PyiTOL

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PyiTOL is a Python CLI tool for automating phylogenetic tree visualization on the iTOL (Interactive Tree Of Life) platform.

Features

Core Features

• 31 iTOL template types (plus external-shape-bubble, a variant of external-shape): color strips, heatmaps, bar charts, pie charts, symbols, binary matrices, gradients, protein domains, connections, and more
• Full API client: upload, export PDF/SVG/PNG/TIFF/EPS/Newick/Nexus/PhyloXML, delete, and batch operations via iTOL batch endpoints
• Taxonomy & monophyly analysis: built-in dendropy integration for monophyletic/paraphyletic/polyphyletic group classification
• Extract taxonomy from tip names: supports GTDB (d__Bacteria;p__Proteobacteria;...), embedded (_d_Bacteria_p_...), NCBI (Genus_species), auto-detection, underscore, and mixed formats
• Monophyly check → conditional styling: check if a taxon is monophyletic, then automatically generate beautification templates
• Color-blind friendly palettes: tol_bright, tol_vibrant, wong, okabeito, and more
• Auto legend positioning: legends placed in bottom-right corner to avoid tree overlap
• Session snapshots & replay: YAML-based operation recording for reproducible workflows
• Newick/Nexus dual format: automatic tree file format detection

Taxonomy Parsing

• Format A (embedded): GB_GCA_0001_d_Bacteria_p_Proteo_c_..._g_Genus
• Format B (semicolon): d__Archaea;p__Thermoproteota;c__Korarchaeia;...
• Mixed mode: auto-detect and unify both formats
• Extensible levels: custom taxonomy level prefixes via --taxonomy-levels
• Special identifiers: LUCA (MRCA of all Bacteria and Archaea), LACA (MRCA of Archaea), LBCA (MRCA of Bacteria), ROOT (all tips in the tree)

Monophyly-Based Clade Collapsing

# Collapse by taxon name (with monophyly check)
pyitol template create-collapse --taxon Cyanobacteriota --rank Phylum \
  --taxonomy tax.csv --tree tree.nwk -o collapse.txt

# Strict mode: terminate on non-monophyletic groups
pyitol template create-collapse --taxon Proteobacteria --rank Phylum \
  --taxonomy tax.csv --tree tree.nwk --strict -o collapse.txt

# Default mode: skip non-monophyletic groups and continue
pyitol template create-collapse --taxon Proteobacteria --rank Phylum \
  --taxonomy tax.csv --tree tree.nwk -o collapse.txt

Real-time Logging

• Stream output, no buffering, direct sys.stdout write with flush
• ISO8601 timestamps: 2025-03-21T10:15:30.123 | INFO | message
• Plain text format, no color output
• --log-file for simultaneous file output
• Log levels: DEBUG, INFO, WARNING, ERROR, CRITICAL

Multi-tree Handling

# Default: prompt user when multiple trees detected
pyitol template create color-strip --tree multi.nwk ...

# Specify processing strategy
pyitol template create color-strip --tree multi.nwk --multi-tree-mode first ...
pyitol template create color-strip --tree multi.nwk --multi-tree-mode last ...
pyitol template create color-strip --tree multi.nwk --multi-tree-mode random ...
pyitol template create color-strip --tree multi.nwk --multi-tree-mode split ...

Input File Validation

# Validate tree, sequence, and taxonomy files
pyitol validate --tree tree.nwk --taxonomy tax.csv --sequence seqs.fasta

# Deep validation: bracket balance, branch lengths, duplicate names, alphabet
pyitol validate --tree tree.nwk --alphabet DNA

Validation includes:

• Trees: bracket balance, negative branch lengths (CRITICAL), empty names (ERROR), duplicate tips (ERROR), multi-root (CRITICAL)
• Sequences: alphabet detection (DNA/RNA/protein), ID uniqueness, length consistency
• Adversarial protection: malicious characters (control/bidi), circular dependencies, empty files

Self-test Mode

# Run self-test: verify dependencies, sample parsing, monophyly logic
pyitol self-test

Outputs [PASS]/[FAIL] table verifying:

• Third-party dependency imports and versions
• Internal module imports
• Newick tree parsing
• Embedded taxonomy extraction
• Monophyly detection logic
• Malicious character detection

Installation

pip install pyitol

With conda (recommended for bioinformatics users):

conda create -n pyitol python=3.12
conda activate pyitol
pip install pyitol

From source:

git clone https://github.com/ZengZichao/PyiTOL.git
cd PyiTOL
pip install -e ".[dev]"

Install optional dependencies (memory monitoring):

pip install pyitol[memory]

Docker Deployment

PyiTOL provides a Docker image for isolated environments:

# Build from the included Dockerfile
docker build -t pyitol:latest .

# Run with mounted data directory
docker run -v $(pwd)/data:/data pyitol:latest \
  pyitol template create color-strip --tree /data/tree.nwk \
  --taxonomy /data/tax.csv --column Phylum -o /data/output.txt

The Docker image includes all runtime dependencies and is suitable for HPC clusters or cloud platforms.

