Introduction

Endosymbiosis, wherein cells of one organism live within the cells (or organs) of another, has evolved many times across the tree of life, in a wide diversity of taxa, and commonly involves intimate interaction between organisms from different kingdoms of life [1]. By enabling species to gain entirely novel traits, such evolutionary mergers of formerly independent species have had a major role in evolutionary innovation [2]. Notable examples of symbiosis-mediated innovations include the gain of autotrophy [3] and gain of nitrogen fixation [4]. Such innovations have permitted symbiotic organisms to invade new ecological zones [5] and have led to the formation of entirely new biomes, such as coral reefs. Consequently, endosymbioses underpin the function of many varied ecosystems, spanning terrestrial, freshwater and marine habitats [6]. By opening up new ecological opportunities, endosymbioses can thus act as key innovations that, over evolutionary timescales, can catalyze diversification and fuel adaptive radiation [7–9], although not always [10].

Besides their role in biodiversity, endosymbioses have also enabled the evolution of more complex organisms [11] by allowing the compartmentalization of functions into specialized structures or organs, thereby increasing organismal multifunctionality and modularity [12]. Most fundamentally, this is evident in the symbiotic origin of the organelles of eukaryotic cells, which perform specialized metabolic functions that would be inefficient (or impossible) if performed in the bulk cytoplasm. Such improved efficiency is believed to have provided the necessary energetic potential for the evolution of larger, multicellular organisms [13]. While eukaryotic organelles are an extreme example, the benefits of compartmentalization are evident more generally across diverse endosymbioses [12].

Despite the ecological and evolutionary importance of endosymbioses, their origins remain poorly understood and difficult to explain. Although many extant endosymbioses appear mutually beneficial, this need not have been the case at their origin [14]. Evolutionary theory suggests that explaining the origin of endosymbiosis through mutualism is challenging because it not only requires that symbiosis be beneficial for both parties, but also that the fitness interests of these unrelated species be aligned, such that whatever increases the fitness of one partner necessarily benefits the other [15]. These conditions are unlikely to be met in newly established partnerships. Accordingly, theory suggests that mutual benefit may be an improbable mechanism for explaining the origin of endosymbiosis [15]. Exploitation, on the other hand, offers a potentially more plausible route for the formation of endosymbiosis [16], and either the host or the endosymbiont can engage in exploitation of their partner. In this Essay, we focus principally on the situation where hosts exploit beneficial endosymbionts. Here, the host benefits at the symbiont’s expense and, provided hosts exert sufficient control over the beneficial symbionts to enforce interaction, the origin of endosymbioses can then be explained entirely from the self-interest of the host species. Once established, and seemingly regardless of the mode of origin (i.e., by mutualism or exploitation by hosts), hosts then often establish a degree of vertical transmission (such that endosymbionts are passed from mother to daughter at cell division) or mechanisms of endosymbiont selection (such that noncooperative endosymbionts can be excluded). This increased fidelity between symbiotic partners sets the scene for the evolution of dependence, whereby adaptation to the symbiotic state is associated with the loss of free-living (Box 1) ability [17]. The particular combination of vertical transmission and dependence necessarily creates strong alignment of the fitness interests of symbiotic partners [18].

The purpose of this Essay is to explore the evolutionary processes driving such adaptation to the symbiotic state and the concomitant loss of free-living fitness, which ultimately leads to increased dependence and alignment of the fitness interests of symbiotic partners.

Conceptualizing endosymbiosis as niche adaptation

Obligate endosymbionts often have highly reduced genomes, far smaller than those of free-living relatives, encoding fewer functions and containing higher numbers of pseudogenes (Box 1) [19]. As such, the evolution of obligate endosymbionts has typically been viewed through a lens of genome degradation: a progressive loss of function driven by random mutations accumulating due to genetic drift (Box 1) within small populations that are strictly clonal [20]. While genome degradation is likely to be a major part of the story of obligate endosymbiont evolution, the same is often not the case for facultative symbionts (Box 1) or those without strict vertical transmission, such as Rhizobia, which commonly retain comparatively large genomes. Nonetheless, the prevailing view that progressive genome degradation driven by genetic drift predominates endosymbiont evolution misses potentially important selection-driven evolutionary processes occurring earlier during the establishment of symbiosis. This limited perspective likely arises because most of what we know of endosymbiont evolution comes from studies of long-established interactions, where signals of earlier evolutionary events are likely to have been lost amid the noise of subsequent genetic drift.

