Effects of factors associated with the season of a fire on germination of species forming soil seedbanks in the fire-prone Hawkesbury Sandstone region of Sydney, Australia.

Paul Bengt Thomas B.Sc., The University of Adelaide B. App. Sc. (Hons), The University of Adelaide

A thesis submitted for the degree of Doctor of Philosophy University of Western Sydney October, 2004

i Declaration

This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text

I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying

Paul Bengt Thomas October 2004

ii Acknowledgements

I would like to thank my supervisors, Charles Morris and Tony Auld for their help and interest throughout this project. This project has benefited from discussions with many people, especially Tony Haigh, and from the technical assistance of Burhan Aramiji. I gratefully acknowledge the help with identification of plant species by staff at the Sydney Botanical Gardens. This work was conducted with the financial assistance of an Australian Postgraduate Award, as part of an ARC SPIRT Grant, and the New South Wales National Parks and Wildlife Service as the industry partner. Seeds were collected under New South Wales National Parks and Wildlife Service Scientific Investigation Licence Number B2063. I would like to thank Chris, Clint, Justin, Matthew and Steve for believing in me. Thank you Stephanie for your love and support during this project.

iii

Declaration...... ii Acknowledgements...... iii Abstract ...... v Chapter 1. General introduction...... 1 Chapter 2. Effects of fire-related germination cues ...... 10 2.1 Introduction...... 10 2.2 Methods...... 18 2.3 Results...... 30 2.4 Discussion ...... 56 Chapter 3. Effects of pre- and post-fire temperature...... 68 3.1 Introduction...... 68 3.2 Methods...... 79 3.3 Results...... 101 3.4 Discussion ...... 159 Chapter 4. Effects of pre-fire hydration status...... 170 4.1 Introduction...... 170 4.2 Methods...... 176 4.3 Results...... 185 4.4 Discussion ...... 205 Chapter 5. Effects of post-fire water...... 211 5.1 Introduction...... 211 5.2 Methods...... 221 5.3 Results...... 236 5.4 Discussion ...... 272 Chapter 6. Effects of fire on germination of aged seed...... 286 6.1 Introduction...... 286 6.2 Methods...... 291 6.3 Results...... 293 6.4 Discussion ...... 306 Chapter 7. General discussion...... 309 References:...... 314 Appendix 1...... 345

iv Abstract

Fire is a recurrent disturbance that removes above ground vegetation in many locations throughout the world, including the Sydney region. Many species in fire-prone locations, and most species in the Sydney region, form soil seedbanks and regenerate through post-fire germination. However, a germination response is determined by the fire regime acting as a selective pressure over a sufficient period of time, rather than a single fire. The components of the fire-regime are intensity, season, type and frequency. The natural fire- regime is dominated by warm-season fire, but management burning is conducted in cooler seasons. Cool season burning produces lower levels of germination than warm season fires in a number of locations with Mediterranean-type climate, but the effects of cool season burning on species composition in the relatively aseasonal Sydney region is unknown. An experimental approach was adopted to address this lack of knowledge. Fire can be simulated using heat shock and smoke (fire cues), and the seasonal factors of temperature and water- availability can be reproduced in the laboratory. I have investigated the effect of various combinations of heat shock and smoke, of various pre-and post-fire cue temperatures, of pre- fire cue hydration status, of various post-fire cue water availabilities, and of accelerated aging before application of fire cues on germination of a number of species forming soil seedbanks in the Sydney region. A degree of primary dormancy was overcome in most species by the combination of heat shock and smoke in the current investigation. Fire intensity is expected to influence germination, as germination of most species was increased by the combination of heat shock and smoke within a narrow heat shock range. Consequently, both post-fire germination and a residual soil seedbank are expected, with the residual soil seedbank providing a buffer against short fire-return intervals. Season of fire is also expected to influence species composition because both temperature and water availability affected germination in experiments. Ambient temperature affected germination directly, and possibly through secondary dormancy. Germination of a similar proportion of species was favoured by the ambient soil temperature during cooler seasons, as was favoured by warmer season temperature, as was favoured by neither temperature. Generally the effect of temperature was of low magnitude. When temperature had a major effect, then the fire-related germination cues extended the range of ambient temperature over which germination could occur, but was sub-compensatory for optimal temperature. Thus, a similar proportion of species would be expected to germinate over

v different season of fire, but species composition would be expected to change with repeated single-season fire. The fire-related germination cues were promotive for a finite duration of post- treatment incubation, thus species that are not favoured by the ambient temperature at the time of the fire are likely to not germinate, rather than merely delay germination until a change of season. When water was intermittently available prior to continuous hydration, then the influence of temperature was strengthened in the single species investigated. The fire-related germination cues followed by dry storage prior to hydration probably caused secondary dormancy. This contrasts with species from Mediterranean-type climates (where the natural fire season and season with adequate rainfall for seedling recruitment are separated by months) where the promotive effects of the fire-related germination cues are retained during dry storage. The Sydney region is aseasonal for rainfall. Rainfall adequate for seedling recruitment probably occurs in most months of most years. An induction of secondary dormancy by fire-related germination cues followed by low water availability could prevent germination within unfavourable microsites. The fire-related germination cues cause seeds to germinate more rapidly for a given water availability, and to require more water availability for germination to occur. The seeds that accumulate sufficient hydration to germinate whilst the fire-related cues remain promotive will be seeds in favourable microsites. The most important aspect of microsite quality is the degree of contact between seed and soil, which is largely determined by the size of the soil particles next to the seed. Soil particle sizes were determined and the potential for great variability in water availability at the scale of the seed size noted. Whilst all the seed of species that are stimulated to germinate across a wide range of heat shock levels could potentially germinate after any one fire, the restriction of germination to more favourable microsites would ensure both post-fire germination and a residual soil seedbank. As noted above, the residual soil seedbank provides a buffer against short fire-return intervals. The heat shock tolerance of organisms has always been reduced by hydration in previous studies, usually extremely so across many different systems, including seeds. In keeping with all previous studies, the heat shock tolerance of dry habitat species was greatly reduced by prior hydration. In contrast, the first positive responses to heat shock whilst hydrated were found in wet habitat species, and the tolerance of wet habitat species to heat shock whilst hydrated was greater than in all previous studies of the interaction for eukaryotic organisms. Seeds within wet habitat occasionally experience high levels of heat shock whilst hydrated, and the differential tolerance of the interaction could account for the segregation of

vi wet and dry habitat species that form water-permeable soil seedbanks in fire-prone environments. Consequently, fire when soil is wet or dry could be considered different fire types. Post-fire residual soil seedbanks buffer species against local depletion or extinction due to unfavourable fire regimes. Given that species composition may depend on fire season, a residual seedbank is crucial to buffer species against local depletion or extinction due to repeated consecutive fires in an unfavourable season. The relative contributions of old and young seed to post-fire germination are unknown. Germination of seeds that were rapidly aged under conditions of high temperature and humidity was increased by the fire-related germination cues even though apparent viability was greatly reduced. If naturally aged seeds are also heat shock tolerant and germinate in response to the fire-related germination cues, then post-fire recovery is less dependent on the more recently produced seed. Seasonally related factors likely to affect post-fire germination of many species in the Sydney region have been identified. The level of heat shock experienced by a seed during fire can be predicted by its depth of burial and the intensity of fire at that location, and the germination response of the seed at that location can be predicted by its response to the combination of heat shock and smoke. Both pre- and post-fire temperatures are also predicted to affect germination of soil-stored seed, and temperature effects may be greatly exacerbated by water availability. The size of soil particles immediately adjacent to a seed is expected to largely determine its level of water availability, and thus the probability that it will germinate. The interaction between ambient temperature and the level of heat shock may affect germination. Soil water content, and thus seed moisture content at the time of a fire may interact with the level of heat shock to affect both germination and survival of a seed. The age of a seed may also affect its germination response to fire. The above factors are predicted to affect the germination of species differently, and thus season of fire is expected to alter species composition. For example, dormancy of Kunzea ambigua is expected to be broken regardless of depth of burial or intensity of fire, but germination is predicted to be considerably higher following autumn fire compared with other seasons, particularly if post- fire water availability is low. Kunzea ambigua is predicted to be eliminated from areas where soil moisture is high at the time of fire, and the proportion of recently produced seed that germinates after fire is predicted to be greater than the proportion of older seed that germinates. Such predictions can be readily field-tested.

vii Chapter 1. General introduction

Fire regime

Crown fire shrublands experience recurrent stand-replacing fires in many locations throughout the world. A substantial fraction of the species in fire-prone locations form soil seedbanks and regenerate through post-fire germination. Most species in the Sydney region have poorly dispersed seed and regenerate from an in situ soil seedbank (Auld 1994). Germination is necessarily linked to the passage of fire, through a response to fire-related cues. However, a germination response is not determined by an individual fire, but by the fire regime, the components of which are intensity, season, type and frequency (Gill 1975). The impact of the fire regime, and in particular the role of fire season on germination is better known for regions with a Mediterranean-type climate than for the relatively aseasonal Sydney region, and is better known for serotinous species than for species that form soil seedbanks. The effect of the season of fire is unknown for the majority of species in fire-prone plant communities of southeastern Australia. Thus, management or hazard reduction burning that is currently being conducted in the Sydney region during cooler months cannot be assessed against the aim of management of vegetation for biodiversity, the conservation of all species in a plant community. The differences between the natural summer-dominated fire regime (Luke & McAuthur 1978; McLoughlin 1998) and burning in cooler months have not previously been investigated for the Sydney region, although burning across different cooler seasons altered species composition (Spring cf Autumn; Clark 1988). The impact on plants with soil seedbanks was investigated because they are the functional group most likely to be affected by fire season. Various aspects of the fire regime were investigated within the current study, with an emphasis on elucidating the fire-related germination cues for a number of species, how the seasonal components of temperature and water availability interact with these cues, and whether the cues still operate when the seasonal components are changed after a period of time. A laboratory-based study can generate species-specific predictions regarding the effects of seasonally dependent environmental variables, and these predictions can be field-tested (Grime et al. 1981). If the results of this study indicate that there is likely to be a seasonal effect of fire, then management of over 500,000 ha of reserved bushland in the Sydney area requires field-testing of the laboratory-based predictions.

1

Fire Intensity

Fire intensity varies as an average across individual fires, and across the landscape within individual fires (Christensen & Kimber 1975; Fox 1978; Hobbs & Atkins 1988; Atkins & Hobbs 1995). Also, the heat generated above ground is rapidly attenuated with increasing soil depth (Auld 1986a; Raison et al. 1986; Bradstock & Auld 1995). Thus, the heat experienced by seed within soil is highly variable. Whilst there is a general relationship between the average intensity of a fire and soil temperature across a landscape, the major determinant of heat experienced by a seed is its depth of burial and the fuel quantity, type and spatial array, and degree of combustion at that location (Bradstock et al. 1992; Bradstock & Auld 1995). Seed experiences the heat transfer as a shock, due to its sudden and disturbing physical and physiological effects. Seed is concentrated in the upper layer of soil, which is also the zone from which seed reserves allow seedling emergence (Bond et al. 1999). A wide range of heat is generated by fire within the zone of seed location, and the germination response of seed across a heat shock range largely determines a species population dynamics. A narrow response zone for heat shock in the seed can result in both germination and a residual seedbank (Tozer 1998). A species response to heat shock is likely to reflect the typical heat generated by fires within its habitat. Excessive heat shock can result in seed death or dormancy. However, investigation across the appropriate range of heat shock has only been adequate for leguminous species within Australia (e.g. Auld 1986a; Auld & O’Connell 1991), with one exception (Auld et al. 1993). Combustion products such as smoke also provide a reliable cue of fire-passage, and promote germination of species from both fire-prone (Brown 1993; Dixon et al. 1995; Roche et al. 1997a, b) and non fire-prone regions (Drewes et al. 1995; Pierce et al. 1995; Thomas & van Staden 1995). Seeds in the soil experience both heat shock and smoke during a fire, but few investigations of the interactive effect of heat and smoke on germination of species forming soil seedbanks in the Sydney region have been conducted (Kenny 2000; Morris 2000; Kenny et al. 2001; Willis et al. 2003). No study has investigated the interaction between these factors over a range of levels of both factors; work to date has typically used one high temperature in the heat shock zone and one level of smoke application. However, an investigation of the interactive effects of heat shock and smoke over a range of levels is essential to understanding the effect of fire on germination from the soil seedbank, and formed part of the current study.

2 The effect of fire-related germination cues has not commonly been assessed for multiple populations within a species (Auld & O’Connell 1991; Kenny et al. 2001). However, populations may have different responses due to random factors such as genetic drift (Cowling 1987), or different selective pressures due to different fire-regimes at different locations (Parker & Kelly 1989). Fire regimes may be different due to random factors, particularly over the short-term, or due to interactions between fire and landscape features such as topography. Populations in different habitats may have experienced different selective factors, including different fire-regimes. The response of a population to fire-related germination cues may be most meaningfully interpreted in relation to the fire regime of the habitat in which it is located. Inter-population variability in response to fire-related cues and climactic factors was addressed for a number of species in the current study.

Fire Season

The season of a fire can potentially affect post-fire germination from soil seedbanks. The critical factors affecting germination are temperature and water availability (Mayer & Poljakoff-Mayber 1989). Seedling survival is frequently limited by water availability, however water may be available for long enough to allow germination, but not for subsequent seedling establishment in an unfavourable season. Both the resource storage and gathering capacity of seedlings are limited, hence they are particularly vulnerable to environmental stresses (Watt 1978, 1982; Hassanyar & Wilson 1978; Fulbright et al. 1984; Frasier 1987). Seed dormancy prevents germination when conditions are favourable for germination but not for seedling survival (Vleeshouwers et al. 1995). Because temperature is a more reliable indicator of prolonged water availability, dormancy is strongly temperature dependent (Vleeshouwers et al. 1995). The temperature prevailing before a fire, and the subsequent changes in temperature due to both the changed conditions and the changing of seasons after a fire may affect dormancy, hence germination from the soil seedbank. Because 1) there is strong selective pressure for species that rely on post-fire seedling recruitment to germinate 2) recruitment is enhanced within a post-fire environment (Bond 1984) 3) the correlation between temperature and water availability is relatively weak in the Sydney region (Nix 1982),

3 the null hypothesis that post-fire germination is independent of ambient temperature, an indicator of season, is worth investigating. Season of fire strongly affected regeneration of serotinous species from seed that is deposited on the soil surface after fire. Seedling establishment of serotinous species is higher following summer-autumn fire compared to fire in winter or spring (Bond et al. 1984; McMahon 1984; Cowling & Lamont 1987; Midgley 1989). Winter or spring fires are of lower intensity than summer fire, hence fewer seeds are released (Bradstock & Myerscough 1981; Enright & Lamont 1989), and those that are released encouter low levels of moisture that limit germination during the following summer. High summer temperatures may cause death (Cowling & Lamont 1987) or dormancy (Specht et al. 1958; Bond 1984; Bradstock 1985; Gill & McMahon 1986; Cowling & Lamont 1987) of seed that is also subject to predation whilst on the soil surface (Bond 1984; Cowling & Lamont 1987). Seasonal effects of temperature and moisture availability are present in the Sydney region, but are probably less pronounced than in Mediterranean-type climatic regions. Also, the seasonal effects of temperature and moisture are less pronounced for seed within the soil than seed that is on the soil surface (le Maitre & Midgley 1992). Seed release and seed predation are factors affecting post-fire germination of serotinous species, not species that form soil seedbanks. Seasonal effects on regeneration from the soil seedbank were considerably more pronounced within Mediterranean-type climatic regions (Horton & Kraebel 1955; Boucher 1981; Bond 1984; McMahon 1984; le Maitre 1988; Hobbs & Atkins 1990; de Lange & Boucher 1993b; Roche et al. 1998) than within the Sydney region (Clark 1988). Burning across different cooler seasons (Spring cf Autumn) affected species composition in the Sydney region (Clark 1988). Although the fires were replicated twice, environmental factors were not manipulated in the field and so correlations alone were used to assess any relationship between seasonal factors and germination (Clark 1988). Both fire-related germination cues and seasonal factors can be finely controlled within the laboratory, allowing testing of hypotheses about relationships between the two. Results derived from laboratory experiments can be used to make predictions about germination expected following fire in any season rather than the particular season(s) in which a field study was conducted; this approach was taken in the current study (Grime et al. 1981). Field studies critically test the predictions of laboratory-based models, including the degree to which the factors investigated account for the field germination, and should be the next step of investigation in this area. The co-incidence of results from a laboratory and field-based study is strong evidence for deterministic effects of season of fire.

4 Water availability is more limiting of germination at sub- or supra-optimal temperatures (e.g. Sharma 1976; Weerakoon & Lovett 1986). However, water availability in the Sydney region is not strongly seasonal; evaporation is seasonal, but rainfall is only slightly seasonal. Therefore, post-fire germination might be independent of the interaction between temperature and water availability, but this null hypothesis must be tested. Three contrasting scenarios can be imagined. If the influence of temperature is largely independent of water availability, then post-fire germination is more a consequence of season per se rather than a particular season. If the influence of water availability is largely independent of temperature, then post-fire germination is poorly related to season (only to the degree that water availability is dependent on season). If the influence of water availability and temperature are interdependent, then post-fire germination is more a consequence of water availability in a particular season rather than season (i.e. ambient temperature) per se. The fire-related cues may exacerbate, ameliorate or not influence the effects of the seasonal factors. There is a strong selective pressure for a fraction of the seedbank of species that rely on post-fire seedling recruitment for local persistence to remain dormant and buffer against short fire-return intervals (Bell et al. 1995; Bradstock et al. 1997). Ways in which both germination and dormancy could be assured include a germination response within a narrow heat shock range that restricts the number of seeds that are stimulated in any single fire (Auld 1987; Auld & O’Connell 1991; Tozer 1998). Species that are stimulated to germinate across a wide heat shock range must have properties that restrict germination of a fraction of the seedbank. Various mechanisms, including a range of dormancy levels could result in the maintenance of a residual seedbank (Roberts 1972a). However, there is probably a strong selective pressure for mechanisms that produce germination in high quality microsites, where the probability of subsequent seedling survival is high (Bewley & Black 1982a; Rice 1985; Fenner 1995). The most important parameter of the safe-site is soil moisture availability (Cook 1979), and it is a factor that has been strongly associated with germination in the field (Battaglia & Reid 1993). The interaction between water availability and fire-related germination cues has not previously been investigated, and was addressed in the current study. If fire-related germination cues reduce the amount of water availability required for germination of a population (median base water potential), then the distribution of these cues within the soil, rather than soil moisture dynamics will largely determine which seeds do and do not germinate.

5 Time is a component of water availability, and germinability of seed in microsites with higher water availability is maintained for longer (Battaglia & Reid 1993). Fire-related germination cues could increase post-fire germination by reducing the duration of hydration that is required for germination of a population (mean hydrotime requirement), whilst a range in the amount of water availability required for germination is functionally equivalent to a range of dormancy levels. A study of the possible mechanisms whereby fire-related germination cues could influence the effect of water availability on post-fire germination has not previously been undertaken. A reduction in the amount of water availability required for germination of a population would be consistent with the regulation of germination by fire- related germination cues per se, whilst a reduction in either the variance of the amount of water availability required for germination of a population, or in the mean duration of hydration that is required for germination of a population would be consistent with a strong environmental regulation of post-fire germination. Water availability is determined by the degree of contact between a seed and soil and by the conductance of water in this zone (Collis-George & Hector 1966; Dasberg & Mendel 1971; Hadas & Russo 1974b); the former factor is considered more important (Bewley & Black 1982a). The resistance of water flow to the seed imposed by the seed-soil interface conditions increased as the seed wetted area, or the soil water conductivity, or both decreased (Hadas 1974, 1976; Hadas & Russo 1974b). The resistance to water flow to the seed increased as the coarseness of the soil texture increased, resulting in a decrease in the rate and final percentage germination (Hadas 1974, 1977). An approximate measure of the degree of contact between the seed and soil particles can be determined from the relative sizes of these entities (Collis-George & Hector 1966), and an approximate measure of hydraulic conductivity can be determined from soil texture (Young & Nobel 1986). However, variability in soil water content (Garrido et al. 1999) and the pathway of soil water flow (Garrido et al. 2001) have been measured by fibre optics at a scale likely to determine germination of an individual seed in the soil, and the change in water availability through time has determined whether or not a seed would remain germinable (Battaglia & Reid 1993). Post-fire germination and retention of a seedbank must be considered in context of both the average and variance in climate and soil texture, and the duration of water availability relative to the duration over which cues remain active, and interactions between these factors. The results of previous studies and the current study may allow the construction of a preliminary theoretical model that could readily be improved and tested.

6 The effect of season of fire on regeneration is confounded by the effect of season on intensity in field studies (Gill 1975). Investigation of fire-related germination under laboratory conditions allows a decoupling of season and intensity effects. Although investigations under laboratory conditions are limited by the assumptions that the important seasonal factors are included and that additional field-related factors are not overly important, decoupling of season and intensity effects is not feasible under field conditions. It is not feasible to record heat shock at enough locations to replicate particular levels of heat shock within and between seasons over replicate seasons. The results from laboratory investigations could be tested in the field using constructed seedbanks, a blow-torch and thermocouples, but the laboratory investigation provides the best first line of attack for the questions to be addressed. The influence of season of fire per se on germination can be, but has not previously been investigated independently of fire intensity.

Fire Type

Fire type as a component of the fire regime is distinguished as above- and below- ground (Gill 1975; 1981). Because the relationship between heat shock and hydration is extremely negative (Just 1877; Sweeney 1956; Altman & Benson 1960; Ghaley & Taylor 1982), it is appropriate to also distinguish between fire in wet and dry habitat. The interaction between heat shock and hydration has previously been inappropriately investigated because physiological factor(s) such as time of hydration have not been included in investigations, probably because the negative interaction has been attributed to physical factors. The conclusive findings from inappropriate investigations have probably precluded further investigation, despite the fact that the water-permeable seed of wet habitat species is subject to high levels of heat shock whilst hydrated. Seed is inevitably dispersed across the landscape on an evolutionary time scale, hence the segregation of wet and dry habitat species requires explanation. The absence of wet habitat species in dry habitat has been attributed to the low tolerance of seedlings to drought (Clarke et al. 1996; Myerscough et al. 1996). The absence of dry habitat species in wet habitat remains unexplained, but could be due to the low tolerance to heat shock of hydrated seed of dry habitat species. This question was investigated in the current study.

7 Fire Frequency

Fire frequency is stochastic, although the probability of fire increases with increasing fuel load, which increases with increasing time since fire until it reaches an asymptote where the rate of production equals the rate of decay (Raison et al. 1986). The effect of fire frequency on serotinous obligate seeders has been most thoroughly studied because such species do not retain a post-fire residual seedbank but require seedling regeneration from in situ seed for local persistence (Gill 1981; Noble & Slatyer 1980; Kruger 1983). In contrast, obligate seeders with soil seedbanks can retain a post-fire residual seedbank that ensures local persistence should fire recur before plants can replenish the soil seedbank (Bradstock et al. 1997). Whilst seed persists within soil for many years (Auld et al. 2000), the contribution of old and new seed to post-fire regeneration is not necessarily in proportions equal to the number in each cohort. The relative contribution of old and new seed to post-fire regeneration is not known and was addressed using an accelerated aging technique in the current study. A number of important gaps in our current knowledge of seasonal effects on soil seedbanks, and how to address these gaps, have been identified. The germination response of seed within soil in the Sydney region to fire-related cues such as heat shock and smoke is currently unknown or poorly known. Whether the season of fire affects the germination response of such seed is also unknown. The hydration status of water-permeable seed in the soil at the time of fire is affected by its position in the landscape, and whether the tolerance of hydrated seed to fire is affected by the habitat of a species has not previously been addressed. Also, whether the germination response of soil-stored seed depends on its age at the time of fire is not known. In order to understand how a flora might respond to such factors it is necessary to study the germination response of a wide range of plant species. Because the effect of season of fire on germination is confounded by the effect of season on intensity, it is necessary to decouple these factors in factorial experiments where factors related to season, such as the ambient temperature and factors related to intensity, such as the level of heat shock are combined. It can be determined whether the combination of heat shock and hydration has a different effect on seed of wet and dry habitat species, which may contribute to habitat segregation. Also, the response of aged seed to fire-related germination cues can be investigated to estimate the potential contribution of long-stored seed to post-fire germination.

8 Investigations

Given the gaps in our knowledge identified above, the following investigations were undertaken. Whether there is a germination response to the fire-related cues, heat shock and smoke, and the form of these responses was investigated for a number of species and populations within some species forming soil seedbanks in the Sydney region. A wide range of heat shock encompassing that generated by fire within the zone of seed location in soil and a range of durations of aerosol smoke was applied to seeds. Dose-related germination responses were interpreted in the context of species habitat, and of ensuring both a residual seedbank and germination (Chapter 2). The interactions between fire-related germination cues and both pre- and post- treatment temperatures, and alteration of temperatures during incubation were investigated for a number of species and populations (Chapter 3). The effect of hydration and dehydration prior to treatments was investigated for a number of species and populations, and interpreted in the context of whether they were from wet and dry habitats (Chapter 4). The relationships between fire-related germination cues and post-treatment water availability at different incubation temperatures were investigated for a number of species. Water availability was determined for soil from wet and dry habitats, and responses of species to the fire-related germination cues were interpreted in the context of their habitat. The optimum response to fire ensures both germination in favourable microsites, and a residual seedbank. The attainment of this outcome was interpreted in the context of the duration over which the promotive effect of the cues was retained, and variability in water availability to small seed in coarse soil. Also, the interaction between the level of heat shock, post-treatment water availability and incubation temperatures was investigated for one species, thus separating the effects of fire intensity and season (Chapter 5). An indication of the contribution to post-fire regeneration of seed that has resided in soil for a long period of time was determined by applying a range of heat shock levels and smoke to artificially aged seed (Chapter 6). Findings are briefly summarized and potential experiments to further investigate some findings are discussed (Chapter 7).

9 Chapter 2. Effects of fire-related germination cues

2.1 Introduction

Overview

The question of whether fire-related germination cues influence germination of species forming soil seedbanks in the fire-prone Sydney region is investigated in this section.

Post-fire Regeneration from Soil-stored Seed

The initial floristic composition model (Egler 1954; Connell & Slatyer 1977) best represents vegetation succession after fire in many fire prone regions throughout the world, including sclerophyll woodland communities in south-eastern Australia (Purdie & Slatyer 1976). The model states that species composition immediately after a disturbance is dependent on propagules that have either dispersed from elsewhere or have persisted through the disturbance at the site, or upon vegetative resprouting from organs surviving the disturbance. Immediately after a disturbance, there is a pulse of recruitment or regrowth under conditions of little competition for resources. Recruitment slows after the initial pulse, since once an individual plant is established it is very difficult to displace (Noble & Slatyer 1981). Seeds of species from Australian fire-prone vegetation communities that rely on germination from an in situ soil seedbank to regenerate post-fire are likely to be dormant and responsive to cues associated with fire.

A dormant seed will not germinate in an environment of adequate water and temperature usually favourable for germination (Mayer & Poljakoff-Mayber 1989). Dormancy allows the accumulation of the seedbank that is required if adult plants, killed by fire, are to be replaced in situ. Many species in fire prone regions throughout the world regenerate after stand-replacing fire through germination from an in situ seedbank. The in situ seedbank is the source of most post-fire recolonisation from seed in Australian fire-prone plant communities (Purdie & Slatyer 1976; Purdie 1977; Whelan 1986; Keith et al. 2002), and large numbers of seeds of many species remain viable and dormant in the soil of these

10 communities (Carroll & Ashton 1965; Hodgkinson et al. 1980; Vlahos & Bell 1986; Wang 1997). Seedbanks of shrub and sedge species in the Sydney region are generally long lived (Auld et al. 2000), with an estimated 74% of species in fire-prone vegetation of this region having a persistent soil seedbank, as compared to 15% of the species with a transient soil seedbank (Auld 1994). Whilst the dormancy status of seed may vary independently of fire, the strongly pulsed pattern of post-fire germination from soil-stored seed is attributed to fire (Auld & Tozer 1995). A pulse of post-fire germination from the soil seedbank has been recorded and attributed to the passage of fire for many species in fire prone vegetation worldwide (e.g. Whelan & Main 1979; Arianoutsou & Margaris 1981; Keeley 1987; Parker & Kelly 1989; Bell et al. 1993; Meney et al. 1994; Menges & Kohfeldt 1995; Benwell 1998).

Inter-fire germination is not favourable when few individuals establish and contribute to future generations before they are killed by subsequent fire (Specht et al. 1958; Tozer & Bradstock 1997). Inter-fire seedling establishment is limited by factors such as predation and competition for resources, whilst the post-fire environment is often conducive to seedling survival due to a reduction in the levels of these factors (Bond 1984). In southwestern Western Australia, only seedlings that arise in the first one or two years following a fire subsequently survive (Whelan & Main 1979; Cowling & Lamont 1987; Bell et al. 1989; Meney et al. 1994). Smoke treated species from this environment emerge earlier (Dixon et al. 1995; Roche et al. 1997a), and with greater vigour than untreated seeds (Roche et al. 1997a, 1998), which is advantageous because the combination of sufficient soil moisture and ambient temperatures conducive to seedling survival is of brief duration (Bell et al. 1993). Levels of seedling recruitment in Sydney region heathland in the first post-fire year may exceed that in subsequent years by orders of magnitude (Keith 1991, unpublished), and germination and seedling survival was increased in burnt plots in a dry sclerophyll eucalypt forest in southeastern Australia (Purdie 1977).

11 Fire-related Germination Cues

One way in which fire stimulates germination is by reducing seed dormancy, hence the dormancy - breaking mechanism(s) must overcome the factor(s) that impose dormancy. Dormancy entails constraint(s) that reside in the embryo itself (embryo dormancy) or belong to the enclosing structures (coat-imposed dormancy), that prevent growth of the embryonic axis (Bewley & Black 1982a). Covering structures can act as a light filter, interfere with the movement of water or gases, contain or prevent the removal or oxidation of inhibitors, or mechanically restrict embryo growth. Also, the embryo may be insufficiently developed, or the interaction between sensitivity to hormones and levels of hormones present may prevent germination. A working hypothesis explaining the roles of hormones in the regulation of seed dormancy and germination is that gibberellic acid, abscisic acid and kinetin are primary, preventative and permissive respectively (Khan 1968; 1971).

Dormancy can be induced, strengthened, weakened and broken by a number of external factors that interact with each other and with the physical and physiological state of the seed. Factors include light quality and quantity; constant, fluctuating and extreme temperatures; moisture availability, oxygen, chemicals, hormone balance, and the duration and timing of exposure and receptivity of the seed to these factors. The levels of dormancy of seeds of many species forming soil seedbanks in fire-prone regions are lowered and germination occurs in response to a variety of fire-related cues (Baskin & Baskin 1998). Fire- related cues, including heat shock (Keeley et al. 1985; Bell et al. 1987; Keeley 1987; Auld & O’Connell 1991; Keith 1997; Gilmour et al. 2000), combustion products including smoke (Brown 1993; Dixon et al. 1995; Roche et al. 1997a, b; Keeley & Fotheringham 1998a), elevated nutrient levels (Thanos & Rundel 1995), altered light spectral composition (Roy & Arianoutsou-Faraggitaki 1985), altered soil temperature regimes (Brits 1986) and removal (Preston et al. 2002) or overriding of allelopathic substances (Krock et al. 2002) have been found to increase germination when applied singly, or in combination. The effects of smoke, heat shock, and the combination of these fire-related germination cues will now be considered in turn.

12 Smoke

Although a compound present in smoke that promotes germination has been found (Flematti et al. 2004), the way in which smoke promotes germination is unknown. Smoke may overcome a germination inhibitor (Egerton-Warburton & Ghisalberti 2001), have a scarification effect (Keeley & Fotheringham 1997; 1998a, b; Egerton-Warburton 1998; Adkins et al. 2003), or have a chemical / hormonal effect. Smoke has the hormone-like property of interaction with, and substitution for other hormones to overcome a light requirement for germination (Drewes et al. 1995; Thomas & Van Staden 1995; Plummer et al. 2001). Smoke may influence the pool of active hormones, or it may sensitise seeds so that the levels of endogenous hormones normally too low to trigger germination become active/promotive, or it may increase membrane permeability so that hormones pass more easily to active sites, which would explain the increased sensitivity to different hormones (van Staden et al. 2000).

Smoke also has the hormone-like property of dose dependence (Brown 1993; Keith 1997; Plummer et al. 2001; Willis et al. 2003), with smoke-promoted germination increasing to a maximum and then decreasing at high durations or concentrations, which can be inhibitory (Baxter et al. 1994; Brown & van Staden 1994; Dixon et al. 1995; Drewes et al. 1995; Roche et al. 1997a; Light et al. 2002; Flematti et al. 2004) or lethal (Keeley & Fotheringham 1998a).

Heat shock

The mechanism by which heat shock breaks the dormancy of seeds with water- impermeable hard seed coats is well known (Cavanagh 1980; Morrison et al. 1992), but the way in which heat shock increases germination of species with water-permeable seed coats is unknown. The effect of heat shock on germination is also dose-dependent (Keeley et al. 1985; Keeley 1987; Auld & O’Connell 1991; Auld et al. 1993). Heat shock of between 40 and 120°C, applied for between 1 and 120 minutes to seeds within an oven, broke dormancy within 35 species of eastern Australian Fabaceae (Auld & O’Connell 1991). Germination tended to a maximum at 80°C and was reduced due to seed death at 120°C. Duration of treatment had little effect, except where seeds were killed by the treatment. Germination was

13 stimulated within narrow ranges of heat shock for species with water-impermeable (Auld & O’Connell 1991) or water-permeable seeds (Auld et al. 1993). Heat shock is rapidly attenuated with soil depth, and a range of temperatures have been recorded in surface soil, where most of the soil seedbank is found, during the passage of low to medium intensity fire (Auld 1986a; Raison et al. 1986; Bradstock & Auld 1995)

Heat shock and smoke combined

In the field, typically seeds will experience fire-related cues in combination. However, whilst the effects of heat shock and smoke as single cues have frequently been investigated, the germination response to the combination of these cues is poorly known. Factorial combination of germination cues is an acknowledged way of investigating this question, as it allows assessment of the germination response to each cue applied singly and in combination. If the combination of cues results in a germination response, then the way in which the cues combined can be assessed e.g. independent, synergistic, obligatory combination. Only the factorial design will allow assessment of the way the cues combine (Underwood 1997). Detection of the underlying dormancy mechanisms requires application of these cues both in isolation and in combination.

Previous work has shown that heat shock and smoke can combine in a number of ways to affect germination. The increases in germination of Australian species following the combined application of heat and smoke have been independent and additive (Keith 1997; Kenny 2000; Morris 2000), synergistic (Gilmour et al. 2000; Kenny 2000) and obligatory combinatorial, where a germination increase only occurs following combined heat and smoke application (Gilmour et al. 2000; Morris 2000; Kenny et al. 2001). Within chaparral vegetation, the combination of heat shock and combustion products produced a synergistic increase for a shrub (Keeley 1987), a herbaceous perennial, and a fire annual species (Keeley et al. 1985). The combination of heat shock and charate produced a synergistic increase in germination of three heat-stimulated chaparral species, and a decrease in one heat-stimulated species (Odion 2000). Germination of two other chaparral species were reduced by heat shock, and reduced further still by the combination of heat shock and charate (Odion 2000). The prevalence of these types of interactions has not been addressed in any fire-prone region, despite their natural co-occurrence.

14 A more accurate assessment of the germination response of a soil seedbank to fire requires application of a combination of heat shock and smoke, rather than these cues in isolation. Also, these cues must be applied over the range of doses that a soil seedbank encounters during the passage of fire. This approach has been adopted in my study. Although buried seed is likely to experience heat shock prior to smoke during the passage of fire, the order of application of these two cues may also affect germination.

Light Both the quantity and quality of light may be altered within a burnt environment, and both these factors may influence germination from the soil seedbank. The removal of canopy cover will increase the intensity and duration of light reaching the soil surface and because this light is not leaf-filtered, it has a higher red : far red ratio which may promote germination (Smith 1973). An increase in the red : far red ratio greatly increased germination of the Mediterranean fire-promoted Sarcopoterium spinosum (Roy & Arianoutsou-Faraggitaki 1985). Light-promoted germination occurs only at shallow (< 4 mm) soil depth (Koller et al. 1964; Woolley & Stoller 1978; Van der Meijen & van der Waals-Kooi 1979; Bliss & Smith 1985; Tester & Morris 1987). Seed at shallow depth would probably be consumed by fire or experience lethal temperature (Purdie 1977). However, the rate of erosion occur following wildfire in the Sydney region increased as much as a thousandfold (Dyson 1966; Blong et al. 1982) and several millimetres of surface soil derived from Hawkesbury Sandstone were removed (Blong et al. 1982). Light-enhanced germination may occur as a secondary outcome of fire.

Range of dormancy levels

Not all seeds are expected to germinate following fire except for the species that form transient soil seedbanks (Meney et al. 1994). Species that form persistent soil seedbanks may retain residual seed because not all seed receives the required cues. Possibly, there is no combination of cues that stimulate all seed to germinate because the level of dormancy in a population of seeds can range from deeply dormant to non-dormant; the distribution of dormancy levels in a population is considered to be normal (Roberts 1972a) and seeds with strong dormancy may not respond. More dormant seeds could buffer a species against

15 possible loss of earlier cohorts (Bell et al. 1995; Bradstock et al. 1997) before they are fire- resistant, or have sufficiently replenished the seedbank. Heathland in the Sydney region can have an inter-fire period as short as 18 months (Bradstock et al. 1997), but between 3 and 4 years are required for half of the obligate seeder species to become reproductively mature, and possibly up to 10 years are required before all such species are mature in this environment (Benson 1985; Keith 1991, unpublished), depending on site quality and post-fire rainfall (Bradstock & O’Connell 1988). At least three subsequent reproductive seasons are required in most obligate seeders before significant seedbank accumulation occurs (Keith et al. 2002), hence a mechanism that ensures a post-fire residual seedbank is required to buffer against local extinction due to frequent fire.

Aims

How germination of undestorey species forming soil seedbanks in the Sydney region is affected by fire-related cues such as heat shock and smoke is poorly known or unknown. Whether and how these cues combine to affect germination has been infrequently investigated for the flora of any region. Also, the effect of the combination of a range of levels of these cues has not been investigated for the flora of any region. The effect of high levels of heat shock (eg 200°C) has rarely been investigated, and even less frequently in combination with a combustion product such as smoke. The effect of light on post-fire germination of species forming soil seedbanks in the Sydney region is also unknown. Whether there is a relationship between species habitat and their germination response to fire-related cues has received little attention in the Sydney region.

This section of the study examined the effect of heat shock and smoke on germination of seeds of 22 species with poorly-known germination requirements, from three major plant families in the Sydney region of south-eastern Australia. These species have a range of adult responses to fire, and occur in habitats of varying fire-proneness. The number of species investigated allows for the investigation of patterns of responses to the fire-related cues, and whether these patterns are related to Family, adult plant regenerative response or habitat.

Section I. A range of heat shock temperatures was combined with a range of smoke levels to determine whether a single cue only, or both cues would produce a germination

16 response. If both cues produced a germination response, then the experimental design allowed anassessment of whether there was an independent or interactive combination of the two signals.

Section II. The range of heat shock was extended for seven species to simulate temperatures experienced during more extreme fire. The higher levels of heat shock were applied to test for the range of heat shock tolerance and the upper thermal limit. The seven species had high, moderate or low levels of germination following 100°C heat shock and a range of patterns of response to the combination of heat shock and smoke. One species had become less responsive to lower levels of heat shock during storage.

Section III. Seeds of a number of species that had been treated with combinations of fire- related germination cues and incubated in darkness were subsequently transferred into light to assess whether germination of any species would increase. An increase in germination subsequent to transferral into light would be indicative that the species was light responsive.

The influence of bulked applications of heat shock and smoke on the germination of nine species, and the effect of such a procedure on the analyses was investigated (Thomas et al. 2003).

The germination responses of these species to these fire-related germination cues are discussed in terms of their persistence in a fire-prone landscape.

17 2.2 Methods

Study Species, Seed Collection and Storage

Seeds from 22 species commonly found in the Sydney region on soils derived from Hawkesbury Sandstone were collected from a number of locations and habitats ranging from wet cliff face to dry heath (Table 2.1; Appendix I). For five species, more than one population was sampled. All species were shrubs or sub-shrubs, with the exception of the sedge, Gahnia sieberiana.

Seeds or seed capsules were collected from tens of plants at a single site into cotton bags during seed release. Seeds were stored in these bags at approximately 25˚C until the seed was processed prior to treatment application. Seed was extracted from capsules by sieving, and likely viable seed was separated from nonviable seed and chaff by size and density differentiation, by floatation, and finally by examination under a dissecting microscope. Seeds were dissected and those seeds with endosperm that fully filled the seedcoat and that were moist throughout the entire endosperm were deemed viable. The external appearance of seeds seeds deemed viable was different from those deemed nonviable. More dense seeds were more frequently viable, thus density differentiation was undertaken prior to visual examination. This likely viable seed was surface sterilized with 1% sodium hypochlorite for ten minutes, rinsed with distilled water, and thence handled using sterile procedures. Seeds were then stored in glass jars in darkness at approximately 25˚C until experiments were performed (Table 2.1).

Seeds were extracted from capsules because most seed is within soil at any moment in time. All seed of many species would be within soil at the time of fire because they are shed before the fire-prevalent season (Table 2.1), however species such as Kunzea ambigua retain seed capsules in the canopy throughout much of the summer fire season. Kunzea ambigua seeds survive within heated capsules, and viable seed has been recovered from burnt K. ambigua shrubs (Judd & Ashton 1991), however most seeds of such species would reside in the soil and so it was considered appropriate to treat extracted seeds.

18 Table 2.1. Study species, habitat, mean seed weight (n = 100), time of seed collection and time of application of experimental treatments. Species arranged alphabetically within

Family, and chronologically within experiments.

Family Species Pop Habitat Weight Collection Experiment Section (mg) time time

Cyperaceae Gahnia sieberiana 1 Rivers edge 0.979 Dec 00 I Mar-00 seed May 02 II

Epacridaceae Dracophyllum secundum 1 Rock ledge 0.030 Oct 00 I Jan-00

2 Sept 01 I Dec-99 May 02 II

Epacris coriacea Moist heath 0.038 Nov 00 I Jul-00 May 02 II

Epacris crassifolia 1 Wet cliff face 0.015 Sept 01 I Jan-00

2 Sept 01 I Jun-00 April 02 III

Epacris longifolia Run-on area 0.086 Sept 01 I Jun-00 April 02 III

Epacris microphylla var. microphylla Moist heath 0.029 Sept 01 I Jun-00 April 02 III

Epacris microphylla var. rhombifolia Wet heath 0.028 Sept 01 I Apr-00 April 02 III

Eparis muelleri Wet cliff face 0.026 Sept 01 I Jun-00 April 02 III

Epacris obtusifolia 1 Wet heath 0.032 Nov 00 I Jun-00

3 Moist heath 0.022 May 02 II Jul-00

Epacris paludosa 1 Wet heath 0.034 Sept 01 I Apr-00 April 02 III

19 Family Species Pop Habitat Weight Collection Experiment Section (mg) time time

Epacris pulchella Wet heath Sept 01 I Jun-00 April 02 III

Sprengelia monticola Wet cliff face 0.015 Dec 00 I Jan-00

Woollsia pungens 1 Wet heath 0.039 Sept 01 I Jan-00 April 02 III

Myrtaceae Baeckea diosmifolia Moist heath 0.048 Nov 00 I Mar-00

Baeckea brevifolia Rock platform 0.085 Dec 01 I Sep-00

Baeckea imbricata 1 Wet heath 0.035 Nov 00 I Jun-00

2 Moist heath 0.028 Dec 01 I Sep-00 May 02 II

3 Moist heath 0.031 Dec 01 I Apr-00

Baeckea linifolia 1 Creek line 0.051 Dec 01 I Jun-00

2 Creek line Dec 01 I Jun-00

Baeckea ramosissima ssp. ramosissima DSF 0.190 Dec 01 I Feb-00

Baeckea utilis Swamp 0.082 Dec 01 I Apr-00

Calytrix tetragonia Dry heath 0.385 Dec 01 I Jan 00

Kunzea ambigua Dry heath 0.039 Oct 00 I Mar-00 Jan 02 II

Kunzea capitata 1 Moist heath 0.049 Oct 00 I Jul-00 May 02 II

Micromyrtus ciliata 1 Dry heath 0.055 Dec 01 I Apr-00

20 Section I Experimental Designs

Seeds were treated with factorial combinations of heat shock (25°C (= control), 50, 75 and 100°C) and smoke exposure (0, 5, 10 and 20 minutes aerosol smoke). Because only one oven and one smoking device was used, it can only be inferred that differences were due to the levels of heat shock or smoke applied rather than peculiarities of these pieces of equipment. However, independence of application of heat shock and smoke exposure was achieved, in contrast with previous work on seed germination that has used these two treatments (Morrison & Morris 2000). Typically, heat shock or smoke has been applied to all the seeds in a treatment simultaneously, with replication of seed containers (punnets, petri dishes) occurring within this single application (Morrison & Morris 2000). Replicates (the seed containers) are not independent using this protocol because of the single application of the treatment. Independent replication of the treatment was achieved in two different ways.

a) Bulked applications

Twelve petri dishes, each containing 10 seeds, were treated with each combination of heat shock and smoke for nine species (Table 2.1). The twelve petri dishes within any one heat shock level were divided into two batches, and each batch of six petri dishes was heated at different times in the same oven. Within each heat shock x smoke combination, the seeds from each heat application were subdivided into two batches of three petri dishes. The two batches were smoked separately, with fresh fuel for each application. There were n = 3 replicates (petri dishes), each with 10 seeds, within each smoke application. Over the two smoke applications and two heat applications, there were 12 replicates per smoke x heat combination. This experimental design allows formal testing for differences between applications within treatments (Thomas et al. 2003). A cost of this procedure is that it results in a weaker test of the terms of interest (heat shock, smoke and their interaction) when the application terms cannot be pooled out. As applications within treatments were generally not significant, but also because the application terms could not be pooled out in many cases (Thomas et al. 2003), it was decided that in subsequent work replicates (individual petri dishes) would be treated independently.

21

b) Single, independent applications

Independent applications of heat shock and smoke were applied to 19 populations. Twelve replicate seed containers, each with 10 seeds, were treated with each level of heat shock and smoke. There were 160 independent applications and, as comparisons between species were not made in the analysis, nine Myrtaceae and ten Epacridaceae species or populations were treated simultaneously as a time-saving measure.

Experimental Treatments

Section I Heat shock and smoke application

To apply heat shock, seeds were heated for five minutes in a fan-forced oven, on open glass petri dishes that were closed when removed from the oven. This temperature range (50- 100°C) and duration applied have been recorded at shallow depth in Hawkesbury Sandstone derived soil, during the passage of low to medium intensity fire (Auld 1986a; Bradstock & Auld 1995), and temperature of soil and seeds of the study species are probably equal (Hungerford 1990).

Seeds were smoked on open glass petri dishes in a glass chamber within a fume cabinet. Smoke was produced in a bee keeper’s burner by burning dry fine fuel litter from a eucalypt forest. This fuel was changed between each smoke application. An electric air-pump forced the smoke was out of the bee keeper’s burner, through a condensing tube, which both cooled and dried the smoke, and into the smoking chamber. The smoking chamber would rapidly fill with dense smoke, which circulated and exited through an outlet hole, and was then extracted by the fume cabinet. The laboratory was thus kept free of smoke, and so smoke contamination of seed was avoided. Species were placed in the same position within the chamber for each application. For seeds that received both treatments, heat shock was applied before smoke. In a pilot study of four species (five populations) that responded to the combination of added heat shock and smoke, the order of application of heat shock (50, 75 or 100°C) and smoke was not important (data not shown). 22 Treated seeds were transferred to plastic 9cm petri dishes on 1 layer of Whatman No. 1 filter paper, moistened with distilled water and immediately double wrapped with aluminium foil to exclude light. Seeds were germinated at a constant 25°C in darkness, as it was considered that, in the field, seed on the surface is likely to be consumed by the passage of fire.

Section II

High intensity heat shock and smoke

The effect of 125, 150, 175 and 200°C heat shock intensities with and without 10 minutes of smoke was assessed for seven species (Table 2.1). Treatments were applied independently to three replicates of twenty seeds of each species, and the seven species were treated simultaneously. Heat shock was applied for five minutes, however the temperature was reduced when the oven was opened, and this decrease in temperature increased with higher temperatures. The duration of 200°C heat shock applied ranged from close to the full five minutes for one replicate, to only one minute after rising from approximately 175 to 200°C over the first four minutes for another replicate. Species were incubated at the temperature regime previously determined as most favourable for germination (Chapter 3). The Kunzea species were incubated at 15°C, Gahnia sieberiana at a diurnally fluctuating regime of 25°C for 20 hours and 35°C for four hours, and the other species were incubated at 25°C.

Section III

Light following dark incubation

After 7 months, germination of the Epacridaceae species within the single, independent application treatment had tapered off. The replicates treated with combinations of 25°C (= control) and 75°C heat shock and 0 and 10 minutes smoke, and with the combination of 50°C heat shock and 0 minutes smoke were then transferred into an 8 hour diurnal cool white light regime and regularly monitored. These species and prior treatments were chosen as a random sub-sample, that include the control and combinations of fire-related cue treatments. 23

Assessment of Germination

Seeds were regularly checked under safe light (Drewes et al. 1995) and scored as a germinant and removed when a 1mm long radicle had emerged (Mott & Groves 1981). The experiment was ended when the rate of germination was very low (bulked application experiment: average 164 days, standard error 7 days). Germination was calculated as a percentage of initial seeds per petri dish.

Data Analysis

Section I

Effects of heat shock and smoke

When each replicate was treated independently, final germination data were analysed using a two-way ANOVA, with the terms heat shock and smoke as fixed factors. When replicates were nested within treatment applications, a four-way mixed-model ANOVA was used to analyse final germination data. The mixed-Model ANOVA had the term heat shock as a fixed factor, heat application nested in heat shock, smoke level as a fixed factor, and smoke application nested within heat shock x heat application x smoke level (Tables 2.4, 2.5, 2.8). Homogeneity of variances was assessed using Cochran’s Test and transformations performed when required for analyses in this Chapter.

For comparison amongst means subsequent to ANOVA, the Sums of Squares were decomposed and orthogonal Planned Comparisons of smoked and unsmoked means and Trend Analysis across heat shock levels was undertaken.

Heat shock x smoke interaction significant

Planned comparisons within the interaction (Table 2.2) followed the analysis of Keppell et al. (1992). A choice was made to make two comparisons within each level of heat shock; 1 unsmoked means vs pooled smoked means; amongst the smoke means. The first comparison tested for whether there is a smoke effect per se; the second comparison tested whether the various durations of smoke differed (smoke dose effect). The form of differences amongst smoked means was visually assessed.

24 The interaction Sums of Squares and degrees of freedom (= 9; Table 2.2) were pooled with the Main Effects smoke Sums of Squares and degrees of freedom (= 3) to give a total Sums of Squares and 12 degrees of freedom for these comparisons (Keppell et al. 1992). Within each heat shock level (i.e. amongst 4 means) there were 3 degrees of freedom; comparison 1 used 1 degree of freedom, comparison 2 used 2 degrees of freedom. The Sums of Squares and degrees of freedom summed across 4 heat shock levels equalled the sum of the interaction and smoke Main Effects Sums of Squares and degrees of freedom (Table 2.2).

A final Planned Comparison (amongst unsmoked means across heat shock levels) was used as a subset of the Sums of Squares and 3 degrees of freedom of the heat Main Effects term (heat Main Effects means were not compared because the interaction was significant) (Table 2.2). If a significant difference was detected, then Trend Analysis was used to test for the shape of the relationship.

Main effects significant

To compare smoke effects when the smoke term was significant and the interaction term was not, smoked and unsmoked means were pooled across all heat shock levels, and a planned comparison between smoked and unsmoked means was then made.

To compare heat shock effects when the heat shock term was significant and the interaction term was not, the trend across means of pooled smoke treatments within heat shock levels was assessed. Trend analysis was used to detect linear or quadratic relationships across means of heat shock levels. Trend analysis was considered appropriate because a change in response has frequently been observed across heat shock levels, and because heat shock can be finely controlled in a laboratory.

25 Table 2.2. Decomposition of Sum of Squares and orthogonal Planned Comparisons of a) smoked and unsmoked means, b) smoked means, and Trend Analysis across heat shock levels for Baeckea brevifolia subsequent to ANOVA (heat shock x smoke significant).

Baeckea brevifolius arcsin (%)

ANOVA Source SS df MS F n,12 P F vs Heat 10681.1 3 5. Amongst unsmoked means 1318.3 3 439.4 3.254 N.S.

Smoke 3875.4 3

heat x smoke 2632.4 9 292.5 2.170 0.0265 residual

1. Amongst 25C means 3561.8 3 1187.3 8.791 <0.001 1a. 25C: Unsmoked vs smoked 682.7 1 682.7 5.055 <0.05 1b. 25C amongst smoked means 2879.1 2 1439.6 10.659 <0.0025 2. Amongst 50C means 2719.4 3 906.5 6.712 <0.01 2a. 50C: Unsmoked vs smoked 192.8 1 192.8 1.428 N.S. 2b. 50C amongst smoked means 2526.6 2 1263.3 9.354 <0.005 3. Amongst 75C means 82.9 3 27.6 0.205 N.S. 3a. 75C: Unsmoked vs smoked 39.9 1 39.9 0.296 N.S. 3b. 75C amongst smoked means 43.0 2 21.5 0.159 N.S. 4. Amongst 100C means 143.7 3 47.9 0.355 N.S. 4a. 100C: Unsmoked vs smoked 35.4 1 35.4 0.262 N.S. 4b. 100C amongst smoked means 108.3 2 54.2 0.401 N.S. total decomposed SS 6507.9 12 residual error 23770.5 176 135.1

Interaction means (n=12) compared by Planned Comparisons above

Heat (°C) Smoke (minutes) 25 50 75 100 0 14.6 7.5 5.2 0.0 5 11.1 8.5 7.3 1.7 10 26.4 15.3 6.0 0.0 20 32.4 26.4 8.6 4.2

26 Response categories

To summarise and highlight patterns in the germination responses of the 28 populations to the fire-related cues, two different classification systems were used. The first classification system is useful for an investigation of the types of germination responses that occur in response to fire-related cues, whilst the second classification system highlights the effect of fire intensity on germination responses.

