NEW INSIGHTS IN THE ECOLOGY AND EVOLUTION OF PLANT NITROGEN LIMITATION

Under increasing additions of reactive nitrogen (N) to the planet via anthropogenic N deposition and excess fertilization, some plant species will thrive while others will not. This may seem counterintuitive, as the growth of most plants is thought to be limited by soil N, but recent evidence shows that excess N can reduce plant community composition, alter plant-microbial interactions, and lead to fundamental alterations in plant growth and fitness. Yet, we lack the ability to predict which plant species will be winners or losers in soil N enrichment scenarios. The primary goal of my dissertation was to examine variation in plant growth responses to N enrichment and whether ecological and evolutionary factors explain such variation. These factors, according to current literature, should include aspects of past evolution such as phylogeny and evolutionary differentiation in resource use traits, nutrient co-limitation, and interactions with root-associated microbes. Because variation in plant responses to soil N enrichment challenges the paradigm in ecology that productivity of all plants is N-limited or N co-limited, a second goal of my dissertation was to determine how this and other recent work changes our understanding of the terrestrial N and carbon (C) cycles and feedbacks between soil N gradients and evolution under global change.In my first chapter, I used a global dataset of plant biomass responses to N fertilization and evolutionary models to show that species vary in the direction and magnitude with which they respond to N enrichment (with more than one in four species responding negatively or neutrally), and that two aspects of past evolution (phylogenetic relatedness and selection associated with constraints on resource use) govern responses to N enrichment. In my second and third chapters, I implemented two greenhouse fertilization experiments and subsets of the 30 functionally diverse tree species within the genus Eucalyptus that are native to Tasmania, Australia. The main result from these experiments was that phylogenetic patterns in biomass responses to N enrichment are associated with phylogenetic variation in root function (specific root length and interactions with ectomycorrhizal fungi), but not co-limitation by phosphorus (despite the fact that Tasmanian eucalypts occur across strong soil phosphorus gradients). In my fourth chapter, I reviewed how this and other current research challenges long-held and fundamental assumptions regarding the source, plant use, and microbial transformations of N and provides insights into eco-evolutionary feedbacks and C cycling under global change. Overall, my dissertation has used major theories in plant ecology and evolution to explain the variation in plant responses to global change, and synthesized research that highlights new understanding of the drivers and consequences of terrestrial N cycling

The evolution of thermal performance in native and invasive populations of Mimulus guttatus

The rise of globalization has spread organisms beyond their natural range, allowing further opportunity for species to adapt to novel environments and potentially become invaders. Yet, the role of thermal niche evolution in promoting the success of invasive species remains poorly understood. Here, we use thermal performance curves (TPCs) to test hypotheses about thermal adaptation during the invasion process. First, we tested the hypothesis that if species largely conserve their thermal niche in the introduced range, invasive populations may not evolve distinct TPCs relative to native populations, against the alternative hypothesis that thermal niche and therefore TPC evolution has occurred in the invasive range. Second, we tested the hypothesis that clines of TPC parameters are shallower or absent in the invasive range, against the alternative hypothesis that with sufficient time, standing genetic variation, and temperature-mediated selection, invasive populations would re-establish clines found in the native range in response to temperature gradients. To test these hypotheses, we built TPCs for 18 native (United States) and 13 invasive (United Kingdom) populations of the yellow monkeyflower, Mimulus guttatus. We grew clones of multiple genotypes per population at six temperature regimes in growth chambers. We found that invasive populations have not evolved different thermal optima or performance breadths, providing evidence for evolutionary stasis of thermal performance between the native and invasive ranges after over 200 years post introduction. Thermal optimum increased with mean annual temperature in the native range, indicating some adaptive differentiation among native populations that was absent in the invasive range. Further, native and invasive populations did not exhibit adaptive clines in thermal performance breadth with latitude or temperature seasonality. These findings suggest that TPCs remained unaltered post invasion, and that invasion may proceed via broad thermal tolerance and establishment in already climatically suitable areas rather than rapid evolution upon introduction

Evolutionary History and Novel Biotic Interactions Determine Plant Responses to Elevated CO2 and Nitrogen Fertilization

