Molecular phylogenetics

Molecular phylogenetics (pronounced /məˈlɛkjʊlər faɪlɵdʒɪˈnɛtɪks/) is the analysis of hereditary molecular differences, mainly in DNA sequences, to gain information on an organism's evolutionary relationships. The result of a molecular phylogenetic analysis is expressed in a phylogenetic tree. Molecular phylogenetics is one aspect of molecular systematics, a broader term that also includes the use of molecular data in taxonomy and biogeography.

History of molecular phylogenetics

Further information: History of molecular evolution

The theoretical frameworks for molecular systematics were laid in the 1960s in the works of Emile Zuckerkandl, Emanuel Margoliash, Linus Pauling and Walter M. Fitch. Applications of molecular systematics were pioneered by Charles G. Sibley (birds), Herbert C. Dessauer (herpetology), and Morris Goodman (primates), followed by Allan C. Wilson, Robert K. Selander, and John C. Avise (who studied various groups). Work with protein electrophoresis began around 1956. Although the results were not quantitative and did not initially improve on morphological classification, they provided tantalizing hints that long-held notions of the classifications of birds, for example, needed substantial revision. In the period of 1974–1986, DNA-DNA hybridization was the dominant technique.

Techniques and applications

Every living organism contains DNA, RNA, and proteins. Closely related organisms generally have a high degree of agreement in the molecular structure of these substances, while the molecules of organisms distantly related usually show a pattern of dissimilarity. Conserved sequences, such as mitochondrial DNA, are expected to accumulate mutations over time, and assuming a constant rate of mutation provide a molecular clock for dating divergence. Molecular phylogeny uses such data to build a "relationship tree" that shows the probable evolution of various organisms. Not until recent decades, however, has it been possible to isolate and identify these molecular structures.

The most common approach is the comparison of homologous sequences for genes using sequence alignment techniques to identify similarity. Another application of molecular phylogeny is in DNA barcoding, where the species of an individual organism is identified using small sections of mitochondrial DNA. Another application of the techniques that make this possible can be seen in the very limited field of human genetics, such as the ever more popular use of genetic testing to determine a child's paternity, as well as the emergence of a new branch of criminal forensics focused on evidence known as genetic fingerprinting.

Theoretical background

Early attempts at molecular systematics were also termed as chemotaxonomy and made use of proteins, enzymes, carbohydrates and other molecules which were separated and characterized using techniques such as chromatography. These have been largely replaced in recent times by DNA sequencing which produces the exact sequences of nucleotides or bases in either DNA or RNA segments extracted using different techniques. These are generally considered superior for evolutionary studies since the actions of evolution are ultimately reflected in the genetic sequences. At present it is still a long and expensive process to sequence the entire DNA of an organism (its genome), and this has been done for only a few species. However it is quite feasible to determine the sequence of a defined area of a particular chromosome. Typical molecular systematic analyses require the sequencing of around 1000 base pairs. At any location within such a sequence, the bases found in a given position may vary between organisms. The particular sequence found in a given organism is referred to as its haplotype. In principle, since there are four base types, with 1000 base pairs, we could have 4¹⁰⁰⁰ distinct haplotypes. However, for organisms within a particular species or in a group of related species, it has been found empirically that only a minority of sites show any variation at all and most of the variations that are found are correlated, so that the number of distinct haplotypes that are found is relatively small.

In a molecular systematic analysis, the haplotypes are determined for a defined area of genetic material; ideally a substantial sample of individuals of the target species or other taxon are used however many current studies are based on single individuals. Haplotypes of individuals of closely related, but supposedly different, taxa are also determined. Finally, haplotypes from a smaller number of individuals from a definitely different taxon are determined: these are referred to as an out group. The base sequences for the haplotypes are then compared. In the simplest case, the difference between two haplotypes is assessed by counting the number of locations where they have different bases: this is referred to as the number of substitutions (other kinds of differences between haplotypes can also occur, for example the insertion of a section of nucleic acid in one haplotype that is not present in another). Usually the difference between organisms is re-expressed as a percentage divergence, by dividing the number of substitutions by the number of base pairs analysed: the hope is that this measure will be independent of the location and length of the section of DNA that is sequenced.

An older and superseded approach was to determine the divergences between the genotypes of individuals by DNA-DNA hybridisation. The advantage claimed for using hybridisation rather than gene sequencing was that it was based on the entire genotype, rather than on particular sections of DNA. Modern sequence comparison techniques overcome this objection by the use of multiple sequences.

Once the divergences between all pairs of samples have been determined, the resulting triangular matrix of differences is submitted to some form of statistical cluster analysis, and the resulting dendrogram is examined in order to see whether the samples cluster in the way that would be expected from current ideas about the taxonomy of the group, or not. Any group of haplotypes that are all more similar to one another than any of them is to any other haplotype may be said to constitute a clade. Statistical techniques such as bootstrapping and jackknifing help in providing reliability estimates for the positions of haplotypes within the evolutionary trees.

Limitations of molecular systematics

Molecular systematics is an essentially cladistic approach: it assumes that classification must correspond to phylogenetic descent, and that all valid taxa must be monophyletic.

Molecular systematics often uses the molecular clock assumption that quantitative similarity of genotype is a sufficient measure of the recency of genetic divergence. Particularly in relation to speciation, this assumption could be wrong if either some genotypic modification acted to prevent interbreeding between two groups of organisms, or genetic modification proceeded at different rates in different subgroups of the organisms.

In animals, it is often convenient to use mitochondrial DNA for molecular systematic analysis. However, because in mammals mitochondria are inherited only from the mother, this is not fully satisfactory, because inheritance in the paternal line might not be detected.

Molecular phylogenies can be affected by myriad problems, including long branch attraction, saturation, and taxon sampling problems: this means that strikingly different results can be obtained by applying different models to the same dataset.

See also

• molecular evolution
• computational phylogenetics
• PhyloCode
• Microbial phylogenetics

Further reading

• Felsenstein, J. 2004. Inferring phylogenies. Sinauer Associates Incorporated. ISBN 0-87893-177-5.
• Hillis, D. M. & Moritz, C. 1996. Molecular systematics. 2nd ed. Sinauer Associates Incorporated. ISBN 0-87893-282-8.
• Page, R. D. M. & Holmes, E. C. 1998. Molecular evolution: a phylogenetic approach. Blackwell Science, Oxford. ISBN 0-86542-889-1.
• Soltis, P.S., Soltis, D.E., and Doyle, J.J. (1992) Molecular systematics of plants. Chapman & Hall, New York. ISBN-0-41202-231-1.
• Soltis, P.S., Soltis, D.E., and Doyle, J.J. (1998) Molecular Systematics of Plants II: DNA Sequencing. Kluwer Academic Publishers Boston, Dordrecht, London. ISBN-0-41211-131-4.

External links

• NCBI - Systematics and Molecular Phylogenetics