First classification system

The first classification system divided responses within a hierarchy of statistical analyses (Table 2.3). The first demarcation was between populations affected by the interaction between heat shock and smoke, and those responding to heat shock and smoke as main effects only.

Those populations affected by the interaction between heat shock and smoke were subdivided according to the nature of the interaction. A synergistic interaction occurred when the increase in germination following application of both cues was greater than the combined increases due to the germination cues applied singly. Other responses to the combination of cues were an increase in germination due to smoke without added heat shock (smoke alone category), or to smoke only within added heat shock levels. This last category is termed obligatory combination (previously ‘unitive’ Kenny 2003, unpublished), where the combination of cues is obligatory for a germination response, and species were further subdivided into the highest heat shock level at which a positive germination response to smoke occurred. Another category consists of a complex interaction not previously reported, where germination was increased either by smoke within a moderate heat shock level, or by the highest heat shock level without smoke.

Those populations responding to heat shock and smoke as main effects did so in an independent and additive manner, and were subdivided into those with a positive and those with a negative heat shock response.

27 Table 2.3. Essential criteria used to allocate species to the response categories of the first classification system.

Response Significant Smoke response within heat shock Heat shock category ANOVA response term

Heat shock (°C) 25 50 75 100

Synergistic Interaction 0 or + + + + + + +

Smoke alone Interaction + + / 0 / - + / 0 / - + / 0 / -

Obligatory Interaction 0 +ve response within one or combination more heat shock levels

Complex Interaction 0 + 0 / - Quadratic effect

Independent and Main +ve independent of heat shock, &/or + / - independent additive effects of smoke

28 Second classification system

The second classification system demarcated populations solely by a combination of the range of heat shock levels over which the germination response to smoke was positive, and the highest heat shock level at which the germination response to smoke was positive (Table 2.9). To this end, planned comparisons of smoke x heat shock interactions were also used for populations when the main effects rather than the interaction term was significant for the purpose of including these populations within a table showing the heat shock levels within which smoke was significant. For these species, heat shock by smoke interaction means were analysed as for the interaction analysis.

Section II

Effect of high heat shock and smoke

The effect of high heat shock levels on final percentage germination was assessed using a 2 way ANOVA with heat shock and smoke as fixed terms.

Section III

Effect of light

Percentage germination of transferred seeds are reported i.e. the starting population comprised seeds that had not germinated during dark incubation. Statistical comparisons between percentage germination in darkness and light were not possible due to lack of independence, hence informal, visual comparisons were made.

29 2.3 Results

Section I

General Overview

Most species responded to the fire-related germination cues (21 populations out of 28, Table 2.4-5), and germination of most of these populations (17 out of 21) showed a significant heat shock and smoke interaction (heat x smoke interaction significant, Table 2.4). Heat shock frequently reduced germination, increasingly so as the heat level increased. High levels of heat shock occasionally increased germination. The effect of smoke was almost always positive within some or all heat shock levels. Generally, the positive interaction between smoke and the lower heat shock levels resulted in more germination than within the control treatment. At the higher heat shock levels, smoke tended to compensate for the adverse effect of heat, such that germination was comparable to the control treatment.

Heat shock and smoke interaction significant

Synergistic effect

Heat shock and smoke had a synergistic effect on Kunzea capitata; the increase in germination was greater than an addition of the effects of both cues when they were applied singly (Table 2.4, Category 2.1; Fig 2.1). Kunzea capitata did not respond to heat shock applied singly, smoke by itself resulted in a small but significant increase in germination, but the combination of these cues produced the major response.

Smoke alone effect

The positive effect of smoke was confined to the no added heat shock treatment in only Baeckea brevifolia (Table 2.4, Category 2.2; Fig. 2.2). Also, germination of B. brevifolia decreased with increasing heat shock levels. Germination of another two species, Baeckea 30 imbricata population 2 and Epacris microphylla was increased by smoke alone, but also by smoke within added heat shock levels (Table 2.4, Category 2.2; Fig 2.2).

Obligatory combination effect

There were 12 other populations with positive interactions between smoke and heat shock restricted to the added heat shock levels only. Three such interactions occurred at the 50°C heat shock, five interactions occurred at 75°C heat shock, three interactions occurred at both 50°C and 75°C heat shock, and one interaction occurred at both 50°C and 100°C heat shock. Three of the seven species with positive interactions between smoke and heat shock within 75°C heat shock had a complex response and so they were placed in a separate category (see below).

Obligatory combination effect within 500C heat shock

Germination of Baeckea utilis and both B. linifolia populations, was positively affected by smoke within only the 50°C heat shock treatment (Table 2.4, Category 2.3; Fig 2.3). The unusually low level of germination of unsmoked B. linifolia population 1 seeds within the 50°C heat shock treatment contributed to this result.

Obligatory combination effect within 750C heat shock

Germination of two species was increased by smoke solely within the 75°C heat shock treatment (Table 2.4, Category 2.4; Fig 2.4). The positive effect of smoke may have countered the negative effect of 75°C heat shock on germination of Dracophyllum secundum population 2. Germination of E. microphylla var. rhombifolia was low until transferral into light (Section III).

31 Obligatory combination effect within 500C and 750C heat shock

Smoke had a positive effect on germination within both the 50°C and 75°C heat shock treatments for three species (Table 2.4, Category 2.5; Fig 2.5). Whilst smoke increased germination of Epacris longifolia within moderate heat shock levels, smoke had a negative effect on germination of this species within the highest heat shock level, as was the case for E. pulchella within the complex response category. Unlike the complex response, the highest heat shock level did not increase germination of unsmoked E. longifolia seeds.

Obligatory combination effect within 1000C heat shock

Germination of smoked Baeckea imbricata population 1 seeds was increased by 50°C and 100°C heat shock (Table 2.4, Category 2.6; Fig 2.6). Germination was negatively affected by increasing heat shock levels, however, germination of smoked seeds within the 100°C heat shock level was comparable to the control because of the positive effect of smoke.

Complex effect

The quadratic term was significant for heat shock alone means for three species. The ‘shape’ of the response was that germination of unsmoked seeds was higher within the 25°C (i.e. control treatment) and 100°C heat shock levels than the intermediate heat shock levels. Germination was also increased by smoke within the 75°C heat shock levels (Table 2.4, Category 2.7; Fig 2.7).

Negative effect of smoke

Smoke had a negative effect on germination of four species within one of the four heat shock levels. There was no indication that three of these occurrences were other than chance, and the frequency of 5% of the significant planned comparisons is the expected frequency of Type I error. High smoke dose had a negative effect on germination of unheated Woollsia pungens seeds (next section), hence the negative effect of smoke within the 50°C heat shock may be biologically significant (Table 2.4, Category 2.8; Fig 2.8).

32 Table 2.4. Effects of heat shock, smoke, applications of these treatments (bulked application design), and interactions between these factors on % germination of the 17 study populations for which the interaction between the main effects was significant; ANOVA P-values are shown. Factors are nested within factors shown in parentheses.

Figure No. Source Heat shock Smoke H apps ‡ S apps (S x H H x S S x H Residual & = H levels = S (H) apps (H)) apps (H) Category† bulked applications df 3 3 4 32 9 12 128 F H apps S x H apps S apps Residual S x H S apps vs apps single independent df 3 3 9 176 applications F Residual Residual Residual vs 2.1 K. capitata (1) ! 0.0093 < 0.0001 0.7882 < 0.0001 0.0015 0.9993 2.2 B. brevifolia < 0.0001 < 0.0001 0.0265 2.2 B. imbricata (2) § < 0.0001 < 0.0001 0.0111 2.2 E. microphylla < 0.0001 0.0639 0.0010 2.3 B. linifolia (1) 0.0012 0.1859 0.0001 2.3 B. linifolia (2) § 0.0394 0.3453 0.0479 2.3 B. utilis 0.0006 0.2595 0.0123 2.4 D. secundum (2) < 0.0001 0.0542 < 0.0001 2.4 E. microphylla var. 0.2204 0.0171 0.0203 rhombifolia§ 2.5 B. diosmifolia 0.0402 0.4983 0.0531 0.7560 0.0430 0.1334 2.5 E. longifolia < 0.0001 < 0.0001 < 0.0001 2.5 E. muelleri 0.1762 0.0006 0.0003 2.6 B. imbricata (1) 0.0058 0.0007 0.8830 0.0002 0.0396 0.8921 2.7 E. crassifolia (1) < 0.0001 0.5885 0.0182 2.7 E. paludosa (1) < 0.0001 < 0.0001 0.0174 2.7 E. pulchella 0.0002 0.0359 < 0.0001 2.8 W. pungens < 0.0001 0.5147 0.0103

†Interaction significant:

Synergistic response 2.1 Positive response within 25°C heat shock Smoke alone response 2.2 Positive response within 25°C heat shock Obligatory combination response 2.3 Positive response within 50°C heat shock 2.4 Positive response within 75°C heat shock 2.5 Positive response within both 50°C and 75°C heat shock 2.6 Positive response within both 50°C and 100°C heat shock Quadratic heat shock response (complex effect) 2.7 Positive response either to smoke within 75°C heat shock, or to 25°C and 100°C heat shock Only a negative response to smoke 2.8 Negative response within 50°C heat shock

‡applications !population § analysis of transformed data

33 Kunzea capitata (population 1)

80 usm < sm *** usm < sm *** usm < sm *** 70

60

50 usm < sm ** 40

30

20 % germination (+/- SE) % germination

10

0 25 50 75 100

Heat Shock (°C)

Figure 2.1: Mean final germination plotted against temperature of heat shock for Kunzea capitata (population 1). Smoke levels are indicated as 0 smoke (2), 5 minutes (), 10 minutes (), 20 minutes (). Significant Planned Comparisons between unsmoked and smoked treatments are indicated above the heat shock level (usm = unsmoked; sm = smoked). Bars = S. E s

34 a) Baeckea brevifolia

40 heat: -ve linear *** usm < sm *** 35

30

25

20

15

10

5

0 25 50 75 100

b) Baeckea imbricata (population 2)

80 heat: -ve linear *** usm < sm *** 70 usm < sm *** usm < sm ** 60 usm < sm ***

50

40

30

20

10 % germination (+/- SE) % germination 0 25 50 75 100

c) Epacris microphylla

90 usm < sm * usm < sm * 80

70

60

50

40

30

20

10

0 25 50 75 100

Heat Shock (°C)

Figure 2.2: Mean final germination plotted against temperature of heat shock for a) Baeckea Nobrevifolia treatment, b) B. effect imbicata (population 2) †, c) Epacris microphylla. Significant Trend Analysis across heat shock levels within unsmoked treatments are indicated (heat = heat shock). Analysis and Symbols as Figure 2.1 † Analysis of transformed data; back-transformed data shown in Figure 35 a) Baeckea linifolia (population 1)

50 usm < sm ***

45

40

35

30

25

20

15

10

5

0 25 50 75 100

b) B a e cke a lin ifo lia (population 2)

18 usm < sm *

16

14

12

10

8

6

4

2 % germination (+/- SE) % germination 0 25 50 75 100

c) Baeckea utilis

45 usm < sm *

40

35

30

25

20

15

10

5

0 25 50 75 100

Heat Shock (°C)

Figure 2.3: Mean final germination plotted against temperature of heat shock for a) Baeckea linifolia (population 1), b) B. linifolia (population 2)†, c) B. utilis. Analysis and Symbols as Figures 2.1 & 2.2

36 a) Dracophyllum secundum (population 2)

70 heat: -ve linear ***

60

50

40 usm < sm **

30

20

10

0 25 50 75 100

b) Epacris microphylla var rhombifolia

16 usm < sm **

14 % germination (+/- SE) % germination 12

10

8

6

4

2

0 25 50 75 100

Heat Shock (°C)

Figure 2.4: Mean final germination plotted against temperature of heat shock for a) Dracophyllum secundum (population 2), b) Epacris microphylla var. rhombifolia† Analysis and Symbols as Figures 2.1 & 2.2

37 a) Baeckea diosmifolia

40 usm < sm * usm < sm *

35

30 usm > sm* 25

20

15

10

5

0 25 50 75 100

b) Epacris longifolia

90 heat: -ve linear * 80 usm < sm * usm < sm ** usm > sm ** 70

60

50

40

30

20

% germination (+/- SE) % germination 10

0 25 50 75 100

c) Epacris muelleri

30 usm < sm ** usm < sm **

25

20

15

10

5

0 25 50 75 100

Heat Shock (°C)

Figure 2.5: Mean final germination plotted against temperature of heat shock for a) Baeckea diosmifolia, b) Epacris longifolia, c) E. muelleri. Analysis and Symbols as Figure 2.1

38 Baeckea imbricata (population 1) heat: -ve linear * 70 usm < sm ***

60

50 usm < sm * 40

30

20 % germination (+/- SE) % germination 10

0 25 50 75 100

Heat Shock (°C)

Figure 2.6: Mean final germination plotted against temperature of heat shock for Baeckea imbricata (population 1). Analysis and Symbols as Figure 2.1.

39 a) Epacris crassifolia (population 1)

35 heat: quadratic ***

30

25 usm < sm * 20

15

10

5

0 25 50 75 100

b) Epacris paludosa (population 1)

60 heat: quadratic *** usm < sm ** 50

40

30

20

10 % germination (+/- SE) % germination

0 25 50 75 100

c) Epacris pulchella

50 heat: quadratic ** 45 usm < sm *** usm > sm ** 40

35

30

25

20

15

10

5

0 25 50 75 100

Heat Shock (°C)

Figure 2.7: Mean final germination plotted against temperature of heat shock for a) Epacris crassifolia, b) E. paludosa (population 1), c) E. pulchella. Analysis and Symbols as Figure 2.1

40 Woollsia pungens heat: -ve linear *** 16

14 usm > sm * 12

10

8

6

4 % germination (+/- SE) % germination

2

0 25 50 75 100

Heat Shock (°C)

Figure 2.8: Mean final germination plotted against temperature of heat shock for Woollsia pungens. Analysis and Symbols as Figure 2.1

41 Heat shock and smoke significant as Main Effects

Germination of four species was affected by heat shock and smoke in an independent and additive manner (Main Effects significant; Table 2.5). Added heat shock increased germination of two species and decreased germination of two species. Heat shock of 75°C and above increased germination of Gahnia sieberiana, and added heat shock increased the global germination of Kunzea ambigua (Table 2.5, Category 2.9; Table 2.6; Fig 2.9). Germination of Baeckea imbricata (population 3) and Micromyrtus ciliata (population 1) decreased with increasing heat shock levels (Table 2.5, Category 2.10; Table 2.6, Fig 2.10). Smoke increased global germination of all four species (Tables 2.5, 2.6; Figs 2.9, 2.10).

Table 2.5. Effects of heat shock, smoke, applications of these treatments (bulked application design), and interactions between these factors on % germination of the 4 study populations for which main effects were significant; ANOVA P-values are shown. Factors are nested within factors shown in parentheses.

Figure No. Source Heat shock Smoke H apps ‡ S apps (S x H H x S S x H Residual & = H levels = S (H) apps (H)) apps (H) Category† bulked applications df 3 3 4 32 9 12 128 F H apps S x H apps S apps Residual S x H S apps vs apps single independent df 3 3 9 176 applications F Residual Residual Residual vs 2.9 G. sieberiana (1) ! 0.0002 0.0223 0.6733 0.0033 0.4089 0.8427

2.9 K. ambigua 0.0399 0.0252 0.1546 < 0.0001 0.3709 0.0688

2.10 B. imbricata (3) § 0.0002 0.0003 0.2360

2.10 M. ciliata (1) § < 0.0001 < 0.0001 0.4976

†Main effects significant: 2.9 Positive heat shock response 2.10 Negative heat shock response

‡applications !population § analysis of transformed data

42 Table 2.6: Comparison of mean (+ standard error) % germination of four study species for heat shock and smoke treatments. Means within the one treatment followed by different letters are significantly different at P = 0.05.

G. sieberiana (1) K. ambigua B. imbricata †(3) M. ciliata †(1) Heat shock level 25 14 (± 1.8) a 49 (± 3.2) a 11 (± 2.0) c 41 (± 2.7) c (0 C) 50 11 (± 1.5) a 84 (± 2.2) b 7 (± 1.5) b 30 (± 3.1) bc

75 51 (± 2.3) b 64 (± 4.7) ab 7 (± 1.3) b 30 (± 2.8) b

100 53 (± 3.0) b 76 (± 3.5) ab 2 (± 0.8) a 16 (± 2.7) a

Smoke level 0 26 (± 3.4) a 51 (± 5.2) a 2 (± 0.7) a 11 (± 2.0) a (minutes) 5 34 (± 4.1) ab 69 (± 3.5) b 8 (± 2.0) b 32 (± 2.9) b

10 31 (± 3.3) ab 79 (± 2.4) b 8 (± 1.4) b 39 (± 2.9) b

20 38 (± 3.7) b 74 (± 3.1) b 9 (± 1.5) b 36 (± 2.9) b

† analysis of transformed data; back-transformed data shown

43 a) Gahnia sieberiana (population 1) usm < sm *** 70 heat: +ve linear *** 60

50

40

30

20

10

0 25 50 75 100

b) Kunzea ambigua usm < sm *** 100 heat: +ve linear ***

% germination (+/- SE) % germination 90

80

70

60

50

40

30

20

10

0 25 50 75 100 Heat Shock (°C)

Figure 2.9: Mean final germination plotted against temperature of heat shock for a) Gahnia sieberiana (population 1), and b) Kunzea ambigua. Results of Planned Comparisons of Main Effect means are shown above each Figure for unsmoked vs smoked means; Trend Analysis of heat shock means (usm = smoked; sm = smoked; heat = heat shock). Symbols as Figure 2.1

44 a) Baeckea imbricata (population 3) usm < sm *** 25 heat: -ve linear *

20

15

10

5

0 25 50 75 100

b) Micromyrtus ciliata (population 1) usm < sm *** 70

% germination (+/- SE) % germination heat: -ve linear *** 60

50

40

30

20

10

0 25 50 75 100

Heat Shock (°C)

Figure 2.10: Mean final germination plotted against temperature of heat shock for a) Baeckea imbricata (population 3) †, b) Micromyrtus ciliata (population 1)†. Analysis as Figure 2.9. Symbols as Figures 2.1 & 2.2

45 Smoke dose effect

Smoke dose affected germination of 16 out of the 21 populations that had smoke as a significant term (Table 2.7). Smoke dose (amongst smoke means) was significant in 21% of cases; there were 18 significant smoke dose effects out of 84 independent tests (21 populations x 4 heat shock levels). High smoke dose increased germination in 11 cases, and reduced germination in 7 cases (Table 2.7). There was no association between heat shock levels and whether the high smoke dose increased or reduced germination. Also, there was no association between whether smoke per se affected germination (first test), and whether smoke dose was significant within a heat shock level (heat shock x smoke significant). Smoke dose was significant in 7 cases where differences between pooled smoke and unsmoked germination was not significant, and smoke dose was significant in 8 cases where differences between pooled smoke and unsmoked germination was significant (Table 2.7). Smoke dose is likely to have had a biological effect on germination of Baeckea brevifolia, Epacris paludosa, E. m. var. rhombifolia and Woollsia pungens. The positive dose effect was strong and repeated for B. brevifolia (Table 2.7, Fig 2.2) and E. paludosa (Table 2.7, Fig 2.7), and a repeated positive trend was apparent for E. m. var. rhombifolia (Table 2.7, Fig 2.4). Germination of W. pungens was reduced by smoke within a 50°C heat shock, hence a negative effect of high smoke dose within a 25°C heat shock is not unlikely (Table 2.7, Fig 2.8). Other smoke dose effects were of lesser magnitude and do not have consistency across heat shock levels, hence do not appear to be of biological consequence.

46 Table 2.7. Planned Comparisons of smoke dose effect within heat shock levels for populations affected by smoke as a main effect or an interaction. Populations are firstly sorted by the lowest heat shock level at which a smoke dose effect occurred, and secondly sorted by the range of heat shock levels within which a smoke dose effect occurred. Species responding positively to higher smoke doses are listed first, and species responding negatively to higher smoke doses are listed secondly. One species responding positively and negatively to higher smoke doses is listed between these categories.

Figure No. & Population Smoke dose effect at heat shock level (°C) Category (Table 2.4) 25 50 75 100 2.3 B. utilis + ‡ 2.2 B. brevifolia ++ ++ ‡ 2.5 B. diosmifolia + 2.7 E. paludosa (1) + ‡ + 2.6 B. imbricata (1) + ‡ 2.10 B. imbricata (3) + 2.4 E. m. var. rhombifolia ++ 2.5 E. muelleri ++

2.9 K. ambigua ++ --

2.8 W. pungens - ‡ 2.5 E. longifolia - ‡ 2.4 D. secundum (2) - ‡ 2.2 E. microphylla - 2.1 K. capitata (1) - 2.2 B. imbricata (2) -

Sum number of positive effects 3 3 5 0 Sum number of negative effects 2 1 3 1

†population ‡ smoke dose effect significant when smoke vs no smoke not significant

47 No treatment effect

Epacris coriacea and Epacris obtusifolia exhibited low dormancy, with high levels of germination across all treatments, none of which significantly affected germination (Table 2.8, Category 11). Overall mean germination was 97% for E. coriacea and 91% for E. obtusifolia.

Germination of Calytix tetragonia was uniformly c. 40% across all treatments. Seed coats appearing to hold seed were used in the experiment. Approximately 48% of such coats did not contain seed. Germination of this species was rapid and uniform, hence it is probable that most seed germinated. Sprengelia monticola exhibited a moderate level of dormancy, with germination across all treatments less than 30%. This species showed no response to heat or smoke (Table 2.8). Germination of Epacris crassifolia population 2 was low, until transferral into light (Section VI). No treatment effects were significant. Two Baeckea ramosissima ssp. ramosissima seeds germinated at 250C, and a further 3 seeds germinated when, after an average of 90 days, seeds were transferred to incubation temperatures of either 15 or 200C. There was no pattern of germination related to the treatments.

48 Table 2.8. Effects of heat shock, smoke, applications of these treatments (bulked application design), and interactions between these factors on % germination of the 7 study populations for which the main effects and interactions between main effects were not significant; ANOVA P-values are shown. Factors are nested within factors shown in parentheses.

Category† Source Heat shock = Smoke levels H apps S apps (S x H x S S x H Residual H = S ‡ (H) H apps (H)) apps (H) bulked df 3 3 4 32 9 12 128 applications F H apps S x H apps S apps Residual S x H apps S apps vs single df 3 3 9 176 independent F Residual Residual Residual applications vs 2.11 B. ramosissima negligable negligable negligable ssp. ramosissima germination germination germination 2.11 C. tetragonia 0.8372 0.3361 0.2857

2.11 D. secundum (1) 0.8059 0.2514 0.1530 0.0017 0.5586 0.2290 ! 2.11 E. coriacea 0.4258 0.8312 0.0444 0.2565 0.6028 0.5909

2.11 E. crassifolia (2) 0.0642 0.7819 0.5018 2.11 E. obtusifolia (1) 0.8059 0.2218 0.1889 0.2055 0.1333 0.7404 2.11 S. monticola 0.9208 0.3184 0.4201 0.0006 0.8325 0.6382

†No effect: 2.11

‡applications !population

49 Replicate populations within species

Most of the replicate populations within species were separated into different response categories (Table 2.12), however, visual inspection reveals similar patterns of response within the species. The response to the fire-related cues of Dracophyllum secundum population 2, and Epacris crassifolia population 1 were minor, hence the overall findings reveal a negligible fire response in these species. The germination patterns of the Baeckea linifolia populations were very similar (Fig 2.3), as were those of the B. imbricata populations (Figs 2.2, 2.6, 2.10). The general response was of heat shock reducing and smoke increasing germination, with an overall strong germination response to fire.

Alternative classification of species

A clear pattern of a response to smoke occurring most frequently within the moderate heat shock levels (50 - 750C) was evident. Germination of over half of the populations that responded to the fire-related cues increased when smoke was applied within the 750C heat shock level. Also, over two thirds of the positive responses to smoke occurred within the 50 and 750C heat shock levels (Table 2.9).

Half of the populations responded positively to smoke within only one heat shock level and may be considered to have narrow requirements for fire-stimulated germination. One quarter of the populations had a positive response to smoke within multiple heat shock levels, not including the highest heat shock level, and may be considered to have intermediate requirements for fire-stimulated germination. One quarter of the populations had a positive response to smoke within the highest heat shock level and may be considered to have broad requirements for fire-stimulated germination. Generally, the populations that responded to smoke alone also responded to smoke across the broadest heat shock range.

50 Table 2.9. Planned Comparisons of smoke effect within heat shock levels for populations affected by fire-related germination cues as main effects or an interaction. Populations are firstly sorted by the highest heat shock level at which a positive effect of smoke occurred, and secondly sorted by the range of heat shock levels within which the effect of smoke was positive.

Figure No. & Population Smoke effect at heat shock level (°C) Category (Table 2.4) 25 50 75 100 2.2 B. brevifolia +

2.3 B. linifolia (1) † + 2.3 B. linifolia (2) + 2.3 B. utilis +

2.4 D. secundum (2) + 2.7 E. crassifolia (1) + 2.4 E. m. var. rhombifolia + 2.7 E. paludosa (1) + 2.7 E. pulchella + - 2.9 G. sieberiana (1) + 2.5 B. diosmifolia - + + 2.5 E. longifolia + + - 2.5 E. muelleri + + 2.2 E. microphylla + + 2.10 B. imbricata (3) + + +

2.6 B. imbricata (1) + + 2.2 B. imbricata (2) + + + + 2.9 K. ambigua + + + + 2.1 K. capitata (1) + + + + 2.10 M. ciliata (1) + + + +

2.8 W. pungens -

Sum number of positive effects 7 12 15 5

†population

51 Section II

High heat shock and smoke effect

In contrast with the very high mortality of Acacia seeds following 1200C heat shock, five of the seven species had more than negligible germination following 1250C heat shock (Fig 2.10). The 1500C heat shock did not reduce germination of four species, 1750C heat shock did not reduce germination of two species, and half of the Kunzea ambigua seeds germinated following 2000C heat shock (Fig 2.11). Smoke did not compensate for the adverse affect of high heat shock, and had a negative effect on germination of Kunzea capitata within the highest heat shock level (Table 2.10, Fig 2.11a).

Interestingly, smoke had a major role in increasing germination within moderate heat shock levels (Section I) but did not within these high heat shock levels.

Table 2.10. Effects of high heat shock (125 - 200°C), smoke, and interactions between these factors on % germination of 7 study species; ANOVA P-values are shown.

Source Heat shock Smoke Heat shock x Smoke Residual df 3 1 9 16 F vs Residual Residual Residual K. capitata (1)‡ < 0.0001 0.1457 0.0434 K. ambigua < 0.0001 0.1484 0.2472 E. coriacea < 0.0001 0.9934 0.0610 B. imbricata (2)† < 0.0001 0.6139 0.6375 E. obtusifolia (3) 0.0484 0.6885 0.6175 D. secundum (2) neglible germination neglible germination neglible germination G. sieberiana (1) no germination no germination no germination †population ‡transformed data

52 a) Kunzea capitata (population 1)

100 a m a m a m a m a m a m

90

80 b m 70

60

50

40 b n

30 % germination (+/- SE) % germination 20

10

0 un smoked smoked un smoked smoked un smoked smoked un smoked smoked

125°C heat shock 150°C heat shock 175°C heat shock 200°C heat shock

Treatment

Figure 2.11: Mean final germination of a) Kunzea capitata (population 1)† seeds treated with 125, 150, 175 or 200°C and 0 or 10 minutes smoke. Mean germination within the one smoke level followed by different letters in the range a – b are significantly different at P = 0.05; means within the one heat level followed by different letters in the range m – n are significantly different at P = 0.05. Bars = S. E.s †Analysis of transformed data; back transformed data shown in Figure

53 b) c) Kunzea ambi gua Epacris coriacea a a a 100 25 a a 90 80 b 20 70

60 15

50

40 10

30 20 5 b b 10

0 0 125 150 175 200 125 150 175 200

d) Baeckea imbri cata (popul ati on 2) e) Epacris obtusi folia (population 3) a 30 a 6 a 25 5 % germination (+/- SE) % germination

20 4

15 3 b ab b

10 b b 2

5 1

0 0 125 150 175 200 125 150 175 200

Heat Shock (°C)

Figure 2.11 continued: Mean final germination of b) Kunzea ambigua, c) Epacris coriacea, d) Baeckea imbricata (population 2), e) E. obtusifolia (population 3) seeds treated with 125, 150, 175 or 200°C and 0 or 10 minutes smoke. Species arranged in order of decreasing levels of germination. Unsmoked and smoked means within each heat level were pooled and comparisons amongst heat levels then made by SNK procedure. Pooled heat level means followed by different letters in the range a – b are significantly different at P = 0.05. Bars = S. E.s

54 Section III

Effects of light

Additional seeds of all 8 species germinated after they were transferred into light (Table 2.11). Germination in the light was uniform across heat shock or smoke treatments (data not shown). The increase in germination of half of the species examined was notable.

Table 2.11. Percentage germination of seeds not treated with added heat shock or smoke and incubated in darkness, or of seeds that did not germinate in darkness and that were then transferred into light, or of seeds that were not treated with added heat shock or smoke and were incubated in light without prior incubation in darkness. Germination of transferred seeds was averaged over fire-related cue treatments because they did not affect germination.

Treatment: Initial % germination in % germination of darkness transferred seeds E. crassifolia (1) 12 92

E. muelleri 7 72

E. paludosa 28 47

E. microphylla var. rhombifolia 4 86

E. crassifolia (2) 2 78

E. microphylla 51 25

E. longiflora 58 7

E. pulchella 18 3

W. pungens 8 < 1

55 2.4 Discussion

Germination of most species was increased by the combination of heat shock and smoke, and only within specific heat shock levels for many species. However, these specific heat shock levels were only slightly predictable on the basis of habitat, and smoke dose responses were also only tentatively related to habitat. The form of the interaction between heat shock and smoke frequently changed over the levels of heat shock, with the loss of a smoke affect at higher heat shock levels. The level of heat shock had a greater effect on germination than the level of smoke that was applied. The frequent high levels of germination of seeds in light following their failure to germinate in darkness indicate that light may be an important factor affecting post-fire germination.

Interaction between heat shock and smoke

In contrast with other fire-prone floras, in which germination of roughly a third of the studied species is stimulated by either moderate heat shock or by charred wood/smoke alone (Keeley & Baer-Keeley 1999), these cues in isolation affected germination of only a few of the study species forming soil seedbanks in the Sydney region. The combination of fire- related cues was required to stimulate germination of many species from within the Sydney region, and many positive interactions between heat shock and smoke only occurred within specific heat shock levels. Although inherent variation in data was often high, and may have resulted in instances of Type I error, the high frequency of significant positive interactions between heat shock and smoke within specific heat shock levels indicates that such a response is probably common. The range of heat shock tolerance has been extended to 200°C, and the effect of smoke at high heat shock levels was frequently overridden. The need to examine a range of dormancy breaking cues and levels within cues in order to more accurately assess the germination response to fire is apparent.

Interactions between heat shock and smoke have been investigated using one (Keith 1997; Clarke et al. 2000; Gilmour et al. 2000; Kenny 2000; Morris 2000; Kenny et al. 2001; Willis et al. 2003) or two (Tieu et al. 2001) levels of added heat shock between 80 and 120°C. This study has revealed that such investigations require expanding on two counts, as illustrated in Figure 2.12. Firstly, if the interaction takes a complex form, it requires greater

56

Smoked Smoked Unsmoked onse onse p Unsmoked

(‘complex’ response) Germination res Germination Smoked

Response zone most frequently investigated Low High (25°C) Heat shock level (200°C) Figure 2.12: Germination responses to combinations of heat shock and smoke found in the present study, that would not have been found by applying heat shock solely within the most frequently investigated zone, or by applying a single heat shock level. Heat shock applied within the current study ( ), and most frequently within previous investigations ( ). Smoked and unsmoked seeds within a response category have the same line format. ( ) Obligatory combination, positive response within 50°C (Fig 2.3, Category 2.3) ( ) Quadratic heat shock response (Fig 2.7, Category 2.7)

57 ( ) from a composite of Figs 2.11a and 3.9.4. 57 sampling of the response zone of heat shock to detect it. Use of one heat shock level within the response zone assumes a uniform response to the interaction within the response zone. Secondly, an experiment using heat shock in the higher range of temperatures would have missed the positive germination response of Baeckea linifolia and B. utilis which occurred only at a moderate heat shock level, and may have recorded a negative response at the higher levels. Detection of response types requires sampling across many levels of the cues.

Interpretation of heat shock response

The germination response to heat shock must be interpreted in the context of 1) the level of heat shock that is likely to be generated within a particular habitat, 2) the soil depth to which heat shock penetrates, and the depth from which seedlings can emerge, and 3) the very important requirement for a post-fire residual seedbank and the less important requirement for post-fire germination to buffer against the possibility of local extinction. A post-fire residual seedbank is required to buffer against short fire-return intervals and post-fire germination is required to allow in situ seed production.

Heat shock within particular habitats

For the species with a positive germination response confined to the low heat shock levels, their location in the landscape probably ensures that they do not experience excessive heat shock. Baeckea brevifolia occurs on rocky outcrops where fuel load is low and discontinuous, hence fire is patchy and intensity is reduced (Benson 1985; Whelan et al. 1996; Clarke 2002). Baeckea linifolia and B. utilis occur in locations where the soil is likely to be wet during fire, and although thermal conductivity increases with moisture content, so does the specific heat content and consequently heat shock is lower within the upper soil layer (Aston & Gill 1976; Campbell et al. 1995). Heat shock as low as 500C reduced germination of Woollsia pungens in the current study. Germination of this species was also reduced by 800C heat shock (Kenny et al. 2001). Epacris crassifolia (population 1) germination was also inhibited by 500C and higher heat shock levels in the current study, however, unlike W. pungens, this species grows on wet cliff faces and so encounters a low frequency of fire. The effect of low intensity heat shock has rarely been investigated, however, heat shock of 40°C

58 (Auld et al. 1993), 50°C (Enright & Kintrup 2001), and 55°C (Warcup 1980) have increased germination of non-legume temperate Australian species.

Heat shock variability and a post-fire residual seedbank

Because the quantity, type and spatial array of fuel is variable, and the degree of combustion ranges from none to complete at small (Bradstock et al. 1992; Bradstock & Auld 1995) and large scale (Christensen & Kimber 1975; Fox 1978; Hamilton et al. 1991; Whelan et al. 1996), and because of variable topography and wind speed and direction (Albini & Anderson 1982), the heat released into soil is spatially variable (Christensen & Kimber 1975; Hobbs & Atkins 1988; Atkins & Hobbs 1995). Also, heat shock is rapidly attenuated with soil depth, hence the level of heat shock within the range of soil depths from which the study species can emerge is highly variable across the landscape. Consequently, the range of heat shock levels at which the study species experience a positive interaction with smoke will occur at the depth from which they can emerge at only some points on the landscape during any one fire, irrespective of its overall intensity.

Recruitment of species that respond positively within a narrow heat shock range is likely to be limited by the lack of coincidence of seed and appropriate heat shock. The maximum depth from which seeds used in the current study (excepting Gahnia sieberiana) could emerge, based on seed volume (Bond et al. 1999) is in the range 7 – 20 mm (average 10 mm, SE 0.56). During high intensity fire in southwestern Western Australia sclerophyll communities, heat shock at 20 mm depth ranged from over 500°C for 10% of a burnt area, to negligible for another 10% (Hobbs & Atkins 1988; Atkins & Hobbs 1995). Seed in patches of extreme heat shock are probably killed within the zone from which seedlings can emerge and are possibly stimulated to germinate at too great a depth to allow emergence. In contrast, during low intensity fire, maximum temperatures below 100°C are predicted in very shallow soil, and maximum temperatures of about 50°C are predicted for soil depths less than 20 mm (Auld 1986a). Although low intensity fires generate sufficient heat shock to stimulate germination of seed throughout the soil depth from which seedlings can emerge, a greater area is likely to experience no or insufficient heat shock. Patchiness in germination due too high or too low levels of heat shock is likely to follow any particular fire.

59 Species composition of germinants from the soil seedbank was determined by local heat shock intensity during the fire (Atkins & Hobbs 1995), and correlations between heat shock temperature and germination from the soil seedbank have also been determined at small scale following fires in the chaparral (Rice 1993; Odion 2000; Odion & Davis 2000), and fynbos (Bond et al. 1990). Species forming soil seedbanks in the Sydney region were also stimulated to germinate within narrow ranges of heat shock (Auld et al. 1993). Species with specific heat shock requirements are possibly ensured of a residual soil seedbank within the zone from which seedlings can emerge because the heat shock requirements are only met within patches of this zone. In contrast, species with more general heat shock requirements may have other requirements such as incubation temperature and water availability that regulate germination (Chapter 5).

Post-fire ambient temperature

In the middle of a summer day, in an open area in the Sydney region that has been burnt, soil surface temperature can exceed 600C, and can reach 500C at 10 mm depth (Auld & Bradstock 1996). Thus, seeds that did not experience high levels of heat shock during the fire may experience high temperatures after the fire, resulting in stimulation of germination of a greater proportion of the seedbank. Germination of Kunzea ambigua was stimulated by 500C heat shock, however, prolonged periods of high temperature when moisture is limiting may induce dormancy in this and other species (Chapter 3). Also, the smoke response of K. ambigua is indicative of a strong response to fire per se, rather than to changed environmental conditions that would only follow a summer fire. Whilst Gahnia sieberiana seeds may encounter high temperatures when the edge of a riverbank is exposed, such temperatures were too low in the current study and did not stimulate germination in a previous study (Auld et al., unpublished data 1996). Smoke may compensate for sub-optimal heat shock, also confining germination of this species to a post-fire environment.

Spatial distribution of smoke

In contrast with heat shock, there were few clear cases of smoke duration, or dose effect in this study. Whilst most species responded to the combination of heat shock and smoke which is a nearly unequivocal indicator of the passage of fire, germination of Baeckea

60 brevifolia, the only species with a positive smoke effect confined to the no added heat shock level, appears to have increased with increasing smoke dose. Baeckea brevifolia occurs on rocky outcrops where fuel load may be low and fire passage patchy, hence the heat shock produced when plants combust may not reliably reach nearby seeds in the soil. Smoke alone is probably also an unreliable indicator of the close passage of fire, as drifted smoke can stimulate germination in unburnt areas (Preston & Baldwin 1999). On rocky outcrops, increasing smoke doses may be associated with increasing proximity of seed to the passage of fire, hence removal of proximate competition.

Epacris microphylla var. rhombifolia and E. paludosa probably responded positively to high smoke dose. Both species occur in swamp habitat where soil water is frequently both slowly moving and near saturation. If the smoke that is generated by fire enters into solution of soil water, then less smoke will out-gas to the atmosphere. The concentration of smoke is likely to remain high for a long period of time if the water is slow moving or stagnant. Germination of species that occur in swamp habitat may possibly be more tolerant of high smoke dose than dry habitat species (e.g. Kunzea ambigua, Chapter 5). Because smoke solution may flow to unburnt areas, a high concentration of smoke may again be associated with increasing proximity of seed to the passage of fire, hence removal of proximate competition.

Germination of Woollsia pungens was reduced by smoke within one heat shock level and by higher smoke dose within another heat shock level. Adult W. pungens plants have been killed by fire, with post-fire recruitment from a soil seedbank (Benson 1985; Benwell 1998), although resprouting from the base has been recorded following a medium intensity fire (Fox 1988). As resprouting capacity is limited and the seed has no special dispersal mechanism, germination from an in situ seedbank would be required for replacement of adult plants killed by fire. The mechanism whereby germination of W. pungens is stimulated by fire remains unknown.

Whilst the full range of variation in smoke dose in the field is not known, smoke dose as short as 30 seconds stimulated germination of Kunzea ambigua, and one hour of smoke did not inhibit germination of Dracophyllum secundum (data not shown). Smoke concentration is probably very poorly correlated with soil depth during or after a fire. Spatial variation in the quantity, type, spatial array, moisture content and degree of combustion of fuel is likely to produce some spatial variability in the concentration of smoke generated. The concentration 61 of smoke that settles on the soil surface may be influenced by the height above the soil at which combustion occurred and wind speed during combustion. The concentration of smoke at greater soil depths may increase with increasing fire intensity because of more complete combustion of above-ground biomass, and because dry heating of buried organic matter may produce the bioactive compound(s) produced by the dry heating of plant material (Baxter et al. 1994; Jager et al. 1996). In contrast with heat shock, the active smoke constituents penetrated through 8 cm of washed river sand, and penetration was affected by subsequent watering (Kenny 2003, unpublished).

No Treatment Effect

The lack of response to heat and smoke of Sprengelia monticola and Epacris crassifolia population 2 possibly reflects the low frequency at which these species are likely to encounter fire because they grow on wet rock faces. Interestingly, germination of E. crassifolia is probably stimulated by light.

Neither the percentage, nor the time course of germination of Epacris coriacea or E. obtusifolia was affected by heat or smoke. High levels of E. obtusifolia post-fire germination have been recorded (Benwell 1998), with heat shock of over 60°C required for maximum germination (Auld et al., unpublished data 1996). Positive germination responses of Epacris obtusifolia to smoke or smoked water have been reported (Roche et al. 1997a; Gilmour et al. 2000). Other soft-seeded species in the genus Epacris have shown a positive germination response to smoke (Gilmour et al. 2000). Responses to heat shock and/or smoke may have been apparent if these two populations had higher levels of dormancy. Similarly, the Calytrix tetragonia population investigated was probably non-dormant, so even if it had a positive response to smoke as has previously been found (Kenny et al. 2001), this response would not have been apparent. In contrast, Baeckea ramosissima ssp. ramosissima was highly dormant and required either different cues or additional factors to stimulate germination.

62 Dormancy in the field

Heat shock and smoke reduce dormancy levels of many species that form soil seedbanks in the fire-prone Sydney region. However, there are likely to be other processes operating within the soil, and these results from the laboratory are interpreted as relative differences between species and treatments. For example, the dormancy status of seed in the soil may vary independently of fire. A fraction of most persistent seed populations are released in a non-dormant state (Auld & O’Connell 1991; Dixon et al. 1995; Keith 1996; Auld et al. 2000), and seeds of shrub species in the Sydney sandstone communities generally gradually decay independently of fire from dormant to non dormant whilst in the soil (Auld 1987; Auld et al. 1993), and dormancy patterns may vary within and between seed crops of the same species (Auld et al. 2000), and may change over time as seeds age (Morrison et al. 1992; Roche et al. 1997a). Notably, some species with long-lived fire-related secondary dormancy also have a constant fraction in which dormancy is enforced in the field (Edwards & Whelan 1995; Auld et al. 2000). Field-enforced dormancy of Kunzea ambigua (Auld et al. 2000) would prevent the high levels of germination of this, and possibly other species, within the control treatment.

However, when germination levels within the control were not extremely high, an increase in germination within the heat shock and smoke treatments was evident for many species. This increase in germination is probably due to germination of the more dormant fraction of the population, and indicates that dormancy is fire-related. The species with low levels of dormancy generally have no or low resprouting capacity, so are totally or highly reliant on seed germination for persistence of a population. However, dormancy is not required for the persistence of seed in soil (Thompson et al. 2003).

Repeatability of studies

Repeatability across previous studies

The response to heat shock and smoke across the multiple populations of the current and a previous study (Kenny et al. 2001) was variable but broadly comparable (Table 2.12). Generally species either did or did not have a fire-related germination response, and different populations responded to different combinations of cues. The increased germination of 63 Kunzea ambigua in response to both heat shock and smoke has been found previously, although the interaction between them was significant in the previous study (Kenny et al. 2001). Similarly, Epacris microphylla responded positively to smoke alone, and to the interaction in both these studies, however heat shock alone also increased germination in the previous study (Kenny et al. 2001). The low germination of Gahnia sieberiana in the current study was consistent with previous observations for this species (Murray 1994, Auld et al. 2000), as was the strong germination response to the two higher heat shock temperatures (Kenny et al. 2001). However, the increase in germination of Gahnia sieberiana only occurred in a previous study when both cues were applied (Kenny et al. 2001), while the current study found the increase due to heat shock and smoke to be independent and additive. Also, germination of Kunzea capitata increased equally with heat shock, smoke, and the combination of these cues (Kenny et al. 2001), while the current study found both cues necessary for an increase in germination. Heat shock had a negative effect on germination of Woollsia pungens across studies, however the interaction also reduced germination within the current study. The different Baeckea imbricata populations in the current study had different responses to the cues, whilst no effect was found in a previous study (Kenny et al. 2001). The cues did not affect germination of Calytrix tetragonia in the current study, whilst smoke increased germination in a previous study (Kenny et al. 2001).

Variability within populations in the germination response to heat shock or smoke have been reported previously, but not for the combination of these cues. Significant population / heat shock intensity / heat shock duration interactions were found for all four Acacia species where two populations were studied (Auld & O’Connell 1991). The range of heat shock that broke dormancy of Darwinia biflora was dependent on site and season (Auld et al. 1993). Burchardia umbellata from southeast eastern Australian grasslands germinated readily without any treatment (Lunt 1995; Morgan 1998), whilst seed of similar viability from southwest western Australian forests required charate (Bell et al. 1987) or smoke (Dixon et al. 1995) to stimulate germination.

64 Table 2.12. Response of multiple populations to fire-related germination cues. Comparisons made between populations within the current study and populations across the current and Kenny et al. (2001) study. Categories are explained in Tables 2.3, 2.4, 2.5, 2.8, and in text.

Species Population Category Category No. B. imbricata 1 Obligatory combination 2.6 2 Smoke alone 2.2 3 Smoke +ve; Heat shock -ve 2.10 Kenny et al. 2001 No effect 2.11

B. linifolia 1 Obligatory combination 2.3 2 Obligatory combination 2.3

C. tetragonia current study No effect 2.11 Kenny et al. 2001 Smoke alone 2.2

D. secundum 1 No effect 2.11 2 Obligatory combination; Heat shock -ve 2.4

E. crassifolia 1 Quadratic heat shock response 2.7 2 No effect 2.11

E. microphylla current study Smoke +ve; Interaction +ve 2.2 Kenny et al. 2001 Smoke +ve; Heat shock & Interaction +ve 2.2

G. sieberiana 1 Smoke +ve; Heat shock +ve 2.9 Kenny et al. 2001 Obligatory combination 2.4

K. ambigua current study Smoke +ve; Heat shock +ve 2.9 Kenny et al. 2001 Smoke +ve; Heat shock & Interaction +ve 2.2

K. capitata current study Synergistic 2.1 Kenny et al. 2001 Smoke +ve; Heat shock & Interaction +ve 2.2

W. pungens current study Heat shock –ve; Interaction –ve 2.8 Kenny et al. 2001 Heat shock –ve; Smoke alone 2.2

65 Repeatability within the current study

Within this study, the response of different populations of a species to heat shock and smoke were different, but the patterns were generally comparable (Table 2.12). There was a consistent trend of decreasing germination of the Baeckea imbricata populations with increasing heat shock and notably, population 3 had both the lowest level of germination and least tolerance of the highest heat shock level. Smoke increased germination of all populations, and the positive effect of smoke became less stimulatory with increasing heat shock. Germination of the two Baeckea linifolia populations was increased by the combination of 50°C heat shock and smoke, and overall germination within heat shock levels decreased with increasing heat shock intensity. Smoke had no or only a slight effect on germination the two Dracophyllum secundum populations, whilst the higher heat shock levels inhibited germination of only population 2. Also, only Epacris crassifolia population 1 was negatively affected by heat shock, possibly because they had thinner testas than population 2 (Cocks & Stock 1997). Epacris crassifolia has positive photoblastic seeds, and the higher level of germination of population 1 in darkness when heat shock was not inhibitory is also consistent with thinner testas (Karssen 1970a, b).

Gross differences between the response of Gahnia sieberiana population 1 to heat shock and smoke were apparent across sections of this study, due to storage effects. Germination of this population decreased with duration of storage, finally becoming totally dormant (Section II). During the transition to dormancy, seed initially became more responsive to smoke, and non-responsive to all bar the highest heat shock level (Chapter 3). Actinotus helianthii also became totally dormant during storage (Chapter 3), and the dormancy of many species increased following storage for more than 3 years (Chapter 4, Section II).

Gross differences between the response of different populations of a species to heat shock and smoke were apparent across sections of this study. Germination of smoked Gahnia sieberiana population 2 seeds was reduced by the 75°C and 100°C heat shock, and germination of unsmoked seed was reduced by the 100°C heat shock, the opposite response to population 1 seeds (Chapter 3, Section II). Also, germination of Epacris obtusifolia populations 2 and 3 was greatly increased due to a strong interaction between heat shock and smoke (Chapter 3, Section I), in contrast to the germination of population 1 (this and other Chapters). 66 The reason for the differences in responses of different populations of a species within this study and between studies is unknown. Differences in methodology and pre-treatment storage could contribute to variability in results between studies. Differences in results within this study indicate large effects due to differences in the environment in which seeds were produced, because fire-induced population fragmentation and unique selective forces following each fire is predicted to result in the rapid evolution of isolated populations into distinct and specialised species (Cowling 1987), and because en masse postfire germination reduces the evolutionary buffer provided by a persistent seed bank, and therefore may increase endemism and narrow adaptation (Parker & Kelly 1989).

Populations may have more similar responses when the seed of multiple years is considered. Currently, results are too variable to allow rigid classification of species germination responses to fire-related germination cues, although a correlation between the fire regime of particular habitats and species germination responses is apparent.

Effects of light

The stimulation of germination by light in some species probably indicates that post- fire germination of some species is dependent on erosion. The effects of light are more fully explored in Chapter 3.

Implications within the current study

A germination response within a narrow heat shock range may ensure both that some germination occurs in response to fire, and that a residual seedbank of non-stimulated seed remains. The possibility that post-fire water availability may ensure a residual seedbank in species that respond across a broad heat shock range is considered in Chapter 5. Because the level of heat shock had a greater effect than the level of smoke applied, a range of heat shock levels but only the intermediary smoke dose (10 minutes) was applied in subsequent investigations.

67 Chapter 3. Effects of pre- and post-fire temperature

3.1 Introduction

Overview

Temperature is probably the most reliable indicator of season, but its affect on germination of species forming soil seedbanks in the Sydney region is unknown. The question of whether storage or incubation temperature affects germination, and whether the fire-related cues interact with temperature to influence germination of this flora is investigated in this chapter. Effects of changes in temperature were explored because temperature changes across seasons and within burnt locations. Also, the effect of light on germination is further investigated in this chapter. Relationships between responses to simulated season of fire and Family, adult plant regenerative response or habitat of species were investigated.

Temperature and germination

Temperature is probably the most important environmental variable linking germination and conditions suitable for seedling establishment because temperature plays a pivotal role in determining seed germination, and natural selection favours environmental cuing mechanisms that decrease the probability of encountering unacceptable growth conditions following germination (Angevine & Chabot 1979). Temperature affects the timing of germination through its separate effects on dormancy and germination. Dormancy acts to restrict the range of temperature over which seeds have the capacity to germinate. This range is characteristic for a species, with clear minimum and maximum temperatures between which seeds can germinate (Thompson 1970), and it is reasonable to assume that these germination responses are adaptive (Thompson 1973). In regions where rainfall is highly seasonal, a number of studies have shown that maximum germination occurs in the temperature range corresponding to when soil moisture is adequate for sufficient time to allow seedling establishment. Southern temperate Australia has a Mediterranean-type climate, with cool, wet winters and hot, dry summers. Germination of understorey species from Eucalyptus marginata forest of south-western Western Australia tend toward a maxima at 15˚C, a temperature that coincides with the more reliable rainfalls

68 from the winter frontal storms, and very little germination occurred above 23˚C (Bell & Bellairs 1992; Bell 1994; Bell et al. 1995). Peak germination responses at lower temperatures in jarrah forest species may reflect an advantage of early germination to maximize the length of establishment prior to the summer drought (Bell & Bellairs 1992). Optimum temperatures for germination were slightly higher for understorey species of the south-western Western Australia sandplain kwongan, where winters are warmer (Bell & Bellairs 1992). Species from regions where moisture is more available during warmer months have higher optimum temperatures for maximum germination (Tothill 1977; Mott 1978; Lodge & Whalley 1981; McKeon 1985; Bell & Bellairs 1992; Maze et al. 1993). The maximum temperature at which three Eucalyptus (Bell & Bellairs 1992) and three Banksia species (Sonia & Heslehurst 1978) germinated reflected a cline in the seasonal moisture availability where they occur. In addition, establishment success of a number of species was greater following germination in the season with the temperature that enhanced germination (Ross 1976; Lodge 1981; Lodge & Whalley 1981). If there are seasonal affects on germination from the soil seedbank in the Sydney region, temperature can be identified a priori as a probable controlling mechanism.

Temperature and dormancy

Dormancy is a seed characteristic, the degree of which defines what conditions should be met to make the seed germinate (Vleeshoumers et al. 1995). Dormancy is a mechanism that prevents germination when conditions are favourable for germination but not for seedling survival. Dormancy strengthens as the environment becomes less conducive to seedling survival and, as dormancy strengthens, the range of environmental conditions over which germination is possible becomes narrower. Conversely, dormancy weakens as the environment becomes more conducive to seedling survival and, as dormancy weakens, there is a widening of the environmental conditions within which germination can proceed (Vegis 1963). The factors that weaken dormancy are factors associated with an environment that is favourable for seedling survival (ibid). Consequently, germination is concentrated within favourable seasons and the seeds that germinate are more frequently within locations favourable for seedling survival (Fenner 1995). When such germinants subsequently have higher reproductive success than germinants in microsites where dormancy is less frequently broken (Rice 1985), then cueing of germination has adaptive value (Antonovics 1976).

