Data and scripts from: Modeling site-and-branch-heterogeneity with GFmix
Data files
Jun 12, 2026 version files 47.63 MB
-
All_Simulations.zip
45.16 MB
-
bin.zip
1.49 MB
-
functions.zip
18.39 KB
-
IdentifyHeterogeneity.R
4.83 KB
-
MakePlots.R
50.64 KB
-
Nucleomorphs.zip
218.59 KB
-
QuickBinomial.R
1.04 KB
-
README.html
648 KB
-
README.md
25.83 KB
-
RunGFmix.R
4.72 KB
-
ShellScripts.zip
6.18 KB
-
SimulateHeteroData.R
2.84 KB
Abstract
Phylogenetic trees are often inferred from protein sequences sampled from diverse taxa across the tree of life. The compositions of these amino acid sequences may be heterogeneous across both sites and branches, particularly if deep phylogenetic divergences are the focus. Under some conditions, failure to model this compositional heterogeneity can lead to phylogenetic artefacts. However, the computational cost of phylogenetic inference with models accounting for compositional heterogeneity can be prohibitive. The originally proposed site-and-branch-heterogeneous GFmix model accounts for changing relative frequencies of G, A, R, and P (GARP) vs. F, Y, M, I, N, and K (FYMINK) amino acids resulting from extreme variation in G+C content among taxa. This GFmix model modifies a fitted site-heterogeneous profile mixture model in a branch-specific manner using parameters that reflect branch-specific amino acid compositions. This approach has been shown to improve likelihoods and reduce compositional artefacts. However, the original implementation of the model includes constraints which may sacrifice accuracy for computability and is limited to modeling variation in GARP/FYMINK composition. Here we investigate the properties of the original GFmix model in greater depth and present several improvements to the model. The improved GFmix models permit fewer constraints on branch-specific composition parameters, allow modeling of user-defined compositional heterogeneity, and provide for full maximum-likelihood optimization of parameters. We have also developed new methods for detecting compositional heterogeneity directly from sequence data. Analyses of simulated site-and-branch-heterogeneous data indicates that the improved GFmix models better estimate branch-specific compositions and branch lengths in heterogeneous trees. We applied the various versions of the GFmix model to a real dataset with known compositional heterogeneity artefacts. We find that the most complex GFmix model with full maximum likelihood parameter optimization consistently supports the correct tree over the artefactual tree with improved likelihoods.
Note: unzip all zip files included in this repository before continuing!
Also note: the formal implementation of the maximum-likelihood GFmix model and a G/F-class selection script will be available from https://www.mathstat.dal.ca/~tsusko/software.html.
Description of the data and file structure
This Dryad deposit contains simulated and real phylogenetic data and scripts used in the analyses described in McCarthy et al (2026), Syst Biol. More information is provided in the README.
Manuscript abstract as follows:
A brief description of each file in the dataset is provided below. For more detailed information, consult the sections following this one.
- All_Simulations.zip: A zip file itself containing four zip files, which contain simulated multiple sequence alignment data generated under different conditions.
- The main Simulations.zip file contains data for the generation and analysis of 100 simulated 16-taxon site-and-branch-heterogeneous multiple sequence alignments and corresponding downsampled 4-taxon alignments. Data in this folder is arranged as follows for all replicates, using replicate 001 as an example:
- 001/: Contains simulating and downsampling tree files (in Newick format), AliSim input files for simulating sites under each individual class of the C20 mixture model under compositionally-homogeneous and heterogeneous conditions (in Nexus and Newick format), and the output generated from these files (one FASTA and log file per mixture class).
- 001_downsampled_iqtree/: Contains output files for LG+C20+G fitting for 4-taxon alignment using IQTree.
- 001_downsampled_optimization: Contains output files for compositional heterogeneity identification methods for 4-taxon alignment (see later sections of this README).
- 001_downsampled_performance: Contains output files for fitting LG+C20+G+{GARP/FYMINK} for 4-taxon alignment using various implementations of GFmix model.
- 001_iqtree/: Contains output files for LG+C20+G fitting for 16-taxon alignment using IQTree.
- 001_optimization/: Contains output files for compositional heterogeneity identification methods for 16-taxon alignment.
- 001_performance/: Contains output files for fitting LG+C20+G+{GARP/FYMINK} for 16-taxon alignment using various implementations of GFmix model.