Verify Installation

After installation, run the following commands to verify:

# Check version
pyitol --version

# Run self-test (verifies dependencies, sample parsing, core logic)
pyitol self-test

Expected output:

pyitol 1.0.0
license: MIT
...

  PyiTOL Self-Test Results
  =======================================================
  [PASS] Import typer (v0.25.1)
  [PASS] Import pandas (v2.3.3)
  [PASS] Parse sample Newick (4 tips)
  [PASS] Monophyly detection (G1=mono, G2=mono)
  ...
  All checks passed.

Quick Start

Local Template Generation (No API Key Required)

The fastest way to get started is to generate iTOL template files locally from your tree and taxonomy data — no API key or internet connection required:

# Generate a color-strip template from a Newick tree and taxonomy table
pyitol template create color-strip --tree tree.nwk --taxonomy tax.csv --column Phylum -o colorstrip.txt

This produces a plain-text iTOL template file (colorstrip.txt) that you can upload manually to iTOL via drag-and-drop.

Validate Input Files

Before generating templates, validate your input files for common issues:

pyitol validate --tree tree.nwk --taxonomy tax.csv

This checks tree format, bracket balance, duplicate tip names, and taxonomy consistency. For deeper validation including sequence alphabet detection:

pyitol validate --tree tree.nwk --taxonomy tax.csv --sequence seqs.fasta --alphabet DNA

Version Info

# Display version, Git hash, dependency versions and licenses
pyitol --version

Output example:

pyitol 1.0.0
license: MIT
date: 2026-08-13
git: abc1234
dependencies:
  typer: 0.25.1 (MIT)
  pandas: 2.3.3 (BSD-3-Clause)
  dendropy: 4.6.4 (BSD-3-Clause)
  numpy: 2.4.4 (BSD-3-Clause)
  ...

Optional: Upload to iTOL

The following steps require a free iTOL API key. If you only need local template generation, you can skip this section.

API Key Configuration

Three methods supported (highest to lowest priority):

# Method 1: Command-line (temporary)
pyitol task upload --tree tree.nwk --api-key YOUR_KEY

# Method 2: Environment variable
export ITOL_API_KEY=YOUR_KEY

# Method 3: Key file
pyitol task upload --tree tree.nwk --api-key-file /path/to/key.txt

Credential security / 凭据安全

• Never commit key files to version control. Patterns *.key, itolapi.key, and .itolapi.key are already listed in .gitignore, but a key file can still leak through other channels — do not place it in cloud-synced folders (e.g. cloud-drive sync directories) or shared directories.
• Prefer ~/.config/pyitol/ (mode 600) or your system keychain for storing key files, rather than the project root.
• Session snapshots and logs automatically redact key material (***REDACTED***), so snapshots are safe to share.
• If a key may have been exposed (committed, synced, or shared), rotate it immediately in your iTOL account settings.

Upload and Export PDF

# One-step: upload → render → export PDF locally
pyitol task upload-and-export \
  --tree tree.nwk \
  --config template1.txt \
  --config template2.txt \
  --api-key YOUR_KEY \
  --dataset-name "MyTree" \
  --format pdf \
  --dpi 300 \
  --output ./result.pdf \
  --wait 30

Supported formats: pdf, svg, png, tiff, eps, newick, nexus, phyloxml. Options: --dpi, --width, --height.

The upload-and-export command also supports --parameter for passing iTOL export parameters (e.g., datasets_visible, display_mode, background), consistent with the task export command:

# Control dataset visibility and rendering via --parameter
pyitol task upload-and-export \
  --tree tree.nwk \
  --config template1.txt --config template2.txt \
  --api-key YOUR_KEY --dataset-name "MyTree" \
  --format png --dpi 300 --output ./result.png --wait 30 \
  --parameter datasets_visible=0,1 \
  --parameter display_mode=2 \
  --parameter background=ffffff

Taxonomy & Monophyly Analysis

Extract Taxonomy from Tip Names

# GTDB format: d__Bacteria;p__Proteobacteria;...;s__Escherichia_coli
pyitol taxonomy extract-from-names --tree tree.nwk --format gtdb -o taxonomy.csv

