Build maximum likelihood phylogenetic trees using IQ-TREE2 and RAxML-ng. Use when inferring publication-quality trees with model selection, ultrafast bootstrap, or partitioned analyses from sequence alignments.
Reference examples tested with: IQ-TREE 2.2+
Before using code patterns, verify installed versions match. If versions differ:
<tool> --version then <tool> --help to confirm flagsIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
"Infer a maximum likelihood tree from my alignment" → Build publication-quality ML trees with automatic substitution model selection and ultrafast bootstrap support assessment.
iqtree2 -s alignment.fa -m MFP -B 1000 (IQ-TREE2)raxml-ng --all --msa alignment.fa --model GTR+G --bs-trees 100 (RAxML-ng)Build maximum likelihood phylogenetic trees with automatic model selection and ultrafast bootstrap.
# Simple ML tree with automatic model selection
iqtree2 -s alignment.fasta -m MFP -B 1000 -T AUTO
# -s: input alignment
# -m MFP: ModelFinder Plus (automatic model selection + tree inference)
# -B 1000: 1000 ultrafast bootstrap replicates (minimum recommended for publication)
# -T AUTO: automatic thread detection
| File | Description |
|---|---|
.treefile | Best ML tree in Newick format |
.iqtree | Full analysis report with model parameters |
.log | Run log |
.contree | Consensus tree with bootstrap support |
.splits.nex | Bootstrap splits in Nexus format |
.model.gz | Model parameters |
.bionj | Initial BIONJ tree |
.mldist | ML distance matrix |
.ckp.gz | Checkpoint file for resuming |
# ModelFinder only (no tree inference)
iqtree2 -s alignment.fasta -m MF
# Use specific model
iqtree2 -s alignment.fasta -m GTR+G4 -B 1000
# Test only specific models
iqtree2 -s alignment.fasta -m MF -mset GTR,HKY,K2P
# Protein models
iqtree2 -s protein.fasta -m MFP -B 1000 -st AA
| Model | Parameters | Use Case |
|---|---|---|
| JC | Equal rates | Very simple, rarely appropriate |
| K2P/K80 | Ti/Tv ratio | Simple, some rate variation |
| HKY | Ti/Tv + base freq | Moderate complexity |
| GTR | 6 rates + base freq | Most general, recommended default |
| +G4 | Gamma rate variation | 4 discrete rate categories |
| +I | Invariant sites | Sites that never change |
| +R4 | FreeRate model | More flexible than Gamma |
# Standard ultrafast bootstrap (UFBoot2)
# B>=1000: Minimum for publication. Use 10000 for final analyses.
iqtree2 -s alignment.fasta -m GTR+G4 -B 1000
# Standard bootstrap (slower but more accurate for small datasets)
iqtree2 -s alignment.fasta -m GTR+G4 -b 100
# SH-aLRT test (fast approximate likelihood ratio test)
iqtree2 -s alignment.fasta -m GTR+G4 -alrt 1000
# Both UFBoot and SH-aLRT
iqtree2 -s alignment.fasta -m GTR+G4 -B 1000 -alrt 1000
| UFBoot | SH-aLRT | Interpretation |
|---|---|---|
| >= 95 | >= 80 | Strong support |
| 80-94 | 70-79 | Moderate support |
| < 80 | < 70 | Weak support |
For multi-gene datasets with different evolutionary rates:
# Create partition file (partitions.nex)
cat > partitions.nex << 'EOF'
#nexus
begin sets;
charset gene1 = 1-500;
charset gene2 = 501-1200;
charset gene3 = 1201-1800;
charpartition mine = HKY:gene1, GTR:gene2, GTR+G:gene3;
end;
EOF
# Run partitioned analysis
iqtree2 -s concat.fasta -p partitions.nex -m MFP -B 1000
# Edge-linked partition model (proportional branch lengths)
iqtree2 -s concat.fasta -q partitions.nex -m MFP -B 1000
# Edge-unlinked (independent branch lengths per partition)
iqtree2 -s concat.fasta -Q partitions.nex -m MFP -B 1000
# Simple ML tree with GTR+G
raxml-ng --all --msa alignment.fasta --model GTR+G --bs-trees 100
# --all: ML search + bootstrapping
# --msa: input alignment
# --model: substitution model
# --bs-trees: number of bootstrap replicates
# DNA models
raxml-ng --msa alignment.fasta --model GTR+G4+I
# Protein models (automatic detection)
raxml-ng --msa protein.fasta --model LG+G8+F
# Check alignment and determine model
raxml-ng --check --msa alignment.fasta --model GTR+G
| File | Description |
|---|---|
.raxml.bestTree | Best ML tree |
.raxml.support | Tree with bootstrap support values |
.raxml.bootstraps | All bootstrap trees |
.raxml.mlTrees | All ML trees from search |
.raxml.log | Analysis log |
.raxml.rba | Binary alignment (for restart) |
# Multiple ML searches (find global optimum)
# --tree pars{10}: 10 starting parsimony trees recommended for thorough search
raxml-ng --msa alignment.fasta --model GTR+G --tree pars{10} --prefix ml_search
# Constrained tree search
raxml-ng --msa alignment.fasta --model GTR+G --tree-constraint constraint.tre
# Site likelihoods for topology tests
raxml-ng --sitelh --msa alignment.fasta --model GTR+G --tree candidate.tre
| Feature | IQ-TREE2 | RAxML-ng |
|---|---|---|
| Model selection | Built-in ModelFinder | External (ModelTest-NG) |
| Ultrafast bootstrap | Yes (UFBoot2) | No |
| Standard bootstrap | Yes | Yes |
| Partition models | Extensive | Good |
| Speed | Faster for UFBoot | Faster for standard BS |
| Memory | Lower | Higher |
| Checkpointing | Yes | Yes |
# IQ-TREE2 with reduced memory
iqtree2 -s large.fasta -m GTR+G -B 1000 -T 4 -mem 8G
# Use approximate NNI search
iqtree2 -s large.fasta -m GTR+G -B 1000 -fast
# RAxML-ng with parsimony starting trees
raxml-ng --msa large.fasta --model GTR+G --tree pars{5} --threads 8
# IQ-TREE2: AU test comparing trees
iqtree2 -s alignment.fasta -m GTR+G -z trees.nwk -n 0 -zb 10000 -au
# Output interpretation:
# p-AU < 0.05: Reject tree
# p-AU >= 0.05: Cannot reject tree
# IQ-TREE2: Enforce monophyly constraint
iqtree2 -s alignment.fasta -m GTR+G -g constraint.tre -B 1000
# Constraint file format (Newick with taxa to constrain):
# ((Human,Chimp),Gorilla);
# 1. Check alignment
iqtree2 -s alignment.fasta -m GTR+G -n 0
# 2. Find best model
iqtree2 -s alignment.fasta -m MF -T AUTO
# 3. Full analysis with best model
iqtree2 -s alignment.fasta -m GTR+I+G4 -B 1000 -alrt 1000 -T AUTO
# 4. Visualize result
cat alignment.fasta.treefile
# IQ-TREE2: Resume from checkpoint
iqtree2 -s alignment.fasta -m GTR+G -B 1000 --redo-tree
# RAxML-ng: Resume
raxml-ng --msa alignment.fasta --model GTR+G --redo
# Set random seed for reproducible results
# seed=12345: Any fixed seed ensures reproducibility across runs
iqtree2 -s alignment.fasta -m GTR+G -B 1000 --seed 12345
raxml-ng --msa alignment.fasta --model GTR+G --seed 12345 --bs-trees 100