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•  Multiple sequence alignment is used in the study of phylogenetics

Phylogeny refers to an evolutionary line of descent and can be determined by comparing sequences in different species

  • Variations in these sequences (either nucleotide or amino acid sequences) will occur due to mutations
  • The greater the differences between the sequences, the longer the time span since the two species had a common ancestor

Cladograms are tree diagrams where each branch point represents the splitting of two new groups from a common ancestor

  • Each branch point (node) represents a speciation event by which distinct species are formed via divergent evolution
  • In cladograms, the length of each branch is the same – the branches do not infer an evolutionary time scale

Phylograms are similar to cladograms, however branch lengths may differ according to the length of time since speciation

  • Unlike cladograms, phylograms do infer the amount of evolutionary time separating two species

Phylogram construction requires comparison of sequences that have a constant rate of mutation (‘evolutionary clocks’)

  • Mitochondrial DNA is a useful source as it is maternally derived, has a known mutation rate and lacks recombination 

Phylogenetic Analysis – Cladogram versus Phylogram

cladogram vs phylogram

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•  Use of software to construct simple cladograms and phylograms of related organisms using DNA sequences

Phylogeny trees can be constructed from DNA sequences by using two online resources:

  • GenBank – a genetic database that serves as an annotated collection of DNA sequences
  • Clustal Omega – an alignment program that compares multiple sequences of DNA



Constructing Phylogeny Trees

Step 1:  Select a gene or protein common to a range of selected organisms

  • Examples of molecules which are ubiquitously found in many animals include haemoglobin and cytochrome c

Step 2:  Copy the molecular sequence (DNA or amino acid) for each of the selected organisms

  • Use the GenBank database to identify relevant DNA or amino acid sequences
  • Sequences can be collated in a Word document and then saved as a document in plain text format (.txt) 
  • Before each sequence, designate a species name preceded by a forward arrow (e.g. '>Human’ or ‘>Chimpanzee’)

Step 3:  Run a multiple alignment and generate a phylogeny tree (cladogram)

  • Use Clustal Omega align multiple DNA or amino acid sequences for comparison
  • Clustal Omega can generate branched phylograms after a sequence alignment is completed (select ‘Phylogenetic Tree’)

Example:  Upload the following plain text files to compare nucleotide and amino acid sequences from different species:

  • CHRNE  –  Cholinergic receptor epsilon (nicotinic) for various species (nucleotide sequence)
  • HBA  –  Haemoglobin alpha chain from various species (amino acid sequence)