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.
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An older and superseded approach was to determine the divergences between the genotypes of individuals by DNA-DNA hybridisation.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.
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.
==References==
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