Fork me on GitHub

Phylogenetic inference with q2-phylogeny

Note

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

Inferring phylogenies

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

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

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

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

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

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

  1. generate a sequence alignment within QIIME 2

  2. mask the alignment if needed

  3. construct a phylogenetic tree

  4. root the phylogenetic tree

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

Sequence Alignment

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

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

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

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

Next, download the data:

Please select a download option that is most appropriate for your environment:
wget \
  -O "rep-seqs.qza" \
  "https://data.qiime2.org/2024.5/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2024.5/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 5276 -N 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9hhpfi7y -n q2 


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

Inference[0]: Time 0.445919 CAT-based likelihood -1238.367206, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1388.324233 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9hhpfi7y/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1388.324233 .... 

Final GAMMA-based Score of best tree -1387.295763

Program execution info written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9hhpfi7y/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9hhpfi7y/RAxML_bestTree.q2

Overall execution time: 0.945790 secs or 0.000263 hours or 0.000011 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 5276 -N 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp9hhpfi7y -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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc -n q2 


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

Inference[0]: Time 0.445124 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.361216 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.372414 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.506146 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.359165 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/tmpec9okgcc/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc/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/tmpec9okgcc/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc/RAxML_bestTree.q2

Overall execution time: 2.626330 secs or 0.000730 hours or 0.000030 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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpec9okgcc -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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_ -n q2bootstrap 



Time for BS model parameter optimization 0.038806
Bootstrap[0]: Time 0.126345 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.084095 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.076588 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.066749 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.078104 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.082360 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.079280 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.072513 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.081542 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.083448 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.059471 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.092599 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.094918 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.076764 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.082724 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.080427 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.084211 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.075659 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.078610 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.079841 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.068442 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.066881 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.075705 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.087709 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.073922 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.075211 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.078369 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.098726 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.092350 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.062138 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.079374 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.080081 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.084779 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.075293 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.071521 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.085974 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.069025 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.090573 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.072082 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.069700 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.081511 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.082157 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.078478 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.091237 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.068891 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.079002 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.071558 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.094811 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.061832 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.083249 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.096285 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.069757 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.080236 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.070619 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.081884 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.085968 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.071861 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.066043 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.097196 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.076993 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.066744 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.076456 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.091567 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.074499 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.068866 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.069171 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.087040 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.084557 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.067417 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.066949 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.074440 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.072071 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.063435 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.071725 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.064653 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.069508 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.078544 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.096857 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.079776 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.083818 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.072770 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.078856 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.092755 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.069348 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.073163 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.078523 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.068165 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.073833 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.079675 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.077943 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.084461 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.089994 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.031337 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.090776 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.069809 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.067407 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.078878 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.084542 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.030799 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.040988 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 7.736158 seconds
Average Time per Rapid Bootstrap 0.077362 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 3.229555 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: 1.611794 seconds
Thorough ML search Time: 0.427618 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 5.272924 secs or 0.001465 hours