69 The high temperature inhibition of germination of species from Mediterranean- climactic regions is attributed to an adaptation against germination following chance summer rain (Thanos & Georghiou 1988), or a drought-avoiding syndrome (Pierce & Moll 1994). Although seedling survivorship was greater within burnt than unburnt areas (Whelan 1977, unpublished), mortality due to desiccation (Whelan & Main 1979; Lamont et al. 1991; Lamont & Witkowski 1995; Richards & Lamont 1996) and competition for moisture between seedlings (Lamont et al. 1993) was very high during their first summers. Germination cued to winter rains would be a benefit in the survival of seedlings in a habitat with harsh summer conditions (Bell 1999). Whilst rainfall is less seasonal in the Sydney region, evaporation is higher during summer, particularly in burnt areas, and the soil derived from Hawkesbury Sandstone is generally well drained. Mortality of post-fire seedlings in the Sydney region was attributed to moisture stress, and soil moisture was more frequently limiting during summer (Auld 1987). Seed dormancy may increase during summer if moisture availability strongly influences seedling survival.

Temperature and secondary dormancy

Factors that reduce dormancy are not required for germination itself, but prime the seed to subsequently respond to conditions that support germination (Bewley & Black 1982a). The degree of dormancy may cycle through seasons with the changes in temperature; this dormancy, subsequent to primary dormancy when seed is shed, is known as secondary dormancy. Dormancy is alleviated during the season preceding the period with favourable conditions for seedling development, then a period of germinability is followed by the induction of secondary dormancy in the season preceding the period with harmful conditions for plant survival (Kaarsen et al. 1988). An annual pattern of change in dormancy of buried seed populations has frequently been found for annual species, and also occurs in some perennial species (Zohar et al. 1975; Baskin & Baskin 1988; Auld et al. 1993, 2000). A seasonal dormancy cycle occurred in buried seeds of Grevillea caleyi and possibly Darwinia biflora, two species that form soil seedbanks in the Sydney region. The proportion of non- dormant seed was high in late summer, when conditions presumably favour seedling recruitment of these species (Auld et al. 2000). The most favourable period for germination in a Mediterranean-type climate is autumn; hence dormancy may be reduced by high summer temperatures and increased

70 following low winter temperatures. However, such a pattern was not found for understorey species from southwestern Western Australia (Bellairs & Bell 1990; Bell & Bellairs 1992).

Fire and dormancy

Factors associated with fire, including heat shock and smoke, may lower the level of seed dormancy, resulting in increased germination. The reduction of dormancy due to smoke is also apparent when the range of conditions under which a species will germinate is increased following smoke application. Germination of the fire climax grass Themeda triandra is increased by fire (Lock & Milburn 1971), and the increased germination produced by smoke extract also occurred within a temperature that was sub-optimal for germination (Baxter et al. 1994). However, whilst aerosol smoke increased germination of a shrub forming soil seedbanks in the Sydney region, smoke did not interact with incubation temperatures (Willis et al. 2003). Whilst dormancy may be influenced by factors associated with fire, dormancy status is determined by a number of simultaneously operating factors, and the strength of influence of these factors reflects their independent and interacting impact on seedling survival. Hence, dormancy status will probably remain high during seasons when moisture supply is inadequate for seedling survival regardless of any other factors. The permissive effect of smoke on germination is only realised under favourable external conditions. Fire and/or smoke applied during summer and/or autumn produce much higher levels of germination than following winter and/or spring applications in the Mediterranean-type climates of southwestern Western Australia (Roche et al. 1998), south-eastern Australia (Tolhurst & Oswin 1992; Tolhurst 1996), and South Africa (Boucher 1981; le Maitre 1988; de Lange & Boucher 1993b), where the trend of maximum regeneration following autumn fires also applies in all-year rainfall areas (Midgley 1989). Also, the percentage survival of seedlings was higher following the autumn rather than spring smoke treatment in southwestern Western Australia (Roche et al. 1998). Post-fire germination of a South African species (de Lange & Boucher 1993b), and of chaparral species, also within a Mediterranean-type climate, is delayed until the first rainy seasons of late winter or early spring, regardless of when fire occurs (Horton & Kraebel 1955; Keeley 1977; Keeley 1991). The timing of post-fire germination is determined by environmental conditions, and they can be altered by fire.

71 Post-fire soil temperature regimes

An environmental condition that can be altered by fire is the diurnal temperature regime of an area. The diurnal range of temperature experienced by a seed increases when a free water, or foliage, or litter layer is removed, and seedling survival may be enhanced under these conditions (Thompson et al. 1977; Thompson & Grime 1983). Low seasonal air temperature is strongly correlated with the breaking of dormancy of the South African Proteaceae, Leucospermum cordifolium (Brits & Van Niekerk 1986). However, L. cordifolium and L. cuneiforme only germinate after fire. Fire removes the vegetative cover, and hence the close to continuous shading within unburnt fynbos, and consequently the soil temperature of the burnt area is increased. The optimum minimum and optimum maximum diurnal temperatures for germination of these species occurs only in burnt areas, and then only in winter (Brits 1986). Ants bury the myrmecochorous seeds, and the optimal maximum temperature within the range of soil depths at which the seeds are buried occurs in late autumn to early winter (Brits 1987). The optimum minimum and maximum diurnal temperatures are slightly lower for germination of another Proteaceae, Serruria florida, but all species germinate at the same time because the climate where S. florida occurs is slightly cooler (Brits 1987). Similarly, germination of a number of other fynbos species were promoted by alternating temperature regimes that occur during autumn and early winter (Pierce & Moll 1994). The season of a burn has a large influence on germination from the soil seedbank in Mediterranean-type climates. Germination from the soil seedbank in the Sydney region may be less influenced by the season of a burn because temperatures are less extreme and rainfall is relatively constant across months. However, both the temperature regime and soil moisture status may be altered when an area is burnt, and the changes in these conditions may be influenced by components of the fire, including season.

Influence of fire on soil temperature regimes in the Sydney region

Average air temperature for Sydney and Katoomba, in the Upper Blue Mountains are shown in Figure 3.1 (Bureau of Meteorology 2003). Relationships between maximum daily air and soil temperature have been determined through a depth profile of soil derived from Hawkesbury sandstone in the Sydney region (Auld & Bradstock 1996). Within unburnt open woodland near Sydney, during sunny days of January 1993, maximum soil surface

72 temperature was predicted to be 40˚C. The amelioration of temperature with soil depth was considerable, as the maximum temperature at 10 mm depth was predicted to be 31˚C. The maximum daily air temperatures in Sydney during this period were between 21 and 37˚C, hence, 10 mm of soil had an insulating effect. The average diurnal temperature range was predicted to be about 10˚C at 10 mm depth, and negligible at 15 mm depth within an unburnt environment (Auld & Bradstock 1996). Maximum soil temperatures during December of 1990 were much higher in the same area following an intense summer fire. Both the air temperature, and the minimum soil temperature were highly comparable, but the maximum soil temperature was substantially higher because it was subjected to longer and more direct solar irradiance after the litter and vegetation cover had been removed (Raison et al. 1986), and because the soil had a higher absorbance coefficient due to the reduction in albedo (Walker et al. 1983). The maximum soil surface temperature of the burnt site was predicted to be 70˚C, and temperatures exceeding 60˚C at midday in summer have been recorded on exposed surfaces of soil derived from Hawkesbury sandstone in the Sydney region (Bradstock 1985, unpublished). The insulation by 10 mm soil depth was predicted to reduce the maximum soil temperature to 52˚C. The average diurnal temperature range of the surface soil was predicted to be greater than 40˚C, and about 25˚C at 10 mm depth within the burnt environment (Auld & Bradstock 1996). In contrast, a low intensity fire removes less litter and canopy cover and causes less change in albedo. Also, lower temperatures, shorter days and lower meridian values in winter result in less irradiance and smaller increase in soil temperatures. The maximum soil surface temperature following a low intensity winter fire in a closed heath near Sydney, during sunny days of August 1992, was predicted to be 26˚C (Auld & Bradstock 1996). The maximum soil temperature at 10 mm depth was predicted to be 19˚C, whilst the maximum daily air temperatures in Sydney during this period were between 13 and 17˚C. The loss of insulation may result in lower minimum temperatures. The minimum soil temperature of about 8˚C recorded at 6 mm depth after the winter fire is the same as the average minimum air temperature for Sydney during August (Auld & Bradstock 1996). However, minimum soil surface temperatures of 0 - 5˚C in burnt areas have been recorded at night in midwinter in the Sydney region (Bradstock 1985, unpublished). Most germination in the Sydney region occurs following fire, therefore the soil temperature following fire may influence germination from the soil seedbank. Maximum temperatures at around 10 mm depth in soil derived from Hawkesbury Sandstone in the Sydney region are comparable to maximum air temperatures, and are in the range 15 to 30˚C

73

a) Mean monthly rainfall (mm)

300 Mean monthly evaporation (mm) 30 Mean daily maximum temperature (°C)

250 25

200 20

150 15

100 10

50 5

0 0

and evaporation (mm) b)

300 30

250 25

200 20 Mean daily maximum temperature (°C) Mean monthly rainfall rainfall Mean monthly

150 15

100 10

50 5

0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Month

Figure 3.1. Mean daily maximum temperature and mean monthly rainfall and evaporation at a) Sydney airport (coastal), b) Katoomba (inland, 1040 m ASL).

74 (Auld & Bradstock 1996). A low to moderate intensity fire will cause change in the soil temperature regime, with a possible slight extension of the temperature range. The seasonal temperature at the time of a low to moderate intensity fire will probably influence dormancy and germination more than a slight change in temperature subsequent to the fire. In contrast, whilst the seasonal temperature at the time of a high intensity summer fire may affect seed germination through its effect on dormancy, both dormancy and germination are likely to be influenced by the relatively high post-fire soil temperature.

Influence of fire on soil moisture regimes in the Sydney region

Whilst rainfall in the Sydney region does not have a strong seasonal pattern, the rate of evaporation is highest in summer (Figure 3.1). The frequency of moist surface-soil days in the Sydney region, predicted on the basis of long-term daily rainfall and monthly evapotranspiration data, was substantially greater in winter than in summer months (Bradstock & Bedward 1992). For example, up to 30 moist surface-soil days are predicted for June in 24% of years, whilst no more than eight moist surface-soil days are predicted for January in 100% of years. The frequency of moist surface-soil days in summer is probably lower still following fire as the rate of evaporation is likely to be discontinuously greater during summer in an area that has recently experienced high intensity, extensive fire, than under any other set of conditions. Higher temperature, greater wind speeds, and removal of a greater amount of organic matter, which enhances the moisture-holding capacity of soil, contribute to this increase in evaporation. Evaporation and high temperatures in shallow soil probably reduced the levels of post-fire seedling emergence due to increased desiccation (Grant et al. 1996; Tozer 1998). High summer temperature was predicted to prevent seedling emergence of two Banksia species in over 50% of years during late spring and summer (Bradstock 1985, unpublished), however the water relationships of large seeds on the soil surface are particularly poor compared with small and/or buried seed. Limiting soil moisture (Auld et al. 1993) or high temperatures in themselves may influence germination, and/or dormancy of soil seedbanks in the Sydney region. The historical (McLoughlin 1998) and current (Conroy 1996) peak activity of fires in the Sydney region is spring-midsummer, which concurs with predictions based on climate (Luke & McAuthur 1978). Therefore, it is probable that most species are adapted to warm season fire. However, heathlands remain fire-prone throughout much of the year due to well- aerated fine fuel, the tendency for dead foliage to persist on some plant species, flammable

75 terpenes and waxes present in the foliage of some shrubs, and the direct exposure of fuel particles to wind and solar radiation in the absence of tree canopies. They are able to carry fire within relatively short time following substantial rain (Keith et al. 2002), hence species may also be adapted to cool season fire. Whether the germination of any or many species is different across cool or warm season fires is currently unknown. Prescribed burning in the Sydney region is predominantly conducted in the cooler autumn-winter season (April-July) (McLoughlan 1998), with unknown effects on species composition. Fire within prescribed burning conditions altered species composition in the Sydney region (Clark 1988). Plots of dry sclerophyll forest were burnt by low to medium intensity fire in autumn and spring, and reburnt in the same seasons after six years. Of the 37 species monitored, eight had population numbers consistently below pre-fire levels irrespective of season, seven had better recovery within the autumn burn plots, eight had better recovery within the spring burn plots, and fourteen had population numbers equal to or greater than pre-fire values across seasons. Three species were consistently absent from the spring burn plots, and two from the autumn burn plots. Following the second fire, three additional species were lost for at least one year from the spring burn plots, and seven from the autumn burn plots. Temperature and rainfall may explain some variation more than the season of burning per se (Clark 1988), however the possibility of species-specific responses to season of a fire was apparent.

Aims

The effect of season of fire on germination from the soil seedbank in the Sydney region is currently poorly known. Temperature is a reliable indicator of season in the Sydney region, and is a priori probably important in determining species germination response to season. The effects of the combination of temperatures that occur in different seasons within the Sydney region, and fire-related cues on the germination of a number of species forming soil seedbanks in a number of habitats (Appendix 1) were investigated. A common storage temperature and different incubation temperatures were used in Sections I – IV, and different storage and incubation temperatures were used in Sections V – VI. Section I Comparisons were made between germination of seeds of 20 species (29 populations) with a range of growth forms, adult plant regenerative responses and from a range of habitats, that were stored at 25°C, treated with a range of heat shock levels, with and without smoke

76 application, and incubated at 15 or 25°C; prescribed burning that is predominantly conducted in the autumn-winter season was simulated in the former case (25°C storage, 15°C incubation), and the spring-midsummer peak activity of fires was simulated in the latter case (25°C storage, 25°C incubation). Both the simulated seasonal temperatures (here called temperatures of incubation), and potential interactions between these temperatures and fire- related germination cues were investigated (Table 3.1, Section I; Figs 3.2.1-5). The null hypothesis was that temperature of incubation would not affect germination, nor was there an interaction between temperature of incubation and fire-related germination cues. Section II As greater temperature fluctuation occurs in the surface soil in a post-fire environment, one species was treated with fire-related cues and incubated within a diurnal temperature regime of 20 hours at 25°C and 4 hours at 35°C. Two populations of the species were treated (Table 3.1, Section II; Fig 3.3). The null hypothesis was that the incubation temperature regime would not affect germination, nor was there an interaction between the temperature regime and fire-related germination cues. Sections III and IV Seeds of 11 species (12 populations), stored at 25°C and treated with fire-related cues, were incubated at a broader range of temperatures (15 to 35°C) because such temperature extremes occur in shallow soil of the Sydney region (Table 3.1, Sections III and IV; Figs 3.4- 5). The null hypothesis was that temperature of incubation would not affect germination, nor was there an interaction between temperature of incubation and fire-related germination cues. Section V The pre- and post-fire temperature regimes may be different if the fire occurs at a change of season and/or due to fire-altered properties of the burnt environment. The germination response of a species may be influenced by an interaction between the fire- related germination cues, the physiological status of the seed at the time of the fire, as determined by the pre-fire temperature regime, and by the post-fire temperature regime. Comparisons were made between germination of seeds of 18 species (19 populations) stored at 15 or 35°C, treated with combinations of heat shock and smoke, and then incubated at the same temperature, or at 25°C (Table 3.1, Section V; Fig 3.6-7). The null hypothesis was that neither storage temperature, nor temperature of incubation would affect germination, nor were there any interactions between storage temperature, temperature of incubation or fire-related germination cues.

77 Section VI The effects of storage and incubation temperatures were investigated in combination with 75°C heat shock for Baeckea imbricata (Sections I, IV, V), however different germination responses occurred when 50°C heat shock was applied (Chapter 2). The effects of 15, 25 or 35°C storage and incubation temperatures were investigated in combination with 50°C heat shock for Baeckea imbricata (Table 3.1, Section VI; Fig 3.8). The null hypothesis was that none of the three factors, smoke, storage temperature, or temperature of incubation would affect germination, and that there were no interactions between these three factors. Section VII Whether seed coats contained water-soluble inhibitors, and the effect of seed coats on heat shock tolerance were investigated for one species, and the effect of seed coats on germination of another species was also examined (Table 3.1, Section V). The null hypothesis was that the removal of seed coats would not affect germination. Subsequent handling (Stage II) At the end of the incubation period as germination tapered off, seeds that had not germinated were transferred from less to more favourable incubation temperature and light conditions, as determined by higher levels of germination within the current and / or previous experiments. Conditions such as temperature and light change over time, hence seeds may experience such changes in the field. Also, post-transferral germination provides an indication of whether viability was affected by prior treatment (Harper & White 1974). The possibility that secondary dormancy was induced by various treatments is explored by reference to post- transferral germination within the Discussion. The major question was whether temperatures that occur in surface soil of the Sydney region influence germination, and whether the fire-related germination cues interact with pre- and/or post-fire temperature. The fire-related germination cues may either ameliorate or exacerbate the temperature effects on germination, thus decreasing or increasing respectively the seasonal effects of fire, or they may have no effect.

78 3.2 Methods

Section I 25˚C Storage, Heat Shock +/- Smoke; 15 or 25˚C Incubation

Seeds of 19 populations stored at 25˚C were treated with factorial combinations of heat shock (25˚C (= control), 50, 75 or 100˚C for 5 minutes), and aerosol smoke (0 or 10 minutes) and incubated at 15 or 25˚C (Table 3.1, Section I; Table 3.2; Figs 3.2.1-2). An additional 10 populations were treated with fewer combinations of heat shock and smoke because insufficient seed was available for all treatments. Seven of the ten species were treated with factorial combinations of 25˚C (= control) or 75˚C heat shock for 5 minutes, and 0 or 10 minutes of aerosol smoke within both incubation temperatures (Table 3.1, Section I; Table 3.2; Fig 3.2.3). For one further species, the germination response for the full range of heat shock and smoke doses had been characterised within the 25˚C incubation temperature (Chapter 2) so only the 25˚C (= control) and 75˚C heat shock with and without smoke was applied within the 25˚C incubation temperature, while the full range of heat shock with and without smoke was applied within the 15˚C incubation temperature (Table 3.1, Section I; Table 3.2; Fig 3.2.4). For two further species about which nothing was known, the full range of heat shock with and without smoke was applied within the 25˚C incubation temperature, while only the 25˚C (= control) and 75˚C heat shock with and without smoke was applied within the 15˚C incubation temperature (Table 3.1, Section I; Table 3.2; Fig 3.2.5). Six replicates of each species were treated with heat shock and smoke independently, one replicate of each species within each independent application of a treatment. A replicate was one petri dish with 10 seeds. Within the incubation temperatures, the replicates within a treatment were equally divided between two temperature-controlled cabinets (four exceptions in Table 3.3.3). Germination was monitored as previously described (Chapter 2) until it had tapered off; germination up to this point was called Stage I. Ungerminated seeds were then transferred into different incubation conditions, called Stage II (see Figs 3.2.1-3.8).

79 Table 3.1. Overview of experiments: storage temperature, heat shock and smoke, and incubation temperature treatments applied to study species.

Section Storage Heat shock Smoke Incubation No. Table Figure Temperature Temperature pop†

I 25 25 - 100 0, 10 15, 25 19 25 25, 75 0, 10 15, 25 7 25 25 - 100 0, 10 15 25, 75 0, 10 25 1 25 25 - 100 0, 10 25 25, 75 0, 10 15 2 3.3.1- 3.9.1 – 5 3.12.4 Σ = 29

II 25 25 - 100 0 25 / 35‡ 1 - 3.13.1 25 25 - 100 0, 10 25 / 35‡ 1 - 3.13.2

III 25 25, 75 0, 10 15, 25, 35 3 - 3.14

IV 25 25 0 15, 35 75 10 15, 35 9 3.4.1 3.15

V a) 15 25, 75 0, 10 15, 25 19 3.5.1 3.16.1 – 4 b) 35 25, 75 0, 10 25, 35 19 3.6.1 3.17.1 – 2

VI 15 50 0, 10 15, 25 25 50 0, 10 15, 35 35 50 0, 10 25, 35 1 - 3.18 VII Effects of seed coat (see text) 2

† Number of populations ‡ 20 / 4 hours at 25 / 35°C

80 Table 3.2. Species (populations) stored at 25°C, treated with heat shock (between 25 and 100°C for five minutes) and smoke (0 or 10 minutes) and incubated at 15 or 25°C.

Heat shock 25 –100 25, 75 25 – 100 25, 75 25, 75 25 – 100

Incubation temperature 15, 25 15, 25 15 25 15 25

Species G. bellidifolia (1) B. utilis M. ciliata G. ovata

G. heterophylla B. brevifolia G. dimorpha

K. ambigua B. imbricata (4)

K. capitata (1) E. paludosa (2)

K. capitata (3) G. buxifolia

D. secundum G. sericea

E. paludosa (1) B. ramosissima

E. obtusifolia (3)

K. capitata (2)

B. imbricata (2)

B. linifolia

S. nuda

E. obtusifolia (2)

B. imbricata (3)

G. acanthifolia

G. decurrens

A. helianthi

G. bellidifolia (2)

H. purpurea

Σ populations 19 7 1 2

81 Fig 3.2.1: Storage temperature, fire-related germination cues, and conditions under which seeds of 19 populations were incubated.

Storage Temp 25

Germination Cues:

Heat shock 25 50 75 100

Smoke 0 10

Incubation Temp 15 25 15 25

End Stage 1 (Analysis)

End Stage 2

Light Light 15°C, dark

82 Fig 3.2.2: Storage temperature, fire-related germination cues, and conditions under which seeds of G. acanthifolia were incubated.

Storage Temp 25

Germination Cues:

Heat shock 25 75 50 100

Smoke 0 10 0 10 0 10 0 10

Incubation Temp 15 25 15 25 15 25 15 25

End Stage 1 (Analysis)

Light Coat removed Light

End Stage 2

Light Dark

83 Fig 3.2.3: Storage temperature, fire-related germination cues, and conditions under which seeds of 7 species were incubated.

Storage Temp 25

Germination Cues:

Heat shock 25 75

Smoke 0 10 0 10

Incubation Temp 15 25 15 25

End Stage 1 (Analysis)

84 Fig 3.2.4: Storage temperature, fire-related germination cues, and conditions under which seeds of M. ciliata (population 2) were incubated.

Storage Temp 25

Germination Cues:

Heat shock 25 75 50 100

Smoke 0 10 0 10 0 10 0 10

Incubation 15 25 15 25 15 15 Temperature

End Stage 1 (Analysis)

85 Fig 3.2.5: Storage temperature, fire-related germination cues, and conditions under which seeds of G. dimorpha var. dimorpha and G. ovata were incubated.

Storage Temp 25

Germination Cues:

Heat shock 25 75 50 100

Smoke 0 10 0 10 0 10 0 10

Incubation 15 25 15 25 25 25 Temperature

End Stage 1 (Analysis)

86 Subsequent handling (Stage II)

Seeds of some species within particular incubation conditions were subsequently transferred into different incubation conditions. Ideally, half of the replicates within a particular treatment would have been left within that treatment as a control. However, at least twice as many replicates would have been required as were available due to the high level of variability between replicates. Instead, all replicates were transferred as it was assumed that no more germination would have occurred within the initial treatment. The assumption that no more germination would have occurred was based on observations from other experiments within the current study. Most experiments were not ended until long after germination had tapered off, and there was never resurgence of germination. Two Kunzea capitata populations (2 and 3) were transferred from a supra-optimal 25˚C to a 15˚C incubation temperature after 107 days, as it was apparent that no further germination would occur at the higher temperature. The coats were removed from Grevillea acanthifolia seeds that had been treated with 25˚C (= control) and 75˚C heat shock with and without smoke and incubated at 25˚C after 67 days because seed coats reduced germination of other Grevillea species (Edwards & Whelan 1995; Morris 2000; Morris et al. 2000; Pickup et al. 2003). Four seeds from each replicate were then incubated in the light and five seeds remained in the dark. The G. acanthifolia seeds that had been treated with 50 and 100˚C heat shock with and without smoke and incubated at 25˚C were transferred into the light at this time because germination trials on Grevillea species are generally carried out in the light (e.g. Vaughton 1998; Kenny 2000; von Richter et al. 2001). Seeds of G. acanthifolia at 15˚C were also transferred into the light after 89 days Actinotus helianthi seeds were transferred into light at 67 days within both the 15 and 25˚C incubation temperatures because no seeds had germinated at this time, and light promoted germination of laboratory-stored A. helianthi seed at higher incubation temperatures (Lee & Goodwin 1993). All experiments were ended before 200 days after their commencement, and germination was calculated as a percentage of initial seeds per petri dish.

87 Data analysis

Germination data were analysed using a three way fixed factor ANOVA with the terms heat shock, smoke and incubation temperature. Preliminary analyses were carried out with the full ANOVA model, which has the term cabinet nested within incubation temperature. The full analyses were problematical; while cabinet was not significant as a main effect, interactions between cabinet and other factors were significant at a rate higher than expected under Type I error, and these interactions were not consistent. Analyses were carried out using the average germination within each incubation temperature (cabinet) x heat shock x smoke treatment as a single replicate to avoid sacrificial pseudoreplication (Hurlburt 1984). The mean square among treatments was a common estimate of variance, hence standard error of cell means was calculated as the square-root of the mean square within samples divided by the sample size (n = 2) (Underwood 1997). When cabinets were not replicated (four cases) or germination only occurred within one incubation temperature, then data was analysed within incubation temperature using a two way fixed factor ANOVA with the terms heat shock and smoke (Table 3.3.3). When a fire- related cue was significant within only one of the incubation temperatures, then this was considered to be consistent with an interaction between incubation temperature and the fire- related cue(s). In all analyses in this thesis, homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. When transformations were unsuccessful in removing heterogeneity of variances, then alpha was reduced and comparisons among means were not undertaken. Lower adjustments of alpha reflect more extreme heterogeneity, i.e. if heterogeneity was significant at the 0.05 level, then alpha was reduced to 0.01 and if heterogeneity was significant at the 0.01 level, then alpha was reduced to 0.001 . Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995). Contrasts amongst means were carried out for the highest order interaction that was significant for each factor; lower order affects are not independent in such cases and cannot be interpreted (Underwood 1997). If no interaction terms were significant, then Main Effects were examined. Species were placed in response groups according to ANOVA results. The highest order significant interactions were grouped together, then lower order significant interactions, and then significant main effects.

88 Section II 25˚C Storage, Heat Shock +/- Smoke; 25˚C or 25 / 35˚C Incubation; Gahnia sieberiana populations 1 & 2

Six replicates of ten seeds of Gahnia sieberiana population 2 that had been stored at 25˚C were treated with factorial combinations of 25°C (= control), 50, 75 or 100°C heat shock for five minutes and 0 or 10 minutes of aerosol smoke and incubated within a 25˚C or a 20/4 hour 25/35˚C temperature regime. Three replicates of 20 seeds of G. sieberiana population 1 were subject to the same heat shock and incubation temperatures, but were not smoked (Table 3.1, Section II; Fig 3.3). In this and subsequent Sections, due to limited numbers of cabinets, a choice had to be made between replicating cabinets for a smaller number of species or not replicating cabinets and screening more species; as very little is known about the effect of the factors investigated on germination of species from the Sydney region, the latter option was taken.

Data analysis

The effect of heat shock on germination of G. sieberiana population 1 within the two different incubation temperatures was analysed using one way ANOVAs. Germination of G. sieberiana population 2 at each incubation temperature was analysed using two way fixed factor ANOVAs with the terms heat shock and smoke.

89 Fig 3.3: Storage temperature, fire-related germination cues, and conditions under which seeds of G. sieberiana (populations 1 and 2) were incubated.

Storage Temp 25

Germination Cues:

Heat shock 25 50 75 100

Smoke 0 10† 0 10 0 10 0 10

Incubation 25 25/35 25 25/35 Temperature

End Stage 1 (Analysis)

† population 2 only smoked

90

Section III 25˚C Storage, 15, 25 or 35˚C Incubation; Heat Shock +/- Smoke

Calytrix tetragonia and Schoenus brevifolius seeds were stored at 25˚C, then treated with factorial combinations of 25˚C (= control) or 75˚C heat shock for 5 minutes, and 0 or 10 minutes of aerosol smoke. S. brevifolius seeds were treated both within their coats, and with their coats removed (Table 3.1, Section III; Fig 3.4). Six replicates of each species were treated with heat shock and smoke independently, one replicate of each species within each independent application of a treatment. These seeds were incubated at 15, 25 or 35˚C in a single cabinet for each temperature.

Subsequent handling

After 60 days, little or no germination had occurred for a considerable period of time so half of the replicates within the 15 and 35˚C incubation temperatures were transferred to a 25˚C incubation temperature. Germination increased within transferred replicates and not within the other replicates. The other replicates were transferred to 25˚C at 127 days. As no germination of the S. brevifolius seeds within coats had occurred when half of the replicates were first transferred into a 25˚C incubation temperature, the top of the seed coat was cut off every seed at the time of the second transfer. The numbers of ungerminated seeds were determined after about 250 days since treatment. Germination was calculated as a percentage of initial seeds per petri dish.

Data analysis

Final percentage germination data within an incubation temperature were analysed using a two way fixed factor ANOVA with the terms heat shock and smoke. Analyses were carried out before (Stage I) and after transferrals across incubation temperatures (Stage II), and alpha was reduced to 0.01 to control experiment-wide error.

91 Fig 3.4: Storage temperature, fire-related germination cues, and conditions under which seeds of 3 populations were incubated at different stages; each stage analysed separately.

Storage Temp 25

Germination Cues:

Heat shock 25 75

Smoke 0 10 0 10

Incubation Temp 15 25 35 15 25 35

End Stage 1 (Analysis 1)

T1† T1 T2‡ T2‡

End Stage 2 (Analysis 2) 25 25

† Timing of transferral

‡ Top cut off S. brevifolius coat at time of transferral

92 Section IV 25˚C Storage, Heat Shock + Smoke or Control; 15 or 35˚C Incubation

Seeds of nine species were stored at 25˚C, then treated with 75˚C heat shock for 5 minutes plus 10 minutes of aerosol smoke, or with 25˚C heat ‘shock’ plus no smoke as a control, and then incubated at either 15 or 35˚C in a single cabinet for each temperature (Table 3.1, Section IV; Fig 3.5).

Subsequent handling

When little or no germination had occurred for a considerable period of time, or the rate of germination had declined such that it was apparent that little or no further germination would occur, then seeds were transferred from both incubation temperatures into a 25˚C incubation temperature. Seeds were first transferred at 60 and 61 days, and the second transfer was at 127 days for all species incubated at 35˚C, and for six species temperatures incubated at 15˚C. Germination of three species, Epacris coriacea, E. obtusifolia and Kunzea ambigua, continued for longer within the 15˚C incubation temperature, hence seeds were first transferred from 15 to 25˚C at 100 days, and the second transfer was at 166 days.

Data analysis

Germination was compared between treatments within temperatures of incubation (because cabinets were not replicated), and separate comparisons were made for germination before (Stage I) and after transferral across incubation temperatures (Stage II). Final germination data before transferral across incubation temperatures were analysed using one way ANOVAs to assess the effect of the ‘fire treatment’. Final germination data within the final temperature of incubation were analysed using two way mixed model ANOVAs. ‘Fire’ was a fixed factor, and the time of transferral across incubation temperatures was a random factor. To control experiment-wide error, alpha was divided by the number of ANOVAs conducted (Underwood 1997), thus it was reduced to 0.01 for a number of species initially incubated at 15°C.

93 Fig 3.5: Storage temperature, fire-related germination cues, and conditions under which seeds of 9 species were incubated at different stages; each stage analysed separately.

Storage Temp 25

Germination Cues:

Heat shock 25 75

Smoke 0 10

Incubation Temp 15 35 15 35

End Stage 1 (Analysis 1)

T1† T1 T2 T2

End Stage 2 (Analysis 2)

Incubation Temp 25 25

† Timing of transferral

94 Section V a) 15˚C Storage; Heat Shock +/- Smoke; 15 or 25˚C Incubation b) 35˚C Storage; Heat Shock +/- Smoke; 25 or 35˚C Incubation; 19 populations

Seeds of 19 populations were stored in the dark for 42 days at 15 or 35˚C before they were treated with factorial combinations of heat shock (25˚C (= control) or 75˚C for 5 minutes), and aerosol smoke (0 or 10 minutes) (Table 3.1, Sections Va, b; Figs 3.6-7). Twelve replicates of each species were treated independently, one replicate of each species within each independent application of a treatment. Six replicates within each treatment were then incubated at the temperature at which they had been stored, and the other six replicates from each storage temperature were incubated at 25˚C in a single cabinet for each temperature.

Subsequent handling

After 109 days, all seeds incubated at 35˚C were transferred to an incubation temperature diurnal regime of 20 hours at 25˚C and 4 hours at 35˚C. Gahnia melanocarpa, G. sieberiana and Schoenus brevifolius seeds that had been stored and incubated at 15˚C were transferred to 25˚C after 105 days, and then into the 25/35˚C diurnal regime after a further 126 days. Half of the Calytrix tetragonia, G. melanocarpa and G. sieberiana replicates that had been stored at 15˚C and incubated at 25˚C were transferred into the 25/35˚C diurnal regime after 129 days and the other half were transferred after 171 days. Half of the Kunzea ambigua and K. capitata seeds that had been stored at 15˚C and incubated at 25˚C were transferred into a 15˚C incubation temperature after 101 days and the other half were transferred into a 35˚C incubation temperature after 174 days. Baeckea imbricata, B. linifolia and Calytrix tetragonia seeds that had been stored and incubated at 15˚C were transferred to 25˚C after 163 days. Caustis flexosa, Epacris crassifolia, E. paludosa, Juncus continuus, Restio gracilis and Woollsia pungens seeds that had been stored at 15˚C and incubated at 15 and 25˚C were transferred into the light after 163 and 166 days respectively. Half of the Caustis flexosa, Juncus continuus and Restio gracilis replicates that had been stored and incubated at 35˚C before being transferred into the 25/35˚C diurnal regime, were subsequently transferred into the light after 151 days and the other half were transferred after 188 days. Dracophyllum

95 Fig 3.6: Storage temperature, fire-related germination cues, and conditions under which seeds of 19 populations were incubated at different stages; each stage analysed separately.

Storage Temp 15

Germination Cues:

Heat shock 25 75

Smoke 0 10 0 10

Incubation Temp 15 25 15 25

End Stage 1 (Analysis 1)

T1† T2

End Stage 2

Light 25C 25/35C 35C Light 15C

End Stage 3

25/35C

† Timing of transferral

96 Fig 3.7: Storage temperature, fire-related germination cues, and conditions under which seeds of 19 populations were incubated at different stages; each stage analysed separately.

Storage Temp 35

Germination Cues:

Heat shock 25 75

Smoke 0 10 0 10

Incubation Temp 25 35 25 35

End Stage 1 (Analysis 1)

End Stage 2

25/35C

T1† T2

End Stage 3

Light Light

† Timing of transferral

97 secundum and Epacris paludosa seeds within the 25/35˚C diurnal regime were transferred into the light after 209 days. Few experiments were ended before 100 days, many continued for more than 350 days, and most ran for over 200 days. End of treatment germination was calculated as a percentage of initial seeds per petri dish.

Data analysis

Stage I data was analysed using a two way fixed factor ANOVA with the terms heat shock and smoke. Informal, visual comparisons were made between germination at the two incubation temperatures for seeds stored at a single temperature (because cabinets were not replicated within temperature). When a fire-related cue was significant within a single incubation temperature, then an interaction between incubation temperature and the fire- related cue(s) was inferred.

Section VI Baeckea imbricata treated with 50˚C heat shock

Because the interaction between heat shock and smoke was positive within the 50˚C heat shock treatment (Chapter 2), seeds of Baeckea imbricata were stored at 15˚C or 35˚C for 28 days, or at 25˚C long-term, then treated with 50˚C heat shock for 5 minutes, and 0 or 10 minutes of aerosol smoke. Seeds that were stored at 15˚C were incubated at 15˚C or 25˚C, seeds that were stored at 25˚C were incubated at 15˚C or 35˚C, and seeds that were stored at 35˚C were incubated at 25˚C or 35˚C (Table 3.1, Section VI; Fig 3.8).

Subsequent handling

After 55 days, seeds that were incubated at 35˚C were transferred into a diurnal temperature regime of 20 hours at 25˚C and 4 hours at 35˚C.

98 Data analysis

The effect of smoke was assessed within each incubation temperature by comparing final percentage germination data using one-way ANOVAs. For seeds that were transferred, α was reduced to 0.01 to control experiment-wide error.

Fig 3.8: Storage temperature, fire-related germination cues, and conditions under which seeds of B. imbricata were incubated at different stages; each stage analysed separately.

Storage Temp 15 25 35

Germination Cues:

Heat shock 50 50 50

Smoke 0 10 0 10 0 10

Incubation Temp 15 25 15 25/35 25 35

End Stage 1 (Analysis 1)

End Stage 2 (Analysis 2) 25/35C

99

Section VII

Effect of seed coat on dormancy of Actinotus helianthi

No Actinotus helianthi seeds had germinated in Section I, however removal of the pericarp and testa, incubation at 25˚C, and in light all increased germination of this species (Lee & Goodwin 1993). The pericarp and testa were removed from A. helianthi seed, and three replicates of five naked seeds were treated with ten minutes smoke and incubated in darkness at 25˚C. Another three replicates of five naked seeds were not smoked and incubated in the light or darkness at 25˚C.

Effect of seed coat on dormancy of Schoenus brevifolius

As only one Schoenus brevifolius seed within its coat had germinated at 25˚C, but seeds without coats germinated readily within a 25˚C incubation temperature, the presence of inhibitors within the seed coat was investigated. The apex was removed from sixty seed coats of S. brevifolius. Three replicates of 10 seeds received no further treatment. The other three replicates of 10 seeds were placed in nylon mesh and water was rapidly run past the seeds for 5 hours to leach away any potential inhibitors within their coats. Seeds were then incubated in darkness at 25˚C. No germination precluded data analysis.

Effect of seed coat on heat shock tolerance of Schoenus brevifolius

To assess the effect of coats on high heat shock tolerance of Schoenus brevifolius seeds, 100°C heat shock was applied for five minutes and 0 or 10 minutes of smoke were applied to seeds within or removed from their coats. Treatments were applied to three replicates of 10 seeds, that were then incubated in darkness within a 20/4 hour 25/35°C diurnal temperature regime.

Data analysis Data were analysed using a two way fixed factor ANOVA with the terms seed coat and smoke.

100 3.3 Results

Section I 25˚C Storage; Heat Shock +/- Smoke; 15 or 25˚C Incubation temperatures; 29 populations

The aim was to assess whether temperature of incubation per se, or in interaction with fire-related cues would affect germination of a large number of populations from a range of habitats.

Overview

Temperature of incubation played a major role in controlling germination, with two- thirds of the populations showing temperature effects. Temperature significantly affected germination of 18 out of 26 populations (Tables 3.3.1-4; three populations showed no germination). Germination was affected in ten cases by interactions between temperature of incubation and the fire-related germination cues (Table 3.3.1-2), and another two such interactions were inferred (Table 3.3.3). Temperature was significant as a main effect for a further four populations (Table 3.3.4). Germination of another three species only occurred within one incubation temperature (Table 3.3.3). Germination of four populations was affected by the fire-related germination cues but not temperature (Table 3.3.5). Of the species that germinated, only three were not affected by any factor (Tables 3.3.3, 3.3.5).

101 Interaction between incubation temperature, heat shock and smoke

Germination of 6 species: Goodenia bellidifolia ssp. bellidifolia (population 1), G. heterophylla, Kunzea ambigua, K. capitata population 1 and population 3, and Baeckea utilis, was affected by the interaction between incubation temperature, heat shock and smoke (Table 3.3.1; Figs 3.9.1 – 3.9.6). A common pattern in the data was that smoke both increased total germination, and resulted in more uniform germination across a wider range of heat shock. Germination of unsmoked seeds was more variable; consequently germination of unsmoked seeds was more frequently different across heat shock and incubation temperature levels, resulting in the second-order interaction observed. Germination of Kunzea capitata (population 3) was increased by the highest level of heat shock or by smoke within the more favourable temperature, and was uniformly negligible at 25°C (Table 3.3.1; Fig 3.9.5). The germination response of Baeckea utilis was different to the general pattern; smoke increased germination only for unheated seeds incubated at 25°C (Fig 3.9.6). Incubation temperature preferences were apparent, with Goodenia heterophylla, Kunzea ambigua, K. capitata population 1 and population 3 favoured by 15°C, and Baeckea utilis favoured by 25°C.

102 Table 3.3.1. Germination of species affected by the interaction between all three factors. Seeds were stored at 25°C, treated with factorial combinations of heat shock (between 25 and 100°C for 5 minutes) and smoke (0 or 10 minutes), then incubated at 15 or 25°C. ANOVA P-values.

Temper- Smoke Heat S x H T x S T x H T x S x ature (T) (S) Shock H pop† df‡ (H)

G. heterophylla A <0.0001 <0.0001 <0.0001 0.0016 0.2577 0.0020 0.0458

G. bellidifolia 1 A 0.5161 0.0086 0.0001 0.2159 0.4996 0.6054 0.0454

K. ambigua A 0.0175 <0.0001 0.0014 0.0015 0.1001 0.0005 0.0025

K. capitata 1 A <0.0001 <0.0001 0.0013 0.0001 0.4432 0.0002 0.0018

3 A <0.0001 <0.0001 0.0002 0.0013 <0.0001 0.0001 0.0009

B. utilis B 0.0007 0.1373 0.2588 0.0066 0.0263 0.1631 0.0105 residual A df 1 1 3 3 1 3 3 16

B df 1 1 1 1 1 1 1 8

† population ‡ degrees of freedom categories A and B at the bottom of the Table

103 a) 15°C

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Figure 3.9.1: Mean final germination of Goodenia bellidifolia ssp. bellidifolia seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E.s. Significant terms in ANOVA: Incubation temperature x Heat shock x Smoke (Table 3.3.1). Results of post-hoc comparisons amongst means shown in Figure above is given below: Incubation temperature Heat shock level

/ smoke level 25°C 50°C 75°C 100°C 15°C / unsmoked a, m, x a, m, x a, m, y a, m, x / smoked a, m, x a, m, xy a, m, xy a, n, y

25°C / unsmoked a, m, x a, m, x a, m, y b, m, y / smoked a, n, x a, m, x a, m, x a, m, x Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Different letters within the range x-z are different across heat shock levels

104 a) 15°C

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Figure 3.9.2: Mean final germination of Goodenia heterophylla seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E.s Significant terms in ANOVA: Incubation temperature x Heat shock x Smoke (Table 3.3.1). Mean % germination and results of post-hoc comparisons amongst means listed below: Incubation temperature Heat shock level

/ smoke level 25°C 50°C 75°C 100°C 15°C / unsmoked b, m, x b, m, y a, m, yz b, m, z / smoked b, n, x a, m, x a, m, x a, m, x

25°C / unsmoked a, m, x a, m, y a, m, z a, m, y / smoked a, n, x a, n, y a, m, y a, n, y Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Different letters within the range x-z are different across heat shock levels

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Figure 3.9.3: Mean final germination of Kunzea ambigua seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E.s Significant terms in ANOVA: Incubation temperature x Heat shock x Smoke (Table 3.3.1). Results of post-hoc comparisons amongst means shown in Figure above is given below: Incubation temperature Heat shock level

/ smoke level 25°C 50°C 75°C 100°C 15°C / unsmoked a, m, y a, m, x b, m, z a, m, z / smoked a, n, x a, n, x b, m, x a, m, x

25°C / unsmoked a, m, x b, m, y a, m, x a, m, y / smoked a, n, x a, m, x a, n, x a, m, x Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Different letters within the range x-z are different across heat shock levels

106 a) 15°C

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Figure 3.1.4: Mean final germination of Kunzea capitata (population 1) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E.s Significant terms in ANOVA: Incubation temperature x Heat shock x Smoke (Table 3.3.1). Results of post-hoc comparisons amongst means shown in Figure above is given below: Incubation temperature Heat shock level

/ smoke level 25°C 50°C 75°C 100°C 15°C / unsmoked b, m, y a, m, x b, m, z b, m, z / smoked b, n, x a, n, x a, m, x b, m, x

25°C / unsmoked a, m, x b, m, y a, m, y a, m, y / smoked a, n, x a, n, x a, n, x a, n, x Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Different letters within the range x-z are different across heat shock levels

107 a) 15°C

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Figure 3.9.5: Mean final germination of Kunzea capitata (population 3) seeds incubated at a) 15 () or b) 25°C (), subsequently transferred into 15°C, plotted against heat shock and smoke treatments. Bars = S. E.s Significant terms in ANOVA: Incubation temperature x Heat shock x Smoke (Table 3.3.1). Results of post-hoc comparisons amongst means shown in Figure above is given below: Incubation temperature Heat shock level

/ smoke level 25°C 50°C 75°C 100°C 15°C / unsmoked a, m, x a, m, x a, m, x b, m, y / smoked b, n, x b, n, y b, n, xy b, m, xy

25°C / unsmoked a, m, x a, m, x a, m, x a, m, x / smoked a, m, x a, m, x a, m, x a, m, x Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Different letters within the range x-y are different across heat shock levels

108 a) 15°C

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Figure 3.9.6: Mean final germination of Baeckea utilis seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E.s Significant terms in ANOVA: Incubation temperature x Heat shock x Smoke (Table 3.3.3). Results of post-hoc comparisons amongst means shown in Figure above is given below: Incubation temperature Heat shock level

/ smoke level 25°C 75°C 15°C / unsmoked a, m, x a, m, x / smoked a, m, x a, m, x

25°C / unsmoked a, m, x a, m, x / smoked b, n, y a, m, x Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Different letters within the range x-y are different across heat shock levels

109 Interaction between incubation temperature and heat shock

Germination of two species, Epacris paludosa (population 1) and Dracophyllum secundum (population 1) was affected by the interaction between incubation temperature and heat shock (Table 3.3.2). Results for Epacris paludosa (population 2) were also consistent with such an interaction (Table 3.3.3). Germination of Epacris paludosa (population 1) generally increased with increasing levels of heat shock (Fig 3.10.1). Smoke increased germination within the moderate heat shock levels (50 or 75˚C), but the highest heat shock level increased germination of unsmoked seeds. The pattern of increasing germination with increasing levels of heat shock was less variable and more pronounced within the higher incubation temperature (Fig 3.10.1). The high heat shock level also increased germination of E. paludosa (population 2) within the higher incubation temperature (Fig 3.10.2). A difference between the populations was that germination of population 1 was greater within the higher incubation temperature, while germination of population 2 was greater within the lower incubation temperature Germination of Dracophyllum secundum was negligible at 15˚C, and much greater when incubated at 25˚C; the effects of heat shock were variable within this incubation temperature (Fig 3.10.3). A temperature preference of 25˚C was apparent for Dracophyllum secundum but not for Epacris paludosa due to differences between populations.

Interaction between incubation temperature and smoke

Germination of two species, Epacris obtusifolia (population 3) and Kunzea capitata (population 2) was affected by the interaction between incubation temperature and smoke (Table 3.3.2). Incubation temperature affected the germination of unsmoked Epacris obtusifolia seeds, with 15˚C incubation being less favourable, but smoke overrode this temperature affect so that germination of smoked seeds was similar across incubation temperatures. Smoke apparently increased germination of Kunzea capitata seeds within the more favourable 15˚C incubation temperature, but not at 25˚C. Interestingly, smoke appeared to increase germination when seeds were transferred from 25 into 15˚C.

110 Results for Baeckea imbricata (population 4) were consistent with an interaction between incubation temperature and smoke (Table 3.3.3). Smoke increased germination of this population within the 15˚C incubation temperature. A 15 and 25˚C temperature preference was apparent for Kunzea capitata and Epacris obtusifolia respectively.

Table 3.3.2. Germination of species affected by an interaction between temperature of incubation and heat shock or smoke. Seeds were stored at 25°C, treated with factorial combinations of heat shock (25 to 100°C for 5 minutes) and smoke (0 or 10 minutes) and incubated at 15 or 25°C. ANOVA P-values.

Temper- Smoke Heat S x H T x S T x H T x S residual ature (S) Shock x H (T) (H) df 1 1 3 3 1 3 3 16 pop† E. paludosa 1 0.0004 0.0081 0.0003 0.0051 0.9062 0.0158 0.6159

D. secundum 1 <0.0001 0.2598 0.0071 0.1355 0.2598 0.0033 0.1862

E. obtusifolia 3 0.0002 <0.0001 0.0368 0.0032 0.0226 0.6243 0.8303

K. capitata‡ 2 <0.0001 <0.0001 0.0153 0.0082 <0.0001 0.0708 0.4918

† population ‡ α reduced to 0.01 due to heterogeneity of variances

111 Table 3.3.3. Germination of 7 populations that were stored at 25°C, treated with factorial combinations of heat shock (25 to 100°C for 5 minutes) and smoke (0 or 10 minutes), then incubated at 15°C or 25°C. ANOVA P-values.

15°C 25°C

Heat Smoke H x S Heat Smoke H x S shock (S) shock (S) (H) (H)

pop† df‡ B. imbricata 4 A 0.2002 0.0015 0.2757 0.1990! 0.3957 0.8802

E. paludosa 2 A 0.5410 0.5090 0.3926 0.0128 0.9032 0.3561

G. dimorpha B No germination 0.0473 0.0280 0.4549 var dimorpha§

G. acanthifolia§ B No germination 0.0525 0.2598 0.1789

B. ramosissima C 0.4998 0.0575 0.1677 No germination

G. buxifolia A 0.8798 0.1975 0.8798 0.2127 0.3001 0.9051

G. sericea A 0.0746 0.1008 0.3144 0.1470!! 0.1470 0.1470 residual residual A df 1 1 1 8 1 1 1 8

B df 3 1 3 8

C df 1 1 1 4

† population ‡ degrees of freedom categories A, B and C at the bottom of Table § transformed data ! α reduced to 0.01 due to heterogeneity of variances !! α reduced to 0.001 due to heterogeneity of variances

112 a) 15°C

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Figure 3.10.1: Mean final germination of Epacris paludosa (population 1) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E.s Significant terms in ANOVA: Incubation temperature x Heat shock, Smoke x Heat shock (Table 3.3.2). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Incubation Heat shock level temperature 25 50 75 100

15°C (12 ± 3.1) a, mn (8 ± 3.5) a, m (13 ± 5.8) a, mn (22 ± 4.0) a, n 25°C (13 ± 2.7) a, m (15 ± 7.3) a, m (38 ± 4.4) b, n (31 ± 5.2) a, n Different letters within the range a-b are different across incubation temperatures Different letters within the range m-o are different across heat shock levels Smoke level Heat shock level 25 50 75 100 Unsmoked (8 ± 1.6) a, mn (4 ± 2.5) a, m (18 ± 8.3) a, n (32 ± 5.2) a, o Smoked (17 ± 1.9) a, m (20 ± 5.3) b, m (33 ± 7.3) b, n (22 ± 3.5) a, m Different letters within the range a-b are different across smoke levels Different letters within the range m-o are different across heat shock levels

113 a) 15°C

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Figure 3.10.2: Mean final germination of Epacris paludosa (population 2) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: b) Heat shock for 25°C incubation (Table 3.3.3). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below:

Heat shock: 75°C (23 ± 4.6) > 25°C (3 ± 3.3)

114 a) 15°C

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Figure 3.10.3: Mean final germination of Dracophyllum secundum (population 1) seeds incubated at a) 15

() or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature x Heat shock (Table 3.3.2). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Incubation Heat shock level temperature 25°C 50°C 75°C 100°C 15°C (0 ± 0.0) a, m (1 ± 0.8) a, m (2 ± 1.1) a, m (3 ± 1.8) a, m 25°C (33 ± 3.9) b, n (22 ± 4.1) b, m (37 ± 4.9) b, n (20 ± 3.7) b, m Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across heat shock levels

115 a) 15°C

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Figure 3.10.4: Mean final germination of Epacris obtusifolia (population 3) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature x Smoke, Heat shock x Smoke (Table 3.3.2). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Incubation Smoke levels temperature Unsmoked Smoked 15°C (5 ± 1.4) a, m (46 ± 4.3) a, n 25°C (26 ± 4.1) b, m (53 ± 5.1) a, n Different letters within the range a-b are different across incubation temperatures Different letters within the range m-n are different across smoke levels Smoke level Heat shock level 25 50 75 100 Unsmoked (11 ± 4.4) a, m (20 ± 8.8) a, m (9 ± 4.5) a, m (23 ± 9.2) a, m Smoked (50 ± 4.7) b, n (63 ± 3.6) b, n (52 ± 6.5) b, n (34 ± 2.7) a, m Different letters within the range a-b are different across smoke levels Different letters within the range m-n are different across heat shock levels

116 a) 15°C

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Figure 3.10.5: Mean final germination of Kunzea capitata (population 2) seeds incubated at a) 15 () or b) 25°C (), subsequently transferred into 15°C, plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature x Smoke, Heat shock x Smoke (Table 3.3.2). Mean % germination (± SE) listed below (post-hoc comparisons amongst means not conducted due to heterogeneity of variances)

Incubation Smoke levels temperature Unsmoked Smoked 15°C (54 ± 4.6) (82 ± 2.3) 25°C (4 ± 2.9) (2 ± 1.0)

Smoke level Heat shock level 25 50 75 100 Unsmoked (23 ± 11.7) (24 ± 13.9) (26 ± 14.9) (43 ± 18.6) Smoked (40 ± 20.2) (45 ± 24.3) (43 ± 24.6) (41 ± 23.3) 117 a) 15°C

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Figure 3.10.6: Mean final germination of Baeckea imbricata (population 4) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: a) Smoke at 15°C only (Table 3.3.3). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Smoke: 10 minutes (38 ± 4.2) > 0 minutes (14 ± 3.7)

118 Incubation temperature as a main effect

In cases where temperature was significant as a main effect, species could be classified by the temperature at which greater germination occurred. Germination of two species was greater when incubated at 25˚C, with no Goodenia dimorpha var dimorpha (Table 3.3.3; Fig 3.11.1) or Grevillea acanthifolia seeds germinating at 15˚C in darkness (Table 3.3.3; Fig 3.11.2). Germination of Grevillea acanthifolia was apparently stimulated by by light within the 25˚C, but not the 15˚C incubation temperature (Fig 3.11.2). Germination of 5 species: Baeckea imbricata (population 2), B. linifolia (population 2), Goodenia ovata, Stackhousia nuda (Table 3.3.4; Figs 3.11.3-6) and Baeckea ramosissima ssp. ramosissima (no germination at 25˚C, Table 3.3.3) was greater within a 15˚C incubation temperature. Although germination was frequently low, temperature preferences were generally pronounced.

