Ecophysiology of the Tree Fern Species Dicksonia Antarctica Labill and Cyathea Australis (R

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Ecophysiology of the tree fern species Dicksonia antarctica Labill and Cyathea australis (R. Br.) Domin

Liubov Vladimirovna Volkova

Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy

October 2009 Department of Forest and Ecosystem Science Melbourne School of Land and Environment The University of Melbourne

Produced on archival quality paper Abstract

Predictions of global warming and associated climate change indicate widespread increases in light intensities, temperatures, and the frequency and severity of droughts in south-eastern Australia. Understanding the ability of plants to respond and acclimate to these events is essential to predict species survival and potential impacts on biodiversity.

This study focuses on two tree fern species – Dicksonia antarctica and Cyathea australis – two iconic understorey species of south-east Australian forests. These tree ferns belong to different families and are of contrasting origins, yet often grow together in south-eastern Australia, typically in shade, often along waterways. Their ecological importance is evident in the high epiphytic diversity on their trunks (ferns, mosses, bryophytes, liverwort etc), and the provision of nursery sites for many tree and shrub species. Both species are decreased by timber harvesting practices such as clearcut logging, with deaths continuing for up to five years in the post-harvest environment. Understand- ing the relative roles of changing light, water, and temperature in these ongoing declines is essential to conserving both tree fern populations and their dependent biota.

The Thesis encompasses three controlled experiments and a field study. In the controlled experiments, the tree ferns were acclimated to contrasting growth light environments (shade or moderate light) and then exposed to an environmental stress (i.e. light, heat, water deficit). The field study examined relationships between environmental variables (i.e. light, temperature, plant water status) and photosynthetic capacity parameters of the tree ferns in their natural environment. Stress responses and acclimation potential of photosynthetic traits, water relation parameters, and frond traits of the tree ferns were studied using infra-red gas analysis, pigment determination techniques, and stable isotope methods.

It was hypothesised that, consistent with their contrasting origins and micro-site preferences, the two tree fern species would possess different physiological characteristics

-i- and therefore respond differently to environmental stresses. It was also hypothesised that plants grown under contrasting light environments would have different reactions to and recoveries from environmental stresses.

Overall, plants were able to sustain and recover from high light stress, while interactive effects of high light and heat were most detrimental to tree fern performance. Both species were susceptible to water stress, either alone or in combination with high light. The hypothesised different responses of the two species (associated with their different origins) were not confirmed, and reaction to and recovery from stress was mainly unaffected by growth light environment. Both species had low acclimation potential to any of the applied environmental stresses. Overall, findings from this study indicate that combined effects of high light and heat most likely cause ongoing decline of tree ferns in post-harvest environments, and that the distribution of tree ferns will most likely contract under future climate scenarios of higher light, increased temperatures, and decreased water availability.

-ii- Declaration

This is to certify that:  the thesis comprises only my original work towards the PhD except where indicated in the Preface,  due acknowledgement has been made in the text to all other material used,  the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliogra- phies and appendices

Liubov Volkova

-iii- Preface

The climate chamber experiment, Chapter 3, was undertaken in Champenoux, France using facilities of the Institut National Reserche Agronomique (INRA). I planned, conducted the research, evaluated and presented the data. The results of the study were presented at the International Eco-Fizz conference, 2007 (a poster) and published in the scientific journal Functional Plant Biology (Volkova L, Tausz M, Bennett LT, Dreyer E, 2009. „Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree-fern Dicksonia antarctica’). M. Tausz and L.T. Bennett are the supervisors of my PhD work. Erwin Dreyer, the fourth co-author of the publication was a hosting party in INRA and supervised my activities. Professor Dreyer is also an honorary staff member of the Department of Forest and Ecosystem Science at The University of Melbourne.

Chapter 4 (high light and water stress experiment) has been submitted for publication in co-authorship with my supervisors and Dr. Andrew Merchant (the University of Syd- ney). I declare that the execution of the experiment, data evaluation and presentation were solely my own work, and that A. Merchant gave useful tips on the experimental design and helped to organise the isotopic analysis of my samples.

Chapters 2 and 5 are written in co-authorships with my supervisors M. Tausz and L.T. Bennett who helped with usual supervisory roles.

-iv- Acknowledgements

Personal financial support for this study was provided by a Melbourne Research Schol- arship. Expenses related to research activities (i.e. field study and construction of the controlled experiments) were partly covered by a research agreement with the Victorian Department of Sustainability and Environment (TA30874).

I am personally grateful to my University supervisors, Ass. Prof. Michael Tausz and Dr Lauren Bennett, for their patience, ongoing support and encouragement during my study. I admire Michael for his ability to think globally and to give me confidence that everything is possible. I admire Ren for her strong personality, always prompt responses; ability to carefully examine every detail; her great friendship and care when I needed it. She was (and is) the Woman, who made me deeply respect women in science. I am indebted to Erwin Dreyer (INRA, France) for his great deal of support and advice during my candidature; his personal friendship is very precious to me. I would like to thank Andrew Merchant for his advice and support throughout my study. My thanks to Chris Western for his patience, always good advice and for being my personal Counsel- lor at difficult times. I acknowledge staff and students at Creswick campus for their support. Thanks particularly to Thomas Wright for his friendship and ongoing help, and Raymond Dempsey for his help in the field. Thanks also to Matt Lee and Najib Ahmady for always providing reliable and timely results, and my thanks to all others.

I am grateful to my family: husband, Fedor Torgovnikov, for his patience, support and help during my study. His ability to fix equipment and build constructions for my experiments was priceless. His patience with my often bad moods due to problems with experiments and understanding my difficulties made me able to finish this study. I thank my daughter, Katerina Torgovnikova, for her help with watering and re-potting plants and her patience with “always busy mum”. I am thankful to my parents-in-law, and most of all, I want to thank my mum, Svetlana Volkova, for teaching me to never give up and always reach my targets.

-v- Table of Contents

Abstract ...... i Declaration ...... iii Preface ...... iv Acknowledgements ...... v Table of Contents ...... vi List of Figures ...... xi Chapter 1. Introduction ...... 1 1.1. Environmental stresses: light, temperature and water deficit ...... 1 1.2. Fundamental effects of high irradiance in interaction with high temperature or drought on plants ...... 1 1.3. Two tree ferns of contrasting origin ...... 3 1.4. The tree ferns in mountain ash forests of south-eastern Australia ...... 5 1.5. Current knowledge of tree fern ecophysiology ...... 6 1.6. Thesis aims and outline ...... 8 Chapter 2. Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica, and Cyathea australis, acclimated to different light intensities...... 11 (i) Abstract ...... 11 2.1. Introduction ...... 11 2.2. Materials and methods ...... 13 2.2.1. Plant material ...... 13 2.2.2. Experimental design ...... 14 2.2.3. Light environment of measured frond ...... 15

2.2.4. Maximal quantum yield of photochemistry (Fv/Fm) ...... 16 2.2.5. Gas exchange measurements ...... 16 2.2.6. Plant water status ...... 17 2.2.7. Frond traits ...... 17 2.2.8. Artificial sunfleck experiment ...... 18 2.2.9. Statistical analyses ...... 19

-vi- 2.3. Results ...... 19 2.3.1. Light environment of the tree ferns ...... 19

Maximum quantum yield of photochemistry (Fv/Fm) ...... 20 2.3.2. Photosynthetic capacity parameters ...... 20 2.3.3. Plant water status ...... 26 2.3.4. Frond traits ...... 26 2.3.5. Artificial sunfleck experiment ...... 29 2.4. Discussion ...... 31 2.4.1. Species overview ...... 31 2.4.2. Acclimation to growth light environment ...... 31 2.4.3. High light stress ...... 33 2.4.4. Acclimation to new light environment ...... 33 2.5. Summary ...... 34 Chapter 3. Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. .. 35 (ii) Abstract ...... 35 3.1. Introduction ...... 35 3.2. Material and methods ...... 39 3.2.1. Plant material ...... 39 3.2.2. Climate chamber conditions and experimental design ...... 39

3.2.3. Frond temperature (Tfrond) ...... 40

3.2.4. Maximal quantum yield of photochemistry (Fv/Fm) ...... 40 3.2.5. Gas exchange measurements ...... 41 3.2.6. Frond nitrogen and chlorophyll content ...... 42

3.2.7. Critical temperature (Tc) ...... 42 3.2.8. Total tissue osmolality ...... 43 3.2.9. Pigments and tocopherol determination ...... 43 3.2.10. Statistical analysis ...... 44 3.3. Results ...... 45

3.3.1. Frond temperature (Tfrond) ...... 45

-vii- 3.3.2. Maximum quantum yield of PS II (Fv/Fm) and photosynthetic capacity parameters ...... 46

3.3.3. Critical temperature (Tc) ...... 52 3.3.5. Carotenoids and α-tocopherol ...... 53

3.3.6. Correlations between Tc and biochemical parameters ...... 57 3.4. Discussion ...... 58 3.4.1. Effect of high irradiance, high temperature and their interaction on photosynthetic capacity parameters of D. antarctica ...... 58 3.4.2. Membrane stability of D. antarctica measured via critical temperature 60 3.4.3. Xanthophyll cycle carotenoids, pigments and α-tocopherol ...... 61 3.5. Summary ...... 63 Chapter 4. Interactive effects of high light and water deficit on the tree fern species Dicksonia antarctica and Cyathea australis ...... 65 (iii) Abstract ...... 65 4.1. Introduction ...... 65 4.2. Materials and methods ...... 68 4.2.1. Plant material ...... 68 4.2.2. Experimental design ...... 68

4.2.3. Maximum quantum yield of PSII (Fv/Fm) ...... 70 4.2.4. Photosynthetic capacity ...... 70 4.2.5. Frond water relations ...... 72 4.2.6. Stable isotope analysis ...... 72 4.2.7. Relative extractable soil water, REW ...... 72 4.2.8. Statistical analysis ...... 73 4.3. Results ...... 74

4.3.1. Maximum quantum yield of PS II (Fv/Fm) ...... 74 4.3.2. Photosynthetic capacity ...... 74 4.3.3. Frond survival ...... 79 4.3.4. Time course of stomatal conductance during 5 days without water ...... 79 4.3.5. Frond water relations ...... 81

-viii- 4.3.6. Intrinsic water use efficiency (calculated as Amax/gs, WUEi) and stable carbon isotope composition (δ13C) ...... 83 4.4. Discussion ...... 85 4.4.1. Pre-treatment period – species differences and effect of light ...... 85 4.4.2. Water deficit and light interactions ...... 86 4.4.3. Rewatering period ...... 88 4.5. Summary ...... 89 Chapter 5. Seasonal variations in photosynthesis of the tree ferns Dicksonia antarctica and Cyathea australis in wet sclerophyll forests of Australia ...... 91 (iv) Abstract ...... 91 5.1. Introduction ...... 91 5.2. Materials and methods ...... 94 5.2.1. Study site and sampling design ...... 94 5.2.2. Tree fern measurement schedule ...... 95 5.2.3. Mean irradiance on measured fronds ...... 96

5.2.4. Maximal quantum yield of photochemistry (Fv/Fm) ...... 96 5.2.5. Gas exchange measurements ...... 96 5.2.6. Frond water potential ...... 97 5.2.7. Frond traits ...... 97 5.2.8. Statistical analysis ...... 98 5.3. Results ...... 99 5.3.1. Relationships between photosynthesis, growth irradiance and temperature ...... 99 5.3.2. Water status parameters ...... 103 5.3.3. Diurnal measurements ...... 103 5.3.4. Stomatal density ...... 106 5.4. Discussion ...... 106 5.4.1. Comparisons between the two tree fern species ...... 106 5.4.2. Light as a limiting factor to tree fern photosynthetic performance ...... 107 5.4.3. Temperature as a limiting factor to tree fern photosynthetic performance ...... 109

-ix- 5.4.4. Effects of plant water status and water relation parameters on tree fern photosynthetic performance ...... 110 5.4.5. Stomatal density ...... 111 5.5. Summary ...... 113 Chapter 6. Ecophysiology of two tree fern species and implications for their future management. General discussion and conclusions ...... 115 6.1. Species overview ...... 115 6.2. Overview of light, temperature, and water availability as stresses on tree fern physiology ...... 117 6.3. Practical implications and future directions ...... 119 REFERENCES ...... 123

-x- List of Figures

Figure 2.1 Photosynthetic capacity parameters of the tree ferns D. antarctica and C. australis grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure)...... Figure 2.2 Mesophyll capacity parameters of the tree ferns D. antarctica and C. australis grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and tree months (long-term exposure) ...... 24

Figure 3.1. Time course of maximum quantum efficiency of PSII and chlorophyll content of high irradiance and shaded D. antarctica during three successive temperature treatments ...... 47 Figure 3.2 Stomatal conductance versus light-saturated rate of net photosynthesis for high irradiance and shaded D. antarctica ...... 51

Figure 3.3 Time course of critical temperature Tc D. antarctica across the experiment ...... 52 Figure 3.4 α -Tocopherol content of high irradiance and shaded D. antarctica under three temperature ...... 56 Figure 3.5 Critical temperature versus xanthophyll zeaxanthin of high irradiance and shaded D. antarctica during three temperature treatments ...... 57

Figure 4.1 Weather conditions during the experiment ...... 73 Figure 4.2 Light saturated net photosynthesis and stomatal conductance of water deficit and control D. antarctica and C. australis under high and moderate light in three successive experimental periods (pre-treatment, water deficit and rewatering) .... 78 Figure 4.3. Time course of stomatal conductance of water deficit D. antarctica and C. australis grown under high and moderate light with decreasing relative extractable soil water and increasing number of days without water ...... 80

-xi- Figure 4.4 Stable isotope composition and intrinsic water use efficiency and of the tree ferns D. antarctica and C. australis under high and moderate light in three successive experimental periods ...... 84

Figure 5.1 Location of the tree ferns at the study area ...... 95 Figure 5.2 Relationships between photosynthetic capacity parameters and frond traits of the tree ferns D. antarctica and C. australis and environmental variables ...... 101 Figure 5.3 Climate conditions during diurnal course measurements in summer and winter ...... 104 Figure 5.4 Relationships between photosynthesis, stomatal conductance and water pressure deficit based on leaf temperature ...... 105 Figure 5.5 Light response curves of the tree ferns D. antarctica and C. australis in summer and winter ...... 106 Figure 5.6 Stomatal density of the tree ferns D. antarctica and C. australis from light- exposed and shaded habitats...... 112

-xii- List of Tables

Table 2.1 Relative irradiance, Isum (i.e. the fraction of penetrating irradiance in the photosynthetically active spectral region) of the tree ferns growing under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure) ...... 20 Table 2.2 Photosynthetic capacity parameters of the of the tree ferns D. antarctica and C. australis grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure) ...... 23 Table 2.3 Predawn frond water potentials and frond traits of the tree ferns D. antarctica and C. australis grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure) ...... 27 Table 2.4 Dynamic responses of photosynthesis to an artificial sunfleck ...... 30

Table 3.1 Temperature of D. antarctica fronds exposed to high irradiance and under shade ...... 60 Table 3.2 Photosynthesis and frond traits of D. antarctica exposed to high irradiance and under shade before and during three successive temperature treatments ...... 49 Table 3.3 Pigment content and osmolality of D. antarctica fronds exposed to high irradiance and under shade during three successive temperature treatments ...... 54

Table 4.1 Chlorophyll fluorescence and photosynthetic capacity variables of water deficit and control tree ferns (D. antarctica and C. australis) grown under high and moderate light during three successive experimental periods ...... 76 Table 4.2 Frond water relations of water deficit and control tree ferns (D. antarctica and C. australis) grown under high and moderate light during three successive experimental periods ...... 100

-xiii- Table 5.1 Significance of the effect of fixed factors (species and season) and of covariates (ANCOVA) on photosynthetic capacity parameters and frond traits of the tree ferns D. antarctica and C. australis ...... 100 Table 5.2 Photosynthetic capacity and water relation parameters of the tree ferns D. antarctica and C. australis in summer and winter...... 102

-xiv- Chapter 1. Introduction

1.1. Environmental stresses: light, temperature and water deficit

Predictions of global warming and associated climate change indicate widespread increases in light intensities, temperatures, and the frequency and severity of droughts in south-eastern Australia (Hennessy et al. 2007). Understanding likely responses of plants to future threats is critical to land management, and some of the main challenges for conservational biology will be to anticipate environmental change and to adjust management approaches accordingly (Rossetto 2008). To achieve this aim, it is crucial to understand the mechanisms of plants to cope with such changes in environmental factors, and to understand the limitations of their coping capacity.

1.2. Fundamental effects of high irradiance in interaction with high temperature or drought on plants

Exposure of plants to high levels of irradiance often leads to photoinhibition and photo- oxidative stress. Photoinhibition is a decline in the quantum yield of photosynthesis. The primary sites of light damage are associated with components located in the thylakoid membranes of chloroplasts (Havaux et al. 1996). Primary damage occurs within the reaction centre of photosystem II (PSII), with associated photoinhibition effects such as decreases in photosynthetic yield, bulk pigment loss with photo-oxidation, loss of enzyme activity (including Rubisco), and, eventually, even cell death (Long et al. 1994). Photo-oxidative stress is caused by the toxic effects of reactive oxygen species (ROS) produced in the photosynthetic apparatus under high irradiance (Niyogi 2000). Plants have developed a number of adaptive mechanisms that allow the photochemical apparatus to cope with rapid changes in light. For instance, when leaves are exposed to strong light that is saturating for photosynthesis, the xanthophyll zeaxanthin is rapidly and reversibly formed by violaxanthin de-opoxidation in bright light via the intermedi- ate antheraxanthin (e.g. Demmig-Adams and Adams 2006).

-1-

Photoinhibition alone is rarely responsible for plant mortality and the plant may recover and become fully acclimated (Lovelock et al. 1994). However, when, in addition to high irradiance, leaves are exposed to other environmental stress factors such as high temperature or drought, there can be sustained reductions in the efficiency of photosynthetic energy conversion and inhibition of repairs to photodamaged PSII (Murata et al. 2007).

Interactive effects of high light and increased temperature are widely discussed in plant physiology literature (e.g. Havaux et al. 1991, Kirchgeßner et al. 2003, Dieleman and Meinen 2007). While some authors suggest that these effects are detrimental for plants because photosynthesis is particularly sensitive to inhibition by heat stress due to labile components in the photosynthetic apparatus (Salvucci and Crafts-Brandner 2004), others insist that high light alleviates negative effects of high temperatures on plants (Ha- vaux et al. 1991).

Water supply is among the most important factors limiting plant species distribution (Howard 1973). The primary effects of water stress on photosynthesis have been com- prehensively discussed (e.g. Flexas et al. 1998), with stomatal conductance among the earliest responses that protect plants from extreme water loss. Decreases in intercellular

CO2 concentrations (Ci) after stomatal closure during water stress may induce down- regulation of photosynthetic apparatus to match available carbon substrate and decreased growth (Chaves et al. 2003). A number of drought effects are mediated by an excess of absorbed light energy in the photosynthetic apparatus, leading to an imbalance between electron transport and electron consumption and causing photoinhibition and photo-oxidative stress (Flexas et al. 1999). Hence, an interactive effect of high light and drought can be fatal to plants (Levitt 1980, Lovelock et al. 1994).

Effects of high light, heat and water deficit on plant performance have been extensively studied over recent decades, yet these studies have been mainly focused on productive, overstorey tree species (e.g. oak, Eucalyptus species, Acacia species etc). Understorey species, with low commercial value, have received much less attention, in spite of their

-2- specific situation, growing under relatively low irradiance, but experiencing occasional exposure to high irradiance through sunflecks or removal of overstorey (e.g. Pearcy 1988, Durand and Goldstein 2001). Hence, results on overstorey trees cannot easily be generalised for these species. Growing concern about biodiversity protection in productive forests and increasing commitment to sustainable forest management is contributing to rising interest in the understorey component of forests.

1.3. Two tree ferns of contrasting origin

Ferns, or pteridophytes, are the largest and most complex group of flowerless plants that reproduce by spores developed in sporangia on the underside of leaves or fronds (Large and Braggins 2004). Some ferns have adopted the tree growth form and are thus called tree ferns. Most tree ferns belong to the families Dicksoniaceae and Cyatheaceae (Large and Braggins 2004). Members of the family Cyatheaceae are the most widespread tree ferns, with many species showing high degrees of local endemism. Centres of diversity include the Great Antilles, Central America, the Andes, Madagascar, Malesia (i.e. including Indonesia, Philippines and New Guinea). The family Dicksoniaceae has high diversity in Indonesia and New Guinea, with some species found in isolated pockets including St Helena Island and the Fernandez Islands off the coast of Chile (Large and Braggins 2004).

The tree ferns Dicksonia antarctica Labill. and Cyathea australis (R. Br.) Domin are iconic and ecologically important understorey plants of Australian forests. Observations by Ashton (2000) indicated that trunks of tree ferns were favourable sites for the establishment of most woody species in wet sclerophyll forest dominated by Eucalyptus regnans F. Muell. Tree ferns, particularly D. antarctica, formed an impressive understorey and were associated with numerous species of ground and epiphytic ferns. Studies by Lindenmayer et al. (1994) found abundance of the mountain brushtail possum Tricho- surus caninus Ogilby increased with numbers of C. australis and D. antarctica. The dead fronds of C. australis were favorite sites for Exoneura bicolor bees (Blows and Schwarz 1991). Crimson Rosella (Platycercus elegans) birds value sori of D. antarctica

-3- as an energy rich food, and sori account for 20-30% of the birds‟ diet in autumn and winter (Magrath and Lill 1983). Both tree fern species are also popular horticulture commodities for domestic and international markets; for example, in 2003/2004 more than 50,000 trunks of D. antarctica were exported from Tasmania (Davies 2005).

Both D. antarctica and C. australis are widespread in the temperate zones of Australia (McCarthy 1998). The most significant habitats for tree ferns are rainforest (cool and warm temperate) and wet sclerophyll forests, particularly in the deepest, least disturbed sheltered gullies (Department of Natural Resources and Environment 2002). D. antarctica is common in wet forest and often dominates moist, shady gullies, where it fre- quently grows in extensive stands. C. australis‟ s habitat ranges from dark gullies to dry forest fringes and creek banks in quite open areas (McCarthy 1998). Observations indicate that the two tree fern species have overlapping but divergent micro-site preferences. For example, a study in south-east Australian wet sclerophyll forest found that tree ferns were more likely to be C. australis than D. antarctica with increasing distance from a stream (Dignan and Bren 2003).

D. antarctica and C. australis belong to contrasting floristic elements of the Australian vegetation. While D. antarctica is believed to be endemic to Australia and derived directly from the original Gondwanan flora, C. australis is considered to be an intrusive species of the Indo-Malayan flora (Barlow 1994). These different origins combined with indications of different micro-site preferences suggest the two tree ferns would have different physiological adaptations to environmental stresses.

During their lifetime, tree ferns can be periodically exposed to the harsh conditions of post-wildfire environments, which are characterised by increased light intensities and leaf temperatures, and consequently increased evapotranspiration and water loss. Effects of these conditions on tree fern physiology have not been studied, but are indicated by poor survival and ongoing decline of both D. antarctica and C. australis after clearcut logging in mountain ash (E. regnans) forest (Ough and Murphy 2004). It was found that only 11-17 % of D. antarctica and C. australis survived one year after clearcut logging,

-4- and of those remaining, up to 40% of D. antarctica and 65% of C. australis were not expected to survive another five years (Ough and Murphy 2004). In contrast, much higher rates of regeneration and survival of the tree ferns were recorded after wildfires (Ought 2001).