What might these selection-driven evolutionary processes be and what kinds of signals would they leave behind in endosymbiont populations? For endosymbionts, the transition to endosymbiosis is one commonly involving a shift from an exterior free-living to an interior within-host environment, which is conceptually equivalent to what any population experiences when colonizing a new ecological niche. As such, to predict the potential evolutionary pathways for endosymbionts upon forming a symbiosis, we can borrow from the body of theory exploring evolutionary diversification and ecological specialization [21,22]. Formally, ecological specialists display a negative genetic correlation (Box 1) across ecological niches, such that adaptation to one niche is associated with reduced fitness in an alternative, contrasting niche, termed a cost of adaptation [23]. Crucially, negative genetic correlations between fitness in free-living and within-host niches due to costs of adaptation can build up through distinct genetic mechanisms, with different consequences for the rate and repeatability of evolutionary trajectories. Below, we place the origin of endosymbiosis within this evolutionary diversification framework.

It is highly probable that the new within-host niche of nascent endosymbionts will vary from their ancestral free-living niche along multiple axes. Key axes include environmental conditions, resources, and external threats. For example, pH and osmolarity are likely to be key environmental variables; within-host environments are likely to be less variable than exterior environments, and these factors may be at very different levels between free-living and within-host niches. The identity and variability of nutrients and energy sources (for example, organic versus inorganic nitrogen) will also differ between free-living and within-host environments. Furthermore, the presence, identity, variability, and virulence of biotic threats (including predators, viruses, and phages) will also differ between free-living and within-host niches; although, within-host niches are not without threats, notably the host immune response. Consequently, if sufficiently contrasting, these free-living and within-host niches are likely to impose divergent selection, favoring distinct sets of traits and adaptations in each. If we assume that the ancestor was well-adapted to the original exterior environment, then adaptation to the within-host niche is unlikely to improve free-living fitness, but may reduce it if there is a cost of adaptation [23]. Such a cost of adaptation would arise when a positive direct response to selection in terms of improved within-host fitness is accompanied by a negative indirect response to selection in terms of reduced free-living fitness. This causes a negative genetic correlation between fitnesses in the free-living and within-host niches.

A cost of adaptation can arise through 2 principle genetic mechanisms: mutational decay or a trade-off arising from antagonistic pleiotropy (Fig 1) [23]. The mechanism most commonly implicated in the evolution of symbioses, as explained above, is mutational decay. Here, the new endosymbiont adaptations selected in the within-host niche themselves have no detrimental effect on free-living fitness, but other traits crucial for free-living undergo mutational decay owing to disuse. Mutations can accumulate in the genes encoding such traits, which although (nearly) neutral in the within-host niche, cause progressive loss of fitness in the free-living niche. Such conditionally deleterious mutations (Box 1) can become fixed by genetic drift alone and are not mechanistically linked to the selective process driving endosymbiont within-host adaptation. Alternatively, the adaptive mutations that improve endosymbiont fitness in the within-host niche could themselves directly reduce fitness in the free-living niche, termed antagonistic pleiotropy. Here, endosymbiont within-host adaptation is mechanistically linked to the loss of free-living fitness, and the consequent negative genetic correlation is known as a trade-off.

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Fig 1. Trade-offs versus mutational decay in the evolution of endosymbiosis.

Under trade-offs, beneficial mutations (green line) in the intracellular niche cause reduced fitness in the extracellular niche through antagonistic pleiotropy (AP). Under mutational decay, beneficial mutations (blue line) in the intracellular niche have no effect on extracellular fitness (non-AP), and negative genetic correlations build up over longer timescales though accumulation by genetic drift of (nearly) neutral mutations that are conditionally deleterious in the extracellular niche (dotted line).