Table 3.3.4. Germination of species affected by temperature of incubation. Seeds were stored at 25°C, treated with factorial combinations of heat shock (between 25 and 100°C for 5 minutes) and smoke (0 or 10 minutes) and incubated at 15 or 25°C. ANOVA P-values.

Temper- Smoke Heat S x H T x S T x H T x S x ature (T) (S) Shock H pop† df‡ (H) B. imbricata 2 A 0.0015 <0.0001 0.0177 0.3827 0.6222 0.1094 0.7060

B. linifolia 2 A 0.0073 0.0045 0.1389 0.0782 0.4465 0.1494 0.6581

G. ovata B 0.0236 0.2490 0.0374 0.8166 0.6532 0.7069 0.3393

S. nuda A 0.0018 0.0693 0.1063 0.5468 0.0841 0.1063 0.6236 residual A df 1 1 3 3 1 3 3 16

B df 1 1 1 1 1 1 1 8 † population ‡ degrees of freedom categories A and B at the bottom of the Table

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Figure 3.11.1: Mean final germination of Goodenia dimorpha seeds incubated at a) 15 () (no germination) or b) 25°C (), plotted against heat shock and smoke treatments. Analysis of transformed data (only within 25°C); back-transformed data shown. Bars = S. E. s Significant terms in ANOVA: Smoke, Heat shock (Table 3.3.3). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Smoke: 10 minutes (6 ± 2.6) > 0 minutes (2 ± 1.6)

Heat shock level 25 50 75 100 (3 ± 2.0) ab (0 ± 0.0) a (4 ± 2.6) ab (10 ± 3.3) b Different letters within the range a-b are different across heat shock levels

120 a) 15°C

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Figure 3.11.2: Mean final germination of Grevillea acanthifolia seeds incubated in darkness at a) 15 (no germination) or b) 25°C (), plotted against heat shock and smoke treatments. Additional germination of seeds transferred into light (2) is also shown. NB an additional treatment of decoating seeds within the 25 and 75°C heat shock treatments resulted in no added germination in darkness, and no difference between germination of seeds in coats and decoated seeds was apparent in the light, hence no such distinction is made in the Figure. Analysis of germination within only the 25°C incubation temperature (Table 3.3.3). Bars = S. E. s

121 a) 15°C

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Figure 3.11.3: Mean final germination of Baeckea imbricata (population 2) seeds stored at 25°C, then incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature, Heat shock, Smoke (Table 3.3.4). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below:

Incubation temperatures: 15°C (45 ± 4.6) > 25°C (24 ± 3.1)

Heat shock level 25 50 75 100 (46 ± 5.5) b (43 ± 8.3) b (35 ± 6.4) ab (30 ± 6.9) a Different letters within the range a-b are different across heat shock levels

Smoke: 10 minutes (53 ± 3.5) > 0 minutes (24 ± 3.1)

122 a) 15°C

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Figure 3.11.4: Mean final germination of Baeckea linifolia (population 2) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature, Smoke (Table 3.3.4). Mean % germination and results of post-hoc comparisons amongst means listed below:

Incubation temperature: 15°C (10 ± 2.1) > 25°C (5 ± 1.1) Smoke: 10 minutes (11 ± 1.8) > 0 minutes (5 ± 1.4)

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Figure 3.11.5: Mean final germination of Goodenia ovata seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock (25°C and 75°C only within 15°C incubation temperature; analysis only within 25°C and 75°C heat shock) and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature, Heat shock (Table 3.3.4). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below:

Incubation temperature: 15°C (14 ± 2.1) > 25°C (7 ± 2.1)

Heat shock: 25°C (7 ± 1.7) < 75°C (14 ± 2.5)

124 a) 15°C

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Figure 3.11.6: Mean final germination of Stackhousia nuda seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Incubation temperature (Table 3.3.4). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below:

Incubation temperature: 15°C (2 ± 0.6) > 25°C (0.2 ± 0.0)

125 No incubation temperature effects

Of the 24 populations that germinated, five were not affected by incubation temperature (Table 3.3.5), and the germination of another two were consistent with no temperature effect (Table 3.3.3). Of these seven, germination of four populations was affected by fire-related germination cues (Table 3.3.5). These four populations, and another six populations that were affected by the fire-related cues independently of temperature are considered below (Figures are within various response categories).

Interaction between heat shock and smoke

Germination of Epacris paludosa population 1, E. obtusifolia population 3 and Kunzea capitata population 2 was affected by the interaction between heat shock and smoke (Table 3.3.2). Smoke increased germination of E. paludosa (Fig 3.10.1) and E. obtusifolia (Fig 3.10.4) and apparently increased germination of K. capitata (Fig 3.10.5) within the lower heat shock levels, but not within the highest heat shock level. The highest heat shock level increased germination of unsmoked seeds of all three species, thus germination was similar to the, ‘complex effect’ outlined in Chapter 2.

Heat shock as a main effect

Heat shock had a positive effect on germination of three species and a negative effect on the germination of two populations. Higher levels of heat shock increased germination of Goodenia ovata (Table 3.3.4; Fig 3.11.5), Epacris obtusifolia (population 2) (Table 3.3.5; Fig 3.12.1) and Goodenia dimorpha var dimorpha (Table 3.3.3; Fig 3.11.1). Germination of Baeckea imbricata population 2 (Table 3.3.4; Fig 3.11.3) and population 3 (Table 3.3.5; Fig 3.12.2) was lowest within the highest level of added heat shock.

Smoke as a main effect

Smoke increased germination of 6 populations: Baeckea imbricata population 2 (Table 3.3.4; Fig 3.11.3) and population 3 (Table 3.3.5; Fig 3.12.2), Epacris obtusifolia (population 2) (Table 3.3.5; Fig 3.12.1), B. brevifolia (Table 3.3.5; Fig 3.12.3), Micromyrtus ciliata (Table 3.3.5; Fig 3.12.4), and B. linifolia (population 2) (Table 3.3.4; Fig 3.11.4)

126 Table 3.3.5. Germination of species not affected by temperature of incubation. Seeds were stored at 25°C, treated with factorial combinations of heat shock (between 25 and 100°C for 5 minutes) and smoke (0 or 10 minutes) and incubated at 15 or 25°C. ANOVA P-values.

Temper- Smoke Heat S x H T x S T x H T x S ature (T) (S) Shock x H pop† df‡ (H)

E. obtusifolia 2 A 0.2770 <0.0001 0.0235 0.0769 0.2335 0.6841 0.4711

B. imbricata 3 A 0.9650 0.0006 0.0125 0.3168 0.4983 0.2478 0.9622

B. brevifolia B 0.3641 0.0368 0.0671 0.2148 0.5796 0.5796 0.8522

M. ciliata 2 B 0.1783 0.0002 0.5067 0.5674 0.9965 0.2687 0.3982

G. decurrens A 0.0531 0.6102 0.9835 0.9462 0.5119 0.7835 0.7097

A. helianthi A No germination G. bellidifolia 2 A No germination H. purpurea A No germination residual A df 1 1 3 3 1 3 3 16

B df 1 1 1 1 1 1 1 8

† population ‡degrees of freedom categories A and B at the bottom of the Table

127 a) 15°C

70

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70

60 % germination (+/- SE) % germination 50

40

30

20

10

0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 50°C heat shock 75°C heat shock 100°C heat shock

Figure 3.12.1: Mean final germination of Epacris obtusifolia (population 2) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Smoke, Heat shock (Table 3.3.5). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Smoke: 10 minutes (39 ± 3.7) > 0 minutes (7 ± 1.6)

Heat shock level 25 50 75 100 (15 ± 4.7) a (26 ± 7.8) ab (31 ± 7.9) b (21 ± 7.1) ab Different letters within the range a-b are different across heat shock levels

128 a) 15°C

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25°C heat shock 50°C heat shock 75°C heat shock 100°C heat shock

Figure 3.12.2: Mean final germination of Baeckea imbricata (population 3) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Smoke, Heat shock (Table 3.3.5). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Smoke: 10 minutes (8 ± 1.0) > 0 minutes (3 ± 0.9)

Heat shock level 25 50 75 100 (8 ± 1.7) b (5 ± 2.0) ab (8 ± 1.1) b (2 ± 0.8) a Different letters within the range a-b are different across heat shock levels

129 a) 15°C

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b) 25°C

30 % germination (+/- SE) % germination 25

20

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25°C heat shock 75°C heat shock

Figure 3.12.3: Mean final germination of Baeckea brevifolia seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Smoke (Table 3.3.5). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Smoke: 10 minutes (12 ± 3.6) > 0 minutes (3 ± 1.5)

130 a) 15°C

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0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 50°C heat shock 75°C heat shock 100°C heat shock

Figure 3.12.4: Mean final germination of Micromyrtus ciliatata (population 2) seeds incubated at a) 15 () or b) 25°C (), plotted against heat shock (25°C and 75°C heat shock only within 25°C incubation temperature; analysis only within 25°C and 75°C heat shock) and smoke treatments. Bars = S. E. s Significant terms in ANOVA: Smoke (Table 3.12.4). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Smoke: 10 minutes (74 ± 3.2) > 0 minutes (46 ± 2.5)

131 Actinotus helianthi

No Actinotus helianthi seeds germinated at either 15 or 25˚C, when incubated in darkness or following transferral into light.

Summary of Section I

Germination of most species was affected by temperature of incubation, and the interaction between temperature of incubation and fire-related germination cues was significant in almost half of the cases where germination occurred. Thus species respond to seasonal changes in temperature, and season of fire probably affects germination. The null hypothesis that temperature of incubation would not affect germination, nor was there an interaction between temperature of incubation and fire-related germination cues was generally rejected.

Section II 25˚C Storage; Heat Shock +/- Smoke; 25˚C or 25 / 35˚C Incubation temperatures. Gahnia sieberiana populations 1 & 2

The aim was to assess whether the incubation temperature regime (constant or diurnally fluctuating temperature) or fire-related cues, or the combination of these factors would affect germination of a single species (two populations)..

There was a stark contrast between the germination responses of the two Gahnia sieberiana populations to both incubation temperature regimes and heat shock (Fig 3.13.1-2). Germination of population one increased in the alternating compared with the constant incubation temperature regime, whereas germination of population two was not affected. Germination of population one increased within the highest heat shock level when incubated at 25˚C, whereas the higher heat shock levels reduced germination of population two within both incubation temperature regimes (Fig 3.13.1-2). Smoke increased germination of population two when incubated at 25˚C (Fig 3.13.2).

132 a) 25°C

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30 % germination (+/- SE) % germination

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25°C heat shock 50°C heat shock 75°C heat shock 100°C heat shock

Figure 3..13.1: Mean final germination of unsmoked Gahnia siberiana population 1 seeds incubated at a)

25°C (), b) () 20/4hr 25/35°C, plotted against heat shock treatments. Bars = S. E. s

Significant terms in ANOVA: a) Heat shock at 25°C (F 3, 8 = 9.24, P = 0.0056). Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Heat shock level 25 50 75 100 3 (± 1.9) a 6 (± 3.1) a 7 (± 1.8) a 23 (± 4.4) b Different letters within the range a-b are different across heat shock levels

133 a) 25°C

35

30

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35

30 % germination (+/- SE) % germination

25

20

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unsm sm unsm sm unsm sm unsm sm

25°C heat shock 50°C heat shock 75°C heat shock 100°C heat shock

Figure 3.13.2: Mean final germination of Gahnia siberiana population 2 seeds incubated at a) 25°C (), b) () 20/4hr 25/35°C, plotted against heat shock and smoke treatments. Bars = S. E. s

Significant terms in ANOVA: a) Heat shock (F 3, 40 = 5.03, P = 0.0047), Smoke (F 1, 40 = 6.22, P = 0.0169) Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Heat shock level 25 50 75 100 14 (± 4.2) b 16 (± 4.4) b 10 (± 3.7) b 0 (± 0) a Different letters within the range a-b are different across heat shock levels

Smoke: 10 minutes (14 ± 3.0) > 0 minutes (6 ± 2.3)

Significant terms in ANOVA: b) Heat shock (F 3, 40 = 10.80, P < 0.0001) Mean % germination (± SE) and results of post-hoc comparisons amongst means listed below: Heat shock level 25 50 75 100 20 (± 3.9) b 22 (± 4.2) b 8 (± 2.5) a 0 (± 0) a Different letters within the range a-b are different across heat shock levels 134 Summary of Section II

An effect of incubation temperature regime on germination of Gahnia sieberiana (population 1) was inferred, as was an interaction between the temperature regime and a fire- related germination cue, therefore the null hypothesis was not supported.

Section III 25˚C Storage; Heat Shock +/- Smoke; 15, 25 or 35˚C Incubation temperatures; 3 populations

The aim was to assess the effects of fire-related cues, a wide range of incubation temperatures, and interactions between these factors on the germination of two species (three populations). .

The 35˚C incubation temperature apparently had a negative affect on the germination of both species when compared to germination at 15 or 25˚C (Fig 3.14). Neither heat shock nor smoke affected germination of either Calytrix tetragonia or decoated Schoenus brevifolius seeds within any incubation temperature (analysis not shown). Only one intact Schoenus brevifolius seed that had been continuously incubated at 25˚C germinated.

Subsequent transferral to 25˚C

When seeds were transferred from 35˚C into the 25˚C incubation temperature, then germination of C. tetragonia was similar across all temperatures (Fig 3.14c). In contrast, few decoated S. brevifolius seeds that were incubation at 35˚C germinated following transferral into 25˚C (Fig 3.14f). Incubation at 35˚C may have induced secondary dormancy (see Discussion for reasoning). No intact S. brevifolius seed germinated subsequent to transferral or removal of the apex of its coat. Timing of the transferral did not affect germination (analysis not shown).

135 a) Calytrix tetragonia d) Schoenus brevifolius (decoated)

60 25

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5 10 % germination (+/- SE) % germination 0 0 c) f)

60 25

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0 0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.14: Mean final germination of Calytrix tetragonia seeds stored at 25°C, and incubated at a) 15°C (), then at 25°C (), b) 25°C, c) 35°C (█), then at 25°C, and of Schoenus brevifolius seeds (without coats) stored at 25°C, and incubated at d) 15°C, then at 25°C, e) 25°C, f) 35°C, then at 25°C, plotted against heat shock (25°C or 75°C for 5 minutes) and smoke (0 or 10 minutes) treatments. Bars = S. E. s

136 Section IV 25˚C Storage; Heat Shock + Smoke or Control; 15 or 35˚C Incubation temperatures; 9 species

The aim was to assess the effects of fire-related cues, and cool (15˚C) or warm (35˚C) incubation temperatures, and interactions between these factors on the germination of a larger number (nine) of species.

The 35˚C incubation temperature apparently had a negative affect on the germination of all species, with almost no germination occurring regardless of fire treatments (Table 3.4.1; Fig 3.15). Comparing germination across the 15 and 35˚C incubation temperatures suggests an interaction between fire-related cues and incubation temperatures; the fire treatment only increased germination within the lower incubation temperature (Table 3.4.1; Fig 3.15; the experimental design did not allow formal testing of this hypothesis.) Most species germinated at 15˚C within the control treatment, and the fire treatment significantly increased germination of Epacris obtusifolia, Kunzea ambigua and K. capitata (Table 3.4.1; Fig 3.15). The fire treatment clearly stimulated germination of Gahnia sieberiana, and because no seeds germinated within the control treatment no analysis was conducted (or possible) (Fig 3.15).

Table 3.4.1. Germination of 9 species stored at 25°C, following control, or 75°C heat shock

and 10 minutes of aerosol smoke treatments. Comparison between treatments for seeds

incubated at 15 or 35°C; ANOVA P-values shown Population 15°C 35°C E. obtusifolia 1 <0.0001 No germination K. ambigua 0.0432 No germination K. capitata 1 0.0017 Negligible germination G. sieberiana 1 No test Negligible germination B. imbricata 1 0.1449 Negligible germination E. coriacea 0.1131 No germination D. secundum 1 No germination No germination S. monticola 1 No germination No germination W. pungens 1 Negligible germination No germination

137 a) Epacris obtusifolia d) Gahnia sieberiana

100 25

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40 6 30 4 20 2 10 % germination (+/- SE) % germination 0 0 c) Kunzea capitata f) Epacris coriacea

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Heat shock: 25 75 25 75 25 75 25 75 Smoke: 0 10 0 10 0 10 0 10

15°C initial post- 35°C initial post- 15°C initial post- 35°C initial post- treatment temperature treatment temperature treatment temperature treatment temperature

Figure 3.15: Mean final germination of seeds incubated at 15 () or 35°C (█), plotted against heat shock and smoke treatments (25°C and 0 minutes or 75°C and 10 minutes) for a) Epacris obtusifolia (population 1), b) Kunzea ambigua, c) Kunzea capitata. d) Gahnia sieberiana (population 1), e) Baeckea imbricata (population 1), f) Epacris coriacea. Bars = S. E. s

138 Subsequent transferral to 25˚C

When seeds were transferred into the 25˚C incubation temperature, then germination of most species increased (Table 3.4.2). Germination of the Kunzea species was favoured by the 15˚C incubation temperature (Section I), so the non-response when transferred from this temperature is not suprising. Germination of Gahnia sieberiana was probably greater within the fire treatment, but no germination in the control meant that analysis could not be conducted. Germination of all other species was not affected by prior treatments (analysis not shown), although variability was high due to the two different times of transferral. In general, more germination resulted from the earlier rather than the later transfer into a 25˚C incubation temperature, however no clear pattern in the interactions between time of transfer and treatment was evident (analysis not shown). The low number of replicates and high variability in germination limited the power of this comparison.

Table 3.4.2. Mean final % germination of 7 species stored at 25°C, following control, or 75°C heat shock and 10 minutes of aerosol smoke (‘fire’) treatments, when incubated at 15 or 35°C (Stage 1), and following transferral into 25°C (Stage 2). Only species with notable germination in Stage 2 presented.

Initial treatment 15°C 35°C pop Stage control ‘fire’ control ‘fire’ E. obtusifolia 1 1 52 94 0 0 2 88 95 32 42

K. ambigua 1 77 95 0 0 2 77 95 33 10

K. capitata 1 1 45 90 0 3 2 50 92 10 8

G. sieberiana 1 1 0 15 0 2 2 0 25 13 25

B. imbricata 1 1 3 10 3 0 2 17 13 23 17

E. coriacea 1 58 76 0 0 2 86 93 65 78

D. secundum 1 1 0 0 0 0 2 19 15 0 2

139 Summary of Sections III & IV

Results were consistent with a strong effect of incubation temperature on germination of all species, and with frequent interactions between temperature and the fire treatment, therefore the null hypothesis was not supported.

Section V (a) 15˚C Storage; Heat Shock +/- Smoke; 15 or 25˚C Incubation temperatures; 19 species

The aim was to assess the effects of storage at a different temperature (15˚C c.f. 25 or 35˚C), fire-related cues, temperatures of incubation (15 or 25˚C), and interactions between these factors on the germination of a large number (19) of species.

Different effects of heat shock and smoke at different incubation temperatures

In contrast to when seeds were stored at 25˚C (Section I), germination of only three of the 15 species with more than negligible germination was affected in a manner consistent with an interaction between incubation temperature, heat shock and smoke (Table 3.5.1). Added heat shock and smoke decreased germination of Epacris obtusifolia within the 25˚C incubation temperature, whereas smoke increased germination within the 15˚C incubation temperature (Table 3.5.1; Fig 3.16.1). Added heat shock and smoke increased germination of Gahnia sieberiana within the 15˚C incubation temperature, while added heat shock increased germination within the 25˚C incubation temperature (Table 3.5.1; Fig 3.16.1). Smoke increased germination of Baeckea linifolia (population 1) within both incubation temperatures, while added heat shock decreased germination within the 25˚C incubation temperature (Table 3.5.1; Fig 3.16.2).

Different effects of heat shock at different incubation temperatures

Different response patterns to fire-related cues at different temperatures of incubation affected germination of another 4 species considered below (Table 3.5.1). An interaction between heat shock and incubation temperature was inferred for Epacris microphylla var.

140 rhombifolia (Table 3.5.1). Added heat shock decreased germination within the 25˚C incubation temperature (Fig 3.16.2).

Different effects of smoke at different incubation temperatures

Different response patterns to smoke at different incubation temperatures affected germination of Baeckea imbricata (population 1), Kunzea ambigua and Sprengelia monticola (Table 3.5.1). Smoke strongly increased germination of B. imbricata and K. ambigua within the 15˚C incubation temperature, and S. monticola within the 25˚C incubation temperature (Fig 3.16.3). Temperature preferences were apparent for six species, with germination of Kunzea ambigua, Epacris crassifolia and K. capitata favoured by the lower incubation temperature, and Epacris microphylla var. rhombifolia, Sprengelia monticola and Dracophyllum secundum favoured by the higher incubation temperature.

Smoke as a main effect

Smoke increased germination of 5 species at both incubation temperatures (Table 3.5.1); Baeckea linifolia (Fig 3.16.2), Dracophyllum secundum, Epacris crassifolia, E. paludosa and Kunzea capitata (Fig 3.16.4). Whilst smoke increased germination of D. secundum and E. crassifolia within both incubation temperatures, smoke appears not to have increased germination to a comparable extent as observed in the more favourable incubation temperature i.e. it was sub-compensatory.

141 Table 3.5.1. Germination of 19 populations that were stored for 42 days at 15°C, treated with factorial combinations of heat shock (25 or 75°C for 5 minutes) and smoke (0 or 10 minutes), then incubated at 15°C or 25°C in darkness. ANOVA P-values.

15°C 25°C

Heat Smoke H x S residual Heat Smoke H x S residual shock (S) shock (S) (H) (H)

df 1 1 1 20 1 1 1 20 pop† E. obtusifolia 1 0.4349 0.0268 0.6945 0.2648‡ 0.1317 0.0335

G. sieberiana 1 <0.0001 0.0680 0.0256 0.0084 0.0567 0.8245

B. linifolia 1 0.2405 0.0002 0.2844 0.0144 0.0004 0.3366

E. m var rhombifolia 0.8551 0.0610 0.2551 0.0168 0.9896 0.6972

B. imbricata 1 0.2829 0.0008 0.0850 0.8180 0.0979 0.8180

K. ambigua 0.3209 <0.0001 0.6456 0.2841 0.7564 0.7564

S. monticola 0.1588! 0.1588 0.1588 0.3100 0.0015 0.6827

D. secundum 1 0.1504 <0.0001 0.1504 0.2314 0.0481 0.5291

E. crassifolia 1 0.1037 <0.0001 0.1037 0.7306 0.0239 0.7306

E. paludosa ‡ 1 0.3108 0.0123 0.5335 0.2793 <0.0001 0.7985

K. capitata 1 0.3926 0.0001 0.9720 0.5339 0.0203 0.1573

C. tetragonia 0.5771 0.1240 0.5084 0.7838 0.0530 0.4474 C. flexosa 0.8005 0.2290 0.9602 0.4646‡ 0.2335 0.1993 S. brevifolius (without coat) 0.2393 0.1393 0.6363 0.0753 0.3016 0.9679 W. pungens 1 0.1540 0.4674 0.4285 0.0786§ 0.5689 0.5689 R. gracilis Negligible germination Negligible germination S. brevifolius (within coat) Negligible germination No germination J. continuus No germination No germination G. melanocarpa No germination No germination

† population ‡ transformed data § α reduced to 0.01 due to heterogeneity of variances ! α reduced to 0.001 due to heterogeneity of variances

142 a) 15°C Epacris obtusifolia (population 1) c) 15°C Gahnia sieberiana (population 1) 100 35

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b) 25°C Epacris obtusifolia (population 1) d) 25°C Gahnia sieberi ana (population 1) 100 35

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25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.16.1: Mean final germination of seeds stored at 15°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 15 () or 25°C (). Germination plotted against heat shock and smoke treatments for a, b) Epacris obtusifolia (population 1), c, d) Gahnia sieberiana (population 1). Bars = S. E. s Interaction between heat shock, smoke and incubation temperature inferred as heat shock x smoke significant within single incubation temperature

143 a) 15°C Baeckea linifolia (population 1) c) 15°C Epacri s microphylla var rhombifolia 90 100

80 90 80 70 70 60 60 50 50 40 40 30 30 20 20

10 10

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b) 25°C Baeckea linifolia (population 1) d) 25°C Epacri s microphylla var rhombifolia 90 100

80 90 80 70 70 60 60 50 50 40 40 30 30 20 20

10 10

0 0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.16.2: Mean final germination of seeds stored at 15°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 15 () or 25°C (). Germination plotted against heat shock and smoke treatments for a, b) Baeckea linifolia (population 1), c, d) Epacris microphylla var.rhombifolia. Bars = S. E. s Interaction between heat shock, smoke and incubation temperature inferred for B. linifolia as heat shock x smoke significant within single incubation temperature Interaction between heat shock and incubation temperature inferred for E. m. var.rhombifolia as heat shock significant within single incubation temperature

144 Baeckea imbricata (population 1) Kunzea ambigua Sprengelia monticola a) 15°C c) 15°C e) 15°C 40 100 18

90 35 16 80 14 30 70 12 25 60 10 20 50 8 40 15 6 30 10 4 20 5 10 2

0 0 0

Baeckea imbricata (population 1) Kunzea ambigua Sprengelia monticola b) 25°C d) 25°C f) 25°C 40 100 18

90 35 16 80 14 30 70 % germination (+/- SE) % germination 12 25 60 10 20 50 8 40 15 6 30 10 4 20 5 10 2

0 0 0 unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.16.3: Mean final germination of seeds stored at 15°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 15 () or 25°C (). Germination plotted against heat shock and smoke treatments for a, b) Baeckea imbricata (population 1), c,

145 d) Kunzea ambigua, e, f) Sprengelia monticola. Bars = S. E. s Interaction between smoke and incubation temperature inferred as smoke significant within single incubation temperature a) 15°C Dracophyllum secundum (population 1) c) 15°C Epacris crassifolia (population 1) 60 60

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b) 25°C Dracophyllum secundum (population 1) d) 25°C Epacris crassifolia (population 1) 60 60

% germination (+/- SE) % germination 50 50

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0 0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.16.4: Mean final germination of seeds stored at 15°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 15 () or 25°C (). Germination plotted against heat shock and smoke treatments for a, b) Dracophyllum secundum (population 1), c, d) Epacris crassifolia (population 1). Bars = S. E. s Smoke significant within both incubation temperatures

146 e) 15°C Epacris paludosa (population 1) g) 15°C Kunzea capitata (population 1) 35 100

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f) 25°C Epacris paludosa (population 1) h) 25°C Kunzea capitata (population 1) 35 100

90 30

% germination (+/- SE) % germination 80

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0 0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.16.4 continued: Mean final germination of seeds stored at 15°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 15 () or 25°C (). Germination plotted against heat shock and smoke treatments for e, f) Epacris paludosa (population 1), g, h) Kunzea capitata (population 1). Bars = S. E. s Smoke significant within both incubation temperatures

147 Germination following transferral of species into more favourable conditions

When ungerminated seeds were transferred into conditions more favourable for germination, the increase in germination was frequently substantial (Table 3.5.2). Smoke increased germination of Kunzea ambigua only after transferral from 25 into 15°C, and increased germination of K. capitata before and after transferral from 25 into 15°C. Previous incubation temperatures affected germination of transferred Restio gracilis, and Schoenus brevifolius seeds within coats (Table 3.5.2). Thus, conditions long after the ‘fire’ apparently affected germination of some species. Fire-related cues increased germination of five species within initial incubation conditions, but the effect was gone following transferral into conditions more favourable for germination (Table 3.5.2). Germination of another three populations germinated uniformly across initial treatments, and increased uniformly following transferral (Table 3.5.2).

148

Table 3.5.2. Mean final % germination of 12 populations that were stored for 42 days at 15°C, treated with factorial combinations of heat shock (25 or 75°C for 5 minutes) and smoke (0 or 10 minutes), then incubated at 15°C or 25°C in darkness (Stage 1), and following transferral into more favourable conditions (Stage 2, 3).

Initial treatment Temperature 15°C 25°C Heat shock 25 25 75 75 25 25 75 75 Smoke 0 10 0 10 0 10 0 10

pop stage stage K. ambigua 1 30 88 39 92 1 4 5 2 2 2† 93 90 95 92 2‡ 34 74 35 50

K. capitata 1 1 60 97 53 90 1 3 28 8 13 2† 85 98 87 90 2‡ 5 48 8 28

R. gracilis 1 0 0 0 3 1 0 0 3 2 2† 83 88 77 82 2† 8 33 22 37

S. brevifolius 1 0 0 2 0 1 0 0 0 0 (within coat) 2! 24 9 24 20 2! 56 38 37 39

B. linifolia 1 1 37 66 33 65 2§ 58 71 70 68

D. secundum 1 1 0 18 0 18 2§ 29 35 41 32

E. crassifolia 1 1 0 27 0 43 1 0 7 0 5 2† 84 81 92 83 2† 87 88 85 83

E. paludosa 1 1 7 21 2 19 1 2 18 3 22 2† 23 48 20 35 2† 25 53 42 42

G. sieberiana 1 1 3 2 13 28 1 0 8 12 18 2§ 5 13 13 33 2! 50 53 65 55 3! 53 55 53 53

C. flexosa 1 13 20 15 21 1 13 3 15 10 2† 51 37 53 55 2† 53 70 57 62

J. continuus 1 0 0 0 0 1 0 0 0 0 2† 65 72 80 73 2† 92 97 85 97

S. brevifolius 1 9 15 13 23 1 3 8 12 17 (without coat) 2! 62 66 46 53 2! 48 67 53 47

† light ‡ 15°C ! 20/4 h at 25/35°C § 25°C

149 Section V (b) 35˚C Storage; Heat Shock +/- Smoke; 25 or 35˚C Incubation temperatures; 19 species

The aim was to assess the effects of storage at a different temperature (35˚C c.f. 15 or 25˚C), fire-related cues, temperatures of incubation (25 or 35˚C), and interactions between these factors on the germination of a large number (19) of species.

Germination at 35˚C

The high incubation temperature had a strong negative effect on germination of most species, as of the 19 species, only Baeckea linifolia and Calytrix tetragonia had germination of note at 35˚C (Table 3.6.1). Smoke increased germination of B. linifolia only within the 25˚C incubation temperature, consistent with an interaction between incubation temperature and smoke (Table 3.6.1; Fig 3.17.1). Germination of both species was reduced within the 35˚C incubation temperature (Fig 3.17.1).

Germination at 25˚C

Germination of four species was affected by the fire-related cues within the 25˚C incubation temperature (Table 3.6.1; Fig 3.17.2). Germination of Kunzea ambigua was increased by the combination of added heat shock and smoke. Germination of Epacris microphylla var rhombifolia, Gahnia sieberiana and Kunzea capitata was increased by smoke. Added heat shock reduced germination of Epacris microphylla var rhombifolia (Table 3.6.1; Fig 3.17.2).

150 Table 3.6.1. Germination of 19 populations that were stored for 42 days at 35°C, treated with factorial combinations of heat shock (25 or 75°C for 5 minutes) and smoke (0 or 10 minutes), then incubated at 25°C or 35°C in darkness. ANOVA P-values.

25°C 35°C

Heat Smoke H x S residual Heat Smoke H x S residual shock (S) shock (S) (H) (H)

df 1 1 1 20 1 1 1 20 pop† B. linifolia 1 0.1705‡ 0.0002 0.9089 0.2861 0.3937 0.6060

C. tetragonia 0.1583 0.5787 0.0746 0.6604 0.5644 0.9780

K. ambigua‡ 0.0032 0.0001 0.0001 No germination

E. m var 0.0197 0.0197 0.4080 No germination rhombifolia

G. sieberiana 1 0.9840 0.0016 0.9840 No germination

K. capitata! 1 0.3493 0.0001 0.1259 No germination

B. imbricata 1 0.9446 0.3151 0.3310 Negligible germination C. flexosa‡ 0.5214 0.9080 0.0588 No germination D. secundum 1 0.6279 0.1362 0.1455 No germination§ E. obtusifolia‡ 1 0.3122 0.0635 0.4386 No germination E. paludosa ‡ 1 0.3491 0.0704 0.9384 No germination§ S. brevifolius (without coat) 0.7323 0.1262 0.7842 No germination

E. crassifolia 1 Negligible germination No germination§ R. gracilis Negligible germination No germination W. pungens 1 Negligible germination No germination§

G. melanocarpa No germination No germination J. continuus No germination No germination S. brevifolius (within coat) No germination No germination S. monticola No germination No germination§

† population ‡ transformed data ! α reduced to 0.001 due to heterogeneity of variances § also, no germination following transferral into 20/4 h at 25/35°C

151

a) 25°C Baeckea linifolia (population 1) c) 25°C Calytrix tetragonia 60 60

50 50

40 40

30 30

20 20

10 10

0 0

b) 35°C Baeckea linifolia (population 1) d) 35°C Calytrix tetragonia 60 60

50 50 % germination (+/- SE) % germination

40 40

30 30

20 20

10 10

0 0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.17.1: Mean final germination of seeds stored at 35°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 25 () or 35°C (█). Germination plotted against heat shock and smoke treatments for a, b) Baeckea linifolia (population 1), c, d) Calytrix tetragonia. Bars = S. E. s Interaction between smoke and incubation temperature inferred for Baeckea linifolia as smoke significant within single incubation temperature

152 a) Kunzea ambigua c) Gahnia sieberiana (population 1)

70 30

60 25

50 20

40 15 30

10 20

5 10

0 0

b) Epacri s microphylla var rhombifolia d) Kunzea capitata (population 1)

14 30

12 25 % germination (+/- SE) % germination

10 20

8 15 6

10 4

5 2

0 0 unsm sm unsm sm unsm sm unsm sm

25°C heat shock 75°C heat shock 25°C heat shock 75°C heat shock

Figure 3.17.2: Mean final germination of seeds stored at 35°C, treated with heat shock (25 or 75°C) and smoke (0 or 10 minutes), then incubated at 25°C () (no germination at 35°C). Germination plotted against heat shock and smoke treatments for a) Kunzea ambigua b) Epacris microphylla var rhombifolia, c) Gahnia sieberiana (population 1), d) Kunzea capitata (population 1). Bars = S. E. s

153 Transferral into more favourable conditions

When ungerminated seeds were transferred into conditions more favourable for germination, the increase in germination was frequently substantial (Table 3.6.2). Warm season conditions are probably better represented by diurnal fluctuations in temperature rather than a continuous high temperature, as markedly depressed germination under warm, wet conditions has not been noted in the field. Fire-related cues increased germination of Kunzea ambigua and K. capitata before and after transferral from 25°C, and increased germination only after transferral from 35°C (Table 3.6.2). Fire-related cues also affected germination of Epacris paludosa and Restio gracilis after transferral from 35°C (Table 3.6.2). Thus, conditions long after the ‘fire’ affected germination of some species. Germination of nine populations was unaffected by previous fire-related cues (Table 3.6.2).

Summary of Section V

Results were consistent with an effect of storage temperature on germination of many species. Temperature of incubation frequently affected germination and, in general, results were consistent with interactions between temperature of incubation and fire-related germination cues. Interactions between storage temperature, temperature of incubation and fire-related germination cues were also possible(see discussion). The null hypothesis that neither storage temperature, nor temperature of incubation would affect germination, nor were there any interactions between storage temperature, temperature of incubation or fire-related germination cues was not supported.

154 Table 3.6.2. Mean final % germination of 13 populations that were stored for 42 days at 35°C, treated with factorial combinations of heat shock (25 or 75°C for 5 minutes) and smoke (0 or 10 minutes), then incubated at 25°C or 35°C in darkness (Stage 1), and following transferral into more favourable conditions (Stage 2).

Initial treatment Temperature 25°C 35°C Heat shock 25 25 75 75 25 25 75 75 Smoke 0 10 0 10 0 10 0 10

pop stage stage K. ambigua 1 3 4 0 47 1 0 0 0 0 2† 11 46 13 53 2‡ 25 41 23 22

K. capitata 1 1 0 17 2 10 1 0 0 0 0 2† 0 27 2 23 2‡ 18 37 28 37

R. gracilis 1 0 2 0 0 1 0 0 0 0 2! 3 12 13 18 2! ‡ 7 25 8 30

E. paludosa 1 0 0 0 0 2! ‡ 13 17 8 17

G. sieberiana 1 1 0 0 0 0 2‡ 53 62 50 60

B. linifolia 1 1 12 14 16 26 2‡ 45 51 46 56

C. tetragonia 1 5 7 7 9 2‡ 25 34 31 39

S. brevifolius 1 0 0 0 0 (without coat) 2‡ 48 38 44 47

S. brevifolius 1 0 0 0 0 (within coat) 2 ‡ 64 63 58 48

J. continuus 1 0 0 0 0 1 0 0 0 0 2! 92 98 88 95 2! ‡ 93 100 97 97

C. flexosa 1 0 0 0 0 2! ‡ 20 15 27 23

E. obtusifolia 1 1 0 0 0 0 2‡ 20 27 25 23

B. imbricata 1 1 0 0 0 2 2‡ 36 37 18 41

† 15°C ‡ 20/4 h at 25/35°C ! light

155 Section VI 15, 25 or 35˚C Storage; 50˚C Heat Shock +/- Smoke; 15, 25 or 35˚C Incubation temperatures: Baeckea imbricata (population 1).

The aim was to assess whether a wide range of storage temperatures (15 to 35˚C), smoke, a wide range of incubation temperatures (15 to 35˚C), or interactions between these three factors would affect germination of heat shock treated seeds of a single species.

The interplay of storage and incubation temperatures and smoke became apparent when these factors were varied for Baeckea imbricata (population 1) seeds treated with 50˚C heat shock (Fig 3.18). Incubation temperature appeared to have a stronger overall affect on germination, which was highest at 15˚C, less at 25˚C (depending on storage temperature), and zero at 35˚C incubation temperature. Seeds incubated at 35˚C only germinated when subsequently transferred into the fluctuating incubation temperature regime. Smoke increased germination of seeds that were incubated at 15˚C, although when seeds were stored at 15˚C then the effect was apparent only as a trend (Fig 3.18). Smoke also increased germination of seeds that were stored at 25˚C and incubated at 35˚C, after subsequent transferral into the fluctuating incubation temperature regime (P = 0.0046, data not shown).

156 a) 15°C storage; 15°C incubation b) 15°C storage; 25°C incubation

80 80

70 70

60 60

50 50

40 40

30 30

20 20

10 10

0 0 c) 25°C storage; 15°C incubation d) 25°C storage; 35°C incubation

80 80

70 70

60 60

50 50

40 40

30 30

20 20

10 10 % germination (+/- SE) % germination

0 0 e) 35°C storage; 25°C incubation f) 35°C storage; 35°C incubation

80 80

70 70

60 60

50 50

40 40

30 30

20 20

10 10

0 0 Unsmoked Smoked Unsmoked Smoked

Figure 3.18: Mean final germination of Baeckea imbricata (population 1) seeds stored at a, b) 15°C, c, d) 25°C, or e, f) 35°C, treated with 50°C heat shock and smoke (0 or 10 minutes), then incubated at a, c) 15 () or b, e) 25°C (), or d, f) 35°C (no germination). Germination plotted against smoke treatments. Bars = S. E. s Effect of smoke: ANOVA P-values a† b c d e f 0.0022 0.3589 0.0261 0.6976 †α reduced to 0.001 due to heterogeneity of variances

157 Section VII

The aim was to assess whether the removal of seed coats would affect germination directly (2 species), or due to a change in heat shock tolerance (1 species).

Effect of seed coat on dormancy of Actinotus helianthi

No Actinotus helianthi smoked or unsmoked seeds germinated following removal of pericarp and testa and incubation in the light or dark at 25˚C.

Effect of seed coat on dormancy of Schoenus brevifolius

No Schoenus brevifolius seeds germinated within a 25°C incubation temperature following leaching or control treatments.

Effect of seed coat on heat shock tolerance of Schoenus brevifolius

Seed coats protect Schoenus brevifolius seeds from excessive heat shock. No decoated S. brevifolius seeds germinated when treated with 100°C heat shock with or without smoke, and they were susceptible to fungal attack, whereas an average of 36% of intact seeds germinated (F 1, 9 = 14.13, P = 0.0055).

Summary of Section VII

The removal of seed coats did not affect germination of Actinotus helianthi seeds. The removal of seed coats did not affect germination of Schoenus brevifolius directly, but did so indirectly by reducing heat shock tolerance of the exposed seeds. The null hypothesis that the removal of seed coats would not affect germination was partly rejected.

158 3.4 Discussion

Storage temperature, temperature of incubation and the fire-related germination cues frequently affected germination, and interactions between these factors were frequently apparent. If the current results also occur in the field, then season affects the post-fire species composition for species forming soil seedbanks in the Sydney region. Notably, germination was greatly reduced when seeds were stored or incubated at 35°C, hence post-fire germination may be limited by ambient temperature during summer. Most species germinated within both 15 and 25°C incubation temperatures, indicating that substantial post-fire germination may occur in any season other than summer, but germination of about one-third of the study species was favoured by ‘cool-season fire’, and about one-third were favoured by ‘warm- season fire’. Effects of the more moderate temperatures (15 or 25°C) were generally of low magnitude, unless the inferred induction of secondary dormancy (see below) occurs in the field, and is not overcome by the fire-related cues. If such secondary dormancy occurs in the field, then the potential for changes in community composition is substantially increased.

Incubation temperatures and fire-related germination cues

The temperatures of incubation frequently influenced the effects of heat shock and smoke on germination, however the patterns of response to the two cues were broadly comparable across temperature. Smoke extended the temperature range over which germination may be expected for two species, but did not stimulate germination to the same maximum as occurred within the more favourable temperature i.e. smoke was ‘sub – compensatory’.

Incubation temperatures

Following intense summer fire that exposes soil to high levels of solar irradiance, the diurnal temperature at 10 mm depth is predicted to range between 52 and 27°C (Auld & Bradstock 1996). If the germination response of seeds to such a temperature regime was adequately represented by the germination response of seeds that were incubated at 35°C, then regeneration through resprouting is likely to predominate following such fire. However, whilst the total heat load may be approximated by a constant 35°C, temperature fluctuation per se may increase germination. Germination of seeds that were stored and incubated at

159 35°C was substantial increased following transferral into 20 hours at 25°C and 4 hours at 35°C. Although maximum temperatures within shallow soil are considerably higher, and are maintained for longer, it is possible that some germination would occur within the fluctuating temperature regime following intense summer fire. The number of species with an optimum incubation temperature of 15˚C was approximately equal to the number of species with higher germination at 25˚C. Eight species had greater germination at 15 than 25˚C and eleven species had greater germination at 25 than 15˚C. Populations of Baeckea imbricata and Epacris paludosa, had greater germination within the different temperatures and so these two species were not counted as favoured by either temperature. Either a 15 or 25˚C incubation temperature did not favour germination of another nine species. Either a 15 or 25˚C incubation temperature did not favour germination of two more populations, however, other populations of these species were favoured by one temperature over the other.

Life-history categories and favourable incubation temperatures

Species were classified along a continuum of mode of post-fire recovery from obligate seeding to strongly resprouting adult plants (Naveh 1975). Approximately the same number of species at any one point along this continuum had more favourable incubation temperatures of 15 and 25˚C (Table 3.7). Also, no family group was contained within one of these categories. There may be a weak association between wetter habitats and a more favourable higher incubation temperature, but not exclusively so (Table 3.7).

160 Table 3.7. More favourable incubation temperature for study species that have been classified along a resprouter- seeder regenerative continuum (NSW NPWS database).

More Regenerative strategy favourable incubation RESPROUTER RESPROUTER SEEDER / SEEDER / SEEDER temperature / seeder RESPROUTER resprouter

15°C Baeckea linifolia Kunzea capitata Baeckea Goodenia ovata Epacris (W) (M) ramosissima (D) crassifolia (W) (D)

Goodenia Stackhousia Kunzea ambigua heterophylla (D) nuda (W) (D)

15 = 25°C Caustis flexosa Calytrix Baeckea brevifolia Grevillea (W) tetragonia (M) (D) buxifolia (D)

Goodenia Micromyrtus Woollsia pungens bellidifolia (W) ciliata (W) (M)

Grevillea sericea (M)

25°C Baeckea utilis Goodenia Dracophyllum Grevillea (M) dimorpha (D) secundum (M) acanthifolia (W)

Gahnia Epacris coriacea Sprengelia sieberiana (M) (M) monticola (W)

Juncus continuus Epacris obtusifolia (W) (W)

Schoenus brevifolius (W)

D = dry habitat; M = moist habitat; W = wet habitat

161 Effect of season of fire on community composition

Excluding results from the simulated summer, if these results translate into the field thena similar proportion of species will germinate from the soil seed bank across the season of a burn, however the understorey community composition will change across the season of a burn. The change in community composition is not predictable on the basis of reseeder or resprouter strategies, or Family, and the proportional representation of these factors are not expected to change across the season of a burn. The differences between germination due to 15 or 25°C incubation temperatures were generally minor in magnitude, hence burning would have to be repeated many times in a single season to substantially change species composition. A fraction of the community may have poor germination and hence low levels of in situ seed production when fire is repeated in a single season, and the capacity for a single fire in a different season to renew the above ground community probably diminishes with the number of single season fires. The crucial element then is the capacity of species to persist in the seedbank. Persistence requires that soil-stored seed remain viable until a fire occurs in a different season. Given the dominance of warm season fire over the landscape (Conroy 1996; Bradstock & Cary 2001), the number of species favoured or even dependent on low temperature for germination following application of fire-related germination cues is surprising. If these results translate into the field, then either substantial regeneration from the soil seedbank for some species may be confined to after relatively infrequent cool season fires, or germination following warm season fires is delayed for these species.

Inferred induction of secondary dormancy

The induction of secondary dormancy is apparent if seeds that were initially non- dormant are kept under unfavourable conditions and do not germinate when transferred into favourable conditions (Crocker 1916). This hypothesis was explored in the current study using the following criteria. Firstly, if seed was stored at a single temperature, treated or not treated with fire-related cues, and incubated at different temperatures, in some cases there was higher germination at one of the temperatures. If germination of seeds transferred from the less favourable to the more favourable incubation temperature did not increase to a similar level to those in the favourable temperature, then the reduction in final germination is consistent with the induction of secondary dormancy during incubation at the less favourable

162 temperature (Fig 3.19.1). The reference point(s) for the initial level of dormancy were 1) germination within the more favourable incubation temperature of the current experiment, and / or 2) germination within experiments that were conducted prior, concurrently, and / or subsequently to the current experiment. Interactions between the fire-related cues and possible secondary dormancy were also explored by visual inspection of the data. Differences were looked for between the relative effect of the fire-related cues on non-dormant seed and on seed that was inferred to have secondary dormancy. The increase in germination of seed treated with fire-related cues relative to their control was compared across non-dormant seed and seed that was inferred to have secondary dormancy. If the relative increase in germination was greater for seed that was inferred to have secondary dormancy, then the fire-related cues were inferred to have reduced the induction of secondary dormancy. Secondly, if batches of seed were stored at different temperatures, incubated at one temperature and germination of one batch was reduced, then the reduction in final germination is consistent with the induction of secondary dormancy at the less favourable storage temperature (Fig 3.19.2). The reference point(s) for the initial level of dormancy, and the interactions between the fire-related cues and possible secondary dormancy were inferred as above. If both of the above scenarios occurred (Figs 3.19.1-2), then the reduction in final germination is consistent with the induction of secondary dormancy during both storage and incubation. When the reference point for the initial level of dormancy was 2) germination within experiments that were conducted prior, concurrently, and / or subsequently to the current experiment, then an alternative explanation would be that secondary dormancy had been induced during laboratory storage in the time between experiments. However, in most cases the reference points for exploring secondary dormancy prior to the current experiment were from experiments conducted before, concurrent and after the current experiment. It is considered extremely unlikely that the initial dormancy of the population would have been different for concurrent experiments. In other cases, the reference points for assessing secondary dormancy prior to the current experiment were from experiments conducted before and after the current experiment. Again it is considered extremely unlikely that the initial dormancy of the population would have changed just while the current experiment was being conducted. In the minority of cases, the reference point for assessing dormancy prior to the current experiment was from an experiment conducted 2 months before the current

163 experiment. It is considered unlikely that the initial dormancy of the population would have so suddenly increased during laboratory storage. Factors such as changes in seed viability could give similar patterns to those observed, however, conditions were not so extreme as to expect a change in viability. Also, an increase in germination following transferral of seeds into more favourable conditions and further increase in germination following transferral of seeds into even more favourable conditions indicates that viability was not reduced in many cases. When germination increases to a uniform level following transferral of seeds into more favourable conditions, then secondary dormancy is likely to explain the initial reduction in germination. Germination patterns consistent with the induction of secondary dormancy were apparent for some species. Examples consistent with the induction of secondary dormancy during incubation at unfavourable temperatures include Epacris obtusifolia and Dracophyllum secundum (Fig 3.19.1 Schema). When these two species were incubated at 35°C, although germination increased when they were transferred into a more favourable 25°C, final germination was apparently less than when seeds were incubated at 25°C from the outset (Table 3.8). Examples consistent with the induction of secondary dormancy during dry storage at unfavourable temperatures include Kunzea ambigua and K. capitata (Fig 3.19.2 Schema). When these two species were stored at 15°C, and then incubated at 25°C, germination was apparently less than when seeds were stored and incubated at 25°C (Table 3.9).

Secondary dormancy and fire-related germination cues

The heat shock and smoke cues that increased germination generally also reduced the degree of inferred secondary dormancy that was induced in a species. However, the effect of the cues on inferred secondary dormancy was small relative to the effect of transferral to a more favourable temperature of incubation. For example, smoke did not overcome the inferred secondary dormancy of Kunzea ambigua due to dry storage at 15˚C (see above) until these seeds were transferred to the more favourable 15˚C incubation temperature (Table 3.5.2), whereas inferred secondary dormancy was overcome in unsmoked seeds by the more favourable temperature (Fig 3.16.3c, Table 3.5.2). Similarly, the inferred secondary dormancy of K. capitata due to dry storage at 15˚C (see above) was also overcome in unsmoked seeds by the more favourable 15˚C incubation temperature, whilst smoke only partially overcame dormancy at the less favourable 25˚C (Fig 3.16.4, Table 3.5.2).

164 Fig 3.9.1. Schema showing (inferred) induction of secondary dormancy during incubation at a less favourable incubation temperature Post-fire incubation

Fire-related germination cues

Increasing germination

More favourable temperature

Pre-fire storage at a single temperature No secondary dormancy

Less favourable temperature Secondary dormancy induced during incubation

Transferral across incubation temperatures

Table 3.8. Comparison between % germination of E. obtusifolia and D. secundum seeds incubated at different temperatures; induction of secondary dormancy during incubation at 35°C inferred (see text)

Species Storage Incubation Treatment % germination % germination Figure Duration‡ Temp Temp (Stage I) Stage I Stage II (25°C)

E. obtusifolia 14 25 35 Control 0 32 3.15a Fire† 0 42

14 25 25 Fire 90 5.4.4

16 25 25 Control 87 6.1a Fire 88

D. secundum 14 25 35 Control 0 0 - Fire 0 2

14 25 25 Fire 44 -

16 25 25 Control 40 6.9a Fire 40

† 75°C heat shock and 10 minutes smoke ‡ months

165 Fig. 3.19.2. Schema showing (inferred) induction of secondary dormancy during storage at an unfavourable temperature

Post-fire incubation

Increasing germination Fire-related germination cues

No secondary dormancy

Pre-fire storage at two Single incubation temperature different temperatures

Secondary dormancy induced during storage

Table 3.9. Comparison between % germination of K. ambigua and K. capitata seeds stored at different temperatures; induction of secondary dormancy during storage at 15°C inferred (see text)

Species Storage Incubation Treatment % Figure Duration‡ Temperature Temperature germination

K. ambigua 19 15 25 Control 4 3.16.3d Fire† 2

19 25 25 Control 40 6.6a Fire 90

K. capitata 19 15 25 Control 3 3.16.4h Fire 13

19 25 25 Control 10 6.5a Fire 50

† 75°C heat shock and 10 minutes smoke ‡ months

166 If secondary dormancy occurs in the field and is only partially overcome by fire- related cues, then it will influence post-fire species composition. The degree of secondary dormancy changes through the seasons, therefore its effect on post-fire species composition will depend on the season of fire. The occurrence of secondary dormancy in the field should be investigated.

Wet habitat and fluctuating soil temperature

Germination of three species was favoured by a 25/35˚C diurnal temperature regime. Not surprisingly, germination of two of these species was clearly greater at 25 than at a 15˚C incubation temperature. All of these species occur on the edge of watercourses, where the greater conductance of wet soil allows above ground temperature fluctuation to penetrate more deeply than through dry soil. Temperature fluctuation would indicate to a buried seed that the soil was not submerged in water, that seed was close to the surface, and an absence of vegetation (Thompson 1974; Thompson et al. 1977) such as after a fire (Raison et al. 1986). Interestingly, the fluctuating temperature requirement for germination of Schoenus brevifolius was absolute, and was presumably required to overcome inhibitors within the seed itself. The coat protects the seed from the deleterious effects of higher levels of heat shock, and probably reduces other hazards such as physical damage or predation. If seed coats break down in the field, then presumably seeds germinate under a wider range of temperatures rather than waiting in a vulnerable state for more favourable conditions.

Intra-population habitat effects

The reason for the different responses of the two Gahnia sieberiana populations to high heat shock and the fluctuating incubation temperature is unknown, but may reflect the different habitats of the populations. Population 2 was situated in a heavily shaded, very moist section of wet sclerophyll forest, hence it is unlikely to experience fire, particularly high intensity fire that would transfer high heat shock through the soil and open the canopy, thus exposing soil to high levels of solar radiation. Population 1 was situated in an open environment on the edge of a dry sclerophyll forest, hence it is likely to experience high intensity fire that would transfer high heat shock through the soil and expose soil to high levels of solar radiation. A population-specific fire response has been related to the frequency of fire across habitats. The fire-related germination cue charate strongly stimulated

167 germination of a population of a shrub from the fire-prone chaparral but only weakly stimulated germination of a population from the relatively fire-free desert (Jones & Schlesinger 1980). Also, substituting charate for heat shock, a similar pattern was found in a chaparral species; alternating temperature increased germination of one of two populations, and charate increased germination within the constant but not the alternating temperature regime (Keeley 1991). Because the contrast between species fire responses across habitats has been limited to single populations within the different habitats, the contrast is tentative.

