Experiment 03PhylogeneticsTree Inference
Molecular Phylogenetics & Tree Reconstruction: Neighbor-Joining, Maximum Likelihood & Bootstrap Resampling
Duration: 4 Hours • Computational Evolution Lab
🎯 Experimental Aim & Learning Objectives
To construct distance-based (Neighbor-Joining) and character-based (Maximum Likelihood) phylogenetic trees from orthologous molecular sequences; select optimal nucleotide/amino acid substitution models using Bayesian Information Criterion (BIC); apply outgroup rooting; and test branch reliability using Felsenstein non-parametric bootstrap resampling ($N = 1000$ replicates).
🔬 Theoretical Foundation & Algorithmic Principles
Molecular phylogenetics reconstructs the evolutionary genealogical histories of species and gene families:
- Evolutionary Substitution Models: Observed pairwise distance must be corrected for multiple substitutions at the same site (saturation). The Kimura 2-Parameter (K2P) model distinguishes transitions ($A leftrightarrow G, C leftrightarrow T$) from transversions ($A/G leftrightarrow C/T$). For protein sequences, the JTT, WAG, or LG substitution models with gamma-distributed rate variation across sites ($+Gamma$) are standard.
- Saitou-Nei Neighbor-Joining (NJ) Algorithm: A bottom-up star-decomposition clustering method. It calculates the $Q$-matrix from pairwise distances:
$$Q(i, j) = (n – 2) cdot d(i, j) – sum_{k=1}^n d(i, k) – sum_{k=1}^n d(j, k)$$
The pair $(i, j)$ with the minimum $Q$-value is merged into a new composite node, and branch lengths from the node are computed. NJ produces an unrooted additive tree where branch lengths correspond to evolutionary change. - Felsenstein Bootstrap Resampling: Tests clade statistical robustness. The alignment columns (sites) are resampled with replacement to create 1000 pseudo-alignments. A tree is built for each. The percentage of trees supporting each bipartition is the bootstrap proportion:
$$text{Support} ge 70% implies text{Moderately supported}; quad ge 95% implies text{Statistically robust}.$$
Figure 1.3: Evolutionary distance matrix reduction via the Saitou-Nei Neighbor-Joining (NJ) clustering algorithm, yielding a resolved phylogram with Felsenstein bootstrap support percentages.
🧪 Computational Tools & Data Sets
- Software: MEGA 11 (Molecular Evolutionary Genetics Analysis), PhyML 3.3, IQ-TREE 2 (ultrafast bootstrap), and FigTree/iTOL tree visualizers.
- Sequence Dataset: Primate mitochondrial Cytochrome b (Cytb) gene sequences: Human (Homo sapiens), Chimpanzee (Pan troglodytes), Gorilla (Gorilla gorilla), Orangutan (Pongo abelii), and Gibbon (Hylobates lar, designated outgroup).
📋 Step-by-Step Computational Protocol
- Sequence Import & Model Selection: Open MEGA 11. Load aligned Cytb FASTA. Select
Models → Find Best DNA/Protein Models (ML). Identify the model with lowest BIC (e.g., Kimura 2-Parameter + Gamma, $K2P + G$). - Distance Matrix Computation: Compute Pairwise Distance matrix under the K2P model with Gamma parameter $alpha = 0.5$. Note shortest evolutionary distance between Human and Chimpanzee ($d approx 0.016$).
- Neighbor-Joining Tree Construction: Click
Phylogeny → Construct/Test Neighbor-Joining Tree. Set Test of Phylogeny:Bootstrap method, No. of Replications =1000. Model:K2P + G. - Outgroup Rooting & Display: In the Tree Explorer, select the Gibbon branch and click
Re-root Tree. Toggle between Cladogram and Phylogram (where branch lengths represent substitutions per site). - Maximum Likelihood Validation: Construct a comparative Maximum Likelihood tree using IQ-TREE with 1000 ultrafast bootstrap replicates:
iqtree2 -s primate_cytb.fasta -m MFP -B 1000 -T AUTO
📊 Representative Phylogenetic Topology & Clade Support
Newick Tree Format:((((Homo_sapiens:0.015, Pan_troglodytes:0.018)98:0.012, Gorilla_gorilla:0.022)100:0.035, Pongo_abelii:0.058)100:0.024, Hylobates_lar:0.082);
Clade Confidence Assessment:
• Hominini Node (Human + Chimpanzee sister relationship): 98% Bootstrap Support. Confirms closest evolutionary lineage divergence.
• Homininae Node ((Human + Chimpanzee) + Gorilla): 100% Bootstrap Support.
• Root divergence to Gibbon outgroup: Total branch length = $0.082$ substitutions per site.
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