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Phylogenetic inference with q2-phylogeny

Note

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

Inferring phylogenies

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

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

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

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

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

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

  1. generate a sequence alignment within QIIME 2

  2. mask the alignment if needed

  3. construct a phylogenetic tree

  4. root the phylogenetic tree

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

Sequence Alignment

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

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

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

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

Next, download the data:

Please select a download option that is most appropriate for your environment:
wget \
  -O "rep-seqs.qza" \
  "https://data.qiime2.org/2021.8/tutorials/phylogeny/rep-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2021.8/tutorials/phylogeny/rep-seqs.qza" > \
  "rep-seqs.qza"

Run MAFFT

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

Output artifacts:

Reducing alignment ambiguity: masking and reference alignments

Why mask an alignment?

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

Note

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

How to mask alignment.

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

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

Output artifacts:

Reference based alignments

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

Note

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

Construct a phylogeny

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

  1. Phylogeny for the faint of heart - a tutorial

  2. Molecular phylogenetics - principles and practice

  3. Phylogenetics - An Introduction

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

  1. FastTree

  2. RAxML

  3. IQ-TREE

Methods

fasttree

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

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

Output artifacts:

Tip

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

raxml

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

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

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid hill-climbing mode

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


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

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 1485 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9yosdnmq/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpyf5dsimo -n q2 


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

Inference[0]: Time 0.740693 CAT-based likelihood -1242.802351, best rearrangement setting 5


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

Inference[0] final GAMMA-based Likelihood: -1387.988361 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpyf5dsimo/RAxML_result.q2


Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.988361 .... 

Final GAMMA-based Score of best tree -1387.260435

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpyf5dsimo/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpyf5dsimo/RAxML_bestTree.q2

Overall execution time: 1.399497 secs or 0.000389 hours or 0.000016 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 1485 -N 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9yosdnmq/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpyf5dsimo -n q2

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

Output artifacts:

Perform multiple searches using raxml

If you’d like to perform a more thorough search of “tree space” you can instruct raxml to perform multiple independent searches on the full alignment by using --p-n-searches 5. Once these 5 independent searches are completed, only the single best scoring tree will be returned. Note, we are not bootstrapping here, we’ll do that in a later example. Let’s set --p-substitution-model GTRCAT. Finally, let’s also manually set a seed via --p-seed. By setting our seed, we allow other users the ability to reproduce our phylogeny. That is, anyone using the same sequence alignment and substitution model, will generate the same tree as long as they set the same seed value. Although, --p-seed is not a required argument, it is generally a good idea to set this value.

qiime phylogeny raxml \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-substitution-model GTRCAT \
  --p-seed 1723 \
  --p-n-searches 5 \
  --o-tree raxml-cat-searches-tree.qza \
  --verbose

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid hill-climbing mode

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


Executing 5 inferences on the original alignment using 5 distinct randomized MP trees

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-ehb_944i/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz -n q2 


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

Inference[0]: Time 0.608137 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.490775 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.509965 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.650553 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.491457 CAT-based likelihood -1261.321621, best rearrangement setting 5


Conducting final model optimizations on all 5 trees under GAMMA-based models ....

Inference[0] final GAMMA-based Likelihood: -1388.324037 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_result.q2.RUN.4


Starting final GAMMA-based thorough Optimization on tree 4 likelihood -1387.557031 .... 

Final GAMMA-based Score of best tree -1387.385075

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz/RAxML_bestTree.q2

Overall execution time: 3.573405 secs or 0.000993 hours or 0.000041 days

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

Command: raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-ehb_944i/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpexq8m2uz -n q2

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

Output artifacts:

raxml-rapid-bootstrap

In phylogenetics, it is good practice to check how well the splits / bipartitions in your phylogeny are supported. Often one is interested in which clades are robustly separated from other clades in the phylogeny. One way, of doing this is via bootstrapping (See the Bootstrapping section of the first introductory link above). In QIIME 2, we’ve provided access to the RAxML rapid bootstrap feature. The only difference between this command and the previous are the additional flags --p-bootstrap-replicates and --p-rapid-bootstrap-seed. It is quite common to perform anywhere from 100 - 1000 bootstrap replicates. The --p-rapid-bootstrap-seed works very much like the --p-seed argument from above except that it allows anyone to reproduce the bootstrapping process and the associated supports for your splits.

As per the RAxML online documentation and the RAxML manual, the rapid bootstrapping command that we will execute below will do the following:

  1. Bootstrap the input alignment 100 times and perform a Maximum Likelihood (ML) search on each.

  2. Find best scoring ML tree through multiple independent searches using the original input alignment. The number of independent searches is determined by the number of bootstrap replicates set in the 1st step. That is, your search becomes more thorough with increasing bootstrap replicates. The ML optimization of RAxML uses every 5th bootstrap tree as the starting tree for an ML search on the original alignment.

  3. Map the bipartitions (bootstrap supports, 1st step) onto the best scoring ML tree (2nd step).

qiime phylogeny raxml-rapid-bootstrap \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-seed 1723 \
  --p-rapid-bootstrap-seed 9384 \
  --p-bootstrap-replicates 100 \
  --p-substitution-model GTRCAT \
  --o-tree raxml-cat-bootstrap-tree.qza \
  --verbose

stdout:

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

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



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



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

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


Alignment has 157 distinct alignment patterns

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

RAxML rapid bootstrapping and subsequent ML search

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



Executing 100 rapid bootstrap inferences and thereafter a thorough ML search 

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

Likelihood of final tree will be evaluated and optimized under GAMMA

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

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




RAxML was called as follows:

raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-5eczinth/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o -n q2bootstrap 



