Download Introduction Cooperative interactions, or mutualisms, are ubiquitous

Introduction
Cooperative interactions, or mutualisms, are ubiquitous in nature and essential in the
evolutionary diversification of life (Stachowicz, 2001). They range from obligate endosymbioses
such as the mitochondria in our own cells, to diffuse facultative interactions among free-living
species, such as those found between most plants and their pollinators. They have greatly
facilitated the lateral acquisition of novel traits and structures, and have generated the evolution
of life as we know it (Bronstein, Alarcón, & Geber, 2006; Douglas, 2010). And yet, the study of
mutualisms as non-trivial biological phenomena is a recent development in biology relative to
the study of antagonistic interspecific interactions (e.g. competition, predation). However, in the
past 50 years mutualistic interactions have received increased recognition as drivers of the major
ecological and evolutionary processes that unite the three domains of life (McFall-Ngai, 2008).
Even as the study of mutualism becomes more common however, its definition remains
unsettled (Klepzig et al., 2009). A brief treatment of terminology is therefore in order, as
biologists have yet to converge on an accepted and consistent lexicon for the study of positive
species interactions. Part of this difficulty arises from the lack of formal vocabulary
distinguishing between inter- and intraspecific positive interactions, which differ in biologically
fundamental ways. In general, 'cooperation' and 'cooperative interactions' are most useful for
describing positive interactions among individuals of the same species, such as those between
human societies, eusocial insects, social mammals, and shoaling fish. Intraspecific cooperation
is something of a special case though, because the cooperating individuals are closely related to
one another, at least relative to other species. In contrast, interspecific mutualisms (the focus of
this dissertation) involve individuals that are not closely related and therefore kin selection—one
mechanism known to promote cooperative behaviors among closely related individuals—cannot
operate. Similarly, and perhaps most importantly, interspecific mutualisms are controlled by the
genomes of two distinct entities such that opportunities for conflict and tradeoffs among partners'
interaction strategies can lead to complex evolutionary dynamics and interaction outcomes (Boza
et al., 2012). However, the distinction made here between cooperation and mutualism is by no
means formally accepted within the symbiology research community, but is a general
observation gleaned from the relevant literature. Finally, the term symbiosis, which is perhaps
the most widely recognized term among non-scientific audiences, was first used by Anton de
Bary in 1879 to refer to any association among individuals of different species, typically
involving persistent contact between individuals (Sapp, 1994). This therefore includes a wide
variety of interspecific interactions, including pathogenic and parasitic interactions as well as
positive interactions. Douglas (2010) points out that this original definition of symbiosis is
therefore so broad as to be generally unuseful: a catchall phrase that doesn't necessarily
distinguish symbiosis from any other type of biological interaction. However, it remains a useful
term for communicating with general audiences, and considering the fluidity of most species
interactions in grading between positive and negative associations (as discussed below), the term
symbiosis is perhaps still appropriate not in spite of its generality, but because of it.
At a bare minimum, mutualistic interactions describe associations among two or more
individuals from which all participants derive a net benefit. As is to be expected in the highly
conditional realm of the living world, however, this is rarely straightforward. A net benefit
occurs when the costs of interacting are outweighed by the benefits received, but the cost-benefit
ratios for many organisms are known to be highly dependent on the biotic and abiotic context in
which the interaction occurs (Bronstein, 1994; Kiers et al., 2010). The outcomes of many
positive species interactions, especially facultative mutualisms, depend on external conditions
such that an interaction can be mutually beneficial under certain conditions, but commensal or
even antagonistic in different environments. External factors such as climate, community
composition, and resource availability may therefore have profound effects on the dynamics,
outcomes, and persistence of many mutualisms. This has led to the recognition that conditional
mutualisms—interactions that can grade from mutualistic to commensal or antagonistic with
varying external factors—are more the general rule than the exception. Yet despite the high
degree of variability in the nature and outcomes of positive species interactions, they remain
persistent and highly influential features in the ecology and evolution of all organisms.
Ecological Significance of Mutualism
Positive species interactions are found in all ecosystems and play a principal role in
determining the distribution and abundance of organisms at both small and large spatiotemporal
scales (Strauss & Irwin, 2004). While abiotic forces determine a species’ fundamental niche,
biotic factors contribute to its realized niche and actual distribution. Thus, in contrast to
antagonistic interactions (i.e. competition, predation), positive interactions can lead to the
expansion and/or shifting of a species' realized niche relative to its fundamental niche, due to the
potential for mutualists to ameliorate abiotic and biotic stressors (Chase & Leibold, 2003).
Furthermore, mutualistic interactions can have profound effects on population structure,
community dynamics, and ecosystem functioning. As an example, we can consider foundation
species such as land plants and corals. Mutualists of foundation species can influence diverse
functional traits of their hosts, including metabolic capabilities, nutritional status, hormonal
pathways, and defensive chemistry. By altering the functional traits of foundation species,
mutualists can play a role in determining niche breadth, community composition, relative
abundance, and ecosystem productivity and nutrient dynamics (Friesen et al., 2011). Finally, the
presence of symbioses can contribute to complex indirect interaction networks in which
mutualistic associations have cascading effects on associations between other species in the
community; for example, mutualists can alter the interspecific interactions between their hosts
and the host's competitors, mutualists, predators, herbivores, and pathogens via effects on host
functional traits (Friesen et al., 2011; Utsumi, Kishida, & Ohgushi, 2010).
