Patterns of Water Use by the Riparian Tree Melaleuca Argentea in Semi-Arid Northwest Australia

Patterns of water use by the riparian tree Melaleuca argentea in semi-arid northwest Australia

Elizabeth Helen McLean

BSc (Hons)

This thesis is presented for the degree of Doctor of Philosophy

The University of Western Australia

School of Plant Biology

2014

ABSTRACT

This thesis examines the water use physiology of the riparian tree Melaleuca argentea, and the ways in which this species may respond to anthropogenic disturbances to hydrologic processes. The research investigated patterns of water use by M. argentea in relation to key aspects of the riparian environment, including in response to flooding and drought as well as to groundwater drawdown.

Study sites focused on the remote Pilbara region of northwest Australia (encompassing ~500,000 km2, ~1700 km north of Perth, the nearest major city). The Pilbara is currently undergoing rapid economic development and population growth due to expansion in the mining sector. There are concerns that intensification of mining efforts, which often involves dewatering for extraction of ore below the water table, and abstraction of water to supply regional populations and industry could produce undesirable environmental outcomes of a long-term or even permanent nature. However, comparatively little is known of the functioning and especially the water requirements of many of the key species of the Pilbara and the ecosystems in which they occur. M. argentea is considered an obligate phreatophyte and is confined to riparian zones where there is permanent surface and near-surface water. M. argentea is thus very likely to be vulnerable to changes in groundwater levels, recharge patterns and altered water dynamics that may arise through abstraction and dewatering. Clearly, an understanding of tree water use patterns of M. argentea will provide much-needed information for ecosystem management, while also contributing to our fundamental understanding of riparian tree water use physiology along ephemeral waterways and their resilience to both natural and imposed dynamics in water availability.

Extreme vapour pressure deficits (VPD) and highly variable rainfall are typical of the Pilbara region, but M. argentea typically has access to an unlimited supply of water. Accordingly, water use patterns of M. argentea trees as determined by sap flow measurements appeared to be largely driven by the atmospheric variables of VPD, wind speed and light intensity. However, multiple regression analysis revealed that 20 to 30% of seasonal variation in water use was not directly attributable to seasonal climatic

i fluctuations. Results suggest that M. argentea utilises internal water storage over both diurnal and seasonal cycles, perhaps due to rates of water transport reaching their maximum limit under high evaporative demand.

While M. argentea mostly occurs where there is access to permanent water, the distribution of water in riparian zones in arid and semi-arid environments nevertheless exhibits considerable spatial and temporal variability. M. argentea displayed plastic root- level strategies to maintain high rates of resource capture under heterogeneous and dynamic distributions of water around the root system. Sap flow measurements in the field suggested that roots redistribute water from the saturated zones into drier regions, maintaining fine root function in the dry zones for periods of at least six months. When the spatial distribution of water was manipulated in glasshouse experiments, the roots rapidly compensated for drying of one-third of the root system by increasing fine root hydraulic conductance, aquaporin content and growth in the hydrated zone. I suggest that these compensatory responses are likely to occur in a wide range of other species, particularly those adapted to heterogeneous environments. During partial root zone drying of M. argentea there was no evidence of root-derived signals inducing stomatal closure, as stomatal conductance, water use (measured gravimetrically) and tissue abscisic acid (ABA) content remained unchanged. Plastic root-level strategies and internal water storage appear to enable M. argentea to maintain high rates of water and nutrient uptake and transpiration, and therefore high rates of photosynthesis and growth, in an environment characterised by high VPD above ground and heterogeneity below ground.

Since M. argentea is confined to the wettest positions in the landscape, it was expected this species would be highly sensitive to disturbances that reduce water availability, such as groundwater drawdown, but tolerant of disturbances increasing water availability, such as prolonged flooding during discharge of water into creeklines. In a glasshouse experiment exposing M. argentea seedlings to a variety of hydrologic conditions, the seedlings were highly tolerant of flooding; rates of photosynthesis and most aspects of shoot growth were undiminished after eight weeks of waterlogging, although total leaf area was slightly lower than in drained plants. Roots responded plastically to waterlogging, with reduced total root biomass but proliferation of shallow roots. In contrast, under even a mild water stress, shoot growth of M. argentea seedlings rapidly ceased and greater biomass was ii allocated to roots, and leaf shedding soon occurred under more severe water stress. M. argentea seedlings are therefore unlikely to establish in locations without abundant water supply, or where near-surface water availability is reduced, while rates of recruitment could increase in flooded locations under groundwater discharge.

The responses of larger trees to groundwater abstraction were explored at abstraction and control sites at the Yule River Borefield on the coastal plain of a large ephemeral river, 50 km southwest of Port Hedland, in northwest Australia. The water table at the abstraction site fell by 4.3 m over a 13-month period, compared with a 1.5 m decline over the same period at an upstream control site. Large, mature trees were generally tolerant of the abstraction, reflected by only small increases in leaf δ13C and slightly reduced sap flow rates relative to the control trees, primarily when the depth to the water table exceeded 5.5 m. However, smaller, younger trees appeared more susceptible to lowering of the water table, most likely due to reduced access to water as a result of their shallower root systems. This finding highlights the potential vulnerability of M. argentea recruitment to long-term reductions in water availability in the riparian zone while also demonstrating some resilience of more mature trees to the range of conditions experienced throughout the study period.

Overall, M. argentea displays highly plastic root-level responses to heterogeneous water availability and to waterlogging, facilitating high rates of water use and growth in the riparian wetland habitats of the Pilbara. Mature M. argentea trees also appear to tolerate groundwater drawdown of at least several metres, most likely by employing the same plastic root strategies to access deeper water. A particularly useful finding is that M. argentea can also withstand short periods of severe drought, by adopting a ‘waiting’ strategy of ceasing growth and shedding leaves to avoid moisture loss, a state from which they can then recover. Nevertheless, M. argentea populations are unlikely to thrive under large and prolonged reductions in water availability. This study clearly demonstrates the need for careful consideration of the complex responses of riparian tree species to changes in water availability, when planning water management in the riparian environments of the Pilbara.

iii

ACKNOWLEDGEMENTS

This PhD would not have been possible without the assistance and advice I received from numerous people, and I am immensely grateful to them all.

Thank you most of all to my supervisors, Pauline Grierson, Martha Ludwig and Mark Adams, for their guidance throughout this PhD from project conception to the final thesis editing. To my primary supervisor Pauline, thank you for keeping me on track while also allowing me the independence to pursue the research topics and directions of my choosing. Through the breadth of your knowledge I always found fresh perspectives and new ideas during our discussions. To Martha, thank you of course for your expert advice, but thank you also for your general encouragement, support and friendly chats, even in topics outside your field.

I thank Rio Tinto Pty Ltd and BHP Billiton for collaboration and support of much of the work contained in this thesis, funded through an Australian Research Council Industry Linkage grant (LP0776626). Thanks particularly to Tim Eckersley, Sally Madden, Sam Luccitti and Nicole Gregory at Rio Tinto, and to Stephen White and Esme Winks at BHP, for their advice and assistance, including in the organising of field sites, sampling trips at Marillana Creek, and sharing datasets. I also thank the Water Corporation of Western Australia and the Department of Water for collaboration and support of the work at the Yule River. Thanks go especially to Jacquie Bellhouse and Andrew Jones at the Water Corporation, and to Mike Baimbridge at the Department of Water, for their assistance with data collection in the field and many useful discussions. I am also grateful to have received a Prescott postgraduate scholarship from the University of Western Australia.

Thank you to Sebastian Pfautsch, Mike Kemp, Tim Bleby and Steve Burgess for extensive advice and practical assistance with sap flow equipment, installation and calculations. I also acknowledge Robert Willows and Tony Jerkovic at Macquarie University for conducting the abscisic acid assays presented in Appendix C. To the many people who helped me in the field, lab and glasshouse – chiefly Tom Wright, Chloe Flaherty, Steve Naughton, Bruno Rosado, Gerald Page, Rachel Argus, Doug Ford, Kate Bowler and Mark Waters – thank you for your diligence and companionship during long iv hours of repetitive work, and in the heat of the Pilbara summer. I am grateful to the workshop and glasshouse staff, especially Rob Creasy and Ray for their help in solving the problems surrounding intricate split-root experiments on trees in huge pots. Thanks to all the members of the ecosystems research group for your friendship, especially Gerald, Doug, Ali and Alex with whom I have shared the PhD journey. I will remember these years most of all for the coffees and lunches by the river. Thanks to Gerald Page for the many helpful lunchtime discussions of plant physiology, among other topics.

And finally, thank you to my family for your patience, understanding and interest in my work. Most of all to Mark – thank you for your seemingly endless patience and love, and undying IT technical support. This has of course been only one small part of our life together, but your willingness to move to the other side of the world and to support me throughout this PhD means more to me than you know.

v STATEMENT OF CANDIDATE CONTRIBUTION

This thesis is my own work, except where otherwise acknowledged, and has been substantially completed during the course of my enrollment for the degree of Doctor of Philosophy at the University of Western Australia. It has not previously been accepted for any other degree, at this or any other institution.

This thesis contains published work; Chapter 5 was published as: McLean E.H., Ludwig M. & Grierson P.F. (2011) Root hydraulic conductance and aquaporin abundance respond rapidly to partial root-zone drying events in a riparian Melaleuca species. New Phytologist 192: 664–675. I was the major contributor to this paper, and completed the data collection, analysis and manuscript preparation. My co-authors (also my PhD supervisors) provided methodological, technical and editorial advice. I have permission from all co-authors to include this work in my thesis.

CONTENTS

CHAPTER ONE: General introduction ...... 1 ECOHYDROLOGY OF the DRYLANDS of NORTHWEST AUSTRALIA ...... 3 Patterns of rainfall and recharge across the Pilbara ...... 4 Hydrologic disturbance and management of groundwater resources in the Pilbara ...... 6 ECOPHYSIOLOGY OF RIPARIAN TREES IN DRYLAND REGIONS ...... 9 OBJECTIVES OF THIS THESIS ...... 13

CHAPTER TWO: Seasonal patterns of water use by the riparian tree Melaleuca argentea in semi-arid NW Australia ...... 15 INTRODUCTION ...... 15 MATERIALS & METHODS ...... 18 Study sites ...... 18 Meteorological data ...... 20 Tree water use ...... 20 Canopy physiological measurements ...... 22 Statistical analysis ...... 22 RESULTS ...... 23 Seasonal variation in tree water use and meteorological conditions ...... 23 Diurnal patterns of tree water use physiology across seasons ...... 24 Relationships of tree daily water use with meteorological conditions ...... 26 The effects of site, rainfall and season on tree water use ...... 30 Tree hydraulic conductance ...... 33 DISCUSSION ...... 34 Factors determining water use of M. argentea ...... 34 Seasonality of water use in M. argentea ...... 36

CHAPTER THREE: Quantifying water fluxes at tree and stand scales from sap flow measurements in the riparian tree Melaleuca argentea ...... 43 INTRODUCTION ...... 43 MATERIALS & METHODS ...... 47 Study sites ...... 47 Sap flow measurement ...... 47

vii

Tree sapwood area ...... 48 Tree hydraulic conductance ...... 48 Analysis of variation in sap flow ...... 48 RESULTS ...... 49 Sap flow variation within the sapwood ...... 49 Temporal variation in sap flow ...... 52 Variation between trees and sites ...... 53 DISCUSSION ...... 57

CHAPTER FOUR: Redistribution of water through root systems of a semi-arid riparian tree during a flooding-drying cycle in an intermittent waterway...... 64 INTRODUCTION ...... 64 METHODS ...... 66 Study site ...... 66 Meteorological measurements ...... 66 Sap flow measurements ...... 66 Statistical analysis ...... 69 RESULTS ...... 69 Patterns of root sap flow with drying conditions ...... 69 Root responses to rainfall ...... 72 DISCUSSION ...... 76

CHAPTER FIVE: Root hydraulic conductance and aquaporin abundance respond rapidly to partial root-zone drying events in a riparian Melaleuca species ...... 80 INTRODUCTION ...... 80 MATERIALS & METHODS ...... 83 Plant material and growth conditions ...... 83 Experimental design ...... 83 Stomatal conductance and tissue water status ...... 85 Plant water use ...... 85 Root hydraulic conductance ...... 86 Xylem sap osmolarity ...... 86 Root growth...... 87 Protein extraction and immunoblotting ...... 87 Statistical analysis ...... 88 RESULTS ...... 89 viii

Stomatal conductance and leaf water status were unaffected by PRD ...... 89 Survival of the dried root portion ...... 90

Water uptake and Lp of the hydrated roots increased during PRD ...... 91 Growth of fine roots in the hydrated root portion ...... 94 Abundance of PIP1 and 2 aquaporins in the hydrated roots was altered by PRD ...... 94 DISCUSSION ...... 94 Adaptive responses to heterogeneous water supply ...... 94 Adjustments in root hydraulic conductance during PRD ...... 97 Conclusions ...... 100

CHAPTER SIX: Morphological and physiological adjustments across a hydrologic gradient by seedlings of a semi-arid riparian Melaleuca ...... 101 INTRODUCTION ...... 101 MATERIALS & METHODS ...... 104 Plant material and growth conditions ...... 104 Experimental design ...... 104 Plant growth and biomass ...... 105 Morphology and anatomy of leaves and roots ...... 105 Plant physiological status ...... 107 Statistical analysis ...... 108 RESULTS ...... 108 Seedling growth and biomass allocation ...... 108 Seedling leaf shedding and survival ...... 111 Morphological responses...... 112 Physiological responses ...... 114 DISCUSSION ...... 118 Growth and responses under saturated conditions ...... 118 Drought responses ...... 121 Implications for response thresholds under field conditions ...... 124

CHAPTER SEVEN: Response of Melaleuca argentea trees to ground water abstraction from the coastal plain aquifer of a large ephemeral river ...... 126 INTRODUCTION ...... 126 MATERIALS & METHODS ...... 129 Study site ...... 129 Groundwater levels and abstraction ...... 131 ix

Soil moisture ...... 131 Isotope analysis of xylem sap, ground and soil water ...... 133 Tree water use ...... 134 Leaf physiological measurements ...... 134 Statistical analysis ...... 135 RESULTS ...... 135 Water table recession with groundwater abstraction ...... 135 Tree water uptake from soil, surface and ground water sources ...... 135 Tree water use ...... 136 Leaf physiology of M. argentea in relation to lowering of groundwater ...... 138 DISCUSSION ...... 139 Tree water use patterns in response to abstraction ...... 143 Effects of abstraction on small trees ...... 144 Implications for vegetation management under increased abstraction ...... 145

CHAPTER EIGHT: General discussion: temporal and spatial patterns of water use by Melaleuca argentea ...... 146 ADAPTATIONS OF M. ARGENTEA ...... 146 Extreme VPD and temperature conditions ...... 146 Heterogeneous belowground environment ...... 150 ECOSYSTEM SCALE IMPLICATIONS OF WATER USE PATTERNS ...... 155 POTENTIAL IMPACTS OF ALTERED WATER REGIMES ON M. ARGENTEA ...... 157 CONCLUSION ...... 160

REFERENCES ...... 161

APPENDIX A: Verification of methods used to measure stomatal conductance in the mid-canopy of mature trees ...... 193

APPENDIX B: Supplementary information for Chapter Five ...... 196

APPENDIX C: Tissue ABA content during partial root zone drying (Chapter Five) ...... 200

APPENDIX D: Examples of micrographs used for anatomical measurements in Chapter Six ...... 202

CHAPTER ONE: General introduction

The aim of this thesis is to increase understanding of the patterns and constraints in water use by the tree Melaleuca argentea W. Fitzg. (silver leaved paperbark or cadjeput), in the semi-arid Pilbara region of northwest Australia. In particular, I sought to characterise the water use physiology of M. argentea and to assess the potential impacts of hydrological disturbances upon this species. The thesis thus examines aspects of the ecohydrological functioning of riparian ecosystems of the Pilbara.

Rivers and streams in arid and semi-arid regions (drylands) provide oases of surface and groundwater within an otherwise water-limited environment. The relative abundance and accessibility of water in these riparian zones support ecosystems of high biodiversity and productivity, including large trees (Sabo et al., 2005; Sheldon et al., 2010; Figure 1.1). However, relatively little is known about the ecological water requirements and water use characteristics of riparian vegetation across northern and inland Australia.

The Pilbara is remote and mostly sparsely populated, but is currently experiencing rapid population growth and development of resource industries. The region contains internationally significant iron ore deposits, as well as smaller deposits of other metals including gold, copper and manganese, and offshore oil and gas reserves (Department of Regional Development and Lands & Pilbara Development Commission, 2011). The ongoing development is increasing the demand for water supplies, as well as impacting on hydrologic regimes across the region (Abbott et al., 2010; Department of Water, 2010). Consequently, there is an urgent need for a clearer understanding of the Pilbara’s riparian vegetation, and its links with the local hydrology, to guide the management of water resources. Among the currently unknown aspects are the resilience of key species to water abstraction, and their physiological functioning against the natural background of episodic floods and droughts.

Melaleuca argentea occurs widely throughout northern Australia, and is one of the key riparian tree species in the Pilbara region (Figure 1.1c–e). M. argentea is found exclusively in locations with permanent water either at or close to the surface (Masini, 1988). The

1 Chapter One

M. (b) Dense riparian trees. trees at a permanent pool in trees (c & d) A stand of M. argentea argentea Karajini National Park. Dales Gorge, trees fringing the wide, sandy river bed of sandy river the wide, fringing trees River, Yule plain sectionthe coastal of the in Photographed river. intermittent a large months after a flooding June 2009, a few with temporaryevent, surface pools still (e) Young bed. in the river present Figure 1.1: Waterways in the Pilbara in the Pilbara Waterways 1.1: Figure woody supportingregion, dense, tall, including stands of Mela- vegetation, argentea leuca along (a) Marillana Creek vegetation (visible along the base of hills), and Tinto Rio (photograph Creek Wolli Weeli Pty Ltd). (b) (e) (d) (c) (a) 2 General introduction presence of M. argentea is also linked with places of cultural significance, as sites of permanent water in particular are vital to indigenous identities, beliefs and livelihoods across the Pilbara (Barber & Jackson, 2011). Owing to its dependence on permanent water, M. argentea might be expected to be highly vulnerable to changes in hydrologic regimes associated with regional development. An understanding of the water requirements and functioning of this species may therefore be of particular value in ecological monitoring, and in identifying critical thresholds of hydrological disturbance.

This chapter outlines the processes that determine patterns of temporal and spatial water availability in the Pilbara, and the ways in which the hydrology may be affected by the disturbances associated with regional development, such as groundwater abstraction, and mining of ore bodies below the water table. I then provide an overview of the current understanding of the water use physiology of riparian trees in dryland zones, with particular reference to M. argentea. This background provides context for the experimental rationale that is developed in chapters 2–7 of this thesis, including the potential applications of this research to improve water management strategies amidst rapid development of semi-arid and arid regions of Australia.

ECOHYDROLOGY OF THE DRYLANDS OF NORTHWEST AUSTRALIA

Ecohydrology refers to the understanding of the hydrologic mechanisms that underlie the distribution, structure and functioning of ecosystems, including the effects of biotic processes, such as tree water use, on the water cycle (Rodriguez-Iturbe, 2000; Nuttle, 2002). The availability of water to plant roots, as soil moisture and accessible groundwater, is one of the key links between climate, hydrology and vegetation. Water availability is a major determinant of terrestrial vegetation structure and productivity on a global scale (Box & Fujiwara, 2009), but the coupling between water and vegetation is particularly close in dryland environments (Caylor et al., 2006; Jackson et al., 2009b). Drylands are drought prone regions, defined by an annual potential evaporation rate that exceeds annual precipitation (Newman et al., 2006). Hot dryland regions, including northwest Australia, are also typically subject to extreme variability in rainfall patterns both within and among years (Schwinning & Sala, 2004; Bunn et al., 2006).

3 Chapter One

Patterns of rainfall and recharge across the Pilbara Droughts, floods and violent storms are not exceptional events across northwest Australia, rather they are integral features of the region with major roles in shaping the vegetation. In the Pilbara, temperatures and vapour pressure deficits (VPD) can be extreme, and rainfall is highly erratic (Figure 1.2). Rainfall patterns are dominated by tropical low pressure systems and cyclones between December and April, with smaller amounts contributed by thunderstorms (Leighton, 2004). Rainfall averages 200–350 mm per year, but inter-annual variation is considerable, for instance, ranging from 44 mm to 630 mm total annual rainfall at Port Hedland during 1943–2012 (Bureau of Meteorology, 2013; Figure 1.2). Long periods of little rainfall are punctuated by large, episodic rainfall events, during which over 100 mm commonly falls within a single day. High evaporation rates also contribute to the aridity of the region, with annual potential evaporation up to 3000 mm, exceeding mean annual rainfall by as much as ten-fold (Dogramaci et al., 2012).

120 (a) 40 (b) 400

100 35 Temperature (°C) 30 300 80 25 60 20 200 15

Rainfall (mm) 40 Rainfall (mm) 10 100 20 5 0 0 0 JFMAMJJASOND 2005 2006 2007 2008 2009 2010

Figure 1.2: Average climatic conditions and inter-annual rainfall variability recorded at Port Hedland, in the Pilbara region. (a) Mean monthly rainfall (bars) and temperature minima and maxima (lines), over the period 1948–2011. (b) Actual monthly rainfall during 2005–2010. Data from the Australian Bureau of Meteorology.

As is common in warm dryland regions, most waterways in the Pilbara are ephemeral, typically with short-lived, high velocity flows, occurring only following large rainfall events (Johnson, 2004; Young & Kingsford, 2006). The Pilbara contains seven major river catchments, with numerous smaller creeklines (Department of Water, 2010; Figure 1.3). High volume and velocity floods erode material from the rugged inland ranges, much of which is deposited on the coastal plains, producing extensive floodplains with deep, duplex soils. On the coastal plain, riverbeds can be very wide (up to 10 km), with braided

4 General introduction channels. Waterways in the ranges tend to be more confined (typically less than 1 km wide), often within deep rocky gorges, although shallower sections with floodplains are also present (Haig, 2009; McKenzie et al., 2009).

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Figure 1.3: Major waterways of the Pilbara region. Modified from Department of Water (2010) and Van Vreeswyk et al. (2004). Locations of the field sites used for this study are indicated in red.

Groundwater is present throughout the Pilbara region. Beneath the coastal floodplains groundwater typically occurs within the alluvium, often with underlying calcrete or limonite, while aquifers in the ranges may be of porous calcrete or pisolite, unconsolidated sedimentary rock (valley fill of alluvium and colluvium), or fractured sedimentary and igneous rock (Johnson & Wright, 2001; Johnson, 2004). Groundwater recharge occurs primarily through floods caused by large rainfall events, with little or no contribution from rainfall events of less than 20 mm (Dogramaci et al., 2012). Recharge is typically rapid; flows through the waterways saturate the shallow alluvium of valleys, gorges and coastal plains within days, followed by seepage into fractured aquifers within weeks (Dogramaci et al., 2012). The extent of aquifer recharge depends on both the size and duration of the flows. Groundwater levels decline naturally between recharge events, through discharge and evapotranspiration, with recession most rapid when the aquifer is at

5 Chapter One its highest level, and slowing as the depth to groundwater increases (Antao & Braimbridge, 2009).

Along sections of many waterways, a saturated zone is commonly present within metres of the riverbed surface, and surface pools may also feature. Most pools are superficial and disconnected from the groundwater; they are maintained only by ongoing rainfall, and dry out during longer drought periods (Fellman et al., 2011; Dogramaci et al., 2012). Other pools are fed by groundwater springs and thus are essentially permanent, such as the Millstream pools on the Fortescue River (Antao & Braimbridge, 2009). A single creekline may contain both superficial pools largely isolated from the water table, and pools with consistent input from shallow alluvium water (Fellman et al., 2011).

The shallow groundwater and ‘waterholes’ of the Pilbara’s riparian zones often support unique ecosystems, including large trees and dense vegetation, in stark contrast with the surrounding landscape. Most of the vegetation of the Pilbara is xerophytic, displaying a range of adaptations to the predominantly arid conditions; spinifex grassland (hummock grasslands dominated by Triodia) is the most common vegetation type, with large areas also occupied by open Acacia shrublands and low woodlands (Beard, 1975; McKenzie et al., 2009). Floodplains are mostly colonised by grasses, and by mangroves on the coastal flats (McKenzie et al., 2009). Most waterways of the Pilbara support woodlands of Eucalyptus camaldulensis and E. victrix, while M. argentea is present only in locations with permanent surface or shallow ground water, where the trees may form denser forests and closed woodlands (Masini, 1988; McKenzie et al., 2009). Waterways also support unique endemics, such as the Millstream fan palm (Livistonia alfredii), a relict species restricted to a few waterhole sites within the Pilbara (Antao & Bambridge 2009). Many of the wetland and pool ecosystems of the Pilbara are of high conservation value and are classified as nationally significant, such as the Millstream pools, the gorges of Karijini National Park, and the Ramsar nominated Fortescue Marsh (Environment Australia, 2001; Jackson et al., 2009a).

Hydrologic disturbance and management of groundwater resources in the Pilbara Many fast-growing populations around the world are located in dryland regions, including in southwestern North America, northern Africa, northern China and southern India, creating particular challenges in sourcing and managing water with scarce and 6 General introduction unpredictable supplies (Batchelor et al., 2003; Newman et al., 2006; Scanlon et al., 2006; Mays, 2009). Securing water supplies frequently involves hydrological disturbance, in the form of groundwater abstraction, and the modification of river flow patterns. Most of the species that inhabit the wetland and riparian zones of drylands, including large riparian trees, are highly dependent on the ground and surface water and could not otherwise survive in an arid environment, and thus can be highly sensitive to any disturbance affecting native patterns of water availability (Ridolfi et al., 2006).

The potential effects of hydrological disturbance on dryland riparian ecology are clearly demonstrated in rivers with long histories of extensive modification. Reducing flow variability, to provide more consistent, secure water supplies and to limit destructive floods, induces common sets of ecological changes (Poff et al., 2007; Poff & Zimmerman, 2010). For example, in semi-arid southwestern USA, dams and diversions are present on most waterways (Stromberg, 2001), leading to reduced riparian biodiversity, declines in native Populus tree species due to recruitment failure, and facilitating invasion by exotic Tamarix tree species (e.g. Busch & Smith, 1995; Tiegs et al., 2005; Williams & Cooper, 2005; Merritt & Poff, 2010). On the Murray River in southeastern Australia, the construction of dams and numerous weirs has reduced flow magnitudes to the lower reaches, and has permanently flooded some previously ephemeral wetlands, while preventing flooding to others (Kingsford, 2000; Walker, 2006). Among the documented ecological effects of these altered hydrologic conditions are lack of recruitment by native trees such as Eucalyptus largiflorens, invasion by exotic weeds, migration of plants normally confined to floodplains into the channel regions, and declining numbers of water birds, native fish and invertebrates (e.g. George et al., 2005; Walker, 2006; Catford et al., 2011). It is clear that the native flow patterns, including the sizes, velocities and frequencies of floods, are integral aspects of riparian environments, and are vital in maintaining riparian ecosystem functioning and biodiversity.

Given the scarcity of permanent surface water across northwest Australia, groundwater is the primary water source used to supply residential and industry needs. Ongoing industry and population growth across the Pilbara will require sourcing of additional water supplies, expected to be achieved primarily through further groundwater developments (Haig, 2009; Department of Water, 2010). Greatest demand for secure and

7 Chapter One reliable water supply is concentrated in towns along the Pilbara coast, including Karratha, Dampier and Port Hedland (Figure 1.2). A large proportion of the water is thus sourced from borefields on the coastal river floodplains, where groundwater typically occurs within the alluvium (Johnson, 2004). In the western Pilbara, water is also obtained from the Millstream dolomite aquifer, located south of Karratha on the Fortescue River, and from Harding Dam, south of Roebourne on the Harding River (Antao & Braimbridge, 2009).

In the Hamersley Basin of the inland Pilbara, water abstraction occurs mainly in association with iron ore mining. Mining is increasingly focused on ore bodies located below the water table that occur as paleoalluvial deposits beneath modern day drainage lines. Localised hydrological disturbances are created by the abstraction of large volumes of groundwater to access the ore, and the subsequent disposal of the water elsewhere in the landscape (Abbott et al., 2010). Since iron ore mining occurs inland, the long distances make it impractical to use ore dewatering abstraction for town water supplies on the coast. While some of the water is utilised by mining operations, the volumes abstracted are usually far in excess of onsite requirements (Abbott et al., 2010). In 2008 approximately 60 GL of water in total was consumed in the Pilbara, while a further 66 GL of groundwater was released to the environment during ore dewatering; both these figures are expected to double by 2030 (Department of Water, 2010).

The riparian and wetland ecosystems of the Pilbara, most of which are considered groundwater dependent, are likely to be highly vulnerable to groundwater alterations. Declines in the water table during abstraction, and the long term flooding of riparian zones during discharge of excess water, are both disturbances that can adversely impact associated riparian vegetation (Hatton & Evans, 1998). However, the nature of responses to disturbance, and the degree of tolerance and resilience to different disturbance types, are still largely unknown for most of the Pilbara’s riparian species.

Although their frequency and magnitude is increasing, the impacts of groundwater alterations in the Pilbara remain fairly localised, with many of the Pilbara’s waterways still largely unmodified, in contrast with most other semi-arid regions of the world. The floodwaters of some dryland rivers such as the Euphrates, Nile and Huang He (Yellow) rivers, have been harvested and managed since ancient times; however the past 150 years have seen the management of rivers on an unprecedented scale (Petts, 2005). The flow

8 General introduction patterns have now been substantially altered in the majority of dryland rivers, through dams, diversions, abstraction, and complete flow regulation, to sustain irrigated agriculture, domestic and industry water supplies, and hydroelectric plants (Kingsford et al., 2006). The riparian zones of the Pilbara have been impacted by widespread grazing and trampling by livestock, and invasion of exotic weeds (Van Vreeswyk et al., 2004; Grice, 2006), but the flow patterns and ecosystems of many waterways are still relatively close to their ‘wild’ state (Halse et al., 2007; Department of the Environment, 2008). The Pilbara region therefore provides increasingly rare opportunities to study the natural dynamics of semi-arid riparian systems.

ECOPHYSIOLOGY OF RIPARIAN TREES IN DRYLAND REGIONS

Understanding water fluxes through trees is a crucial component in developing an understanding of the ecohydrological functioning of riparian systems (Asbjornsen et al., 2011). Trees can transpire large volumes of water, with estimates of up to 200 litres per day in a range of species, for trees of approximately 20 m height (e.g. Wullschleger et al., 1998; Schaeffer et al., 2000; Gazal et al., 2006). The extensive root systems of trees can also redistribute water throughout soil profiles, linking deeper water with surface soil layers and the atmosphere (Domec et al., 2010). Consequently, trees substantially influence whole ecosystem productivity, patterns of evapotranspiration (ET), runoff, groundwater levels, and climates (Lee et al., 2005; Amenu & Kumar, 2008; Bonan, 2008; Scott et al., 2008; Domec et al., 2010).

Plant water use is determined by water availability to the roots, the evaporative demand of the atmosphere, and the hydraulic resistance and capacitance of the flow pathway through the soil, plant tissues, and leaf boundary layer (e.g. Sperry et al., 2003). The leaves, fine roots and rhizosphere are frequently the most vulnerable points in the flow pathway, where cavitation is most likely to occur under high flux rates (Sperry et al., 2002; Sperry et al., 2003; Brodribb et al., 2007). The factors that limit transpiration rates ultimately place limits on the rates of carbon assimilation and growth. Such limitations are most commonly discussed with respect to the availability of water to the roots, particularly in dryland environments (e.g. Gazal et al., 2006; O'Grady et al., 2009; Du et al., 2011). Drought resistant plants are generally able to withstand more negative xylem water

9 Chapter One potentials (Ψ), and thus to draw water out of drier (more negative Ψ) soils. However, water is relatively abundant in most riparian zones, and most riparian tree species have relatively low cavitation thresholds and thus are sensitive to dry conditions. For instance, all riparian Populus species of the northern hemisphere appear to be reliant on shallow groundwater, and with reduction in water availability display rapidly increasing water stress responses, initially stomatal closure and reduced photosynthesis, progressing to canopy loss and branch abscission (e.g. Cooper et al., 2003; Rood et al., 2003; Hultine et al., 2010). Due to the similarity in habitat type, M. argentea is likely to exhibit similar water stress response patterns to riparian trees in other dryland regions.

In undisturbed habitat, trees such as M. argentea typically have constant access to abundant water, while the canopy may be exposed to extreme VPD conditions. Under these conditions, very high rates of transpiration would be expected, with water use patterns driven largely by VPD. However, the water must be transported efficiently from the soil reservoir to the leaf surfaces, and under some conditions, the total transport capacity may become the limiting factor in water use (Tyree, 2003). Transpiration rates may therefore be restricted at extreme levels of VPD via stomatal control, to prevent water potentials reaching a critical threshold, even when soil water content is high. For example, transpiration appeared restricted when VPD exceeded 1.2 kPa in montane tropical rainforest trees in Ecuador (Motzer et al., 2005), and stomatal conductance and photosynthesis began to decrease at VPD greater than 2 kPa in irrigated orange trees in Spain (Martin-Gorriz et al., 2011). However, limitations on water use imposed by transport capacity can be difficult to distinguish from those of soil water availability, since high VPD conditions often co-occur with soil drought. The study of M. argentea in undisturbed conditions, with confirmed presence of shallow groundwater, provides an opportunity to examine the physiology of tree water use under extreme evaporative demand, but with unlimited water availability.

In addition, any changes over time in fine root and canopy surface areas also influence water use patterns, by affecting the quantities of water that can be taken up from the soil and lost from the leaf surfaces. Many of the well-studied northern hemisphere riparian species are winter-deciduous, while most Australian species are evergreen, including M. argentea. As a result, the temporal patterns of leaf and fine root flushing and senescence

10 General introduction can differ markedly, creating very different seasonal water use dynamics (e.g. Eamus et al., 2000; Goodrich et al., 2000; Hultine et al., 2004; Scott et al., 2004; Zeppel & Eamus, 2008). Therefore, while the water use physiology of M. argentea is expected to be similar in many respects to other semi-arid riparian trees worldwide, some differences in water use patterns are also expected reflecting differing phenology, with growth and transpiration continuing year-round.

Some estimates of water use have been made for M. argentea in the wet-dry tropics of northern Australia, near Darwin (O'Grady et al., 2006a). This study found considerable variation in tree water use among three different sites along the Daly River, but no significant seasonal variation in water use. However, water use patterns and physiological functioning of M. argentea have not been evaluated in the semi-arid Pilbara region, where the environment can be quite different, with an overall drier climate, more variable rainfall and flooding patterns, and greater seasonal fluctuations in VPD. Thus the patterns of water use and physiological functioning of M. argentea in the Pilbara may show greater seasonal variability. With increasing pressures on water resources in the Pilbara, the ability to calculate ecosystem water requirements and hydrological budgets will be important to manage and allocate water appropriately. There is therefore a need to quantify water use patterns for key species like M. argentea, including defining the relationships with climatic variables, and factors such as tree size.

Hydraulic redistribution (HR) of water through tree roots is another important water flux mediated by trees, occurring when the root system spans a gradient of soil water potential, leading to passive transfer of water from wetter to drier soil portions (Burgess et al., 1998). These fluxes can be particularly significant in semi-arid environments, where groundwater may be present at depth while surface soil layers are dry, and many tree species have both deep and shallow roots which link these soil layers (Ryel et al., 2008; Scott et al., 2008; Bleby et al., 2010; Hao et al., 2010). HR can allow root survival in dry soil, thereby increasing the total root surface area, enabling greater total tree water use and greater nutrient availability (Domec et al., 2004; Bauerle et al., 2008; Armas et al., 2012). Water transferred to shallow layers can also be used by shallow rooted plants, so can influence productivity and functioning of the whole ecosystem (Brooks et al., 2006; Hawkins et al., 2009). For example, HR by Pinus taeda in North Carolina, USA, maintained moisture in

11 Chapter One the surface soil for longer periods of the year, increasing the total transpiration by both the trees and understorey species by 30-50%, and leading to substantially higher ecosystem productivity (Domec et al., 2010). With their permanent access to groundwater, mature M. argentea trees may redistribute water to surface layers during the long rainless periods, when surface soils are dry. This redistribution is likely an important process both in the patterns of water flux through the trees themselves, and more broadly in the ecohydrology of the Pilbara’s riparian ecosystems.

Although water may be relatively abundant in riparian zones compared with the surrounding landscape, riparian trees must nevertheless contend with the high spatial and temporal heterogeneity in water and nutrient availability that characterises dryland waterways. Plant responses to, and impacts upon, environmental heterogeneity are currently topics of much research interest, owing to the increasing realisation that heterogeneous resource distributions can have consequences beyond those that might be predicted based solely on studies conducted under homogeneous conditions (e.g. Hodge, 2010; García-Palacios et al., 2012). Plant root density in riparian zones is usually highly variable in both time and space, reflecting responses of plants to heterogeneity in resource distribution (Kiley & Schneider, 2005). Trees lining dryland streams can also be subject to high energy floods that move large amounts of material and alter channel topography, scouring some zones and depositing sediment and debris in others (Naiman & Decamps, 1997; Friedman & Lee, 2002). M. argentea is frequently found along central channels where groundwater is close to or above the surface of the river bed, and where resource heterogeneity and flood velocities are typically greatest. Portions of M. argentea root systems are frequently exposed, so that prostrate trees destabilised by flood erosion are common along channel banks. In other locations M. argentea trunks are partially buried in sediment, and the trees often produce roots along the submerged stems. M. argentea also commonly forms extensive root mats within surface pools left by flood waters, which then die back as the pools dry. Due to the high levels of resource heterogeneity and disturbance, M. argentea might be expected to display plastic and opportunistic responses to the availability of water around the root system.

The nature of groundwater and surface flows are integral to the ecology of riparian and wetland environments. With changes to the water regime, such ecosystems appear to

12 General introduction be vulnerable to abrupt shifts in vegetation state (Ridolfi et al., 2006). Knowledge of critical thresholds to identify when abrupt and potentially irreversible ecological change may occur is highly desirable for optimal water management, although true physiological thresholds for say, tree survival, are rarely defined (Dodds et al., 2010; Oliveira, 2013). Nonetheless, there is increasing interest in identification and use of thresholds for the purposes of early intervention and preventative management actions (Groffman et al., 2006; Dodds et al., 2010). As a key overstorey species in the wetland environments of the Pilbara, the responses of M. argentea to disturbances may have flow-on effects for understorey species and for whole ecosystem functioning, and this species may be considered an early indicator of any local change in water status. As outlined above, many riparian ecosystems of the Pilbara are subject to abstraction of underlying groundwater, while others are subject to increased flows and prolonged flooding, where excess water is discharged. Given its wet habitat, I expected that M. argentea would be sensitive to prolonged drought and to declines in groundwater levels during abstraction, but would be tolerant of prolonged flooding. Identification of physiological tolerance thresholds in M. argentea would be valuable in water management strategies in the Pilbara’s riparian ecosystems.

OBJECTIVES OF THIS THESIS

The general objective of this thesis was to characterise the temporal and spatial patterns of water use by M. argentea trees in the Pilbara, and to assess the ways these patterns may be affected by disturbances to the native water regime.

I examined water use in M. argentea trees through sap flow measurements at two contrasting sites in the Pilbara. I sought to assess the major factors controlling the observed patterns of water use, such as atmospheric conditions and aspects of tree hydraulic physiology (Chapter 2). I also aimed to characterise the patterns of water use over diurnal and seasonal cycles, within different parts of the sapwood, and across tree sizes, information that will be required to calculate ecosystem scale water budgets, and ecological water requirements (Chapters 2 & 3).

Responses of M. argentea to spatiotemporal heterogeneity in water availability were also investigated, with the aim of assessing characteristics of the root system that enable the species to thrive in highly changeable riparian habitats. Sap flux was measured in deep and

13 Chapter One shallow roots of M. argentea trees during a 12-month period to assess temporal variation in water fluxes, and assess whether redistribution of water through tree roots may be an important process in the Pilbara’s riparian ecosystems (Chapter 4). Controlled glasshouse experiments were also conducted, using a split-root set up to mimic the heterogeneous water distribution of riparian zones. The experiments aimed to determine whether compensatory responses are displayed by M. argentea during drying of parts of the root system, a situation that can occur during the flooding and drying cycles of ephemeral waterways (Chapter 5).

Finally, I examined aspects of the tolerance and response of M. argentea to flooding, drought and groundwater abstraction, including potential threshold responses with decreasing water availability. Seedling responses to water availability were investigated in controlled experiments, with the aim of understanding the possible effects of altered water regimes on recruitment patterns (Chapter 6). Responses of trees to groundwater abstraction were assessed through a case study at the Yule River borefield, on the coastal plain region of the Pilbara. This study aimed to evaluate the changes in tree water use physiology that occur with increasing groundwater drawdown (Chapter 7).

Each experimental chapter of this thesis is presented as a self-contained paper, and as such, a small amount of repetition is inevitable across the body of work presented. Chapter 8 provides a general discussion of the outcomes of this research, their relevance to the ecological understanding and water management of semi-arid northwest Australia, and suggests avenues for further research.

