Evidence for Demographic Shift in Woody Plant Species Under a Changing Climate in Southwest Australian Mediterranean-Type Ecosystems

Evidence for demographic shift in woody plant species under a changing climate in southwest Australian Mediterranean-type ecosystems.

Ebony Cowan

Bachelor of Science Honours in Environmental Science

This thesis is presented for the degree of Bachelor of Science Honours, School of Veterinary and Life Sciences, of Murdoch University, 2018.

I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. Ebony Cowan 22th October 2018

Abstract

Climate change is projected to cause reduced rainfall, increased temperatures and more frequent fires for many parts of the world, particularly in Mediterranean-type ecosystems (MTEs). Such changes may profoundly impact species demography and ultimately their persistence. However, the impact of climate change on seed demography is not well documented. The MTE region of southwest Australia is a renowned plant biodiversity hotspot and has experienced significant warming and drying over the past 40 years. An iconic and important portion of the region’s biodiversity are serotinous species which retain a canopy seed bank in woody fruits and offer a powerful opportunity to study climate impacts; these species often retain their lifetime reproductive effort as woody fruits which may be quantified, rather than most other plant species which disperse their seeds regularly upon ripening. The drivers of changes to the size of the seed store in serotinous species is unknown, but potential mechanisms include changes in seed/fruit production, seed viability or cone opening.

Therefore, these factors were quantified for a selection of 11 serotinous species in the region and compared to data collected in the past (1983-2004) to quantify possible climate change impacts.

Total viable seed production was variable between species and time frames, with 54% of species showing a reduction in seeds produced (reductions of 4-76%). This result was driven by a reduction in fruit production in 57% of species surveyed, while seed viability did not change. Across a broad climatic gradient, there was more rapid cone opening in serotinous species (i.e. seed release into the inter-fire period) in mesic sites compared to xeric sites, with cones also opening at a younger age than they did in the past. This suggests that climate change is influencing the size of the overall seed store in serotinous species, with changes to fruit production and cone opening being most

iii significant. This threatens the long-term persistence of these species under a changing climate, with less seeds available for post-fire recruitment leading to potential population collapse. These findings are applicable to many serotinous species in MTEs globally and warrant further investigation.

Contents Abstract ...... iii List of tables ...... vii List of figures ...... viii Acknowledgments ...... ix 1. Introduction ...... 1 1.1 Thesis objectives ...... 3 1.2 Thesis structure ...... 5 2. Literature review: Assessing evidence for demographic shift in MTEs ...... 7 2.1 Introduction ...... 7 2.2 Objectives and scope ...... 8 2.3 Methods for searching the literature ...... 10 2.4.1 Evidence for demographic shift at different sites in MTEs ...... 10 2.4.2 Evidence for demographic shift in response to a drought event in MTEs ...... 13 2.4.3 Evidence for demographic shift in non-MTEs ...... 16

2.5 The effects of enhanced CO2 on cone production ...... 16 2.6 Factors influencing serotiny and the role of fire regimes ...... 18 2.6.1 Wet-dry cycles in imitating natural post-fire seed release ...... 19 2.6.2 Seed morphology across dominant serotinous genera in MTEs ...... 20 2.7.1 Evidence for fruit opening across a climatic gradient ...... 21 2.7.2 Evidence for fruit opening in response to drought ...... 24 2.7.3 Evidence for fruit opening in non-MTE regions ...... 26 2.8 Synthesis of findings and future directions ...... 27 3. Methods ...... 30 3.1 Study area...... 30 3.1.1 Location and land use ...... 30 3.1.2 Climate ...... 31 3.1.3 Geology ...... 33 3.1.4 Topography ...... 34 3.1.5 Vegetation ...... 35 3.1.6 The role of fire ...... 35 3.2 Study methods ...... 38 3.2.1 Previous data ...... 38 3.2.2 Site selection ...... 41 3.2.3 Focal species ...... 43 3.3 Fieldwork methods ...... 45 3.3.1 Fruit counts ...... 45 3.3.2 Fruit collection ...... 47

3.3.3 Banksia fruit collection ...... 47 3.3.4 Cone processing ...... 47 3.3.5 Seed counts and germination trials ...... 48 3.4 Standardized Precipitation Evapotranspiration Index (SPEI) ...... 49 3.5 Data analysis ...... 50 4. Results ...... 53 4.1 Viable seed production over time ...... 53 4.2 Mechanisms influencing seeds produced ...... 57 4.2.1 Changes in fruits per plant ...... 57 4.2.2 Changes in seed viability ...... 59 4.2.2.1 Changes in seed viability with cone age ...... 61 4.3 Changes in cone opening rates ...... 62 4.3.1 Changes in cone opening across a climatic gradient ...... 63 4.3.2 Changes in cone opening across different time frames ...... 63 4.4 Standardized Precipitation Evapotranspiration Index (SPEI) ...... 64 5. Discussion ...... 67 5.1 Evidence for interval squeeze ...... 67 5.1.1 How has climate change influenced seed production?...... 68 5.1.2 How has climate change influenced fruit production? ...... 69 5.1.3 How has climate change influenced seed viability? ...... 71 5.2 Changes in fruit opening ...... 72 5.2.1 How has climate change affected cone opening across a climate gradient? ...... 72 5.2.2 How has climate change affected cone opening across different time periods? ...... 73 5.3 Factors influencing variation in serotiny ...... 74 5.3.1 Fire regimes and serotiny ...... 75 5.3.2 Plant trade-offs and serotiny ...... 76 5.4 Limitations and future implications ...... 78 6. References ...... 81 7. Appendices ...... 98 7.1 Characteristics for ageing of Banksia tricuspis cones ...... 99 7.2 Fruit production summary for plants surveyed in 2018 ...... 100 7.3 Total viable seed production per plant surveyed in 2018...... 102

List of tables

Table number and title Page 2.1: Characteristics of the five global MTEs. 8 2.2: Evidence for changes in cone production in MTEs across different 11 climate conditions. 2.3: Evidence for demographic shift in MTEs from a drought event or drying 14 climates. 2.4: Evidence for cone opening across a climatic gradient. 22 2.5: Evidence for cone opening due to drought conditions. 25 3.1: Location and climate data for three locations spanning the study region. 32 3.2: Soil pH and concentrations of major nutrients for the three kwongan 34 substrate types surveyed near Eneabba. 3.3: Summary of studies used to provide a comparison on the seed production 39 of serotinous species near Eneabba. 3.4: Focal species examined in this study, and the nature of previously 40 published reports on their various demographic attributes. 3.5: Locations, substrates and ages (time since fire) of study sites and plots. 43 3.6: Focal species, their fire response strategy and family. 44 3.7: Number of individual plants per species measured for fruit counts at each 46 site (with year of last fire in parentheses) near Eneabba, Western Australia 2018. 4.1: The difference in seed production (%) for a selection of serotinous 56 species surveyed in the past (between 1985 and 2004) and 2018 near Eneabba, Western Australia. 4.2: The cone age for 50% of follicles to open in the Banksia species 63 surveyed near Eneabba, Western Australia. 4.3: The average December SPEI for the vegetation ages of sites surveyed 65 both in the past and 2018 showing the mean difference in SPEI per site and between time frames. 7.1: Cone characteristics that assisted in the aging of Banksia tricuspis cones. 99 7.2: Field data summary of species surveyed near Eneabba, Western Australia 100 in 2018. 7.3: Laboratory data summary, including viable seeds per plant for species 102 surveyed near Eneabba, Western Australia in 2018.

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List of figures

Figure number and title Page 1.1: ‘Interval squeeze’ conceptual model (Enright et al. 2015). 2 1.2: Demographic attributes potentially influenced by demographic shift due 4 to climate change that will be investigated as part of this study. 2.1: Worldwide distribution of MTEs (Keeley et al. 2012). 8 3.1: Map of the SWAFR within Australia, with conservation areas 31 highlighted in grey (Hopper and Gioia 2004). 3.2: Map of approximate location of past data collection sites near Eneabba, 39 Western Australia. 3.3: Map of both past and 2018 study sites near Eneabba, Western Australia. 42 3.4: Different fruit types of serotinous species surveyed. 44 3.5: Eneabba 12 month SPEI from 1960 to 2015 (Global SPEI Database 49 2017). 4.1: Changes in the mean number of viable seeds per plant in Myrtaceae 54 species from 1986-2004 (blue) compared to 2018 (pink) for a selection of serotinous species surveyed near Eneabba, Western Australia. 4.2: Changes in the mean number of viable seeds per plant in Proteaceae 55 species from 1986-2004 (blue) compared to 2018 (pink) for a selection of serotinous species surveyed near Eneabba, Western Australia 4.3: Changes in the fruits produced per plant in 1987-2004 (blue) compared 58 to 2018 (pink) for a selection of serotinous species on sandy substrates surveyed near Eneabba, Western Australia. 4.4: Changes in the percentage of viable seeds between 1987-2001 (blue) 60 compared to 2018 (pink) for a selection of serotinous species surveyed on sandy substrates near Eneabba, Western Australia. 4.5: Changes in the percentage of viable seeds from 1985 to 2018 for 61 different aged Banksia tricuspis cones surveyed at Lesueur National Park, Western Australia. 4.6: Changes in the rate of cone opening for Banksia species surveyed in the 62 region surrounding Eneabba, Western Australia. 4.7: Scatterplot of the percent difference in SPEI and viable seeds per plant 65 production between the time frames surveyed.

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Acknowledgments

First and foremost, I would like to thank my supervisors Neal Enright and Joe Fontaine.

Neal, I am so grateful for your willingness to have me come to work with you on such an exciting project. I am thankful to you for organising Joe to be co-supervisor on this project as well. You have both been consistently extremely encouraging, patient and full of guidance, and for this I am very grateful for. Your passion for the conservation of plants is truly inspiring and I’m privileged to have been able to work with you both on such an exciting project. Thanks must also be extended to Joe, for his help and guidance in data analysis. Without either of you this project would not have been possible.

A massive thank you also must go out to field assistant Billi Veber, without her data for this project would have been much harder to collect. Thank you for your patience in plant I.D., and for being a great friend and source of encouragement this entire year.

Additionally, to the volunteers who assisted with both field and laboratory work, I am extremely grateful for the work you all put in even when the task was repetitive. You all cut down my work hours substantially. I am also grateful for the friendships that have formed, and the encouragement you have all provided.

Thanks must also be extended to the Environmental Science technical staff for their assistance in laboratory work and sample processing.

I would like to thank my friends and family, particularly my dad for supporting my move to WA, but also for the constant source of encouragement and motivation.

Finally, I would like to thank Murdoch University, the Chevron Harry Butler Institute and Men of the Trees/Fyfe Family for financial support, and the Department of

Biodiversity, Conservation and Attractions for allowing me to carry out this research.

1. Introduction

The relationship between fires, climate change and the persistence of vegetation is a complex one regardless of the ecosystem or vegetation community. Under a changing climate, where many parts of the world are expected to become warmer and drier, flora and fauna will be affected and respond in myriad ways. Of particular concern for woody plant species in biodiverse Mediterranean-type ecosystems (MTEs) is the possibility of

‘Interval squeeze’ as climate conditions, and associated fire regimes, potentially become more unfavourable to the persistence of many plant species (IPCC 2018; Enright et al.

2015; Figure 1.1). ‘Interval squeeze’ is made up of three interacting factors;

‘demographic shift’, ‘post-fire recruitment shift’ and a ‘fire interval shift’, which together are projected to reduce the window of time relative to recurrence of fire within which a species is able to self-replace and persist (Enright et al. 2015; Figure 1.1).

‘Demographic shift’ refers to changes in established plant demography due to a warming and drying climate, such as plants taking longer to reach reproductive maturity, producing fewer seeds and/or increased levels of mortality (Allen et al. 2010;

Enright et al. 2015). The second factor is ‘post-fire recruitment shift’, where an increasing probability of warmer, drier conditions after fire will create conditions unfavourable for seedling recruitment (Enright et al 2015). Finally, a ‘fire interval shift’ is likely as fires become more frequent due to a warmer and drier climate (Finnigan et al. 2009; Mortiz et al. 2012; Enright et al. 2015). Species may be killed or damaged by fire prior to reaching reproductive maturity or having developed a sufficient viable seed bank for self-replacement, leading to reduced (or no) recruitment (Enright et al. 2015).

Combined, these three factors will cause an ‘interval squeeze’ where the window of time between successive fires available for a plant to reach reproductive maturity will be squeezed, causing the potential extinction of many plant species globally (Enright et al. 2015; Figure 1.1). 1

Figure 1.1: ‘Interval squeeze’ conceptual model (Enright et al. 2015). A significant threat to natural environments and a key factor in the ‘interval squeeze’ model is climate change due to anthropogenic activities (IPCC 2018; Thomas et al.

2004). Changes in atmospheric concentration of greenhouse gases is driving alterations to weather patterns including more frequent extreme weather events such as floods, droughts and consequently fires (IPCC 2018). In relation to long-term average change, large parts of the world have experienced warming whilst precipitation is more varied across regions (Diffenbaugh & Field 2013; IPCC 2018).

Of particular concern in the face of climate change are MTEs which are characterised by cool wet winters and hot dry summers with frequent fire recurrence (Keeley et al.

2012). MTEs have high species diversity and endemism (Cowling et al. 1996; Myers et al. 2000) which is part of the reason they have been identified as one of the most at risk vegetation communities to climate change (Thomas et al. 2004). Under current climate change projections, MTEs are likely to experience a reduction in rainfall, increasing

2 temperatures and more frequent fires (Moritz et al. 2012; Diffenbaugh and Field 2013;

IPCC 2018).

Fire is a key disturbance in MTEs (Keeley et al. 2012; Moritz et al. 2012), and plants have evolved strategies to encourage their survival after a fire (Pausas & Keeley 2009).

These include re-sprouting (fire surviving species) after a fire from epicormic buds or lignotubers (Bradstock et al. 2012; Clarke et al. 2013b), which promotes the resistance of a species to disturbances (Enright et al. 2014). Alternatively, non-sprouters (fire killed species) persist after a fire through recruitment from seeds (Lamont et al. 1991;

Enright et al. 2014). Seeds are stored either in a soil seed bank or in the canopy of the plant (serotiny; Lamont et al. 1991). Serotiny is rare in most ecosystems but is a dominant feature of fire prone vegetation in MTEs, especially in Australia where seeds are stored in woody fruits in the canopy for extended periods of time until seed release is cued by fire (Lamont et al. 1991). Through studying serotinous species, due to the retention of their fruits they show changes in seed demography and storage that may be occurring due to climate change, thus serving as a climate change indicator. Changes in seed production could be occurring in a variety of plant species globally, but due to serotinous plants holding their seeds for multiple years, they provide a rare opportunity to access demography at any stage of a plants life, which is unmatched in most other plant species.

1.1 Thesis objectives Given potential impacts in demographic attributes of plants as climate changes, this thesis aims to quantify changes in seed demography for a selection of serotinous plants in Western Australia, testing the ‘demographic shift’ component of the ‘interval squeeze’ model. Some evidence for demographic shift has already been identified in the serotinous shrub Banksia hookeriana in Western Australia, where between 1987 and

2012, for plants surveyed at the same age and same site, there has been a 50% reduction

3 in the number of cones produced (Enright et al. 2015). This study builds on this work.

Through using a collection of other published works by comparing past and present data collected between 1983 and 2018, evidence of changes in plant demography due to climate change can be evaluated. Changes in total viable seed production can be influenced by various demographic attributes, including fruit production, premature opening of fruits, and seed viability (Figure 1.2), which will be evaluated in this study.

Globally, few studies have been conducted investigating the impact of climate change on seed production and storage in serotinous plants. This thesis aims to determine if the seed production change observed in B. hookeriana is occurring for other serotinous species in the MTE shrublands surrounding Eneabba, Western Australia due to climate change.

Figure 1.2: Demographic attributes potentially influenced by demographic shift due to climate change that will be investigated as part of this study. This study addresses the following research questions and tests the associated hypotheses with expected hypotheses posited by the interval squeeze model of Enright et al. (2015):

1. How has seed production and associated attributes changed in a selection of

serotinous species in Western Australia due to climate change?

Null Hypothesis

a) Seed, fruit production and viability have not changed over time in

response to a change in climate conditions.

2. How has the opening rate of Banksia cones changed along a climatic gradient

and over time?

Null Hypothesis

a) Cone opening is not occurring at younger cone ages than that in the

past.

b) Seeds will not be released at a younger cone age at sites with

different climate conditions

1.2 Thesis structure This thesis is presented in the following five chapters, followed by references and appendices:

Chapter 1: Introduction: A brief outline of the importance of MTEs for

plant diversity, along with the potential impacts of climate change and

fire regime change on these vegetation communities is provided, along

with research aims and associated hypotheses.

Chapter 2: Literature review: Evidence for changes in seed production

and seed storage in MTEs across the world is presented and reviewed.

Drought and different climatic gradients are investigated to assist in

understanding how climate change is likely impacting demographic

attributes in serotinous species in MTEs.

Chapter 3: Methods: Outlines the steps taken to analyse past and current evidence for changes in seed production and storage in serotinous plants in Western Australia. Descriptions of the study area, species, and site characteristics are provided to further develop an understanding of the role of serotinous species and the factors that influence their occurrence in the study region.

