Statistical Analyses
All statistical analyses were performed using R Statistical Software
(version 4.2.1, R Core Team 2022). Linear mixed models (LMM) were first
used to examine the effects of rarity, phylogenetic relatedness of
conditioned soil (soil origin), N enrichment, and fungicide application
on total plant biomass, ECM colonization, and DSE colonization (“lmer”
function in “lme4” package, R) (S1 Table ). The LMM yielded an
interactive effect between rarity and soil origin, however, there was no
main effect of fungicide nor interactive effects between rarity/soil
origin and other fixed effects on total biomass. On the other hand,
fungicide demonstrated a main effect on ECM colonization and DSE
colonization. To account for the effects of N enrichment (low vs. high)
and fungicide (live cultures vs. fungicide) application on total
biomass, ECM colonization, and DSE colonization, N enrichment was
utilized as a fixed effect and fungicide application was accounted for
in the error structure of all linear mixed models as a random effect.
Specifically, to address Hypothesis 1 and 2 that total plant biomass,
ECM colonization, and DSE colonization vary based on the rarity of plant
host species and the phylogenetic origins of conditioned soil inoculum
under different levels of N enrichment, linear mixed models were used to
determine the singular and interactive effects of rarity, soil origin,
and N enrichment on the total biomass, ECM colonization, DSE
colonization of eucalypts accounting for a blocked design in the error
structure of the model. Tukey HSD post-hoc analyses were completed for
all significant results (“ghlt” and “cld” functions in “multcomp”
package, R).
Additionally, standardized plant-soil feedback mean effect sizes
(Hedges’ g) were calculated for the total biomass and percent ECM
colonization of plant species grown in conspecific versus same lineage
soils, and conspecific versus opposite lineage soils (i.e., the soils
include rare and common species within and across lineages) on the basis
of the 2nd generation of plants that varied in level
of rarity, described above. Effect sizes were calculated using the
Hedges’ g statistic as it accounts and corrects for variance that may be
introduced by unequal or small sample sizes (Hassan et al. 2022).
Positive values of Hedges’ g indicated positive plant-soil feedbacks in
which the total biomass or percent ECM colonization was greater in soils
conditioned by similar or opposite lineages compared to conspecific
soils. These types of positive feedbacks in biomass and mycorrhizal
colonization mediated by phylogenetically similar soil conditions
represent a home-field advantage in which phylogenetically similar plant
species readily share microbial mutualists due to a shared evolutionary
history and symbiosis of host-associated microbial communities (Anacker
et al. 2014; Fitzpatrick et al. 2018; Kohl 2020; Kazarina et al. 2023).
On the other hand, negative values of Hedges’ g indicate negative
plant-soil feedbacks in which the total biomass or percent ECM
colonization was lower in soils conditioned by similar or opposite
lineages compared to conspecific soils. These types of negative
feedbacks in biomass and mycorrhizal colonization mediated by
phylogenetically similar or dissimilar soil conditions are indicative of
a disruptive effect on plant growth and inform the maintenance of trait
variation within plant communities. Hedges’ g values that were not
significantly different than 0 were representative of a neutral or lack
of plant-soil feedback. Therefore, the Hedges’ g values associated with
each comparison along the rarity gradient represented a measure of both
the strength and direction of microbially mediated plant-soil feedbacks.
The strength of plant-soil feedbacks was assessed by the magnitude of
the effect size, while the direction of the feedbacks was determined by
the sign of the effect size. Pearson product-moment correlations between
the effect sizes of total biomass and ECM colonization along a rarity
gradient were also analyzed to further understand the significance and
direction of feedbacks.
Results
In support of Hypothesis 1 that total plant biomass, ECM colonization,
and DSE colonization are related to the rarity of plant host, rare
species displayed significantly lower biomass, but higher rates of ECM
colonization than common counterparts when grown in a common garden
environment across all soil conditions. (Table 1 ;Figure 1 ). For example, the rarest species had 62% more ECM
colonization while maintaining 88% less total plant biomass than common
counterparts (Figure 1). The rarest species also demonstrated 64%
greater DSE colonization in soil than common species, however, this
difference was not significant at α=0.05. Further, the relationship
between total plant biomass and ECM colonization was significantly
dependent on soil N enrichment (Table 1). Eucalypt species across all
rarity levels grown in high N conditions demonstrated a 37 % increase
in biomass but 11 % decrease in ECM colonization than species under low
N conditions (Figure 2 ). Consequently, high N conditions
significantly facilitated higher levels of plant biomass but lower ECM
colonization, while low N conditions significantly supported lower
levels of plant biomass but higher ECM colonization. Additionally,
rarity and N treatment demonstrated a significant interaction to affect
the ECM colonization of Tasmanian eucalypts (Table 1; Figure 2). The
disparity between rare versus common biomass and ECM colonization was
more prevalent under high N enrichment, as demonstrated by a 166%
increase in biomass and a 64% decrease in ECM colonization from rare to
common species under high N conditions, compared to a 149% increase in
biomass and a 27% decrease in ECM colonization from rare to common
species under low N conditions.
