School of Earth and Planetary Science Functional ecology of calcrete aquifers in arid zone Western Australia Mattia Saccò This thesis is presented for the Degree of Doctor of Philosophy Of Curtin University January 25th, 2020 DECLARATION I hereby declare that this thesis is my own work, except where otherwise stated with respect to manuscript contributions, and contains no material published prior to or outside of the PhD enrollment period. I also certify that this thesis provides original and significant contribution to the field of groundwater ecology. This work has not been submitted in any form for another degree or diploma at any other tertiary institution. Information derived from the published and unpublished work of others has been acknowledged in the text. Details on authors’ contributions, per each one of the published and submitted manuscripts composing this thesis is detailed after each chapter title. Signed, Mattia Saccò January 25th, 2020 II COPYRIGHT NOTICES Notice 1 Under the Copyright Act 1968, this thesis must be used only under the normal conditions of scholarly fair dealing. In particular no results or conclusions should be extracted from it, nor should it be copied or closely paraphrased in whole or in part without the written consent of the author. Proper written acknowledgement should be made for any assistance obtained from this thesis. Notice 2 I certify that I have made all reasonable efforts to secure copyright permissions for third- party content included in this thesis and have not knowingly added copyright content to my work without the owner's permission. III ABSTRACT Groundwaters host vital resources - 97% of unfrozen freshwater worldwide - playing a key role in the near future of humanity. Stygofauna (subterranean obligate aquatic invertebrates), together with microbes, are crucial actors in shaping and maintaining the organic matter cycles in these environments, which are characterized by low energy and scarce carbon availability. However, our knowledge about how these ecosystems function is limited, and subterranean environments are increasingly exposed to anthropogenic impacts and climate change-related processes. In order to dig into groundwater ecological dynamics, we investigated arid zone calcrete stygofaunal and microbial patterns linked with contrasting rainfall periods (low rainfall (LR), dry season; high rainfall (HR), wet season), through an interdisciplinary design composed of multivariate analyses, hydrology, isotopic ecology and genetics. Our results from multivariate investigations indicate that stygofaunal niches are closely linked to the hydrodynamic conditions influenced by different rainfall regimes (LR and HR). Isotopic evidence depicted stygofaunal tendencies towards opportunistic and omnivorous habits, typical of an ecologically tolerant community, shaped by bottom-up controls linked with changes in carbon flows. Biochemical data suggest that the inflow of rainfall under HR is responsible for increased nutrient concentrations in the system and dissolved organic carbon pulses. Metabarcoding data confirmed that the HR regime, and its subsequent terrestrial carbon incorporation, triggers a cascade effect driven by microbes (organic matter processors), copepods and amphipods (biofilm grazers), which is finally transferred to the aquatic beetles (top predators). This study provides baseline biochemical and ecological data for stygofaunal interactions in calcretes. Further long-term investigations, incorporating broader ecological perspectives, will help to understand the impacts associated with climate change and anthropogenic pressures on one of the most threatened and underrated ecosystems in the world. IV ACKNOWLEDGEMENTS Curtin University sits on the traditional land of the Whadjuk Noongar people, and I would like to acknowledge their continuing connection to the land, waters and culture of their country. I offer my respects to their Elders, past, present and emerging. This thesis is the result of an unforgettable journey that could not have been possible without the support of many people. First, I would like to thank my main supervisor Alison Blyth for ‘bringing’ me down under and giving me a unique opportunity to grow both personally and professionally. Thank you, Alison, for putting up with my loudness and messiness. Your guidance played a crucial role in achieving this professional milestone in my life. I would also like to thank my other supervisor Bill Humphreys. Thank you, Bill, for your constant willingness to give me your priceless help and scientific wisdom. My other two supervisors Bill Bateman and Kliti Grice also greatly contributed to my research with support and advice. I gratefully acknowledge the Australian Research Council and the Australian Institute of Nuclear Science and Engineering for supporting this research. Special thanks go all the coauthors of the published (and submitted) manuscripts and all the colleagues, technicians, professors, consultants that contributed to this research. You all made it possible that this interdisciplinary study came to reality. I would like to thank my friends spread all around the world for making me feel at home wherever I travelled and lived. You have been a crucial pillar during the last four years. Foremost, I specially thank my Italian and Australian families for their love. This thesis is dedicated to you. Thank you for always being there, for encouraging me daily and for bearing with me during hard times. V TABLE OF CONTENTS Thesis overview ..................................................................................................................... 1 Chapter 1| Background and significance ................................................................................. 3 1.1 Ecological dynamics in groundwaters ................................................................................ 3 1.1.1 Groundwater habitats ............................................................................................. 3 1.1.2 Temporal and spatial patterns in groundwater ecosystems ................................... 4 1.1.3 Subterranean biotic communities ............................................................................ 6 1.2 Methodological challenges and novel approaches ........................................................... 9 1.3 Research gaps and thesis question .................................................................................. 11 1.3.1 Research gaps in groundwater ecology ................................................................. 11 1.3.2 Groundwater ecology in Australia ......................................................................... 12 1.3.3 Thesis question: Functional ecology of calcrete aquifers in arid zone Western Australia ......................................................................................................................... 13 References ............................................................................................................................. 14 Chapter 2 | New light in the dark - a proposed multidisciplinary framework for studying functional ecology of groundwater fauna.............................................................................. 22 Abstract .................................................................................................................................. 23 2.1 Introduction ..................................................................................................................... 25 2.2 Stygofauna vs Environment: diving into groundwater dynamics .................................... 27 2.3 Beyond the (subterranean) big picture ........................................................................... 29 2.3.1 Stygofaunal diversity ............................................................................................. 30 2.3.2 Pressures on groundwater ecosystems ................................................................. 33 2.3.3 Biogeochemical patterns ....................................................................................... 34 2.3.3.1 Biotic/abiotic transitions ............................................................................. 34 2.3.3.2 Trophic dynamics ......................................................................................... 36 2.4 Filling (groundwater) voids: CSIA, 14C and DNA analysis ................................................ 37 2.4.1 CSIA: deciphering food webs ................................................................................. 38 2.4.2 Radiocarbon dating (14C): digging into the path of carbon .................................. 42 2.4.3 eDNA and DNA metabarcoding: the new biomonitoring frontiers........................ 46 2.5 The statistical linkage: Bayesian mixing models .............................................................. 48 2.6 Conclusions ...................................................................................................................... 51 Acknowledgements ............................................................................................................... 51 References ............................................................................................................................. 51 Chapter 3 | Stygofaunal community trends along varied rainfall conditions: deciphering ecological niche dynamics of a shallow calcrete in Western Australia .................................... 68 Abstract .................................................................................................................................