# Embedded format: GB_GCA_0001_d_Bacteria_p_Proteo_c_..._g_Genus
pyitol taxonomy extract-from-names --tree tree.nwk --format embedded -o taxonomy.csv

# NCBI format: Genus_species
pyitol taxonomy extract-from-names --tree tree.nwk --format ncbi -o taxonomy.csv

# Auto-detect format
pyitol taxonomy extract-from-names --tree tree.nwk --format auto -o taxonomy.csv

# Custom taxonomy levels
pyitol taxonomy extract-from-names --tree tree.nwk \
  --taxonomy-levels "d:Domain,p:Phylum,c:Class,o:Order,f:Family,g:Genus,s:Species" \
  -o taxonomy.csv

Monophyly Check → Conditional Styling

# Check if Escherichia is monophyletic
# If yes → generate branch coloring template
# If no → warn and exit
pyitol taxonomy check-and-style \
  --tree tree.nwk \
  --taxonomy tax.csv \
  --taxon Escherichia \
  --rank Genus \
  --action color-branch \
  --color "#e41a1c" \
  --output clade.txt

# Using special identifiers
pyitol taxonomy check-and-style \
  --tree tree.nwk \
  --taxonomy tax.csv \
  --taxon LUCA \
  --rank Domain \
  --action color-branch \
  --output luca.txt

# Without taxonomy file → auto-extract from tip names
pyitol taxonomy check-and-style \
  --tree tree.nwk \
  --taxon Salmonella \
  --rank Genus \
  --action highlight \
  --output highlight.txt

Supported styling types:

• color-branch: branch coloring
• highlight: label background highlighting
• color-strip: outer color strip

Monophyly Check

# Check monophyly of all genera
pyitol taxonomy monophyly --tree tree.nwk --taxonomy tax.csv \
  --rank Genus --output monophyly_results.csv

# Check special identifiers
pyitol taxonomy monophyly --tree tree.nwk --taxonomy tax.csv \
  --taxa LUCA,LACA,LBCA,ROOT --output special_monophyly.csv

# Extract taxonomy summary
pyitol taxonomy extract --tree tree.nwk --taxonomy tax.csv \
  --rank Genus --output taxonomy_summary.csv

# List available taxonomy ranks
pyitol taxonomy ranks --taxonomy tax.csv

Template Examples

# Color strip - categorical coloring by taxonomic rank (legend auto-positioned)
pyitol template create color-strip --tree tree.nwk --taxonomy tax.csv \
  --column Phylum --palette tol_bright -o strip.txt

# Heatmap - numeric data visualization
pyitol template create heatmap --tree tree.nwk --taxonomy tax.csv \
  --columns "gc_content,genome_size" --gradient "#ffffcc,#800026" -o heatmap.txt

# Bar chart - quantitative comparison
pyitol template create simple-bar --tree tree.nwk --taxonomy tax.csv \
  --column genome_size --bar-color "#3c5484" -o bar.txt

# Binary matrix - presence/absence
pyitol template create binary --tree tree.nwk --taxonomy tax.csv \
  --columns "amr_genes,virulence_factors" -o binary.txt

# Connection lines between nodes (JSON format)
pyitol template create connections --connections connections.json -o connections.txt

# Branch gradient - continuous coloring
pyitol template create branch-gradient --tree tree.nwk --taxonomy tax.csv \
  --column gc_content --gradient "#313695,#a50026" -o gradient.txt

# Domain architecture
pyitol template create domains --data-file domains.json -o domain.txt

# Multi-column bubble external shape (numeric column visualization)
pyitol template create external-shape-bubble --tree tree.nwk --taxonomy tax.csv \
  --columns "gc_content,genome_size,gene_count" -o bubble.txt

# Bundle multiple templates at once
pyitol template bundle --tree tree.nwk --taxonomy tax.csv \
  --config '[{"type":"color-strip","column":"Phylum"},{"type":"heatmap","columns":"gc,genome_size"}]' \
  --output-dir ./templates/

Output File Structure

PyiTOL generates plain text template files that can be directly uploaded to iTOL:

Output Type	Format	Description
Template files (.txt)	iTOL standard	Contains dataset header and data rows, ready for iTOL
Taxonomy table (.csv)	CSV/TSV	ID column + taxonomy rank columns
Monophyly results (.csv)	CSV	Contains group/status/lca_node/member_count columns
Session snapshots (.yaml)	YAML	Operation history, supports replay

Template file structure:

DATASET_COLORSTRIP
SEPARATOR TAB
DATASET_LABEL Phylum
COLOR #ff0000
...
DATA
TaxonA	#4477AA	Proteobacteria
TaxonB	#EE6677	Firmicutes

Workflow

┌─────────────┐     ┌─────────────┐     ┌─────────────┐
│  Input Files │     │   Taxonomy  │     │  Templates  │
│             │     │   Parsing   │     │  Generation │
│ tree.nwk    │────>│ From names  │────>│ color-strip │
│ tax.csv     │     │ or table    │     │ heatmap     │
│ seqs.fasta  │     │             │     │ bar/pie/... │
└─────────────┘     └─────────────┘     └─────────────┘
                           │                   │
                           v                   v
                    ┌─────────────┐     ┌─────────────┐
                    │  Monophyly  │     │  Upload to  │
                    │   Check     │     │    iTOL     │
                    │             │     │             │
                    │ monophyly   │     │ upload      │
                    │ check       │     │ export PDF  │
                    └─────────────┘     └─────────────┘

Typical workflow:

1. Prepare input: Phylogenetic tree (Newick/Nexus) + taxonomy table (CSV/TSV)
2. Parse taxonomy: Auto-extract from tip names or use external table
3. Monophyly check: Verify if taxa are monophyletic
4. Generate templates: Create iTOL visualization templates based on taxonomy
5. Upload & export: Upload to iTOL platform and export PDF/SVG/PNG

Color-Blind Friendly Palettes

Palette	Colors	Source
tol_bright	#4477AA #EE6677 #228833 #CCBB44 #66CCEE #AA3377 #BBBBBB	Paul Tol
tol_vibrant	#EE7733 #0077BB #33BBEE #EE3377 #CC3311 #009988 #BBBBBB	Paul Tol
wong	#000000 #E69F00 #56B4E9 #009E73 #F0E442 #0072B2 #D55E00 #CC79A7	Wong (2011)
okabeito	#E69F00 #56B4E9 #009E73 #F0E442 #0072B2 #D55E00 #CC79A7 #000000	Okabe & Ito (2008)
ibm	#648FFF #785EF0 #DC267F #FE6100 #FFB000	IBM Design

Usage: --palette wong

Session Snapshots & Reproducibility

# After a workflow, session snapshot is saved automatically
pyitol task upload --tree tree.nwk --config template.txt
# Snapshot saved to ~/.pyitol/session_logs/

# Replay a previous session
pyitol replay --session session.yaml

# Show suggested commands without executing
pyitol replay --session session.yaml --dry-run

Large-scale Data Handling

# Low memory mode (for large datasets)
pyitol --low-memory template create color-strip --tree large_tree.nwk ...

# Verbose logging (shows memory usage etc.)
pyitol --verbose template create color-strip --tree large_tree.nwk ...

# Log to file
pyitol --log-file pyitol.log template create color-strip --tree tree.nwk ...

• Trees with >10,000 tips trigger INFO log about resource requirements
• Use --verbose for DEBUG-level memory usage (requires psutil)

Output File Management

# Default: error if output file exists (all subcommands are now protected)
pyitol template create color-strip --tree tree.nwk -o output.txt

# Force overwrite (unified entry 'template create' only)
pyitol template create color-strip --tree tree.nwk -o output.txt --force

# Skip existing files (unified entry 'template create' only)
pyitol template create color-strip --tree tree.nwk -o output.txt --no-clobber

All standalone subcommands (e.g., create-color-strip, create-heatmap, etc.) now check if the output file already exists to prevent accidental overwrites. To overwrite, use the unified entry template create with --force.

Graceful Interrupt

Press Ctrl+C to gracefully terminate:

• Outputs current processing progress
• Closes all open file handles
• Deletes incomplete temporary files
• Exit code 130

Error Codes

Exit Code	Meaning	Description
0	Success	Operation completed successfully
1	Runtime Error	Internal error, dependency issue, API failure
2	Parameter Error	Invalid CLI arguments, validation failure
3	Data Error	Input file format/content error
130	User Interrupt	SIGINT (Ctrl+C) received

See Error Codes Documentation for details.