Time for BS model parameter optimization 0.052836
Bootstrap[0]: Time 0.170693 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.114613 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.101337 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.087394 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.104698 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.107978 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.105473 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.096073 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.108550 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.113641 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.082432 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.123133 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.128292 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.105271 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.107896 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.107426 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.102315 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.093886 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.104308 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.112749 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.104153 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.091879 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.102365 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.124415 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.103297 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.104028 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.103310 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.125896 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.132631 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.092505 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.110425 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.113720 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.117549 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.104713 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.099534 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.122348 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.095410 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.122528 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.096021 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.094323 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.107938 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.103487 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.104179 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.124561 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.091953 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.104642 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.090662 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.132739 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.086274 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.114540 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.128531 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.093288 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.109733 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.097501 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.114626 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.116471 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.093083 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.097340 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.129783 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.107996 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.093773 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.107486 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.122397 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.099876 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.097966 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.095521 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.121193 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.116252 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.090777 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.094549 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.100864 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.095459 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.087437 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.099370 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.088464 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.095693 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.108129 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.133323 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.104505 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.107982 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.098936 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.107928 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.123401 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.095919 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.110003 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.108519 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.094783 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.094824 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.107831 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.103259 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.112158 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.104051 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.041227 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.116447 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.092708 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.094375 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.106999 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.120879 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.044797 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.058183 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 10.500647 seconds
Average Time per Rapid Bootstrap 0.105006 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 4.142738 seconds

Slow ML Search 0 Likelihood: -1387.994678
Slow ML Search 1 Likelihood: -1387.994678
Slow ML Search 2 Likelihood: -1387.994676
Slow ML Search 3 Likelihood: -1387.994650
Slow ML Search 4 Likelihood: -1387.994685
Slow ML Search 5 Likelihood: -1388.092954
Slow ML Search 6 Likelihood: -1388.182551
Slow ML Search 7 Likelihood: -1388.182563
Slow ML Search 8 Likelihood: -1388.182547
Slow ML Search 9 Likelihood: -1387.994723
Slow ML optimization finished

Slow ML search Time: 2.205195 seconds
Thorough ML search Time: 0.573052 seconds

Final ML Optimization Likelihood: -1387.204993

Model Information:

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

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


ML search took 6.926079 secs or 0.001924 hours

Combined Bootstrap and ML search took 17.427136 secs or 0.004841 hours

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



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 17.436997 secs or 0.004844 hours or 0.000202 days

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

Command: raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-5eczinth/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -w /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpacovj62o -n q2bootstrap

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

Output artifacts:

Tip

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

iqtree

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

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-4onk1yuf/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree -nt 1
Seed:    494065 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:01:57 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-4onk1yuf/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.003 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: -1389.617
Optimal log-likelihood: -1388.792
Rate parameters:  A-C: 0.36565  A-G: 2.36143  A-T: 2.13526  C-G: 1.23580  C-T: 3.29735  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: 1.401
Parameters optimization took 1 rounds (0.007 sec)
Time for fast ML tree search: 0.074 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.602     45  2895.204     2919.846     3046.673
  2  GTR+F+I       1401.121     46  2894.242     2920.134     3049.077
  3  GTR+F+G4      1387.358     46  2866.716     2892.608     3021.551
  4  GTR+F+I+G4    1387.726     47  2869.452     2896.633     3027.653
  5  GTR+F+R2      1382.364     47  2858.729     2885.910     3016.930
  6  GTR+F+R3      1382.420     49  2862.840     2892.718     3027.773
 16  SYM+G4        1387.134     43  2860.269     2882.528     3005.006
 18  SYM+R2        1383.095     44  2854.189     2877.621     3002.292
 29  TVM+F+G4      1388.357     45  2866.713     2891.356     3018.182
 31  TVM+F+R2      1383.789     46  2859.578     2885.470     3014.413
 42  TVMe+G4       1387.122     42  2858.245     2879.368     2999.616
 44  TVMe+R2       1383.080     43  2852.159     2874.418     2996.896
 55  TIM3+F+G4     1391.377     44  2870.754     2894.186     3018.857
 57  TIM3+F+R2     1385.912     45  2861.825     2886.468     3013.294
 68  TIM3e+G4      1390.358     41  2862.715     2882.738     3000.720
 70  TIM3e+R2      1385.918     42  2855.836     2876.959     2997.207
 81  TIM2+F+G4     1393.635     44  2875.270     2898.702     3023.373
 83  TIM2+F+R2     1387.681     45  2865.362     2890.005     3016.831
 94  TIM2e+G4      1396.795     41  2875.589     2895.613     3013.594
 96  TIM2e+R2      1391.574     42  2867.148     2888.270     3008.519
107  TIM+F+G4      1390.363     44  2868.726     2892.158     3016.829
109  TIM+F+R2      1384.933     45  2859.866     2884.509     3011.335
120  TIMe+G4       1394.002     41  2870.005     2890.028     3008.010
122  TIMe+R2       1389.000     42  2862.000     2883.123     3003.371
133  TPM3u+F+G4    1392.306     43  2870.611     2892.870     3015.348
135  TPM3u+F+R2    1387.329     44  2862.659     2886.091     3010.762
146  TPM3+F+G4     1392.306     43  2870.611     2892.870     3015.348
148  TPM3+F+R2     1387.329     44  2862.659     2886.091     3010.762
159  TPM2u+F+G4    1394.536     43  2875.071     2897.330     3019.808
161  TPM2u+F+R2    1389.054     44  2866.107     2889.539     3014.210
172  TPM2+F+G4     1394.536     43  2875.071     2897.330     3019.808
174  TPM2+F+R2     1389.054     44  2866.107     2889.539     3014.210
185  K3Pu+F+G4     1391.405     43  2868.811     2891.070     3013.548
187  K3Pu+F+R2     1386.385     44  2860.769     2884.201     3008.872
198  K3P+G4        1394.023     40  2868.046     2887.005     3002.685
200  K3P+R2        1389.010     41  2860.020     2880.043     2998.025
211  TN+F+G4       1394.039     43  2874.077     2896.336     3018.814
213  TN+F+R2       1388.239     44  2864.479     2887.911     3012.582
224  TNe+G4        1396.796     40  2873.593     2892.552     3008.232
226  TNe+R2        1391.590     41  2865.180     2885.204     3003.185
237  HKY+F+G4      1394.954     42  2873.909     2895.032     3015.280
239  HKY+F+R2      1389.609     43  2865.217     2887.476     3009.954
250  K2P+G4        1396.829     39  2871.658     2889.589     3002.931
252  K2P+R2        1391.598     40  2863.197     2882.156     2997.836
263  F81+F+G4      1405.747     41  2893.495     2913.518     3031.500
265  F81+F+R2      1400.802     42  2885.604     2906.727     3026.975
276  JC+G4         1407.653     38  2891.306     2908.243     3019.213
278  JC+R2         1402.856     39  2883.713     2901.644     3014.986
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree.model.gz
CPU time for ModelFinder: 0.611 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.644 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1383.080
Optimal log-likelihood: -1383.077
Rate parameters:  A-C: 0.22953  A-G: 1.98475  A-T: 1.54898  C-G: 0.78167  C-T: 1.98475  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.713,0.411) (0.287,2.461)
Parameters optimization took 1 rounds (0.005 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.004041 sec (of wall-clock time) 0.004009 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000083 sec (of wall-clock time) 0.000087 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1396.842
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.086 second
Computing log-likelihood of 98 initial trees ... 0.082 seconds
Current best score: -1383.077

