Phylogenetics

Phylogenetics

Overview

Phylogenetic analysis reconstructs the evolutionary history of biological sequences (genes, proteins, genomes) by inferring the branching pattern of descent. This skill covers the standard pipeline:

1. MAFFT — Multiple sequence alignment 2. IQ-TREE 2 — Maximum likelihood tree inference with model selection 3. FastTree — Fast approximate maximum likelihood (for large datasets) 4. ETE3 — Python library for tree manipulation and visualization

Installation:

# Conda (recommended for CLI tools)
conda install -c bioconda mafft iqtree fasttree
pip install ete3

When to Use This Skill

Use phylogenetics when:

• Evolutionary relationships: Which organism/gene is most closely related to my sequence?
• Viral phylodynamics: Trace outbreak spread and estimate transmission dates
• Protein family analysis: Infer evolutionary relationships within a gene family
• Horizontal gene transfer detection: Identify genes with discordant species/gene trees
• Ancestral sequence reconstruction: Infer ancestral protein sequences
• Molecular clock analysis: Estimate divergence dates using temporal sampling
• GWAS companion: Place variants in evolutionary context (e.g., SARS-CoV-2 variants)
• Microbiology: Species phylogeny from 16S rRNA or core genome phylogeny

Standard Workflow

1. Multiple Sequence Alignment with MAFFT

import subprocess
import os

def run_mafft(input_fasta: str, output_fasta: str, method: str = "auto",
               n_threads: int = 4) -> str:
    """
    Align sequences with MAFFT.

    Args:
        input_fasta: Path to unaligned FASTA file
        output_fasta: Path for aligned output
        method: 'auto' (auto-select), 'einsi' (accurate), 'linsi' (accurate, slow),
                'fftnsi' (medium), 'fftns' (fast), 'retree2' (fast)
        n_threads: Number of CPU threads

    Returns:
        Path to aligned FASTA file
    """
    methods = {
        "auto": ["mafft", "--auto"],
        "einsi": ["mafft", "--genafpair", "--maxiterate", "1000"],
        "linsi": ["mafft", "--localpair", "--maxiterate", "1000"],
        "fftnsi": ["mafft", "--fftnsi"],
        "fftns": ["mafft", "--fftns"],
        "retree2": ["mafft", "--retree", "2"],
    }

    cmd = methods.get(method, methods["auto"])
    cmd += ["--thread", str(n_threads), "--inputorder", input_fasta]

    with open(output_fasta, 'w') as out:
        result = subprocess.run(cmd, stdout=out, stderr=subprocess.PIPE, text=True)

    if result.returncode != 0:
        raise RuntimeError(f"MAFFT failed:\n{result.stderr}")

    # Count aligned sequences
    with open(output_fasta) as f:
        n_seqs = sum(1 for line in f if line.startswith('>'))
    print(f"MAFFT: aligned {n_seqs} sequences → {output_fasta}")

    return output_fasta

# MAFFT method selection guide:
# Few sequences (<200), accurate: linsi or einsi
# Many sequences (<1000), moderate: fftnsi
# Large datasets (>1000): fftns or auto
# Ultra-fast (>10000): mafft --retree 1

2. Trim Alignment (Optional but Recommended)

def trim_alignment_trimal(aligned_fasta: str, output_fasta: str,
                            method: str = "automated1") -> str:
    """
    Trim poorly aligned columns with TrimAl.

    Methods:
    - 'automated1': Automatic heuristic (recommended)
    - 'gappyout': Remove gappy columns
    - 'strict': Strict gap threshold
    """
    cmd = ["trimal", f"-{method}", "-in", aligned_fasta, "-out", output_fasta, "-fasta"]
    result = subprocess.run(cmd, capture_output=True, text=True)
    if result.returncode != 0:
        print(f"TrimAl warning: {result.stderr}")
        # Fall back to using the untrimmed alignment
        import shutil
        shutil.copy(aligned_fasta, output_fasta)
    return output_fasta

3. IQ-TREE 2 — Maximum Likelihood Tree

def run_iqtree(aligned_fasta: str, output_prefix: str,
                model: str = "TEST", bootstrap: int = 1000,
                n_threads: int = 4, extra_args: list = None) -> dict:
    """
    Build a maximum likelihood tree with IQ-TREE 2.

