Using Molecular Evidence in Classification (OCR A-Level Biology A): Revision Notes

Using Molecular Evidence in Classification

Development of molecular phylogeny

Until the mid-twentieth century, taxonomists classified organisms using data from anatomy, morphology, behaviour, physiology and cell structure. The development of new technologies enabled scientists to study macromolecules, particularly the sequences of amino acids in proteins and nucleotides in DNA. This led to the emergence of molecular phylogeny – a branch of biology that uses molecular data to determine evolutionary relationships between organisms.

Using antibodies to compare species

Early molecular phylogeny research (beginning in 1904) used antibodies to detect similarities between proteins in different species. This method exploits the specificity of antibody-antigen binding.

Methodology

The technique involves several steps:

1. Blood is taken from a species (e.g., humans) and processed to obtain serum by removing cells and preventing clotting
2. This serum is injected into a test animal such as a rabbit or mouse
3. After approximately one week, the test animal produces an immune response – B cells specific to the foreign proteins divide to form plasma cells
4. These plasma cells secrete antibodies against the injected proteins
5. Blood is taken from the test animal and processed to create an anti-serum containing these antibodies
6. The anti-serum is then mixed with serum from various species

Interpretation of results

When the anti-serum is mixed with serum from different species, the degree of precipitation indicates the similarity between proteins:

• Higher precipitation = greater similarity between proteins
• Lower precipitation = fewer shared proteins or more divergent protein structures

Protein sequencing

An alternative approach involves directly comparing the amino acid sequences of specific proteins. Cytochrome c has been particularly useful for this purpose because it:

• Occurs in many organisms where it plays a key role in respiration
• Is located in the inner membranes of mitochondria in eukaryotes
• Consists of a single polypeptide of approximately $100$ amino acids
• Has a conserved structure across diverse species

Reading amino acid sequences

To interpret protein sequences, you need to understand both the three-letter and one-letter codes for amino acids. These codes are standardized across biology. The table below also shows the DNA codons (on the coding strand) that specify each amino acid.

Constructing phylogenetic trees from protein data

Researchers align amino acid sequences from multiple species and compare them position by position, identifying similarities and differences. The number of differences indicates evolutionary distance – species with fewer differences shared a more recent common ancestor.

This phylogenetic tree based on cytochrome c sequences clearly reveals the three main kingdoms of eukaryotes: fungi, animals and plants. Such molecular trees generally agree closely with classification systems based on morphology and anatomy, providing independent evidence for common descent and evolutionary relationships.

DNA hybridisation

DNA molecules consist of two complementary strands held together by hydrogen bonds between base pairs. DNA hybridisation exploits this structure to compare genetic similarity between species.

Principle

• Heating DNA to approximately $90°C$ breaks the hydrogen bonds between bases, separating the two strands
• When single-stranded DNA from two different species is mixed and allowed to cool, the strands attempt to bond together
• However, genetic differences between species mean the match is imperfect
• More imperfect matches have weaker hydrogen bonding and require less heat to separate the strands
• Closer matches require more heat to break the bonds

Applications

DNA sequencing

DNA sequencing has become the most powerful method for determining evolutionary relationships. It provides more detailed and comprehensive data than either antibody comparisons or protein sequencing.

Advantages over protein sequencing

DNA sequencing offers several key advantages:

Detection of mutations: DNA sequencing can identify:

• Silent mutations: changes to base pairs that produce the same amino acid due to code degeneracy
• Neutral mutations: changes that alter the amino acid but not the functional properties (e.g., substituting one non-polar amino acid for another)
• Functional changes that have become fixed in populations

Greater accessibility: Modern sequencing technology makes it easier to sequence DNA than proteins, and databases contain extensive sequence information for comparison.

Stability: DNA is more stable than protein, allowing sequencing of genetic material from extinct organisms such as:

• Mammoths (extinct approximately $4500$ years ago)
• Neanderthals (remains dated to $38000$ years ago)

Choice of genes for sequencing

Different genes evolve at different rates, making them suitable for answering different evolutionary questions:

Mitochondrial DNA:

• Does not contain non-coding sequences within genes, making alignment easier
• Not combined with histone proteins ('naked' DNA)
• Present in multiple copies per cell
• Has a higher mutation rate, useful for studying closely related organisms
• Inherited maternally with relatively little change
• Extensively sequenced for many species

Housekeeping genes:

• Code for proteins required for fundamental cellular processes common to all organisms
• Provide useful comparative data across diverse species

Non-coding DNA sequences

In eukaryotes, much nuclear DNA does not code for proteins. These non-coding sequences have various functions or may have no known function. They are relatively free to accumulate mutations without affecting protein structure, and therefore provide valuable information about evolutionary relationships and genetic drift.

Case study: marsupials and placentals

Researchers compared rRNA gene sequences from mitochondria in eight mammalian species – four marsupials (including the extinct thylacine) and four placentals. The table shows nucleotide positions 29-41 in aligned sequences:

Gene barcoding

DNA sequencing enables species identification through gene barcoding – using specific gene sequences as unique identifiers for species. Researchers:

1. Sequence particular genes from identified specimens
2. Publish these sequences as 'barcodes'
3. Deposit barcodes in databases for public access
4. Use barcodes to identify unknown specimens

Databases for molecular phylogeny

Molecular sequence data is stored in publicly accessible online databases, enabling researchers worldwide to share and compare information:

The Tree of Life (http://tolweb.org)

• Provides information on biodiversity, characteristics of organism groups and evolutionary history

NCBI Taxonomy Database (www.ncbi.nlm.nih.gov/taxonomy)

• Contains information on approximately $10\%$ of described species
• Linked to DNA and protein sequence databases
• Part of the National Center for Biotechnology Information

Animal Diversity Web (http://animaldiversity.ummz.umich.edu)

• Curated by University of Michigan
• Contains data on animal natural history, distribution, classification and conservation

The National Herbarium of The Netherlands (http://vstbol.leidenuniv.nl)

• Largest single plant database
• Records of over $2$ million specimens

Encyclopedia of Life (EoL) (http://eol.org)

• Aims to provide a web page for every species
• Aggregates information from museums, scientific societies and experts
• Single, easy-to-use online portal

Classification systems and molecular evidence

Molecular evidence has transformed how biologists classify organisms:

Confirms traditional classifications: Molecular phylogenetic trees often agree with classification systems based on morphology and anatomy, providing independent verification of evolutionary relationships.

Reveals hidden relationships: Molecular data can show connections not apparent from structure alone, such as the closer relationship between fungi and animals than between fungi and plants.

Enables comparison of similar species: DNA sequencing can distinguish between closely related species that lack obvious structural differences.

Case study: Australasian cicadas

Researchers investigated whether cicadas migrated to New Zealand from Australia by sequencing mitochondrial DNA from $14$ species across Australia, New Zealand and New Caledonia.

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