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Evidence Project Final Report

 Note In line with the Freedom of Information Project identification Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. 1. Defra Project code BD1451 The Evidence Project Final Report is designed to capture the information on 2. Project title the results and outputs of Defra-funded Diversification of grassland through the manipulation of research in a format that is easily plant- interactions publishable through the Defra website An Evidence Project Final Report must be completed for all projects. 3. Contractor Lancaster Environment Centre  This form is in Word format and the organisation(s) boxes may be expanded, as appropriate. Lancaster University Lancaster LA1 4YQ  ACCESS TO INFORMATION The information collected on this form will be stored electronically and may be sent

to any part of Defra, or to individual 54. Total Defra project costs £ researchers or organisations outside (agreed fixed price) Defra for the purposes of reviewing the st project. Defra may also disclose the 5. Project: start date ...... 1 April 2009 information to any outside organisation acting as an agent authorised by Defra to st process final research reports on its end date ...... 31 march 2013 behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 11 6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...... YES NO (a) When preparing Evidence Project Final Reports contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow. Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer. In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. (b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary 7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work. Here we report on the findings of a two-year extension to objective 2 of BD1451, a cross-site mesocosm study set up to test how plants modify soil microbial communities and ultimately botanical diversity under different environmental conditions. This study tested the hypothesis that certain plant species facilitate changes in the soil microbial which feedback to encourage colonization of late successional species and hence promote grassland diversity restoration. To achieve this, a series of mesocosm experiments were established across a range of climatic and soil conditions in 2004, including variations in , at three sites in the north-east, south-east and south-west of England.

Results from the first four years of this study were reported in the Final Report of BD1451. At this time (2008), we found that the introduction of facilitator species into species-poor swards had promoted fungal growth in soil, and we expected that this would lead to enhanced colonization of late successional species in coming years. However, at the time of reporting, insufficient time had elapsed for the late successional species to become established across treatments and sites. As result, a two-year extension of this study was funded to examine longer-term responses to the experimental treatments; we report on the findings of this extension here.

In 2011, we found that the presence of facilitator species significantly reduced the fungal to bacterial PLFA across all sites and , indicating that these plants had promoted the growth of bacteria more than fungi. This finding was opposite to what was found in 2008, when the facilitators significantly increased the of fungi relative to bacteria, as initially predicted. This switch in the response of the microbial community to facilitator species was associated with changes in soil conditions, although these effects were site specific. Facilitator species had no effect on any of the measured soil nutrients at Reading, and, in general, the addition of Rhinanthus to swards had no effect on soil microbial communities or nutrient levels, aside from increasing soil concentrations at the Newcastle site. In sum, these findings suggest that, in the longer term, the addition of facilitator species has shifted the microbial community to one that is more dominated by bacteria, and at certain sites, this has increased the concentrations of some nutrients in soil, albeit in an inconsistent way. Facilitator species increased overall plant at North Wyke, in both alluvial and

EVID4 Evidence Project Final Report (Rev. 06/11) Page 2 of 11 clay soils, but not at Newcastle. This response, however, was mostly attributed to the presence of the facilitator species themselves; despite increasing overall plant species richness at North Wyke, the addition of facilitator plants suppressed the Shannon diversity and evenness of the plant community. The addition of facilitator species also strongly suppressed the biomass and species richness of the late successional species at Newcastle and North Wyke. This response was presumably a consequence of increased competitive of the facilitator species, which suppressed the growth of late successional species.

Contrary to expectations, the addition of Rhinanthus had no impact on overall plant species richness or diversity across sites and soil treatments, and didn’t affect the colonization of late successional species. Across all sites, one of the strongest determinants of plant and evenness was soil type, both being greater in clay than alluvial soil at Newcastle and North Wyke. Likewise, the biomass and richness of late successional species was affected by soil type, but the effect was in the opposite direction: biomass and richness of late successional species were both greater in alluvial than clay soils, which was most likely due to more intense in clay soil.