Chapter 4: Results: Evaluates the results of this study, particularly changes in seed production and mechanisms that may influence this over time for all species with available past data. Evidence for changes in rates at which cones are opening and changes in climate conditions across the region are also analysed.

Chapter 5: Discussion: Interprets the findings of this study and places it within the context of the current literature, as well as aiming to help determine why potential changes may occur, and the role climate change will have in this. Limitations of this study and potential future research directions are also discussed.

2. Literature review: Assessing evidence for demographic shift in MTEs

2.1 Introduction Warmer and drier conditions, coupled with more frequent fire are expected to affect

MTEs globally, with the interval squeeze model (Enright et al. 2015) highlighting this.

Anthropogenic activities since the 1750s have seen increases of 40%, 20% and 150% in atmospheric carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) respectively, which is driving alterations to weather patterns including more frequent extreme weather events such as floods, droughts and consequently fires (IPCC 2013). In relation to long-term average change, there has been a consistent increase in global average temperatures with a 0.72C rise between 1951 and 2012, whilst shifts in precipitation have varied across regions (IPCC 2013; Australian Bureau of Meteorology

2018). Further increases in temperature and rainfall variability are expected globally, impacting flora and fauna in diverse ways.

Whilst restricted to only five regions globally making up less than 5% of the earth’s surface (Figure 2.1), MTEs have a total plant diversity of around 47,000 species comprising approximately 20% of total global floral diversity with high rates of endemism (Cowling et al. 1996; Myers et al. 2000; Table 2.1). This extremely high plant diversity, coupled with a changing climate makes MTEs one of the most at risk ecosystems from climate change, with understanding of associated impacts essential for their management (Thomas et al. 2004).

Figure 2.1: Worldwide distribution of Mediterranean Type Ecosystems (Keeley et al. 2012).

Table 2.1: Characteristics of the five global Mediterranean Type Ecosystems.

Western South Mediterranean United States Africa Basin Australia Central Chile

Vegetation chaparral fynbos, matorral, kwongan, matorral types a renosterveld maquis, mallee garrigue

Total 98 575 68 175 1 286 950 430 875 111 725 vegetated area (km2) a

Natural fire 40-60 10-20 20-50 10-15 Fire free frequency (years) b

Native flora 4300 8550 25000 8000 2400 taxa b

Dominant Anacardiaceae Asteraceae Anacardiaceae Asteraceae Anacardiaceae plant Ericaceae Ericaceae Cistaceae Ericaceae Asteraceae families a Fabaceae Fabaceae Ericaceae Fabaceae Fabaceae Fagaceae Proteaceae Fabaceae Myrtaceae Fagaceae Rhamnaceae Rhamnaceae Fagaceae Proteaceae Lauraceae Rosaceae Rutaceae Rhamnaceae Rhamnaceae a= from Keeley et al. (2012) b= Kruger (1983), Fox (1994) and Hobbs et al. (1995) 2.2 Objectives and scope This review will focus on the MTEs in the Mediterranean Basin, South Africa, southwestern Australia and the western United States (Figure 2.1). Central Chile is 8 excluded as natural fires are not a major disturbance here (Cowling et al. 1996; Keeley et al. 2012) and shrubs present do not show strong adaptations to fire with little post-fire recruitment (Montenegro et al. 2003). Investigating the ‘demographic shift’ component of the ‘interval squeeze’ model is the aim of this review. It focuses on changes in serotinous fruit/cone stores and factors causing seeds to be released in MTE regions due to climate change or climate related events, i.e. drought. Drought was chosen to be an appropriate phenomenon to investigate as it is already occurring and is likely to become more severe over time (IPCC 2018). Evidence from non-MTE areas is acknowledged to further assist in understanding how serotinous plant demography is impacted under a warmer and drier climate.

Focusing on cone/fruit production and seed release in serotinous species will allow for an investigation into the impacts of climate change on these traits. However, particularly well researched demographic impacts attributed to climate change include an increase in plant mortality (Lloret et al. 2004; Allen 2010; Ruthrof et al. 2015) and changes to seed germination and recruitment success (Fay & Schultz 2009; Walck et al. 2010). For many serotinous genera found in MTEs, increased tree and crown mortality coincided with times of increased temperatures and decreased rainfall (van Mantgem et al. 2009;

Camarero et al. 2015; Ruthrof et al. 2015). However, effects from climate change on seedling germination or dormancy are more varied globally and among genera

(Quintana et al. 2004; Mustart et al. 2012; Enright et al. 2014). Water stress can decrease the chance of successful germination as well as reduce the time seeds are dormant and encourage them to be released prematurely (Walck et al. 2010; Chamorro et al. 2017), suggesting that drier climates may reduce the successful germination and recruitment of species. There is considerable evidence for these phenomena, and as these are not the focus of the research proposed here, the effects of climate change on mortality and seed germination/dormancy will not be focused on.

2.3 Methods for searching the literature To find appropriate evidence, databases ‘Scopus’ and ‘Google Scholar’ were used, as well as looking at the references and citations of relevant papers. To maximise the detection of published evidence, there was not a time restriction on the publishing date of the papers. Search terms used included; ‘drought’, ‘climate change’, ‘seed’, ‘seed bank’, ‘cone’, ‘fecundity’, ‘seed release’, ‘demography’ and ‘Mediterranean’. MTE regions were also used as search terms as well as genera that have a lot of serotinous species within these regions. Serotinous species are at great threat from both changing climatic conditions and fire frequencies, and the analysis of their seed stocks can help to gain an understanding of the impacts of these threats which could be applied to other species across the globe.

2.4.1 Evidence for demographic shift at different sites in MTEs Varying climatic conditions in MTEs allows for the comparison of seed production between sites, assisting in determining how species cope with different climate variables such as rainfall and temperature. From this, potential impacts of climate change can be inferred to assist in understanding how species will respond to a warmer and drier climate (Table 2.2).

Table 2.2 Evidence for changes in cone production in Mediterranean Type Ecosystems across different climate conditions.

Species Location and Climate Findings Evidence

Pinus Yeste and Calasparra, SE Female cones per tree: Gonzalez- halepensis Spain Yeste: 0.23 ± 0.07 Ochoa et Average between 1985 and Calasparra: 0.19 ± 0.17 al. 2004 2005 Yeste: 15.03°C, 498mm Female cones per tree: De Las Calasparra: 16.5°C, 275 mm Yeste: 4.69 ± 2.70 Heras et al. Calasparra: 0.47 ± 0.19 2007

Female cones per tree: Moya et al. Yeste=0.3 2008a Calasparra=0.1

Six sites in SE Spain No consistent trend in female cone Moya et al. Average rainfall/yr from 1984 production between sites 2008b and 2004=798 mm in Cardona Cardona vs Calasparra= 0.4 vs 0.3 Yeste (most mesic site), 290 mm in vs Calasparra= 2.3 vs 0.4 Calasparra (most xeric site)

Tunisia, northern Africa A decrease in total cone production as Ayari et al. Annual averages for 1997- climate became more xeric 2011 2006 of 486 mm and 17.7°C Variable cone production between sites, Ayari et al. decreasing as sites became more xeric. 2012 Rainfall influenced cone production at the most mesic and xeric sites

Banksia Western Australia Cone production was lower in xeric site Lamont et menziesii Xeric site=506 mm/yr, mean than mesic site(13.8 vs 24 cones per al. 1994 max temp for summer=38.8°C plant) Mesic site=639 mm/yr, mean Seed production was higher at xeric site max temp for summer 29.8°C than mesic site (140.7 vs 112.3 seeds per plant)

A dominant obligate seeding (i.e. fire-killed, depends on seeds for post-fire recruitment)

serotinous conifer in the Mediterranean Basin is Pinus halepensis Mill. (Aleppo Pine).

It is fast-growing, reaching reproductive maturity when the plant is around five years of

age (Tapias et al. 2001) and relatively resistant to drought due to its deep and

asymmetrical root system able to reach unevenly distributed water and nutrients

(Ganatsas & Spanos 2005). A large landscape-scale fire in 1994 in south-eastern Spain

prompted a number of scientists to examine cone stocks of P. halepensis in the region,

specifically sites around Yeste, in a dry Mediterranean climate area, and Calasparra, in a

11 semi-arid Mediterranean climate area, 70km from each other (Moya et al. 2008a).

Studies were conducted into the effects of forest thinning and cone production at the two sites, but all studies had a control plot which was chosen to be analysed to reflect the least manipulated stands of forest.

Investigating the effects of forestry on cone production, it was mostly found that cone production was higher at sites in more mesic conditions, however, this trend was not consistent across studies (Table 2.2). Gonzalez-Ochoa et al. (2004), De Las Heras et al.

(2007) and Moya et al. (2008a) all found a higher number of female cones per tree at

Yeste, the more mesic site, than Calasparra (Table 2.2). The number of female cones per tree at Calasparra was similar across three studies (0.19 vs 0.47 vs 0.1; Gonzalez-Ochoa et al. 2004; De Las Heras et al. 2007; Moya et al. 2008a) but there was over 10 times higher count of cones per tree at Yeste observed by De Las Heras et al. (2007) (4.69 vs

0.3 & 0.23; Table 2.2). Sites were sampled at different years and seasons which could account for some of the variance, and these findings would begin to suggest that cone production is higher at wetter and cooler sites.

In comparison, Moya et al. (2008b) investigated six sites including Yeste and

Calasparra, along a climatic gradient increasing in aridity and found no consistent trend between cone production and climatic variation. But, between the most mesic and xeric site, cone production was slightly higher at the more mesic site (0.4 vs 0.3 female cones respectively), and between Yeste and Calasparra (2.3 vs 0.4 female cones respectively;

Table 2.2), following the observed trend of more cones being produced in more mesic sites.

Furthermore, P. halepensis is the most widely spread pine tree in Tunisia and Algeria in northern Africa. Across multiple sites in Tunisia, P. halepensis cone production was greater at mesic sites than xeric sites (Ayari et al. 2011; Ayari et al. 2012; Table 2.2).

However, shade, basal area and tree height all had significant effects on cone production for all sites, with rainfall only being significant in influencing cone production at the most mesic and xeric sites (Ayari et al. 2012). This may assist in explaining the differences between sites in Moya et al. (2008b) and significant increase of cones found by De Las Heras et al. (2007). However, as these variables were not analysed, it is hard to determine how much these factors accounted for the variability. As the climate becomes warmer, and rainfall decreases, climates at all sites are likely to become more xeric so cone production may reduce. Plants at the most xeric ends of their distribution may have very high rates or complete cone production failure as these sites become more aridic in their conditions which plants may be unable to adapt to.

The MTEs in southwestern Australia have the highest diversity of serotinous plants globally (Lamont et al. 1991; Keeley et al. 2012). Nevertheless, there are few studies looking at seed production and storage in relation to climate and climate change. One of the dominant plant genera found in Australian MTEs is Banksia. Banksia menziesii R.

Br cone production between road verge and non-road verge sites in two locations with varying climates was analysed (Lamont et al. 1994; Table 2.2). For plants at least 50 m from the road, more cones were produced in the mesic than xeric sites but despite this, the closed follicles per cone count was higher in the more xeric site than the mesic site contributing to a higher seed count at the xeric site (Lamont et al. 1994; Table 2.2). This is similar to findings for P. halepensis regarding cone production, suggesting that water availability plays an important role in cone production in serotinous species (Table 2.2).

Assessing if this trend continues due to a drought event is explored below.

2.4.2 Evidence for demographic shift in response to a drought event in MTEs As droughts become more frequent and longer due to climate change (Dai 2011; IPCC

2018), changes in seed production in plants is likely (Enright et al. 2015; Table 2.3). 13

MTEs are at significant risk of enhanced drought-like conditions in the future from climate change and understanding the effects that drought events have on seed production will assist in ensuring that at risk vegetation communities are managed effectively.

Table 2.3 Evidence for demographic shift in Mediterranean Type Ecosystems from a drought event or drying climates.

Species Location and study Findings Evidence details

Pinus Anoia, NE Spain There was a reduced production of Espelta et al. halepensis Drought in 2005/06 female cones during times of 2011 drought (2.9 vs 10.3)

SE France Production of cones decreased Girard et al. Cooler year in 1999, during times of low annual 2012 drought in 2005 temperatures and drought

Pinus contorta British Columbia Temperature and rainfall were the Lew et al. Many sites over a large climatic variables which had the 2017 region greatest influence on cone production

26 serotinous South Africa Seed stock numbers were mostly Treurnicht et Leucadendron Many sites over a large driven by fire intervals, not al. 2016 and Protea region climatic variables species

Banksia Western Australia Cones per plant reduced: 1987 Enright et al. hookeriana Studies from 1987 to (42.1 ± 7.2), 1994 (4.1 ± 0.3), 2015 2012. 2004 (14.9 ± 0.1) and 2012 (1.9 ± Rainfall reduced by 20% 0.1) since the 1960s and Seeds per plant reduced over time: temperature increase of 16yr old plant:1987 (371), 1994 0.15°C per decade (50), 7yr old plant: 2004 (153) and 2012 (24)

Studies of Pinus halepensis in northern Spain (Espelta et al. 2011) and France (Girard et al. 2012) found that lower cone production coincided with times of less than average rainfall (Table 2.3). During a drought in 2005/6 in Spain, average cone production was

2.9 cones per tree, compared to 10.3 cones during years of average rainfall (Espelta et al. 2011). These findings were consistent with studies in France where cone production decreased during years of less rainfall (0.08 vs 0.34 cones per year; Girard et al. 2012).

Potential reasons for the significant differences between cone numbers at the two sites 14 could be climatic conditions, severity of drought as well as different stand ages when surveying occurred (19 vs 58 years). Similarly, Pinus contorta Douglas ex Louden

(lodgepole pine), a dominant serotinous pine in California in the south and British

Columbia in the north (Koch 1996), was found to have precipitation and temperature influence the production of cones (Lew et al. 2017; Table 2.3). Five year mean precipitation, ten year mean precipitation, five year summer precipitation and five year mean surface temperature were the climatic variables that had the strongest relationship on cone production at all sites, but the extent of this was highly variable (Lew et al.

2017). For example, it was found at one site that increased summer surface temperature led to a significant reduction in seed production (r=-0.62; p=0.01) whilst the relationship with mean winter surface temperature was not as strong (r=-0.51, p=0.03;

Lew et al. 2017). This makes it difficult to draw conclusions on the specific effect of these variables on cone production but shows a similar trend that rainfall effects cone production.

In Western Australia, between the 1960s and 2010, there was a 20% reduction in rainfall and 0.15°C increase in temperature per decade (Eneabba Climate Station No.

08225; Australian Bureau of Meteorology). Between 1987 and 2012, there has been a substantial reduction in both the total cones per plant and the estimated seeds per plant for Banksia hookeriana Meissner (Enright et al. 2015; Table 2.3) likely to be due to a drier and warmer climate, which again reinforces the effect of climate variables on cone and seed production. In South Africa, Treurnicht et al. (2016) investigated the seed stocks of serotinous Leucadendron and Protea species to determine the factors that have the greatest influence on seed production. Across all species, seed numbers were mostly driven by fire intervals rather than climatic factors (r2=0.33 vs r2=0.07 respectively;

Treurnicht et al. 2016). Soil moisture stress, heat and frost days were observed to affect the seed output of 38% of the species sampled, with soil moisture stress and heat having

15 mostly negative effects on fecundity (Treurnicht et al. 2016). This highlights the importance of rainfall and temperature for cone/seed production, but also suggests that fire intervals, which are likely to become shorter, have the most significant effect on seed production for serotinous MTE species.

As climatic conditions become more unfavourable in MTEs due to a hotter and drier climate, understanding which has a greater effect on seed production; climate change or fire regimes, would be beneficial in ensuring appropriate management of species. The impact of climate change and fire intervals on plant demography further complicates the management of serotinous species.

2.4.3 Evidence for demographic shift in non-MTEs For serotinous species in non-MTE regions, similar observations have been made regarding seed production under drier climates. Climate in Arkansas, Mississippi and

Lousiana in the south-eastern United States is similar, but cooler than that of the west coast near California. Pinus taeda L. is a common serotinous species in this region, and seed production in the south-eastern US was investigated focussing on how precipitation affected seed production over multiple years (Cain & Shelton 2000). It was found that amount of precipitation during late summer and early autumn influenced seed production, and in general it was a positive trend (p<0.1, r=0.6) (Cain & Shelton

2000). Across all sites, there was also a negative trend observed between summer temperatures and seed production, with increases in temperature leading to a reduction in cone and seed production (p=0.05, r=-0.35) (Cain & Shelton 2000). These results are comparable with those of Pinus halepensis and Banksia hookeriana as reduced cone production coincided with a reduction in rainfall.