    Args:
        aligned_fasta: Aligned FASTA file
        output_prefix: Prefix for output files
        model: 'TEST' for automatic model selection, or specify (e.g., 'GTR+G' for DNA,
               'LG+G4' for proteins, 'JTT+G' for proteins)
        bootstrap: Number of ultrafast bootstrap replicates (1000 recommended)
        n_threads: Number of threads ('AUTO' to auto-detect)
        extra_args: Additional IQ-TREE arguments

    Returns:
        Dict with paths to output files
    """
    cmd = [
        "iqtree2",
        "-s", aligned_fasta,
        "--prefix", output_prefix,
        "-m", model,
        "-B", str(bootstrap),   # Ultrafast bootstrap
        "-T", str(n_threads),
        "--redo"                # Overwrite existing results
    ]

    if extra_args:
        cmd.extend(extra_args)

    result = subprocess.run(cmd, capture_output=True, text=True)

    if result.returncode != 0:
        raise RuntimeError(f"IQ-TREE failed:\n{result.stderr}")

    # Print model selection result
    log_file = f"{output_prefix}.log"
    if os.path.exists(log_file):
        with open(log_file) as f:
            for line in f:
                if "Best-fit model" in line:
                    print(f"IQ-TREE: {line.strip()}")

    output_files = {
        "tree": f"{output_prefix}.treefile",
        "log": f"{output_prefix}.log",
        "iqtree": f"{output_prefix}.iqtree",  # Full report
        "model": f"{output_prefix}.model.gz",
    }

    print(f"IQ-TREE: Tree saved to {output_files['tree']}")
    return output_files

# IQ-TREE model selection guide:
# DNA:     TEST → GTR+G, HKY+G, TrN+G
# Protein: TEST → LG+G4, WAG+G, JTT+G, Q.pfam+G
# Codon:   TEST → MG+F3X4

# For temporal (molecular clock) analysis, add:
# extra_args = ["--date", "dates.txt", "--clock-test", "--date-CI", "95"]

4. FastTree — Fast Approximate ML

For large datasets (>1000 sequences) where IQ-TREE is too slow:

def run_fasttree(aligned_fasta: str, output_tree: str,
                  sequence_type: str = "nt", model: str = "gtr",
                  n_threads: int = 4) -> str:
    """
    Build a fast approximate ML tree with FastTree.

    Args:
        sequence_type: 'nt' for nucleotide or 'aa' for amino acid
        model: For nt: 'gtr' (recommended) or 'jc'; for aa: 'lg', 'wag', 'jtt'
    """
    if sequence_type == "nt":
        cmd = ["FastTree", "-nt", "-gtr"]
    else:
        cmd = ["FastTree", f"-{model}"]

    cmd += [aligned_fasta]

    with open(output_tree, 'w') as out:
        result = subprocess.run(cmd, stdout=out, stderr=subprocess.PIPE, text=True)

    if result.returncode != 0:
        raise RuntimeError(f"FastTree failed:\n{result.stderr}")

    print(f"FastTree: Tree saved to {output_tree}")
    return output_tree

5. Tree Analysis and Visualization with ETE3

from ete3 import Tree, TreeStyle, NodeStyle, TextFace, PhyloTree
import matplotlib.pyplot as plt

def load_tree(tree_file: str) -> Tree:
    """Load a Newick tree file."""
    t = Tree(tree_file)
    print(f"Tree: {len(t)} leaves, {len(list(t.traverse()))} nodes")
    return t

def basic_tree_stats(t: Tree) -> dict:
    """Compute basic tree statistics."""
    leaves = t.get_leaves()
    distances = [t.get_distance(l1, l2) for l1 in leaves[:min(50, len(leaves))]
                 for l2 in leaves[:min(50, len(leaves))] if l1 != l2]

    stats = {
        "n_leaves": len(leaves),
        "n_internal_nodes": len(t) - len(leaves),
        "total_branch_length": sum(n.dist for n in t.traverse()),
        "max_leaf_distance": max(distances) if distances else 0,
        "mean_leaf_distance": sum(distances)/len(distances) if distances else 0,
    }
    return stats

def find_mrca(t: Tree, leaf_names: list) -> Tree:
    """Find the most recent common ancestor of a set of leaves."""
    return t.get_common_ancestor(*leaf_names)

def visualize_tree(t: Tree, output_file: str = "tree.png",
                    show_branch_support: bool = True,
                    color_groups: dict = None,
                    width: int = 800) -> None:
    """
    Render phylogenetic tree to image.