In conclusion, our findings provide little support for the idea that direct facilitator species promote the restoration of species diversity in mesotrophic grassland, at least in the short term. We did not find consistent effects of Rhinanthus on soil microbial communities or plant diversity, in that effects were highly site specific reflecting its variable establishment. However, as shown in other studies, the establishment and persistence of Rhinanthus varies with soil conditions and , and as a result its effect on vegetation diversity, varies across sites. Therefore, our results do not challenge the general value of Rhinanthus as a tool for diversity restoration; rather, they support the view that its role varies across sites. While drawing conclusions from our study, we should caution that they are derived from mesocosms that do not always reflect what happens in the field. Nevertheless, they reveal some of the mechanisms involved in the way that plants modify soils, and in our case, raise questions over the value of direct facilitator species for promoting grassland diversification, at least in the short timescale of our study.

Project Report to Defra 8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information

EVID4 Evidence Project Final Report (Rev. 06/11) Page 3 of 11 obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:  the objectives as set out in the contract;  the extent to which the objectives set out in the contract have been met;  details of methods used and the results obtained, including statistical analysis (if appropriate);  a discussion of the results and their reliability;  the main implications of the findings;  possible future work; and  any action resulting from the research (e.g. IP, Knowledge Exchange).

Background and objectives In recent years, there has been a growing awareness among ecologists of the importance of plant–soil feedback as a driver of plant community dynamics, especially in the context of plant succession and invasion, and processes such as nitrogen and carbon cycling. By altering the physical, chemical and biological nature of their soil environment, individual plants have the ability to influence their performance relative to their competitors, ultimately leading to changes in plant community composition and diversity. The aim of this study, which formed part of Objective 2 of BD1451, was to quantify the impact of key plant species on the development of fungal dominated soils and resultant impacts on vegetation community development. To do this, we set up, in 2004, a cross-site mesocosms experiment designed to test whether the introduction into species-poor grassland of plant species known to promote soil fungal-to- bacterial biomass ratios, termed facilitators, leads to enhanced plant species diversity in grassland communities commonly subject to management for enhancement of botanical diversity.

Detail on the experimental approach and key results for the period 2004-2008 are given in the Final Report of BD1451. To summarise, we found, over that time period, that the introduction of facilitator species into species-poor swards had promoted fungal growth in soil, and we expected that this would lead to enhanced colonization of late successional species in coming years. However, at the time of reporting, insufficient time had elapsed for the late successional species to become established across treatments and sites. As result, a two-year extension of this study was funded to examine longer-term responses to the experimental treatments; we report here on the findings of this extension.

Experimental approach As noted above, detail on the experiment approach is given in the Final Report of BD1451. Briefly, mesocosms were set up at three sites, and at each site vegetation treatments were established in two contrasting soil types with two levels of soil fertility resulting from long-term management, namely intensively managed fertilized grassland and unimproved grassland with no known history of artificial fertilizer application. At each site, we compared the two most common mesotrophic soil types of the region. For North Wyke, Devon, these were a clay or alluvial soil; at Close House, in the Tyne Valley, we used a clay loam or sandy alluvial soil; and at Reading we used a chalk and neutral soil. For vegetation, all soils were sown with a mix of six grass species typical of agriculturally improved grassland: Lolium perenne, Agrostis capillaris, Poa trivialis, Alopecurus pratensis, Holcus lanatus and Phleum pratense. These plants were allowed to establish for one year before applying the remaining two treatments in September 2005, namely the addition or not of a mixture of seven direct facilitator species (i.e., Lotus corniculatus, Prunella vulgaris, Ranunculus acris, R. bulbosus, Anthoxanthum odoratum, Trifolium pratense, and Plantago lanceolata and the addition or not of the indirect facilitator species Rhinanthus minor. With a randomized design of the 4 treatments and 4 replicates, this led to 16 treatments established within 64 plots at each site. At each of the three sites, the DF and IF plant species were allowed to condition the soils for two years before we added, in September 2007, the same suite of late successional plant species, typical of hay meadow restoration schemes, to each of the 16 different treatments across all three locations: Briza media, Centaurea nigra Galium verum, Knautia arvensis, Leontodon hispidus, Pimpinella saxifraga, Primula veris, Succisa pratensis, and Trisetum flavescens. Additional late successional species, specific to the different soil types were added at each location. Traditional

EVID4 Evidence Project Final Report (Rev. 06/11) Page 4 of 11 hay management was applied to the mesocosms throughout the experiment at each site, i.e., simulated grazing (to a height of 5cm) and trampling in April and September with a July hay cut (to a height of 5 cm). As reported in the Final Report, vegetation and soils were sampled annually between 2004–2008 and soils were sampled in 2005, 2006 and 2008 for soil chemical and microbial analysis, using PLFA. Here, we report on findings from sampling of vegetation and soils in 2011, six years after the introduction of DF and IF species.