2.5 The effects of enhanced CO2 on cone production Climate change has been attributed to significant increases in the amounts of greenhouse gases in the atmosphere, one of these being CO2 (IPCC 2018). As CO2 is

16 essential for the growth of plants through photosynthesis, understanding the effects of enhanced CO2 on plants is useful to ensure appropriate management under a drying and warming climate. Studies have been conducted of enhanced CO2 on Pinus taeda on the east coast of North America. LaDeau & Clarke (2001) and Way et al. (2010) set up two

-1 plots in P. taeda stands, a control plot and one with 200µL L CO2 increase above ambient concentration. Data were collected on stands that were 19 years old and had

been exposed to enhanced CO2 levels for three years (LeDeau & Clarke 2001). Trees in

the CO2 enhanced plot produced three times as many cones and seeds, and began cone production when trees were at a smaller diameter than those in the control plots

(LaDeau & Clarke 2001). Way et al. (2010) reported similar findings. In all years of

measurement, cone production was higher in the elevated CO2 plots than the control

plots (Way et al. 2010). They also found that when moisture stressed, elevated CO2 plots still produced more seeds than the control plots (Way et al. 2010). This suggests

that enhanced CO2 can account for greater seed production than water stress (p<0.001

and p=0.03 respectively), which may be due to the CO2 increasing water use efficiency and buffering against the moisture stress (Way et al. 2010). Similar findings have been found in grassland species although in those cases seed quality and size reduced

(Jackson et al 1994; Huxman et al. 1999). This suggests that the excess CO2 is being used to generate more seeds of high quality in Pinus taeda rather than producing smaller and poorer quality seeds in non-woody species such as grasses (Jackson et al

1994; Huxman et al. 1999). Under this scenario, it may be inferred that woody species such as Pinus may better respond to enhanced CO2 but further studies would need to be conducted to confirm this. Studies using enhanced CO2 treatment allow for an understanding of the effects of CO2 on plants, particularly seed production, and suggests that water stress accounts for greater reduction in seed production than CO2 enhancement (Way et al. 2010).

2.6 Factors influencing serotiny and the role of fire regimes Degree of serotiny is a measure of viable seeds retained in fruits with increasing age of fruits (Cowling & Lamont 1985b; Lamont 1991). Many serotinous fruits open and seeds are released when they are burnt by a fire, but other factors can cause premature opening of fruits, e.g. plant/branch death, and a number of plant and fruit age related impacts including plant senescence, and long-term exposure of fruits to heat and drying, all of which lead to the increased loss of seeds in the inter-fire period (Lamont et al.

1991). Fire cued release allows seeds to establish in a suitable post-fire environment, with maximum chance of successful recruitment due to a nutrient-rich ash bed, lack of competition from other plants and lack of predation from granivores (Lamont et al.

1991; Enright et al. 1998). The ability for serotinous plants to keep seeds enclosed in fruits under a changing climate will be essential for their long-term persistence to ensure they have an adequate seed bank during time of recruitment.

It cannot be ignored the importance that fire intervals have on determining degrees of serotiny (Lamont et al. 1991). Drier areas often are subject to more frequent crown fires rather than surface fires, due to shorter plant heights and drier conditions, which highlights the interaction that fire regimes play in determining degrees of serotiny

(Cowling & Lamont 1985b; Battersby et al. 2017). Many serotinous species have traits that can enhance flammability such as oily and dry leaves, loose bark and retention of dead branches (Bradstock et al. 2012) which can assist in ensuring maximum seed release. For example, some species of Banksia such as B. hookeriana and B. leptophylla

A. S. George retain old florets on cones which not only promotes flammability, but also may protect the seeds from the effects of heating and drying slowing the opening of the follicles (Lamont & Cowling 1984). These adaptations help emphasise that serotiny is a fire-adapted trait in fire prone environments.

In terms of evidence for changes in serotiny across a climate gradient, further reductions in rainfall suggests that serotinous species will have increased proportions of closed follicles leading to a greater seed bank size, but this is an evolutionary timescale response and species are unlikely to be able to respond quickly enough in this respect.

Rather, drying and warming is more likely to lead to increased fruit/cone opening and seed release during the inter-fire period as a direct response to the increased effects of heating and drying.

As explored in the interval squeeze model, drier and warmer conditions from climate change are likely to cause unfavourable post-fire recruitment conditions (Meehl &

Tebaldi 2004; Enright et al. 2015) affecting germination success in species characterised by cohort recruitment after fire (Savage et al. 1996, Buma et al. 2013). Additionally, increases in fire frequencies are likely to cause an immaturity risk due to insufficient growing time for plants to develop a viable seed bank prior to the next fire (Westerling et al. 2011). Combined, these factors may threaten a plant species persistence, and increased release of seeds during the inter-fire period further enhance this threat.

2.6.1 Wet-dry cycles in imitating natural post-fire seed release The relationship between fire, rainfall and drying is significant in ensuring maximum seed release in serotinous species (Cowling & Lamont 1985a). Wet-dry cycles are part of the natural cycle of seed release in highly serotinous Banksia species. The heat of fire scorches cones and causes the follicles to rupture and some, but not all, seeds to be released (George 1981; Cowling & Lamont 1987). Many seeds remain within these ruptured follicles until the onset of winter rains with alternate wetting (from rain) and drying (between rain events) assisting to force seeds out of the follicles as the wings of the separator spread and recurve when drying (Wardrop 1983; Cowling & Lamont

1985a). This assists in ensuring maximum seed release into the post-fire environment for the greatest chance of successful recruitment. Banksia species that had gone through

19 wet-dry cycles released a significantly higher proportion of seeds compared to those that didn’t undergo the treatment highlighting the importance of rainfall after a fire in facilitating seed release (Cowling & Lamont 1985a). In laboratory experiments, wet-dry cycles are a commonly used method to maximise seed release (Lamont & van Leeuwen

1988; Lamont et al. 1994; Cochrane et al. 2016) which reinforces the importance of fire, rainfall and heat for the release of Banksia seeds. Delayed release of seeds allows for maximum potential for recruitment as they are not exposed to the direct heat of the sun or predated by granivores for months prior to germination occurring (Lamont et al.

1991).

2.6.2 Seed morphology across dominant serotinous genera in MTEs Water availability is important for maintaining closed cones and fruits in serotinous species and as climates become drier, there is an increased threat of the premature release of seeds (Causley et al. 2016; Martin-Sanz et al. 2017). Pinus cones are sealed with a resin layer (Cameron 1953), and temperatures between 40-50°C melt this resin so the cones split open and a lowering of moisture causes seed release (Goubitz et al. 2003;

Nathan & Nee’man 2004; Moya et al. 2008c). Protea species have cones with bracts surrounding the seeds and open gradually overtime due to both heat and drying (Rourke

1980). Hakea species have woody fruits with thicker and denser fruit walls in more serotinous species providing greater protection from the heat of fire compared to species with thin walls (Lamont et al. 1991; Lamont & Groom 1998). Again, their fruits will often rupture and release seeds due to a lack of moisture (Enright & Goldblum 1999;

Causley et al. 2016). Seeds of myrtaceous species are small and numerous, and are retained in woody capsules either in clusters or singularly, with a valve which will commonly open when cut off from the plants water supply (Lamont et al. 1991).

Banksia seeds are contained in follicles in a cone and within the follicles there is a

20 woody separator with wings that move together when moistened and spread and recurve when dried (Wardrop 1983; Cowling & Lamont 1985a).

All genera mentioned have the potential for increased seed loss into the inter-fire period during times of moisture stress causing recruitment failure (Richards & Lamont 1996), which is increasingly likely due to climate change (Lamont et al. 1991; Enright et al.

2015). Combined with increasing fire frequencies (Moritz et al. 2012) causing plants to be unable to reach reproductive maturity (Westerling et al. 2011), the availability of seeds at the time of fire is likely to be reduced due to seed loss, which may affect the persistence of plant populations (Enright et al. 2015; Petrie et al. 2017). Species characterised by different types of cones/fruits may vary in their vulnerability to changes in climate, with some losing more seeds during the inter-fire period than others.

This issue is worthy of further investigation, with potential impacts of premature seed release including changes in patterns of species abundance, in dominance hierarchies and even species losses.

2.7.1 Evidence for fruit opening across a climatic gradient There is evidence for changes in degree of serotiny in plants in relation to climate gradients (Table 2.4). Species that occupy a wide range allow for investigations of their demography when exposed to different climate conditions. By looking at populations at xeric ends of their distributions, it can be inferred how a particular species may respond to drier and warmer conditions.

Table 2.4 Evidence for cone opening across a climatic gradient.

Evidence Site location and climate Species Findings

Cowling Western Australia Banksia % of closed 8yr old cones increased and Most mesic site: Kings Park=883 attenuata as sites became more xeric (0 vs 60) Lamont mm/yr, 27.8°C mean max temp Maximum viable cone age increased 1985b Jan as sites became more xeric (4 vs 17 Most xeric site: Northhampton= yrs) 506.7 mm/yr, 33.8°C mean max temp Jan Banksia % of closed 5yr old cones increased prionotes as sites became more xeric (0 vs 45) Maximum viable cone age increased as sites became more xeric (<2 vs 12 yrs)

Banksia % of closed 5yr old cones increased menziesii as sites became more xeric (0 vs 5) Maximum viable cone age increased as sites became more xeric (<2 vs 11 yrs)

Bond South Africa 12 serotinous Plants in wetter areas had a lower 1985 Rainfall varied between 550 and Leucadendron proportion of cones 2> yrs closed 1100 mm/yr and Protea (42%) compared to those from more species xeric sites (66%)

Borchert California, United States Pinus coulteri Mesic site had a higher proportion 1985 Mesic site- Santa Cruz= 794 of open cones per tree than the xeric mm/yr, average summer site (43.4 vs 25%) temp=21°C Xeric site: Santa Ynez=575 mm/yr, average summer temperature=25.7°Cb

Lamont Western Australia Banksia Follicles opened increased as sites et al. Most mesic site= 639 mm/yr, menziesii became more mesic (21.7 vs 65.3%; 1994 mean max temp=29.8°C p=0.0001) Most xeric site= 50 6mm/yr, mean max temp=38.8°C

Moya et Southeast Spain Pinus % of closed cones increased as sites al. 2008b Average rainfall/yr from 1984 halepensis became more xeric (83% vs 98%) and 2004=798 mm in Cardona (most mesic site), 290 mm in Calasparra (most xeric site)

Martin- Central Spain Pinus Mesic site=30% of cones opened at Sanz et Mesic site=475 mm/yr, mean halepensis 10 weeks, xeric site= 7% of cones al. 2017 annual temp=13.6°C open at 10 weeks Xeric site=417 mm/yr, mean annual temp=14.4°C a= National Institute of Water and Atmospheric Research 2003 b=US Climate Data 2018 As sites become more xeric, within SW Australian serotinous species’ geographic ranges, the proportion of closed follicles increases. In Banksia attenuata R.Br. (strongly

22 serotinous), B. prionotes Lindley (weakly serotinous) and B. menziesii (Lamont et al.

1994; Hanley & Lamont 2001) both the degree of serotiny and the maximum age of cones with viable seeds increases along a south to north gradient of decreasing rainfall

(Cowling and Lamont 1985b; Table 2.4). However, across a drying climatic gradient moving inland from Perth, B. attenuata does not show this increasing degree of serotiny as sites become more xeric due to fire intervals not changing (pers.comm. Enright N.

2018). This makes it difficult to determine how climate influences degree of serotiny in

B. attenuata, but other factors impacting this may include fire regimes. Banksia menziesii showed a higher degree of serotiny in more xeric sites, with 65.3% of follicles opened at the most mesic site, as opposed to 21.7% in the most xeric site (p<0.001;

Lamont et al. 1994; Table 2.4) reinforcing that rainfall availability likely does influence degrees of serotiny.

Similarly, Pinus coulteri D. Don in California (Borchert 1985) and P. halepensis in

Spain (Martin-Sanz et al. 2017) showed increased proportion of closed cones as sites became more xeric (Table 2.4), suggesting that for these species, serotiny is related to rainfall, with drier sites showing higher degrees of serotiny. In South Africa, six

Leucadendron and six Protea species were analysed in different regions investigating climatic variability on seed stocks (Bond 1985). However, this study did not provide a comparison within the same species at different regions, so it cannot be inferred how a particular species responds under different climate variables. Plants from cooler areas had a lower degree of serotiny with cones two years and older only contributing to 42% of the total cone reserves, as opposed to 66% in more xeric conditions (Bond 1985).

This suggests, like Cowling & Lamont (1985b), that plants in xeric sites store more seeds as their fruits remained closed for longer, but as varying climate conditions for the same species was not analysed, it is difficult to determine how much of this variation is

23 attributed to climatic variables as plants were from different species with varying ages and life histories.

2.7.2 Evidence for fruit opening in response to drought The effects of drying can lead to the premature opening of serotinous fruits (Lamont et al. 1991) so there is potential for seeds to be released during times of drought. When exposed to short but extremely hot and dry weather events (Sharav events) in Israel,

Pinus halepensis released a higher proportion of seeds compared to moderate and non-

Sharav events (Nathan et al. 1999; Table 2.5). Similarly, in Spain P. halepensis showed an increase in the proportion of cones opening in drought years (18.5%) than in average weather years (4.5%; Espelta et al. 2011; Table 2.5). When cones were detached from plants, severing any possible xylem water flow, there was a higher proportion of open cones after 15 weeks (13% of cones) compared to those attached to the plant (4% of cones; Martin-Sanz et al. 2017).

Table 2.5 Evidence for cone opening due to drought conditions.

Evidence Site and climate conditions Species Findings

Nathan et Israel Pinus Greater seed release al. 1999 Mean rainfall 600 mm/yr, mean hottest halepensis corresponded with more temp 24-26°C severe Sharav events Severe Sharav events: >35°C, <30% (severe Sharav=32.6, relative humidity moderate Sharav=25.1, Moderate Sharav events: >30°C, <35% non-Sharav=2.7 seeds/day) relative humidity

Espelta et North-eastern Spain Pinus More cones opened in the al. 2011 Mean rainfall (2004-2008)=549 mm/yr halepensis drought year (12.1±3.4%) Mean summer temp= 22.36°C and the following year Drought year in 2005 of 202 mm/yr and (25±6.1%) vs non-drought mean summer temp=23.1°C year (average 3.3%)

Causley et Western Australia Banksia Average % of follicles al. 2016 Mean rainfall=489 mm/yr, average attenuata open: control=37.4±23.3, summer temp of 28°C drought=46.5±13.4 Drought was simulated by a branch with more than five cones/fruits was severed Banksia Average % of follicles and then re-attached to the plant carlinoides open: control=8.8±9.0, drought=0.9±2.7

Banksia Average % of follicles hookeriana open: control=10, drought=15

Hakea Average % of follicles psilorrhyncha open: control=10.3±21.0, drought=96.2±8.7

Simulated drought in cones severed from plants was tested in Banksia attenuata, B. carlinoides Meissner (formerly Dryandra carlinoides), B. hookeriana and Hakea psilorrhyncha R. M. Barker (Causley et al. 2016). For all species, a higher proportion of opened follicles was observed in the drought group than control group, except for B. carlinoides which showed the opposite (Causley et al. 2016; Table 2.5). For B. carlinoides, the control group location may not have been ideal conditions for cones to stay closed due to higher moisture availability. The other highly serotinous species, B. hookeriana, had the smallest increase in all Banksia species studied between control and drought induced follicle opening (10% vs 15%; Causley et al. 2016; Table 2.5). This suggests that the more serotinous the plant, the longer the follicles will stay closed and thus contain viable seeds. However, Hakea psilorrhyncha had almost complete follicle 25 opening when detached from a water supply (10.3%±21.0% vs 96.2±8.7% open follicles; Table 2.5) which suggests, that under enhanced drying conditions where water availability is restricted, these fruits will likely open and release seeds into the inter-fire period where there will be minimal successful recruitment (Causley et al. 2016). This suggests that a lack of moisture is a significant driver for cones to open (Espelta et al.

2011; Causley et al. 2016; Martin-Sanz et al. 2017).

However, it is important to note the role that fire intervals play in determining when fruits begin to open, as it has been observed when plants begin to senesce, which can occur when a plant has not been burnt for an extended period of time, a greater proportion of fruits open (Enright & Goldblum 1999; Witkowski et al. 1991). This may coincide with times when plants are allocating less moisture to older cones, so they may be opening from a lack of water availability (Enright & Goldblum 1999). Again, this reinforces the importance of moisture availability in maintaining closed cones and further complicates the relationship between levels of serotiny, climate change and fire regimes.

2.7.3 Evidence for fruit opening in non-MTE regions In non-MTE regions, degrees of serotiny are influenced by climate variables and fire intervals, like that of MTEs and show a similar trend. In New Zealand, total annual rainfall in summer was found to be negatively correlated with serotiny (ρ =-0.299, p<0.001) in Leptospermum scoparium J.R. Forst. & G. Forst (Battersby et al. 2017). In drier and warmer conditions, there was a higher proportion of closed cones than that of more mesic conditions (90% vs 10% of cones closed; Battersby et al. 2017). Areas in the North Island where fires are sporadic had a higher proportion of closed serotinous cones, than those in the South Island where fires are sparse due to open vegetation and cooler climates (Battersby et al. 2017). Banksia serrata L. F. found along the east coast of Australia in a temperate climate was found to have increased proportion of closed

26 cones in sites that were more inland than coastal (30% vs 5% cones open), and temperature and rainfall were not determined to affect this correlation (Whelan 1988).

