Characterising the response of plant-mycorrhizal networks to a changing alpine environment

<p>Anthropogenic climate change effects are particularly acute in alpine ecosystems. New Zealand’s alpine regions are experiencing climatic changes at higher than global mean rates, particularly warming and drying. These communities are also facing increasing rates of invasion by exotic plant species. Notably, multiple drivers of change, such as warming and invasion, have been evidenced to interact and facilitate greater ecosystem change. This is of particular concern as New Zealand's alpine plant communities are unique globally and represent national hotspots of biodiversity. Therefore there is a pressing need to understand how they may be affected by the independent and interactive drivers of global environmental change. Alpine plant species form ubiquitous and obligate symbiotic associations with mutualistic mycorrhizal fungi. Plant-mycorrhiza networks are foundational interactions that underpin the diversity and function of terrestrial communities. Plant-mycorrhiza networks are also particularly sensitive to temperature shifts and plant invasions. In this thesis, I investigate the independent and interactive effects of warming and the presence of an invasive species (Common Heather, Calluna vulgaris) on the fungal community composition and the network of mycorrhiza interactions of alpine plants in Tongariro National Park, New Zealand. I sampled the roots of plant species within the Warming and species Removal in Mountains (WaRM) experiment, a factorial combination of warming and Calluna vulgaris removals (n = 8 per treatment) established in TNP in 2015. The plant community at the site consists of plant species that form either arbuscular mycorrhizas or ericoid mycorrhizas. I selected the three most abundant plant species of each mycorrhizal type at the site scale for sampling in each of the 32 plots. DNA was extracted from plant roots, and the internal transcribed spacer of the fungal rRNA gene was amplified by PCR and sequenced on the Illumina Mi-seq platform. Sequence data was demultiplexed and fungal OTUs were identified using the PIPITS pipeline, referencing the UNITE fungal database. In my second chapter, I consider plant species and treatment effects on the diversity and community composition of mycorrhizal fungi. I found WaRM treatments were significant determinants of mycorrhizal compositions in host plant species. Warming simultaneously increased the mycorrhizal fungal diversity and richness of invasive Calluna vulgaris and reduced that of the native host plant species. In chapter 3, using network analyses from the bipartite package of R, I constructed 32 plant mycorrhizal networks of the plot sampled and calculated metrics pertaining to properties of network structure and robustness at the whole network, trophic-level and species/mycorrhizal fungal OTU scales. I then examined the responses of these metrics to the WaRM treatments. I found that warming significantly reduced the robustness of native plant-mycorrhizal networks and increased the strength of the interaction network associated with invasive C. vulgaris. The removal of C. vulgaris had a secondary effect on how mycorrhizal fungal compositions and interaction networks responded to warming. As a generalist C. vulgaris was critical for the ongoing diversity of ericoid mycorrhizal fungi, particularly under warming. However, C. vulgaris simultaneously suppressed the mycorrhizal interaction- networks of native plant species, which further fragmented under warming. I conclude that warming and the presence of invasive C. vulgaris synergistically reduced and decentralised the native plant-mycorrhizal interactions within the network. In summary, my thesis demonstrates the below-ground interactions of alpine plant communities are destabilising under multiple interacting drivers of global environmental change.</p>

Characterising the response of plant-mycorrhizal networks to a changing alpine environment

