It is possible to differentiated among mutations in ways that go well beyond different types of changes in nucleotides sequence. These can include in terms of mutation impact of other genes, on phenotype, and on fitness, as well as in terms of mutation fixation, mutation impact on evolution generally, and mutation impact on phylogenies. In this section we take a look at these more complicated aspects of changes in nucleotide sequence.
Going beyond ratios of nonsynonymous to synonymous substitutions, an even stronger indicator of positive selection are mutations that give rise to what is known as parallel evolution. With parallel evolution the same mutation, or at least a mutation that results in a change to the same amino acid in a gene's product (re: redundancy in the genetic code), is found in multiple, independently evolving lineages of organisms. This is considered to be stronger evidence of a beneficial effect because it is assumed that independent increases in frequency of the same allele within different populations is more likely to be a consequence of deterministic evolution (natural selection) than of stochastic processes (genetic drift). One way to think about this is that synonymous substitutions represent changes that may not impact phenotype (and thus fixation is likely to be stochastic and therefore due to drift), nonsynonymous substitutions are changes that are more likely to impact phenotype (and thus fixation is more likely to be due to natural selection), and parallel evolution is changes that are likely to impact phenotypes associated with different lineages in the same way (and therefore fixation is even more likely to be deterministic and consequently due to natural selection).
In general, that is, the more times that certain things happen then the more likely we are to assume that there is some underlying reason or bias towards that thing happening (a.k.a., deterministic). With parallel evolution the assumption is that this bias is a selective advantage bestowed by the mutation in question: Mutations that give rise to increased reproductive success simply are more likely to be retained within populations and if those mutations are hit upon independently by more than one lineage of organisms, then they are more likely to be retained by the different lineages if they are beneficial. This multiple retention in turn is described as parallel evolution. Equivalent mutations that appear within multiple lineages that do not lead to increased reproductive success, by contrast, are less likely to be retained by these multiple lineages. There is a bias, therefore, for retention of beneficial alleles within lineages, across multiple lineages this bias can be seen as parallel evolution, and parallel evolution therefore can serve as evidence for positive selection, which in turn is the biased retention by populations of beneficial alleles.
Note that parallel evolution is not convergent evolution. The difference is that with parallel evolution the same character in independent lineages changes and the change is of an equivalent genotype and equivalent manner: Similar continues to be similar though in changing ways due to ongoing genetic change. With convergent evolution, by contrast, different characters change in ways that make them more similar: Dissimilar becomes less dissimilar over the course of ongoing change. Thus, with parallel evolution the assumption is that the background sequence of a gene in which parallel mutations are occurring is similar or even identical whereas with convergent evolution, as viewed in terms of genes, the background sequence of a gene in which change is occurring would not be similar. Especially, parallel evolution is seen in genes already displaying high identity whereas convergent evolution, to the extent it occurs at the level of genes, is seen in genes that do not display high identity.
Note that in observing evidence of parallel or convergent evolution one must always be concerned that the changes in fact may not be independent of each other but instead could be products of horizontal gene transfer from one lineage to another. That is, generally, in everyday life, one way by which two things can become more similar is by purchasing or otherwise obtaining the same parts for both items. This does represent a biased acquisition or retention of a given character/item, and therefore can serve as evidence of positive selection. It does not, however, serve as the biased retention within populations of independently acquired point mutations. Instead, in biological systems, horizontal gene transfer serves as a means by which otherwise potentially unique mutations that have arisen within one lineage of organisms can find their way into an otherwise unrelated second lineage of organisms.
It is also possible for two lineages to display mutations that are similar but nonetheless not identical. Typically these are seen, minimally, as mutations that occur in the same codon, giving rise to changes in amino acids in the gene product, but with these changes giving rise to non-identical amino acids rather than identical changes. Such mutations can be suggestive that changes in the focus amino acid have impacts on protein function, and this inference may be particularly valid if these mutations arise quickly within populations that are under relatively strong selection. An alternative explanation, however, can be that the amino acid position in question has little impact on protein function and therefore that substitutions at that site have only an only neutral impact on fitness. Thus, rapid accumulation of these "coincidental" (Chattopadhyay et al., 2009) mutations in multiple lineages can be seen as a sign of positive selection whereas gradual accumulation may instead be the product instead genetic drift, that is tiny, gradual changes in protein structure.
The environmental influence on the phenotype is sometimes referred to in today's parlance as the 'envirome' (Butte and Kohane, 2006). The environmental influence on the phenotype is sometimes referred to in today’s parlance as the 'envirome' (Butte and Kohane, 2006) . More traditionally, it has been regarded as one aspect of 'context,' a term that includes both external and internal influences on the activity and phenotypic effects of the gene or sequence under consideration. The importance of context for gene expression has been recognized since the beginning of formal genetics. Developmental plasticity studies date back at least to the nineteenth century, when butterfly morphs, originally assigned to species status, were recognized to be different forms of one temperature-sensitive species (Morgan, 1907) . … Although most scientists may intellectually agree that context is of basic importance in gene expression, it is still far more common in practice to carry out genetic screens rather than context screens, not only because they are usually easier to implement but also because they give seemingly more clearcut results and conform to the prevailing expectation of the primacy of genes. — quoted from Atallah and Larsen (2009) (p. 123)
Mutations in certain regions of chromosomes, e.g., such as in pseudogenes, don't affect organism fitness – or, at least, are less likely to affect fitness – because these regions represent extra DNA, which therefore is not closely tied with the generation of phenotype. There also exist mutations that affect phenotype under some conditions but not or less so under others. That is, these mutations are conditional. More generally, the impact of mutations on fitness as well as on phenotype may vary as a function of environment as well as the rest of an organism's genotype. In microbial genetics, conditionally expressed mutations, such as conditionally lethal mutations, have historically been important in establishing associations between genotype and phenotype, i.e., which genes encode which characters (and how). An important example is auxotrophic mutations, which are approximately neutral in their fitness impact during growth within relatively rich media, but lethal or at least prevent organism growth within more minimal media.