Similarly, there is evidence for lower serotiny in Pinus rigida Mill. (north-eastern US;

Givnish 1981) and Pinus torreyana Parry ex Carrière (southern US; McMaster and

Zedler 1981) in coastal sites. This may be due to different fire types in some coastal areas characterised by lower intensity surface fires with longer fire intervals and fewer crown fires (McMaster & Zedley 1988; Whelan et al 1988). Once again, this highlights the importance of fire regimes in determining degrees of serotiny and suggests that globally, degree of serotiny is characterised by warmer temperatures and fire intervals.

2.8 Synthesis of findings and future directions There is large variation in the effects of a drying climate on cone and seed production in

MTEs globally. Evidence from a number of species and regions suggest that lower cone production coincides with times of reduced rainfall. This is likely to be problematic for species as the climate becomes more xeric and fires become more frequent (IPCC 2018) which may lead to their failed persistence (Enright et al. 2015). For Banksias, it appears that rainfall likely has a significant effect on the proportion of closed follicles but depending on the species, this is either a negative or positive effect. Degree of serotiny seems to be a crucial factor in determining this effect which is significantly influenced by fire intervals (Cowling & Lamont 1985b; Pausas et al. 2004). Again, this highlights the important relationship between fire intervals, climate change and degree of serotiny.

However, Hakea species in Australia showed almost complete fruit opening from moisture stress (Causley et al. 2016) which may be critical in determining the persistence of these species in a warmer and drier climate and highlights the necessity of fire regimes in ensuring this species has a viable seed stock. From this, it may be suggested that Hakeas are most at risk of recruitment failure due to climate change as almost all seeds are released in the inter-fire period when exposed to a restricted water

27 supply where successful recruitment is low. Similarly, studies in South Africa by

Treurnitche et al. (2016) found soil moisture stress, heat and frost days affected the seed output of 38% of species, with no consistent trend and these factors are likely to influence cone production in other serotinous species globally.

For MTEs in general, it seems that rainfall does play a role in determining the number of cones and seeds produced, but the effects of this are variable between species.

Increased cone production seems to coincide with increases in rainfall but the influence of this is not significant across all species. Due to this variation in findings, it is difficult to form a general conclusion on what is likely to happen for species in these regions as the climate becomes more warmer and drier, and experiences more frequent fires. Furthermore, as effects of climatic variables was different across species and regions, this highlights the importance of other non-climate related factors influencing cone production and opening such as plant community structure or fire frequencies.

Knowing the role these factors play will be crucial in understanding the complexities of a vegetation community and how to best manage it.

Between studies, there are inconsistent methodologies and outcomes which make it difficult to identify a clear overall trend for changes in seed production due to climate change in MTEs. This highlights large knowledge gaps in this area of study and the urgency for studies quantifying the effects of climate change on serotinous species. For the variety of serotinous genera and species in MTEs globally, it is concerning that only a small proportion of these have been investigated. On an ecosystem scale, it is therefore difficult to determine how a vegetation community will be affected by climate change in terms of demography. Studies that compare present with past data where substantial climatic changes have occurred would be beneficial. Also, studies where predicted future climate scenarios are used to show changes in seed stocks would also be useful. In doing so, knowledge will be gained on what is happening to the seed 28 stocks of serotinous woody species in these regions, some of which are threatened. This can assist to ensure that appropriate management strategies are implemented to promote their persistence in the future.

Through comparing upon data collected between 1983 and 2004, seed stocks and storage for a selection of species from Myrtaceae and Proteaceae families from the southwest biodiversity hotspot in Western Australia will be quantified, and viability will be examined. The results from this will provide a valuable understanding in how species have coped with significant changes in climatic conditions and will allow for an understanding of how they may respond in the future when climate conditions become more unfavourable. This evidence will be applicable to serotinous species across the world and provide a baseline for future work to be conducted to quantify seed stock changes and viability in the long-term. Findings will assist in ensuring that appropriate management strategies are conducted sooner, rather than later to help ensure serotinous plant species survival, and help to indentify the most significant threats to their long- term persistence under changing climate and fire regimes.

3. Methods

3.1 Study area The central objective of this study was to evaluate evidence for change in overall canopy seedbank size, as well as potential changes to fruit production, seed viability and accelerated fruit opening rates due to climate change. Evidence of depressed seed production for one species, the fire killed shrub Banksia hookeriana, from 1987-2012 and its relationship with climate was reported by Enright et al. (2015). This study expands on this effort, encompassing a broader range of species and evidence types from the same region.

3.1.1 Location and land use The southwest of Australia is a globally recognized biodiversity hotspot due to its high levels of endemic biodiversity and threats to species persistence from land use changes and climate change (Myers et al. 2000). It is part of the Southwest Australian Floristic

Region (SWAFR) which has around 7,380 species of vascular flora, 49% of these being endemic to the region with 2,500 species of conservation concern (Hopper & Gioia

2004). Part of the only recognized biodiversity hotspot in Australia, the SWAFR occupies approximately 302,627 km2 and extends from Shark Bay in the north, diagonally to the west of Esperance in the south mirroring the Mediterranean climate zone (Figure 3.1; Myers et al. 2000; Hopper & Gioia 2004). Large parts of the region are dominated by heathland type vegetation communities, locally known as ‘kwongan’ which has been defined as a ‘type of country, sandy and is open without timber sized trees but with scrubby vegetation’ (Beard 1976). The kwongan vegetation type corresponds to shrub-dominated systems found in other MTEs (fynbos, chaparral, garrigue and matorral; Keeley et al. 2012). Common land uses include mining, farming, and towns, which have resulted in extensive land clearing (Miller et al. 2007), causing kwongan to be restricted mostly to National Parks, reserves and unallocated crown land

(Coates et al. 2014). The areas of remnant kwongan are well-recognised wildflower tourism hotspots, whilst also being utilised for the exportation of seeds and wildflowers

(Hopper & Lambers 2014).

Figure 3.1: Map of the Southwest Australian Floristic Region with conservation areas highlighted in grey (Hopper & Gioia 2004).

3.1.2 Climate The region is characterised by a Mediterranean-type climate, with hot dry summers and cool wet winters. The study was centred around Eneabba (-29.820574, 115.266418) approximately 270 km north of Perth. Over the last 50 years, Eneabba has had a mean annual temperature of 27.9°C, with January temperatures averaging 36.4°C (Australian

Bureau of Meteorology 2018). Annual rainfall for this time period is approximately 486 mm with almost 70% of this falling between May and August creating drought-like conditions in the summer months (Table 3.1; Australian Bureau of Meteorology 2018).

Eneabba is located in a transitional climate zone of increasing aridity, with climate becoming more xeric the further north travelled. Annual precipitation ranges from 550.9

31 mm south of Eneabba (near Jurien) and decreases to 416.2 mm further north (near

Arena) and sites within this study span this climate gradient (Table 3.1).

Table 3.1: Location and climate data for three locations spanning the study region.

Jurien Eneabba Mt Adams

Latitudea -30.298895 -29.820574 -29.405546

Longitudea 115.042216 115.266418 115.088245

Mean annual rainfall (mm) 550.9b 486.9b 416.2d

Mean maximum temp (°C) 24.9c 27.9c 28.2e

Mean January temp (°C) 30.0c 36.4c 37.3e

Data sourced from the Australian Bureau of Meteorology 2018). a= coordinates of the approximate location of townships b= average 1964-2017 (Jurien and Eneabba climate station) c= average between 1972-2017 (Jurien and Eneabba climate station) d= average between 1980-2017 (Arena climate station) e=average between 1998 and 2017 (Morawa Airport)

Mediterranean-type climates are at high risk from climate change (Keeley et al. 2012) and since the 1970s, climate change has seen a 20% reduction in rainfall and a 0.15°C per decade rise in mean annual temperature at Eneabba (Australian Bureau of

Meteorology 2018). Similar changes have also occurred in Perth, with a decline in rainfall leading to one third less of the average amount of water flowing into the water supply system between 1997 and 2005 than that between 1911 and 1974 (Bates et al.

2008). The changes in rainfall in the region is largely due to a decline in very high rainfall years with less rainfall occurring in winter (England et al. 2006; Bates et al.

2008). Across Australia similar changes in climate are occurring, with the number of hot days and nights increasing leading to a warmer climate for much of the country

(Williams et al. 2009). This increased aridity is likely also to lead to an increase in fire frequency and severity due to drier fuels and lengthened periods of high fire hazard weather (Finnigan et al. 2009; Clarke et al. 2013a).

3.1.3 Geology The study region is located in the northern part of the Swan Coastal Plain and the southern end of the Geraldton Sandplains, Western Australia (Department of the

Environment and Energy 2012). Moving eastwards from the coast, substrates change from limestone, to sandplain and then to laterite, with the Gingin Scarp running approximately north-south marking the boundary between sandplains and laterite substrates (Geological Survey of Western Australia 1990; Enright et al. 2007).

Limestone substrates were formed in the mid to late Pleistocene (less than 2.6M BP) from both aeolian and marine deposits (Playford et al. 1976) and are comprised of unconsolidated sands over Tamala Limestone (Tapsell et al. 2003; Enright et al. 2007).

Large parts of the sandplains are shoreline, marine, beach, lagoon and dune deposits from the early Pleistocene (2.6Ma BP), that are rich in heavy metals including rutile, zircon, and leucoxene (Playford et al. 1976). Laterite areas are composed of high concentrations of iron and aluminium oxides formed in the Miocene (6-23 Ma BP;

Geological Survey of Western Australia 1990).

Limestone substrates are of variable depth with a layer of sand over the limestone of 0-

50 cm deep (Tapsell et al. 2003). The sandplains are made up of an undulating dune swale sequence due to the rate of material deposited exceeding the capacity for this material to be removed (Wyrwoll et al. 2014). During the last glacial maximum

(approximately 20-30 kyBP), Australia had temperatures 4-7°C lower, 50% less rainfall and sea levels 120-150 m lower than present (Krauss et al. 2006; Lambeck & Chappell

2001). This drier climate is likely to have resulted in less vegetation cover, therefore causing the dunes to be more mobile as nothing was present to hold the sand in place

(Krauss et al. 2006). Lateritic substrates are composed of a five-metre thick layer of concentrated iron and aluminium oxides, with a thin sand layer above in some places, and clay below (Hnatiuk & Hopkins 1981; Playford et al. 1976).

Combined with a lack of glaciation, volcanic and mountain building activity for at least the past 50 million years (Geological Survey of Western Australia 1990) long-term weathering has produced substrates with low levels of nutrients and a slightly acidic pH

(Enright et al. 2007; Wyrwoll et al. 2014; Table 3.2). These factors not only influence the vegetation present but are also thought to be some of the main drivers of the high plant species diversity in the region (Hopper & Gioia 2004; Lambers et al. 2010;

Laliberte et al. 2013).

Table 3.2: Soil pH and concentrations of major nutrients for the three kwongan substrate types surveyed near Eneabba.

Exchangeable Total N Total S Colwell P Colwell K Site Soil pH Ca (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Crest 6.2 ± 0.0 8.9 ± 0.3 3.5 ± 0.1 4.0 ± 0.2 2.9 ± 0.1 31.3 ± 1.0 Laterite 6.1 ± 0.0 19.6 ± 0.8 4.8 ± 0.2 4.7 ± 0.2 4.0 ± 0.2 50.8 ± 1.8 Limestone 6.7 ± 0.0 34.1 ± 1.9 7.4 ± 0.3 6.5 ± 0.9 4.0 ± 0.1 55.6 ± 1.7 Data are means ± SE Source: Enright et al. 2007

3.1.4 Topography The region is dominated by sand, laterite and limestone substrates, each with different depths and water availability, which influences the vegetation found. The sandplain is made up of undulating dunes (sands up to 10 m deep) and swales (<1 m deep) with an average of 0.5-1 km between adjacent dunes (Groenveld et al. 2002). The deep sands of dunes act as an effective water store in summer allowing taller and deeper rooted plants to grow, while the shallow sands of swales (underlain by a layer of less permeable clay) have limited water holding capacity (Enright et al. 2007). Unlike the dune, the soil profiles of swales dry out quickly in summer. Lateritic sites generally have a layer of shallow sand on top of them and similarly do not hold water well (Bettenay 1984;

Enright et al. 2007). Limestone sites are covered with sands of variable depths thereby influencing local patterns of water and root penetrability.

All substrates have variable, but generally very low, concentrations of key nutrients due to their weathering and the elements present (Table 3.2) and this, combined with

34 substrate depth and water penetrability influences the vegetation that can grow at these sites (Enright et al. 2007). Taller statured plants (shrubs and small trees) such as

Banksias and Xylomelum generally have deeper roots and are found on dunes, while smaller statured plants (sub-shrubs) occur across all substrates (Bellairs & Bell 1990,

Hnatiuk & Hopkins 1981; Enright et al. 2007). This creates strong differentiation in vegetation structure between dunes, with shrubs and small trees 2 – 4 m high over sub- shrubs to 1 m, and swales, dominated by low sub-shrub layer <1 m in height.

3.1.5 Vegetation

Highly diverse kwongan shrubland vegetation originally covered 27% of the southwest region (Beard 1984). It is dominated by sclerophyll leaved plants, with many species dependent on fire for their survival, whilst also being adapted to dry and nutrient-poor conditions as seen in other MTEs globally (Beard 1976; Cowling et al. 1996; Hopper &

Lambers 2014; Groom & Lamont 2015). Dominant plant families include Proteaceae,

Myrtaceae, Fabaceae, Orchidaceae, Ericaceae and Asteraceae. The high levels of diversity in this region are unmatched in most parts of the world, with 131 plant species having been found within a 1000 m2 plot near Eneabba (Hopkins & Hnatiuk 1981). This plant diversity reflects the evolutionary adaptations to survive in such a hostile environment, as restricted water and nutrient availability may increase diversity by reducing growth rates and competitive exclusion (Groom & Lamont 2015).

Furthermore, climate stability, landscape age, restricted soil nutrients and fire regimes have caused great diversity in survival responses to fire, drought, restricted nutrient availability, pollinators and herbivores (Groom & Lamont 2015).

3.1.6 The role of fire

Like other MTEs fire is a key disturbance factor in the SWAFR, and plants have evolved various responses to help maximise their chance of long-term persistence in a

35 fire prone environment (Keeley et al. 2012; Miller & Dixon 2014). Common fire response strategies in kwongan vegetation are resprouting (i.e. fire surviving) or reseeding/non-sprouting (i.e. fire killed). Through these strategies, resistance (capacity to survive a disturbance, associated with resprouters) and resilience (capacity to recover to pre-disturbance levels through recruitment, associated with non-sprouters) are enhanced to maintain biodiverse ecological communities (Enright et al. 2014).

Non-sprouting and resprouting species have employed various trade-offs in response to fire intervals, growth time and seed production to ensure their persistence. Populations of non-sprouting plants recruit from seed primarily in the first year after fire, and therefore are single aged cohorts, with their times of recruitment and longevity equal to or less than that of the fire interval (Miller & Dixon 2014). Resprouting species on the other hand can survive multiple fires through resprouting epicormically or basally, creating populations comprised of different aged cohorts recruited after successive fires

(Clarke et al. 2013b, Miller & Dixon 2014). Suitable fire intervals are a key factor that influences the persistence of non-sprouters, as under shortened fire intervals, species may not have enough time to form a sufficient seed bank to ensure they are able to self- replace after the next fire (Enright et al. 2014). Fire intervals that are too long may see non-sprouters begin to senesce as they are often faster growing and take a shorter period of time to reach reproductive maturity compared to resprouters (Miller & Dixon 2014).

Additionally, seed production between resprouters and non-sprouters can vary greatly, with non-sprouters generally producing more seeds as this is their only method of persistence, while resprouters can survive multiple fires and do not rely solely on seeds for persistence (Bellairs & Bell 1990; Enright et al. 2007). The resource allocation that high seed production in a relatively short time period takes, may at least partly explain why non-sprouters experience the effects of senescence earlier than resprouters (Pausas et al. 2004). Conversely, resprouting species allocate more resources to ensure their

36 regeneration buds, e.g. lignotubers (an underground storage of starch and other nutrients they resprout from after fire) are maintained (Pate et al. 1990; Bond & Midgley 2001;

Clarke et al. 2013b). These trade-offs allow for a vegetation community that is rich in both non-sprouting and resprouting species.

Fire has been a major disturbance type in the region since the Pliocene (2.5-3.6Ma BP;

Hassell & Dodson 2003), with natural fires being caused by lightning (Bell 2001). It is difficult to determine how often fires occurred prior to human arrival, but a dry and warm climate, combined with fire adapted vegetation suggests that fires were more frequent in kwongan than in many other Australian plant communities (Abbot 2003).

Fires have likely become more frequent in the presence of Indigenous Australians, but details of any altered fire regime are difficult to confirm (Enright et al. 2012), with studies based on fire scars on grass trees (Xanthorrhoea spp.) suggesting most areas were burnt every 3-5 years (Ward et al. 2001), whilst others suggest they were burnt at intervals greater than four years (Abbott 2003). Issues arise with these estimates however as large proportions of the local flora require more than five years to form a viable seed bank to ensure their long-term persistence (Enright et al. 2005; Burrows et al. 2008; Wilson et al. 2014). Contemporary fire regimes over the past 40 years for the entire SWAFR region range from 13-40 years, with fire frequencies generally increasing as rainfall declines (Enright et al. 2012). By analysing LandSat satellite imagery, over the past 30 years it has been estimated that fires have occurred roughly every 13-20 years around Eneabba (Enright et al. 2005; Enright et al. 2012). These fires include both naturally started fires and fuel reduction burns. However, regardless of the fire frequency, it is clear that fire has played a significant role in the evolution and adaptations of the taxa present (Miller & Dixon 2014).