    Args:
        t: ETE3 Tree object
        color_groups: Dict mapping leaf_name → color (for coloring taxa)
        show_branch_support: Show bootstrap values
    """
    ts = TreeStyle()
    ts.show_leaf_name = True
    ts.show_branch_support = show_branch_support
    ts.mode = "r"  # 'r' = rectangular, 'c' = circular

    if color_groups:
        for node in t.traverse():
            if node.is_leaf() and node.name in color_groups:
                nstyle = NodeStyle()
                nstyle["fgcolor"] = color_groups[node.name]
                nstyle["size"] = 8
                node.set_style(nstyle)

    t.render(output_file, tree_style=ts, w=width, units="px")
    print(f"Tree saved to: {output_file}")

def midpoint_root(t: Tree) -> Tree:
    """Root tree at midpoint (use when outgroup unknown)."""
    t.set_outgroup(t.get_midpoint_outgroup())
    return t

def prune_tree(t: Tree, keep_leaves: list) -> Tree:
    """Prune tree to keep only specified leaves."""
    t.prune(keep_leaves, preserve_branch_length=True)
    return t

6. Complete Analysis Script

import subprocess, os
from ete3 import Tree

def full_phylogenetic_analysis(
    input_fasta: str,
    output_dir: str = "phylo_results",
    sequence_type: str = "nt",
    n_threads: int = 4,
    bootstrap: int = 1000,
    use_fasttree: bool = False
) -> dict:
    """
    Complete phylogenetic pipeline: align → trim → tree → visualize.

    Args:
        input_fasta: Unaligned FASTA
        sequence_type: 'nt' (nucleotide) or 'aa' (amino acid/protein)
        use_fasttree: Use FastTree instead of IQ-TREE (faster for large datasets)
    """
    os.makedirs(output_dir, exist_ok=True)
    prefix = os.path.join(output_dir, "phylo")

    print("=" * 50)
    print("Step 1: Multiple Sequence Alignment (MAFFT)")
    aligned = run_mafft(input_fasta, f"{prefix}_aligned.fasta",
                         method="auto", n_threads=n_threads)

    print("\nStep 2: Tree Inference")
    if use_fasttree:
        tree_file = run_fasttree(
            aligned, f"{prefix}.tree",
            sequence_type=sequence_type,
            model="gtr" if sequence_type == "nt" else "lg"
        )
    else:
        model = "TEST" if sequence_type == "nt" else "TEST"
        iqtree_files = run_iqtree(
            aligned, prefix,
            model=model,
            bootstrap=bootstrap,
            n_threads=n_threads
        )
        tree_file = iqtree_files["tree"]

    print("\nStep 3: Tree Analysis")
    t = Tree(tree_file)
    t = midpoint_root(t)

    stats = basic_tree_stats(t)
    print(f"Tree statistics: {stats}")

    print("\nStep 4: Visualization")
    visualize_tree(t, f"{prefix}_tree.png", show_branch_support=True)

    # Save rooted tree
    rooted_tree_file = f"{prefix}_rooted.nwk"
    t.write(format=1, outfile=rooted_tree_file)

    results = {
        "aligned_fasta": aligned,
        "tree_file": tree_file,
        "rooted_tree": rooted_tree_file,
        "visualization": f"{prefix}_tree.png",
        "stats": stats
    }

    print("\n" + "=" * 50)
    print("Phylogenetic analysis complete!")
    print(f"Results in: {output_dir}/")
    return results

IQ-TREE Model Guide

DNA Models

Protein Models

Tip: Use -m TEST to let IQ-TREE automatically select the best model.

Best Practices

• Alignment quality first: Poor alignment → unreliable trees; check alignment manually
• Use `linsi` for small (<200 seq), `fftns` or `auto` for large alignments
• Model selection: Always use -m TEST for IQ-TREE unless you have a specific reason
• Bootstrap: Use ≥1000 ultrafast bootstraps (-B 1000) for branch support
• Root the tree: Unrooted trees can be misleading; use outgroup or midpoint rooting
• FastTree for >5000 sequences: IQ-TREE becomes slow; FastTree is 10–100× faster
• Trim long alignments: TrimAl removes unreliable columns; improves tree accuracy
• Check for recombination in viral/bacterial sequences before building trees (RDP4, GARD)

