500

Seed germination and dormancy in south-western Australian fire ephemerals

and burial as a factor influencing seed responsiveness to smoke

μm 500 μm

500 μm 500 μm

Katherine S. Baker BNatRes(Hons)

This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia

School of Plant Biology

Faculty of Natural and Agricultural Sciences The University of Western Australia

2006

Species on title page from top left clockwise; Actinotus leucocephalus, Tersonia cyathiflora, Codonocarpus cotinifolius, Alyogyne huegelii.

ABSTRACT

Fire ephemerals are pioneer species that germinate in large numbers after fire and

generally live for between six months and four years. Seeds produced during the short

life span of these plants persist in the soil seedbank until a subsequent fire. This study

examined the dormancy characteristics and germination requirements of ten Australian

fire ephemeral species from five families.

Seeds of four species germinated at one or more incubation temperatures in the

laboratory, indicating that a proportion of their seedlots were non-dormant at the time of

testing. Austrostipa compressa and Austrostipa macalpinei (Poaceae) produced >80%

germination at 10ºC and Alyogyne hakeifolia and Alyogyne huegelii (Malvaceae)

produced 30-40% and 35-50% germination respectively at 10 to 25ºC. In each of the

Alyogyne species approximately 50% of seeds were impermeable to water, but

scarification did not enable germination of all viable seeds suggesting that seeds which

did not germinate, may have possessed physiological dormancy as well as physical

dormancy. Remaining species had water permeable seeds. Actinotus leucocephalus

(Apiaceae) seeds had underdeveloped embryos and therefore morphophysiological

dormancy, and all remaining species, Codonocarpus cotinifolius, Gyrostemon

racemiger, Gyrostemon ramulosus and Tersonia cyathiflora (Gyrostemonaceae) and

Grevillea scapigera (Proteaceae) possessed seeds with fully developed embryos and

physiological dormancy.

Further laboratory treatments were used to break dormancy or stimulate

germination. At 20ºC scarification (manual or acid) was one of the optimal treatments

for the two Austrostipa species, Alyogyne huegelii, Actinotus leucocephalus and

Grevillea scapigera. A combination of heat (70ºC for one hour) and smoke water were

among the optimal treatments for Actinotus leucocephalus and Codonocarpus

cotinifolius, and enhanced germination beyond the control in the two Austrostipa

species and Alyogyne huegelii. Gibberellic acid induced maximum germination of

Tersonia cyathiflora. Although many of the treatments induced germination,

Codonocarpus cotinifolius, Grevillea scapigera, Gyrostemon racemiger, Gyrostemon

ramulosus and Tersonia cyathiflora retained a high proportion of filled but

ungerminated seeds (>60%) after all treatments.

Following twelve months of soil burial, germination of Actinotus leucocephalus,

Codonocarpus cotinifolius, Gyrostemon racemiger, Gyrostemon ramulosus and

Tersonia cyathiflora seeds was enhanced by smoke water, whereas germination of

i

Grevillea scapigera seeds was enhanced by a heat treatment (70ºC for one hour).

Germination of both Alyogyne species declined after six months of winter burial but was

enhanced by heat treatments after a further six months of summer burial. Actinotus

leucocephalus and Tersonia cyathiflora seeds exhibited annual dormancy cycling over

two years of burial. Dormancy was alleviated over summer, allowing seeds of both

species to germinate in smoke water when seeds were exhumed in autumn, and re-

imposed over winter, suppressing germination in spring. In Actinotus leucocephalus

these dormancy changes were induced in the laboratory by warm (≥15ºC) and cold

(5ºC) temperatures, alleviating and re-imposing dormancy, respectively. Wetting and

drying seeds stored at 37ºC further accelerated the rate of dormancy release. This

dormancy cycling would increase the likelihood of seeds germinating when moisture

availability in south-western Australia is greatest for seedling survival. It also explains

the variation in germination response to smoke water observed in many species. Thus

under natural conditions dormancy levels of fire ephemerals were altered during soil

storage which enabled them to respond to fire-related cues such as heat and smoke

water, and germinate in autumn. This information will assist in the use of these species

in land rehabilitation and ornamental horticulture, and in the conservation of rare or

endangered fire ephemerals.

ii

PUBLICATIONS ARISING FROM THIS THESIS

Baker, K.S., Steadman, K.J., Plummer, J.A. Merritt, D.J. and Dixon, K.W. (2004)

Seed dormancy of two Australian Gyrostemonaceae fire ephemerals. in Ed. M.