3.2 Study methods Through comparing present with past data on seed production and storage for serotinous plant species in the region, the effect of climate change on plant demographic attributes, in particular seed/fruit production, seed viability and storage were able to be analysed.

Previously published evidence shows that for the serotinous species Banksia hookeriana, a decline in cone production has occurred for plants of the same age and at the same site over time, with these differences being likely due to climate change

(Enright et al. 2015). By surveying a selection of serotinous species in Western

Australia, determining if this pattern is occurring for other species can be evaluated.

3.2.1 Previous data

To investigate seed production in serotinous species in the Eneabba region under a changing climate, studies between 1983 and 2004 were compared (Table 3.3). These studies formed the basis for study design, species selection and vegetation age (time since last fire; Figure 3.2). By trying to match these factors, it allows for a direct comparison between sample dates, where factors that are different between sites over time are mostly climate related, and not soil or plant age (time since fire) related. This allows the comparison between past data collection and 2018 to be as accurate as possible.

All papers/data used for this comparison provided an estimate of the total number of seeds produced per plant. In addition, data on fruits produced, opening rates and seed viability were also available for some species (Table 3.4). For the Banksia species surveyed, changes in cone attributes based on cone age were assessed as well as changes along a climatic gradient for B. attenuata (Table 3.4). All results however, provide evidence to evaluate if seed production and storage in serotinous plants is changing - potentially due to climate change.

Table 3.3: Summary of studies used to provide a comparison on the seed production of serotinous species near Eneabba. Site age is years since last fire

Paper Site area Topography Site age (years) Cowling and Lamont 1985b Mt Adams, Jurien Sandy dune and 15 swale sequence Bell et al. 1987 Junction of Jurien East Sandy swale 16 Road and Brand Highway Lamont and van Leeuwen Lesueur National Park Limestone 18 1988 Enright and Lamont 1989 Eneabba Sandy dune and 15 swale sequence Bellairs and Bell 1990 Eneabba Deep sand and 19 Laterite Enright et al. 2007 Eneabba Sandy dune and 12-20 swale sequences

Figure 3.2: Map of approximate location of past data collection sites near Eneabba, Western Australia.

Table 3.4: Focal species examined in this study, and the nature of previously published reports on their various demographic attributes.

Viability Opening Geographic Species Evidence Seeds/plant Fruit/plant Viability with age with age variation

Banksia attenuata Cowling & Lamont ✓ ✓ 1985b

Enright & Lamont 1989 ✓ ✓ ✓

Bellairs & Bell 1990 ✓

Banksia hookeriana Cowling & Lamont 1985b

Enright & Lamont 1989 ✓ ✓

Bellairs & Bell 1990 ✓

Enright et al. 2007 ✓ ✓ ✓

Banksia tricuspis Lamont & van Leeuwen ✓ ✓ ✓ ✓ 1988

Beaufortia elegans Bell et al. 1987 ✓

Bellairs & Bell 1990 ✓

Enright et al. 2007 ✓ ✓ ✓

Eremaea Bellairs & Bell 1990 ✓ beaufortioides Enright et al. 2007 ✓ ✓ ✓

Eremaea violacea Bellairs & Bell 1990 ✓

Hakea Bell et al. 1987 ✓ psilorrhyncha Bellairs & Bell 1990 ✓

Enright et al. 2007 ✓ ✓ ✓

Leptospermum Bellairs & Bell 1990 ✓ spinescens Enright et al. 2007 ✓ ✓ ✓

Melaleuca scabrum Bellairs & Bell 1990 ✓

Melaleuca Bellairs & Bell 1990 ✓ leuropoma Enright et al. 2007 ✓ ✓ ✓

Xylomelum Bellairs & Bell 1990 ✓ angustifolium

3.2.2 Site selection

Site selection was based on the location of the previous studies to ensure that factors such as different topography or soil type did not influence seed production. This also helped to minimise the chance of measuring slightly different plant communities (e.g. with different resource availability) to what was studied previously. As the exact location of plots within sites was not usually specified in the comparison studies, the general area or reserve was used (Figure 3.3). Fire maps from the Department of

Biodiversity, Conservation and Attractions, as well as other relevant authorities were used to assess the fire history of the sites based on time since last fire. Sampled sites were as close to the same postfire age as possible to the comparison studies to ensure plants were at the same stage of their lifecycle. However, site ages for Lesueur National

Park and cone aging for B. attenuata were unable to be matched to that of past surveys.

Site ages ranged from 7 to 35 years (Table 3.5) and were all in kwongan vegetation except for the Jurien AltaMare site which was a Banksia woodland ecosystem. This change in ecosystem structure is primarily due to the differences in rainfall (higher) and substrate (limestone; higher nutrient) characteristics. For some sites, multiple plots were established to obtain a sufficient sample size of the selected species.

Figure 3.3: Map of both past and 2018 study sites near Eneabba, Western Australia.

Table 3.5: Locations, substrate and ages (time since fire) of study sites and plots.

Site/Reserve Plot name Substrate Latitude Longitude Site age (years) Beekeepers East High Dune 8 Dune -29.735253 115.226552 20 Unallocated High Dune 8.5 Dune -29.735829 115.232621 20 crown land Coomallo Nature Coomallo Sand -30.229492 115.400583 16 Reserve Jurien AltaMare AltaMare Sand -30.298879 115.114487 23 Conservation Park Lesueur National Lesueur Carpark Limestone -30.162716 115.199757 35 Park Lesueur South site Limestone -30.151263 115.185446 7 Lesueur North site Limestone -30.150768 115.187973 7 South Eneabba SE Crest plot 1 Dune -29.876957 115.249666 16 (SE) Nature SE Crest plot 2 Dune -29.877375 115.249897 16 Reserve SE Laterite South Laterite -29.985517 115.287061 16 plot 1 SE Laterite South Laterite -29.985201 115.290936 16 plot 2 SE Laterite North Laterite -29.959641 115.275443 16 plot 1 SE Laterite North Laterite -29.985201 115.287061 16 plot 2 SE Sand Sand -29.833631 115.262926 28 Yardanogo Mt Adams plot 1 Dune -29.405913 115.126064 25 Nature Reserve Mt Adams plot 2 Dune -29.407158 115.108162 16 Mt Adams plot 3 Dune -29.406628 115.091906 7

3.2.3 Focal species All species surveyed are serotinous allowing for measurements to be conducted at any

time of the year due to the retention of fruits in the plant canopy for several to many

years (Lamont 1991). Species were selected based on the availability of past data (Table

3.4) and whether or not they were found at survey sites in a sufficient population size to

provide an accurate estimate of their canopy seed stores. All species were from the

Proteaceae or Myrtaceae plant families (Table 3.6), these being dominant in many

vegetation communities throughout Australia. Fire response strategies of species

included both non-sprouters (i.e. fire killed) and resprouters (i.e. fire surviving; Table

3.6). All species were shrubs, with most being sub-shrubs less than 1.5 m tall.

Xylomelum angustifolium at up to 5 m was the tallest species. None of the species

surveyed are of conservation concern in Western Australia, except for Banksia tricuspis,

which is a priority 4 species (rare or near threatened; Department of Parks and Wildlife

2017; Western Australian Herbarium 2018). Some species have undergone a name

change since the previous studies, including Hakea psilorrhyncha R. M. Barker

(formerly Hakea obliqua) and Melaleuca leuropoma Craven/M. systena Craven

(formerly M. acerosa). Melaleuca systena and M. leuropoma occur on different

substrate types (M. systena on laterite and M. leuropoma on sandplains), but are

otherwise identical and the two are referred to collectively as M. leuropoma here to

allow for data matching to the earlier studies. Fruit type between species was also

variable, which influences fruit opening rates and seed storage (Figure 3.4).

Table 3.6: Focal species, their fire response strategy and family.

Species Family Fire response strategy Banksia attenuata R. Br Proteaceae Resprouter Banksia hookeriana Meisnner Proteaceae Non-sprouter Banksia tricuspis Meisnner Proteaceae Resprouter Hakea psilorrhyncha R. M. Barker Proteaceae Resprouter Xylomelum angustifolium Meisnner Proteaceae Resprouter Beaufortia elegans Schauer Myrtaceae Non-sprouter Eremaea beaufortioides Benth Myrtaceae Resprouter Eremaea violacea F. Muell Myrtaceae Resprouter Leptospermum spinescens Endl Myrtaceae Resprouter Melaleuca scabrum R. Br Myrtaceae Resprouter Melaleuca leuropoma Craven Myrtaceae Resprouter

Cone Nuts in a cluster Small fruit Large fruit Banksia tricuspis Beaufortia elegans Eremaea violacea Xylomelum Banksia Hookerian Melaleuca leuropoma Eremaea beaufortioides angustifolium Banksia attenuata Melaleuca scabrum Leptospermum spinescens Hakea psilorrhyncha

Figure 3.4: Different fruit types of serotinous species surveyed.

Through surveying a selection of both non-sprouting and resprouting species, changes

in seed production and storage can be compared between different fire responses. This

44 assists in understanding how species with different responses to fire have responded to climate change (e.g. through altering the proportion of fruits that remain closed, or the number of seeds produced per plant). For plant functional groups that are sensitive to more frequent fires (i.e. non-sprouters) as projected from climate change, determining changes in their reproductive effort will allow for appropriate management of these species.

3.3 Fieldwork methods

Fruit counts were conducted in March and April 2018, while the harvesting of fruit samples occurred mostly in late June 2018. Appropriate permits to collect fruits were obtained from the Department of Biodiversity, Conservation and Attractions Parks and

Wildlife Service WA (Scientific or Other Prescribed Purposes License no. SW019715;

Regulation 4 Authority no. CE005811). All surveys took place at least 15 m from any disturbed area such as a road, track or fire break to reduce the chance of edge effects influencing the data collected.

3.3.1 Fruit counts

A target of 50 individual plants from each species at each site were surveyed to gather fruit production data. However for some species this was not achieved due to low density of focal plant individuals (Table 3.7). Data collection used a nearest neighbour, plotless approach, where once in a representative area, one focal plant was found and assessed and then the next (nearest neighbour) plant was found and surveyed. This next nearest neighbour approach was used to allowed for the achievement of the desired sample size, whilst also ensuring there was no bias in sampling as all focal plants within an area where surveyed. All species regardless of their fire response strategy had the height to the tallest live part of the plant recorded. All resprouter species sampled also had canopy dimensions (longest axis and an orthogonal axis) measured (See appendix

7.2). Canopy area or volume is a proxy for age in resprouting species as the age of

resprouters cannot be determined directly.

Table 3.7: Number of individual plants per species measured for fruit counts at each site (with year of last fire in parentheses) near Eneabba, Western Australia 2018.

Beekeepers East Lesueur Mt South Jurien Coomallo Unallocated National Adams South South Eneabba AltaMare Nature Crown Park (1993, Eneabba Eneabba Nature Conservation Reserve Land (1983 & 2002 & Crest Laterite Reserve Park (1995) (2002) (1998) 2011) 2011) (2002) (2000) (1990) Banksia attenuata 26 53 90 52 71 Banksia hookeriana 51 46 Banksia tricuspis 90 Beaufortia elegans 50 51 51 98 Eremaea 52 102 beaufortioides Eremaea violacea 8 12 67 Hakea psilorrhyncha 50 52 103 Leptospermum 4 17 106 spinescens Melaleuca scabrum 100 Melaleuca leuropoma 50 52 103 Xylomelum 50 52 angustifolium

For each plant surveyed, the number of closed (seed containing) and open (seeds

released) fruits were recorded. At each site, a minimum of 10 individual Banksia

attenuata plants had all cones assigned an age in years. Bankisa attenuata produces

node scars on branches most years showing stem growth allowing cones to be aged

(Lamont 1985). Banksia tricuspis cones could not be aged in this manner but were

classified as one, two, three and four years and older, as described in Lamont and van

Leeuwen (1988). The extent of fading/weathering, proportion of open follicles, location

of the cone (i.e. terminal or not) and the presence of leaves were factors used to

determine cone age (Appendix 7.1). The number of open and closed follicles per cone

were recorded to provide a measure of seed storage on the plant and of seed release

during the inter-fire period. 30 individual B. attenuata plants also had a count of cones

with all open follicles, all closed follicles and mixed (both and open and closed

follicles) done per plot. For B. tricuspis site ages were unable to be matched, however,

46 by surveying cone crops by their age, potential changes in cone productivity between

2018 and 1985 still could be determined. At each plot at least 30 B. tricupsis plants had all cones assigned an age between 1 year and 4 years and older, with open and closed follicle counts being recorded for at least two cones per age class for each individual plant surveyed.

3.3.2 Fruit collection

Fruits were harvested off all focal plant species from sites in which they occurred. Fruits from each plant were bagged individually on the day collected so that seed counts and viability tests could be conducted. For all species except Banksias, between 15 and 30 plants were harvested. For each plant harvested, height and canopy dimensions were recorded. Plants harvested were representative of the population in terms of size and did not have any signs of canopy death or poor health.

3.3.3 Banksia fruit collection

As Banksia tricuspis is a priority four species, only 10 plants at the carpark site and five plants from the north and south sites had cones collected. One cone was obtained for each age class: one year, two year, three year and four, year and older cones. Banksia attenuata cones were collected from AltaMare and High Dune sites from ~30 individual plants and stratified into young (1-2yrs), intermediate (3-4yrs) and old (>4yrs). These age classes were chosen due to less accuracy in the age estimation using node counts as well as limitations with the previous data that were being compared. Cones were not harvested from all sites where cone count data were collected as little change in viability is likely across a relatively small geographical region with similar climatic conditions.

3.3.4 Cone processing

Fire is required to release the seeds from the closed cones of many banksia species

(Cowling & Lamont 1985a). All Banksia cones and Xylomelum fruits collected were

47 burnt using a flame torch until all or most of their follicles ruptured and seeds began to fall out. Wet-dry cycles were then used for Banksia cones to mimic subsequent events of rainfall and drying, and facilitate seed release from ruptured cones, replicating the natural environment after fire (Cowling & Lamont 1985a). Once burnt, cones were immersed in water for roughly one minute and then left to dry, assisting the separator to spread and recurve and encouraging seed release (Wardrop 1983; Cowling & Lamont

1985a). Wetting and drying was repeated until all seeds were released and the number of firm (potentially viable with a developed embryo), aborted (no embryo and therefore not potentially viable) and predated (seeds that had been eaten/damaged by insects) were recorded. To help speed up the process of seed release, where necessary cones were put into a drying oven at 40°C for two hours after they had been burnt and immersed in water. All other focal species released their seeds due to the effects of drying when detached from the plant. To facilitate the rapid release of seeds, fruits were placed into a drying oven for 48 hours at 40°C.

3.3.5 Seed counts and germination trials

The number of seeds produced per fruit was counted, as was the number of nuts per clusters for a selection of myrtaceous species as appropriate. Equal weights of organic material from small fruited species that included non-seed fractions was placed into petri dishes with at least two replicates for each age class, site and species. 20 seeds of the larger seeded species (Hakea psilorrhyncha and Xylomelum angustifolium) were placed into petri dishes in the same manner as smaller seeded species. Banksia species had a minimum of three replicates petri dishes per age class per site. Each petri dish was lined with two pieces of Whatmans 1 Filter Paper and kept moist using distilled water every 2-3 days. To control the growth of fungi, Mancozeb (5g/L) was sprayed on seeds as necessary. Seeds were rinsed, and filter paper changed if required. Samples were kept in a growth cabinet at a constant temperature of 18°C, and germination trials run for 30 48 days (Enright et al. 2007). A seed was classed as viable when the radicle was at least

5mm long and the proportion of seeds that germinated were recorded. Small seeded species petri dishes were checked by binocular microscope to ensure accurate counts of the number of germinated seeds per petri dish.

3.4 Standardized Precipitation Evapotranspiration Index (SPEI) The Standardized Precipitation Evapotranspiration Index (SPEI) combines precipitation, potential evapotranspiration (evaporation and transpiration) and temperature to provide a measurement of drought severity, and the potential imbalance occurring in a region based on levels of precipitation and rate of evaporation/transpiration (Vicente-Serrano et al. 2009). A greater level of evapotranspiration than precipitation results in a negative

SPEI value which causes water stress in plants, and they experience drought-like conditions. For the region around Eneabba, since the year 2000, there has been a consistent negative SPEI suggesting drought-like conditions have been occurring, while prior to this, the SPEI fluctuated depending on season and year (Figure 3.5).

Jul-64 Jul-73 Jul-82 Jul-91 Jul-00 Jul-09

Jan-60 Jan-69 Jan-78 Jan-87 Jan-96 Jan-05 Jan-14

Oct-93 Oct-66 Oct-75 Oct-84 Oct-02 Oct-11

Apr-62 Apr-71 Apr-80 Apr-89 Apr-98 Apr-07 -1

-2

-3

Figure 3.5: Eneabba 12 month SPEI from 1960 to 2015 (Global SPEI Database 2017). Negative values show a greater level of evapotranspiration than precipitation (i.e. moisture stress), and a positive value shows the opposite to this.

