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Conserving New Zealand’s endemic carnivorous patrickensis through restoration of a functioning ecosystem

A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

at Lincoln University by Benjamin R. Waterhouse

Lincoln University 2014

Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. Abstract

Conserving New Zealand’s endemic carnivorous land snail Powelliphanta patrickensis through restoration of a functioning ecosystem

by Benjamin R. Waterhouse

As of 2014, New Zealand’s largest active opencast mine, covering approximately 2500 hectares, and located on the West Coast of the has been granted an extension that will further impact the diverse range of native and endemic flora and fauna in the area. Powelliphanta patrickensis, a locally endemic carnivorous land snail, will be subject to restoration and relocation programmes as a result of habitat loss. Mining of this type requires the complete removal of ecosystems, including vegetation, soil and rock to access the mineral resources beneath, impacting a range of and ecological processes. Soil and rock are then stored in large, mixed stockpiles that can become depleted in volume, organic matter and nutrients. A number of restoration measures are already undertaken on site including: vegetation direct transfer (VDT), where intact sods of topsoil, flora and fauna are lifted and transplanted to a holding site or site requiring restoration; addition of biosolids to stockpiled soils to restore organic matter and nutrient levels in stockpiled soil; and spread of untreated soil stockpiles. The objectives of this thesis were to test the efficacy of the current restoration methods and further develop ecological restoration practices following mining to inform decision-making concerning restoration, with an initial focus on P. patrickensis.

Molecular analysis of P. patrickensis diet showed that this species has a relatively generalist feeding behaviour consisting predominantly, if not exclusively, of endemic earthworms. Notable seasonal variation occurred between summer and winter diet composition, and comparison to the closely related but allopatric species showed a strong difference in diet composition. Such analyses support the notion that flagship species do not necessarily represent the needs of other, even closely related, species.

The impact of biosolids amendment on a key earthworm species (present in 22% and 77% of P. patrickensis and P. augusta diets respectively) was assessed in a glasshouse experiment. Biosolids

ii addition led to total earthworm mortality, compared to significantly lower mortality rates in pots containing ‘unmodified’ native and untreated stockpiled soil. Biosolids, however, doubled plant growth compared to controls, and both biosolids and earthworms benefited soil community and function.

Subsequent work investigated the response of various other ecosystem processes to VDT, biosolids amendments and stockpiled soil. Soil microbial activity, biomass, community-level physiological profile and functional diversity were examined in a field setting. VDT was the most effective restoration technique, followed by biosolids treatment, and then stockpiled soil spreads. Reconciling results with studies of the effects of restoration protocols on earthworm species richness and abundance led to the recommendation that VDT is a preferable restoration type, and that P. patrickensis should be relocated only to VDT or undisturbed native sites when being translocated.

Responses of other ecosystem functions, including plant-arbuscular mycorrhzal fungal (AMF) interactions and their effects on plant growth and soil microbial communities, to restoration practices were examined in glasshouse experiments. It was found not only that native vegetation was sensitive to the AMF species present, but also that biosolids disrupted colonisation of plant roots by AMF.

Rather than pursue a ‘flagship’ or ‘keystone’ species approach focusing on a single species, this thesis used the legal, conservation and media attention that P. patrickensis attracted, to investigate a range of ecosystem-wide processes that inform site restoration. Recommendations for restoration should be supported by strong empirical evidence, and this thesis has provided a basis for further work on complex ecological interactions associated with restoration of opencast mining.

Keywords: AMF, biomass production, CLPP, conservation, diet, diversity, DNA, earthworms, ecological restoration, ecosystem function, food web, functional diversity, mine reclamation, molecular, mollusc, mycorrhizae, native, opencast mining, plant growth, Powelliphanta, rehabilitation, richness.

iii Acknowledgements

Undertaking a PhD is tricky business, and I would like to express my sincere gratitude to a number of people who enabled my completion of this research.

First and foremost, my supervisors Steve Wratten and Stéphane Boyer for their continued support, intellectual stimulation, guidance and conversation. Both of my supervisors have operated and open-door policy of which I have taken full advantage and the student-supervisor dynamic was the best I could have hoped for. Thank you.

I have been lucky to enlist the help of a number of other advisors whose expertise has taught me a range of skills and enriched my work and experience. Namely, Dr. Andrew Holyoake: one of the most talented and patient molecular scientists I know; Dr. Hayley Ridgway and Dr. Eirian Jones, my mycorrhizal and soil biological science gurus; Dr. Karen Adair who was my go-to for soil biology and multivariate statistics (and a roof over my head following my arrival in New Zealand).

My work on an active opencast mine would not have been possible without the continued and impeccable support of Mark Hamilton and his team at MBC Contracting Ltd., the onsite mine environmental consultants. Paul Weber and Phil Rossiter were my main contacts at Solid Energy New Zealand Ltd. who arranged site access and have great knowledge of the processes and environmental issues on site. Barry Walker and Robbie Blankenstein were extremely generous with onsite support, access, information and general interest in my project.

Technical and laboratory support was provided by Roger Cresswell (soil assays and soil laboratory practice), Brent Richards (nursery and glasshouse experiments), Dave Saville (statistics), and Celine Blonde (DGGE), who put up with my innumerable requests for advice and equipment.