In support of Hypothesis 2 that total plant biomass, ECM colonization,
and DSE colonization are related to the phylogenetic origin of
conditioned soil, there was a significant interaction between plant
rarity and soil origin; rare species maintained significantly lower
biomass but higher rates of ECM colonization in same lineage and
opposite lineage soils compared to conspecific soils (Table 1;Figure 3 ). For example, the rarest species, grown in soil
conditioned by phylogenetically similar species demonstrated a 114%
increase in ECM colonization, but a 94 % decrease in total biomass
compared to conspecific conditioned soils. These patterns were also
consistent in species grown in phylogenetically distant soil which
demonstrated a 96 % increase in ECM colonization, but a 71 % decrease
in total biomass compared to conspecific conditioned soils. On the other
hand, common species displayed similar patterns, however, to a much
lesser extent; ECM colonization increased by 26%-78% and total biomass
decreased by 25%-52% in conspecific relative to distantly related soil
inoculum and conspecific to closely related soil inoculum, respectively.
DSE colonization did not vary significantly by phylogenetic origin of
conditioned soil across all rarity levels. These results from a common
garden suggest that rare species may maintain distinct and critical
tradeoffs between aboveground performance traits and belowground
associations with fungal mutualists not prevalent in common species
(i.e., there is reduced performance but increased reliance on ECM in any
different soil than that conditioned by the same species in rare
eucalypts).
Consistent with Hypothesis 3, that the divergence in plant total biomass
in conspecific versus soils conditioned by other species is driven by
rates of ECM colonization, biomass exhibited strong negative plant-soil
feedbacks in conditioned soil relative to conspecific soil, while rates
of ECM colonization demonstrated simultaneous strong positive plant-soil
feedbacks (Figure 4 ). These results suggest that species grown
in soil conditioned by other species maintain lower biomass but stronger
associations with mycorrhizal mutualists when compared to the same
species grown in monoculture. Furthermore, the effect size of ECM
colonization directly affected the effect size of total biomass in
phylogenetically distant conditions with a significant negative
correlation (r^2 = -0.83) to show that performance traits, such as
biomass, may be a function of ECM colonization acting differently among
species varying in rarity (Figure 5 & Table 2 ). Further, the
strength of the relationship between belowground mycorrhizal feedbacks
and aboveground plant traits was significantly affected by the
phylogenetic relatedness of conditioned soil. While both conspecific to
same lineage and conspecific to opposite lineage treatments demonstrated
similar patterns in the relationship between total biomass and rates of
ECM colonization, the differences in feedbacks in conspecific to same
lineage treatments displayed a moderate negative relationship (r^2 =
-0.58). Differences underlying the significance of conspecific to same
lineage and conspecific to opposite lineage feedbacks may be due
inadequate sample size. These results suggest that differences in ECM
colonization not only drive aboveground feedbacks differently in rare
versus common species, but also more strongly in distantly related
species. Taken together, these results suggest that microbially mediated
belowground feedbacks function to drive aboveground feedbacks in
performance traits critical for species survival. Furthermore, if the
continued survival of rare species is dependent on the maintenance of
low biomass via belowground microbial associations, then the legacy
effects of soil conditioned by species varying in rarity and
phylogenetic relatedness are expected to exert strong control over
future plant community phenology, composition, and biodiversity at the
population level.
