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Sex-Specific Interactions in Arbuscular Mycorrhizas in an Ecological Community Context

Periodic Reporting for period 1 - SIAM (Sex-Specific Interactions in Arbuscular Mycorrhizas in an Ecological Community Context)

Reporting period: 2015-04-13 to 2017-04-12

The aim of the project was to explore and understand plant – arbuscular mycorrhizal (AM) fungal interactions in a more realistic multi-trophic context, by investigating how sex-specific interactions between AM fungi and sexually dimorphic plants impact plant pollinators and herbivores.

Understanding the importance of belowground organisms for plant growth, community dynamics, and ecosystem processes is fundamental. One of the most significant components of belowground biota is the AM fungi growing in symbiosis with roots of most land plants. Since their advent 360 million years ago, AM have been tightly linked to plant life history evolution. The effects of AM fungi on plant fitness have been extensively studied in sexually monomorphic plants, but plant populations with separate sexes are relatively common in nature. Moreover, most available studies on this topic have been exclusively focused on two trophic levels, neglecting the fact that plants live in complex multi-trophic scenarios, where both mutualistic and antagonistic relationships interact both above- and below-ground. Therefore this aimed to explore and understand plant – AM fungal interactions in a more realistic multi-trophic context.

Implementation of this project had scientific importance for the improvement of our understanding of complex multi-trophic interactions in plants and has potentially enormous economic significance for agriculture as it includes pollination ecology and herbivory. Besides the scientific importance of the proposed project itself, this project significantly enhanced the applicant’s career by acquiring up-to-date techniques and other significant skills and provided expertise on mycorrhizal fungi research for the host institution.

The overall research objectives of this action were:
- To demonstrate if plant roots from different sexes are colonised by different AM fungi
- To determine the effect of AM fungi on floral traits and attractants to pollinators and herbivores
The work performed and the results achieved so far for the two work packages (WP) of this project are detailed below.

WP1. AM fungi in roots of plants with different sex. This WP aimed to demonstrate if plant roots from different sexes are colonised by different AM fungal communities by
identifying and comparing AM fungal communities colonizing roots 1a) from different sexes in several plant species; and 2a) along a latitudinal gradient using two focal plant species.

Roots from more than 700 different plant individuals from 23 different species (Asparagus officinalis, Beta vulgaris, Bryonia dioica, Cirsium arvense, Echium vulgare, Geranium sylvaticum, Glechoma hederacea, Humulus lupulus, Knautia arvensis, Lycopus europaeus, Mercurialis perennis, Oregano vulgare, Plantago coronopus, Plantago lanceolata, Plantago maritima, Rumex acetosa, Rumex acetosella, Silene dioica, Silene latifolia, Succisa pratensis, Tammus communis, Thymus polytrichum, Urtica dioica) were collected and microscopically analysed to check for AMF colonisation after trypan blue staining. This analysis confirmed that several species are indeed non-mycorrhizal in the field (e.g. Silene dioica, Silene latifolia, Urtica dioica, Rumex acetosa, Rumex acetosella).

Following visual estimation of AMF presence in roots, AMF DNA was isolated from about 260 root samples and identified in part of the samples (n = 120) using next generation sequencing. The rest of the samples (n =140) are being currently sequenced. Once all the sequencing data is obtained, we will analyse it to demonstrate if plant roots from different sexes are colonised by different AM fungal communities.

WP2. AM fungal-plant-animal interactions. This WP aimed to determine the effects of AM fungi on floral traits and attractants to pollinators and herbivores by examining 2a) how AM fungi affect pollinators; and 2b) how AM fungi affect herbivores. For this WP, an experimental population composed of 210 Knautia arvensis plants inoculated with increasing levels of AM fungal diversity was established at the beginning of the Fellowship. The plan was to grow the plants until flowering and then collect and analyse floral volatiles to investigate whether plants with different sexes differ in their volatile composition and how AM fungi may affect these compounds. Unfortunately, only 3 plants have flowered so far even though we expect them to flower during this summer so we can finally carry out the volatile collection. Nevertheless, we have collected data on plant growth and survival in response to different AM fungi and we have tried the methods.

At this point, we do not have the results to demonstrate if plant roots from different sexes are colonised by different AMF as samples are currently being sequenced. Similarly, we are still analysing volatile samples to determine the effect of AM fungi on floral traits and attractants to pollinators and herbivores. We expect to obtain all results within the next few months. Then, data will be published as soon as possible in suitable high-ranked international peer-reviewed journals and made available in data repositories to ensure maximum dissemination. All obtained genetic sequences will become available in GenBank and submitted to MaarjAM database. Results will be presented at international and national conferences. Press releases and articles in non-specialised press will be written to disseminate even further the results gained.
The impacts of this fellowship are numerous. For the Fellow, besides the direct impact that the acquisition of competencies will have on my future career, this project has given the opportunity to secure a lecturer position at the University of Lincoln, my host institution. The host institution has also benefited of this Fellowship as I have been giving seminars, working with students, and giving lectures in the field of plant and fungal ecology, field relatively underrepresented before I arrived.

This research project investigated a very novel research topic: whether the relationship between AM fungi and plants is sex-specific and what are the consequences of such interactions for herbivores and pollinators aboveground. Therefore, this project has high potential for scientific breakthroughs. The description of AM fungal effects on plant performance together with the demonstration of the mechanisms behind such effects will provide the platform to develop a complete understanding of the role of AM fungi in plant life history traits and breeding system evolution. The outcome of this project will thus provide the magnitude and the mechanistic role of AM fungi in plant evolution, which will change our understanding of the role of these important microbes and surely will open new horizons for future frontier research in evolutionary and environmental biology.
Experimental setup using Knautia arvensis investigating AMF effects on pollinators and herbivores