Knowledge of water use patterns in M. argentea will contribute to the fundamental understanding of the water use physiology of riparian trees. Insights into riparian ecohydrology of key indicator species such as M. argentea gained in the Pilbara, particularly from a more mechanistic perspective, are also likely to provide much needed baseline or reference information that will inform the development of ‘best practice’ water management strategies not only in the Pilbara, but in dryland regions worldwide.

CHAPTER TWO: Seasonal patterns of water use by the riparian tree Melaleuca argentea in semi-arid NW Australia

INTRODUCTION

Tree water use can vary over annual cycles in response to seasonal climatic fluctuations. Studies of seasonality in tree water use physiology have mostly focused on temperate or dry- tropical regions with clearly defined and regular hydroclimatic seasons (e.g. Greco & Baldocchi, 1996; Ford et al., 2004; Kelley et al., 2007; Zeppel & Eamus, 2008; Kunert et al., 2010). In such environments, trees require physiological strategies to cope with the seasonal extremes in temperature or water availability. For example, dry-tropical and Mediterranean deciduous species shed their leaves to avoid transpiration during the dry season, while co-occurring evergreen species maintain water transport to their canopy year- round through more conservative water use and hydraulic properties such as greater cavitation resistance (e.g. Eamus, 1999; Baldocchi et al., 2010; Fu et al., 2012). There has been less study of seasonal responses of trees in more arid regions, partly because rainfall and water availability are less predictable both in space and time. However, trees within the riparian zones of dryland regions usually co-occur with groundwater and thus can experience relatively predictable and constant water availability (e.g. O'Grady et al., 2006b). Melaleuca argentea is one such tree species in the semi-arid Pilbara region of northwest Australia. Temperatures and vapour pressure deficits (VPD) in arid climates often display predictable seasonal fluctuations; the water use and physiology of riparian trees in these regions might therefore be expected to display corresponding seasonal patterns.

Tree water use is determined primarily by water availability to the roots, the evaporative demand of the atmosphere, and the hydraulic resistance and capacitance of the flow pathway through the soil, plant and leaf boundary layer (e.g. Sperry et al., 2003). Theoretically, transpiration rates should be high under conditions of abundant water supply and exposure to high VPD, with transpiration largely defined by atmospheric conditions until the limit of safe hydraulic function is approached (Brodribb, 2009). The soil and roots are often weak points in the flow pathway (Sperry et al., 1998), but where

15 Chapter Two trees have roots in the saturated zone, such as many riparian trees, xylem hydraulic conductivity (K) may be the primary limiting factor for water use (Brodribb, 2009). In tall trees, transpiration can be limited by the rate at which water can be transported from the soil reservoir to the leaves, rather than by water availability or demand (Goldstein et al., 1998; Williams et al., 2001). In riparian zones, where trees are not limited by water availability, transpiration rates might also be limited by transport rates under extreme VPD. Although K can vary with xylem anatomy, the rates of sap flux in riparian trees in high VPD environments, such as Populus and Salix species in semi-arid Arizona, USA, typically fall within the same range of flux rates observed in non-riparian species (Wullschleger et al., 1998; Schaeffer et al., 2000). Over diurnal cycles, transpiration is maintained within safe limits by the adjustment of stomatal aperture, leading to a ‘saturation’ of transpiration above a certain VPD (Goldstein 1998, Brodribb 2003). For example, in well-watered Populus fremontii, rates of sap flow saturated at VPD between 4 and 6 kPa (Gazal et al., 2006). In addition, trees can utilise internal water storage as a buffer over diurnal cycles to partly overcome the limitation of xylem K (Scholz et al., 2011). Eucalyptus victrix, a riparian tree that often co-occurs with M. argentea in the Pilbara region, displayed withdrawal and refilling of stem water stores over diurnal cycles, even with access to shallow ground water (Pfautsch et al., 2011). Thus, while M. argentea occurs only with access to a shallow water table, water use might nonetheless be restricted by tree K and/or capacitance under the extreme VPD conditions that can occur during the middle of the day in the summer months (frequently greater than 5 kPa).

Over annual cycles, seasonal fluctuations in the major driving variables of evaporative demand and soil water availability often largely explain water use patterns in evergreen trees, such as coniferous species and eucalypts (e.g. Oren et al., 1999; Ford et al., 2004; Zeppel et al., 2004; Small & McConnell, 2008). However, these seasonal water use patterns are also modified by tree adjustments in canopy cover, as well as by variation in hydraulic resistance and capacitance. Leaf area is clearly a major determinant of water use. For example, many of the well-studied forests of the northern hemisphere, including semi- arid riparian systems in the south-west of the USA, consist largely of deciduous trees in which seasonal water use patterns tend mostly to reflect the patterns of leaf flushing and senescence (e.g. Goodrich et al., 2000; Scott et al., 2004). The canopy cover of evergreen

16 Seasonal patterns of water use by Melaleuca argentea trees can also vary seasonally as part of a species’ water use strategy. For instance, in the savannas of northern Australia, Eucalyptus species display patterns of canopy thinning and flushing, coinciding with the pronounced wet and dry seasons, where canopy cover may vary by as much as 50% in any given year (Duff et al., 1997; O'Grady et al., 1999). Xylem hydraulic conductivity can also vary seasonally, typically due to xylem embolism induced by water stress during dry periods or during freeze-thaw cycles in climates with cold winters (e.g. Magnani & Borghetti, 1995; Tognetti et al., 2004). While the techniques used for measurement of xylem embolism and recovery have recently come into question, researchers nevertheless agree that embolism does occur at least under conditions of severe water stress and freezing (Cochard & Delzon, 2013; Wheeler et al., 2013). Stem water storage can further significantly influence seasonal water use patterns in many species, by enabling the tree to draw upon internal reserves during dry periods (e.g. Zweifel & Häsler, 2001; Baker et al., 2002; Betsch et al., 2011). However, riparian trees such as M. argentea in the Pilbara occur in conditions that are favourable throughout the year i.e., with warm temperatures and a perennial water supply. It might therefore be expected that M. argentea would display little seasonal variation in leaf area, K or capacitance as it would be well buffered against seasonal variation in water availability. Without seasonal variation in these physiological factors, or in water availability to the roots, then the seasonal variation in atmospheric demand would remain as the sole factor driving patterns of tree water use across the year.

The objective of this chapter was to characterise temporal patterns of water use by M. argentea, in the semi-arid Pilbara region of NW Australia, by determining the relationships between tree water use and meteorological conditions. I measured tree sap flow, canopy physiology (leaf water potential and stomatal conductance) and meteorological conditions over periods of at least 12 months, at two contrasting and spatially separated sites within the Pilbara, enabling a detailed analysis of tree water use patterns over time. Aspects of canopy physiology were also examined at intervals, including leaf water potential, stomatal conductance and canopy cover. I expected that due to its warm habitat and constant access to groundwater, M. argentea trees in undisturbed habitat would show little seasonal adjustment in canopy cover, K or capacitance, and would be relatively insensitive to additional pulses of surface soil moisture. I therefore hypothesised that seasonal patterns of

17 Chapter Two

Melaleuca water use would strongly reflect variations in atmospheric conditions (largely VPD), perhaps with some limitation of water use at extreme VPD, as has been observed in other well-watered trees over diurnal cycles. Without the complicating factors of seasonal variations in water supply, or in tree physiological or morphological adjustments, I anticipated that the relationships between water use and atmospheric variables would remain constant over annual cycles.

MATERIALS & METHODS

Study sites Sites were located on two intermittent waterways in the Pilbara region of north-west Australia. The first site was at the Yule River (20.656° S 118.295° E), at a near-coastal location and the second site at Marillana Creek (22.721° S, 118.958° E), in the inland Hamersley Basin (Figure 2.1). The two sites are geographically separated (approximately 250 km apart), located within two distinct sub-regions of the Pilbara, with differing underlying hydrogeology. By conducting the study at these contrasting sites, more confidence can be placed in the broader applicability of the findings. At both sites, Melaleuca argentea co-occurred with Eucalyptus camaldulensis and E. victrix, two other common riparian tree species. The Yule River site consisted of a narrow, dense band of trees bordering a wide, sandy riverbed, with vegetation beyond the river bank composed largely of grassland. At the Marillana Creek site, the riparian vegetation was confined within the (usually dry) creek bed, between steep, rocky banks, and the trees clustered along deeper sub-channels. Spinifex grassland and open Eucalyptus woodland surrounded the creekline.

The climate of the Pilbara region is classed as semi-arid to arid and is characterised by highly variable rainfall, with extreme temperatures (up to 49 °C) and VPD (up to 10 kPa) during November to March (Australian Bureau of Meteorology data). Cooler temperatures occur between June and August (daily minima typically 5–18 °C and maxima 18–32 °C). Cyclones and other low pressure systems can pass through the region during December to April, bringing rainfall of up to 400 mm over a few days that results in flooding; such large events typically occur every 4–6 years. In contrast, small, localised rainfall events (25 mm or less) can occur throughout the year (Dogramaci et al., 2012). River flows are intermittent

18 Seasonal patterns of water use by Melaleuca argentea

Waterway Study site 100 km Major road Town

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W F ort e escu s e t Riv

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Figure 2.1: Study site locations. Modified from Department of Water (2010) and Van Vreeswyk et al. (2004). and highly unpredictable, reflecting the variable rainfall patterns. The coastal subregion of the Pilbara (containing the Yule River site) is slightly warmer and more humid compared with the inland subregion (containing the Marillana Creek site). Winter (June–August) temperatures inland are typically 3–6oC cooler than the coast, while summer (December– February) temperatures are similar between subregions (minima typically 20–29oC and maxima 32–42oC), but relative humidity is greater on the coast (45–60%) than inland (20– 40%). Seasonal periods are referred to in this chapter as: summer (December–February), autumn (March–May), winter (June–August) and spring (September–November).

Just prior to this study, cyclone activity resulted in substantial flows in most Pilbara waterways during the December 2008–Feb 2009 summer. Large surface pools were present in the riverbeds within 10 m of the study trees at both sites when this study commenced (in March 2009 at Yule River, and in June 2009 at Marillana Creek), but had dried down by November 2009. Five M. argentea trees were selected across a size range of 100–500 mm 19 Chapter Two diameter at breast height (1.2 m), within a 900 m2 area at each study site. Measurements were conducted on the same ten trees throughout the study.

Meteorological data At the Yule River, weather stations were installed approximately 18 km NW of the study site (HOBO; Onset Computer Corporation, Bourne, MA, USA during March–September 2009; and ET107; Campbell Scientific Inc., Logan, Utah, USA during September 2009– May 2010). Stations were positioned in grassland on a flat area near the top of the main river channel bank, more than 100 m from any trees. Temperature, humidity, light intensity, wind speed, wind direction and rainfall data were logged half hourly by the HOBO station, and hourly by the ET107 station. Due to problems with the light sensors, daily solar radiation data modelled from satellite images were also obtained from the Australian Bureau of Meteorology (http://www.bom.gov.au/climate/data-services/) for the entire period.

At Marillana Creek a weather station (ET107; Campbell Scientific Inc., Logan, UT, USA) was installed in the open at the top of the creek bank, approximately 300 m from the study trees, between September 2009 and September 2010. Data were logged as for the Yule River site. For the period June–September 2009, daily data were provided by Rio Tinto Pty Ltd., from a weather station approximately 16 km ESE of the study site at the Yandicoogina Mine.

Vapour pressure deficit was calculated from the temperature and humidity readings according to Howell et al. (1995), as:

VPD = (1 – RH/100) 0.6108 e17.27 T / (T + 237.3) where VPD has units of kPa, RH is the relative humidity (%) and T is the temperature (°C).

Tree water use Tree water use patterns were determined with sap flow probes (HRM-30; ICT International, Armidale, NSW, Australia), via the heat ratio method (Burgess et al., 2001). Probes were installed into the main stem of each tree at 1.2 m height. Bark was first peeled back from the insertion sites, to leave a 5 mm layer over the cambium, then holes were drilled 0.6 mm apart using a drill guide (ICT International, Armidale, NSW, Australia) 20 Seasonal patterns of water use by Melaleuca argentea following the grain of the wood, and the probes inserted. Sensor units and probes were covered with bubble wrap and insulation foil to minimise effects of ambient temperature fluctuations. Data were obtained from a single measurement point positioned at 7.5 mm depth into the sapwood, which corresponded to the approximate centre of the sapwood band. Heat pulses were set at 30 min intervals, and the data processed to heat pulse velocity

(Vh) values by the “Smart Logger” (SL5; ICT International, Armidale, NSW, Australia). Sap flow was logged continuously at the Yule River site from 29th March 2009 to 12th May 2010, and at Marillana Creek from 18th July 2009 to 13th September 2010, apart from three periods at Marillana Creek, when equipment was damaged by livestock despite being fenced (1st–22nd September 2009; 20th June–22nd July 2010; and 26th July–1st Sept 2010).

All calculations of Vh, and conversion to sap flow velocity (Vs) were performed according to methods of Burgess et al. (2001) and Bleby et al. (2004). For each probe, the minimum value of Vh recorded on nights with low VPD and wind speed was set to zero (after filtering of the datasets), to correct baseline errors caused by slight probe misalignment. Wood properties (density and volumetric water content) were measured on blocks of sapwood 2–5 cm3 in size, cut from five trees at each site. Volume was measured by the displacement weight of water by fresh samples. Fresh and dry mass were determined by weighing before and after drying at 60 °C. Mean values for sapwood properties were then determined for each site to calculate Vs. Wounding was examined in additional blocks of sapwood cut from three probe insertion sites from trees at Marillana Creek at the end of the study period; sapwood was shaved back and the diameter of darkened wood around the insertion site measured under a dissecting microscope. The mean diameter of 0.22 cm was used in all Vs wounding corrections. Wood cores were also taken from each study tree with an increment borer (Haglöf, Långsele, Sweden), to assess wood appearance and sapwood depth. Surfaces of the fresh cores were shaved back and stained with methyl orange (1% w/v in 4% v/v methanol); the sapwood stained deep red and the heartwood yellow.

The Vs data presented here were collected from the same positions within the same trees over time as my objective for this chapter was to assess temporal patterns of water use in detail. Spatial variation in Vs and volumetric quantification of water use in M. argentea are examined separately in Chapter 3.

21 Chapter Two

Canopy physiological measurements Leaf measurements were conducted on sunlit, outer canopy leaves cut from 5–7 m height with extendable clippers. At each time point, measurements were made on 2–3 leaves per tree, each from branchlets cut separately from the canopy, then averaged to give a single value for each parameter per tree. Leaf water potential (Ψl) was measured with a Scholander-type pressure chamber (PMS instruments, Albany, OR, USA) immediately after cutting from the trees. Stomatal conductance (gs) was measured with a leaf porometer (Decagon Devices, Pullman, WA, USA) immediately after severing from the tree. Testing showed that if the measurement was complete within 2 min of excision, the mean gs did not differ between excised and attached leaves, although the variance of gs was greater for excised leaves (Appendix A).

Statistical analysis Statistical analysis was performed in SAS version 9.2 (SAS Institute Inc.; Cary, NC, USA). Mixed-effect ANOVA was used to test for site and seasonal differences in water use, canopy cover and nocturnal sap flow, with the individual trees as a random effect. ANCOVA was used to test for site and seasonal differences in the relationships between tree water use and meteorological parameters.

For multiple regression analysis, fixed effects (meteorological variables) were selected using the least absolute shrinkage and selection operator (LASSO) method (PROC GLMSELECT), using minimum cAIC as the model selection criterion (Flom & Cassell, 2007). The selected fixed effects were then fitted in a mixed model (PROC MIXED) including a random effect for the individual tree subjects over time (Fernandez, 2007). R2 statistics were computed from models containing the fixed effects only, both with and without individual tree subjects also included as fixed effects, to assess the amount of variation explained by the model when among-individual variation was included, and when among-individual variation was eliminated.

The selected meteorological models were then used to test the significance of the effects of recent rainfall (considered here as a proxy for moisture of upper soil layers), site and season, after controlling for the meteorological differences. These additional factors were included individually in the models as fixed effects, and their significance assessed by the resulting change in cAIC values, log-likelihood tests, and type-3 F-tests.

22 Seasonal patterns of water use by Melaleuca argentea

RESULTS

Seasonal variation in tree water use and meteorological conditions Patterns of most meteorological variables were similar between the Yule River and Marillana Creek study sites during the study period (Figure 2.2). Seasonal variation in VPD and temperature was pronounced with lowest values in June–July (daily maxima 0.8– 3.5 kPa) and highest November–February (daily maxima 2.5–8.2 kPa). Marillana Creek received five times more rainfall than the Yule River site during the study period (160 mm over 517 total measurement days, c.f. 26 mm over 423 days). No cyclones occurred during the study period and all rainfall events were relatively small with the largest 37 mm occurring in November 2009 at Marillana Creek.

5 (a)

4 Figure 2.2: Seasonal patterns of water use 3 by Melaleuca argentea trees, and the major driving meteorological (m day-1) s 2 V variables during the study period. (a) Daily sap flow velocity V( s) at 1 Yule River each study site, aver- Marillana Creek aged by month. Error bars are standard error, 0 (b) calculated to include 8 8 variance both between days and between the VPD five measured trees.(b)

6 6 max Daily solar irradiance (kPa) and maximum VPD at each study site, aver- 4 4 aged by month. Closed symbols; Yule River site, open symbols; Maril- 2 2 lana Creek site. Irradiance (kWh m-2 day-1)

0 0 JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC Month

23 Chapter Two

Rates of tree water use varied seasonally, with daily Vs lowest in June and highest in December at both sites (p<0.0001; Figure 2.2a). Water use patterns reflected the seasonal variation in the major driving variables of solar irradiance and VPD (Figure 2.2). Although seasonal patterns were similar between the two sites, Vs was higher at the Yule River site than at Marillana Creek throughout the year. The seasonal fluctuation in water use was greater at Marillana Creek; mean daily Vs in June was only 50% of Vs in December at

Marillana Creek, but 70% of the December Vs at the Yule River (Figure 2.2a).

Diurnal patterns of tree water use physiology across seasons

Diurnal patterns of Vs, Ψl and gs were assessed separately on days in mid-winter, late spring and late autumn (Figure 2.3). Vs increased rapidly in the mornings, coinciding with sunrise and stomatal opening at both sites and on all measurement dates. Stomatal conductance

(gs) peaked mid-morning, declining toward midday. In November (late spring) at both sites, a second peak in gs then occurred late afternoon, while in cooler months only the mid-morning peak occurred. VPD was greatest mid-afternoon, and so was higher during the afternoon than in the mornings. Stomatal conductance therefore correlated negatively with VPD during much of the day, which meant a fairly constant Vs was maintained throughout the middle of the day (Figure 2.3). This plateauing of sap flow generally occurred once VPD exceeded ~3 kPa. Minimum Ψ l occurred between midday and 3 pm, but remained above -2 MPa on all dates measured. Minimum VPD usually occurred a few hours before dawn, and sap flow frequently continued at low rates throughout the night, reaching a minimum sometime between midnight and dawn. Nocturnal sap flow at Marillana Creek ranged between 16 and 38% of the total flow over the 24 hr period compared to between 9 and 41% at Yule River. Predawn Ψ l was close to zero on all measured dates.

The relationships between Vs and the driving variables of irradiance and VPD displayed marked hysteresis over diurnal cycles during much of the year (Figure 2.4). On most days in winter and spring, Vs was lower during the morning than at the same light intensity in the afternoon (anti-clockwise hysteresis), most likely due to the higher VPD conditions in the afternoon driving higher water use (Figure 2.4a, d). However, the degree of hysteresis between Vs and irradiance decreased during the summer and autumn (Figure 2.4b, c, e, f), i.e., rates of afternoon water use were not much higher than in the mornings,

24 Seasonal patterns of water use by Melaleuca argentea

(a) June 2009 November 2009 May 2010

1200 6 VPD (kPa)

800 4

400 2 Irradiance (W m-2) 0 0 g s (mmol m-2 s-1) 300 20 200 10 (cm h-1)

s 100 V 0 0

-1 (MPa) leaf Ψ -2

0:006:0012:00 18:00 0:006:00 12:00 18:00 0:006:00 12:00 18:00 0:00 Time of day (h)

(b) November 2009 July 2010

1200 g 6 VPD (kPa)

800 4

400 2

Irradiance (W m-2) 0 0 s (mmol m-2 s-1) 20 300

200 10

(cm h-1) 100 s V 0 0

-1 (MPa) leaf Ψ -2

0:006:0012:00 18:00 0:006:00 12:00 18:00 0:00 Time of day (h) Figure 2.3: Diurnal patterns of irradiance, vapour pressure deficit (VPD), tree sap flow velocity (Vs), stomatal conductance (gs) and leaf water potential (Ψ) at (a) Yule River and (b) Marillana Creek sites. Each vertical column of panels shows values measured over a single 24 h period during the month indicated at the top of the column. Values of Vs, Ψ and gs are means ± standard error of five trees.

25 Chapter Two despite the higher VPD in the afternoons. By contrast, Vs displayed clockwise hysteresis with VPD on most days year round (Figure 2.4g–l), meaning that water use was higher during the morning than at the same VPD in the afternoon on any given day.

Maximum rates of sap flow reached each day were largely consistent across sites and seasons. However, the number of hours that maximum rate was maintained during the middle of the day differed between summer (on average 10.5 h per day) compared to winter (on average 7 h per day). Thus, daily total sap flow was linearly correlated with both daily irradiance and VPD (Figure 2.5).

Relationships of tree daily water use with meteorological conditions

Daily Vs was significantly correlated with daily values of nearly all meteorological variables measured (Figure 2.5a–d, Table 2.1). Many of the meteorological variables were highly correlated with one another (not shown), but the variables giving the strongest correlations with Vs were the average daily VPD, its interaction with average wind speed (VPDavg x uavg), solar irradiance, and maximum temperature (Table 2.1). The correlation between Vs and recent rainfall (falling within the previous 10 days) was significant though weak at Marillana Creek. Nocturnal sap flow also correlated with meteorological conditions at both sites. The best correlates for nocturnal Vs were the average nocturnal VPD (giving positive correlations, with R2 of 0.32 at the Yule River and 0.24 at Marillana Creek), and nocturnal

2 VPDavg x uavg (giving positive correlations, with R of 0.34 at the Yule River and 0.25 at

Marillana Creek). The maximum and total daily Vs differed between the two sites, with trees at the Yule River having higher rates of water use for a given value of a meteorological parameter (such as VPD or light intensity), with site differences more pronounced at lower

Vs (Figure 2.5). For example, daily Vs was on average 1.8 times greater at the Yule River than Marillana Ck on days with maximum VPD of 2 kPa, and 1.3 times greater at the Yule River on days with maximum VPD of 8 kPa.

The relationship between daily Vs and VPD differed among seasons (Figure 2.5a, b). The range of VPD was similar between spring and autumn, but the regressions of VPD with daily Vs differed between these two periods; at the Yule River the intercept of the relationships differed significantly (p<0.0001), and at Marillana Creek the slope of the relationships differed (p=0.0005). Tree water use was greater during the spring (season of increasing VPD), than during autumn (season of decreasing VPD), i.e., clockwise hysteresis

26 Seasonal patterns of water use by Melaleuca argentea

SPRING SUMMER AUTUMN (a) YR (b) YR (c) YR 30 pm

pm 20 am am 10 am

0 (cm h-1)

s (d) MC (e) MC (f) MC V 25

20 pm pm pm 10 am am 5

0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Irradiance (W m-2)

SPRING SUMMER AUTUMN (g) YR (h) YR (i) YR 30 am am 20 pm pm pm 10

0 (cm h-1)

s (j) MC (k) MC (l) MC V 25 am 20 am pm 10 pm pm 5

0 024602460246 VPD (kPa)

Figure 2.4: Patterns of hysteresis over the diurnal cycle in the relationship between sap flux velocity (Vs) and the major driving variables of (a–f) light intensity and (g–l) vapour pressure deficit (VPD). Data are from representative days in mid spring (left hand panels), summer (centre panels) and autumn (right hand panels), for trees at the Yule River (YR; upper panels in each block), and Marillana Creek (MC; lower panels in each block). The arrows indicate the direction of hysteresis. Values are means +/- standard error of five trees.

27 Chapter Two

Yule River Marillana Creek

5 (a) (b) R2= 0.38 R2= 0.84 4 R2 = 0.32 3 R2= 0.82 (m day-1) s

V 2 Summer Autumn 1 Winter Spring 0 024680246810 VPD (kPa)

5 (c) (d)

3 (m day-1) s

V 2

0 024680246810 Irradiance (kWh m-2 day-1)

(e) R2= 0.73 (f) 16 R2= 0.64

12 (cm h-1) s

8 R2= 0.91

Nocturnal V 4 R2= 0.84 0 0 1234 015 234 Nocturnal VPD (kPa)

Figure 2.5: Comparisons of sap flux velocity (Vs) between seasons. Relationships of (a & b) total daily Vs with vapour pressure deficit (VPD),(c & d) total daily Vs with solar irradiance, and (e & f) nocturnal Vs averaged over the dark period with nocturnal VPD, at (a, c & e) the Yule River and (b, d & f) Marillana Creek. Each datapoint is a single day of the study period, and Vs values are means of five trees. Where relationships differed significantly between seasons, regression lines and their R2 values are shown for spring and autumn.

28 Seasonal patterns of water use by Melaleuca argentea

Table 2.1: Correlations between tree daily sap flow and individual meteorological variables. Pearson coefficients (r) and probability values (p) are provided. VPD; vapour pressure deficit, T; temperature, RH; relative humidity, u; wind speed, R; solar irradiance, max; daily maximum, min; daily minimum, avg; daily mean of hourly measurements, tot; daily total, 1 day; value for the same day, 10 days; total during the previous 10 days.

Meteorological Yule River Marillana Creek Sites pooled parameter r p r p r p

VPDmax 0.373 <.0001 0.397 <.0001 0.301 <.0001

VPDmin 0.322 <.0001 0.355 <.0001 -0.023 0.1905

VPDavg 0.422 <.0001 0.398 <.0001 0.157 <.0001

Tmax 0.347 <.0001 0.390 <.0001 0.364 <.0001

Tmin 0.179 <.0001 0.378 <.0001 0.300 <.0001

Tavg 0.293 <.0001 0.399 <.0001 0.326 <.0001

RHmax -0.240 <.0001 -0.175 <.0001 0.206 <.0001

RHmin -0.299 <.0001 -0.251 <.0001 -0.132 <.0001

RHavg -0.328 <.0001 -0.220 <.0001 0.122 <.0001

umax 0.213 <.0001 0.193 <.0001 0.142 <.0001

umin 0.251 <.0001 0.182 <.0001 0.086 <.0001

uavg 0.383 <.0001 0.268 <.0001 0.175 <.0001

umax x VPDmax 0.337 <.0001 0.365 <.0001 0.277 <.0001

uavg x VPDavg 0.462 <.0001 0.407 <.0001 0.193 <.0001

Rtot 0.390 <.0001 0.427 <.0001 0.370 <.0001

Rain1day -0.040 0.0945 -0.084 0.0014 -0.112 <.0001 Rain10days -0.004 0.8641 0.074 0.0048 -0.069 <.0001

between VPD and Vs exists at both diurnal and seasonal scales. In contrast, the regression

between irradiance and daily Vs did not differ among seasons (p>0.05; Figure 2.5c, d). The

seasonal difference in the relationship between Vs and VPD was evident during both the

light and dark periods, with greater Vs in spring than autumn throughout the diurnal cycle.

Nocturnal Vs was also greater in spring than autumn when averaged to a per-hour-of- darkness basis, to account for the seasonal difference in day length (Figure 2.5e, f). However, the proportional contribution of nocturnal flow to the total daily flow over a 24 h period remained similar across the year, ranging from 6 to 48% of daily sap flow at both sites. The proportional contribution of nocturnal flow increased with nocturnal VPD, but this relationship did not vary seasonally (not shown).

29 Chapter Two

Multiple regression analysis was used to assess the extent to which meteorological conditions could explain temporal patterns and site differences in tree water use. To assess whether the observed seasonal differences in the relationships of Vs with VPD were due simply to other meteorological factors varying between sites and seasons, or whether tree physiological or growth responses are involved, I first constructed meteorological models based on atmospheric variables, then added other variables individually (site and season). These multiple regression models also allowed testing of whether the apparent effect of rainfall (or lack thereof) on tree water use was related to changes in surface soil moisture, or due to the influence of other meteorological effects that tend to accompany periods of rainfall, such as reduced light intensity and cooler temperatures. Given daily Vs was linearly related to the daily values of the meteorological variables, I employed linear regression to construct the models.

All variables shown in Table 2.1, except for rainfall, were considered as fixed effects in the regression analysis, as well as interactions between the VPD, wind speed and light parameters (29 variables in total). Reduced models were selected with the LASSO procedure (Flom & Cassell, 2007), and the resulting models are shown in Table 2.2. Greater relative importance cannot necessarily be attributed to the variables listed in Table 2.2, owing to the problems inherent in any model selection process and the high degree of multicollinearity amongst the predictor variables (Graham, 2003; Bolker et al., 2009). Rather, the selected models are considered here to be adequate meteorological descriptions for the tree water use patterns, for the purposes of testing the effects of further factors. Overall, I found that meteorological conditions explained 67–80% of the temporal variation in tree water use when among-tree variation was accounted for by including the tree individuals as additional factors in the models (Table 2.3). However, the variability in water use among individual trees was large and when not accounted for, the amount of variation explained by the meteorological conditions was only 23–42%.

The effects of site, rainfall and season on tree water use The two sites still differed significantly in rates of water use after controlling for meteorological differences; the addition of site as a fixed-effect variable significantly improved the pooled site model (Table 2.4a). The Vs of trees at the Yule River site was on

30 Seasonal patterns of water use by Melaleuca argentea

p > F 0.7084 0.2257 0.0687 0.0049 0.0610 0.0003 0.3871 0.0046 0.0409 0.1006 0.3337 0.5902 0.2001 0.0302 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

perature (°C), RH; relative 31 ± 80 48 ± 26 -68 ± 24 6.1 ± 1.1 2.4 ± 2.8 3.2 ± 1.5 3.6 ± 2.2 4.5 ± 3.5 7.8 ± 1.7 -3.0 ± 3.1 -1.3 ± 2.3 -5.5 ± 2.5 12.3 ± 1.6 -11.4 ± 9.4 -15.8 ± 2.5 1.04 ± 0.56 1.74 ± 0.18 -0.83 ± 0.23 -2.90 ± 0.22 -2.69 ± 0.95 Co-efficient Sites pooled

tot max avg tot max avg max avg

x u x u

x u x u x R

max min avg max max min min avg avg min avg

Estimates for the intercept and regression Estimates for the intercept

max min avg tot max avg max min Effect Intercept VPD VPD VPD T T RH RH RH u u R VPD VPD VPD VPD VPD VPD VPD VPD g; daily mean of hourly measurements, tot;

p > F 0.4173 <.0001 <.0001 <.0001 <.0001 <.0001 D; vapour pressure deficit (kPa), T; tem

) with meteorological variables. 22.7 ± 1.4 -1 -161 ± 172 4.39 ± 0.27 0.94 ± 0.21 2.30 ± 0.59 -1.15 ± 0.13 Co-efficient Marillana Creek

), max; daily maximum, min; minimum, av

-2 min avg tot max min

Effect Intercept T RH RH u R

pe-3 F-test probabilities are provided. VP

p > F for tree daily sap flow (cm day 0.0243 0.6351 0.5305 0.5305 0.0332 0.0112 0.5661 0.2622 0.0685 0.0226 <.0001 <.0001 <.0001 <.0001 <.0001

), R; solar irradiance (kWh m -1

5.9 ± 1.2 5.5 ± 8.7 6.1 ± 2.8 4.4 ± 1.1 241 ± 57 -2.9 ± 1.2 -1.7 ± 2.9 -9.6 ± 8.6 -2.7 ± 1.2 13.4 ± 2.0 13.8 ± 7.6 1.05 ± 0.22 -0.34 ± 0.72 -2.27 ± 0.25 -0.28 ± 0.44 Co-efficient

; windspeed (m s ; windspeed (m max min avg min max avg min u

u u

u u u

x x x u x u min x x x

max min avg avg max min avg

x max avg Yule River tot tot max min avg (a) Effect Intercept T RH RH u u R VPD VPD VPD VPD VPD VPD VPD R

total. Table 2.2: Multiple regression models Table 2.2: humidity (%), coefficient parameters ± standard errors, and ty

31 Chapter Two

Table 2.3: Proportion of variance explained by the multiple regression models of tree daily sap flow shown in Table 2.2. R2 values were computed including among tree variation (accounting for meteorological effects only), and also with among tree variation accounted for (by including tree individuals as factors in the model; meteorological + tree effects).

Meteorological Meteorological + effects tree effects Marillana Creek 0.23 0.79 Yule River 0.27 0.67 Sites pooled 0.42 0.80

Table 2.4: Tests for differences in tree sap flow (a) between sites and (b) after rainfall, after adjusting for meteorological factors. Site or recent rainfall (mm falling within the previous 10 days) was added as a fixed-effect variable to the meteorological models shown in Table 2.2. Where significant, the regression coefficient estimate and standard error are shown. ΔcAIC is the reduction in score when the additional factor is added to the model. The log likelihood ratio (LLR) and type-3 F-test (p>F) probabilities are also provided.

(a) Effect of site Coefficient Δ cAIC LLR p > χ2 p > F Yule River relative to 134 ± 51 15.1 0.0005 0.0343 Marillana Creek

(b) Effect of rainfall Yule River - 3.6 0.0544 0.1423 Marillana Creek - 1.1 0.2942 0.1388

average 1.34 ± 0.51 m day-1 faster than trees at Marillana Creek, if all meterological variables were identical between the sites. However, recent rainfall had no significant effect on sap flow at either Marillana Creek or Yule River once the other meteorological variables were controlled for, indicating no effect of surface soil moisture on rates of tree water use (Table 2.4b).

Seasonality in tree water use was still highly significant (p<0.0001) after accounting for meterological and site effects (Table 2.5). The residuals from the pooled site multiple regression model, averaged over each month, are shown in Figure 2.6. The actual water use was less than predicted by the model during February to May, and higher than predicted during July to December. Water use adjusted for site and meteorological differences was also significantly lower in all autumnal months compared to spring months.

32 Seasonal patterns of water use by Melaleuca argentea

Table 2.5: Tests for seasonal differences in tree water use after adjusting for meteorological variables. Month was added as a fixed-effect variable to the meteorological models shown in Table 2.2, with site and rainfall variables also included where significant (Table 2.4). ΔcAIC is the reduction in score when month is added to the model. The log likelihood ratio (LLR) and type-3 F-tests (p>F) for the effect of month are also provided.

Δ cAIC LLR p > χ2 p > F

Yule R 219.2 <0.0001 <0.0001 Marillana Creek 450.7 <0.0001 <0.0001 Sites pooled 492.3 <0.0001 <0.0001

Figure 2.6: Difference 30 d between the measured tree de de sap flux (Vs), and the sap flux predicted by a multiple 20 cd ce cd regression model of mete- ac 10 orological and site effects. Values are residual daily Vs

(cm day-1) ab s averaged by month (mean ± V 0 standard error). Negative b b b values indicate lower rates of -10 b sap flux than predicted by the Residual model, and positive values -20 higher rates than predicted. Different letters indicate -30 significant differences JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC between months.

Tree hydraulic conductance Since seasonal variation in tree sap flow was not fully explained by meteorological conditions, whole tree hydraulic conductance (K) was examined for evidence of seasonal variation. Changes in factors such as canopy cover, quantity of conducting sapwood or root absorptive surface area can alter tree K and affect rates of tree water use. There was no

detectable variation in tree K seasonally, as the relationship between Vs and Ψl across the diurnal cycle remained constant, with data points from different seasons falling within the same line (examples in Figure 2.7).

33 Chapter Two

30 20 (a) (b)

15 20 V s (cm h-1) 10 (cm h-1) s V 10 June July November 5 May November

0 0 -1.5-2.0-2.5 -0.5-1.0 00-1.5-2.0 -0.5-1.0

Ψleaf (MPa)

Figure 2.7: Examples of relationships between sap velocity (Vs) and leaf water potential

(Ψleaf), used to assess whole tree hydraulic conductance. Data are shown for (a) one tree at the Yule River in winter (June 2009), spring (November 2009) and autumn (May 2010), and (b) one tree at Marillana Creek in winter (July 2010) and spring (November 2009).

DISCUSSION

Factors determining water use of M. argentea My results confirm that water use by M. argentea in the Pilbara is primarily determined by atmospheric evaporative demand, with up to 79% of day to day variation explained by atmospheric conditions. Pulses of surface soil moisture resulting from sporadic rainfall events had no discernible effect on rates of sap flow. Pre-dawn Ψl was also close to zero throughout the year and these findings collectively confirm that water use was not limited by availability of water to the roots. These findings are consistent with observations of other tree species in similar environments. For example, transpiration was strongly related to meteorological factors and unaffected by rainfall in Populus deltoides forests above a shallow water table, on the semi-arid Middle Rio Grande river system, New Mexico (Cleverly et al., 2006). In contrast, where trees do not have access to permanent water, particularly in semi- arid and arid regions, rates of tree water use typically increase following precipitation events (e.g. Hubbard et al., 1999; Zeppel et al., 2008; Du et al., 2011).

As water supply was constant, this study provided an opportunity to more closely examine the effects of atmospheric variables on tree water use over time. Evaporative demand, wind speed and direction, temperature and solar irradiance can all influence

34 Seasonal patterns of water use by Melaleuca argentea transpiration rates, but since these are all inter-correlated, it is generally difficult to determine which factors might be the most important drivers of water use (Leng et al., 2006). However, VPD was the variable that best correlated with sap flow in M. argentea, suggesting that this may be the primary driver of tree water use. Wind speed may play an additional role, since the interaction between VPD and wind speed explained sap flow patterns significantly better than VPD alone. Tree water use is frequently influenced by wind speed, as under conditions of low air movement, transpiration can be inhibited by the buildup of a humid boundary layer around the leaves (e.g. Chu et al., 2009; Zheng & Wang, 2012). Leaf boundary layer conductance may therefore be a significant factor in the water use of M. argentea, as is commonly the case for broad-leafed species (Meinzer et al., 1997a; Cleverly et al., 2006). My results also indicate that atmospheric controls on tree water use may have differed between the two study sites. Water use by trees at Marillana Creek appeared more closely linked to meteorological drivers compared to the Yule River site (79 % compared to 67%) despite the more complex multiple regression equation used to describe water use in the Yule River trees. Relationships between climate and water use by other riparian tree species have been shown previously to differ between sites (Cleverly et al., 2006). Reasons for such variation among stands may relate to differing site characteristics, but remain largely unknown.

While seasonal patterns of sap flow were largely explicable by atmospheric variables in all the M. argentea trees examined, the absolute rates of sap flow were highly variable among individuals. Daily sap flow varied by two- to three-fold among trees that were growing under the same environmental conditions. Daily rates of sap flow also differed substantially between the two study sites. Inter-tree and site variability in water use by M. argentea trees has been previously noted by O’Grady et al. (2006a), on the Daly River in the wet tropics of Northern Australia. Likely reasons for inter-tree variability both within and among sites include tree size (such as differences in sapwood area and foliage area), and variation in sap flow rates within different regions of the sapwood (e.g. Gebauer et al., 2008; Zeppel & Eamus, 2008).

In the Pilbara region, M. argentea grows in extreme VPD conditions, while having constant access to a reliable water supply, and the rates of sap flow were therefore relatively

-1 high. The maximum observed site averages of Vs in the present study were 23 cm h and

35 Chapter Two

3.9 m day-1 at Marillana Creek, and 31 cm h-1 and 5.2 m day-1 at the Yule River. These rates are similar to those measured on M. argentea trees in the wet tropics of the Northern Territory (O'Grady et al., 2006a). The sap flow rates of M. argentea are also within the range of values reported for riparian trees in other semi-arid regions. For example, studies in semi-arid Arizona, USA, recorded peak flow rates for Populus species with access to permanent water of 15–35 cm h-1, and for Salix species up to 60 cm h-1 (Schaeffer et al., 2000; Gazal et al., 2006). Nocturnal sap flow in M. argentea was also similar to rates reported for semi-arid poplars (10–25% of total daily water use; Gazal et al. 2006). Water use and water use physiology of semi-arid riparian trees therefore appears to be similar across the continents.

Seasonality of water use in M. argentea I found a distinct seasonal pattern in the water use of M. argentea in the semi-arid Pilbara, largely corresponding with seasonal fluctuation in atmospheric conditions. This result is in contrast to O’Grady et al. (2006), who did not observe any significant seasonal variation in M. argentea water use in the wet-dry tropics at the Daly River. I attribute this partly to climate differences between regions; water use was lower during the cool winter months in the Pilbara, while the VPD in the tropics can be highest during the ‘winter’ dry season (June to August). The long period of continuous measurement over this study also allowed me to analyse water use over time in much greater detail. O’Grady et al. (2006) collected sap flow measurements during three two-week periods across the year, which may not have been sufficient to detect any subtle seasonal patterns that may have been present. Seasonal climatic fluctuations in the Pilbara clearly drive seasonal variation in the water use of M. argentea.

Although water use by M argentea was largely explained by meteorological variation, the ‘shape’ of the relationships of water use with the major driving variables changed across the year, contrary to expectations. Hysteresis was evident in the relationships over both diurnal and seasonal cycles. M. argentea water use exhibited counter-clockwise hysteresis with irradiance on most days in winter and spring, with the extent of hysteresis decreasing during summer and autumn, and moving to clockwise hysteresis on a few days in summer.