Discussion
Species rarity is often considered an unfavorable ecological response to
environmental factors (Wamelink et al. 2014; Irl et al. 2017). Recent
studies, however, have demonstrated that ecological and evolutionary
factors interact to drive divergent patterns in aboveground traits
between rare and common species (Boyd et al. 2022; Nytko et al. in
review ). Much less consideration has been given to potential
belowground mechanisms driving the aboveground divergence of traits and
subsequent plant-soil feedbacks in rare species. This omission is
important as species do not live in isolation; community context and
soil legacy affects are meaningful and likely have important feedbacks
that can reinforce rarity over time. Our results show that 1) the total
biomass and ECM colonization of a species is dependent on the rarity of
the plant host; 2) the total biomass and ECM colonization of a species
is also affected by the phylogenetic relatedness and N enrichment of
conditioned soil; and 3) differences in belowground ECM colonization
drive aboveground trait divergence and larger plant-soil feedbacks in
rare versus common species. Taken together, the results suggest that
rare plants may more heavily utilize mycorrhizal associations in
phylogenetically distant communities under low N conditions, than common
species, even while maintaining lower biomass levels for continued
persistence across the landscape. Overall, rare plant species, while
having reduced biomass aboveground, may maintain rich belowground
mycorrhizal, rather than non-mycorrhizal, fungal communities conducive
to nutrient acquisition and high potential for adaptation under climatic
change. Results such as these have not commonly been considered for rare
species.
Plant performance is often measured using a variety of functional traits
such as life-form, dispersal, seed biology, and phenotypic response to
environmental conditions (Pywell et al. 2003, Bragion et al. 2018;
Hanisch et al. 2020). However, without an understanding of the
mechanisms underlying functional trait expression, predictions of trait
response to climate and associated changes in ecosystem function are
limited. For example, our results demonstrate that rare species overall
maintain lower biomass than common species across all soil conditions.
Under current measures of plant species’ success, lower productivity is
often considered ecologically unfavorable due to associations between
lower biomass and decreased fecundity (Poorter et al. 2015; Younginger
et al. 2017). However, our results also indicate that the aboveground
trait values of rare species may not be representative of an
unsuccessful survival strategy, as is often seen in common species.
Instead, lower biomass in rare species may reflect a unique tradeoff in
which more resources are allocated to belowground mycorrhizal symbionts
for nutrient uptake and pathogen protection that allow for continued
persistence (Poot & Lambers 2003; Bothe et al. 2010; Soudzilovskaia et
al. 2019; Tedersoo & Bahram 2019). Furthermore, the trade-off between
ECM fungal colonization and aboveground biomass is dependent on the
phylogenetic relatedness of conditioned soil in which plants were grown
and the rarity level of plant host, such that the rarest species
experience the strongest negative feedbacks in growth but strongest
increase in ECM colonization when grown in soil conditioned by
phylogenetically distant species. These conclusions support and expand
upon the findings of Kempel et al. (2018) and Jiang et al. (2022) to
demonstrate the microbial mechanisms underlying plant-soil feedbacks in
plant communities varying in rarity and phylogenetic distance.
While these patterns may be mediated by the unique functional traits of
rare species varying in phylogenetic relatedness, an alternative
hypothesis may suggest that the characteristic low biomass of rare
species is a function of ECM parasitism in low N environments. While
these results are consistent with parasitic patterns in belowground
fungi, as well as the functional equilibrium model of resource
allocation under N enrichment (Johnson et al. 2008), it is also unusual
to observe parasitism of mycorrhizal fungi in low N environments in
which mutualisms are necessary for nutrient acquisition (Johnson 1993;
Johnson et al. 2008). Consequently, the biomass of rare plant species
may be representative of: 1) N-limitation; 2) allocation to belowground
resources for limiting micronutrients; 3) the maintenance of valuable
functional traits rather than biomass production in nutrient-lacking
environments. Although the effects of soil nutrient conditions on
productivity in rare versus common plant species need to be studied
further, these results suggest that the ability of a rare species to
survive and persist in expanding range and niche space may be
microbially mediated, particularly via previous plant communities.
Specifically, rare plant species may benefit geographically,
competitively, and functionally from mycorrhizal mediated soil legacy
effects, to facilitate the persistence and potential expansion of rare
species in phylogenetically distant, and to a lesser extent
phylogenetically similar, communities rather than common species under
climate change.
Predicting the future spatial and temporal persistence of rare species
is key to conservation and management, as well as maintaining ecological
function (Kunin & Gaston 1997). Species’ rarity can be predicted by
prominent negative plant-soil feedbacks (Ke et al. 2015; Klironomos
2002), however, predictions of rare species persistence are limited
without a mechanistic understanding of these plant-soil feedbacks.
Studies examining the mechanistic causes of rarity are often ecological
in focus, however, recent research has demonstrated that the
phylogenetic origin of plant species and the microbial community of
previously conditioned soil can interact to affect the strength and
direction of mycorrhizal mediated feedbacks (Segnitz et al. 2020). For
example, Woolbright et al. (2014) suggested that novel interactions with
ECM fungi may influence the ability of relict species to migrate,
persist, or outperform common counterparts, while maintaining lower
levels of genetic variability, in atypical habitats (Chung et al. 2015).
