Testing for microbe-mediated adaptation
Reciprocal transplant experiments can be used to understand how microbes
affect patterns of plant adaptation and to distinguish between
microbe-mediated local adaptation and microbe-mediated adaptive
plasticity. Reciprocal transplants can be employed exactly as in the
traditional design to study local adaptation and plasticity (Clausenet al. 1948) with the added element of manipulating microbes
(Fig. 2).
In classical reciprocal transplant experiments, seeds from one
population are transplanted both into a different habitat and replanted
into their natal habitat. Populations are often chosen because one or
more environmental variable is demonstrably different between
populations (e.g. serpentine vs. non-serpentine soils, Wrightet al. 2006 or presence or absence of a competing species, Lau
2006). A reciprocal transplant to test for microbe-mediated adaptation
would involve transplanting seeds into different habitats and providing
those plants with local or foreign microbes. Although it can be
difficult or impossible to completely exclude microorganisms, sterilized
control plants should be included to definitively attribute effects to
microbes (e.g. , if microbe-mediated local adaptation is strong,
then patterns of local adaptation will be much weaker or not evident
when microbes are absent).
A statistical model to understand interactive contributions of plant and
microbes to plant local adaptation includes some measure of plant
fitness (e.g. germination,
survival, fecundity, or ideally an integrated fitness metric
encompassing all life history stages [ASTER models] Geyer et
al. 2007; Shaw et al. 2008) as a response variable and habitat
type, plant source, microbial source, and sterilization as fixed effects
(Table 1). While this design potentially results in a four-way
interaction, with large enough sample sizes and patience, the complexity
of this and other interactions in the models can be understood,
especially through hypothesis testing with planned comparisons. The
figures presented here assume that patterns of adaptation are largely
attributable to habitat-specific microbes (Fig. 1B, C, D), however in
the text we also discuss the importance of significant sterilization
terms.
A three-way interaction between plant source, microbial source, and
habitat (GpxGmxE) would indicate
microbe-mediated local adaptation, if home genotypes have higher fitness
with their home microbes in their home habitat (Fig. 3A). Whereas, an
interaction between plant source and microbial source
(GpxGm) may indicate that plant
genotypes do best with their own microbes, regardless of habitat and are
locally adapted to their natal microbes (Fig. 3B). For example, Petipaset al. (2020) found that germination of Hypericum
perforatum sourced from limestone barrens was highest when transplanted
with microbes from limestone barrens into limestone habitats, a pattern
consistent with microbe-mediated local adaptation
(GpxGmxE). However, survival of H.
perforatum from limestone barrens was highest when transplanted with
microbes from limestone barrens regardless of transplant site, a pattern
suggesting that plants are locally adapted to their natal microbial
communities (Gp x Gm), rather than
indicating a pattern of microbe-mediated local adaptation.
An interaction between habitat and microbial source may be indicative of
microbe-mediated adaptive plasticity. In this case, local microbes are
responsible for locally adapted plant phenotypes; however, plant source
populations may be equally adept at utilizing local microbes
(i.e. they exhibit plasticity in microbe facing traits, allowing
them to attract, retain, regulate relevant local microbes; Fig. 3C). An
example of a trait underlying these adaptive patterns is
microbe-mediated flowering time, where plants only achieve the optimal
flowering time phenotype in association with local microbes (Fig. 3D).
Significant sterilization terms can also indicate microbe-mediated local
adaptation and microbe-mediated adaptive plasticity. For example, where
hosts strongly control microbiome composition (e.g. Gehring et al.
2017), microbe-mediated local adaptation would be evident as a
Gp x E x sterilization effect (rather than
GpxGmxE), where plant local adaptation
is only observed in the presence of microbes, but the microbial effects
are primarily driven by plant genotype specific recruitment from a
common soil source. This may still be considered microbe-mediated local
adaptation because the relative importance of different microbes varies
across context and the observed plant adaptation is still due to
interactions with relevant microbes. However, in this case, the plant
nearly entirely controls the composition of those microbes. Similarly,
sterilization could indicate microbe-mediated adaptive plasticity when
the environment elicits plastic shifts in plant phenotypes that in turn
influence microbial communities in ways that feedback to affect plant
fitness. Because the sterilization effect could also simply indicate the
benefits or costs of association with microbial communities, inferring
microbe-mediated adaptive plasticity from a significant sterilization
effect is challenging and would require: 1) showing that shifts in plant
phenotypes are associated with changes to the microbial community, and
2) these changes to the microbial community are associated with shifts
in plant traits and/or fitness. The potential for this type of
interaction is exemplified by host-mediated microbiome engineering
approaches that use plant phenotypes to select adaptive microbiomes
(Swenson et al. 2000; Panke-Buisse et al. 2015, 2017). In
one example, wheat was cyclically exposed to drought and in each cycle
the microbiomes associated with the most drought tolerant seedlings were
moved to the next cycle. After six cycles, a plant phenotype mediated
drought microbiome was selected that deferred drought symptoms in wheat
seedlings by five days (Jochum et al. 2019).
Additionally, microbe-mediated adaptation necessarily involves a
complicated experimental design and multiple players each with the
capacity of becoming locally adapted and exhibiting trait plasticity.
Consequently, there are many patterns that can emerge from these
interactions, which requires careful experimental planning (including of
experimental tests) and careful interpretation of the resulting data.
Finally, although we have discussed microbe-mediated local adaptation
and microbe-mediated adaptive plasticity largely independently, they are
not mutually exclusive.
The same caveats and considerations that apply to typical reciprocal
transplant experiments also apply to microbe-mediated adaptation
transplant experiments, including the relevant spatial scale, which
aspects of fitness to measure, and how long the experiment should be
monitored (Cheplick 2015). The latter is especially important given that
contamination in the field is likely to erode microbial treatments over
time. For tests of microbe-mediated adaptation, additional care should
be taken to determine relevant microbes, proper controls, and the
feasibility of different microbial transplant designs (Box 3, Fig. 4).