Light, habitat and fire-related germination cues

The presence of light-stimulated species in a fire-prone environment highlights the fact that high levels of erosion can occur post-fire. Ash particles within soil pores (Mallik et al. 1984) probably result in low levels of light penetration through soil, hence ‘post-fire’ incubation in darkness before transferral into light probably simulates the change in field conditions. Small-scale pilot studies indicated that most of the study species have a neutral light response, and that prior dark incubation was not required by the species with a light response. Species with germination that was increased by light, but generally not by the fire- related germination cues all occur in habitat where soil movement is highly likely following disturbance. Caustis flexosa, Epacris paludosa, Juncus continuus and Restio gracilis occur in waterways, where flowing water is likely to cause soil movement, and Epacris crassifolia and E. muelleri occur on wet cliff faces where gravity is likely to exacerbate erosion. Both the amount of light reaching the soil surface, and the amount of erosion increase in a post-fire environment, hence germination may be indirectly linked to fire. A post-fire residual soil seedbank would be ensured because erosion would expose only a fraction of the seedbank to light. A response to light would also allow germination following other disturbances that are also associated with a resource enhanced environment that is favourable to seedling survival (Fenner 1980; Orozco-Segovia et al. 1993), and is possibly an ancestral trait that pre-dates the fire-prone nature of the current habitat. Most of the light-responsive species occur in environments where water is frequently not limiting, so although they may germinate near the soil surface, they would only infrequently encounter the hazard of a soil surface drying more rapidly than the expanding seedling hypocotyl. Light did not increase germination of the wet habitat species Baeckea linifolia, hence the light response was not entirely predictable on the basis of habitat.

168 Germination of Kunzea ambigua was also increased in light (current study), and following disturbance (Temple & Bungey 1980), but it occurs in environments where a rapidly drying soil surface is a frequently encountered hazard, particularly during warmer months. If the massive reduction in germination due to fluctuating water availability under warm temperature conditions in darkness (Chapter 5) also occurs in light, then the probability of desiccation of a seedling from a surface-germinating seed would be greatly reduced. Presumably germination of K. ambigua due to disturbance occurs only during cooler months, when such a hazard would be less frequently encountered. The germination response of Epacris crassifolia, E. paludosa, Kunzea ambigua and K. capitata to smoke or light, factors both associated with disturbance, has been found in previous studies (Drewes et al. 1995; Thomas & van Staden 1995; van Staden et al. 1995; Gardner et al. 2001).

169 Chapter 4. Effects of pre-fire hydration status

4.1 Introduction

Overview

The question of whether the pre-‘fire’ hydration status of soil seedbanks in the fire- prone Sydney region influences post-‘fire’ germination or viability, and whether wet and dry habitat species are affected differently is investigated in this section.

Negative effect of pre-fire moisture

Post-fire germination from a soil seedbank is potentially affected by the hydration status of the seed before the fire. Fire in the wet season and ‘hazard reduction’ burning have been associated with large reductions in germination from soil seedbanks (Horton & Kraebel 1955; Shea et al. 1979; Parker 1987; Portlock et al. 1990). The reduction in germination from soil seedbanks following fire when the soil is moist may be due to the death of species with water-permeable seeds. The negative correlation between heat shock tolerance and hydration is very strong, probably due to the massively increased susceptibility of proteins to denaturation whilst in an environment of free water (Warth 1985). The denaturation of solid egg albumin due to heat increased with the twelfth power of sorbed water (Altman & Benson 1960). The capacity of spores to survive heat decreased markedly with increased free water (Murrell & Scott 1957; Gerhardt & Marquis 1989; Setlow & Setlow 1995), and bacterial endospores survive approximately 1,000-fold longer in dry heat than in moist heat (Fox & Eder 1969). Survivability is the only conferred attribute that is vital to all of the organisms that form spores (Sussman 1969). Decreased heat shock tolerance of seeds when in a hydrated state was first determined by Just (1877), and survival was markedly lower for hydrated seeds in species with water-permeable seeds from the fire-prone chapparal (Sweeney 1956; Parker 1987). All sixteen herbaceous species germinated after air-dry seeds were treated with 5 minutes of 150°C or more heat shock, and were killed by 80°C when hydrated (Sweeney 1956). However, temperate wet heath can carry fire within 24 hours after substantial rain and when the dead fuel has a surface moisture content of up to 70% (Marsden-Smedley &

170 Catchpole 1995). Water-permeable seeds track soil moisture (Thompson 2000), hence seeds in wet habitat could receive heat shock whilst hydrated.

Pre-fire hydration across habitats

Importantly, the intense fires associated with extreme weather generally occur when the soil moisture of dry habitats is low, hence the seeds of dry habitat species usually have relatively low moisture contents when they experience high intensity fire. In contrast, wet habitats are restricted and surrounded by large areas of dry habitats, and intense fires from dry habitat continue through wet habitat regardless of the soil moisture level. Thus, seeds of wet habitat species can encounter high levels of heat shock when they are hydrated and when they are dehydrated. Although the level of heat shock is reduced when fuel (Luke & McArthur 1978) or soil (Nakshabandi & Kohnke 1965; Aston & Gill 1976; Valette et al. 1994; Campbell et al. 1995) including soil derived from Hawkesbury Sandstone (Beadle 1940) is wet, high levels of heat shock are generated during the intense fires associated with extreme weather. It could be postulated that the heat shock tolerance of species from wet habitats (currently unknown for any flora) would remain high regardless of hydration status. In contrast, the heat shock tolerance of species from dry habitats would be expected to increase as the seed and its surroundings dry out because the heat that the seed may encounter also increases as the surroundings become dry. If there is differential tolerance of heat shock whilst hydrated between species from wet and dry habitats, then this factor may contribute to wet/dry habitat segregation for species forming water-permeable soil seedbanks. Periodical waterlogging of the soil in wet habitat produces physiological disturbance in plants due to anaerobic conditions and associated phytotoxin activity (Armstrong 1981). Morphological adaptations to waterlogging are important, and ecophysiological adaptations are essential (Specht 1981). Differential tolerances of waterlogging due to morphological and ecophysiological adaptations contribute to the segregation of wet and dry habitat species (Bannister 1964; Armstrong & Boatman 1967; Jones 1971; Bolton 1977; Meney et al. 1990). Toposequences of plant communities correlating with soil moisture and nutrient gradients have been recorded for upland swamps in the Sydney region (Burrough et al. 1977; Keith & Myerscough 1993; Keith 1994), and boundary movements occur due to prolonged wet or dry periods (Buchanan 1980). Superimposed on these edaphic factors are recurring fires that are commonplace in contemporary and fossil records of swamps in the Sydney region (Kodela & Dobson 1988; Johnson 1994; Martin 1994; Mooney et al. 2001). These fires interact with life

171 cycle characteristics to influence community composition (Keith & Bradstock 1994; Keith 1995). A hitherto unexamined ecophysiological adaptation to wet habitat is the capacity of seed of species that regenerate from water permeable soil seedbanks to survive high heat shock whilst hydrated. This capacity would co-occur with other adaptations as a syndrome (sensu Stebbins 1974), however, the capacity of seed to survive high heat shock whilst hydrated forms the first requirement for occupation of wet habitat in a fire-prone environment for species with water permeable seed that regenerate from soil seedbanks.

Duration of hydration prior to heat shock

The interaction between heat shock and hydration has been investigated following greatly differing periods of hydration prior to heat shock. When heat shock has been applied as soon as seeds have been hydrated, then the investigation of the interaction is of a more physical nature. In physical terms, seeds have been fully hydrated and then treated with heat shock temperatures spanning the range of tolerances, or seeds have been hydrated to a range of moisture contents and then treated with heat shock (Robbins & Petsch 1932; Sweeny 1956; Ghaley & Taylor 1982). However when seeds have been hydrated for a considerable period of time, allowing greater numbers of physiological changes to occur prior to heat shock, then the investigation of the interaction is of a more physiological nature. In physiological terms, seeds were treated with heat shock after being imbibed for a range of durations and correlations determined between heat shock tolerance and the concentration of heat shock proteins present following each imbibition period (Abernethy et al. 1989). Duration of imbibition prior to heat shock is potentially an important factor that was investigated.

Post-fire heat shock tolerance

It is reasonable to propose that heat shock proteins play a major role in thermotolerance (Key et al. 1985), because they increase protein stability. For dry seed, the onset of protein denaturation did not occur until they were heated to 150°C (Golovina et al. 1997) or more (Wolkers et al. 1998), whereas protein denaturation in dry mutant seeds that did not form heat shock proteins commenced at 70°C (Wolkers et al. 1998). Heat shock proteins probably conferred a higher degree of protein stability than drying per se, as the

172 onset of protein denaturation occurred when hydrated seed was heated to 56°C (Wolkers et al. 1998). Factors that convey heat shock tolerance to seed are likely to incur costs, hence these factors are likely to be eliminated by seed within an environment where such tolerance in not required. Seed in a post-fire environment does not require tolerance of high levels of heat shock, hence the factors that convey heat shock tolerance are likely to be eliminated or reduced unless these factors are of benefit to the germinating seed or seedling. Heat shock proteins incur numerous physiological costs (Feder & Hofmann 1999), and heat shock proteins are rapidly eliminated when not required (Welte et al. 1993; Parsell & Lindquist 1994). However, the role of heat shock proteins in refolding of other proteins (Wehmeyer et al. 1996), and the transport, assembly and disassembly of multi-structured units, and degradation of misfolded or aggregated proteins (Gething & Sambrook 1992; Parsell & Lindquist 1994; Bross et al. 1999; Jolly & Morimoto 1999; Gregersen et al. 2001) may be important during germination. The heat shock tolerance of seed that has recently experienced heat shock and that has commenced the process of germination was investigated because it may provide information about the stresses experienced by seed and seedlings in a fire-prone environment, and the mechanisms whereby they tolerate such stresses.

Investigations

Hydration status is an important component of heat shock tolerance, and the interaction between these factors was investigated in physical and in physiological terms within the current study. Time, seed number, and seed quality limited the number of species that were investigated and the detail of the investigation. Section I. The influence of ‘pre-fire’ hydration status was investigated for species ranging from wet to dry habitat, using a range of durations of both hydration and dehydration. Seeds were hydrated for a range of durations (days); some were maintained hydrated, and the others were dehydrated for a range of durations (days). These hydrated or dry seeds were then treated with factorial combinations of heat shock and smoke. Detection of the underlying mechanisms whereby hydration status affects the germination response to heat shock or smoke requires application of these cues both in isolation and in combination. To investigate the possibility that the response of seed to fire whilst hydrated may contribute to habitat segregation, one dry habitat, one moist habitat and three populations of a wet habitat species were used. The null hypotheses

173 were that the combination of fire-related cues and hydration status would not affect germination, and that there would be no relationship between a species habitat and its response to the combination of fire-related cues and hydration status. Section II. The interaction between ‘pre-fire’ hydration and the fire-related cues was investigated for a large number of species using fewer treatments. Due to time constraints, the response of fewer species to more treatments or of more species to fewer treatments could have been investigated. As nothing is known about the response of species forming soil seedbanks in the Sydney region to the combination of fire-related cues and hydration status, the latter option was taken. The null hypotheses were again that the combination of fire-related cues and hydration status would not affect germination, and that there would be no relationship between a species habitat and its response to the combination of fire-related cues and hydration status. Section III. The possibility that seeds need to be hydrated at the time of fire to overcome dormancy was investigated for two species. Two species had not germinated in a number of experiments after being treated with fire-related cues whilst air-dry. These seeds had been dry stored, whereas in the field they would have experienced periods of hydration prior to fire. The null hypothesis was that the combination of fire- related cues and hydration status would not affect germination of dormant seeds. Section IV. The effect of a short period of pre-fire hydration (hours) was investigated for two species. The short period of time would have allowed seed to fully hydrate, thus the effect of water, as a physical agent, would be present. In contrast, little physiological change would have occurred within seed during the short time of hydration. Therefore, if changes in the germination response of these species to the fire-related cues with hydration status (previously observed) were dependent on the major physiological changes leading to germination, then hydration for a short period of time would not affect their response to the fire-related cues. The null hypotheses were that the germination response to fire-related cues of air-dry seed and of fully hydrated seed that had not undergone major physiological changes would not be different, and that there would be no relationship between a species habitat and its response to the combination of fire-related cues and hydration. Section V. The effect of a long period of pre-fire hydration (months) was investigated for one wet-habitat species. Seed in wet habitat is likely to be hydrated for long periods of time prior to fire, hence it is appropriate to examine the germination response to fire- related cues of long-term hydrated seed. The null hypothesis was that the germination

174 response to fire-related cues of air-dry seed and of long-term hydrated seed would not differ. This experiment also allowed an examination of whether the effects of heat shock or smoke were retained for a long period of time in hydrated seed. Because germination of the species was affected by a strong synergistic interaction between heat shock and smoke, seeds that had received heat shock alone or smoke alone prior to long-term hydration would show a strong germination response to the other cue if the original cues was retained. A subsidiary null hypothesis was that the germination response of seed to the combination of heat shock and smoke would not differ if these cues were applied to seeds that had not been hydrated, or if there was a long period of hydration between the applications of the two cues. Section VI. The change in heat shock tolerance following application of fire-related germination cues and subsequent progress toward germination wasinvestigated for two species. Because there must be a substantial period of time between fires in the field, and because the factor(s) that convey heat shock tolerance may interfere with the process of germination, these factor(s) may be degraded for a period of time in seed treated with heat shock and smoke. If these factor(s) are degraded, then the response of seed to heat shock will be different to its response to a second heat shock within a short period of time. The null hypotheses were that the germination response of seed that was progressing toward post-fire germination would not be affected by a second application of heat shock, and that there would be no relationship between a species habitat and its response to the second application of heat shock. Inferred secondary dormancy status of seeds following a number of these treatments was investigated at the end of experiments by transferring seeds across incubation temperatures, and from darkness into light. Importantly, the transferral of seeds allowed an assessment of whether seeds were killed by the initial treatment (Harper & White 1974).

175 4.2 Methods

Hydration and dehydration.

The effect of different durations of hydration and deydration prior to exposure to fire- related cues was investigated. Experimental design for pre-‘fire’ treatment was 1) Pre-fire hydration: unhydrated (0 days) or hydrated (varying number of days; Table 4.1) 2) Pre-fire dehydration: (none, or varying numbers of days; Table 4.1) The longest duration of hydration was slightly less than the time required for first germination of seeds from the population in previous experiments (Chapters 2, 3 & 5). Seeds were hydrated as a batch with reverse osmosis water on a single layer of Whatman No. 1 filter paper at 25°C in darkness. Because the water potential of air-dry seed is very low (generally between –350 and –50 MPa; Roberts & Ellis 1989), water-permeable seeds rapidly imbibe water (Stage I). The rapid uptake of water in the three species investigated, which probably resulted in full hydration within 12 hours (Fig 4.1), is typical of such seed. As the seed water potential increases during imbibition and the gradient for water uptake decreases, the water content asymptotically approaches a plateau level (Stage II). No or little water is imbibed during Stage II, which can last for years (Powell et al. 1984); the metabolic activity that occurs during Stage II ultimately culminates in germination. Rapid water uptake occurs after germination (Stage III), and is associated with growth of the emerging radicle (Bewley & Black 1982b; Bradford 1995). All seeds that were hydrated when treated would be expected to be in Stage II. Seed was dehydrated under the same conditions by draining the excess water and leaving the dishes open to the atmosphere. Seed of the three species investigated returned to their air-dry weight by the end of one-day dehydration. Seed was also subject to a five-day dehydration treatment because the reversion from the physiological status of Stage II to that of seed that had not been hydrated may not have occurred in the time it took to become air- dry. When seed is primed (prolonged Stage II hydration due to low water potential of the medium), then physiological changes associated with Stage II are retained following a return to an air-dry state (Hanson 1973; Dearman et al. 1987; Bray 1995). When wheat embryos were hydrated and then dehydrated for one day, they had lower heat shock tolerance than embryos that had not been hydrated, despite having the same moisture content (Fahey et al.

176 1980). Also, seed that had not been hydrated was subject to a procedural ‘dehydration’ treatment to achieve a balanced design. Seeds within each combination of hydration and dehydration were treated in a single petri dish. Thus, replicates were not independent within hydration and dehydration treatments (Hurlbert 1984). However, in contrast with heat shock, smoke or incubation temperature treatments, it is difficult to imagine that there would have been variability within the combinations of hydration and dehydration treatments if they had been applied to individual replicates. Moreover, the task of hydrating and dehydrating hundreds of petri dishes, each open to the atmosphere and in darkness, would have been logistically very difficult. Insufficient seed precluded hydration and dehydration of replicate batches of seed. Pseudoreplication within each combination of hydration and dehydration treatments should be borne in mind when interpreting results. Hydration and dehydration treatments were combined with fire-related cues as set out below.

Sections I - IV Fire-related cues applied following hydration and dehydration

The details of experimental design and species used are given in Table 4.1 and are summarized below.

Table 4.1, section I a; Factorial combinations of heat shock (25˚C (= control) or 75°C for 5 minutes) and smoke (0 or 10 minutes) were applied to three populations.

Table 4.1, section I b; The heat shock range was extended to 125°C for two of these populations (one a sub-population) to test for lethal effects at higher temperature.

Table 4.1, section I c; The heat shock range was extended to 175°C for two other populations. Seed was hydrated for one day less than the time required for first germination in previous experiments. The two species were thus hydrated for comparable physiological times, but not for the same chronological time (8 cf 16 days). Due to time and seed limitations, seed could not be treated with both the same chronological time and comparable physiological time; the latter was utilised because it was considered to be of far greater biological consequence.

177 Table 4.1. Hydration and dehydration treatments prior to the application of fire-related germination cues to a number of study species

Sect- Species pop† Hydration Dehyd- Heat Smoke n seed Temp- Transfer ion (days) ration shock (mins) erature (post (days) (°C) analysis)

I a E. obtusifolia 2 0, 7, 14 0, 1, 5 25, 75 0, 10 4 25 25

3 0, 7, 14 0, 1, 5 25, 75 0, 10 4 25 25

K. capitata 2 0, 7, 14 0, 1, 5 25, 75 0, 10 4 25 25 15°C, light

I b E. obtusifolia 2a 16 0, 1, 5 125 0, 10 4 10 25

K. capitata 2 16 0, 1, 5 125 0, 10 3 10 25 15°C, light

I c E. obtusifolia 1a 0, 16 0, 1, 5 25-175 0, 10 3 20 25

K. ambigua 0, 8 0, 1, 5 25-175 0, 10 3 20 15 light

II 16 species 0 0 25 0 3 30 25 ‡ (15°C, or (see Table 4.3) 0 0 100 10 3 30 25 ‡ 25/35°C, 16 0 100 10! 3 30 25 ‡ light)

III G. melanocarpa 0, 26 0 25-100 0, 10 3 30 25

W. pungens 1 0, 19 0 25-100 0, 10 3 30 25

IV E. obtusifolia 1a 0, 0.38 0 100 0, 10 4 40 25

K. ambigua 0, 0.38 0 75 0, 10 4 40 15 light

V E. obtusifolia 2 259-300§ 1,14,28 25, 75 0, 10 1 § 15, 25

VI E. obtusifolia 1 7 0 100 0 6 50 25 14 0 25, 100 0 6 50 25

K. ambigua 7 7 25, 75 0 6 50 15 light

† population ! additional unsmoked treatment applied only to hydrated, 100°C heat shock treated K. parvifolia ‡ except E. m. var. rhombifolia; incubated at 15°C § see text and Table 4.4 Pre-treatment of 100°C, or 10 minutes smoke Pre-treatment of 75°C, or 10 minutes smoke

178 0.5

0.45

0.4

0.35

Seed weight (mg) 0.3

0.25 Epacris coriacea Kunzea ambigua Epacris obtusifolia 0.2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Hours hydration

Figure 4.1: Mean weight of Epacris coriacea, Kunzea ambigua and Epacris obtusifolia seeds during hydration in free water. Lines drawn by hand.

179 Table 4.1, section II; The response of wet and dry seeds of sixteen species to 100°C heat shock and smoke was assessed.

Table 4.1, section III; Two species with low or no germination were treated with 25˚C (= control) - 100°C heat shock and smoke (0 or 10 minutes).

Table 4.1, section IV; Two species were hydrated for a short period of time before treatment with a single level of heat shock and smoke (0 or 10 minutes).

Seeds that were dehydrated for 0 days were blot dried with tissue paper immediately before heat shock and smoke treatments. Replicates were double wrapped in aluminium foil, and germination was monitored under safe light conditions as previously described (Chapter 2). Final germination was assessed for all species when germination in darkness had tapered off, except for Kunzea capitata seeds treated with 125°C heat shock (Table 4.1, section I b). Final germination of K. capitata seeds treated with 125°C heat shock was assessed subsequent to transferral of seeds into a 15°C incubation temperature and an 8 hour diurnal cool white light regime. Seeds of all Kunzea species were transferred into light after dark incubation to assess their dormancy and viability status.

Data analysis

When seeds were treated with a range of durations of hydration and dehydration, heat shock and smoke (5 populations), then data were analysed using a four way ANOVA, with hydration, dehydration, heat shock and smoke as fixed factors (Table 4.1, section I a, c). When seeds were hydrated for 16 days, dehydrated for 0 or 5 days and treated with 125°C heat shock (2 species), then data were analysed using a two way ANOVA, with dehydration and smoke as fixed factors (Table 4.1, section I b). Germination of Kunzea capitata was analysed following transferral of seed into a 15°C incubation temperature and 8 hours diurnal cool white light. Only significant P-values are presented. When seeds were not hydrated and treated with 25˚C (= control) or 100°C heat shock or hydrated for 16 days and treated with 100°C heat shock (16 species), then data were analysed using a one way ANOVA, with the three (or four; Kunzea parvifolia) different treatments as fixed factors (Table 4.1, section II).

180 No Gahnia melanocarpa and negligible Woollsia pungens germination precluded data analysis (Table 4.1, section III) When seeds of two species were hydrated for 0 or 9 hours and treated with added heat shock and smoke (0 or 10 minutes), then data were analysed using a two way ANOVA, with hydration and smoke as fixed factors (Table 4.1, section IV). Only significant P-values are presented. Homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

Section V Application or re-application of fire-related cues applied following a long period of hydration.

Ungerminated seeds that had been hydrated for a long period of time provided an opportunity to investigate whether the response to fire-related cues was altered by long-term hydration. Ungerminated seeds of Epacris obtusifolia (population 2) that had been treated with combinations of 25 or 75°C heat shock and 0 or 10 minutes of aerosol smoke and incubated at a range of water potentials (Chapter 5, Fig 5.2.6) were re-treated. Seeds had been incubated for a total of between 259 and 300 days; broken up into between 138 and 151 days at a range of water potentials and then 121 days in free water. The ungerminated seeds were allowed to dehydrate for 1, 14 or 28 days and then treated with combinations of 25˚C (= control) or 75°C heat shock and 0 or 10 minutes of aerosol smoke (Table 4.1, section V; Fig 4.2; Table 4.4). Of major interest was whether long-term hydration altered the response of seeds to the germination cues. Seeds that had been within one container of the control treatment were dehydrated for 14 days. Seeds on the three filter papers within the one container were subsequently treated with smoke, added heat shock or the combination of added heat shock and smoke. Seeds on the three filter papers in the other container of the control treatment were dehydrated for 1, 14 or 28 days and treated with the combination of added heat shock and smoke (Fig 4.2; Table 4.4). Seeds that were initially treated with the combination of added heat shock and smoke were re-treated the same as for the initial control treatment (Fig 4.2; Table 4.4).

181 Also of major interest was whether the heat shock and/or smoke effects from the initial application were retained under certain conditions of incubation temperature and water potentials. Because this population had a strong germination response to the combination of added heat shock and smoke (Chapter 5, Fig 5.2.6), the retention of the original heat shock effect would be evident if germination was markedly higher when smoke was applied to previously heated seeds than when smoke was applied to previously unheated seeds. Similarly, the retention of the original smoke effect could be tested. Seeds on the three replicate filter papers within one container that had previously been treated with heat shock were dehydrated for 1, 14 or 28 days and treated with 10 minutes of aerosol smoke (Fig 4.2; Table 4.4). The three replicates from the other container that had previously been treated with heat shock were also dehydrated for 1, 14 or 28 days, and then treated with 75°C heat shock and 10 minutes of aerosol smoke. Similarly, three previously smoked replicates from the one container were dehydrated for 1, 14 or 28 days and then treated with heat shock only, and the three replicates from the other container were dehydrated for 1, 14 or 28 days and treated with both heat shock and smoke (Fig 4.2; Table 4.4). Seeds were briefly exposed to light during these treatments, then double wrapped in aluminium foil and returned to the same incubation temperature.

Data analysis

As neither the initial water potential regime (n = 5), nor the incubation temperature (n = 2) apparently affected the germination response to subsequent treatments, the average germination across re-treatments (n = 10) is shown (Table 4.4). Germination of the seeds dehydrated for 14 days and re-treated with smoke, added heat shock, or the combination of added heat shock and smoke was informally compared by visual inspection of the data across incubation temperatures and re-treatments. Germination of the seeds within a single re-treatment following dehydration for 1, 14 or 28 days was informally compared by visual inspection of the data across incubation temperatures and durations of dehydration. Germination of the seeds within a single duration of dehydration following different re-treatments was informally compared by visual inspection of the data across incubation temperatures and re-treatments.

182 Fig 4.2. Incubation temperatures, water potentials, and fire-related germination cues initially applied to Epacris obtusifolia (population 2) seeds (Chapter 5), and subsequent dehydration and fire-related cues applied within the current experiment to the previously ungerminated seeds.

Incubation temp 15 25

Water potential 0 -0.3 -0.6 -0.9 0/-0.9

Heat shock 25 75

Smoke 0 10 0 10

Free water (Chapter 5)

Re-treatment (Current experiment)

Dehydration 14 1 14 28 1 14 28 1 14 28 1 14 28 14

Fire-related cues H H&S S H&S H H&S S H&S H&S H H&S S

H = heat shock, S = smoke, H&S = heat shock and smoke 183 Section VI Heat shock applied during the germination process, subsequent to fire-related cues.

To investigate whether heat shock tolerance was reduced in seeds that were stimulated to germinate by fire-related cues, a second heat shock was applied to two species. Kunzea ambigua seeds were treated with 75°C heat shock for 5 minutes or with 10 minutes of aerosol smoke. Seeds were hydrated for 7 days and then dehydrated for 7 days (Table 4.1, section VI). Seeds were dehydrated so the effect of the germination process on heat shock tolerance would not be confounded by the known reduction in heat shock tolerance due to hydration. A second heat shock of 25˚C (= control) or 75°C was then applied for 5 minutes duration. Six replicates of 50 seeds were treated independently and incubated at 15°C (Table 4.1, section VI). Epacris obtusifolia (population 1) seeds were treated with 5 minutes of 100°C heat shock or 10 minutes of aerosol smoke. Seeds were hydrated for 7 days and then blot dried and treated with 5 minutes of 100°C heat shock, or hydrated for 14 days and then blot dried and treated with 5 minutes of 25°C (procedural control) or 100°C heat shock. Six replicates of 50 seeds were treated independently and incubated at 25°C (Table 4.1, section VI). Replicates of both species were double wrapped in aluminium foil, and germination was monitored under safe light conditions as previously described (Chapter 2).

Transferral into light

Kunzea ambigua seeds were transferred into light after 68 days.

Data analysis Data were analysed using a one way ANOVA, with the second treatment as a fixed factor. Homogeneity of variances was assessed using Cochran’s Test. Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

184 4.3 Results

Section I a Fire-related cues applied following periods of hydration and dehydration.

The aim was to assess whether the combination of fire-related cues and hydration status would affect germination, and whether there was a relationship between a species habitat and its response to the combination of fire-related cues and hydration.

Seeds of the wet habitat species Epacris obtusifolia population 2 changed their germination response depending on their hydration status at the time of the ‘fire’ (hydration x heat shock x smoke interaction significant, Table 4.2; Fig 4.3). Most importantly, unsmoked seeds from this population responded positively to 75°C heat shock whilst hydrated. Germination of unsmoked seeds within the added heat shock treatment increased within each level of increasing hydration. Added heat shock only increased germination of air-dry seeds when they were smoked, but increased germination of both smoked and unsmoked seeds when seeds had been hydrated. The response of E. obtusifolia population 3 and Kunzea capitata population 2 to the combinations of treatments was variable; germination was not consistently affected by prior hydration and dehydration treatments (Table 4.2; Figs 4.4 & 5). Smoke increased germination regardless of other treatments, but the effect of heat shock was variable. Viability of K. capitata was not affected by the application of 75°C heat shock to hydrated seeds, as germination increased when seeds were transferred into a 15°C incubation temperature, and to a uniformly high level when transferred into light (data not shown).

185 Table 4.2. Effects of hydration, dehydration, heat shock, smoke, and interactions between these factors on % germination of five study populations. ANOVA P-values are shown.

Source E. E. K. E. K. obtusifolia obtusifolia† ambigua obtusifolia capitata population 2 3 2 1a df df df Hydration (= Hyd) 2 <0.0001 0.1028 0.0011 1 <0.0001 1 <0.0001

Dehydration (= DHyd) 2 0.2395 0.2151 0.1324 2 0.5661 2 <0.0001

Heat (= H) 1 <0.0001 0.9530 0.5006 2 <0.0001 3 <0.0001

Smoke (= S) 1 <0.0001 <0.0001 <0.0001 1 0.7109 1 <0.0001

Hyd x DHyd 4 0.4614 0.0001 0.2170 2 0.0264 2 <0.0001

Hyd x H 2 0.8047 0.9468 0.0165 2 <0.0001 3 <0.0001

Hyd x S 2 0.0096 0.0070 0.0421 1 0.9114 1 0.0198

DHyd x H 2 0.5114 0.4352 0.0745 4 0.0032 6 <0.0001

DHyd x S 2 0.9283 0.4792 0.6790 2 0.3114 2 0.1256

H x S 1 0.6584 0.1357 0.0195 2 0.0004 3 <0.0001

Hyd x DHyd x H 4 0.7943 0.4375 0.3842 4 0.1509 6 <0.0001

Hyd x DHyd x S 4 0.2544 0.7664 0.4269 2 0.3486 2 0.0034

Hyd x H x S 2 0.0376 0.0253 0.3680 2 0.9503 3 0.0032

DHyd x H x S 2 0.0560 0.2743 0.4113 4 0.1344 6 0.2859

Hyd x DHyd x H x S 4 0.4390 0.9886 0.0393 4 0.6871 6 0.3196

residual 108 72 96

† transformed data

186

a) 0 days dehydration d) g)

70 70 70

60 60 60

50 50 50

40 40 40

30 30 30

20 20 20

10 10 10

0 0 0 b) 1 day dehydration e) h)

70 70 70

) 60 60 60 - SE

/ 50 50 50 + ( 40 40 40

30 30 30

20 20 20 ermination

g 10 10 10 % 0 0 0 c) 5 days dehydration f) i)

70 70 70

60 60 60

50 50 50

40 40 40

30 30 30

20 20 20

10 10 10

0 0 0 unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm 25°C 75°C 25°C 75°C 25°C 75°C

0 days hydration 7 days hydration 14 days hydration

Figure 4.3: Mean final germination of Epacris obtusifolia (population 2) seeds. Seeds were hydrated for a - c) 0 days, d - f) 7 days, g - i) 14 days, then dehydrated for a, d, g) 0, b, e, h) 1, or c, f, i) 5 days, then treated with heat shock (25°C or 75°C) and 0 (2), or 10 minutes () smoke. Germination plotted against heat shock and smoke treatments. Bars = S. E.s

187 a) 0 days dehydration d) g)

18 18 18

16 16 16

14 14 14

12 12 12

10 10 10

8 8 8

6 6 6

4 4 4

2 2 2

0 0 0 b) 1 day dehydration e) h)

18 18 18

16 16 16

14 14 14

12 12 12

10 10 10

8 8 8

6 6 6

4 4 4

2 2 2 % germination (+/- SE) % germination

0 0 0 c) 5 days dehydration f) i)

18 18 18

16 16 16

14 14 14

12 12 12

10 10 10

8 8 8

6 6 6

4 4 4

2 2 2

0 0 0 unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm 25°C 75°C 25°C 75°C 25°C 75°C

0 days hydration 7 days hydration 14 days hydration

Figure 4.4: Mean final germination of Epacris obtusifolia (population 3) seeds. Seeds were hydrated for a - c) 0 days, d - f) 7 days, g - i) 14 days, then dehydrated for a, d, g) 0, b, e, h) 1, or c, f, i) 5 days, then treated with heat shock (25°C or 75°C) and 0 (2), or 10 minutes () smoke. Germination plotted against heat shock and smoke treatments. Bars = S. E. s

188 a) 0 days dehydration d) g)

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0 0 0 unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm

25°C 75°C 25°C 75°C 25°C 75°C

0 days hydration 7 days hydration 14 days hydration

Figure 4.5: Mean final germination of Kunzea capitata (population 2) seeds. Seeds were hydrated for a - c) 0 days, d - f) 7 days, g - i) 14 days, then dehydrated for a, d, g) 0, b, e, h) 1, or c, f, i) 5 days, then treated with heat shock (25°C or 75°C) and 0 (2), or 10 minutes () smoke. Germination plotted against heat shock and smoke treatments. Bars = S. E. s

189 Section I b A contrast between the wet habitat Epacris obtusifolia population 2a and the moist habitat Kunzea capitata (population 2) tolerance of heat shock whilst hydrated became apparent when higher heat shock was applied. The 125°C heat shock did not affect germination of Epacris obtusifolia differently when applied to hydrated or dehydrated seed (Fig 4.6a; analysis not shown). In contrast, K. capitata seeds that were hydrated when treated did not germinate (Fig 4.6d). The difference between germination of K. capitata seeds that were hydrated or air-dry when treated became more pronounced after seeds were transferred into a 15°C incubation temperature, and then into light (Fig 4.6d-f). The 125°C heat shock appeared to kill hydrated seed of K. capitata because there was no further germination when seeds within the treatment were transferred into conditions that greatly increased germination in all other experiments. Heat shock tolerance was restored following dehydration for 1 or 5 days, as seeds germinated within the 25°C incubation temperature (Fig 4.6). Germination following transferral into 15°C and light was greater within the 1 and 5 day dehydration treatments (F 2, 12 = 18.70, P = 0.0002; Fig 4.5). In contrast, 175°C heat shock applied to seeds that had not been hydrated did not reduce germination of K. capitata (population 1), and seeds germinated following 200°C dry heat shock (Chapter 2, Fig 2.11).

190 Epacris obtusifolia Kunzea capitata

a) 0 days dehydration d) 0 days dehydration

12 80

70 10

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0 0 b) 1 day dehydration e) 1 day dehydration

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0 0 c) 5 days dehydration f) 5 days dehydration

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6 40

30 4

20

2 10

0 0

125°C heat shock 125°C heat shock 125°C heat shock 125°C heat shock 0 minutes smoke 10 minutes smoke 0 minutes smoke 10 minutes smoke

Figure 4.6. Mean final germination of a - c) Epacris obtusifolia (population 2a), d - f) Kunzea capitata (population 2) seeds hydrated for 16 days, then dehydrated for a, d) 0, b, e) 1 or c, f) 5 days, and then treated 125°C heat shock and smoke (0 or 10 minutes). Seeds were incubated at 25°C in darkness (), then K. capitata seeds were transferred into 15°C (), then into light (2). Germination plotted against dehydration and smoke treatments. Note change of scale on Y-axis from left hand side to right hand side figures. Bars = S. E.

191 Section I c Markedly different results were observed in 1) the upper range of heat shock tolerance whilst hydrated and 2) the range of heat shock tolerance whilst air-dry, of the wet and dry habitat species compared. Pre-hydration increased germination of Epacris obtusifolia population 1a within the 125°C heat shock treatments (Table 4.2; Fig 4.7a-c cf d-f). As observed before, heat shock applied to hydrated seeds increased germination of E. obtusifolia (Table 4.2; Figs 4.3 & 7d). The remarkable tolerance of E. obtusifolia to 125°C heat shock whilst hydrated, observed previously (Fig 4.6), was again evident (Fig 4.7d). The heat shock tolerance of hydrated E. obtusifolia populations 1a and 2a seeds contrasts markedly with the lack of tolerance of the dry habitat species Kunzea ambigua to high levels of heat shock whilst hydrated (Table 4.2; Fig 4.8). Although Kunzea ambigua seeds that had not been hydrated could tolerate heat shock up to 200°C (Fig 4.8; Chapter 2, Fig 2.11), seeds that were hydrated when treated with 125°C did not germinate (Fig 4.8d). Heat shock of 125°C or more probably killed the hydrated Kunzea ambigua seeds because, in contrast with all other experiments, there was no further germination when seeds within the treatment were transferred into light (data not shown). Tolerance of high levels of heat shock was gradually restored following dehydration of Kunzea ambigua seeds. Following one day dehydration, tolerance of 125°C heat shock was fully restored and tolerance of 175°C heat shock was partially restored (Fig 4.8e). Tolerance of K. ambigua seed to 175°C heat shock was fully restored after five days dehydration (Fig 4.8f). In contrast to the increasing heat shock tolerance with dehydration of the Kunzea species (Figs 4.6 & 8), there was a trend of decreasing germination in response to heat shock or smoke with dehydration of E. obtusifolia (population 2) (Fig 4.3).

Summary of Section I

The combination of fire-related cues and hydration status affected germination of most species, and the effect of the combination of these factors was related to the species habitat for the small number of species investigated. The null hypothesis that the combination of fire- related cues and hydration status would not affect germination was rejected. The null hypothesis that there was no relationship between a species habitat and its response to the combination of fire-related cues and hydration was preliminarily rejected.

192 a) 100 0 days dehydration d) 100 0 days dehydration 90 90

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10 10 % germination (+/- SE) % germination 0 0 c) 5 days dehydration f) 5 days dehydration

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0 0

unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm 25°C 75°C 125°C 175°C 25°C 75°C 125°C 175°C 0 days hydration 16 days hydration

Figure 4.7: Mean final germination of Epacris obtusifolia (population 1a) seeds hydrated for 0 days, then dehydrated for a) 0 days, b) 1 day, c) 5 days, or hydrated for 16 days, then dehydrated for d) 0 days, e) 1 day, f) 5 days, then treated with heat shock (25°C, 75°C, 125°C or 175°C) and 0 (2), or 10 minutes () smoke. Germination plotted against heat shock and smoke treatments. Bars = S. E. s

193 a) 0 days dehydration d) 0 days dehydration

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unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm unsm sm 25°C 75°C 125°C 175°C 25°C 75°C 125°C 175°C

0 days hydration 8 days hydration

Figure 4.8: Mean final germination of Kunzea ambigua seeds hydrated for 0 days, then dehydrated for a) 0 days, b) 1 day, c) 5 days, or hydrated for 8 days, then dehydrated for d)

0 days, e) 1 day, f) 5 days, then treated with heat shock (25°C, 75°C, 125°C or 175°C) and 0 (2), or 10 minutes () smoke. Germination plotted against heat shock and smoke treatments. Bars = S. E. s

194

Section II

The aim was to assess whether the combination of fire-related cues and hydration status would affect germination, and whether there was a relationship between a species habitat and its response to the combination of fire-related cues and hydration

Another wet habitat Epacris species, E. longifolia responded positively to the interaction between heat shock (100°C) and hydration (Table 4.3; Fig 4.9). Surprisingly, germination of none of the 16 populations investigated was decreased by the ‘wet’ relative to the ‘dry fire’ treatment (Table 4.3; Figs 4.9 & 10). Added heat shock treatments reduced germination of Dracophyllum secundum (Table 4.3; Fig 4.9), and marginally reduced germination of Baeckea imbricata (Table 4.3; Fig 4.9). Also, Epacris muelleri seeds treated with added heat shock did not germinate when transferred into light (Fig 4.9). Only after seeds of E. paludosa and Kunzea parvifolia were transferred into light was a possible negative effect of the wet heat compared with dry heat apparent (Figs 4.9 & 10). Cross experiment comparisons reveal that most populations investigated here had substantially reduced germination, consistent with a reduction in viability or an increase in dormancy. If populations had entered into secondary dormancy, then the interaction between hydration and fire does not reduce dormancy.

Summary of Section II

The fire-related cues increased germination of hydrated seed of a wet habitat species, therefore the null hypotheses that the combination of fire-related cues and hydration status will not affect germination, and that there is no relationship between a species habitat and its response the combination of fire-related cues and hydration is partly, preliminarily rejected. Because of the inferred secondary dormancy, the seed that was used poorly represented other species, and therefore the experiments were not a fair test of the null hypotheses.

195 Table 4.3. Effects of control treatment, 100°C heat shock and smoke applied to air-dry seed, or 100°C heat shock and smoke applied to seed hydrated for 16 days on % germination of 16 study populations. One-way ANOVA P-values are shown.

Source population Treatment

E. longifolia† 0.0276

D. secundum 1 0.0275

A. helianthii No germination

B. diosmifolia No germination

B. imbricata 2 0.0550

4 0.4616

B. linifolia 1 No germination

2 Negligible germination

E. m. var. rhombifolia‡ No germination

E. muelleri 0.5346

E. paludosa 1 0.3924

E. pulchella 0.5491

G. sieberiana 1 Negligible germination

2 No germination

K. parvifolia § Negligible germination

W. pungens 1 No germination

† transformed data ‡ incubated at 15°C in darkness, then light § an additional treatment of 100°C heat shock and no smoke was applied to K. parvifolia seeds that had been hydrated for 16 days.

196 a) 25 Epacris longifolia b) 25 Epacris pulchella

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25°C heat shock 100°C heat shock 100°C heat shock 25°C heat shock 100°C heat shock 100°C heat shock 0 minutes smoke 10 minutes smoke 10 minutes smoke 0 minutes smoke 10 minutes smoke 10 minutes smoke

Not hydrated Hydrated Not hydrated Hydrated when treated when treated when treated when treated

Figure 4.9: Mean final germination of a) Epacris longifolia, b) E. pulchella, c) Baeckea imbricata (population 2), d) Dracophyllum secundum (population 1), e) E. paludosa, f) E. muelleri plotted against control, or 100°C heat shock and 10 minutes aerosol smoke applied to air dry seeds, or 100°C heat shock and 10 minutes aerosol smoke applied to seeds following 16 days hydration treatments. Seeds were incubated at 25°C in darkness (), then E. paludosa and E. muelleri seeds were transferred into light (2). Bars = S. E.s

197

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Not hydrated Hydrated when treated when treated

Figure 4.10: Mean final germination of Kunzea parvifolia plotted against control treatment, or 100°C heat shock and 10 minutes aerosol smoke applied to air dry seeds, or 100°C heat shock and 0 or 10 minutes aerosol smoke applied to seeds following 16 days hydration. Seeds were incubated at 25°C in darkness (), then transferred into15°C (), then transferred into light (2). Bars = S. E.s

198

Section III

The null hypothesis was that the combination of fire-related cues and hydration status would not affect germination of dormant seeds.

The absence of germination in Gahnia melanocarpa and the low level in Woollsia pungens (c. 2%) also indicate that prior hydration does not prime dormant seeds to respond to fire. The null hypothesis that the combination of fire-related cues and hydration status would not affect germination of dormant seeds was accepted for these two species.

Section IV Fire-related cues applied following a brief period of hydration.

The aim was to assess whether the germination response to fire-related cues of air-dry seed and of fully hydrated seed that had not undergone major physiological changes would differ, and whether there was a relationship between a species habitat and its response to the combination of fire-related cues and hydration.

The response of seeds to fire-related cues following a period of time that probably only just allowed full hydration was very similar to the response of seeds hydrated for many days. Nine hours hydration prior to heat shock and smoke application did not significantly affect germination of Epacris obtusifolia (population 1a) (Fig 4.11), but reduced germination of Kunzea ambigua seeds (F 1, 12 = 13.47, P = 0.0032; Fig 4.11). Thus, heat shock tolerance of hydrated seed increased with increasing wetness of the habitat of three species. Germination of the dry habitat Kunzea ambigua was reduced by 75°C heat shock applied to hydrated seed, 125°C heat shock killed hydrated seed of the moist habitat K. capitata, and germination of the wet habitat Epacris obtusifolia was increased when 125°C heat shock was applied to hydrated compared with dry seed.

Smoke increased germination of K. ambigua across prior hydration treatments (F 1, 12 = 49.08, P < 0.0001; Fig 4.11).

199 Transferral into light

Germination of Kunzea ambigua increased to a uniformly high level when seeds were transferred into light, so although heat shock and smoke following nine hours of hydration reduced germination, the treatment did not reduce viability (data not shown).

Summary of Section IV

Germination of a dry habitat species was reduced by the combination of heat shock and short-term hydration, whilst the effect of smoke was positive regardless of hydration status. In contrast, germination of the wet habitat species was not affected by the combination of fire-related cues and hydration. The response to the fire-related cues was similar following short-term and long-term hydration. The null hypothesis that the germination response to fire- related cues of air-dry seed and of fully hydrated seed that had not undergone major physiological changes would not be different was rejected. The null hypothesis that there would be no relationship between a species habitat and its response the combination of fire- related cues and hydration was preliminarily rejected.

200

a) Epacris obtusifolia; 100°C heat shock 70

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Smoke: 0 minutes 10 minutes 0 minutes 10 minutes

Not Hydrated Hydrated

Figure 4.11: Mean final germination of a) Epacris obtusifolia (population 1a), b) Kunzea ambigua seeds hydrated for 0 or 9 hours, then treated with smoke (0 or 10 minutes) and heat shock;

a) 100°C, then incubated at 25°C in darkness (). b) 75°C, then incubated at 15°C in darkness (). Germination plotted against hydration, heat shock and smoke treatments. Bars = S. E.s

201 Section V Application or re-application of fire-related cues applied following a long period of hydration.

The aim was to assess whether the germination response to fire-related cues of air-dry seed and of long-term hydrated seed would differ, and whether the germination response of seed to the combination of heat shock and smoke would differ if these cues were applied to seed that had not been hydrated or if there was a long period of hydration between the applications of the two cues.

The long period of hydration markedly changed the response of Epacris obtusifolia (population 2) to the fire-related germination cues. Whereas the different treatments applied to laboratory stored seeds resulted in large differences in germination (Fig 4.3a - c; Chapter 5, Fig 5.2.6), the different treatments applied to the long-hydrated seeds of the same population (i.e. not previously treated with heat shock or smoke, Table 4.4, Initial treatment = Control) resulted in uniformly high levels of germination. Germination of seeds within the initial heat shock or smoke alone treatments was also uniformly high, and germination of seeds initially within the heat shock plus smoke treatment was lower, almost certainly because most seeds with lower levels of dormancy had already germinated following the initial treatment (Chapter 5, Fig 5.2.6). Whereas the response of the population to the fire-related germination cues rapidly changed in response to short-term hydration dynamics (Fig 4.3d - i), one day or one month dehydration before treatment did not affect the response of the long-hydrated population to the treatment (Table 4.4). Clearly, a change in seed physiology occurred during long-term hydration. Whether the initial effects of heat shock or smoke were retained could not be assessed because, in contrast to laboratory-stored seed (Fig 4.3a - c; Chapter 5, Fig 5.2.6), these cues applied singly to long-hydrated seed produced similar levels of germination as did the combination of heat shock and smoke (Table 4.4). Germination was also fairly uniform across incubation temperatures, and total germination i.e. following the combination of first (Chapter 5) and second treatments was fairly uniform across all factors.

202 Table 4.4. Percentage germination of Epacris obtusifolia (population 2) seeds that had been dehydrated for various periods of time and treated with fire- related germination cues, having been initially treated with fire- related germination cue and incubated for a long period of time within various water potentials. Germination averaged across previous water potential treatments.

Initial treatment Re- treatment Average % germination Duration of dehydration Cue(s) (days) Control 14 Heat shock 55 Smoke 61 Heat shock x Smoke 58

1 Heat shock x Smoke 59 14 57 28 60

Smoke 1 Heat shock 35 14 41 28 40

1 Heat shock x Smoke 41 14 43 28 43

Heat shock 1 Smoke 48 14 50 28 53

1 Heat shock x Smoke 53 14 50 28 56

Heat shock x Smoke 14 Heat shock 9 Smoke 9 Heat shock x Smoke 12

1 Heat shock x Smoke 11 14 8 28 8

203 Summary of Section V

The germination response of seed to fire-related cues was greatly increased following long-term hydration, and the null hypothesis that the germination response to fire-related cues of air-dry seed and of long-term hydrated seed was not different was rejected. Because the germination response of seed to fire-related cues was greatly increased following long-term hydration, it was not possible to assess whether the effects of heat shock or smoke were retained in seed that was hydrated for a long period of time. The null hypothesis that the germination response of seed to the combination of heat shock and smoke was not different if these cues are applied to seed that had not been hydrated or if there was a long period of hydration between the applications of the two cues was thus not testable.

Section VI Heat shock applied during the germination process, subsequent to fire-related cues.

The aim was to assess whether the germination response of seed that is progressing toward post-fire germination would be affected by a second application of heat shock, and whether there was a relationship between a species habitat and its response to the second application of heat shock

Seeds of Epacris obtusifolia (population 1a) and Kunzea ambigua that had progressed toward ‘post-fire’ germination were not less tolerant of the applied heat shock (data not shown).

Transferral into light

Heat shock during the germination process did not reduced viability of Kunzea ambigua, as germination increased to a uniformly high level when seeds were transferred into light (data not shown).

Summary of Section VI

The second heat shock did not affect germination of seed that was progressing toward post-fire germination, therefore the null hypothesis that the germination response of seed that

204 was progressing toward post-fire germination would not be affected by a second application of heat shock was rejected. The null hypothesis that there would be no relationship between a species habitat and its response to the second application of heat shock was preliminarily accepted.

4.4 Discussion

Species with water-permeable seed that form soil seedbanks in fire-prone wet habitat occasionally experience high levels of heat shock whilst hydrated. Water-permeable soil seedbanks of dry habitat species are unlikely to experience high levels of heat shock whilst hydrated. Differential tolerance of this stress was consistent with species habitat and may explain habitat segregation, although generalisation is limited due to small number of species used.

Interaction between seed hydration and fire-related germination cues

The germination response to the combination of hydration of Epacris obtusifolia and E. longifolia seed and a high level of heat shock was positive; this is the first positive response recorded and is highly significant because all previous responses recorded since Just (1877) have been highly negative. Highly negative responses to the combination of hydration and heat shock have been recorded for germination of bacterial spores (Murrell & Scott 1957; Dunn et al. 1985; Setlow & Setlow 1995), fungal cysts (Fahey et al. 1978; Dunn et al. 1985) and seeds (Robbins & Petsch 1932; Beadle 1940; Sweeny 1956; Fahey et al. 1980; Wolkers et al. 1998). An increase of air-dry seed moisture content of only 2% greatly reduced heat shock tolerance of wheat seed (Ghaley & Taylor 1982). Eukaryotic organisms generally tolerate less than 80°C for 5 minutes whilst hydrated, although less than 10% of fungal spores still germinated after fully hydrated spores were treated with 100°C for 5 minutes (Fahey et al. 1978). The response of a number of species to heat shock whilst hydrated has extended the known upper limit of tolerance for eukaryotic organisms. Germination was affected by interactions between smoke, hydration and dehydration in ways that may indicate the mechanisms involved. Germination of Epacris obtusifolia (population 2) was increased by smoke following the longer duration (14 days) of hydration,

205 and decreased with subsequent dehydration. The physiological changes associated with 14 days hydration possibly allowed smoke to have a greater effect. In contrast, when seeds of this population had been hydrated for a long period of time (259 days or more), the seeds were also more responsive to smoke, but this effect did not disappear following dehydration. Notably, when seed is primed (prolonged Stage II hydration due to low water potential of the medium), then physiological changes associated with Stage II are retained for months or years following a return to an air-dry state (Hanson 1973; Dearman et al. 1987; Bray 1995). Smoke probably interacts with physiological properties of seed that are altered in the short-term by short-term hydration, and that are altered in the long-term by long-term hydration. The combination of smoke and long-term soil storage was required for germination of species forming soil seedbanks in the chaparral, as seed that was dry-stored for a short or a long period of time did not germinate with or without smoke (Keeley & Fotheringham 1998a). Germination of seed that has resided in the soil for a long period of time may be more responsive to fire than recently shed seed. The value in recently shed seed remaining unresponsive and so forming a post-fire residual seedbank may be because such seed has the greatest potential longevity. It is not known whether the physiological state of seeds following the durations of dark incubation that were imposed was comparable to the state of seeds following long-term soil storage, however, both a short period of dark incubation on filter paper and long incubation in soil have been reported to produce the same increased germination response to light in the two heathland species investigated (Pons 1989). The response of Epacris obtusifolia population 2 to fire-related germination cues was dependent on the duration of hydration. At most, two durations of hydration were investigated, and most species were only investigated using a single duration of hydration. Also, the level of dormancy of many populations had apparently increased during laboratory storage prior to the experiments. Even though many species may have been hydrated for a sub-optimal period of time and were also probably entering a state of dormancy, heat shock tolerance of hydrated seeds was greater than found in previous investigations, for Epacris longifolia, E. obtusifolia and E. pulchella, and for E. paludosa and Kunzea parvifolia when transferred into more favourable conditions. The high levels of germination of Epacris obtusifolia following heat shock of 125°C whilst imbibed is remarkable, given that germination of a species from the most heat-resistant life-forms on earth, bacterial endospores (Nicholson et al. 2000), was reduced by 1 log per 30 minutes when subject to 90°C wet heat, but 120°C of dry heat was required to achieve this rate of killing (Setlow & Setlow 1995). Endospores achieve this extreme protection against

206 inactivation by heat by maintaining a dehydrated central protoplast, and when this control is lost, then sensitivity to heat increases by four to five orders of magnitude (Gould & Dring 1975). Seed of each population was hydrated and dehydrated as a batch, therefore there was no test of whether there was anything unusual within a particular batch x hydration and dehydration treatment. Nothing unusual was apparent within the experiments, and directional trends within the data rather than unexplained variation or outliers are consistent with responses due to the experimental treatments. Repeated runs of each experiment will be necessary in future work to overcome the limitations in design of the current study.