Investigating the changes in SPEI for different time periods allows for a greater understanding of how the climate has changed and the physiological stress this has likely put on plants. Based on the vegetation age (time since last fire) of sites surveyed, the average summer SPEI that those plants have been exposed to was determined. The

SPEI calculated for sites was based on that closest to their location for Eneabba, Jurien and Mt Adams with Jurien SPEI being used for Lesueur National Park.

3.5 Data analysis

My overarching objective was to evaluate evidence for the demographic shift portion of the interval squeeze hypothesis. In order to quantify changes in seed demography, both past and present data were first organized into both Microsoft Access (2016) and

Microsoft Excel (2016) spreadsheets. After obtaining component data (closed fruit counts, seeds per fruit, seed viability) viable seeds per plant were calculated for all species and past and present values contrasted. To explore potential mechanisms driving changes in canopy seed banks I used subsets of the broader dataset based on evidence collected in prior work. These evidence types included changes in fruit production, seed viability and cone opening. Finally, to investigate links between the effect of changing climate and seed production, SPEI for the region was analysed to determine its relationship with changes in the canopy seed bank.

For all species and substrates (sand or laterite), viable seeds per plant was quantified from 2018 and contrasted with past data. Means and 95% confidence intervals for all species and sites were calculated for all plant measurements including height and width, fruit counts (closed and open fruits), seeds per fruit, nuts per cluster and germination rate (Appendix 7.2 & 7.3). To determine the average number of viable seeds per plant, the number of closed fruits, seeds per fruit and germination rate were multiplied

(Appendix 7.2 & 7.3). For other evidence types, including fruit production, seed

50 viability and rate of cone opening, data were compared to that of past data collection where available. Cones were separated by age and the proportion of cones with all follicles closed were determined to analyse the extent of opening in Banksia cones. As two B. tricuspis sites were the same age (Lesueur North and South), when presenting results these two sites were averaged. All 2018 data collected was compared to that of past data collection, with all graphs showing means and 95% confidence interval overlap. Lack of overlap in confidence interval error bars were interpreted as evidence of a statistical difference based on p<0.05 (Ramsey & Schafer 2002). As access to the entire data set of past studies were not always available, data reported in papers used to provide a comparison often lacked error estimates.

The percent difference in average viable seed production and fruit production between

2018 and combined past data collected were determined for all species and substrate type surveyed (either sand or laterite). The effect of fire response strategy (resprouter or non-sprouter), and plant family (Myrtaceae or Proteaceae) were analysed as factors in linear models to determine their influence on seed production.

To understand the extent of climate change, average December SPEI for each region surveyed (Jurien, Eneabba and Mt Adams; SPEI Global Database 2018) was sourced.

To determine the effect of this on both past and present vegetation surveyed, the average SPEI were determined for each species and site based on its time since last fire, i.e. if a site was surveyed in 2018 and was 20 years old, the average December SPEI from 1998 to 2017 was determined. Similarly to seed production, the average December

SPEI for both time periods surveyed were analysed using a t-test to determine if there has been significant differences in SPEI over the time periods surveyed. To analyse the effect of climate change on seed production, the percent difference in seed production and SPEI were analysed in a linear model for all species surveyed and sites. All data analysis were conducted in RStudio (RStudio Team 2016). 51

The following chapter presents the results based on the methods described. Changes in viable seeds per plant, followed by mechanisms influencing seed production including fruit production, seed viability and cone opening changes are presented. SPEI changes for the region are then analysed.

4. Results

4.1 Viable seed production over time Mean viable seeds per plant in 2018 was contrasted with past values for all species by site combinations (Figure 4.1 & 4.2). The number of viable seeds produced per plant in

2018 versus earlier records was highly variable both between and within species and among sites, with 91% of species showing no significant change in seed production over time (based on the overlap or lack of confidence interval overlap; Figure 4.1 &

4.2). Lack of error estimates in past work made statistical comparisons impossible. For some species such as Beaufortia elegans on laterite substrates, and Eremaea violacea and Melaleuca leuropoma on sand and laterite substrates, current seed production was very similar to that of 1987 (Figure 4.1). There was no consistent pattern of an increase or decrease in viable seed production between different plant families (r2=0.009,

2 F1,9=0.08, p=0.78) and fire response strategies (r =0.004, F1,9=0.04, p=0.85).

Interestingly, for Beaufortia elegans and Hakea psilorrhyncha surveyed in Coomallo

Nature Reserve south of Eneabba, at the cooler and wetter end of the sampled climate gradient, viable seeds produced per plant was significantly less for both time periods surveyed. Compared to seed production in Eneabba in a warmer and drier location,

Beaufortia elegans produced less seeds, whilst Hakea psilorrhyncha produced significantly more seeds (Figure 4.1 & 4.2).

Figure 4.1: Mean number of viable seeds per plant in Myrtaceae species from 1986-2004 (blue) compared to 2018 (pink) for a selection of serotinous species surveyed near Eneabba, Western Australia. 54

Data in green is from Coomallo Nature Reserve near Jurien, Western Australia. Data are means with 95% confidence interval error bars. Graphs are separated by species and substrate type (either deep sand or laterite). The two measurements from Bellairs and Bell (1990) are from different plots within the same site. Fire response strategy: RS=resprouter; NS= non-sprouter. Legend: past data collection are evidence source; 2018 data collection is site with year of last fire in parentheses.

Figure 4.2: Mean number of viable seeds per plant in Proteaceae species from 1987-2004 (blue) compared to 2018 (pink) for a selection of serotinous species surveyed on sand substrates near Eneabba in Western Australia. Data in green is from Coomallo Nature Reserve near Jurien, Western Australia. Data are means with 95% confidence interval error bars. Fire response strategy: RS=resprouter; NS= non-sprouter The two measurements from Bellairs and Bell (1990) and Enright and Lamont (1989) are from different plots within the same site. Legend: past data collection are evidence source; 2018 data collection is site with year of last fire in parentheses Banksia hookeriana and Hakea psilorrhyncha showed a decline in seed production between time periods surveyed whilst other Proteaceae species surveyed showed an

55 increase in seed production over time (Figure 4.2). Banksia hookeriana had a significant reduction in viable seeds produced between 1987 and 2018 (380 & 362 vs 84 & 90 viable seeds per plant; Figure 4.2), whilst for B. attenuata a slight reduction occurred across all sites but it can’t be determined if this was significant due to lack of error estimates in past work (36 & 58 vs 11.52 & 28.14 & 33.7 viable seeds per plant; Figure

4.2). Banksia tricuspis had variable seed production across sites and time frames for the differently aged cones. Overall seeds per plant was higher in 2018 than in 1985 across all sites surveyed (2018=129, 207, 126 vs 1985=84 viable seeds per plant) but these changes are unlikely to be significant between time frames, and are not significant between sites surveyed in 2018 (Figure 4.2).

Table 4.1: The difference in seed production (%) for a selection of serotinous species surveyed in the past (between 1985 and 2004) and 2018 near Eneabba, Western Australia. A negative percent difference shows a reduction in the number of seeds produced between past data and 2018, whilst a positive percent difference shows an increase in viable seeds produced per plant between past data and 2018. Species are organised by fire response strategy.

Combined past 2018 % Fire viable viable Difference difference response seed seed in seed in seed Species strategy Family plant-1 plant-1 production production Beaufortia elegans non-sprouter Myrtaceae 437.02 472.08 35.06 8.023 Banksia hookeriana non-sprouter Proteaceae 257.18 90.04 -167.14 -64.99 Hakea psilorrhyncha non-sprouter Proteaceae 19.8 18.85 -0.95 -4.8 Eremaea beaufortioides resprouter Myrtaceae 89.55 102.532 13 14.52 Eremaea violacea resprouter Myrtaceae 68.5 100.98 32.48 47.42 Leptospermum spinescens resprouter Myrtaceae 97.63 78.41 -19.22 -19.69 Melaleuca leuropoma resprouter Myrtaceae 1300.56 300.97 -999.59 -76.86 Melaleuca scabrum resprouter Myrtaceae 158 123.18 -24.72 -15.65 Banksia attenuata resprouter Proteaceae 32.5 28.01 -4.49 -13.82 Banksia tricuspis resprouter Proteaceae 84 136.42 52.42 61.2 Xylomelum angustifolium resprouter Proteaceae 61.8 80 19.2 31.07

Analysis of mean percent change in seeds per plant revealed that 54% (6 of 11 species) surveyed had a negative percent change in seed production (seed production reduced), although the extent of this change was highly varaible, whilst 46% (5 of 11 species) had a positive percent change in seed production (seed production increased; Table 4.1). 56

2 There was no evidence of a relationship between plant family (r =0.01, F1,8=0.05,

2 p=0.82), fire response strategy (r =0.04,F1,8=0.33, p=0.59) or plant family and fire

2 response strategy combined (r =0.04 ,F2,7=0.14, p=0.87) in changing the reproductive output from past to present, thus underscoring the wide variation across species and populations (Table 4.1).

4.2 Mechanisms influencing seeds produced

4.2.1 Changes in fruits per plant Comparison of mean closed fruits per plant provided in Enright et al. (2007) yielded a broad pattern of decreased fruit production across most species (Figure 4.3). For

Eremaea beaufortioides, Melaleuca leuropoma, Beaufortia elegans and Hakea psilorrhyncha the lack of confidence interval overlap between most sites revealed strong evidence of a significant decline in fruit production (Figure 4.3). Comparing data collected from Enright & Lamont (1989), Banksia hookeriana showed a reduction in cones produced per plant in of over 50% in 2018 compared to 1987 (averaged 20 vs 42 cones per plant respectively; Figure 4.3). Banksia attenuata on the other hand, had more variable cone production in 2018 than in 1987, with cone production increasing across all sites of comparable age (Figure 4.3). Like that of B. attenuata, cone counts for B. tricuspis between 1985 and 2018 was variable and generally more cones were produced in 2018 (Figure 4.3). Across most sites and all cone ages, there was an increase in the number of cones produced per plant over time, but these changes are unlikely to be statistically significant (Figure 4.3).

2 There is no relationship between fruit production and plant family (r =0.21, F1,4=1.08, p=0.38). All non-sprouters surveyed showed a statistically significant reduction in fruit production between 2004 and 2018, whilst resprouters show high variability (some

57 species have a slight increase or decrease) with no statistically significant difference over time (Figure 4.3).

Figure 4.3: Mean fruits produced per plant in 1985-2004 (blue) compared to 2018 (pink) for a selection of serotinous species on deep sand substrates surveyed near Eneabba, Western 58

Australia. Data are means with 95% confidence interval error bars RS= resprouter,NS= non sprouter The two measurements from Enright and Lamont 1987 are from different plots within the same site. Legend: past data collection are evidence source; 2018 data collection is site with year of last fire in parentheses. 4.2.2 Changes in seed viability Through germination tests, the proportion of total firm seeds produced that were viable was analysed and compared to data collected in 2004 (Enright et al. 2007) and in 1987

(Enright & Lamont 1989). Viability fluctuated slightly (-22% to 20%) between time periods, with generally broad confidence intervals with no significant increase or decrease in most species (Figure 4.4). Eremaea beaufortioides demonstrated a 22% decline in seed viability and L. spinescens an increase of 20% (Figure 4.4).

Figure 4.4: Percentage of viable seeds between 1987-2004 (blue) compared to 2018 (pink) for a selection of serotinous species surveyed on deep sand substrates near Eneabba, Western Australia. Data are means with 95% confidence intervals error bars. RS=resprouter, NS= non-sprouter. Legend: past data collection are evidence source; 2018 data collection is site with year of last fire in parentheses 60

4.2.2.1 Changes in seed viability with cone age

Viability analysis for different aged B. tricuspis cones revealed little change in seed viability between 1985 and 2018 across all sites and all ages, with the exception of cones four years and older (Figure 4.5). Between 1985 and the car park site, the proportion of viable seeds in four years and older cones was more than four times greater in 2018 than that in 1985 (18 vs 82.4% viability respectively; Figure 4.5).

However, for all other aged cones and across sites, there was no statistically significant difference in seed viability between 1985 and 2018, except for the one-year-old cones at the carpark site which produced less viable seeds than the other sites (85 vs 100% of seeds viable; Figure 4.5).

Figure 4.5: Percentage of viable seeds from 1985 to 2018 for different aged Banksia tricuspis cones surveyed at Lesueur National Park, Western Australia. Legend: past data collection are evidence source; 2018 data collection is site with year of last fire in parentheses

4.3 Changes in cone opening rates Cone opening rates for two focal Banksia species were contrasted between past and present data across climatic gradients to assess evidence for increased opening rates and loss of seed stores (Figure 4.6). Degrees of serotiny are generally lower in climates that are more mesic and fires less frequent, with cones releasing their seeds at a younger cone age compared to plants in a more xeric, fire-prone location (Cowling & Lamont

1985b). This was explored for Banksia attenuata and B. tricuspis across different time periods (Figure 4.6), with the cone age where 50% of follicles opened also provided for species surveyed (Table 4.2).

Figure 4.6: Cone opening rate for Banksia species surveyed between 1985-1987 and 2018 in the region surrounding Eneabba, Western Australia. Data are means with 95% confidence interval error bars Legend: past data collection are evidence source; 2018 data collection is site with year of last fire in parentheses

Table 4.2: The cone age for 50% of follicles to open in the Banksia species surveyed near Eneabba, Western Australia. Past years surveying were; Banksia attenuata=1983, Banksia tricuspis= 1985

Species Site Cone age at 50% opening (years) Past Present

Banksia attenuata Mt Adams 9 7 Eneabba 6 5 Jurien 5 2 Banksia tricuspis Lesueur >4 >4 Carpark >4 North & South >3

4.3.1 Changes in cone opening across a climatic gradient The trend of banksia cones opening earlier in more mesic sites than xeric sites was unchanged over the 1983-2018 period for B. attenuata (Figure 4.6). The two drier sites

(Mt Adams and Eneabba) had 100% of follicles on one year old cones closed, while the most mesic site (Jurien) had just 86% of follicles closed, compared to 100% closed in

1985 (Figure 4.6). As cones aged, follicle opening rate was slower and more gradual at

Mt Adams than at Eneabba and Jurien, with a steep decline in the proportion of closed follicles in >5 year old cones in Eneabba, and >2 year old cones in Jurien (Figure 4.6).

Mt Adams had the highest degree of serotiny of the sites surveyed (Table 4.2).

4.3.2 Changes in cone opening across different time frames Comparing from 1983 to 2018, a smaller proportion of follicles remained closed in B. attenuata cones (between 0 and 50% difference; Figure 4.6). Across all sites and most cone ages, seeds were being released at a younger cone age now than they have in the past. At Mt Adams, cones between the ages of 1-5 years show little change, but for cones >6 years old, the proportion of cones with follicles closed is lower in 2018 than in

1983 (Figure 4.6). Similarly, at Eneabba cones >5 years had significantly less follicles closed in 2018 compared to 1983 (Figure 4.6). At Jurien, the most mesic site, across all

63 cone ages there was an increased proportion of follicles open in 2018 than in 1983

(Figure 4.6).

In B. tricuspis, the proportion of closed follicles per cone decreased with increasing cone age, with more than 50% of follicles opened prior to cones reaching four years of age (Figure 4.6). However, the proportion of follicles closed was similar between 1985 and 2018 across all age classes, except for the three year old cones at the North/South site (Figure 4.8).

For B. attenuata, 50% of follicles had opened at a younger cone age in 2018 compared to 1983 for all sites surveyed (Table 4.2). However, B. tricuspis had 50% of follicles open on cones one year younger (>4 vs 3 years) at the North/South site, but the same at the carpark site (both >4 years; Table 4.2)

4.4 Standardized Precipitation Evapotranspiration Index (SPEI) For all sites surveyed, average SPEI experienced for growing periods of plants has become more negative, with 2018 plants experiencing a significantly higher negative

SPEI than those in past periods (t9.13=5.83, p=0.0002; Table 4.3). All changes in SPEI are negative values suggesting that drought overtime has increased, with 53% of species surveyed across either sand or laterite substrate having a reduction in seeds produced, and 47% having an increase (Figure 4.7). The relationship between SPEI difference and seed production difference over time is not significant and do not influence each other

2 (r =0.0002, F1,19=0.004, p=0.95; Figure 4.7).

Table 4.3: The average December SPEI for the vegetation ages of sites surveyed both in the past and 2018 showing the mean difference in SPEI per site and between time frames. SPEI data from SPEI database 2018.

