI: done in 0.0172751 secs using 98.3% CPU
Optimizing NNI: done in 0.0081141 secs using 99.53% CPU
Optimizing NNI: done in 0.00413179 secs using 99.96% CPU
Optimizing NNI: done in 0.01034 secs using 98.79% CPU
Optimizing NNI: done in 0.00707006 secs using 99.84% CPU
Optimizing NNI: done in 0.00771904 secs using 99.31% CPU
Optimizing NNI: done in 0.00681591 secs using 99.55% CPU
Optimizing NNI: done in 0.01209 secs using 99.64% CPU
Iteration 170 / LogL: -1385.304 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.0111802 secs using 99.27% CPU
Optimizing NNI: done in 0.00273108 secs using 99.92% CPU
Optimizing NNI: done in 0.00963616 secs using 99.46% CPU
Optimizing NNI: done in 0.00464392 secs using 99.33% CPU
Optimizing NNI: done in 0.00519013 secs using 99.15% CPU
Optimizing NNI: done in 0.00822711 secs using 99.76% CPU
Optimizing NNI: done in 0.00828791 secs using 99.78% CPU
Optimizing NNI: done in 0.00428295 secs using 99.32% CPU
Optimizing NNI: done in 0.0071559 secs using 99.55% CPU
Optimizing NNI: done in 0.0101869 secs using 99.12% CPU
Iteration 180 / LogL: -1385.309 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.00313497 secs using 99.36% CPU
Optimizing NNI: done in 0.00926089 secs using 99.33% CPU
Optimizing NNI: done in 0.00563383 secs using 99.93% CPU
Optimizing NNI: done in 0.015708 secs using 99.14% CPU
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Optimizing NNI: done in 0.00558996 secs using 99.93% CPU
Optimizing NNI: done in 0.00565505 secs using 99.43% CPU
Optimizing NNI: done in 0.00965214 secs using 99.7% CPU
Optimizing NNI: done in 0.00953698 secs using 99.48% CPU
Optimizing NNI: done in 0.00910211 secs using 99.92% CPU
Iteration 190 / LogL: -1385.304 / Time: 0h:0m:2s (0h:0m:0s left)
Optimizing NNI: done in 0.00541997 secs using 99.72% CPU
Optimizing NNI: done in 0.00868082 secs using 98.92% CPU
Optimizing NNI: done in 0.00988889 secs using 99.95% CPU
Optimizing NNI: done in 0.00396085 secs using 99.98% CPU
Optimizing NNI: done in 0.00855398 secs using 99.97% CPU
Optimizing NNI: done in 0.00864601 secs using 99.65% CPU
Optimizing NNI: done in 0.00578403 secs using 99.65% CPU
Optimizing NNI: done in 0.011658 secs using 99.81% CPU
Optimizing NNI: done in 0.00642109 secs using 99.59% CPU
Optimizing NNI: done in 0.0089829 secs using 99.96% CPU
Iteration 200 / LogL: -1385.315 / Time: 0h:0m:2s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1407.419
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.991
Optimizing NNI: done in 0.0115681 secs using 99.56% CPU
Optimizing NNI: done in 0.00787091 secs using 99.95% CPU
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.303
Optimal log-likelihood: -1385.303
Rate parameters:  A-C: 0.39653  A-G: 1.57282  A-T: 1.00000  C-G: 0.39653  C-T: 1.57282  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.402) (0.277,2.564)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1385.303
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.splits.nex
Total tree length: 6.856

Total number of iterations: 202
CPU time used for tree search: 2.882 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.747 sec (0h:0m:2s)
Total CPU time used: 2.961 sec (0h:0m:2s)
Total wall-clock time used: 2.842 sec (0h:0m:2s)

Computing bootstrap consensus tree...
Reading input file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.splits.nex...
20 taxa and 133 splits.
Consensus tree written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.contree
Reading input trees file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1385.304

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.contree
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot.treefile -m "TPM3{0.396526,1.57282}+FQ+R2{0.723444,0.402241,0.276556,2.56368}" --length 214

Date and Time: Tue Sep 20 15:01:38 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9tvu0rfg/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

Perform single branch tests alongside ufboot

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

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

stdout:

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

Host:    Elizabeths-MacBook-Pro-2.local (AVX512, FMA3, 32 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    907086 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Tue Sep 20 15:01:45 2022
Kernel:  AVX+FMA - 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/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000128031 secs using 81.23% CPU
Alignment most likely contains DNA/RNA sequences
Constructing alignment: done in 0.000226974 secs using 75.34% CPU
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.00136e-05 secs using 89.88% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)
Checking for duplicate sequences: done in 4.1008e-05 secs using 75.6% CPU

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 907086)...