Combined Bootstrap and ML search took 13.009450 secs or 0.003614 hours

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



Found 1 tree in File /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 13.019428 secs or 0.003617 hours or 0.000151 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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -w /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpoq44w8v_ -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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpdf5_sozl/q2iqtree -nt 1
Seed:    686784 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:02:39 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000855923 secs using 17.76% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 0.000487089 secs using 4.106% 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)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.002 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.605
Optimal log-likelihood: -1388.793
Rate parameters:  A-C: 0.37543  A-G: 2.37167  A-T: 2.15334  C-G: 1.24271  C-T: 3.32366  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.400
Parameters optimization took 1 rounds (0.004 sec)
Time for fast ML tree search: 0.041 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         1402.600     45  2895.200     2919.843     3046.669
  2  GTR+F+I       1401.121     46  2894.242     2920.135     3049.077
  3  GTR+F+G4      1387.358     46  2866.716     2892.609     3021.551
  4  GTR+F+I+G4    1387.726     47  2869.452     2896.633     3027.653
  5  GTR+F+R2      1382.364     47  2858.729     2885.910     3016.930
  6  GTR+F+R3      1382.420     49  2862.840     2892.718     3027.773
 14  GTR+F+I+R2    1382.418     48  2860.837     2889.346     3022.403
 15  GTR+F+I+R3    1382.449     50  2864.898     2896.186     3033.197
 25  SYM+G4        1387.134     43  2860.269     2882.528     3005.006
 27  SYM+R2        1383.095     44  2854.189     2877.621     3002.292
 36  SYM+I+R2      1383.227     45  2856.454     2881.097     3007.923
 47  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 49  TVM+F+R2      1383.789     46  2859.578     2885.470     3014.413
 58  TVM+F+I+R2    1383.812     47  2861.625     2888.805     3019.826
 69  TVMe+G4       1387.122     42  2858.245     2879.368     2999.616
 71  TVMe+R2       1383.079     43  2852.159     2874.418     2996.896
 80  TVMe+I+R2     1383.224     44  2854.449     2877.881     3002.552
 91  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 93  TIM3+F+R2     1385.912     45  2861.825     2886.468     3013.294
102  TIM3+F+I+R2   1386.041     46  2864.082     2889.975     3018.917
113  TIM3e+G4      1390.358     41  2862.715     2882.738     3000.720
115  TIM3e+R2      1385.918     42  2855.836     2876.959     2997.207
124  TIM3e+I+R2    1386.073     43  2858.145     2880.404     3002.882
135  TIM2+F+G4     1393.635     44  2875.270     2898.702     3023.373
137  TIM2+F+R2     1387.681     45  2865.362     2890.005     3016.831
146  TIM2+F+I+R2   1387.782     46  2867.564     2893.456     3022.399
157  TIM2e+G4      1396.795     41  2875.589     2895.613     3013.594
159  TIM2e+R2      1391.574     42  2867.148     2888.270     3008.519
168  TIM2e+I+R2    1391.651     43  2869.302     2891.561     3014.039
179  TIM+F+G4      1390.363     44  2868.726     2892.158     3016.829
181  TIM+F+R2      1384.933     45  2859.866     2884.509     3011.335
190  TIM+F+I+R2    1385.016     46  2862.032     2887.925     3016.867
201  TIMe+G4       1394.002     41  2870.005     2890.028     3008.010
203  TIMe+R2       1389.000     42  2862.000     2883.123     3003.371
212  TIMe+I+R2     1389.095     43  2864.190     2886.449     3008.927
223  TPM3u+F+G4    1392.306     43  2870.611     2892.870     3015.348
225  TPM3u+F+R2    1387.329     44  2862.659     2886.091     3010.762
234  TPM3u+F+I+R2  1387.462     45  2864.923     2889.566     3016.392
245  TPM3+G4       1390.374     40  2860.748     2879.708     2995.387
247  TPM3+R2       1385.925     41  2853.851     2873.874     2991.856
256  TPM3+I+R2     1386.070     42  2856.140     2877.263     2997.511
267  TPM2u+F+G4    1394.533     43  2875.067     2897.325     3019.804
269  TPM2u+F+R2    1389.057     44  2866.113     2889.545     3014.216
278  TPM2u+F+I+R2  1389.101     45  2868.201     2892.844     3019.670
289  TPM2+G4       1396.823     40  2873.646     2892.605     3008.285
291  TPM2+R2       1391.578     41  2865.155     2885.178     3003.160
300  TPM2+I+R2     1391.649     42  2867.297     2888.420     3008.668
311  K3Pu+F+G4     1391.381     43  2868.762     2891.021     3013.499
313  K3Pu+F+R2     1386.371     44  2860.742     2884.174     3008.845
322  K3Pu+F+I+R2   1386.425     45  2862.850     2887.493     3014.319
333  K3P+G4        1394.015     40  2868.030     2886.989     3002.669
335  K3P+R2        1389.002     41  2860.004     2880.028     2998.009
344  K3P+I+R2      1389.099     42  2862.197     2883.320     3003.568
355  TN+F+G4       1394.038     43  2874.077     2896.336     3018.814
357  TN+F+R2       1388.241     44  2864.483     2887.915     3012.586
366  TN+F+I+R2     1388.289     45  2866.578     2891.221     3018.047
377  TNe+G4        1396.791     40  2873.582     2892.542     3008.221
379  TNe+R2        1391.586     41  2865.172     2885.195     3003.177
388  TNe+I+R2      1391.666     42  2867.332     2888.454     3008.703
399  HKY+F+G4      1394.951     42  2873.902     2895.024     3015.273
401  HKY+F+R2      1389.609     43  2865.217     2887.476     3009.954
410  HKY+F+I+R2    1389.663     44  2867.327     2890.759     3015.430
421  K2P+G4        1396.825     39  2871.649     2889.580     3002.922
423  K2P+R2        1391.594     40  2863.189     2882.148     2997.828
432  K2P+I+R2      1391.664     41  2865.327     2885.351     3003.332
443  F81+F+G4      1405.743     41  2893.486     2913.509     3031.491
445  F81+F+R2      1400.805     42  2885.611     2906.733     3026.982
454  F81+F+I+R2    1400.908     43  2887.816     2910.075     3032.553
465  JC+G4         1407.650     38  2891.299     2908.236     3019.206
467  JC+R2         1402.858     39  2883.715     2901.646     3014.988
476  JC+I+R2       1402.926     40  2885.851     2904.811     3020.490
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/tmpdf5_sozl/q2iqtree.model.gz
CPU time for ModelFinder: 0.811 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.852 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.925
Optimal log-likelihood: -1385.924
Rate parameters:  A-C: 0.40868  A-G: 1.56206  A-T: 1.00000  C-G: 0.40868  C-T: 1.56206  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.716,0.409) (0.284,2.490)
Parameters optimization took 1 rounds (0.002 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00061202 secs using 93.13% CPU
Computing ML distances took 0.000662 sec (of wall-clock time) 0.000609 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.69549e-05 secs using 119.1% CPU
Computing RapidNJ tree took 0.000128 sec (of wall-clock time) 0.000170 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.820
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.065 second
Computing log-likelihood of 98 initial trees ... 0.050 seconds
Current best score: -1385.924

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.880
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.311
Iteration 10 / LogL: -1385.344 / Time: 0h:0m:1s
Iteration 20 / LogL: -1385.345 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1385.311 / CPU time: 0.263
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1398.051 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.311
UPDATE BEST LOG-LIKELIHOOD: -1385.310
Iteration 40 / LogL: -1385.310 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 50 / LogL: -1385.673 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 60 / LogL: -1385.313 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 70 / LogL: -1385.924 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 80 / LogL: -1385.884 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 90 / LogL: -1385.313 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1385.707 / Time: 0h:0m:1s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 103 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.310
Optimal log-likelihood: -1385.306
Rate parameters:  A-C: 0.39449  A-G: 1.56664  A-T: 1.00000  C-G: 0.39449  C-T: 1.56664  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.721,0.402) (0.279,2.542)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.306
Total tree length: 6.846

Total number of iterations: 103
CPU time used for tree search: 1.143 sec (0h:0m:1s)
Wall-clock time used for tree search: 0.982 sec (0h:0m:0s)
Total CPU time used: 1.971 sec (0h:0m:1s)
Total wall-clock time used: 1.850 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpdf5_sozl/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpdf5_sozl/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpdf5_sozl/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpdf5_sozl/q2iqtree.log

Date and Time: Thu Jun 27 17:02:41 2024
n cores 1
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpdf5_sozl/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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpt6ul5ag_/q2iqtree -nt 1
Seed:    420707 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:02:51 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.00012207 secs using 80.28% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.00136e-05 secs using 79.89% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

