The most challenging of all the major evolutionary transitions appears to be the origin of eukaryotes [78]. Certainly, all evidence suggests a long period in which life on earth was dominated by prokaryotes. What were the obstacles that hindered this transition for 2 billion years? While we may never completely comprehend the answers to this question, there can be no doubt that evolutionary conflict was a serious hurdle. Metabolism in general and chemiosmosis in particular likely had crucial roles in mediating these conflicts.

The advantages of eukaryotic cells are straightforward. Not only are they more complex, but they are also larger than their prokaryotic forebearers. As Bonner [79] points out, “…the reason for non-stop selection for organisms of increased size is that the top of the size scale is an ever-present open niche and has been open during the entire course of organic evolution”. Larger size provides a number of ecological advantages including the exploitation of more and different food resources, more efficient dispersal (e.g., escaping the constraints of low Reynolds numbers), producing more offspring, and escaping predators [80]. Prokaryotes, however, face a conundrum that greatly limits their options for size increase: their energy-converting complexes are found on the cell membrane [81]. If a prokaryotic cell gets larger, there is less surface to convert energy and more volume requiring energy conversion. Despite some exotic exceptions, by-and-large prokaryotes never transcended these surface-to-volume constraints.

One way to circumvent these constraints is to move small energy-converting cells inside a larger complex cell, thus freeing the external membrane from duties related to energy conversion [81,82]. This arrangement allows for the complex cell to increase in size. From this perspective, endosymbiosis is integral to the evolution of eukaryotes [83]. A clever engineering solution for surface-to-volume constraints, however, results in a levels-of-selection nightmare.

This can be better understood by recognizing the fundamental duality of life. All life takes up energy from the environment and typically converts this energy into more useful forms. At the same time, life involves information and replication. There remains a constant tension between these two attributes in all forms of life. In a structured population, a biological unit that relies on its sister units for energy conversion and specializes in replication may be favored. The success of such a unit, however, is frequency dependent [7]. As the proportion of units specializing in replication increases within a group, the availability of the products of energy conversion inexorably decreases. At some point, the lack of these products threatens the entire community. For the community to persist, mechanisms of conflict mediation must evolve.

While these issues prevail throughout the entire history of life, they were particularly acute in the origin of the eukaryotic cell. Two great symbioses—that of the mitochondrion and the plastid—figure prominently in the rise of eukaryotes. Both of these symbioses involve energy-converting lower-level units, with groups of these lower-level units and host cells constituting the higher-level units. In both cases, evolutionary conflict no doubt involved the usual pattern of lower-level units specializing in replication at the expense of distributing the products of energy conversion to the higher-level community.

Mitochondria may have been attendant to the very early stages in eukaryotic origins and were very likely a feature of the last eukaryotic common ancestor (LECA) [84]. Plastids were acquired by some eukaryotes not long after LECA [85,86]. The origin of eukaryotes continues to be one of the most debated topics in all of biology. Considerations have turned to the nature of the original host and metabolic relationships that may have led to the endosymbiosis, topics that continue to attract considerable attention and sometimes sharp disagreements [87,88,89,90,91,92].

In modern eukaryotes, evolutionary conflict is mediated by a number of mechanisms (e.g., the nearly complete loss of organellar genomes). Nevertheless, the evolution of many of these mechanisms required strong selection on the higher-level units. Such selection cannot have molded the initial steps in the symbiosis, which by definition involved only lower-level units. Notably, mitochondria, chloroplasts, and their bacterial relatives convert energy using chemiosmosis, which involves electron flow and proton extrusion as described above.

The discovery of quantum electron transfer in biological systems [93] occurred contemporaneously with the development the chemiosmotic theory [94]. Later work showed that membrane-bound electron carriers could form “super-complexes.” Both quantum electron transfer and super-complex formation result in extremely rapid electron transfer in chemiosmotic processes [95,96]. When chemiosmosis is linked to the soluble reactions that store energy, this rapidity poses problems. For example, the ATP and NADPH produced by chemiosmosis in chloroplasts is stored by RuBisCO and other Calvin Cycle enzymes by reducing carbon dioxide. RuBisCO is thus the most abundant protein on Earth. The channeling of chemiosmosis into slower soluble reactions with potentially limited storage capacity has other effects. As pointed out above, if products cannot be consumed or stored and their accumulation inhibits electron flow, electron carriers become highly reduced, and these electrons may divert to molecular oxygen forming ROS. The chemiosmotic process itself, by separating protons and electrons, is the cause of ROS formation.