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.824
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1382.304
Iteration 10 / LogL: -1382.851 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.316 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.304 / CPU time: 0.436
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1386.105 / Time: 0h:0m:0s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 31: -1382.008
Iteration 40 / LogL: -1382.426 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 50 / LogL: -1382.457 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 60 / LogL: -1382.479 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 70 / LogL: -1382.025 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1383.182 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 90 / LogL: -1382.376 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1382.008 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 110 / LogL: -1382.296 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.005
Iteration 120 / LogL: -1382.426 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1382.399 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 132 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.005
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19295  A-G: 1.84412  A-T: 1.53813  C-G: 0.77355  C-T: 1.84412  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.723,0.408) (0.277,2.547)
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1382.002
Total tree length: 7.115

Total number of iterations: 132
CPU time used for tree search: 2.441 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.258 sec (0h:0m:2s)
Total CPU time used: 2.475 sec (0h:0m:2s)
Total wall-clock time used: 2.288 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree.log

Date and Time: Thu Sep  9 18:02:00 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-4onk1yuf/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpf0l9dx4d/q2iqtree -nt 1

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

Output artifacts:

Specifying a substitution model

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-awb4rziu/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmppr4jod2f/q2iqtree -nt 1
Seed:    816749 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:02:04 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-awb4rziu/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.239 / LogL: -1394.430
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.306 / LogL: -1394.720
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.315 / LogL: -1394.793
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.313 / LogL: -1394.791
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.755
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.309 / LogL: -1394.783
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.305 / LogL: -1394.729
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.307 / LogL: -1394.742
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.308 / LogL: -1394.753
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.312 / LogL: -1394.757
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.430

Parameters optimization took 0.462 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007320 sec (of wall-clock time) 0.007284 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000087 sec (of wall-clock time) 0.000102 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.715
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.091 second
Computing log-likelihood of 98 initial trees ... 0.114 seconds
Current best score: -1394.430

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.264
Iteration 10 / LogL: -1387.735 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.281 / Time: 0h:0m:1s
Finish initializing candidate tree set (1)
Current best tree score: -1387.264 / CPU time: 0.601
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.403 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1395.842 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1387.370 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 60 / LogL: -1387.351 / Time: 0h:0m:2s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 65: -1387.168
Iteration 70 / LogL: -1387.374 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 80 / LogL: -1389.480 / Time: 0h:0m:2s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 90 / LogL: -1387.437 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 100 / LogL: -1387.336 / Time: 0h:0m:3s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 110 / LogL: -1387.168 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 120 / LogL: -1387.339 / Time: 0h:0m:3s (0h:0m:1s left)
Iteration 130 / LogL: -1387.194 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 140 / LogL: -1387.190 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 150 / LogL: -1387.318 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 160 / LogL: -1395.217 / Time: 0h:0m:5s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 166 ITERATIONS / Time: 0h:0m:5s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34761  A-G: 2.33581  A-T: 2.15596  C-G: 1.24168  C-T: 3.23770  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: 1.283
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.167
Total tree length: 7.613

Total number of iterations: 166
CPU time used for tree search: 4.817 sec (0h:0m:4s)
Wall-clock time used for tree search: 4.647 sec (0h:0m:4s)
Total CPU time used: 5.315 sec (0h:0m:5s)
Total wall-clock time used: 5.141 sec (0h:0m:5s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmppr4jod2f/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmppr4jod2f/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmppr4jod2f/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmppr4jod2f/q2iqtree.log

Date and Time: Thu Sep  9 18:02:09 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-awb4rziu/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmppr4jod2f/q2iqtree -nt 1

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

Output artifacts:

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-8bnc51sc/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree -nt 1 -fast
Seed:    303751 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:02:14 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-8bnc51sc/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

---> START RUN NUMBER 1 (seed: 303751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.239 / LogL: -1394.543
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.019, 1.304 / LogL: -1395.286
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.023, 1.362 / LogL: -1395.498
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.029, 1.363 / LogL: -1395.791
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.025, 1.366 / LogL: -1395.635
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.028, 1.364 / LogL: -1395.780
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.023, 1.362 / LogL: -1395.545
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.024, 1.363 / LogL: -1395.603
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.025, 1.363 / LogL: -1395.653
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.026, 1.364 / LogL: -1395.699
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.543

Parameters optimization took 0.317 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007354 sec (of wall-clock time) 0.007299 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000085 sec (of wall-clock time) 0.000090 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.665
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.279
Finish initializing candidate tree set (3)
Current best tree score: -1387.279 / CPU time: 0.047
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.279
Optimal log-likelihood: -1387.259
Rate parameters:  A-C: 0.33583  A-G: 2.26334  A-T: 2.13846  C-G: 1.17736  C-T: 3.29208  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: 1.304
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1387.259
Total tree length: 6.807

Total number of iterations: 2
CPU time used for tree search: 0.092 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.047 sec (0h:0m:0s)
Total CPU time used: 0.450 sec (0h:0m:0s)
Total wall-clock time used: 0.395 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 304751)

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 (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007746 sec (of wall-clock time) 0.015217 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000085 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.770
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.175
Finish initializing candidate tree set (3)
Current best tree score: -1388.175 / 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: -1388.175
2. Current log-likelihood: -1387.953
3. Current log-likelihood: -1387.794
4. Current log-likelihood: -1387.675
5. Current log-likelihood: -1387.586
6. Current log-likelihood: -1387.517
7. Current log-likelihood: -1387.463
Optimal log-likelihood: -1387.420
Rate parameters:  A-C: 0.33192  A-G: 2.28178  A-T: 2.15597  C-G: 1.18364  C-T: 3.30463  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: 1.351
Parameters optimization took 7 rounds (0.025 sec)
BEST SCORE FOUND : -1387.420
Total tree length: 6.741