Results Results from the first four years of this study were reported in the Final Report of BD1451. By 2008, we found that the introduction of facilitator species into species-poor swards had promoted fungal growth in soil, and speculated that this would lead to enhanced colonization of late successional species in coming years. Here we report on the impact of experimental treatments on plant and soil microbial communities, and soil nutrients, across sites, sampled in 2011.

Soil microbial communities and nutrients - In 2011, the presence of direct facilitator species significantly (F1,168 = 3.983, P = 0.048) reduced the fungal to bacterial PLFA across all sites and soils, indicating that these plants had promoted the growth of bacteria more than fungi (Figure 1). This finding was opposite to what was found in 2008, when the direct facilitators significantly increased the biomass of fungi relative to bacteria, as initially predicted. This switch in the response of the microbial community to direct facilitator species was associated with changes in soil conditions, although these effects were site specific (Figure 2): the presence of direct facilitators significantly increased soil C and N content at North Wyke, soil pH at Newcastle and North Wyke, and soil concentrations of calcium and sodium at Newcastle and North Wyke. Facilitator species had no effect on any of the measured soil nutrients at Reading, and, in general, the addition of Rhinanthus to swards had no effect on soil microbial communities or nutrient levels, aside from increasing soil potassium concentrations at the Newcastle site (F1,168=4.116, P=0.044). In sum, these findings suggest that, in the longer term, the addition of direct facilitator species has shifted the microbial community to one that is more dominated by bacteria, and at certain sites, they had increased the fertility of soil.

Figure 1. Effect of direct facilitators on fungal-bacterial PLFA ratio across all three sites.

Plant community responses – Facilitator species increased overall plant species richness at North Wyke, in both alluvial and clay soils, but not at Newcastle or Reading (P<0.001 for the site x facilitator interaction) (Figure 3). This response, however, was mostly attributed to the presence of the direct facilitator species themselves, which also colonized treatments where they had not been added; despite increasing overall plant species richness at North Wyke, the addition of facilitator plants suppressed the species diversity and evenness of the plant community (Figure 4). The same response was detected at Newcastle and Reading, but only in the clay and chalk soil, respectively (Figure 4). Also, the addition of facilitator species suppressed the biomass (P <0.001; Figure 5) and richness of late successional species at all sites (P<0.001; Figure 6). This response was presumably a consequence of increased dominance of the direct facilitator species and hence competition, which suppressed the growth of late successional species. Contrary to expectations, the addition of Rhinanthus had no impact on overall plant species richness or diversity across sites and soil treatments, and didn’t affect the colonization of late successional species. Across all sites, one

EVID4 Evidence Project Final Report (Rev. 06/11) Page 5 of 11 a) b)

c) d)

e) f)

g) h)

Figure 2. Effects of direct facilitator species upon soil nutrients and C in three different sites. Interaction terms between site and facilitator presence for a) pH, b) total N, c) total C, d) Olsen’s P, e) calcium, f) potassium, g) , and h) sodium.

of the strongest determinants of plant species diversity (P = 0.006) and evenness (P = 0.009) was soil type, both being greater in clay than alluvial soil at Newcastle and North Wyke (Figure 4). Likewise, the biomass (P = 0.017) and richness (P <0.017) of late successional species was affected by soil type, but the effect was in the opposite direction: biomass and richness of late

EVID4 Evidence Project Final Report (Rev. 06/11) Page 6 of 11 successional species were both greater in alluvial than clay soils, but did not differ between the chalk and neutral soil at Reading (Figure 5 and 6).

