The physical basis of molecular variation

With the exception of RNA viruses, the hereditary information in all organisms is carried in DNA. Ultimately, differences in any of the molecular markers we study (and of genetically-based morphological, behavioral, or physiological traits) is associated with some difference in the physical structure of DNA, and molecular evolutionists study a variety of its aspects.

Nucleotide sequence

A difference in nucleotide sequence is the most obvious way in which two homologous stretches of DNA may differ. The differences may be in translated portions of protein genes (exons), portions of protein genes that are transcribed but not translated (e.g., introns, 5' or 3' untranslated regions), non-transcribed functional regions (e.g., promoters), or regions without apparent function.

Protein sequence

Because of redundancy in the genetic code, a difference in nucleotide sequence at a protein-coding locus may or may not result in proteins with a different amino acid sequence. Important note: Don't forget that some loci code for RNA that has an immediate function without being translated to a protein, e.g., ribosomal RNA and various small nuclear RNAs.

Secondary, tertiary, and quaternary structure

Differences in amino acid sequence may or may not lead to a different distribution of $\alpha$-helices and $\beta$-sheets, to a different three-dimensional structure, or to different multisubunit combinations.

Imprinting

At certain loci in some organisms the expression pattern of a particular allele depends on whether that allele was inherited from the individual's father or its mother.

Expression

Functional differences among individuals may arise because of differences in the patterns of gene expression, even if there are no differences in the primary sequences of the genes that are expressed.³

Sequence organization

Particular genes may differ between organisms because of differences in the position and number of introns. At the whole genome level, there may be differences in the amount and kind of repetitive sequences, in the amount and type of sequences derived from transposable elements, in the relative proportion of G-C relative to A-T, or even in the identity and arrangement of genes that are present. In microbial species, only a subset of genes are present in all strains. For example, in Streptococcus pneumoniae the ``core genome'' contains only 73% of the loci present in one fully sequenced reference strain [7]. Similarly, a survey of 20 strains of Escherichia coli and one of E. fergusonii, E. coli's closest relative, identified only 2000 homologous loci that were present in all strains out of 18,000 orthologous loci identified [9]

Copy number variation

Even within diploid genomes, there may be substantial differences in the number of copies of particular genes. In humans, for example, 76 copy-number polymorphisms (CNPs) were identified in a sample of only 20 individuals, and individuals differed from one another by an average of 11 CNPs. [8].

It is worth remembering that in nearly all eukaryotes there are two different genomes whose characteristics may be analyzed: the nuclear genome and the mitochondrial genome. In plants there is a third: the chloroplast genome. In some protists, there may be even more, because of secondary or tertiary endosymbiosis. The mitochondrial and chloroplast genomes are typically inherited only through the maternal line, although some instances of biparental inheritance are known.