Additional Resources

• MAFFT: https://mafft.cbrc.jp/alignment/software/
• IQ-TREE 2: http://www.iqtree.org/ | Tutorial: https://www.iqtree.org/workshop/molevol2022
• FastTree: http://www.microbesonline.org/fasttree/
• ETE3: http://etetoolkit.org/
• FigTree (GUI visualization): https://tree.bio.ed.ac.uk/software/figtree/
• iTOL (web visualization): https://itol.embl.de/
• MUSCLE (alternative aligner): https://www.drive5.com/muscle/
• TrimAl (alignment trimming): https://vicfero.github.io/trimal/

IQ-TREE 2 Phylogenetic Inference Reference

Basic Command Syntax

iqtree2 -s alignment.fasta --prefix output -m TEST -B 1000 -T AUTO --redo

Key Parameters

Model Selection

# Full model testing (automatically selects best model)
iqtree2 -s alignment.fasta -m TEST --prefix test_run -B 1000 -T 4

# Specify model explicitly
iqtree2 -s alignment.fasta -m GTR+G4 --prefix gtr_run -B 1000

# Protein sequences
iqtree2 -s protein.fasta -m TEST --prefix prot_tree -B 1000

# Codon-based analysis
iqtree2 -s codon.fasta -m GY --prefix codon_tree -B 1000

Bootstrapping Methods

Ultrafast Bootstrap (UFBoot, recommended)

iqtree2 -s alignment.fasta -B 1000  # 1000 replicates
# Values ≥95 are reliable
# ~10× faster than standard bootstrap

Standard Bootstrap

iqtree2 -s alignment.fasta -b 100  # 100 replicates (very slow)

SH-aLRT Test (fast alternative)

iqtree2 -s alignment.fasta -alrt 1000 -B 1000  # Both SH-aLRT and UFBoot
# SH-aLRT ≥80 AND UFBoot ≥95 = well-supported branch

Branch Support Interpretation

Output Files

Advanced Analyses

Molecular Clock (Dating)

# Temporal analysis with sampling dates
iqtree2 -s alignment.fasta -m GTR+G \
        --date dates.tsv \           # Tab-separated: taxon_name  YYYY-MM-DD
        --clock-test \               # Test for clock-like evolution
        --date-CI 95 \              # 95% CI for node dates
        --prefix dated_tree

Concordance Factors

# Gene concordance factor (gCF) - requires multiple gene alignments
iqtree2 --gcf gene_trees.nwk \
        --tree main_tree.treefile \
        --cf-verbose \
        --prefix cf_analysis

Ancestral Sequence Reconstruction

iqtree2 -s alignment.fasta -m LG+G4 \
        -asr \                      # Marginal ancestral state reconstruction
        --prefix anc_tree
# Output: {prefix}.state (ancestral sequences per node)

Partition Model (Multi-Gene)

# Create partition file (partitions.txt):
# DNA, gene1 = 1-500
# DNA, gene2 = 501-1000

iqtree2 -s concat_alignment.fasta \
        -p partitions.txt \
        -m TEST \
        -B 1000 \
        --prefix partition_tree

IQ-TREE Log Parsing

def parse_iqtree_log(log_file: str) -> dict:
    """Extract key results from IQ-TREE log file."""
    results = {}
    with open(log_file) as f:
        for line in f:
            if "Best-fit model" in line:
                results["best_model"] = line.split(":")[1].strip()
            elif "Log-likelihood of the tree:" in line:
                results["log_likelihood"] = float(line.split(":")[1].strip())
            elif "Number of free parameters" in line:
                results["free_params"] = int(line.split(":")[1].strip())
            elif "Akaike information criterion" in line:
                results["AIC"] = float(line.split(":")[1].strip())
            elif "Bayesian information criterion" in line:
                results["BIC"] = float(line.split(":")[1].strip())
            elif "Total CPU time used" in line:
                results["cpu_time"] = line.split(":")[1].strip()
    return results

# Example:
# results = parse_iqtree_log("output.log")
# print(f"Best model: {results['best_model']}")
# print(f"Log-likelihood: {results['log_likelihood']:.2f}")

Common Issues and Solutions

MAFFT Alignment Guide

# Accurate (< 200 sequences)
mafft --localpair --maxiterate 1000 input.fasta > aligned.fasta

# Medium (200-1000 sequences)
mafft --auto input.fasta > aligned.fasta

# Fast (> 1000 sequences)
mafft --fftns input.fasta > aligned.fasta

# Very large (> 10000 sequences)
mafft --retree 1 input.fasta > aligned.fasta

# Using multiple threads
mafft --thread 8 --auto input.fasta > aligned.fasta