Comparison Years Years Paper Site (past data) SPEI (present data) SPEI Difference Bell et al. 1987 Coomallo Nature 1971-1986 -0.32 2002-2018 -1.16 -0.84 Reserve Bellairs & Bell Beekeepers Nature 1968-1987 -0.17 1998-2018 -0.82 -0.65 1990 Reserve South Eneabba Crest 1968-1987 -0.17 2002-2018 -0.96 -0.79 and Laterite Cowling & Jurien 1968-1983 -0.39 1995-2018 -0.87 -0.48 Lamont 1985b 1968-1983 -0.20 1993-2018 -0.65 -0.45 Mt Adams Plot 1 1968-1983 -0.20 2002-2018 -0.90 -0.70 Mt Adams Plot 2 Mt Adams Plot 3 1968-1983 -0.20 2011-2018 -0.84 -0.64 Enright & South Eneabba Reserve 1972-1987 -0.20 1990-2018 -0.63 -0.42 Lamont 1989 Beekeepers Nature 1968-1987 -0.17 1998-2018 -0.82 -0.65 Reserve Lamont & van Lesueur Car Park 1967-1985 -0.33 1983-2018 -0.63 -0.30 Leeuwen 1988 Lesueur North and 1967-1985 -0.33 2011-2018 -0.79 -0.53 South

Figure 4.7: Scatterplot of the percent differences in SPEI and viable seed per plant production between the time frames surveyed. For serotinous plants surveyed near Eneabba, average seed production between 1983-

2004 and 2018 was variable, with plant family and fire response strategy not

65 influencing this, thus highlighting its variability. However, fruit production has decreased between 2018 and 2004 for most species, whilst seed viability remained unchanged. Across a climate gradient, B. attenuata follicles have opened at cone ages younger than they have in the past, with a higher proportion of them being open in more mesic sites than xeric sites. Climate conditions based on SPEI show that 2018 plants surveyed have experienced more moisture stressed conditions than plants surveyed in prior years, but this does not have a relationship with changes in seed production over time.

5. Discussion

5.1 Evidence for interval squeeze Gaining more evidence for the interval squeeze model (Enright et al. 2015) was one of the main motivators for this study, as changes in seed demography due to climate change in serotinous species is poorly understood. The relationship between increased fire frequency, coupled with warmer and drier conditions is a complex one, and changes to demographic attributes needs to be further analysed. The potential for plants to be burnt prior to reaching reproductive maturity, combined with changes in seed production and storage due to unfavourable climate conditions may lead to collapse of serotinous species populations (Enright et al. 2015). Combined, these factors threaten the persistence of woody serotinous species globally.

A reduction in cone and seed production due to climate change in Banksia hookeriana supports the interval squeeze theory (Enright et al. 2015). Through investigating if this pattern is occurring for multiple serotinous species in the Eneabba region, this study suggests some evidence for the interval squeeze model based on a demographic shift, particularly reductions in seed/fruit production and increases in seed loss during the inter-fire period from increased rates of cone opening. There is partial support for reduced seed production due to climate change based on the results found in this study.

Although seed production was quite variable between time frames, and also between sites and species, over 50% of species surveyed showed a reduction in the average number of seeds produced in 2018 compared to when data was collected between 1987 and 2004. However, there is greater support that climate change reduced fruit production in serotinous species. Over half of the species surveyed had a significant reduction in fruits produced, which similarly also influences total seed production and seed storage on a plant. There was no evidence for changes in seed viability. Although seed viability is not mentioned in the interval squeeze model, it was included in this

67 study for its potential to influence viable seed bank size. Regarding fruit opening however, for the Banksia species surveyed, there is strong evidence for interval squeeze as a greater proportion of seeds are being lost into the inter-fire period where they are unlikely to germinate, compared to in the past. Based on these findings, this study shows partial support for the interval squeeze hypothesis, with greatest support for reduction in fruit production and changes in opening rates with cone/fruit age for the species surveyed. These findings raise concern for the future populations of these species under a warmer, drier and more fire prone climate, and can also be applicable to other serotinous species globally.

5.1.1 How has climate change influenced seed production? For the species surveyed in Eneabba, seed production was variable, over half of all species surveyed had a reduction in average seed production in 2018 compared with earlier published estimates, while the rest showed no change or an increase. Thus, there is no clear evidence to support the contention that reduced seed production is occurring as a consequence of recent warming and drying. However, variability in the results presented here might indicate greater sensitivity to changing climate in some species than others, so individual species responses are worthy of further examination.

Seed production among populations of the same species is known to be quite variable, regardless of climatic conditions (Bellairs & Bell 1990; Henery & Westoby 2001), and this was found to be true in this study as well. This makes it difficult to gauge longer term trajectories of seed production given high variation within and between populations of the same species. Changes in seed production due to changing climatic conditions have been found for serotinous Leucadendron and Protea species in South

Africa (Treurnicht et al. 2016). Soil moisture stress and extreme heat were observed to have the most negative effects on seed production, both of which are well documented impacts associated with drought-like conditions (Sheffield & Wood 2008; Gallant et al.

2013; Treurnicht et al. 2016). Likewise, B. hookeriana plants surveyed near Eneabba between 1987 and 2012 have had a >50% decrease in total seed production, which correlates with reduction in rainfall and increases in temperature in the study region

(Enright et al. 2015). This suggests that increased soil moisture stress, and more excessively hot days due to climate change may play a role in influencing seed production, as found in South Africa.

Climate change impacts on plant growth, survival and seed production among species and regions with variable extents. In some regions, such as those with a temperate climate, there may be increases in growth and reproduction in some species due to warming (Gworek et al. 2007; Caignard et al. 2017), whilst for other species, increased rainfall has lead to increased seed production, with temperature changes having no effect (Burns 2012). From this, it is difficult to determine whether changes in temperatures or changes in rainfall will have the greatest impact on seed production, with a variety of factors likely influencing this such as; ecosystem, weather patterns, changes in climate and plant life history traits/biology. A long-term decline in rainfall and rising temperatures as observed in Eneabba would be expected to lead to reduced seed production, but like some species, such as those in temperate climates mentioned above, changes in climate have had the reverse effect on seed production. However, there are various demographical attributes that play a role in determining the average number of seeds per plant which cannot be ignored. For serotinous plants, some of these include changes in fruit production, seed viability and opening rates of fruits/cones.

5.1.2 How has climate change influenced fruit production? The storage of fruits in serotinous plants for multiple years is essential to the canopy seed bank these plants have at any given time, and a reduction in this will reduce the total amount of seeds available for recruitment. For most species surveyed (62%), a

69 significant reduction occurred in the number of fruits per plant produced between 2004 and 2018 (Figure 4.3). Between the time frames of when data were collected (1983-

2004 vs 2018), climate conditions for the region have been warmer and drier (Australian

Bureau of Meteorology 2018). Since 2000 in the region surrounding Eneabba, there has been a consistently negative average SPEI, but prior to this, it fluctuated between positive SPEI and negative SPEI (SPEI Global Database 2018; Figure 3.5). A negative

SPEI suggests that plants are moisture stressed and experiencing drought-like conditions and extreme heat (Sheffield & Wood 2008; Gallant et al. 2013) which have caused reduced seed (and potentially fruit) production in South African serotinous species (Treurnicht et al. 2016). The plants surveyed in 2018 have only persisted during a time of a negative SPEI imbalance (i.e. consistently moisture stressed) compared to plants surveyed in 2004 and prior, which have experienced fluctuations in SPEI, including many years characterised by non-drought conditions. The constant moisture stress over most of their life-times experienced by plants surveyed in 2018 may help to explain why fruit production has reduced for most of these species and highlights the role climate (particularly rainfall) plays in the production of fruits (Espelta et al. 2011;

Girard et al. 2012).

Drought has been documented to cause a reduction in annual cone production for serotinous species in MTEs around the world which could lead a reduction in the total amount of seeds produced and stored per plant. Evidence for Pinus species in the

Mediterranean Basin, and western United States suggests that cone production is lower during years of drought (Espelta et al. 2011; Girard et al. 2012), with summer precipitation and annual precipitation over the past five years having the greatest effect on cone production (Lew et al. 2017). Similarly, cone production is greater in mesic areas compared to drier areas (Gonzalez-Ochoa et al. 2004; De Las Heras et al. 2007;

Moya et al. 2008b; Ayari et al. 2011) which highlights the importance that water

70 availability and/or temperature may have in cone production. The general trend of fewer cones produced under drier and hotter climates, as determined for many serotinous

MTE species globally is likely applicable to other serotinous species in MTEs, including the species surveyed near Eneabba.

5.1.3 How has climate change influenced seed viability? Across all species examined, there was no evidence of any changes in seed viability from past to present (with the exception of a possible increase in Banksia tricuspis;

Figure 4.4). This suggests that the levels of warming and drying experienced have not lead to changes in the production of viable seed material. Under a warming and drying climate, no changes in seed viability may assist in maximising postfire recruitment as whilst less seeds will be available, a high proportion of them will still be able to germinate. Furthermore, as the sites surveyed are so low in nutrient availability, particularly nitrogen and phosphorus, when a plant allocates nutrients to seed production, it is likely to ensure that seeds produced have the greatest chance of producing offspring and thus invest the necessary nutrients to ensure viability.

Changes to seed viability in response to climate conditions has not been studied in serotinous species, but studies acknowledge the importance of fire in influencing seed viability. In particular, for non-sprouting serotinous species, fire intervals that are too long often lead to substantial reductions in seed viability due to plants beginning to senesce (Enright et al. 1998; Tonnabell et al. 2012). Therefore, when a fire goes through and mass recruitment generally occurs, viable seed availability will be much less due to a greater proportion of seeds no longer being viable, potentially leading to substantial recruitment failure. However, due to the lack of literature exploring this topic, it is difficult to determine why there is no change in seed viability.

5.2 Changes in fruit opening The rate of opening of serotinous cones will determine how many seeds are lost into the inter-fire period where they are unlikely to germinate (Lamont & Enright 2000). Greater release of seeds prematurely due to changes in climate conditions leads to less seeds available after a fire, which has the same effect as reduced seed production. Therefore, through aging cones and analysing the rates of follicle opening across both climatically different time frames, and climatically different sites, impacts of climate related variables can be examined to determine the role these play in seed release for different serotinous species.

5.2.1 How has climate change affected cone opening across a climate gradient? Across a broad climate gradient (550-416 mm average annual precipitation) Banksia attenuata reflected clear evidence of more rapid cone opening in more mesic sites

(Table 4.2). This is consistent with Cowling & Lamont (1985b) for Banksia species surveyed which suggests a higher degree of serotiny at drier, and more fire prone sites than cooler, less fire prone sites. This pattern is observed across all MTE regions, as generally more cones stay closed in drier, fire prone locations compared to wetter, less fire prone locations. For serotinous Pinus halepensis in the Mediterrenean Basin, P. coulteri in the United States, and Leucadendron and Protea species in South Africa, a general trend of a higher proportion of follicles being open in mesic sites compared to xeric sites was observed (Bond 1985; Borchert 1985; Moya et al. 2008b, Martin-Sanz et al. 2017). Similarly, Banksia serrata in eastern Australia, Pinus rigida and P. torreyana in non-MTE regions of the United States, and Leptospermum scaparium in New

Zealand also followed this pattern, where a higher proportion of follicles/cones were open in coastal areas than inland areas (Givnish 1981; McMaster & Zedler 1981;

Whelan et al. 1988; Battersby et al. 2017). Across these studies, higher degrees of serotiny were positively correlated with lower rainfall (Martin-Sanz et a. 2017), and

72 higher temperatures (Battersby et al. 2017). This reinforces that for serotinous species globally, cones hold their seeds for longer at sites that are more xeric and also more fire prone, highlighting that warmer and drier conditions are generally associated with increased serotiny. The warmer and drier conditions experienced at these sites lead to increased fire frequency which also affects degrees of serotiny.

5.2.2 How has climate change affected cone opening across different time periods? Compared to 1983, a greater proportion of seeds were released in 2018 for cones of the same age in Banksia attenuata plants, with more follicles opening as cone age increased

(Figure 4.6). B. tricupsis also had a general trend of more follicles opening as cone age increased, but follicle opening rates were more variable across sites and time frames

(Figure 4.6). Long-term studies comparing cone opening rates over more than 20 years are not documented for serotinous species elsewhere, so it is difficult to fully explore the role that long-term climate change may be having on seed release. Instead studies conducted are focused on a single year or a period of a few years that was drier than average, and have found that a greater proportion of follicles are opening in drier years.

Similarly, Pinus halepensis in Spain and Israel was found to have a higher proportion of cones opening in response to drought events (Nathan et al. 1999; Espelta et al. 2011).

This suggests that under a drying climate, seeds are being released earlier than they have in the past into an unfavourable environment, where recruitment is unlikely due to increased competition for resources (Lamont et al. 1991; Enright et al. 2015).

A lack of access to water has been documented to increase follicle opening in serotinous cones prematurely without fire, but the extent of this depends on individual species.

When detached from a plant, a selection of Western Australian Banksia species had an increased proportion of their follicles open, compared to that of cones still attached to a plant receiving a water supply (Causley et al. 2016). This highlights the importance of 73 an available water source so that plants are able to maintain their closed follicles.

Furthermore, seed extraction for most species surveyed in this study occurred by severing fruits off the plant to release seed material. Again, this suggests that drier climates predicted for MTEs and reduced water access, may lead to increased rates of follicles opening. However, varying degrees of serotiny also play a role in the opening rates of fruits from both fire and restricted access to water (Lamont 1991).

5.3 Factors influencing variation in serotiny Serotiny is a dominant plant trait in MTEs globally (Lamont et al. 1991; Keeley et al.

2012), with the extent that serotiny is a trait adapted to dry climates or recurrent fires having been widely discussed. From this study, it is difficult to determine what plays a greater role in the maintenance of serotiny in the species surveyed, as whilst climate conditions have changed over time which have affected various demographic attributes, fire regimes have not been analysed and some sites surveyed were not of the same vegetation age. However, evidence from other MTEs globally can assist in determining potential factors driving serotiny.

Most evidence suggests that serotiny is driven by fire intervals. For example,

Leptospermum scaparium in New Zealand had a higher proportion of fruits closed at sites that had recurring fire and known fire histories, than those where fire was a rare event (Battersby et al. 2017). The presence of fire, also correlated with site climate conditions, with fire prone sites being located in warmer inland areas, whilst sites with no fire history were located in mesic coastal areas (Battersby et al. 2017). Similar patterns are described for Banksia serrata, Pinus rigida and P. torreyana, where a higher proportion of seeds were released in coastal sites compared to inland sites

(Givnish 1981; McMaster and Zedler 1981; Whelan et al. 1988). However, climate between the sites was not different which suggests that there are other factors that play an important role in determining degrees of serotiny.

Other, specific characteristics may influence serotiny and are often related to fire type.

Changes in plant height occur due to differences in climate with generally taller plants being found in more mesic locations, which correlate with weaker serotiny (Cowling &

Lamont 1985b; Pausas et al. 2004). This pattern is also consistent in some vegetation communities where coastal sites are taller and denser than those inland (McMaster &

Zedler 1981). These differences in plant height and density influence fire regimes which drives serotiny. Therefore, warming and drying climates, coupled with increased fire frequencies may also change future levels of serotiny.

5.3.1 Fire regimes and serotiny The region surveyed near Eneabba is dominated by stand replacing crown fires partly due to the persistence of sclerophyll leaves rich in flammable oils, and dense woody vegetation (Bradshaw et al. 2011; Fontaine et al. 2012). Similarly, the dominant fire type in MTEs are crown fires which are often of high intensity leading to most of the canopy being scorched depending on its height and fuel structure. By scorching most of the canopy this ensures that cones are able to be burnt, which in highly serotinous species is the main way seed release occurs (Lamont et al. 1991). This allows for mass recruitment immediately after a fire when there is little competition and a nutrient-rich ash bed (Lamont & Enright 2000). The plants surveyed in this study are subjected to these fire regimes.

To ensure the persistence of serotinous plant communities in areas with crown fires, long-term seed storage is essential for the ability of these plant communities to be able to self-replace (Lamont & Enright 2000). Due to the lack of successful recruitment in the inter-fire period, the proportion of cones/follicles staying closed is essential to prevent seed loss. Conversely, if this community were dominated by surface fires, there may be a greater release of seeds into the inter-fire period (Lamont & Enright 2000), so the importance of fruit production is more significant than follicles staying closed due to

75 potential recruitment in the inter-fire period (Cowling and Lamont 1985b). Therefore, higher levels of serotiny are favoured in crown fire ecosystems, such as those at

Eneabba, whilst other regimes with a tree and understorey layer have lower levels of serotiny due to the increased potential for inter-fire recruitment and increased portions of the canopy that survive fire (Enright et al. 1998).

5.3.2 Plant trade-offs and serotiny The effects of a warmer and drier climate, combined with more frequent fires in MTEs, will lead to changes in resource allocations and trade-offs. These changes will determine how plants adapt to and respond to changing climate conditions. Serotinous plants either release their seeds from the effects of drying, or from the heat of a fire

(Lamont 1991; Clarke et al. 2010). Fire in MTEs is the dominant disturbance leading to the release of seeds for self-replacement, and thus is well studied, but the role of water availability in maintaining follicles is less documented.

Through trying to understand variables leading to cone opening, older cones in Pinus halepensis have been found to open at lower fire temperatures and have a lower water content than younger cones, which suggests they are more weathered and contain a lower moisture content (Martin-Sanz et al. 2017). But for a selection of serotinous species from South Africa and Australia, it was found that degree of serotiny had no relationship with water content of cones (Cramer & Midgley 2009). Climate change for the region around Eneabba show that water availability has declined, and this may be causing the premature opening of follicles under drier climates, potentially due to a lower moisture content as observed in P. halepensis (Martin-Sanz et al. 2017) and B. attenuata in this study. Instead a larger proportion of a plants resources may be allocated to cones that are of a younger age which generally have increased viability as well as lower predation rates (Cowling & Lamont et al. 1985b; Lamont & van Leeuwen

1988), rather than older cones, assisting to ensure that when a fire goes through, seeds released will be of the highest viability and number to help maximise recruitment.