1.4. The tree ferns in mountain ash forests of south-eastern Australia

Mountain ash forest of south-eastern Australia is a unique wet sclerophyll ecosystem that typically forms an interface between two broad vegetation types, rainforest and dry sclerophyll forest (Campbell and Clarke 2006). These forests are highly prized as water catchments for the Melbourne region, and for flora and fauna conservation and recrea- tion purposes (Attiwill and Fewings 2001). E. regnans, itself, is a valuable timber species, and about 40% of these forests are available for timber harvesting (Bennett and Adams 2004).

The dominant harvesting practice in mountain ash forests includes clearcut, slash burn- ing of debris and remaining vegetation, and seeding with E. regnans seeds (Bennett and Adams 2004). Such harvesting practices cause major disturbance, including physical damage to resprouting plants, changes in soil physical and chemical properties, disturbance to soil stored plant propagules, and sudden exposure of understorey plants to full sunlight (Ough and Murphy 1996).

Reasons for steady declines in tree fern numbers in post harvest environments remain uncertain. Soil disturbance was suggested as a likely major contributor to poor regeneration of tree ferns a decade after clear-felling compared with wildfire regeneration (Ough 2001). Greater survival of tree ferns was recorded in understorey islands (i.e. areas within a coupe where trees can be felled but disturbance to understorey species and soil is minimized) than in logged coupes, but mortality also occurred in understorey islands across all size classes of tree ferns (Ough and Murphy 1998). Apart from soil disturbance, there are other obvious differences between post-wildfire and post-harvesting environments: a fire-killed forest provides much more shade and many more micro-

-5- habitats than the relatively uniform ash-bed created by high intensity regeneration burns after logging (Hickey 1994). Logging also results in sharp edges in the boundary zone, increasing light penetration up to 100% (Dignan and Bren 2003), which can create high light stress for vegetation remaining in buffer zones, including understorey islands.

High light stress gives rise to two other stress factors – heat and drought. Heat, because direct irradiance will also increase leaf temperatures, and drought, because greater leaf temperatures will lead to a greater evaporative demand. As discussed above, interactive effects of these three stresses can be fatal for a plant (Levitt 1980). Thus, it is possible that sudden changes in light intensity, water availability and temperature contribute to D. antarctica and C. australis mortality in the post-harvest environment.

1.5. Current knowledge of tree fern ecophysiology

Little is known about the physiology of tree ferns. It is obvious that tree ferns tolerate a broad range of environmental conditions throughout their life cycle. Periodically disturbed by wildfires, they have evolved under a regime of variable light levels from high (immediately after fires) to low or moderate after canopy re-establishment (Hunt et al. 2002).

Certainly, other studies indicate potential for fern acclimation to different light regimes. For example, New Zealand ferns from contrasting habitats displayed contrasting characteristics in terms of photosynthetic light compensation point, which were tightly correlated with specific frond area (Bannister and Wildish 1982). Frond characteristics (frond surface area, epidermis thickness, palisade/ spongy mesophyll ratio, blade size, petiole length) of a South American Cyathea species were also correlated with the irradiance regime at its local micro-habitat (Arens 1997). During the course of forest ecosystem dynamics including gap formation, bushfires, or forest harvesting, tree ferns may be suddenly exposed to full sunlight. Studies in Hawaii indicated limited capacity of shade- acclimated tree ferns to quickly and efficiently adjust to sudden increase in irradiance

-6- due to gap formation (Durand and Goldstein 2001). However, further studies of high light stress on tree ferns and their rate of recovery are currently lacking.

Effects of high temperature, either alone or with high light, on the physiological performance of tree ferns have also been poorly studied. Tingey et al. (1987) found that photosynthesis of D. antarctica was particularly susceptible to inhibition with increasing temperature and high light; and Nobel et al. (1984) also mentioned negative effects of high temperature on gas exchange of ferns. Moreover, there are indications that tree ferns are very susceptible to temperature increases due to their reticulated vascular system (White and Weidlich 1995), which might not be as efficient in delivering water to fronds as the vascular system of angiosperms (Brodribb et al. 2005). However, more detailed studies, examining acclimation potential of photosynthetic apparatus of tree ferns to temperature increases and its reversibility are lacking, despite the obvious importance of this knowledge to predicting species‟ acclimation potential and survival in the future.

Adequate water supply as an important element for tree ferns can be suggested from their distributional patterns in the forests (mostly along waterways), and is also mentioned in the horticultural literature (e.g. Jones and Clemesha 1993, Large and Braggins 2004). Observational studies indicate that tree ferns can sustain periods of drought if they are protected by canopy. Ashton (2000) observed that despite infrequent but severe drought events, tree fern numbers increased by 80% in the lower strata of wet sclerophyll forests over 48 years. Hunt et al. (2002) also reported that D. antarctica can maintain favourable water relations during short periods of drought if its habitat is limited to sheltered sites. However, these field observations involve potentially confounding effects of shade, temperature and (soil and air) humidity, because more shaded sites are also cooler and moister. Thus, it often remains unresolved whether alleviation of drought stress is a direct effect of lower irradiance – e. g. shading ameliorates drought- related photoinhibition and photo-oxidative stress – or an indirect effect of greater water availability and less evaporative demand in shade.

-7- 1.6. Thesis aims and outline

The principal research objectives of this thesis are the characterisation of physiological responses of D. antarctica and C. australis to varying light conditions, temperature regimes and water availability. There have been no prior studies of the comparative physiology of D. antarctica and C. australis, and there has been little previous examination of the interactive effects of light, temperature and water deficit on tree fern physiology.

In my first experimental study (Chapter 2) I examine effects of high light on photosynthetic capacity parameters of D. antarctica and C. australis in a controlled glasshouse experiment.

In my second experimental Chapter (Chapter 3), I report effects of high light and light by temperature interactions on photosynthetic performance of D. antarctica in a controlled climate chamber experiment. This experiment was based in France, which meant that C. australis could not be included because a European source of this species could not be found.

In my third experimental Chapter (Chapter 4), I examine effects of water deficit either alone or in interaction with high light on the photosynthetic capacity of D. antarctica and C. australis in a semi-controlled, open-air experiment.

In my fourth and final experimental Chapter (Chapter 5) I examine the ecophysiology of both tree fern species under field conditions in the buffer zones surrounding a clearcut mountain ash forest of central Victoria, Australia. Here, I examine relationships of growth irradiance, leaf/air temperatures, plant water status with photosynthesis, frond traits, and water relation parameters of mature tree ferns over two consecutive years.

In my final Chapter 6, I provide an overall discussion of the results and indicate possible implications of my findings.

-8-

Each experimental chapter was written as a stand-alone paper for journal submission. Thus, some repetitions of citations and of text from this Introductory Chapter were in- evitable.

-9-

-10- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Chapter 2. Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica, and Cyathea australis, acclimated to different light intensities.

(i) Abstract

We examined the responses of two tree fern species (Dicksonia antarctica and Cyathea australis) growing under shade or variable light (intermittent shade) to sudden exposure to high light. Steady-state gas exchange as well as dynamic responses of plants to artificial sunflecks indicated that difference in growth light environment had very little effect on the tree ferns‟ capacity to utilise and acclimate to prevailing light conditions. Two weeks of exposure to high light (short-term acclimation) led to decreases in all photosynthetic parameters and more negative predawn frond water potentials, mostly irrespective of previous growth light environment. After three months in high light (long- term acclimation), D. antarctica fully recovered while C. australis previously grown under variable light recovered only partially, suggesting high light stress effects under the variable light environment for this species.

2.1. Introduction

The light environment in the understorey of closed forests is often characterized as a low level of diffuse light punctuated by intense sunflecks resulting from direct-beam solar radiation through holes in the canopy (Pearcy 1988). However, through forest ecosystem dynamics including gap formation and bushfires, or anthropogenic management such as forest harvesting, understorey species can suddenly be exposed to prolonged full sunlight, a stress factor that can contribute to decline in their photosynthetic performance (Levitt 1980). Rapid physiological adjustment to unfavourable levels of irradiance (i.e. acclimation, Lambers et al. 2008) is then required for understorey species survival.

-11- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

The tree ferns Dicksonia antarctica (Labill.) and Cyathea australis (R.Br.) Domin are characteristic and ecologically important understorey plants of south-eastern Australia (Large and Braggins 2004). Even though both species prefer high rainfall wet sclerophyll forests (Jones and Clemesha 1993), they have different micro-site preferences: D. antarctica is common in wet, shady gullies, whereas the often co-occurring species C. australis seems to preferentially grow within the forest or even along forest margins (McCarthy 1998). An observational study confirmed that the greater the distance to the stream the more likely it‟s to encounter C. australis rather than D. antarctica (Dignan and Bren 2003), suggesting a greater dependence of D. antarctica on water availability and shade protection. D. antarctica and C. australis belong to contrasting floristic elements of the Australian vegetation (Gondwanan vs. Intrusive, Tropical; Barlow 1994), which suggests different physiological adaptation potential and supports their distribution patterns within forests.

Periodically disturbed by wildfires, D. antarctica and C. australis have evolved under a regime of variable light levels from high (immediately after fires) to low or moderate after canopy re-establishment (Hunt et al. 2002). Evidence of plasticity in frond mor- phology and anatomy in response to different levels of irradiance was found in a study on South American Cyathea species (Arens 1997). Hunt et al. (2002) also suggested that during the period of regeneration of woody species following fire, D. antarctica may experience prolonged periods of exposure to high light intensities and dry atmos- pheric conditions. Potential of D. antarctica and C. australis to tolerate a broad range of light conditions is also indicated in horticultural publications (Jones and Clemesha 1993; Large and Braggins 2004).

Despite their apparent longer term acclimation potential to variable light conditions, tree ferns seem particularly vulnerable after the formation of large gaps. Studies in Hawaii found that shade-adapted tree ferns were damaged in disturbed areas and forest gaps, because they are unable to adjust quickly or efficiently to high light environments (Du- rand and Goldstein 2001). Moreover, Ough and Murphy (2004) found that only about 11 – 17 % of D. antarctica and C. australis survived one year after clearcut logging in

-12- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press the mountain ash (Eucalyptus regnans F. Muell) forests of the Victorian Central High- lands. Of those remaining, up to 40 % of D. antarctica and 65 % of C. australis would not survive another five years (Ough and Murphy 2004).

The objectives of this study were to elucidate acclimation potential and vulnerability of D. antarctica and C. australis to sudden increases in light. The two species were grown under either full or intermittent shade („variable light‟) in a glasshouse and, after prolonged acclimation, were suddenly exposed to high irradiance. We measured steady- state and dynamic (i.e. sunflecks) photosynthetic responses of the tree ferns acclimated to each light environment in order to test the following hypotheses:

 Responses to light would be different between the two species, with D. antarctica performing better in full shade and being more prone to high light-induced damage;  Sudden exposure to high light would cause limitations in gas exchange (e.g. photoinhibition) in both species in the short term (two weeks), with those acclimated to full shade most strongly affected;  Both tree fern species would have limited capacity to acclimate to high light even in the longer term (three months).

2.2. Materials and methods 2.2.1. Plant material

Ten sporophytes of D. antarctica and ten of C. australis (Fern Acres nursery, King Lake West, Australia) were transplanted into 25-l pots. Potting mix contained (% volume) composted pine bark (30), gravel (45), coarse fern mulch (5), composted mulch (14.5), fine fern mulch (5), „Dynamic lifter‟ (0.16: Yates, Padstow, NSW, Australia), and two types of slow-release fertiliser (0.17 each; Osmocote, Baulkham Hills, NSW, Australia). Before the experiment, tree ferns were propagated from spores and grown in an open-air nursery under a dense canopy that provided ca 70% shade. All plants were about six months old and 20-25 cm tall at the start of the experiment.

-13- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

2.2.2. Experimental design

The experiment ran from December 2005 to July 2006 in a glasshouse at the site of the University of Melbourne‟s Creswick campus, in south-eastern Australia (143º53‟E, 37º25‟S; elevation 392 m above sea level). After the first two weeks, old and fully developed fronds were cut off and only new fronds, which developed under a designated growth light environment, were measured during the experiment.

Plants were randomly assigned to two growth light environments in a fully randomised block design with five replicates (i.e. two species within two treatments per each of five blocks). The two growth light environments – „shade‟ and „variable light‟ – were applied using wavelength neutral shadecloth. Shade allowed ca 20% uniform light penetration; whereas the variable light simulated sunflecks – the shadecloth was cut into 12 cm-wide stripes, and these were alternated with uncovered gaps of the same width (the 12 cm width was based on the 20 min movement of the sun at its zenith). Under direct sun in the glasshouse, the maximum recorded photosynthetic photon flux density (PPFD) was 1900 µmol photons m-2 s-1 at plant height (PAR range, 400-700 nm, measured with a Li-Cor quantum sensor).

Plants were watered twice per day to maintain soils at field capacity throughout the experiment. Relative humidity and air temperature in the glasshouse were maintained using a Humidex I greenhouse climate control system (Nelan Industries Pty. Ltd., Mel- bourne, Australia). Mean conditions throughout the experiment were: 8ºC minimum temperature, 24ºC maximum temperature, and > 60 % relative humidity.

Plants were measured at the end of three periods:

1) „Before exposure‟ (early April 2006): measurement of new fully-developed fronds after four months of growth under the designated light environment (shade or variable light);

-14- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

2) „Short-term exposure‟ (late April 2006): measurements of the same cohort of fronds after two weeks of shade removal (indication of short-term acclimation potential); 3) „Long-term exposure‟ (late July 2006): measurements of a new fully developed cohort of fronds after three months of shade removal (indication of long-term acclimation potential).

In addition, an artificial sunfleck experiment was conducted for two days in late March 2006 (i.e. before exposure and just after full expansion of new fronds). See below for details.

Chlorophyll a fluorescence, predawn water potential, and gas exchange parameters were measured at the end of each of the three experimental periods. Samples for nitrogen and chlorophyll were collected at the same time. All measurements were made on the mid-third of the youngest fully expanded fronds.

2.2.3. Light environment of measured frond

The growth light environment for each plant was calculated from hemispherical photographs. These were taken at the level of each measured frond using a fish-eye lens (Nikon, F- 601, Japan). Black and white negatives were scanned and evaluated using Winphot software (ter Steege 1996). Relative irradiance at the measurement location

(Isum) was calculated according to Niinemets et al. (1998):

Isum = pdif Idif + (1- pdif) Idir Eqn. (1)

Where pdif is the ratio of diffuse irradiance to total irradiance in the photosynthetically active spectral region (400-700nm) above the plant; and Idif and Idir are the factors of diffuse and direct radiation that will penetrate to the measured location relative to the

-15- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press total irradiance above the plant (ter Steege 1996). All parameters were calculated for each day of the experiment, taking into account amount of sunshine hours in Victoria during each month of the experiment (data from the Australian Bureau of Meteorology, http://www.bom.gov.au/jsp/ncc/climate _averages/sunshine-hours/index.jsp, verified 1 September 2009).

2.2.4. Maximal quantum yield of photochemistry (Fv/Fm)

Predawn chlorophyll a fluorescence was measured on overnight dark-adapted leaves with a pulse modulated fluorometer (OS-30p, Opti-Sciences, Hudson, USA). Ground fluorescence (Fo) was obtained with a low intensity modulated light (600 Hz, 650nm, -2 -1 photosynthetic photon flux density PPFD <1 µmol m s ). Maximum fluorescence (Fm) was induced by a saturating flash. Maximum efficiency of PSII was estimated as Fv/Fm

= (Fm - Fo)/Fm, after Maxwell and Johnson (2000).

2.2.5. Gas exchange measurements

Gas exchange parameters were measured using a Li-Cor 6400 gas exchange system, equipped with a 2x3 cm broadleaf chamber (Li-Cor, Lincoln, Nebraska, USA).

A light response curve was generated for each plant at CO2 concentration of 400 µmol mol-1, block temperature 25ºC, air flow rate 400 µmol air s-1, and relative humidity >60%. PPFD was increased stepwise from 0 to 2000 µmol m-2s-1. Fronds were induced in the dark for approximately 10 min and the rate of dark respiration was recorded when stability was reached. PPFD was then increased in 11 successive steps to 2000 µmol m- 2s-1 with two measurements per PPFD level. Measurements were recorded once rates of gas exchange were stable. Apparent quantum yield (ф) and maximum photosynthetic rate Amax were calculated according to Lambers et al. (2008).

An A-Ci curve was generated according to Long and Bernacchi (2003) with some modifications: PPFD 1000 µmol m-2s-1, block temperature 25ºC, air flow rate 400 µmol air s-

-16- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

1 , and relative humidity > 60%. Reference CO2 concentration was increased from 75 to -1 2200 µmol mol in 13 successive steps with two measurements per CO2 concentration. Measurements were recorded once gas exchange parameters were stabilised, which on average took at least 5 min. After finishing the A-Ci curve, illumination in the leaf chamber was turned off, CO2 concentration was decreased to ambient and respiration due to oxidative phosphorylation was recorded after 5 min in the dark.

Using the Farquhar model (Farquhar et al. 1980), maximum carboxylation rate (Vcmax), and maximum electron transport rate (Jmax) were evaluated by fitting A- Ci curves to non-rectangular hyperbolas (as described in Dreyer et al. 2001 and Montpied et al. 2009). Triose phosphate use (TPU) limitation was not included in the model, and corresponding points with decreased Amax at elevated Ci were disregarded (Long and Bernac- chi 2003). The set of primary parameters of Rubisco kinetic properties used here -1 -1 -1 (Kc=327µmol mol , Ko=282600 µmol mol , Γ*=43.7 µmol mol ) are from von Caemmerer et al. (1994).

The frond area enclosed in the chamber for light response and A- Ci curves was marked, detached, scanned and calculated using imaging software (UTHSCSA Image Tool Ver- sion 3, University of Texas, USA). All gas exchange measurements were recalculated on a frond-area basis.

2.2.6. Plant water status

Predawn frond water potential (Ψpredawn) of each plant was measured at the end of each period using a pressure chamber (PMS Corvallis, OR, USA).

2.2.7. Frond traits

Specific leaf area (SLA), needed for calculation of nitrogen and chlorophylls on a frond-area basis, was calculated as the ratio of frond area over frond dry weight (m2 kg-1 dry weight). Fresh frond samples were collected, the frond area scanned and calculated

-17- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press using Scion Image software (Scion Corporation 2000-2001, USA), and frond material then dried at 60ºC for 48 h for dry weight.

Frond samples were analysed for total nitrogen and carbon content using an elemental analyser (LECO CHN-1000, Michigan, USA). Frond samples were dried as described above and ground to a fine powder. Photosynthetic nitrogen use efficiency (PNUE) was calculated as Amax divided by frond nitrogen content (on a frond-area basis).

For measurements of frond chlorophyll content, four frond discs (each diameter 3.75 mm) were collected, immediately immersed in liquid nitrogen, and stored at -80ºC until extraction. Chlorophyll a and b were extracted using 1.8 ml of 100% dimethyl sulphoxide (DMSO). Extracts were heated for 30 min at 65ºC in a dry block heater Termoline L+M (Northgate, Queensland, Australia). The supernatant was then transferred to a spectrophotometer Carry 300 (Varian, The Netherlands). A blank of pure DMSO was used to calibrate the spectrophotometer at zero absorbance. Chlorophyll a, b and total concentrations were calculated according to Wellburn (1994).

2.2.8. Artificial sunfleck experiment

Predawn Fv/Fm was recorded for each dark-adapted plant. A frond was then enclosed in -2 -1 the Li-Cor chamber at PAR 20 µmol m s and photosynthesis rate (A20) and gs20 were recorded once the readings were stable (after at least 5 min). PPFD was then increased to 2000 µmol m-2s-1 in one step, and gas exchange parameters recorded every 10 sec- onds for 20 min. Fv/Fm immediately and 30 min after the sunfleck were recorded using a pulse modulated fluorometer (as above). Experimental conditions were: chamber rela- -2 -1 tive humidity 75-80%, reference CO2 concentration 400 µmol m s and block temperature 25±1ºC.

The following parameters were calculated to characterise the dynamic response of net photosynthesis to a sudden increase in PAR from 20 to 2000 µmol m-2s-1: maximal pho-

-18- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

tosynthesis rate (Amax_ind, calculated from the induction curve) and time to reach 63% of change in photosynthesis (t63%), nomenclature after Tausz et al. (2005).

2.2.9. Statistical analyses

Repeated-measures models of SPSS 15 (SPSS Inc. Chicago, USA) were used for statistical analyses, with light and species as the between-subject factors and period as the within-subject factor (all fixed). Effects of period (before exposure; short-term exposure; long-term exposure), growth light environment (variable light; shade) and species (D. antarctica; C. australis), and period by light by species interactions on each dependent variable were tested. Data for statistical analyses were the values per individual plant at the end of each period. Significant differences between periods were examined by using the repeated contrast function (SPSS 15).

A two-way general linear model (SPSS 15) with growth light environment and species as fixed factors was used to analyse the artificial sunfleck data.

Each dependent variable was checked for normality using the Shapiro-Wilk test and log transformed if assumptions of normality were not satisfied. Data were checked for homogeneity of variance using Cochrane‟s test, and it was ensured by visual examination of scatter plots that means and variances were not correlated across experimental groups.

2.3. Results 2.3.1. Light environment of the tree ferns

Relative irradiance, Isum, did not differ between species within growth light environment (P=0.4, data not shown), confirming randomised block design for the two species. Yet,

Isum in the variable light was 2.5 times greater than Isum of shade (Table 2.1). Shade removal increased growth light intensity almost two-fold for variable light plants, and

-19- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press more than four-fold for shaded plants (Table 2.1). These relatively high levels of irradiance remained until the end of the experiment.

Table 2.1. Relative irradiance, Isum (i.e. the fraction of penetrating irradiance in the photosynthetically active spectral region) of the tree ferns growing under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure). Values are means (n = 5) ± s.e. Affect abbreviations: P, Period; L, Growth light environment.

Differences in Isum between growth light environments for each period were determined using one-way ANOVAs‟. Significance level:***P<0.001.

Isum Growth light environment Effects Variable light Shade L S Before exposure 0.344±0.02 0.134±0.01 *** n.s. (0.4) Short-term exposure 0.656±0.03 0.575±0.04 n.s. (0.2) n.s. (0.1) Long-term exposure 0.617±0.04 0.676±0.05 n.s. (0.9) n.s. (0.4)

Maximum quantum yield of photochemistry (Fv/Fm)

Maximum quantum yield of photochemistry (Fv/Fm) was similar between species and tended to be lower under variable light than shade in the before exposure period (mean across species of 0.76 versus 0.82; Table 2.2). Short-term (two week) exposure to high light led to significant decreases in Fv/Fm of both species irrespective of the growth light environment (to ca 0.70; Table 2.2). After three months of exposure to high light, Fv/Fm partially recovered in previously shaded plants but remained low in plants previously grown under variable light (Period x Light, P<0.001, Table 2.2).

2.3.2. Photosynthetic capacity parameters

Light-saturated rate of net photosynthesis (Amax) and stomatal conductance (gs) at Amax were similar across species and growth light environments in the before exposure period

-20- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

(Figs. 2.1 a, 2.1 b). Both parameters decreased after two weeks of exposure to high light in both species irrespective of the growth light environment, and both parameters recovered after three months of high light exposure, with the exception of Amax in C. australis, which remained low (Figs. 2.1 a, 2.1 b).

Respiration rate in the dark (Rd) was not significantly affected by the growth light environment, species or sudden exposure to high light (Fig. 2.1 c). However, three months after exposure, Rd increased significantly in C. australis previously grown under variable light (Fig. 2.1 c).

Apparent maximum quantum yield (ф) was significantly greater in shade than in variable light plants in the before exposure period, then decreased in previously shaded plants but increased in variable light plants after two weeks of exposure to high light (Table 2.2). After three months of exposure to high light, ф increased to near or greater than the before exposure values in all but previously shade-grown C. australis plants (Table 2.2). Effect of species on ф was insignificant in all periods.