- There are then temporary files generated during GFmix fitting for the 16-taxon and 4-taxon alignments. 001.phy (or 001_downsampled.phy) are alignments in PHYLIP format with 0-indexed sequence headers that conform to the strict 10-character PHYLIP formatting for header names. 001.phy.bfile, 001.phy.wfile, and 001.phy_GARP_FYMINK.dfile define the starting b-parameters, mixture weights, and G/F-class assignments. 001.phy.utreec and 001.phy.utreecu are representations of the corresponding tree for replicate 001 in custom format.
- The three additional zip files are as follows:
- CladeA_Downsampled_Simulations.zip: 13-taxon site-and-branch-heterogeneous simulated data in which Clade A is a single taxon and Clades B-D consist of four taxa each. Folder structure and file types similar to Simulations.zip.
- Homogeneous_Simulations.zip: 16-taxon site-heterogeneous but branch-homogeneous simulated data. Folder structure and file types similar to Simulations.zip.
- sim_topologies.zip: Simulating topologies in Newick format.
- The main Simulations.zip file contains data for the generation and analysis of 100 simulated 16-taxon site-and-branch-heterogeneous multiple sequence alignments and corresponding downsampled 4-taxon alignments. Data in this folder is arranged as follows for all replicates, using replicate 001 as an example:
- bin.zip: Contains binaries and other files required for the various GFmix implementations and related scripts included in this folder. These binaries are called by various analysis scripts and functions provided elsewhere in the dataset.
- File types contained in this folder include C source code and object files (.h, .c, .o, Makefiles) and their corresponding executables. Executables may need to be recompiled. See https://www.mathstat.dal.ca/~tsusko/software.html for more information on compilation.
- functions.zip: Contains R scripts for functions called by the various analysis scripts in this folder.
- IdentifyHeterogeneity.R: see Introduction.
- MakePlots.R: see Introduction.
- Nucleomorphs.zip: Contains alignments, trees and result data for the real data set analysis for the modified Novak et al 2026 data set.
- QuickBinomial.R: see Introduction.
- README.html: HTML version of the README file.
- README.md: Markdown version of the README file.
- RunGFmix.R: see Introduction.
- ShellScripts.zip: shell scripts used to automate analysis of simulated or real data using the scripts in this folder; see Introduction.
- SimulateHeteroData.R: see Introduction.
Introduction
This folder contains R scripts used in several analyses performed in McCarthy et al (2026). There are a number of R scripts in the main folder, of which the following in bold are executable:
- SimulateHeteroData.R: simulate a 16-taxon multiple sequence alignment under a site-and-branch heterogeneous LG+C20+G{0.5}+GFmix{GARP/FYMINK} mixture model, with the option to also generate a 4-taxon down-sampling of the same alignment.
- QuickBinomial.R: perform a quick binomial test on a multiple sequence alignment to determine amino acid heterogeneity to be modelled via GFmix.
- IdentifyHeterogeneity.R: perform a more precise optimization procedure to determine amino acid heterogeneity to be modelled via GFmix.
- RunGFmix.R: run one of four implementations of the GFmix model tested in the manuscript, providing an input alignment, tree, model and preferred amino acid compositional heterogeneity (default: GARP/FYMINK).
To replicate the analysis on simulated data in McCarthy et al (2026), I've provided a number of shell scripts in the "ShellScripts.zip" folder which will automate most of the tasks involved. This is done so sequentially, so expect some of the scripts involving more computationally-intensive parts (e.g. likelihood calculations) to take some time. These scripts should be run from within the main folder. Scripts are listed in the order that their tasks occur in the manuscript:
- Simulate100Alignments.sh: This will generate 100 simulated LG+C20+G+{GARP/FYMINK} alignments using SimulateHeteroData.R. Output alignments will be in the folder Simulations, output AliSim data for simulated alignment 1 will be in the folder Simulations/001, et cetera.
- RunIQTree.sh: This will run IQ-Tree 3 on all simulated and downsampled alignments, fitting an LG+C20+G model. This is a required step for the next two parts. Output for simulated alignment 1 will be in the folder Simulations/001_iqtree/; output for downsampled alignment 1 will be in the folder Simulations/001_downsampled_iqtree, et cetera.
