Linkage is the binding together of two or more genetic loci. This is most easily envisaged in terms of adjacent loci found on the same chromosome where allele separation is possible only given molecular recombination, typically homologous recombination. This separation is observed, for example, during the crossing over step of meiosis. In some organisms actual molecular recombination either is not observed or is only rarely observed (e.g., certain viruses). In these organisms linkage within chromosomes may be absolute or nearly so. There still may be genetic recombination, i.e., reassortment, which allows a separation of at least some loci given outcrossing. Indeed, even given molecular recombination, separation of linked alleles can only be observed, genetically, given outcrossing such that different allele combinations may result.

Given sufficient amounts of both outcrossing and recombination then a state known as linkage equilibrium can be achieved. With linkage equilibrium, within a population, the presence of one allele on a chromosome is not predictive of the presence of another allele. That is, with linkage equilibrium the presence of different alleles at different loci can be described as statistically independent. By contrast, in a state known as linkage disequilibrium, two or more alleles may be more likely to be found together than their relative prevalence in the population would predict. In this case the likelihood of two or more alleles being present on a single chromosome is not statistically independent. Typically, two alleles will more likely be in linkage disequilibrium if they are found more closely together on chromosomes and/or if outcrossing or recombination is rare.

An absence of linkage disequilibrium is evidence of a relative abundance of both outcrossing and recombination (sex) as well as random mating among individuals (i.e., the fifth Hardy-Weinberg assumption). Spatial structure, e.g., geographic boundaries, can inhibit random mating while infrequent sexual phases can allow linked mutations to occur which are not readily separated by homologous recombination. The result can be the coevolution of alleles within the genomes of subpopulations but which may then be readily broken apart via mating with other subpopulations. These linked, coevolved genetic modules include operons as observed in bacteria and the typically adjacent capsid genes observed in phages. Particularly, progeny for which coevolved genetic complexes have been disrupted may display reduced fitness relative to their parents. In higher organisms such animals disruption of coevolved genetic complexes serves as a key post-zygotic isolation mechanisms, one that is important to the process of cladogenesis, i.e., the splitting of one species into two or more. In general, asexuality as well as limited organism mobility can promote linkage disequilibrium, promote within-genome coevolution, and potentially promote the first steps toward speciation. Ongoing outcrossing and environmental mixing, by contrast, promote linkage equilibrium, though potentially at the expense of within-genome coevolution.

Phenotypic Expansion

Genotypes interact with environments through their associated phenotypes. Following outcrossing and recombination, an organism will then be subject to natural selection. What is the nature of new-to-organism phenotypes and, given horizontal gene transfer, what is the fate of newly acquired genetic sequences vis-à-vis natural selection? I begin to consider these questions in this section, though more detailed discussion will be found in the following chapter, covering Genomes and their evolution.

Movement of Coadapted Gene Complexes

The utility of horizontal gene transfer to microbial evolution is three fold: (1) It involves more nucleotides than point mutations and therefore can introduce more novel genetic material per transfer than can individual mutation events, (2) the genetic material received likely has already been subjected to natural selection, and (3) entire metabolic pathways if linked together may be moved simultaneously. Indeed, the linkage of the genes involved in metabolic pathways, forming coadapted gene complexes (i.e., genes involved in epistatic relationships), though likely occurring in part of the sake of co-regulation, also seem to exist so that their movement between organisms can occur as a single unit. In this way the utility of these complexes to a recipient organism can be immediate. Another way of looking at this is that the organism in which a metabolic pathway is discovered, and first characterized, is not necessarily the lineage within which that pathway first evolved.

In addition to more basic metabolic pathways, organisms frequently possess linked genes that have properties indicating that these genes were only recently acquired. Codon usage in particular may differ from that of the rest of the genome (that is, tendencies within genomes are for use of particular codons among multiple synonymous codons encoding individual amino acids, and different organisms will possess different biases in terms of what codons they preferentially use/possess). In addition, sequences such as genomic islands can possess, among the linked genes, genes seemingly involved in horizontal gene transfer process, such as integrases. Other sequences are clearly prophages that have been acquired in the course of lysogenic infection by temperate phages, though often these prophage sequences have degraded over time and therefore are defective with regard to their ability to produce functional phage products. For a number of bacterial species the differences seen between individual strains seem to involve, especially, probable products of horizontal gene transfer, consisting of many more-or-less lineage-unique genes found linked together within genomes.