Light compensation point (LCP) was similar across species and growth light environments in the before exposure period (Table 2.2). Two weeks of exposure to high light led to significant increases of LCP in both species with greater increased in previously shaded than in variable light plants. LCP continued to increase after three months of exposure to high light in all but previously shade-grown D. antarctica (Table 2.2).

The maximal carboxylation rate (Vcmax) as well as the maximal light driven electron flux

(Jmax) did not differ between growth light environments and species in the before exposure period (Figs. 2.2 a, 2.2 b). Two weeks of exposure to high light led to significant decreases in Vcmax and Jmax, irrespective of species and growth light environments. Vcmax remained low even after three months of exposure in all plants and only marginally recovered in D. antarctica previously grown in variable light (P=0.06). In contrast, recovery of Jmax was observed in all plants with exception for C. australis previously grown in variable light (Fig. 2.2 b).

-21- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Photosynthetic nitrogen use efficiency (PNUE) was not affected by the growth light environment but was significantly higher in C. australis in the before exposure period (Table 2.2). Short-term (two week) exposure to high light did not affect PNUE of variable light plants contrasting with a decrease in shade-grown plants. PNUE significantly increased after three months of exposure in all plants, with greater rises in plants previously grown in variable light (Light x Period, P=0.02; Table 2.2).

-22- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyat- heaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Table 2.2. Photosynthetic capacity parameters of the of the tree ferns D. antarctica and C. australis grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure).

Values are means (n = 5) ± s.e. of: Fv/Fm, maximal quantum yield of photochemistry; ф, the apparent maximum quantum yield; LCP, light com-

pensation point; PNUE, photosynthetic nitrogen use efficiency (Amax/nitrogen content). Effect abbreviations: P, Period; L, Growth light environment; S, species. Significance levels:*P≤0.05; **P<0.01; ***P<0.001; x, interactions. Only significant effects and interactions are presented.

Parameters Species Growth light environment Effects Variable light Shade Before ex- Short-term Long-term Before ex- Short-term Long-term ex-

posure exposure exposure posure exposure posure

Fv/Fm D. antarctica 0.78±0.02 0.72±0.03 0.69±0.02 0.83±0.01 0.68±0.02 0.71±0.02 P *** C. australis 0.75±0.02 0.68±0.03 0.68±0.01 0.82±0.01 0.67±0.02 0.73±0.01 L x P **

ф (mol CO2 D. antarctica 0.068±0.005 0.079±0.009 0.096±0.031 0.085±0.008 0.059±0.008 0.047±0.002 L*; L x P * mol-1quanta) C. australis 0.069±0.009 0.112±0.028 0.106±0.008 0.081±0.012 0.065±0.013 0.080±0.012 LCP (µmol m- D. antarctica 9±0 12±3 15±4 12±2 26±6 14±3 P*** 2s-1) C. australis 11±2 12±1 17±3 7±2 15±3 19±5 PNUE (µmol D. antarctica 25±4 30±1 59±11 33±3 25±3 48±4 P ***; S*** -1 - CO2 molN s L x P * 1) C. australis 32±5 33±3 70±5 66±13 23±4 56±8 L x S *

-23- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Shade Variable light

140

) a

-1

s 120

-2 100 P-value of effects

O m

2 80 P ***

(mmol H

max 40

at A 20

g 0 8 b

)

-1 s 6 P-value of effects

-2

m P ***

2 P x L x S * 4

mol CO



( 2

max

0 1.6 1.4 c

)

-1 1.2 s P-value of effects

-2 1.0

2 P x L x S* 0.8 0.6

mol CO



(

d 0.4

R 0.2 0.0

Short_term Long_term Short_term Long_term Before_exposure Before_exposure

Period

Fig. 2.1. a) Stomatal conductance under saturating irradiance; b) maximum photosynthetic rate and c) mitochondrial respiration in the dark of the tree ferns D. antarctica ( ) and C. australis ( ) grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short- term exposure) and three months (long-term exposure). Values are means (n=5) ± s.e. Ef- fect abbreviations: S, Species; L, Growth light environment; P, Period. Significance levels:*P≤0.05; ***P<0.001; x, interactions. Only significant effects and interactions are presented.

-24- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Shade Variable light 40 a

)

-1 s 30 P-value of effects

-2

m P*

mol CO



(

cmax

) 100 b

-1

-2 80

2 P-value of effects 60 P**

mol CO S**



( 40

P x S *

max

J 20

Short_term Long_term Short_term Long_term Before_exposure Before_exposure

Period

Fig. 2.2. a) Maximal carboxylation rate of Rubisco and b) maximal light driven electron flux of the tree ferns D. antarctica ( ) and C. australis ( ) grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and tree months (long-term exposure). Values are means (n=5) ± s.e. Effect abbreviations: S, Species; P, Period. Signifi- cance levels:*P≤0.05; **P<0.01; x, interactions. Only significant effects and interactions are presented.

-25- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

2.3.3. Plant water status

Predawn frond water potentials (Ψ predawn) were similar across growth light environments and species in the before exposure period (-0.3 to -0.4 MPa; Table 2.3). Two weeks of exposure to high light led to small although significant decreases in Ψ predawn in both species and both growth light environments (-0.6 to -1.1 MPa), with full recovery after three months in high light (Table 2.3).

2.3.4. Frond traits

Nitrogen (NA) and carbon (CA) content per frond area were significantly greater in plants grown under variable light than in shade, and in D. antarctica than in C. australis

(Table 2.3). Two weeks of exposure to high light stimulated increases in CA in shade- grown plants and decreases in NA in variable light plants of both species. After three months in high light, NA remained relatively unchanged in shade-grown plants, but decreased significantly in plants previously grown in variable light. In contras, CA was comparable with pre-exposure levels in variable light plants while continued to increase in previously shade-grown plants (Table 2.3).

Total chlorophyll (a + b) and chlorophyll a content per frond area were significantly greater in shade-grown than in variable light plants, and in D. antarctica than in C. australis (Table 2.3). Sudden exposure to high light had no immediate effect on total chlorophyll, which decreased only after three months of high light exposure (Table 2.3). Chlorophyll a/b ratio was significantly greater in D. antarctica for much of the experiment, whereas growth light environment and short-term (two week) exposure to high light had no effect on chlorophyll a/b ratio (Table 2.3).

-26- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Table 2.3. Predawn frond water potentials and frond traits of the tree ferns D. antarctica and C. australis grown under variable light and shade during the before exposure period, and then exposed to high light, and measured after two weeks (short-term exposure) and three months (long-term exposure). Values are means (n = 5) ± s.e. of: Ψ predawn, predawn frond water potential; NA, nitrogen content on a frond area basis; CA, carbon content on a frond area basis; Chl total, total chlorophyll content on a frond area basis; Chl a, chlorophyll a content on a frond area basis; Chl a/b, chlorophyll a/b ratio. Effect abbreviations: P, Period; L, Growth light environment; S, Species. Significance levels:*P≤0.05; **P<0.01; ***P<0.001; n.s., non significant; x, interactions. Only significant interactions are presented.

Parameters Species Variable light Shade Effects Before Short-term Long-term Before Short-term Long-term

exposure exposure exposure exposure exposure exposure

Ψ predawn D. antarctica -0.4±0.1 -1.1±0.2 -0.5±0.1 -0.3±0.1 -0.9±0.2 -0.4±0.0 P *** (MPa) C. australis -0.3±0.1 -0.7±0.1 -0.5±0.1 -0.4±0.1 -0.6±0.1 -0.3±0.2

-2 NA (g m ) D. antarctica 4.0±0.3 3.2±0.1 2.0±0.3 2.7±0.1 2.6±0.1 2.1±0.2 P ***; L ***; S ***; C. australis 3.4±0.5 1.9±0.2 1.5±0.1 1.8±0.2 1.8±0.1 1.5±0.1 L x P **

-2 CA (g m ) D. antarctica 74±5 71±4 72±9 48±5 58±5 75±5 P *; L ***; S * C. australis 62±8 64±6 69±1 39±5 45±5 57±6

Chl total D. antarctica 592±71 581±80 465±83 605±65 740±86 493±34 P *; L *; S ** (µmol m-2) C. australis 433±78 352±52 367±66 567±65 554±103 427±60

-27- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Parameters Species Variable light Shade Effects Before Short-term Long-term Before Short-term Long-term

exposure exposure exposure exposure exposure exposure

Chl a (µmol D. antarctica 454±51 442±64 337±63 454±46 538±60 394±24 P*; L*; S*** m-2) C. australis 321±57 264±40 267±55 422±48 400±72 312±45

Chl a/b D. antarctica 3.3±0.2 3.4±0.2 1.3±0.2 3.1±0.1 2.6±0.1 1.5±0.1 S***; L x P * C. australis 2.6±0.3 3.0±0.3 2.4±0.2 2.9±0.1 2.7±0.2 2.7±0.3

-28- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

2.3.5. Artificial sunfleck experiment

Predawn Fv/Fm was significantly lower in variable light than shade-grown plants (never- theless near 0.80), with the lowest Fv/Fm recorded for C. australis (0.78). Immediately after a sunfleck, Fv/Fm decreased to near 0.50 for both species irrespective of the growth light environment, but partly recovered within 30 min, although recovery of variable light C. australis was least (0.70; Table 2.4). Photosynthesis rate in low light (A20) as well as maximal photosynthesis rate calculated from the induction curve Amax_ind did not differ between species and growth light environments (Table 2.4). Increase in stomatal conductance (∆gs) in response to a sunfleck was insignificant for both species irrespective of the growth light environment (Table 2.4). Time to reach 63% of change in photosynthesis (t63%) did not differ significantly between species and growth light environments (Table 2.4).

-29- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

Table 2.4. Dynamic responses of photosynthesis to an artificial sunfleck. Values are means (n = 5) ± s.e. of: Fv/Fm _pr, Fv/Fm predawn; Fv/Fm _ immed, Fv/Fm immediately after the sunfleck; Fv/Fm _30 min, Fv/Fm 30 min after sunfleck; A20, maximal photosynthesis -2 -1 rate at PAR 20 µmol m s ; Amax _ind, maximal photosynthesis rate calculated from the induction curve; ∆ gs (gsmax–gs20), difference between stomatal conductance at the end of -2 -1 the experiment (gsmax) and at PAR 20 µmol m s (gs20); t 63%, time to reach 63% of change in photosynthesis. Effect abbreviations: S, Species; L, Growth light environment. Significance levels:*P≤0.05; n.s.,-non significant. Only significant interactions are presented.

Parameter Species Light regime P-value of effect Variable Shade

Fv/Fm _pr D. antarctica 0.81±0.01 0.81±0.01 L *, L x S * C. australis 0.78±0.01 0.82±0.00

Fv/Fm _ immed D. antarctica 0.53±0.04 0.49±0.03 n.s. C. australis 0.53±0.04 0.53±0.03

Fv/Fm _30 min D. antarctica 0.76±0.01 0.71±0.02 L x S * C. australis 0.70±0.02 0.72±0.01

A20 D. antarctica 0.51±0.12 0.15±0.18 n.s. (µmol m-2s-1) C. australis 0.42±0.11 0.54±0.13

Amax _ind D. antarctica 6.25±0.29 3.94±0.68 n.s. (µmol m-2s-1) C. australis 5.46±0.89 5.86±0.59

∆ gs D. antarctica 26±7 51±13 n.s. (mmol m-2s-1) C. australis 35±16 29±26 t 63% (s) D. antarctica 27±4 82±25 n.s. C. australis 49±18 22±4

-30- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

2.4. Discussion 2.4.1. Species overview

D. antarctica and C. australis had comparable maximum photosynthetic rates, Amax and mesophyll capacity parameters (i.e. Vcmax and Jmax). Photosynthetic rates were within the -2 -1 range previously reported in the literature for D. antarctica (Amax of 6 – 11 µmol m s

(Hunt et al. 2002; Volkova et. al 2009), while Vcmax and Jmax were within the lowest range of values, measured under similar light intensities and leaf temperatures, among a large number of species reviewed by Wullschleger (1993).

N content per frond area was significantly greater in D. antarctica than in C. australis. Values of up to 4 g m-2 in D. antarctica are beyond the range previously recorded for tree fern species (e.g. Cibotium menziesii (Hook) max 2.1 g m-2, Durand and Goldstein 2001). D. antarctica also had significantly greater chlorophyll a/b ratio, due to its greater chlorophyll a content, which is a characteristic of sun acclimated plants (Arens 1997). This result was at odds with this species‟ putative preference for well-shaded microhabitats (Dignan and Bren 2003). However, potential confounding effects of tree fern age should be considered given that our study involved young sporophytes rather than field-grown adults (Herbinger et al. 2007). Although D. antarctica seems to show attributes of a more light-adapted plant at the leaf scale, differences at the whole-tree scale, such as less hydraulic conductance (typical for trees with small rooting volumes), may contribute to re- stricting this species to wetter and shadier sites (see, for instance, McDowell et al. 2008). In addition, results of a field study on mature tree ferns indicated more similar values in chlorophyll a/b ratios between the two species (Volkova et al, in preparation).

2.4.2. Acclimation to growth light environment

We observed plasticity of frond traits in response to the growth light environment in both species (except for frond shape, which did not differ between the two growth light environments). More carbon per frond area, CA, in plants grown under variable light, indicated greater carbon gain under increased light availability (Oikawa et al. 2006). Shade-grown plants had more total chlorophyll (a + b) per frond area, indicating an enhanced invest-

-31- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press ment of resources to improve light harvesting in low irradiance (Niinemets et al. 1998). Although shade leaves commonly have lower chlorophyll a/b ratio, the growth light environment did not affect chlorophyll a/b ratio in our tree ferns, possibly because plasticity of this parameter is limited in ferns. Due to a lack of data on acclimation of chlorophyll a/b ratio in other tree fern species, we compared our tree ferns with shade adapted Trichomanes ferns, where the chlorophyll a/b ratio did not change after plants were transferred to high light (Johnson et al. 2000). We believe that such a comparison with non- arboreal ferns is appropriate as according to Large and Braggins (2004) „tree fern‟ is an arbitrary term, applied to any ferns with large erect rhizomes.

In terms of photosynthetic capacity parameters both species displayed limited acclimation potential to the growth light environment (i.e. effect of light was significant only for the maximum apparent quantum yield, ф), especially given that all measurements were made on fronds that developed under those environments. Greater ф of shade-acclimated plants probably allowed plants to photosynthesise more efficiently under low light (Seidlova et al. 2009).

Acclimation of plants to variable light did not lead to more efficient use of sunflecks – none of the measured parameters during sunflecks differed significantly from shaded plants. These results are in agreement with findings for the fern Polypodium virginianum L. by Gildner and Larson (1992) and confirm our suggestion of low acclimation potential of the tree fern photosynthetic apparatus to changing light conditions.

Significant decreases in Fv/Fm after the sunfleck treatment indicated engagement of nonphotochemical quenching, which was in agreement with other studies (e.g. Watling et al. 1997). There was no significant stomatal response to the artificial sunfleck, suggesting that stomata were not controlling dynamic photosynthetic response in the tree ferns, as was previously found in a co-occurring tree species Nothofagus cunninghamii Oerst. (Tausz et al. 2005).

-32- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press

2.4.3. High light stress

Irrespective of previous growth light environment, sudden exposure to high light resulted in significant decreases in a number of parameters, namely Fv/Fm, Amax, gs, Vcmax, Jmax and

Ψpredawn in both species. Decreases in photosynthetic capacity parameters (i.e. Amax, Vcmax,

Jmax) are consistent with effects of high light stress (Larcher 2003). The apparent quantum yield of photosynthesis, ф, was the only parameter more strongly affected in shade- acclimated plants than in those grown under variable light, probably indicating that excess irradiance reduced the quantum yield of photosynthesis via inactivation or down- regulation of PSII (Montgomery et al. 2008) – greater decrease in Fv/Fm in shade-grown plants is in agreement with this statement.

Significant decreases in Ψ predawn were not associated with changes in water availability as plants were watered to field capacity of the potting mix at all times and may point towards the effect of increased transpiration due to increase in leaf temperature trigged by sudden exposure to high light (Levitt 1980). Decreases in Ψ predawn in our plants (to -1.1 MPa) possibly indicated hydraulic stress. Ferns are known to have dichotomous branching veins which are not very efficient in delivering water to fronds. Furthermore, vein density is very low in these species (Brodribb et al. 2007).

2.4.4. Acclimation to new light environment

Non-recovery of Fv/Fm after three months of exposure indicated mild, albeit significant, photoinhibition in all plants, consistent with findings of Guo et al. (2006), who observed non-recovery in Fv/Fm of tropical rainforest sub-canopy species after transfer to high light.

However, mild photoinhibition would have very little effect on Amax measured under saturated light (Zhu et al. 2004) and therefore would not contribute markedly to longer-term persistence of the tree ferns under high light conditions.

Photosynthesis (Amax) of D. antarctica but not C. australis recovered after three months exposure to high light. This result is consistent with field observations by Ough and Mur-

-33- This Chapter is published Volkova L, Bennett LT and Tausz M “Effects of sudden exposure to high light on two tree fern species Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) acclimated to different light intensities, Australian Journal of Botany, v.57, issue 7, 2009 In press phy (2004) of greater decline in C. australis than D. antarctica within five years of clearcut logging.

While Vcmax remained low even after three months of exposure to high light in all plants, full recovery (and even further increase of Jmax in D. antarctica) suggest that electron transport and RuBP regeneration capacity, rather than Rubisco activity, were limiting factors to tree fern photosynthesis, as suggested by Wise et al. (2004).

Increases in Rd with prolonged exposure to high light of C. australis pre-acclimated to variable light suggests greater energy loss by this species (Seidlova et al. 2009) and may contribute to the lack of recovery of Amax. These results are consistent with our earlier suggestion and findings by Ough and Murphy (2004) that C. australis appeared to be more vulnerable to high light stress.

2.5. Summary

Regardless of putative differences in origin and observed differences in micro-site preferences, both tree fern species had comparable photosynthetic capacity parameters. Steady- state gas exchange as well as dynamic responses of plants to an artificial sunfleck indicated that difference in the growth light environment had very little effect on the tree ferns‟ capacity to utilise and acclimate to prevailing light conditions. Sudden exposure to high light led to decreases in all photosynthetic parameters and more negative predawn frond water potentials, indicating high light stress responses that were mostly irrespective of the growth light environment. Despite its field preference for shadier and wetter sites, D. antarctica showed greater acclimation capacity to sustained high light stress than C. australis under glasshouse conditions. After three months in high light, D. antarctica fully recovered while C. australis previously grown under variable light recovered only partially, indicating limited capacity of these plants to acclimate to high light, or perhaps suggesting some previous light stress under the variable light environment. Results of this study can be used in planning of forest management practises for better protection of biodiversity.

-34- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Chapter 3. Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica.

(ii) Abstract

Effects of high irradiance and moderate heat on photosynthesis of the tree fern Dick- sonia antarctica were examined in a climate chamber under two contrasting irradiance regimes (900 and 170 µmol photons m-2 s-1) and three sequential temperature treatments (15ºC; 35ºC; back to 15ºC). High irradiance led to decline in predawn quantum yield of -2 photochemistry, Fv/Fm (0.73), maximal Rubisco activity (Vcmax; from 37 to 29 µmol m -1 -2 -1 s ), and electron transport capacity (Jmax; from 115 to 67 µmol m s ). Temperature increase to 35ºC resulted in further decreases in Fv/Fm (0.45) and in chlorophyll bleaching of high irradiance plants, while Vcmax and Jmax were not affected. Critical temperature for thylakoid stability (Tc) of D. antarctica was comparable with other higher plants

(ca 47ºC), and increases of Tc with air temperature were greater in high irradiance plants. Increased Tc was not associated with accumulation of osmotica or zeaxanthin formation. High irradiance increased the xanthophyll cycle pigment pool (V+A+Z, 91 vs. 48 mmol mol-1 chlorophyll-1), de-epoxidation state (56% vs. 4%), and α-tocopherol. Temperature increase to 35ºC had no effect on V+A+Z and de-epoxidation state in both light regimes, while lutein, β-carotene and α-tocopherols increased, potentially contributing to increased membrane stability under high irradiance.

3.1. Introduction

While understorey species of evergreen forests often experience high intensity sunflecks, they are not usually exposed to prolonged periods of high irradiance (Lovelock et al. 1998; Tausz et al. 2005). A protective canopy usually creates a favourable microclimate with more moderate temperature fluctuations and greater air humidity than in the above-canopy atmosphere. However, during the course of forest ecosystem dynam-

-35- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press. ics including gap formation, bushfires, or anthropogenic management such as forest harvesting, understorey species may be suddenly exposed to full sunlight and high temperatures, stress factors that can contribute to temporary decline of these species. Ac- cording to climate-change projections, these factors are likely to become even more significant in the future, as temperatures are predicted to increase, and disturbances in forest canopies may become more frequent (Hennessy et al. 2007).

In the short term, exposure of shade-acclimated plants to high levels of irradiance often leads to photoinhibition and photo-oxidative stress. Photoinhibition alone is rarely responsible for plant mortality and the plant may recover and become fully acclimated. Photo-oxidative stress is caused by the toxic effects of reactive oxygen species (ROS) produced in the photosynthetic apparatus under high irradiance when carbon assimilation is light-saturated (Niyogi 2000). Many plants can, to a certain extent, acclimate to increased irradiance through enhanced dissipation of absorbed light energy in the thylakoids, a process related to the conversion of the light harvesting xanthophyll violaxanthin to the energy quenching zeaxanthin (Demmig-Adams and Adams 2006). Protection against high irradiance can also involve the accumulation of tocopherol (Munné-Bosch 2005), an antioxidant that scavenges toxic ROS and contributes to thylakoid membrane stability.

When, in addition to high irradiance, leaves are exposed to other environmental stress factors such as high temperature, there can be sustained reductions in the efficiency of photosynthetic energy conversion and inhibition of repairs to photodamaged photosystem II (PSII; Murata et al. 2007). Photosynthesis is particularly sensitive to inhibition by heat stress due to labile components in the photosynthetic apparatus (Salvucci and Crafts-Brandner 2004). The thylakoid membrane is one of the main temperature stress targets and changes during acclimation occur at that level (Ducruet et al. 2007). The degree of thermostability of the thylakoids can be estimated by the critical temperature Tc – the temperature threshold above which irreversible damage occurs to PSII (Schreiber and Berry 1977). Tc changes with growing conditions, reflecting thermal acclimation of the photosynthetic apparatus (Ducruet et al. 2007). Chlorophyll fluorescence yield is a

-36- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

sensitive indicator of the state of thylakoids, and can be used to assess Tc in plants as the temperature threshold above which ground level fluorescence (F0) increases (e.g. Froux et al. 2004). The mechanisms underlying acclimatory changes in Tc are still poorly un- derstood, although some results point towards stabilising effects of protective compounds on thylakoids. For example, the xanthophyll zeaxanthin (Havaux and Gruszecki 1993; Havaux and Tardy 1996), as well as increased soluble sugar concentration (Hüve et al. 2006), are believed to have a stabilising effect and shift Tc towards higher temperatures.

Our model understorey species, the tree fern Dicksonia antarctica (Labill., Dick- soniaceae), is known to decline after clearcut logging in Victoria, Australia (Ough and Murphy 2004). These tree ferns are iconic and ecologically significant understorey species in many humid forest types in the Southern Hemisphere, including Australian temperate rain forests and wet sclerophyll (eucalypt) forests (Large and Braggins 2004). They support a large epiphytic diversity on their trunks and provide nursery sites for many tree and shrub species as well as nesting and feeding sites for marsupials, insects and birds (Lindenmayer et al. 1994; Roberts et al. 2005). Decline in D. antarctica numbers is expected to negatively impact on many dependent species and thus maintenance of tree ferns is often an objective of forest management plans (Department of Natural Resources and Environment 2002).