Total number of iterations: 2
CPU time used for tree search: 0.085 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.044 sec (0h:0m:0s)
Total CPU time used: 0.254 sec (0h:0m:0s)
Total wall-clock time used: 0.131 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 305751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.259
2. Current log-likelihood: -1403.078
3. Current log-likelihood: -1398.354
4. Current log-likelihood: -1396.979
5. Current log-likelihood: -1396.262
Optimal log-likelihood: -1395.753
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98595  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007771 sec (of wall-clock time) 0.015217 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000078 sec (of wall-clock time) 0.000076 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.687
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.950
Finish initializing candidate tree set (4)
Current best tree score: -1387.950 / CPU time: 0.087
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.950
2. Current log-likelihood: -1387.817
3. Current log-likelihood: -1387.717
4. Current log-likelihood: -1387.641
5. Current log-likelihood: -1387.582
Optimal log-likelihood: -1387.535
Rate parameters:  A-C: 0.36767  A-G: 2.30152  A-T: 2.10663  C-G: 1.21803  C-T: 3.25857  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: 1.325
Parameters optimization took 5 rounds (0.018 sec)
BEST SCORE FOUND : -1387.535
Total tree length: 7.544

Total number of iterations: 2
CPU time used for tree search: 0.170 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.087 sec (0h:0m:0s)
Total CPU time used: 0.324 sec (0h:0m:0s)
Total wall-clock time used: 0.166 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 306751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007846 sec (of wall-clock time) 0.015393 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000069 sec (of wall-clock time) 0.000073 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.762
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.150
Finish initializing candidate tree set (4)
Current best tree score: -1388.150 / CPU time: 0.062
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: -1388.150
2. Current log-likelihood: -1387.960
3. Current log-likelihood: -1387.822
4. Current log-likelihood: -1387.720
5. Current log-likelihood: -1387.643
6. Current log-likelihood: -1387.582
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.37061  A-G: 2.31378  A-T: 2.12048  C-G: 1.22494  C-T: 3.28291  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: 1.329
Parameters optimization took 6 rounds (0.021 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.519

Total number of iterations: 2
CPU time used for tree search: 0.121 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.062 sec (0h:0m:0s)
Total CPU time used: 0.282 sec (0h:0m:0s)
Total wall-clock time used: 0.145 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 307751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06956  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007655 sec (of wall-clock time) 0.014998 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000075 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.741
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1395.320
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 2: -1388.005
Finish initializing candidate tree set (4)
Current best tree score: -1388.005 / CPU time: 0.040
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: -1388.005
2. Current log-likelihood: -1387.830
3. Current log-likelihood: -1387.694
4. Current log-likelihood: -1387.596
5. Current log-likelihood: -1387.522
6. Current log-likelihood: -1387.466
Optimal log-likelihood: -1387.422
Rate parameters:  A-C: 0.32681  A-G: 2.24701  A-T: 2.11978  C-G: 1.16571  C-T: 3.24537  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.023 sec)
BEST SCORE FOUND : -1387.422
Total tree length: 6.698

Total number of iterations: 2
CPU time used for tree search: 0.078 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.040 sec (0h:0m:0s)
Total CPU time used: 0.239 sec (0h:0m:0s)
Total wall-clock time used: 0.123 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 308751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.459
2. Current log-likelihood: -1403.057
3. Current log-likelihood: -1398.315
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.224
Optimal log-likelihood: -1395.705
Rate parameters:  A-C: 0.23667  A-G: 2.08551  A-T: 1.97696  C-G: 1.07651  C-T: 2.86649  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.035 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007720 sec (of wall-clock time) 0.015146 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000075 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.710
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.973
Finish initializing candidate tree set (3)
Current best tree score: -1387.973 / CPU time: 0.064
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: -1387.973
2. Current log-likelihood: -1387.813
3. Current log-likelihood: -1387.692
4. Current log-likelihood: -1387.599
5. Current log-likelihood: -1387.528
6. Current log-likelihood: -1387.472
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33542  A-G: 2.27104  A-T: 2.14661  C-G: 1.17790  C-T: 3.29174  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: 1.350
Parameters optimization took 6 rounds (0.022 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.765

Total number of iterations: 2
CPU time used for tree search: 0.125 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.064 sec (0h:0m:0s)
Total CPU time used: 0.280 sec (0h:0m:0s)
Total wall-clock time used: 0.144 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 309751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84175  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007654 sec (of wall-clock time) 0.015037 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000075 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.741
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1395.309
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 2: -1388.004
Finish initializing candidate tree set (4)
Current best tree score: -1388.004 / CPU time: 0.040
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: -1388.004
2. Current log-likelihood: -1387.829
3. Current log-likelihood: -1387.694
4. Current log-likelihood: -1387.595
5. Current log-likelihood: -1387.522
6. Current log-likelihood: -1387.466
Optimal log-likelihood: -1387.422
Rate parameters:  A-C: 0.32668  A-G: 2.24644  A-T: 2.11919  C-G: 1.16539  C-T: 3.24437  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.022 sec)
BEST SCORE FOUND : -1387.422
Total tree length: 6.698

Total number of iterations: 2
CPU time used for tree search: 0.078 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.040 sec (0h:0m:0s)
Total CPU time used: 0.238 sec (0h:0m:0s)
Total wall-clock time used: 0.123 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 310751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06956  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.037 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007617 sec (of wall-clock time) 0.014961 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000073 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.741
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1395.320
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 2: -1388.005
Finish initializing candidate tree set (4)
Current best tree score: -1388.005 / CPU time: 0.040
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: -1388.005
2. Current log-likelihood: -1387.830
3. Current log-likelihood: -1387.694
4. Current log-likelihood: -1387.596
5. Current log-likelihood: -1387.522
6. Current log-likelihood: -1387.466
Optimal log-likelihood: -1387.422
Rate parameters:  A-C: 0.32681  A-G: 2.24701  A-T: 2.11978  C-G: 1.16571  C-T: 3.24537  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 6 rounds (0.025 sec)
BEST SCORE FOUND : -1387.422
Total tree length: 6.698