https://doi.org/10.1371/journal.pbio.3002580.g001

Both mutational decay and antagonistic pleiotropy thus create negative genetic correlations among non-symbiotic and endosymbiotic genotype fitnesses across free-living and within-host niches. However, we would expect the dynamics and repeatability of the evolutionary processes driving these negative genetic correlations to differ (Fig 1). Evolution driven by selection on a trade-off should happen much faster than mutational decay by genetic drift. As such, trade-offs would be expected to cause rapid loss of extracellular fitness strongly reinforcing symbiosis, compared to the more progressive and gradual fitness decline caused by mutational decay. Moreover, the order in which mutations are fixed should be more predictable for selection on trade-offs, sequenced according to their fitness effects in the within-host niche, whereas (nearly) neutral mutations may become fixed in any order. By extension, we would expect that selection-driven evolution would result in more repeatable evolutionary outcomes between independent populations (i.e., parallel evolution), but that the outcomes of mutational decay would be closer to random sampling of mutational events per population. However, deviations from randomness, such as mutational biases, and/or mild selective benefits to losing free-living traits (for example, owing to their metabolic cost) could enhance the predictability of mutational decay.

Evidence for the role of fitness trade-offs in symbiosis

The dichotomy we outline between trade-offs and mutational decay is, of course, an oversimplification. Indeed, it is probable that both processes combine to determine the evolution of endosymbionts. Nonetheless, a clear expectation deriving from their comparison is that selective processes driven by trade-offs are likely to dominate the early-stage evolution of endosymbioses, whereas mutational decay will become increasingly dominant in later-stage, longer-established endosymbioses. As such, it is likely that traditional approaches for studying endosymbiont evolution (for example, by comparing genomes of long-established endosymbionts with distant free-living relatives) are unlikely to be able to distinguish between these mechanisms. In this section, we examine the experimental approaches that enable us to study this question (Fig 2) and what data already exist to support a role for trade-offs in the evolution of endosymbiosis.

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Fig 2. Experimental approaches to studying the role of trade-offs in symbiosis.

There are 4 main experimental approaches to studying the role of trade-offs in the evolution of symbiosis: comparative studies of early-stage endosymbioses in nature; experimental evolution of host-shifted endosymbiosis; experimental evolution of endosymbionts without hosts; and experimental evolution of synthetic endosymbioses. The most powerful approach for understanding the origins of symbiosis is to experimentally evolve newly established synthetic symbioses because this directly recapitulates an origin event that is usually lost deep in evolutionary time for most extant symbioses. Created using Biorender.

https://doi.org/10.1371/journal.pbio.3002580.g002

Conclusion and future outlook

Trade-offs are likely to have an important role in the origin of endosymbiosis. Only trade-offs cause rapid loss of free-living fitness through antagonistic pleiotropy as an endosymbiont adapts to its new within-host niche, enforcing the endosymbiotic lifestyle. By contrast, under mutational decay, loss of free-living fitness occurs more gradually, such that nascent endosymbionts could inhabit either niche for a more extended period. These mechanisms are not, however, mutually exclusive and it is likely that early evolutionary events mediated by trade-offs become hidden by subsequent mutational decay in many extant symbioses. This limits our ability to study the role of trade-offs in symbiosis retrospectively, although some systems such as the bacterium Arsenophonus do offer glimpses of the entire transition from free-living to obligate endosymbiont [24].

Instead, experimental evolutionary biologists have found multiple creative ways to study trade-offs prospectively, including studying free-living adaptation, host-shifted associations, and creating entirely synthetic symbioses (Fig 2). Of these approaches, synthetic symbioses offer the greatest potential to study symbiotic origins, because these directly recapitulate this event, which is lost deep in evolutionary time for most natural symbioses. Common mutational targets causing antagonistic pleiotropic trade-offs are regulatory genes, including quorum-sensing systems and global metabolic regulators. Here, a single mutation can have major phenotypic effects through altering the expression of many other genes, exemplified by the E. coli synthetic endosymbionts of stinkbugs, in which mutation of the CCR regulator altered expression of hundreds of genes, remodeling metabolism and altering cell shape [35]. Wholesale changes in the expression of the genome will be likely to relax selection on regions no longer expressed in the within-host niche, enabling their degradation through mutational decay over longer timescales. The apparent importance of regulatory mutations in trade-offs highlights the need to integrate multiple omics data—including transcriptomes, proteomes, and metabolomes—to fully understand how these levels of cellular organization interconnect and manifest as an integrated endosymbiotic phenotype.