Lysogenic Conversion

Bacteria can acquire one or more genes in association with temperate phages. These phages are capable of displaying lysogenic cycles in which the phage genome, now called a prophage, becomes a more or less permanent genetic component of the host bacterium. Perhaps in part because prophages can otherwise be metabolically burdensome, many carry genes that can contribute to host, i.e., lysogen fitness, in a process called lysogenic conversion. Specifically, lysogenic conversion, which some call simply phage conversion, is a prophage-mediated qualitative modification of the phenotype of a host bacterium. Note that I use the word qualitative to distinguish lysogenic conversion from simple "quantitative" declines in host fitness as might occur due either to the above-noted metabolic burden of carrying a prophage or due to a prophage's potential to display productive cycles in which the host bacterium is killed. With lysogenic conversion a bacterium instead acquires some new phenotype, just as one sees upon bacterial acquisition of many plasmids.

Probably the most common qualitative change that a prophage imparts on a host bacterium is the display of immunity against superinfection. This immunity function is mediated against similar or identical phages attempting to infect the same bacterium, resulting in a failure by the second bacterium to successfully infect (and which potentially is a product of soft selection). Alternatively, some of these conversion genes can contribute directly to bacterial pathogenicity by encoding virulence factors, including bacterial exotoxins (such as the toxins responsible for cholera, diphtheria, and E. coli O157:H7 pathogenicity; for an essay addressing more fully just how prophages may benefit from encoding bacterial virulence factors, see Abedon and LeJeune, 2005 ). The number of converting genes a prophage can carry is relatively small, however – as compared with pathogenicity islands or even plasmids – which is in part a consequence of the need for temperate phages to also display productive cycles that produce new phage virions. Temperate phages thus are a highly effective means of gene transfer between bacteria, e.g., such as in comparison with pathogenicity islands, though a cost of that mobility is seen, apparently, in terms of a phage's apparently lower than might be expected potential to carry multiple converting genes.

Outcrossing and Genomic Diversity

The essentials of the modular theory of bacteriophage evolution can be stated as follows: … The product of evolution is not a given virus but a family of interchangeable genetic elements (modules) each of which carries out a particular biological function. Each virus encountered in nature is a favorable combination of modules (one for each viral function) selected to work optimally individually and together to fill a particular niche. Exchange of a given module for another that has the same biological function (e.g., DNA replication) occurs by recombination among a population of different viruses… Viruses in the same interbreeding population can differ widely in any characteristic (including morphology and host range) since these are aspects of the function of individual modules… — David Botstein (1980)

Sex among viruses tends to occur in the course of replication, during the coinfection of a single cell by more than one virus. Such sex, however, is generally not necessary for virus replication since with most viruses coinfection is not required for successful infection. A consequence of this simple truism is that viruses can be both highly recombinogenic and highly promiscuous in their outcrossing. The result, at an extreme, is the exceedingly mosaic nature of the genomes of tailed bacteriophages. As I will suggest in this section, this outcome is part of a pattern where rarity of outcrossing can result in broad experimentation and recombinogenic diversity whereas when outcrossing is very common then less experimentation (greater conservation) may be necessary, resulting in less recombinogenic diversity. Another more general way of saying this is that organisms can be free to be more reckless if only a small proportion of a lineage may be lost to a given activity. That is, sex in some cases can be a potentially high-cost but also high-payoff strategy.

Sex for many eukaryotes not only occurs in conjunction with replication but in fact replication, in the sense of the formation of the next generation, cannot occur without sex. These organisms, that is, are obligately sexual such that sex cannot be avoided by lineages and also in the sense that in fact meiosis is engaged in once per life cycle. For single-celled eukaryotes, by contrast, reproduction can occur via a mitosis that is only episodically interrupted by meiosis. Meiosis results in a lack of clonality that, in turn, interferes with such things as periodic selection, genetic hitchhiking, and clonal interference. For organisms that are not obligately sexual, however, even if gene exchange is possible it may not occur sufficiently often to counter the linkage that leads to these (just-listed) mechanisms of natural selection inefficiency (i.e., periodic selection, etc.). By unlinking genes, sex can allow natural selection to be highly efficient especially in terms of its action upon individual alleles. A relative absence of sex, resulting in greater linkage disequilibrium, can by contrast allow for more efficient evolution of coadapted gene complexes.