CLI Overview

pyitol
├── --version             Display version, Git hash and dependency versions
├── --verbose             Enable verbose logging
├── --quiet               Show only critical info
├── --log-file            Log file path
├── --low-memory          Low memory mode
├── self-test             Run self-test
├── validate              Validate input files
├── config                Configuration file management
├── template              Create and manage iTOL template files
│   ├── create-*          31 template creation subcommands
│   ├── create-collapse   Collapse branches (with --taxon monophyly check)
│   ├── bundle            Batch-generate multiple templates
│   └── validate          Validate template format
├── taxonomy              Taxonomy analysis & monophyly detection
│   ├── ranks             List taxonomy ranks
│   ├── extract           Extract taxonomy info
│   ├── extract-stats     Extract taxonomy statistics
│   ├── extract-from-names  Auto-extract taxonomy from tip names
│   ├── monophyly         Monophyly check (supports LUCA/LACA/LBCA/ROOT)
│   ├── check-and-style   Monophyly check → conditional styling
│   ├── style             Taxonomy style templates
│   ├── convert-binary    Convert to binary matrix
│   └── convert-connect   Convert to connection pairs
├── task                  iTOL API task management
│   ├── upload            Upload to iTOL
│   ├── export            Export from iTOL
│   ├── upload-and-export Upload + export in one step
│   ├── delete            Delete iTOL trees
│   ├── status            Query tree status
│   └── run               Batch task execution
├── tree                  Tree file operations
├── utils                 Utility tools
├── learn                 Reverse-learn from existing templates
└── replay                Replay operation records

Project Structure

PyiTOL/
├── src/pyitol/                 Source code
│   ├── api/                    iTOL API client
│   ├── cli/                    CLI command definitions
│   ├── core/                   Core algorithms (parser, monophyly, taxonomy)
│   ├── templates/              Template generators and schema definitions
│   └── utils/                  Utility functions (color, I/O, session, logging, shutdown)
├── tests/                      Test suite (1702 tests)
│   ├── data/                   Test data files
│   ├── core/                   Core algorithm tests
│   ├── cli/                    CLI command tests
│   ├── templates/              Template system tests
│   ├── utils/                  Utility function tests
│   └── api/                    API client tests
├── examples/
│   └── data/                   Demo data files
├── docs/                       Documentation
│   └── error_codes.md          Error codes reference
├── benchmarks/                 Performance benchmarks
└── pyproject.toml              Project configuration

Documentation

• Quick Start Guide
• Template Type Overview
• Taxonomy Analysis Tutorial
• CLI Reference
• API Guide
• Error Codes Reference
• Contributing

Dependency Licenses

Library	Version	License
typer	>=0.15.0	MIT
rich	~=13.0	MIT
pandas	~=2.0	BSD-3-Clause
dendropy	~=4.6	BSD-3-Clause
numpy	>=1.24	BSD-3-Clause
requests	~=2.32	Apache-2.0
PyYAML	~=6.0	MIT
pydantic	~=2.5	MIT

Citation

If you use PyiTOL in your research, please cite:

• PyiTOL: Zeng, Z. (2026). PyiTOL: reproducible Python workflows for iTOL annotation and taxonomic monophyly assessment. bioRxiv. doi:10.64898/2026.08.27.747471
• iTOL: Letunic, I., & Bork, P. (2021). Interactive Tree Of Life (iTOL) v5. Nucleic Acids Research, 49(W1), W293-W296. doi:10.1093/nar/gkab301
• DendroPy: Sukumaran, J., & Holder, M. T. (2010). DendroPy. Bioinformatics, 26(12), 1569-1571. doi:10.1093/bioinformatics/btq228
• DendroPy 5: Moreno, M. A., Holder, M. T., & Sukumaran, J. (2024). DendroPy 5: a mature Python library for phylogenetic computing. Journal of Open Source Software, 9(101), 6943. doi:10.21105/joss.06943
• itol.toolkit: Zhou, T., Xu, K., Zhao, F., Liu, W., Li, L., Hua, Z., & Zhou, X. (2023). itol.toolkit accelerates working with iTOL by an automated generation of annotation files. Bioinformatics, 39(6), btad339. doi:10.1093/bioinformatics/btad339

If you use the benchmark comparison features (ete3/ete4, optional dependencies):

• ETE 3: Huerta-Cepas, J., Serra, F., & Bork, P. (2016). ETE 3: Reconstruction, analysis, and visualization of phylogenomic data. Molecular Biology and Evolution, 33(6), 1635-1638. doi:10.1093/molbev/msw046

See CITATION.cff for machine-readable citation metadata.

Contact & Support

• GitHub Issues: Report bugs or request features
• GitHub Discussions: Community discussions
• Email: zengzichao@sjtu.edu.cn
• Maintainer: Zichao Zeng

Feel free to reach out through the above channels for questions, suggestions, or contributions.

License

This project is licensed under the MIT License. See NOTICE for third-party copyright and license information.