Arianoutsou and V.P. Papanastasis. Proceedings of the 10th International

Conference on Mediterranean Climate Ecosystems of the World, Greece, April

25-May 1, 2004, Millpress, Rotterdam.

Baker, K.S., Plummer, J.A., Steadman, K.J., Merritt, D.J. and Dixon, K.W. (2005)

Changes in sensitivity to smoke water during burial of a fire ephemeral,

Actinotus leucocephalus (Apiaceae). pp. 133-139 in Ed. S.W. Adkins, P.J.

Ainsley, S.M. Bellairs, D.J. Coates and L.C. Bell. Proceedings of the Fifth

Australian Workshop on Native Seed Biology. Brisbane, Queensland. 21-23

June 2004. Australian Centre for Mining Environmental Research, Brisbane.

Baker, K.S., Plummer, J.A., Merritt, D.J. and Dixon, K.W. (2005) Australian fire

ephemerals have potential as cover crops in land rehabilitation. pp. 263-265 in

Ed. S.W. Adkins, P.J. Ainsley, S.M. Bellairs, D.J. Coates and L.C. Bell.

Proceedings of the Fifth Australian Workshop on Native Seed Biology.

Brisbane, Queensland. 21-23 June 2004. Australian Centre for Mining

Environmental Research, Brisbane.

Baker, K.S., Steadman, K.J., Plummer, J.A. and Dixon, K.W. (2005) Seed

dormancy and germination responses of nine Australian fire ephemerals. Plant

and Soil 277, 345-358.

Baker, K.S., Steadman, K.J., Plummer, J.A., Merritt, D.J. and Dixon, K.W. (2005)

Dormancy release in Australian fire ephemeral seeds during burial increases

germination response to smoke water or heat. Seed Science Research 15, 339-

348.

Baker, K.S., Steadman, K.J., Plummer, J.A., Merritt, D.J. and Dixon, K.W. (2005)

The changing window of conditions that promote germination of two fire

ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora

(Gyrostemonaceae). Annals of Botany 96, 1225-1236.

iii

ACKNOWLEDGEMENTS

Thanks to my supervisors, Dr Kathryn Steadman and Associate Professor Julie

Plummer. Thanks also to Dr Anle Tieu for early discussions on smoke and germination.

Thanks to my fellow office mates for their friendship, particularly Maratree

Plainsirichai, Andreas Neuhaus, Nicole Mann, Anna Klyne, Ni Luh Arpiwi and Aneeta

Pradhan. Thanks also to my laboratory buddies, especially Helen (Hui Liu), Danica

Goggin, Julia Wilson and Ben Croxford. Extra special thanks to Eleftheria Dalmaris for

letting me use her computer to do statistics, Dr Krystyna Haq for her encouragement

and advice on writing, Dr Pieter Poot and Assoc. Prof. David Turner for their help and

advice on statistics, Alan Mullett and Sean Grosse for help with all things computer-

related, Dr Sean Bellairs who helped me collect seeds, John Murphy and Sharon Platten

at the Centre for Microscopy and Microanalysis for their helpfulness and assistance with

the microscopes, and Gary Cass for lending me equipment. The last few years have

been a great opportunity to meet so many people in the Plant Biology Department of

UWA and Kings Park and find out about their different areas of research. Lastly, thanks

to my parents who always believed I could write a thesis.

This research was undertaken with financial assistance from the Robert and

Maude Gledden postgraduate research scholarship and seeds were collected with

permission from the Western Australian Department of Conservation and Land

Management (Licence number SW007276).

STATEMENT OF CANDIDATE CONTRIBUTION

This thesis is my own work and does not contain information generated by persons

other than myself, except where due acknowledgement has been made. The papers

either in press or submitted to journals are primarily my own work and the coauthors are

my supervisors. This thesis has been completed during the course of enrolment at The

University of Western Australia and has not been submitted previously for a degree at

this or any other institution.