Hydration and heat shock proteins

The capacity of seed to withstand the high heat shock associated with the passage of fire has been attributed to unknown physiological rather than physical properties (Bell & Williams 1998). However, the physiological mechanism(s) whereby heat shock promotes germination have been poorly addressed. Heat shock proteins probably conferred the heat shock tolerance of the seeds in the current study, and consequently enhance seed survival during the passage of fire, although investigation is required (Chapter 7). If heat shock proteins did confer the heat shock tolerance of the seeds in the current study, then they are not degraded following the process of germination initiated by the fire-related germination cues. Heat shock proteins and the related late-embryogenesis-abundant proteins are synthesised as a normal part of seed development in many species (Helm & Abernethy 1990; Almoguera et al. 1993; Hernandez & Vierling 1993; Coca et al. 1994; DeRoucher & Vierling 1994; zur Nieden et al. 1995; Wehmeyer et al. 1996). Heat shock proteins confer protection from heat stress (DeRoucher & Vierling 1994), desiccation (Coca et al. 1994; Wehmeyer et al. 1996; Wolkers et al. 1998; Sales et al. 2000), or both stresses (Buitink et al. 2002), possibly due to their protection of the cellular membrane (Li et al. 1982; Sales et al. 2000) and / or cytoskeletal structure (Neumann & Nover 1991). It follows that heat shock proteins may have a role in the frequently observed promotion of germination by heat shock.

Heat shock protein dynamics and habitat

Heat shock proteins may be stored with their active sites hidden inside filaments and released upon heat shock (Trent et al. 1998), or rapidly synthesised in response to heat shock.

207 Dry habitat species may not store heat shock proteins or they may have low capacity to synthesise heat shock proteins whilst they are hydrated. In contrast, results for wet habitat species are explicable if they store heat shock proteins and/or they are capable of synthesising heat shock proteins whilst they are hydrated. If heat shock proteins conferred heat shock tolerance, then their persistence in seeds of wet habitat species is explicable, as high heat shock due to fire can occur in wet habitat when soil moisture, and thus seed hydration is at any level. Heat shock proteins have been found to persist in seeds during imbibition (Coca et al. 1994; DeRoucher & Vierling 1994; zur Nieden et al. 1995; Wehmeyer et al. 1996), and the involvement of small heat shock proteins in long- term thermotolerance has been supported (Al-Niemi & Stout 2002). Maintainance of heat shock proteins has a strong cost-benefit trade-off (Krebs & Loeschcke 1994). Whilst wet habitat species may incur the cost of continually maintaining heat shock proteins (Feder et al. 1992; Krebs & Loeschcke 1994; Krebs & Feder 1997; Silbermann & Tatar 2000), they obtain the benefits that exist at the extreme ends of environmental regimes (Levins 1969; Leroi et al. 1994; Hoffmann 1995). The capacity of seeds to withstand high heat shock whilst imbibed allows wet habitat species to persist in wet environments that have higher productivity (Kruger & Bigalke 1984; Keith 1991, unpublished; Keith & Myerscough 1993; Benwell 1998) and probably higher reproductive capacity (e.g. Bradstock & O’Connell 1988), at least until resources are reduced due to competition (Keith & Bradstock 1994; Keith 1995). In contrast, dry habitats would only experience low intensity fire when the dry habitat is wet. Thus, low concentrations of heat shock proteins are probably sufficient whilst the seeds of dry habitat species are hydrated. Moderate heat shock of hydrated Kunzea seeds reduced germination, but germination in the field would be expected due to the promotive effect of smoke. Also, sufficient heat shock protein was probably present such that viability was not reduced, thus preservation of a soil seedbank in dry habitat would be expected following fire when the soil is wet. Heat shock proteins were induced by desiccation of plants (Almoguera et al. 1993; Alamillo et al. 1995), and fly pupae (Tammariello & Rinehart 1999), and reductions in the levels of heat shock proteins during imbibition have been reported (DeRoucher & Vierling 1994; zur Nieden et al. 1995). A reduction in the concentration of heat shock proteins during the hydration period probably allows more rapid metabolism to occur. The metabolic activity of non-germinating seeds (Villiers 1971, 1974; Villiers & Edgecumbe 1975) of dry habitat species probably needs to be more rapid, because they have less time in a hydrated state than do the seeds of wet habitat species.

208 Heat shock tolerance and habitat segregation

Figure 4.11. Segregation of wet and dry habitat species due to differential heat shock tolerance whilst hydrated

Hot fires that occur Wet when soil moisture is habi higher tat sp ecies high kill the seeds of dry habitat species D r y h Heat shock ab itat tolerance s pec ies Concentration of heat shock proteins? lower

lower higher

Seed hydration (= soil moisture)

The different tolerances of high temperature heat shock whilst imbibed could account for habitat segregation of species with water permeable seed coats that form soil seedbanks, as represented in Figure 4.11. However, the generality of this model is limited by the low number of species and populations examined. Species composition forms a continuum from wet to (periodically) moist to dry habitat (Pidgeon 1938). Seedlings of dry heath species could establish in wet heath when their seeds were buried (Clarke et al. 1996), but the species did not establish seedbanks in the wet habitat (Myerscough et al. 1996), possibly because the seeds are killed by the heat shock of fire when they are hydrated. Wet heath species form seedbanks in dry heath (Myerscough et al. 1996), however their seedlings are unable to survive in dry heath, apparently due to the drier conditions (Clarke et al. 1996; Myerscough et al. 1996). Although the Kunzea species require greater water availability for germination than the wet habitat species (Chapter 5, higher median base water potentials), seedling drought tolerance is not predictable from their germination response to limited water availability (Young et al. 1968; McWilliam & Phillips 1971; Watt 1974; Oomes & Elberse 1976). In a water-limiting environment, the capacity of a seedling to survive is more important than the number that germinate, in determining final recruitment (Thomas & Davis 1989). The

209 seedlings of the dry habitat species probably have the capacity to survive low water availability.

Contrasting heat shock tolerance requirements in wet habitat across fire-regenerative strategies

Notably, serotinous species can inhabit wet habitat without incurring the costs entailed with physiological tolerance of seed to heat shock whilst imbibed because the seedbank is aerial and protected from lethal heating by woody fruiting bodies (Ashton 1986; Bradstock et al. 1994; Judd 1994). Also, resprouting species could potentially inhabit wet habitat without having seed that is physiologically tolerant of heat shock whilst imbibed, because vegetative recovery would allow them to persist through fires that occur whilst their seed is imbibed. Consequently, resprouting species would have more opportunity to evolve seed that is physiologically tolerant of heat shock whilst imbibed, because any single seed produced in the wet habitat with this property will be in a location where it has a selective advantage. In comparison, a seeder species without heat shock tolerance whilst imbibed would require the combination of producing seed with this property, and transport of seed into wet habitat (in either order). The ability to inhabit wet habitat without having seed that is physiologically tolerant of heat shock whilst imbibed, and the greater opportunity to evolve this property may be factors contributing to the higher ratio of resprouter to obligate seeder species in wet habitat. Interestingly, whilst the continual presence of high concentrations of heat shock proteins represents a simpler state than altered concentrations in response to stress, the latter state is universal to numerous kinds of stresses in all organisms investigated (Sorensen et al. 2003), hence the simpler state is probably a derived state.

210 Chapter 5. Effects of post-fire water

5.1 Introduction

Overview

The question of whether fire-related cues affect the hydration requirements for germination of species forming soil seedbanks in the fire-prone Sydney region is investigated in this section. Also, the effect of soil texture on factors affecting water availability to seed of the study species is explored, and the effects that variability in water availability at the seed size scale may have on both post-fire germination and the retention of a residual soil seedbank are discussed.

Post-fire moisture

Notwithstanding the importance of other environmental factors in the control of seed germination, the germination of all seeds is ultimately dependent on the availability of water (Koller & Hadas 1982; Probert 1992). A suitable combination of temperature and water is probably the most crucial factor in determining germination (Mayer & Poljakoff-Mayber 1989), thus it is appropriate to investigate the interaction between temperature and post-fire moisture availability. It has been found that fire-related germination cues increased the range of ambient temperatures over which seeds would germinate (Chapter 3), and it is possible that these cues also increase the capacity of seeds to germinate at lower levels of moisture availability. The mechanism whereby fire-related germination cues influence the dormancy status of seeds, and hence their capacity to germinate at reduced water potentials could be investigated using empirical experimentation in the laboratory and/or field, or using hydrotime modelling. The later is the more physiological approach, and was adopted in the current study.

211 Hydrotime modeling

Within a range of water potentials, there is a fraction of the seed within a population that will germinate, and a fraction that will not germinate because the water potential is too low. Whilst by definition, seed that will germinate is not prevented from germinating by dormancy, a degree of dormancy is present in seed that will germinate, and a wide range of dormancy states extends through both the fractions of a seed population that will and will not germinate. The combination of low water potential and dormancy prevents a seed from germinating, and if the level of dormancy is sufficiently reduced by a germination-promoting treatment, then a seed that previously would not germinate can be made to germinate. Germination occurs when promotive factors within a seed exceed a threshold level of dormancy (Still & Bradford 1997). The deeper the level of dormancy and hence further from the threshold that a seed is, the stronger the germination promoting treatment must be. The degree of dormancy amongst the fraction of seeds that will germinate can be assessed by the germination rate, or inverse of the time to radicle emergence. Increased percentage germination, subsequent to dormancy-breaking treatments, was associated with increased germination rates (Gordon 1973). Even after all seeds had become capable of germination, germination rate could continue to increase with more extended or extreme treatments. A seed with a high dormancy threshold will germinate very slowly, and when the promotive factors do not exceed a seed’s sensitivity threshold, this delay is extended indefinitely and the seed is physiologically dormant (Bradford 1986; Gray et al. 1990; Grappin et al. 2000). A non-dormant seed has the capacity for cell elongation while a dormant seed has not (Bewley & Black 1982a). A seed with a high capacity for germination has the capacity for water uptake, required for cell elongation, at a low water potential. Seed requiring very negative water potential to prevent embryo growth will germinate more rapidly than seed with a less negative water potential threshold. Sensitivity distributions to water potential can be defined for a population of seeds. The base, or threshold, water potential for a given seed can be defined as the water potential that just prevents embryo growth (radicle emergence). Individual seeds vary in their base water potentials, and the variation in base water potential of a population of seeds closely approximates a normal frequency distribution (Gummerson 1986; Bradford 1990; Dahal & Bradford 1990). Seeds with base water potentials more negative than the external water potential germinate, and the lower their base water potential the more rapidly they germinate.

212 The dormancy status of a population of seeds can be quantified in terms of two characteristics, 1. the threshold sensitivity distribution (defined by a mean and standard deviation), and 2. a time constant that relates the external water potential relative to the threshold water potential to the actual time to germination. A hydrotime constant (θH) can then be defined as

θH = (Ψ - Ψb(g)) tg Equation 5.1 where Ψ is the ambient water potential, Ψb(g) is the threshold or base water potential that will just prevent germination of percentage g, and tg is the time required for the percentage g to germinate (the inverse of the rate of germination). As θH is a constant for a given set of conditions, the difference between the ambient Ψ and the Ψb(g) value of a particular percentage g is inversely proportional to the time required for that same percentage to germinate (tg). Seeds with the most negative Ψb values will have the highest germination rates

(shortest tg values), while those with higher Ψb values will have progressively slower germination rates. Since θH is a constant, the difference in germination rates among seeds is based entirely on the variation in their Ψb(g) values (Bradford 1990; Dahal & Bradford 1990, 1994). Treatments that inhibit germination increase the base water potential and/or increase the hydrotime of the population, whilst treatments that promote germination decrease the base water potential and/or decrease hydrotime. Abscisic acid (Ni & Bradford 1992, 1993), sub- optimal (Dahal et al. 1993), supra-optimal (Dutta & Bradford 1994) and both sub- and supra- optimal temperature (Allen et al. 2000) increased the median base water potential, whilst gibberelic acid decreased the median base water potential and reduced its standard deviation in tomato seeds (Ni & Bradford 1993). Stratification promoted germination through a reduction in the time required for germination (tg) during the initial period, whilst subsequent improvements in germination rate and capacity due to longer duration of stratification occurred because of a decrease in the base water potential of an apple embryo population (Bradford 1996). Stratification was not necessary for germination of the embryos, however the amount of time required for germination was predicted to be so long that the embryos were effectively dormant. Therefore, the reduction in time required for germination during the initial period of stratification effectively broke dormancy (Bradford 1996). Priming, where pre-germinative activity is permitted but emergence of the radicle is prevented due to low water availability (Heydecker et al. 1975) or abscisic acid (Finch- Savage & McQuistan 1991), reduced the time required for germination, while base water potential was unchanged or increased slightly in lettuce (Bradford & Somasco 1994) and

213 tomato seeds (Dahal & Bradford 1990). The cell walls of lettuce embryos become more extensible during priming (Carpita et al. 1979; Haigh 1988, unpublished) and, although embryo water uptake was not physically constrained by the enclosing endosperm (Bradford 1990), cell wall degradation and cell separation in the endosperm occurred prior to or coincident with radicle emergence but did not occur under conditions where radicle emergence was inhibited (Georghiou et al. 1983; Haigh 1988, unpublished; Sanchez et al. 1990; Groot & Karssen 1992). Smoke (not nitric oxide, Light & van Staden 2003) also increased germination of lettuce seeds (van Staden et al. 1995; Drewes et al. 1995; Jager et al. 1996; Gardener et al. 2001). Smoke probably increased extensibility of the cell walls of lettuce embryos, and may have increased cell wall hydrolysing enzyme activity in the endosperm opposite the radicle tip (Karssen et al. 1989; Dahal & Bradford 1990; Sanchez et al. 1990; Hilhorst & Karssen 1992; Nonogaki et al. 1992; Bradford et al. 2000). Heat shock and smoke have increased the final percentage germination of the seeds under study when applied singly and in combination (Chapters 2 & 3), and smoke has both increased the rate of germination, and decreased the spread of the germination time-course of Epacris tasmanica (Gilmour et al. 2000) and E. obtusifolia (Roche et al. 1997a). The hypothesis that heat shock and/or smoke may promote germination by causing a decrease in base water potential and/or the amount of time required for germination was investigated in the current study.

Osmotic and matric water potential

Soil water potential is comprised of the addition of osmotic and matric potentials. When soil-moisture diffusivity and seed-soil contact are non-limiting, then osmotic and matric potentials are equivalent (McWilliams & Phillips 1971). Polyethylene glycol (PEG) is a high molecular weight solute that reduces the osmotic potential of a solution. PEG does not enter the seed (Manohar 1966; McWilliams et al. 1970), thus its effect on germination is due to the reduction in availability of water to the seed and there is no adverse effect of contact with PEG. per se (Emmerich & Hardegree 1990). The effect of PEG-induced osmotic potential on germination was equivalent to the effect of soil matric potential when soil texture was fine (McWilliams & Phillips 1971; Young & Evans 1972), however the equivalent matric potentials were more inhibitory when soil texture was coarse (Young et al. 1970; Young & Evans 1972; Hagon & Chan 1977). The texture of the Hawkesbury Sandstone-derived soil is coarse, and the study species have small seeds, hence seed-soil contact is poor and matric

214 potential will largely control the germination process. However, the effects of heat shock and smoke on the amount of time required for germination and the base water potentials of the study species were investigated in the absence of the effects of soil-moisture diffusivity and seed-soil contact on seed water availability. Soil-moisture diffusivity and seed-soil contact were calculated, and their effects on germination were considered in light of a number of factors, including the hydrotime requirements of the study species.

Seed hydration dynamics and post-fire germination in soil

Physiological mechanisms that may cause a decrease in base water potential and/or the amount of time required for germination include osmotic adjustment of the embryo, and a reduction in the endosperm resistance to radicle penetration. Reduction in the endosperm resistance to radicle penetration was produced by gibberelic acid (Groot & Karssen 1987) and, notably, was hydration dependent (Georghiou et al. 1983; Bradford 1990). The degree of hydration of seeds in the soil varies with rainfall and evaporation, and with water retention and transmission properties of the soil. Soil moisture characteristics vary with factors such as topography, microtopography, structure, texture and organic matter content. The relative rates of water loss and gain by a seed determine the success or failure of germination (Harper & Benton 1966). The ratio of the area of contact between seed and soil to the seed surface area is critical in determining the degree of hydration of seeds in the soil (Sedgley 1963; Collis-George & Hector 1966). Soil hydraulic conductivity in the vicinity of the seed is also critical in determining seed water uptake (Hadas 1970; Dasberg & Mendel 1971; Shaykewich & Williams 1971; Ward & Shakewich 1972; Hadas & Russo 1974b). The resistance to water flow to the seed imposed by the seed-soil interface conditions increases as the seed wetted area, or the soil water conductivity, or both, decrease (Hadas 1974, 1976; Hadas & Russo 1974b). The resistance to water flow to the seed increases as the coarseness of the soil texture or structure increases, resulting in a decrease in the rate and final percentage germination (Hadas 1974, 1977). The resistance to water flow to the seed is expected to be highly limiting for germination of the study species within Hawkesbury Sandstone-derived soil. The study species have small seeds that, combined with the coarse soil texture, result in a low area of contact that is greatly reduced as the soil water content is reduced (Collis-George & Hector 1966). The soil derived from Hawkesbury Sandstone is coarse textured and drains rapidly (Bradstock 1985, unpublished), and only the low number of smaller pores will then conduct water (Hillel 1998), hence the resistance to water flow to seeds of the study species is

215 expected to be highly limiting to germination except following rainfall, or in water-enriched locations. An approximate measure of the degree of contact between the seed and soil particles can be determined from the relative sizes of these entities (Collis-George & Hector 1966), and an approximate measure of hydraulic conductivity can be determined from the soils texture (Young & Nobel 1986). The relationship between seed size and soil texture was investigated in the current study and is discussed in the context of ensuring post-fire seedling recruitment and retention of a seedbank.

Intermittent post-fire water availability

Seed hydration in freely draining Hawkesbury Sandstone derived soil is likely to be intermittent, and generally limiting between rainfall events. Fluctuations in soil water potential result in changes in the rate of progress of a seed towards germination. Progress towards germination during adequate hydration was retained during periods of dehydration (Wilson 1973; Heydecker 1973/74; Bradford 1986; Gibson & Bachelard 1986; Allen et al. 1993a, b; Dahal et al. 1993), provided the hydration period was longer than a certain minimum duration (Berrie & Drennan 1971). Also, progress towards germination occurred at water potentials too low to allow germination (priming), such that germination was rapid when water was no longer limiting (Wilson 1972; Heydecker & Coolbear 1977; Khan 1992). The relative lengths of the wet-dry periods affected the number of seeds that germinated (Frasier et al. 1985; Frasier 1989; Gonzalez-Zertuche et al. 2001). Seedling emergence was greatest if, when precipitation was low it was concentrated in a few events and, as precipitation increased, when the intervals between precipitation events were short (Bai & Romo 1995). Adequate cumulative hydration is required before a seed will germinate, and it may be crucial that a seed accumulate this hydrotime within a critical time period. A critical time period is likely to exist if overcoming dormancy and/or preventing the induction of dormancy is dependent on hydration-dependent processes, and/or if a narrow window of opportunity exists for germination. A germination-promoting factor with a short period during which it is effective represents a narrow window of opportunity. The germination-promoting effect of smoke has been retained in dehydrated seed (Baxter & Van Staden 1994; Dixon et al. 1995; Roche et al. 1997a, b; Tieu et al. 1999), for as long as one year (Brown et al. 1998; Brown & van Staden 1998). The retention of a smoke effect in dry storage may be beneficial for seeds from these Mediterranean climatic regions where the fire prevalent season and the season with

216 adequate moisture for seedling recruitment are separated for long periods of time. However, the climate of the Sydney region is relatively aseasonal for rainfall (Nix 1982). Another hydration-dependent process is the change in the concentration of smoke over time. In the absence of water, germination promoting compounds within smoke will be released to the atmosphere as gas (Keeley & Fotheringham 1998a), and the germination- promoting compounds within smoke are leached out as water percolates through soil (Dixon & Barrett 2003; Kenny 2003, unpublished). The interaction between fire-related germination cues and post-fire seed hydration is potentially complex. Both the water potential (between free water and no water), and the times during which water is available were varied in the current study. Whilst post-fire recruitment would enhance local persistence of a population, a residual seedbank is essential to buffer against the possibility of local post-fire extinction due to short fire-return intervals. The factors regulating post-fire germination are discussed in terms of meeting these two requirements, with emphasis on the greater importance of the persistence of a residual seedbank.

Investigations

The following interactions between fire related germination cues and ‘post-fire’ temperature and water availability were investigated. Section I. Interactions between heat shock, smoke and water availability were investigated for two species. Water availability is frequently limiting for germination in the field, and interactions between water availability and fire related germination cues may regulate post-fire germination. It is appropriate to investigate the effect of combinations of heat shock and smoke to understand the underlying mechanism whereby these cues influence water requirements for post-fire germination. The null hypothesis was that germination would not be affected by water availability, heat shock or smoke, or interactions between these factors. Section II a. Interactions between levels of heat shock, ‘post-fire’ incubation temperatures and water availability were investigated for smoked seeds of one species. Smoked seeds were treated with two levels of heat shock and incubated at a range of water potentials at either 15 or 25°C. The interactions between heat shock, ‘post-fire’ incubation temperatures and fluctuating water

217 potential were also investigated. The effects of season of fire on regeneration and of season on fire intensity are confounded in the field, but can be decoupled by a factorial combination of simulated seasons (represented by different temperatures of incubation) and fire intensities (represented by different levels of heat shock). Water availability is another seasonal factor that can be included in such an investigation. The null hypothesis was that germination would not be affected by the level of heat shock, temperature of incubation, water availability, or interactions between these factors. Section II b. Interactions between heat shock, smoke, ‘post-fire’ incubation temperatures and water availability were investigated for a number of species by treating seeds with factorial combinations of heat shock and smoke and incubating them at a range of water potentials at either 15 or 25°C. The interactions between the fire-related germination cues, ‘post-fire’ incubation temperatures and fluctuating water potential were also investigated. Wet and dry habitat species were investigated because the differences in water availability across the habitats may result in differences in the way water requirements for germination are altered by the fire-related cues. A further constraint was that hydrotime modelling requires seed with low level of dormancy. The null hypotheses were that germination would not be affected by heat shock, smoke, water availability, temperature of incubation, or interactions between these factors, and that interactions between these factors would not affect germination of wet and dry habitat species differently. Section III. The effects of the fire-related germination cues on hydrotime requirements were assessed for a number of species. A reduction in the amount of time required for germination and/or standard deviation of the base water potential would be consistent with an increase in exogenous control over germination, whilst a reduction in the median base water potential would be consistent with a decrease in exogenous control over germination. An investigation of the change in water required by seed for germination is necessary in order to understand how post-fire germination may be regulated by water availability. The null hypotheses were that neither heat shock, nor smoke, nor temperature of incubation would affect the hydrotime parameters, and that these factors would not affect the hydrotime parameters of wet and dry habitat species differently.

218 Section IV. The effects of factorial combinations of heat shock and smoke followed by various intermittent hydration cycles prior to continuous incubation at 15 or 25°C on germination of one species was investigated. The effect of high smoke concentration and whether such an effect is altered by the intermittent hydration treatments was also investigated. It is appropriate to investigate the effect of intermittent water availability on germination, as water availability is likely to be intermittent in the field, regardless of season. Also, it is appropriate to investigate the effect of very high concentration of smoke on germination, as high concentration of smoke is likely in certain environments such as swampy areas in the field. The null hypotheses were that neither heat shock or concentration of smoke or water availability or temperature of incubation would affect germination, and that germination would not be affected by interactions between these factors. Section V a. The effect of ‘post-fire drought’ was investigated by treating seeds of a number of species with heat shock and smoke, then dry storing seeds for long durations before incubation. Long periods without rainfall can occur following fire, hence it is appropriate to investigate the effects of heat shock and/or smoke followed by delay before incubation. The null hypothesis was that delayed incubation of seed treated with heat shock and/or smoke would not affect germination. Section V b. Little germination occurred in Section V a, consistent with the possibility that secondary dormancy was induced by ‘post-fire drought’. Fire-related germination cues and changes in incubation conditions were imposed to overcome the inferred secondary dormancy. Of interest were both the depth of the inferred secondary dormancy, and the effectiveness of the various treatments in overcoming the inferred secondary dormancy. Seed in the soil that experiences fire and then drought may be affected by secondary dormancy. Whether the dormant seed germinates when rain eventually occurs may depend on the level of dormancy. It is appropriate to investigate the level of dormancy in seed treated with heat shock and/or smoke followed by delay before incubation to explore the likelihood that post-fire drought will delay rather than prevent germination. The null hypothesis was that the fire-related cues would not affect germination of seeds that were treated with heat shock and/or smoke and then stored dry for 168 days.

219 Section VI. The effect of ‘post-fire drought’ was investigated for smoked and unsmoked seeds of one species using a different level of heat shock than the one that was inferred to have induced secondary dormancy (above). The level of heat shock experienced by seed may influence whether it affected by post- fire drought. The null hypothesis was that germination of seed treated with a low level of heat shock would not be affected by delayed incubation. Dormancy status of seeds following a number of these treatments was investigated at the end of experiments by transferring seeds across water potentials, incubation temperatures, and from darkness into light. Thus, the effect of the fire-related cues in preventing the inferred induction of secondary dormancy during incubation at low water potential was ascertained. Also, whether the fire-related cues continued to promote germination following prolonged incubation was determined. Transferral also allowed a distinction between secondary dormancy and loss of viability to be made (Harper & White 1974). The germination response of a species to factors such as the interaction between fire and seed hydration dynamics is probably related to the characteristics of its habitat. Section VII. The seed:soil particle size ratio has a major influence on seed hydration dynamics, hence germination. The size of both seeds and soil particles were determined. Soil texture and water retention were characterised and related to habitats. Interactions between fire related germination cues and seed hydration dynamics have been compared across species from wet and dry habitats. Interactions between fire-related germination cues, incubation temperature and seed hydration dynamics that could result in both post-fire germination and the retention of a residual soil seedbank to buffer against local extinction due to short fire intervals are discussed in light of findings from the above investigations.

220 5.2 Methods

Section I Fire-related germination cues followed by incubation at various water potentials (0 to -1.5 MPa); Epacris coriacea and Kunzea ambigua

Seeds of Epacris coriacea and Kunzea ambigua were treated with factorial combinations of 25˚C (= control) or 75°C heat shock for 5 minutes followed by 0 or 10 minutes of aerosol smoke. Three replicates of 10 seeds were independently treated, double wrapped in aluminium foil, and incubated at 25°C on a single layer of Whatman No. 1 filter paper in 9 cm plastic petri dishes at water potentials of 0, - 0.5, - 1 and – 1.5 MPa. The required water potentials were produced with 6000 MW polyethylene glycol (PEG), using the formulae of Michel (1983). Producing a separate solution for each petri dish was impractical, hence a single batch of each water potential was produced. Pseudoreplication of petri dish within water potential (Hurlbert 1984) should be borne in mind when interpreting results. Actual water potentials would have been slightly lower than that calculated due to PEG exclusion by the paper (Hardegree & Emmerich 1990). Changes in water potential due to hydration would have been of negligible consequence because of the small ratio of seed to solution volumes. A decrease in solution volume due to evaporation was visible and, because this reduced the water potential, the solutions were changed after 58 days.

Transferrals into free water

After 84 days, it was apparent that no further germination was likely to occur within the lowest water potential, or of K. ambigua within – 1 MPa, so the remaining seeds within these treatment were then transferred to free water. The PEG solution was washed off seeds under safe light conditions. The remaining E. coriacea seeds within the – 1 and – 0.5 MPa treatments, and K. ambigua seeds within the – 0.5 MPa treatment were transferred to free water after 119 days.

221 Data analysis

Final percentage germination within the reduced water potential treatment was analysed using a 3 way ANOVA, with water potential, heat shock, and smoke as fixed factors. Homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. The heterogeneity of variances in the Kunzea ambigua data set was not corrected by transformation. Cochran’s Test was significant at P < 0.05, hence α was adjusted downward to 0.01 to compensate for the increased risk of Type I error and comparisons among means was not attempted because of heterogeneity of variances (Day & Quinn 1989). Unplanned contrasts amongst means were carried out on the Epacris coriacea data set using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

Section II a Effect of heat shock levels applied to smoked Baeckea imbricata seeds, followed by incubation at various water potentials and temperatures.

Seeds of Baeckea imbricata were treated with 50 or 75°C heat shock followed by 10 minutes of aerosol smoke. Six replicates of 20 Baeckea imbricata seeds were incubated in two replicate containers per water potential treatment, in two different incubation cabinets per incubation temperature (15 or 25°C), at water potentials of 0, -0.3, -0.6 and –0.9 MPa. Seeds were placed on boats of single layer Whatman No. 1 filter paper, and these were placed on a single layer sheet of Whatman No. 1 filter paper, which was on a grid suspended within an airtight container of 3.9 L capacity. The 350 ml of water or PEG solution produced the appropriate degree of wetness on the paper surface on which the seeds were incubated. The required water potentials were produced with 6000 MW polyethylene glycol (PEG), using the formulae of Michel (1983). The large volume of PEG solution resulted in negligible change in water potential due to seed hydration, and the solution to paper ratio exceeded 30:1, so any change due to PEG exclusion by the paper was also negligible (Hardegree & Emmerich 1990). The large volume of air within the containers and the frequent opening of the containers would have ensured that atmospheric composition did not appreciably change (Berkat & Briske 1982). The air-tight seal of the containers prevented evaporative loss of water, and containers were only opened within partially enclosed surrounds that were humidified by bubbling air through an open body of water (Berkat & Briske 1982). Containers were double wrapped in aluminium foil and germination was monitored under safe

222 light conditions as previously described (Chapter 2). Germination was monitored every three days whilst the rate of germination was high, then with decreasing frequency as the rate of germination decreased.

Fluctuating water potential

Seeds of Baeckea imbricata were also incubated within a fluctuating water potential treatment. Seeds were treated as previously described, then initially incubated for 10 days at – 0.9 MPa. Seeds were then washed from the PEG boats onto mesh, and the PEG solution washed away using reverse osmosis water under safe light conditions. Seeds were placed on new boats and incubated for four days in free water. Seeds were then transferred from free water into the PEG solution on the same boat, removing only the free water using blotting paper. Four transferral cycles between free water and the PEG solution were performed. The 0.5 grams of water transferred with the boat (x 6 populations – see below) was calculated to reduce the water potential of the solution to -0.77 MPa over the course of the four transferral cycles. Seeds were finally transferred to the PEG solution (-0.77 MPa) for the remainder of the first section of the experiment.

Section II b Effect of fire related germination cues followed by incubation at various water potentials and temperatures (7 populations)

Seeds of seven populations were treated with factorial combinations of 25˚C (= control) or 75°C heat shock for 5 minutes followed by 0 or 10 minutes of aerosol smoke. Six replicates of 50 Kunzea ambigua seeds, 20 K. capitata (population 2) seeds, 40 K. capitata (population 4) seeds, 50 Epacris obtusifolia (population 2) seeds and 20 Baeckea linifolia seeds were incubated as described above (Baeckea imbricata). Kunzea ambigua, K. capitata (population 2), Epacris obtusifolia (population 2) and Baeckea imbricata were incubated at both 15°C and 25°C, K. capitata (population 4) was incubated at 15°C, and B. linifolia was incubated at 25°C. These five populations were also incubated within a fluctuating water potential treatment as described above (Baeckea imbricata). Also, three replicates of 20 Epacris coriacea seeds, and 40 E. obtusifolia (population 1) seeds were incubated in single containers, at 25°C, at water potentials of 0, -0.4, -0.8 and –

223 1.2 MPa. The minimum water potentials were determined as those that allowed a population of seeds to imbibe a weight of water equal to hydration of free water (Bradford 1986).

Data analysis

Final percentage germination before transferral into free water was compared across treatments for Baeckea imbricata using a 3 way ANOVA, with heat shock, incubation temperature and water potential regime as fixed factors. A three way ANOVA, with heat shock, smoke and water potential regime as fixed factors was used to analyse final percentage germination of Baeckea linifolia, Epacris coriacea, E. obtusifolia (population 1) and Kunzea capitata (population 4). Final percentage germination were analysed for Epacris obtusifolia (population 2), Kunzea ambigua and K. capitata (population 2) using 4 way ANOVA, with heat shock, smoke, incubation temperature and water potential regime as fixed factors. Containers were not replicated within cabinets, however there is no reason to suspect that containers per se would have significantly affected germination. Preliminary analyses were carried out with the full ANOVA model, which has the combined cabinet and container term nested within incubation temperature. The full analyses were problematical; while cabinet was not significant as a main effect, interactions between cabinet and other factors were significant at a rate higher than expected under Type I error, and these interactions were not consistent or of major research interest. Analyses were carried out using the average germination within each incubation temperature (cabinet) x water potential x heat shock x smoke treatment as a single replicate to avoid sacrificial pseudoreplication (Hurlburt 1984). The mean square among treatments was a common estimate of variance, hence standard error of cell means was calculated as the square-root of the mean square within samples divided by the sample size (n = 2) (Underwood 1997). Homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. The heterogeneity of variances in the Kunzea ambigua data set was not corrected by transformation. Cochran’s Test was significant at P < 0.01,hence α was adjusted downward to 0.001 to compensate for increased risk of Type I error and comparisons among means was not attempted because of heterogeneity of variances (Day & Quinn 1989). Unplanned contrasts amongst means were carried for all other species out using the Student- Newman-Keuls procedure (Sokal & Rohlf 1995).

224 Species were placed in response groups according to ANOVA results. The highest order significant interactions were initially considered, then lower order significant interactions, and then significant main effects.

Transferral into free water, across incubation temperatures and into light

When germination had decreased to a negligible rate (25°C, 138 days; 15°C, 151 days), the remaining seeds of all replicates were transferred into free water on a double layer of Whatman No. 1 filter paper in 9cm petri dishes. The transferral was performed under safe light, and the petri dishes were double wrapped in aluminium foil. When germination of the Kunzea species within the 25°C incubation temperature had decreased to a negligible rate (179 days), the remaining seeds of all replicates were transferred into a 15°C incubation temperature. When germination of the Kunzea species in darkness had decreased to a negligible rate (continuously within 15°C, 235 days; originally within 25°C, 280 days), the remaining seeds of all replicates were transferred into cool white light.

Data comparison

Germination as a percentage of the number of seeds transferred was informally compared by visual inspection of the data across the initially imposed treatments. Final germination as a percentage of the starting population (i.e. the sum of all germination within the cumulative treatments) was also informally compared by visual inspection of the data across the initially imposed treatments.

225 Section III Effect of fire-related cues on median and standard deviation of base water potentials, and on hydrotime requirements

Provided a population has no or low dormancy within free water, the median and standard deviation of its base water potential, and the mean hydrotime required for germination of any fraction of the population can be calculated. These attributes could be assessed within all heat shock and smoke treatments for Epacris coriacea, E. obtusifolia (population 1), Kunzea ambigua, K. capitata (population 4) and for K. capitata (population 2) within a 15°C incubation temperature. Moderately high levels of dormancy of K. capitata (population 2) within a 25°C incubation temperature greatly reduce the reliability of this assessment. Base water potential and hydrotime could be assessed for Epacris obtusifolia (population 2) within the smoke and the heat shock plus smoke treatments only, as dormancy was very high within the unsmoked treatments. A germination time course was obtained from frequent assessment of germination, and analysed using hydrotime modelling. Proper application of such an analysis requires that the samples at each time point be independent (Finney 1971). However, for practical purposes, as the methods of terminating separate seed samples at each counting interval and scoring cumulative germination in a single sample over time produced identical results (Campbell & Sorensen 1979); the latter method was used. It is assumed that the process of monitoring germination did not influence subsequent responses. The cumulative percentage of total germination was transformed using the logistic equation [ln(g/(100-g)); g = germination], thus converting the normal sigmoid curve into a straight line (Hewlett & Plackett 1978). The mean time until germination was calculated on the basis of total seed germination because this accounts for the progressive delay in germination in relation to the normally distributed maximum lifetimes within a population (Bradford et al. 1993). Germination time courses are skewed toward longer times as maximum germination percentage is approached (Brown & Mayer 1988), whilst the inverse of the days until germination, which is the rate at which seed embryos develop toward germination, has a normal distribution (Campbell & Sorensen 1979; Dahal et al. 1990). The cumulative percentage of final germination was regressed against the germination rate, or inverse of the days until germination. The mean germination rate (logit = 0) was determined from the regression, and the mean germination rate for each water potential was then

226 regressed against the water potentials. The inverse of the slope of this regression is an approximation of the hydrotime. The base water potential for each fraction of the population was determined from the algebraic rearrangement of equation 5.1; Ψb(g) = Ψ – (θH/ tg), where Ψb(g) is the threshold or base water potential that will just prevent germination of percentage g, Ψ is the ambient water potential, θH is a constant, and tg is the time required for that same percentage g to germinate. The base water potentials for each germination percentage g were regressed against the corresponding logit transformation of cumulative germination, with data from all water potential treatments converging about a single regression line. Cumulative germination of 50% of the population corresponded with logit germination = 0, and the median base water potential for the population was determined by rearrangement of the regression equation. The standard deviation (σΨb) of the normal distribution of the base water potential was determined from the equation σ Ψb = 1/[slope * π / √3] (Hewlett & Plackett 1978). Hydrotime and the mean and standard deviation of base water potentials are presented for visual inspection across treatments.

Section IV Effects of intermittent hydration, heat shock and high smoke concentration; Kunzea ambigua

Seeds of Kunzea ambigua were treated with factorial combinations of heat shock (25 or 75˚C for 5 minutes) followed by 0 or 5 minutes of aerosol smoke. The four replicates of ten seeds within a level of hydration were treated independently with heat shock and smoke. Two different treatments were applied to seeds that were to be immediately incubated. Seeds of half of the replicates were treated with smoke and heat shock and then transferred onto filter paper, whilst seeds of the other replicates were treated with smoke and heat shock whilst on the filter paper on which they were incubated. Seeds within all intermittent hydration treatments were also treated with smoke and heat shock whilst on the filter paper on which they were incubated. Because the filter paper was also smoked, the initial smoke concentration within the intermittent hydration treatments was high. Smoke concentration was probably reduced due to vapour emission (Keeley & Fotheringham 1998a) when petri dishes were open and filter papers were dry during dehydration treatments.

227 Following treatment with smoke and heat shock, seeds were then subjected to various combinations of durations of hydration and dehydration within a 15 or 25˚C ambient temperature. Seeds were either incubated immediately and continuously, or hydrated for a total of four days during the first 44 days before being incubated in constantly available water. Seeds were intermittently hydrated within four different regimes. Seeds were either hydrated for the first four days, or for the first two days and then for another two days after 20 days dehydration, or for the first day and then one day after each 10 days dehydration, or for 4 days after 20 days delay.

Transferrals across incubation temperatures and into light

Seeds within the immediate incubation at 15˚C treatment were transferred into light after 60 days. Fifty-four days after being transferred into constantly available water, seeds that had been subject to intermittent hydration within the 15˚C incubation temperature were transferred into light. Seeds that had been immediately incubated at 25˚C treatment were transferred into 15˚C after 62 days, and into light after another 36 days. Sixty days after being transferred into constantly available water, seeds that had been subject to intermittent hydration within the 25˚C incubation temperature were transferred into 15˚C, and then into light after another 73 days.

Data analysis

Because cabinets were not replicated, final percentage germination within each incubation temperature was analysed using a 3-way ANOVA with treatment, heat shock and smoke as fixed factors. Heterogeneity of variance was not corrected by transformation, hence α was adjusted downward to 0.001 in the 15˚C data set, and to 0.01 in the 25˚C data set to guard against increased risk of Type I error, and comparisons among means was not attempted (Day & Quinn 1989).

228 Section V a (Experiment A) Fire-related cues, followed by 0 or 47 days delay before incubation; 19 species

Seeds were subjected to one of three treatments; smoke (10 minutes of smoke), heat shock alone (75˚C for 5 minutes), or both cues combined. Replicates of each species were treated independently, and one replicate of each of the 19 species was treated simultaneously. Six replicates of 10 seeds of each species were then either incubated immediately or stored for 47 days before incubation. Stored and incubated replicates were double wrapped in aluminium foil at 25˚C, and germination was monitored under safe light conditions as previously described (Chapter 2).

Transferral across incubation temperatures and into light

After 134 days, Gahnia sieberiana and Schoenus brevifolius seeds within and without coats within the 47 days delay treatment were transferred into a 20/4 hour 25/35°C diurnal incubation temperature regime. After 181 days, S. brevifolius seeds within and without coats within the 0 days delay treatment were transferred into the more favourable fluctuating incubation temperature regime. At these same times, the Kunzea seeds were transferred into a 15°C incubation temperature, and Juncus continuous and Restio gracilis were transferred into light.

Data analysis

Final germination and the number of ungerminated seeds were determined for all species after about 250 days since hydration, and germination prior to transferral (Stage I) was calculated as a percentage of initial seeds per petri dish. The effect on germination of heat shock and smoke followed by 0 or 47 days delay before hydration was analysed using two way ANOVA (Table 5.5.1, 2; Experiment A). The fire-related germination cue(s) was a fixed factor and was orthogonal to the random factor delay treatments. Germination of Schoenus brevifolius seeds within coats was analysed subsequent to transferral into a 20/4 hour 25/35°C diurnal incubation temperature regime because no germination occurred at 25°C. Germination of Restio gracilis and Juncus continuus seeds was analysed subsequent to transferral into an 8 hour diurnal cool white light regime because no germination occurred in darkness. Data were analysed for Epacris coriacea and E. obtusifolia following germination within the 168 days

229 delay treatment (next section). Homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

Section V a (continued) Fire-related cues, followed by 0, 47 or 168 days delay before incubation; Epacris coriacea and E. obtusifolia

Seeds of Epacris coriacea and E. obtusifolia were treated as above (fire-related germination cues followed by 0 or 47 days storage before incubation), and an additional delay level (168 days storage without any further treatment) was also used.

Data analysis

Final percentage germination was analysed using two way ANOVA. The fire-related germination cue(s) was a fixed factor and was orthogonal to the random factor delay treatments. Homogeneity of variances was assessed using Cochran’s Test. Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

Section V b (Experiments B, C) Fire-related cues, followed by 168 days delay, then re-treatment with fire-related cues before incubation; 16 species

The original design involved no delay, a short or a long delay before incubation. However, germination of all species other than Epacris coriacea and E. obtusifolia was greatly reduced by the 47 days delay treatment and so, rather than investigating the effect of a long delay per se, the design was changed to investigate the effect of smoke and heat shock treatments after this long delay. No Gahnia melanocarpa seeds germinated within the no or 47 days delay treatments, hence this species was not included in the long delay treatment. Seeds of all other species that were treated with heat shock and/or smoke and stored for 168 days were again treated with heat shock and/or smoke immediately before incubation. Although such a short fire-return interval is very unlikely, it is not unreasonable to ask whether any physiological and / or morphological changes induced by the fire-related

230 germination cues would have reverted to an approximate pre-treatment state during the intervening period of time. Germination of twice-treated seed was informally compared by visual inspection of the data with the once-treated seed, assuming that the effects of the shorter and longer delays were similar. To assess whether the same cue that, in combination with ‘drought’ was inferred to have induced secondary dormancy could reduce secondary dormancy, eight populations were treated with the same cue as they had originally received (Experiment B). To assess whether different cue combinations would reduce the inferred secondary dormancy, the seeds of another eight species were subjected to the treatments that they had not originally received (Experiment C). For example, three of the six replicates that were originally treated with only smoke were subsequently treated with only heat shock, and the other three replicates were treated with the combination of heat shock and smoke.

Transferral across incubation temperatures

After 102 days, Gahnia sieberiana and Schoenus brevifolius seeds without coats were transferred into a more favourable 20/4 hour 25/35°C diurnal temperature regime, Restio gracilis and Juncus continuus seeds were transferred into an 8 hour diurnal cool white light regime and the Kunzea species were transferred into a 15°C incubation temperature.

Data analysis

Data were analysed for each species using one way ANOVA. The combination of fire- related germination cue(s) was a fixed factor; for the species where the original treatments were repeated there were three levels (Table 5.5.1; Experiment B), and for the species where different treatment was imposed after the delay, there were six levels (Table 5.5.2; Experiment C). Germination of Schoenus brevifolius seeds within coats was analysed subsequent to transferral into a 20/4 hour 25/35°C diurnal incubation temperature regime because no germination occurred at 25°C. Germination of Restio gracilis and Juncus continuus seeds was analysed subsequent to transferral into an 8 hour diurnal cool white light regime because no germination occurred in darkness. Homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

231

Section VI 50˚C heat shock +/- smoke, followed by 0 or 68 days delay, then re-treatment with fire- related cues before incubation; Baeckea imbricata (population 1)

Because Baeckea imbricata was adversely affected by certain interactions between 75˚C heat shock and delay treatments, and because germination of this species had previously been found to increase following the combination of 50˚C heat shock and smoke (Chapter 2), the interaction between 50˚C heat shock with and without smoke and delayed hydration was investigated for this species. Heat shock of 50˚C with and without smoke was initially applied to B. imbricata, and these seeds were incubated immediately or dry stored for 68 days in darkness at 25˚C. After dry storage, smoke only, 50˚C heat shock only or 50˚C heat shock and smoke were applied to seeds from the two pre-dry storage treatments, and these seeds were incubated at 25˚C in darkness. Six replicates of 10 seeds were independently treated with each combination of treatments.

Data analysis

Final germination was analysed using a two way ANOVA. The initial smoke treatment was a fixed factor and was orthogonal to the random factor re-treatments. Homogeneity of variances was assessed using Cochran’s Test. Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995).

Section VII Soil texture and water retention

In the current study, the germination response of seeds to post-fire water availability has been determined under laboratory conditions. A difference of major importance between laboratory and field conditions is seed/substrate contact, hence soil particle and seed size were determined and contact ratios were calculated. Also, variability in soil water retention that occurs across and within habitats was quantified to provide context for the laboratory- determined influences of water availability over germination.

232 Soil samples

Soil derived from Hawkesbury Sandstone was collected from three sites within a one- kilometre radius. Large loose litter was removed and a block of soil 30 cm square and 2 cm deep extracted from the flat section on a ridge top, from half way down the slope in locations of protruding parent rock, and at the bottom of the slope from the flat section adjacent to the water channel. A soil core was extracted without disrupting the soil structure to assess the bulk density immediately adjacent to the soil taken for soil texture and moisture characterisation.

Soil moisture retention curves

The blocks of soil were air dried, and dry sifted through a 2 mm sieve. Soil samples were sifted through 2 mm mesh to remove the gravel, because gravel greatly reduces water holding capacity (Reinhart 1961). This procedure was considered appropriate because, whilst the percentage soil moisture was slightly underestimated for disturbed samples of sandy loam, the moisture characteristics of sandy loam did not differ significantly when determined for disturbed and undisturbed samples (Salter & Williams 1965a). Twenty five gram soil samples were placed in soil sample retaining rings 1 cm high by 5.5 cm in diameter on wet Cellulose Membranes and saturated with RO water. Three replicate membranes were used until one failed; thereafter duplicate samples were prepared. One sample was placed on each membrane and left for approximately 8 hours in order to ‘connect’ the water films in the sample with those in the membrane. The soil samples were left in a sealed container to minimize evaporation, and re-wetted if not fully saturated. This procedure assures the maximum rate of water flow from the sample during the extraction process. The cellulose membranes were then placed in the Extractor Cylinder (Soilmoisture Equipment Corp., Santa Barbara, CA 93105 U.S.A.) and pressure of between 0.03 and 1.5 MPa applied. After the readings on the outflow buret indicated that equilibrium had been achieved, samples were removed into pre-weighed moisture boxes, sealed and weighed. The boxes were then opened and the samples dried at 105°C until there was no further loss of weight. The boxes were resealed, allowed to cool in a desiccator, and weighed. The difference in weight of the moist sample and the dry sample was divided by the dry weight of the sample and multiplied by 100 to obtain the percentage moisture content of the sample corresponding to each

233 moisture extraction pressure. These values were plotted on a graph and smoothed curves fitted to produce a moisture retention curve for each soil.

Soil texture

The gravel and soil were weighed to determine the percentage gravel by weight. The percentages of soil particles within the Australian System size groups (Sands: 0.02 – 2.0 mm; Silts: 0.002 – 0.02 mm; Clays: <0.002 mm) were determined using the hydrometer method (Gee & Bauder 1986). Duplicate samples of 2 mm sieved soil were placed in Calgon dispersant solutions and mixed with an electric mixer to separate the particles. The solution was washed into a cylinder and thoroughly mixed by repeated inversion of the cylinder. After five minutes when the sand particles had settled out, density of the solution due to suspended silt and clay particles was determined using a hydrometer. After 89 minutes from the start of settling, the density due to suspended clay particles only was measured. Temperature was recorded at the same time as solution density, and corrections were made for both the temperature at the time of density measurements, and for the density of the Calgon solution. The average of the duplicates was calculated, and the texture determined from the texture triangle of the United States Department of Agriculture (Soil Survey Staff 1975). Duplicate soil samples were dry sifted through 0.5 mm, 0.25 mm, and 0.2 mm sieves. The fraction of soil particles with diameter of between 0.5 and 0.25 mm was determined because calculated diameters of the seeds of the study species were within this range. The fractions were oven dried, weighed, averaged, and percentages calculated. The percentage silt and clay (previously determined – see above) was subtracted from the percentage fraction of fine sand. The average soil particle size from each site and location was determined by multiplying the percentage within each size category by the average of the size category and summing these results.

Soil structure

Bulk density was determined by drying the soil sample at 105°C for 5 days, and dividing the dry mass by the internal volume of the extraction cylinder. Bulk density was assessed only for cores not containing gravel.

234 Soil organic matter

Percentage organic matter was determined by the Walkley-Black (1934) method. Duplicate soil samples were ground to less than 0.15 mm diameter, mixed in a 0.5 M potassium dichromate solution, and the soil was digested and organic matter oxidised in concentrated sulphuric acid. The resultant solutions were centrifuged, and the supernatant transferred into a cuvette. The absorbance of a 600 nm wavelength of each solution was determined in a spectrophotometer. The absorbance of sucrose standards were also determined and a standard curve constructed. The amount of carbon present in the soil samples was determined by comparison with the standard curve. The oven dry sample weights were determined by dividing the air dry weight by one plus the moisture content, which was determined by the change in weight following oven drying. The percentage organic matter was calculated as percentage carbon divided by 0.53, assuming that the organic matter of surface soil contains 53% carbon (Broadbent 1953).

Seed size

The diameters (2 x radius, r) of the study species were calculated using the formula r = 3√ (3V/4π) by assuming seeds were spherical and that air-dry weight was equivalent to volume (V). Average air-dry weight was determined from three samples of 100 seeds. All of the study species were only slightly sub-spherical (excepting Calytrix tetragonia which was excluded from the analysis), and they passed through sieve sizes as predicted by this calculation. As comparisons with soil particle size were only approximate, the required level of accuracy was obtained despite the inherent assumptions.

235 5.3 Results

Section I Fire-related germination cues followed by incubation at various water potentials (0 to -1.5 MPa); Epacris coriacea and Kunzea ambigua

The aim was to assess whether germination would be affected by water availability, heat shock, smoke, or interactions between these factors .

Water potential changed both the level of germination, and the effect of the fire- related germination cues (Table 5.1; Figs 5.1.1, 5.1.2). Added heat shock increased germination of Kunzea ambigua in the free water but not within the – 0.5 MPa treatment (Table 5.1; Fig 5.1.2). At lower water potentials the effect of smoke was more pronounced for both Epacris coriacea and Kunzea ambigua. Smoke stimulated germination at water potentials that otherwise prevented germination. A single batch of each water potential was produced, therefore there was no test of whether each water potential was correctly produced. Care was taken with measurements and nothing unusual was apparent within the experiments. Directional trends within the data rather than unexplained variation or outliers are consistent with responses due to the experimental treatments rather than any other causes (Hurlbert 1984).

Tranferral into 0 MPa

A fire-related germination cue was active following prolonged incubation, as final germination of smoked seeds of both species was apparently greater than germination of unsmoked seeds following transferral into free water (Figs 5.1.1, 5.1.2).

Summary of Section I

Reduced water potential had a negative effect on germination and smoke had a large positive effect on germination that increased as water potential decreased. The null hypothesis that germination would not be affected by interactions between water availability and heat shock or smoke was rejected.

236 Table 5.1: Effects of water potential, heat shock, smoke, and interactions between these factors on % germination of Epacris coriacea and Kunzea ambigua. ANOVA P-values are shown

Source df E. coriacea† K. ambigua‡

Water potential (WP) 3 <0.0001 <0.0001

Heat shock (H) 1 0.5723 0.0081

Smoke (S) 1 0.0004 <0.0001

WP x H 3 0.5668 0.0008

WP x S 3 0.0010 <0.0001

H x S 1 0.9308 0.4994

WP x H x S 3 0.8203 0.5770 residual 32

† transformed data ‡ α reduced to 0.01 due to heterogeneity of variances

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Figure 5.1.1: Mean final germination of Epacris coriacea treated with heat shock (25 or

75°C) and smoke (0 or 10 minutes), then incubated at a) 0 MPa (2), or reduced water potentials () of b) – 0.5 MPa, c) – 1 MPa, d) – 1.5 MPa before being transferred into 0 MPa (2). Bars = S. E.s Analysis of transformed data; back-transformed data shown

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Figure 5.1.2: Mean final germination of Kunzea ambigua treated with heat shock (25 or

75°C) and smoke (0 or 10 minutes), then incubated at a) 0 MPa (2), or reduced water potentials () of b) – 0.5 MPa, c) – 1 MPa, d) – 1.5 MPa before being transferred into 0 MPa (2). Seeds within –1.0 and –1.5 MPa water potential treatments were transferred into free water before those within –0.5 MPa (see text). Bars = S. E.s

239 Section II a Effect of heat shock levels applied to smoked Baeckea imbricata seeds, followed by incubation at various water potentials and temperatures.

The aim was to assess whether germination would be affected by the level of heat shock, temperature of incubation, water availability, or interactions between these factors.