Our results not only confirm that rare species on average suffer
stronger negative plant-soil feedbacks on biomass when grown in
conspecific soils, but also show that the strength and direction of
plant-soil feedbacks in rare species are mediated by positive feedbacks
in ECM colonization in both closely and distantly related
plant-conditioned soils. These results suggest that
conspecific-conditioned soils may be lacking adequate levels of
mycorrhizal mutualists, while incurring high levels of plant pathogens
(Hannula et al. 2021). Consequently, rare species productivity is
inextricably tied to belowground mycorrhizal colonization, making rare
species more susceptible (both positively and negatively) to soil
conditioning effects such as legacy and priority effects as well as
above- and belowground species interactions (Nytko et al. in
review ). Therefore, long-lasting microbially mediated legacy effects of
previous plant communities may drive patterns of rare species abundance,
as demonstrated by low abundant species in Anacker et al. (2014), Maron
et al. (2016), and Yan et al. (2022), as well as patterns of rare
species productivity and composition in successional communities.
Biotic interactions shape species’ distributions, functional traits, and
rarity (Cosentino et al. 2023; Kempel et al. 2020; Nytko et al. in
review ; Wisz et al. 2013); however, biotic interactions are commonly
underrepresented in conservation, leading to errors in the estimation of
rare species occurrence and future persistence (Flores-Tolentino et al.
2020). Although the majority of literature regarding the causes and
consequences of rarity is dedicated to the response of species to
abiotic factors, Woolbright et al. (2014) demonstrated the critical
importance of biotic interactions in understanding both the geographic
occurrence and genetic underpinnings of isolated relict species. Our
results expand on the hypotheses proposed in Woolbright et al. (2014),
to demonstrate the critical importance of above-and belowground
interactions in understanding rarity as well as determining functional
trait expression (Kempel et al. 2020). These results suggest that the
expression of phenotypes among populations within the same species are
the consequence of previous plant communities and current feedbacks with
belowground symbionts. Moreover, biotic interactions may drive patterns
of geographic mosaics in rare species by shaping the direction of
co-evolution to establish differences in trade-off strength and
subsequent trait expression not seen in common species. Consequently,
geographically based differences in the availability and abundance of
mycorrhizal symbionts may result in population-related variation in the
expression of traits, long-term species persistence, and conservation
priority. As such, the phenotypes of rare species that we consider to be
a conservation priority may be a consequence of microbially mediated
plant-soil feedbacks.
Conclusions & Implications : The majority of plant species are
rare in any given plant community. Furthermore, the conservation of rare
populations are often characterized by geographic and genetic
parameters, such that a rare population is one of small size, located in
an uncharacteristically atypical area, with limited genetic diversity
(Rabinowitz 1981; Cole 2003) without consideration of the community
context or past evolutionary history of that species. Rare species are
rare for a variety of reasons, but some may maintain lower levels of
biomass and higher rates of ECM colonization to persist. If the
microbially mediated plant-soil feedbacks seen in rare species are
representative of a unique trade-off between allocation to ECM
colonization versus aboveground biomass when growing in intermediately
related plant-conditioned soils, then there are likely to be significant
implications for conservation practices. Although abiotic factors are
important proponents of functional trait expression (Lavorel et al.
2011; Caruso et al. 2020), the long-lasting microbial impacts of
previous plant communities, especially ones that may not strongly alter
soil communities in positive or negative ways for the next generation of
plants, may exert more control over the phenotypic appearance and
persistence of rare species than originally hypothesized. As legacy
effects become increasingly pronounced under climate change, accounting
for above- and belowground biotic interactions will become increasingly
important in spatial distribution modeling (SDM) and predictions of rare
species occurrence in changing communities. Additionally, if species
rarity is dependent on microbial counterparts for the successful
maintenance of lower biomass, rare species management may be improved
through the consideration of belowground colonization via mycorrhizal
mutualists at the population level. These greenhouse results suggest
that the aboveground traits of rare species are not indicative of an
unsuccessful plant strategy, but rather a consequence of previous plant
conditioning effects as well as interactions with current fungal
mutualists that vary by previous plant community inhabitants that need
to be field-tested. Accordingly, the low abundance and biomass
characteristic of rare species may disguise a rich belowground world of
beneficial mutualists, worthy of further investigation into the
contribution of belowground feedbacks to aboveground functional traits
in rare species around the world.