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

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

Parameters optimization took 0.270 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.001127 secs using 88.02% CPU
Computing ML distances took 0.001198 sec (of wall-clock time) 0.001038 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.90871e-05 secs using 127.2% CPU
Computing RapidNJ tree took 0.000174 sec (of wall-clock time) 0.000189 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.870
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.071 second
Computing log-likelihood of 98 initial trees ... 0.065 seconds
Current best score: -1392.870

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
Iteration 10 / LogL: -1387.282 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.267 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1387.265 / CPU time: 0.359
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.348 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 40 / LogL: -1387.282 / Time: 0h:0m:0s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.260
Iteration 50 / LogL: -1387.274 / Time: 0h:0m:1s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 57: -1387.168
Iteration 60 / LogL: -1387.406 / Time: 0h:0m:1s (0h:0m:2s left)
BETTER TREE FOUND at iteration 70: -1387.168
Iteration 70 / LogL: -1387.168 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1412.738 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1387.168 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 100 / LogL: -1387.215 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 110 / LogL: -1387.180 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 120 / LogL: -1387.169 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 130 / LogL: -1387.201 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 140 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 150 / LogL: -1387.191 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 160 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 170 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 171 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34630  A-G: 2.32104  A-T: 2.14178  C-G: 1.23360  C-T: 3.21635  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
Total tree length: 7.605

Total number of iterations: 171
CPU time used for tree search: 2.788 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.654 sec (0h:0m:2s)
Total CPU time used: 3.067 sec (0h:0m:3s)
Total wall-clock time used: 2.939 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpt6ul5ag_/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpt6ul5ag_/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpt6ul5ag_/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpt6ul5ag_/q2iqtree.log

Date and Time: Thu Jun 27 17:02:54 2024
n cores 1
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpt6ul5ag_/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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/q2iqtree -nt 1 -fast
Seed:    458006 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:03:03 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000157833 secs using 57.02% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Analyzing sequences: done in 1.00136e-05 secs using 89.88% CPU

---> START RUN NUMBER 1 (seed: 458006)

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: -1492.20
2. Current log-likelihood: -1404.59
3. Current log-likelihood: -1399.23
4. Current log-likelihood: -1397.83
5. Current log-likelihood: -1397.07
Optimal log-likelihood: -1396.49
Rate parameters:  A-C: 0.24620  A-G: 2.08306  A-T: 1.99581  C-G: 1.06240  C-T: 2.85598  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.432
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000979185 secs using 94.98% CPU
Computing ML distances took 0.001067 sec (of wall-clock time) 0.000970 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.31402e-05 secs using 69.4% CPU
Computing RapidNJ tree took 0.000137 sec (of wall-clock time) 0.000123 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.972
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.188
UPDATE BEST LOG-LIKELIHOOD: -1388.187
Finish initializing candidate tree set (3)
Current best tree score: -1388.187 / CPU time: 0.028
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: -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 (0.014 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.055 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.028 sec (0h:0m:0s)
Total CPU time used: 0.117 sec (0h:0m:0s)
Total wall-clock time used: 0.078 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 459006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.259
2. Current log-likelihood: -1403.078
3. Current log-likelihood: -1398.354
4. Current log-likelihood: -1396.979
5. Current log-likelihood: -1396.262
Optimal log-likelihood: -1395.753
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98595  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000906944 secs using 179% CPU
Computing ML distances took 0.000968 sec (of wall-clock time) 0.001731 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.88486e-05 secs using 83.19% CPU
Computing RapidNJ tree took 0.000175 sec (of wall-clock time) 0.000113 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.173
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / CPU time: 0.041
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 (0.012 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.743

Total number of iterations: 2
CPU time used for tree search: 0.080 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.041 sec (0h:0m:0s)
Total CPU time used: 0.284 sec (0h:0m:0s)
Total wall-clock time used: 0.167 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 460006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1491.925
2. Current log-likelihood: -1402.064
3. Current log-likelihood: -1396.813
4. Current log-likelihood: -1395.392
5. Current log-likelihood: -1394.652
Optimal log-likelihood: -1394.078
Rate parameters:  A-C: 0.27467  A-G: 2.39505  A-T: 2.12238  C-G: 1.21030  C-T: 3.30514  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.388
Parameters optimization took 5 rounds (0.020 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000891924 secs using 197.9% CPU
Computing ML distances took 0.000967 sec (of wall-clock time) 0.001863 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.90871e-05 secs using 79.07% CPU
Computing RapidNJ tree took 0.000140 sec (of wall-clock time) 0.000109 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.807
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.181
Finish initializing candidate tree set (4)
Current best tree score: -1388.181 / CPU time: 0.029
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.181
2. Current log-likelihood: -1387.974
3. Current log-likelihood: -1387.831
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.646
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36872  A-G: 2.32249  A-T: 2.12947  C-G: 1.22911  C-T: 3.29731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.331
Parameters optimization took 6 rounds (0.013 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.508

Total number of iterations: 2
CPU time used for tree search: 0.056 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.029 sec (0h:0m:0s)
Total CPU time used: 0.424 sec (0h:0m:0s)
Total wall-clock time used: 0.241 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 461006)