Figure 3. Influence of direct facilitators on plant species richness in soils at Newcastle, North Wyke and Reading. Data are divided on the basis of original species, facilitators, and late successional species.

Figure 4. Influence of facilitators on plant community evenness in soils at Newcastle, North Wyke and Reading

Discussion In contrast to what was expected on the basis of our past field experiments (Smith et al. 2008),

EVID4 Evidence Project Final Report (Rev. 06/11) Page 7 of 11 direct facilitator species caused a reduction in the success of later successional species, reducing their biomass and richness across all sites. A similar response was also found in the MICROSITES project (BD1459), based at North Wyke, where the establishment of target late successional species was found to be negatively related the of facilitator species in a range of ground disturbance treatments, thereby challenging the notion that that early successional species create a suitable conditions for later successional species. On the contrary, our results, and those from the MICROSITES project, suggest that competitive interactions between facilitator species and sown late successional species may outweigh any positive effects of facilitators on colonisation of the latter via soil conditioning, at least in the short timescale of these studies.

While the direct facilitator species were beginning to show signs of modifying the soil microbial community in favour of fungi during the initial stages of the experiment, as was expected, this response did not persist into the later stages of the experiment: by the end of the experiment, in the addition of direct facilitator species to the sward had caused a reduction in the abundance of fungi relative to bacteria, which was associated with changes in concentrations of some soil nutrients, albeit in a site specific and inconsistent way. The variable response of nutrients to the presence of facilitators might reflect the fact that they performed differently across sites, for example with Plantago lanceolata, Prunella vulgaris, and Trifolium pratense being the most common facilitator species at North Wyke, Reading and Close House, respectively. We do not know the cause of this variability, but it may reflect soil and or climatic effects on the establishment of these plant species.

The response of the soil microbial community to the presence of direct facilitators is also in sharp contrast to what was expected based on previous findings in the field, where they were shown to promote fungal relative to bacterial growth in soil (Smith et al. 2003, 2008). We do not know what caused the reduction in the abundance of fungi relative to bacteria in treatments sown with direct facilitator species in the later stages of the experiment. However, previous studies show that a key driver of such shifts in microbial community composition are increases in the amount or quality or organic matter entering soil, either in the form of plant litter (shoot and roots) or root exudates, which result from plant community change (Orwin et al. 2010; De Vries et al. 2012). Therefore, the change in microbial community composition observed here likely reflects an increase in the quality (e.g. N content) and/or amount of organic matter entering soil due to either the direct facilitators themselves, or their indirect impact on the composition of the plant community. Further studies are needed to elucidate the mechanisms involved.

Surprisingly, Rhinanthus had no consistent impact on soil microbial communities or nutrients, and didn’t affect plant diversity or the colonization of late successional species. This finding is in contrast to what was found earlier on in this experiment, when Rhinanthus presence increased inorganic nitrogen availability in soil, and also to previous studies which show Rhinanthus to promote nutrient cycling in soil (Bardgett et al. 2006). Also, the finding that Rhinanthus had no impact on plant diversity or the success of sown late successional species also in contrast to what has been found previously, where Rhinanthus is commonly used as tool for restoring plant species diversity due to its debilitating effect on the competitive dominance of grasses (Pywell et al. 2004; Bullock and Pywell 2005; Bardgett et al. 2006). This discrepancy, however, likely reflects the high variability in Rhinanthus establishment across sites and soil types in the present study. For example, Rhinanthus failed to establish in some replicate mecososms at North Wyke early on in the experiment, and this may have created a differential soil legacy between replicates during the early stages of this study. Also, there was a tendency for Rhinanthus to establish better on the alluvial soils at Close House and North Wyke than on clay soils, again increasing variation in its capacity to impact vegetation and soil in a consistent manner. Sporadic establishment of Rhinathus, and variable effects on plant biomass and establishment of target species, has also been found in other studies, such as the MICROSITES project, where soil moisture and disturbance were key factors determining its success.

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Figure 5. Influence of facilitator species on the biomass of late successional species different soils at Newcastle, North Wyke, and Reading

Figure 6. Influence of facilitator species on the number of late successional species in different soils at Newcastle, North Wyke, and Reading.