Cooperation is not an automatic outcome of evolutionary interactions because it often involves costs. Selection may thus favor incompatible features of hosts and symbionts [97]. A symbiont may be selected to defect, e.g., by hoarding resources obtained from the host and symbiont community and using these resources for its own replication. While cooperative symbionts at least in part forgo reproduction and share resources with the larger community, defectors may gain a replicatory advantage by sequestering these resources. Yet, by sharing resources with the host, the cooperative symbiont community may establish a durable environment and insure its long-term persistence. Even with these long-term advantages, the higher-level unit (the host and the larger symbiont community) may be exploited by lower-level defectors. While symbiosis can be classified as mutualistic or parasitic (i.e., in bilateral terms), mechanistically these evolutionary interactions are multilateral and multilevel. Defecting symbionts can still arise and flourish even when a host-symbiont community appears to be dominated by mutualistic interactions. Mechanisms of conflict mediation are always necessary to control lower-level defectors. A structured population may mediate conflicts, e.g., if a population is subdivided into many small groups. These groups can lead to kin selection and reciprocity. Further, groups of cooperators can arise purely by chance. In this way, even if cooperation is selected against at the level of the individual, it can still arise and be favored at the level of the group [11].

Remarkably, ROS signaling pathways may serve as arbiters of evolutionary conflict. Chemiosmosis proceeds rapidly and conserves a large proportion of the energetic input, quickly generating products. The products of chemiosmosis may be stored, but storage mechanisms can be slow relative to chemiosmosis. Additionally, storage capacity is not unlimited. When conditions are favorable, chemiosmotic cells and organisms are often confronted with “end-product inhibition” [56,57,58,98], which can have detrimental consequences. As discussed above, an overabundance of product can inhibit electron flow. In the presence of molecular oxygen, this enhances the formation of ROS. Such partially reduced forms of oxygen can have a variety of deleterious effects. To avoid blocking electron flow, the abundant products of chemiosmotic energy conversion must be consumed, stored, or simply gotten rid of. While mechanisms that modulate chemiosmosis are available [99,100], alternatively the products of chemiosmosis can be dispersed into the environment. Because ROS are ephemeral, they are particularly well suited for this role. With the onset of end-product inhibition, ROS would increase, possibly signaling the initiation of the export of chemiosmotic products. As these products were exported, end-product inhibition would lessen, and ROS would rapidly subside.

Crucially, chemiosmosis and its consequential ROS formation can thus under some circumstances favor sharing rather than hoarding the products of energy conversion. Sharing can lead to the formation of groups, whether symbiotic associations or multicellular aggregations. These can be thought of as a by-product of sharing, which in turn is a by-product of chemiosmosis. Thus formed, these groups may be the key to the evolution of cooperation, potentiating kin selection, reciprocity, or group selection. Indeed, the biophysical constraints of chemiosmosis may have provided the initial mediation of conflicts associated with origins of eukaryotic cells and thus allowed the first steps in the formation of complex life on Earth.

Mechanisms that mediate evolutionary conflict typically constrain the variation at the lower level, so that defectors are less likely to evolve, or increase the variation at the higher level, so that cooperative groups are favored by selection [9,10]. ROS signaling pathways may or may not fit this paradigm. If a pathway leads to cell death, it constrains variation at the lower level. On the other hand, if a pathway leads to nutrient export and group formation, this clearly does not constrain lower-level variation. By leading to group formation, it may, however, be viewed as increasing the variation at the higher level. Depending on which pathway(s) are activated, ROS could lead to one or the other. Hence, as further discussed below, ROS are termed “arbiters” of evolutionary conflict to better represent their putative role in initiating a signaling process that resolves conflict and leads to the emergence of a higher-level unit.

As endosymbioses became established and higher-level units emerged, energy-converting lower-level units remained an obstacle to cooperation. Numerous additional mechanisms of conflict mediation subsequently evolved, facilitated by the large populations of higher-level units, which each contained relatively small populations of lower-level units. Purely on the basis of chance, some higher-level units could be formed from cooperative lower-level units. These groups of cooperators could then outcompete cells that included one or many defectors. Nevertheless, under conditions that lead to end-product inhibition, defectors that arise via loss-of-function mutations could still be eliminated from cooperative groups. In this way, unicellular eukaryotes developed robust and stable symbioses.

The biophysics of chemiosmosis, particularly the formation of ROS as a by-product, were likely central to the initial steps of eukaryogenesis, when only lower-level selection was operating. Both quantum electron transfer and super-complex formation contribute to high rates of chemiosmotic processes. Large quantities of products may thus be produced. While several mechanisms can modulate chemiosmosis, alternatively excess product can be released, thus providing a way to protect against an accumulation of end-products and the consequent formation of dangerous by-products such as ROS. Sharing may thus occur with little cost and considerable benefit—"the free lunch you are forced to make” [16]—thus facilitating groups. Such groups can lead to cooperative symbioses. Even after such groups have formed, however, cooperators are always vulnerable to exploitation by defectors, so additional mechanisms of conflict mediation are usually necessary.

While there are examples of prokaryotic aggregations (e.g., biofilms), these pale in comparison to eukaryotic multicellularity (e.g., Conway Morris [101]: “The history of life shouts ‘Look! Once there was bacteria, now there is New York’”). Eukaryotes seems particularly able to form complex, multicellular organisms. Possibly, the mechanisms of cooperation established by the eukaryotic cell may have been repeatedly co-opted during the emergence of eukaryotic multicellularity [102]. In multicellular organisms, the result may be fundamental relationships between nutrient supply, metabolism, and cooperation that are only now beginning to come into focus [16,17,18].