Katherine Sue Baker

December 2006

iv

This thesis is dedicated to my Grandparents who encouraged me to undertake research, and who died within 12 hours of one another on 25th August 2005

v

TABLE OF CONTENTS

Chapter 1 Introduction 1 Outline 3 Literature Review 7

Fire, Mediterranean Ecosystems and Vegetation The Problem: Australian Fire Ephemerals Are Difficult to

Germinate Why Examine the Germination Requirements of these

Australian Fire Ephemerals? Description of the Study Species Requirements for Seeds to Germinate

Possible Methods of Inducing Fire Ephemerals to Germinate

7 13

16

19 32 40

Seedbank Ecology May Provide Insights Into Germination Requirements

52

Aims of this Thesis 63 Chapter 2 Seed dormancy and germination responses of nine Australian fire ephemerals

65

Abstract 67 Introduction 68 Materials and methods 70 Results 74 Discussion 82 Chapter 3 Dormancy release in Australian fire ephemeral seeds during burial increases germination response to smoke water or heat

87

Abstract 89 Introduction 90 Materials and methods 91 Results 95 Discussion 102 Chapter 4 The changing window of conditions that promotes germination of two fire ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae)

107

Abstract 109 Introduction 110 Materials and methods 112 Results 116 Discussion 128 Chapter 5 General Discussion 133 Appendix 147 References 151

Publications Arising From This Thesis 173

vi

LIST OF FIGURES Chapter 1

Figure 1. Season when fires are most likely to occur in different parts of Australia. 12

Figure 2. Western Australian bioclimates based on seasonality and amount of rainfall. 14

Figure 3. The three main botanical provinces of Western Australia (Northern, Eremaean and South-West) and regions within them.

15

Figure 4. Western Australian distribution of the ten fire ephemerals that will be examined in this thesis.

18

Figure 5. Actinotus leucocephalus a) head of umbels surrounded by white woolly bracts, b) mericarps with a persistent calyx, and c) plant growing to approximately 45 cm tall.

19

Figure 6. Flowers (a-d), seeds (e,f) and dehiscent capsules (g,h) of Alyogyne hakeifolia (b,d,e and g) and Alyogyne huegelii (a,c,f and h).

22

Figure 7. Caryopsis of a) Austrostipa compressa and b) Austrostipa macalpinei showing doubly bent awn and twisted column.

25

Figure 8. Grevillea scapigera a) plant showing divided leaves b) flower head and c) seeds. The flat surface of the seeds is shown on the left and the convex, mottled side on the right.

27

Figure 9. Gyrostemon racemiger a) plants growing en masse 1.5 years after fire to over 2 m tall, b) male flowers, and c) dry carpels releasing seeds. Seeds of d) Codonocarpus cotinifolius, e) Gyrostemon ramulosus, f) Tersonia cyathiflora and g) Gyrostemon racemiger. Codonocarpus cotinifolius seedling h) showing leaf form. Tersonia cyathiflora i) female sprawling plant, j) mature indehiscent fruit along stem, k) inside of fruit and l) flower.

30

Figure 10. The relative abundance of plants and seeds over time following a fire in four groups of species classified according to their life history and response to fire: a) monocarpic fire ephemeral, b) polycarpic fire ephemeral, c) seeder and d) resprouter.

56

Chapter 2

Figure 1. Comparison of the effect of a 12 h diurnal light/dark regime and continuous darkness on germination of the nine fire ephemeral species.

76

Figure 2. Cumulative germination at 5ºC, 10ºC, 15ºC, 20ºC and 15/25ºC for the four species (a) Austrostipa compressa, (b) Austrostipa macalpinei, (c) Alyogyne hakeifolia and (d) Alyogyne huegelii that produced >3% germination at one or more temperatures.

77

Figure 3. Effect of red to far-red ratio of 1.1:1 v. 9:1 during the light phase of a 12 h light/dark diurnal cycle at 10ºC and 15/25ºC on (a) Austrostipa compressa and (b) Austrostipa macalpinei.

78

Figure 4. Germination in response to a range of chemical, scarification, smoke water and heat treatments in (a) Austrostipa compressa, (b) Austrostipa macalpinei, (c) Alyogyne hakeifolia, (d) Alyogyne huegelii, (e) Actinotus leucocephalus, (f) Grevillea scapigera, (g) Codonocarpus cotinifolius, (h) Gyrostemon racemiger and (i) Tersonia cyathiflora.

79

Figure 5. Germination of Actinotus leucocephalus and Tersonia cyathiflora seeds incubated at a range of smoke water dilutions for 35 days at 20°C.