To achieve levels of intragenic coevolution approaching those seen with clonal populations, an obligately sexual lineage must be less promiscuous in terms of the range of organisms with which outcrossing may be attempted. That is, coevolution within the genomes of obligately sexual organisms must be able to survive some approximation of linkage equilibrium within their population. Indeed, the scattering of coevolved genes throughout the eukaryotic genome relative to that of bacterial genomes – operons versus a relative lack of co-localization – is highly suggestive that mechanisms other than genetic linkage prevent the dissociation of coadapted genes.

One means of avoiding such dissociation is through inbreeding, i.e., limiting mating to organisms that possess the same coadapted gene complexes including in terms of some approximation of the same underlying alleles. Thus, obligate sexuality such as that seem among animals would be predicted to, and indeed seems to result in gene pools, as equivalent to mating pools, of relatively limited breadth. Gene pools in this case thus consist of populations of similar, interbreeding individuals while mating with less-similar but nonetheless similarly defined populations is relatively unlikely. Furthermore, if the testing of the boundaries between these different populations is fairly common, then the result can be the evolution of adaptations that limit hybridization between populations. Speciation such as within animals thus can be viewed, at least in part, as a consequence of natural selection acting to assure the retention of coadapted gene complexes among progeny despite extensive participation by individual organisms in sexual processes.

In other words, a cost of outcrossing is the potential for disharmony within the resulting recombinant genomes. Therefore, if sex is both frequent and obligatory, then disharmony may be reduced by limiting the breadth of genotypes with which one mates else one's matings may not be productive since the resulting progeny might not be terribly fit. By contrast, organisms for which sex is less frequent, not obligatory, and indeed involving less of the genome per sexual event, can afford to be much less careful in "choosing" what they cross with. Genetic experimentation therefore is much more permissible. In addition, even outcomes that are catastrophic to participants, in a fitness sense, can be permissible so long as such outcomes are uncommon because experimentation is rare, as often is the case with both viruses and bacteria. Alternatively, such outcrossing can be relatively benign if it occurs between closely related organisms such as among clonally related viruses or bacteria or if recombination typically involves only small snippets of DNA.

With obligately sexual eukaryotes, recombination is too efficient (as also seen in viruses) and too frequent (as not seen in viruses), whereas for bacteria recombination resulting from outcrossing is both less efficient and more rare. In terms of the achievement of genomic recombinogenic diversity – that is, variation resulting from genetic recombination – the result is lower levels for obligately sexual eukaryotes, higher levels for bacteria, and highest levels for dsDNA viruses, such as tailed phages. Indeed, and consistent with these musings, highly diversifying sexuality for eukaryotes seems to occur in those situations where recombination is least efficient, e.g., such as in terms of "you are what you eat" or instead as a consequence of the combining together of complete genomes which do not subsequently require extensive recombination, as occurs during the formation through hybridization of new, polyploid plant species or following endosymbiotic events. Both too much sex (i.e., obligately sexuality) and too little sex (i.e., pure clonality), in other words, can increase the efficiency of evolution especially in the short term, but both too much sex and too little sex also can lead to greater evolutionary conservatism (less diversity!) in the longer term.