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: 2.10647  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 (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00108314 secs using 173.9% CPU
Computing ML distances took 0.001177 sec (of wall-clock time) 0.001986 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 78.87% CPU
Computing RapidNJ tree took 0.000138 sec (of wall-clock time) 0.000111 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.031
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: 0.058 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.031 sec (0h:0m:0s)
Total CPU time used: 0.573 sec (0h:0m:0s)
Total wall-clock time used: 0.323 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 462006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.491
2. Current log-likelihood: -1404.606
3. Current log-likelihood: -1399.220
4. Current log-likelihood: -1397.821
5. Current log-likelihood: -1397.063
Optimal log-likelihood: -1396.484
Rate parameters:  A-C: 0.24153  A-G: 2.03298  A-T: 1.94373  C-G: 1.02159  C-T: 2.79340  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.438
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000886917 secs using 197.1% CPU
Computing ML distances took 0.000967 sec (of wall-clock time) 0.001892 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.12328e-05 secs using 70.44% CPU
Computing RapidNJ tree took 0.000181 sec (of wall-clock time) 0.000114 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.204
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.198
UPDATE BEST LOG-LIKELIHOOD: -1388.190
Finish initializing candidate tree set (3)
Current best tree score: -1388.190 / CPU time: 0.030
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.190
2. Current log-likelihood: -1387.968
3. Current log-likelihood: -1387.808
4. Current log-likelihood: -1387.688
5. Current log-likelihood: -1387.597
6. Current log-likelihood: -1387.526
7. Current log-likelihood: -1387.471
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33201  A-G: 2.23761  A-T: 2.11213  C-G: 1.16005  C-T: 3.23466  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 (0.015 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.057 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.030 sec (0h:0m:0s)
Total CPU time used: 0.723 sec (0h:0m:0s)
Total wall-clock time used: 0.403 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 463006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.550
2. Current log-likelihood: -1403.090
3. Current log-likelihood: -1398.358
4. Current log-likelihood: -1396.975
5. Current log-likelihood: -1396.257
Optimal log-likelihood: -1395.748
Rate parameters:  A-C: 0.23785  A-G: 2.06889  A-T: 1.95307  C-G: 1.06292  C-T: 2.77329  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: 1.358
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000967026 secs using 199.3% CPU
Computing ML distances took 0.001064 sec (of wall-clock time) 0.002001 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 6.19888e-05 secs using 88.73% CPU
Computing RapidNJ tree took 0.000168 sec (of wall-clock time) 0.000135 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.197
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / CPU time: 0.043
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.961
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: -1387.469
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.33442  A-G: 2.26003  A-T: 2.13495  C-G: 1.17227  C-T: 3.27212  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 (0.012 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.742

Total number of iterations: 2
CPU time used for tree search: 0.082 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.043 sec (0h:0m:0s)
Total CPU time used: 0.892 sec (0h:0m:0s)
Total wall-clock time used: 0.493 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 464006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.021 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000977993 secs using 169.8% CPU
Computing ML distances took 0.001108 sec (of wall-clock time) 0.001808 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.69413e-05 secs using 81.66% CPU
Computing RapidNJ tree took 0.000144 sec (of wall-clock time) 0.000109 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.184
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.955
Finish initializing candidate tree set (4)
Current best tree score: -1387.955 / CPU time: 0.025
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 (0.012 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.744

Total number of iterations: 2
CPU time used for tree search: 0.048 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.025 sec (0h:0m:0s)
Total CPU time used: 1.026 sec (0h:0m:1s)
Total wall-clock time used: 0.565 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 465006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28732  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000895023 secs using 196.6% CPU
Computing ML distances took 0.000975 sec (of wall-clock time) 0.001864 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.60012e-05 secs using 77.78% CPU
Computing RapidNJ tree took 0.000192 sec (of wall-clock time) 0.000130 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.030
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 (0.014 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.059 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.031 sec (0h:0m:0s)
Total CPU time used: 1.176 sec (0h:0m:1s)
Total wall-clock time used: 0.645 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 466006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.199
2. Current log-likelihood: -1404.591
3. Current log-likelihood: -1399.228
4. Current log-likelihood: -1397.831
5. Current log-likelihood: -1397.074
Optimal log-likelihood: -1396.495
Rate parameters:  A-C: 0.24620  A-G: 2.08306  A-T: 1.99580  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 (0.023 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00089097 secs using 195% CPU
Computing ML distances took 0.000940 sec (of wall-clock time) 0.001808 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.19481e-05 secs using 81.38% CPU
Computing RapidNJ tree took 0.000194 sec (of wall-clock time) 0.000155 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.972
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.188
UPDATE BEST LOG-LIKELIHOOD: -1388.187
Finish initializing candidate tree set (3)
Current best tree score: -1388.187 / CPU time: 0.033
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 (0.015 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.060 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.034 sec (0h:0m:0s)
Total CPU time used: 1.330 sec (0h:0m:1s)
Total wall-clock time used: 0.729 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 467006)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.097
2. Current log-likelihood: -1401.816
3. Current log-likelihood: -1396.523
4. Current log-likelihood: -1395.122
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.818
Rate parameters:  A-C: 0.27163  A-G: 2.41073  A-T: 2.17144  C-G: 1.24911  C-T: 3.27679  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: 1.416
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000952005 secs using 197.5% CPU
Computing ML distances took 0.001025 sec (of wall-clock time) 0.001978 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.69413e-05 secs using 81.66% CPU
Computing RapidNJ tree took 0.000195 sec (of wall-clock time) 0.000105 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.794
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.214
Finish initializing candidate tree set (3)
Current best tree score: -1388.214 / CPU time: 0.024
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: -1388.214
2. Current log-likelihood: -1388.015
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.760
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35522  A-G: 2.35151  A-T: 2.13874  C-G: 1.20261  C-T: 3.36909  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: 1.362
Parameters optimization took 7 rounds (0.014 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.046 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.024 sec (0h:0m:0s)
Total CPU time used: 1.467 sec (0h:0m:1s)
Total wall-clock time used: 0.802 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 7 gave best log-likelihood: -1387.423
Total CPU time for 10 runs: 1.479 seconds.
Total wall-clock time for 10 runs: 0.809 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/q2iqtree.treefile
  Trees from independent runs:   /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/q2iqtree.runtrees
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/q2iqtree.log

Date and Time: Thu Jun 27 17:03:04 2024
n cores 1
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp7xefwcsc/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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp5dhrgi7/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    433087 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:03:14 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000151873 secs using 90.87% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.00136e-05 secs using 79.89% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

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, 1.218 / LogL: -1394.499
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.293 / LogL: -1394.744
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.008, 1.296 / LogL: -1394.738
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.008, 1.294 / LogL: -1394.737
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.766
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.007, 1.297 / LogL: -1394.718
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.299 / LogL: -1394.735
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.301 / LogL: -1394.747
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.304 / LogL: -1394.750
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.761
Optimal pinv,alpha: 0.000, 1.218 / LogL: -1394.499