Evolutionary Significance of Mutualism
While it is clear that mutualisms are of fundamental importance in all ecosystems, their
very existence is puzzling, as the evolution and maintenance of mutualisms has posed one of the
most important and persistent theoretical problems in evolutionary biology. Much of the
language used to describe mutualism implies that these interactions involve deliberately helpful
or altruistic organisms. However it is more accurate to view mutualisms as the reciprocal
exploitation of two or more species that still yield net benefits to all players (Herre et al., 1999).
From the perspective that mutualisms are exploitative interactions, natural selection favors
individuals that can maximize the benefits they receive from an interaction while minimizing the
costs they incur. This phenomenon is often cast within the framework of game theory, in which
individuals receive a benefit according to both their own strategy and the strategy of their
interaction partner. Players receive the biggest payoff by minimizing the costs associated with
cooperating while maximizing the benefits received from their partner. In many cooperation
games, such as the classic Prisoner’s Dilemma (Trivers 1971), cooperation proves evolutionarily
untenable and defection becomes the dominant strategy. Indeed, there is ample evidence of
defection in real biological systems, in which individuals evolve to maximize the rewards they
receive from an interaction while providing reduced or no benefit in return (Douglas, 2008;
Sachs & Simms, 2006). These individuals are typically referred to as exploiters, cheaters, or
parasites of mutualism, and from the perspective that these individuals are more fit relative to
their conspecifics that receive a smaller payoff from the interaction, natural selection should
favor cheaters over cooperators. Understanding the conditions that maintain cooperative
interactions in nature has thus been an area of active theoretical research for decades, with many
factors identified as stabilizing interspecific mutualism, such as spatial structure, partner choice
and game iterations (Axelrod & Hamilton 1981; Boza et al. 2012; Nowak & Sigmund 2000).
Regardless of the specific mechanisms invoked to explain the evolution and maintenance
of mutualistic interactions, their persistence and prevalence across all levels of biological
organization is unequivocal. We now know that mutualism has evolved independently and
repeatedly in many lineages. Furthermore, positive associations are at the root of some of the
most important evolutionary transitions in the history of life. According to endosymbiotic theory,
the evolution of eukaryotes owes its origins to symbiosis, as the key organelles of eukaryotic
cells originated from free-living bacteria that were incorporated into the cell as endosymbionts.
There is ample molecular and biochemical evidence that the mitochondria in eukaryotic cells
developed from proteobacteria, and mitochondria have retained separate DNA that bears closer
resemblance to bacterial DNA—both in size and structure—than the DNA found in the cell
nucleus. Similarly, all plants owe their photosynthetic capabilities to endoymbioisis, as their
chloroplasts developed from cyanobacteria acquired by single-celled prokaryotes. Dr. Ernst
Mayr, one of history's most influential evolutionary biologists, opined that the evolutionary
transition between free-living bacteria to eukaryotic intracellular symbionts represents the single
most evolutionarily important transition in the history of life (Ryan, 2006). Terrestrial plants also
owe their evolutionary success to microbial symbionts: it is widely accepted that early plants
were able to colonize terrestrial environments only with the assistance of symbiotic arbuscular
mycorrhizal fungi that could provide plants with nutrients and protection from desiccation
(Humphreys et al., 2010; Pirozynski & Malloch, 1975). It is difficult to imagine how different
the earth would be today if these ancient symbioses had not been forged. Indeed, the metabolic,
physiological, and nutritional capabilities conferred by early endosymbioses facilitated the very
evolution and proliferation of eukaryotic life.
What characteristics of symbiosis make it so special as to allow for such evolutionary
innovation? Perhaps several explanations could be invoked, but key among them is that
symbiosis provides a mechanism for phenotypic novelty at very fast timescales and without
mutation. According to the Modern Evolutionary Synthesis—the reconciliation and unification
of formerly disparate evolutionary concepts that took place in the 1930's and '40's—organisms
adapt to their environments via the slow process of descent with heritable modification.
However, we now know that vertical transmission is not the only mechanism for the proliferation
of adaptive traits, and that lateral acquisition has allowed traits of great ecological and
evolutionary importance to be gained from phylogenetically distant taxa (Douglas, 2010).
Currently there are two known mechanisms of lateral acquisition of phenotypic novelty:
horizontal gene transfer among bacteria, and symbiosis. Horizontal gene transfer allows
unrelated bacteria to exchange genes that confer diverse functions, and has been an important
mechanism in the evolution of antibiotic resistance and metabolic capabilities. In symbioses,
however, entire organisms are acquired rather than individual genes, such that a single
evolutionary event can confer novel metabolic capabilities, physiological thresholds, defensive
capabilities, and more. Relative to other evolutionary processes, mutualisms provide
opportunities for substantial evolutionary innovation over incredibly fast timescales, obviating
many of the challenges associated with crossing adaptive valleys through incremental changes.