- RunModels.sh and RunModelsDownsampled.sh: these will run each implementation of GFmix separately on all simulated and downsampled alignments. Output for simulated alignment 1 will be in the folders Simulations and Simulations/001_performance/; output for downsampled alignment 1 will be in the folders Simulations and Simulations/001_downsampled_performance/, et cetera.
- OptimizeGF.sh and OptimizeGFDownsampled.sh: these will run each G-class / F-class identification and optimization routine separately on all simulated and downsampled alignments. Output for simulated alignment 1 will be in the folders Simulations and Simulations/001_performance/; output for downsampled alignment 1 will be in the folders Simulations and Simulations/001_downsampled_performance/, et cetera.
- SummarizeOptims.sh: This will summarize the results of each optimization for all simulated and downsampled alignments into TSV files. Output for all simulated alignments will be OptSummary.txt, and output for all downsampled alignments will be OptDownSummary.txt.
Previously generated simulations analyzed in the paper can be found in "All_Simulations.zip".
Finally, there is a plotting script - MakePlots.R - that will produce calculations and plots similar to those in the manuscript using much of the output generated by the above shell scripts. I recommend interacting with this script via RStudio or some other IDE and not on the command line.
More information on the main R scripts themselves follows below.
SimulateHeteroData.R
Usage
$ SimulateHeteroData.R -aln <alignment_name> -fmt <phylip | fasta> [-downsample] [-trees]
Command-line options
-aln (default: test): Filename for the output alignment(s). Can be provided with or without a filename extension. Will also be the name of an output folder containing all input files for AliSim and log files generated in the background. Supports writing output to new folders - e.g., "simulations/test" will also create a folder called "simulations" if the latter doesn't exist.
-fmt (default: phylip): Format of output alignment(s). Either sequential PHYLIP or FASTA permitted.
-downsample: If provided on the command line, script will also generate downsampled 4-taxon alignment of the original 16-taxon alignment in the same format.
-trees: If provided, output simulating tree (and downsampled tree if "-downsample" is provided) without branch lengths for use as user fixed tree for IQTREE (-te option).
Output
- A 16-taxon 10,000-site alignment in FASTA or PHYLIP format simulated under a LG+C20+G{0.5}+GFmix{GARP/FYMINK} model.
- If option "-downsample" is specified, a 4-taxon 10,000-site alignment downsampled from 1).
- A folder of the same name as 1) containing all input files for AliSim and output log files from AliSim. If "-trees" is provided, this folder with also contain the simulating tree (and downsampled tree if "-downsampled" is provided) in Newick format.
Details
This script simulates a 16-taxon alignment under a site-and-branch heterogeneous LG+C20+G{0.5}+GFmix{GARP/FYMINK} mixture model, with the option to also generate a 4-taxon down-sampling of the same alignment. Simulation is performed using the AliSim component of the IQTREE software package.
As simulating branch-heterogeneity is not currently a native function of AliSim, the branch-heterogeneity component of the simulation is performed using a custom procedure. This procedure is detailed in the Supplementary Information of McCarthy et al (2026).
This script is used to generate 16-taxon simulated alignments to assess 1) the performance of different implementations of the GFmix model and 2) the performance of different G/F class identification methods in McCarthy et al (2026). The down-sampled 4-taxon alignments are used to perform similar assessments under more sparse taxon sampling conditions.
QuickBinomial.R
Usage
$ QuickBinomial.R -aln <alignment_file> -fmt <phylip | phylip-interleaved | fasta> -out <output_file>
Command-line options
-aln (required): Path to multiple sequence alignment file.
-fmt (required): Format of MSA file - sequential or relaxed PHYLIP (default), interleaved PHYLIP or FASTA.
-out: Path to output file; print output to screen if not provided.
Output
The output of this script is a tab-separated table containing three columns as in the simplified example below:
AA Z-score Assignment
A 10.29 G-class
D 1.73 O-class
N -6.4 F-class
where AA is an amino acid, Z**-score** is the result of the binomial test for that amino acid, and Assignment indicates whether that amino acid is assigned to the G-, O-, or F-class for GFmix.