Parameters optimization took 0.289 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000930071 secs using 97.52% CPU
Computing ML distances took 0.001015 sec (of wall-clock time) 0.000979 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.7895e-05 secs using 93.21% CPU
Computing RapidNJ tree took 0.000168 sec (of wall-clock time) 0.000227 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.880
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.066 second
Computing log-likelihood of 98 initial trees ... 0.069 seconds
Current best score: -1392.880

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.268
Iteration 10 / LogL: -1387.284 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.284 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1387.268 / CPU time: 0.360
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.392 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 40 / LogL: -1387.373 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 50 / LogL: -1387.564 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.267
Iteration 60 / LogL: -1387.587 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 70 / LogL: -1387.377 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.262
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 80: -1387.172
Iteration 80 / LogL: -1387.172 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1387.335 / Time: 0h:0m:1s (0h:0m:1s left)
BETTER TREE FOUND at iteration 94: -1387.168
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 100 / LogL: -1387.440 / Time: 0h:0m:2s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 110 / LogL: -1387.385 / Time: 0h:0m:2s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 120 / LogL: -1388.427 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 130 / LogL: -1387.170 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 140 / LogL: -1387.189 / Time: 0h:0m:2s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 150 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 160 / LogL: -1387.205 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1387.203 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 180 / LogL: -1387.186 / Time: 0h:0m:3s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 190 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 195 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34848  A-G: 2.32802  A-T: 2.14972  C-G: 1.23812  C-T: 3.23041  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: 1.282
Parameters optimization took 1 rounds (0.003 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: 7.615

Total number of iterations: 195
CPU time used for tree search: 3.206 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.105 sec (0h:0m:3s)
Total CPU time used: 3.549 sec (0h:0m:3s)
Total wall-clock time used: 3.457 sec (0h:0m:3s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp5dhrgi7/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp5dhrgi7/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp5dhrgi7/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp5dhrgi7/q2iqtree.log

Date and Time: Thu Jun 27 17:03:17 2024
n cores 1
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpp5dhrgi7/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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpa3zmvgtz/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    619631 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:03:27 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000105858 secs using 85.96% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
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)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.605
Optimal log-likelihood: -1388.793
Rate parameters:  A-C: 0.37543  A-G: 2.37167  A-T: 2.15334  C-G: 1.24271  C-T: 3.32365  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.400
Parameters optimization took 1 rounds (0.004 sec)
Time for fast ML tree search: 0.042 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         1402.600     45  2895.200     2919.843     3046.669
  2  GTR+F+I       1401.121     46  2894.242     2920.135     3049.077
  3  GTR+F+G4      1387.358     46  2866.716     2892.609     3021.551
  4  GTR+F+I+G4    1387.726     47  2869.452     2896.633     3027.653
  5  GTR+F+R2      1382.364     47  2858.729     2885.910     3016.930
  6  GTR+F+R3      1382.420     49  2862.840     2892.719     3027.773
 14  GTR+F+I+R2    1382.418     48  2860.837     2889.346     3022.403
 15  GTR+F+I+R3    1382.449     50  2864.899     2896.187     3033.197
 25  SYM+G4        1387.134     43  2860.269     2882.528     3005.006
 27  SYM+R2        1383.095     44  2854.189     2877.621     3002.292
 36  SYM+I+R2      1383.227     45  2856.454     2881.097     3007.923
 47  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 49  TVM+F+R2      1383.789     46  2859.578     2885.470     3014.413
 58  TVM+F+I+R2    1383.812     47  2861.625     2888.805     3019.826
 69  TVMe+G4       1387.122     42  2858.245     2879.368     2999.616
 71  TVMe+R2       1383.079     43  2852.159     2874.418     2996.896
 80  TVMe+I+R2     1383.224     44  2854.449     2877.881     3002.552
 91  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 93  TIM3+F+R2     1385.912     45  2861.825     2886.468     3013.294
102  TIM3+F+I+R2   1386.041     46  2864.082     2889.975     3018.917
113  TIM3e+G4      1390.358     41  2862.715     2882.738     3000.720
115  TIM3e+R2      1385.918     42  2855.836     2876.959     2997.207
124  TIM3e+I+R2    1386.073     43  2858.145     2880.404     3002.882
135  TIM2+F+G4     1393.635     44  2875.270     2898.702     3023.373
137  TIM2+F+R2     1387.681     45  2865.362     2890.005     3016.831
146  TIM2+F+I+R2   1387.782     46  2867.564     2893.456     3022.399
157  TIM2e+G4      1396.795     41  2875.589     2895.613     3013.594
159  TIM2e+R2      1391.574     42  2867.148     2888.270     3008.519
168  TIM2e+I+R2    1391.651     43  2869.302     2891.561     3014.039
179  TIM+F+G4      1390.363     44  2868.726     2892.158     3016.829
181  TIM+F+R2      1384.933     45  2859.866     2884.509     3011.335
190  TIM+F+I+R2    1385.016     46  2862.032     2887.925     3016.867
201  TIMe+G4       1394.002     41  2870.005     2890.028     3008.010
203  TIMe+R2       1389.000     42  2862.000     2883.123     3003.371
212  TIMe+I+R2     1389.095     43  2864.190     2886.449     3008.927
223  TPM3u+F+G4    1392.306     43  2870.611     2892.870     3015.348
225  TPM3u+F+R2    1387.329     44  2862.659     2886.091     3010.762
234  TPM3u+F+I+R2  1387.462     45  2864.923     2889.566     3016.392
245  TPM3+G4       1390.374     40  2860.748     2879.708     2995.387
247  TPM3+R2       1385.925     41  2853.851     2873.874     2991.856
256  TPM3+I+R2     1386.070     42  2856.140     2877.263     2997.511
267  TPM2u+F+G4    1394.533     43  2875.067     2897.325     3019.804
269  TPM2u+F+R2    1389.057     44  2866.113     2889.545     3014.216
278  TPM2u+F+I+R2  1389.101     45  2868.201     2892.844     3019.670
289  TPM2+G4       1396.823     40  2873.646     2892.605     3008.285
291  TPM2+R2       1391.578     41  2865.155     2885.178     3003.160
300  TPM2+I+R2     1391.649     42  2867.297     2888.420     3008.668
311  K3Pu+F+G4     1391.381     43  2868.762     2891.021     3013.499
313  K3Pu+F+R2     1386.371     44  2860.742     2884.174     3008.845
322  K3Pu+F+I+R2   1386.425     45  2862.850     2887.493     3014.319
333  K3P+G4        1394.015     40  2868.030     2886.989     3002.669
335  K3P+R2        1389.002     41  2860.004     2880.028     2998.009
344  K3P+I+R2      1389.099     42  2862.197     2883.320     3003.568
355  TN+F+G4       1394.038     43  2874.077     2896.336     3018.814
357  TN+F+R2       1388.241     44  2864.483     2887.915     3012.586
366  TN+F+I+R2     1388.289     45  2866.578     2891.221     3018.047
377  TNe+G4        1396.791     40  2873.582     2892.542     3008.221
379  TNe+R2        1391.586     41  2865.172     2885.195     3003.177
388  TNe+I+R2      1391.666     42  2867.332     2888.454     3008.703
399  HKY+F+G4      1394.951     42  2873.902     2895.024     3015.273
401  HKY+F+R2      1389.609     43  2865.217     2887.476     3009.954
410  HKY+F+I+R2    1389.663     44  2867.327     2890.759     3015.430
421  K2P+G4        1396.825     39  2871.649     2889.580     3002.922
423  K2P+R2        1391.594     40  2863.189     2882.148     2997.828
432  K2P+I+R2      1391.664     41  2865.327     2885.351     3003.332
443  F81+F+G4      1405.743     41  2893.486     2913.509     3031.491
445  F81+F+R2      1400.805     42  2885.611     2906.733     3026.982
454  F81+F+I+R2    1400.908     43  2887.816     2910.075     3032.553
465  JC+G4         1407.650     38  2891.299     2908.236     3019.206
467  JC+R2         1402.858     39  2883.715     2901.646     3014.988
476  JC+I+R2       1402.926     40  2885.851     2904.811     3020.490
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/tmpa3zmvgtz/q2iqtree.model.gz
CPU time for ModelFinder: 0.825 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.858 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1385.925
Optimal log-likelihood: -1385.924
Rate parameters:  A-C: 0.40868  A-G: 1.56206  A-T: 1.00000  C-G: 0.40868  C-T: 1.56206  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.716,0.409) (0.284,2.490)
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 0.000590801 secs using 97.66% CPU
Computing ML distances took 0.000654 sec (of wall-clock time) 0.000625 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.00407e-05 secs using 79.89% CPU
Computing RapidNJ tree took 0.000128 sec (of wall-clock time) 0.000108 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.820
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.073 second
Computing log-likelihood of 97 initial trees ... 0.062 seconds
Current best score: -1385.924