I would like to thank my funding bodies: Miss E. L. Hellaby Indigenous Grassland Research Trust as my primary funding source; The Bio-Protection Research Centre in Lincoln University, New Zealand; and Solid Energy New Zealand Limited. Without this financial support, the project would not have happened.

I have made numerous new friends and connections while living in New Zealand, and I am grateful for the good times had with all of them. The laboratory can become a lonely place, and having friends around for conversation and company is invaluable… even when they encourage the use of contaminated primers from the bin, Jack Lee.

Finally, I would like to thank my partner Lara for her long-suffering support, tolerance and hot meals that kept the engine running throughout. iv Table of Contents

Abstract ...... ii Acknowledgements ...... iv Table of Contents ...... v List of Tables ...... viii List of Figures ...... ix

Chapter 1 Introduction ...... 1 1.1 General introduction ...... 1 1.2 Impacts of opencast mining...... 4 1.3 Ecological Restoration ...... 5 1.3.1 Restoring ecosystem function ...... 5 1.3.2 Soil restoration ...... 6 1.3.3 Earthworms and biosolids ...... 8 1.4 Current research ...... 8

Chapter 2 Pyrosequencing of prey DNA in faeces of carnivorous landsnails to facilitate ecological restoration and relocation programmes ...... 11 2.1 Abstract...... 11 2.2 Introduction ...... 12 2.3 Methods ...... 15 2.3.1 Site location and sample collection ...... 15 2.3.2 Morphological analysis ...... 15 2.3.3 Molecular analysis ...... 15 2.3.4 Library preparation...... 17 2.3.5 Sequence analysis...... 17 2.4 Results...... 18 2.4.1 Morphological analysis ...... 18 2.4.2 Molecular analysis ...... 19 2.5 Discussion ...... 23 2.5.1 Diet composition for P. patrickensis...... 23 2.5.2 Diet comparison between P. patrickensis and P. augusta ...... 24 2.5.3 Consequences for habitat restoration and snail conservation ...... 24 2.5.4 Limitations of the molecular approach ...... 25 2.6 Conclusion ...... 26

Chapter 3 Using municipal biosolids in ecological restoration: what is good for plants and soil may not be good for endemic earthworms ...... 27 3.1 Abstract...... 27 3.2 Introduction ...... 28 3.3 Methods ...... 30 3.3.1 Sampling and experimental design ...... 30 3.3.2 Plant biomass ...... 31 3.3.3 Earthworm mortality and biomass ...... 31 3.3.4 Soil dry weight ...... 32 3.3.5 Dehydrogenase activity ...... 32 v 3.3.6 Microbial community-level physiological profile and functional diversity ...... 33 3.3.7 Soil chemistry and pH ...... 34 3.3.8 Statistical analysis ...... 34 3.4 Results...... 34 3.4.1 Plant biomass ...... 34 3.4.2 Earthworm mortality and biomass ...... 35 3.4.3 Dehydrogenase activity ...... 35 3.4.4 Microbial community-level physiological profile and functional diversity ...... 38 3.4.5 Soil chemistry & pH ...... 38 3.5 Discussion ...... 41 3.5.1 Earthworm mortality and biomass ...... 41 3.5.2 Plant growth ...... 42 3.5.3 Microbial community-level physiological profiles and functional diversity ...... 43 3.6 Conclusion ...... 44

Chapter 4 Advanced mine restoration protocols facilitate early recovery of soil microbial biomass, activity and functional diversity ...... 45 4.1 Abstract ...... 45 4.2 Introduction ...... 46 4.3 Methods ...... 48 4.3.1 Experimental design and site selection ...... 48 4.3.2 Soil collection ...... 49 4.3.3 Soil microbial biomass ...... 49 4.3.4 Dehydrogenase activity ...... 49 4.3.5 Community-level physiological profile and functional diversity ...... 49 4.3.6 Statistical analyses ...... 50 4.4 Results ...... 50 4.4.1 Soil microbial biomass ...... 50 4.4.2 Soil dehydrogenase activity ...... 50 4.4.3 Community level physiological profile and functional diversity ...... 52 4.5 Discussion ...... 53 4.6 Conclusion ...... 55

Chapter 5 Re-establishing plant-AMF symbioses is dependent on AMF source community and disrupted by high nutrient levels ...... 56 5.1 Abstract ...... 56 5.2 Introduction ...... 57 5.3 Methods ...... 59 5.3.1 Soil preparation ...... 59 5.3.2 Preparation of AMF spore inoculum ...... 60 5.3.3 Plant preparation ...... 60 5.3.4 Experimental design ...... 61 5.3.5 Plant biomass and AMF root colonisation ...... 61 5.3.6 Soil community analysis ...... 61 5.3.7 Dehydrogenase activity ...... 62 5.3.8 Community-level physiological profile and functional diversity ...... 62 5.3.9 Statistical analysis ...... 62 5.4 Results ...... 63 5.4.1 Plant biomass ...... 63 5.4.2 Root colonisation ...... 63

vi 5.4.3 CLPP, functional diversity a