<p>Anthropogenic climate change effects are particularly acute in alpine ecosystems. New Zealand’s alpine regions are experiencing climatic changes at higher than global mean rates, particularly warming and drying. These communities are also facing increasing rates of invasion by exotic plant species. Notably, multiple drivers of change, such as warming and invasion, have been evidenced to interact and facilitate greater ecosystem change. This is of particular concern as New Zealand's alpine plant communities are unique globally and represent national hotspots of biodiversity. Therefore there is a pressing need to understand how they may be affected by the independent and interactive drivers of global environmental change. Alpine plant species form ubiquitous and obligate symbiotic associations with mutualistic mycorrhizal fungi. Plant-mycorrhiza networks are foundational interactions that underpin the diversity and function of terrestrial communities. Plant-mycorrhiza networks are also particularly sensitive to temperature shifts and plant invasions. In this thesis, I investigate the independent and interactive effects of warming and the presence of an invasive species (Common Heather, Calluna vulgaris) on the fungal community composition and the network of mycorrhiza interactions of alpine plants in Tongariro National Park, New Zealand. I sampled the roots of plant species within the Warming and species Removal in Mountains (WaRM) experiment, a factorial combination of warming and Calluna vulgaris removals (n = 8 per treatment) established in TNP in 2015. The plant community at the site consists of plant species that form either arbuscular mycorrhizas or ericoid mycorrhizas. I selected the three most abundant plant species of each mycorrhizal type at the site scale for sampling in each of the 32 plots. DNA was extracted from plant roots, and the internal transcribed spacer of the fungal rRNA gene was amplified by PCR and sequenced on the Illumina Mi-seq platform. Sequence data was demultiplexed and fungal OTUs were identified using the PIPITS pipeline, referencing the UNITE fungal database. In my second chapter, I consider plant species and treatment effects on the diversity and community composition of mycorrhizal fungi. I found WaRM treatments were significant determinants of mycorrhizal compositions in host plant species. Warming simultaneously increased the mycorrhizal fungal diversity and richness of invasive Calluna vulgaris and reduced that of the native host plant species. In chapter 3, using network analyses from the bipartite package of R, I constructed 32 plant mycorrhizal networks of the plot sampled and calculated metrics pertaining to properties of network structure and robustness at the whole network, trophic-level and species/mycorrhizal fungal OTU scales. I then examined the responses of these metrics to the WaRM treatments. I found that warming significantly reduced the robustness of native plant-mycorrhizal networks and increased the strength of the interaction network associated with invasive C. vulgaris. The removal of C. vulgaris had a secondary effect on how mycorrhizal fungal compositions and interaction networks responded to warming. As a generalist C. vulgaris was critical for the ongoing diversity of ericoid mycorrhizal fungi, particularly under warming. However, C. vulgaris simultaneously suppressed the mycorrhizal interaction- networks of native plant species, which further fragmented under warming. I conclude that warming and the presence of invasive C. vulgaris synergistically reduced and decentralised the native plant-mycorrhizal interactions within the network. In summary, my thesis demonstrates the below-ground interactions of alpine plant communities are destabilising under multiple interacting drivers of global environmental change.</p>

Common Mycorrhizal Networks Asymmetrically Contribute to Increased N and P Acquisition by Millet/Chickpea Mixture

Aim Cereal/legume intercropping is known to increase yield, partly because of increased nitrogen (N) and phosphorus (P) acquisition. The aim of this paper was to investigate the role of common mycorrhizal networks (CMNs) in overyielding by the crop species mixture and to find out if the effect of a CMN depends on which of the two species was colonized by AM fungi.Methods Microcosms with two compartments were used, separated by a 30-μm nylon mesh. Both compartments contained either chickpea or millet, in monoculture or mixed. One or none of the two compartments was inoculated with the AMF species Funneliformis mosseae. The plant in the inoculated compartment was referred to as the AMF donor, and the plant in the neighboring, non-inoculated compartment as the AMF receiver. Results Inoculation in one compartment resulted in mycorrhiza formation in the other compartment, providing evidence for the formation of CMNs. Inoculation of chickpea in the mixture increased N and P acquisition and biomass of both chickpea (AMF donor) and millet (AMF receiver), whereas inoculation of millet increased biomass of chickpea (AMF receiver) only, but did not increase N or P acquisition by any of the two species. Chickpea as AMF donor had higher numbers of phosphate-solubilizing bacteria in its rhizosphere compared to chickpea as receiver. The shoot N:P ratio of chickpea as AMF donor was lower than as receiver. Conclusion Our study demonstrated asymmetry in nutrient gains by a mixture of cereal and a legume, dependent on which plant species was the AMF donor or receiver. This suggests that initiating mycorrhizal networks by legumes in intercropping could be an important factor contributing to the magnitude of the intercropping effect.