A major frontier in global change research is predicting how multiple agents of global change will alter plant productivity, a critical component of the carbon cycle. Recent research has shown that plant responses to climate change are phylogenetically conserved such that species within some lineages are more productive than those within other lineages in changing environments. However, it remains unclear how phylogenetic patterns in plant responses to changing abiotic conditions may be altered by another agent of global change, the introduction of non-native species. Using a system of 28 native Tasmanian Eucalyptus species belonging to two subgenera, Symphyomyrtus and Eucalyptus, we hypothesized that productivity responses to abiotic agents of global change (elevated CO2 and increased soil N) are unique to lineages, but that novel interactions with a nonnative species mediate these responses. We tested this hypothesis by examining productivity of 1) native species monocultures and 2) mixtures of native species with an introduced hardwood plantation species, Eucalyptus nitens, to experimentally manipulated soil N and atmospheric CO2. Consistent with past research, we found that N limits productivity overall, especially in elevated CO2 conditions. However, monocultures of species within the Symphyomyrtus subgenus showed the strongest response to N (gained 127% more total biomass) in elevated CO2 conditions, whereas those within the Eucalyptus subgenus did not respond to N. Root:shoot ratio (an indicator of resource use) was on average greater in species pairs containing Symphyomyrtus species, suggesting that functional traits important for resource uptake are phylogenetically conserved and explaining the phylogenetic pattern in plant response to changing environmental conditions. Yet, native species mixtures with E. nitens exhibited responses to CO2 and N that differed from those of monocultures, supporting our hypothesis and highlighting that both plant evolutionary history and introduced species will shape community productivity in a changing world

Wooliver et al. 2018 fungal sequencing data

Relative abundances and fungal guilds of fungal OTUs identified in conspecific soil inocula used in the Wooliver et al. 2018 greenhouse experiment. Soils were harvested from trees of 16 Eucalyptus species growing in two Tasmanian common gardens and pooled at the species level, then sequenced for fungal DNA using next-generation sequencing

Wooliver et al. 2018 greenhouse data

Individual plant height (in centimeters), total biomass (in grams), and number of roots colonized by ectomycorrhizal (ECM), arbuscular mycorrhizal (AM), dark septate endophytic (DSE), and non-filamentous fungi out of 50 fields of view. Individuals include 15 Tasmanian Eucalyptus species within the subgenus Symphyomyrtus that comprise two phylogenetic lineages: the alpine white, black, and yellow gums (lienage 1) and the white and blue gums (lineage 2). Individuals were arranged in a randomized block design and received factorial treatments of low vs. high nitrogen enrichment, fungicide vs. live soils, and four soil inocula (control potting mix, soils conditioned by conspecific trees, soils conditioned by trees within the same lineage, and soils conditioned by trees within the opposite lineage)

Data from: Soil fungi underlie a phylogenetic pattern in plant growth responses to nitrogen enrichment

1. Under increasing anthropogenic nitrogen (N) deposition, some plant species will thrive while others will not. Previous work has shown that plant phylogeny can predict these responses, and that interactions with mycorrhizal fungi are a mechanism that drives variation in plant responses to N enrichment. Yet, much of this work has ignored the roles of other root-associated fungi and whole soil fungal communities in driving these responses. 2. We tested whether soil fungi mediate responses of plant growth and plant-soil feedbacks (between close and distant plant relatives) to N enrichment by implementing a greenhouse experiment in which we applied factorial treatments of N fertilization, host-specific soil inocula, and fungicide to 15 eucalypt tree species that co-occur on the island state of Tasmania, Australia and form two phylogenetic lineages within the subgenus Symphyomyrtus. 3. Conspecific-conditioned soil fungi enhanced growth responses to N enrichment for plants within one lineage (lineage 1) but depressed growth responses to N enrichment for plants within another lineage (lineage 2). Lineage-specific shifts in ectomycorrhizal (ECM) colonization were consistent with previous evidence that more vs. less successful strategies under N enrichment are those where carbon allocation to mycorrhizal fungi is reduced vs. maintained, respectively. The latter was also accompanied by a stronger reduction in root colonization of non-filamentous fungi (of unknown function) under N enrichment. Plant-soil feedbacks were neutral for lineage 1 but negative for lineage 2 (i.e., greater growth in soils conditioned by opposite vs. same lineage individuals), but were not altered by N enrichment or fungicide. Lineage-level differences in root colonization suggest that these feedbacks could be driven by differential plant responsiveness to dark septate endophytes and non-filamentous fungi, the colonization of which seemed to benefit plant growth. 4. Our results confirm that interactions with soil fungi (ECM fungi in particular) underlie phylogenetic patterns in tree species’ growth responses to N enrichment and may thus influence which plants win or lose under future N deposition scenarios. Yet, we provide some of the first evidence (albeit from controlled rather than natural conditions) that N deposition may not play a strong role in shifting plant-soil feedbacks

Linear mixed effects model results of eucalypt productivity (total, aboveground and belowground; TB, AGB, and BGB, respectively) and biomass allocation (root to shoot ratio; R∶S) across CO₂, soil N, species pair type (monoculture vs. mixture with the non-native <i>E. nitens</i>) treatments and native species subgenus (N = 190).

