Evolution Sequence Variation Within and Between Species

A fundamental concept in molecular evolution is that the sequence variation observed within a single species is fundamentally different, in origin and evolutionary fate, from the sequence differences observed between species. Within-species variation represents new mutations that have arisen recently and have not yet been screened by natural selection over long periods, whereas between-species differences represent mutations that have already become fixed in the population of one species or another. This distinction can be understood clearly by examining a well-studied human gene, BRCA1, which shows both extensive documented variation within humans and a long history of divergence across mammalian species.

1. BRCA1 as a Model Gene

1.1 Biological Role

BRCA1 is a gene associated with hereditary breast and ovarian cancer in humans. Women carrying mutations in this gene have a significantly elevated risk of developing cancer. The normal function of the BRCA1 protein is believed to be the repair of oxidative damage to DNA. When this repair function is disrupted, DNA damage accumulates in somatic cells, which can eventually lead to cancer.

1.2 Documented Mutations Within the Human Species

The Human Gene Mutation Database catalogues disease-associated mutations in human genes. For BRCA1, several hundred distinct disease-associated mutations have been documented, distributed along the entire length of the gene. These mutations fall into several categories:

• Single nucleotide substitutions – account for roughly one-third of the documented mutations.
• Small deletions – removal of one or a few nucleotides from the coding sequence; slightly more common than single-nucleotide substitutions.
• Gross deletions and insertions – larger-scale structural changes, observed less frequently.
• Inversions – a small number of cases where a segment of sequence is reversed in orientation.

1.3 Splice-Site Mutations

BRCA1, like many human genes, has a complex intron/exon structure, consisting of 24 exons. Correct production of the functional BRCA1 protein therefore depends on accurate splicing of the messenger RNA. Mutations occurring at splice sites can severely disrupt this process in two main ways:

1. A mutation may destroy an existing splice site, causing an entire exon to be omitted from the final protein.
2. A mutation may create a new, incorrect splice site (referred to as activation of a cryptic splice site), leading to abnormal processing of the mRNA.

Both mechanisms can produce large-scale errors in the resulting protein, and splice-site mutations form a significant proportion of disease-causing BRCA1 mutations.

2. Nature of Mutations: Deleterious, Neutral, and Advantageous

Since genes have been refined over long periods of natural selection, they are generally efficient at performing their biological role. This has important consequences for the type of mutations we expect to observe:

• Deleterious mutations: Most new mutations that arise in a functioning gene reduce its fitness relative to the original sequence. Mutations found in disease databases are typically deleterious mutations severe enough to produce a visible disease phenotype. Because natural selection acts against them, such mutations usually remain rare in the population.
• Nearly neutral mutations: Some mutations have only a minor effect on gene function, with no obvious consequence for the individual. Although such effects appear negligible in the short term, they can be significant over evolutionary time scales, and natural selection tends to keep even slightly deleterious variants at low frequency.
• Advantageous mutations: A small proportion of mutations increase the fitness of the sequence. Such mutations must occur occasionally, since without them, a gene could never have evolved its functional form in the first place. However, advantageous mutations are expected to be considerably rarer than deleterious ones, so a random sample of mutations observed in a population will mostly be deleterious in nature.

3. Comparing BRCA1 Across Species

To study evolutionary change, the BRCA1 gene has been sequenced and aligned across many mammalian species. This comparison reveals systematic patterns that are different from the mutation patterns observed within a single species.

3.1 DNA-Level Alignment: Synonymous Substitutions

When the DNA sequences of BRCA1 from multiple mammals are aligned, most of the substitutions observed between species turn out to be synonymous changes — that is, changes in the DNA sequence that do not alter the amino acid encoded, because of redundancy in the genetic code. Key observations include:

• Synonymous substitutions occur overwhelmingly at the third position of the codon, since this position is most tolerant of change without altering the encoded amino acid.
• As a result, third-position sites evolve more rapidly than first- and second-position sites.
• For amino acids that have exactly two synonymous codons, the two alternative bases at the redundant position are typically either both purines or both pyrimidines. Since a change between two purines (or two pyrimidines) is a transition, this explains why synonymous substitutions are more often transitions than transversions.
• Consequently, transitions are observed more frequently than transversions in molecular sequence comparisons generally.
• Not all substitutions at redundant positions are transitions; occasionally a transversion is also synonymous, if the amino acid in question has four synonymous codons (i.e., any base change at that position remains silent).