In extreme cases, symbiosis can generate not only novel traits but novel organisms. Through the
process of symbiogenesis a distinct organism can be formed through the merging of two
symbionts in a heritable association, such that the evolutionary fates of the mutualists are tied
and natural selection acts jointly on the symbiotic pair rather than individually on the constituent
partners (Margulis, 2008). Even for less extreme cases in which symbiotic organisms retain their
distinct species identities, the hologenome theory of evolution has been put forth to advocate for
the consideration of the holobiont—the animal or plant plus all of its associated microbiota—as
the appropriate unit of selection in evolution (Zilber-Rosenberg & Rosenberg, 2008). Not
surprisingly, symbiogenesis and the hologenome theory of evolution present considerable
challenges to the conceptual framework of the Modern Synthesis, and current evolutionary and
ecological theory will need to be reconsidered in light of what is now known about symbiology
and microbiology (McFall-Ngai et al., 2013). This represents both an opportunity and challenge
for the study of mutualism, however, which contains its own conceptual and practical
shortcomings as well.
Significance of Multiple Mutualisms
One major limitation of previous studies of mutualism is that they typically cast
mutualisms as strictly pairwise interactions, often between closely coevolved species (Afkhami
et al., In preparation). While symbioses certainly can include strictly bipartite associations, it is
increasingly recognized that tight pairwise interactions are far less common in nature than
associations between sets of interacting partners (e.g. “mutualist guilds,” Stanton 2003). The
effect of having multiple functionally equivalent partners available (versus one partner in
pairwise interactions) is unclear, and outcomes likely depend on the functional diversity of
available mutualists. In the simplest case, multiple available partners could belong to the same
functional group and therefore provide the same service to their host in exchange for the same
reward. The availability of multiple mutualists may be beneficial if it allows the members to
select the best partner—the partner that provides the greatest benefit while conferring the lowest
cost. Alternatively, having more functionally redundant partners could result in greater costs
while providing no additional benefit. Similarly, if there is competition among several partners
for access to one mutualistic partner (i.e. a host), then a “Tragedy of the Commons” scenario
may arise, where partners exploit the host at unsustainable levels leading to reduced benefit (or
harm) to the host (Douglas, 2010). On the other hand, associating with multiple partners at the
same time may be preferable if each partner provides complementary benefits (Stachowicz &
Whitlatch, 2005). In functionally diverse multiplayer mutualisms, interaction guilds often
involve the asymmetric exchange of different services between partners, frequently accompanied
by evolutionary tradeoffs (Bronstein et al., 2006). The result is that non-additive effects emerge
from interactions involving multiple concurrent mutualists, and examples of both synergy and
conflict can be found in many multiplayer mutualisms. Considering the prevalence of nonadditive effects in multiplayer mutualisms, it is likely that most pairwise studies fail to capture
the true dynamics—and therefore the significance—of most mutualisms. These studies could
either underestimate the biological prevalence and importance of mutualism (if there are
complementarities or synergetic effects), or they could overestimate the importance of mutualism
(if there are conflicts among subsets of mutualists; Afkhami et al., In Preparation).
Given these pitfalls, it may appear surprising that both empirical and theoretical research
has focused primarily on pairwise interactions. There is of course, the very valid explanation that
pairwise interactions are much simpler to conceptualize, manipulate, and analyze, and therefore
provide the most tractable starting point for mutualism research. But it is also true that many
interactions are studied as pairwise because they at first appear to be pairwise to the casual (or
even not so casual) observer. In particular, it is easy to dismiss or overlook microscopic
organisms as participants in these interactions, but with recent advances in microbial ecology it
is increasingly clear that microscopic organisms are common participants in the mutualisms
between macroscopic organisms. These oft-overlooked players include rhizobia, mycorrhizae,
algae, enteric microbes, epiphytic and endophytic fungi, and many others. Embracing the
prevalence and complexity of multispecies mutualism, including those involving microscopic
organisms, not only improves our understanding of the role of mutualism in natural systems, but
presents opportunities for the reconsideration of current ecological and evolutionary principles
that have been largely based on macroecological observations (Little et al., 2008; McFall-Ngai et
al., 2013).
While much of the previous and current research in this area is focused on the ways in
which microscopic symbionts affect host traits, the role of microbes in determining the
magnitude and/or direction of interactions among free-living organisms is only now beginning to
be explored. This dissertation examines the role of symbiotic bacteria in mediating macroscopic
ecological community properties and host traits. A community ecology approach is taken in all
research chapters to address an overarching question: how do microscopic symbiotic organisms
alter the macroscopic interactions of their free-living hosts? Additionally, an organismal theme
unifies the research chapters, which investigate associations among plants, insects, and
bacteria—three organismal groups known to be functionally and numerically dominant in all
terrestrial ecosystems. It is my hope that this work contributes to the growing appreciation of the
significance of mutualism in nature, and that it highlights the need for revised ecological and
evolutionary thinking in light of symbiosis at both micro- and microscopic scales.
With ever-growing gratitude to my symbionts…
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