81

vii

Chapter 3 Figure 1. Mean monthly minimum and maximum air temperature (ºC) and monthly rainfall

(mm, bars) between January 2002 and March 2003. 93

Figure 2. Filled seed (mean ± se) at the time of burial and after 6 and 12 months of burial in burnt and unburnt soil for each species. (A) Actinotus leucocephalus; (B) Alyogyne hakeifolia; (C) Alyogyne huegelii; (D) Grevillea scapigera; (E) Codonocarpus cotinifolius; (F) Gyrostemon racemiger; (G) Gyrostemon ramulosus; and (H) Tersonia cyathiflora.

96

Figure 3. Germination (mean ± se) as a percentage of filled seeds for (A-D) Actinotus leucocephalus; (E-H) Alyogyne hakeifolia; (I-L) Alyogyne huegelii; (M-P) and Grevillea scapigera when treated with water (A, E, I, M), smoke water (B, F, J, N), heat (C, G, K, O) and heat and smoke water (D, H, L, P). Seeds were stored in the laboratory at a constant 15ºC, or buried in burnt or unburnt soil for 6 and 12 months.

98

Figure 4. Germination (mean ± se) as a percentage of filled seeds for the four Gyrostemonaceae species. (A-D) Codonocarpus cotinifolius; (E-H) Gyrostemon racemiger; (I-L) Gyrostemon ramulosus; and (M-P) Tersonia cyathiflora when treated with water (A, E, I, M), smoke water (B, F, J, N), heat (C, G, K, O) and heat and smoke water (D, H, L, P). Seeds were stored in the laboratory at a constant 15ºC, or buried in burnt or unburnt soil for 6 and 12 months.

99

Figure 5. Seed moisture content (% fresh weight, mean ± se) of each species following exhumation in spring 2002 from the burnt and unburnt sites, and after air dry storage in the laboratory at 15ºC.

100

Figure 6. Germination (mean ± se) of the eight species when placed in soil (A) and (B) given an aerosol smoke treatment. Aerosol smoke treatments were performed in autumn 2002 and autumn 2003. Filled bars indicate germination during the first winter season and the open bars represent germination over the second winter in the soil.

101

Chapter 4

Figure 1. Germination (mean ± se) of Actinotus leucocephalus (A-D) and Tersonia cyathiflora (E-H) seeds following 6, 12, 18 and 24 months of burial in nylon mesh bags in recently burnt or unburnt soil, or laboratory storage at 15ºC. Germination was assessed by incubating seeds at 20ºC in water (A,C,E,G) or smoke water (B,D,F,H) for 35 days without preheating (A,B,E,F) or following pretreatment at 70ºC for 1 h (C,D,G,H).

117

Figure 2. Number of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds filled (mean ± se) after burial in nylon mesh bags for 6, 12, 18 and 24 months in unburnt and recently burnt soil.

118

Figure 3. Germination (mean ± se) of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds at a range of incubation temperatures (10, 15, 20, 25, 30ºC) following 24 months of burial in nylon mesh bags in unburnt soil or laboratory-storage at 15ºC. Seeds were incubated in water or smoke water for 35 days.

119

Figure 4. Germination (mean ± se) of Actinotus leucocephalus (A,B) and Tersonia cyathiflora (C,D) seeds under an alternating light/dark regime (12 h light/12 h dark) or continuous darkness for 35 days. Seeds were incubated in water (A,C) or smoke water (B,D) at 20ºC. Prior to testing seeds had been stored in the laboratory (15ºC) or buried in nylon mesh bags in unburnt soil for 24 months.

120

Figure 5. Germination (mean ± se) of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds following 24 months of burial in nylon mesh bags in burnt bushland or laboratory-storage (15ºC) when incubated at 20ºC for 35 days in water, smoke water (sw), sw plus 30 μM GA3, 30 μM GA3, 30 μM GA3 plus 10 mM KNO3, or 10 mM KNO3. Additional seeds were soaked in H2SO4 for 1 min prior to incubation in water or smoke water.

121

viii

Figure 6. Environmental Scanning Electron Microscope images of the surface of Actinotus leucocephalus seeds following laboratory-storage (A,B), burial in the unburnt site for 24 months (C,D), and surface sterilization following burial in the unburnt site for 18 months (E,F). For each pair of images the whole dispersal units is shown (A,C,E) and then the mid-section of the seeds is captured at higher magnification (images B,D and F respectively).