A phenomenon related to that of conditional mutations is phenotypic plasticity. Here the phenotype associated with a given genotype varies as a function of environment. This variation may be detrimental (i.e., declines in fitness given a change in environment) or instead beneficial (i.e., increases in fitness given such change), though those distinctions may simply be a matter of perspective, that is, depending on whether the higher-fitness or instead the lower-fitness conditions represent the baseline. For microorganisms, a typical example of phenotypic plasticity would be reductions in growth rates given nutrient deficiencies, or the establishment of a more durable state under certain conditions such as starvation. One additionally could include physiological modifications under this heading of plasticity in phenotype, such as enzyme induction. Returning to conditional mutations, these are mutations that provide one phenotype under one circumstance, such as an approximation of the wild-type phenotype, but another phenotype, the mutant phenotype, under a different set of circumstances, such as different temperatures.
Microorganism gene expression, as well as phenotype, even given constancy in gene expression, thus can vary from environment to environment including in ways that are adaptive. Alternatively, mutations may occur that impact organism phenotype, and fitness, in one environment but not another. Indeed mutations can even improve fitness in one environment at the expense of their impact on fitness in another. An example of the latter could be mutations that have the dual effect – i.e., are pleiotropies – of inhibiting sporulation in one environment while improving vegetative growth in another.
Many conditional mutations can be described as having a neutral impact on fitness within certain environments but a detrimental impact in other environments. An important means by which balanced polymorphisms may occur, that is the persistence of more than one allele at a given locus within a population, is as a result of just such conditional mutation accumulation. Nonetheless, rare neutral mutations that are beneficial given environmental change may be key to the long-term survival of species. The accumulation of mutations independent of selection, i.e., neutral mutations, also is important to the establishment of molecular clocks, which serve as a means of estimating the timing of divergence of reproductively isolated populations in molecular evolution studies.
An additional complication on the generation of phenotype from genotype is the potential for different loci to interact. This phenomenon is called epistasis and is typically observed in terms of a mutation in one locus affecting the expression of an allele found in a different locus. A related concept is that of the compensatory mutation. Here a mutation in one locus, which is either deleterious or lethal, becomes less detrimental as a consequence of a mutation found in a different locus. In molecular genetics such compensatory mutations can be described as pseudoreversions, that is, a restoration of a wild-type phenotype or some approximation of wild type via mutation in a locus that differs from the locus in which the original mutation to no longer wild type occurred. By contrast, an actual reversion would be a mutation in the same locus, and strictly this would be a mutation that is an actual reversal or some approximation thereof of the original mutation. Alternatively, it is possible for a second mutation in a different location in the same gene also to restore the wild-type phenotype (which also is an example of a pseudoreversion).
It is possible for multiple mutations either to display similar compensating phenotypes or to work together towards effecting a single compensating phenotype. These would be, respectively, multiple routes towards repair of a function versus instead a single repair involving or requiring multiple mutations (which is possible as well). On the other hand, while these compensating mutations may work together to restore fitness losses associated with a detrimental mutation, in fact the resulting fitness gains may dependent on the existence of the original mutation. That is, without the original damaging mutation the compensating mutations may fail to positively affect fitness. Furthermore, the compensatory mutation in the absence of the original mutation could very well have a detrimental impact on fitness. An important example of such epistatic interactions between different loci, where one or more mutations improves fitness in response to changes which have occurred elsewhere in an organism's genome, are mutations that compensate for the acquisition of antibiotic resistance (see "Evolution of Resistance", below, which considers a number of issues relevant to the evolution of antibiotic resistance in bacteria). All of the above can be restated and generalized as the fitness of a given allele being conditional as a function not just of an organism's environment but also of the rest of an organism's genotype.
The reason that compensatory mutations can be readily found at different loci is a consequence, in microorganisms, of the relatively low likelihood of recombination, that is linkage disequilibrium that results from a lack of obligate sexuality. In other words, the breaking up of allelic combinations is an important consequence of sex, but if sex is not required for reproduction then two alleles can coevolve even if they otherwise are not closely associated within a chromosome (Levin and Bergstrom, 2000) . In short, genes and their products interact, and either due to restoration of gene product-gene product interactions or because of an acquired metabolic redundancy, it is possible for changes in one gene to compensate for detrimental changes in a different gene.
Certain mutations, typically not neutral in their effects, can impact multiple phenotypic traits simultaneously, a phenomenon known as pleiotropy. A variation on the idea of pleiotropy is that a given allele can have different impacts on fitness in different environments, i.e., such as considered above in terms of conditional mutations. One illustration, known as antagonistic pleiotropy, is a situation in which a specific mutation provides positive fitness benefits in one environment, or set of circumstances, but gives rise to negative fitness effects under other circumstances (in the literature this term can be seen, for example, in reference to the genetics of aging, where an allele that is helpful in youth can become harmful as an organism ages). Pleiotropy thus can be used as a description of multiple phenotypic effects associated with one mutation in one individual in one environment, or just a single phenotypic effect such as fitness that is associated with one individual but which varies oppositionally (i.e., antagonistically) in terms of its fitness impact in different environments. As noted, this variance in phenotype as a function of environment cam be essentially an example of phenotypic plasticity, though in case of pleiotropy, as so described, the plasticity specifically is the phenotype that is associated with a specific allele and in fact may be simply Darwinian fitness as a phenotype.