Systematics, Molecular | Research & Encyclopedia Articles

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Molecular phylogeny Summary

Systematics, Molecular

Molecular systematics is the use of molecules to determine classification systems and relationships. For hundreds of years botanists used morphology, or overall appearance, to identify and classify plants. Morphological systematics has been important for the basic understanding of plant evolution and relationships; however, it has limitations. One limitation to morphology in plants is homology. Homology assumes that two similar structures have the same evolutionary origin. In other words, the trait arose in an ancestor and was passed down to its descendants. Homology in plant morphology is frequently very difficult to resolve since plant structures can become modified into other forms (e.g., spines of cacti are modified leaves).

Just as a botanist may compare the shape of a leaf between two different plants, molecular systematists compare molecules. Molecules have an advantage over morphology in two aspects. First, homology is usually much easier to determine in molecules than in morphology. Second, molecules tend to provide many more pieces of information than can be gained from morphology. A scientist studying morphology may compare one hundred traits, but a scientist using molecules will compare several hundred to several thousand traits depending on the technique.

Early molecular systematics began with micromolecules. The earliest of these studies can be traced as far back as the 1880s, but much of the work was conducted between the 1950s and 1970s. Micromolecules are small molecules mostly responsible for colors, scents, and chemical defenses of plants. Chemicals found in different plants are identified and compared across species for similarities. Species sharing compounds are presumed to be more closely related. Later botanists used macromolecules, which are proteins and nucleic acids. Much of the work on proteins was conducted in the 1970s and consisted of determining the order of amino acids in specific proteins (protein sequencing) or determining whether different populations or species of plants had different forms of specific enzymes (isozyme variability). Other protein-based studies utilized principles of serology and created antibodies for protein extracts that were compared to extracts from a different species. The degree to which the antibodies of one plant matched the proteins of a another plant provides an estimate of how closely the two plants are related.

Studies began to use deoxyribonucleic acid (DNA) in the late 1960s and 1970s with DNA-DNA hybridization. This method uses the principle that DNA is a double-stranded molecule and that high temperatures (greaterthan 80°C) can cause all of the DNA to become single-stranded. When cooled, the DNA resumes its double-stranded nature (re-annealling) and the temperature at which it becomes completely double-stranded is an indication of how similar the strands of DNA are. In this method, DNA from two plants is combined and heated. If all of the DNA is from closely related plants, the re-annealling temperature is high. If the DNA is from two distantly related plants it is lower. The re-annealling temperature is an estimate of how similar the plants are. The closer the temperatures are to the re-annealling temperature of a single plant, the more closely the plants are assumed to be related.

A. Restriction site map for five plants labeled A to E. The numbers above the bars represent the distance between restriction sites (in units of one thousand nucleotides), which are the numbers in the circles. For plant A, the nucleotide sequence that is recognized by this restriction enzyme is enlarged and the areas where the enzyme cuts the DNA are shown with black carats. B. A drawing of what a gel would look like after restriction enzymes had cut the DNA for the five plants in A. Each bar represents a fragment and the fragment sizes are shown on the sides. C. A phylogeny for the five plants based on the restriction site variation shown. The sites are marked on the tree, where they are present for all plants farther up the tree.

A. A portion of an aligned sequence showing fifteen consecutive nucleotides for five plants labeled A to E. B. The phylogenetic tree generated from the data in A. Bars along the branches indicate where changes in the DNA sequence serve to unite two or more plants together. For example, since only plants B and D have the nucleotide C at position 6, this serves to put theses two plants in the same group.

During the 1980s botanists made comparisons of DNA between plants using restriction site analysis. Scientists used restriction enzymes that cut DNA into fragments of various lengths. These enzymes cut the DNA at specific combinations of nucleotides every time this combination of sequences is encountered. The fragments are separated by size using gel electrophoresis and visualized by a probe that matches specific regions of the DNA. Comparing fragment sizes, it is possible to determine whether a specific restriction site is present or absent in any given species. The presence of a restriction site in two or more plants implies that the plants with the site have a more recent common ancestor. Restriction site data are capable of producing hundreds of sites depending on the numbers of enzymes that are used. Most botanists use the DNA from the chloroplast since it is smaller in comparison to other regions of the genome and a number of probes are available.

During the late 1980s and the 1990s molecular systematists made a shift to comparing DNA sequences. A specific gene or DNA region is selected and the order of nucleotides of that gene are determined (sequencing). DNA sequencing was made easier by the polymerase chain reaction (PCR), which allows millions of copies of a gene to be made (amplification) from a single copy. Once a gene is amplified, it is relatively easy to sequence. Nucleotide sequences generated from the same gene of different plants can be compared, or aligned. The traits that are compared are the nucleotides that occur at each aligned position in the gene. As with restriction sites, the shared presence of a specific nucleotide at a specific site in two or more plants is assumed to mean that these plants share a more recent common ancestor.

Many botanists utilize a gene called ribulose bis-phosphate carboxylastoxygenase, large subunit, abbreviated rubisco, found in the chloroplast DNA. This gene is functionally important for plants as it encodes the enzyme that allows plants to make CO₂ into complex molecules. Because it is so important, changes in the gene sequence are infrequent, allowing botanists to use these changes to answer questions about relationships and origins of flowering plants. Recently, botanists have expanded the number of genes studied. Many genes are now used depending on the taxonomic level of interest. Genes with lesser, or no, function evolve quickly and are useful to compare species or populations. Genes with functional constraints are more useful to compare genera or families.

It is common for scientists to use several genes for a study. As more genes are added, it strengthens the results by adding more data, and from genes that may evolve differently. One important example of this is the comparisonof nuclear genes to chloroplast genes. Chloroplasts usually are inherited through a single parent (the mother), whereas nuclear genes are inherited from both parents. If a botanist studies only chloroplast genes, it is possible that only the maternal lineage will be resolved. This is especially critical in plant groups that are known to hybridize. To counter this potential error, many botanists look at chloroplast genes in conjunction with nuclear genes. Common nuclear genes that are used are the ribosomal ribonucleic acid (RNA) genes.