Furthermore, resource partitioning in serotinous South African fynbos species in regard to follicle opening have been investigated (Tonnabel et al. 2012), but these have little direct relationship to changes in climatic conditions and water availability. The plant life history traits and biology of these species are quite similar to Australian serotinous species, particularly Proteaceae species, so these findings are likely to be applicable to some of the species surveyed at Eneabba. It was found in fire prone ecosystems containing species with moderate-high levels of serotiny, that once plants have established and are likely to persist under normal growing conditions, the proportion of resources allocated to seed production, and in particular growth, begins to decrease, whilst seed maintenance begins to increase (i.e. resources are being directed towards keeping cones closed; Tonnabel et al. 2012). Therefore, if a fire goes through when plants have reached reproductive maturity, the greatest number of seeds will be available, thus maximising recruitment success (Enright et al. 1998; Tonnabel et al.

2012)

But, fire intervals are predicted to become shorter for MTEs (Moritz et al. 2012), which is likely to influence plant resource allocations. For plants to adapt to this and ensure self-replacement in the future, resources allocated to seed production may increase, whilst those allocated to seed maintenance may reduce (Tonnabel et al. 2012). This would be beneficial to plants under shortened fire regimes but the extent of this is variable depending on the time it takes for a plant to reach reproductive maturity. For example, more frequent fires may select for individuals that start to produce seeds at a younger age, but due to increased fire frequency they may be smaller in size, thus having less resources and producing fewer seeds. This may help to protect against immaturity risk as at the time of fire (and recruitment) and ensure they have a viable 77 seedbank. Or, for plants that take longer to reach reproductive maturity, by producing more seeds, instead of maintaining seeds, this may ensure that there is enough time under reduced fire regimes to maximise seed production to ensure self-replacement

(Tonnabel et al. 2012). However, whilst it is understood how fire regimes influence resource partitioning in serotinous species, there is little understanding of how different climatic conditions, in particular restricted access to water, determine the trade-offs a plant will use to ensure its persistence into the future. Therefore, determining the combined effects of more frequent fire regimes and warmer and drier climates on degree of serotiny needs to be examined, and separating the impacts of these to changes in serotiny is essential.

5.4 Limitations and future implications Whilst this study does provide some evidence for the interval squeeze model based on demographic attributes surveyed, findings need to be treated with caution. The use of past data for comparison purposes was essential for this study, however differences in sampling design and site selection could potentially lead to differences in results.

Furthermore, lack of data regarding standard errors for past data collection dates makes it difficult to determine to extent of changes between them and 2018 data. Additionally, due to the variation in sampling design between studies, as well as lack of complete information on how past studies were conducted, determining a sampling method that was relatable to all of these different sampling designs was difficult. Therefore, the sampling methods used here were based around getting the data required to compare to past studies, as well as within resource and time allocations.

One major limitation to this study was that for some sites surveyed, vegetation age was not similar to that of the previous studies. This was particularly relevant to the aging of

Banksia cones. For B. attenuata plants surveyed, site ages varied 8-10 years from that when surveyed in 1983, whilst B. tricuspis site ages were either 11 or 12 years different.

There is little doubt that these differences in site ages have an impact on the total cone/seed production per plant, with plant maturity/senescence potentially influencing this. But as cones were aged, follicle opening rates may only be different if plants are beginning to experience senescence, so the role of this needs to be acknowledged.

Furthermore, potential error in cone age estimations due to a lack of experience with the stem node cone aging methodology may be possible.

It is difficult to determine specific reasons why all of the changes observed in this study may have occurred. Nonetheless, this is a starting point in beginning to understand the role that climate change has in influencing plant demography, particularly seed demography in serotinous plants. Determining this is vital for the persistence of serotinous species, particularly non-sprouting species which rely on seeds for recruitment. If past data are available for demographic attributes in other serotinous species, there should be a re-census of them to determine any potential changes in demography due to climate change. As various demographic attributes are related and play a role in a plants persistence which determine plant community structure, studies on different plant demographical behaviour such as mortality or time to reproductive age in response to changes in climate conditions need to occur. By analysing various responses to climate change, a holistic understanding of the effect of climate change on serotinous plants can be gained, which in turn will help to ensure these species are able to persist in the future.

The findings in this study suggest that changes in plant demography in serotinous species in Western Australia due to a warmer and drier climate are occurring. However, the extent of this is variable depending on attribute studied. Seed production over time, was variable between species, but in general there was a pattern of a decrease in seed production due to a warmer and drier climate. Fruit production however had significantly reduced for most species surveyed, alongside with cones in Banksia 79 species releasing their seeds at earlier ages than that in the past. This provide evidence for potential population collapse due to climate conditions becoming warmer and drier, however the importance of fire intervals cannot be ignored. Whilst further research is essential to better understand the effects of climate change on seed demography, this study provides a starting point in beginning to understand the role changing climates have on seed demography and how plants may need to adapt to this to ensure their persistence.

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7. Appendices

7.1 Characteristics for ageing of Banksia tricuspis cones Whilst collecting field data on Banksia tricupsis cones, different cone characteristics were used to age them. These were mostly based off weathering.

Table 7.1: Cone charactersitcs that assisted in the aging of Banksia tricuspis cones.

Cone age Age descriptors Example cone

One year - Fluff on cones - No follicles open - Leaves at base - Terminal

Two year - Grey in colour - Drier leaves at base - No or very few follicles open - Not terminal

Three year - Darker grey than one and two year old cones - Multiple follicles open, with most brown inside - Located further down the branch - No leaves

Four years and - Almost black in older colour - Most follicles open and are dark grey rather than brown inside - Peeling back of grey on most follicles - Located further down the branch

7.2 Fruit production summary for plants surveyed in 2018 Field data for all species is shown below for all sites surveyed. Species are organised by

plant family.

Table 7.2: Field data summary of species surveyed near Eneabba, Western Australia in 2018. Data are means with 95% confidence intervals.

Fire % Site Site Height Width Closed Species Family response Open Fruits closed Codes* age (cm) (cm) Fruits strategy fruits

Banksia attenuata Proteaceae Resprouter Jurien 23 190 ± 20 4.0 ± 2.4 6.1 ± 2.5 39.6 SER 28 129 ± 6 1.3 ± 0.7 4.4 ± 1.4 22.8

SEC 16 137 ± 7 186 ± 18 19.1 ± 6.1 6.7 ± 1.9 74 HD 20 130 ± 63 188 ± 41 3.8 ± 1.2 5.1 ± 1.8 42.7 MARPlot1 25 155 ± 13 14.7 ± 4.8 7.9 ± 2.4 65 MARPlot2 16 119 ± 5 12.8 ± 3.0 0.5 ± 0.4 96.2 MARPlot3 7 96 ± 5 5.1 ± 1.3 0 100

Banksia hookeriana Proteaceae Non-sprouter SER 28 158 ± 7 12.3 ± 2.3 5.3 ± 1.1 66.1 HD1 20 147 ± 7 12.1 ± 2.6 5.0 ± 1.3 70.8

MARPlot1 25 184 ± 15 19.8 ± 5.2 4.9 ± 2.0 71 MARPlot2 16 117 ± 5 18.4 ± 4.2 1.6 ± 1.3 92 MARPlot3 7 81 ± 7 4.7 ± 2.4 0 100

Hakea psilorrhyncha Proteaceae Non-sprouter SEC 16 195 ± 11 5.9 ± 2.9 1.1 ± 0.9 84.3

Coom 16 219 ± 17 24.0 ± 6.1 11.5 ± 4.7 67.6

SELn 16 234 ± 12 10.1 ± 3.8 0.6 ± 0.5 94.4 SELs 16 184 ± 10 21.3 ± 6.6 1.3 ± 0.5 94.2 Xylomelum angustifolium Proteaceae Resprouter SEC 16 376 ± 19 264 ± 23 50.2 ± 11.4 1.2 ± 0.6 97.7

HD 20 334 ± 16 318 ± 29 37.3 ± 11.7 3.3 ± 2.2 91.9

Beaufortia elegans Myrtaceae Non-sprouter SEC 16 89 ± 4 107.7 ± 22.4 62.8 ± 24.4 63.2

HD 20 86 ± 4 108.1 ± 22.3 132.8 ± 31.6 44.9

Coom 16 76 ± 4 31.1 ± 5.7 37.5 ± 11.1 45.6

SELn 16 64 ± 3 57.3 ± 15.8 32.5 ± 12.2 63.8

SELs 16 69 ±4 70.9 ± 20.9 45.3 ± 13.7 61.1

Eremaea beaufortioides Myrtaceae Resprouter SEC 16 90 ± 5 74 ±12 7.4 ± 3.8 8.0 ± 3.7 48.1

HD 20 93 ± 6 111 ± 13 160.2 ± 46.2 14.2 ± 5.7 91.7 SELn 16 49 ± 3 72 ± 8 15.7 ± 5.9 3.9 ± 2.3 80.1 SELs 16 82 ± 6 80 ± 9 43.3 ± 10.3 8.5 ± 2.6 83.6 Eremaea violacea Myrtaceae Non-sprouter SEC 16 35 ±4 58 ± 11 22.6 ± 18.7 8.5 ± 6.9 72.7 HD 20 52 ± 21 86 ± 32 62.4 ± 42.1 8.8 ± 8.5 86.9 SELn 16 49 ± 6 89 ± 12 122.2 ± 83.2 17.3 ± 11.6 87.6 SELs 16 44 ± 3 72 ± 5 44.1 ± 18.7 7.6 ± 6.6 85.3 58 ± 30 22 ± Leptospermum spinescens Myrtaceae Non-sprouter SEC 16 82 ± 23 6.5 ± 9.4 77.2 47.5 HD 20 72 ± 12 56.5 ± 18 20.5 ± 14.2 10.4 ± 10.2 66.3 SELn 16 72 ± 6 40 ± 5 21.1 ± 10.1 12.7 ± 5.8 62.4 SELs 16 75 ± 5 31 ± 4 12.9 ± 3.9 5.6 ± 2.5 69.7 Melaleuca leuropoma Myrtaceae Non-sprouter SEC 16 52 ± 3 56 ± 6 13.9 ± 6.4 12.9 ± 4.8 51.9

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HD 20 50 ± 3 71 ± 6 40.8 ± 16.2 27.6 ± 9.1 60.5 SELn 16 39 ± 3 35 ± 3 14.1 ± 5.7 2.8 ± 1.1 84.4 SELs 16 47 ± 3 58 ± 5 78.6 ± 20.1 31.3 ± 9.0 71.5

Melaleuca scabrum Myrtaceae Non-sprouter SELn 16 34 ± 2 60 ± 7 20.6 ± 5.0 7.6 ± 1.9 73 SELs 16 27 ± 2 50 ± 6 6.1 ± 2.1 3.5 ± 1.2 63.5 *= Substrate type; SEC/HD=sand, SELs/n=Laterite (SE=South Eneabba)

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7.3 Total viable seed production per plant surveyed in 2018

Summary of seed demography for all serotinous species surveyed in 2018 near

Eneabba, Western Australia, with species organised by plant family (mean ± 95%

confidence intervals)

Table 7.3 Laboratory data summary, including viable seeds per plant for species surveyed near Eneabba, Western Australia in 2018.

Time since fire Seeds Follicles/Nuts Germination Viable seeds Species Site (years) follicle/fruit-1 cone/cluster-1 rate (%) plant-1 Banksia attenuata (P;RS) Jurien 35 2 ± 0 6.4 ± 0.5 77.2 ± 8.6 25.2 ± 17.3 SE Reserve 28 2 ± 0 6.6 ± 0.3 83.9 ± 9.4 11.5 ± 4.3 SE Crest 16 2 ± 0 6.6 ± 0.3 83.9 ± 9.4 28.14 ± 5.31 High Dune 20 2 ± 0 6.6 ± 0.3 83.9 ± 9.4 33.8 ± 10.4 Mt Adams 1 25 2 ± 0 7.1 ± 0.4 83.9 ± 9.4 136.1 ± 41.7 Mt Adams 2 16 2 ± 0 7.1 ± 0.4 83.9 ± 9.4 119.2 ± 34.8 Mt Adams 3 7 2 ± 0 7.1 ± 0.4 83.9 ± 9.4 47.1 ± 9.2 Banksia hookeriana (P;NS) SE Reserve 28 2 ± 0 7.3 ± 0.7 96.3 ± 6.1 84.9 ± 17.4 High Dune 20 2 ± 0 7.3 ± 0.7 96.3 ± 6.1 90 ± 19.4 Mt Adams 1 25 2 ± 0 7.3 ± 0.7 96.3 ± 6.1 164.8 ± 58.6 Mt Adams 2 16 2 ± 0 7.3 ± 0.7 96.3 ± 6.1 152.4 ± 43.2 Mt Adams 3 7 2 ± 0 7.3 ± 0.7 96.3 ± 6.1 38.9 ± 10.8 Banksia tricuspis (P;RS) Carpark 35 2 ± 0 24.3 ± 15.2 85.2 ± 6.7 129 ± 42.2 North 7 2 ± 0 17.1 ± 10.9 100 ± 0 207.5 ± 68.5 South 7 2 ± 0 20.6 ± 11.9 93.9 ± 12.1 126.5 ± 37.7 Hakea psilorrhyncha (P;NS) SE Crest 16 2 ± 0 1 ± 0 70 ± 21.5 8.2 ± 4.0 Coomallo 16 2 ± 0 1 ± 0 55 ± 21.8 26.4 ± 6.7 SE Laterite North 16 2 ± 0 1 ± 0 80 ± 21.3 16.7 ± 6.1 SE Laterite South 16 2 ± 0 1 ± 0 80 ± 21.3 34.1 ± 10.6 Xylomelum angustifolium (P;RS) SE Crest 16 2 ± 0 1 ± 0 95* ± 0 95.3 ± 21.7

High Dune 20 2 ± 0 1 ± 0 95* ± 0 70.8 ± 22.2 Beaufortia elegans (M;NS) SE Crest 16 8.5 ± 3.7 14.8 ± 1.9 93.7 ± 8.7 854.5 ± 177.8 High Dune 20 5.2 ± 1.4 13.7 ± 1.8 92.4 ± 6.9 517 ± 106.4 Coomallo 16 7.9 ± 2.2 15.6 ± 0.3 91 ± 16.1 222.5 ± 72.6 SE Laterite North 16 4.2 ± 1.7 15.7 ± 1.1 96.8 ± 4.2 231.5 ± 63.8 SE Laterite South 16 4.2 ± 1.7 15.7 ± 1.1 96.8 ± 4.12 286.6 ± 84.3 Eremaea beaufortiodes (M;RS) SE Crest 16 1.8 ± 0.5 1 ± 0 72.1 ± 8.3 9.7 ± 4.9 High Dune 20 3.0 ± 0.4 1 ± 0 56.4 ± 10.1 274.6 ± 79.2 SE Laterite North 16 2.9 ± 0.5 1 ± 0 74.8 ± 7.2 33.8 ± 12.7 SE Laterite South 16 2.9 ± 0.5 1 ± 0 74.8 ± 7.2 92.4 ± 21.9 102

Eremaea violacea (M;RS) SE Crest 16 1.2 ± 0.4 1 ± 0 67.9 ± 16.3 34.7 ± 28.7 High Dune 20 1.2 ± 0.4 1 ± 0 67.9 ± 16.3 52 ± 35.1 SE Laterite North 16 2.6 ± 0.4 1 ± 0 84.4 ± 41.7 233.2 ± 158.8 SE Laterite South 16 2.6 ± 0.4 1 ± 0 84.4 ± 41.7 84.1 ± 35.7 Leptospermum spinescens (M;RS) SE Crest 16 8.6 ± 1.2 1 ± 0 53.7 ± 23 101.5 ± 219.8 High Dune 20 8.6 ± 1.2 1 ± 0 53.7 ± 23 94.6 ± 67.1 SE Laterite North 16 8.1 ± 1.5 1 ± 0 42.8 ± 16.7 72.5 ± 34.7 SE Laterite South 16 8.1 ± 1.5 1 ± 0 42.8 ± 16.7 44.7 ± 13.7 Melaleuca leuropoma (M;RS) SE Crest 16 13.4 ± 5.3 11.5 ± 1.9 89.9 ± 6.4 162.3 ± 75.5 High Dune 20 10 ± 2.6 8.9 ± 1.1 80.9 ± 20.8 331.9 ± 131.5 SE Laterite North 16 9.9 ± 1.9 11.9 ± 0.9 77.6 ± 4.3 107.9 ± 43.5 SE Laterite South 16 9.9 ± 1.9 11.9 ± 0.9 77.6 ± 4.3 601.8 ± 154.1 Melaleuca scabrum (M;RS) SE Laterite North 16 10.3 ± 2. 12.3 ± 0.3 89.9 ± 6.4 190.4 ± 46.3

SE Laterite South 16 10.3 ± 2.0 12.3 ± 0.3 89.9 ± 6.4 55.9 ± 19.1 *Data from Enright et al. (2007) M=myrtaceae, p=proteaceae; RS=resprouter, NS=non-sprouter Sites: Substrate type with year of last fire, SEC/HD=sand, SELs/n=Laterite, SE=South Eneabba

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