These changes in diurnal hysteresis suggest that Vs was increasingly limited by water availability to the canopy during summer and autumn. Diurnal hysteresis of water use with

36 Seasonal patterns of water use by Melaleuca argentea respect to VPD and solar irradiance is commonly observed, and can be explained by the time lag that exists between the irradiance and VPD conditions, stomatal responses to light, as well as changes in the availability of water to the leaf surfaces over the diurnal cycle (e.g. Zeppel et al., 2004; Zheng & Wang, 2012). With abundant water availability, if stomatal conductance is primarily a function of light intensity, then the fact that maximum light levels occur at midday, while VPD peaks mid-afternoon, will generate clockwise hysteresis of water use with VPD and counter-clockwise hysteresis with light (Zeppel et al., 2004). At a given VPD, the light intensity and therefore gs is greater during the morning than at the same VPD in the afternoon, driving greater Vs in the increasing phase of VPD than the decreasing phase generating clockwise hysteresis. Similarly, at a given light intensity, the

VPD is lower in the morning than in the afternoon, driving higher Vs during the decreasing phase of light intensity than during the increasing phase, resulting in counter-clockwise hysteresis. However, if water use is also influenced by a reduction of water supply over the daylight period, such as that caused by depletion of water in the rhizosphere or in internal water stores, or through increasing xylem embolism over the course of the day, then gs will tend to be higher in the morning, than at the same VPD or light intensity in the afternoon. Thus, diurnal regulation of transpiration by a combination of both light intensity and water limitation ought to lead to a reduced afternoon water use, manifesting as an increase in the extent of the clockwise hysteresis with VPD, and a reduction in the extent of the counter-clockwise hysteresis with light, eventually moving into clockwise hysteresis with light, as the severity of water deficit increases.

A seasonal shift in the diurnal pattern of hysteresis between water use and irradiance was seen in M. argentea despite continuous availability of water to the roots. Strong counter-clockwise hysteresis with irradiance has been shown in a well irrigated Populus hybrid in Washington, USA (Meinzer et al., 1997b), and also in numerous tree species in wet, high humidity tropical forests in Costa Rica (O'Brien et al., 2004) and Indonesia (Horna et al., 2011). Patterns of hysteresis with light also show clear changes during drought. For example, water use by Eucalyptus crebra in northwest New South Wales, Australia, displayed counter-clockwise hysteresis with irradiance during the wetter summer, but little or no hysteresis during a prolonged drought (Zeppel et al., 2004). Water use by Haloxylon ammodendron trees in arid China also displayed counter-clockwise hysteresis with

37 Chapter Two irradiance after rainfall, shifting to clockwise hysteresis under dry conditions (Zheng & Wang, 2012). The similar pattern seen in M. argentea, of counter-clockwise hysteresis of water use with irradiance in the winter and spring, moving to no hysteresis or clockwise hysteresis in summer and autumn indicates seasonal changes in the supply of water to the leaf surfaces, such as through a limitation in the transport capacity of the trees.

Water use by the M. argentea trees exhibited clockwise diurnal hysteresis of Vs with VPD year round, with no clear patterns evident in the extent of hysteresis. Similarly, clockwise hysteresis in the relationship between water use and VPD appears almost ubiquitous across species, and the extent of the hysteresis is usually not clearly attributable to water availability. For example, hysteresis with respect to VPD did not alter in response to changing soil water content in Eucalyptus globulus in Tasmania, Australia (O'Grady et al., 2008). Hysteresis with VPD also did not vary with water availability in any of four tree species planted in an urban park in Liaoning Province, China (Chen et al., 2011). However, hysteresis did increase during the dry season in E. tetrodonta and E. miniata in northern Australia (O'Grady et al., 1999). The extent of diurnal hysteresis with VPD appears to depend most strongly on the level of VPD itself, perhaps reflecting more complex regulation of stomatal and leaf conductance by combined factors of VPD, light and water availability (O'Grady et al., 2008; Guyot et al., 2012). Hysteresis in the relationship between water use and irradiance therefore appears to be a more useful indicator of water limitation than hysteresis with respect to VPD.

In the present study, hysteresis was also observed over the annual cycle in the water use of M. argentea trees with respect to VPD, but not irradiance. Water use patterns over annual cycles might display patterns of hysteresis for similar reasons as over diurnal cycles; the annual peaks in irradiance and VPD are offset in a similar manner as seen diurnally, and water availability can also vary seasonally. Therefore, by the same reasoning as applied to diurnal cycles, the lack of hysteresis in the relationship between Vs and irradiance suggests restricted water use during the summer and autumn (the period of decreasing irradiance). The annual patterns of hysteresis were still present after accounting for other meteorological factors; compared with that predicted from meteorological conditions, tree water use was higher in spring and early summer, declined from mid-summer and through autumn, then began to increase in the winter. Seasonal differences in the relationships of

38 Seasonal patterns of water use by Melaleuca argentea tree water use with VPD were also observed in E. crebra which were attributed to the marked seasonal differences in soil water content and plant water status (Zeppel et al., 2004). Similarly, in M. argentea, both seasonal patterns, and the seasonal variation in diurnal patterns, suggest that water use by M. argentea was restricted during summer and autumn.

The apparent water deficit during parts of the year observed in this study may be related to limitations in the rate of water transport to the canopy. The seasonal variation in tree water use patterns are unlikely to be due to changes in water availability to the roots due to soil drying, since the M. argentea trees appeared to be accessing a perennial shallow water table at both study sites, and there was no discernible tree response to small rainfall events. Sap flow readings can decline over time due to the wounding response of xylem tissue (Hatton et al., 1995; Hogg & Hurdle, 1997). However, a wounding artefact is unlikely to have produced the observed patterns of Vs, since the pattern was consistent across study sites, regardless of whether the sequence of measured seasons ran from autumn 2009 to autumn 2010 (Yule River), or winter 2009 to spring 2010 (Marillana Creek). In addition, progressive wounding over the study period should not affect diurnal patterns, and so would not explain the seasonal variation in diurnal hysteresis.

Alternative explanations for the seasonal response here are changes to tree hydraulic conductance, which may occur with phenology of growth (canopy cover and conducting xylem tissue in particular), or changes in tree capacitance. While not measured directly at my sites during the time of the study, long-term monitoring at Weeli Wolli Creek, upstream of Marillana Creek, showed no seasonal changes of canopy cover of M. argentea (Figure 2.8; Rio Tinto Pty Ltd, unpublished data). This is in contrast with data from locations experiencing groundwater abstraction, where clear reductions in canopy cover were detectable in M. argentea trees over the same period (Rio Tinto Pty Ltd, unpublished data). Canopy cover of M. argentea therefore appears not to alter as a regular seasonal adjustment at undisturbed locations, but partial canopy loss may occur as a safety mechanism under water stress. There were also no visible growth rings in the wood of the studied M. argentea trees, indicating relatively continuous growth, similar to many wet tropical forest trees (Martı́nez-Ramos & Alvarez-Buylla, 1998). Seasonal variation in production and loss of leaves and conducting xylem are therefore unlikely at the sites used

39 Chapter Two in the present study. Likewise, no difference was detectable in whole tree resistance of M. argentea among seasons, either in the present study, or by O’Grady et al. (2006a) in the wet-dry tropics. Seasonal changes in tree K that were not detectable from the measurements of Vs and Ψ conducted in the present study or by O’Grady et al. (2006a) cannot be entirely ruled out. However, changes in the use of internal water stores by M. argentea across the year appear to be the most likely explanation for the seasonal variation in water use patterns observed here.

0.7 a a a a 0.6 Figure 2.8: Leaf area index (LAI) of Melaleuca argentea trees at undis- 0.5 turbed sites on Weeli Wolli Ck, across seasons (Rio Tinto Pty Ltd, unpub- 0.4 lished data). Values are relative changes over time in canopy cover, 0.3 determined at long term monitoring points by digital cover photography 0.2

LAI (cover proportion) LAI (cover (Macfarlane et al., 2007). Values are 0.1 means ± standard error of eight to ten trees, and did not differ significantly 0 between seasons. JUN OCT DEC MAR 2010 2010 2010 2011

My results suggest that M. argentea may utilise water storage over annual cycles. M. argentea is likely to have a reasonably high wood capacitance, due to its moderate to low wood density (Borchert & Pockman, 2005). Eucalyptus victrix, a co-occurring riparian species in the Pilbara region, has been shown to utilise internal water storage over diurnal cycles, despite constant access to groundwater, to meet the water transport demands of the high VPD environment (Pfautsch et al., 2011), and the same process might occur in M. argentea. Seasonal fluctuation in stem water storage is commonly observed in tree species subjected to seasonal drought, where water withdrawn from internal reserves during dry periods and replenished during wetter months enables a greater total water use (e.g. Loustau et al., 1996; Baker et al., 2002; Domec et al., 2005; Betsch et al., 2011). Although the M. argentea trees had a constant water supply, under the extreme VPD conditions during parts of the diurnal and annual cycles the rate of water transport appeared to 40 Seasonal patterns of water use by Melaleuca argentea become the limiting factor in tree water use; high capacitance might partly overcome this limitation.

Internal water stores are depleted during daylight hours and refilled during the dark period, but full replenishment does not always occur by dawn (Bucci et al., 2004; Scholz et al., 2007). Nocturnal sap flow comprises refilling as well as nocturnal transpiration (Phillips et al., 2010). The correlation between nocturnal Vs and VPD in M. argentea indicates night time transpiration losses, however, flows still occurred on nights when VPD was close to zero, suggesting that parts of the nocturnal flow were due to refilling. Nocturnal Vs was also greater during spring than autumn for any given VPD, suggesting the capacity to replenish stores may be lower in summer and autumn. High VPD and rates of water use during summer may progressively deplete internal stores in M. argentea, despite constant access to groundwater. The lower VPD during winter may provide a respite that allows the trees to refill under conditions of reduced leaf level water loss. However, further data would be needed to verify seasonal changes in water storage and capacitance in M. argentea, such as sap flow measurements from sensors distributed along the trunk and upper branches (Meinzer et al., 2004b; Phillips et al., 2009), measurements of changes in wood water content over time and/or diurnal changes in stem circumference (Čermák et al., 2007; Betsch et al., 2011).

Overall, the relationships between temporal patterns in water use and meteorological factors (light, VPD and wind speed) were more complex than expected. I observed distinct seasonal variation in water use patterns, which was not accounted for by seasonal meteorological variation. With access to a perennial water supply, but in a high VPD environment, rates of water transport appear to limit the water use of M. argentea during high demand periods, over both diurnal and annual cycles. Stored water likely enables the higher rates of transpiration by the trees during spring and early summer. However, I hypothesise that the long periods of high VPD progressively deplete water stores, leading to reduced rates of water use during late summer and autumn. The short period of low VPD conditions in the winter may allow recharging of stored water. Nonetheless, water use by M. argentea in the Pilbara region can be described to a large extent by the meterological variables of light, VPD and wind speed, as would be expected for a tree with constant access to groundwater. Water use patterns over time are thus relatively predictable, which

41 Chapter Two might allow relatively simple methods of calculating total water fluxes through trees over annual and multi-annual scales. However, there was considerable unexplained variation in sap flow rates among trees and between the study sites. Further study of this variability would clarify spatial patterns in tree water use, and aid in quantifying the hydrological fluxes through trees.

CHAPTER THREE: Quantifying water fluxes at tree and stand scales from sap flow measurements in the riparian tree Melaleuca argentea

INTRODUCTION

Volumetric estimates of tree water use at the scale of whole trees and stands are important in both assessing vegetation water requirements for resource management, and for understanding tree and ecosystem functioning. For instance, whole tree water use measurements are important in understanding physiological controls on transpiration, such as the roles of stomata and hydraulic architecture (Meinzer et al., 2013). Furthermore, trees usually play a dominant role in transpiration and redistribution of water within ecosystems, and so tree water fluxes are an important component of most ecosystem water budgets (Baird & Maddock, 2005; Eamus et al., 2006b; Domec et al., 2010). However, few estimates of water requirements or fluxes have been made for any of the tree species of northern or inland Australia.

Sap flow measurements are often used to obtain accurate point measurements of rates of water flow through trees, which may then be scaled up to the level of whole trees and stands (e.g. Schaeffer et al., 2000; Doody & Benyon, 2010; Tateishi et al., 2010). However, for such scaled estimates to be accurate, the sampling must adequately encompass the spatial and temporal variation in sap velocity (Fiora & Cescatti, 2006; Ford et al., 2007; Kume et al., 2010; Tsuruta et al., 2010). Failure to account for the variability can lead to significant margins of error in water use estimates. For example, estimates of whole-tree water use by the mangrove Avicennia marina varied by up to 240% depending on where sap flow sensors were located within the sapwood (Van de Wal et al., 2013).

Spatially, sap flow varies within trees throughout different regions of the sapwood, between trees within a stand, and between stands. Within the sapwood, flow velocities are generally higher in the younger, outer wood and decrease moving inward toward the heartwood, as vessels become increasingly blocked by tyloses with age. The shape of this radial flow distribution appears to be species specific, with both linear and curved patterns observed depending on properties such as the pattern of vessel distribution across the sapwood (Cohen et al., 2008; Gebauer et al., 2008). The relationship between sap flow 43 Chapter Three velocity and radial depth generally needs to be verified for each species, if water use estimates are to be accurate, by placing sensors at multiple depths. Sap flow also varies azimuthally, around the tree stem circumference, in a manner that is usually less consistent among trees than radial variation (Cohen et al., 2012). Azimuthal variation may relate to variation in vessel anatomy and the positioning of major roots and branches (e.g. Tateishi et al., 2008; López-Bernal et al., 2010). Accounting for sapwood variation is crucial in obtaining volumetric estimates of water use at the whole tree scale.

When seeking to scale up water use to the stand and landscape scales, variability in flux among trees and between stands can become more important than within tree variation. Variation in tree size, quantity of conducting sapwood area, and canopy area can all contribute to significant differences in water use between trees. For instance, in Japanese cedar stands, between-tree variation was identified as the most important level to account for in estimating stand water use (Kumagai et al., 2005; Shinohara et al., 2013). In twelve trees within one stand, sap flux density varied more than 2.6-fold between trees for unknown reasons, with the variation not explained by factors such as tree size (Kumagai et al., 2005). In Pinus strobus plantations in North Carolina, USA, variation between different stands in tree sapwood area and stand density was the source of greatest variability in estimating catchment scale water fluxes (Ford et al., 2007). A continuing challenge in estimating landscape scale water fluxes is to understand and quantify the variability in water use among trees and locations.

For stand and landscape scale tree water use estimates, universal scaling relationships have been proposed based on factors such as tree structure, stand density, and moisture availability (e.g. Enquist, 2002; Meinzer, 2003; Meinzer et al., 2004a; Zeppel & Eamus, 2008; O'Grady et al., 2009), but these require significant validation. Inherent species differences in properties such as stand structure and sensitivity to water deficit may mean that these scaling relationships do not always hold, and species and/or site specific relationships may be required (Aranda et al., 2012). In addition, the convergence of water use among species tends to be related to the constraint of limited soil moisture availability (e.g. Meinzer et al., 2004a; Kelley et al., 2007; O'Grady et al., 2009). Thus, it remains unclear whether the proposed universal scaling relationships might apply to species in wetland-type ecosystems, where water is constantly available. Direct measurements of

44 Quantifying water use by Melaleuca argentea individual tree sap flow are therefore valuable to determine patterns and variability of tree water use, particularly in non-water-limited environments.

Temporally, sap flow varies over diurnal and seasonal cycles as well as inter-annually, with changes in environmental conditions. For instance, transpiration is regulated by stomata in response to irradiance and water availability, and total tree water use also depends on the canopy area, which can display phenological cycles in response to seasonal fluctuation in temperature or water availability (e.g. Hutley et al., 2001; Gazal et al., 2006; Zheng & Wang, 2012). At the stand and landscape scales, water flux estimates for time periods of a year or longer are frequently required. Measurements taken during shorter time windows distributed across the year are commonly scaled up to annual fluxes, for example measurements may be taken for a few weeks during the wet and dry seasons (e.g. Irvine et al., 2004; O'Grady et al., 2006a; Zeppel et al., 2008). However, this approach has rarely been tested. It might also be possible to estimate tree water use over time from environmental conditions such as atmospheric demand and soil water availability, since these factors are the main drivers of tree water use (Granier et al., 2000). If species and/or site specific relationships can be determined, monitoring of the appropriate environmental variables might allow ongoing estimates of tree water flux. For species and sites where water use is driven largely by atmospheric demand, then it might be possible to accurately estimate tree water use from meteorological data alone, which is usually widely available and easily accessed.

Water resources throughout the world are under increasing demand from human populations, requiring greater degree of water management and accounting, in order to balance competing human and ecological requirements (e.g. Arthington et al., 2010; Shen & Chen, 2010). In the semi-arid Pilbara region of northwest Australia, rapid population growth and groundwater perturbations related to mining activity are placing increasing pressure on the riparian ecosystems (Department of Water, 2010). However, the region remains relatively remote and there has been limited quantitative assessment of vegetation water requirements against the background of extreme hydroclimatic variability (Leighton, 2004; Dogramaci et al., 2012). M. argentea is one of the dominant riparian tree species in the Pilbara region, found only in locations with perennial surface or near-surface water. Rates of water use by this species are relatively high (Chapter 2), leading to high vegetation

45 Chapter Three water requirements. In some locations, water use by M. argentea is therefore likely to contribute significantly to total ecosystem hydrological fluxes. Volumetric estimates of tree water use would be valuable in assessing vegetation water requirements. Obtaining such volumetric estimates from sap flow measurements requires characterisation of the spatial and temporal patterns of sap flow variability, and validation of approaches to extrapolate localised measurements obtained over short time periods to larger spatial and temporal scales.

In Chapter 2, I demonstrated that sap flow of M. argentea was highly variable among trees, both within and among sites, a finding consistent with studies undertaken on this same species in the tropics (O'Grady et al., 2006). My objective here was to assess spatial variation in sap flow of M. argentea, including within and between tree variation. I examined patterns of sap velocity within the sapwood and across tree sizes, as well as tree allometric ratios, to identify relationships that could facilitate water use estimates at whole tree and stand scales, from fewer point measurements of sap flow. I also demonstrated in Chapter 2 that sap flow of M. argentea is strongly related to the atmospheric evaporative demand, with no detectable influence of variation in soil moisture content, due to constant access to a shallow water table. I generated regression models from meteorological variables at two contrasting sites within the Pilbara region, which explained up to 80% of the daily variation in tree sap flow (Chapter 2). In the present study, I hypothesised that accurate estimates of total annual tree sap flow could be derived from these models. In the present study, I extracted annual estimates of sap flow from these existing models, and compared these with the measured values. I also tested whether these models may be broadly applicable, or whether different models would need to be determined for different stands of trees, by applying the models to a different stand of trees from which they were derived. In addition, the long period of continuous sap flow measurements (more than 12 months) at each site allowed me to test the approach of scaling up sap flow measurements from short time windows to total annual flows. Four-week windows were selected at evenly spaced time points across the year, and the scaled estimates compared with the measured total annual sap flow. Characterisation of spatial sap flow variability, and validation of temporal scaling approaches, will assist in determining the extent of sap flow sampling required to

46 Quantifying water use by Melaleuca argentea obtain acceptable estimates of water use by M. argentea at the scale of whole trees, stands and landscapes.

MATERIALS & METHODS

Study sites The study was conducted at undisturbed locations on the Yule River and Marillana Creek, two intermittent waterways in the Pilbara region of NW Australia as described in Chapter 2. Measurements were conducted on the same ten Melaleuca argentea trees as the study presented in Chapter 2.

Sap flow measurement Sap flow measurements were performed with heat pulse velocity probes via the heat ratio method (HRM30; ICT International, Armidale, NSW, Australia), as described in Chapter 2. The measurement of wood properties, and conversion of pulse velocity to sap velocity

(Vs) are also described in Chapter 2.

Variation in Vs within the sapwood was assessed in four trees at Marillana Creek. In two trees, multiple sensors were installed on the eastern side of the stem at 1.2 m height, with measurement points positioned at 5 mm depth increments into the sap wood from

2.5 mm to 27.5 mm, to assess radial variation in Vs. In another two trees at Marillana Creek, three or four sensors were installed at evenly spaced positions around the stem circumference, to assess azimuthal variation. Measurements were recorded every 30 min for two weeks during July 2010. To estimate volumetric sap fluxes, the cross-sectional sap wood area (As; see next section) was divided into sections (concentric radial bands or azimuthal quadrants) delineated by the midpoints between adjacent sensor points.

Volumetric flow was then calculated as the product of Vs and the As of the section in which the sensor point was located (Burgess et al., 2001). Total tree water use (Q) was estimated as the sum of the volumetric fluxes of all sap wood sections in the tree.

The variation in Vs among trees, and over time, was quantified from the data presented in Chapter 2. Briefly, sensors were installed in five trees at the Yule River and five at Marillana Creek, and data collected from a measurement point positioned at 7.5 mm

47 Chapter Three depth (the approximate centre of the sap wood band). Vs measurements were recorded every 30 min for approximately 12 months.

Tree sapwood area

The cross-sectional sapwood area (As) at 1.2 m height was determined for each study tree as a measure of tree size relevant to water use, and for estimation of volumetric fluxes (see previous section). The sapwood depth (ws) was determined from 3–4 wood cores taken at equally spaced positions around the tree circumference with an increment borer (Haglöf, Långsele, Sweden). Surfaces of the fresh cores were shaved back and stained with methyl orange (1% w/v in 4% v/v methanol); the sapwood stained deep red and the heartwood yellow. The position of the colour change corresponded well with the boundary of conducting sap wood as determined by adjusting the depths of the sap flow probes. Bark depth (wb) was measured with a needle-type gauge at a minimum of four points around the tree, and overbark diameter (d) measured with a diameter-tape. The cross-sectional sapwood area was determined from the above measurements assuming circularity of the stem cross section:

2 2 As = π(½d – wb) – π(½d – wb – ws)

Tree hydraulic conductance Conductance of the whole tree hydraulic pathway (K) was calculated as the slope of the linear part of the relationship between Vs and leaf water potential (Ψl), obtained from the data presented in Chapter 2 (examples in Figure 2.8).

Analysis of variation in sap flow Statistical analysis was performed in SAS version 9.2 (SAS Institute Inc.; Cary, NC, USA). The effects of within-sapwood variation in sap flow on the calculation of volumetric tree water use was assessed, by comparing the total tree water use calculated using a single measurement point, with that calculated using the weighted average of all measurement points (positioned at intervals radially or azimuthally). Relationships of As with sap flow, K,

Ψl and tree diameter were assessed by linear regression, and differences in relationships between seasons and sites tested by ANCOVA.

Extrapolation of water use over time from shorter time periods was tested, by assuming the measurements from equally spaced selected months across the year were 48 Quantifying water use by Melaleuca argentea representative of an equal portion of the year. Estimation of water use from meteorological data was also tested. Models of daily tree sap flow based on meteorological variables for each site were described in Chapter 2. When used to derive daily estimates of water use, the models gave slight over- or under-estimates depending on the season (Chapter 2). In the present chapter, I assessed these models for their utility in obtaining water use estimates integrated across longer periods of time, by comparing the measured monthly and annual total sap flow with the values predicted by the models. I also tested whether these meteorological models may be applicable to different sets of trees from those from which they were derived, by using the model produced for one study site to predict water use at the other. The fixed (meteorological) effects from the models were used for these tests, and tree As was also added as an additional fixed effect, to account for any effect of tree size on rates of water use. The accuracy of the water use estimates derived from calculated and extrapolated values are reported as percentage deviance from the actual measured values of sap flow.

RESULTS

Sap flow variation within the sapwood

Across the radial sapwood profiles Vs was highest toward the outer sapwood, and showed and almost linear decrease to zero toward the central heartwood. However in one tree Vs peaked at 7.5 mm depth, while in the other tree highest Vs was recorded by the sensor at 2.5 mm depth (Figure 3.1). The linear portion of the decline across the radial profile was significantly steeper in tree 1 than tree 2 (p=0.038). The proportional contribution of sap flow at each depth remained constant over time, over diurnal cycles (Figure 3.1c, d) and over the two week measurement period (Figure 3.1e, f).

Azimuthally, Vs varied both over time and between trees (Figure 3.2). In tree 3, morning sap flow was greatest on the northern and eastern sides of the stem, midday flow was greatest on the eastern side, and afternoon flow on the southern side (Figure 3.2a). Over the course of a day the eastern side contributed the most to water use and the western side the least. In contrast, in tree 4 morning flow was greatest on the eastern side, while midday and afternoon flow were greatest on the southwest (Figure 3.2b). Over the course of a day, the southwestern side contributed the most to sap flux and the northern the least.

49 Chapter Three

There

Tree 1 Tree 2 18 (a) (b) 16 8:00 14 12:00 12 18:00 10 0:00 total daily

(cm h-1) 8 s V 6 4 2 0 (c) (d) 0.5

0.4

0.3

0.2

0.1

Contribution to total (proportion) total to Contribution 0 0 5 10 15 20 25 0 5 10 15 20 25 30 Radial depth (mm)

Figure 3.1: Variation in sap flow velocity (Vs) with radial depth (0 is the cambium) into the sapwood. (a & b) Vs across the radial profile, and(c & d) the proportional contribution of each radial position to the total sapflux across the profile (weighted by the sapwood area of each radial band), in (a & c) tree 1 and (b & d) tree 2. Data shown are from July 24th 2010, as a representative example, for mid-morning, midday, mid-afternoon, midnight and for the cumulative sap flux over 24 hours (total daily).

was an approximately 2-fold difference in Vs between the fastest and slowest azimuths during the middle of the day, in both trees. The proportional contribution to flux of each azimuth therefore varied over the course of a diurnal cycle (Figure 3.2c, d), although the overall diurnal pattern was similar throughout the sapwood, and between days (Figure 3.2e).

Due to the variation in Vs throughout the sapwood of these study trees, calculating total tree water use (Q) from measurements at a single sensor point would be inaccurate for many of the points. Daily Q values calculated as the average of multiple sensor points

(weighted by the As of the azimuth or radial band in which each was located) are compared

50 Quantifying water use by Melaleuca argentea with Q calculated from each sensor point alone in Table 3.1. The values presented in Table 3.1 are for a single day as an example, but similar results were obtained for the other days

Tree 3 Tree 4 40 (a) (b)

20 (cm h-1) s V

0 (c) (d) 0.6

0.5

0.4 8:00 0.3 12:00 0.2 18:00 0:00 0.1 total daily Contribution to total (proportion) total to Contribution 0 NESWNESW Azimuthal position (compass direction)

Figure 3.2: Variation in sap flow velocity (Vs) with azimuthal position around the stem circumference, at 7.5 mm depth into the sapwood. (a & b) Vs with azimuthal position, and (c & d) the proportional contribution of each azimuth to the total sap flow around the stem, in(a & c) tree 3 and (b & d) tree 4. Data shown are from July 24th 2010, as a representative example, for mid-morning, midday, mid-afternoon, midnight and for the cumulative sap flow over 24 hours (total daily).

during the two week measurement period. Failing to account for radial variation when calculating Q in these study trees resulted in potentially larger errors (over- or underestimates of up to 70% of the weighted average) than failing to account for azimuthal variation (which lead to estimates differing from the weighted average by up to 35%).

Sensors positioned in the radial band of fastest Vs yielded the least accurate single-point estimates of Q.

51 Chapter Three

Temporal variation in sap flow

Although the absolute Vs and diurnal Vs patterns varied among different regions of the sapwood, the relative patterns of daily Vs over time remained consistent throughout the

Table 3.1: Comparison of estimates of whole tree water use (litres) during a single representative day (24th July 2010), calculated as the weighted average of multiple sensor positions or from single sensor points. Sensors were placed at (a) 3 radial depths or (b) 3–4 azimuthal positions within the sapwood.

(a) radial Sensor position (mm depth) weighted 2.5 7.5 12.5 average Tree 1 calculated water use 21.1 12.2 33.6 17.5 % difference from weighted average -42 59 -17 Tree 2 calculated water use 14.5 24.6 13.0 7.9 % difference from weighted average 70 -10 -46

(b) azimuthal Sensor position weighted N E S W average Tree 3 calculated water use 29.1 30.1 34.5 32.1 20.0 % difference from weighted average 3 19 10 -31 Tree 4 calculated water use 117.1 76.1 109.4 157.3 (SW) % difference from weighted average -35 -7 34

sapwood. The measurements taken at a single point within each of the ten study trees therefore appear to be valid for assessment of temporal variation in Vs, over the scale of days and months.

The accuracy of estimating total annual sap flow by extrapolating from shorter measurement periods was tested by extrapolating from the sap flow data during April and October only (beginning and end of the dry season), or during January and July only (mid- summer and mid-winter), or from data during all four of these months. The estimates deviated from the measured annual sap flow by at most 16% for Marillana Creek trees and 9.8% for the Yule River trees (Table 3.2). Using January and July measurements gave slightly better estimates than using April and October measurements, particularly for the Yule River trees. Using quarterly measurements did not yield substantially better estimates than using bi-annual data.

52 Quantifying water use by Melaleuca argentea

The possibility of quantitatively estimating sap flux over time from meteorological variables was assessed using linear regression models previously determined for these study trees (Chapter 2). Estimates for individual months deviated from the measured values by up to 5% at the Yule River, and by up to 16% at Marillana Creek. However, over the year as a whole the estimates deviated from the measured by less than 3% at both sites (Table 3.3).

Table 3.2: Accuracy of estimates of annual sap flow (% difference from the measured value), derived by extrapolating from measurements taken during selected months. The total range of accuracies of five trees (range) and the mean ± standard deviation of accuracies of five trees (mean) are provided. Negative values indicate under-estimates, positive over-estimates.

Yule River Marillana Creek Selected months Range Mean Range Mean April & October -8.4–9.8 2.3 ± 7.4 5.8–15.4 10.0 ± 3.5 January & July -5.3–5.2 0.2 ± 4.6 -1.3–16.1 9.2 ± 6.6 January, April, July & October -1.5–4.1 1.2 ± 2.4 3.6–15.8 9.6 ± 4.3

Table 3.3: Accuracy of estimating monthly and annual sap flow (% difference from the measured value) from meteorological variables, through linear multiple regression models. Negative values indicate underestimates, positive values overestimates. YR; Yule River, MC; Marillana Creek. See text for details of the models used.

J F M A M J J A S O N D Annual YR 1.6 -4.4 -2.4 -1.5 -5.1 -4.8 -2.0 -0.5 -1.4 -2.3 -0.2 -0.8 -2.42 MC -1.1 15 5.8 9.3 2.5 -1.8 -8.6 -5.1 14 -16 -9.2 -8.4 -2.00

Variation between trees and sites Sap flow velocity varied considerably between trees, both within and between sites, as measured at a single point near the centre of the sapwood band in each tree. Tree size appeared to partially, but not completely, explain the difference among trees. Daily sap flow varied approximately 2-fold among trees during cooler months, and 3-fold during hotter months (Figure 3.3a). Daily flow was significantly correlated with tree size (As) on most days during the hottest months (e.g. 10th Jan 2010; p=0.040, R2=0.427), but not during cooler months (e.g. 24th July 2009; p=0.291, R2=0.138). On all days the correlation was positive, i.e. flow was greater in larger trees. Cumulative sap flow over the

53 Chapter Three course of a year did not correlate significantly with tree size (p=0.09), although there may be a trend toward greater annual flow in larger trees (Figure 3.3b). The annual sap flow at the Marillana Creek site (during July 2009–June 2010) varied nearly 3-fold among trees, with values ranging from 454 to 1304 m year-1 (Figure 3.3b). Annual flow at the Yule River (during April 2009–March 2010) varied 1.5-fold among trees, ranging from 1197 to 1772 m year-1. Since all these inter-tree comparisons were measured at a single point within each tree, and within-sapwood variation can also be considerable (as described above), clearly part of the among-tree variation in Vs will be due to the within-sapwood component of the variability. However, the correlation between daily sap flow and tree size during warmer months indicates a likely additional among-tree component of variation, related to the size of the tree.

6 2000 (a) (b) 5 1600 4 R2 = 0.41 (sites combined) 1200 3 (m year-1) (m day-1)

s 800 s V 2 V Marillana Ck 400 1 Yule River 0 0 0 100 200 300 0 100 200 300

As (cm2)

Figure 3.3: Correlation between sap flow velocity (Vs) and tree size (cross sectional sapwood area; As). (a) Daily sap flow of trees at the Yule River (closed symbols) and Marillana Creek (open symbols), on a representative day in winter (July 2009; blue) and in summer (January 2010; red). (b) Annual sap flow measured over April 2009−March 2010 at the Yule River, and August 2009−July 2010 at Marillana Creek. Regression lines are shown for significant relationships (p<0.05).

Tree Vs was correlated more strongly with As than with stem diameter. Nonetheless, stem diameter may still provide an acceptable estimate of As, since the two were strongly correlated in the studied M. argentea trees (Figure 3.4a). However, the relationship between diameter and As differed between the study sites, with As increasing more steeply with diameter in the Yule River trees than at Marillana Creek (p=0.002). The width of the sapwood band (ws) tended to be greater at the Yule River than Marillana Creek (p=0.02),

54 Quantifying water use by Melaleuca argentea explaining the greater As of the Yule River trees (Figure 3.4b). However ws did not correlate with tree diameter (p=0.63), while bark depth did increase significantly with diameter (p=0.01).

6 (a) (b) bark sapwood 300 5 y = 7.51x - 90.81

R2 = 0.96 Width (cm) 4 R² = 0.56 200 (sites combined) 3 (cm2) s A 2 100 y = 4.20x - 40.35 1 R2 = 0.88 0 0 02010 3040 500 10 20 3040 50 60 Diameter (cm)

Figure 3.4: Relationships of tree stem dimensions at 1.2 m height, for trees at the Yule River (closed symbols) and at Marillana Creek (open symbols). (a) Correlation between stem diameter and sapwood cross sectional area (As), (b) correlations of stem diameter with bark width (triangles), and sapwood width (circles). Regression lines are shown for the significant relationships (p<0.05) of diameter withA s at each site, and with bark depth for sites pooled.

Whole tree hydraulic conductance (K) varied widely among individuals, from 6 to 31 cm h-1 MPa-1, but did not relate clearly to tree size (Figure 3.5a), and so appears not explain the higher Vs in larger trees. Likewise, leaf water potential (Ψl), which could drive higher Vs, appeared not to vary with tree size at either site (Figure 3.5b). However, Vs displayed a plateau of high flow during the middle of the day (Chapter 2, Figure 2.3), and the length of the plateau phase was correlated with tree size (Figure 3.5c). Thus, larger trees sustained low Ψl and high flow rates for longer periods. In addition, nocturnal flows were greater in larger trees (Figure 3.5d), although the contribution of nocturnal flow was much smaller than that of the daytime plateau phases.

Inter-tree variation in Vs was considerable within each site, but Vs also differed between the two study sites, with Yule River trees generally having higher flow rates than trees at Marillana Creek, even after accounting for meteorological and tree size differences between the sites. While Vs was largely predictable from meteorological conditions in M. argentea, the regression models that were created for Vs against meteorological parameters

55 Chapter Three

40 0 (a) (b) -0.5 30 Ψ Midday

-1.0

20 leaf

-1.5 (MPa) (cm h-1 MPa-1) tree

K 10 -2.0

0 -2.5 (c) (d) 12 R2=0.88 120 V Nocturnal R2=0.62 R2=0.50 11 100

10 R2=0.74 80 s

R2=0.76 (cm night-1) 9 60

plateau length (h) plateau 8 R2=0.32 40 s-max V 7 20 R2=0.93 6 0 0 100 200 300 0 100 200 300

As (cm2)

Figure 3.5: Relationships between water use physiology and tree size (sapwood area; As). (a) Whole tree hydraulic conductance (Ktree), (b) midday leaf water potential (Ψleaf ), (c) length of the daytime plateau phase of maximum sap flux velocity V( s-max), (d) total nocturnal sap flow. Data are from Yule River trees (closed symbols) and Marillana Creek trees (open symbols), in November (red) and June/July (blue symbols). Regression lines are shown for significant relationships (p<0.05). differed between the two study sites (Chapter 2). When the meteorological models generated for each site were used to estimate sap flow at the other site, the predictions were much less accurate (Table 3.4). The model produced from the Yule River data generated a

predicted annual sap flow that deviated only 7.6% from the measured value when it was applied the same data set. However, when the Yule River model was applied to the Marillana Creek meteorological data, the predicted annual sap flow for the Marillana Creek trees deviated 36% from the measured value. Similarly, the Marillana Creek model produced an annual sap flow estimate that deviated by 13% when re-applied to the Marillana Creek data, but deviated 27% when applied to the Yule River data. Many of the individual month estimates were even less accurate, deviating from the measured values by up to 128%. 56 Quantifying water use by Melaleuca argentea

Table 3.4: Accuracy of estimates of monthly and annual sap flow (% difference from the measured value), derived from regression models of meteorological variables that were produced for trees at a different study site. Negative values indicate underestimates, positive values overestimates. See text for details of the models used.

J F M A M J J A S O N D Annual Yule River (YR) model YR* -0.8 16 13 27 32 40 9.7 7.9 36 -11 -13 -12 7.60 MC+ -20 -28 -27 -20 -25 -16 -19 -24 -29 -28 -23 -23 -36.5 Marillana Creek (MC) model MC* -19 -18 -15 0.0 -7.3 -10 -7.0 -11 -14 -15 -17 -18 -12.8 YR+ 17 40 34 54 78 83 128 19 81 6.0 12 5.0 27.0 *Predictions generated for the same data the model was derived from, as controls. +Predictions generated for data from the other study site, as a test of potential applicability of the model to a new site

DISCUSSION

Rates of sap flow in M. argentea appear to be highly spatially variable at multiple scales, from within-tree variation throughout the sapwood, to variation between trees and sites. Sap flow also varied over time, but the temporal variation was more predictable, and estimates derived from meteorological data or extrapolation from shorter time periods are likely to be adequate, especially for estimates of total sap flux over longer periods (e.g. annual fluxes). Further examination of spatial variation in water use in this species would be required in order to fully characterise and understand the basis for spatial patterns, and to obtain sufficiently accurate estimates of stand and ecosystem water fluxes.

Flow rate varied across the radial sapwood profile in M. argentea, showing a linear decrease from the cambium to the heartwood in one tree, while in another tree Vs peaked at a depth of approximately 7 mm, then appeared to decrease linearly to the heartwood. Both these types of radial pattern are commonly observed in other tree species (e.g. Cohen et al., 2008; Gebauer et al., 2008). The formation of tyloses increasingly blocks vessels with age, reducing the conductance toward the heartwood. The temporary increase in conductance with age that occurs in the peaked radial pattern may be related to morphological changes

57 Chapter Three of the pit membranes (Gartner & Meinzer, 2005). Given that the slope of the radial flow profile differed between the two M. argentea trees in our study, further data would be required to establish whether there may be common patterns across trees in the radial profile that would allow total flux estimates from fewer sensor points. For example, the radial decline in Vs can vary with tree size in some species (Delzon et al., 2004). However, the width of the sapwood band did not appear to alter with tree size in M. argentea, and was only 15–30 mm, which is quite narrow compared with most other species. Some Eucalyptus species also have sapwood widths in the range 15–40 mm, which also remain constant with age (e.g. Miranda et al., 2009; Pfautsch et al., 2010). In contrast, sapwood widths of species including Populus fremontii, Populus canescens, Fagus sylvaticus, Acer pseudoplatanus and Pinus taeda usually increase with tree size and are typically 70–200 mm (Nadezhdina et al., 2002; Ford et al., 2004; Gebauer et al., 2008; Hultine et al., 2010). Although the patterns of radial flow require further analysis, the relatively uniform sapwood width among trees should simplify future sampling strategies and flux calculations.

As well as the usual decrease across the radial profile, I also observed an approximately two-fold variation in flow rates around the circumference of each measured tree. While some species show negligible azimuthal variation in sap flow, such as Pinus taeda (Domec et al., 2010), two-fold azimuthal variation may be broadly typical of a wide range of other species, including Quercus glauca (Tateishi et al., 2008) and Chamaecyparis obtusa (Tsuruta et al., 2010). This azimuthal variation might be related to the positions of large roots and branches, and coupling of certain stem sapwood portions with sections of the root system and canopy (Čermák et al., 2008). In addition, M. argentea stems are frequently asymmetrical and non-vertical as a result of the high levels of physical disturbance in the riparian zone, which can lead to variation in vessel anatomy and K around the stem (e.g. Vertessy et al., 1997). Azimuthal variation has been previously highlighted as a factor that can cause large errors in estimates of tree water use, and remains an ongoing methodological challenge (e.g. Saveyn et al., 2008; Tsuruta et al., 2010; Cohen et al., 2012). It is not clear whether there may be simple ways of predicting azimuthal variation, and thus for tree scale water use measurements might need to be assessed individually on every tree. When quantifying stand scale water use, varying the azimuthal

58 Quantifying water use by Melaleuca argentea positioning of sensors among measured trees might help to overcome any systematic over- or under-estimation due to aspect (Shinohara et al., 2013).

