Interactions among Multiple Mutualists & links to underlying molecular mechanisms

In nature, most organisms interact simultaneously with multiple mutualistic species (e.g., plants with pollinators, microbial symbionts, and ants), however mutualism studies have traditionally focused on bipartite interactions between a single partner and host. Bipartite studies will underestimate the importance of mutualism for ecological and evolutionary dynamics if complementarity occurs among functionally different partners, and overestimate it if different partners are in conflict. In a synthesis paper (Afkhami et al. Ecology 2014), we merged two important, but disparate, research approaches (network and consumer-resource models) to define the multiple mutualist effects – MMEs – that occur when a focal species has multiple partner mutualists.

2014-07-04 16.09.05

Medicago truncatula with small nodules 2.5 weeks after inoculation with rhizobia.

2014-07-03 15.45.46

Medicago plants at 2.5 weeks: Left is a plant grown with both rhizobia and mycorrhizal fungi and right is a plant grown with neither.

In recent research, we have begun to integrate this conceptual framework with genomic approaches to provide new insights into the functional genomic basis of how organism balance and interact with multiple mutualists.

For example, we have used factorial experiments, genome-wide association mapping, and RNAseq with the tri-partite mutualism between Medicago truncatula, rhizobia, and arbuscular mycorrhizal fungi to (1) identify genes uniquely expressed in the presence of multiple mutualists and (2) to detect polymorphisms associated with plant fitness in response to multiple mutualists. Our first paper looking at MME identified pervasive genome-wide effects of multispecies microbial effects on gene expression, including genes whose expression responds non-additively to multiple mutualists (Afkhami & Stinchcombe Molecular Ecology 2016), and we are in the revision process on the first association mapping paper.  To see a great article on our Molecular Ecology paper click here.  We have also expanded this work in several new directions including how multispecies mutualisms impact coexpression network structure of their host plants, which has been led by University of Miami undergrad Sathvik Palakurty and is accepted for publication in Molecular Ecology. Further, we are pursuing the interaction between the primary symbionts influences the microbiome (collaboration with grad student Colleen Friel) and the role of microRNAs in these interactions (University of Miami ULINK program collaboration).

Impact of Mutualisms on Species Distributions and the Niche


Figure 2 from Afkhami et al 2014 Ecology Letters:  (A) Species distribution models demonstrated the geographic differentiation between fungal-associated (E+) and fungal-free (E-) Bromus laevipes. Habitat suitability for B. laevipes ranged from high (red) to low/unsuitable (blue). (B) E+ B. laevipes occupied a different climatic niche than E- populations, including uniquely occupying dry habitats (low values of PC3). Endophyte-associated changes to the climatic niche occurred along PC2-4, which explained ca. 45% of the variation in California’s climate. Filled circles = E+ populations, open circles = E- populations.

Understanding species distributions is a central goal of ecology and evolution,
and critical to predicting the consequences of climate change. Recent work has produced new theory on the roles of biotic interactions in shaping species’ distributions, but these effects are rarely investigated over large geographic scales. Unlike antagonistic species interactions which contract a species’ range, positive species interactions have the unique potential to ameliorate biotic and abiotic stressors, and may thus expand species niches, resulting in larger species ranges.

In a recent paper, I demonstrated that associating with a facultative mutualist can increase the range of a native species by thousands of square kilometers, or approximately 20% of the entire host range (Afkhami et al. Ecology Letters 2014). Combining (1) range-wide field observations from ~100 populations, (2) species distribution models, (3) 10 common garden experiments across the species range, and (4) greenhouse experiments, I showed that endophytic fungi can ameliorate drought stress and allow their grass host (Bromus laevipes) to grow in drier regions unoccupied by grasses that lack this partner. Range divergence between B. laevipes plants with and without endophytes was comparable to species-level divergence among congeners (other native Bromus), indicating that the mutualists’ effects were biologically significant.  Collaborators on this project were  Sharon Strauss and Pat Mcintyre. (To see this great article on our paper click here.)

Alterations to species niches and/or ranges caused by mutualisms have the potential to play an important role in the evolution and diversification of species. Mutualist-generated niche differentiation could pave the way for speciation, if selection diverges across niches, leading to reduced gene flow. Collaborators and I are examining how association with mutualistic nitrogen-fixing bacteria affects diversification rates across ~20,000 species of legumes, the third most speciose family of plants (Afkhami et al., 2018).