The reasons for poor survival and ongoing decline of D. antarctica after logging remain uncertain (Ough and Murphy 2004), but exposure to high irradiance, combined with increased air and frond temperatures, could be contributing factors. Periodically disturbed by wildfires in their natural habitat, D. antarctica are exposed to a broad range of irradiance during their lifetime (Hunt et al. 2002), which suggests that this species is able to at least partly acclimate to different levels of irradiance. Certainly, other studies indicate potential for fern acclimation to different light regimes. For example, New Zealand ferns from contrasting habitats displayed contrasting characteristics in terms of photosynthetic light compensation point, which were tightly correlated with specific frond area (Bannister and Wildish 1982). Frond characteristics (frond surface area, epidermis

-37- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press. thickness, palisade/spongy mesophyll ratio, blade size, petiole length) of a South American Cyathea species (another important tree fern genus) were also correlated with its local irradiance (Arens 1997). However, other studies suggest limited capacity of shade-acclimated tree ferns to efficiently adjust to increased irradiance (Durand and Goldstein 2001). To our knowledge, only a few studies have examined effects of high temperature, either alone or with high irradiance on the physiological performance of tree ferns: Tingey et al. (1987) found that photosynthesis of D. antarctica was particularly susceptible to inhibition with increasing temperature and high light; and Nobel et al. (1984) also mentioned negative effects of high temperature on gas exchange of ferns. D. antarctica’ s natural distribution in Australia is limited to the temperate zone (McCarthy 1998), characterised by cool to warm conditions (Köppen classification Aus- tralian Bureau of Meteorology, http://www.bom.gov.au/iwk/climate_zones, verified 13 August 2009), perhaps indicating that the species has limited potential for acclimation to temperature increases (such as after clearcut logging, but potentially also due to climate change), making it susceptible to ongoing decline.

In this study, we investigated the responses of D. antarctica to high irradiance, moderately high temperature (+35ºC), and a combination of both under fully controlled climate chamber conditions. Measuring Tc together with a number of variables related to photosynthesis, chlorophyll fluorescence, and chloroplast pigments, we addressed the following specific questions:

 Are photosynthetic parameters of D. antarctica adversely affected by a) high irradiance; b) high temperatures; and c) their interactions?

 Does membrane stability (measured via critical temperature, Tc,) increase in D. antarctica fronds with increased temperature (indicative of an acclimation to high

temperature), and if yes, are Tc changes associated with accumulation of osmotica or zeaxanthin formation?  Do other potentially protective thylakoid compounds, such as carotenoids and tocopherol, change in relation to high irradiance and high temperature?  Are effects of high temperature on the above parameters reversible?

-38- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

3.2. Material and methods 3.2.1. Plant material

One-year-old sporophytes of Dicksonia antarctica Labill (HSK Gardening and Leisure Avon Dassett, UK) were transplanted into 10-l pots, containing a mixture of sand and peat (50/50 v/v) and 40g slow release fertiliser (Nutricote 100, Chisso-Asahi Fertilizer Co. Ltd, Tokyo, Japan, N/P/K, %, 13/13/13) per pot. The plants were grown under uniform sunlit conditions in a naturally illuminated glasshouse at INRA, Champenoux, France (48º44‟N, 6º14‟E) for two months in spring 2007. At the end of this period, the plants were transferred to a climate chamber (Chambre Phytotronique STRADER, An- gers, France) at Champenoux.

3.2.2. Climate chamber conditions and experimental design

Irradiance in the climate chamber was provided by two types of 400 W lamps (HQI Philips (mercury halide) and SONT Philips (sodium halide) Koninklijke Philips Elec- tronics N.V., Eindhoven, The Netherlands) and resulted in a photosynthetic photon flux density (PPFD) of 900 µmol photons m-2s-1 at plant height (PAR range, 400-700 nm, measured with a Li-Cor quantum sensor). Relative humidity was 70-80%, air temperature was controlled to ± 0.5ºC (see temperature treatments below), and photoperiod was 16 h day-1.

The tree ferns were randomly assigned to two experimental groups (n = 7 in each). One group was shielded by a wavelength-neutral shade mesh (17% light transmission, PPFD: 170 µmol photons m-2s-1) – „shade‟, the other exposed to full light (900 µmol photons m-2s-1) – „high irradiance‟. Such levels are representative of irradiance conditions in the open on overcast winter and clear summer days respectively, at typical D. antarctica field sites in mountain ash forests of Victoria, Australia. Plants were kept well-watered at all times and were rotated daily at random within their designated irradiance regime.

-39- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

A sequence of temperature treatments was applied as follows: (1) 10 days at 15ºC day and night; (1a) 3 days at 25°C day and night (to avoid heat shock); (2) 12 days at 35°C/25°C day/night (typical hot summer days for D. antarctica in the field); (3) 10 days at 15°C day and night (to check reversibility of temperature effects).

Chlorophyll a fluorescence and critical frond temperature were measured for each individual every 1 to 2 days throughout the experiment. Photosynthesis was recorded from net CO2 uptake (A) versus intercellular CO2 concentration (A-Ci curves) on the first day of the experiment and at the end of each temperature treatment. Samples for nitrogen (N) content, xanthophyll analyses, and osmolality were taken at the end of each of the three temperature treatments. All measurements were made on the mid-third of the youngest fully-expanded fronds that were of healthy appearance (i.e. not discoloured).

Total chlorophyll content was measured for each individual every 1 to 2 days throughout the experiment; measurements were made over the entire plant irrespective of frond condition.

3.2.3. Frond temperature (Tfrond)

Frond temperature (as 10 random points across the entire plant) was measured twice during the 35ºC temperature treatment (beginning/end) at predawn and midday, and once at the end of the experiment (during the second 15ºC temperature treatment), using an IR laser thermometer (Raynger PM, Raytech Inc, Santa Cruz, CA, USA).

3.2.4. Maximal quantum yield of photochemistry (Fv/Fm)

Fv/Fm was derived from chlorophyll a fluorescence measured on dark-acclimated fronds (at the end of the „night‟ period) with a modulated fluorometer (PAM 2000, Heinz Walz

-40- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

GmbH, Effeltrich, Germany). Maximum quantum yield of PSII was estimated as Fv/Fm

= (Fm–F0)/Fm, after Maxwell and Johnson (2000).

3.2.5. Gas exchange measurements

Gas exchange parameters were measured using a Li-Cor 6400 gas exchange system, equipped with a 2x3 cm broadleaf chamber (Li-Cor, Lincoln, Nebraska, USA). All gas exchange measurements were conducted at the reference frond temperature of 25ºC. For -2 -1 each plant, an A-Ci curve was generated at PPFD 1000 µmol m s , frond temperature 25ºC, air flow rate 400 µmol air s-1, and relative humidity (RH)>60%. Photosynthesis -1 was induced at a CO2 mole fraction of 50 µmol mol for 15-20 min prior to measurements to ensure maximal stomatal opening and maximal activity of Calvin cycle en- -1 zymes. CO2 mole fraction was then increased in 13 successive steps to 2200 µmol mol with two measurements at each step. After finishing the A-Ci curve, illumination in the -1 leaf chamber was turned off, CO2 mole fraction was decreased to 400 µmol mol and respiration rate was recorded after 5 min in the dark. The frond area enclosed in the Li- Cor chamber was marked, photographed and calculated using imaging software

(UTHSCSA Image Tool Version 3, University of Texas, USA). Values for Amax and gs -1 at ambient CO2 (400 µmol mol ) were derived from these curves.

Using a biochemical photosynthetic model Farquhar (Farqhuar et al. 1980), apparent

(i.e. assuming that mesophyll conductance to CO2, gi, is infinite) maximum carboxylation rate (apparent Vcmax), and the maximum apparent rate of electron transport (apparent Jmax) were estimated by fitting the A-Ci curves to the model as described in Mont- pied et al. (2009). Triose phosphate use (TPU) limitation was not included in the model; points with decreasing A at high CO2 mole fractions were disregarded. A set of primary -1 parameters of Rubisco kinetic properties used herein, Kc= 327 µmol mol , Ko = 282600 µmol mol-1, Γ* = 43.7 µmol mol-1, were taken from von Caemmerer et al. (1994).

Then, gi was estimated with the curve fitting approach introduced by Ethier and

Livingston (2004) and described by Montpied et al. (2009) and real Vcmax and Jmax were

-41- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

computed based on the A-Cc (chloroplastic CO2 mole fraction); therefore, only corrected

Vcmax and Jmax (i.e., under the hypothesis of finite gi) are given and discussed in this study.

3.2.6. Frond nitrogen and chlorophyll content

Frond samples were analysed for nitrogen (N) content using an elemental analyser (NCS 2500, CE instrument Thermo Quest, Milano, Italy). Samples were dried at 60ºC for 48 h (to determine dry weight) and then ground to a fine powder. Frond area of fresh samples was scanned and calculated using Scion Image software (Scion Corporation 2000-2001, USA), and these data used to calculate N content on a frond area basis.

Frond chlorophyll content was estimated from transmittance values measured with the Minolta SPAD-502 Chlorophyll meter (Minolta, Illinois, USA; hereafter „SPAD‟). Val- ues were the mean of two to three separate pinnules per plant (randomly selected irrespective of frond colour).

Total chlorophyll (a + b) was also measured by HPLC (see Pigments and tocopherol determination).

3.2.7. Critical temperature (Tc)

Critical temperature was estimated in vivo from the sharp rise of basal chlorophyll a fluorescence under increasing temperature (Schreiber and Berry 1977). Disks of tree fern pinnules were placed into a temperature-controlled aluminium body, with the fibre- optics of the fluorometer (PAM 2000, Walz, Effelrich, Germany) pointing at the sample. Ground fluorescence (F0) was induced with a red diode at low PPFD of about 1 µmol m-2s-1. Temperature of the aluminium body was increased gradually (1°C min-1) from 20°C to 60°C. F0 was continuously recorded and critical temperature (Tc) was estimated graphically at the beginning of the heat-induced fluorescence rise (Froux et al. 2004).

-42- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

3.2.8. Total tissue osmolality

Total tissue osmolality was measured using freeze-point depression from hot water extracts of dried frond tissue (Callister et al. 2006). Approximately 60 mg of dried ground frond tissue (as prepared for N analysis) was placed in a 2-ml polypropylene vial to which 1.6 ml of deionised water was added. The samples were placed in a water-bath at 90ºC for 60 min. The samples were left to cool to room temperature, centrifuged at 10,000 x g for 2 min and 1 ml of the supernatant was transferred to a 1.7-ml polypropylene vial. Osmolality of the solution was measured using an OSMOMAT 030 cryoscopic osmometer (Gonotec, Berlin, Germany).

3.2.9. Pigments and tocopherol determination

Frond discs (3.75 mm diameter) were collected at midday at the end of each of the three temperature treatments and immediately frozen in liquid nitrogen. Samples were freeze- dried, sealed with silicagel in airtight plastic bags, and kept at -20ºC until analysis.

Four discs per plant were ground in a Matrix Mill (Retsch MM301, Germany) at the temperature of liquid nitrogen. To avoid the presence of traces of acid in the acetone used for the extraction, 0.5 g l-1 of calcium carbonate were added to samples prior to grinding (García-Plazaola and Becerril 1999). The resulting powder was extracted with 0.5 ml of ice-cold acetone, homogenised and centrifuged at 4ºC for 1 min at 15000 x g. The pellets were re-extracted as described above to a combined sample volume of 1 ml. Extracts were stored in sealed vials at -20ºC until analysis. Prior to injection, samples were centrifuged at 4ºC for 20 min at 15000 x g and the clean supernatant was transferred into HPLC vials.

HPLC separation of chloroplast pigments and tocopherols was according to the methods given in Tausz et al. (2003). Chromatographic conditions were:

-43- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

 Pigments: 25 x 4.6 mm Spherisorb ODS 25 µm column. Gradient: solvent A: acetonitrile: methanol: water = 100:5:10 (v/v/v), solvent B: ethylacetate: acetone = 1:2 (v/v), 10% B to 70% B in 17 min, hold at 70% B for 5 min, return to 10% B in 5 min. Flow rate 1 ml min-1. The injection volume was 20 µl, photometric detection at 440 nm.  α-Tocopherol: 25 x 4.6 mm Spherisorb ODS 25 µm column. Solvent 100% methanol isocratic. Flow rate 1 ml min-1. Injection volume was 20 µl. Fluorescence detection excitation 295 nm, emission 325 nm.

Acetone, acetonitrile, methanol and ethyl acetate were of HPLC grade and water was deionized. A standard of α-tocopherol was purchased from Sigma (Sigma-Aldrich, Cas- tlehill, NSW, Australia); standards for carotenoids and chlorophyll a and b were prepared as follows: several generic extracts were prepared in 100% acetone and measured at three wavelengths in the spectrophotometer (Varian UV/V 300, USA). Using the equations of Lichtenthaler (1987), chlorophyll a, b and total carotenoid concentrations were calculated at a spectrophotometer resolution range of 1 – 4 nm. The same extracts were then re-run in the HPLC and conversion factors for chlorophyll a, b and total carotenoids were calculated, disregarding the minor differences in carotenoid absorption coefficients at the wavelength in question.

3.2.10. Statistical analysis

Repeated measures models of SPSS 15 (SPSS Inc. Chicago, USA) were used for statistical analysis, with irradiance as the between-subject factor and temperature as the within-subject factor (both fixed). Effects of irradiance (high irradiance, shade), and 3 levels of temperature (15ºC, 35°C, back to 15ºC), and irradiance by temperature interactions on each dependent variable were analysed. Data for statistical analyses were the values per individual plant at the end of each temperature treatment. Photosynthetic parameters, measured before the start of the experiment were not used in the model.

-44- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

3.3. Results

3.3.1. Frond temperature (Tfrond)

Tfrond did not differ among plants at the beginning of the experiment (data not shown).

After the temperature increased to 35ºC, Tfrond was similar in shaded and high irradiance plants at predawn (below 25°C) but differed on average by 1.5ºC at midday (around

35°C vs. 33.5°C, Table 3.1). By the end of the treatment at 35ºC, Tfrond of shaded plants was on average 0.7ºC cooler at predawn, and 3.3ºC cooler at midday (Table 3.1).

Table 3.1. Temperature of Dicksonia antarctica fronds (Tfrond) exposed to high irradiance and under shade. Values are means ± s.e. (n = 7 plants, ten measurements per plant) at predawn and midday on the first (1) and last (12) days of two temperature treatments (35ºC and back to 15ºC). P values indicate significance of difference between high irradiance and shaded plants within temperature treatments (Student‟s t-test)

35ºC Back to 15ºC High Differ- High P Differ- P irradi- Shaded ence irradi- Shaded ence ance ance Pre- dawn Day 1 24.3±0.1 24.2±0.1 0.1 0.30 15.3±0.0 16.0±0.0 -0.7 <0.001 Day 12 23.0±0.2 22.3±0.2 0.7 0.02

Midday Day 1 34.9±0.6 33.4±0.3 1.5 0.02 19.7±0.4 17.2±0.4 2.5 <0.001 Day 12 34.6±0.2 31.3±0.5 3.3 0.002

-45- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

3.3.2. Maximum quantum yield of PS II (Fv/Fm) and photosynthetic capacity parameters

Predawn Fv/Fm remained close to the optimum value of 0.83 in shaded plants across all temperature treatments. In contrast, Fv/Fm declined after the first day of exposure to high irradiance (Fig. 3.1 a). The 35ºC treatment resulted in further decreases of Fv/Fm (Fig.

3.1 a). After return to 15°C, a partial recovery of Fv/Fm was detected.

-46- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Fig. 3.1. a) Time course of maximum quantum efficiency of PSII and b) chlorophyll content of high irradiance (open symbols) and shaded (closed symbols)D. antarctica during three successive temperature treatments(delineated by dotted lines). Values are means of n = 7 (± s.e.). P-values indicate significance of effects of irradiance (I), temperature (T), and irradiance by temperature interaction (I x T).

-47- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Photosynthetic parameters were comparable among all plants at the start of the experiment (Table 3.2, „before‟). Ten days of high irradiance (at 15ºC) resulted in decreases in both light-saturated rate of net photosynthesis at a reference temperature of 25°C, 25 Amax , and corresponding stomatal conductance gs (Table 3.2; Fig. 3.2). Increasing the temperature to 35ºC induced stomatal opening in all plants (Table 3.2). However, while

Amax of all shaded plants also increased (Table 3.2. Fig. 3.2), Amax was less responsive in high irradiance plants (Fig. 3.2). Nonetheless, all changes in gs and Amax were fully reversible upon return to 15ºC (Table 3.2).

When measured at the reference temperature of 25°C, maximal carboxylation rate,

Vcmax, and maximal light-driven electron flux Jmax decreased in response to increased irradiance. Both were insensitive to temperature treatments regardless of irradiance regime (Table 3.2). Mesophyll conductance to CO2, gi, was highly variable, probably owing to the low accuracy of the fitting procedure used. As a result, no significant effects of irradiance and temperature could be detected (Table 3.2).

Nitrogen content per frond area (g m-2) was not affected by either irradiance or temperature (Table 3.2). Photosynthetic nitrogen use efficiency (PNUE), calculated as Vcmax/N, was lower under high irradiance than in shade and remained such until the end of the experiment. Changes in temperature did not affect PNUE (Table 3.2).

Total chlorophyll content (in SPAD units) decreased at 35ºC under high irradiance and remained low thereafter (Fig. 3.1 b). A similar albeit not significant effect was detected for chlorophyll a + b in HPLC extracts (Table 3.2). It should be noted that visually damaged fronds were avoided for HPLC analyses, while SPAD measurements were made across entire fronds. Chlorophyll a/b ratios were similar between irradiance regimes during the 15ºC temperature treatment. With temperature increase to 35ºC, chlorophyll a/b ratios decreased in high irradiance plants in contrast to a significant increase in shaded plants (Table 3.2). With temperature return to 15ºC, chlorophyll a/b ratios of high irradiance plants recovered to the initial values.

-48- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Table 3.2. Photosynthesis and frond traits of D. antarctica exposed to high irradiance and under shade before and during three successive 25 temperature treatments. Values are means ± s.e. (n = 7) of Amax , light-saturated net CO2 assimilation rate at 25°C; gs, stomatal conductance under saturating irradiance at 25°C; NA, nitrogen content on a frond area basis; PNUE, photosynthetic nitrogen use efficiency 25 25 (Vcmax /NA) at 25°C; Chl total, total chlorophyll content in SPAD units and on a frond area basis; Chl a/b, chlorophyll a/b ratio; Vcmax , 25 maximal carboxylation rate of Rubisco at 25°C; Jmax , maximal light driven electron flux at 25°C; gi, mesophyll conductance to CO2 measured at 25°C; Effect abbreviations: I, Irradiance; T, temperature; I x T, irradiance by temperature interaction; Significance levels:*P≤0.05; **P<0.01; ***P<0.001; n.s., non significant; n.d., no data

Parameter Temperature treatments Significance of Irradiance regime effects (P) Before 15ºC 35ºC Back to 15ºCº

High irradiance 6.0±0.5 3.7±0.5 5.1±0.6 3.9±0.4 25 -2 -1 Amax (µmol CO2 m s ) I***; T* Shade 6.4±0.4 6.4±0.8 7.6±0.3 5.6±0.5

25 High irradiance 82±11 57±6 108±7 50±6 gs at Amax -2 -1 I*; T*** (mmol H2O m s ) Shade 80±7 80±14 152±12 70±5

High irradiance 37.0±2.0 29.3±2.7 23.2±2.8 29.1±3.3 I*** 25 -2 -1 Vcmax (µmol CO2 m s ) Shade 36.0±1.0 37.1±2.9 40.6±3.8 34.5±4.2

High irradiance 115±6 68±9 50±7 85±11 I** 25 -2 -1 Jmax (µmol CO2 m s ) Shade 105±6 85±10 86±12 98±10

-49- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Parameter Temperature treatments Significance of Irradiance regime effects (P) Before 15ºC 35ºC Back to 15ºCº

High irradiance 155±64 141±53 143±115 115±25 n.s. -2 -1 gi (mmol CO2 m s ) Shade 115±35 222±63 294±195 259±112

High irradiance n.d. 16.9±1.0 15.9±0.6 17.4±0.7 -2 NA (g m ) n.s. Shade n.d. 16.5±0.7 16.0±0.8 15.5±2.1

High irradiance n.d. 204±21 201±24 175±13 PNUE (V 25/N),(µmol cmax I** mol-1 N-1m-2) Shade n.d. 278±18 311±30 300±41

High irradiance n.d. 49.1±1.7 39.3±2.3 34.1±2.1 I**; T***; Chl total (SPAD units) I x T*** Shade n.d. 48.9±0.4 47.1±0.5 47.3±1.0

Chl total (a + b) High irradiance n.d. 782±142 721±111 675±90 -2 n.s. (µmol m ) Shade n.d. 810±63 914±127 843±81

High irradiance n.d. 2.36±0.05 2.29±0.05 2.41±0.07 I**; T**; Chl a/b I x T*** Shade n.d. 2.46±0.05 2.71±0.05 2.66±0.07

-50- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Fig. 3.2. Stomatal conductance, gs versus light-saturated rate of net photosynthesis Amax for high irradiance (on the right) and shaded (on the left) D. antarctica measured at the standardised temperature of 25ºC during three temperature treatments: 15ºC (open circle), 35ºC (closed diamond) and back to 15ºC (open triangle). Values are means of n = 6 (high irradiance) and n = 7 (shaded).

-51- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

3.3.3. Critical temperature (Tc)

During the 15°C treatment, critical temperature for photochemistry (Tc) was similar under shade and high irradiance (means of 47.5ºC and 47.2ºC respectively; Fig. 3.3). In- crease in temperature resulted in significant rises of Tc that were greatest under high irradiance. Tc started to decrease after return to 15°C, although pre-treatment values were not reached by the end of the experiment (Fig. 3.3).

Fig. 3.3. Time course of critical temperature, Tc of high irradiance (open symbols) and shaded (closed symbols) D. antarctica across the experiment. Values are means of n = 7 (± s.e.). P-values indicate significance of effects of irradiance (I), temperature (T), and irradiance by temperature interaction (I x T).

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3.3.4. Total tissue osmolality

Total tissue osmolality was not affected by irradiance but decreased significantly during the 35ºC treatment (Table 3.3). It increased to close to the original values after return to 15°C.

3.3.5. Carotenoids and α-tocopherol

Neoxanthin and lutein contents (per mol total chlorophyll) were significantly greater under high irradiance, whereas α- and β-carotene contents remained comparable to shaded plants (Table 3.3). Temperature increase to 35ºC led to significant increase in β- carotene and lutein in high irradiance plants. These pigments tended to remain high on return to 15ºC, although α-carotene significantly decreased under high irradiance (Table 3.3).

The xanthophyll cycle pigment pool (i.e., Violaxanthin, Antheraxanthin, and Zeaxan- thin, V+A+Z) was significantly greater under high irradiance for the whole experiment. Changes in temperature did not affect V+A+Z under either irradiance regime (Table 3.3). The de-epoxidation state of xanthophylls (expressed as (0.5A+Z)/(V+A+Z)) was greater under high irradiance than shade (on average 44% vs. 3%; Table 3.3). It decreased by ca 32% under high irradiance upon return to 15°C, but was insensitive to temperature under shade. Small amounts of lutein-epoxide were detected (Table 3.3), but they were not affected by light, temperature and their interactions.

Content of α-tocopherol was significantly greater under high irradiance than shade (Fig. 3.4). Whereas α-tocopherol was invariant to temperature changes in shaded plants, α- tocopherol increased in high irradiance plants with temperature increase to 35ºC, and remained high after the temperature returned to 15ºC; due to the lack of a high light-low temperature control, we cannot clearly separate the high irradiance and high temperature effects on these changes.