Details
This script performs a simple binomial test of two proportions for a multiple sequence alignment to determine which amino acids are compositionally heterogeneous across sequences for GFmix (Baker et al 2024, Williamson et al 2025). The counts of all 20 amino acids are tabulated for the input MSA file. A Pearson's χ2 test is then performed on these counts, and the residuals of this test are extracted. These residuals are converted into a distance matrix and used to hierarchically cluster sequences using UPGMA. The resulting dendrogram is cut in two to provide the two groups of sequences needed for the binomial test.
The binomial test of two proportions measures the difference in the number of amino acids across the two groups of sequences as a Z-score. Amino acids with Z > 1.96 are assigned as "G-class" amino acids for GFmix. Amino acids with Z < -1.96 are assigned as "F-class" amino acids for GFmix. The G- and F-classes are the classes of amino acids expected to be compositionally heterogeneous across sequences. All remaining amino acids are assigned as "O-class" amino acids for GFmix and are expected to be compositionally homogeneous across sequences.
IdentifyHeterogeneity.R
Usage
$ IdentifyHeterogeneity.R -aln <alignment_file> -fmt <phylip | phylip-interleaved | fasta> -crit <bi | chi | ssf | ogf> -out <output_file> [-group <group_file> -iqtree <iqtree_file> -tree <tree_file> -root <root_file> -mix <mixture_file>]
Command-line options
The following options apply to all criteria:
-aln (required): Path to multiple sequence alignment file.
-fmt (required): Format of MSA file - sequential or relaxed PHYLIP (default), interleaved PHYLIP or FASTA.
-crit (default = chi): Criterion used to identify heterogeneous G/F classes. bi = Binomial test, chi = χ² test, ssf = sum-of-squares calculation from SSF implementation of GFmix, ogf = likelihood calculation from OGF implementation of GFmix. If bi is selected, the binomial test will proceed as in QuickBinomial.R above unless a groupfile is specified via -group. All other criteria will first perform a binomial test to get a starting G/F, and then perform an optimization of G/F class identification such that G/F returns a minimum (ssf) or maximum (chi, ogf) criterion value.
-group (optional): Path to file listing all taxa in 0-index numerical taxon IDs order to be assigned to group 1 for the binomial test. All other taxa in the alignment are assigned to group 2. The test will then proceed as outlined in QuickBinomial.R above.
-out: Path to output file; print output to screen if not provided.
For tree/model-dependent criteria SSF and OGF, the following options are required:
-iqtree: Path to IQTREE report file, used to extract mixture weights and alpha from initial branch-homogeneous model fitting.
-tree: Path to IQTREE tree file in Newick format.
-root: Path to file specifying all 0-index numerical taxon IDs on one side of the root split of the tree.
-mix: Path to mixture frequency file defined in PAML format. See e.g. /bin/C10.aafreq.dat and similar files for examples.
Output
The output of this script is a tab-separated table containing nine columns as in the example below:
G O F Criterion Optimizer Iterations Iteration time Optimization time Swap times
INKSFYC LW THQDMPGERVA -297403.457148472 GFmix 1 20.5378649234772 20.5381350517273 6.69813680648804,7.07171702384949,6.13255596160889,6.89132213592529,6.14467668533325,7.21630525588989,7.22756910324097,6.21991729736328,6.12342095375061,7.5722804069519,7.48150897026062,6.68500685691833,6.30993390083313,7.1081178188324,6.22749519348145,6.92600727081299,6.91627287864685,6.21400308609009,6.30487322807312,6.70887923240662,6.33306813240051,5.87276887893677
INKFYC SLW THQDMPGERVA -297304.861787549 GFmix 2 20.2848722934723 40.8244557380676 6.12418437004089,6.02716970443726,7.67252635955811,6.2017548084259,6.25790357589722,7.46998977661133,7.21854448318481,6.35003447532654,7.22700953483582,7.18159770965576,6.07644891738892,6.88717651367188,6.00872111320496,6.13095831871033,6.70149421691895,6.65720152854919,6.71095514297485,6.04676532745361,6.84607219696045,7.07174921035767,5.90395402908325,6.1761839389801,6.59189343452454
INKFYC SLWD THQMPGERVA -297282.982375572 GFmix 3 20.5320293903351 61.3579752445221 6.47557616233826,7.09872388839722,7.14146637916565,7.24875950813293,6.0775625705719,7.11838126182556,6.19135355949402,6.33416557312012,6.1560595035553,7.79260945320129,6.31776738166809,7.06242704391479,5.99902677536011,6.79112243652344,7.17432618141174,7.04719638824463,6.07590007781982,6.27450609207153,6.37050318717957,6.73439121246338,6.42640733718872,6.36274027824402,5.88187694549561,5.69265222549438