NOTE: 1 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.239 / LogL: -1394.544
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.340 / LogL: -1394.887
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.353 / LogL: -1394.887
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.352 / LogL: -1394.871
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.836
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.862
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.884
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.346 / LogL: -1394.826
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.838
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.841
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.544

Parameters optimization took 0.307 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000981808 secs using 96.35% CPU
Computing ML distances took 0.001053 sec (of wall-clock time) 0.000998 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 82.45% CPU
Constructing RapidNJ tree: done in 6.10352e-05 secs using 88.47% CPU
Computing RapidNJ tree took 0.000131 sec (of wall-clock time) 0.000104 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.870
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.080 second
Computing log-likelihood of 97 initial trees ... 0.082 seconds
Current best score: -1392.870

Do NNI search on 20 best initial trees
Optimizing NNI: done in 0.0188529 secs using 186.8% CPU
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
Optimizing NNI: done in 0.01982 secs using 137.7% CPU
Optimizing NNI: done in 0.017731 secs using 91.44% CPU
Optimizing NNI: done in 0.0147111 secs using 95.25% CPU
Optimizing NNI: done in 0.012063 secs using 98.39% CPU
Optimizing NNI: done in 0.0119851 secs using 98.98% CPU
Optimizing NNI: done in 0.011714 secs using 98.99% CPU
Optimizing NNI: done in 0.0124371 secs using 96.8% CPU
Optimizing NNI: done in 0.012373 secs using 98.45% CPU
Optimizing NNI: done in 0.022414 secs using 96.34% CPU
Iteration 10 / LogL: -1388.153 / Time: 0h:0m:0s
Optimizing NNI: done in 0.0116489 secs using 97.75% CPU
Optimizing NNI: done in 0.012085 secs using 97.81% CPU
Optimizing NNI: done in 0.0130119 secs using 97.11% CPU
Optimizing NNI: done in 0.0261559 secs using 94.11% CPU
Optimizing NNI: done in 0.023701 secs using 98.06% CPU
Optimizing NNI: done in 0.0237889 secs using 96.86% CPU
Optimizing NNI: done in 0.020153 secs using 98.9% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Optimizing NNI: done in 0.0226152 secs using 97.91% CPU
Optimizing NNI: done in 0.019486 secs using 97.85% CPU
Optimizing NNI: done in 0.022258 secs using 98.36% CPU
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1387.259 / CPU time: 0.536
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
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Optimizing NNI: done in 0.0043509 secs using 97.54% CPU
UPDATE BEST LOG-LIKELIHOOD: -1387.257
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Iteration 50 / LogL: -1387.273 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1411.150
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Iteration 90 / LogL: -1387.347 / Time: 0h:0m:2s (0h:0m:2s left)
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UPDATE BEST LOG-LIKELIHOOD: -1387.256
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Iteration 100 / LogL: -1396.540 / Time: 0h:0m:2s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1411.150
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
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Iteration 140 / LogL: -1387.384 / Time: 0h:0m:3s (0h:0m:1s left)
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Iteration 150 / LogL: -1387.506 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1411.150
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Iteration 170 / LogL: -1387.346 / Time: 0h:0m:4s (0h:0m:0s left)
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UPDATE BEST LOG-LIKELIHOOD: -1387.255
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UPDATE BEST LOG-LIKELIHOOD: -1387.255
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Iteration 200 / LogL: -1387.346 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1410.459
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
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TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.255
Optimal log-likelihood: -1387.252
Rate parameters:  A-C: 0.32906  A-G: 2.26160  A-T: 2.13970  C-G: 1.17320  C-T: 3.29054  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.318
Parameters optimization took 1 rounds (0.003 sec)
BEST SCORE FOUND : -1387.252

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.045 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.splits.nex
Total tree length: 6.749

Total number of iterations: 202
CPU time used for tree search: 4.417 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.338 sec (0h:0m:4s)
Total CPU time used: 4.840 sec (0h:0m:4s)
Total wall-clock time used: 4.770 sec (0h:0m:4s)

Computing bootstrap consensus tree...
Reading input file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.splits.nex...
20 taxa and 160 splits.
Consensus tree written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.contree
Reading input trees file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.428

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.contree
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.log

ALISIM COMMAND
--------------
--alisim simulated_MSA -t /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/q2iqtreeufboot.treefile -m "GTR{0.32906,2.2616,2.1397,1.1732,3.29054}+F{0.243216,0.181967,0.319196,0.255621}+I{3.38041e-07}+G4{1.31785}" --length 214

Date and Time: Tue Sep 20 15:01:50 2022
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/ee47f656-b8e5-4849-86d0-6dc5d5b7686a/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpl7mo0tok/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!