In M. argentea, the contribution of each radial depth remained fixed over diurnal cycles, and between days over the measured period of two weeks. However, the contribution of azimuthal portions varied over the course of the day, most likely related to changing irradiance levels on different parts of the canopy, although the azimuthal contributions to total daily water use remained constant over the two week measurement period. In some species, patterns of sap flow within the sapwood can vary diurnally and seasonally (Ford et al., 2004; Fiora & Cescatti, 2006). For instance, in Abies alba, Picea abies and Fagus sylvatica the inner sapwood increases its relative contribution during high demand periods, both diurnally and seasonally (Fiora & Cescatti, 2006; Saveyn et al., 2008). However, other studies indicate that in most species the radial sap flow pattern is likely to remain fairly constant over time, showing little variation in response to evaporative demand or soil water availability, except under severe stress (Caylor & Dragoni, 2009; Dragoni et al., 2009). The present study of M. argentea only observed within-sapwood patterns over a few weeks, but contributions of each measured sapwood portion were constant during this time. Nonetheless it is possible that azimuthal patterns could vary seasonally, with sun positions changing patterns of irradiance and shading across the canopy. It is also not known whether within-sapwood patterns might alter with severe stresses such as major hydrological disturbance. However, it appears that variation in sapwood contributions over time is unlikely to be a significant source of error in quantifying whole tree water use in this species, at least in its normal habitat.

A wide variation in Vs was observed among the individual M. argentea trees in this study. This was also noted by O’Grady et al. (2006a), who found inter-tree variation to be much greater in M. argentea than in the co-occurring Corymbia bella, along the Daly River in the wet tropics of Northern Australia. Nonetheless, similar variation in Vs (of two to three fold) among trees has been observed in other species, such as Eucalyptus regnans in southeastern Australia (Vertessy et al., 1997). Part of the observed variation in M. argentea will be due to the within-sapwood variation, since most trees were sampled only at a single point within the sapwood. However differences in Vs among M. argentea trees also appears

59 Chapter Three to be related to tree size, and variation in the allometric ratios between tree diameter, As and canopy area might also contribute to the variability (e.g. Cohen et al., 2012).

Sap flow velocity tended to increase with tree size (trunk diameter and As) in M. argentea, a finding that is unusual given the relatively small size of the studied trees (less

2 than 50 cm diameter, and As 0.03 m ). Flow velocity frequently varies with tree size, as xylem K, and leaf area to sapwood area ratio (Al:As) change with tree height and age, due to altered flow path lengths and tortuosity, and effects of gravity (McDowell et al., 2002). Total tree water use shows sigmoidal relationships with tree diameter and sapwood area, in a variety of angiosperm and gymnosperm species (Meinzer et al., 2005). Across an As of

2 approximately 0.1–0.4 m , water use generally increases disproportionately with As, (i.e. Vs increased with tree size), while above and below this size range, changes in water use slows and saturates (i.e. Vs remains constant or decreases with size) (Meinzer et al., 2005).

Observations in other studies also appear to correspond with these ranges of As. Sap velocity

2 increased with tree size in Populus fremontii of As up to 0.21 m , while in Salix goodingii

2 with As less than 0.06 m , Vs decreased with tree size (Schaeffer et al., 2000). In small

2 Eucalyptus crebra and Callitris glaucophylla trees (As of 0.001–0.03 m ), Vs and Al:As did not vary with diameter or height (Zeppel & Eamus, 2008). The M. argentea trees in the present

2 study also had As less than 0.03 m , yet daily sap flow increased with As during hotter parts of the year.

However, the increase in Vs with tree size did not appear to be related to differences in tree K in M. argentea, but rather was due to larger trees maintaining maximal rates of flow for longer periods during the middle of the day. Larger trees often have greater internal water storage capacity (e.g. Phillips et al., 2003; Scholz et al., 2011), which may explain the greater daily water use of larger trees in M. argentea. Greater internal storage capacity is associated with higher total rates of water use, due to the maintenance of high rates of transpiration for longer portions of the diurnal and annual cycles (e.g. Goldstein et al., 1998; Meinzer et al., 2003), as observed in M. argentea. The higher rates of nocturnal flow in larger M. argentea trees are also consistent with depletion and replenishment cycles of greater magnitude in these trees. Seasonal changes in capacitance, or a lower requirement to utilise water stores during low VPD periods (Chapter 2), could also explain the seasonal

60 Quantifying water use by Melaleuca argentea difference in the relationship between daily Vs and tree size, with a clear relationship present during summer, but much less pronounced in winter.

The inter-tree variation in M. argentea observed in the present study and in O’Grady et al. (2006) will also partly reflect within sapwood variation, since most of the trees were sampled at only a single position. Accounting for variation within the sapwood, through more intensive sampling within each tree, might therefore resolve some of the observed noise in the relationship between tree size and sap flow rates. If the relationship between Vs and tree size can be further clarified through increased sampling, the relationship could be used to scale estimates of tree water use. In addition, Vs depended upon tree size primarily during hotter months, and accounting for tree size appears to be less important for cumulative estimates over longer time periods (e.g. annual flux). A robust linear relationship between As and tree diameter was observed in this study, indicating that simple tree diameter measurements may be a useful way of adjusting stand scale water use estimates.

The trees at the Yule River displayed significantly higher rates of sap flow than trees at Marillana Creek, even after accounting for meteorological conditions and tree sizes. Variation in water use by M. argentea was also high among the study sites of O’Grady et al. (2006), where sites were within 50 km of one another. The observed among site variation could simply be a part of the inter-tree and within sapwood variation discussed above, and thus inter-site variation might be resolved by better accounting for tree and sapwood variation through increased sampling. For example, in Eucalyptus regnans in southeastern Australia, sap velocity varied two to three fold among individuals, but total stand water use varied by less than 20% among sites (Vertessy et al., 1995; Vertessy et al., 1997). A sample size larger than the four to five M. argentea trees used in the present study and by O’Grady et al. (2006) is likely to be needed. Ten to fifteen trees were needed to accurately estimate stand scale water use of a Chamaecyparis obtusa plantation in Japan (Kume et al., 2010) and at least 20 trees were required in a Pinus strobus plantation (Ford et al., 2007). In natural ecosystems containing multiple species and variable tree sizes and stand densities, the required numbers of trees may be even higher. Nonetheless, the different relationships of Vs with meteorological conditions at each study site suggest some difference in tree functioning (Chapter 2). The different relationships of tree diameter with As at each site

61 Chapter Three also suggests a site difference in tree allometry. Differences may relate to specific site conditions, or to genetic differences between trees on different waterways. Different responses to atmospheric conditions among locations has been observed in other species, for example in Populus deltoides in New Mexico (Cleverly et al., 2006). If variation in tree structure and physiology exists among stands of M. argentea, it may be necessary to sub- sample each location in order to estimate tree water use, unless underlying explanations for the variation can be uncovered.

Temporal variation in Vs was well characterised in the M. argentea trees (Chapter 2). Our results show that extrapolating from shorter periods of measurements distributed across the year could produce reasonable estimates for annual water fluxes in M. argentea in undisturbed conditions. On average, extrapolating from two or four sampling periods, each one month long, overestimated annual sap flow by a few per cent at the Yule River, and by approximately 10% at Marillana Creek. Two sampling windows spaced six months apart are likely to be sufficient, as four sampling windows did not improve the estimates at either site. Using daily meteorological parameters to estimate annual sap flow via regression models was even more accurate, leading to underestimates of only 2–3%. However, the high spatial variation in flow rates means that the meteorological models were not readily applicable to other locations, and so may not be a practical method of estimating tree water use. In general, cumulative annual flows appear to be predictable enough in M. argentea to allow reasonable estimates of tree water requirements to be derived from shorter windows of Vs measurements, or from meteorological measurements. However, water use estimates for shorter periods than one year may need to account for the additional seasonal component of variation in Vs. Tree water requirements may vary seasonally in a manner that is not entirely related to meteorological conditions, due to factors such as the potential influence of internally stored water (Chapter 2).

While the temporal component of sap flow variation has been well quantified, the present study has only touched upon the spatial aspects of sap flow variability in M. argentea. The sample sizes were small and thus I have not thoroughly assessed the extent of spatial variation, or the underlying explanations. Nonetheless, the findings demonstrate that M. argentea is among the more heterogeneous species in sap flow rates, and more spatially intensive sampling would be required to properly establish a basis for accurately

62 Quantifying water use by Melaleuca argentea quantifying water use in this species. In particular, further investigation of azimuthal variation, and of the relationship between tree size and flow rates, would contribute to a more complete understanding of tree physiology, and improve our ability to estimate hydrological fluxes at stand and landscape scales.

CHAPTER FOUR: Redistribution of water through root systems of a semi-arid riparian tree during a flooding-drying cycle in an intermittent waterway

INTRODUCTION

In dryland environments, soil moisture frequently varies throughout the soil profile. Along intermittent, dryland waterways in particular, water may be present at depth, while surface soil layers are dry. Plant root systems frequently transfer water from wet to dry soil regions, a passive process driven by water potential (Ψ) gradients, known as hydraulic redistribution (HR) (Burgess et al., 1998). HR appears to be an almost ubiquitous phenomenon, occurring whenever root systems span gradients of soil water potential, during periods that are not dominated by upward fluxes to above ground parts. In dryland areas, trees often have strongly dimorphic root systems comprising both deep and shallow roots; these deep roots can transport groundwater or soil water at depth into dry surface soils, a process referred to as hydraulic lift (HL) (Ryel et al., 2008; Lubczynski, 2009; Bleby et al., 2010). There is growing evidence that HR can have substantial hydrological and ecological impacts in some ecosystems, particularly those that are seasonally dry (e.g. Lee et al., 2005; Domec et al., 2010; Prieto et al., 2012).

Hydraulic lift can increase total ecosystem evapotranspiration and productivity via a number of processes. First, nutrient release and microbial activity are usually concentrated in the surface soil layers (~ top 20 cm or so) and can be strongly limited by moisture when soils are dry (e.g. Grierson & Adams, 2000; McIntyre et al., 2009). HL can ‘irrigate’ surface horizons in dry periods, thereby enhancing nutrient availability to the trees themselves as well as to other plants (Aanderud & Richards, 2009; Armas et al., 2012). HL also increases root longevity in the dry surface soils by maintaining root hydration (Bauerle et al., 2008), which in turn allows the tree to rapidly take advantage of resource pulses, such as those following rainfall events. Second, HL can increase the total root system water uptake capacity, increasing transpiration by as much as 30–50% in some cases (e.g. Caldwell & Richards, 1989; Scott et al., 2008; Domec et al., 2010). Third, HL in some cases supplies water to understorey plants with shallower root systems that are unable to access deeper

64 Redistribution of water through root systems during a flooding and drying cycle water directly (Hawkins et al., 2009; Pang et al., 2013). Consequently, HL by trees lining ephemeral waterways may play an important role in resource capture by the trees themselves, and in resource availability in the ecosystem as a whole.

Along the intermittent waterways that are common in dryland regions, the distribution of water can fluctuate considerably over time. The Pilbara region of northwest Australia is characterised by prolonged dry periods of up to several years, punctuated by often extreme episodic flooding from cyclone activity (Chapter 1). Groundwater is commonly present along waterways within ten metres of the ground surface, and is recharged during flooding then usually progressively recedes during dry conditions (Dogramaci et al., 2012). Given these conditions it might be expected that the root system of the phreatophytic riparian tree, Melaeluca argentea, would redistribute deeper groundwater to drier surface soil layers, and that this would maintain function of the surface roots during dry periods.

Patterns of sap flow in different roots can be used to demonstrate redistribution of water within the root system. HL manifests as acropetal flows (“reverse” flows away from the stem base) in shallow lateral roots, combined with basipetal flows (water uptake) in the descending roots with access to deeper water. In phreatophytes with constant access to groundwater, HL sap flow patterns commonly occur whenever surface soils are dry (e.g. Prieto et al., 2010). However, HL typically ceases if the surface soils are wet; for instance in Quercus fusiformis, Bumelia lanuginosa and Prosopis glandulosa trees accessing deep water, sap flow in surface lateral roots changed from acropetal to basipetal flows following rainfall, and the rates of basipetal flows in deep roots concurrently decreased (Bleby et al., 2010). If the deeper soils are not saturated, and surface soil moisture becomes greater than the moisture present at depth, hydraulic descent can occur with basipetal flows through surface roots and acropetal flows in deep roots (e.g. Hultine et al., 2004; Yu et al., 2013). Changes in the soil moisture content of surface soils can clearly influence root water fluxes, even in trees accessing deeper water supplies.

In this study, I used heat ratio method (HRM) probes to measure sap flows in lateral roots, roots descending to the groundwater, and in trunks of M. argentea trees on an intermittent creek, during 14 months following a major flooding event. I sought to examine the dynamics of root spatial flows over the cycle of wetting and drying, and to

65 Chapter Four assess the potential signficance of HL in the riparian ecosystems of the Pilbara. I hypothesised that M. argentea would utilise floodwater immediately post-flooding via shallow lateral roots. I also expected that as the surface soils subsequently dried out, water from deeper in the soil profile would be redistributed into surface layers through these shallow roots.

METHODS

Study site The study took place at an undisturbed section of Marillana Creek (22.72° S, 118.96° E), an intermittent waterway in the Pilbara region of northwest Australia (Chapter 2; Figure 2.1). Groundwater levels in the catchment fluctuate considerably between cyclonic recharge events. Water level records from a bore ~1 km from the study site for the period 1991– 2004 show levels ranged from 5.4 to 17.1 m below the surface both within and among years (Figure 4.1; data provided by Rio Tinto Pty Ltd). Following cyclonic rainfall and flooding in 1996, 1999 and 2002, the groundwater level rose by 7–8 m. Unfortunately there was no permanent monitoring bore adjacent to my site. However, cyclonic rainfall resulted in substantial river flows and replenished groundwater in the region in February 2009, with surface pools still apparent at the site when the study commenced in July 2009 (Figure 4.2). There were no further major rainfall events during 2009 or 2010. Based on personal observations at the site over the study period and general understanding of this and similar creeks in the area (Rio Tinto, unpublished data), ground water levels at the study site receded by between 4 and 7 m over the 14 month duration of the study.

Meteorological measurements As described in Chapter 2, a weather station (ET107; Campbell Scientific Inc., Logan, UT, USA) was installed at the top of the creek bank, approximately 300 m from the study trees. Rainfall, temperature, relative humidity, light intensity, wind speed, and wind direction were logged hourly.

Sap flow measurements Three M. argentea trees along the bank of the main channel of the creek were selected for sap flow measurements. The base of some large, downward angled (descending) roots were

66 Redistribution of water through root systems during a flooding and drying cycle

0 400

2 350

4 300

6 (mm) Rainfall 250

8 200 10 150 12

100 14 Depth to groundwater level (m) level groundwater Depth to

16 50

18 0 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Figure 4.1: Groundwater level fluctuations and rainfall ~1 km from the study site from 1991 to 2004.

(a) (b)

Figure 4.2: The main channel of Marillana Creek, beside the study trees. (a) The start of the study period (July 2009), five months after a flood, with surface pools still present.(b) The end of the study period (October 2010), twenty months after the flood event.

already exposed on the channel side of the trees due to flood erosion (Figure 4.3) and were excavated further where necessary. A small lateral root of each tree was also located by excavating soil on the bank side of the trees. Sap flow was measured via the heat ratio method with sensor units (HRM-30; ICT International, Armidale, NSW, Australia)

67 Chapter Four installed and operated as described in Chapter 2. Probes were inserted into a descending root and a lateral root in each tree, within 30 cm of their joining with trunk. An additional probe was also installed in the main trunk of each tree at 1.2 m height above the ground. For each probe set, data were obtained from a single measurement point, positioned near the centre of the sapwood band. The sizes of the instrumented roots and stems are shown in Table 4.1. Sap flow data were logged from 19th July 2009–12th September 2010, apart from periods when equipment was damaged by livestock (1st September–23rd October 2009, 20th June–23rd July 2010 and 26th July–1st September 2010).

Figure 4.3: Two of the study trees at Marillana Creek, showing the base of large decending roots instrumented with sap flow probes.The stem and root bases on the creek channel side of the trees cwere already exposed due to previous flood scouring.

Table 4.1: Sizes of the roots and stems fitted with sap flow sensors. Overbark diameter 2 (d; cm) and sapwood cross-sectional area (As; cm ) at the sap flow measurement positions.

d As Tree 1 stem 48.8 166.2 descending root 16.1 58.4 lateral root 5.3 8.55

Tree 2 stem 43.5 131.3 descending root 18.9 99.7 lateral root 3.9 5.11

Tree 3 stem 36.6 131.7 descending root 9.4 39.7 lateral root 3.1 3.30

68 Redistribution of water through root systems during a flooding and drying cycle

The calculation of heat pulse velocity (Vh) and its conversion to sap flow velocity (Vs) were performed as described in Chapter 2 (Burgess et al., 2001; Bleby et al., 2004). To correct for small misalignments of the probe needles, the zero flow baseline was determined for each root at the end of the study period, by completely severing the roots from the tree, while continuing to log sap flow for a further 12 hours. For the stem sap flow measurements, the zero flow baselines were set to the minimum value of Vh recorded on nights with low VPD and wind speed, after filtering of the datasets. Root acropetal flows are displayed as negative values, and basipetal flows as positive values.

To determine sapwood width (ws) and cross sectional area (As), cross sectional discs were cut from the roots through the probe insertion sites. The sapwood-heartwood boundaries were determined by the distinct colour transition, and where necessary by staining the wood with methyl orange (1% w/v in 4% v/v methanol), which stains the sapwood deep red and the heartwood yellow. The root discs were imaged in a flatbed scanner, and As measured from the images using Image J (Abramoff et al., 2004). Stem ws and As were determined from wood cores, as described in Chapter 3.

Statistical analysis Relationships between daily total sap flow and vapour pressure deficit (VPD) were assessed via linear regression for each tree separately. The effects of recent rainfall on flow rates, alone and in combination with VPD, were also assessed through linear models. Relationships are reported as significant where p<0.05. All analyses were performed in R version 2.13.0 (R Development Core Team, 2010).

RESULTS

Patterns of root sap flow with drying conditions The surface lateral roots of trees 1 and 2 functioned as expected; they contributed to water uptake at the beginning of the study while surface soils were wet, then as surface soils dried with increasing time since the flood, displayed net acropetal (negative) flows on most days (Figure 4.4a, b). The acropetal flows were often substantial, reaching a maximum of 1 m per day in tree 1 and 3.5 m per day in tree 2, approaching some of the basipetal velocities seen in the stems and descending roots. However, the due to the much smaller size of the surface laterals compared with the descending roots (Table 4.1), any water effluxed from 69 Chapter Four the lateral roots is likely to be only a small fraction of the total quantity of water taken up by the tree. Net flow rates in the surface laterals then declined to zero in tree 1 by 12 months after the flood and in tree 2 by 15 months after the flood.

The lateral root of tree 3 behaved somewhat differently; this root showed mostly basipetal flows, providing net contribution to water uptake (Figure 4.4c). The pattern and rates of flow in this root were similar to the flows in the stem and descending root of the same tree, during much of the study period. For instance, flows of up to 5 m day-1 were recorded in December 2009. However, unlike stems and descending roots, the rates of flow in the lateral of tree 3 declined over the study period and eventually reached zero flow by May 2010, approximately 15 months after the flooding event (Figure 4.4c). While this root arose at right angles to the base of the stem and appeared similar those instrumented on the other trees, the flow pattern suggests this root likely descended close to the saturated zone. The tree 3 lateral might therefore be better classified as an intermediate root, in between a deep descending root and a surface lateral.

The flow patterns in the large descending roots corresponded closely with those in the main stem of the same tree. Both diurnal and seasonal patterns of descending roots and stems largely followed the patterns of vapour pressure deficit (Figure 4.4; see also Chapter 2). The high rates of basipetal flow in the descending roots throughout the study (up to 7 m day-1 in tree 1, 3 m day-1 in tree 2 and 6 m day-1 in tree 3 during the highest VPD months) clearly demonstrate that these roots are the primary source for tree water uptake and have constant access to an abundant water supply, most likely groundwater.

Flows in the laterals of all three trees had decreased to close to zero, with little or no diurnal flow pattern evident, by 15 months after the flooding event (Figure 4.4). Zero flow was recorded from the lateral of tree 1 from mid February 2010 onward. The absorbing roots of the lateral of tree 1 may have died at that point, since flow in this root did not respond to any rainfall events after mid-February (root responses to rainfall are described in the next section below). However, the root wood appeared to be alive at the point of probe insertion, based on the moisture content and pale, fresh appearance of the sapwood when it was excised at the end of the study. Zero flow was recorded in the lateral roots of trees 2 and 3 from mid-May 2010, however both of these laterals responded to rainfall in

70 Redistribution of water through root systems during a flooding and drying cycle

7 (a) Tree 1 stem 6 descending root lateral root 5 4 3 (m day-1)

s 2 V 1 0 -1

(b) Tree 2 3 2 1 0 (m day-1) s -1 V -2 -3

6 (c) Tree 3 5 4 3 2 (m day-1)

s 1 V 0 -1 -2 8 (d) 7

15 6 VPD (kPa) 5 10 4 3 Rainfall (mm) 5 2 1 0 0 AUG SEP OCT NOV DEC JAN FEB MAR APR MAY

Figure 4.4: Daily sap flow velocity (Vs) in stems, descending roots and lateral roots of three Melaleuca argentea trees. Measurements were conducted over July 2009− June 2010, as the site dried out following flooding in early 2009. Panel(d) shows daily rainfall (bars) and minimum and maximum vapour pressure deficit (VPD; lines) over the same period. Negative root Vs values indicate net acropetal flows (away from the stem base).

71 Chapter Four

September, four months later, suggesting maintenance of at least some of the absorptive roots. The lateral of tree 3 was contributing to tree water uptake during most of the study, until May 2010 (Figure 4.4c) when its initial water source may have no longer been present, i.e. the water table may have receded beyond the range of this root.

The acropetal (negative) flows through the surface lateral roots of trees 1 and 2 showed clear diurnal patterns during the early and middle parts of the study period, but were frequently negative throughout most or all of the diurnal cycle (Figure 4.5). Maximum (or least negative) flows occurred between mid-morning and midday. The diurnal cycling in these surface laterals shows that the acropetal flows were inhibited but not entirely prevented by high transpiration rates in the canopy. However, on the scale of total daily flows, the flows through the surface lateral roots appeared not to be affected by VPD or transpiration (Figure 4.6). During the periods of active lateral root flows that were not influenced by recent rainfall (omitting periods of zero flow and the twenty days following rainfall events from the dataset), surface lateral root flows were in fact negatively correlated with VPD and stem sap flow, with greater acropetal flow occurring on days with higher VPD and rates of stem flow (Figure 4.6a). During the same time periods, sap flow in the stems and in the roots involved in water uptake were positively correlated with VPD, and with each other (Figure 4.6b–d). Similar results were obtained whether nocturnal, daytime, or total daily flows were considered (not shown). The apparent negative correlation of surface lateral root flow with VPD and stem flow is probably due to the fact that both VPD and acropetal flows ‘co-incidentally’ decreased over the course of the study period. The decline in lateral root flows may be related to the elapsed time since the last large (cyclonic) rainfall event, and the associated decline in surface soil moisture and fine root biomass over time. Thus the effects of soil moisture and root surface area may have masked the effects of VPD on surface lateral root flow over the longer term.

Root responses to rainfall While there were no major rainfall events during the study period, numerous rain showers of <40 mm occurred (Figure 4.4d). Responses in lateral root Vs were apparent following the three largest of these rainfall events (all >10 mm within a 7 day period; Figure 4.7). The first of the larger rainfall events was 37 mm over nine days, in November 2009, the second 17 mm in a single day in February 2010, and the third 32 mm over two days, in September

72 Redistribution of water through root systems during a flooding and drying cycle

40 (a) 30

20 (cm h-1)

s 10 V

-10 Figure 4.5: Diurnal patterns of sap (b) flow velocity (Vs) in stems (black), 10 descending roots (blue) and lateral roots (red) of Melaleuca argentea 0 trees. Data is shown from two different trees in panels (a) and (b) from (cm h-1)

s the 3rd and 4th January 2010 as an V -10 example, with vapour pressure deficit (VPD) and solar irradiance for -20 the same period shown in panel (c). Irradiance (W m-2) (W Irradiance (c) 1000 6 800 4 600 400 2 VPD (kPa) 200 0 0 0:00 12:00 0:00 12:00 0:00

2010. There were no responses in sap flow to any of the smaller showers that occurred during the study.

Following the first (November) rainfall event, the lateral root of tree 1 switched from net acropetal to basipetal flow, with positive daily net flows peaking at 1 m day-1, and positive flows throughout most of the diurnal cycle for approximately ten days after the event (Figure 4.4a, 4.7a). However, there was no detectable response in the lateral of tree 1 to either of the subsequent rainfall events; zero flows were recorded for this root for the remainder of the study (Figure 4.7b, c). The lateral root of tree 2 responded to all three events with basipetal flow during the days immediately following the rainfall of up to 4 cm h-1 and 3 m day-1, then flows gradually returned to net acropetal or zero (Figure 4.7a– c). The lateral of tree 3 was already displaying net positive flows at the time of the first rainfall event, and there was no detectable response to rainfall (Figure 4.7a). After the

73 Chapter Four

1 (a) shallow lateral roots (b) intermediate lateral root R2 = 0.52 5 0 4 -1 3

-2 Tree 1 2 Total daily V Tree 2 R2 = 0.33 -3 Tree 3 1 (m day-1)

s R2 = 0.61 0

-4 s

(c) descending roots (d) main stems (m day-1) 7 5 6 R2 = 0.38 4 Total daily V Total 5 R2 = 0.05 4 3 3 2 2 R2 = 0.60 1 1 R2 = 0.08 R2 = 0.18 R2 = 0.68 0 0 0123450123456 VPD VPD

Figure 4.6: Relationships between net daily sap flow velocity V( s) and average daily vapour pressure deficit (VPD). Flow rates in (a) Shallow lateral roots of trees 1 and 2, and (b) root of tree 3 intermediate between a shallow lateral and a deep descending root, (c) deep descending roots, and (d) main stems. Data were excluded from periods of zero flow activity in the lateral roots, and from periods affected by rainfall pulses. Note differing y-axis scales.

second (February) and third (September) rainfall events the lateral of tree 3 responded with initial acropetal flow immediately after rainfall, followed by a period of net water uptake (Figure 4.7b, c). The initial acropetal flows in the lateral of tree 3 were most rapid during the middle of the day, when the strong negative water potentials generated in the canopy by transpiration ought to prevent large acropetal flows. The explanation for these measurements is unknown, but the values are assumed to be erroneous, possibly generated by rainwater affecting the sensor unit.

In descending roots and stems, water uptake appeared to increase slightly following the February (second) rainfall event, (Figure 4.4, 4.7). For instance, in the descending root

-1 of tree 1 Vs initially increased from 1.4 to 3.6 m day , then over the next ten days declined

-1 to approximately 2 m day . In the descending root of tree 2 Vs increased from 1 to 1.7 m day-1 immediately after the rainfall, then declined to approximately 1.2 m day-1 over the next ten days. The increased uptake occurred mainly through longer duration of the

74 Redistribution of water through root systems during a flooding and drying cycle

Rainfall (mm) 8 4 0 16 12 131211109876 stems tree 1 tree 2 tree 3 rainfall Date (September 2010) Date ainfall is also displayed in the ainfall is also displayed stems descending roots descending Date (February- March 2010) March (February- Date 20 21 22 23 24 25 26 27 28 1 2 3 4 5 25242322212019181716151413121110987654 stems lateral rootslateral roots lateral roots lateral descending roots descending Vertical columns display responses to rainfall events in November 2009, in November rainfall events to responses display columns Vertical pulses. small rainfall to ) in response s Date (November 2009) (November Date (a)(d) (b) (e) (c) (f) (g) (h) 8 6 4 2 0 0 0

-2 -4

20 30 20 10

s (cm h-1) (cm V Figure 4.7: Changes in sap flow velocity ( V 4.7: Changes in sap flow Figure upper panels. Data from descending roots in Spetember 2010 is missing due to equipment failure. 2010 is missing due to in Spetember roots descending Data from upper panels. February 2010 and September 2010, in lateral roots (upper panels), descending roots (centre panels) and stems (lower panels). R (lower panels) and stems (centre roots (upper panels), descending roots 2010 and September 2010, in lateral February

75 Chapter Four

plateau phase of sap flow during the middle of the day, but also elevated rates of nocturnal flow, particularly in tree 3 where nocturnal minima increased from 4.5 to 10.4 cm h-1 in the descending root and from 7 to 15 cm h-1 in the stem (Figure 4.7e, g). There were no discernible responses in stem or descending root flows to either of the other two rainfall events (November 2009 and September 2010), apart from reduced flow rates while the rain was actually falling.

DISCUSSION

Water uptake occurred through the lateral roots of the M. argentea trees at the beginning of the study (July and August 2009) as hypothesised, when surface pools were present in the creek bed around the base of the study trees. Also as expected, substantial acropetal flows commenced in the surface lateral roots of two trees by November 2009, when surface pools had dried down. However, over the following eight months the magnitude of flows in the lateral roots diminished, as the site dried down further with increasing time since the last cyclonic recharge event. The acropetal flows clearly indicate that surface fine roots were maintained in dry surface soils, for a period of at least six months. However, the surface roots were not maintained indefinitely, and the internal transfer of water to these roots eventually ceased. During the period of acropetal flows and to some extent during the zero flow period, the lateral roots showed dramatic responses to relatively minor rainfall (15–40 mm), evidenced by a transitioning to net water uptake for the ten to twenty days following rainfall. Overall, this study clearly demonstrates the temporal variation in patterns of root sap flow that can occur during the multi-year flooding and drying cycles, and suggest that hydraulic lift could be an important process in the riparian ecosystems of the Pilbara.

The patterns of HL in M. argentea appear largely typical of those observed in other phreatophytes. Redistribution of water from deeper reservoirs to surface roots is very commonly observed when groundwater is accessible to deep roots while surface soils are relatively dry. The rates of acropetal flow in the surface laterals of M. argentea were similar to those seen in other studies, for instance, 1–3 cm hr-1 acropetal flows were reported in Eucalyptus camaldulensis (Burgess et al., 1998), Fraxinus velutina and Juglans major (Hultine et al., 2003b). HL generally ceases if groundwater becomes inaccessible (Brooksbank et al., 2011) or if surface soils are re-wet by rainfall (Millikin Ishikawa & Bledsoe, 2000; Hultine

76 Redistribution of water through root systems during a flooding and drying cycle et al., 2004; Oliveira et al., 2005; Bleby et al., 2010), and HL also ceased in M. argentea for short periods after rainfall pulses.

However, HL in M. argentea also declined and eventually ceased as the site became drier over the course of the study period, despite constant access to groundwater by the deeper roots and dry surface soils. As surface soil moisture declines from high to moderate soil water content, HL usually increases in trees with access to groundwater, due to the increasing water potential (Ψ) differential between the deep and shallow portions of the root system (e.g. Meinzer et al., 2004a; Brooks et al., 2006). However, HL can be inhibited if surface soils become very dry, due to declining soil conductivity, and increasing root xylem cavitation as water potentials become more negative, leading eventually to fine root mortality (Warren et al., 2007; Neumann & Cardon, 2012). In Retama sphaerocarpa shrubs accessing groundwater, HL was greatest at intermediate Ψsoil, decreasing as Ψsoil fell below -4 MPa (Prieto et al., 2010). Similarly, in Acacia tortillis with access to groundwater during the dry season in East Africa, HL ceased when surface soil Ψ fell below -5 MPa, and more HL occurred during a wetter year than in a particularly dry year (Ludwig et al., 2003). In the present study, saturation of surface soil layers by the cyclonic rainfall in early 2009 may have triggered surface fine root proliferation and surface water uptake, then as the soil moisture decreased, acropetal flows began to take place. The acropetal flows may have diminished and eventually ceased, as the soil dried beyond a critical threshold.

Another somewhat unusual finding in my study was that acropetal flows in the lateral roots were frequently observed throughout the diurnal cycle. This indicates that Ψ of the surface roots were able to “compete” with the canopy for water, even under high rates of upward stem flow. Acropetal root flows typically occur at night, facilitated by low transpiration rates and leaf Ψ closer to zero, but can occur at any time the Ψ of the dry root portions and/or surrounding soil becomes more negative than the canopy. A few studies have reported HR concurrent with daytime transpiration, including HL in Blepharocalix salicifolius in a Brazilian savanna (Scholz et al., 2002), and hydraulic descent in Juglans major in the Chihuahuan desert, Arizona (Hultine et al., 2003a), but it seems to be a fairly rare occurrence. Our data from M. argentea demonstrate that HL can continue throughout the diurnal cycle for prolonged periods, even under extreme VPD (at times exceeding 7 kPa) and high rates of transpiration. Leaf Ψ in M. argentea usually do not fall below -2.5

77 Chapter Four

MPa despite high VPD (Chapter 2, 6), due to their constant access to groundwater and low xylem cavitation resistance. These high Ψleaf, combined with high clay content in the surface soils (which can require high pressures to release stored water), most likely enabled the continuous and substantial hydraulic lift by the M. argentea trees in this study.

Even after long periods of zero flow in the surface lateral roots, immediate responses to rainfall were still observed in two of the M. argentea trees. The immediate response suggests that some of the fine roots were maintained, and therefore fine root cavitation and mortality is unlikely to have been the sole cause of the cessation of lateral root sap flows. Surface roots may have survived through internal transfer of water within the root system, but low soil hydraulic conductivity or loss of contact between roots and soil may have prevented measureable effluxes of water from the roots into the soil (Richards & Caldwell, 1987; Nobel & Cui, 1992). Alternatively, cell membrane water channels (aquaporins) appear to play a role in modulating HR, although the extent of their role is unknown (McElrone et al., 2007; Prieto et al., 2012). It is possible that efflux from the surface roots of M. argentea toward the end of the study might have been prevented by reduced quantities or gating of aquaporins, but this requires further investigation.

Hydraulic redistribution can sometimes benefit trees by increasing their total water use capacity; transfer of water into dry soil portions can then enable the entire root system to participate in uptake during high demand periods (Ryel et al., 2002; Domec et al., 2010). However, in other cases HR contributes little or nothing to the total plant water use (Hultine et al., 2003b). It cannot be discerned from sap flow data alone whether water was effluxed to the soil, or only rehydrated the root tissues themselves. In the present study, there was a total net loss of water from the two surface lateral roots over 8 months of measurements, and net losses on most days, with daytime flows rarely positive in the absence of rainfall inputs. If any water was effluxed to the surface soils in the present study, it was not recaptured during high demand periods, and so HR appears not to play a role in the water use strategy of M. argentea. Any water effluxed by the shallow roots of the M. argentea trees may instead have evaporated, or been consumed by understorey plants.

The surface laterals of M. argentea in the present study switched from net acropetal flows to net water uptake immediately following rainfall events, which is likely to facilitate opportunistic nutrient capture. Even if water is not actually effluxed to the soil, internal

78 Redistribution of water through root systems during a flooding and drying cycle transfer of water can maintain surface roots (e.g. Bauerle et al., 2008), enabling a rapid response to take advantage of any nutrient pulses. In dryland environments in general, release of nutrients can be limited by water availability, and precipitation typically occurs in short pulses which tend to trigger a pulse of nutrients at the surface (Ivans et al., 2003; Ryel et al., 2008). Similar patterns of lateral root flow and responses to rainfall have been observed in other semi-arid phreatophytes, including Prosopis velutina in Arizona (Hultine et al., 2004) and Populus euphratica in northwest China (Yu et al., 2013). HL during dry periods might therefore be important for nutrient capture in a wide range of semi-arid riparian trees, including M. argentea. Nonetheless, the extent to which HL actually improves plant nutrient status has not been well established, and warrants further study to determine whether HL might play a major role in nutrient uptake (Hodge, 2010; Prieto et al., 2012).

Hydraulic lift by M. argentea might prolong the periods for which surface roots and surface soil moisture are maintained in the Pilbara’s intermittent waterways. However, HL did not continue indefinitely in the present study, despite dry surface soils and access to groundwater, as the soil at the study site appeared to become too dry for HL to continue. The extent and duration of HL by M. argentea will most likely depend on the surface soil type, which can vary considerably in the Pilbara’s riparian zones, ranging from coarse gravels to finely textured clays (Johnson, 2004). Fine textured soils can typically reach more negative Ψsoil, so are likely to support higher rates of HL initially, but are also more likely to reach values of Ψsoil too low for HL to occur. Further analysis of fine root conductance and mortality, and of soil water content and matric potential surrounding surface roots, will clarify the extent to which HR by M. argentea contributes to whole ecosystem evapotranspiration throughout the Pilbara.

CHAPTER FIVE: Root hydraulic conductance and aquaporin abundance respond rapidly to partial root-zone drying events in a riparian Melaleuca species*

INTRODUCTION

Partial root-zone drying (PRD) occurs when a part of the root system dries out, while water remains available to other portions. Plant responses to PRD have been studied primarily in agricultural settings, in attempts to reduce irrigation while maintaining crop yields and quality (Davies et al., 2002; Kang & Zhang, 2004). However, heterogeneity of water availability around root systems is a far more general phenomenon, occurring widely in natural environments. River banks provide an extreme example of a heterogeneous environment, although most if not all plants may experience some degree of heterogeneity of water supply throughout their lifespan. Riparian trees can be subject to natural cycles of drying and re-wetting of portions of their root systems, especially in arid and semi-arid environments where stream flows are often intermittent or strongly seasonal (Dettinger & Diaz, 2000). This heterogeneous water availability around root systems, both spatially and temporally, can result in frequent PRD events.

Partial root-zone drying often results in partial stomatal closure, leading to increased water use efficiency. In some cases, stomatal closure appears to be due to a chemical signal from the drying roots, and not simply water deficit, since the stomata re-open when the dried root portion is severed (Gowing et al., 1990; Liu et al., 2001). Increased abscisic acid (ABA) content of the dried roots and shoot xylem sap and increased xylem sap pH have been observed during stomatal responses to PRD, and are considered likely signalling mechanisms (Davies et al., 2002; Dodd et al., 2008). However, the extent of the stomatal response to PRD varies considerably. For example, the stomatal conductance (gs) of Citrus aurantium subject to PRD was ~70% that of fully watered control plants (Zekri & Parsons,

1990), while in another study the gs of Acer pseudoplatanus saplings under PRD declined to ~30% of controls (Khalil & Grace, 1993). In other experiments using Lupinus cosentinii,

*Chapter published as: McLean EH, Ludwig M, Grierson PF (2011). Root hydraulic conductance and aquaporin abundance respond rapidly to partial root-zone drying events in a riparian Melaleuca species. New Phytologist 192: 664-675. 80 Partial rootzone drying in a riparian Melaleuca

Quercus robur, Betula pendula, Olea europaea, Phaseolus vulgaris and Persea americana, no stomatal response to PRD was observed; instead gs and rates of water use consistent with pre-PRD conditions were maintained (Gallardo et al., 1994; Fort et al., 1997; Fort et al., 1998; Centritto et al., 2005; Wakrim et al., 2005; Neuhaus et al., 2007). While differences in PRD response can be partly explained by the variable conditions used within each study, some of the variation may also reflect species differences (e.g. Croker et al., 1998). I hypothesise that differences among species in PRD reflect adaptations to environmental heterogeneity or homogeneity. If this hypothesis is correct, I would expect gs and water use to be unaffected by PRD in species commonly subjected to natural PRD events, including some riparian trees, provided adequate water is available to other parts of the root system. If a plant is able to maintain water uptake via the remaining hydrated zones, the best adaptive strategy may be to continue water use, and therefore photosynthesis and growth, at a constant rate.

The mechanisms by which plants maintain water uptake via only a portion of the root system are unclear. Even when total water use is reduced in response to PRD, the uptake rates of the wet roots can increase, thus compensating partly for reduced uptake from the dried parts of the root system (Lawlor, 1973; Zekri & Parsons, 1990; Kang et al., 2003; Mingo et al., 2004). The increased flow rates in the wet root portion during PRD may result solely from changes in hydrostatic gradients, or the roots might also adjust their hydraulic conductance (Lp) according to demand. Kang et al. (2003) found that Pyrus communis tree roots may have increased Lp in response to PRD, although this result was inferred from sap flux data and not measured directly. However, there are many examples of roots rapidly adjusting Lp to match demand under other circumstances. Partial root excision in wheat plants resulted in increased Lp of the remaining roots (Vysotskaya et al.,

2004). Lp is known to fluctuate diurnally, in synchrony with transpirational demand (Henzler et al., 1999; Beaudette et al., 2007; Vandeleur et al., 2009). Shading can also induce a reduction in fine root Lp (McElrone et al., 2007). If plants do increase Lp under PRD, then the adjustment must be rapid, and localised to the watered root portion.

Root water uptake occurs via parallel apoplastic, symplastic and transcellular pathways (Steudle, 1994). The relative conductance of these pathways depends heavily on the extent of hydrophobic apoplastic barriers and the nature of the gradients involved (i.e.,

81 Chapter Five hydrostatic or osmotic), but the apoplast is generally considered the path of least resistance

(Steudle & Peterson, 1998; Steudle, 2000). Rapid adjustment in apparent Lp can either result directly from changes in the gradients driving water flow, by changing the contribution of each flow pathway (Steudle, 2000), or through changes in the permeability of the root tissues themselves. Although anatomical changes can affect apoplastic Lp over longer timescales (Enstone et al., 2003), the only known means of rapid permeability adjustment is via transcellular Lp, through changes in the quantity or activity of root cell plasma membrane intrinsic protein (PIP) aquaporins. There is growing evidence that short- term changes in Lp may be generated primarily through aquaporins (Maurel et al., 2010).