3.2 Protein-Level Alignment: Conservative Substitutions

When comparing BRCA1 protein sequences (rather than DNA) across species, it is evident that not all amino acid substitutions occur with equal likelihood. Substitutions between amino acids with similar chemical properties — for example, between two basic amino acids, or among several hydrophobic amino acids — occur more frequently than substitutions between chemically dissimilar amino acids. Such substitutions are termed conservative changes, since they tend to preserve the physicochemical character, and hence the folding and function, of the protein. Methods for quantifying the relative likelihood of different amino acid substitutions are addressed separately using amino acid substitution matrices.

4. Insertions and Deletions (Indels) in Cross-Species Alignments

Alignments of BRCA1 sequences across mammals also reveal several indel (insertion/deletion) events, which provide valuable evolutionary information:

• A deletion of nine bases (three amino acids) is shared by a specific group of mammals that includes species such as the dugong, hyrax, aardvark, and tenrec. This shared deletion was used as molecular evidence to establish that these morphologically very different species belong to a single evolutionary group (the superorder Afrotheria, which also includes golden moles, elephant shrews, and elephants). This example illustrates how molecular sequence data can reveal evolutionary relationships that are not obvious from outward physical appearance.
• A separate, smaller deletion (three bases) is found only in the opossum.
• A further deletion (six bases) is present in all the mammals studied except the wombat and the opossum — both of which are marsupials, in contrast to the remaining species, which are eutherian (placental) mammals. This pattern is consistent with the deep evolutionary split between marsupials and eutherians.

4.1 Ambiguity of Deletion versus Insertion

Whenever a gap appears in a sequence alignment, it is not always possible to determine with certainty whether the gap represents a deletion in one lineage or an insertion in the other. In some cases, contextual evidence makes one interpretation more plausible (for example, assuming that shorter sequences arose from longer ancestral sequences through deletion). In other cases — such as the marsupial/eutherian difference described above — the alignment alone cannot indicate whether the change originated as a deletion in the eutherian ancestor or an insertion in the marsupial ancestor.

4.2 Indels and Reading Frame

A notable feature of all three indels described above is that each involves a number of bases that is a multiple of three. This means that each indel removes or adds whole codons, without causing a frameshift in the reading of the downstream sequence. Small in-frame indels, particularly those that do not disrupt regions of secondary structure, may have only a minor effect on protein function and can behave as nearly neutral changes, similar to conservative amino acid substitutions.

In contrast, indels that are not multiples of three cause a frameshift, altering the reading frame of all downstream codons and typically producing a severely disrupted, non-functional protein. Frameshift-causing indels are known to occur in humans, as documented in disease mutation databases, and presumably occur at similar rates in other species. However, such mutations are rarely, if ever, observed in between-species comparisons, because they are highly deleterious and are eliminated by natural selection before they can become fixed in a population.

5. Key Distinction: Within-Species Variation versus Between-Species Divergence

The overall comparison of BRCA1 within humans and across mammalian species highlights a central principle of molecular evolution:

• Sequence variants within a species are, for the most part, recently arisen mutations. The majority of these are deleterious and are expected to be eventually eliminated by natural selection; they have not yet been “tested” over long evolutionary time.
• Sequence differences between species reflect mutations that have already become fixed in the population of one species or another. Because deleterious mutations are unlikely to reach fixation, the differences observed between species are predominantly either neutral or advantageous mutations that were not eliminated by selection.

All homologous sequences compared across species, such as BRCA1 in different mammals, are understood to have descended from a single common ancestral sequence, gradually diverging over time as different mutations became fixed independently in each evolutionary lineage. This raises an important theoretical question — the relative contribution of neutral mutations versus advantageous (positively selected) mutations to overall molecular evolution — which is addressed through the theoretical frameworks of coalescence theory and the process of fixation of mutations in populations.

Conclusion

The BRCA1 gene provides a clear illustration of how sequence variation differs in nature between the within-species and between-species levels. Within humans, most documented mutations are deleterious, disease-associated changes, including nucleotide substitutions, small and gross indels, inversions, and splice-site defects, all kept rare by natural selection. Across species, however, the fixed differences observed reflect predominantly neutral or advantageous changes, showing characteristic patterns such as a bias toward synonymous and transition substitutions, conservative amino acid replacements, and in-frame indels that reveal deep evolutionary relationships, as seen in the shared Afrotherian deletion. This contrast between within-species and between-species variation forms the conceptual basis for understanding how mutations become fixed in populations over evolutionary time.