Total number of iterations: 2
CPU time used for tree search: 0.078 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.040 sec (0h:0m:0s)
Total CPU time used: 0.244 sec (0h:0m:0s)
Total wall-clock time used: 0.125 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 311751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.491
2. Current log-likelihood: -1404.606
3. Current log-likelihood: -1399.220
4. Current log-likelihood: -1397.821
5. Current log-likelihood: -1397.063
Optimal log-likelihood: -1396.484
Rate parameters:  A-C: 0.24153  A-G: 2.03298  A-T: 1.94373  C-G: 1.02159  C-T: 2.79340  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.438
Parameters optimization took 5 rounds (0.040 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007855 sec (of wall-clock time) 0.015391 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000061 sec (of wall-clock time) 0.000065 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1400.067
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.199
Finish initializing candidate tree set (3)
Current best tree score: -1388.199 / CPU time: 0.058
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.199
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.809
4. Current log-likelihood: -1387.688
5. Current log-likelihood: -1387.597
6. Current log-likelihood: -1387.527
7. Current log-likelihood: -1387.472
Optimal log-likelihood: -1387.427
Rate parameters:  A-C: 0.33146  A-G: 2.22541  A-T: 2.09978  C-G: 1.15373  C-T: 3.21464  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.358
Parameters optimization took 7 rounds (0.027 sec)
BEST SCORE FOUND : -1387.427
Total tree length: 6.730

Total number of iterations: 2
CPU time used for tree search: 0.113 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.058 sec (0h:0m:0s)
Total CPU time used: 0.289 sec (0h:0m:0s)
Total wall-clock time used: 0.148 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 312751)

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

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.038 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007935 sec (of wall-clock time) 0.015602 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000071 sec (of wall-clock time) 0.000075 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1399.762
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.150
Finish initializing candidate tree set (4)
Current best tree score: -1388.150 / CPU time: 0.063
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: -1388.150
2. Current log-likelihood: -1387.960
3. Current log-likelihood: -1387.822
4. Current log-likelihood: -1387.720
5. Current log-likelihood: -1387.643
6. Current log-likelihood: -1387.582
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.37061  A-G: 2.31378  A-T: 2.12048  C-G: 1.22494  C-T: 3.28291  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: 1.329
Parameters optimization took 6 rounds (0.021 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.519

Total number of iterations: 2
CPU time used for tree search: 0.123 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.063 sec (0h:0m:0s)
Total CPU time used: 0.284 sec (0h:0m:0s)
Total wall-clock time used: 0.146 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 1 gave best log-likelihood: -1387.259
Total CPU time for 10 runs: 2.914 seconds.
Total wall-clock time for 10 runs: 1.667 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree.treefile
  Trees from independent runs:   /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree.runtrees
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree.log

Date and Time: Thu Sep  9 18:02:15 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 10 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-8bnc51sc/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpws32nroe/q2iqtree -nt 1 -fast

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

Output artifacts:

Single branch tests

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

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

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

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

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-b_6bg39d/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_k41ymgr/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    331302 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:02:20 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-b_6bg39d/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

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

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

Parameters optimization took 0.476 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007419 sec (of wall-clock time) 0.007369 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000086 sec (of wall-clock time) 0.000100 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.692
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.093 second
Computing log-likelihood of 98 initial trees ... 0.114 seconds
Current best score: -1394.499

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1394.449
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1387.268
Iteration 10 / LogL: -1387.283 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.283 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.268 / CPU time: 0.614
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1387.253
Iteration 30 / LogL: -1387.280 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 40 / LogL: -1387.355 / Time: 0h:0m:1s (0h:0m:2s left)
BETTER TREE FOUND at iteration 46: -1387.244
Estimate model parameters (epsilon = 0.100)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 50 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:4s left)
Iteration 60 / LogL: -1387.168 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 70 / LogL: -1387.351 / Time: 0h:0m:2s (0h:0m:2s left)
BETTER TREE FOUND at iteration 71: -1387.168
Iteration 80 / LogL: -1387.354 / Time: 0h:0m:2s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 90 / LogL: -1387.214 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 100 / LogL: -1387.443 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 110 / LogL: -1396.418 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 120 / LogL: -1414.225 / Time: 0h:0m:4s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 130 / LogL: -1387.203 / Time: 0h:0m:4s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 140 / LogL: -1387.170 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 150 / LogL: -1387.169 / Time: 0h:0m:4s (0h:0m:0s left)
Iteration 160 / LogL: -1387.421 / Time: 0h:0m:5s (0h:0m:0s left)
Iteration 170 / LogL: -1387.192 / Time: 0h:0m:5s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 172 ITERATIONS / Time: 0h:0m:5s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34795  A-G: 2.33030  A-T: 2.15644  C-G: 1.23843  C-T: 3.22661  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.285
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.167

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...
0.057 sec.
Total tree length: 7.601

Total number of iterations: 172
CPU time used for tree search: 5.349 sec (0h:0m:5s)
Wall-clock time used for tree search: 5.170 sec (0h:0m:5s)
Total CPU time used: 5.914 sec (0h:0m:5s)
Total wall-clock time used: 5.731 sec (0h:0m:5s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_k41ymgr/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_k41ymgr/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_k41ymgr/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_k41ymgr/q2iqtree.log

Date and Time: Thu Sep  9 18:02:26 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-b_6bg39d/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp_k41ymgr/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

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

Output artifacts:

Tip

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

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

stdout:

Plugin warning from phylogeny:

iqtree is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-6mm9a7nj/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    426693 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:02:30 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-6mm9a7nj/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.281
2. Current log-likelihood: -1389.723
Optimal log-likelihood: -1388.882
Rate parameters:  A-C: 0.33811  A-G: 2.30745  A-T: 2.15809  C-G: 1.19223  C-T: 3.30164  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.033
Gamma shape alpha: 1.423
Parameters optimization took 2 rounds (0.013 sec)
Time for fast ML tree search: 0.061 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.278     46  2866.557     2892.449     3021.392
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.422
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 16  SYM+G4        1387.155     43  2860.310     2882.568     3005.047
 18  SYM+R2        1382.244     44  2852.489     2875.921     3000.592
 29  TVM+F+G4      1388.425     45  2866.850     2891.493     3018.319
 31  TVM+F+R2      1382.482     46  2856.965     2882.857     3011.799
 42  TVMe+G4       1387.122     42  2858.244     2879.367     2999.615
 44  TVMe+R2       1382.298     43  2850.596     2872.855     2995.333
 55  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 57  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
 68  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
 70  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
 81  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
 83  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
 94  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
 96  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
107  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
109  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
120  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
122  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
133  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
135  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
146  TPM3+F+G4     1392.567     43  2871.134     2893.393     3015.871
148  TPM3+F+R2     1386.368     44  2860.736     2884.168     3008.839
159  TPM2u+F+G4    1395.282     43  2876.564     2898.823     3021.301
161  TPM2u+F+R2    1388.115     44  2864.231     2887.663     3012.334
172  TPM2+F+G4     1395.282     43  2876.564     2898.823     3021.301
174  TPM2+F+R2     1388.115     44  2864.231     2887.663     3012.334
185  K3Pu+F+G4     1392.067     43  2870.133     2892.392     3014.870
187  K3Pu+F+R2     1385.124     44  2858.247     2881.679     3006.350
198  K3P+G4        1394.798     40  2869.597     2888.556     3004.236
200  K3P+R2        1388.380     41  2858.761     2878.784     2996.766
211  TN+F+G4       1394.627     43  2875.254     2897.513     3019.991
213  TN+F+R2       1386.824     44  2861.648     2885.079     3009.750
224  TNe+G4        1397.746     40  2875.492     2894.452     3010.131
226  TNe+R2        1391.135     41  2864.270     2884.293     3002.275
237  HKY+F+G4      1395.753     42  2875.505     2896.628     3016.876
239  HKY+F+R2      1388.692     43  2863.383     2885.642     3008.120
250  K2P+G4        1397.751     39  2873.502     2891.433     3004.775
252  K2P+R2        1391.217     40  2862.434     2881.394     2997.073
263  F81+F+G4      1406.484     41  2894.968     2914.991     3032.973
265  F81+F+R2      1400.605     42  2885.210     2906.333     3026.581
276  JC+G4         1408.433     38  2892.866     2909.803     3020.773
278  JC+R2         1403.022     39  2884.045     2901.976     3015.318
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree.model.gz
CPU time for ModelFinder: 0.656 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.659 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1382.298
Optimal log-likelihood: -1382.296
Rate parameters:  A-C: 0.21620  A-G: 2.01102  A-T: 1.56832  C-G: 0.77577  C-T: 2.01102  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.406) (0.278,2.543)
Parameters optimization took 1 rounds (0.004 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.004139 sec (of wall-clock time) 0.004114 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000084 sec (of wall-clock time) 0.000100 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1396.935
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.092 second
Computing log-likelihood of 97 initial trees ... 0.081 seconds
Current best score: -1382.296

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.296
Iteration 10 / LogL: -1382.305 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.311 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.296 / CPU time: 0.435
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 24: -1382.087
Iteration 30 / LogL: -1382.174 / Time: 0h:0m:0s (0h:0m:3s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 31: -1382.004
Iteration 40 / LogL: -1382.396 / Time: 0h:0m:0s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.003
Iteration 50 / LogL: -1382.031 / Time: 0h:0m:0s (0h:0m:3s left)
Iteration 60 / LogL: -1382.003 / Time: 0h:0m:0s (0h:0m:2s left)
Iteration 70 / LogL: -1382.013 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1382.004 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1382.004 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 100 / LogL: -1382.070 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 110 / LogL: -1382.093 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 120 / LogL: -1382.345 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 130 / LogL: -1382.007 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 140 / LogL: -1382.348 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 150 / LogL: -1382.023 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 160 / LogL: -1382.013 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1382.091 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.002
Iteration 180 / LogL: -1382.049 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 190 / LogL: -1382.092 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.002
Iteration 200 / LogL: -1382.018 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 210 / LogL: -1382.374 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 220 / LogL: -1382.886 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 230 / LogL: -1382.002 / Time: 0h:0m:2s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 232 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.002
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19088  A-G: 1.83909  A-T: 1.53607  C-G: 0.77021  C-T: 1.83909  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.724,0.409) (0.276,2.554)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.002
Total tree length: 7.108

Total number of iterations: 232
CPU time used for tree search: 3.011 sec (0h:0m:3s)
Wall-clock time used for tree search: 2.824 sec (0h:0m:2s)
Total CPU time used: 3.042 sec (0h:0m:3s)
Total wall-clock time used: 2.851 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree.mldist
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree.log

Date and Time: Thu Sep  9 18:02:33 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-6mm9a7nj/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp9cpcccif/q2iqtree -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