<p>In a greenhouse experiment, 28 native Tasmanian eucalypt species within two subgenera (S), <i>Symphyomyrtus</i> and <i>Eucalyptus</i>, were treated with factorial combinations of ambient or elevated CO₂ (C; 420 or 700 ppm, respectively) and low or high soil N (N; 3 or 30 kg/ha/mo), and paired with a conspecific or a non-native (<i>E. nitens</i>) individual (M). In these models, whole-pot biomass measurements and ratios of root to shoot biomass were averaged for each native species in each treatment combination, cube root transformed, and blocked by species. P values are shown in bold and are significant at α≤0.05.</p>δ<p>TB, total biomass; AGB, aboveground biomass; BGB, belowground biomass; R∶S, root to shoot ratio; M, species pair type (native species monoculture vs. mixture with <i>E. nitens</i>); C, CO₂ treatment (420 or 700 ppm); N, nitrogen treatment (3 or 30 kg ha⁻¹ mo⁻¹).</p><p>Linear mixed effects model results of eucalypt productivity (total, aboveground and belowground; TB, AGB, and BGB, respectively) and biomass allocation (root to shoot ratio; R∶S) across CO₂, soil N, species pair type (monoculture vs. mixture with the non-native <i>E. nitens</i>) treatments and native species subgenus (N = 190).</p

Linear mixed effects model results of subgenus-level eucalypt productivity (total, aboveground and belowground; TB, AGB, and BGB, respectively) and biomass allocation (root to shoot ratio; R∶S) across CO₂, soil N, and species pair type (monoculture vs. mixture with the non-native <i>E. nitens</i>).

<p>In a greenhouse experiment, 28 native Tasmanian eucalypt species within two subgenera (S), <i>Symphyomyrtus</i> and <i>Eucalyptus</i>, were treated with factorial combinations of ambient or elevated CO₂ (C; 420 or 700 ppm, respectively) and low or high soil N (N; 3 or 30 kg/ha/mo), and paired with a conspecific or a non-native (<i>E. nitens</i>) individual (M). In these models, whole-pot biomass measurements and ratios of root to shoot biomass were averaged for each native species in each treatment combination, cube root transformed, and blocked by species. P values are shown in bold and are significant at α≤0.05.</p>δ<p>TB, total biomass; AGB, aboveground biomass; BGB, belowground biomass; R∶S, root to shoot ratio; M, species pair type (native species monoculture vs. mixture with <i>E. nitens</i>); C, CO₂ treatment (420 or 700 ppm); N, nitrogen treatment (3 or 30 kg ha⁻¹ mo⁻¹).</p><p>Linear mixed effects model results of subgenus-level eucalypt productivity (total, aboveground and belowground; TB, AGB, and BGB, respectively) and biomass allocation (root to shoot ratio; R∶S) across CO₂, soil N, and species pair type (monoculture vs. mixture with the non-native <i>E. nitens</i>).</p

Productivity responses to global change scenarios are contingent upon species evolutionary history and novel biotic interactions.

<p>Overall, monocultures (pairs of conspecific individuals) of species in the subgenus <i>Symphyomyrtus</i> (top right panel) in elevated CO₂ conditions exhibit the strongest responses to N. On average, these monocultures produce 126% more biomass than all other species pairs in high N and elevated CO₂ treatments (1.301±0.205 g and 0.576±0.061 g, respectively). Above- and belowground biomass follow similar patterns. Error bars represent ±1 SEM.</p

Evolutionary History and Novel Biotic Interactions Determine Plant Responses to Elevated CO₂ and Nitrogen Fertilization

<div><p>A major frontier in global change research is predicting how multiple agents of global change will alter plant productivity, a critical component of the carbon cycle. Recent research has shown that plant responses to climate change are phylogenetically conserved such that species within some lineages are more productive than those within other lineages in changing environments. However, it remains unclear how phylogenetic patterns in plant responses to changing abiotic conditions may be altered by another agent of global change, the introduction of non-native species. Using a system of 28 native Tasmanian <i>Eucalyptus</i> species belonging to two subgenera, <i>Symphyomyrtus</i> and <i>Eucalyptus</i>, we hypothesized that productivity responses to abiotic agents of global change (elevated CO₂ and increased soil N) are unique to lineages, but that novel interactions with a non-native species mediate these responses. We tested this hypothesis by examining productivity of 1) native species monocultures and 2) mixtures of native species with an introduced hardwood plantation species, <i>Eucalyptus nitens</i>, to experimentally manipulated soil N and atmospheric CO₂. Consistent with past research, we found that N limits productivity overall, especially in elevated CO₂ conditions. However, monocultures of species within the <i>Symphyomyrtus</i> subgenus showed the strongest response to N (gained 127% more total biomass) in elevated CO₂ conditions, whereas those within the <i>Eucalyptus</i> subgenus did not respond to N. Root:shoot ratio (an indicator of resource use) was on average greater in species pairs containing <i>Symphyomyrtus</i> species, suggesting that functional traits important for resource uptake are phylogenetically conserved and explaining the phylogenetic pattern in plant response to changing environmental conditions. Yet, native species mixtures with <i>E. nitens</i> exhibited responses to CO₂ and N that differed from those of monocultures, supporting our hypothesis and highlighting that both plant evolutionary history and introduced species will shape community productivity in a changing world.</p></div