Although our findings do not support the idea that facilitator species promote plant diversity restoration through modifying soil, they do illustrate the power of changes in plant communities to modify soil microbial communities and properties, with potential feedback consequences for ecosystem services such as carbon and nutrient cycling. They also reveal that facilitator plants can strongly influence mineral nutrient concentrations, such as K and Na, promoting their availability across sites. The consequences of this for and grassland restoration are not known, nor are the mechanisms involved; but, they presumably relate to differences in organic matter supply to soil, from either plant litter or root exudates, which lead to changes in microbial activities that control the availability of nutrients in soil. Further more detailed assessment of the effects of treatments on the functional capacity of microbial communities would be required to probe the mechanisms involved, which is beyond the scope of this study.

A key finding to emerge from our study is the importance of soil type as a factor influencing the restoration of plant species diversity and the role of plant-soil interactions in this process. Although it is not possible to change soil type, these findings suggest that the mechanisms that underpin grassland diversification vary across soil types; hence, recommendations for restoring grassland diversity will likewise need to take into account differences in soil conditions, as also highlighted from the results of our national grassland survey reported in the Final Report of BD1451. We suggest that further research is needed to determine the role of variations in soil type in controlling plant-soil interactions and their role in grassland diversity restoration.

In conclusion, our findings, especially when combined with those of the MICROSITE project, provide little support for the idea that direct facilitator species promote the restoration of species diversity in mesotrophic grassland, at least in the short term. We did not find consistent effects of

EVID4 Evidence Project Final Report (Rev. 06/11) Page 9 of 11 Rhinanthus on soil microbial communities or plant diversity, in that effects were highly site specific reflecting its variable establishment. However, as shown in other studies, the establishment and persistence of Rhinanthus varies with soil conditions and disturbance, and as a result its effect on vegetation diversity, varies across sites (Ameloot et al. 2005; Hellstrom et al. 2011). Therefore, while our results do not challenge the general value of Rhinathus as a tool for diversity restoration, they support the view that its value varies across sites. While drawing conclusions from our study, we should caution that they are derived from mesocosms that do not always reflect what happens in the field. Nevertheless, they reveal some of the mechanisms involved in the way that plants modify soils, and in our case, raise questions over the value of direct facilitator species for promoting grassland diversification, at least in the relatively short timescale of our study.

References:

Ameloot, E., Verheyen, K. & Hermy, M. (2005) Meta-analysis of standing crop reduction by Rhinanthus spp. and its effect on vegetation structure. Folia Geobotanica, 40, 289-310. Bardgett, R. D., Smith, R. S., Shiel, R. S., Peacock, S., Simkin, J. M., Quirk, H. & Hobbs, P. J. (2006) Parasitic plants indirectly regulate below-ground properties in grassland . Nature 439, 969-972. Hellström, K., Bullock, J.M. & Pywell, R.F. (2011) Testing the generality of hemiparasitic plant effects on mesotrophic grasslands: a multi-site experiment. Basic and Applied , 12, 235-243. Orwin, K., Buckland, S., Johnson, D., Turner, B., Smart, S., Oakley, S. & Bardgett, R (2010). Linkages of plant traits to soil properties and the functioning of temperate grassland. Journal of Ecology, 98, 1074-1083. Pywell, R. F., Bullock, J. M., Walker, K. J., Coulson, S. J., Gregory, S. J. & Stevenson, M. J. (2004) Facilitating grassland diversification using the hemiparasitic plant Rhinanthus minor. Journal of , 41, 880-887. Smith, R. S., Shiel, R. S., Bardgett, R. D., Millward, D., Corkhill, P., Evans, P., Quirk, H., Hobbs, P. J. & Kometa, S. T. (2008) Long-term change in vegetation and soil microbial communities during the phased restoration of traditional meadow grassland. Journal of Applied Ecology, 45, 670-679. Smith, R. S., Shiel, R. S., Bardgett, R. D., Millward, D., Corkhill, P., Rolph, G., Hobbs, P. J. & Peacock, S. (2003) Soil microbial community, fertility, vegetation and diversity as targets in the restoration management of a meadow grassland. Journal of Applied Ecology, 40, 51-64.

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References to published material 9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.

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