-53- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Table 3.3. Pigment content and osmolality of D. antarctica fronds exposed to high irradiance and under shade during three successive temperature treatments. Values are means ± s.e. (n = 7) of: osmolality; carotenoids (on a chlorophyll basis): lutein, neoxanthin and xanthophyll pool (i.e. Violanxanthin, Antheraxanthin, Zeaxanthin, V+A+Z), α- and β-carotene, lutein-epoxide and the de-epoxidation state of violaxanthin = (0.5A+Z)/(V+A+Z). Effect abbreviations: I, Irradiance; T, temperature; I x T, irradiance by temperature interaction; Signifi- cance levels:*P≤0.05; **P<0.01; ***P<0.001; n.s. –not significant

Temperature treatments Significance of effects Irradiance regime (P) 15ºC 35ºC Back to 15ºC

High irradiance 1.56±0.06 1.33±0.04 1.45±0.05 Osmolality (mosmol g d.w.-1) T*** Shade 1.55±0.02 1.40±0.03 1.51±0.01

High irradiance 91.9±9.2 118±15.0 113.7±15.9 V+A+Z (mmol mol-1 chl-1) I*** Shade 47.8±1.7 45.8±1.2 45.5±1.2

High irradiance 56.6±3.4 53.1±7.7 21.4±4.7 De-epoxidation (%) I***; T***; I x T*** Shade 4.4±1.1 2.3±0.8 1.6±0.6

High irradiance 263±9 339±25 388±29 Lutein (mmol mol-1 chl-1) I***; T**; I x T** Shade 185±4 186±3 198±6

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Temperature treatments Significance of effects Irradiance regime (P) 15ºC 35ºC Back to 15ºC

High irradiance 61.3±2.6 65.9±3.8 69.3±3.6 Neoxanthin (mmol mol-1 chl-1) I*** Shade 45.2±1.9 46.2±1.1 48.4±2.0

High irradiance 11.1±2.3 24.2±3.4 5.8±1.5 α-carotene (mmol mol-1 chl-1) I*; T***; I x T** Shade 16.1±2.6 24.1±2.4 20.9±1.8

High irradiance 62.2±7.8 94.6±8.7 89.3±7.0 β-carotene (mmol mol-1 chl-1) I***; T** Shade 44.7±5.7 59.4±5.3 59.3±3.3

Lutein-epoxide High irradiance 5.2±1.7 6.3±1.4 6.2±2.2 n.s. -1 -1 (mmol mol chl ) Shade 3.4±0.4 3.9±0.4 4.3±0.3

-55- This Chapter is published Volkova L, Tausz M, Bennett LT, Dreyer E (2009) Interactive effects of high irradiance and moderate heat on photosynthesis, pigments, and tocopherol in the tree fern Dicksonia antarctica. Functional Plant Biology In press.

Fig. 3.4 α - Tocopherol content of high irradiance (open bars) and shaded (closed bars) D. antarctica under three temperature treatments. Values are means of n = 7 (± s.e.). P- values indicate significance of effects of irradiance (I), temperature (T), and irradiance by temperature interaction (I x T).

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3.3.6. Correlations between Tc and biochemical parameters

No significant correlation was detected between Tc and osmolality, tocopherol, and α- and β-carotenes (correlations not shown). Correlations between Tc and zeaxanthin, presented as Z/(Z+V) (after Havaux and Gruszecki 1993) were also not significant, either for each irradiance treatment separately or for the combined data (Fig. 3.5).

Fig. 3.5. Critical temperature, Tc versus xanthophyll zeaxanthin (expressed as Z/(Z+V); after Havaux and Gruszecki, 1993) of high irradiance (open symbols) and shaded (closed symbols) D. antarctica during three temperature treatments: 15ºC (circle), 35ºC (diamond) and back to 15ºC (triangle). Each point represents an individual measurement. Relationships were also non-significant when data were separated by irradiance treatment. Z, zeaxanthin; V, violaxanthin.

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3.4. Discussion 3.4.1. Effect of high irradiance, high temperature and their interaction on photosynthetic capacity parameters of D. antarctica

Photosynthetic capacity of D. antarctica in this study was within ranges reported in the 25 literature. Maximum light saturated rates of net photosynthesis (Amax ) were comparable with values reported for the same species: 6 – 10.8 µmol m-2s-1 (Nobel et al. 1984) -2 -1 25 and 8.3 µmol m s (Hunt et al. 2002). Maximum carboxylation rates, Vcmax (the 25 maximal in vivo Rubisco activity), and the maximum rate of electron transport, Jmax , at the reference temperature of 25°C were comparable to some shade tolerant tree species, such as silver fir (Abies alba Mill, Robakowski et al. 2002), and mesophyll conductance, gi, corresponded to typical values of evergreen trees among the species reviewed by Ethier and Livingston (2004).

Photosynthetic capacity of D. antarctica was adversely affected by high irradiance. 25 Jmax decreased with respect to shaded plants, and this decrease was paralleled by a decrease of maximum quantum yield of PSII, Fv/Fm, indicating a moderate but chronic photoinhibition (Table 3.2; Tallon and Quiles 2007). High irradiance also led to de- 25 creases in Amax , due to both reduced stomatal conductance gs and decreased photosyn- 25 thetic capacity, i.e., Rubisco activity and Jmax . A number of processes may result in deactivation of Rubisco – for example, remobilisation and export of nitrogen from the leaves, interruption in the electron transport chain, or the presence of reactive oxygen species (ROS). In our case, deactivation of Rubisco in high irradiance plants was not related to a remobilisation of frond nitrogen – the N content per frond area was not affected by irradiance. This can be seen also through the decline of photosynthetic nitrogen use efficiency (PNUE). However, high irradiance alone did not affect total chlorophyll content and chlorophyll a/b ratios, indicating that photoprotective mechanisms were efficient enough to avoid chlorophyll degradation.

High temperature had no negative effects on photosynthetic capacity of D. antarctica 25 25 under shade. Increasing temperature to 35ºC even stimulated Amax . Increases in Amax

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were in this case solely due to increases in gs, as we found no effects of temperature on 25 25 photosynthetic capacity (Vcmax , Jmax ). The increase in chlorophyll a/b ratio in shaded plants with increasing temperature perhaps indicates temperature-stimulated resynthesis of photosynthetic reaction centres relative to light-harvesting antenna complexes, which commonly coincides with other changes in pigment composition, e.g. lutein, β-carotene (Haldimann 1999).

The interactive effect of high irradiance and high temperature led to severe photoinhibition in agreement with earlier findings (Berry and Björkman 1980). Temperature in- 25 crease stimulated gs yet without commensurate increases in Amax , indicating that meta- 25 bolic limitations (e. g. Rubisco activity) governed Amax (consistent with the findings of e.g. Law and Crafts-Brandner 1999 on Rubisco activation). Many studies have shown a negative effect of moderate heat and photoinhibition on the activation of Rubisco mediated by an activase (for details see Salvucci and Crafts-Brandner 2004). In our study, we did not measure the activity of Rubisco-activase, but found no increase in photosynthetic capacity to increased temperatures. Prolonged photoinhibition and heat interaction resulted in decreases in chlorophylls as in numerous other studies (e.g. Lambers et al. 2008). Decreases in chlorophyll a/b ratio indicated that under prolonged light stress, chlorophylls were destabilised with chlorophyll a being more sensitive than chlorophyll b (Yamamoto et al. 2008).

25 A return of temperature to 15ºC induced stomatal closure thus reversing Amax to the initial values at 15°C, as found by Ghouil et al. (2003). The only partial recovery of

Fv/Fm in high irradiance plants demonstrated detrimental interactive effects of high irradiance and temperature on Fv/Fm, suggesting that degradation processes (i.e. bleaching of chlorophylls or photo-degradation of thylakoid complexes) did not allow rapid recovery of Fv/Fm (Ottander et al. 1995). The fact that Vcmax and Jmax remained non- responsive possibly underlined the high sensitivity of Rubisco to high irradiance in D. antarctica.

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In summary, photosynthetic capacity and photosynthetic nitrogen use efficiency were rapidly affected by exposure to high irradiance under 15°C, while chlorophylls and predawn Fv/Fm declined further under the combination of high irradiance and high temperature.

3.4.2. Membrane stability of D. antarctica measured via critical temperature

Critical temperature, Tc, recorded in D. antarctica was ca 47ºC, comparable with a range of overstorey species, such as Quercus petraea (46.7ºC; Dreyer et al. 2001). Comparable data for other tree fern species are currently lacking.

An increase in air temperature induced an increase in Tc, as found by many authors (e.g.

Dreyer et al. 2001). This increase in Tc was larger under high irradiance than shade, co- inciding with a 3.3°C higher midday frond temperature under high irradiance. Previ- ously published data suggest that an increase in Tc by 1ºC (as found in our study) requires an increase in ambient temperatures of about 10ºC (Froux et al. 2004). We therefore believe that the larger rise in Tc in high irradiance plants was not solely caused by the difference in frond temperature, but directly related to high irradiance effects, which resulted in enhanced thermostability of thylakoid membranes.

Contrary to findings by Hüve et al. (2006), increased thermostability of the thylakoid membranes was not associated with the accumulation of osmotically active substances. In our study, osmotically active solutes even decreased when temperature increased. It may be argued that the hot water extract method results in artefacts, because some cell wall or other material can be brought into solution as a result of the grinding and extraction procedures (Callister et al. 2006). Yet, this method is widely used (e.g. Merchant et al. 2006) and moreover, a tight correlation was found among methods even though the absolute results were different (Callister et al. 2006). Our observation therefore seems reliable.

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With subsequent temperature decrease to 15ºC, we found a tendency for Tc to return to the initial values, even though this return was not complete. The observed lag confirms the hysteresis found by Froux et al. (2004), when the increase in Tc with increasing temperature is faster than relaxation from this effect after temperature decrease.

3.4.3. Xanthophyll cycle carotenoids, pigments and α-tocopherol

Exposure of D. antarctica to high irradiance alone resulted in almost two-fold increase in xanthophyll cycle carotenoids (V+A+Z), both on a chlorophyll as well as on a frond area basis. A high de-epoxidation state was also recorded (50% vs. 2-4% in the shade). Values for V+A+Z were within the range of values presented for other species (Thayer and Björkman 1990). The maximum de-epoxidation state of the xanthophyll cycle was lower than maximum values measured in epiphytic ferns (Tausz et al. 2001). An increase was also recorded in pools of other carotenoids such as neoxanthin and lutein in response to high irradiance. Values were within the range reported for other ferns, e.g. a range of epiphytic species (Tausz et al. 2001) or the tree fern Cyathea microdonta (Ma- tsubara et al. 2009). These carotenoids (e.g. neoxanthin) may preserve PSII from photo- inactivation and protect membrane lipids from photo-oxidation by ROS (North et al. 2007). The observed increase in lutein (located primarily in both the proximal and distal light harvesting centres of PSI and PSII) is probably associated with the acclimation of antennae to increasing irradiance (Senger et al. 1993). As antenna size is usually reduced in response to increasing irradiance, it is also likely that an increasing fraction of these carotenoids is not bound to antenna proteins. High irradiance also induced increases in α–tocopherols, known for their protective function in thylakoids, consistent with findings elsewhere (e.g. García-Plazaola and Becerril 1999; Munné-Bosch 2005).

Increased temperature stimulated an increase in Tc in shaded plants but without simultaneous accumulation of zeaxanthin, which is contrary to observations by Havaux and

Gruszecki (1993) and Havaux and Tardy (1996). Irrespective of an increased Tc, high temperature had no effect on concentration of α-tocopherol in shaded plants. According to our results, it is therefore unlikely that increased membrane stability as measured by

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Tc is directly and generally dependent on zeaxanthin or α-tocopherol. There was some coincidence of increased Tc with increases in α- and β-carotenes in shaded plants, which may have reflected changes in carotenoid synthesis rates. Carotenes may also contribute to an overall increase in membrane stability, as proposed by Tausz et al. (2001).

Interactions between irradiance and temperature had no additional effect on the xanthophyll cycle pool and de-epoxidation state of high irradiance plants. In contrast to V+A+Z, the combination of high irradiance with high temperature led to further increases in the amount of lutein and β-carotene, which remained greater until the end of experiment. It is not clear from our experiment whether sustained concentrations of carotenoids indicated their role as a last resort in membrane photoprotection under extreme stress, or simply reflected their superior stability under such conditions. In contrast to shaded plants, temperature increase stimulated an almost two-fold increase in α- tocopherol in high irradiance plants. Although these results for high irradiance plants appear to support findings of Llusià et al. (2005) – who suggested that increased tolerance to high temperatures might be at least partly due to an increase in α-tocopherol – our results for shaded plants indicate that changes in Tc can occur independently of changes in α-tocopherol.

Temperature return to 15°C did not affect total V+A+Z pool of high irradiance plants, but the de-epoxidation state decreased by 30% to remain significantly higher than in shaded plants. De-epoxidation of the xanthophyll cycle is driven by an acidic thylakoid lumen, which can be the consequence of an imbalance between electron transport and electron consumption (Demmig-Adams and Adams 2006). It seems that heat related changes in the photosynthetic apparatus lead to a relaxation of the pH gradient upon temperature decrease, despite the continuation of high irradiance. This may be related to a sustained decrease in light use efficiency as suggested by persistently low Fv/Fm values, and other, as yet unexplained, changes in the photosynthetic membrane. Such further changes were also expressed in the observed change in Tc, possibly in combination with an increased electron consumption rate upon relaxation of the high temperature. Temperature decrease did not affect concentration of carotenoids except for α-carotene.

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Significant decreases in α-carotene in high irradiance plants can be explained in terms of its ease of oxidation to lutein under conditions of oxidative stress (Senger et al. 1993), or its conversion to β-carotene to increase scavenging of free radicals in core complexes under conditions of stress (Kirchgeßner et al. 2003).

In summary, increased thylakoid stability in D. antarctica observed during our experiment could not be explained by any of the measured changes in pigments or α- tocopherol, although they may all play partial roles. Alternative or additional explana- tions may involve presence of certain heat shock proteins or changes in the composition of membrane lipids, as suggested by Sinsawat et al. (2004). Discrepancies with earlier literature may be related to the fact that pigment changes are fast responses to temperature increases (in the order of one day; as e. g. in Havaux and Tardy 1996), while we investigated longer term acclimation (12 days) to high temperature. Longer term acclimation of plants to rising temperatures is also related to the appearance of polar lipids with saturated fatty acids causing a decrease in membrane fluidity (Zsófi et al. 2009). In parallel with an increased threshold temperature for thermal inactivation of PSII (Down- ton et al. 1984) this can increase thylakoid stability. The difference between the rate of acclimation and de-acclimation supports this hypothesis: the fast rise may be initially due to rapid changes in pigments that are later completed by slower changes in lipid composition. The reversal of these changes may be slower, which would be the cause for a slow return to initial levels of stability. However, our data do not allow confirma- tion of this speculation.

3.5. Summary

High irradiance caused chronic photoinhibition (measured as sustained decrease in maximum PSII quantum efficiency), and decreases in all photosynthetic capacity parameters of D. antarctica. Whilst we observed some acclimation in terms of increases in protective carotenoids, which may have sufficed to avoid chlorophyll degradation, similar or even decreasing chlorophyll a/b ratios indicated limited short-term acclimation potential of D. antarctica fronds to high irradiance. Temperature alone appeared to have

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Chapter 4. Interactive effects of high light and water deficit on the tree fern species Dicksonia antarctica and Cyathea australis

(iii) Abstract

We examined the responses of two tree fern species (Dicksonia antarctica and Cyathea australis) growing under moderate and high light regimes to short-term water deficit followed by rewatering. Under adequate water supply, morphological and photosynthetic characteristics differed between species. D. antarctica, although putatively the more shade and less drought adapted species, had greater chlorophyll a/b ratio, and greater water use efficiency and less negative δ13C. Both species were susceptible to water deficit regardless of the light regime showing significant decreases in photosynthetic parameters (Amax, Vcmax, Jmax) and stomatal conductance (gs) in conjunction with decreased relative frond water content (RWC) and predawn frond water potential (Ψ predawn). Stomatal conductance under moderate light responded later and at lower soil water content. More shaded D. antarctica seemed to be most vulnerable to drought as evidenced by greatest decreases in Ψ predawn, and lowest stomatal conductance and photosynthetic rates. Both tree fern species were able to recover after short but severe water stress.

4.1. Introduction

Drought is considered to be one of the most important factors limiting plant performance (e. g. Ribas-Carbo et al. 2005). Shade grown plants are potentially less tolerant to reduced soil moisture than light grown plants because survival in light limiting environments generally requires a large leaf area, which can only be supported under moist conditions (Smith and Huston 1989). This is supported by a number of studies e. g. Val- ladares and Pearcy (2002), Valladares and Niinemets (2008), which have found that shade and drought tolerance cause conflicting requirements for biomass investment.

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Shade tolerance favours foliage for efficient light capture, whereas drought tolerance requires predominant investment in roots for efficient water uptake.

During periods of insufficient water supply, plants may be exposed to light conditions in excess of their ability to use in photosynthetic fixation. The resulting imbalance between electron transport and consumption leads to photoinhibition and photo-oxidative stress (Flexas et al. 1999). Thus, an interactive effect of high light and water deficit can be more detrimental than water deficit alone (Levitt 1980, Lovelock et al. 1994). Val- ladares and Pearcy (2002) observed that the capacity to withstand severe drought was not enhanced in the shade but was decreased due to increased below-ground competition for water with established trees. Furthermore, photoinhibition becomes relatively more important for carbon gain in shade than in sun due to the relatively more important effect of low photochemical efficiency under low light following sunflecks (Valladares and Pearcy 2002).

The tree ferns Dicksonia antarctica (Labill.) and Cyathea australis (R.Br.) Domin are ecologically important (Lindenmayer et al. 1994, Roberts et al. 2005) understorey species of south-eastern Australian forest ecosystems, including wet sclerophyll forests and cool temperate rainforests characterised by mild to warm summers with occasionally short periods of droughts and high temperatures (Australian Bureau of Meteorology, 2006). D. antarctica and C. australis, are believed to have different micro-habitat preferences as D. antarctica typically dominates wet, shady gullies while C. australis is more common along forest margins (McCarthy 1998). An observational study confirmed that the greater the distance to the stream the more likely it‟s to encounter C. australis than D. antarctica (Dignan and Bren 2003). D. antarctica and C. australis belong to contrasting floristic elements of the Australian vegetation (Gondwanan vs. Tropical, Groves 1994) thus physiological adaptation to micro-habitats (if they exist) are likely to have arisen during the contrasting phytogeographical history of these species.

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During their lifetime, tree ferns can be periodically exposed to harsh conditions of post- wildfire environments, characterised by increased light intensities and leaf temperatures, and consequently increased evapotranspiration and water loss. Direct effects of these conditions on tree fern physiology have not been studied, but are indicated by poor survival and ongoing decline of both D. antarctica and C. australis after clearcut logging (Ough 2001). Understanding negative effects of excess light and water deficit on tree fern persistence is of increasing importance given predictions of more frequent drought and fire events in climate change scenarios relevant to south-eastern Australia (Hennessy et al. 2007).

Both D. antarctica and C. australis are able to tolerate short periods of drought if some shade is available. Despite infrequent but severe drought events, tree fern numbers increased by 80% in the lower strata of wet sclerophyll forests over 48 years (Ashton 2000). Hunt et al. (2002) also reported that D. antarctica can maintain favourable water relations during short periods of drought, if its habitat is limited to sheltered sites. This apparent ameliorating effect of shade on drought might simply be due to reduced water loss from soil and plants, rather than a direct effect of light intensity. Observational field studies usually have to accept such confounding effects between shade, temperature and (soil and air) humidity, because more shaded sites are also cooler and moister. Thus, it often remains unresolved whether alleviation of drought stress is a direct effect of lower irradiance – e. g. shading ameliorates drought-related photoinhibition and photo- oxidative stress – or an indirect effect of greater water availability.

In this study, we compared interactive effects of short-term water deficit and high light on the physiological performance of two tree ferns – one (D. antarctica) putatively less drought/light tolerant than the other (C. australis) – by applying short-term but severe water deficit treatment to potted individuals under semi-controlled conditions. We analysed key variables relevant to photosynthetic capacity (gas exchange, chlorophyll a fluorescence), and plant water relations (water potential, osmolality, carbon isotope discrimination) during a drought-rewatering cycle. Our research hypotheses were:

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 The tree fern species will display contrasting physiological characteristics, owing their different origins and microclimate preferences, where D. antarctica will be more susceptible to water deficit;  Water deficit will be detrimental for high light exposed tree ferns, while shade will ameliorate the negative effects of water deficit;  The tree ferns will be able to recover after short but severe water stress, with those in shade recovering faster.

4.2. Materials and methods 4.2.1. Plant material

Twenty sporophytes of Dicksonia antarctica (Labill.) and twenty of Cyathea australis (R.Br. Domin) (Fern Acres nursery, King Lake West, central Victoria, Australia) were transplanted two months before the experiment into spacious 25-l pots, containing (% volume) composted pine bark (30), gravel (45), coarse fern mulch (5), composted mulch (14.5), fine fern mulch (5), „Dynamic lifter‟ (0.16, Yates, Padstow, NSW, Australia), and two types of slow-release fertiliser (0.17 each) Osmocote Baulkham Hills, NSW, Australia (18+4.8+8.3 mg) and (16+3.5+10+1.2 mg). Before the experiment plants grew in an open-air nursery under the dense canopy, providing ca 70% of shade. All plants were two and a half years-old at the start of the experiment.

4.2.2. Experimental design

The experiment ran from February to April 2008 under the prevailing summer to autumn weather at Creswick Campus of the Melbourne School of Land and Environment, The University of Melbourne, Victoria, Australia (143º 53‟ E, 37º 25‟ S; 392 m above sea-level).

Ten plants of each species were randomly assigned to two light regimes – “moderate light” (35% of ambient light intensity by a wavelength neutral shade-cloth) and “high light” (70% of ambient light intensity). We choose 35% of ambient light as most repre-

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sentative for field conditions of south-eastern Australia (personal observations), while 70% of ambient light was the required minimum (derived from previous experiments, unpublished) to protect plants from high light stress. Maximum photosynthetic photon flux density (PPFD, 400 – 700 nm, measured with a Li-Cor PAR sensor, Li-Cor, USA) at noon on clear sunny days was on average 600 µmol photons m-2 s-1 in moderate light and 1000 µmol m-2s-1 in high light at frond height, (ambient maximum PPFD ca 1800 µmol m-2s-1). Measurements with a Solarmeter (Ultraviolet Radiometer 5.0, So- lartech Inc.) confirmed that UV-A and UV-B filtration was similar to that in the PAR range (30% and 60% filtration, respectively). Weather conditions were recorded by a weather station (Tain™ Electronics, Melbourne, Australia).

Within each light regime, 5 plants per species were randomly assigned to two water treatments: - watered daily to field capacity for the entire experiment (control) and water-deficit (deficit). Plants were divided into five blocks, each containing one plant per species and four treatments (i.e. all combinations of “control” and “deficit” with “moderate light” and “high light”). All measurements (as below) within one block were made on the same day, with a one day lag in the application of water deficit treatments for consecutive blocks. Plants were rotated randomly within their designated light regime three times per week to minimise differences in light and temperature conditions.

Plants were measured: (1) After 20 days of acclimation to designated light regimes, all plants regularly watered to field capacity – “pre-treatment” period; (2) After 10 days of treatment – “water deficit” period (deficit plants not watered for 5 days then maintained at 10% of field capacity for next 5 days); (3) Twenty days of watering to field capacity – “rewatering” period; Additional shade cloth (to reach 50% of ambient light intensity) was placed over high light plants to avoid damage in extremely hot weather (>35°C) during the first 10 days of the rewatering period (Fig. 4.1).

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Each plant was measured for chlorophyll a fluorescence, predawn water potential and gas exchange at the end of each of the three experimental periods. In addition, stomatal conductance (gs) was measured daily for the first five days of water deficit period from 8 to 11.30 a.m. Samples for chlorophyll content, osmolality and stable isotopes were taken in the morning at the end of each of the three periods. All measurements were made on the mid-third of the youngest fully expanded fronds.