The response of Baeckea imbricata to the level of added heat shock was strongly dependent on the incubation temperature. (T x H sig., Table 5.2; Fig 5.2.1). The 75°C heat shock resulted in more germination than the 50°C heat shock within the 15°C incubation temperature. In contrast, no difference in germination between heat shock levels occurred within the 25°C incubation temperature (Table 5.2; Fig 5.2.1).

Summary of Section IIa

The confounded effects of season of fire on regeneration and of season of fire on intensity were decoupled, and the lower level of heat shock applied to seed incubated at the lower temperature resulted in less germination across levels of water availability. The null hypothesis that germination would not be affected by interactions between the level of heat shock, temperature of incubation or water availability was rejected.

Section II b Fire-related germination cues followed by incubation at various water potentials and temperatures; 7 populations

The aim was to assess whether germination would be affected by heat shock, smoke, water availability, temperature of incubation, or interactions between these factors, and whether these factors would affect germination of wet and dry habitat species differently.

Interactions between fire-related germination cues, temperature and water potential

The fire-related germination cue(s) affected germination of another 5 out of 7 populations in interaction with the seasonal factor(s) of temperature and water potential (Table 5.2). Across three Kunzea populations, a common pattern was that the negative effect

240 of decreasing water potential was substantially ameliorated by smoke (Table 5.2; Fig 5.2.2 – 4), and smoke ameliorated the negative effect of a low water potential in Epacris obtusifolia (population 1) (Table 5.2; Fig 5.2.5). The complexity of the interactions can be seen for example in added heat shock increasing germination of Kunzea ambigua within the fluctuating water potential treatment when seeds were incubated at 15°C, but not at 25°C (Table 5.2; Fig 5.2.2). Added heat shock also increased germination of Kunzea capitata (population 4) within the fluctuating water potential treatment (only incubated at 15°C) (Table 5.2; Fig 5.2.4). Smoke alone greatly increased germination of Epacris obtusifolia (population 2) within fluctuating water potential treatments, but not within the lowest water potential (Table 5.2; Fig 5.2.6). The combination of added heat shock and smoke was most effective in stimulating germination, including germination within the lowest water potential. Smoked E. obtusifolia seeds were possibly more able to respond to the brief periods of free water when incubated at the higher rather than lower temperature (Fig 5.2.6).

Main effects of fire-related germination cues and water potential

Germination of Baeckea linifolia and Epacris coriacea were not affected by interactions between factors (Table 5.2). Germination of Baeckea linifolia decreased with decreasing water potential, and within the added heat shock treatment (Table 5.2; Fig 5.2.7). Notably, germination was not increased within the fluctuating water potential treatment relative to the lowest water potential. Germination of Epacris coriacea decreased within the lowest water potential and smoke caused a small increase (Table 5.2; Fig 5.2.8).

Summary of Section IIb

Smoke generally increased germination, particularly within lower levels of water potential, regardless of species habitat. The null hypothesis that germination would not be affected by interactions between heat shock or smoke and water availability or temperature of incubation was rejected. The null hypothesis that interactions between these factors would not affect germination of wet and dry habitat species differently was accepted.

241

Table 5.2. Effects of incubation temperature, water potential, heat shock, smoke, and interactions between these factors on % germination of eight study populations. ANOVA P-values are shown.

Source df† K. K. K E. B. B. df† E. E. ambigua‡ capitata capitata obtusifolia§ imbricata! linifolia obtusifolia coriacea population 2 4 2 1 Temperature 1 0.0001 <0.0001 0.0001 <0.0001 (= T) Water 4 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0012 3 <0.0001 <0.0001 potential (= WP) Heat shock 1 <0.0001 0.0724 0.0051 <0.0001 <0.0001 0.0091 1 0.5627 0.1638 (= H) Smoke (= S) 1 <0.0001 <0.0001 <0.0001 <0.0001 0.1956 1 0.0982 0.0070

T x WP 4 0.0020 0.0425 <0.0001 0.1197

T x H 1 0.0005 0.5988 0.0185 0.0001

T x S 1 0.0003 0.1925 0.1980

WP x H 4 0.0001 0.5684 0.3176 0.0043 0.1438 0.4700 3 0.7440 0.6919

WP x S 4 <0.0001 0.0003 0.0001 <0.0001 0.4490 3 0.0304 0.8885

H x S 1 <0.0001 0.0941 0.0450 <0.0001 0.6996 1 0.8303 0.4943

T x WP x H 4 0.0582 0.0968 0.1451 0.1493

T x WP x S 4 <0.0001 0.0002 0.0124

T x H x S 1 0.3533 0.2111 0.0124

WP x H x S 4 0.0001 0.0454 0.0683 0.1720 0.4387 3 0.2138 0.5000

T x WP x H 4 0.0002 0.0103 0.1039 x S † residual degrees of freedom: K. ambigua, K. capitata (2), E. obtusifolia (2) = 40 K. capitata (4), Baeckea imbricata, B. linifolia = 20 E. obtusifolia (1), E. coriacea = 32 ‡ α reduced to 0.001 due to heterogeneity of variances §transformed data ! all seeds smoked

242 a) 15°C

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Figure 5.2.1: Mean germination of Baeckea imbricata population 2 smoked seeds treated with 50°C (), or 75°C () heat shock, and incubated at a) 15°C b) 25°C plotted against the five different water potential regimes (0, -0.3, -0.6, -0.9 MPa and a fluctuating water potential regime (see text))

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Figure 5.2.2: Mean germination of Kunzea ambigua seeds treated with combinations of heat shock and smoke, and incubated at a) 15°C, b) 25°C plotted against the five different water potential regimes (0, -0.3, -0.6, -0.9 MPa and a fluctuating water potential regime (see text))

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

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Figure 5.2.3: Mean germination of Kunzea capitata (population 2) seeds treated with combinations of heat shock and smoke, and incubated at a) 15°C, b) 25°C plotted against the five different water potential regimes (0, -0.3, -0.6, -0.9 MPa and a fluctuating water potential regime (see text))

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

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Figure 5.2.4: Mean germination of Kunzea capitata (population 4) seeds treated with combinations of heat shock and smoke and incubated at 15°C, plotted against incubation water potential (0, -0.3, -0.6, -0.9 MPa and a fluctuating water potential regime (see text))

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

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Figure 5.2.5: Mean germination of Epacris obtusifolia (population 1) seeds treated with combinations of heat shock and smoke, plotted against the four different water potential regimes (0, -0.4, -0.8, -1.2 MPa)

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

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Figure 5.2.6: Mean germination of Epacris obtusifolia (population 2) seeds treated with combinations of heat shock and smoke, and incubated at a) 15°C, b) 25°C plotted against the five different water potential regimes (0, -0.3, -0.6, -0.9 MPa and a fluctuating water potential regime (see text))

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

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Figure 5.2.7: Mean germination of Baeckea linifolia population 2 seeds treated with combinations of heat shock and smoke, plotted against water potential (0, -0.3, -0.6, - 0.9 MPa and a fluctuating water potential regime (see text))

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

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Figure 5.2.8: Mean germination of Epacris coriacea seeds treated with combinations of heat shock and smoke, plotted against the four different water potential regimes (0, -0.4, -0.8, -1.2 MPa)

Combinations of heat shock and smoke are indicated as 25°C heat shock (25), 0 minutes smoke (0) (2), 25°C heat shock, 10 minutes smoke (10) (), 75°C heat shock (75), 0 minutes smoke (), 75°C heat shock, 10 minutes smoke ().

250 Transferral of seeds from reduced water potential into 0 MPa

A fire-related germination cue remained active following prolonged incubation, and this effect was dependent on incubation temperature for two species. When seeds of all Kunzea populations were transferred from the reduced water potential into 0 MPa, markedly more smoked than unsmoked seeds germinated. Smoke increased germination of Kunzea ambigua only within the more favourable 15°C incubation temperature. In contrast, smoke markedly increased germination of transferred Epacris obtusifolia (population 2) seeds within the less favourable 15°C incubation temperature, and germination of unsmoked seeds was greater within the more favourable 25°C incubation temperature. For all species, the lower the original water potential, the higher the level of germination amongst the transferred seeds within 0 MPa.

Transferral of seeds from a 25°C into a 15°C incubation temperature

The inferred interaction between smoke and incubation temperature, with the germination stimulating effect of smoke having less longevity at 25°C, was again apparent for Kunzea ambigua. In contrast to the effect of transferring K. ambigua seeds from reduced water potential into free water, markedly more unsmoked than smoked seeds germinated following transferral from the 25°C into the 15°C incubation temperature. Most germination occurred only within the previously low water potentials. Germination at this stage was very similar across the initial 25 and 15°C incubation temperatures. Negligible Kunzea capitata (population 2) seeds germinated following transferral from the 25°C into the 15°C incubation temperature.

Transferral of seeds from darkness into light

Viability was not reduced during the experimental treatments, as a very high percentage of seeds of all Kunzea populations germinated following transferral into light. Final germination was very similar across all treatments, in marked contrast to the large differences between previous treatments.

251 Section III Effect of fire-related cues on median and standard deviation of base water potentials, and on hydrotime requirements

The aim was to assess whether heat shock, smoke, or temperature of incubation would affect the hydrotime parameters, and whether these factors would affect the hydrotime parameters of wet and dry habitat species differently.

The fire-related cues changed all three water requirement parameters for germination, and all three changes would indicate an increase in external control over germination. Consequences of these changes are that seeds would 1) germinate more rapidly for a given level of water availability, but 2) require a higher level of water availability, with 3) less variability in this requirement following treatment with the fire-related cues. Added heat shock and smoke greatly reduced the hydrotime requirements of all but one species for which data were available (Epacris coreacea being the exception) (Table 5.3). The fire-related cues: 1) reduced the hydrotime requirement by an average of 50% for the five populations for which an accurate calculation could be performed. A decrease in the hydrotime requirement of Epacris coreacea would probably have been detected if lower water potentials had been used. 2) increased the median base water potential of the five populations with one exception (Kunzea ambigua treated with added heat shock and smoke combined) by an average of 17% (Table 5.3). 3) reduced the standard deviation of the base water potentials by an average of 49% for the five populations Both the increase in median base water potential and the reduction in the standard deviation of the base water potentials would have acted to increase the hydrotime requirement. Therefore the calculated reduction in hydrotime requirement in response to the fire-related cues under-represents the actual change.

252 Table 5.3. Hydrotime (θHT) and median base water potentials (Ψb MPa) (standard deviation in parenthesis) for populations treated with factorial combinations of heat shock (25°C or 75°C for 5 mins) and aerosol smoke (0 or 10 mins) and incubated at 15°C or 25°C.

Treatment Population Temperature Control Smoke Heat shock Heat shock (°C) and Smoke

† E. coriacea 25 θHT 35.70 40.24 40.64 33.84 -1.91 (0.228) -1.70 (0.206) -1.92 (0.227) -1.87 (0.163) Ψb

E. obtusifolia 25 θHT 151.33 92.87 67.15 66.17 (1) ‡ -3.49 (0.787) -2.86 (0.501) -2.22 (0.312) -2.29 (0.230) Ψb

E. obtusifolia 15 θHT 90.11 56.11 (2) ‡ -2.19 (0.179) -1.67 (0.111) Ψb

25 θHT 91.02 77.19 -2.47 (0.215) -2.45 (0.194) Ψb

K. ambigua 15 θHT 31.72 13.51 17.68 12.39 -1.33 (0.128) -1.12 (0.058) -1.28 (0.079) -1.17 (0.067) Ψb

25 θHT 34.89 19.75 15.15 20.75 -1.26 (0.157) -1.21 (0.049) -0.95 (0.048) -1.36 (0.073) Ψb

‡ K. capitata (2) 15 θHT 40.69 19.74 17.41 18.59 -1.59 (0.157) -1.22 (0.075) -1.10 (0.075) -1.21 (0.077) Ψb

25 θHT 16.42 22.02 18.32 -1.15 (0.076) -1.21 (0.174) -1.32 (0.123) Ψb

‡ K. capitata (4) 15 θHT 25.87 13.33 14.80 14.25 -1.24 (0.129) -1.09 (0.093) -1.02 (0.105) -1.15 (0.086) Ψb † accuracy of analysis limited due to insufficiently low water potentials ‡ population NB. Cells blank when germination at 0 MPa not sufficient to allow analysis

253 Summary of Section III

Heat shock and smoke substantially altered all three hydrotime parameters in the same manner for both wet and dry habitat species, whilst temperature of incubation had no consistent effect. The null hypotheses that neither heat shock, nor smoke would affect the hydrotime parameters were rejected. The null hypotheses that temperature of incubation would not affect the hydrotime parameters, and that heat shock, smoke and temperature of incubation would not affect the hydrotime parameters of wet and dry habitat species differently were accepted.

Section IV Effects of intermittent hydration and high smoke concentration; Kunzea ambigua

The aim was to assess whether heat shock or concentration of smoke or water availability or temperature of incubation would affect germination, and whether germination would be affected by interactions between these factors.

The small differences in germination of Kunzea ambigua between incubation temperatures seen under constant hydration in previous experiments were greatly exacerbated under hydration conditions more closely approximating the field. Intermittent hydration greatly reduced germination only within the 25°C incubation temperature, and germination was not increased by the fire-related germination cues within these treatments (Table 5.4; Fig 5.3a, b). Germination within the 15°C incubation temperature was increased by the combination of heat shock and smoke (Table 5.4; Fig 5.3a). Germination of Kunzea ambigua was also reduced within the supra-optimal incubation temperature when seeds were treated with smoke on paper and immediately incubated (3 way interaction significant, Table 5.4; Fig 5.3b). For many seeds that were treated on paper the radicle elongated through the ruptured seed coat, but did not emerge through the enclosing endosperm.

254 Transferral of seeds from a 25°C into a 15°C incubation temperature

When seeds were transferred from a 25°C into a 15°C incubation temperature, a moderate to large increase in germination occurred within all treatments. Within each treatment, total germination of seeds following transferral from a 25°C incubation temperature was similar to germination of seeds that were within a 15°C incubation temperature from the outset (Fig 5.3c).

Transferral of seeds from darkness into light

Germination within all treatments was approximately 90% following transferral into light, with one notable exception. Seeds that were smoked on paper and immediately incubated at 15°C did not germinate when transferred into light.

Summary of Section IV

An inferred interaction between water availability and temperature of incubation had a massive effect on germination, and the fire-related cues, including smoke concentration, and the temperature of incubation also affected germination. The null hypotheses that neither heat shock or concentration of smoke or water availability or temperature of incubation would affect germination, and that germination would not be affected by interactions between these factors was not supported.

255 Table 5.4. Effects of treatments (smoke concentration and intermittent hydration before continuous incubation), heat shock, smoke, and interactions between these factors on % germination of Kunzea ambigua. ANOVA P-values shown.

Source df 15°C† 25°C‡

Treatment§ 5 <0.0001 <0.0001

Heat shock 1 0.7686 0.4985

Smoke 1 <0.0001 0.0002

Treatment x Heat shock 5 0.0031 0.4817

Treatment x Smoke 5 0.0036 0.3233

Heat shock x Smoke 1 0.0006 0.0133

Treatment x Heat shock x Smoke 5 0.0515 <0.0001 residual 72

† α reduced to 0.001 due to heterogeneity of variances

‡ transformed data; α reduced to 0.01 due to heterogeneity of variances

§ smoke concentration and intermittent hydration before continuous incubation (see text)

256 a)

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Figure 5.3: Mean germination of Kunzea ambigua seeds treated with combinations of heat shock (25°C or 75°C) and smoke (0 or 5 minutes) applied to seeds only (seeds), or to both seeds and filter paper (paper), and incubated at: a) 15°C () b) 25°C () c) 25°C (= b), then transferred into 15°C following post-treatment hydration / dehydration (h/d) periods of 0, 0, 4/40, 2/20/2/20, 1/10/1/10/1/10/1/10, or 0/20/4/20 days before constant hydration Bars = S. E.s.

257 Section V a Fire-related cues followed by 0 or 47 days delay before incubation; 17 species

The aim was to assess whether delayed incubation of seed treated with heat shock and/or smoke would affect germination.

The 47 day delay between the fire-related cues and hydration reduced germination of nine species that were previously found to be positively affected by heat shock and smoke treatments followed by immediate hydration (Chapter 2) (Tables 5.5.1, 2 experiment A; Figs 5.4.1 – 3; no G. melanocarpa germination). Germination within the 47 day delay treatment averaged only 28% (SE 7.5) of that within the no delay treatment across the 7 species affected by the delay as a main effect. Effect of the delay was greater than the effects of the fire-related cues per se (Table 5.5.1, 2; Figs 5.4.1 – 3). Germination of Baeckea linifolia, Restio gracilis and Schoenus brevifolius seeds without coats was different across the fire-related cue treatments (Tables 5.5.1, 2).

258 Table 5.5.1. Effects of 0 or 47 days delay before incubation of seeds treated with heat shock, smoke or heat shock and smoke on % germination (Experiment A); and effect of re- application of the original treatment following 168 days delay on % germination of 8 species (Experiment B); ANOVA P-values shown

Experiment A Experiment B Species 0 vs 47 Treatment Interaction Residual Treatment Residual days delay within 168 days delay

F vs residual interaction residual residual df 1 2 2 30 2 15

K. capitata <0.0001† 0.5603 0.0111 0.0270†

R. gracilis§ <0.0001 0.0488 0.6018 0.0449

S.brevifolius 0.8453 0.0261 0.9035 0.0073 (- coat) K. ambigua <0.0001† 0.3298 0.2939 0.3135†

G. sieberiana 0.0001 0.1980 0.1658 0.1040

W. pungens 0.0382 0.6107 0.1966 No test

J. continuus§ 0.5173 0.6411 0.5227 0.2231†

S.brevifolius 0.7175 0.0856 0.8963 0.6760 (+ coat)‡

† transformed data ‡analysis following transferral into 20/4 hour 25/35°C incubation temperature regime § analysis following transferral into 8 hour diurnal light

259 Table 5.5.2. Effects of 0 or 47 days delay before imbibition of seeds treated with heat shock, smoke or heat shock and smoke on % germination (Experiment A); and effect of application of the treatments that were not originally applied, following 168 days delay on % germination of 8 study species (Experiment C); ANOVA P-values shown

Experiment A Experiment C Species 0 vs 47 Treatment Interaction Residual Treatment Residual days delay within 168 days delay

F vs residual interaction residual residual df 1 2 2 30 5 12

B. linifolia 0.4552† 0.0091 0.9782 0.3361

D. secundum 0.0119 0.3743 0.3587 0.0334†

E. paludosa 0.0010 0.3661 0.0903 0.0047

B. imbricata <0.0001† 0.2262 0.2698 0.0048

E. crassifolia 0.0007† 0.1623 0.4960 Negligible germination C. tetragonia 0.5426 0.5918 0.4851 No test

C. flexosa 0.8093 0.8083 0.5598 0.0611

E. microphylla 0.0723 0.5134 0.4607 0.0963 ssp rhombifolia

† transformed data

260 a) K. ambigua 0 days delay d) K. capitata 0 days delay

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Figure 5.4.1: Mean final germination of a - c) Kunzea ambigua and d - f) Kunzea capitata (population 1) treated with heat shock (25°C or 75°C) and smoke (0 or 10 minutes), then incubated a, d) immediately, b, e) following 47 days or c, f) 168 days delay. Seeds within the 168 days delay treatment were re-treated with the same cues immediately before incubation.

Seeds within delay treatments were incubated at 25°C (), then transferred into 15°C (). Germination plotted against heat shock and smoke treatments. Bars = S. E.s.

261 a) E. paludosa 0 days delay d) R. gracilis 0 days delay

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Figure 5.4.2: Mean final germination of a – c) Epacris paludosa and d – f) Restio gracilis in light, following dark incubation. Seeds were treated with heat shock (25°C or 75°C) and smoke (0 or 10 minutes), then incubated a, d) immediately, b, e) following 47 days or c, f) 168 days delay. Epacris paludosa seeds within the 168 days delay treatment were re-treated with 10 minutes smoke (), 75°C heat shock (), or 75°C heat shock and10 minutes smoke (). Restio gracilis seeds within the 168 days delay treatment were re-treated with the same cues immediately before incubation. Germination plotted against heat shock and smoke treatments. Bars = S. E.s

262 a) Gahnia sieberiana; 0 days delay d) Schoenus brevifolius; 0 days delay

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Figure 5.4.3: Mean final germination of a – c) Gahnia sieberiana (population 1) and d –f)

Schoenus brevifolius seeds without coats treated with heat shock (25°C or 75°C) and smoke (0 or 10 minutes), then incubated a, d) immediately, b, e) following 47 days or c, f) 168 days delay. Seeds within the 168 days delay treatment were re-treated with the same cues immediately before incubation.

Seeds were incubated at 25°C (), then transferred into 25°C / 35°C (), except a) G. sieberiana 0 days delay. Germination plotted against heat shock and smoke treatments. Bars = S. E. s.

263 Section V a (continued) Fire-related cues, 0, 47 or 168 days delay before incubation; Epacris coriacea and E. obtusifolia In contrast to the nine species with germination strongly inhibited by the delay treatment, germination of Epacris coriacea was not affected by either of the delay treatments. Germination of E. obtusifolia was reduced by the 47 day delay between the fire-related cues and hydration, thus this treatment reduced germination of all ten species that were previously found to be positively affected by heat shock and smoke treatments followed by immediate hydration. However, germination of E. obtusifolia was only moderately reduced by the 47 and

168 days delay treatments (F 2,45 = 14.89, P < 0.0001; Fig 5.4.4).

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Figure 5.4.4: Mean final germination of Epacris obtusifolia (population 1) treated with

heat shock (25°C or 75°C) and smoke (0 or 10 minutes), then incubated immediately (2), or following 47 days (), or 168 days () delay. Germination plotted against heat shock

and smoke treatments. Bars = S. E.s

Summary of Section V a

The delay treatment generally caused a substantial reduction in germination, hence post-fire drought is likely to reduce germination. The null hypothesis that delayed incubation of seed treated with heat shock and/or smoke would not affect germination was rejected.

264 Section V b Fire-related cues, 168 days delay before re-treatment with fire-related cues, then incubation; 16 species

The aim was to assess whether the fire-related cues would affect germination of seeds that were treated with heat shock and/or smoke and then stored dry for 168 days.

The reduction in germination within the delayed hydration treatment (Section V a above) was consistent with the induction of secondary dormancy. Re-treatment with the fire- related germination cues apparently overcame the inferred secondary dormancy in three species. Germination of Kunzea ambigua (Fig 5.4.1), Epacris paludosa (Fig 5.4.2) and Restio gracilis (Fig 5.4.2) was visibly increased by one or more re-treatments relative to germination within the short-delay. The different re-treatments affected germination of six species (Table 5.5.1, experiment B; Table 5.5.2, experiment C). Most notably, two applications of heat shock reduced germination of Dracophyllum secundum (Table 5.5.2; data not shown) and Schoenus brevifolius seeds without coats (Table 5.5.1; Fig 5.4.3).

Transferral across incubation temperatures

The combination of a more favourable incubation temperature and the re-application fire-related germination cues overcame the inferred secondary dormancy in the Kunzea species (Fig 5.4.1). The combination of a more favourable incubation temperature and the re-application of smoke apparently also increased germination of Gahnia sieberiana (Fig 5.4.3). The detrimental effect of two heat shock treatments on germination of Schoenus brevifolius seeds without coats mentioned above may have been due to mortality, as fewer seeds that had been twice treated with heat shock apparently germinated within the more favourable temperature regime (Fig 5.4.3). In contrast, germination of the twice-treated Schoenus brevifolius seeds in their coats was apparently not different across treatments within a 25/35°C diurnal temperature regime (data not shown).

265 Summary of Section V b

The inferred secondary dormancy that was induced by delayed incubation was partly overcome by the fire-related cues, and more so by the combination of cues and more favourable incubation conditions. A high level of secondary dormancy is predicted for the soil seedbank following post-fire drought, which prevents rather than delays germination. The null hypothesis that the fire-related cues would not affect germination of seed that was treated with heat shock and/or smoke and then stored dry for 168 days was only partly rejected.

Section VI 50˚C heat shock +/- smoke, followed by 0 or 68 days delay, then re-treatment with fire- related cues before incubation; Baeckea imbricata (population 1)

The aim was to assess whether germination of seed treated with a low level of heat shock would be affected by delayed incubation.

The effect of ‘post-fire drought’ was not dependent on heat released by the ‘fire’, as 50 and 75˚C heat shock followed by a delay before re-treatment with fire-related cues and incubation had comparable effects on germination of Baeckea imbricata (population 1). The 50˚C heat shock followed by 68 days delay and re-treatment with fire-related cues before incubation reduced germination (F 3,40 = 11.13, P < 0.0001; data not shown).

The level of heat shock experienced by seed did not alter the effect of delayed incubation, therefore the effect of post-fire drought may not be dependent on fire intensity. The null hypothesis that germination of seed treated with a low level of heat shock would not be affected by delayed incubation was rejected.

266 Section VII Soil texture and water retention

Soils ranged from sandy loam to sand (Table 5.6; Fig 5.5). All soil was characterised by a predominance of coarse sand, and low values for bulk density, silt, clay and organic matter. Soil moisture ranged from nearly 50% down to less than 5% at ‘field capacity’ (Figure 5.6). Soil moisture retention was negatively correlated with percentage coarse sand and positively correlated with percentage silt and organic carbon, as expected (Salter et al. 1966). The available water decreased markedly from sand to loamy sand because almost all water in sand is freely draining, whereas more than half of the water in saturated loamy sand is held at between field capacity and 0.8 MPa tension (Salter & Williams 1965b). Soil moisture was greatly reduced by low extraction pressure, and the pattern of moisture retention was similar across samples due to the common predominant influence of coarse sand (Figure 5.6). Soil moisture retention was highest for the run-on samples, and lowest for the samples mid-slope as expected due to the particle distribution determined by erosion. The contrast would have been even greater if the soil had not been sifted, because percentage soil moisture was slightly underestimated for disturbed samples of sandy loam, but not for sand (Salter & Williams 1965a), and because the percentage of gravel within the soil matrix was greatest mid-slope, and gravel greatly reduces soil moisture content (Reinhart 1961). Interestingly, the samples from flat ridge tops are indicative of inherent variability across locations where erosion is not as predominant. The variability along the moisture retention profiles of soil from ridge tops was almost as great as that between run-on and mid- slope samples (Figure 5.6). The percentage moisture of ridge-top soil was highest at site 3, lower at site 1 due to higher bulk density and lower organic matter, and lower again at site 2 due to higher bulk density, lower organic matter and coarser soil texture (Table 5.6).

Soil particle size

When averaged across all samples, 42% of the soil weight was comprised of particles in the 0.5 - 0.25 mm diameter range (Table 5.6). The average soil particle size ranged from 0.18 mm at the bottom of site 1, to 0.54 mm at the middle of site 1, and averaged 0.36 mm (SE 0.04) across all site x locations.

267 Table 5.6. Hawkesbury Sandstone derived soil texture, structure, and organic matter characteristics for three topographic locations at three study sites. Soils arranged in order of decreasing water-holding capacities (see Figure 5.6).

Soil type Site Topographic Gravel as a Soil matrix % composition Bulk % (fabric) location % of soil density Organic matrix matter > 5 < 5 <2 <0.5 <0.25 <0.02 silt clay mm mm mm mm mm mm

Sandy loam 1 Bottom 0 0.2 5.0 27.8 4.6 36.4 8.5 17.6 0.98 7.3 (porphric)

Loamy 3 Top 0.2 3.3 17.6 34.4 6.8 24.9 4.9 11.4 0.71 8.4 sand (plectic)

Loamy 2 Bottom 0.8 0.6 14.5 43.1 6.4 19.5 4.5 11.9 1.07 5.5 sand (plectic)

Loamy 3 Bottom 0.8 0.2 9.8 42.8 7.6 25.0 4.5 10.3 0.83 7.8 sand (plectic)

Loamy 1 Top 2.7 0.5 5.4 46.3 8.1 24.1 5.7 10.4 1.06 5.7 sand (plectic)

Sand 1 Middle 12.2 11.2 30.5 40.0 3.9 15.8 1.9 7.8 1.73 3.8 (chlamydic)

Sand / 3 Middle 7.7 1.4 27.9 40.3 4.6 15.8 2.0 9.4 1.21 3.6 loamy sand (chlamydic) Sand 2 Middle 5.4 0.9 22.6 43.7 5.9 17.5 1.5 8.9 1.19 2.9 (chlamydic)

Sand 2 Top 0.2 4.5 7.7 56.9 6.9 18.2 3.4 6.9 1.13 3.6 (chlamydic)

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Figure 5.5: Texture of Hawkesbury Sandstone derived soil (<2 mm) at three topographic locations (ridge top = top; mid-slope = mid; bottom flat = run-on) at three study sites (1 – 3). Soils arranged in order of decreasing water-holding capacities (see Figure 5.6). Particle size categories are >0.5 mm (█), <0.5 mm (), <0.25 mm (), >0.02 mm (), silt <0.02 mm (||||), clay <0.002 mm (2).

269 50 sandy loam (bottom site 1) loamy sand (top site 3) 45 loamy sand (bottom site 2) loamy sand (bottom site 3) 40 loamy sand (top site 1) sand (middle site 1) sand / loamy sand (middle site 3)

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10

5

0 0.01 0.06 0.11 0.16 0.21 0.26 0.31 0.36 0.41 0.46 0.51 0.56 0.61 0.66 0.71 0.76 0.81 0.86 0.91 0.96 1.01 1.06 1.11 1.16 1.21 1.26 1.31 1.36 1.41 1.46

Extraction pressure (- MPa)

Figure 5.6. Soil moisture extraction curves for Hawkesbury Sandstone derived soil at 3 topographic locations at 3 sites.

270 Seed size

Seed diameters calculated for a number of study species ranged from 0.31 to 0.72 mm (Table 5.7). The average calculated seed diameter was 0.44 mm (SE 0.02).

Table 5.7. Calculated diameters of seeds of 23 study populations.

Species Population Diameter (mm)

B. diosmifolia 0.45

B. brevifolia 0.55

B. imbricata 1 0.38

2 0.38

3 0.39

B. linifolia 1 0.46

2 0.46

B. ramosissima .ssp. ramosissima 0.72

B. utilis 0.54

D. secundum 1 0.39

E. coriacea 0.42

E. crassifolia 0.31

E. longifolia 0.55

E. microphylla. var. rhombifolia 0.38

E. muelleri 0.37

E. obtusifolia 1 0.40

E. paludosa 1 0.40

E. pulchella 0.56

K. ambigua 0.42

K. capitata 0.46

M. ciliata 0.47

S. monticola 0.31

W. pungens 1 0.45

271 5.4 Discussion

Three separate findings indicated a high degree of exogenous control over post-fire germination. Firstly, the fire-related cues increased the degree of exogenous control over post- fire germination by reducing the hydrotime requirement for germination, by reducing the variance of the median base water potential, and by increasing the median base water potential. Secondly, the effect of temperature of incubation on germination was more pronounced when water availability was intermittent, and thus more representative of field conditions. Thirdly, ‘post-fire drought’ greatly reduced germination and possibly induced secondary dormancy. A high degree of exogenous control may restrict post-fire germination to favourable microsites, resulting in both some germination and the retention of a residual soil seedbank to buffer against the risk of local extinction.

Water potential and fire-related germination cues

The absolute and relative germination-promoting effects of the fire-related cues became increasingly apparent at lower water potentials. In absolute terms, the difference in final germination between treatments became greater, and in relative terms, the difference between the promotive effects of the individual cues when applied singly, and when applied in combination became greater with decreasing water potential. The effect sizes were small, but these differences are likely to be important in the field, where water availability is frequently limited. Reduced water availability also exacerbated differences between germination across incubation temperatures. Most notably, high levels of germination following warm season fire would be predicted for Kunzea ambigua from experiments performed under benign laboratory conditions, but low levels of germination would be predicted from experiments performed under conditions more closely resembling the field. Again, a requirement for field testing of hypotheses derived from laboratory results is apparent.

272 Influence of fire-related germination cues on hydrotime and the median and variance of base water potential

Increased germination was associated with a reduction in the hydrotime requirement caused by the fire-related germination cues for all species where hydrotime could be accurately assessed. The increase in median base water potential due to the fire-related germination cues may arise because a reduction in base water potential represents a risky strategy (sensu Kigel 1995), reducing both the residual seedbank and the probability of seedling establishment. Also, Hawkesbury Sandstone derived soils have as a rule, a low percentage loss of organic matter and negligible reduction of water-retaining capacity due to fire (Beadle 1940). In contrast, the variance in base water potential was reduced, hence any spread in germination over levels of water availability was determined more by extrinsic than innate factors.

Water requirements for germination across habitats

Water requirements are only meaningful in the context of water availability. The Kunzea species inhabit soils high in sand content, characterised by poor water retention and rapid drying. Kunzea seeds had high median base water potentials with intermediate to low variance, and low hydrotime requirements characteristic of species from such an environment (Allen et al. 2000). Consequently, species from such environments rapidly germinate when water is not limiting but are strongly inhibited from germination at reduced water potentials (Allen et al. 2000). The germination responses of the Kunzea species are ecologically significant because they tend to minimize post-germination seedling losses (Mott 1972; Matthews 1976; Evans & Etherington 1990). In contrast, the high hydrotime requirements of the wet habitat species would restrict germination to within wet habitat by preventing a germination response to short-term water availability. Baeckea linifolia is confined to wet habitat and the lack of an increase in germination within the fluctuating water potential treatment may be due to a high hydrotime requirement. Slow germination can be as effective as dormancy in preventing germination under unfavourable conditions (Newman 1963). A repeated hydration and dehydration treatment characteristic of a dry habitat resulted in germination of a species from dry sandy soil heathland that was both greater and markedly more rapid than that of the wet heathland species (Pons 1989). Because prolonged incubation at low water availability induced

273 secondary dormancy (Khan & Karssen 1980; Khan et al. 1980/81; Khan & Samimy 1982), the low base water potential of wet habitat species may be required for them to maintain sufficient hydration whilst making an assessment of the long-term availability of water in their habitat.

Physiological mechanisms whereby germination is increased and decreased

The increase in median base water potential and reduction in hydrotime required for germination produced by the fire-related cues has also been caused by priming (Dahal & Bradford 1990; Bradford & Somasco 1994). Priming promotes radicle growth (Carpita et al. 1979; Haigh 1988, unpublished), and reduces resistance to embryo expansion (Gibson & Bachelard 1986). Hormones mediate these changes (Karssen et al. 1989; Dahal & Bradford 1990; Sanchez et al. 1990; Hilhorst & Karssen 1992; Nonogaki et al. 1992; Bradford et al. 2000), and smoke sensitises seeds to growth hormones (Thomas & van Staden 1995; van Staden et al. 1995; van Staden et al. 2000; Schwachtje & Baldwin 2004). The fire-related germination cues may have reduced hydrotime requirements through interactions with endogenous hormones. Progression towards germination depends on the balance between the stimulatory effects of gibberelic acid and the repressive effects of abscisic acid (Koorneef & Van der Veen 1980; Koorneef et al. 1982; Karssen et al. 1983; Hilhorst & Karssen 1992; Grappin et al. 2000; Gomez-Cadenas et al. 2001). Smoke followed by continuous water availability reduced the level of abscisic acid in seeds of a post-fire annual (Schwachtje & Baldwin 2004), however, drought stress increased the level of abscisic acid in seeds (Leung & Giraudat 1998; Seo & Koshiba 2002), thus inhibiting germination (Kim et al. 2003). Abscisic acid dramatically lowered the ability of seeds to cope with osmotic stress (Liptay & Schopfer 1983; Schopfer & Plachy 1984). The reduced germination of smoked seeds under drought conditions in the current study may have been mediated by abscisic acid. Exogenous abscisic acid produced elongation without radicle emergence (Leubner- Metzger et al. 1995; Leubner-Metzger & Meins 2000; Krock et al. 2002). The elongation without radicle emergence of the Kunzea ambigua seeds may have been because the excessive smoke (Keeley & Fotheringham 1997; Light et al. 2002; Willis et al. 2003) made the seeds highly sensitive to abscisic acid (van Staden et al. 1995). Smoke may also sensitise seeds to the growth promoting hormone gibberelic acid (Thomas & van Staden 1995; van Staden et al. 1995; Schwachtje & Baldwin 2004).

274 Mechanisms whereby gibberelic acid promotes germination (Groot & Karssen 1987; Chen & Bradford 2000; Nonogaki et al. 2000; Wu et al. 2001) are also hydration dependent (Georghiou et al. 1983; Bradford 1990). In the current study, the increased germination of smoked seeds when water was not limiting may have been mediated by gibberelic acid.

Temperature, water potential and fire-related germination cues

Heat shock and smoke had the same effect as a growth regulator that increased germination across germination-inhibiting incubation temperatures and water potentials (Kaufmann & Ross 1970). The effect of less favourable incubation temperatures became more pronounced with decreasing water potentials as expected (Sharma 1976; Dahal et al. 1993; Dutta & Bradford 1994; Alvarado & Bradford 2002). Although germination of Kunzea seeds was greater within the lower incubation temperature, the difference was not associated with differences in hydrotime requirements or median base water potentials. The greater germination of Epacris obtusifolia (population 2) seeds within the higher incubation temperature was associated with lower median base water potential. Although the hydrotime requirement of seeds treated with the combination of added heat shock and smoke was low within the suboptimal incubation temperature, germination was most likely reduced due to the high median base water potential. A complex interaction between incubation temperatures, fire-related germination cues, hydrotime requirement and median base water potential affected germination of Epacris obtusifolia (population 2). The complex interaction between incubation temperature and heat shock that affected germination of smoked Baeckea imbricata (population 2) seeds could result in germination from different depths of burial across different seasons. During warmer seasons, when ambient temperature stays above 20°C in the upper 4 cm of soil (Auld & Bradstock 1996) and fire is likely to generate 50°C heat shock at least to this depth (Auld 1986a), then germination is likely to be stimulated throughout a wide depth profile. High midday temperature in shallow soil following warm season fire (Auld & Bradstock 1996) might reduce germination in shallow soil, and thus the probability of seeding desiccation. In contrast, ambient temperature stays below 20°C in the upper 4 cm of soil during cooler seasons (Auld & Bradstock 1996), but fire is likely to generate 75°C heat shock only within the top 1 cm (Auld 1986a; Auld & Bradstock 1996). Particularly if low temperature physiologically limits seedling emergence, then such depth-detection may prevent fatal germination. Also, germination in shallow soil is less likely to result in seedling desiccation in winter than in

275 summer. Detection of the depth of burial may be mediated by integration of the heat shock level and ambient temperature information. Ambient temperature is probably sensed through the membrane (Meyer 1986), and the integration of information may also occur at the membrane (Hilhorst 1998, Hallett & Bewley 2002).

Retention of cues over durations of incubation at low water potential

Incubation at low water potentials was inferred to have induced a degree of secondary dormancy in all species of the current study, as germination following transferral into free water remained lower than germination of seeds that were continuously incubated in free water (Khan 1960; Khan & Karssen 1980). High levels of germination following transferral of Kunzea seeds into light and re-treatment of Epacris obtusifolia (Chapter 4) indicate that viability was not affected by prior treatments. The fire-related germination cues reduced the degree of inferred dormancy induced in some populations, and the effect was influenced by the duration of incubation. When both Epacris coriacea and Kunzea ambigua seeds were transferred from low water potential into free water, the germination response was influenced by an interaction between the duration of incubation at low water potential, and smoke treatment. The germination of seeds transferred after a short duration at low water potential was increased by prior smoke treatment (Section I), whereas the smoke effect was not present after a longer duration at low water potential (Section II). These results suggest that the germination promoting effect of the fire-related cues may decay over time, under incubation conditions. Growth-promoting hormones prevent the induction of secondary dormancy that occurs during prolonged incubation at low water potential (Khan & Karssen 1980; Khan et al. 1980/81; Khan & Samimy 1982), possibly by reducing the restraint on embryo expansion (Hemmat et al. 1985). Because smoke sensitises seeds to hormones, sensitivity to water availability could arise in smoked seeds. The fire-related germination cues probably advanced the germination process, with subsequent advancement dependent on hydration, and reversion during dehydration.

276 Germination within the fluctuating water potential treatment

The germination pulses associated with increased water availability within the fluctuating water potential regime were expected (Roberts & Potter 1980; Roberts 1984). Also, the rapid re-establishment of the block to germination by water stress when seeds were transferred from water into PEG E.G. solutions has been reported (Hegarty & Ross 1980/81; Dell’Aquilla 1992). Germination within the fluctuating water potential treatment was similar to that at the intermediate water potentials, probably because they experienced similar hydrotimes. Hydrotime modelling has predicted germination under variable field conditions (Finch-Savage & Phelps 1993; Finch-Savage et al. 1998, 2000; Allen et al. 2000; Durr et al. 2003). The fluctuating water potential and intermittent water availability treatments had markedly different effects on germination of Kunzea ambigua; water availability in soil derived from Hawkesbury Sandstone is better represented by the intermittent water availability treatment.

Influence of fire-related germination cues on germination of Kunzea ambigua within the intermittent water availability treatments

Secondary dormancy was probably induced by intermittent watering alone i.e. in the absence of fire-related germination cues, within the supra-optimal incubation temperature. Fluctuating water conditions can cause secondary dormancy (Hegarty 1978; Bewley & Black 1982a), and the induction of secondary dormancy can depend on the incubation conditions. Unless prior incubation was within alternating temperature and/or light, then secondary dormancy was induced by a period of dehydration during the germination process; consequently deeply buried seed is expected to be dormant and surface seed to remain germinable (Vincent & Cavers 1978). Similarly, Kunzea ambigua seeds may be mostly dormant during warm seasons due to the influence of intermittent hydration in the field. Hydration in the field is also dependent on the soil matrix surrounding the seed, and germination was reduced by the combination of a poor water supply matrix and less favourable incubation temperature (Nutile & Woodstock 1967). The reduction in germination due to intermittent watering within the supra-optimal incubation temperature was not overcome by the fire-related germination cues, hence germination following warm-season fire may be very low relative to germination following cool-season fire. Winter burning is predicted to favour germination of Kunzea ambigua. In

277 contrast, dry summers are stochastic in occurrence (Bradstock & Bedward 1992), hence their effect on germination of K. ambigua is unpredictable in the short-term. Small amounts of rainfall during a dry summer may be important for germination of K. ambigua, as intermittent hydration at the supra-optimal temperature prevented the induction of deep dormancy that was inferred within the 47 days drought treatment. An interaction between seasonal temperature and rainfall is predicted to strongly influence post-fire germination.

Influence of fire-related germination cues on germination when followed by drought

Germination of all but one species that were otherwise stimulated to germinate by heat shock and smoke was reduced when seeds were not hydrated soon after receiving these treatments. Gemination was stimulated by transferral into more favourable incubation temperatures and by re-treatment with the fire-related germination cues, hence the delay before hydration probably induced secondary dormancy. In the field, post-fire richness and density of native seedlings were both positively associated with rainfall in the first 6 months (Ross et al. 2004), and germination of fire-stimulated species was highly reduced when smoke was applied in an unfavourable season with subsequent low rainfall (Roche et al. 1998). Also, low post-fire rainfall probably induced dormancy in a fire-stimulated species (Keith 2002), and low post-fire rainfall has been observed to result in reduced recruitment through germination in the Sydney region (Auld pers. comm. 2002). Fire followed by drought can alter species composition (Jacks 1984, unpublished in Frazer & Davis 1988), and obligate seeders are more affected than resprouters (Frazer & Davis 1988; Saruwatari & Davis 1989; Thomas & Davis 1989). Whilst seedling survival is more important than the number of seedlings produced (Frazer & Davis 1988; Thomas & Davis 1989), different levels of germination may initiate a change in species composition. Fire followed by drought had little or no inhibitory affect on germination of the obligate seeders Epacris obtusifolia and E. coriacea respectively, and these two species had the lowest base water potentials and highest ‘post-fire’ hydrotime requirements. Species with such characteristics may predominate when fire is followed by drought. Nevertheless, a fraction of each population germinated when the fire-related germination cues were followed by a delay before hydration, despite the level of seed hydration being lower for a longer duration than would be encountered in the field. Similar levels of germination of Kunzea ambigua occurred at 25°C when seeds were treated with fire- related germination cues and intermittently hydrated for 4 out of 44 days before continuous

278 incubation (5%) as when post-treatment seeds were not hydrated for 47 days before incubation (2%). The similar levels of germination indicate that once the level of hydration passes below a threshold, the fraction of the population that will germinate does not continue to decrease. The probability of a population persisting would be enhanced if a fraction of the soil seedbank germinated following all or most fires.

Contrasts between the influence of ‘post-fire drought’ across seed types, and across climates

The application of fire-related germination cues followed by dry storage possibly induced secondary dormancy in Grevillea seeds from the Sydney region. A one week delay between the application of aerosol smoke and water reduced germination of Grevillea seeds, compared with the immediate application of water (Kenny 2003, unpublished). Smoke was applied to buried seeds and, when watering was delayed, seeds were transferred into petri dishes before watering. The reduction in germination due to delayed watering may have been caused by a reduction in the amount of smoke reaching seeds because the smoke was not washed into the soil, but for a given amount of smoke adsorption, the positive effect of the smoke may increase with increasing hydration status of the seed. The reduction in germination of the water-permeable seeds of the study species due to ‘post-fire drought’ contrasts markedly with the progress towards germination of heat shocked leguminous seeds during four weeks of dry storage (Martin & Cushwa 1966). Also, the inferred induction of dormancy in species from the Sydney region where rainfall is only slightly seasonal (Nix 1982) contrasts with species from Mediterranean climatic regions where the germination-promoting effect of aerosol smoke (Roche et al. 1997a) or aqueous smoke extracts (Baxter & Van Staden 1994; Brown et al. 1994; Tieu 1999), was retained in dehydrated seed for as long as one year (Brown & van Staden 1998). The retention of a smoke effect in dry storage may be beneficial for seeds from these Mediterranean climatic regions where the fire prevalent season and the season with adequate moisture for seedling recruitment are temporarily separated by long periods of time (De Lange & Boucher 1993b; Roche et al. 1997b). Because rainfall adequate for germination is frequently likely in the month following fire in the Sydney region regardless of season (Bradstock & Bedward 1992), a different response to post-fire hydration dynamics of seed may have evolved which ensures the retention of a soil seedbank.

279 Fire-related germination cues, soil water dynamics and germination in the field

The response to post-fire hydration dynamics may be a mechanism that ensures a fraction of the population germinates and a fraction remains as a residual soil seedbank. The duration over which the fire-related germination cues remain active is probably extended for seeds with more favourable post-fire hydration dynamics. More favourable hydration dynamics occur in more humid microsites, and when seeds under an intermittent watering regime were subsequently subject to no water stress, then germination remained depressed in the low humidity microsites, but final germination was not affected by the watering history in the high humid microsites (Battaglia & Reid 1993). Emergence in the field was also depressed when planting was followed by periods of dry conditions prior to adequate moisture for germination, and the dry conditions were determined by both microsite and seasonal weather patterns (Battaglia & Reid 1993). Thus, each seed ‘summed its environmental experience’ (Trewavas 1988; Dubrovsky 1996), and the population was divided on the basis of their hydration history into seeds that would and would not germinate when conditions became favourable, resulting in germination that was largely confined to favourable microsites. Because the fire-related germination cues stimulate germination for a limited period of time, and germination is influenced by post-fire water availability, germination is probably confined to favourable microsites (or submicrosites; as the mass of a seed is characteristically compressed into the most economically compact space, it exists within a submicroenvironment, Koller 1972). After embryo growth has begun, the expanded tissues can be damaged by desiccation and unable to resume growth (Berrie & Drennan 1971; Hegarty 1977; Schopfer et al. 1979; Schopfer & Plachy 1984). An expanding radicle must keep pace with the decending soil water zone (Hillel 1972; Koller 1972), and susceptibility to death remains high if water availability is inadequate before root formation (Hassanyar & Wilson 1978; Watt 1978, 1982; Senaratna & McKersie 1983; Fulbright et al. 1984; Auld 1986b; Frasier 1987; Tozer 1998; Leprince et al. 2000). If fatal germination is less likely in a microsite with long-term favourable moisture characteristics, then germination is closely coupled with the probability of successful recruitment. Most importantly, the confinement of germination to favourable microsites also ensures that a residual seedbank will be retained in the unfavourable locations.

280 Fire-related germination cues, residual seedbanks and local extinction

Residual seedbanks buffer populations against local extinction due to fire-return intervals that can be as short as 18 months in the Sydney region (Bradstock et al. 1997) and, to a lesser extent, due to poor recruitment (Lamont et al. 1991; Keith 1995, 1996). Whilst depletion of the non-leguminous seedbank following one fire may reduce recruitment after a subsequent fire (Clark 1988; Cary & Morrison 1995), residual post-fire soil seedbanks are present and do buffer against short fire-return intervals (Bradstock et al. 1997; Vaughton 1998). Germination-promoting stimuli associated with fire that are received by only a fraction of the soil seedbank, such as a narrow range of heat shock (Chapter 2) or light (Chapters 2 & 3), could ensure a residual seedbank. However, cool season fire is predicted to result in a non- dormant Kunzea soil seedbank, and non-germination of non-dormant seed is probably due to variability in water availability at the microsite scale. Microtopography (Evans & Young 1972; Adamson et al. 1983; Battaglia & Reid 1993), and texture of the soil surrounding seed (Hagon & Chan 1977) largely determine water availability at the microsite scale.

Hydraulic conductivity of Hawkesbury Sandstone-derived soil

Microsite quality for the study species is likely to vary enormously due to their small seed size and the coarse soil texture. The inherent coarse texture of the Hawkesbury Sandstone derived soil was responsible for the rapid decrease in moisture content with external pressure. There were substantial differences in water retention across samples of different texture because the more coarse textured soil is more freely draining. Species segregation on soil derived from Hawkesbury Sandstone can be caused by differences in water retaining capacity that are due to the coarseness of soil texture rather than poor drainage or soil depth (Davis 1941). Hydraulic conductivity is related to the total cross sectional area available for flow, and because the largest pores empty first, and because coarse textured soil contains a larger proportion of large pores, the hydraulic conductivity of the more coarse textured soil decreases more rapidly with decreasing soil water content. Also, the pathway becomes more torturous when soil water is conducted through the few small pores. Soil hydraulic conductivity calculated for a sandy loam was 5.4 x 10-13 m2 s-1 Pa-1 when at 20% moisture content (field capacity), decreased by an order of magnitude when at 12% moisture content (–0.1 MPa soil water potential), and decreased by another order of magnitude when at 8% moisture content (–0.35 MPa soil water potential) (Young & Nobel

281 1986). Soil hydraulic conductivity would be even lower in loamy sand, and lower again in sand. Seed water uptake from soil decreased markedly over a very short distance and was negligible beyond 1 cm (Dasberg 1971), thus the differences in hydraulic conductivity due to texture in the immediate vicinity of a seed are likely to greatly affect germination (Williams & Shaykewich 1971; Ward & Shaykewich 1972; Hadas & Russo 1974a).

Soil-seed contact area; Hawkesbury Sandstone-derived soil and study species

The size of the study species seed is similar to a large percentage of the Hawkesbury Sandstone derived soil particles thus, whilst the contact points between soil water and seeds are few and minute in area (Hadas 1974), the area of contact is especially low due to the high soil grain to seed size ratio (Hadas & Russo 1974b). The area of contact is also reduced by lower matric potential, and the matric potential of the surface soil derived from Hawkesbury Sandstone is rapidly lowered because it is freely draining. Considering only the sand of similar size to the study species seed, the fractional wetted areas of the seeds are approximately 20% at –0.01 MPa, 2% at –0.1 MPa and 0.2% at –1.0 MPa matric potential. Assuming the seed was surrounded by only the fine sand particles (0.25 mm diameter), the fractional wetted areas are approximately 65% at –0.01 MPa, 8% at – 0.1 MPa and 0.8% at –1.0 MPa matric potential (Collis-George & Hector 1966). The water uptake rate, germination rate and final germination percentage of seed is greater within less coarsely textured soil (Young et al. 1970; Young & Evans 1972; Currie 1973; Hadas 1974; 1976; 1977; Hagon & Chan 1977). High variability in seed water uptake occurred at high matric potential because of variability in the contact area between seed and soil particles (Josiah et al. 1994), emphasising the extreme importance of differences in the texture of soil immediately adjacent to seeds.

Soil water variability within and across habitats

The expected pattern of soil derived from Hawkesbury Sandstone having coarse texture on slopes (Erskine & Melville 1983), and less coarse texture in fans (Erskine & Melville 1983) and dells (Melville & Fitzpatrick 1983) was found in the current study. However, variability within topographic units was also present, including the full range of soil water properties across the three ridge tops.

282 Except after rain, the flow of water into seeds of the study species within Hawkesbury sandstone derived soil will be greatly limited by conductance and contact area (Collis-George & Hector 1966; Hadas & Russo 1974b). Conductance and seed-soil contact area may be highly variable depending on the location of the seed relative to the sand grains because coatings of clay and silt on sand grains broaden and coalesce where adjacent sand grains approach each other (Brewer 1979). The microclimate in the zone that influences seed water status can vary substantially, and affect subsequent germination (Harper et al. 1965; Harper & Benton 1966; Sheldon 1974; Potts 1986; Battaglia & Reid 1993). Soil water is highly variable at a small scale (Bond & Harris 1964; Burrough 1983; Arp & Krause 1984), and has been measured at the grain of sand scale (Garrido et al. 1999). Also, the pathway of water flow will influence water availability at the scale of a seed because flow is not uniform but preferential (Bond 1964; Garrido et al. 2001). Germination is affected by much less than the variability in soil water that exists in the field (Bachelard 1985; Battaglia 1993), and the water available from soil derived from Hawkesbury Sandstone to the study seeds probably varies greatly relative to the influence that this factor has on germination.