INKFYC SLWDE THQMPGRVA -297276.69162113 GFmix 4 20.3952906131744 81.754460811615 6.04374837875366,7.05961346626282,7.38548278808594,7.17769002914429,7.29365706443787,6.13704299926758,6.17062592506409,7.14532494544983,6.16820573806763,6.10510230064392,6.04489731788635,6.00628995895386,6.63558959960938,6.07484149932861,6.78895688056946,6.74252796173096,7.01699233055115,6.12856340408325,7.04669499397278,6.23496556282043,6.46155619621277,5.88197803497314,6.36025285720825,5.79745721817017,6.30330538749695
INKFYC SLWDE THQMPGRVA -297276.69162113 GFmix 5 20.8720452785492 102.627875566483 7.0819718837738,6.12668251991272,6.08473467826843,6.30716323852539,6.73491859436035,7.09119820594788,6.09684181213379,6.89634132385254,7.12700629234314,7.22654986381531,7.6830906867981,6.22978925704956,7.17844367027283,6.18623828887939,6.95319533348083,6.15571475028992,6.19138741493225,6.15588641166687,7.05212783813477,6.83798933029175,6.10110259056091,7.00507116317749,5.7416775226593,5.94215822219849,6.5214831829071,6.53870368003845
where G, O and F are the optimum G/O/F classes at a given step in the optimization procedure, Criterion is the criterion value, Optimizer is the criterion being optimized, Iterations is the current iteration of the optimization procedure, Iteration time is the time in seconds for that iteration in the optimization procedure, Optimization time is the time in seconds to date for the optimization procedure and Swap times are the list of times in seconds for assessment of each candidate G/O/F class for a given iteration in the optimization procedure. The final line in this file is the optimized G/O/F class for the input tree/model under a given optimization criterion.
Details
This script is used to determine the most compositionally-heterogeneous G/F classes for a given alignment (with optional information from a corresponding tree and protein evolution model) for modeling with GFmix. In McCarthy et al (2026), this script was used to assess the performance of different identification criteria on 16- and 4-taxon simulated data, where the simulating G/F = GARP/FYMINK and the OGF-based identification method was applied to a real algal nucleomorph dataset.
The binomial criterion operates as in the binomial test of two proportions implemented in QuickBinomial.R. The χ² test criterion uses the binomial criterion as a starting point and then seeks to assign G/O/F classes for an alignment that maximizes χ² through an optimization procedure detailed in the main text of McCarthy et al (2026). Both criteria only require an alignment as input and optionally a group file, which manually assigns the groups used in the binomial test of two proportions.
The SSF and OGF-based criteria use the SSF and OGF implementations of the GFmix model and thus require additional tree and model information. Both criteria use the same binomial-based starting point and optimization procedure as the χ² test criterion. See the main text of McCarthy et al (2026) for evaluation and discussion of the performance of each criterion.
RunGFmix.R
Usage
$ RunGFmix.R -aln <alignment_file> -fmt <phylip | phylip-interleaved | fasta> -type <ogf | ssf | gfp | gff> -mix <mixture_file> -iqtree <iqtree_file> -tree <tree_file> -root <root_file> [-out <out_file> -conv -params <params_file> -gclass <GARP> -fclass <FYMINK>]
Command-line options
-aln (required): Path to multiple sequence alignment file.
-fmt (required): Format of MSA file - sequential or relaxed PHYLIP (default), interleaved PHYLIP or FASTA.
-type: Implementation of GFmix to use. OGF = original implementation in which branch-composition parameters are estimated using a hierarchical procedure, SSF = implementation in which branch-composition parameters are optimized to reduce the sum-of-squares difference between branch and model frequencies, GFP = maximum-likelihood implementation in which branch-composition parameters are optimized, GFF = maximum-likelihood implementation in which all tree and model parameters are optimized.
-iqtree: Path to IQTREE report file, used to extract mixture weights and alpha from initial branch-homogeneous model fitting.
-tree: Path to the IQTREE tree file in Newick format.
-root: Path to file specifying all 0-index numerical taxon IDs on one side of the root split of the tree.