4.2.3. Maximum quantum yield of PSII (Fv/Fm)

Maximum quantum yield of PSII (Fv/Fm) was measured at predawn with a pulse modulated fluorometer (OS-30p, Opti-Sciences, Hudson, USA). Ground fluorescence (F0) was obtained with a low intensity modulated light (600 Hz, 650nm, PPFD<1 µmol m-2s- 1 ). Maximum fluorescence (Fm) was induced by a saturating flash. Maximum quantum yield of PSII was estimated as Fv/Fm = (Fm–F0)/Fm, nomenclature after Maxwell and Johnson (2000).

4.2.4. Photosynthetic capacity

Gas exchange was measured with a Li-Cor 6400 portable photosynthesis measurement device, equipped with a 2x3 cm broadleaf chamber with red-blue LEDs (Li-Cor, Lin- coln, Nebraska, USA). All gas exchange measurements were conducted at the reference frond temperature of 25ºC. For each plant, an A-Ci curve was generated after Long and Bernacchi (2003) with some modifications: PPFD was 1000 µmol m-2 s-1, frond temperature was 25ºC, air flow rate was 400 µmol air s-1, and relative humidity (RH)>60%. -1 CO2 mole fraction was increased in 13 successive steps from 50 to 2200 µmol mol s with two measurements at each step. After finishing the A-Ci curve, illumination in the -1 leaf chamber was turned off, CO2 mole fraction was decreased to 400 µmol mol and respiration rate was recorded after 5 min in the dark. The frond area enclosed in the chamber was marked, detached, scanned and calculated using Scion Image programme (Scion Corporation 2000-2001, USA). Gas exchange measurements were then recalculated for real frond area.

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Intrinsic water use efficiency (WUEi) was calculated as Amax/gs; where Amax and gs were -1 measured at the conditions mentioned above and reference CO2 of 397±1 µmol mol s .

Using the Farquhar model (Farqhuar et al. 1980), maximum carboxylation rate (Vcmax), and maximum electron transport rate (Jmax) were evaluated by fitting A-Ci curves to the model, as described in Dreyer et al. (2001). Triose phosphate use (TPU) limitation was not included in the model, and corresponding points with decreased A at elevated Ci were disregarded. The set of primary parameters of Rubisco kinetic properties used -1 -1 -1 herein (Kc=327µmol mol , Ko=282.6 mmol mol , Γ*=43.7 µmol mol ) are from von Caemmerer et al. (1994).

A time course of gs versus days of water withheld was measured using the Li-Cor 6400 gas exchange system, with chamber conditions as above and a reference CO2 concentration of 397±1 µmol mol-1.

For measurements of frond chlorophyll content, four frond discs (3.75 mm diameter) were immediately immersed in liquid nitrogen and then stored at -80ºC until extraction. Chlorophyll a and b were extracted using 1.8 ml of 100% dimethyl sulphoxide (DMSO). Extracts were heated for 30 min at 65ºC in a dry block heater (Termoline L+M, Northgate, Queensland, Australia). The supernatant was then transferred to a microplate reader (Tecan GmbH, Austria). Blank microplates were scanned and their absorbance was deducted from final measurements. Chlorophyll a, b and total were calculated according to Wellburn (1994). The absorbance of 200 μl of sample in a microplate was converted into a 1 cm pathlength, and then corrected using correction coefficients (Warren 2008). Correction coefficients for chlorophyll a and b were calculated from 20 samples using regressions of initial measurement against repeated measurement in a spectrophotometer (Carry 300, Varian, The Netherlands) at the same wavelengths (all regressions were highly significant P<0.001). Leaf mass per area (LMA), needed to calculate total chlorophyll on a frond area, was calculated as dry weight/frond area (g m-2).

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4.2.5. Frond water relations

Predawn frond water potential (Ψ predawn) of each tree fern was measured using a pressure chamber (PMS Corvallis, OR, USA) at the end of each of the three periods.

Concentrations of osmotically active solutes (osmolality) were measured via freeze- point depression from fresh sap extracts using an OSMOMAT 030 cryoscopic osmometer (Gonotec, Berlin, Germany), nomenclature by De Costa et al. (2007).

Relative water content (RWC) was determined as follows: pinnae were detached and weighed (fresh weight), then floated in water for ca 5 h in the dark to reach full hydra- tion. Pinnae were blotted dry with tissue paper and weighed (saturated weight), then dried at 60ºC for 48 h and weighed again (dry weight). RWC (%) was calculated as: (fresh weight – dry weight)/(saturated weight – dry weight) x 100.

4.2.6. Stable isotope analysis

Due to rapid turnover of the soluble carbon pool, we tracked differences in stable isotope composition (δ13C) of a hot water extract (e.g. Warren et al. 2007). Hot water extracts were prepared according to Callister et al. (2006). Extract sub-samples (100 µl) were dried in tin capsules at 50ºC for 48 h, and δ13C determined by IRMS (EuroVector, IsoPrime Mass Spectometer, Manchester, UK) with Dumas flash combustion. Analysis of δ13C (in ‰ units) was against a tertiary standard (Acetanilide: δ13C =33.44‰ and C=71.09%), which was calibrated against a PDB (Pee Dee Belemnite) standard.

4.2.7. Relative extractable soil water, REW

Relative extractable soil water (REW), was calculated as the soil moisture content at each day of water deficit divided by soil moisture content at field capacity minus soil moisture content at permanent wilting point (after Bogeat-Triboulot et al. 2007). For

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this, each pot of water deficit treated plants was weighed at field capacity and at the end of each day of water withheld. Potential differences in evaporation from the pot surface between light regimes was taken into account by placing a plant-free pot in each of the light regimes and weighed at the same time as treated plants (Merchant et al. 2006). Soil moisture content at field capacity was determined gravimetrically by oven drying soils at 105ºC for 24 h.

4.2.8. Statistical analysis

Effects of species (D. antarctica, C. australis), light (moderate, high), water treatment (control, deficit), and their interactions on dependent variables were analysed using the general linear models of SPSS (SPSS Inc. Chicago, USA). Variables were graphically checked for deviations from normality, homogeneity of variances was tested using Levene‟s test, and means and variances were not correlated across treatments. Each period (pre-treatment, water deficit, rewatering) was analysed separately.

Pre-treatment Drought RewateringRewatering 55 100 C (extra (shade o 50 shade removal) 45 on high light plants) 80 40 35 60 30 25 20 40

15 Daily at 9 a.m.,RH % 10 20

Maximal daily air temperature, 5 0 0 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

DOY Fig. 4.1 Weather conditions during the experiment. Dotted grey line is maximum daily air temperature (ºC), black line - daily relative humidity (RH, %) at 9 a.m. Vertical lines indicate stages of the experiment for Block 1.

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4.3. Results

4.3.1. Maximum quantum yield of PS II (Fv/Fm)

Even during the pre-treatment period, both species had lower Fv/Fm than what is considered an optimum value for healthy fronds (0.83, Maxwell and Johnson 2000, Table 4.1).

Fv/Fm did not differ between species but was significantly higher (P<0.001) in moderate light plants during the pre-treatment and the water deficit periods (Table 4.1). Fv/Fm was not affected by water treatment in the water deficit period regardless of the light regime, but was significantly greater in control than deficit plants in the rewatering period.

Drought stressed D. antarctica in moderate light had significantly lower Fv/Fm in the rewatering period (Table 4.1).

4.3.2. Photosynthetic capacity

Light saturated net photosynthesis (Amax) and stomatal conductance (gs) at Amax were significantly greater in C. australis than D. antarctica in the pre-treatment and the water deficit periods, but not the rewatering period (Fig. 4.2). Water deficit resulted in a significant reduction in Amax and gs in both species (water treatment P<0.001), with marginally higher, albeit non-significant, values under high light. Amax and gs recovered with rewatering in both species, and under both light regimes (Fig. 4.2).

The maximum carboxylation rate, Vcmax, and the maximum rate of electron transport,

Jmax, did not differ significantly between species and light regimes in the pre-treatment period (Table 4.1). However, water deficit caused significant decreases in both variables for both species – an effect that may have been most pronounced for D. antarctica under moderate light, although increasingly inaccurate Ci estimates at very low gs made impossible to construct meaningful A-Ci curves (i.e. missing estimates for Vcmax Jmax;

Table 4.1). Vcmax and Jmax recovered to near control values with rewatering for both species, although Jmax was significantly lower in C. australis in this period (Table 4.1).

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Total chlorophyll content per frond area (µmol m-2) did not significantly differ between species, light regimes, and water treatments across all experimental periods (Table 4.1). Chlorophyll a/b ratios were significantly greater in D. antarctica than C. australis in the pre-treatment period (P=0.01, Table 4.1). However, this difference was not detected for the remainder of the experiment, with chlorophyll a/b ratios of both species largely invariant to light regimes and water treatments (Table 4.1).

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Table 4.1 Chlorophyll fluorescence and photosynthetic capacity variables of water deficit and control tree ferns (D. antarctica and C. australis) grown under high and moderate light during three successive experimental periods. -2 -1 Values are means (n=5) ± s.e. of: Fv/Fm, maximum quantum yield of PSII, Vcmax, maximal carboxylation rate of Rubisco (µmol m s ); -2 -1 -2 Jmax, maximal light driven electron flux (µmol m s ); Chl total (µmol m ), total chlorophyll content on a frond area basis, Chl a/b, chlorophyll a/b ratio. Effect abbreviations: L, Light regime; W, Water treatment; S, Species; x, interaction. Significance levels:* P<0.05; ** P<0.01; *** P<0.001; n.s., non significant, n/d, none detected.

High light Moderate light Significance of effects, P Water deficit Rewatering Water deficit Rewatering Pre- Water Rewa- Variable Species Pre- Pre- treat- defi- tering treatment control deficit control deficit treatment control deficit control deficit ment cit D. ant- 0.72±0.0 0.76±0.0 0.73±0.0 0.78±0.0 0.73±0.0 0.78±0.0 0.81±0.0 0.78±0.0 0.77±0.01 0.64±0.0 arctica W*** Fv/Fm L*** L*** C. aus- SxL** 0.73±0.0 0.75±0.0 0.75±0.0 0.74±0.0 0.68±0.0 0.75±0.0 0.78±0.0 0.78±0.0 0.79±0.01 0.75±0.0 tralis D. ant- 36.4±5 30.4±4 16.9±3 39.8±4 37.6±6 37.9±3 26.4±5 n/d 40.0±1.8 37.3±4 arctica Vcmax n.s. W** n.s. C. aus- 33.3±4 38.0±3 21.0±4 34.3±2 36.5±4 50.5±8 33.0±3 26.0±3 45.7±3 38.2±3 tralis D. ant- 106±13 141±35 98±13 154±19 141±35 111±8 88±20 n/d 145±12 139±25 arctica Jmax n.s. W** S*** C. aus- 91±11 96±8 53±10 101±7 90±6 144±27 83±6 69±10 109±7 103±12 tralis

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High light Moderate light Significance of effects, P Water deficit Rewatering Water deficit Rewatering Pre- Water Rewa- Variable Species Pre- Pre- treat- defi- tering treatment control deficit control deficit treatment control deficit control deficit ment cit D. ant- 382±30 277±14 218±12 288±12 219±13 372±31 275±40 253±31 251±36 268±25 arctica Chl total n.s. n.s. n.s. C. aus- 349±15 285±66 242±18 252±14 222±23 354±23 256±13 219±18 328±65 261±25 tralis D. ant- 4.0±0.1 3.5±0.1 3.4±0.2 3.5±0.1 3.5±0.2 4.1±0.1 3.4±0.2 3.4±0.2 3.4±0.1 3.5±0.2 arctica Chl a/b S** n.s. n.s. C. aus- 3.9±0.1 2.8±0.3 3.4±0.1 3.4±0.1 3.0±0.4 3.8±0.1 3.4±0.1 3.2±0.2 3.0±0.3 3.5±0.1 tralis

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High light Moderate light 300 Pre- Water Re- treatment deficit watering 250

)

-1 s 200 -2 S* S** n.s. W** 150

(mmol m (mmol

g 100

0 12 Pre- Water Re- 10 treatment deficit watering

)

-1

-2 8

mol m mol 6



( S* S*** n.s. W**

max max 4

0 pre-treatment water deficit rewatering pre-treatment water deficit rewatering Period Period Fig. 4.2. Stomatal conductance and light saturated net photosynthesis of water deficit (hatch) and control (solid) D. antarctica (white bars) and C. australis (grey bars) under high and moderate light in three successive experimental periods (pre-treatment, water deficit and rewatering). Values are means n=5 (± s.e.). Effect abbreviations: S, Species; W, Water treatment; none of interactions were significant. Signifi- cance levels:* P<0.05; ** P<0.01; *** P<0.001; n.s., non significant

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4.3.3. Frond survival

Approximately 1/3 of fronds of water deficit plants survived the water deficit period – a proportion that did not significantly differ between species and light regimes (high light: 33±7% for D. antarctica and 24±4% for C. australis; moderate light: 27±4% for D. antarctica and 35±6% for C. australis).

4.3.4. Time course of stomatal conductance during 5 days without water

Stomatal response to withholding water did not differ between species but was affected by light regime (Fig. 4.3). Water deficit plants under high light decreased gs by almost 50% relative to controls after one day without water, at relative extractable soil water (REW) of ca 80% (Fig. 4.3). In contrast, plants under moderate light maintained initial stomatal conductance for 1 day longer and decreased gs at lower REW (ca 65%; Fig.4.3).

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High light Moderate light

gs control gs control 100

D. antarctica D. antarctica 80

0 gs control gs control 100

(% of controls)

g 80 C. australis C. australis

5 days of water withheld 40

0 100 80 60 40 20 100 80 60 40 20 REW, % REW,%

day 1 day 2 day 3 day 4 day 5 day 1 day 2 day 3 day 4 day 5 Days without water Days without water

Fig. 4.3. Time course of stomatal conductance of water deficit D. antarctica and C. australis grown under high and moderate light during first five days of water deficit. Where gs, stomatal conductance, and REW, relative extractable soil water. Values are means (n=5) ± s.e.

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4.3.5. Frond water relations

Significant differences in frond water relations were detected between species in the pre-treatment period. D. antarctica had significantly lower Ψ predawn, RWC and significantly greater osmolality (Table 4.2). With the exception of Ψ predawn, these species differences were not detected during the water deficit period, although the difference in osmolality was reinstated after rewatering.

Frond water relations of both species were significantly affected by water treatments.

Water deficit significantly decreased Ψ predawn and RWC of deficit relative to control plants of both species under both light regimes (Table 4.2). These effects were reversed in the rewatering period. Osmolality was not affected by water deficit; however, a significant species by water treatment interaction was detected in the rewatering period (i.e. increased osmolality of control D. antarctica relative to water deficit plants, and the opposite trend in C. australis; Table 4.2).

Light regimes did not significantly affect frond water relations of either species, apart from incomplete Ψ predawn recovery of deficit D. antarctica under moderate light in the rewatering period (Light x Water; P=0.04; Table 4.2).

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Table 4.2. Frond water relations of water deficit and control tree ferns (D. antarctica and C. australis) grown under high and moderate

light during three successive experimental periods. Values are means (n=5) ± s.e. of: Ψ predawn, predawn frond water potential (MPa); osmolality (osmol kg-1); RWC, relative water content (%). Effect abbreviations: L, Light regime; W, Water treatment; S, Species; x, interaction. Significance levels:* P<0.05; ** P<0.01; *** P<0.001; n.s., non significant.

High light Moderate light Significance of effects, P Water deficit Rewatering Water deficit Rewatering Pre- Wa- Re- Variable Species Pre- Pre- treat- ter watering treatment control deficit control deficit treatment control deficit control deficit ment defi cit D. antarc- S ** S * LxW* -0.3±0.0 -0.7±0.2 -1.3±0.2 -0.3±0.1 -0.3±0.1 -0.3±0.0 -0.5±0.1 -2.5±0.3 -0.2±0.1 -0.4±0.1 tica W* Ψ predawn ** C. -0.2±0.0 -0.5±0.0 -1.2±0.4 -0.4±0.1 -0.3±0.0 -0.2±0.0 -0.5±0.0 -1.3±0.4 -0.2±0.0 -0.3±0.1 australis S** n.s. S*** D. antarc- 0.69±0.0 0.62±0.1 0.61±0.0 0.79±0.1 0.68±0.0 0.71±0.0 0.60±0.0 0.68±0.1 0.79±0.0 0.72±0.0 SxW** tica Osmolality C. 0.64±0.0 0.63±0.0 0.60±0.0 0.56±0.0 0.66±0.0 0.62±0.0 0.58±0.0 0.62±0.0 0.63±0.0 0.65±0.0 australis D. S* W* n.s. 95±0 94±1 92±2 97±0 96±1 94±1 95±2 93±1 96±0 96±1 antarctica RWC C. aus- 97±1 97±1 89±3 97±0 97±1 97±0 94±1 90±5 97±1 94±1 tralis

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4.3.6. Intrinsic water use efficiency (calculated as Amax/gs, WUEi) and stable carbon isotope composition (δ13C)

During the pre-treatment period, WUEi was significantly greater in D. antarctica and was not affected by light regime. Water deficit treatment significantly increased WUEi of deficit relative to control plants irrespective of the light regime and species. With rewatering, WUEi was greater in C. australis than D. antarctica (Fig. 4.4).

δ13C was significantly more negative in C. australis across the experiment (Fig. 4.4). During water stress period, δ13C of deficit plants under high light were comparable with pre-treatment values, but δ13C became more negative in control plants under moderate light, particularly in D. antarctica (Light x Water interaction, P=0.02; Fig. 4.4). This effect was not detected in the rewatering period, when δ13C within species was comparable across water treatments and light regimes (Fig. 4.4).

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High light Moderate light

Pre- Water Re- -5 treatment deficit watering

)

-1 -10

-2 S *** S *** S* -15 W ** L x W *

(mmol m (mmol

C -20



-25

-30

100 Pre- Water Re- treatment deficit watering

) 80

-1

S * W ** S * 60

mol mol mol



(

i 40

WUE

0 Pre-treatment water deficit rewatering Pre-treatment water deficit rewatering Period Period 13 Fig. 4.4. Stable isotope composition, δ C and intrinsic water use efficiency, WUEi and of water deficit (hatch) and control (solid) D. antarctica (white bars) and C. australis (grey bars). Values are means (n=5); ± s.e. Effect abbreviations: S, Species; W, Water treatment; L, Light regime; L x W, light by water treatment interaction. Significance levels:* P<0.05; ** P<0.01; *** P<0.001

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4.4. Discussion 4.4.1. Pre-treatment period – species differences and effect of light

Photosynthetic capacity of both species were within range for tree fern species reported in the literature: 6 – 10.8 µmol m-2s-1 (Nobel et al. 1984) and 8.3 µmol m-2s-1 (Hunt et al. 2002) for D. antarctica, or even similar to some middle storey canopy species in these types of forests, e.g. Nothofagus cunninghamii (Tausz et al. 2005). Maximum carboxylation rates, Vcmax (the maximal in vivo Rubisco activity), and the maximum rate of electron transport, Jmax at the reference temperature of 25°C were within the lowest values among the large number of species reviewed by Wullschleger (1993).

13 D. antarctica had greater WUEi that together with its less negative δ C in the pre- treatment period suggests that photo-assimilation may be comparatively less susceptible to changes in water status.

Chlorophyll a/b ratios were also significantly higher in D. antarctica, a result inconsis- tent with this species‟ putative preference for well shaded microhabitats. Normally, chlorophyll a/b ratios are lower in shade acclimated than sun foliage because of greater chlorophyll b contents (Hoober et al. 2007). For D. antarctica, we observed greater chlorophyll a content while b was comparable between species (data not shown). How- ever, our study involved young sporophytes and it must be noted that these species differences might change at later stages in the tree fern life cycle.

Both species displayed low acclimation potential to changes in light regime - neither photosynthetic capacity (except Fv/Fm) nor water relation variables were affected by a nearly two-fold difference in maximum irradiance. Reduction in Fv/Fm usually indicates down-regulation of PSII (i.e. photoinhibition, Savitch et al. 2000), yet because Fv/Fm was measured at predawn, the down-regulation should be relaxed, indicating either damage of PSII or sustained down-regulation of some type. Down-regulation or damage of PSII in high light plants may affect maximum light use efficiency of photosynthesis, and likely depress photosynthetic performance - yet effects of light regimes on Amax were non significant, suggesting small differences in Fv/Fm under moderate and high

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light had minimal effect on Amax. These differences would become more relevant for photosynthesis during low light periods and therefore integrated carbon gain (Zhu et al. 2004) – an issue not addressed in this study.

4.4.2. Water deficit and light interactions

Both species were susceptible to short-term water stress. Deeper shade did not ameliorate the negative effects of water deficit, and there is no evidence to suggest that water deficit in combination with high light was more detrimental to plant function. The absence of significant water treatment by species interactions indicates that both species responded to water deficit in a similar way. The only parameter insensitive to water deficit was Fv/Fm - while this is in agreement with some studies (e.g. Epron and Dreyer

1992), others showed that drought reduced predawn Fv/Fm more in shade than in sun (Valladares and Pearcy 2002). In our study, plants under moderate light had consistently higher predawn Fv/Fm than high light plants even under water deficit, indicating full recovery of PSII overnight.

We detected contrasting stomatal behaviour under the different light regimes - plants growing under moderate light closed stomata later and at lower relative extractable soil water content. This indicated slower stomatal response under lower light, a result consistent with the findings of Roberts et al. (1984) where stomata of bracken fern from the most shaded understorey level were less sensitive to soil moisture treatments than those from more irradiated levels. Surprisingly, even at REW of 30%, stomatal conductance was not close to zero, possibly indicating limited stomatal control or high residual cu- ticular conductance (which was not distinguished from stomatal conductance in our measurements).

Decreases in gs by ca 70%, relatively to the pre-treatment values indicated that stress was rather severe – the result we aimed at. However, a straightforward definition of drought stress severity as given by Flexas and Medrano (2002) – that is, moderate stress -2 -1 intensity at gs <150 and severe at <50 mmol H2O m s – appeared to be unsuitable in our study. Ferns seem to have generally low stomatal conductance (Doi et al. 2006,

Hunt et al. 2002) and in our study, gs of D. antarctica rarely exceeded 150 mmol H2O

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m-2s-1 even with adequate watering. The severity of drought stress was also confirmed by the decrease in mesophyll capacity parameters of photosynthesis - both Vcmax and

Jmax decreased significantly after ten days of drought, confirming findings by Bota et al. (2004) that impaired Rubisco activity and/or RuBP regeneration do not limit photosynthesis until drought is severe and A and gs are strongly depressed.

Reduced values of gs and Amax in D. antarctica under moderate light resulted in increased error in the computation of Ci, therefore it was not possible to accurately construct A-Ci curves for these plants and measure Vcmax and Jmax values. This precluded a formal significance test of the result. D. antarctica under moderate light also had greater decreases in Ψ predawn which most probably reflect conditions close to the point of turgor loss in some fronds, and it can be speculated that D. antarctica under moderate light was most severely affected by water deficit treatment, possibly as a consequence of greater water loss due to the slower stomatal closure under moderate light.