Management After Windstorm Affects the Composition of Ectomycorrhizal Symbionts of Regenerating Trees but Not Their Mycorrhizal Networks

Due to ongoing climate change, forests are expected to face significant disturbances more frequently than in the past. Appropriate management is intended to facilitate forest regeneration. Because European temperate forests mostly consist of trees associated with ectomycorrhizal (ECM) fungi, understanding their role in these disturbances is important to develop strategies to minimize their consequences and effectively restore forests. Our aim was to determine how traditional (EXT) and nonintervention (NEX) management in originally Norway spruce (Picea abies) forests with an admixture of European larch (Larix decidua) affect ECM fungal communities and the potential to interconnect different tree species via ECM networks 15 years after a windstorm. Ten plots in NEX and 10 plots in EXT with the co-occurrences of Norway spruce, European larch, and silver birch (Betula pendula) were selected, and a total of 57 ECM taxa were identified using ITS sequencing from ECM root tips. In both treatments, five ECM species associated with all the studied tree species dominated, with a total abundance of approximately 50% in the examined root samples. Because there were no significant differences between treatments in the number of ECM species associated with different tree species combinations in individual plots, we concluded that the management type did not have a significant effect on networking. However, management significantly affected the compositions of ECM symbionts of Norway spruce and European larch but not those of silver birch. Although this result is explained by the occurrence of seedlings and ECM propagules that were present in the original forest, the consequences are difficult to assess without knowledge of the ecology of different ECM symbionts.

Shooting the messenger: Identifying the mycorrhizal species transferring carbon between neighboring trees

&lt;p&gt;EMF play an important role in forests around the globe, by improving tree nutrition and water supply, as well as connecting different tree species through common mycorrhizal networks (CMN's). However, the extent to which EMF control resource sharing within these networks has not yet been thoroughly addressed. We constructed a simple network of tree-fungus-tree and monitored carbon flow from a &lt;sup&gt;13&lt;/sup&gt;CO&lt;sub&gt;2&lt;/sub&gt; labeled donor tree to the final recipient. &amp;#160;DNA Stable Isotope Probing (DNA-SIP) of ectomycorrhizal root tips was used to identify the main fungal symbionts involved in carbon transfer among trees. We used pairs of inter and intra-specie Pinus halepensis and Quercus calliprinos saplings, and examined the carbon dynamics for 40 days within the leaf, stem and root tissues. The peak of &lt;sup&gt;13&lt;/sup&gt;C in the roots of the donor trees was around day 4 post labeling, while the recipient roots peaked at day 9 with observed differences between pairs. The intrinsic tree carbon pool, and not the tree species identity, was the main factor governing carbon transfer between trees. Finally, we were able to identify the main fungal symbionts enriched with &lt;sup&gt;13&lt;/sup&gt;C. Our results add the &quot;missing piece of the puzzle&quot; by linking specific mycorrhizal species to carbon transfer within CMN's.&lt;/p&gt;

A Small Amount of Nitrogen Transfer from White Clover to Citrus Seedling via Common Arbuscular Mycorrhizal Networks

Few studies have examined if perennial leguminous cover crops are able to transfer nitrogen (N) via common mycorrhizal networks (CMNs) to neighboring fruit trees; the gradient of such N transfer could affect the N nutrition of both plants. Using separated three-column chambers to grow plants in a greenhouse, 99 atom% 15N as (15NH4)2SO4 was applied to leaves of white clover (Trifolium repens L.) and 15N was then traced in neighboring citrus (Citrus sinensis (L.) Osbeck) seedlings interconnected by an arbuscular mycorrhizal fungus (AMF, Rhizophagus intraradices). A range of 66.85–68.74% mycorrhizal colonization in white clover (mycorrhizal and/or Rhizobium trifolii inoculated) and 19.29–23.41% in citrus (non-mycorrhizal inoculated) was observed after 12 months of AMF inoculation in the white clover, indicating a successful CMN linkage was established between these two plant species. This CMN establishment resulted in significant increases in biomass, N accumulation, and 15N content of citrus when accompanied with nodulated and mycorrhizal fungus colonized white clover. N transfer from white clover to citrus was significantly greater under nodulation plus mycorrhization (46.23 mg N per pot, 1.71% of N transferred) than under non-inoculated control (4.36 mg N per pot, 0.21% of N transferred), and higher than sole mycorrhization (36.34 mg N per pot, 1.42% of N transferred). The percentage of N in citrus derived from white clover under nodulated/mycorrhization was 1.83–1.93%, and was highest in leaves (3.31%), moderate in stems (2.47%), and lowest in roots (0.41%) of citrus. In summary, results from this experiment demonstrated that nearly 2.0% of N transferred from white clover to citrus via CMN. Further studies are needed to quantify N transfer between white clover and citrus by other routes, including soil or root exudation pathways.