iqtree-ultrafast-bootstrap

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

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

stdout:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-kw3l_x7l/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    360141 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:02:38 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-kw3l_x7l/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.281
2. Current log-likelihood: -1389.723
Optimal log-likelihood: -1388.882
Rate parameters:  A-C: 0.33811  A-G: 2.30746  A-T: 2.15809  C-G: 1.19223  C-T: 3.30165  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.033
Gamma shape alpha: 1.423
Parameters optimization took 2 rounds (0.013 sec)
Time for fast ML tree search: 0.061 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 286 DNA models (sample size: 214) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.278     46  2866.557     2892.449     3021.392
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.611     47  2855.222     2882.402     3013.422
  6  GTR+F+R3      1380.659     49  2859.317     2889.195     3024.250
 16  SYM+G4        1387.155     43  2860.310     2882.568     3005.047
 18  SYM+R2        1382.244     44  2852.489     2875.921     3000.592
 29  TVM+F+G4      1388.425     45  2866.850     2891.493     3018.319
 31  TVM+F+R2      1382.482     46  2856.965     2882.857     3011.799
 42  TVMe+G4       1387.122     42  2858.244     2879.367     2999.615
 44  TVMe+R2       1382.298     43  2850.596     2872.855     2995.333
 55  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 57  TIM3+F+R2     1384.431     45  2858.861     2883.504     3010.330
 68  TIM3e+G4      1390.540     41  2863.080     2883.103     3001.085
 70  TIM3e+R2      1385.228     42  2854.456     2875.578     2995.827
 81  TIM2+F+G4     1394.180     44  2876.360     2899.792     3024.463
 83  TIM2+F+R2     1386.234     45  2862.469     2887.112     3013.938
 94  TIM2e+G4      1397.742     41  2877.483     2897.507     3015.488
 96  TIM2e+R2      1391.117     42  2866.235     2887.357     3007.606
107  TIM+F+G4      1390.802     44  2869.603     2893.035     3017.706
109  TIM+F+R2      1383.206     45  2856.411     2881.054     3007.880
120  TIMe+G4       1394.796     41  2871.592     2891.616     3009.597
122  TIMe+R2       1388.300     42  2860.600     2881.723     3001.971
133  TPM3u+F+G4    1392.567     43  2871.134     2893.393     3015.871
135  TPM3u+F+R2    1386.368     44  2860.736     2884.168     3008.839
146  TPM3+F+G4     1392.567     43  2871.134     2893.393     3015.871
148  TPM3+F+R2     1386.368     44  2860.736     2884.168     3008.839
159  TPM2u+F+G4    1395.282     43  2876.564     2898.823     3021.301
161  TPM2u+F+R2    1388.115     44  2864.231     2887.663     3012.334
172  TPM2+F+G4     1395.282     43  2876.564     2898.823     3021.301
174  TPM2+F+R2     1388.115     44  2864.231     2887.663     3012.334
185  K3Pu+F+G4     1392.067     43  2870.133     2892.392     3014.870
187  K3Pu+F+R2     1385.124     44  2858.247     2881.679     3006.350
198  K3P+G4        1394.798     40  2869.597     2888.556     3004.236
200  K3P+R2        1388.380     41  2858.761     2878.784     2996.766
211  TN+F+G4       1394.627     43  2875.254     2897.513     3019.991
213  TN+F+R2       1386.824     44  2861.648     2885.079     3009.750
224  TNe+G4        1397.746     40  2875.492     2894.452     3010.131
226  TNe+R2        1391.135     41  2864.270     2884.293     3002.275
237  HKY+F+G4      1395.753     42  2875.505     2896.628     3016.876
239  HKY+F+R2      1388.692     43  2863.383     2885.642     3008.120
250  K2P+G4        1397.751     39  2873.502     2891.433     3004.775
252  K2P+R2        1391.217     40  2862.434     2881.394     2997.073
263  F81+F+G4      1406.484     41  2894.968     2914.991     3032.973
265  F81+F+R2      1400.605     42  2885.210     2906.333     3026.581
276  JC+G4         1408.433     38  2892.866     2909.803     3020.773
278  JC+R2         1403.022     39  2884.045     2901.976     3015.318
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TVMe+R2
Best-fit model: TVMe+R2 chosen according to BIC

All model information printed to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.674 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.680 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 360141)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1382.298
Optimal log-likelihood: -1382.296
Rate parameters:  A-C: 0.21620  A-G: 2.01102  A-T: 1.56832  C-G: 0.77577  C-T: 2.01102  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.406) (0.278,2.543)
Parameters optimization took 1 rounds (0.007 sec)
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.005066 sec (of wall-clock time) 0.005007 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000095 sec (of wall-clock time) 0.000089 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1396.935
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.090 second
Computing log-likelihood of 98 initial trees ... 0.082 seconds
Current best score: -1382.296

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1382.296
Iteration 10 / LogL: -1382.305 / Time: 0h:0m:0s
Iteration 20 / LogL: -1382.595 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1382.296 / CPU time: 0.612
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 25: -1382.087
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 27: -1382.007
Iteration 30 / LogL: -1382.421 / Time: 0h:0m:0s (0h:0m:5s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.006
Iteration 40 / LogL: -1382.426 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 50 / LogL: -1382.090 / Time: 0h:0m:1s (0h:0m:4s left)
Log-likelihood cutoff on original alignment: -1406.976
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 60 / LogL: -1382.004 / Time: 0h:0m:1s (0h:0m:4s left)
Iteration 70 / LogL: -1382.092 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 80 / LogL: -1382.005 / Time: 0h:0m:1s (0h:0m:3s left)
BETTER TREE FOUND at iteration 81: -1382.004
UPDATE BEST LOG-LIKELIHOOD: -1382.004
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 90 / LogL: -1382.196 / Time: 0h:0m:1s (0h:0m:4s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 100 / LogL: -1382.115 / Time: 0h:0m:2s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1406.355
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.997
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 110 / LogL: -1382.014 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 120 / LogL: -1382.005 / Time: 0h:0m:2s (0h:0m:3s left)
Iteration 130 / LogL: -1382.014 / Time: 0h:0m:2s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 140 / LogL: -1382.099 / Time: 0h:0m:3s (0h:0m:3s left)
Iteration 150 / LogL: -1382.445 / Time: 0h:0m:3s (0h:0m:2s left)
Log-likelihood cutoff on original alignment: -1407.273
Iteration 160 / LogL: -1382.090 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 170 / LogL: -1382.004 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 180 / LogL: -1382.041 / Time: 0h:0m:3s (0h:0m:2s left)
Iteration 190 / LogL: -1382.004 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 200 / LogL: -1382.040 / Time: 0h:0m:4s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1407.287
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
Iteration 210 / LogL: -1382.095 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 220 / LogL: -1382.012 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 230 / LogL: -1382.016 / Time: 0h:0m:4s (0h:0m:1s left)
Iteration 240 / LogL: -1382.004 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 250 / LogL: -1382.025 / Time: 0h:0m:5s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1407.287
Iteration 260 / LogL: -1382.004 / Time: 0h:0m:5s (0h:0m:0s left)
Iteration 270 / LogL: -1382.039 / Time: 0h:0m:5s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1382.004
Iteration 280 / LogL: -1382.098 / Time: 0h:0m:5s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 282 ITERATIONS / Time: 0h:0m:6s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1382.004
Optimal log-likelihood: -1382.002
Rate parameters:  A-C: 0.19393  A-G: 1.85549  A-T: 1.54650  C-G: 0.77829  C-T: 1.85549  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.725,0.409) (0.275,2.555)
Parameters optimization took 1 rounds (0.005 sec)
BEST SCORE FOUND : -1382.002
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.splits.nex
Total tree length: 7.114

Total number of iterations: 282
CPU time used for tree search: 6.144 sec (0h:0m:6s)
Wall-clock time used for tree search: 5.975 sec (0h:0m:5s)
Total CPU time used: 6.255 sec (0h:0m:6s)
Total wall-clock time used: 6.086 sec (0h:0m:6s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.splits.nex...
20 taxa and 136 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1382.003

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot.log

Date and Time: Thu Sep  9 18:02:45 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-kw3l_x7l/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmpbzt86z52/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

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

Output artifacts:

Perform single branch tests alongside ufboot

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

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

stdout:

Plugin warning from phylogeny:

iqtree-ultrafast-bootstrap is deprecated and will be removed in a future version of this plugin.
IQ-TREE multicore version 2.1.4-beta COVID-edition for Mac OS X 64-bit built Jun 24 2021
Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung,
Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams.