RWC can affect Rubisco activity and photochemistry of plants in response to drought. According to Flexas et al. (2006) Rubisco activity remains essentially unaffected by wa- -2 -1 ter stress until gs drops below 50 mmol H2O m s regardless of species. While our results on D. antarctica support this suggestion, gs of C. australis varied from less than 50 -2 -1 to above 100 mmol H2O m s , yet Rubisco activity (measured via Vcmax) decreased significantly. Despite a significant decrease in RWC under water deficit, RWC of both species remained around 90%. This corresponds to ca 30% of the cases reviewed by Flexas and Medrano (2002), where decrease in Rubisco activity was associated with high RWC between 90-100%. Possibly, our inability to detect significant osmotic adjustment in this study suggests that RWC is maintained in these species by a combination of alternative factors. High solute levels in frond tissues may provide „pre-emptive‟ protection against rapid changes in water status, a concept that would require additional investigation given the lack of data on tree fern chemistry and physiology. If water conservation measures such as early and rapid stomatal closure fail, fronds may be left without strong tissue level tolerance mechanisms. In our study, this was indicated by necroses of two-thirds of fronds by the end of the water deficit period. In agreement

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with their ecological distribution, tree ferns may be unable to tolerate a major drop in RWC instead relying on an „avoidance‟ strategy of frond death.

We observed significant increases in WUEi under severe water stress in line with the stomatal responses. This was accompanied by significant reductions in photosynthetic activity. For carbon isotopic composition, the general lack of significant changes between treatments may be due to low photosynthetic activity limiting the contribution of new assimilates to the water extractable pool (Dawson et al. 2002). The observed significant δ13C shift in control plants under moderate light during the rewatering period (when air temperatures increased for ca 10ºC) could be related to stomatal closure in response to high air temperature while Rubisco remained active. Indeed, our other study of heat and light interactions indicate that moderate light may have mediated effects of heat on control plants (Volkova et al. 2009). An increase in air temperature can also explain relatively large changes for some variables within a species between pre-treatment and control (Fv/Fm, Ψ predawn, RWC etc).

Overall, there were some indications that shade exacerbated drought stress effects in D. antarctica (i.e. mesophyll capacity parameters, Ψ predawn). These findings are consistent with results elsewhere indicating that shaded plants can be more susceptible to drought, although the mechanisms explaining such an effect in other studies – such as increased below-ground competition, greater reduction in predawn Fv/Fm (Valladares and Pearcy 2002) – were apparently not applicable to our study.

4.4.3. Rewatering period

Both species of tree fern recovered from short-term water deficit with rewatering (i.e. treatment effect was non significant at this stage for all parameters but Fv/Fm). Recovery of plants was mostly unaffected by light regime, however, due to an unexpected heat wave we had to put an additional shade protection over high light plants, which resulted in rather small differences in PPFD between light regimes during first 10 days of rewatering, and probably smoothed potential differences during recovery.

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Decreases in Fv/Fm in the rewatering period were presumably mediated by further increase in ambient air temperatures (Galle et al. 2007). We speculate that the greater decrease in Fv/Fm of deficit plants was an after-effect of the treatment, which may have made them more sensitive to the following conditions.

Species differences in Ψ predawn, RWC, Amax, gs, chlorophyll a/b during the pre-treatment period were no longer evident in the rewatering period. This could be partly explained by effects of more extreme temperatures combined with different cohorts of fronds, but might also reflect some subtly different responses in frond physiology between the two species triggered by the treatments.

4.5. Summary

D. antarctica and C. australis displayed contrasting physiological characteristics some- times contrary of what would have be expected from species‟ different origin and mi- 13 cro-site preferences - greater WUEi, chlorophyll a/b ratios and less negative δ C for D. antarctica would indicate this species as more drought and light tolerant. Results of our study also suggest that shade does not ameliorate drought effects on C. australis and D. antarctica. However, both species were resilient to short-term severe water stress, their ability to restore physiology of surviving fronds and re-sprout after considerable loss of fronds undoubtedly plays a role for both species exposed to more extreme changes in environmental conditions. These findings are consistent with their distribution in temperate forest systems that are subject to seasonal droughts and rapid changes in forest canopy structure.

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Chapter 5. Seasonal variations in photosynthesis of the tree ferns Dicksonia antarctica and Cyathea australis in wet sclerophyll forests of Australia

(iv) Abstract

Steady state and dynamic responses of two tree fern species of contrasting origins, Dicksonia antarctica (Gondwanan) and Cyathea australis (Pan-tropical), were studied over two consecutive years under field conditions in wet sclerophyll forest of south- eastern Australia. Irrespective of their different origins and micro-site preferences, there were no significant differences in photosynthetic performance between the two species. Growth irradiance (from open sites to dense canopy cover) had very little effect on photosynthetic rates of the tree ferns. Both species performed better in winter than in summer, when photosynthetic rates reached higher values under similar irradiance and leaf temperatures. However, at the same leaf temperature, Fv/Fm was significantly lower in winter than in summer, suggesting some cold-induced limitation in PSII efficiency indicating persistent photoinhibition associated with cold winter mornings. Both species displayed seasonal acclimation in a number of measured photosynthetic parameters and frond traits. Acclimation of stomatal density to spatial variation in growth irradiance seemed limited in both species, although stomatal pattern differed between species. Be- cause there were no significant differences between the two species in photosynthetic parameters, both species could be described by the same carbon gain and water use models at the leaf-scale.

5.1. Introduction

The tree ferns Dicksonia antarctica (Labill) and Cyathea australis (R. Br.) Domin are well known Australian representatives of the fern genera Cyathea and Dicksonia, which include most tree fern species worldwide. Cyathea has a broad global distribution,

-91- This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia whereas Dicksonia is diverse throughout Indonesia and New Guinea, and can be found in isolated pockets including off the coast of Chile (Large and Braggins 2004).

Little is known about the photosynthesis and stomatal conductance of D. antarctica and C. australis in their natural habitats. This knowledge gap effectively excludes tree ferns from long-term carbon uptake and energy flux calculations, and limits the validity of predictions of their survival under climate change. Yet, these tree ferns often form the dominant understorey component of wet sclerophyll forests in south-eastern Australia (Ough and Murphy 1996). These forests support as much as 1,053 tonnes carbon ha-1 in above-ground living biomass (Keith et al. 2009), although the contribution of tree ferns remains unknown. Tree ferns of the genus Cyathea account for 33% of above-ground biomass in dwarf forests of Puerto Rico (Weaver 2008). Understorey species of evergreen forests world wide contribute an average of 49% to ecosystem respiration (Mis- son et al. 2007).

D. antarctica and C. australis are species of great ecological importance (Lindenmayer et al. 1994, Roberts et al. 2005). Both species are broadly distributed in the wetter parts of south-eastern Australia, with D. antarctica dominating wet, shady gullies, while C. australis can also extend to forest margins (McCarthy 1998). Although D. antarctica and C. australis can grow together in wet sclerophyll forests, previous studies have indicated they have different micro-habitat preferences. Dignan and Bred (2003) found greater probability of a tree fern being C. australis than D. antarctica with increasing distance from a stream, suggesting greater drought tolerance of C. australis. The postulated difference in origin of the two species – Gondwanan for D. antarctica compared with pan-tropical for C. australis (Page and Clifford 1981) – also suggests different physiological adaptation potential consistent with their observed distribution patterns within forests.

During their lifetime, tree ferns of south-eastern Australia can be periodically exposed to the harsh conditions of post-wildfire environments, characterised by increased irradiance and leaf temperatures, and consequently stronger evapotranspiration and water

-92- This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia loss. Sensitivity to high temperatures, high irradiance and decreased water could potentially limit growth and distribution of tree ferns in the field, although there are few field data to evaluate this theoretical model.

Ferns, as other plants (angiosperms or gymnosperms), have displayed a capacity to acclimate to changing irradiance. For example, leaf characteristics (frond surface area, epidermis thickness, palisade/spongy mesophyll ratio, blade size, petiole length) of a South American Cyathea species were found to be correlated with local irradiance (Arens 1997). However, stomatal density, which might be expected to increase with increasing light intensity (Casson and Gray 2008), was found to have limited plasticity in the tree fern Cyathea caracasana in response to light environment (Arens 1997).

To evaluate relationships between physiological performance of the tree ferns and prevailing environmental conditions, we analysed diurnal and seasonal trends in gas exchange during summer and winter over two consecutive years using pairs of D. antarctica and C. australis at contrasting micro-habitats (shaded creek-side and more exposed rocky knoll). The objectives of this study were:

 To determine if D. antarctica and C. australis showed differences in morphological and physiological frond traits among contrasting micro-habitats in the field. The postulated difference in their origin in particular leads us to hypothesise that the species would have different seasonal acclimation to temperature, with D. antarctica having higher photosynthesis in winter, and C. australis favouring summer;  To determine whether light, temperature and plant water status limited physiological performance in the field;  To examine acclimation of stomatal density in the two tree ferns; that is, would stomatal density differ between the two species and/or between high light exposed and shaded habitats?

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5.2. Materials and methods 5.2.1. Study site and sampling design

Our study site was established in mountain ash (Eucalyptus regnans F. Muell) forest in the Victorian Central Highlands (145‟42ºE 37‟35ºS, elevation 450 m). The average annual rainfall of a representative weather station (Healesville, 145,53ºE 37.68ºS, elevation 131 m) is 1021 mm. October is the wettest (106 mm) and January is the driest (58 mm) month on average. Mean maximum temperature of the hottest month (February) is 26ºC and mean minimum of the coldest month (July) is 4ºC (Australian Bureau of Me- teorology, 2009 www.bom.gov.au/climate /averages/cdo/about/about-stats.shtml, verified August 2009).

The experimental design involved tree ferns on the margins of a logging coupe that was clearcut in late 2003 and early 2004, then slash-burnt in March 2004. The coupe is bounded by the Acheron River to the south, and a creek to the east. Retained undis- turbed vegetation buffer zones were maintained along the waterways in accordance with the Code of Forest Practices (Department of Natural Resources and Environment, 1996), and were 200 m wide along the Acheron River and 20 m along the creek.

We selected eight mature individuals of each of two species, Dicksonia antarctica (La- bill.) and Cyathea australis (R.Br.) Domin. To cover the full range of local environmental conditions evenly for both species, tree ferns were selected in pairs (one from each species) growing in close vicinity (Fig. 5.1). Such pairs were selected from the most exposed sites at the fringe of the clearing on a rocky knoll (elevation up to 490 m above sea level, proximity to the creek 49 - 100 m) to the most sheltered sites in buffer zones (average elevation 450 m) near waterways (ca 2 m).

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C D D C; D D D C C C C D

Road C C D D

Creek

Unlogged area

Fig. 5.1. Location of the tree ferns at the study area. Where D is D. antarctica and C. is C. australis

5.2.2. Tree fern measurement schedule

Each plant was measured for pre-dawn chlorophyll a fluorescence, predawn/midday water potential, gas exchange measurements (diurnal and maximal net CO2 assimilation rate Amax) in winter (August-September) and summer (December-January) over two consecutive calendar years (2006-2008). Gas exchange measurements were made over two consecutive days during each season per year. Diurnal measurements were made once in summer (December 2006) and winter (August-September 2007). Frond samples for nitrogen and chlorophyll content were collected at the same time as gas exchange measurements. All measurements were made on the mid-third of the youngest fully expanded fronds of similar north-facing orientation.

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Samples for stomatal density determination were collected in January 2007. For this analysis only, fronds that were most representative of the tree fern‟s light environment were collected; that is, most light-exposed fronds of tree ferns growing in the open, and most shaded fronds of shade-grown tree ferns. Orientation (e.g. north facing) was not taken into account.

5.2.3. Mean irradiance on measured fronds

Mean daily photosynthetic photon flux density (daily PPFD) was estimated from hemispherical photographs (Nikon 601 F-601, Japan, fisheye lens), which were taken from the position of each measured frond. Black and white negatives were scanned and evaluated using Winphot software (ter Steege 1996). Calculated irradiance (summer/winter) ranged from 40/17 mol m-2 d-1 for a tree fern growing in open habitat to 13/3 mol m-2 d-1 for a tree fern growing under dense canopy near the creek.

5.2.4. Maximal quantum yield of photochemistry (Fv/Fm)

Maximal quantum yield of photochemistry (Fv/Fm) was measured at predawn (5 a.m. in summer, 6 a.m. in winter) with a pulse modulated fluorometer (OS-30p, Opti-Sciences,

Hudson, USA). Ground fluorescence (F0) was obtained with a low intensity modulated -2 -1 light (600 Hz, 650nm, PPFD<1 µmol m s ). Maximum fluorescence (Fm) was induced by a saturating flash. Maximum efficiency of photosystem II (PSII) was estimated as

Fv/Fm = (Fm–F0)/Fm, nomenclature after Maxwell and Johnson (2000).

5.2.5. Gas exchange measurements

Light-saturated rates of net photosynthesis (Amax) and gs at Amax were measured using a Li-Cor 6400 portable photosynthesis measurement system, equipped with a 2x3 cm broadleaf chamber with red-blue LEDs (Li-Cor, Lincoln, Nebraska, USA). PPFD was 1500 µmol m-2 s-1 (determined as saturating by preliminary light response curves), air -1 -1 flow rate was 400 µmol air s , reference CO2 concentration 400 µmol mol , and leaf

-96- This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia temperature and relative humidity were kept at ambient values. The frond area enclosed in the Li-Cor chamber was marked, photographed and calculated using imaging software (UTHSCSA Image Tool Version 3, University of Texas, USA). Diurnal courses of gas exchange under ambient conditions were measured using the Li- Cor transparent chamber. Air/leaf temperature and water pressure deficit based on leaf temperature (VPD) were also recorded.

5.2.6. Frond water potential

Predawn frond water potential was chosen as a measure of plant water status, because it is an appropriate estimate for the water availability in the soil reached by the roots (Jones 1992).

Predawn (Ψ predawn) and midday (Ψ midday) frond water potentials were measured using a pressure chamber (PMS Corvallis, OR, USA) at predawn 5 a.m./6 a.m. and midday 12 p.m./1 p.m. (summer/winter, respectively).

5.2.7. Frond traits

Specific leaf area (SLA) was calculated as the ratio of frond area over frond dry weight 2 -1 (m kg dry weight). Fresh frond samples were collected, the frond area scanned and calculated using Scion Image software (Scion Corporation 2000-2001, USA), and frond material was then dried at 60ºC for 48 h for dry weight. Different cohorts of fully- expanded fronds produced in summer and winter, were used for calculation of SLA.

Frond samples were dried as described above and ground to a fine powder, then analysed for total nitrogen and carbon content using an elemental analyser (LECO CHN- 1000, Michigan, USA). Photosynthetic nitrogen use efficiency (PNUE) was calculated as Amax divided by frond nitrogen content (on a frond area basis).

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For measurements of frond chlorophyll content, four frond discs (diameter of 3.75 mm each) were collected, immediately immersed in liquid nitrogen, and stored at -80ºC until extraction. Chlorophyll a and b were extracted using 1.8 ml of 100% dimethyl sulphoxide (DMSO). Extracts were heated for 30 min at 65ºC in a dry block heater Termoline L+M (Northgate, Queensland, Australia). The supernatant was then transferred to a spectrophotometer Carry 300 (Varian, The Netherlands). A blank of pure DMSO was used to calibrate the spectrophotometer at zero absorbance. Chlorophyll a, b and total were calculated according to Wellburn (1994).

Fresh frond material (from 3-4 fronds per tree fern) was analysed for stomatal density by variable pressure scanning electron microscopy (VP-SEM, model Leo 1450 VP; Leo Electron Microscopy Inc., NY, USA). A flat cross-section of both sides of each frond was scanned and stomatal density was calculated for four randomly chosen views (giv- ing 12 to 16 stomatal density estimates per tree fern).

5.2.8. Statistical analysis

A general linear model of SPSS 15 (SPSS Inc. Chicago, USA) was used to analyse effects of species (D. antarctica, C. australis) and season (summer, winter) on each dependent variable using the following covariates: estimated mean daily PPFD, leaf temperature during gas exchange measurement, Tleaf, and predawn frond water potential Ψ predawn (as a measure of plant water status).

Every variable was checked for normality using the Shapiro-Wilk test, and log transformed if the assumption of normality was not satisfied. Relationships of covariates with dependent variables were visually checked for linearity and differences in slopes (see Fig. 5.2). In this analysis, the effect of season would only be significant if seasonal differences were over and above the variation explained by seasonal differences in growth irradiance, temperature, and water status as defined by the covariates.

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For diurnal course measurements we used boundary-line analysis in a scatter- plot of all data points of each parameter against the environmental variable in question. Functions for boundary line analysis were chosen according to González-Rodríguez et al. (2001) and Larcher (2003), with accuracy of fits evaluated using r2 (in all fits, r2 >0.85).

5.3. Results 5.3.1. Relationships between photosynthesis, growth irradiance and temperature

Predawn quantum yield of photochemistry Fv/Fm never decreased below 0.7. It was negatively related to Tleaf (significant covariate effect in Table 5.1), but did not differ between the two species. It was significantly higher in summer, despite a negative relationship with Tleaf, reflecting a flatter slope in their relationship in summer than in winter (Fig 5.2).

Tleaf was positively correlated with the light-saturated rate of net photosynthesis Amax and stomatal conductance gs, while growth irradiance was significantly correlated to

Amax, but not gs (Table 5.1, Fig. 5.2). Amax and gs were similar for both species (Table 5.1), and were significantly higher in winter than in summer (Fig. 5.2).

Nitrogen content per frond area, NA was correlated with growth irradiance and Tleaf (Ta- ble 5.1). NA was significantly greater in D. antarctica than in C. australis, and in winter than in summer (Fig. 5.2). Photosynthetic nitrogen use efficiency PNUE (Amax/NA) did not vary significantly between species and seasons and was not significantly affected by any of the tested covariates (Table 5.2).

SLA was inversely related to growth irradiance (Table 5.1). In addition, C. australis displayed significantly greater specific leaf area, and SLA was significantly greater in summer than in winter (Fig. 5.2; Table 5.1).

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None of the covariates had a significant effect on total chlorophyll or chlorophyll a/b (Table 5.2). Total chlorophyll content (on an area basis) was significantly greater in D. antarctica and in winter, while chlorophyll a/b ratios were comparable between species and seasons (Table 5.2).

Table 5.1 Significance of the effect of fixed factors (species and season) and of covariates (ANCOVA) on photosynthetic capacity parameters and frond traits of the tree ferns D. antarctica and C. australis

Where: Fv/Fm, maximal quantum yield of photochemistry; Amax, light saturated rates of net photosynthesis; gs, stomatal conductance at Amax; SLA, specific leaf area; NA, frond nitrogen content on an area basis. Significance levels:*P<0.05; **P<0.01; ***P<0.001; n.s., non significant; (+) positive relationship; (-) inverse relationship. No interactive effects were significant

Effect and covari- Fv/Fm Amax gs SLA NA ates

Effect Species n.s.(0.1) n.s (0.9) n.s. (0.1) * * Seasons *** ** * ** ** Covariate Mean daily PPFD n.s. (0.08) (+)** n.s. (0.6) (-)* (+)** (mol m-2d-1)

Tleaf (ºC) (-)*** (+)** (+)* n.s. (0.2) (+)*

Ψ predawn (MPa) (+)* n.s. (0.4) n.s. (0.8) n.s. (0.08) n.s. (0.5)

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16 16 14 14

) ) 12 12

-1

-2 -2 10 10 8 8

mol m mol



( ( 6 6

max

A 4

A 4 2 2 0 0 7 0.25

6 0.20 5

)

-1

)

-2 4 -2 0.15

(g m (g A 3 0.10

(mol m (mol

s 2 g 0.05 1

0 0.00 25 0.86 0.84 20 0.82

) -1 0.80

kg 15

/F 0.78

F 10 0.76 SLA (m SLA 0.74 5 0.72 0.70 0 0.68 0 10 20 30 40 0 10 20 30 40 -2 -1 T (oC) Daily mean PPFD (mol m d ) leaf

Fig. 5.2. Relationships between photosynthetic capacity parameters and frond traits of the tree ferns D. antarctica and C. australis and environmental variables

Where: Amax - light saturated rates of net photosynthesis; gs, stomatal conductance at

Amax; Fv/Fm, maximum quantum yield of photosystem II; SLA, specific leaf area; NA, frond nitrogen content on an area basis. D. antarctica (triangle) and C. australis (circle) in winter (open symbols) and summer (closed symbols). Regressions are indicated by dashed lines in summer, and solid lines in winter. All regressions were highly significant (P<0.01). See Table 5.1 for significance of effects and relationships.

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Table 5.2. Photosynthetic capacity and water relation parameters of the tree ferns D. antarctica and C. australis in summer and winter. Values are means (n = 32) ± s.e. of: PNUE, photosynthetic nitrogen use efficiency, Chl total, total chlorophyll content on a frond area basis; Chl a/b, chlorophyll a/b ratio; WUEi, intrinsic water use efficiency; Ψ predawn and Ψ midday, predawn and midday frond water potentials. Significance levels: **P<0.01; ***P<0.001; n.s., non-significant; n/a, non applicable; (+) positive relationship (covariates tested were mean daily PPFD, Tleaf, Ψ predawn). No interactive effects were significant.

Parameter Summer Winter Effects D. antarctica C. australis D. antarctica C. australis Species Seasons Covariates PNUE 39±5 37±7 33±6 51±11 n.s. (0.3) n.s. (0.7) n.s.

-1 -1 -1 (µmol CO2 mol N s ) Chl total (µmol m-2) 390±51 279±27 725±88 496±58 ** ** n.s. Chl a/b 3.0±0.1 3.1±0.2 3.5±0.4 3.1±0.1 n.s. (0.7) n.s. (0.7) n.s. 1 Ψ predawn (MPa) -0.6±0.1 -0.5±0.1 -0.1±0.0 -0.1±0.0 n.s. (0.5) *** n/a

Ψ midday (MPa) -1.1±0.1 -1.2±0.1 -0.5±0.1 -0.3±0.1 n.s. (0.9) *** n.s.

WUEi n.s. (0.2) n.s. (0.8) 125±16 109±12 62±6 64±12 PPFD* (+) -1 -1 (µmol CO2 mol H2O ) 1-t-test

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5.3.2. Water status parameters

Predawn frond water potential (Ψ predawn) did not differ between species but was significantly more negative in summer (t-test, P<0.001, Table 5.2). Fv/Fm was positively related to Ψ predawn (Table 5.1). Midday frond water potential (Ψ midday) was not related to any of the tested covariates. It was similar between species but significantly more negative in summer than in winter (Table 5.2).

Growth irradiance had a significant positive effect on intrinsic water use efficiency

WUEi (Table 5.2). WUEi did not vary between species and seasons once covariates were accounted for (Table 5.2).

5.3.3. Diurnal measurements

Instantaneous values of PPFD, leaf/air temperature, and leaf-to-air vapour pressure deficit (VPD) at the measured fronds did not differ between species within season (t-test P>0.1, data not shown). These variables differed significantly between seasons, with mean daily PPFD, air temperature, and VPD in summer double that in winter (Fig.5.3).

Boundary-line analysis showed near-linear relationships between A and gs in summer for both species, while in winter these relationships approached a saturation curve with -2 -1 A reaching saturation at gs of ca 0.15 mol m s in both species (Fig. 5.4 a). VPD became limiting to gs only at values greater than 1 kPa, which occurred only in summer (Fig. 5.4 b). Optimal leaf temperature for photosynthesis in winter was on average 15ºC for D. antarctica and 20ºC for C. australis, while in summer, A continued to rise at leaf temperatures above 30ºC in both species (Fig. 5.4 c).

From light response curves fitted to the data of the diurnal measurements (Fig. 5.5),

Amax did not differ significantly between species but was significantly (P<0.05) higher in summer than in winter (mean 17.4 vs. 8.6 µmol m-2s-1 respectively). Light saturation was reached at about 1100 in winter and nearly 1500 µmol photons m-2s-1 in summer.

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Summer Winter 100

RH (%) RH 40

o 20

T air ( T air 10

0 2000

)

-1

s -2 1500

mol m mol

 1000

500

PPFD ( PPFD 0 9:00 11:00 1:00 3:00 5:00 9:00 11:00 1:00 3:00 5:00 Time (h)

Fig. 5.3. Climate conditions during diurnal course measurements in summer and winter. Lines indicate means.