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.880
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.311
BETTER TREE FOUND at iteration 5: -1385.309
Iteration 10 / LogL: -1385.336 / Time: 0h:0m:1s
Iteration 20 / LogL: -1385.343 / Time: 0h:0m:1s
Finish initializing candidate tree set (3)
Current best tree score: -1385.309 / CPU time: 0.293
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1385.928 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Iteration 40 / LogL: -1385.309 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 50 / LogL: -1385.835 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 60 / LogL: -1385.339 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1385.310 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1385.875 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1385.506 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1385.311 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 110 / LogL: -1385.527 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 120 / LogL: -1385.310 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Iteration 130 / LogL: -1385.506 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 140 / LogL: -1385.310 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 150 / LogL: -1385.309 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.309
Iteration 160 / LogL: -1385.310 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1385.811 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 180 / LogL: -1385.310 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 190 / LogL: -1385.310 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1385.314 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 206 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.309
Optimal log-likelihood: -1385.305
Rate parameters:  A-C: 0.39448  A-G: 1.56670  A-T: 1.00000  C-G: 0.39448  C-T: 1.56670  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.721,0.402) (0.279,2.542)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1385.305
Total tree length: 6.847

Total number of iterations: 206
CPU time used for tree search: 1.672 sec (0h:0m:1s)
Wall-clock time used for tree search: 1.573 sec (0h:0m:1s)
Total CPU time used: 2.521 sec (0h:0m:2s)
Total wall-clock time used: 2.457 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpa3zmvgtz/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpa3zmvgtz/q2iqtree.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpa3zmvgtz/q2iqtree.mldist
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpa3zmvgtz/q2iqtree.log