Transient increases in root Lp during early drought may be due to increased PIP permeability (Hose et al., 2000), reduction of root Lp under anoxia can occur through proton gating of PIPs by cytosolic acidification (Tournaire-Roux et al., 2003), and the reduction and subsequent recovery of root Lp during chilling is associated with changes in expression and phosphorylation states of PIPs (Aroca et al., 2005). If rapid root Lp adjustments do occur in the wet root portion during PRD, aquaporins may be involved.

The riparian tree Melaleuca argentea is widespread across northern Australia, in locations where permanent shallow ground water is accessible. Following flood events, M. argentea commonly grows extensive aquatic root mats within temporary pools that form in the creek beds. Sap flux studies have shown that these aquatic roots can provide the majority of the water used by the tree, especially when adjacent bank soils are relatively dry (Graham, 2001). However, as the pools dry out, the aquatic roots die back and water uptake must occur via different parts of the root system. Given the natural frequency of drying events, I hypothesised that M. argentea would not respond to PRD with stomatal closure but would maintain water uptake by compensating with other parts of the root system. I also hypothesised that such a compensatory effect would involve rapid increases in root hydraulic conductance, which may be mediated by changes in aquaporin content or permeability. I tested these hypotheses with seedlings of M. argentea in a split-root system, mimicking the conditions frequently observed in the field with a portion of the roots in soil, and a portion in an aquatic pool. This design allowed us to test the effects of draining the aquatic pool on the shoot, and on the remaining hydrated part of the root system.

82 Partial rootzone drying in a riparian Melaleuca

MATERIALS & METHODS

Plant material and growth conditions Seedlings of Melaleuca argentea W.Fitzg. were collected from an intermittent creek in the semi-arid Pilbara region of Northwestern Australia (22.9114° S, 119.2139° E). Seedlings were planted into pots of river sand with slow release fertiliser (OsmocoteR Native Gardens, Scotts, Baulkam Hills, Australia), and grown for several months before transplanting into split-root pots for PRD experiments. Genetically identical plants were generated for Experiment 3 (below) by taking stem segment cuttings from a single seedling. Cuttings were grown for 9 months before transplanting into split-root pots. Seedlings of ~1 m height and 1–2 cm basal stem diameter were used in all experiments. Experiments took place between October 2008 and May 2010, in a glasshouse at the University of Western Australia (31.9828° S, 115.7997° E).

Experimental design The PRD responses of M. argentea were tested in three experiments. Experiment 1 assessed field collected seedlings for stomatal responses, tissue water status and root growth responses to PRD over a 14-day period. Water stress treatments were also conducted as comparisons to verify the sensitivity of our measurements. Four treatments were included, with five to six replicate plants in each; a PRD treatment, PRD with restricted water (PRD- RW), complete root drying (CRD) and two fully watered control treatments. Experiment 2 examined survival and recovery of the dried root portion after 28 days of PRD-RW treatment, to determine whether rapid root death may explain a lack of signalling from the drying roots. The control treatment consisted of six replicates, and the PRD-RW eight replicates, four of which were re-wet at 28 days for the recovery treatment. Finally,

Experiment 3 tested for rapid responses in root Lp and aquaporin expression. Genetically identical plants derived from cuttings were used to reduce variability among replicates. Plants were treated with PRD for either 24 or 48 h, or remained fully watered as controls.

Four replicate plants of each treatment were measured for root Lp, and a second set of four replicates harvested for aquaporin analysis. Treatments were applied across a three-week period to allow measurements and sample collection to be conducted on all plants between 11:00 and 14:00 hours. Conditions within the glasshouse for each experiment are shown in

83 Chapter Five

Table 5.1. Details of the treatments for all experiments are described at the end of this section.

Table 5.1: Glasshouse conditions during the partial root-zone drying experiments. Values give the total range of daily observations during the experimental periods. See text for descriptions of experiments.

Experiment 1 Experiment 2 Experiment 3 Maximum temperature (°C) 28–42 27–36 24–29 Minimum temperature (°C) 12–21 18–24 8–13 Maximum relative humidity (%) 45–71 nd 57–67 Minimum relative humidity (%) 26–49 nd 28–44 Maximum PAR (μmol m-2 s-1) 690–1451 1000–1600 800–1332 nd; not determined

Split-root pots consisting of two 15 litre compartments were used for Experiments 1 and 2, for Experiment 3 two 7 litre compartments were used, as the plants were required to be transportable. For all experiments, one compartment of each pot was fully sealed and filled with a diluted modified Hoagland’s nutrient solution (Hoagland & Arnon, 1950) to form the aquatic compartment, and the second compartment had drainage holes in the base and was filled with river sand to form the soil compartment. The moisture release properties of the river sand as determined in a pressure chamber are shown in Appendix B*. Approximately one third of the root system was placed in the aquatic compartment, with the remaining two thirds including the taproot in the soil compartment. Soil compartments were watered with ¼ strength modified Hoagland’s solution. The dilution of the Hoagland’s solution in the aquatic compartment was calculated from the field capacity of the river sand (~200 ml l-1), so that the quantity of nutrients per volume was equal between the two compartments. The aquatic compartments were replenished daily, and the solution changed every 7–10 days. Soil surfaces were covered with a 1 cm layer of white plastic beads, and aquatic compartments covered tightly with white plastic to minimise evaporation and light penetration. Plants were allowed to establish in the split-root pots for 3–5 weeks before the start of treatment, by which time there was copious new root growth in both compartments.

*Appendix B was included with the published version of this chapter as Online Supporting Information 84 Partial rootzone drying in a riparian Melaleuca

Plants were allocated randomly across treatments, and treatments were begun at midday, by draining the aquatic compartments. The soil compartments of PRD plants were watered twice daily with nutrient solution. For the PRD-RW treatment, the soil compartment was watered only once per day, which allowed the water content to fall below 80% (v/v) of field capacity. M. argentea is a riparian species that has highest rates of growth and transpiration under semi-saturated conditions; 80% of field capacity of river sand would thus restrict water for this species. For the CRD treatment, the aquatic compartment was drained, and watering of the soil compartment ceased. Two groups of controls were used, with soil compartments watered either once per day as controls for PRD-RW, or twice per day as controls for PRD. The moisture content of the soil compartments during Experiment 1 is shown in Appendix B. The aquatic compartments of control plants remained filled and maintained as described above.

Stomatal conductance and tissue water status

Stomatal conductance (gs) was measured on the abaxial surface of young, mature, exposed leaves with a leaf porometer (Decagon Devices, Pullman, WA, USA). At each sampling, measurements were taken on three leaves per plant then averaged to give a single value for each plant. Leaf water potential (Ψl) was measured with a Scholander-type pressure chamber (PMS Instruments, Albany, OR, USA). A single mature leaf from the upper half of the plant was cut and the balancing pressure determined immediately. Measurements of gs and Ψl were conducted in all experiments to verify consistency of plant response to PRD.

Leaf and fine root (< 2 mm diameter) water content were determined from 0.2–1 g samples collected mid-morning. Roots were washed clean of sand and surface dried thoroughly on paper towel. Samples were weighed immediately upon harvesting, then frozen in liquid nitrogen and freeze-dried. Samples were re-weighed and original moisture contents calculated, before processing of the samples for other analyses.

Plant water use Plant water uptake was measured as the change in pot weight over the time intervals between scheduled watering. Details of the method used to determine uptake from each compartment are provided in Appendix B. Water uptake is expressed relative to pre-

85 Chapter Five treatment values, to normalise for variation in plant size. Water uptake measurements were conducted in all experiments to verify consistency of plant response.

Root hydraulic conductance Hydraulic conductance of roots (denoted L prior to normalisation against root surface area) in both split-root pot compartments was measured with a High Pressure Flow Meter (HPFM; Dynamax Inc., Houston, TX, USA), using the “transient” method (Tyree et al., 1995). All measurements of L were conducted between 11:00 and 14:00. Portions of roots of diameter up to 10 mm were excavated in each soil compartment, the selected root cut under water, and the cut end attached to an HPFM coupling with a watertight seal. Transient pressure ramps were applied immediately. Water flow into the root and applied pressure were logged every 3 s while applied pressure was increased at a rate of 3–7 kPa s-1, from 0 to 550 kPa. At least three successive ramps were applied to each root. L was calculated as the slope of the linear region (between 200 and 550 kPa for most measurements) of the relationship between flow and applied pressure. Successive ramps applied to the same root gave similar values of L, and no consistent differences among samples in the direction of change between successive ramps were observed. The temperature of the laboratory during measurement periods remained between 15.5 and 16.5 °C.

The roots measured for L were separated from the rest of the root system and their fine root portions (<2 mm diameter) dried at 60 °C. The values of root L were normalised against fine root dry mass as a proxy for root surface area, to yield values of Lp. Correlation between fine root dry mass and surface area was established by scanning samples of fine roots and analysing with WinRhizo software (Regent Instruments Inc, Quebec City, Canada), prior to drying (Appendix B).

Xylem sap osmolarity Xylem sap was collected between 12:00 and 13:00 h on sampling days to assess the osmotic gradients from soil solution to xylem sap. Branchlets approximately 20 cm in length were cut from the seedlings and inserted into a Scholander-type pressure chamber. Bark was removed from the end 5 mm of the stump, to avoid contamination with phloem contents. The stem was then pressurised to no more than 0.4 MPa above the balancing pressure

86 Partial rootzone drying in a riparian Melaleuca using a single pressurisation, to avoid dilution of the sap with cellular water (Berger et al., 1994). The first droplet of xylem fluid was discarded and the cut surface blotted clean to remove cellular contents of wounded cells. A few drops of fluid were then allowed to flow into a vial, which was sealed and frozen at -80 °C until analysis. Osmolarity of 20 μl samples was determined by freezing point depression with a Fiske 210 micro-osmometer (Advanced Instruments Inc, Norwood, MA, USA).

Root growth Photographs of roots in the soil compartments of the split-root pots were taken through windows cut into the pot walls, to assess growth rates of the hydrated root portion. Windows were kept sealed between measurement timepoints to prevent roots from being exposed to light. Each photograph was taken of the same region, at the mid-point of each pot (50 cm depth), and cropped to a final image for analysis of 10–20 cm2. All fine roots visible in the images were traced in Adobe Illustrator (Adobe Systems Inc, San Jose, CA, USA), and the tracings analysed in WinRhizo (Regent Instruments Inc, Quebec City, Canada) to obtain root length measurements.

Protein extraction and immunoblotting Treatments were staggered so that all samples for protein analysis could be harvested on the same day, between 12:00 and 13:00 h. Frozen fine root tissue was ground under liquid nitrogen and mixed with protein extraction buffer (50 mM HEPES-KOH pH 7.1, 1 mM

EDTA, 1 mM dithiothreitol, 5 mM MgCl2, 1 mM PMSF, 1% polyvinylpolypyrrolidone (w/v)) using 200 μl of buffer per 100 mg of tissue powder. Samples were further homogenised in the buffer, then sodium dodecyl sulphate (SDS) added to 2% (w/v). The samples were heated to 95 °C for 5 min and clarified by centrifugation at 12 000 x g. Total protein concentration of the samples was determined using a bicinchoninic acid quantification kit (Pierce, Rockford, IL, USA), with bovine serum albumin as a standard, according to the manufacturer’s instructions.

Specificity of primary antibody binding was tested by immunoblot analysis (Appendix B). Protein samples were separated on 12% polyacrylamide gels (SDS-PAGE) with molecular mass standards (GE Healthcare, http://www.gelifesciences.com) and then transferred to Hybond C nitrocellulose membrane (GE Healthcare,

87 Chapter Five http://www.gelifesciences.com) by semi-dry electroblotting (Bio-Rad, http://www.bio- rad.com) for 1.5 h at 0.54 mA cm-2. Membranes were blocked for 1 h in 5% (w/v) skim milk powder in TBS-T (Tris-buffered saline pH 7.9 containing 0.1% (v/v) Tween 20), then incubated overnight at 4 °C with either anti-PIP1 (AS09487; Agrisera, Vännäs, Sweden) or anti-PIP2 (AS09491) at 1:1000 dilution in blocking buffer. Membranes were then washed in TBS-T, incubated with anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare, http://www.gelifesciences.com) at 1:3000 dilution in TBS-T for 1 h, and washed again in TBS-T with a final wash in TBS. The signal was detected with chemiluminescence substrate (ECL; Pierce, Rockford, IL, USA) according to the manufacturer’s instructions, and imaged with a LAS3000 LumiImager (Fujifilm Corp., Tokyo, Japan).

Immunoassays for quantification were performed as dot-blots (Heinicke et al., 1992). Samples were diluted to a uniform total protein concentration, and 2 μl of each applied to a grid on Hybond C nitrocellulose membrane (GE Healthcare, http://www.gelifesciences.com). One sample was used to create a standard curve, against which the relative signal intensity of other samples was measured. Antibody binding and detection were performed as described for the immunoblots. The dot-blots were imaged with dark and flat frame corrections (Gassmann et al., 2009), with several images taken across a range of exposure times. Densitometry was performed with ImageJ software (Abramoff et al., 2004). Three replicate dot-blots were quantified with each primary antibody, and the relative signal intensities averaged for each sample.

Statistical analysis Differences between control and treatment groups were analysed by repeated measures

ANOVA for time series data (gs, Ψl, tissue water content, water uptake and root growth), and by single-factor ANOVA at selected time points. Differences among treatments in Lp and PIP expression were analysed by single-factor ANOVA with Tukey’s HSD. Analysis was performed in Microsoft Excel 2003 and SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Differences are reported as significant where p<0.05.

88 Partial rootzone drying in a riparian Melaleuca

RESULTS

Stomatal conductance and leaf water status were unaffected by PRD

The gs of the PRD plants did not differ significantly from the controls at any measurement

point during 14 days of treatment (Figure 5.1a, c). However, gs began to decrease after about 24 h in the restricted water supply treatments, in which either less frequent watering allowed soil moisture content to fall below 80% of field capacity each day (PRD-RW), or watering of the soil ceased completely (CRD) (Figure 5.1b). After the initial decrease, gs of

the PRD-RW plants recovered to control levels at about day 7, while gs of the CRD plants continued to decrease (Figure 1d).

800 (a) (b) Control PRD PRD-RW 600 CRD

400

200

0 (c) (d) 800 (mmol m-2 s-1) s g 600

400

200

0 0122436480 2 4 6810 12 14 Time after treatment (h) Time after treatment (days)

Figure 5.1: Stomatal conductance (gs) of Melaleuca argentea seedlings under partial root- zone drying (PRD) and water stress treatments. (a) gs of control and PRD plants during the first 48 h of treatment. The PRD treatment was applied at midday (time zero, indicated by the arrow), by draining the aquatic portion of the root system. Black bars on the x-axis indicate dark periods. Values are means ± SE of five plants.(b) Midday gs of the same plants shown in (a), over 14 days of treatment. (c) gs of control plants, PRD plants with restricted water supply to the wet roots (PRD-RW) and completely droughted plants (CRD) during the first 48 h of treatment. (d) Midday gs of the same plants shown in (b), over 14 days of treatment. Values are means ± SE of six plants.

89 Chapter Five

Even with restricted water supply to the wet root portion (PRD-RW), the Ψl of PRD plants did not differ from the controls at any measurement point, with midday Ψl remaining between -0.7 and -1.5 MPa, and predawn Ψl between -0.05 and -0.5 MPa

(Figure 5.2a, b; only PRD-RW and control treatments shown). Predawn Ψl of the droughted (CRD) plants was significantly lower than controls by day 7 (Figure 5.2a), at which point soil water content in the CRD treatment had fallen to 38 ± 3% (v/v) of field capacity. However, midday Ψl of the CRD plants was lower than controls from 24 h of treatment Figure 5.2b). Leaf water content was also unaffected by PRD-RW, but decreased slightly in the CRD plants and was lower than controls by day 14 (Figure 5.2c).

0.0 (a)

(MPa) -0.5 leaf -1.0 Control PRD-RW Figure 5.2: Leaf water status of plants -1.5 CRD Predawn ψ Predawn under partial root-zone drying (PRD) with restricted water supply, and -0.5 (b) drought treatment. (a) Predawn leaf water potential (Ψl) of control, PRD -1.0 with restricted water supply to the wet (MPa) roots (PRD-RW) and completely leaf -1.5 droughted (CRD) plants. (b) Midday Ψl of control, PRD-RW and CRD plants. (c) -2.0 Leaf water content (LWC) of control, Midday ψ PRD-RW and CRD plants. Values are means ± standard error, n=6 for Ψl, (c) n=5 plants for leaf water content. 80 Where error bars are not visible they are smaller than the symbols. 60 LWC (%) LWC

40 0 0 2 4 6 8 10 12 14 Time after treatment (days)

Survival of the dried root portion Water content of fine roots in the hydrated zone remained the same as the control in PRD- RW plants, but was lower than the control in CRD plants at day 14 (Figure 5.3a). The drying aquatic roots rapidly lost water content relative to the control in both PRD-RW and

90 Partial rootzone drying in a riparian Melaleuca

CRD treatments (Figure 5.3b). However, at least some of the dry roots were able to survive for as long as four weeks because when re-wet after 28 days of PRD-RW, aquatic root water uptake resumed within 24 h, and returned to control levels within days (Appendix B). Water uptake by roots in the soil compartment concurrently began to decline, also returning to control levels.

(a) Figure 5.3: Fine root water content in 90 Melaleuca argentea seedlings during 80 partial root-zone drying (PRD). Seed- 70 lings had roots divided between an Control aquatic compartment and a soil com- 60 PRD-RW partment. The PRD treatment was CRD 50 applied by draining the aquatic compart- 0 ment, while the soil compartment (b) 100 remained well watered. (a) Moisture content of the soil roots of control, PRD 80 and completely droughted (CRD) plants. Root water content (%) content Root water 60 (b) Moisture content of roots in the 40 aquatic compartment of control, PRD 20 and CRD plants. All values are means ± 0 standard errors of four plants. Where 02468101214 error bars are not visible they are smaller Time after treatment (days) than the symbols.

Water uptake and Lp of the hydrated roots increased during PRD In untreated (control) plants eight weeks after transplanting, the dry mass of fine roots in the aquatic compartment was 32%, and in the soil compartment 67% (SE 2%, n=4) of the total fine root biomass. However, just prior to the start of treatments, water uptake via the aquatic roots was 58% (SE 5%, n=17) of the total plant water use. Therefore, draining the aquatic compartments to induce PRD dried one third of the root system, but amounted to removing nearly two thirds of the plants’ water supply.

Total water use by PRD plants did not differ significantly from controls at any measurement point during 14 days of treatment (Figure 5.4a). During the first 5 h after the aquatic compartments were drained, there was a significant decline in the total water uptake by PRD-RW plants relative to the control, but by 24 h total water uptake by PRD- RW plants had increased and did not differ significantly from controls for the remainder of

91 Chapter Five the experiment (Figure 5.4b). Water uptake by the CRD plants also declined during the first 5 hours of treatment, and continued to fall thereafter (Figure 5.4b).

(a) 120 Figure 5.4: Water uptake during partial root-zone drying (PRD). (a) 100 Change in the total water uptake by 80 control and PRD plants, relative to 60 pre-treatment water uptake. Values Control PRD-RW 40 are means ± standard error of five PRD CRD plants. (b) Change in total plant water 20 uptake relative to pre-treatment water 0 uptake, by control plants, PRD plants (b) 120 with restricted water supply to the remaining wet part of the root system 100 (PRD-RW), and completely droughted 80 plants (CRD). Values are means ± stan- Total water use (% of control) water Total 60 dard error of six plants. (c) Water 40 uptake from the hydrated compartment by control and PRD plants, 20 relative to pre-treatment uptake, 0 02468101214 during the first 48 h of treatment. The Time after treatment (days) PRD treatment was applied at midday (at time zero), and water uptake measured as the change in pot mass 500 (c) during 4–5 h time intervals. Values are 400 Control plotted at the mid-points of the mea- PRD 300 surement intervals, and the start and end points of the intervals are 200 indicated by vertical dashed lines. The 100 black bars on the x-axis indicate dark 0 periods. Values are means ± standard 012243648 Soil use (% of control) water error of four plants. Time after treatment (h)

Water uptake by roots in the hydrated soil compartment of the PRD plants did not differ from those of control plants during the first 5 hours of treatment. However, by 24 h, uptake by the hydrated root portion of the PRD plants had increased. By day 2, uptake was on average more than three-fold greater than the control plants’ uptake from that compartment (Figure 5.4c). Since the roots in the soil compartment of control plants were on average responsible for 42% of the total plant water uptake (see above), the trebling of their uptake rate under PRD enabled the total water uptake of the PRD plants to remain at control levels.

92 Partial rootzone drying in a riparian Melaleuca

Midday Lp did not differ significantly between roots growing in the aquatic and soil compartments in plants that did not experience drying (control treatment; Figure 5.5).

After 24 h of partial drying, the Lp of the wet soil roots was on average more than three-fold that of the control, the same magnitude of change as seen in the rates of water uptake by those roots. However, after 48 h of partial drying, Lp of the wet soil roots had returned to control levels. The Lp of the dried aquatic roots of PRD plants was not significantly affected by the treatment (Figure 5.5). Xylem sap osmolarity was also unchanged by the PRD treatment (Table 5.2), so the radial osmotic gradient across the roots (between the external root medium and the xylem) is unlikely to have differed between treatments.

50 Aquatic roots Soil roots Figure 5.5: Root hydraulic conduc- 40 b tance (Lp) during partial root-zone drying (PRD) treatment. Seedlings had 30 roots divided between two pot compartments, an aquatic and a soil com-

MPa-1 g-1 ) partment. The PRD treatment was

20 applied by draining the aquatic com- a partment, while the soil compartment (ml s-1 p

L remained well watered. Values are a a 10 means ± standard error of four plants. a a Different letters indicate a significant difference (p<0.05). 0 Control 24 h 48 h Treatment

Table 5.2: Osmolarity (mOsmol kg-1) of xylem fluid collected at midday from stems of partial root-zone dried (PRD) and control plants. Values are means ± SE of five plants.

Time after treatment Treatment 24 h 48 h 96 h PRD 4.8 ± 0.5 4.6 ± 0.4 3.4 ± 0.3 Control 4.6 ± 0.8 4.4 ± 0.5 5.0 ± 0.9

93 Chapter Five

Growth of fine roots in the hydrated root portion The rate of root length increase in the wet soil compartments as measured at transparent pot windows is shown in Figure 5.6. The difference between control and PRD root growth was not significant; however there is an overall trend in the data. Root growth rate in the wet compartments of PRD plants was slightly higher during the first two days of treatment, then returned to the growth rate seen in control plants.

Control 4 PRD Figure 5.6: Fine root growth rate in the watered root portion during 2 partial root-zone drying (PRD). Root growth rates measured in control and 0 PRD plants, at windows in the pot

walls. Values are means ± standard error of four plants. Root growth (mm cm-2 day-1) Root growth -2 0–1 1–2 2–4 4–7 7–14 Time interval (days after treatment)

Abundance of PIP1 and 2 aquaporins in the hydrated roots was altered by PRD The relative abundance of aquaporin proteins in fine root samples was determined by immunoassays using antibodies generated against conserved regions of Arabidopsis thaliana PIP1 or PIP2 sequences. Binding of the antibodies to M. argentea total protein samples was highly specific (Appendix B). After 24 h of PRD, there was a small but significant increase in the total PIP1 pool in the hydrated root portion, compared with the control treatment (Figure 5.7a). By 48 h of partial drying, PIP1 content in the hydrated roots was more variable, and was not significantly different from either the control or the 24 h level. Total PIP2 content did not differ from the control at 24 h of PRD treatment, but at 48 h of PRD PIP2 levels had declined relative to 24 h (Figure 5.7b).

DISCUSSION

Adaptive responses to heterogeneous water supply M. argentea responded to PRD with a rapid increase in water uptake by the remaining hydrated roots, which allowed leaf function and water status to remain unchanged. This

94 Partial rootzone drying in a riparian Melaleuca

2 (a) PIP1 (b) PIP2 b Figure 5.7: Abundance of PIP aquapo- ab rins in the watered root portion 1.5 during partial root-zone drying (PRD). ab Relative abundance of (a) PIP1 and (b) a a 1 PIP2 aquaporin subfamily proteins in b untreated (control) plants, and plants subjected to 24 and 48 h of PRD. Values 0.5

Relative abundance are means ± standard error of four plants. Different letters indicate a 0 significant difference (p<0.05) within Control 24 h 48 h Control 24 h 48 h each panel. Treatment

manner of responding to PRD events most likely represents an adaptation to the highly variable environments in which M. argentea is found. I used a well-watered, sandy substrate in this study to minimise the influence of changes in soil conductance that occurs with soil drying, particularly in clays (Nobel & Cui, 1992). Our experimental soil conditions are also consistent with those in which M. argentea naturally occurs. However, the lower gs observed when water supply was restricted to the wet root portion of M. argentea clearly shows how the wet roots were unable to compensate below a certain threshold of soil water availability. Many agricultural PRD experiments use deficit rates of irrigation (below the evapotranspiration rate of untreated plants) and have in fact measured ‘drought’ response, making it difficult to determine to what extent the plants are responding to hydraulic or non-hydraulic signals. It is likely that more species than currently recognised would in fact fully compensate for PRD events if soil conductance and water content around the wet root portion were not limiting.

In cases where leaf responses to PRD are not observed, the plant is clearly displaying root-level compensatory adjustments. Drying a portion of the roots of M. argentea seedlings induced an increased Lp in the remaining hydrated roots within 24 h, which may have mediated the rapid increase in the rate of water uptake by those roots. An increase in fine root growth in the wet soil compartment may have provided longer-term compensation. Observations of root growth in other species during PRD have yielded variable findings; however, increased wet-zone root growth under PRD was observed in oak saplings and peach trees, along with little or no gs response (Fort et al., 1997; Goldhammer et al., 2002; Abrisqueta et al., 2008). Partial root-zone drying also weakly stimulated root growth in

95 Chapter Five

Citrus aurantium, corresponding with a mild stomatal response (Zekri & Parsons, 1990;

Mingo et al., 2004). Increased root Lp and fine root growth in wet zones may be important means by which species adapted to soil moisture heterogeneity compensate for partial drying events.

Production of a signal for stomatal closure by the dried roots (most likely ABA, or changes in xylem sap pH that then induce changes in ABA concentrations or compartmental distribution within leaf cells) appears to be the primary PRD response in some plants. Observations of stomatal re-opening after severing of dried roots demonstrate that the wet roots were capable of compensating, but were prevented from doing so through signalling of stomatal closure (Gowing et al., 1990; Croker et al., 1998; Liu et al., 2001). An increase in ABA is frequently, although not always, detectable in the drying roots, xylem sap and leaves during stomatal responses to PRD (e.g., Neales et al., 1989; Liu et al., 2001). In contrast, in PRD studies where no stomatal response was detected, ABA content did not change, suggesting that in some cases the signal is simply not produced by the drying roots (Gallardo et al., 1994; Fort et al., 1997; Fort et al., 1998; Wakrim et al., 2005). Consequently, while I did not measure ABA*, I argue it is likely that in M. argentea seedlings under PRD, the dry roots did not generate any changes in ABA. Species in which the absence of stomatal PRD responses has been observed still produce an ABA efflux from roots under drought, so do not lack the ability to produce or respond to ABA (Gallardo et al., 1994; Fort et al., 1997; Fort et al., 1998). Rather, it seems that in some species, the extent of root ABA signalling is dependent upon the integrated soil moisture across the whole root system, or on interactions with shoot responses to water stress. The response of a species to soil moisture heterogeneity most likely has implications for the way in which it responds to water stress.

The variations in PRD response that have been observed among plants may in part be due to adaptations to soil moisture heterogeneity. The numerous crop species with strong leaf-level PRD responses may have adapted to relatively homogeneous soil conditions during their domestication. In homogeneous soil environments, localised soil drying might indicate imminent drought, making root signalling to conserve soil water a

Measurements of ABA were completed subsequent to the publication of this chapter, which confirmed our expectations. The ABA results are presented in Appendix C. 96 Partial rootzone drying in a riparian Melaleuca useful adaptation. Stomatal conductance and growth of cultivated lettuce (Lactuca sativa) declined when upper soil layers were dried, while a wild lettuce (L. serriola) native to spatially heterogeneous soil habitats did not show any leaf level responses to the treatment (Gallardo et al., 1996). It might also be expected that large plants would encounter greater heterogeneity simply due to the scale of their root systems, and indeed many of the species reported to respond weakly or not at all to PRD are trees. Croker et al. (1998) observed a weak, negative correlation between PRD stomatal response and drought sensitivity in six temperate tree species. Drier environments tend to be more heterogeneous in a range of soil properties (Jackson & Caldwell, 1993; North & Nobel, 2000), so adaptation to soil moisture heterogeneity might account for the weaker PRD stomatal responses in some drought tolerant species. Contrary to our hypothesis, Salix dasyclados, the only other riparian tree examined under PRD to date, showed a 30–40% reduction in gs during PRD (Liu et al., 2001). However, S. dasyclados is native to regions where waterways are considerably more consistent than in northern Australia, the region where M. argentea occurs. More data are needed on the PRD responses of species with well-described habitats before any firm conclusions can be made as to the adaptive nature of the responses; however, our study with M. argentea provides a clear example.

Adjustments in root hydraulic conductance during PRD Our results show that increased water uptake by the hydrated roots during PRD, rather than simply being a passive process, may involve an active change in the Lp of the hydrated roots. Adjustments of root Lp could occur in any species that compensates fully or partly for partial drying events via the hydrated roots. In our study, the hydrated root portion was required to triple its previous water uptake in order to fully compensate for the partial drying. Notably the measured Lp of these roots was also approximately three times that of the control, suggesting a causal relationship between the increase in water uptake and the increase in Lp. However the Lp of the hydrated roots then returned to control values after 48 h, while the water uptake rate remained high. Increased fine root growth during the first few days of PRD may have contributed enough to water uptake capacity to allow the decline in Lp. Root growth in the soil compartment had certainly occurred by 48 h, and although not significantly greater in the PRD plants than the controls, our measurements were made via transparent windows which do not always accurately reflect total root

97 Chapter Five growth (Glinski et al., 1993). Water use from the hydrated compartment increased within hours of partial drying, but total water uptake was not identical to control levels until day 4, suggesting some gradual continuing adjustment, which would be consistent with the growth of new roots.

Changes in aquaporin content were detected in the hydrated root portion, which corresponded with the measured root Lp. PIP1 abundance increased after 24 h of PRD, followed by a decline in PIP2s and possibly also PIP1s by 48 h. The magnitude of change in PIP levels was small, but could still be sufficient to account for the observed changes in

Lp. Individual PIP isoforms vary in their location, conductance to water and response to a given treatment, suggesting that each may have a slightly different biological function (Jang et al., 2004; Hachez et al., 2006; Galmés et al., 2007). Since the PIP antibodies used in this study will have detected many isoforms, the signal of change from one or a few isoforms will be diluted within the total pool. The identity, location within the root, and the proportion of proteins in un-gated states in our study are all unknown, and await further examination. Nonetheless, the small change in total PIP protein levels observed in this study could be physiologically significant, and the correspondence with Lp suggests that changes in PIP levels could be involved in root responses to PRD.

PIP2s generally have high water permeability, while PIP1s often have little or no measurable permeability (Chaumont et al., 2000; Kaldenhoff et al., 2008). However, interaction between PIP1 and PIP2 isoforms in the formation of hetero-tetramers can dramatically increase total permeability (Fetter et al., 2004; Mahdieh et al., 2008;

Vandeleur et al., 2009). A correlation between root Lp and PIP1 levels with no change in PIP2 was observed in bean and grapevine. ABA application to the roots of bean (Phaseolus vulgaris) plants increased both Lp and the PIP1 protein pool (Aroca et al., 2006). In grapevine (Vitis vinifera), VvPIP1;1 is proposed to regulate water transport to match demand diurnally and under water stress via its interaction with VvPIP2;1, which did not change in transcript levels (Vandeleur et al., 2009). A similar mechanism may operate in M. argentea during response to PRD, with PIP1 and 2 isoforms interacting to produce the observed effects on Lp. The difference between the PIP1 and PIP2 subfamilies in the direction and timing of their response hints at the complexity that may exist at the molecular level in root responses to PRD.

98 Partial rootzone drying in a riparian Melaleuca

In our study, stomata of M. argentea did not close during PRD and there was increased water uptake from the wet roots. Lp and aquaporin adjustments in the wet roots under PRD raises the question of how these responses are signalled. Although ABA can upregulate Lp and PIP abundance (Mariaux et al., 1998; Hose et al., 2000; Aroca et al., 2006), it is unlikely to be the signalling mechanism in this case since ABA concentrations appear not to change in wet roots under PRD, whether or not stomatal responses occur (e.g. Zhang et al., 1987; Fort et al., 1997; Fort et al., 1998). PIP abundance and gating can be regulated by several other stimuli, including Ca+, pH and pressure pulses (Wan et al., 2004; Luu & Maurel, 2005). Direct hydraulic ‘signalling’ may be the dominant mechanism of controlling water use, particularly in woody plants (Brodribb, 2009). Consequently, the hydraulic pressure effects of shoot demand may act directly to modulate aquaporin levels and Lp in the hydrated roots of M. argentea.

The results presented here indicate a possible role for aquaporins in root water uptake under a sudden increase in transpirational demand. The widely accepted composite transport model (CTM) predicts that under the strong hydrostatic gradients produced by transpiration, water flow should be largely apoplastic, with aquaporin-mediated flow playing a lesser role (Steudle, 2000). Under the CTM, observations of rapid adjustment of root Lp to match transpirational demand are explained by changes in the apoplastic contribution as a direct result of gradient changes (Steudle & Frensch, 1996; Steudle & Peterson, 1998). However, as Vandeleur et al. (2009) argue, gradient induced changes in flow pathways cannot account for differences in Lp as measured under consistent gradients and conditions. Transient pressure ramps by HPFM apply the same hydrostatic pressures in each measurement, and the osmotic gradient appears consistent since xylem sap osmolarity and applied nutrient solutions did not differ between PRD and control treatments. HPFM measurements can themselves affect the osmotic gradients within the root, since water is injected into the root xylem, potentially polarising solutes (Knipfer et al., 2007). However, the consistent Lp observed between successive pressure ramps in our experiment indicates that the degree of polarisation caused by measurements was likely to be small, and similar among treatments. If the driving forces are the same, the remaining possibility to explain a difference in Lp is a change in the permeability of the root tissues themselves. The rapid but not immediate response in the rate of root water uptake in our

99 Chapter Five study also argues against an entirely passive mode of compensation, but rather is consistent with a process requiring several hours for its induction. Adjustments in aquaporin quantity or permeability are the only currently known mechanisms by which rapid and reversible changes in tissue permeability can occur (Maurel et al., 2010), and may explain our observations; however, I cannot entirely rule out other explanations.

There are several other lines of evidence suggesting that aquaporins may at times play a larger role in root water uptake than is usually supposed. In some plants, purely apoplastic root radial flow appears not to be possible, requiring water to enter cell-to-cell pathways (Knipfer & Fricke, 2010). Numerous studies using aquaporin inhibitors point to a substantial contribution of aquaporins to total root Lp in a wide range of species and conditions (Javot & Maurel, 2002). Transgenic approaches have also revealed significant aquaporin contributions, with one Arabidopsis isoform even appearing to increase hydrostatic root Lp, but not osmotic Lp (Martre et al., 2002; Siefritz et al., 2002; Javot et al., 2003; Postaire et al., 2010). The levels of aquaporin transcripts in roots also peak at midday, corresponding with the time of highest transpiration (Henzler et al., 1999; Beaudette et al., 2007; Vandeleur et al., 2009). Whether aquaporins are in general important during high rates of water uptake is not yet clear, but evidence is growing that they play a significant part.

Conclusions Plant responses to PRD events vary enormously as recorded in the literature; some of the variation may be due to species adaptations to soil moisture heterogeneity or homogeneity. Species appear to fall onto a continuum in PRD response, from those responding primarily at leaf level, to those responding mainly via root-level compensation. Some degree of compensatory increase in water uptake from the wet-zone during PRD occurs in many species, and our results with M. argentea show that this may involve short-term increases in root Lp, possibly via changes in aquaporin levels. Our findings form part a growing body of evidence that aquaporin-mediated water flow may be significant when root water uptake rates are high.

100

CHAPTER SIX: Morphological and physiological adjustments across a hydrologic gradient by seedlings of a semi-arid riparian Melaleuca

INTRODUCTION

The availability of water and the depth to the underlying water table in and around dryland streams can vary considerably, both spatially and temporally (Lite et al., 2005; Larned et al., 2010). Recruitment of riparian trees in such environments generally occurs only after flood events, since the flood waters disperse propagules and deposit sediments providing the most optimal conditions for germination, establishment and survival (Scott et al., 1997; Pettit & Froend, 2001; Friedman & Lee, 2002; Stromberg et al., 2010a). The few years after germination is a critical phase, as the availability of water during this establishment period can be the major determinant of survival to maturity (Horton & Clark, 2001; Dixon, 2003). In intermittent waterways, water availability typically declines post-recruitment due to the recession of surface and ground water (Donovan & Ehleringer, 1991; Rood et al., 2003). Riparian tree seedlings must therefore grow rapidly during the initial saturated conditions, to quickly establish a large and deep enough root system to follow the water table downward as the system dries (Mahoney & Rood, 1998).

The vulnerability of riparian tree recruitment is made most apparent where water regimes have been altered by human activities. For instance, dams on the Kootenai river in western North America have reduced the recruitment rate of Populus species, by both altering channel hydraulics and bed sediment distribution, as well as increasing the rate of water table recession during seedling establishment periods (Burke et al., 2009). Riparian and wetland ecosystems appear particularly susceptible to abrupt changes in community composition, occurring at particular thresholds of change to the water regime (Ridolfi et al., 2006). Such threshold shifts were observed in the semi-arid riparian systems of Arizona, USA; when the depth to groundwater and surface flow frequency fell below particular values, recruitment of native Salix and Populus seedlings was reduced, and replaced by exotic Tamarix ramosissima seedlings (Lite & Stromberg, 2005; Merritt & Poff, 2010). These changes in recruitment and survival patterns are likely in turn to have further ecological effects. Understanding the tolerance of trees, particularly at the seedling stage,

101 Chapter Six across the range of water availability conditions is important in understanding a species ability to establish in the riparian environment, and the likely responses to anthropogenic changes to the water regime.

Plant responses to water availability can also occur in a threshold pattern with decreasing water availability, related to thresholds of physiological stress (Hacke & Sauter, 1996; Nardini et al., 2001; Froend & Drake, 2006). Soil waterlogging and drought create different types of stresses to the root system, and thus require different root responses to tolerate the stress. However, leaf-level responses to drought and waterlogging can be similar since the shoot can effectively experience water stress under both conditions due to a lack of water in one case, and impaired root functioning in the other. Under waterlogged conditions the major stress is usually lack of oxygen in the root zone, and associated changes in soil chemistry (Kozlowski, 1997; Pezeshki, 2001). The major adaptive traits associated with waterlogging tolerance are production of shallow and adventitious roots, which have better access to oxygen at the surface of the flood water, as well as aerenchyma formation, which allows oxygen transport within the root system (Justin & Armstrong, 1987; Kozlowski, 1997; Pimenta et al., 1998). In contrast, in dry soils, the production of a deeper root system and allocation of greater biomass to root growth are frequently adaptive (e.g. Leiva & Fernandez-Ales, 1998; Bell & Sultan, 1999). Suberisation of the root endodermis is often observed in dry conditions, and can provide a barrier to water loss from the root into the soil (e.g. North & Nobel, 1998). These adaptations to soil drought and waterlogging would be expected to increase the survival and growth rates of tree seedlings that display them in response to the conditions in which they are growing.

There appears to be a tradeoff between waterlogging and drought tolerance, at least for angiosperm trees and shrubs, which may be due to the different adaptive responses required (Niinemets & Valladares, 2006). For example, Muehlenbeckia florulenta, a shrub common to semi-arid floodplains of Australia, is more tolerant of drying than flooding, and seedlings correspondingly showed plastic adjustments in many traits during drought, while under flooded conditions seedling growth ceased and morphological changes were not observed (Capon et al., 2009). Most tree species are vulnerable to waterlogging, and some gain a tolerant status only because they survive flooded periods in a dormant state (Parolin & Wittmann, 2010). However, species that must not only persist but also grow rapidly

102 Seedling responses across a hydrologic gradient in a riparian Melaleuca during flooded periods must possess particularly strong adaptations to waterlogging. An ability to achieve high rates of growth and transpiration in wet conditions is often associated with poorer drought tolerance. For example, seedlings of Salix viminalis and Salix triandra had higher mortality rates during a three week drought, but higher rates of growth and transpiration when well watered, compared with more drought tolerant Salix alba and Populus nigra (Van Splunder et al., 1996). Riparian species can be subject to both drought and flooding, particularly at the seedling stage. However, adaptive traits enabling rapid growth in waterlogged or moist conditions following floods, necessary for establishment, may preclude a strong adaptation to drought stresses.