-mix: Path to mixture frequency file defined in PAML format. See, e.g., /bin/C10.aafreq.dat and similar files for examples.
-out: Path to output file; print output to screen if not provided.
-params: Path to params file, which outputs likelihood, alpha, tree parameters, and mixture weights to file.
-conv: convert sequence IDs to 0th-index sequential numerical IDs.
-gclass: G-class for GFmix modeling (default = GARP).
-fclass: F-class for GFmix modeling (default = FYMINK).
Output
The output of this script is a tab-separated table containing ten columns as in the example below:
Alignment Mixture +F Tree Method G F Likelihood Runtime NewGFmixInfo
Nucleomorphs/Downsampled.phy bin/C20.aafreq.dat FALSE Nucleomorphs/Downsampled_NM_Apart_LG+C20+G/Downsampled_NM_Apart_LG+C20+G.treefile OGF THQMPGRVA FYCINK -297276.69162113 5.65325570106506 NA
where Alignment is the input alignment file, Mixture is the input mixture file, +F indicates whether the observed frequency class (+F) was included in the base model, Tree is the input tree file, Method is the implementation of GFmix used, G and F are the G and F classes modeled GFmix, Likelihood is the likelihood of the tree given the model and alignment, Runtime is the time taken to evaulate the likelihood and NewGFmixInfo is additional information taken from the output of the GFP or GFF implementations of GFmix which use maximum-likelihood parameter optimization.
Details
This script applies one of the four GFmix model implementations - OGF, SSF, GFP, or GFP - to a given alignment, tree, and branch-homogeneous protein evolution model. See McCarthy et al (2026) for details on these implementations and the GFmix model more generally. Each implementation requires input from IQTREE to extract information (i.e., alpha, mixture weights) for the base branch-homogeneous model, as well as information on how to root the tree (as IQTREE by default outputs unrooted trees). By default, RunGFmix.R runs GFmix with G/F = GARP/FYMINK, but any valid G/F may be provided - including G/F classes determined by IdentifyHeterogeneity.R above.
As each implementation of the GFmix model expects taxon IDs to be 0th-index numerical IDs, it is usually necessary (and advised) to convert sequence IDs to 0th-index sequential IDs via the -conv option. The -params option may be used to output a file containing the tree (in custom UTREE format), tree and model parameters, and final likelihood under a given model. It is advised to use this option if the user wants to compare estimated parameters under different GFmix implementations.
When RunGFmix.R is called with either the GFP or GFF implementation of GFmix, the output of the L-BFGS-B optimization algorithm will be printed to the screen. This may be redirected to a specific file for logging purposes using 2> <logging_file> on the command-line. See e.g. RunModels.sh for an example of such a redirect.
Important notes
These scripts have been tested primarily using MSAs in sequential, relaxed, or interleaved PHYLIP format. FASTA-formatted MSAs should also be compatible, but have not been as extensively tested.
The functions.zip and bin.zip folders contain R/C++ files with internal functions used across the various scripts in this repository. Nucleomorphs.zip contains the real data examined in the manuscript. README.md and README.html are versions of this README.
Installation
These scripts have been written and tested on remote and desktop Linux environments and should also work on macOS. Usage on Windows has not been tested and is not advisable. All scripts must be located in the same directory. If the scripts are not already executable on your operating system, please use chmod +x on each of the executable scripts to enable run permission.
These scripts depend on the parallel library, which is included in most R installations. MakePlots.R has additional dependencies which can usually be installed for the user if the script is opened through RStudio.
References
McCarthy, Susko, Harada, and Roger (2026). Modeling site-and-branch-heterogeneity with GFmix. Syst Biol.
Williamson, K et al (2025). A robustly rooted tree of eukaryotes reveals their excavate ancestry. Nature 640, 974–981. https://doi.org/10.1038/s41586-025-08709-5
Baker, BA et al. (2024). Expanded phylogeny of extremely halophilic archaea shows multiple independent adaptations to hypersaline environments. Nat Microbiol 9, 964-975. https://doi.org/10.1038/s41564-024-01647-4
Munoz-Gomez, S et al (2022). A site-and-branch-heterogeneous model on an expanded dataset favor mitochondria as sister to known Alphaproteobacteria. Nat Ecol Evol 6, 253-262. https://doi.org/10.1038/s41559-021-01638-2