Host:    ghost.mggen.nau.edu (AVX, 16 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9cigc9i6/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    501359 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Sep  9 18:02:49 2021
Kernel:  AVX - 1 threads (8 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 8 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9cigc9i6/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta ... Fasta format detected
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.001 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 501359)...

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, 1.282 / LogL: -1392.553
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.377 / LogL: -1392.829
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.391 / LogL: -1392.898
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.388 / LogL: -1392.889
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.853
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.384 / LogL: -1392.879
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.007, 1.379 / LogL: -1392.828
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.382 / LogL: -1392.844
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.383 / LogL: -1392.849
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.008, 1.384 / LogL: -1392.858
Optimal pinv,alpha: 0.000, 1.282 / LogL: -1392.553

Parameters optimization took 0.477 sec
Computing ML distances based on estimated model parameters...
Computing ML distances took 0.007758 sec (of wall-clock time) 0.007728 sec(of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Computing RapidNJ tree took 0.000082 sec (of wall-clock time) 0.000091 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1398.750
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.095 second
Computing log-likelihood of 98 initial trees ... 0.113 seconds
Current best score: -1392.553

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.261
Iteration 10 / LogL: -1387.282 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:1s
Finish initializing candidate tree set (2)
Current best tree score: -1387.261 / CPU time: 0.806
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1387.258
Iteration 30 / LogL: -1387.277 / Time: 0h:0m:1s (0h:0m:9s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.256
Iteration 40 / LogL: -1390.679 / Time: 0h:0m:1s (0h:0m:7s left)
Iteration 50 / LogL: -1387.351 / Time: 0h:0m:2s (0h:0m:6s left)
Log-likelihood cutoff on original alignment: -1406.020
UPDATE BEST LOG-LIKELIHOOD: -1387.256
Iteration 60 / LogL: -1387.350 / Time: 0h:0m:2s (0h:0m:5s left)
Iteration 70 / LogL: -1387.350 / Time: 0h:0m:2s (0h:0m:5s left)
Iteration 80 / LogL: -1387.350 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 90 / LogL: -1387.329 / Time: 0h:0m:3s (0h:0m:4s left)
Iteration 100 / LogL: -1387.351 / Time: 0h:0m:3s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1406.020
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
Iteration 110 / LogL: -1387.562 / Time: 0h:0m:3s (0h:0m:3s left)
Iteration 120 / LogL: -1396.573 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 130 / LogL: -1387.354 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 140 / LogL: -1387.350 / Time: 0h:0m:4s (0h:0m:2s left)
Iteration 150 / LogL: -1387.352 / Time: 0h:0m:5s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1405.826
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 160 / LogL: -1387.371 / Time: 0h:0m:5s (0h:0m:1s left)
Iteration 170 / LogL: -1387.350 / Time: 0h:0m:6s (0h:0m:1s left)
Iteration 180 / LogL: -1387.350 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 190 / LogL: -1387.529 / Time: 0h:0m:6s (0h:0m:0s left)
Iteration 200 / LogL: -1387.351 / Time: 0h:0m:7s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1407.433
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.987
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 210 / LogL: -1387.261 / Time: 0h:0m:7s (0h:0m:3s left)
Iteration 220 / LogL: -1387.350 / Time: 0h:0m:7s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.255
Iteration 230 / LogL: -1387.350 / Time: 0h:0m:8s (0h:0m:2s left)
Iteration 240 / LogL: -1387.350 / Time: 0h:0m:8s (0h:0m:2s left)
Iteration 250 / LogL: -1387.352 / Time: 0h:0m:8s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1407.433
Iteration 260 / LogL: -1389.338 / Time: 0h:0m:9s (0h:0m:1s left)
Iteration 270 / LogL: -1387.357 / Time: 0h:0m:9s (0h:0m:1s left)
Iteration 280 / LogL: -1387.350 / Time: 0h:0m:9s (0h:0m:0s left)
Iteration 290 / LogL: -1387.352 / Time: 0h:0m:10s (0h:0m:0s left)
Iteration 300 / LogL: -1387.352 / Time: 0h:0m:10s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1407.433
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
TREE SEARCH COMPLETED AFTER 300 ITERATIONS / Time: 0h:0m:10s

--------------------------------------------------------------------
|                    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: 0.33717  A-G: 2.27752  A-T: 2.15656  C-G: 1.18247  C-T: 3.31123  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: 1.315
Parameters optimization took 1 rounds (0.004 sec)
BEST SCORE FOUND : -1387.252

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.056 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.splits.nex
Total tree length: 6.749

Total number of iterations: 300
CPU time used for tree search: 10.320 sec (0h:0m:10s)
Wall-clock time used for tree search: 10.158 sec (0h:0m:10s)
Total CPU time used: 10.965 sec (0h:0m:10s)
Total wall-clock time used: 10.803 sec (0h:0m:10s)

Computing bootstrap consensus tree...
Reading input file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.splits.nex...
20 taxa and 160 splits.
Consensus tree written to /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.contree
Reading input trees file /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.426

Analysis results written to: 
  IQ-TREE report:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.contree
  Screen log file:               /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/q2iqtreeufboot.log

Date and Time: Thu Sep  9 18:03:00 2021
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/qiime2-archive-9cigc9i6/a9a9164d-6850-4eb0-b2e6-a72fdc481658/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/9h/268zfwl56h37jwt5qv866jcr0000gp/T/tmp8w4sxwx7/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!