Melaleuca argentea is a tree species common to riparian habitats across Northern Australia, including the Pilbara region. M. argentea, like many other Melaleuca tree species, occurs in the wettest parts of the riparian zone (Masini, 1988) and might therefore be expected to be tolerant of flooding, but sensitive to drought. In Pilbara waterways undergoing groundwater drawdown (Chapter 1), M. argentea is likely to be one of the earliest and most severely affected species, and consequently may be a useful ‘indicator’ species for broader ecological monitoring of these areas and for setting thresholds for ecological water requirements. However, the adaptive characteristics that enable M. argentea seedlings to both establish and survive remain poorly described. In the previous chapter (Chapter 5) I showed that the roots of M. argentea seedlings can display rapid responses to changes in water availability around the root system to maintain high rates of water uptake. To further elucidate the tolerance and responses of seedlings across the full range of soil water conditions, I grew seedlings under a range of water availability treatments, from complete waterlogging to severe drought, in controlled glasshouse experiments. Growth rates were evaluated as a measure of seedling performance, and the likelihood of successful establishment under those conditions. A range of leaf and root physiological and morphological characteristics, considered to be adaptive responses to waterlogging and/or drought, were also examined across the gradient of treatments. I then compared the functional responses between waterlogging and drought conditions, and assessed thresholds of water availability for various responses, and sequence in which they occurred across the gradient. I hypothesised that (i) M. argentea seedlings would show greater tolerance and morphological and physiological response during waterlogging than

103 Chapter Six under drought stress, and (ii) that seedling physiological performance and growth rate would decline in threshold-type patterns with decreasing water availability.

MATERIALS & METHODS

Plant material and growth conditions Melaleuca argentea W. Fitzg. seedlings were collected from the streambed of Weeli Wolli Creek in the semi-arid Pilbara region of Western Australia (22.91° S, 119.21° E), and transferred to a glasshouse at the University of Western Australia (31.985° S, 115.820° E). Seedlings were planted into 5 litre pots of river sand with slow release fertiliser mixed through (3 ml l-1; OsmocoteR Native Gardens, Scotts, Baulkam Hills, Australia), and allowed to recover from transplanting for at least six weeks under well watered, drained conditions. Experiment 1 was conducted in April–May 2008, and Experiment 2 in March– May 2010. Glasshouse conditions during the experimental periods are provided in Table 6.1.

Table 6.1: Glasshouse conditions during the experimental periods. Values give the total range of daily observations; see text for descriptions of experiments.

Experiment 1 Experiment 2 Maximum temperature (°C) 19 –33 28–42 Minimum temperature (°C) 8 –18 12–21 Maximum relative humidity (%) 64 –85 45–71 Minimum relative humidity (%) 24 –69 26–49 Maximum PAR (μmol m-2 s-1) 264–1206 552–1161

Experimental design Experiment 1 (hydrologic gradient) characterised morphological and physiological responses across a range of soil water content. Four treatments were established, 100% saturation (fully waterlogged), 80% of sand saturation by volume (partially waterlogged), 50% of saturation (close to field capacity) and 20% of saturation (low water), with six replicates (the moisture release properties of the river sand used in this study are provided in Appendix B, Table B-1). Seedlings were 40–64 cm in height, and were divided across the four treatments so that each treatment contained a similar range of seedling sizes. To impose the treatments, the pots were enclosed in plastic bags, which were fastened around 104 Seedling responses across a hydrologic gradient in a riparian Melaleuca the base of the stem to prevent water loss by evaporation, and each pot placed inside a larger container. Plants in the 20%, 50% and 80% saturation treatments were watered to weight every 48 h. For the 100% saturation treatment, the outer container was filled with water, maintained within 2 cm of the sand surface. Plants were positioned randomly on the glasshouse benches, and rotated weekly. Treatments were maintained for eight weeks, after which final measurements were taken and plants were harvested for biomass measurements.

Experiment 2 subjected plants to progressive drought; watering of seedlings ceased, and sand was allowed to dry out, while control plants remained watered to field capacity. For the first seven days of treatment, the soil water content of the droughted plants was adjusted by weight so that soil water content remained approximately equal among individuals. After the first week, soil was allowed to dry out naturally. The droughted plants were re-watered after eight weeks of treatment to ascertain survival.

Plant growth and biomass Plant height was measured weekly by running a measuring tape along the longest stem from base to apical meristem. Leaf elongation was determined from measurements of the distance between tip and the junction of the petiole with the stem taken at 24 h intervals with digital calipers of three immature leaves per plant on the apical stem. Leaf elongation rates were then averaged to give a single value per plant for each time point. Stem basal diameter was determined with digital calipers, as the average of two measurements taken per stem at 90° angles. After eight weeks, all plants from Experiment 1 were harvested and the leaves, stems and washed roots of each plant dried at 60 °C and weighed for total biomass and biomass allocation ratios. Leaf area of each plant was measured with an optical leaf area meter (LI-3100C; LI-COR, Lincoln, NE, USA) before drying. In Experiment 2 (progressive drought), cardboard sheets were taped around the base of each pot in a cone shape to catch fallen leaves. Leaves that had fallen from the plant or were visibly dead (entire leaf dull coloured, shrivelled and brittle) were removed every 1–2 days, dried at 60 °C and weighed to measure the rate of leaf shedding.

Morphology and anatomy of leaves and roots Leaf morphological characteristics were measured in Experiment 1 at the end of the eight weeks treatment, on young, fully expanded leaves. Terminal leaves were tagged at the start

105 Chapter Six of treatment to ensure that leaves collected for analysis had developed during the treatment period. Specific leaf area (SLA) was calculated from the area of fully hydrated leaves measured in an optical leaf area meter (LI-COR, Lincoln, NE, USA), and their mass after drying at 60 °C. Thickness of leaves and of individual tissue layers were measured by taking transverse sections by hand across the centre of fresh leaves. Leaf sections were imaged through a light microscope fitted for epifluorescence (Zeiss, Oberkochen, Germany), under both brightfield and ultraviolet (UV) illumination (example images are shown in Appendix D). Measurements were taken from the resulting images using ImageJ software (Abramoff et al., 2004), at ~2 mm from the midvein on each side; the cuticle was distinguishable by its blue fluorescence under UV light, and other tissues were clearly visible under both types of illumination. The cuticle, epidermis and pallisade mesophyll layers were not consistently thicker on the adaxial or abaxial side, so for each tissue type the values were averaged to give a single value per leaf. Leaf density was calculated by dividing leaf specific dry mass (mass per leaf surface area) by the leaf thickness, to yield values of dry mass per volume of fresh tissue.

Stomatal and leaf trichome density were determined by photographing leaf surface epifluorescence under UV light. Images were taken of both adaxial and abaxial surfaces near the centre of each leaf, for 3 fields of view per leaf surface. The guard cells and trichomes fluoresced blue against the red chlorophyll background (Appendix D), enabling the numbers of stomata and trichomes in each image to be counted with ImageJ (Abramoff et al., 2004). Stomatal and trichome densities were determined in the same manner for an additional series of leaves collected along the main stems of four separate seedlings, to assess effects of developmental age.

At the end of the treatment period, pots were cut open and the soil brushed away where necessary, to examine the macro-morphological characteristics of the root systems. Root endodermal suberisation and aerenchyma were assessed in roots of diameter 0.6–1.6 mm. Transverse sections were taken by hand at 4–6 cm from the root tip. Sections were photographed under UV illumination through a light microscope fitted for epifluorescence (Zeiss, Oberkochen, Germany) to view the endodermis, which fluoresced blue. As verification, several sections were also stained with Sudan red III or Sudan black B (lipophilic dyes); the stained portions of the endodermis corresponded to the portions

106 Seedling responses across a hydrologic gradient in a riparian Melaleuca fluorescing blue under ultraviolet (not shown). The extent of suberisation was measured as the proportion of the endodermal ring showing fluorescence; up to a value of 200% (two complete suberised rings, the maximum observed). Example images are shown in Appendix D. Aerenchyma was measured as the area occupied by air spaces as a proportion of the total cortical area (Sojka, 1988), in bright field images of the same sections.

Plant physiological status

Shoot water potential (Ψshoot) was determined with a Scholander-type pressure chamber

(PMS instruments, Albany, OR, USA). In Experiment 1, Ψshoot was measured at midday at the end of the eight week treatment period; the top of the main stem with 4–6 leaves attached was cut from each seedling, and the balance pressure determined immediately. In

Experiment 2, Ψshoot was determined both predawn and midday at time points throughout the eight week drought period; measurements were conducted on individual leaves (Ψleaf) until the majority of leaves had been shed by the droughted plants, after which point Ψstem was measured for terminal branchlets in both the drought and control treatments. Leaf relative water content (RWC) was determined in Experiment 1 at the end of the treatment. Young, fully expanded leaves were cut between 11:00 and 12:00, weighed immediately for fresh weight, then immersed in water for 24 h, surface dried and weighed for re-hydrated weight, and then dried at 60 °C for dry weight.

Gas exchange was measured with an open flow infra-red gas analyser (LI-6400; LI- COR, Lincoln, NE, USA) with a broad leaf chamber. Chamber conditions were controlled

-2 -1 at 380 ppm CO2, 1500 umol m s PAR, 25 °C and 40–60% relative humidity, and leaf gas exchange recorded once stabilised within the chamber (typically within 5 min). The leaf portion contained within the chamber was then traced onto a sheet of paper, and the area measured in an optical leaf area meter (LI-3100C; LI-COR, Lincoln, NE, USA).

Leaf and root samples were analysed for carbon isotope ratio, and total C and N contents (%). Leaf 13C is used as an indicator of intrinsic water use efficiency integrated

12 over the life span of the leaf; RuBisCO preferentially utilises CO2, but as stomatal conductance decreases, CO2 within the sub-stomatal cavities and leaf tissues becomes

13 limiting, forcing increased fixation of CO2 (Dawson et al., 2002). Young, fully expanded leaves and fine root samples collected at the end of treatments were dried at 60 °C and ground to a fine powder in a ball mill. Samples were analysed at the West Australian 107 Chapter Six

Biogeochemistry Centre, using an Automated Nitrogen-Carbon Analyser Mass Spectrometer (Europa Scientific Ltd., Crewe, UK). 13C contents are reported as delta values in parts per thousand (‰), relative to the Vienna PeeDee Belemnite international standard. External error (standard deviation) of the analysis was <0.15‰. The C and N contents are given as percentages by weight, with external error of analyses <1.5% for C and <0.5% for N.

Statistical analysis For each parameter measured, the treatments were compared by ANOVA with Tukey’s HSD, performed with SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). For each analysis, homogeneity of variance among groups was examined with Levene’s test, and residuals were tested for departure from a normal distribution using a Shapiro-Wilk test. Differences are reported as significant where p<0.05.

RESULTS

Seedling growth and biomass allocation Shoot growth (height, biomass accumulation, leaf area, stem basal diameter, and stem and leaf elongation rates) in the hydrologic gradient experiment (Experiment 1) was least in the low water (20% of saturation) treatment (Figure 6.1). Shoot growth in the 20% treatment also showed a different pattern over time, where height increase was relatively rapid until week three and then slowed; by week six, shoot growth had virtually ceased. In contrast, shoot height in the 50, 80 and 100% soil saturation treatments all increased at similar rates of ~32 cm over 8 weeks, compared to only 13.4 cm in the 20% treatment (Figure 6.1a).

The greatest increase in total biomass and leaf area occurred in the 80% saturation treatment; this treatment had 8.5 g greater dry mass and 0.04 m2 greater leaf area than the 50 or 100% treatments by week eight (Figure 6.1b, c). Leaf elongation rate after eight weeks of treatment were similar between the 80 and 100% treatments (3.3 mm day-1) but lower under the 50% treatment (2.2 mm day-1) and lower still under the 20% treatment (0.3 mm day-1) (Figure 6.1d). The basal stem diameter showed a linear relationship with soil water content, with the greatest diameter at 100% saturation (Figure 6.1e).

108 Seedling responses across a hydrologic gradient in a riparian Melaleuca

400 (a) 20% 50% 300 80% 100% 200

100 Height increase (mm) Height increase 0 0102030405060 Time (days) 0.25 30 (b) root whole plant 60 Whole plant dry mass (g) (c) leaf 0.20 stem B BC Leaf area ( m2) B BC C 20 b C 40 0.15 A b c b AA ab bc 0.10 10 b a 20 a ab ab 0.05 a Leaf/ root/ stem dry mass (g) 0 0 0 Basal diameter increase (mm) (d) b b Stem elongation (mm day-1) (e) 10 10 stem C 3 D leaf ab 8 8 B 2 6 B 6 B 4 B A 4 1 A 2 2 a

Leaf elongation (mm day-1) 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 Soil water content (% of saturation) Soil water content (% of saturation)

Figure 6.1: Growth of Melaleuca argentea seedlings over eight weeks across a range of soil water content. (a) Cumulative increase in shoot height from the start of treatment (day zero). (b) Biomass after eight weeks treatment of whole seedlings, and of each component organ. (c) Total leaf area of seedlings after eight weeks. (d) Rates of leaf and stem elongation measured at the end of eight weeks. (e) Increase in stem basal diameter from the start of treatment. Note dual scales in panels (b) and (d). Values are means ± standard error of six plants. Different letters indicate significant differences (p<0.05) between treatments within a measured parameter. Within each panel, upper case letters refer to the parameter shown in open symbols, lower case letters the parameter shown in closed symbols.

109 Chapter Six

The relative biomass allocation of the seedlings after eight weeks differed significantly among treatments (Figure 6.2). The 80% treatment had the greatest total biomass of both roots and shoots (Figure 6.1b). However, the 20% treatment had the highest proportional allocation to roots, with these organs comprising 43% of total dry mass (Figure 6.2a). The lowest root:total biomass ratio occurred at 100% saturation where roots made up only 28% of plant dry mass. Specific leaf area increased with soil water content until the 80% treatment, and did not differ between the 80 and 100% treatments, indicating greater investment in the leaf tissue produced under lower water availability (Figure 6.2b). Leaf nitrogen content was lowest in the 100% saturation treatment at 1.7% (w/w), compared with 2.7% in the three lower water treatments (Figure 6.2c). In contrast, the nitrogen content of roots was slightly higher in both the 100% and 20% treatments (1.3 and 1.4%) than in the 50 and 80% treatments (1.1 and 1.0%).

50 160 (a) (b) 150 40 A SLA (cm2 g-1) B 140 30 B B C B 130 20 AB 120 10 A 110 Root dry mass (% of total) 0 (c) (d) 1.5

3 Root N content (%) A A A A A AB 1.0 2 B

B 1 0.5 Leaf N content (%)

0 0 0 20 40 60 80 100 0 20 40 60 80 100 Soil water content (% saturation) Figure 6.2: Resource allocation by seedlings after eight weeks treatment across a range of soil water content. (a) Root biomass as a proportion of total plant biomass. (b) Specific leaf area of young mature leaves that had developed during the treatment. (c) Nitrogen content of leaves (% w/w). (d) Nitrogen content of roots (% w/w). Values are means ± standard error of six plants, values labelled with the same letter are not significantly different (p>0.05).

110 Seedling responses across a hydrologic gradient in a riparian Melaleuca

Seedling leaf shedding and survival In the progressive drought treatment of Experiment 2, shoot growth not only ceased, but seedlings also shed leaves. Seedlings first began to shed leaves after 13 days of soil drying, when soil water content was ~12% of saturation. By 35 days, when soil water content was less than 3% of saturation, the seedlings had lost all leaves (Figure 6.3). No leaf shedding was observed in the control (field capacity) treatment during the experimental period, or in any of the treatments in Experiment 1 (data not shown).

50 100 Cumulative leaf mortality (% of total)

40 80

30 60

20 40

10 20 Soil water content (% saturation) Soil water content 0 0 0 1020304050 Time (days)

Figure 6.3: Leaf shedding by Melaleuca argentea seedlings during severe drought treatment, as soil water content declined from field capacity (50% of saturation) to undetect- able levels. Leaf mortality values are the cumulative mass of dead leaves as a percentage of the total plant leaf mass, mean ± standard error of six plants.

All plants survived the eight week treatment periods, including the progressive drought treatment (Experiment 2). Although all droughted seedlings in Experiment 2 were leafless at five weeks of treatment, they all produced new leaves when re-watered after eight weeks of drought. However, re-sprouting took up to one month to occur; the first shoot buds appeared 22 days after re-watering, and buds were apparent on all plants by 32 days after re-watering (data not shown). The new shoots were produced solely from the thickest parts of the stem near the base of the seedlings, suggesting that thinner branches were killed by the drought treatment.

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Morphological responses The density, thickness and water content of leaves differed between treatments in Experiment 1. In both the 80 and 100% soil saturation treatments, leaf water content was 80% (w/w) at the end of the eight weeks treatment, declining to 78% leaf water content in the 50% soil saturation treatment, and 74% at 20% soil saturation (Figure 6.4a). Leaf tissue was denser under the two lower soil water content treatments (0.23 g cm-3), compared with the two higher soil water treatments (0.19 g cm-3). The thickest leaves occurred in the 100% treatment at 0.40 mm, and the thinnest of 0.35 mm in the 50% treatment, with the 20% and 80% treatments intermediate (Figure 6.4b). The variation in leaf thickness appeared to be mainly due to differences in the spongy mesophyll thickness; the palisade mesophyll thickness did not differ between treatments (Figure 6.4c). Leaf epidermal thickness also did not differ significantly across the soil water gradient, while there was a small but significant increase in cuticle thickness with decreasing soil water content from 4.4 mm to 6.2 mm (Figure 6.4d).

Leaf trichome and stomatal densities also differed between treatments (Figure 6.4e). Trichome density was lowest under the 20% soil water treatment, while stomatal density peaked in the 50% treatment, with fewer stomata under both 20% and 100% saturation. Leaf size (of the same leaves measured for trichome and stomatal density) tended toward smaller in both the 20% and 100% treatments, but the difference was not statistically significant (Figure 6.4a), suggesting that the differing trichome and stomatal densities were not simply due to differences in the extent of leaf expansion between treatments.

A series of leaves from along the stems of a separate set of seedlings (growing at field capacity (i.e. ~50% soil saturation)) was examined to determine changes in surface morphology with leaf age (Figure 6.4f). Immature, actively expanding leaves had more densely packed trichomes and stomata, and densities reduced with age. The leaves designated as the ‘youngest mature leaves’ (leaf 1) in the age series were at a similar developmental stage to those collected from Experiment 1, and indeed densities of stomata and trichomes were similar to the 50% treatment of Experiment 1. However, older leaves had slightly lower densities, suggesting that although the leaves sampled in Experiment 1 were not expanding in length, some lateral expansion may still have been occurring. Nonetheless the ratio of stomata to trichomes remained similar throughout leaf

112 Seedling responses across a hydrologic gradient in a riparian Melaleuca

82 0.45 (a) (b) (g Leaf densitycm-3) 10 a Leaf size (cm2) size Leaf 0.25 80 B 0.40 a AB AB B B 8 78 0.35 AB 0.20 a 6 76 a a A a 4 0.30 b b Hydrated LWC (%) LWC Hydrated 74 A Leaf thicknessLeaf (mm) 0.15 0 20406080100 0 20406080100 Soil water content (% saturation) Soil water content (% saturation)

12 200 (c) spongy mesophyll (d) A A A palisade 10 A A 8 A 150 A A 6 a a a a 100 a a a 4 a cuticle Layer thickness ( μ m) Layer Layer thickness ( μ m) Layer 2 epidermis 50 0 0 20406080100 0 20406080100 Soil water content (% saturation) Soil water content (% saturation)

25 120 Trichomes (per mm2) Trichomes (per (e) (f) mm2) Trichomes (per 600 600 b b 20 100 b 80 500 15 400 60 A 10 40 400 AB 200 5 AB 20 Stomata (per mm2) Stomata (per mm2) a B 300 0 0 0 0 20406080100 -2 -1 1 2345 Soil water content (% saturation) Leaf number along stem

Figure 6.4: Leaf morphology and anatomy of seedlings under a range of soil water contents. (a) Leaf water content (LWC) following hydration to full turgor, (b) leaf thickness and density, (c) thickness of leaf spongy and palisade mesophyll layers, (d) thickness of leaf epidermis and cuticle. Leaf stomatal and trichome density of (e) youngest fully expanded leaves on seedlings across a range of soil water content, and (f) leaves across an age sequence on seedlings at 50% soil water saturation (leaves are numbered according to their relative position along the stem, where leaf 1 is the youngest fully expanded leaf, similar to the leaves used for the measurements in the other panels). Values are means ± standard error of four to six plants. Different letters indicate significant differences (p<0.05) between treatments within a measured parameter. Within each panel, upper case letters refer to the parameter shown in open symbols, lower case letters to the parameter shown in closed symbols.

113 Chapter Six development and aging (Figure 6.4f), while the ratios clearly differed between the soil water content treatments (Figure 6.4c, d). While trichome numbers were similar between the 50%, 80% and 100% saturation treatments, the 80% and 100% plants had fewer stomata, suggesting that partial and complete soil saturation triggered formation of leaves with fewer stomata. In the 20% treatment, however, both trichome and stomatal densities were lower than in the 50% treatment, which is explained most simply by the 20% treatment leaves being older; since growth rates were greatly reduced in the low water plants, the ‘youngest mature leaves’ were undoubtedly older in terms of actual time than in the higher water treatments.

Adjustments in root morphology were also evident across the soil water gradient. The 100% soil saturation plants produced a dense root mat in the top of the pot, and numerous upward growing roots with tips just protruding from the surface of the flooding water (Figure 6.5a, c). All roots in the lower half of the pots had blackened and died by the end of the eight weeks. The 80% soil saturation plants had numerous fine roots near the sand surface, including a few upward growing root tips protruding from the surface, and roots emerging from the stem base, but also maintained a large quantity of roots in the lower half of the pot (Figure 6.5b, d). The quantity of surface roots decreased markedly with decreasing sand water content, with roots in the 20% treatment concentrated in the lower portion of the pot (Figure 6.5c–f).

The extent of endodermal suberisation in fine roots at 5 cm from the root tip was lower in the 100% treatment, compared with the 20% and 50% soil water content treatments (Figure 6.6a). Endodermal suberisation did not differ between the 20% and 50% treatments. The quantity of root aerenchyma was similar between the 50%, 80% and 100% treatments, and lower in the 20% treatment, although the only significant difference in aerenchyma was between the 20% and 100% treatments (Figure 6.6b).

Physiological responses

The midday Ψshoot became more negative with decreasing soil water content. In Experiment 1, there were small but significant differences between all treatments, leading to a linear relationship between soil water content and Ψshoot from -1.50 MPa in the 20% treatment, to -0.64 MPa in the 100% treatment (Figure 6.7a). Similarly, leaf RWC increased slightly

114 Seedling responses across a hydrologic gradient in a riparian Melaleuca

(a) 100% (b) 80%

1 cm 1 cm

1 cm

Figure 6.5: Root macro-morphology of seedlings grown under soil water conditions ranging from 100% saturation (waterlogged) to 20% of saturation (low water). (a) Surface roots of a fully waterlogged seedling, with upward growing root tips protruding from the soil surface. (b) Above-ground surface roots emerging from the stem of a partially waterlogged seedling (80% soil saturation). (c−f) Seedling root systems exposed by cutting away pot walls, illustrating the variation in root quantity and depth across the range of soil water content treatments (100, 80, 50 and 20% of soil saturation).

with soil water content, from 85.0% RWC in the 20% treatment, to 89.3% RWC in the

100% treatment (Figure 6.7a). In Experiment 2, midday Ψleaf was more negative than the control by day 4 (soil moisture 29% of saturation), and predawn Ψleaf after day 7 (19% of saturation) (Figure 6.7b). At 14 days of progressive drying (soil 8% of saturation), corresponding with the initiation of leaf shedding, midday and predawn Ψleaf were -2.2

MPa and -1.4 MPa respectively. The Ψleaf of control (field capacity) plants remained above -1.2 MPa at all timepoints. Once leaf loss was complete in the droughted plants at day 35, the water potential of stems was -6.5 MPa at predawn, and below -7 MPa (the detection

115 Chapter Six

(a) 160

120 A A 80 B Figure 6.6: Root anatomy of seedlings under AB 40 a range of soil water contents. (a) Percentage of endodermal ring suberised (up to a maxi-

Endodermal suberisation (%) 0 mum of 200%, indicating two complete suber- (b) 20 A ised rings). (b) Root air space (as a percentage A of the root cortex cross sectional area). Values A 15 are means ± SE of four plants, values labelled with the same letter are not significantly differ- 10 ent (p>0.05). A 5 Cortical air space (%)

0 0 20 40 60 80 100 Soil water content (% saturation)

limit of the pressure chamber) at midday, and below -7 MPa at both predawn and midday thereafter.

Leaf gas exchange parameters measured in Experiment 1 at the end of the eight week treatment period are shown in Figure 6.8. There were no detectable differences in the rate of carbon assimilation (A) between treatments. However, transpiration (E) and stomatal conductance (gs) were significantly affected by soil water content. At midday, E and gs were highest in the 80% treatment and lowest in the 20% treatment (Figure 6.8a), although earlier in the morning (08:00) and in the later afternoon (16:00) there were no differences among treatments (not shown). As a result, the lowest photosynthetic water use efficiency (WUE) occurred at 80% soil saturation throughout most of the day, and highest WUE under 20% saturation (Figure 6.8b). The leaf δ13C indicated a similar pattern of WUE, although it was a less sensitive measure; δ13C was higher in the 20% treatment, but did not differ significantly among the three higher water treatments (Figure 6.8b). Leaf gas exchange was measured weekly during the treatment period, and the relative differences among treatments remained similar throughout (not shown).

116 Seedling responses across a hydrologic gradient in a riparian Melaleuca

0 (a) 92 Figure 6.7: Shoot water status of Mela-

b Leaf RWC (%) -0.4 b leuca argentea seedlings under a range ab 88 of soil water content. (a) Shoot water a -0.8 D potential (ψ) and leaf water content C 84 relative to full hydration (RWC) of seedlings after eight weeks of treatment at -1.2 B constant soil water content. Different A 80 Shoot ψ (MPa) letters indicate significant differences -1.6 between treatments within a parameter. 76 0 20406080100 (b) Shoot ψ of seedlings during eight Soil water content (% saturation) weeks of progressive drought (soil allowed to dry out over time) or control (field capacity; ~50% Time (days) saturation) treatment. 0 2 4 7 14 21 35 0 The x axis of panel (b) (b) displays the soil water -1 content of the droughted plants, -2 with the times since -3 the start of treatment indicated above the graph. All values are -4 Control predawn means ± standard Shoot ψ (MPa) Control midday -5 error of six plants, Drought predawn where error bars are Drought midday -6 not visible they are smaller than the -7 50 40 30 20 10 0 symbols. Drought treatment soil water content (% saturation)

20 (a) AA

A 8 E (mmol m-2 s-1) 16 A Figure 6.8: Leaf gas exchange param- 6 12 eters of seedlings after eight weeks c under a range of soil water content bc 4 8 b conditions. Instantaneous rates of (a) a carbon assimilation (A), transpiration 4 2 (E), and (b) intrinsic water use efficiency A (μmol CO2 m-2 s-1) CO2 m-2 A (μmol (WUE), at midday under light satura- 0 0 tion, and (b) leaf 13C content. Values are (b) A mean ± standard error of six plants. 5 -31 B δ13C (‰) Different letters indicate significant 4 a BC differences (p<0.05) between treat- C 3 ments within a measured parameter. -32 Within each panel, upper case letters 2 b b b refer to the parameter shown in open 1 symbols, lower case letters the parameter shown in closed symbols. 0 -33 WUE (μmol CO2. mol H2O-1) CO2. mol WUE (μmol 0 20406080100 Soil water content (% saturation) 117 Chapter Six

DISCUSSION

Growth and responses under saturated conditions M. argentea seedlings are clearly highly tolerant of saturated soil conditions, with the highest growth rates observed in partially waterlogged (80% saturation) soil. Under complete waterlogging (100% saturation) the seedlings also performed well, although some aspects of growth were reduced relative to partial waterlogging, most likely due to a limitation in root growth or functioning. As hypothesised, seedlings in the 80% and 100% soil saturation treatments displayed numerous adjustments, including substantial change to root system morphology with production of shallow and negatively gravitropic roots. The morphological and physiological responses observed most likely enable this species to grow rapidly under waterlogged and semi-waterlogged conditions, which is vital for establishment in intermittent waterways.

Unlike non-flood-tolerant species (e.g. Pezeshki & Anderson, 1996; Glenz et al., 2006), M. argentea seedlings were able to continue growth, and showed no signs of water stress during eight weeks of stagnant waterlogging. Leaf RWC, Ψ and rates of photosynthesis in waterlogged seedlings were all higher or no different from those in the ‘optimal’ partial waterlogging conditions. Species of Salix and Populus inhabit equivalent habitats to M. argentea in the northern hemisphere, and might be expected to show similar levels of flooding tolerance. Seedlings of S. gracilistyla in southeast Asia, and S. nigra in North America appear to be less flood tolerant than M. argentea, displaying severely reduced growth and rates of photosynthesis under stagnant waterlogging relative to drained conditions, and in some cases inhibited growth under partial waterlogging also (Pezeshki et al., 1998; Li et al., 2004; Nakai et al., 2009). On the other hand, S. exigua may be more tolerant than M. argentea, as seedling growth increased during complete waterlogging (Amlin & Rood, 2001). Most poplars are less tolerant, for instance waterlogging greatly reduced growth in seedlings of P. angustifolia, P. balsamifera and P. deltoides, and mature P. balsamifera and P. deltoides were killed by long term inundation in Alberta, Canada (Amlin & Rood, 2001). In seedlings of fourteen lines of North American poplar hybrids (hybrids among P. trichocarpa, P. deltoides, P. nigra, and P. maximowiczii), photosynthesis decreased during seven weeks of waterlogging, and plant height, leaf area and biomass were reduced in most genotypes, although a few performed well (Gong et al., 2007). M. argentea

118 Seedling responses across a hydrologic gradient in a riparian Melaleuca seedlings therefore appear to have flooding tolerance equivalent to some of the most tolerant willows and poplars.

Many other Melaleuca species are also highly tolerant of waterlogging, and some may be considered semi-aquatic trees or shrubs (e.g. Lockhart, 1996). Establishment of seedlings of M. quinquenervia, native to tropical northern Australia, was optimal in very moist to saturated soils (Myers, 1983), and transpiration rates of mature M. quinquenervia, were unaffected by a seasonal inundation period of several months (McJannet, 2008). An ability to continue such high rates of transpiration and growth during waterlogging is relatively rare among tree species (Parolin & Wittmann, 2010). The success of M. quinquenervia as a highly invasive weed in wetlands of Florida, USA, is attributed largely to its rapid and dense root growth, and ability to rapidly produce deep roots to follow a receding water table (Myers, 1983; Lopez-Zamora et al., 2004). Seedlings of M. cajuputi, native to far northern Australia and tropical regions of Asia, also showed no difference in rates of photosynthesis or shoot growth during 19 days of root hypoxia, compared with aerated controls, although gs was reduced (Kogawara et al., 2006). M. argentea appears to share with other wetland Melaleuca species a plastic and rapidly responding root system, which plays a major role in flooding tolerance.

The M. argentea seedlings displayed a range of root-level responses to waterlogging. Waterlogged seedlings had a shallow root system and wide stem base, responses common to flood adapted species due to higher oxygen levels near the surface of waterlogged soil (Pezeshki et al., 1998; Glenz et al., 2006; Myster, 2009). The M. argenta seedlings also produced negatively gravitropic roots, similar those produced by the highly flood tolerant M. quinquenervia (Sena Gomes & Kozlowski, 1980). The proportion of root cortical airspace (aerenchyma) in M. argentea was low, remaining at only ~15% of the cross sectional area in the field capacity, partially and fully waterlogged seedlings. Aerenchyma frequently makes up 40–60% of the root cortical cross section in wetland plants, including in Salix viminalis, and tropical wetland grasses (Baruch & Mérida, 1995; Jackson & Attwood, 1996). However, production of extensive aerenchyma may be less common in trees due to development of secondary thickening, for instance most central Amazonian floodplain trees do not form appreciable root aerenchyma during waterlogging, even evergreen species that actively grow during long flooded periods (De Simone et al., 2002b).

119 Chapter Six

Increased levels of enzymes involved in anaerobic metabolism may be an alternate strategy to compensate for low levels of root porosity (Benz et al., 2007). In a comparison of two flood tolerant Amazonian tree species, the highly aerenchymatous Salix martiana produced low levels of alcohol dehydrogenase (ADH), while Tabernaemontana juruana produced little aerenchyma but higher levels of ADH (De Simone et al., 2002a). Other highly flood tolerant Melaleuca species such as M. cajiputi produce high levels of ADH under hypoxia (Yamanoshita et al., 2005), and M. argentea may also adopt this strategy, reducing the requirement for root aerenchyma.

Shoot morphological responses were also observed in M. argentea during waterlogging which may be adaptations for shoot submergence. These included leaf and stem elongation, and production of lower density leaves with a thicker spongy mesophyll layer and lower stomatal density. While trees are highly unlikely to be fully submerged by floods in semi-arid Australia, their seedlings may be, so these responses could be important for young plants. Leaf responses can be critical during shoot submergence, for example Melaleuca halmaturorum displays few leaf responses to flooding, and seedlings are highly resistant to anoxic soil conditions, but susceptible to shoot submergence (Denton & Ganf, 1994). Ethylene production in the roots during flooding can signal shoot elongation, a response thought to aid the plant in escaping flood waters (e.g. Voesenek et al., 2003). In seedlings of the semi-aquatic M. quinquinervia, increased height growth under submerged conditions was associated with improved survival rates (Lockhart et al., 1999). The thicker, lower density spongy mesophyll suggests development of airspaces allowing internal oxygen transport through the leaves; this response is observed during waterlogging in small plant species that experience shoot submergence, such as the chrysanthemum Dendranthema zawadskii (Yin et al., 2010), although leaf airspace formation appears to be less common in flood tolerant tree species (e.g. Herrera et al., 2009). Lower stomatal density may be advantageous during shoot submergence. At the Mapire River in Venezuela, partial submergence reduced stomatal density in a few tree species, such as Eschweilera tenuifolia, although not in other species (Herrera et al., 2009). In heterophyllous semi-aquatic species, including M. quinquenervia and Ranunculus flabellaris, leaves produced underwater have fewer stomata than those produced in air (Young & Horton, 1985; Lockhart, 1996). The

120 Seedling responses across a hydrologic gradient in a riparian Melaleuca leaf and shoot responses seen in M. argentea might confer benefits under soil waterlogging, and indicate that the seedlings may also be tolerant of shoot flooding.

Drought responses The results of this study highlight the high water requirements of M. argentea in early stages of establishment. As hypothesised, seedlings displayed fewer adaptive drought responses compared with responses to waterlogging, and were clearly susceptible to drying of the substrate. Below soil water content of 80% saturation, growth decreased and leaf Ψ and RWC indicated declining water status. Under more severe water stress, the seedlings appeared to rapidly enter a leafless, dormant state, in which they were able to survive at least eight weeks without water. Rather than ‘tolerating’ drought, the plants appeared to adopt a ‘waiting’ strategy, which may give the seedlings the best chance of survival in the event that the drought is short lived, and abundant water returns.

Despite the cessation of growth and the significant decreases in shoot water status (RWC and Ψ), the M. argentea seedlings maintained high rates of carbon assimilation under low soil water content (20% of soil saturation). A small further decline in soil water content, to 10-15% of soil saturation in the severe drought experiment, triggered a sudden, rapid decline in Ψleaf, and onset of leaf shedding. Although the severe drought was a progressive treatment, and thus may not be entirely comparable with the constant soil water gradient experiment (e.g. Hukin et al., 2005), it appears that the M. argentea seedlings maintained carbon assimilation rates with decreasing soil water, down to a point very close to the threshold of ‘catastrophic’ xylem cavitation, and therefore displayed very limited stomatal control of water loss. Some stomatal response was evident since gs decreased and WUE increased with decreasing soil water content. However, species with sensitive stomatal control or efficient osmo-regulation show little change in leaf RWC or Ψ with moderate reductions in water availability (Gonzalez-Rodriguez et al., 2005). Drought tolerant trees such as olive (Olea europa) typically also display a tight correlation between gs and photosynthetic rate with decreasing water availability, indicating stomatal limitation of both water loss and carbon gain (e.g. Guerfel et al., 2009). The M. argentea seedlings did not show these types of physiological responses, instead displaying several morphological responses to drought, including canopy shedding.

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Many riparian Salix and Populus species also readily shed leaves and branches under drought. Canopy abscission is due to hydraulic failure of the leaf, petiole or stem xylem, and early canopy loss during drought is therefore seen in species with low cavitation resistance (Tyree et al., 1994; Nardini et al., 2001; Salleo et al., 2002). Leaf shedding was observed in seedlings of a range of Salix species at predawn Ψleaf just below -1 MPa (Savage & Cavender-Bares, 2011), similar to the point at which M. argentea seedlings shed leaves in this study. Rapid leaf drop reduces water loss and can prevent the development of more extensive xylem cavitation, leading to improved rates of re-sprouting upon rewetting (Brodribb & Cochard, 2009; Savage & Cavender-Bares, 2011). For example, Populus balsamifera in Alberta, Canada, displayed rapid leaf and branch senescence during moderate groundwater drawdown over a period of 2 years, but tree growth rate returned to control levels within a year of the drawdown ceasing (Amlin & Rood, 2003). Carbon starvation can also lead to drought mortality, often acting in combination with hydraulic failure, since avoiding hydraulic failure by closing stomata occurs at the cost of carbon assimilation. A stronger reduction of growth than of photosynthesis during early water stress, as seen in the M. argentea seedlings, is associated with accumulation of carbohydrates, which can then sustain cellular respiration as drought severity increases (McDowell, 2011; Mitchell et al., 2013). The M. argentea seedlings all survived three weeks without water in a leafless state, and re-sprouted within four weeks of re-watering. The maintenance of gs and photosynthesis through early drought, allowing ‘precocious’ shedding of leaves and branches, may represent a drought survival strategy for many riparian tree species (Rood et al., 2000), including M. argentea. By retaining moisture and carbohydrate reserves and preventing further drought damage, the trees may increase their chance of survival and recovery if favourable conditions return.

Several leaf-level responses to drought were observed in the M. argentea seedlings; SLA and leaf hydrated water content decreased, and cuticle thickness increased slightly. However, the strongest drought responses to water stress were observed in the roots. Resource allocation to roots increased under drought; root:shoot ratio, root depth and root nitrogen content all increased below 80% soil saturation. Melaleuca species are known to accumulate nitrogenous compounds (proline and analogues) during drought as osmo- protectants (Naidu et al., 1987); while osmolytes were not measured in this study, the

122 Seedling responses across a hydrologic gradient in a riparian Melaleuca increased nitrogen levels could indicate osmo-protection of root tissues during water stress (see further discussion below). The extent of root endodermal suberisation was also greater in the lower water treatments, a common response which can prevent water loss from the roots into dry soils (North & Nobel, 1998; Enstone et al., 2003). Root growth is crucial for semi-arid riparian tree seedlings, in order to maintain contact with groundwater as it recedes. Many trees species increase investment in roots and rooting depth during drought (Osunubi & Davies, 1981; Bongarten & Teskey, 1987; Abrams, 1990), but by no means all species do so (Osunubi & Davies, 1981; Pallardy & Rhoads, 1993; Joslin et al., 2000). The protection of root tissues via osmotic and anatomical adjustments, and increased allocation to roots by the M. argentea seedlings are clear adaptive responses to low water.

While M. argentea seedlings displayed some adaptive responses to drought, several responses common in more drought tolerant species were lacking. Increased stomatal density under water stress, which provides greater control of transpiration, is seen in drought tolerant Quercus species and olive (Abrams, 1990; Bosabalidis & Kofidis, 2002), but was not observed in M. argentea. Olive leaves also increased trichome density under water stress to ~100 trichomes per mm2, which can reduce water loss and reflect heat and radiation (Guerfel et al., 2009), while M. argentea leaves had 20 or fewer trichomes per mm2. Increased leaf osmolyte concentration is another commonly observed response to water stress, including in Eucalyptus astringens in semi-arid parts of south-west Australia (Arndt et al., 2008). Some other Melaleuca species are adapted to dry conditions in arid parts of Australia, such as M. filifolia, M. scabra and M. globifera, and these species accumulate proline analogue compounds as osmo-protectants, but the most effective osmolytes were absent from drought susceptible species including M. argentea (Naidu et al., 2000). Leaf nitrogen content of the seedlings in the present study did not change with decreasing SWC, indicating that changes in proline based osmolytes with water stress is unlikely. Even in the absence of nitrogenous osmo-protectants, increases in leaf nitrogen content are frequently associated with improved drought tolerance, including in many Salix species and in Quercus suber (Aranda et al., 2005; Weih et al., 2011). This response in leaf nitrogen was not observed in the M. argentea seedlings.