Conflict in Positive Interactions

Vertically transmitted symbionts (i.e., passed parent to offspring) associate with some of the most ecologically dominant species on Earth, and their fixation has

Ecology Letters Nov 15 cover.1-page-001

Cheaters must prosper: Reconciling theoretical and empirical perspectives on cheating in mutualism. (Ecology Letters, 2015)

led to major evolutionary transitions (e.g., chloroplasts). Theory predicts that vertically-transmitted symbionts will be highly mutualistic because the fitness of the symbiont and host are coupled. However, partner conflict still exists in these intimate associations.

(1) Jenn Rudgers and I documented the first example of imperfect vertical transmission in the widespread symbiosis between plants and fungal endophytes (Afkhami & Rudgers American Naturalist 2008), which has since been identified as a common phenomenon and a central cause of variation in endophyte frequencies in nature (Rudgers, Afkhami et al. Ecology 2009).

(2) In a collaboration with Jenn Rudger’s lab, we documented subtle conflict due to the maternal inheritance of symbiosis, which biased host sex allocation toward maternal seed production at a cost to the host (Gorischek, Afkhami et al. American Naturalist 2013). To our knowledge this is the first example of a
symbiont altering sex allocation in plants.

(3) As part of an NCEAS working group, I have been collaborating on a novel
synthesis of the empirical and theoretical literature on cheating in mutualisms (Jones, Afkhami, et al. Ecology Letters 2015) and (Gould et al, Ecology Letters, In Review).

Microbial Mutualists as Community Players

Keystone species are important for understanding the maintenance of biodiversity and alternative stable states, as well as in guiding decisions on the conservation and restoration of ecosystems. While studies of keystone species have focused on highly visible macro-organisms, I have shown that microfungal endophytes can function as keystone mutualists. A 3-year field manipulation of endophyte in a native Californian grass showed that high endophyte abundance dramatically increased plant community diversity and altered community composition by suppressing a dominant invasive plant. The quantitative effects of endophytes on community diversity was on par with textbook examples of keystone species, such as starfish in the intertidal. Moreover, endophytes increased plant community diversity in 5 additional common gardens spanning >400km across the host grass range, demonstrating the keystone role of these fungi occurs on a range-wide scale (Afkhami & Strauss, Ecology, 2016). I have also found that endophyte effects on plant communities cascade up to higher trophic levels, such as predatory arthropods (in collaboration with Sharon Strauss and Megan Anderson).

Water bowls to collect aerial arthropods in the community experiment plots.

Setting up pitfall traps to catch ground-dwelling arthropods in our community plots.

Surveying plant community diversity, density, and composition.

Demography of Mutualisms across Species Ranges

Common garden of Bromus laevipes at McLaughlin Reserve in the Coast Range of California.

Many studies of mutualisms quantify their short term fitness effects or the rewards exchanged among partners. To fully understand the consequences of mutualisms for long-term persistence of its participants, one must quantify fitness effects across the life cycle – because mutualists can have opposing effects at different life history stages. As a next step in investigating mutualism’s effect on species distributions, my lab will study demography across the species range. In 2009, I manipulated fungal endophyte abundance across five common gardens of plants from 11 Bromus laevipes populations. I have gathered 6 years of plant performance data (e.g., growth, reproduction, and survivorship) with which we will build integral projection models. To our knowledge, these gardens are the only set of replicated endophyte manipulations spanning a species’ native range, and have the potential to reveal complex, range-wide demographic effects of symbiosis.

Mutualistic Rewards = Fitness ?

We are interested in whether rewards traded among mutualist partners (in this case natural enemy defense) actually translate into meaningful fitness benefits for partners. Further, we are also interested in endophyte conferring protection to their hosts because it is another way in which they could impact communities, species ranges, and other ecological and evolutionary processes.


Natural enemy damage on B. laevipes observed in the field.

We know endophytes can deter natural enemies, reducing damage to their hosts for some species (e.g., Afkhami & Rudgers Environ. Entomology 2009), and I have recently found in quantitative surveys of damage and long term common gardens that endophytes in B. laevipes clearly deter enemies in their natural habitats. Further, genetic and chemical evidence showed that endophytes in B. laevipes produce a diversity of anti-herbivore alkaloids (Charlton, Craven, Afkhami et al. FEMS Microbiology Ecology 2014).  After documenting that the reward was conferred, I have also set up and collected 3 years of data on a field experiment looking at whether endophyte’s ability to deter enemies causes (or is only correlated with) increases in plant fitness.


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