Date and Time: Thu Jun 27 17:03:30 2024
n cores 1
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmpa3zmvgtz/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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    332051 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:03:41 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.000110865 secs using 84.79% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.00136e-05 secs using 79.89% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1390.589
Optimal log-likelihood: -1389.805
Rate parameters:  A-C: 0.32695  A-G: 2.17649  A-T: 2.01742  C-G: 1.14417  C-T: 3.04933  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.035
Gamma shape alpha: 1.401
Parameters optimization took 1 rounds (0.004 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         1405.417     45  2900.834     2925.477     3052.303
  2  GTR+F+I       1403.836     46  2899.673     2925.565     3054.508
  3  GTR+F+G4      1388.331     46  2868.663     2894.555     3023.498
  4  GTR+F+I+G4    1388.709     47  2871.418     2898.598     3029.618
  5  GTR+F+R2      1382.562     47  2859.124     2886.305     3017.325
  6  GTR+F+R3      1382.602     49  2863.203     2893.081     3028.136
 14  GTR+F+I+R2    1382.708     48  2861.416     2889.925     3022.983
 15  GTR+F+I+R3    1382.679     50  2865.358     2896.647     3033.657
 25  SYM+G4        1388.448     43  2862.897     2885.156     3007.634
 27  SYM+R2        1384.041     44  2856.081     2879.513     3004.184
 36  SYM+I+R2      1384.179     45  2858.357     2883.000     3009.826
 47  TVM+F+G4      1389.408     45  2868.815     2893.458     3020.284
 49  TVM+F+R2      1384.278     46  2860.555     2886.448     3015.390
 58  TVM+F+I+R2    1384.278     47  2862.557     2889.737     3020.758
 69  TVMe+G4       1388.431     42  2860.861     2881.984     3002.232
 71  TVMe+R2       1384.070     43  2854.141     2876.400     2998.878
 80  TVMe+I+R2     1384.207     44  2856.414     2879.846     3004.517
 91  TIM3+F+G4     1392.277     44  2872.555     2895.987     3020.658
 93  TIM3+F+R2     1385.911     45  2861.822     2886.465     3013.291
102  TIM3+F+I+R2   1386.045     46  2864.089     2889.981     3018.924
113  TIM3e+G4      1391.664     41  2865.328     2885.351     3003.333
115  TIM3e+R2      1386.836     42  2857.673     2878.795     2999.044
124  TIM3e+I+R2    1386.991     43  2859.982     2882.241     3004.719
135  TIM2+F+G4     1395.130     44  2878.260     2901.692     3026.363
137  TIM2+F+R2     1388.182     45  2866.364     2891.007     3017.833
146  TIM2+F+I+R2   1388.319     46  2868.637     2894.529     3023.472
157  TIM2e+G4      1398.824     41  2879.647     2899.671     3017.652
159  TIM2e+R2      1393.017     42  2870.033     2891.156     3011.404
168  TIM2e+I+R2    1393.107     43  2872.215     2894.474     3016.952
179  TIM+F+G4      1391.782     44  2871.563     2894.995     3019.666
181  TIM+F+R2      1385.369     45  2860.738     2885.381     3012.207
190  TIM+F+I+R2    1385.497     46  2862.994     2888.887     3017.829
201  TIMe+G4       1396.021     41  2874.043     2894.066     3012.048
203  TIMe+R2       1390.461     42  2864.922     2886.044     3006.293
212  TIMe+I+R2     1390.568     43  2867.136     2889.394     3011.872
223  TPM3u+F+G4    1393.267     43  2872.534     2894.793     3017.271
225  TPM3u+F+R2    1387.637     44  2863.275     2886.706     3011.377
234  TPM3u+F+I+R2  1387.756     45  2865.513     2890.156     3016.982
245  TPM3+G4       1391.670     40  2863.340     2882.300     2997.980
247  TPM3+R2       1386.891     41  2855.783     2875.806     2993.788
256  TPM3+I+R2     1387.026     42  2858.051     2879.174     2999.422
267  TPM2u+F+G4    1396.100     43  2878.200     2900.459     3022.937
269  TPM2u+F+R2    1389.793     44  2867.586     2891.018     3015.689
278  TPM2u+F+I+R2  1389.878     45  2869.756     2894.399     3021.225
289  TPM2+G4       1398.841     40  2877.682     2896.641     3012.321
291  TPM2+R2       1393.061     41  2868.123     2888.146     3006.128
300  TPM2+I+R2     1393.137     42  2870.275     2891.398     3011.646
311  K3Pu+F+G4     1392.937     43  2871.874     2894.133     3016.611
313  K3Pu+F+R2     1387.034     44  2862.068     2885.500     3010.171
322  K3Pu+F+I+R2   1387.111     45  2864.221     2888.864     3015.690
333  K3P+G4        1396.021     40  2872.041     2891.001     3006.680
335  K3P+R2        1390.496     41  2862.992     2883.015     3000.997
344  K3P+I+R2      1390.601     42  2865.202     2886.325     3006.573
355  TN+F+G4       1395.486     43  2876.972     2899.231     3021.709
357  TN+F+R2       1388.575     44  2865.151     2888.583     3013.254
366  TN+F+I+R2     1388.715     45  2867.429     2892.072     3018.898
377  TNe+G4        1398.826     40  2877.651     2896.611     3012.290
379  TNe+R2        1393.014     41  2868.027     2888.051     3006.032
388  TNe+I+R2      1393.104     42  2870.208     2891.331     3011.579
399  HKY+F+G4      1396.476     42  2876.951     2898.074     3018.322
401  HKY+F+R2      1390.188     43  2866.375     2888.634     3011.112
410  HKY+F+I+R2    1390.281     44  2868.561     2891.993     3016.664
421  K2P+G4        1398.841     39  2875.683     2893.614     3006.956
423  K2P+R2        1393.061     40  2866.123     2885.082     3000.762
432  K2P+I+R2      1393.137     41  2868.274     2888.297     3006.279
443  F81+F+G4      1406.476     41  2894.952     2914.975     3032.957
445  F81+F+R2      1401.149     42  2886.297     2907.420     3027.668
454  F81+F+I+R2    1401.264     43  2888.528     2910.787     3033.265
465  JC+G4         1408.763     38  2893.527     2910.464     3021.434
467  JC+R2         1403.897     39  2885.793     2903.724     3017.066
476  JC+I+R2       1403.935     40  2887.870     2906.829     3022.509
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/tmp63_bmzhr/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.820 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.846 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 332051)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1386.891
Optimal log-likelihood: -1386.887
Rate parameters:  A-C: 0.39145  A-G: 1.51425  A-T: 1.00000  C-G: 0.39145  C-T: 1.51425  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.718,0.396) (0.282,2.538)
Parameters optimization took 1 rounds (0.002 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000716925 secs using 94.85% CPU
Computing ML distances took 0.000767 sec (of wall-clock time) 0.000719 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.69413e-05 secs using 81.66% CPU
Computing RapidNJ tree took 0.000160 sec (of wall-clock time) 0.000147 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.864
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.064 second
Computing log-likelihood of 98 initial trees ... 0.049 seconds
Current best score: -1386.887