123 Chapter Six

Implications for response thresholds under field conditions Changes in population structure or physiological functioning of dominant species can have implications for whole ecosystem function (Xu & Zhou, 2011). In particular, identification of response thresholds (abrupt shifts which occur across a small change in conditions) can be important in ecosystem monitoring and management (e.g. Polasky et al., 2011), since some changes may be irreversible or require prolonged recovery times, even after removal of the environmental stressor (Ridolfi et al., 2006). A series of response thresholds was evident in the M. argentea seedlings, moving from the wettest to driest ends of the soil water gradient. Many parameters displayed a threshold between 80 and 50% saturation, including reductions in leaf elongation rate and leaf water content at full turgor, and increases in leaf tissue density and endodermal suberisation. Further reduction in soil water from 50 to 20% produced an abrupt increase in root:shoot ratio, a reduction in the quantity of root aerenchyma, and cessation of shoot height increase. With a further decrease in soil moisture to ~15% of saturation, the threshold for a rapid decline in shoot water potential and leaf shedding was reached. Abrupt physiological responses to decreasing water availability are usually associated with xylem cavitation, occurring at distinct threshold water potentials (Horton et al., 2001a), and the threshold responses in leaf elongation, shoot height and leaf shedding are most likely directly linked with water potential (Cosgrove, 1986; Salleo et al., 2002).

Partially saturated soils appear to be the ‘ideal’ condition for M. argentea seedlings, likely to facilitate the maximum rates of tree recruitment. This is consistent with the observation that M. argentea is frequently found in partially flooded locations in the field, close to central river channels where pools of surface water form. The tolerance of waterlogging is most likely attributable to the numerous adaptive responses that were observed in both the roots and shoot. In contrast, the seedlings displayed fewer adaptive responses to water stress, corresponding with poor drought tolerance and rapid decline in performance with decreasing soil water content. However, the ‘non-tolerance’ of water stress may in itself be considered an adaptive response to survive temporary drought. Nonetheless, M. argentea clearly could not readily establish in persistently dry conditions, while under conditions of increased water availability, such as during discharge of excess water into a creekline (Chapter 1), recruitment rate may increase.

124 Seedling responses across a hydrologic gradient in a riparian Melaleuca

The point at which drought causes whole-plant death was not reached in this study, although irreversible damage was caused to some branches. Since M. argentea appears to enter dormancy during severe drought, the survival rate will be a function of the length of the drought, and may also vary with factors such as temperature and tree size (McDowell et al., 2008; Adams et al., 2009). Thinner stems and branches are often more vulnerable to cavitation, and have greater surface area to volume ratios for evaporation to occur (Sperry & Ikeda, 1997). Large M. argentea trees, with thick layers of bark, might therefore survive a relatively long drought period in a dormant state. However, the relationship between tree size and drought survival in a dormant state appears to vary among species and sites, possibly depending on factors such as root depth, canopy exposure and water requirements, and in some cases larger trees are more susceptible (Van Nieuwstadt & Sheil, 2005). Survival can also depend upon the extent of carbohydrate reserves, the ability to mobilise the reserves, and the rate of depletion during the dormancy period (McDowell et al., 2008; Sala et al., 2010). High temperatures frequently increase cellular respiration rates, and may also increase the desiccation rate due to evaporation, and so could reduce drought survival times (Pinheiro & Chaves; Chaves et al., 2002; Adams et al., 2009). The ability of trees to survive a period of drought is a complex process, however, the early leaf shedding response of M. argentea might enable the species to survive relatively long periods of severe drought.

Some of the morphological and physiological responses that occurred at specific points along the soil water gradient may be useful in understanding the extent of ‘stress’ experienced by M. argentea trees following hydrologic perturbation. For example, leaf Ψ and canopy loss are frequently used to monitor tree ‘health’. Leaf Ψ appears to respond to water availability in a linear fashion, which may belie the larger responses occurring in other parameters. On the other hand, leaf loss may not occur until an advanced level of water stress, and progresses rapidly below a threshold of water availability, so cannot provide advance warning of severe stress in M. argentea. However, the trait response patterns observed in this study would need to be verified under field conditions, and for mature trees.

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CHAPTER SEVEN: Response of Melaleuca argentea trees to ground water abstraction from the coastal plain aquifer of a large ephemeral river

INTRODUCTION

In many arid and semi-arid regions of the world, groundwater is exploited to support agriculture, industry and direct human consumption. As riparian trees are generally dependent on shallow groundwater for survival and growth, abstraction can result in declining tree health, including canopy loss and eventual mortality (Cooper et al., 2003; Rood et al., 2003). Seedling recruitment along semi-arid ephemeral rivers is also reliant on the episodic availability of surface and near-surface water (Mahoney & Rood, 1998). Under groundwater abstraction these conditions occur less frequently, as complete groundwater recharge takes longer and requires larger volumes of rainfall. Groundwater abstraction can therefore lead to long term changes in tree population structures (Smith et al., 1991; Williams & Cooper, 2005; Evans, 2007). As described in earlier Chapters, regional growth in northwest Australia is placing greater demands on groundwater resources (Department of Water, 2010). For example, the Yule River coastal plain aquifer, 50 km to the southwest of the Port Hedland township, is currently under abstraction of up to 6.5 GL per year for town and industry water supplies, and an increase of 2 GL per year in abstraction is proposed. Current management of impacts on the groundwater dependent ecosystems is based upon historical groundwater level records, but an increased abstraction regime would require revision of the minimum groundwater levels that are set as the ‘trigger’ to reduce abstraction pressure (Baimbridge, 2010). Relatively little is known about the ecological water requirements of the groundwater dependent riparian vegetation of northwest Australia, and thus there is a need to quantify vegetation responses to reduced groundwater levels.

The sensitivity of riparian tree species to declining groundwater varies. For example, Melaleuca argentea, a tree common to the waterways of northern Australia, appears to have a low tolerance to drought (Masini, 1988; Chapter 6), and to show signs of decline earlier than co-occurring Eucalyptus camaldulensis and E. victrix (unpublished data). Similarly, along rivers in semi-arid Arizona, USA, Salix goodingii suffered more severe canopy dieback

126 Responses of Melaleuca argentea to groundwater abstraction and greater rates of mortality than the co-occurring Populus fremontii when the depth to groundwater fell below 3 m, while Tamarix chinensis in the same areas was more tolerant of groundwater decline than either S. goodingii or P. fremontii (Horton et al., 2001b). The differential responses between species may relate to differences in drought tolerance, and differences in ability to maintain contact with a receding water table via root elongation responses.

The rate of groundwater decline is a critical factor in the survival and response of dependent vegetation (Eamus et al., 2006a). Provided the decline is gradual enough, trees may be able to compensate for seasonal fluctuation in groundwater levels, and for additional groundwater declines due to various human activities, via root elongation to follow the water table. Numerous controlled rhizopod studies have been conducted with tree seedlings and cuttings under simulated groundwater decline, in order to estimate the maximum rates of root elongation, and therefore the maximum tolerable rate of groundwater decline. Trees accustomed to large groundwater fluctuations may show greater tolerance of drawdown, for example cuttings of Populus balsamifera, native to mountain regions where water table depths often change abruptly, survived 10 cm day-1 simulated drawdown, while cuttings of P. deltoides and P. angustifolia, native to floodplains with more stable water tables, were mostly killed by the rapid drawdown (Kranjcec et al., 1998). Recession rates of 1–2 cm day-1 appear tolerable for seedlings of a wide range of riparian species (e.g. Alnus incana, Populus fremontii, Salix exigua, S. gooddingii), with rates of >3 cm day-1 frequently leading to higher mortality rates, and reduced growth of any surviving seedlings (Segelquist et al., 1993; Hughes et al., 1997; Amlin & Rood, 2003; Stella & Battles, 2010). However sensitivity can clearly vary even among co-occuring species, e.g. Tamarix chinensis showed no change in survival rate with drawdown of >4 cm day-1, while S. gooddingii can show increased mortality at any rate of drawdown above zero (Horton & Clark, 2001).

If groundwater recession exceeds the tolerable rate, or if groundwater levels fall below maximum rooting depths, the impact on riparian and floodplain vegetation can be equated to gradually increasing drought stress (Eamus et al., 2006a). Decline of groundwater levels will dry an increasing portion of the root system; in the early stages the tree may be able to compensate by taking up water via deeper roots (Chapters 4 & 5), but eventually a critical

127 Chapter Seven root volume will be dried, beyond which the tree can no longer compensate. Decreasing leaf water potential (Ψl), reduced stomatal conductance (gs) and reduced rates of transpiration are early symptoms of water stress, progressing to canopy loss and mortality with increasing stress (e.g. Horton et al., 2001c; Cooper et al., 2003). While in well watered environments the vapour pressure deficit (VPD) of the air, and the soil and tree hydraulic conductance (K) are the main drivers of gs and transpiration rate, in drying soil the soil matric potential (Ψsoil), becomes the major determinant of gs (Brodribb, 2009; Chapter 2). Stress responses during groundwater recession frequently occur in a threshold pattern at a particular depth to groundwater (DGW), most likely corresponding to the root system depth (Horton et al., 2001b; Chapter 6). Since juveniles frequently have shallower root systems, they can be exposed to greater variation in water availability in dry environments (Donovan & Ehleringer, 1991). It is therefore likely that larger trees will be able to access water from deeper in the soil profile than smaller trees, and so the critical extent of root drying under drawdown will be reached earlier for small trees. Thus, at a given DGW, small trees may be exposed to lower Ψsoil, and so display greater water stress than larger trees.

Along the coastal plain regions of the Yule River, DGW fluctuates considerably within and between years (Department of Water, unpublished data), due to the highly episodic rainfall patterns characteristic of the region, which in turn leads to episodic surface flows and aquifer recharge. There are also likely to be high rates of evaporative water loss from the aquifer through the wide, sandy river bed exposed to the high VPD conditions (see Chapter 2). The potential impacts of groundwater abstraction therefore need to be placed within the context of the natural range of water levels within the aquifer. M. argentea is one of the dominant riparian tree species along the Yule River. Although this species appears sensitive to small reductions in water availability (Chapter 6), trees on the Yule River coastal plain may be relatively tolerant of groundwater abstraction, due to their natural exposure to large fluctuations in water level. However abstraction causing water levels to fall beyond a critical threshold of DGW would still be expected to induce drought stress.

In this Chapter, I aimed to define the relationships between changing DGW and physiological functioning of M. argentea trees growing along the Yule River, a large

128 Responses of Melaleuca argentea to groundwater abstraction ephemeral river in semi-arid NW Australia. I expected that (i) groundwater abstraction would increase DGW; (ii) that lowering of the water table would impact negatively upon M. argentea trees and (iii) the degree of impact would depend on tree size, with smaller trees having reduced access to deeper portions of the soil profile, and thus showing signs of water stress with smaller DGW than larger trees. I assessed tree physiological functioning and the depth of water accessed by the trees, during one year of groundwater abstraction from the Yule River aquifer. Trees in close proximity to the abstraction bores were compared with those at an upstream location exposed only to the natural seasonal groundwater fluctuations.

MATERIALS & METHODS

Study site The Yule River coastal plain aquifer is located in the Pilbara region of northwest Australia, where the river traverses the Port Hedland coastal plain ~20 km from the river mouth estuary (Figure 7.1). Water extracted from the aquifer forms one of the water supply sources for the town of Port Hedland, ~50 km to the NE. Due to increasing population and demand for water, in 2009–2010 a trial was conducted to assess the ability of the riparian vegetation to tolerate an increased rate of abstraction from the Yule River borefield.

Soils within the borefield are layered alluvial sands, silts and clays. The control site surface soil is a pindan sand-clay to 1 m depth, then sand below 1m, with shingle and sand layers at 1–4 m and 5–6 m depths. The impact site has a sandy surface soil to 4 m depth, then shingle and sand at 4–7 m, and clay and sand below 7 m (data provided by the Water Corporation, Western Australia). The climate of the region is semi-arid, characterised by high temperature and VPD conditions for much of the year, and highly variable and episodic rainfall patterns. Meteorological data was recorded during the study period by a weather station located at the impact site, and additional daily solar radiation data were obtained from the Australian Bureau of Meteorology, as described in Chapter 2.

The Yule River is ephemeral, with intermittent flows reflecting the highly variable rainfall patterns of the Pilbara. The region can be subject to cyclone activity in the December–April period, which can bring large rainfall events and flooding; river flows and ground water recharge occur only after these events. Depth to the groundwater in the Yule

129 Chapter Seven

Port Hedland

Karratha YULE RIVER

YULE BOREFIELD

Impact

Control

Study site Long term monitoring bores Production bore Watercourse

5 km West Coastal Hwy North

Figure 7.1: Location of the Yule River borefield and study sites. Modified from maps provided by the Water Corporation of Western Australia.

130 Responses of Melaleuca argentea to groundwater abstraction

River borefield fluctuates, as water levels decline naturally between episodes of recharge. The section of the Yule River within the borefield consists of a wide riverbed of alluvial sand, bordered by narrow bands of riparian vegetation, with broad floodplains beyond the river bed vegetated by grassland and shrub-steppe. The production bores are located mostly within the northern half of the borefield. A control site was established upstream, on the river bank near the southern boundary of the borefield at 20.6563° S 118.2948° E, and an impact site near to the northern-most production bores at 20.5372° S 118.1750° E, ~20 km from the control site (Figure 7.1). The control site is described in Chapter 2. The impact site consisted of a wider band of riparian vegetation (~200 m), with sparser canopy cover and trees less densely distributed, compared with the control site. The dominant riparian tree species of Eucalyptus camaldulensis, E. victrix, and Melaleuca argentea were present at both sites. Five M. argentea trees were selected at each site, encompassing a diameter range (at 1.2 m height) of 140–500 mm. All measurements were conducted on these same ten trees throughout the study.

Groundwater levels and abstraction Cyclone activity during December 2008–February 2009 generated significant flows in the Yule River, which recharged the aquifer (data provided by the Department of Water, Western Australia). The pumping trial began in early April 2009, with abstraction from the northern-most bores near the impact site given priority. However, pump failure meant that several times the southern bores were also used by the Water Corporation in order to meet the water supply requirements of Port Hedland, and the total rate of abstraction was lower than planned. Hourly values of DGW were provided by the Department of Water, measured at monitoring bores installed at the control and impact sites, fitted with sensors and loggers. Historical water table depths were also obtained from the Department of Water, for bores near to each site (Figure 7.2).

Soil moisture Soil moisture data were provided by the Water Corporation, Western Australia. Soil moisture through the profile to the water table was measured monthly with a neutron moisture probe at each study site. Soil cores were also drilled at each site in July and November 2009, and the volumetric water content measured on samples from 0.25 m depth increments were used to calibrate the neutron probe readings.

131 Chapter Seven

Days after start of pumping 0 100 200 300 400

0 AMMJJAS ODJFMAN 1 (a) 2 3 4 DGW (m) 5 Control Impact 6 7

6 (b) 4 2 0 -2 -4 Rate of decline (cm day-1) -6

Year 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 0 (c) 1

DGW (m) 5

6 Control Impact 7 Control- long term Impact- long term 8

Figure 7.2: Groundwater levels and rates of decline at the Yule Borefield study sites.Depth to groundwater (DGW) is shown relative to the ground surface at each study site. (a) Ground- water levels during the pumping trial study period. (b) Daily rate of groundwater decline during the study period (negative values indicate a water table rise). (c) Groundwater levels over ten years; the study period (shown in (a) and (b)) is indicated with vertical grey lines. Solid lines show data from the bores at the study sites installed in March 2005, dashed lines show data from long term monitoring bores, located within 2.5 km of the study sites, but further away from the river bed.

132 Responses of Melaleuca argentea to groundwater abstraction

Isotope analysis of xylem sap, ground and soil water Isotope analysis was used to identify the depth of the water within the soil profile accessed by the trees. The isotopic composition of water usually varies through the soil profile, due to evaporation at the surface enriching surface and near-surface soil water in the heavier isotopes. Water uptake by plant roots is generally indiscriminate with respect to isotopic composition (Dawson & Ehleringer, 1991). The isotopic signature of tree xylem water can therefore be compared to the isotopic signatures of soil and groundwater, to assess the likely source water depth used by the trees.

Samples of xylem sap were collected from the ten study trees (five at each site) in March, June and November 2009, and May 2010. The sap was vacuum extracted in the field; branchlets of ~1 cm diameter were cut from ~5 m height above the ground in the early morning, all leaves removed, and the bark removed from the first 5 mm of the cut end to prevent contamination with phloem sap. Xylem contents were collected in 2 ml vials, which were sealed well and kept refrigerated or in an ice-box during transportation and storage until analysis.

Soil cores were drilled at each site in July 2009 and early December 2009, and soil samples collected at 0.6 m depth increments to a total depth of 3.6 m. Additional soil samples were collected by hand auger in November 2009, at 0.1–0.2 m depth increments to a total of 1.6 m. Soil was tightly packed into 30 ml vials immediately after drilling or augering, and kept well sealed and refrigerated or in an ice-box until analysis. Drilling and augering took place within the riparian tree bands, close to the study trees, near the base of the bank to the main riverbed. Groundwater samples were taken from the monitoring bores at each site, in November 2009 and May 2010. Water from surface pools was also sampled in June 2009 (pools had dried down by November 2009).

Soil water was cryogenically distilled, and the 2H and 18O content of all water samples determined by mass spectrometry (L1115-I Cavity Ring-down Spectrometer, Picarro, Santa Clara, USA) at the West Australian Biogeochemistry Centre. Isotopic contents are expressed as delta values in parts per thousand (‰) relative to the Vienna Standard Mean Ocean Water international standard. The external error (standard deviation) for δ2H analysis was ~2‰ for distilled samples, and <1‰ for other samples. The external error for δ18O analysis was ~0.2‰ for distilled samples, and <0.1‰ for liquid samples.

133 Chapter Seven

Tree water use Sap flow sensors (HRM 30; ICT International, Armidale, NSW, Australia) were installed on 28th March 2009, just before the start of the pumping trial in early April. A single sensor was installed in the stem of each of the ten study trees (five at each site), at 1.2 m height, as described in Chapter 2. Heat pulse velocity was recorded every half hour from 29th March 2009 until 12th May 2010, except for the period 15th March–12th May 2010 at the impact site, when battery failure resulted in the half hourly measurements being recorded only between 08:00 and 18:00 on each day (when equipment was powered by solar panel directly). I was therefore unable to calculate total daily sap flow for this period at the impact site, but was still able to determine maximum daytime flow rates. The methods of calculating sap flow velocity (Vs) and the measurement of sapwood cross sectional area are also described in Chapter 2.

Leaf physiological measurements Leaf measurements were conducted in March, June and November 2009, and May 2010, on the five study trees at each site. Leaf water potential (Ψl) was measured with a Scholander-type pressure chamber, as described in Chapter 2. Leaf 13C was measured in young, mature leaves collected from the outer canopy, ~5 m height above the ground. Leaf 13C is used as an indication of intrinsic water use efficiency integrated over the life span of

12 the leaf; RuBisCO preferentially utilises CO2, but as stomatal conductance decreases, CO2 within the sub-stomatal cavities and leaf tissues becomes limiting, forcing increased fixation

13 of CO2. Leaf material was dried at 55 °C, ground to a fine powder in a ball mill, and samples analysed in a Nitrogen-Carbon Analyser Mass Spectrometer (Europa Scientific Ltd., Crewe, UK), at the West Australian Biogeochemistry Centre. 13C contents are given as delta values in parts per thousand (‰), relative to the Vienna PeeDee Belemnite international standard. External error (standard deviation) of the analysis was <0.15‰. For comparison with the 13C values, stomatal conductance was also measured with a leaf porometer (Decagon Devices, Pullman, WA, USA) at both sites in November 2009 and May 2010, as described in Chapter 2. Leaf specific area was determined from the same samples as used for isotopic analysis; a hole punch was used to cut discs of known size from ten leaves of each sample, and the discs weighed after drying at 55 °C.

134 Responses of Melaleuca argentea to groundwater abstraction

Statistical analysis Statistical analyses were performed with R version 2.13.0 (R Development Core Team, 2010). Linear mixed-effect models (lme4) were used to assess differences in tree sap flow, leaf 13C, and xylem sap 2H and 18O. Comparisons were made between the sites, both over time and over DGW. A random effect of tree individuals was included in the analysis when it significantly improved the model (as determined by comparing models based on AIC scores and χ2 log-likelihood tests). The effects of site, days after start of trial, DGW and tree size on the response variable (sap flow, leaf 13C or isotopic signature) were tested by comparing nested models differing in a single variable based on AIC scores and χ2 log- likelihood tests. Effects are reported as significant where the χ2 log-likelihood test yielded a p value of <0.05, relative to the simpler model.

RESULTS

Water table recession with groundwater abstraction The amount of groundwater abstraction was less than planned; as a result the range of groundwater levels observed during the study was mostly within the levels previously recorded in the aquifer. At the impact site, the water table declined 4.3 m, from 2.4 m DGW at the start of the trial (March 2009) to 6.7 m DGW in May 2010 (Figure 7.2a). The water table at the control site also declined during the study period, but only by 1.5 m, from 0.5 to 2.0 m DGW. The daily rates of decline increased toward the end of the study period, the maximum recorded was 6 cm day-1 (Figure 7.2b). Cyclonic rainfall just before the start of the study filled the aquifer to levels that were one of the highest recorded during the previous ten years (Figure 7.2c). By the end of the study period, the water table at the impact site had fallen just below the previous ten-year minimum.

Tree water uptake from soil, surface and ground water sources The δ18O of the ground and soil water remained similar over time at each site (Figure 7.3a). Groundwater δ18O values differed by 2.3‰ between the upstream control site (at -7.1‰) and impact site (at -9.4‰), indicative of evaporation from the sandy riverbed as the groundwater flows downstream underground. Soil water δ18O varied with depth below the surface, with a similar range of values at both sites, of -13.0 to 3.5‰. Surface pools were present at the control site at the start of the study, with δ18O of -6.3 to -5.2‰. Volumetric

135 Chapter Seven soil water content in the upper 4 to 5 m also remained very similar over time at both sites (Figure 7.3b). At the control site, soil water content ranged from 5% (vol/vol) near the surface to 25% at 5 m depth, while at the impact site surface soil moisture was less than 2%, increasing to 20% in a high moisture layer at 3.7 m, then returning to 4% by ~4.5 m depth. At depths below 4.5 m, soil water content decreased over time with the increasing DGW, with much more pronounced changes over time and depth at the impact site (Figure 7.3b).

Tree xylem sap δ18O at the control site (mean -6.4‰) was generally similar to that of the groundwater at that site (-7.1‰), while at the impact site, the sap was slightly more enriched (mean -7.2‰) compared to groundwater (-9.4‰) (Figure 7.3a). At the end of the study period at the impact site, δ18O was also significantly more enriched in the sap of smaller trees, while larger trees had sap isotopic signatures closer to that of groundwater (p=0.022). There was no significant effect of tree size on sap δ18O at the earlier time points at the impact site (March, June and November 2009), or at any time point at the control site. δ2H was also analysed for each water sample, yielding results very similar to those obtained with δ18O (not shown).

Tree water use Tree water use reflected a lowering of the water table, even though groundwater levels were mostly within the historic range (Fig. 7.2). Tree daily Vs did not differ between sites at the start of the pumping trial (March and April 2009; p=0.95). However, water use at the impact site decreased significantly over the study period relative to the control site (p=0.003; Figure 7.4a). Water use at both sites fluctuated over the study period, reflecting seasonal patterns in VPD (Chapter 2), with lower rates of sap flow during winter (~3 m day-1 at the control site and ~1.5 m day-1 at the impact site) than in summer (up to 5 m day-1 at the control site and 3 m day-1 at the impact site) (Figure 7.4a). The proportion of sap flow occurring during the dark period was consistently slightly lower in trees at the impact site throughout the study period (on average lower by 4.5%, with nocturnal flow ranging from 10–41% of total daily flow at the control site, and 7–37% at the impact site), but there was no detectable effect of groundwater drawdown on nocturnal sap flow rates

(not shown). The maximum rates of sap flow velocity reached each day (Vs-max) were much more stable over time than the total daily sap flow (Vs) (Figure 7.4b). The Vs-max was lower

136 Responses of Melaleuca argentea to groundwater abstraction at

CONTROL SITE IMPACT SITE (a) 300 tree March 2009 250 xylem June 2009 ) 2 Nov 2009 200 May 2010 150

100

50 Sapwood area (cm 0 1 2 3 4

Depth (m) 5 6 source water 7 -15 50-5-10 -15 50-5-10 δ18O (‰) δ18O (‰) (b) 0 March 2009 2 July 2009 Dec 2009 4 April 2010

6 Depth (m) 8

10 0 20 40 60 80 0 20 40 60 80 100 SWC (% (vol)) SWC (% (vol))

Figure 7.3: Soil and ground water sources used by the trees at the Yule River control (lefthand panels) and impacted (righthand panels) study sites during the pumping trial period. (a) Isotopic signature (δ18O) of water extracted from tree xylem (upper panels) and from soil (lower panels, open symbols), and of groundwater obtained from bores (lower panels, closed symbols). Values are plotted against tree size (sapwood area) for xylem water, and against depth below the ground surface for source water. (b) Soil water content (SWC) with depth below the ground surface.

the impact site throughout the study (p=0.002; Figure 7.4b). At the impact site, there was a significant interaction effect between DGW and tree size on Vs-max, with smaller trees having lower Vs-max at greater DGW (p<0.001). At the control site there was no detectable effect of either DGW or tree size on Vs-max (p=0.12 for tree size, p=0.51 for DGW). Effects of tree

137 Chapter Seven size were not detectable on Vs at either site. There appeared to be an inflection point in the

Vs-max values at the impact site at ~day 230 (November 2009), after which the Vs-max began to decline. The inflection point corresponds to DGW of 5.5 m, a decline of 3.1 m since the start of the study, a depth change that was never reached by the groundwater at the control site. Toward the end of the study period, when groundwater levels at the impact site were at their lowest, fluctuations in the groundwater level at that site appear to be reflected in the tree Vs-max, with a lag time of several days (Figures 7.2, 7.4).

7 (a) 6 Control Impact 5

3 (m day-1) s V 2

0 35 (b) 30 25 20 (cm h-1) 15 s-max V 10 5 0 A MJJASON DJFMAM

0 100 200 300 400 Days after start of pumping

Figure 7.4: Tree sap flux at control and impact sites during the pumping trial study period. (a) Daily sap flux velocity V( s), and (b) daily maximum sap flux velocity V( s-max). Values are means (bold lines) and standard errors (pale lines) of five trees. The x-axis displays the time elapsed since the start of the pumping trial study period, as well as letters indicating the months of the year (from March 28th 2009 to May 12th 2010).

Leaf physiology of M. argentea in relation to lowering of groundwater

In March 2009, before the start of the pumping trial, there were no differences in Ψl evident between the sites or between trees of different size, but as the trial progressed the

138 Responses of Melaleuca argentea to groundwater abstraction predawn Ψl decreased in the smallest trees at the impact site (Figure 7.5). There were no significant changes in the predawn Ψl of the larger trees, or in the midday Ψl of trees of any size. The whole tree K, estimated from the linear portion of the relationships between Ψl and Vs, are shown in Figure 7.6 (see also Figure 2.7 in Chapter 2 for examples of the Ψl by

Vs relationships). The K of trees at the impact site decreased over the trial period, particularly in the smaller trees, while K did not change at the control site. The impact site trees also had greater K than the control site trees at the beginning of the study period.

The patterns of leaf δ13C signatures over time differed between sites (p=0.006, Figure 7.7a); δ13C increased over time at the impact site (p=0.026), and did not change significantly at the control site (p=0.11). There was no effect of tree size on δ13C detectable at either site (p=0.67 control site; p=0.24 impact site). Trees of all sizes at the impact site therefore appear to have increased their intrinsic water use efficiency progressively over the study period. Stomatal conductance (gs) measured November 2009 and May 2010 also revealed differences between sites (Figure 7.7b). The gs of trees at the impact site was lower throughout the day, and at the mid-morning time points there was also a significant interaction effect between site and tree size (p=0.0023 Nov 2009, p=0.0481 May 2010). At the impact site, small trees had lower mid-morning gs than larger trees (p=0.003 Nov 2009, p=0.034 May 2010), while there was no effect of tree size at the control site at those same times (p=0.151 and 0.099). There was no detectable effect of tree size at any other time of day at either site. There was also no detectable difference in leaf specific area between sites, or over time (data not shown).

DISCUSSION

The DGW in the Yule River borefield appeared to be increased by the abstraction regime; levels fell by 4.3 m (2.4–6.7 DGW) at the impact site, and by only 1.5 m (0.5–2.0 DGW) at the control site, over the 13 months of this study. However the impact site has clearly experienced a history of greater groundwater fluctuation than the control site, so it is difficult to determine the extent to which the increase in DGW is due to abstraction, as opposed to ‘natural’ fluctuation patterns. Nonetheless, greater DGW appears to have mildly impacted physiological functioning of M. argentea trees near the production bores, compared with trees at the upstream control site, with small trees more strongly affected

139 Chapter Seven than larger trees. By the end of the study period, the small trees at the impact site had lower

Tree sapwood area at 1.2 m height (cm2) 0 100 200 300 0 100 200 300 0 0 -0.2 March 2009 -0.5 -0.4 -1.0 -0.6 Control -0.8 -1.5 Impact -1.0 -2.0 -1.2 -1.4 -2.5 March 2009 0 0 -0.2 -0.5 -0.4 -1.0 -0.6 leaf waterMidday potential (ψ; MPa) -0.8 -1.5 -1.0 -2.0 -1.2 -1.4 -2.5 June 2009 June 2009 0 0 -0.2 -0.5 -0.4 -0.6 -1.0 -0.8 -1.5

Predawn leaf water potential (ψ; MPa) potential leaf water Predawn -1.0 -2.0 -1.2 -2.5 -1.4 November 2009 November 2009 0 0 -0.2 -0.5 -0.4 -0.6 -1.0 -0.8 -1.5 -1.0 -2.0 -1.2 -1.4 -2.5 May 2010 May 2010

Figure 7.5: Variation in leaf water potential (ψ) with tree size during the pumping trial study period. Predawn ψ is shown in the lefthand panels, and midday ψ in the righthand panels; note difference in y-axis scales.

140 Responses of Melaleuca argentea to groundwater abstraction

June 2009 November 2009 May 2010 70 (a) Control (b) Impact 60 50 40 30 20

K (ml. h-1. cm-2. MPa-1) 10 0 0 100 200 300 0 100 200 300 Tree sapwood area at 1.2 m height (cm2)

Figure 7.6: Relationship between whole tree conductance (K) and tree size (sapwood area) for trees at the (a) control and (b) impact sites. K was estimated from the linear part of the relationship between sapflux and leaf water potential.

-30 (a) Control -31 Impact

-32 δ13C (‰) -33

-34 March June November May 2009 2009 2009 2010

400 (b) Nov 2009 (c) May 2010

300

200 (mmol m-2 s-1) s

g 100

0 6:00 12:00 18:00 0:00 6:00 12:00 18:00 0:00 Time of day (h) Time of day (h)

Figure 7.7: (a) Foliage carbon isotope ratio (δ13C) and (b) instantaneous stomatal conductance (gs) of study trees at the control (closed circles) and impact sites (open circles).

141 Chapter Seven

mid-morning gs, lower predawn water potentials, and sap isotopic signatures that indicated increased use of soil water and therefore reduced access to the groundwater. In all trees at the impact site, water use was reduced, intrinsic water use efficiency increased, and whole tree hydraulic conductance reduced. These observations correspond with the finding that

M. argentea seedlings with lower water availability have lower gs and transpiration rates than seedlings under ‘optimal’ conditions of partial saturation (Chapter 6). However, the midday Ψl of trees at the impact site remained at control levels throughout the study, so the physiological changes I observed appear to have been a mild response only. Changes in leaf morphological traits associated with increasing water stress, such as reduction in specific leaf area (e.g. Smith et al., 1991; Chapter 6), were also not detectable during drawdown at the Yule River. The decreasing whole tree K at the impact site was most likely due to cavitation of xylem vessels, or resistance at the soil-root interface, or both (Sperry et al., 1998; Tognetti et al., 2004). In either case, the trees appear to have responded to the lower

K with reduction in gs, which maintained midday Ψl at control levels.

Plants can also respond to water stress with leaf drop to reduce canopy area, thereby reducing transpiration and increasing the water potential of the remaining leaves (e.g. Eamus et al., 2000; Cooper et al., 2003). I observed no obvious leaf loss or deterioration of the canopy over time at the impact site, however it is possible that some leaf area adjustments also occurred in addition to the reduction in gs. However, the extent of groundwater recession at the impacted Yule River site was relatively large, at an increase in DGW of 4.3 m over 13 months; similar and smaller declines in groundwater have resulted in much more severe effects in other riparian tree species. Populus balsamifera on Willow Creek in Alberta, Canada, suffered water stress under drawdown of 2.5 m over 2 years, with widespread early leaf abscission (Amlin & Rood, 2003). On the South Platte River in Colorado, USA, Populus deltoides suffered crown dieback of up to 30% following acute groundwater drawdown of 2 m over a period of a few weeks (Cooper et al., 2003). P. deltoides on Coal Creek in Colorado, USA, showed a threshold response with a decline of just 1m, from 2.5 to 3.5m DGW; canopy desiccation and branch loss was evident within weeks, and was associated with higher mortality rates in subsequent years (Scott et al., 1999). Populus fremontii and Salix gooddingii on the Hassayampa River in Arizona showed

142 Responses of Melaleuca argentea to groundwater abstraction a threshold response when groundwater dropped below about 3m depth from the surface, with increased canopy dieback and mortality at greater depths (Horton et al., 2001b). M. argentea trees at the Yule River therefore appear to have relatively deep root systems for phreatophytic riparian trees, with rates of water use remaining unchanged to 5.5 m DGW (a decline of 3.1 m since the start of the study). As the water table fell beyond 5.5 m, water use began to decline, primarily in small trees. In large trees there was no change in leaf water potential and little change in sap flow rates beyond 6.5 m DGW.

The rates of water table decline during abstraction at the Yule River borefield were also quite high toward the end of the study period. The maximum observed rate was 6 cm day-1, although that was not maintained for longer than a couple of days at a time, and was interspersed with declines of 2- 4 cm day-1 and small rises in the water table. Recession rates of >3 cm day-1 are considered high for many riparian tree species, at least for seedlings, and can lead to higher mortality rates and reduced growth (Segelquist et al., 1993; Hughes et al., 1997; Amlin & Rood, 2003; Stella & Battles, 2010). While the tolerance of some species to more rapid drawdown can be related to their greater drought tolerance (e.g. Horton & Clark, 2001), in the case of M. argentea which appears to be drought sensitive, tolerance of drawdown may be due to their rapid root growth rates (Chapter 6), or due to constitutively deep root systems. Nonetheless, the small M. argentea trees impacted by drawdown may have been unable to elongate roots rapidly enough, and suffered effects of mild water stress, while the larger trees’ root systems may have been deep enough or elongated rapidly enough to compensate. The type of substrate also affects survival and growth; a given rate of decline is more severe in coarser soils (Mahoney & Rood, 1992; Hughes et al., 1997). The layers of clay-type soils at the Yule River sites may have mitigated the decline to some extent, slowing the recession of the non-saturated fringe.

Tree water use patterns in response to abstraction Changes over time in the daily maximum rates of sap flow appear to be a highly sensitive indicator of changes in water availability. Toward the end of the study there was a striking mirroring of the small variations in groundwater depth, and the variations in daily Vs-max. The lag time of several days could reflect the lag in the rate at which the unsaturated capilliary fringe zone retreats as the saturated zone declines. Smaller trees had greater reduction in Vs-max, although all trees at the impact site had slightly reduced water use, and

143 Chapter Seven increased intrinsic water use efficiency. Larger trees may have reduced their water use by shortening the plateau period of high water use during the day, rather than through reduction in Vs-max (Chapter 2).

The large M. argentea trees at the impact site reduced water use and increased water use efficiency, despite a lack of any other symptoms of water stress, including no change in predawn Ψl. M. argentea appears to reduce its water use, through partial stomatal closure, in response to relatively small reductions in water availability (Chapters 4 and 5). Stomata are generally ‘inefficient’ if water is abundant, with greater losses of water for each incremental increase in carbon assimilation, and even increasing water use without any change in the observed assimilation rate (Thomas et al., 1999). For example, Salix babylonia in NSW, Australia, used substantially more water when standing within the stream, compared with nearby trees on the river bank (Doody & Benyon, 2010). Eucalyptus camaldulensis over a shallow aquifer near the Murray River, SE Australia, had slightly increased growth rates following an experimental flood treatment while Ψl was unchanged, suggesting that the trees were utilising the additional water, although they were not previously water stressed (Bacon et al., 1993). Populus fremontii trees on the San Pedro River in Arizona, USA, along an intermittent reach with ground water 3.5–5 m below the surface had rates of Vs that were less than half those of trees at a site with shallower ground water, within 3.5 m of the surface (Gazal et al., 2006). Trees with abundant water are likely to use more, even though trees at slightly ‘drier’ locations may still have access to an adequate supply.

Effects of abstraction on small trees The smaller M. argentea trees at the impact site in our study did appear to show early signs of water stress. Predawn Ψl were reduced, and isotopic signatures indicated greater uptake of soil water by small trees with groundwater recession, demonstrating their reduced access to the water table. Rates of sap flow and gs were reduced in all trees at the impact site, but to a greater extent in small trees. Similar differences among tree size classes under drier conditions have been observed in other riparian systems. Smith et al. (1991) found that changes in riparian tree morphology and physiology in the Sierra Nevada resulting from stream diversion were more pronounced in juveniles than in the mature trees, and

Donovan & Ehleringer (1991) found that Ψl, gs and photosynthesis differed between juvenile and reproductive plants of several woody species to a much greater extent at a dry

144 Responses of Melaleuca argentea to groundwater abstraction location than at a wet location. Mature riparian Populus trees have been shown to survive quite dramatic changes in hydrological regime, such as river damming and regulation; rather the problem is often that recruitment of seedlings cannot take place under the new conditions (Williams & Cooper, 2005). Sustained low groundwater levels can therefore lead to mortality of young trees, and alter the community structure (Smith et al., 1991).

Implications for vegetation management under increased abstraction As shown elsewhere, when water is abundant M. argentea consumes large quantities, but the species is sensitive to small reductions in water availability, even in the absence of water ‘stress’ (Chapters 5 & 6). The maximum daily rates of sap flow reached by the trees appears to be particularly sensitive, and this parameter has the potential to be used as an indicator of groundwater levels, or as an ‘early warning signal’ of likely vegetation water stress.

The mature M. argentea trees in this study tolerated a relatively large recession in groundwater. However, the Yule River site has historically been subjected to large fluctuation in groundwater levels, both within and between years. The trees may therefore be unusually tolerant of drawdown, and it is not known whether M. argentea would show lower tolerance in locations with more consistent historical water table levels.

Due to the more pronounced effect of DGW on smaller trees, it is possible that increased water abstraction could adversely affect the persistence of M. argentea populations in the long term. Under increased abstraction, conditions supporting recruitment may become less frequent, since the ability of seedlings to establish will require recharge events large enough to replace abstracted water, and refill the aquifer from its reduced levels. Establishment of young trees may also be impaired by higher rates of drawdown during the juvenile phase. However, the effects of abstraction on the Yule River riparian ecosystem as a whole will depend on how widespread the drawdown effects are. If the effects of abstraction are relatively localised and populations of M. argentea are therefore able to thrive at nearby unimpacted locations, the total impact is likely to be minor.

145

CHAPTER EIGHT: General discussion: temporal and spatial patterns of water use by Melaleuca argentea

This thesis demonstrates the patterns of water use by Melaleuca argentea trees at several scales, from fluxes within different portions of a tree, to seasonal and site differences in tree water use. Many of the observed patterns and physiological responses are clearly explicable in terms of the adaptation of M. argentea to the riparian environments of the Pilbara, which combine harsh atmospheric conditions above ground with highly heterogeneous and changeable conditions below ground. The water use physiology of this species is adapted to the sub-zones of highest water availability and highest disturbance within the riparian environments of northern Australia. High rates of water transport enable high rates of growth and photosynthesis in the water abundant, high VPD environment. Nonetheless, the limitation in water use apparent under extreme VPD points toward the fundamental limits and tradeoffs involved in plant water transport. The plastic root responses of M. argentea enable rapid adjustment to the disturbance and shifts in resource distribution that commonly occur around the root system. These compensatory responses include a previously undescribed mechanism involving rapid increase in the aquaporin levels and hydraulic conductance of fine roots. The plasticity and waterlogging tolerance of the root system allow these trees to thrive in the flood and disturbance prone river channels, which provide resource rich sites in an otherwise harsh environment.

The findings of this research reveal new understanding of the water use physiology and root system plasticity of M. argentea, which will in turn inform more accurate estimates of the ecological water requirements of riparian systems in the Pilbara. These results also considerably expand the relatively few studies made of riparian tree species in arid and highly dynamic environments elsewhere in the world.

ADAPTATIONS OF M. ARGENTEA

Extreme VPD and temperature conditions In northwest Australia, atmospheric vapour pressure deficit (VPD) is typically high (greater than 3 kPa; e.g. Lio et al., 2004; Bobich et al., 2010) and often extreme in the austral

146 General discussion summer, frequently exceeding 7 kPa, with maximum temperatures often greater than 40 °C (Chapter 2). Under such conditions, very high rates of transpiration are expected for leaf cooling and to meet the evaporative demand necessary to maintain photosynthesis and growth. I observed maximum rates of sap flow in M. argentea in the range of 30–40 cm h-1 and up to 5 m day-1, similar to rates observed in other semi-arid riparian trees including Populus and Salix species in Arizona, USA (Schaeffer et al., 2000; Gazal et al., 2006). While these values are indeed relatively high, they are still within the range reported for many other species, and even trees growing in cooler environments with much lower VPD can at times reach similar sap velocities (e.g. Zang et al., 1996; Wullschleger et al., 1998). I also found evidence that the water use of M. argentea did not always fully meet the evaporative demand of the atmosphere. Tree hydraulic conductance (K) varied over diurnal cycles during summer, with partial stomatal closure observed during the middle part of the day, and the relationship between water use and VPD also varied over annual cycles, most likely due to seasonal variation in tree capacitance and/or K (Chapter 2). The rate and volume of water transported to the leaf surface therefore appears to place a limit on the water use of M. argentea, despite abundant water availability to the roots.