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Cyathea australis Dicksonia antarctica 16 14 a 12

)

-1 10

-2 8

mol m mol 6



(

A 4 2 0

0.00 0.05 0.10 0.15 0.200.00 0.05 0.10 0.15 0.20 -2 -1 gs (mol m s )

0.20 b

)

-1 0.15

-2 0.10

(mol m (mol

s g 0.05

0.00 0 1 2 3 4 5 6 0 1 2 3 4 5 6 VPD (kPa) 16

14 c 12

)

-1 10

-2 8

mol m mol 6



(

A 4 2 0

0 10 20 30 40 0 10 20 30 40 T (oC) leaf Fig. 5.4. Relationships between photosynthesis, stomatal conductance and water pressure deficit based on leaf temperature. Where: (a) Photosynthesis (A) versus stomatal conductance (gs); (b) gs vs. water pressure deficit based on leaf temperature (VPD); (c)

A vs. leaf temperature (Tleaf) of the tree ferns C. australis (on the left) and D. antarctica (right) in summer (open circles) and winter (closed triangles). Boundary-line fits are indicated by dashed lines in summer and dotted lines in winter (for a and c only).

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0.20

0.15

-1

-2 0.10 5 10 15 20 25 30 35 40

, mol m

s g 0.05

0.00 5 10 15 20 25 30 35 40

T ,oC leaf This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia

Cyathea australis Dicksonia antarctica 16

-1

-2

m 8

molCO



A, 0

-4 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 PAR, mol photons m-2s-1

Fig. 5.5. Light response curves of the tree ferns D. antarctica and C. australis in summer and winter. Where: open circles – summer; closed circles – winter. Boundary-line fits are indicated by dashed lines in summer and dotted lines in winter (r2 >0.9 in all fits).

5.3.4. Stomatal density

Fronds of both species had no stomata on their adaxial (upper) surface. D. antarctica displayed a more regular stomatal pattern in contrast to the random distribution of stomata in C. australis (Fig. 5.6). Stomatal density did not differ significantly between species (P=0.08) and was not correlated with growth irradiance (r =-0.007, P>0.9).

Leaf hairs were only observed on fronds of C. australis. Growth irradiance marginally affected leaf hair density (P=0.06), which varied from 22 leaf hairs mm-2 on high light exposed fronds to 11 leaf hairs mm-2 on shaded fronds, with an overall average of 13 hairs per mm-2.

5.4. Discussion 5.4.1. Comparisons between the two tree fern species

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Regardless of their putatively different origins and distribution patterns within forests, the majority of photosynthetic parameters did not differ between our two tree fern species. Light saturated rates of net photosynthesis (Amax) were similar and within the range reported previously for D. antarctica (Hunt et al. 2002, Volkova et al. 2009). Specific leaf areas of D. antarctica and C. australis were in the range of SLAs for humid temperate and tropical forests (Reich et al. 1999).

Both D. antarctica and C. australis performed better in winter, when photosynthetic rates reached higher values under similar irradiance and leaf temperatures. Both higher stomatal conductance, through less diffusive resistance to CO2, and greater frond N content, via increased carboxylation capacity dependent on N-rich proteins such as Rubisco (Niinemets and Tenhunen 1997), could jointly enable greater assimilation rates.

Chlorophyll a/b ratios did not vary between seasons, suggesting low acclimation of both tree ferns for this parameter, consistent with previous observations of ferns in the genus Trichomanes (Johnson et al. 2000).

5.4.2. Light as a limiting factor to tree fern photosynthetic performance

Under our field conditions light-saturation of photosynthesis was recorded at above 1100 µmol m-2s-1, considerably higher than previously reported for D. antarctica (600 µmol m-2s-1 for plants grown under natural illumination in a glasshouse, and 150 µmol m-2s-1 for plants grown in a gully, Hunt et al. 2002). This might reflect physiological acclimation to open canopy conditions created after logging (Kursar and Coley 1999), or differences between studies related to plant and frond age.

Growth irradiance (measured via mean daily PPFD) had stimulating effects on Amax and

NA confirming results with other tree species (e.g. González-Rodríguez et al. 2001,

Oliveira and Peñuelas 2004, Niinemets 2007). Increase in WUEi (Amax/gs) with increasing growth irradiance possibly reflected the strong relationship between light and Amax.

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Linear relationships between A and gs, as observed in summer indicate that photosynthesis was limited only by gs (Jones 1992), which, in turn, tended to decrease with in- -2 -1 creases in VPD. In winter, A reached a plateau at ca gs 0.15 mol m s , indicating additional non-stomatal limitation of A (such as amount or activity of Rubisco or the rate of electron transport, Massonnet et al. 2007).

In contrast to results from many other species (e.g. Romero and Botía 2006, Tazoe et al.

2009), gs at Amax (which reflects longer term response of gs to growth irradiance) was insensitive to mean daily PPFD, indicating a lack of acclimation in gs to observed growth irradiance for our tree fern species. In addition, there was no significant relationship between instantaneous gs and instantaneous PPFD (as measured during the diurnal measurements, P=0.9). This is in contrast to common knowledge that increasing light induces gs until a saturation point is reached (Larcher 2003), a relationship commonly used in models of stomatal responses. Such results may suggest poor responsiveness of tree ferns‟ stomata to light and confirm observations of Doi and Shimazaki (2008), when stomatal conductance of the fern Adiantum capillus-veneris showed much higher light responsiveness when the light was applied to the lower leaf surface, where stomata were situated, than when it was applied to the upper surface (as in our case). Similar non-responsiveness of stomata to instantaneous light was found for Vicia faba (Mott et al. 2008), leading to the conclusion that mesophyll signals play decisive roles in pre- conditioning stomatal response to light.

The absence of significant relationship between the growth irradiance and Fv/Fm indicates that PSII of both species fully recovered overnight regardless of received mean daily PPFD.

Inverse relationship between the growth irradiance and SLA is consistent with the view that sun leaves are more sclerophyllous (Groom and Lamont 1997, Niinemets 2007). However, in contrast to the opinion that higher level of sclerophylly (i.e. decreased SLA) results either from combinations of less water availability and high light intensities or from nutrient impoverished soils (for details see Groom and Lamont 1997), we

-108- This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia found lower SLA in winter‟s than summer‟s frond cohorts, when light intensities were lower and water availability was greater. In agreement with Turner (1994) we can only conclude that increase in sclerophylly was not related to increased drought tolerance or acclimation to drier conditions of the tree ferns.

Overall, the growth irradiance stimulated photosynthetic capacity and nitrogen alloca- tion in fronds, yet, it had little effect on stomata or PSII in these tree fern species.

5.4.3. Temperature as a limiting factor to tree fern photosynthetic performance

Both tree fern species displayed broad temperature optima for net photosynthesis similar to a wide range of C3 plants reviewed by Kattge and Knorr (2007). Optimum temperatures were higher in summer, confirming acclimation potential of the tree ferns to higher growth temperatures, consistent with our previous study (Volkova et al. 2009).

Inverse relationship of Tleaf with Fv/Fm reflects the sensitivity of PSII to high temperatures, which is often cited as the most heat-sensitive component of photosynthesis in temperate species (e.g. Berry and Björkman 1980). Over and above the effect of seasonal differences in Tleaf, season also had a significant additional effect on Fv/Fm – at the same Tleaf, Fv/Fm was significantly lower in winter than in summer (Fig. 5.2) – suggesting some cold-induced limitation in PSII efficiency. Reduction of Fv/Fm during winter is confirmed by numerous other studies (see Wittmann et al. 2007), and may be related to either chilling-induced photo-degradation of PSII components, or overnight retention of de-epoxidised xanthophylls (Adams and Demmig- Adams 1994). Furthermore, Larcher and Nagele (1992) demonstrated that photosynthetic capacity of Fagus sylvatica stems decreased in winter and even short-term rewarming treatments could not restore it to summer values. Thus, we may suggest that in winter, the tree ferns may experience persistent photoinhibition of PSII induced by cold morning temperatures that are common for this study site.

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Significant positive relations between NA and temperature might reflect changes in soil nutrient mineralisation and thus N availability with temperature. In addition, a study by Muller et al. (2009) indicated that temperature rather than irradiance primarily determined changes in NA in natural conditions for broad-leaved evergreen species.

Evaporative cooling through increased stomatal conductance and associated transpiration implies a negative relationship between Tleaf and gs (see Snider et al. 2009). In contrast, we observed a positive relationship of Tleaf with gs, which may indicate that in our study area gs is affected by low rather than high temperatures. Stomatal closure can also occur when the water supply from the roots is restricted because of low temperatures in the root zone (Davies et al. 1982).

5.4.4. Effects of plant water status and water relation parameters on tree fern photosynthetic performance

Plant water status (measured via Ψ predawn) showed no significant relationship with any of the measured parameters except Fv/Fm. Apparently, slightly, albeit significantly, more negative Ψ predawn in summer (-0.05 MPa) relative to winter (-0.01 MPa) was not enough to impose significant limitations on any of the measured variables. In agreement with their ecological distribution in moist sites, tree ferns may be unable to tolerate a larger drop in water potential, instead relying on an „avoidance‟ strategy involving frond loss, as indicated in another open-air study by the first author (yet unpublished data). Sensi- tivity of Fv/Fm to plant water status suggests that the efficiency of the photosystem likely decreased due to reduced efficiency of the light-harvesting and antenna complexes to deliver quanta to reaction centres (Wright et al. 2009).

No effect of season on intrinsic water use efficiency (WUEi) indicates prodigal or water spending strategy by both species – the strategy, appropriate for plants that are subject to droughts of short duration (Passioura 1982). Ferns are known for poor efficiency in water transport through the leaf (Sack and Holbrook 2006), and together with the ob-

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served low acclimation potential of WUEi to seasonal fluctuations illustrates why many fern species are confined to moist environments (Franks and Farquhar 1999).

5.4.5. Stomatal density

Stomatal patterns but not density differed between our two tree fern species. In contrast to many angiosperms, where stomatal density is higher in leaves from sunny habitats (Larcher 2003), stomatal density of both tree fern species did not correlate with the growth irradiance. A similar lack of relationship between stomatal density and the growth irradiance was observed for tree ferns of Cyatheaceae family by Arens (1997), and for whole fern communities by Kessler et al. (2007). Our results indicate that tree ferns (Pteridophyte) lack acclimation in terms of changing stomatal density in response to their light environment.

Leaf hairs, known to reduce leaf temperature due to their reflection function (Lambers et al. 2008) or to protect against UV-B radiation (Grammatikopoulos et al. 1994), were found only in C. australis, possibly reflecting its microclimate preferences and assumed Pan-tropical origin (i.e. adaptation to greater light intensities).

-111- This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia

Dicksonia antarctica High light exposed habitat Shaded habitat a b

Cyathea australis High light exposed habitat Shaded habitat c d

Fig. 5.6. Stomatal density of the tree ferns (a, b) D. antarctica and (c, d) C. australis from (a, c) light-exposed and (b, d) shaded habitats.

-112- This Chapter is submitted for publication Volkova L, Bennett LT and Tausz M “Seasonal variations in photosynthesis of the tree ferns Dick- sonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia

5.5. Summary

Regardless of different origins and micro-site habitats, there were no significant differences in terms of photosynthetic performance between D. antarctica and C. australis, and both tree fern species had greater photosynthetic capacity in winter. Low temperatures appeared to be most limiting factor on plant performance under field conditions. Plant water status did not vary markedly and had no effect on any of the measured parameters. Similar to other plants, both species of tree ferns displayed seasonal acclimation in a number of measured photosynthetic parameters and frond traits (i.e. Fv/Fm,

Amax, gs, NA, chlorophyll total, SLA). Acclimation of stomatal density to spatial variation in growth irradiance among micro-sites seemed limited in both species, while stomatal pattern differed between species.

Because there were no significant differences between the two species in photosynthetic parameters, both species can probably be described by common carbon gain and water use models at the leaf scale.

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Chapter 6. Ecophysiology of two tree fern species and implications for their future management. General discussion and conclusions

6.1. Species overview

The maximum amount of carbon fixed per unit leaf area and time or Amax (Taiz and Zeiger 2002) is believed to be low in ferns compared with many other plants (Nobel et al. 1984). In fact, both tree fern species in all of my studies had maximal photosynthetic rates similar to a range of tree species in comparable ecosystems (e.g. middle-storey canopy species Nothofagus cunninghamii Oerst (Tausz et al. 2005), Canarian laurel forest tree species Persea indica (González-Rodríguez et al. 2002), or Laurus azorica (González-Rodríguez et al. 2001, Chapters 2-5).

According to von Caemmerer and Farquhar (1981), changes in Amax reflect changes in both stomatal conductance (gs) and mesophyll capacity parameters (i.e. Vcmax and Jmax). Stomatal conductance was greater in C. australis than D. antarctica at the beginning of the water stress experiment (Chapter 3), but was comparable between two species in all other experiments (Chapters 2 and 5, with an average of 100 mmol m-2s-1). The maximum carboxylation rate, Vcmax, or in vivo apparent Rubisco activity, was comparable in both species across all experiments (Chapters 2, 4), while the maximum rate of electron transport, Jmax, was consistently higher in D. antarctica than C. australis (Chapters 2,

4). Overall, both species had relatively low values of Vcmax and Jmax comparable with some shade tolerant tree species, such as silver fir (Abies alba Mill, Robakowski et al. 2002, Chapters 2-5).

13 Stable isotope composition, δ C, is related to the ratio of internal to external CO2 concentrations (Ci/Ca), and therefore can be a useful tool in assessing intrinsic water use 13 efficiency, WUEi (Dawson et al. 2002). Lowest δ C assumes greater WUE (Farquhar 13 et al. 1989). δ C values of both tree fern species were within the ranges reported for C3 plants (-20 to -35‰, Dawson et al. 2002). However, despite lower δ13C in C. australis‟s

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fronds (closer to -30‰, Chapter 4), it was D. antarctica that had significantly greater instantaneous (i. e. measured by gas exchange) WUEi. Values for WUEi in juveniles of -1 both species (range of 43-80µmol CO2 mol H2O; Chapter 4) were comparable with those recorded for mature tree ferns in the field study (Chapter 5), yet they were double those recorded for an evergreen herbaceous fern of the family Adiantaceae Adiantum reniforme var. sinensis (Liao et al. 2008).

Specific leaf area (SLA) is an index of sclerophylly (Lamont et al. 2002) and tends to be higher in tropical and humid temperate forests (Reich et al. 1999). D. antarctica had consistently lower SLA than C. australis across several experiments (e.g. 8.6 vs. 10.4 m2kg-1 respectively). However, compared to a broad range of species, both species had SLA similar to native Hawaiian tree ferns of the genus Cibotium (C. chamissoi), grown in semi-wet to wet forests (Durand and Goldstein 2001).

Concentrations of chlorophylls were comparable between tree fern species in the first glasshouse experiment (high light stress), but were greater in D. antarctica than C. australis in other experiments involving older plants (Chapters 4, 5). Total chlorophyll concentrations (per leaf area) of both species were comparable with a wide range of tree species (e.g. shade grown Fagus sylvatica (Lichtenthaler 2007). Absolute values of total chlorophyll concentrations only significantly differed in the second experiment, which examined high light and heat interactions (Chapter 3). This could be due to the different methods used to determine chlorophyll concentrations (HPLC vs. LECO), and possibly due to plant age (Louis et al. 2009). Chlorophyll a/b ratio was comparable between the two species in mature, field plants (Chapter 5), but was consistently higher in D. antarctica than C. australis in juvenile plants (Chapters 2, 4) due to greater chlorophyll a concentrations.

Overall, my project found that the photosynthetic characteristics of D. antarctica and C. australis were comparable and within reported ranges for a number of plant types. Thus, the different origins of the two species, and their apparent preference for different mi-

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cro-sites, do not appear to have resulted in a divergence of their physiological responses to a range of environmental stresses.

6.2. Overview of light, temperature, and water availability as stresses on tree fern physiology

My results in Chapter 2 (high light stress) indicated immediate down-regulation of photosynthetic capacity parameters by tree ferns in response to high light, but acclimation to high light after prolonged (three month) exposure. These results are consistent with the view that high light alone does not have detrimental effect on photosynthesis and plants can often fully recover (Levitt 1980, Lovelock et al. 1994).

In Chapter 3, I examined interactive effects of high light and moderate heat on photosynthetic capacity of D. antarctica. Here, combination of high light and moderate heat led to negative effects including chlorophyll bleaching and severe photoinhibition

(measured as a decrease in Fv/Fm<0.4). Given the comparable responses to stresses by both tree fern species in all other studies (see above), it is reasonable to assume that high light with moderate heat would be equally problematic for C. australis. This result of detrimental effects of light by temperature interactions supports findings of many other studies (e.g. Al-Khatib and Paulsen 1999, Montgomery et al. 2008), although it is in contrast to Havaux et al. (1991), who stated that high light could alleviate negative effects of high temperatures.

In Chapter 4, I studied interactive effects of high light and water deficit on photosynthetic capacity and water relation parameters of both species D. antarctica and C. australis. It appeared that both species were susceptible to water deficit either alone or in interaction with high light. This funding was in contrast with Levitt (1980) stating that interactive effect of high light and water deficit can be more detrimental than water deficit alone. Stomatal response to water withheld was slower in shade-grown than in high light exposed plants, confirming the findings with bracken fern where stomata from the most shaded understorey level were less sensitive to soil moisture treatments

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than those from more irradiated levels (Roberts et al. 1984). Probably as a result of it there were some indications that shade did not ameliorate but rather intensified drought effects on D. antarctica, the result confirming observation of Valladares et al. (2002) who stated that the capacity to withstand severe drought was not enhanced in the shade but decreased due to increased below-ground competition for water with established trees.

The focus of Chapter 5 was on mature tree ferns growing in their natural environment, the mountain ash forests of the Victorian Central Highlands, Australia. Measurements of photosynthetic capacity as well as water relations and frond parameters over two consecutive years indicated that both species, irrespective of their different origins and apparent differences in micro-site preferences, performed better in winter than in summer. Low light and low temperatures were limiting factors for the tree ferns performance in the field. In contrast to many reports in the literature (e.g. Romero and Botía 2006, Tazoe et al. 2009), stomatal conductance of the tree ferns did not correlate with growth irradiance but was positively correlated only with temperature. Plant water status (Ψ predawn) had no effect on any of the measured parameters (except maximal quantum yield of photochemistry, Fv/Fm), possibly because decreases in Ψ predawn to - 0.05 MPa were not enough to impose limitations. Another interpretation is that tree ferns lack the ability to adjust to decreased water availability and cannot endure more pronounced drops in tissue water potentials. This may also explain observations in both, field and glasshouse, plants, where ferns shed fronds under more severe water deficit.

Both species showed characteristics of a water spending strategy irrespective of season

(summer or winter), i.e. WUEi in summer, when temperature was higher and less water available, was comparable to WUEi in winter, a season characterised by lower temperatures and greater rainfalls. Such prodigal strategy is common and effective for plants that only occasionally experience drought (Passioura 1982). Using this interpretation, results of this field Chapter were consistent with indications of low acclimation of the tree ferns to water availability found in the controlled experiment involving juvenile plants (Chapter 4). Tree ferns growing at open sites showed little difference in stomatal

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density from those growing under dense canopy cover, indicating a lack of acclimation of stomatal density in the tree ferns to growth irradiance. This is in contrast to observations on many other tree species (Larcher 2003), but consistent with findings of Arens (1997) on the tree fern Cyathea caracasana.

6.3. Practical implications and future directions

Despite their ecological importance as dominant understorey species in Australia‟s wet sclerophyll forests, the physiology of D. antarctica and C. australis has previously been under-examined. Understorey plants like these two species may account for as much as 33% of living above-ground biomass (Weaver 2008), and, in evergreen forests worldwide, contribute on average 49% of total ecosystem respiration (Misson et al. 2007). Thus, the paucity of knowledge about tree fern physiology not only limits our ability to predict potential impacts of climate change on their distribution but also excludes a significant component of temperate forests from total carbon balance calculations (i.e. by excluding a significant portion of potential carbon sinks and sources).

Two experiments and one observational field study (Chapters 2, 4 and 5) indicated that D. antarctica and C. australis are fairly similar with respect to (eco)physiological and morphological characteristics. Correspondingly, growth and carbon sequestration models could use a common set of parameters for both species, which would greatly sim- plify the task of including tree ferns in process-based carbon accounting or environmental risk assessment models. Moreover, because D. antarctica and C. australis belong to the two main tree fern families Dicksoniaceae and Cyatheaceae – arguably the most important tree fern families worldwide – the knowledge created in this thesis will be a sound starting point for understanding the ecophysiology of other tree ferns across a potentially broad range of environmental conditions.

The problem of poor survival and ongoing decline of the tree fern numbers after clearcut logging (Ought and Murphy 1998) was one of the main inspirations for this study. My experimental results, particularly Chapter 3 (high light and high temperature), indi-

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cate detrimental effects of combined high light and moderate temperatures on tree fern health, which should be considered when designing logging configurations. For example, understorey islands should be designed to minimise tree fern numbers near edges where they will be exposed to greater light intensities and consequently higher temperatures. This could involve consideration of a minimum distance to edge for tree fern populations of considerable numbers.

The results of Chapter 4 (high light and water deficit) reiterated the importance to tree ferns of retaining buffer zones along waterways. This experiment also indicated that retention of understorey islands in the middle of logging coupes away from waterways, might not lead to substantial improvement in the protection of tree ferns in logging coupes under climate change predictions of increasing drought frequency. Indeed, my results indicated that shade led to slower stomatal responses in D. antarctica thereby intensifying drought stress. Thus, design of understorey islands should take tree fern needs for access to water into consideration, and should thus be placed on lower rather than upper slopes.

Climate change predictions indicate that the severity and frequency of both droughts and fire will increase in south-eastern Australia (Hennessy et al. 2007), leading to changes in species distributions (see Fitzpatrick et al. 2008). Thus, species better adapted to drought and fire could expand their range, while other elements of native vegetation might be at risk (Watt et al. 2009). Findings from my study indicate that the range of tree ferns in south-eastern Australia may contract under a more irradiated (i.e. due to fire-induced canopy death), warmer, and drier climate. In addition, the current divergence in micro-site preferences between the two studied species might become less evident. That is, current localised extensions of C. australis to forest margins might contract to more shaded, moister sites to be closer to the current distribution of D. antarctica. This emphasises the need for buffer zones along waterways, which under future climates could be considered refugia for tree ferns – that is, isolated areas of habitat that retain the environmental conditions that were once widespread, Stewart and Lister 2001).

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While my study provides useful indications of tree fern responses to environmental stresses, it was limited to photosynthetic capacity parameters at the leaf-level scale. To increase accuracy of carbon models, future studies should consider dynamics of frond development, including frond longevity, and the potentially different physiology of fronds of different age cohorts. In addition, precise micro-climate data will be another essential component for improved modelling of tree fern ecophysiology and of understorey plants in general. Modelling at landscape to regional scale would be of particular benefit to conservation goals, but scaling errors need to be considered.

Overall, my study revealed that both species of the tree ferns might be vulnerable in the future because of their low acclimation potential to environmental stresses. These findings highlight the need to develop more flexible conservation policies to maintain tree fern communities under optimal conditions where they can be most resilient to both predicted and unexpected future changes.

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Volkova, Liubov Vladimirovna

Title: Ecophysiology of the tree fern species Dicksonia antarctica Labill and Cyathea australis (R. Br.) Domin

Date: 2009

Citation: Volkova, L. V. (2009). Ecophysiology of the tree fern species Dicksonia antarctica Labill and Cyathea australis (R. Br.) Domin. PhD thesis, Dept. of Forest and Ecosystem Science, Melbourne School of Land and Environment, The University of Melbourne.

Persistent Link: http://hdl.handle.net/11343/37893

File Description: Ecophysiology of the tree fern species Dicksonia antarctica Labill and Cyathea australis (R. Br.) Domin

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