Small, water-permeable, soil-stored seeds

The trade-off between fewer larger or more, smaller seeds has been demonstrated for the Hawkesbury Sandstone vegetation (Henery & Westoby 2001). The greater seed dispersal of more, smaller seed reduces spatial and temporal variation in success (Venable & Brown 1988). Increasing seedbank dormancy also reduces spatial and temporal variation in success (Venable & Brown 1988; Kalisz & McPeek 1993) however dormancy is not necessary for maintenance of a persistent seedbank (Thompson et al. 2003), and environmental factors can cause intermittent germination of non-dormant seed (Cavers et al. 2000) thus providing insurance against the loss of a cohort (Harper & McNaughton 1960; Salisbury 1961). Because of their small size, seed of the study species almost certainly experience very high variability in microsite quality. It can be postulated that seeds in poor microsites would remain ungerminated because the balance of germination promoting to inhibiting factors is unfavourable over the period that the fire-related cues are active. Ungerminated seed buffers against the risk of local extinction. Notably, the fire-related germination cues reduced the variance of the median base water potential, thus increasing the influence of the microsite over germination. Possible

283 interactions between microsites and post-fire weather over successive fires are illustrated in Figure 5.7. Whether a seed that has been stimulated to germinate by fire-related germination cues actually germinates is determined by the exogenous factors of microsite and post-fire weather. The endogenous factor of a range of seed dormancy levels is not required to ensure a residual seedbank in Figure 5.7, however, it would add to the variability in the likelihood of germination of a given seed. Post-fire erosion of soil derived from Hawkesbury Sandstone (Blong et al. 1982; Humphries & Mitchell 1983), and inter-fire soil movement due to the combination of rainwash and biotic factors such as lyrebird (Adamson et al. 1983), anteater (Mitchell 1988), and ant and worm activity (Humphries 1981; Humphries & Mitchell 1983) could result in frequent alteration of microsite quality. However, the slight movement of a seed and/or of soil particles in its vicinity could also alter the seed/soil contact and thus microsite quality. The regulation of the fraction of seeds that can germinate by environmental conditions rather than by innate dormancy is characteristic of a predictable environment (Venable & Lawlor 1980; Freas & Kemp 1983; Clauss & Venable 2000) such as the Sydney region (Nix 1982).

284 Figure 5.7. A schematic representation of the potential for the degree of soil / seed contact to determine soil seedbank dynamics (see text). Three successive fires are followed by germination of some seed and a residual seedbank, despite no seedbank replenishment.

First fire followed by low rainfall:

Germination

Second fire followed by high rainfall:

Germination

Third fire and redistribution of fine soil particles:

Germination Residual soil = seed seedbank

= coarse soil particle

= fine soil particle

285 Chapter 6. Effects of fire on germination of aged seed

6.1 Introduction

Overview

The question of whether species forming soil seedbanks in the fire-prone Sydney region continue to germinate in response to fire-related cues after seed has been acceleration aged is investigated in this section.

Sub-populations within a soil seedbank

A persistent soil seedbank consists of surviving seeds from a series of crops, and is thus multi-aged. Different levels of seed dormancy and/or vigour may be associated with different ages, where vigour is a manifestation of the ability to survive a series of environmental stresses during germination (Abdul-Baki 1980). Also, seeds of different ages may differ in their physical and physiological status. These different sub-populations may respond differently to germination cues associated with fire, and thus germination from each sub-population may not be equally proportional to their numbers in the seedbank.

Seed aging and death

Seed aging, evident in increasing deterioration with age, precedes death, the probability of which increases with age (Ellis & Roberts 1981). Seed mortality curves often show an initial period of stability preceding a dynamic rate of cumulative mortality (Bernal- Lugo & Leopold 1998). This period of initial stability may be lacking in a species, or following long-term storage under poor storage conditions (Crocker & Barton 1957 in Bernal- Lugo & Leopold 1998), or progressively decrease with increasing levels of accelerated aging (Berjak & Villiers 1972a; Kraak & Vos 1987; Argerich et al. 1989; Tarquis & Bradford 1992). Hence, the rate-controlling processes may change during seed aging, or differences in the deteriorative mechanisms may exist between the early and later stages of deterioration (Bernal-Lugo & Leopold 1998). Levels of aging are required to investigate the effects of cumulative changes.

286 During the initial period when the rate of seed deterioration is very low, degenerative events accumulate with age, but are countered by repair processes. Similar cytological damage occurred in imbibed dormant seeds and dry stored seeds (Villiers 1972), however, the level of damage was greater in the dry stored seeds and they had substantially less longevity (Villiers 1972; Villiers 1974). Imbibed, non-germinating seeds carry on a variety of activities (Villiers 1971), allowing repair of some damage (Villiers 1974; Villiers & Edgecumbe 1975). Repair processes occur soon after imbibition of aged seeds (Rao et al. 1987; Davison et al. 1991), and germination is delayed whilst accumulated cytological damage is repaired in seeds that have been rapidly aged (Berjak & Villiers 1972a), dry stored (Berjak et al. 1986), or stored whilst imbibed and dormant (Villiers 1972). Both the rate of damage, and the rate of repair increase with increasing levels of seed hydration, and if seeds are alternately dried and imbibed, damage occurring during the dry period is rapidly repaired when seeds are imbibed (Villiers & Edgecumbe 1975). Aging reduces the capacity of seeds to repair damage (e.g. Osborne 1980, 1983; Sung & Chiu 1995; Bailly et al. 1996). Equivalent damage was far more consequential in aged than young seeds (Tarasenko et al. 1965), probably because the capacity to repair this damage becomes impaired in dry stored (Elder et al. 1987), and artificially aged seed (Vazquez- Ramos et al. 1988; Vazquez et al. 1991; Elder & Osborne 1993). Heat shock reduced protein synthesis in aged wheat (Dell’Aquila & Di Turi 1996), and wheat and barley seeds relative to unaged seeds (Dell’Aquila et al. 1998). Also, protein synthesis, including synthesis of an enzyme required for germination (Livesley & Bray 1993) was reduced following heat shock applied to low vigour relative to high vigour wheat seeds (Helm et al. 1989; Livesley & Bray 1993). Protein synthesis may be required to repair membrane damage (Bewley 1986). Progressive membrane deterioration, accompanied by damage to repair mechanisms has been considered adequate to provide an explanation for the decline in vigour and loss of seed viability with age (Smith & Berjak 1995). Cell membrane integrity is likely to be critical if, as proposed by Mayer (1986), membranes are the site at which water potential, light and temperature are sensed, and the changes occurring in the membrane determine the seed’s response to its environment. Cell membrane structure has been associated with the regulation of dormancy and germination using many lines of evidence (Hilhorst 1998, Hallett & Bewley 2002). Cell membranes are affected similarly by both long term and accelerated aging. Deteriorative changes in membranes occur during aging (e.g. Hallam 1973; Villiers 1973; Dawidowicz-Grzegorzewska & Podstolski 1992; Smith & Berjak 1995), and were associated

287 with viability loss (Parrish & Leopold 1978; Kole & Gupta 1982; Basra et al. 2003; Krishnan et al. 2003). Mitochondria of the root tip cells were amongst the first organelles to show damage in maize seeds when age-accelerated (Berjak & Villiers 1972a, b, c), and when aged in the long term (Berjak et al. 1986). Membrane repair occurred following imbibition in embryos from intermediate aging stages, but this repair did not occur in more severely aged seeds, and total degeneration of the protoplast followed (Berjak & Villiers 1972c). The degeneration of structure of imbibed seeds over time is probably similar to that occurring in natural situations in the soil (Villiers 1980) and, as the processes that occur during aging may depend on the level of seed hydration (Vertucci 1993), deterioration under humid conditions may be more similar to aging in soil than deterioration under dry storage conditions. The rate of decline in seed viability can be predicted from the moisture content and temperature at which they are stored (Roberts 1973). The relative effects of seed water potential (Ellis et al. 1989; Roberts & Ellis 1989) and temperature (Dickie et al. 1990) on longevity apply over a diverse range of species. Thus, similar deterioration is likely across species subject to accelerated aging, where seeds are stored under high moisture content and high temperature conditions. Whilst the degenerative changes within an aging seed are cumulative and predictable, the consequences of these changes may not be so predictable. The rate of germination may increase with lower levels of accelerated aging, followed by a decrease with increasing levels of aging that coincides with a reduction in viability (Gelmond et al. 1978; Perl et al. 1978; Welbaum & Bradford 1991). Experimental manipulation of levels of aging is required to investigate the effects of cumulative changes. Accelerated aging was used in the current study. The effect of the actual age of the seed on germination can not be predicted from the current study because the amount of actual time and the storage conditions required to produce damage comparable to that induced by the aging treatments used is not known. Instead, it was assumed that, all else being equal, the response of seeds subjected to the more extreme aging treatment was more representative of older seeds in the soil seedbank. However, the level of damage accumulated by seeds in the soil seedbank is also likely to vary across space and time. For example, the ratio of rates of damage to repair will probably vary across seasons, and be maximal when soil is hot and dry, however, these conditions are variable across and through the soil profile. The level of damage in a seed population, which may vary with age, season and spatial distribution, may interact with fire- related germination cues to determine what fraction of the population germinates following a fire.

288 Post-fire species composition will be influenced by the quantity of soil-stored seed of constituent species, and their quality, particularly if quality affects the seed response to fire. Species with more transient seedbanks would be less buffered against the effect of a reduction in seed quality, whether it occurs during seed production or during storage in the soil. The regeneration of some obligate seeding species from fire-prone regions may be highly dependent on the most recent seed production, due to high rates of seed decay (Keeley 1977; Zammit & Zedler 1988; Pierce 1990, unpublished; Musil 1991; Pierce & Cowling 1991; Meney et al. 1994), and post-fire recruitment may be reduced due to seedbank depletion (Bond 1980). Whilst 74% species in the Sydney region have persistent seedbanks, 15% have more transient seedbanks (Auld 1994). Although most species had high longevity, a half-life of 0.4 years, assuming an exponential rate of decay was determined for a heath species from the Sydney region (Auld et al. 2000). Pre-fire combinations of a recent seed crop of low quality and environmental conditions that are damaging to the seed may dramatically reduce recruitment of a species with a short-lived seedbank. This effect would be exacerbated by factors such as fire before maturation of the next year’s seed crop and by short fire return intervals. If the increase in germination due to fire related cues is negated due to aging, then the population is less buffered against this deterioration in quality. The heat shock tolerance and capacity to repair heat-induced damage may be reduced in aged seeds. Heat shock reduced both the amount and the rate of germination of artificially aged wheat (Dell’Aquila & Di Turi 1996), and wheat and barley seeds (Dell’Aquila et al. 1998) relative to unaged seeds. Also, given that aging is highly disruptive of seed membrane structures, and that it is likely that smoke affects membrane permeability or receptor sensitivity (van Staden et al. 1995, 2000), fewer old seeds may germinate in response to the fire-related cues. However, soil storage prior to the application of charcoal (Parker 1987) or smoke increased the germination of many species (Dixon et al. 1995; Roche et al. 1997a, b; Keeley & Fotheringham 1998a). The combination of smoke and soil storage rather than aging per se was required for germination in one study (Keeley & Fotheringham 1998a), but the other studies did not include laboratory storage as a treatment, hence it is not possible to separate out aging effects from soil effects. There may be selective value in older seed becoming more responsive to fire-related germination cues, because they contribute less to the potential longevity of a post-fire residual seedbank.

289 Aims

This section of the study examined the effect of heat shock and smoke on germination of seeds that had been subject to accelerated aging. A substantial fraction of the seed of species forming persistent soil seedbanks will be aged prior to experiencing fire, hence it is appropriate to investigate the response of aged seed to fire-related germination cues. The number of species investigated allows for the detection of patterns of response to the treatments. Accelerated aging was used instead of soil storage due to time constraints, but a limitation of the study is the assumption that these forms of aging are comparable.

The null hypotheses were 1) heat shock or smoke would not affect total germination across levels of accelerated seed aging, 2) accelerated aging would not affect the rate of germination of seeds that were treated or not treated with heat shock or smoke. The implications of deterioration in seed quality on the post-fire regenerative potential of a number of species forming soil seedbanks in the Sydney region are discussed.

290 6.2 Methods

Seeds of nine species were first subject to accelerated aging and then treated with factorial combinations of heat shock and smoke. Three levels of accelerated aging were imposed; no aging consisted of dry storage in the laboratory; moderate aging consisted of 4 aging cycles and severe aging of 8 cycles. An aging cycle consisted of placing seeds on dry filter paper in an open petri dish in a sealed container with ample free water to ensure 100% relative humidity at 35˚C. These conditions were maintained for three days, then free water was added to the petri dish for one day. The free water was then drained away and the petri dish was transferred out of the humid environment into 35˚C dry air for two days. At the end of the aging process, the final cycle was ended with the humidity treatment i.e. seeds were not subsequently hydrated and dehydrated. This protocol assumes that the deleterious effects of aging were imposed during the high temperature and high humidity phases of storage, whilst the effects of changing moisture contents, which possibly resulted in some repair processes, were expected to more closely represent field conditions. Accelerated aging of two severities was applied to a batch of seeds of each species, i.e. there was no replication within treatments. However, the effect of aging was similar across the two different severity treatments for all nines species, indicating that none of the accelerated aging treatments were aberrant. Six replicates of ten seeds within each level of aging were treated with factorial combinations of heat shock ranging from 25˚C (= control) to 100˚C and 0 or 10 minutes of smoke and incubated at 25˚C. Replicates of each species were treated independently, and one replicate of each of the nine species was treated simultaneously. Seeds were incubated in the dark and monitoring of germination was as previously described (Chapter 2). Germination was monitored approximately weekly when the rate of germination was high. Monitoring frequency declined proportionally with germination rate, until seedlings of abnormal appearance that failed to elongate appeared and it was apparent that further germination of normal seedlings would be negligible. Final germination and the number of ungerminated seeds were determined for species within a 25˚C incubation temperature after about 250 days since treatment. Germination of seedlings of normal appearance was calculated as a percentage of the initial population of seeds per petri dish.

291 Transferrals across incubation temperatures

After 140 days, it was apparent that no further germination of the Kunzea species would occur at a 25˚C incubation temperature, and seeds that had been moderately aged were transferred to 15˚C for a further 140 days. After about 250 days, moderately and severely aged Gahnia sieberiana seeds were transferred into a diurnal temperature regime of 20/4 hours at 25/35˚C for a further 100 days. These incubation temperature regimes were better for germination, as determined in Chapter 3.

Data analysis

The effects of aging, heat shock and smoke were analysed using three way fixed factor ANOVAs. Homogeneity of variances was assessed using Cochran’s Test and transformations carried out as required. The heterogeneity of variances in the Kunzea ambigua data set was not corrected by transformation. Cochran’s Test was significant at P < 0.01, hence α was adjusted downward to 0.001 to compensate for increased risk of Type I error and comparisons among means was not attempted because of heterogeneity of variances (Day & Quinn 1989). Unplanned contrasts amongst means were carried out using the Student-Newman-Keuls procedure (Sokal & Rohlf 1995). Species were grouped according to their germination response to the experimental factors.

Mean time until germination

The mean time until germination was assessed by logistic analysis, as detailed in Chapter 5. Mean time until germination was considered the most appropriate parameter for comparison across aging treatments because it makes use of the central part of the logit curve, which is subject to least error, and because more frequent monitoring of germination would have been required to assess the time until first germination. Differences in mean time to germination were compared informally (Gelmond et al. 1978) across aging treatments and, when insufficient germination occurred, comparisons were made through visual inspection of the time course of germination.

292 6.3 Results

Accelerated aging affected germination of all 9 species, either as an interaction with the fire-related cues (8 species) or as a main effect (1 species) (Table 6.1). Germination of six species was greatly reduced by the aging treatments. Although variance was high when germination was greatly reduced within the aging treatments, responses to the fire-related cues were still apparent in many cases.

Age x heat shock x smoke significant

Germination of Epacris obtusifolia and Sprengelia monticola was affected by the interaction between all three factors (Table 6.1; Figs 6.1, 2). Aging did not greatly reduce germination of these two species, or cause a notable change in their responses to the fire- related cues.

Age x smoke significant

Smoke had a positive effect within at least one aged population of four species (Table 6.1). Smoke increased germination of the unaged and the severely aged Gahnia sieberiana (Table 6.1; Fig 6.3), Baeckea imbricata (Table 6.1; Fig 6.4), and Kunzea capitata seeds (Table 6.1; Fig 6.5). Probably because germination of the aged Kunzea seeds was very low and highly variable, smoke reduced germination of the moderately aged Kunzea ambigua seeds (Table 6.1; Fig 6.6). Also, germination of smoked seeds of both K. ambigua and K. capitata were lower within the moderately than the severely aged treatments, however these effects were not present when seeds were transferred into a 15ºC incubation temperature.

Age x heat shock significant

Aging did not reduce heat shock tolerance of seeds. Germination of unaged Baeckea diosmifolia seeds was increased by moderate heat shock (Table 6.1; Fig 6.7). Germination of severely aged Epacris coriacea seeds was moderately reduced within the 75ºC heat shock treatment (Table 6.1; Fig 6.8), possibly due to Type I error. Germination of unaged Kunzea ambigua seeds was greater than germination of aged seeds within all heat shock levels (Table 6.1; Fig 6.6). Added heat shock increased germination of unaged K. ambigua seeds, and

293 germination within the 75ºC heat shock treatment was greater than germination within the 100ºC heat shock treatment. The highest heat shock level increased germination of G. sieberiana across aging levels (Table 6.1; Fig 6.3). However, the response to heat shock decreased with increasing levels of aging. Germination of unaged seeds was increased by both 75 and 100ºC heat shock, germination of the moderately aged seed was increased by only the 100ºC heat shock, and germination of severely aged seeds treated with 100ºC heat shock was greater than germination within only the 25ºC heat shock treatment (Table 6.1; Fig 6.3).

Smoke significant

Smoke produced small increases in germination of Baeckea diosmifolia and Epacris coriacea (Table 6.1; Figs 6.7, 8).

Age significant

Gemination of Dracophyllum secundum was more adversely affected by the severe than the moderate aging treatment (Table 6.1; Fig 6.9). Germination of all species was lowered by the aging treatments, generally more so by severe aging, and the difference between aged and unaged seeds was greater than between the moderate and severe aging levels (Figures 6.1 – 9).

294 Table 6.1. Effects of aging, heat shock, smoke, and interactions between these factors on % germination of nine study species; ANOVA P-values are shown.

Aging Heat Smoke H x S A x H A x S A x H Residual (A) shock level x S (H) (S) df 2 3 1 3 6 2 6 120

E. obtusifolia <0.0001 0.0001 0.0097 0.0512 0.0055 0.4600 0.0331

S. monticola† 0.0179 0.7971 0.1925 0.7782 0.1066 0.0704 0.0143

G. sieberiana† <0.0001 <0.0001 <0.0001 0.6215 <0.0001 0.0182 0.5902

B. imbricata† <0.0001 0.3129 <0.0001 0.5924 0.1241 0.0135 0.8101

K. capitata <0.0001 0.4602 <0.0001 0.9118 0.1377 <0.0001 0.2793

K. ambigua‡ <0.0001 <0.0001 0.0427 0.8519 <0.0001 <0.0001 0.4370

B. diosmifolia† <0.0001 0.5677 0.0088 0.6804 0.0049 0.7913 0.8882

E. coriacea <0.0001 0.8585 0.0349 0.8796 0.0341 0.1723 0.1956

D. secundum <0.0001 0.1679 0.0706 0.7760 0.1774 0.0511 0.5256

†transformed data ‡ variances in the data were heterogeneous, hence α was adjusted downward to 0.001

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Figure 6.1. Mean final germination of a) unaged, b) moderately aged, c) severely aged Epacris obtusifolia seeds plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Bars = S. E.s. Significant terms in ANOVA: Age x Heat shock x Smoke (Table 6.1). Results of post-hoc comparisons amongst means shown in Figure above is given below: Age Heat shock level / smoke level 25°C 50°C 75°C 100°C unaged / unsmoked b, m, x b, m, x b, m, x a, m, x / smoked b, m, x b, m, x b, m, x a, m, x Mod aged / unsmoked a, m, x a, m, xy a, m, xy a, m, y / smoked a, m, x a, m, x b, n, x a, m, x Sev aged / unsmoked a, m, xy b, m, x a, m, x a, m, y / smoked ab, n, y a, m, y a, m, x a, m, y Different letters within the range a-b are different across age levels Different letters within the range m-n are different across smoke levels Different letters within the range x-y are different across heat shock levels

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Figure 6.2. Mean final germination of a) unaged, b) moderately aged, c) severely aged Sprengelia monticola seeds plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Analysis of transformed data; back-transformed figures. Bars = S. E.s. Significant terms in ANOVA: Age x Heat shock x Smoke (Table 6.1). Results of post-hoc comparisons amongst means shown in Figure above is given below: Age / smoke level Heat shock level 25°C 50°C 75°C 100°C Unaged / unsmoked a, m, x a, n, x a, m, x b, m, x / smoked a, m, xy a, m, x a, m, y a, m, y Mod. aged / unsmoked a, m, x a, m, x a, m, x b, m, x / smoked a, m, x b, m, x a, m, x a, m, x Sev. aged / unsmoked a, m, x a, m, x a, m, x a, m, x / smoked a, m, x ab, m, x a, m, x a, n, x Different letters within the range a-b are different across age levels Different letters within the range m-n are different across smoke levels Different letters within the range x-y are different across heat shock levels

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Figure 6.3. Mean final germination of a) unaged, b) moderately aged, c) severely aged Gahnia sieberiana seeds incubated at 25°C () [aged seeds later transferred into 20/4 hours at 25/35°C ()] plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Analysis within 25°C incubation temperature. Analysis of transformed data; back-transformed data shown. Bars = S. E.s. Significant terms in ANOVA: Age x Heat shock, Age x Smoke (Table 6.1). Mean % germination (S.E.) and results of post-hoc comparisons amongst means listed below: Age level Heat shock level 25 50 75 100 Unaged 13 (3.7) b, m 9 (1.9) b, m 42 (4.2) b, n 54 (6.0) c, n Moderately aged 1 (0.8) a, m 2 (1.1) a, m 4 (1.9) a, m 19 (4.0) b, n Severely aged 2 (3.5) a, m 3 (2.6) ab, mn 5 (1.4) ab, mn 10 (1.1) b, n Different letters within the range a-b are different across aging levels Different letters within the range m-o are different across heat shock levels Smoke level Age level Unaged Moderately aged Severely aged Unsmoked 24 (4.5) a, o 6 (2.3) a, n 1 (0.7) a, m Smoked 35 (5.1) b, n 7 (2.1) a, m 9 (2.1) b, m Different letters within the range a-b are different across smoke levels Different letters within the range m-o are different across aging levels

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Figure 6.4. Mean final germination of a) unaged, b) moderately aged, c) severely aged Baeckea imbricata seeds plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Analysis of transformed data; back-transformed data shown. Bars = S. E.s. Significant terms in ANOVA: Age x Smoke (Table 6.1). Mean % germination (S.E.) and results of post-hoc comparisons amongst means listed below: Smoke level Age level Unaged Moderately aged Severely aged Unsmoked 15 (2.3) a, n 8 (2.6) a, m 8 (1.7) a, m Smoked 40 (4.5) b, o 10 (2.9) a, m 17 (2.2) b, n Different letters within the range a-b are different across smoke levels Different letters within the range m-o are different across aging levels

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Figure 6.5. Mean final germination of a) unaged, b) moderately aged, c) severely aged Kunzea capitata seeds incubated at 25°C () [moderately aged seeds later transferred into 15°C ()], plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Analysis within 25°C incubation temperature. Bars = S. E.s. Significant terms in ANOVA: Age x Smoke (Table 6.1). Mean % germination (S.E.) and results of post-hoc comparisons amongst means listed below: Smoke level Age level Unaged Moderately aged Severely aged Unsmoked 9 (2.3) a, m 8 (3.0) a, m 4 (1.2) a, m Smoked 42 (3.8) b, o 3 (1.1) a, m 16 (3.0) b, n Different letters within the range a-b are different across smoke levels Different letters within the range m-o are different across aging levels

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Figure 6.6. Mean final germination of a) unaged, b) moderately aged, c) severely aged Kunzea ambigua seeds incubated at 25°C () [moderately aged seeds later transferred into 15°C ()], plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Analysis within 25°C incubation temperature. Bars = S. E.s. Significant terms in ANOVA: Age x Heat shock, Age x Smoke (Table 6.1). Mean % germination (S.E.) and results of post-hoc comparisons amongst means listed below: Age level Heat shock level 25 50 75 100 Unaged 41 (3.2) b, m 80 (3.9) b, no 85 (2.9) b, o 73 (3.3) b, n Moderately aged 2 (1.1) a, m 5 (3.4) a, m 0 (0) a, m 4 (1.9) a, m Severely aged 1 (0.9) a, m 8 (3.9) a, n 1 (0.9) a, mn 4 (2.3) a, mn Different letters within the range a-b are different across aging levels Different letters within the range m-o are different across heat shock levels Smoke level Age level Unaged Moderately aged Severely aged Unsmoked 64 (4.0) a, n 5 (1.9) a, m 3 (1.9) a, m Smoked 76 (4.1) b, n 0.4 (0.4) a, m 5 (1.6) a, m Different letters within the range a-b are different across smoke levels Different letters within the range m-o are different across aging levels

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Figure 6.7. Mean final germination of a) unaged, b) moderately aged, c) severely aged Baeckea diosmifolia seeds plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Analysis of transformed data; back-transformed data shown. Bars = S. E.s. Significant terms in ANOVA: Age x Heat shock, Smoke (Table 6.1). Mean % germination (S.E.) and results of post-hoc comparisons amongst means listed below: Age level Heat shock level 25 50 75 100 Unaged 18 (3.2) b, mn 27 (3.8) b, n 29 (4.8) b, n 14 (3.4) b, m Moderately aged 6 (1.5) a, m 7 (1.9) a, m 1 (0.8) a, m 3 (1.9) a, m Severely aged 3 (1.8) a, m 3 (1.3) a, m 4 (2.6) a, m 7 (1.9) ab, m Different letters within the range a-b are different across aging levels Different letters within the range m-o are different across heat shock levels

Smoke: 10 minutes (12 ± 1.6) > 0 minutes (8 ± 1.3)

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Figure 6.8. Mean final germination of a) unaged, b) moderately aged, c) severely aged Epacris coriacea seeds plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Bars = S. E.s. Significant terms in ANOVA: Age x Heat shock, Smoke (Table 6.1). Mean % germination (S.E) and results of post-hoc comparisons amongst means listed below: Age level Heat shock level 25 50 75 100 Unaged 91 (2.4) a, m 88 (2.4) a, m 93 (1.9) b, m 88 (3.3) a, m Moderately aged 82 (2.1) a, m 83 (4.0) a, m 89 (3.6) b, m 87 (2.6) a, m Severely aged 84 (3.3) a, n 79 (4.0) a, mn 70 (5.1) a, m 80 (3.5) a, mn Different letters within the range a-b are different across aging levels Different letters within the range m-o are different across heat shock levels

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Figure 6.9. Mean final germination of a) unaged, b) moderately aged, c) severely aged Dracophyllum secundum seeds plotted against heat shock (25, 50, 75 or 100°C) and smoke (0 or 10 minutes) treatments. Bars = S. E.s. Significant terms in ANOVA: Smoke (Table 6.1). Mean % germination (S.E.) and results of post-hoc comparisons amongst means listed below: Unaged (43 ± 2.2) > Moderately aged (20 ± 2.0) > Severely aged (11 ± 1.4)

304 Transferred seeds

No differential patterns of mortality or dormancy due to fire-related cues were apparent when seeds were transferred into more optimal conditions. The germination response of aged G. sieberiana seeds when transferred into a 20/4 hour 25/35ºC diurnal temperature regime was the same as seeds in all other experiments. Germination increased such that it was uniform across all smoke and heat shock treatments, and the high heat shock treatment produced the same level of germination as within the optimal temperature regime (Fig 6.3). The increase in germination of the moderately aged Kunzea species when transferred into the optimal 15ºC incubation temperature was also uniform across treatments (Figs 6.5, 6). A differential pattern of mortality or dormancy due to severity of aging was apparent, as germination of the severely aged G. sieberiana seeds remained lower than the moderately aged population subsequent to transferral into the optimal temperature regime (Fig 6.3).

Rate of germination

Aging increased the rate of germination of three species and decreased the rate for two species. The time until 50% germination decreased with increased levels of aging in Epacris coriacea, E. obtusifolia and Sprengelia monticola. The aging treatment had little effect on the percentage germination of these populations. The average time until 50% germination of E. coriacea decreased from 53 days for unaged seeds to 52 days for moderately aged seeds to 39 days for severely aged seeds. Germination of 50% of E. obtusifolia took 64, 57, and 47 days, with increasing levels of aging, and germination of 50% of S. monticola took 94, 79, and 52 days, with increasing levels of aging. The time until 50% germination was greater for the aged Baeckea imbricata and Dracophyllum secundum populations. Half of the unaged B. imbricata population had germinated after 25 days, whilst this level of germination of the aged populations took an average of 21 days longer. The average time until 50% germination of D. secundum was 63 days for the unaged population and 86 days for the aged seeds. Visual inspection of the germination time course reveals that for the species that had their rate of germination increased by the aging treatment, this treatment reduced the time until the commencement of germination, but did not increase the rate of germination once commenced. When germination was delayed by the aging treatment, this result was influenced by the delay before the commencement of germination for E. obtusifolia and S.

305 monticola, but was due to a decrease in the rate of germination of E. coriacea. The time until 50% germination of the Epacris species was generally reduced by smoke across heat shock and aging levels. Smoke also generally reduced the time until 50% germination of unaged B. imbricata, K. ambigua and S. monticola seeds, although too few aged K. ambigua seeds germinated to allow an assessment of their rate of germination.

6.4 Discussion

Seeds of reduced physiological integrity, or quality, were still responsive to fire- related germination cues. If this result also occurs in the field, then populations are less reliant on recent seed production for post-fire regeneration.

The aging treatments reduced germination of all species. Despite the severity of the aging treatment, apparent from the decreased germination of six species by over 50%, aged seeds of four species responded positively to smoke and decreased tolerance of heat shock was not evident for any species. Therefore, seeds that are approaching the limits of their longevity following soil storage are still likely to germinate in response to the passage of fire, albeit in reduced numbers. Assuming that the response of the aged seed is comparable to the response of seed aged in soil, then the actual level of post-fire germination is likely to be between the extremes of the unaged and aged levels, because in other work, field emergence was more accurately predicted using seeds that had been rapidly aged than by standard germination testing (Matthews 1980; Egli & TeKrony 1995). This is consistent with reports suggesting that vigour tests, including rate of germination, may be better predictors of field emergence in stress conditions (Perry 1970; Delouche & Baskin 1973; Burris 1976; Johnson & Wax 1978). The percentage germination of aged seed may be reduced by stress (MacKay 1972; Ellis & Hanson 1974; Ellis & Roberts 1980), such as sub- and supra-optimal temperatures (Ellis & Roberts 1981; Ellis et al. 1987; Argerich & Bradford 1989). Smoke increased germination of an aged population of three species, however, the level of germination of these smoked populations was still very low relative to non-aged seeds. The large increase in variation following the aging treatments was expected because, as the viability of a population declines, so does its mean performance, whilst the standard

306 deviation remains constant (Ellis & Roberts 1981). Consequently, aging that resulted in low levels of germination was associated with inconsistent responses to the fire-related germination cues across the degrees of aging.

Rate of germination

The increase in time to germination of the remaining viable seeds (Ellis & Roberts 1980, 1981; Roberts 1986; Argerich et al. 1989; Tarquis & Bradford 1992; Bradford et al. 1993) was expected for the less vigorous populations. Such populations may produce abnormal seedlings that are susceptible to stress until the initial damage has been repaired (Roberts 1972b; Argerich & Bradford 1989). The reduction in time taken for germination of the vigorous populations following aging, has been previously found (Gelmond et al. 1978; Perl et al. 1978; Welbaum & Bradford 1991). The increased rate of germination of some species due to smoke has been found in previous studies (Brown et al. 1994; Dixon et al. 1995; Roche et al. 1997a; Kenny 2000). However, the effect of smoke on the rate of germination was independent of seed aging, thus smoke does not enhance the repair process that occurs before germination. Other growth promoters have only marginally increased vigour or viability of aged seed (Huber & McDonald 1982; Priestly 1986).

Contribution of aged seeds to post-fire recruitment

Within the limitations of this study, it can be concluded that the germination response to fire is probably proportional to the physiological integrity, or quality of the seed population. The older or more damaged fraction will contribute proportionately less to post fire recruitment relative to their numbers. However, fire probably stimulates germination of seeds that are approaching the limits of their longevity. Whilst these results highlight the importance of the quality of population of seed as compared to absolute numbers, the retention of a fire response is a buffer against a decline in quality. The quality of a seed population will be affected by both the growing conditions when it was produced, and conditions during subsequent soil storage, hence post-fire recruitment may vary between years due to variation in environmental conditions. Also, the quality of a population will reflect the most recent conditions in the soil and these conditions vary both within and between seasons, hence post-fire recruitment may vary due to variation

307 in seasonal seed quality. The timing of a fire may be important if seed quality is affected by the conditions recently preceding it, and this may vary between and within years. Differences in seed quality have been implicated in determining the levels of post-fire recruitment within a population (de Lange & Boucher 1993a). The seedling to seed ratio within one area was disproportionately higher than the ratio of the rest of the population, although differences in microhabitat conditions may have contributed to this phenomenon (de Lange & Boucher 1993a). Higher quality seeds, in terms of protection from heating, produced higher levels of germination following heat shock of an obligate seeding species (Delgado et al. 2001). A major factor contributing to the reduction in germination of the aged seeds is likely to be an associated disruption of the seed membrane structure. As the effect of aging generally did not reduce the relative response of seeds to smoke, the disruption of the seed membrane structure by aging is likely not to have been antagonistic to the possible effects that smoke has on membrane permeability or receptor sensitivity (van Staden et al. 1995, 2000). Also, the tolerance of aged seed to high heat shock levels indicates that this possible stress is not overly deleterious to seed survival. Germination of most of a suite of species forming seedbanks in the Sydney region were not adversely affected by burning in three consecutive years, indicating that heat shock did not reduce tolerance of seeds to subsequent heat shock events (Bradstock et al. 1997).

308 Chapter 7. General discussion

Effects of the fire regime, the components of which are intensity, frequency, type and season, on germination of species forming water-permeable soil seedbanks in the Sydney region were investigated within the current study. Fire-related cues affected germination of such species, and these effects were interpreted in terms of fire intensity and frequency. The effects on germination of ambient temperature and water availability in combination with fire- related cues were investigated and interpreted in terms of the seasonal and frequency components of the fire regime. The interaction between fire season and fire intensity was also explored by applying these factors to seeds. Fire type was expanded to include whether fire occurs when soil is wet or dry, and the effect of fire type on germination of water-permeable seed was investigated using the combination of fire-related cues and hydration. Whether physiological deterioration of seed due to accelerated aging alters the effect of fire-related cues on germination was also investigated and interpreted in terms of fire frequency. Fire-related germination cues of heat shock and smoke affected germination of species forming water-permeable soil seedbanks in the Sydney region. The positive germination response to the combination of heat shock and smoke often occurred within a narrow range of heat shock. If these results occur in the field, then only a fraction of the soil seedbank will be stimulated to germinate following any one fire. Similarly, light will stimulate germination of only the seed fraction that is exposed by post-fire erosion, and the residual soil seedbank will buffer against a short fire-return interval. Interactions between ambient temperature and fire-related cues frequently affected germination, however the effect size was generally small. The effect of temperature on germination was greatly increased by secondary dormancy, and by limiting water availability. Water availability to seeds after they receive fire-related cues may be a key factor regulating germination from the soil seedbank. The fire-related cues decreased the amount of time until germination and increased the amount of water required for germination. Seeds that do not receive sufficient water in the immediate post-fire period are likely to become dormant and thus form a residual soil seedbank, whilst post-fire germination occurs from seeds in favourable microsites. If these results occur in the field, then variability in seed microsite quality may ensure both post-fire germination and a residual soil seedbank to buffer against short fire-return intervals. Also, if these results occur in the field, then germination will be affected more by the particular season of a fire rather than season per se.

309 The effect of ambient temperature on germination was dependent on the level of heat shock applied to seed that was incubated within a number of water potentials. If this result, found for the single species investigated, also occurs in the field then the effect of season of fire on germination is dependent on the intensity of the fire. The hydration status of seed affected its tolerance of heat shock, and seed of wet habitat species and a dry habitat species were affected differently by this combination of factors. Germination of wet habitat species was increased if seed was hydrated when treated with heat shock, whereas the heat shock tolerance of a dry habitat species was greatly reduced by seed hydration. If this result occur in the field, then the potential is apparent for fire type i.e. whether fire occurs when soil is wet or dry, to contribute to habitat segregation. Water permeable seed of dry habitat species that disperses into wet habitat is likely to be killed by the high intensity fires that continue from dry habitat through wet habitat. Seed that had been subject to physiological deterioration during accelerated aging was still responsive to fire-related germination cues. If this result occurs in the field, then seed that has resided for a period of time in the soil will contribute to post-fire regeneration. Regeneration is thus less dependent on the more recently produced seed, hence the level of seed production in the season prior to fire and whether those seeds are shed before the fire is less important. A persistent seedbank that remains responsive to fire-related germination cues is an effective buffer against short fire-return intervals. ‘Event-dependent’ rather than ‘interval-dependent’ effects of fire on population regulation have been emphasised in the current study. Event-dependent effects are generally far less predictable, and the greatest intrinsic uncertainty in fire-driven dynamics occurs in woody vegetation with heterogenous fuel (Bond & van Wilgen 1996), such as Hawkesbury Sandstone vegetation. Germination responses within a narrow range of heat shock, seasonal effects of temperature, and the influence of pre-fire seed hydration and post-fire rainfall over germination are all event-dependent.

Physiological considerations

Several physiological considerations arose from the current study. Differences in heat shock protein dynamics could account for the separation of species with water-permeable seed that form soil seedbanks in wet and dry habitat. The dynamics of heat shock proteins in wet and dry habitat species through hydration cycles could be assessed. Heat shock protein concentration is expected to remain high in wet habitat species regardless of hydration status.

310 In contrast, heat shock protein concentration is expected to increase as seeds of dry habitat species dehydrate and decrease as seeds are hydrated. Heat shock can separate a population into two distinct fractions; those with heat shock tolerance that is probably conferred by heat shock proteins will germinate, whereas those without will die. The current study provides evidence that, if heat shock proteins do confer heat shock tolerance, then they are retained following an initial heat shock. Thus, a comparison can be made between the heat shock proteins in the germinated and the dead seeds. Physiological properties can vary over orders of magnitude and so they must be assessed for individual seeds for correlations to be found (Dahal et al. 1994; Still et al. 1997; Still & Bradford 1997). Lines of investigation into the physiological mechanism(s) whereby heat shock and smoke affect germination are apparent within the current study. Germination responses to smoke were greatly increased by heat shock, by short-term hydration provided seed was hydrated at the time of smoke application, or by long-term hydration regardless of whether seed was hydrated at the time of smoke application. Species with strong (e.g. synergistic) interactions between heat shock and smoke / 3-methyl-2H-furo[2,3-c]pyran-2-one (Flematti et al. 2004) could be treated with heat shock, and the physiological changes assessed. These physiological changes may revert with time. The germination-promoting effect of smoke on these heat-treated seeds may also become less pronounced with time. Correlations between changes in the physiological status of heat shock treated seed and its germination response to smoke through time could be assessed. A similar assessment could be made where seeds were treated with smoke prior to heat shock. A similar procedure could be used to investigate the physiological interaction between hydration and heat shock or smoke. The physiological changes of interest are ones that rapidly revert following short-term hydration and that are retained following long-term hydration. Different combinations of short-, medium-, and long-term hydration and dehydration could be used to generate different levels of the aforementioned physiological changes. Correlations between changes in the physiological status of hydration treated seed and its germination response to heat shock or smoke could be assessed. Examples of changes in physiological properties include changes to membrane structure or permeability, changes in hormone concentration or sensitivity or changes in cell wall extensibility.

311 Management considerations

If results of the current study occur in the field, then several management issues follow. Three lines of evidence indicate that the soil seedbank has the capacity to buffer against unfavourable fire events, such as fires followed by limiting soil moisture availability or short fire-return intervals. Firstly, seeds survive the levels of heat shock generated in the surface soil during management fires. Secondly, seed that has resided in the soil for a period of time is likely to contribute to post-fire regeneration. Thirdly, post-fire germination and a residual soil seedbank is likely to occur because germination is frequently increased only within a narrow range of heat shock levels. Also, water availability to seed in the soil is likely to produce post-fire germination of seeds in favourable microsites and a residual soil seedbank in unfavourable microsites. The larger effect of post-fire moisture availability than temperature on germination indicates that the effects of fire on regeneration are likely to be stochastic. An assessment of the degree to which post-fire moisture availability affects germination will require investigation of seed in soil. The effect of microsite quality on the physiological status of seed must be determined i.e. whether a given amount of soil water causes seed to remain germinable or to become dormant, and whether the size of the soil particles surrounding a seed affects its physiological status. The effect of climate on soil moisture status has been determined (Bradstock & Bedward 1992). The effect of climate on soil moisture status can be determined for microsites of different quality, and the frequency of the different quality microsites can be measured in the field. The consequence of a particular post-fire climate on germination from the soil seedbank could then be determined. The probability of different soil moisture dynamics within any month can be determined using long-term climate data (Bradstock & Bedward 1992). The probability of different levels of post-fire germination within any month could be determined using the aforementioned data. All that can be said without this information is that autumn or winter fire may result in higher levels of germination from the soil seedbank because soil moisture is less likely to be limiting than following spring or summer fire. Fire management involves a choice of soil moisture level at the time of a burn, and that choice may affect species composition at a local level. A prolonged dry period may result in a temporary reduction in the soil moisture content of wet or moist habitats. Fire may be lit during a prolonged dry period under cool, calm conditions that allow fire containment. Such a fire may allow the encroachment of dry habitat species into wetter habitat. Conversely, fire

312 that is lit when the soil moisture level is high in dry habitat may result in a depletion of the soil seedbank of dry habitat species due to mortality. Such fire may be considered severe, due to its effects on the biota (Borchert & Odion 1995). The current study has highlighted a number of ways in which fire management decisions may be affecting species composition in the Sydney region. Whilst results from the current study indicate the direction in which community composition may be altered, determination of the magnitude of alteration requires experiments such as those just mentioned. If changes in community composition are of low magnitude, then the residual soil seedbank may allow a favourable fire to reverse changes caused by a number of consecutive unfavourable fires. If changes in community composition are of a higher magnitude, then changes may be of a more permanent nature. The magnitude of a change in the abundance of a species is proportional to that species initial abundance and its capacity to recover from the perturbation. The potential for large changes in the abundance of a species following unfavourable fire(s) is evident in the current study. Unfavourable fire(s) may threaten the viability of rare species.

Recommendations

The interaction between hydration and heat shock tolerance of the Sydney soil seedbank should be investigated as a matter of priority. Because heat shock can kill hydrated seed, fire that occurs when soil moisture is high may remove both above ground vegetation and the soil seedbank i.e. it can cause local extinction. Although some seed may not experience lethal temperature due to patchiness in fire intensity and because of deep burial, the depletion of a soil seedbank may cause a large reduction in the viability of a local population, particularly if the population was small or if the fire coincided with other unfavourable events (e.g. high levels of predation, short fire-return intervals). Rare species are at particular risk from such fires, particularly if the species has a limited distribution and thus many sub-populations are affected by the single fire event. Investigations into the seasonal effects of fire on species forming soil seedbanks in the Sydney region should include considerations of secondary dormancy, and especially the effect of post-fire water availability. The effect of variability in microsite quality should be investigated as a factor that may regulate soil seedbank dynamics in many cases rather than dormancy per se.

313 Appendix 1. Location, habitat, weight and time of seed collection and treatment. Species arranged alphabetically within Family, and chronologically within experiments.

Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time `Apiaceae Actinotus helianthi Blackheath‡ 6272900 Ridge top 0.85 May 01 3 I 8930-1S 251900 Jan-00 decoated Jan 03 3 VII

Aug 03 4 II

Cyperaceae Caustis flexuosa Narabeen Creek bank 0.94 Aug 01 5 V 9130-I-S 339000 6268900 Apr-00 Oct 01 3 V Jan 03 3 VII

Gahnia melanocarpa Royal N P Rivers edge 18.29 Aug 01 5 V 9129-IV-N 320700 6228000 Mar-00 achne Oct 01 3 V Oct 01 6 Oct 03 4 III

Gahnia sieberiana 1 Royal N P Rivers edge 9.792 Dec 00 2 I 9129-IV-N 320700 6228000 Mar-00 seed July 01 3 II Aug 01 3 IV Aug 01 5 V Oct 01 3 V

Oct 01 6 May 02 2 II Aug 03 4 II

345 345

Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Gahnia sieberiana (continued) 2 Blackheath‡ Damp gully Feb 01 3 II 8930-1S 251000 6272500 Jan-00 Aug 03 4 II

Schoenus brevifolius Royal N P Moist heath 1.80 Aug 01 3 III 9129-IV-S 328100 6221400 Feb-00 Aug 01 5 V Oct 01 3 V Jan 03 3 VII

Epacridaceae Dracophyllum secundum 1 Wentworth Falls‡ Rock ledge 0.30 Oct 00 2 I 8930-1S 256700 6265000 Jan-00 Aug 01 3 IV Aug 01 5 V Oct 01 3 V Oct 01 6 Feb 02 3 I Aug 03 4 II

2 Blackheath‡ Rock ledge Sept 01 2 I 8930-1S 250850 6273000 Dec-99 May 02 2 II

Epacris coriacea Blue Mountains N P Moist heath 0.38 Nov 00 2 I 8930-1S 256700 6265000 Jul-00 Aug 01 3 IV Aug 01 5 V Oct 01 6 Dec 01 5 I May 02 2 II June 02 5 II, III

346 346 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Epacris crassifolia 1 Wentworth Falls‡ Wet cliff face 0.15 Aug 01 5 V 8930-1S 256700 6265000 Jan-00 Sept 01 2 I Oct 01 3 V April 02 2 III

Epacris crassifolia 2 Blackheath‡ Wet cliff face Sept 01 2 I 8930-1S 250800 6275400 Jun-00 April 02 2 III

Epacris longifolia Ku-ring-gai Chase N P 0.86 Sept 01 2 I 9130-4-S 336000 6273000 Jun-00 April 02 2 III Aug 03 4 II

Epacris microphylla var microphylla Newnes Junction Moist heath 0.29 Sept 01 2 I 8931-III-S 243600 6292700 Jun-00 April 02 2 III

Epacris microphylla var rhombifolia Kanangra Boyd N P Wet heath 0.28 Aug 01 5 V 8930-III-S 226700 6240400 Apr-00 Sept 01 2 I Oct 01 3 V April 02 2 III Aug 03 4 II

Eparis muelleri Blackheath‡ Wet cliff face 0.26 Sept 01 2 I 8930-1S 250800 6275400 Jun-00 April 02 2 III Aug 03 4 II

347 347 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Epacris obtusifolia 1 Royal N P Wet heath 0.32 Nov 00 2 I 9129-IV-N 321800 6223200 Jun-00 Aug 01 3 IV Aug 01 5 V Oct 01 3 V Oct 01 6 April 02 2 III June 02 5 II, III

1a 0.25 Aug 03 4 Ic Oct 03 4 VI Nov 03 4 IV

2 Ku-ring-gai Chase N P Moist heath 0.35 Feb 02 3 I 9130-1-S 338000 6274800 Jun-00 May 02 5 II, III March 03 4 V April 03 4 Ia

2a Aug 03 4 Ib

3 Wentworth Falls‡ Moist heath 0.22 Feb 02 3 I 8930-1S 256700 6265000 Jul-00 May 02 2 II April 03 4 Ia

348 348 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Epacris paludosa 1 Kanangra Boyd N P Wet heath 0.34 Sept 01 2 I 8930-III-S 226700 6240400 Apr-00 Aug 01 5 V Oct 01 3 V Feb 02 3 I April 02 2 III Aug 03 4 II

2 Newnes Junction Wet heath May 02 3 I 8931-III-S 243600 6292700 Jun-00

Epacris pulchella Royal N P Wet heath Sept 01 2 I 9129-IV-N 321800 6223200 Jun-00 April 02 2 III Aug 03 4 II

Spengelia monticola Blue Mountains N P Wet cliff face 0.15 Dec 00 2 I 8930-1S 255500 6265200 Jan-00 Aug 01 3 IV Oct 01 3 V Oct 01 6

Woollsia pungens 1 Blackheath‡ Wet heath 0.39 Aug 01 3 IV 8930-1S 250800 6275400 Jan-00 Sept 01 2 I April 02 2 III Aug 01 5 V Oct 01 3 V Oct 03 4 III

2 Ku-ring-gai Chase N P Dry heath 0.16 Augt 03 4 II 9130-4-S 336000 6273000 Sep-00

349 349 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Goodeniaceae Goodenia bellidifolia ssp bellidifolia 1 Newnes Junction Heath Feb 02 3 I 8931-III-S 243600 6292700 Jan-00

2 Kings Tableland‡ 0.51 Feb 02 3 I 8930-1S 256600 6262900 Jan-00

Goodenia decurrens Blackheath‡ DSF 0.53 Feb 02 3 I 8930-1S 250800 6275400 Jan-00

Goodenia dimorpha var augustifolia Royal N P Dry heath 0.14 Feb 02 3 I 9129-IV-S 321000 6220600 Feb-00

Goodenia heterophylla Blue Mountains N P DSF 0.91 Feb 02 3 I 8930-1S 248000 6263000 Jan-00

Goodenia ovata Kurrajong DSF 0.65 Feb 02 3 I 8930-1S 251000 6272500 Jan-00

Juncaceae Juncus continuus Kings Tableland Wet Heath 0.36 Aug 01 5 V 8930-1S 256600 6262900 Jan-00 Oct 02 3 V

Lamiaceae Hemigenia purpurea Royal N P DSF Feb 02 3 I 9129-IV-N 322800 6227500 Feb-00

Myrtaceae Baeckea diosmifolia Garigal N P 6268000 Moist heath 0.48 Nov 00 2 I 9130-4-S 335500 Mar-00 Oct 01 6

Aug 03 4 II

Baeckea brevifolia Ku-ring-gai Chase N P Dry heath rock 0.85 Dec 01 2 I 9130-1-N 342000 6282600 Sep-00 platform Feb 02 3 I

350 350 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Baeckea imbricata 1 Royal N P Wet heath 0.35 Nov 00 2 I 9129-IV-N 321800 6223200 Jun-00 Aug 01 3 IV Aug 01 5 V Oct 01 3 V Oct 01 5 VI Oct 01 6 Dec 01 3 VI May 02 5 IIa

2 Ku-ring-gai Chase N P Moist heath 0.28 Dec 01 2 I 9130-1-S 338000 6274800 Sep-00 Feb 02 3 I May 02 2 II Aug 03 4 II

3 Royal N P Moist heath 0.31 Dec 01 2 I 9129-IV-S 322600 6221000 Apr-00 Feb 02 3 I

4 Ku-ring-gai Chase N P 0.30 Feb 02 3 I 9130-1-S 338000 6276500 Mar-00 Aug 03 4 II

Baeckea linifolia 1 Blackheath‡ Creek line 0.51 Aug 01 5 V 8930-1S 250800 6275400 Jun-00 Oct01 3 V Dec 01 2 I May 02 5 II Aug 03 4 II

351 351 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Baeckea linifolia (continued) 2 Royal N P Creek line Dec 01 2 I 9129-IV-N 321800 6223200 Jun-00 Feb 02 3 I Aug 03 4 II

Baeckea ramosissima ssp ramosissima Royal N P DSF 1.90 Dec 01 2 I 9129-IV-N 322800 6227500 Feb-00 Feb 02 3 I

Baeckea utilis Kanangra Boyd N P Swamp 0.82 Dec 01 2 I 8930-III-S 226700 6240400 Apr-00 Feb 02 3 I

Calytrix tetragonia Royal N P Dry heath 3.85 Aug 01 3 III 9129-IV-S 322500 6222500 Jan-00 seed Aug 01 5 V Oct 01 3 V Dec 01 2 I

Kunzea ambigua Ku-ring-gai Chase N P DSF 0.39 Oct 00 2 I 9130-1-S 336000 6273000 Mar-00 Aug 01 3 IV Aug 01 5 V Oct 01 3 V Oct 01 6 Dec 01 5 I May 02 2 II May 02 5 II, III Dec 02 3 I Aug 03 4 Ic Sept 03 5 IV Oct 03 4 VI Nov 03 4 IV

352 352 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Kunzea capitata 1 Wentworth Falls‡ Moist heath 0.49 Oct 00 2 I 8930-1S 256700 6265000 Jul-00 Aug 01 3 IV Aug 01 5 V Oct 01 3 V Oct 01 6 Feb 02 3 I May 02 2 II

2 Kings Tableland‡ Moist Heath 0.49 Feb 02 3 I 8930-1S 256600 6262900 Jun-00 May 02 5 II, III April 03 4 Ia

Aug 03 4 Ib

3 Ku-ring-gai Chase N P Feb 02 3 I 9130-I-N 342000 6282600 Jun-00

4 Wentworth Falls‡ Moist heath 0.49 May 02 5 II, III 8930-1S 256700 6265000 Sep-00

Kunzea parvifolia Kanangra Boyd NP Aug 03 4 II 8930-III-S 228000 6237050 Apr-00

Micromyrtus ciliata 1 Duffys Forest 0.55 Dec 01 2 I 9130-4-S 332300 6273800 Apr-00

2 Ku-ring-gai Chase N P Moist Heath 0.70 Feb 02 3 I 9130-1-S 338000 6274800 Jun-00

Proteaceae Grevillea acanthifolia Newnes Junction Creek line 18.0 Feb 02 3 I 8931-III-S 243600 6292700 Jan-00

Grevillea buxifolia ssp buxifolia Ku-ring-gai Chase N P DSF 61.5 Feb 02 3 I 9130-4-S 336000 6273000 Feb-00

353 353 Family Species Pop Location Map N S Habitat Weight Collection Experiment Chapter Section (mg) time time Grevillea sericea Duffys Forest DSF 25.2 Feb 02 3 I 9130-4-S 332300 6273800 Apr-00

Restionaceae Restio gracilis Ku-ring-gai Chase N P Wet Heath 0.95 Aug 01 5 V 9130-1-S 338000 6276500 Mar-00 Oct 01 3 V

Stackhousiaceae Stackhousia nuda Newnes Junction Swamp edge Feb 02 3 I 8931-III-S 243600 6292700 Jan-00

354 354

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