Isambert and Stein (2009) provide an interesting counter perspective to that provided above. In their view, organisms have tendencies to endogenously increase their DNA content through genomic duplication mechanisms. For organisms that are constrained by selection in terms of their genome size, e.g., such as prokaryotes, but also viruses, this natural increase must be countered by tendencies toward deletion mutations, i.e., see Lawrence et al. (2001) . Less-frequently used genes will not be under strong pressure for retention, and thus will tend to be lost from lineages via these deletions. Countering this tendency to lose genes is horizontal gene transfer. As they suggest, "prokaryotes must remain under strong selection pressure in order to maintain the long-term evolutionary adaptation of their 'mutualized' gene pool." Indeed: "We propose that the abundance of horizontal gene transfers in free-living prokaryotes is a simple but necessary consequence of two opposite effects: i) their apparent genome size constraint and ii) the underlying expansion dynamics of their genome through gene duplication-divergence evolution…" A problem with this argument, however, is that it suggests that there is selection for horizontal gene transfer in prokaryotes (an arguable assumption), and further that this selection (seemingly) might be maintained for the group (i.e., group selection). I would suggest, instead, that bacteria might be able to "get away" with their relatively small genome sizes, given the proposed tendencies, because they have difficulty escaping horizontal gene transfer, an argument that in fact is quite consistent with prevailing ideas that, in the absence of sex, bacterial genomes tend to degrade (Lawrence, 2005) . As it seems likely that small genome sizes and horizontal gene exchange were present in living things perhaps even before there, technically, were living things, presumably bacteria simply have never fully abandoned these tendency.

Why be Sexual?

Explanations for the utility of sexuality abound. These range from microevolutionary utility (i.e., various fitness benefits) to greater macroevolutionary efficiency (i.e., greater rates of speciation and species survival). Sex, for example, can interfere with Muller's ratchet, as well as clonal interference. It also can limit the potential for deleterious alleles to hitchhike to fixation on otherwise well adapted genomes. Indeed, sex serves to counter the tendency of periodic selection to reduce population diversity, plus is a means by which beneficial alleles may make their way into the same genomes without multiple, independent mutation events.

While sex in obligately sexual eukaryotes may serve to limit the breadth of beneficial alleles that may be acquired sexually, at the same time non-meiotic sexual processes in eukaryotes, such as "You are what you eat", can provide similar sexual benefits as those seen in the much less sexual bacteria. Sexuality may also serve to increase progeny genetic diversity during transitions to new environments, such as prior to spore or cyst formation in response to a declining suitability of environments, or in response to the faster evolution of especially mutational diversity among parasites. Finally, recombination, specifically, likely serves in many or most organisms as a mechanism of repair especially of double-stranded nucleic-acid damage.

Note that the question of why be sexual also can be differentiated into to sub-questions. The first considers the question of why sex evolved in the first place while the second addresses the question of why sex is maintained. One can also use the phrase "origin of sex" when referring to what less ambiguously might be described instead as an "origin of gender". In addition, it is important to not confuse the utility (or lack thereof) of sexual processes with the obvious Darwinian utility of replication, which of course can often be found intimately associated with sexual processes.

In answer to the first question, why be sexual, it can be relevant to distinguish between the outcrossing and the recombination aspects of sex, where recombination almost certainly represents a mechanism of DNA repair whereas outcrossing in some instances, e.g., such as that seen in bacteria and viruses, may not even represent an adaptation so much as a consequence of factors that exist for reasons other than outcrossing. Among these other reasons include the accidental coinfection of the same cell (by viruses), the virus-mediated movement of cell DNA (transduction), movement of genes via conjugative plasmids, and, with the most controversy, the potential that transformation represents a means of DNA feeding by bacteria rather than an intentional mechanisms of DNA acquisition for genetic means (Redfield, 1993; 2001). Note that few people argue that sex arose for purposes of increasing genetic variation in individuals. This latter mechanism nevertheless is routinely invoked as a reason for the maintenance of sex by populations.

Microbes can be useful models for studying the evolution of sexuality. Xu (2004) provides a list of the benefits of employing microorganisms for such studies, many of which are applicable to more than just study of the evolution of sex (Elena and Lenski, 2003) . These microbe characteristics include that microbes are inexpensive to work with, that they can be grown to high densities easily, that they are readily amendable to long-term storage, that the degree of sexuality displayed (i.e., frequency of outcrossing) can both vary greatly between systems and may be experimentally controlled, that genetic manipulation such as via genetic engineering is often easily achieved, and that measurement of properties such as may be seen with versus without outcrossing can be easily and accurately accomplished. That is, microbes are easy to work with and have properties that are not only useful and interesting but analogous and even homologous to those studied, often through greater effort, in more familiar but nevertheless larger and less experimentally tractable organisms such as animals, plants, and macrofungi.