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.310
UPDATE BEST LOG-LIKELIHOOD: -1385.308
Iteration 10 / LogL: -1385.340 / Time: 0h:0m:1s
Iteration 20 / LogL: -1385.331 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1385.308 / CPU time: 0.366
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1385.307
Iteration 30 / LogL: -1385.936 / Time: 0h:0m:1s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.306
UPDATE BEST LOG-LIKELIHOOD: -1385.306
Iteration 40 / LogL: -1385.689 / Time: 0h:0m:1s (0h:0m:2s left)
BETTER TREE FOUND at iteration 41: -1385.306
Iteration 50 / LogL: -1385.307 / Time: 0h:0m:1s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1409.009
Iteration 60 / LogL: -1385.308 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 70 / LogL: -1385.517 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.306
UPDATE BEST LOG-LIKELIHOOD: -1385.306
Iteration 80 / LogL: -1385.502 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1385.690 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.306
Iteration 100 / LogL: -1385.306 / Time: 0h:0m:2s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1409.009
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.994
Iteration 110 / LogL: -1385.839 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 120 / LogL: -1385.689 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 130 / LogL: -1385.306 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 140 / LogL: -1385.631 / Time: 0h:0m:2s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.306
Iteration 150 / LogL: -1385.501 / Time: 0h:0m:2s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1409.853
Iteration 160 / LogL: -1385.380 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 170 / LogL: -1385.306 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 180 / LogL: -1385.641 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 190 / LogL: -1385.306 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 200 / LogL: -1385.308 / Time: 0h:0m:3s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1410.424
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
Iteration 210 / LogL: -1385.308 / Time: 0h:0m:3s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.306
Iteration 220 / LogL: -1385.309 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 230 / LogL: -1385.527 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 240 / LogL: -1386.614 / Time: 0h:0m:4s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 242 ITERATIONS / Time: 0h:0m:4s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.306
Optimal log-likelihood: -1385.305
Rate parameters:  A-C: 0.39602  A-G: 1.57582  A-T: 1.00000  C-G: 0.39602  C-T: 1.57582  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.400) (0.278,2.554)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.305
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.splits.nex
Total tree length: 6.876

Total number of iterations: 242
CPU time used for tree search: 3.265 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.195 sec (0h:0m:3s)
Total CPU time used: 4.142 sec (0h:0m:4s)
Total wall-clock time used: 4.102 sec (0h:0m:4s)

Computing bootstrap consensus tree...
Reading input file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.splits.nex...
20 taxa and 167 splits.
Consensus tree written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.contree
Reading input trees file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1385.904

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.contree
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/q2iqtreeufboot.log

Date and Time: Thu Jun 27 17:03:45 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp63_bmzhr/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 2.3.4 COVID-edition for MacOS Intel 64-bit built Jun 18 2024
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, Olga Chernomor,
Heiko Schmidt, Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    elizabethsmbp7.nau.edu (AVX512, FMA3, 32 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/qiime2/elizabethgehret/data/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    765254 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Jun 27 17:03:55 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.00019002 secs using 76.83% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.71661e-05 secs using 87.38% 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)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 765254)...

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: -1394.543
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.342 / LogL: -1394.886
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 0.292 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000897884 secs using 98.45% CPU
Computing ML distances took 0.000988 sec (of wall-clock time) 0.000961 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.81334e-05 secs using 74.64% CPU
Computing RapidNJ tree took 0.000118 sec (of wall-clock time) 0.000113 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.870
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.069 second
Computing log-likelihood of 98 initial trees ... 0.065 seconds
Current best score: -1392.870

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
UPDATE BEST LOG-LIKELIHOOD: -1387.260
Iteration 10 / LogL: -1387.260 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1387.260 / CPU time: 0.480
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 30 / LogL: -1387.257 / Time: 0h:0m:0s (0h:0m:5s left)
Iteration 40 / LogL: -1387.347 / Time: 0h:0m:1s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.256
Iteration 50 / LogL: -1387.538 / Time: 0h:0m:1s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1408.237
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 60 / LogL: -1387.347 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 70 / LogL: -1387.346 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1387.346 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1387.346 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 100 / LogL: -1387.255 / Time: 0h:0m:2s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1409.202
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.994
Iteration 110 / LogL: -1387.345 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 120 / LogL: -1387.315 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 130 / LogL: -1387.345 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 140 / LogL: -1387.359 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 150 / LogL: -1387.421 / Time: 0h:0m:3s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1408.933
Iteration 160 / LogL: -1390.643 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 170 / LogL: -1387.345 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 180 / LogL: -1387.372 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 190 / LogL: -1387.536 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 200 / LogL: -1387.608 / Time: 0h:0m:4s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1409.473
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
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: 0.32905  A-G: 2.26158  A-T: 2.13969  C-G: 1.17321  C-T: 3.29053  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 (0.002 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...
0.047 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.splits.nex
Total tree length: 6.749

Total number of iterations: 202
CPU time used for tree search: 3.887 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.829 sec (0h:0m:3s)
Total CPU time used: 4.272 sec (0h:0m:4s)
Total wall-clock time used: 4.227 sec (0h:0m:4s)

Computing bootstrap consensus tree...
Reading input file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.splits.nex...
20 taxa and 143 splits.
Consensus tree written to /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.contree
Reading input trees file /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.428

Analysis results written to: 
  IQ-TREE report:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.contree
  Screen log file:               /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/q2iqtreeufboot.log

Date and Time: Thu Jun 27 17:04:00 2024
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/0939b2e0-fc3c-45de-a03a-b611fc36b0d3/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/gt/s61zzgwx7gz_npzjm4gxfp3w0000gn/T/tmp_zkr0khg/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!