At low to moderate evaporative demand (generally defined as VPD of 0–3 kPa), transpiration usually increases with VPD, both within individual trees over diurnal and annual cycles, and across climate gradients (e.g. Dragoni et al., 2009). However, transpiration rates reach an ‘upper limit’ under higher VPD (often at 2–3 kPa), maintained by reducing stomatal conductance (gs) and often leading to reductions in photosynthetic rate, as documented in a wide variety of species and environments (e.g. Meinzer et al., 1993; Granier et al., 1996; Hogg & Hurdle, 1997; Oren et al., 2001; Lio et al., 2004; Woodruff et al., 2010). High VPD often correlates with drought conditions both temporally and spatially, and so the reduced gas exchange is often attributable to soil drought (Chaves et al., 2002). However, the ‘saturation’ of transpiration at high VPD is also seen when water is not limiting, such as in irrigated orange trees in semi-arid Spain (Martin-Gorriz et al., 2011), rainforest trees in the Ecuadorian Andes (Motzer et al., 2005), after rainfall in semi-arid forests of the Loess Plateau region of China (Du et al., 2011), and in riparian trees in north-west Australia (Pfautsch et al., 2011, Chapter 2). Trees native to low VPD environments may show transpiration saturation at lower VPD than those native

147 Chapter Eight to high VPD regions. In Sequoia sempervirens, native to the relatively mild, humid climate of coastal California, USA, transpiration rate saturated at VPD of less than 1 kPa when grown under higher VPD conditions with irrigation in the Los Angeles basin, USA (Litvak et al., 2011). In contrast, transpiration rates of M. argentea in northwest Australia saturated at VPD of approximately 3.5 kPa (Chapter 2). Nonetheless, 3.5 kPa is still a relatively low VPD in the context of the extreme atmospheric conditions that commonly occur in the Pilbara. Transpiration rates therefore appear in general to be limited at high VPD, even in trees with access to abundant water, and native to environments where high VPD is common.

Water transport can be limited by constraints on hydraulic architecture and vessel structure. It would be expected that the optimal strategy in an environment with both high demand and high availability of water would be to maximise tree transpiration and hydraulic conductance (K). Indeed, xylem specific K (Ks) may be a key trait that maintains plant water balance; across climatic zones Ks increases with soil water availability and with temperature (i.e., with both availability and demand) (Gleason et al., 2012). The increase in Ks across climate gradients is primarily due to increasing vessel diameter, which may also increase the vulnerability to embolism (Zanne et al., 2010; Gleason et al., 2012). Many studies indicate such a tradeoff between K and embolism vulnerability, on both theoretical and empirical grounds (e.g. Pockman & Sperry, 2000; Wheeler et al., 2005; Hacke et al., 2006). However, some studies have found contradictory evidence, observing a positive correlation between transport capacity and cavitation resistance (e.g. Domec et al., 2005;

Bucci et al., 2013). In trees experiencing seasonal drought, Ks and transpiration rates might be constrained during the wet season by the requirement for higher resistance to embolism during dry periods, such as in several Eucalyptus species in northern Australian savannas (Eamus et al., 2000). However, trees growing under conditions of constant high water availability, such as M. argentea, are not constrained by seasonal water deficit. When drought does occur, M. argentea adopts a strategy of rapid leaf loss and dormancy, indicating low cavitation resistance (Chapter 6). The high rates of water use and low drought tolerance of M. argentea are consistent with a vascular structure enabling high K but low cavitation resistance.

148 General discussion

The vascular structure of trees is also constrained by mechanical strength requirements, since an increase in vessel lumen area is associated with structural weakness (Enquist, 2003). Riparian trees require high wood compression and shearing strengths, as the environment is frequently subject to high energy flood disturbance (Naiman et al., 2005; Niklas & Spatz, 2010). Tree hydraulic architecture and xylem structure appear in general to be consistent with the maximisation of transport efficiency within the constraints of mechanical strength (McCulloh & Sperry, 2005). In addition, xylem embolism could still pose risks to transport when water is abundant, during periods of extreme evaporative demand, if Ks is constrained by strength requirements. A combination of wood mechanical strength and resistance to cavitation may therefore be the major limiting factors in the rate of water transport by trees such as M. argentea.

Capacitance is also likely to play a role in the water transport strategy of riparian trees. High wood and tissue capacitance can allow the use of internal water storage to prevent highly negative water potentials developing (Meinzer et al., 2009). Capacitance may thus partly overcome any tradeoff that exists between hydraulic efficiency and safety (Sperry et al., 2008), though would probably not ameliorate a tradeoff between efficiency and strength. Low wood density (low dry mass per volume) is associated with high sapwood capacitance (Hultine et al., 2003b; Gartner & Meinzer, 2005; Scholz et al., 2007), but lower mechanical strength (Niklas & Spatz, 2010; Zanne et al., 2010). M. argentea wood is of intermediate density at 0.55 g cm-3, perhaps reflecting a balance between these conflicting requirements. Both M. argentea and the co-occurring E. camaldulensis in wet habitats in northwest Australia appear to draw upon internal water stores (Pfautsch et al., 2011 Chapter 2). Diurnal cycles of dehydration and rehydration were observed in E. camaldulensis (Pfautsch et al., 2011), and my results with M. argentea trees suggested that internal stores were depleted and replenished over annual cycles (Chapter 2). Semi-arid riparian trees on other continents may also utilise capacitance, for example the invasive Tamarix chinensis in southwestern USA displayed diurnal changes in stem wood water content (Moore et al., 2008). Many trees in the seasonally dry tropics have high capacitance, which appears to allow these species to retain their leaves during the dry season, and to increase their transport capacity (Goldstein et al., 1998; Meinzer et al., 2003; Borchert & Pockman, 2005). Stem water storage appears to be a common strategy in trees

149 Chapter Eight exhibiting high rates of water use, reducing their vulnerability to cavitation without compromising transport efficiency, but the extent of capacitance might be limited by mechanical strength requirements in some cases.

The tradeoffs and limitations among the various functions of wood tissues (water transport efficiency, resistance to embolism, water storage capacity, mechanical strength and resistance to decay) remain only partly resolved, but will be better clarified with further study of wood biomechanical and hydraulic properties (Chave et al., 2009; Fournier et al., 2013). Compilation of wood trait databases across species and biomes is currently underway, with the aim of improving understanding of the inter-relationships among wood properties and functions (Kattge et al., 2011; Choat et al., 2012). The increasing availability of technologies such as nuclear magnetic resonance, microtomography and synchrotron x-ray sources are also allowing in-planta analysis of wood functioning, including time-lapse imaging of xylem water flow (e.g. Holbrook et al., 2001; Windt et al., 2006; Kim & Lee, 2010; Page et al., 2012; Brodersen et al., 2013). Similarly, changes in wood micro- and nano-structures occurring under mechanical stresses can be examined in detail and in real time using these imaging approaches (Müller, 2009; Fernandes et al., 2011). These developments will help to resolve questions of the maximum limits to hydraulic functioning, and species such as M. argentea will provide good examples for analysis of vascular systems operating near the upper limits of water transport capacity.

Heterogeneous belowground environment Large and long-lived species in intermittent riparian environments, such as M. argentea, encounter especially heterogeneous belowground conditions. All plants are exposed to heterogeneity of numerous properties within their root-zone and canopy, but riparian environments are particularly heterogeneous at multiple scales (Naiman et al., 2005; Pettit & Naiman, 2005). Riparian heterogeneity is due primarily to the fluvial processes creating cycles of disturbance and resource availability, and to the interactions between this resource heterogeneity and biotic processes such as vegetation type and microbial activity (Naiman et al., 2005). Highly variable flow patterns, typical of intermittent waterways in semi-arid regions such as the Pilbara, further exacerbate the degree of spatial and temporal heterogeneity. The basis of most plant responses to belowground heterogeneity is the efficient use of available nutrient and water resources. When patches of water and nutrients

150 General discussion are encountered, roots are able to detect and respond to them, however species appear to vary widely in the extent to which they exploit patches (e.g. Huang et al., 1994; Gallardo et al., 1996; Einsmann et al., 1999; Kembel & Cahill, 2005; Hodge, 2006; Neatrour et al., 2007; Mommer et al., 2011). Semi-arid riparian trees have a high demand for water to maintain transpiration and growth in the high VPD environment, and therefore are expected to utilise resource patches. The high degree of root system plasticity in response to changes in the distribution of water observed in M. argentea (Chapters 4, 5, 6) most likely constitutes a strategy to maintain high rates of resource capture.

M. argentea displayed rapid root growth and physiological response times within patches of high water availability, while simultaneously maintaining roots in previously rich patches for up to several months, even after the water source was no longer present (Chapters 4 & 5). Heterogeneous resource distribution can theoretically increase plant performance in responsive species such as M. argentea, by allowing smaller investments in roots into the rich patches only (foraging precision) (Hutchings & John, 2004; Magyar et al., 2007). However, high foraging precision is only likely to be advantageous if conditions change slowly relative to the response time of the roots, and/or if resource distributions are relatively predictable (Alpert & Simms, 2002; Dunbabin et al., 2004; Hutchings & John, 2004). A permanently accessible water table is a highly predictable element of the habitat in which M. argentea occurs (Masini, 1988), and accordingly trees appear to readily invest in large roots which tap this water source, providing the majority of the water to the tree, at least during drier periods (Chapter 4, 7). Upper soil layers can repeatedly experience pulses of water and nutrients, and depressions in the creek bed forming temporary pools are likely to refill with further rainfall. Correspondingly, M. argentea trees maintained surface roots in the field for several months despite relatively little rainfall, and aquatic root mats were partially maintained for at least 4 weeks after draining of the pools in glasshouse experiments (Chapters 4 & 5). At the same time, high root growth rates can be important in effectively exploiting ephemeral patches (Kembel & Cahill, 2005). Rapid root growth and physiological responses (such as changes in root hydraulic conductivity) are also likely to be beneficial in the event of dramatic shifts in resource patterns, such as those following a destructive flood.

151 Chapter Eight

Water was the focus of the present study, however, nutrient availability will undoubtedly play a role in the root responses of M. argentea. The relative importance of water versus nutrients on root responses varies among studies reported in the literature, and the effects of water and nutrients can also interact (Pregitzer et al., 1993; Coleman, 2007; Zhou & Shangguan, 2007). In pot experiments, precise foraging for nutrients is commonly reported, including in riparian trees such as Nyssa aquatica and Fraxinus pennsylvanica (Blair & Perfecto, 2004; Neatrour et al., 2007). However, root foraging patterns can be imprecise with respect to water in riparian trees with access to abundant water. For example, fine root distribution did not closely reflect the distribution of water in Populus fremontii seedlings grown in containers with vertical heterogeneity of water availability (Snyder & Williams, 2007). Similarly, in this study when M. argentea seedlings were planted into split-root containers mimicking access to an aquatic pool, the proportion of fine roots in the aquatic pool remained unchanged eight weeks after planting, despite the higher water availability in that compartment (Chapter 5). In contrast, rapid proliferation of roots into aquatic pools by M. argentea has been observed in the field (personal observations; Graham, 2001). Natural surface pools can also be nutrient rich, and the lack of proliferation in the controlled experiment may have been due to the homogeneous distribution of nutrients between compartments, while water supply was already adequate. The primary response of riparian trees in water-abundant conditions may be to nutrient availability, since nutrients are usually in more limited supply. Nonetheless, both water and nutrient uptake will preferentially occur in the most densely rooted zones, regardless of the primary response trigger.

My research has demonstrated considerable plasticity of root behavior in M. argentea, but much remains unknown about how roots detect and signal their responses to soil resources, especially to water (hydrotropism) (Cassab et al., 2013). My study illustrates how the lack of a chemical signal (in this case ABA; Appendix C) can itself be an important part of the signalling process, generating flow-on effects to other parts of the plant. The apparent absence of a root-to-shoot signal under partial root zone drying in M. argentea led to demand-driven compensatory responses in the remaining wet part of the root system (Chapter 5). Many plant responses to water are generated largely through non-chemical, hydraulic means, for example water potential and cell turgor pressure can directly affect

152 General discussion stomatal conductance and growth (Brodribb, 2009; Kroeger et al., 2011). Genomic approaches have revealed a multitude of gene expression responses to hydrostatic pressures in bacteria and yeast (Ishii et al., 2005; Bravim et al., 2012), demonstrating the potential for direct, molecular effects of hydrostatic pressure on cell functioning. The water flow capacity of aquaporin channels also appears to be directly modulated by turgor pressure, which most likely induces conformational changes (Wan et al., 2004). Further investigation of gene expression patterns and protein functioning in response to hydrostatic pressure in plant roots will improve the understanding of root water detection and response at the molecular level.

Under conditions of spatial moisture heterogeneity, water may be passively transferred through root systems from wet to dry soil regions (hydraulic redistribution; HR). The acropetal flows in surface lateral roots of M. argentea during dry periods demonstrate the transfer of groundwater through the root system into shallow roots residing in the dry surface soil layers (Chapter 4). HR clearly improves root survival in dry soil portions (Domec et al., 2004; Bauerle et al., 2008), most likely enabling the almost instantaneous resumption of water uptake observed following rewetting, such as in pear (Pyrus communis) roots under alternating drip irrigation (Kang et al., 2003), and in M. argentea roots both in the field following rainfall (Chapter 4) and in glasshouse experiments during partial root-zone drying and rewetting (Chapter 5). Although the occurrence of HR is very widespread, there are a few documented cases of plants not displaying HR despite moisture gradients across their root system, and so HR may not necessarily be an inevitable consequence of heterogeneity. Species showing little or no HR activity, such as Quercus margaretta in South Carolina, USA, appear to rapidly shed drying roots, preventing loss of water into the dry soil, a mechanism which could itself be beneficial if total water availability is limited (Ludwig et al., 2003; Espeleta et al., 2004; Prieto et al., 2012). Differences among species in HR activity may be related to root hydraulic conductivity; low conductivity may reduce the rate of HR, leading to fine root mortality in dry soils, which would further prevent HR (Domec et al., 2004; Espeleta et al., 2004). The substantial HR observed in M. argentea may thus be an indication of high root system hydraulic conductance, likely to be an adaptive trait given the requirement for high rates of water transport, and given that water availability is rarely limiting across the root system as

153 Chapter Eight a whole. HR might also benefit M. argentea by enabling increased nutrient uptake from surface soil layers, either as a side effect of high root conductance, or as an adaptive trait in its own right.

The floods that bring pulses of water and nutrients also cause physical disturbance, through erosion and deposition of sediments along river channels. Riparian trees are dependent upon this disturbance for recruitment, since their propagules can generally only establish in bare, moist sediment beds (e.g. Stromberg et al., 2010b). However, established trees must then also withstand the impacts of burial in sediment and inundation, or alternatively scouring of sediment and uprooting; both types of disturbance can lead to tree mortality (Friedman & Auble, 1999; Bornette et al., 2008; Asaeda et al., 2010). For highly waterlogging tolerant trees such as M. argentea, the greatest threat to survival appears to be sediment erosion, which may cause the destruction or drying out of part of the root system, impacting upon the anchorage role of the roots, as well as reducing the capacity for resource capture (Kazda & Schmid, 2009; Asaeda et al., 2010). M. argentea roots were able to rapidly compensate for part of the root system drying out by increasing rates of uptake from other portions of the root system (Chapter 5), a response which may aid recovery from flood scouring.

Plastic and responsive growth patterns appear to be key traits in disturbance-prone riparian sites, in particular deeply anchored root systems combined with an ability to rapidly re-sprout both shoots and adventitious roots from stumps (Bornette et al., 2008). For example, Melaleuca leucadendra in the Burkedin River catchment in NE Australia displays greater incidence of re-sprouting and clonal growth at higher disturbance locations than at low disturbance sites (Chong, 2008). The most disturbance prone positions in the riparian zone appear to be occupied by specialised species that display these highly plastic strategies. For example, tree species were strongly segregated within the Koike Lake caldera in Japan; species with life histories well adapted to frequent disturbance such as Neolitsea aciculata and Quercus gilva occurred within the river channels, while other species were confined to more stable zones (Ito et al., 2006). M. argentea in the Pilbara region is confined to the wettest locations closest to the river channels, and possesses a relatively deep root system, an ability to readily re-sprout following drought, as well as rapid production of shallow root mats in flooded soils and surface pools (Chapter 5, 6, 7). The ability of M.

154 General discussion argentea to plastically alter growth and response patterns of both above and below ground structures is likely to be important for survival in disturbance-prone riparian locations.

ECOSYSTEM SCALE IMPLICATIONS OF WATER USE PATTERNS

Responses of trees to their environment have effects at the ecosystem scale. Transpiration by riparian trees can be a significant element of the total hydrological budget in some cases, particularly in dryland regions with high VPD (Scott et al., 2000; Dahm et al., 2002). For instance, total evapotranspiration (ET) from riparian vegetation was estimated at 20–33% of total losses from a riparian corridor in the Middle Rio Grande in semi-arid New Mexico, USA (Dahm et al., 2002). Water fluxes through tree roots can be particularly important in ecosystem functioning, not only through their influence on total ET, but also through their effects on soil processes and understorey vegetation (Prieto et al., 2012). Soil moisture heterogeneity and tree root processes can also have large impacts upon critical ecosystem parameters such as catchment scale water runoff and soil carbon storage (e.g. Ren & Henderson-Sellers, 2006; Zheng & Wang, 2007; Ren et al., 2008). Knowledge of the spatial and temporal patterns of water uptake and root functioning, such as those investigated in this thesis, can therefore be valuable in land and water management by providing insights into ecosystem scale processes.

A striking finding of this study was the high variability in water fluxes through M. argentea trees, at multiple scales. Quantifying and explaining such spatiotemporal heterogeneity is a central issue in modelling ecosystem scale fluxes, and in the fundamental understanding of ecosystem functioning (Asbjornsen et al., 2011). While quantifying hydrological fluxes is critical in heavily populated areas (Schmandt, 2002; Jury & Vaux, 2007), determining aquifer fluxes and budgets in the less intensively managed Pilbara region is nonetheless important in assessing likely impacts and recovery rates of hydrological perturbations (Kirkpatrick & Dogramaci, 2010). For Pilbara waterways, the contribution of tree water use to total hydrological fluxes is likely to vary widely between locations. In areas such as the Yule River coastal plain, with a shallow aquifer beneath wide sandy river beds, evaporation is likely to be the major component of ET and transpiration may be relatively negligible. However, at Marillana Creek in the Hamersley Ranges, ET accounts for an estimated 20% of losses from the aquifer and the contribution of

155 Chapter Eight transpiration is likely to be significant though remains unquantified (Kirkpatrick & Dogramaci, 2010). Temporal variation in sap flow by M. argentea in undisturbed habitat may be largely predictable from meteorological variables, and over annual scales may also be sufficiently estimated from short periods of measurements (Chapter 3). However, spatial variation in flux rates were considerable, for reasons that remain unclear, and more intensive sampling is required to resolve spatial patterns of water use in M. argentea among sapwood regions, trees, stands and waterways.

Soil moisture is a key parameter in models of ecosystem water fluxes, and tree responses to soil moisture and moisture heterogeneity can therefore be critical. Under heterogeneous soil moisture, M. argentea root systems compensated for partial root zone drying (Chapter 5), and also transferred water through the root system from wet to dry regions (Chapter 4); both of these processes are likely to affect ecosystem water use. In some tree species, moisture heterogeneity such as drying of the surface soil layers is expected to reduce water use due to stomatal responses to partial root zone drying (Guswa, 2005; Zheng & Wang, 2007). However, the findings reported in this thesis indicate that water use by mature M. argentea trees in natural habitat is likely to be unaffected by surface soil moisture, and to remain high throughout a wide range of conditions, as the root system readily adjusts according to the spatial water distribution (Chapter 2, 5). Hydraulic lift may also effect ecosystem net productivity and water use, since transfer of water to the surface through tree roots can supply water to understorey vegetation, and increase rates of soil evaporation and nutrient cycling (e.g. Brooks et al., 2002; Peñuelas & Filella, 2003; Scott et al., 2008; Hawkins et al., 2009; Domec et al., 2010; Armas et al., 2012). For example, using deuterium as a tracer, water transfer was observed from deep layers into surface soils by Pinus nigra, where it was taken up by small Quercus ilex trees and sprouts (Peñuelas & Filella, 2003). Hydraulic lift by a grass in controlled experiments increased the rate of surface organic matter decomposition and plant nitrogen uptake (Armas et al., 2012). Through the transfer of deep water to the surface, the presence of M. argentea is likely to increase ecosystem ET during the early months of dry periods, and may extend the length of time that water is available in the upper soil layers (Chapter 4). However, the period of hydraulic lift may be of limited duration, and is therefore unlikely to indefinitely sustain understorey plants and soil processes throughout dry periods.

156 General discussion

POTENTIAL IMPACTS OF ALTERED WATER REGIMES ON M. ARGENTEA

Riparian trees are adapted to and dependent upon the natural water flow regimes under which they have developed. In many parts of the world, alterations to flows caused by human activities such as dam construction, water abstraction or modification of channel morphologies are frequently deleterious to tree health and ecosystem integrity (Lytle & Poff, 2004). Consequently, we might expect perturbations to natural flows to pose significant threats to riparian vegetation in northwest Australia. In parts of the Pilbara, iron ore deposits are mined from below the water table, which requires abstracting groundwater that must then be discharged elsewhere (Abbott et al., 2010; see also Chapter 1). The release of this excess water into what are normally intermittent waterways with only occasional surface water has raised concern over the effects of prolonged flooding of the riparian vegetation. Prolonged inundation has occurred in other parts of the world upstream of dams and caused widespread mortality and lack of regeneration in riparian species. For example, dam enlargement on the Strymon River in northern Greece led to extensive loss of waterlogged Salix alba woodlands (Crivelli et al., 1995). However, M. argentea is highly tolerant of long term waterlogging (Chapter 6), and increased flows are unlikely to cause major declines in this species. Indeed, greater water availability might in some cases lead to greater recruitment and establishment, faster growth rates and expansion of M. argentea populations. Elsewhere, inundation of trees following reservoir construction in Arkansas, USA, lead to increased growth of the more waterlogging tolerant Quercus lyrata, with concurrent mortality of less tolerant species including Q. phellos and Q. nuttallii (King et al., 1998). Although some Melaleuca species can be highly invasive (Lopez-Zamora et al., 2004), and M. argentea might conceivably invade new areas during riparian inundation, M. argentea is also very drought sensitive, and these new recruits would be unlikely to persist once discharge ceased at the completion of mining and groundwater returned to pre-mining levels. Some other riparian species in the Pilbara may be adversely affected by inundation, but effects on M. argentea appear to be of little ecological concern.

In contrast, groundwater drawdown resulting from dewatering of iron ore deposits, and through abstraction for town and industry water supplies (Chapter 1) is likely to cause declines in health and survival of M. argentea in some cases (Chapters 6 & 7). However, some mature M. argentea trees appear to have relatively deep and/or responsive root

157 Chapter Eight systems, which may be partly dependent on site; for instance trees at the Yule River were able to tolerate groundwater abstraction leading to a greater than 4 m recession over 13 months (Chapter 7). However, whether this is typical of M. argentea trees throughout the Pilbara is far from clear, since the levels of the Yule River aquifer show considerable natural fluctuations of greater than 5 m, and so the trees may have been pre-adapted to the large and rapid recession rates. Likewise, the maximum rates and extent of drawdown that would tolerable for M. argentea are currently unknown.

The sequence of responses with decreasing water availability that were observed in M. argentea might be useful in predicting the approach of major drought stress at sites impacted by drawdown. While M. argentea is highly adaptable provided water is available to some part of the root system, the species has very limited resistance to a limitation in water availability across the root system as a whole. If groundwater levels fall below the range of the root system, an abrupt and rapid decline in tree health would be expected (Chapter 6). Other riparian tree species subject to groundwater abstraction show similar threshold-type patterns of decline, for example a groundwater decline of just 1 m at Coal Creek in Colorado, USA, lead to widespread canopy desiccation and branch loss in Populus deltoides within weeks, and was associated with higher tree mortality in subsequent years (Scott et al., 1999). An imminent collapse in tree health can therefore be difficult to predict, with little visible early indication. However, after foliage loss M. argentea appears to survive for some time in a dormant state; in a glasshouse experiment droughted seedlings recovered when re-watered 45 days after initiation of leaf shedding (Chapter 6). The length of time the trees might survive without water while dormant in the field is unknown, and warrants further investigation. The physiological parameters of leaf water potential and sap flow velocity are able to provide quite sensitive indications of early drought stress (Chapter 6, 7). These parameters are more demanding and expensive to monitor than canopy cover and visual assessments of canopy health. In addition, the values at which major xylem dysfunction occurs in M. argentea have not yet been ascertained under field conditions, and in the case of sap flow velocity the threshold values are likely to vary widely among individuals (Chapter 3). Further calibration under field conditions is therefore required, before these parameters can be used to accurately monitor for impending ecosystem ‘stress’ and decline.

158 General discussion

Mild levels of inundation and abstraction that are tolerated well by mature trees can still impact upon recruitment, leading to loss of trees from the impacted area in the longer term. Of particularly concern for recruitment is the homogenisation of flow regimes and elimination of the peak flood events, as is often the case on dammed and regulated rivers. One thoroughly documented example is that of the Populus fremontii and Salix gooddingii forests along the semi-arid rivers of southwestern North America, where distinct cohorts of trees clearly correspond to past major floods, and forests are currently in decline along highly regulated rivers where large floods no longer occur (Stromberg et al., 2010b). However, such intensive regulation and dam construction is uncommon in the Pilbara, and so even in impacted waterways, extreme flood events can still occur largely unimpeded. Tree recruitment is therefore unlikely to be eliminated completely from impacted waterways. Nonetheless, groundwater abstraction could reduce the frequency of successful recruitment, by reducing water availability to seedlings during the early establishment phase (Chapter 7).

Hydrological perturbations can alter the quantities of water used by riparian vegetation. Stand and ecosystem scale water flux estimates based on values at unimpacted sites might therefore be inaccurate for impacted areas. Transpiration rates of M. argentea seedlings were reduced under both drought and stagnant waterlogging of the entire root system (Chapter 6). Rates of water use also decreased as groundwater declined during abstraction at the Yule River (Chapter 7). The water use of most other riparian trees also appears to vary with water availability. Riparian trees in southwestern USA, such as Populus deltoides and Tamarix ramosissima, can show substantial natural inter-annual variation in water use, which appears to relate at least partly to the depth to the water table (Devitt et al., 1998; Dahm et al., 2002). P. fremontii and Salix exigua near a reservoir in Utah, USA, reduced water use during draining of the reservoir, with rates of water use recovering as the reservoir re-filled (Hultine et al., 2010). On the Ain River in France, water use by Populus nigra and Fraxinus excelsior responded to inter-annual variations in river flows (Singer et al., 2012). Intermittent flooding can increase water use by riparian trees, but longer term inundation can in some species lead to reductions in water use (Akeroyd et al., 1998; Parolin & Wittmann, 2010). The apparent sensitivity of the rates of water use by M.

159 Chapter Eight argentea to water availability suggest that anthropogenic variation in water regimes could significantly influence the transpiration rate of this species.

CONCLUSION

M. argentea clearly has high water requirements, and is likely to be adversely affected by reductions in water availability, either through changes in recharge regime due to shifts in climate patterns, or occurring through groundwater drawdown. My findings illustrate the need to better characterise the considerable spatial variation in tree water fluxes, if accurate volumetric estimates of ecosystem water requirements and hydrological fluxes are to be derived. I also observed a sequence of responses in M. argentea with decreasing water availability that with further calibration under field conditions, has the potential to provide sensitive methods to monitor for impending ecosystem water ‘stress’ and decline. Young trees are likely to be more strongly affected by hydrological perturbations. Accordingly, the processes of recruitment and establishment should be key considerations in managing impacts if groundwater drawdown is spatially widespread, regardless of the severity of the drawdown in terms of water table depth and rate of recession. Water management plans for the riparian environments of the Pilbara, and of riparian environments more generally, should carefully consider the often complex responses of dominant riparian tree species.

160

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192

APPENDIX A: Verification of methods used to measure stomatal conductance in the mid-canopy of mature trees

Introduction

Stomatal conductance (gs) was measured throughout this thesis using a leaf porometer. When conducting field measurements on mature trees, it was not possible to access leaves within the mid-canopy directly, so the viability of taking measurements from severed foliage was tested, to determine whether leaves sampled from the mid-canopy with pole clippers could provide a reasonable estimate of intact gs. While measurements from severed branches can be acceptable, the response of stomata to excision varies among plant species, and the utility of such measurements should therefore be tested for each species (Santiago & Mulkey, 2003; Miyazawa et al., 2011). In addition, once M. argentea leaves are severed from the canopy it can be difficult to identify the abaxial and adaxial surfaces, since the appearance of leaves is very similar on each surface, and mature leaves frequently attach to the stem in an almost vertical orientation. Therefore, differences in gs and stomatal density between the two leaf surfaces were also tested, to assess the importance of standardising or accounting for leaf surface during measurement of gs.

Materials & methods Testing took place on six seedlings collected from Weeli Wolli Creek, upstream from Marillana Creek, and growing in pots in a glasshouse at the University of Western

Australia. The gs measurements were taken with a leaf porometer (Decagon Devices, Pullman, WA, USA), at midday, on sunlit, young, mature leaves, with air temperatures recorded at the leaf surfaces during measurements of 28–32 °C. Stomatal density was measured from leaf surface images taken through a microscope (method detailed in Chapter 6, example image in Appendix D).

Results & Discussion Figure A-1 shows the comparison between repeated measurements taken on leaves attached to the plants (control), on excised single leaves, and on leaves attached to excised branchlets. Stomatal conductance declined slightly with repeated measurement in the control leaves. Leaves on excised branchlets gave similar values to the control, when

193

measurements were conducted within minutes of severing from the plant. However, when single leaves were severed, their gs increased during the five minutes after severing that was examined in this experiment. A transient increase in gs (the ‘wrong-way’ response) is commonly observed following perturbations of hydraulic supply or demand, including leaf severing, and is most likely due to a decline in epidermal and guard cell turgor passively causing a widening of the stomatal aperture (Buckley, 2005; Powles et al., 2006). Following the transient ‘wrong-way’ response, the gs then declines in the expected ‘right-way’ response, attributed to active guard cell closure through osmo-regulation. Severing individual leaves in our test experiment clearly induced the stomatal ‘wrong-way’ response, and the duration of our experiment was probably too short to observe the eventual decline in gs, which may not occur until more than 10 minutes after severing (e.g. Hoshika et al., 2011). Severing of branchlets, however, appears to have buffered the effect on stomata, yielding gs values similar to attached leaves.

200 control branchlet excised leaf excised

150

100

0 since time zero (mmol m-2 s-1) time zero since s -50

-100 Change in g

-150

-200 0:00 1:00 2:00 3:00 4:00 5:00 6:00 Time (minutes)

Figure A-1: Stomatal conductance (gs) of leaves measured repeatedly at time intervals, while either remaining attached to the plant (control), or after severing of from the plant at time zero. Leaves were severed either at the petiole (leaf excised) or as a branchlet (branchlet excised). Values of gs at each time point were subtracted from the pre-severing value for that leaf (for severed treatments), or from the first measurement made for that leaf (control treatment). Values are mean ± standard error of four to six leaves, each leaf from a different plant.

194 Appendices

The gs and stomatal density were slightly higher on the adaxial than the abaxial leaf surfaces in four out of the five leaves tested. However the differences were small; much smaller than the differences observed among the individual ‘equivalent’ leaves, and indeed non-significant in paired t-tests (p > 0.05; Table A-1).

-2 -1 Table A-1: Difference in (a) stomatal conductance (gs; mmol m s ) and (b) stomatal density (stomata mm-2) between adaxial and abaxial leaf surfaces of Melaleuca argentea seedlings.

Leaf*

(a) gs 1 2 3 4 5 adaxial 373.2 484.8 313.0 287.9 432.9 abaxial 244.0 483.9 392.7 284.6 347.8 difference 129.2 0.9 -79.7 3.3 85.1

(b) stomatal density 1 2 3 4 5 adaxial 444.3 308.9 499.0 355.1 360.5 abaxial 502.5 305.2 473.6 354.5 356.6 difference -58.2 3.7 25.4 0.6 3.9 *Each leaf was from a different plant, and a different set of leaves were used in parts (a) and (b).

Conclusions

All field measurements of gs presented in this thesis were therefore conducted by severing branchlets from the canopy, and completing the measurement as quickly as possible after cutting; generally within 60 seconds. Measurements were performed on either side of a leaf, which aided in minimising the delay between cutting and measurement, since time was not spent attempting to identify the surfaces.

195

APPENDIX B: Supplementary information for Chapter Five

The contents of this appendix were included as Online Supporting Information with the published version of Chapter 5.

Table B-1: Moisture release properties of the river sand used in this study. Values are means ± standard error of three replicates.

Applied pressure (MPa) Soil water content (% v/v) 0.001 33.9 ± 0.4 0.005 21.0 ± 0.6 0.01 1.52 ± 0.09 0.03 1.44 ± 0.04 0.1 1.32 ± 0.06 1.5 1.1 ± 0.03

100 Figure B-1: Water content of the soil 80 compartments during treatment. Plants were subjected to partial root 60 Control zone drying (PRD), PRD with restricted PRD water (PRD-RW), complete root drying 40 PRD-RW (CRD) or remained fully watered as CRD controls. Treatments were initiated at 20 time 0 by draining the aquatic com- SWC (% field capacity) partment. Values are means ± standard 0 024 6810 12 14 error of five to six plants. Time after treatment (days)

1200

1000 Figure B-2: Relationship between fine ) 2 800 root surface area and dry mass for Melaleuca argentea seedlings. Fine 600 roots were sampled from 12 plants, each point is a sample from a single 400 R2= 0.87 plant. Due to the strong linear correla- Surface area (cm tion, dry mass was used as a proxy for 200 fine root surface area in the calculation of root hydraulic conductance (Lp). 0 0 0.5 1.0 1.5 2.0 Dry mass (g)

196 Appendices

Method of measuring of plant water uptake from each split-pot compartment Plant water uptake was measured as the change in pot weight over the time intervals between scheduled watering. During each watering, the soil compartments were watered to capacity, and the aquatic compartments filled to a consistent level so that water use since the previous watering could be calculated by subtraction from the fully hydrated weights. In order to accurately refill aquatic compartments, a hole was drilled in each compartment 3 cm from the top, and sealed over with waterproof tape. The pots were then filled to above the level of the hole, allowed to drain for 2 min, and the holes re-sealed. Through this method the refilling of the aquatic compartments was reproducible to within 5 ml (2- 4% of daily plant uptake from this compartment). Other methods, such as refilling to a line marked in the pots, resulted in levels of error that were at times ~20% of the daily plant water uptake from the compartment. At each measurement time, each split-pot unit was weighed just before scheduled watering, then the aquatic compartments refilled as described, and the pots weighed again. Water uptake from the soil compartments could then be calculated by subtracting the weights after aquatic refilling, from the weight of the fully hydrated pot. Water uptake from the aquatic compartment was calculated by subtracting the weights before aquatic refilling from the weights after refilling. Rates of evaporation were also accounted for, by subtracting the water loss over the same time period from pots without plants. Water uptake was measured in all plants over several days just prior to the start of treatments, and water use during the treatment periods expressed as a change relative to the pre-treatment water use, in order to normalise variation due to differences in plant sizes.

197

(a) PIP1 (b) PIP2

kDa 12 1 2 3

97 66 66 45 45

30 30

Figure B-3: Specificity of PIP antibodies againstMelaleuca argentea fine root total protein samples. Proteins were extracted from fine roots collected from the hydrated zone of plants subjected partial root-zone drying, and from control plants. Total protein (12–15 μg) was separated by SDS-PAGE, blotted onto nitrocellulose membrane, and incubated with antibodies against (a) PIP1 or (b) PIP2 subfamily proteins. The positions of the size marker bands are shown beside the images. The multiple bands in each lane appear to represent the mono-, di- and trimeric forms of PIP proteins, as demonstrated by the single trimer-sized band when the reducing agent dithiothreitol was added to a sample at a 100-fold lower concentration than usual (panel (a), lane 2). Expected size of the monomers is 23–31 kDa (Maurel et al., 2008). Lanes in panel (b) are three different root protein samples.

198 Appendices

800 (a) Aquatic roots

600

400 Control 200 PRD-RW Rewet PRD-RW

0 (b) Soil roots

Water uptake (ml) 200

100

0 22 24 26 28 30 32 34 Time after PRD treatment (days)

Figure B-4: Recovery of root water uptake following re-wetting of the dried root portion after 28 days of partial root-zone drying (PRD). Saplings of Melaleuca argentea had roots divided between an aquatic compartment and a soil compartment. The PRD treatment was applied by draining the aquatic compartment while the soil compartment remained watered, although total water supply was restricted, inducing water stress and a reduction in total water use by treated plants relative to controls. Water uptake from (a) aquatic compartments and (b) soil compartments is shown for control (untreated) plants, plants under PRD with restricted water (PRD-RW), and plants under PRD-RW treatment for 28 days followed by re-wetting of the dried aquatic compartment (PRD-RW re-wet; re-wetting point is indicated by arrows). Values are means ± standard error of four to six plants. The rapid resumption of water uptake by the aquatic roots is evidence that some fine roots survived the PRD treatment.

199

APPENDIX C: Tissue ABA content during partial root zone drying (Chapter Five)

Measurements of abscisic acid (ABA) were completed subsequent to the publication of Chapter 5.

100 800 (a) Leaves (e) Leaves 80 600 60 Control PRD 400 40 Drought 20 200

0 0

(b) Aquatic roots (f) Aquatic roots mass) ABA (ng g-1 dry 140 250 120 100 200 80 150 60 100 40 50

ABA (ng g-1 dry mass) 20 0 0 (c) Soil roots (g) Soil roots 140 400 120 100 300 80 60 200 40 100 20 0 0 Control 24 h 48 h 1414 Treatment Days after treatment 80 4 (d) Xylem sap (h) Xylem sap ABA (ng g-1 fresh mass) 60 3 40 2

1 20 ABA (ng g-1 fresh mass) ABA (ng g-1 fresh 0 0 12 1414 Days after treatment Days after treatment

200 Appendices

 Figure previous page: Abscisic acid (ABA) content of leaves, roots and xylem sap in Melaleuca argentea seedlings under fully watered (control), partial root zone drying (PRD) and drought treatments. Seedlings had roots divided between an aquatic compartment and a soil compartment. The PRD treatment was applied by draining the aquatic compartment while the soil compartment remained watered, and the drought treatment applied by draining the aquatic compartment and also ceasing watering of the soil compartment. ABA was measured by enzyme-linked immunosorbent assay, in freeze dried material from a subset of plants from experiments 1 (panels d−g) and 3 (panels a−c); full descriptions of experiments are provided in the main text of Chapter 5. Values are means ± standard error of one to three plants, note differing axis scales in each panel.

Plants under PRD treatment showed no significant change in ABA levels relative to controls in any examined tissue (a−d). Under drought treatment, little or no change in ABA levels were observed during the first few days of treatment, but marked increases in ABA were seen in leaves and sap by the14th day of drought (e, h). Results from roots of droughted plants were less clear, but drought did appear to induce smaller increases in root ABA, at least at some timepoints (f, g). PRD in M. argentea therefore appears not to elicit an ABA root-to- shoot signal from the drying portion of the roots, in contrast with findings in many other plant species. However, M. argentea still clearly possesses the capacity to generate ABA in response to water stress, as elevated levels were detected during drying of the entire root system. See also the discussion of expected signalling mechanisms in the main text of Chapter 5.

201

APPENDIX D: Examples of micrographs used for anatomical measurements in Chapter Six

(a) (b) EP

VB GC T OG

PM SM PM

A EN EN

ST ST

(e) (f)

EN ST ST

Examples of micrographs used for measurement of leaf and root morphological and anatomical features. (a) Leaf transverse section showing midvein vascular bundle (VB), pallisade mesophyll (PM), spongy mesophyll (SM) and epidermis (EP). (b) Leaf surface epifluorescent image under ultraviolet illumination; stomatal guard cells (GC) and trichomes (T) fluoresce blue, oil glands (OG) appear black. Transverse sections of roots under brightfield illumination, illustrating (c) absence and (d) presence of air spaces (A) within the cortex. The stele (ST) and endodermis (EN) are also indicated. Epifluorescent images of root transverse sections under ultraviolet light, illustrating (e) endodermis with two rings of cells with suberised walls and (f) endodermis with unsuberised regions. Lignin and suberin in xylem and endodermal cell walls fluoresce blue. All images are fresh, unstained tissue, sectioned by hand. Scale bars 100 μm. 202