123

Figure 7. Environmental Scanning Electron Microscope images of the surface of Tersonia cyathiflora seeds following laboratory-storage (A,B), burial in the unburnt site for 24 months (C,D), and burial in the unburnt site for 24 months and soaking in H2SO4 for 1 min (E,F). For each pair of images the whole dispersal units is shown (A,C,E) and then the mid-section of the seeds is captured at higher magnification (B,D and F respectively).

124

Figure 8. Germination (mean ± se) of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds following burial in an unburnt area for 4 months over summer in nylon mesh bags or sealed foil packets. Seeds were incubated at 20ºC for 35 days in water or smoke water (sw), with or without a 1 h 70ºC heat or 1 min H2SO4 pretreatment.

125

Figure 9. Germination (mean ± se) of Actinotus leucocephalus in water and smoke water at 20ºC following laboratory storage of seeds for 1, 2 and 3 months at air-dry storage at 20/50ºC (A), air-dry storage at 37ºC (B), a weekly regime of air-dry storage at 37ºC for 5 days, imbibition of seeds on wet absorbent sponge for 1 day at 20ºC followed by one day of air-drying at 20ºC (C), 5ºC stratification (D), or air-dry storage at 20/50ºC after 2 months of 5ºC stratification (E).

127

LIST OF TABLES Chapter 1

Table 1. Families with monocarpic or polycarpic fire-ephemeral taxa in the chaparral, kwongan and fynbos.

10

Table 2. Main distinguishing characteristics of non-dormant seeds and the five dormancy classes.

37

Chapter 2

Table 1. Date and provenance of seed collection for each of the species studied. 70Table 2. Percentage (mean ± se) of filled seeds, and percentage of filled seeds that were

viable, possibly viable or dead according to the tetrazolium chloride test. 75

Chapter 3

Table 1. Seed collection date and provenance for the species studied. 92

Chapter 5

Table 1. Dormancy classification and optimal germination treatment for 10 fire ephemeral species at the time of initial testing.

137

Appendix

Table 1. Fire ephemerals in Australia. 149

ix

x

Chapter 1

OUTLINE

This thesis provides an examination of the seed dormancy characteristics and

germination requirements of ten fire ephemerals from south-western Australia. Fire

ephemerals are a group of plant species that predominantly germinate after fire, are

short-lived (living for less than one year to up to ten years) and are inferred to exist

only as seeds in the soil seed bank until a subsequent fire.

Firstly, a review of the literature will consider the relationship between fire and

vegetation in Mediterranean climate ecosystems globally, before examining in greater

detail one functional group of plants, fire ephemerals. Under natural conditions these

species germinate after fire but Australian fire ephemerals are often difficult to

germinate ex situ. An understanding of the germination requirements of these seeds

would be beneficial to land rehabilitation programs and the propagation of rare and

potentially valuable horticultural species. For germination to proceed, seeds must be

viable, non-dormant, and able to imbibe water. Suitable temperatures and germination

stimulants may also be required before seeds germinate. Methods of assessing seed

viability, the nature of germination stimulants and different dormancy classification

schemes, as well as the relative benefits of each, are examined. A range of possible

germination treatments, with a focus on fire-related cues, are outlined, which may

stimulate germination, or bypass dormancy and provide insights into the kinds of

dormancy present in the seeds. Under natural conditions, fire ephemeral seeds are

inferred to persist in the soil for long periods of time between fires. Classifications of

seedbank longevity will be discussed, as well as methods of assessing seed persistence.

Possible influences of seed burial on subsequent dormancy and germination will also be

considered. The literature review will conclude with an outline of the overall aims of

this thesis which are to 1) better understand the ecological and physiological basis of

the dormancy mechanism(s) in seeds of southwestern Australian fire ephemerals, 2)

develop effective methods of removing dormancy in these seeds in the laboratory and

field.

Three experimental chapters then follow in an investigation of these aims. Each

chapter has been published as a paper and is in the format of the respective journal. The

first experimental chapter, ‘Seed dormancy and germination responses of nine

Australian fire ephemerals’, has been published in Plant and Soil. As the title suggests,

the effect of a range of germination treatments are examined in this chapter. Some of

3

Chapter 1

these treatments are linked to fire, such as heat and smoke, whereas others, like

exogenous gibberellic acid application, do not occur under natural conditions but may

induce germination. These tests will indicate whether seeds can germinate with or

without the application of specific germination stimulants, or whether seeds are

dormant. The response to these germination tests can also be used to determine what

type of dormancy seeds may possess and answer the question of whether seeds of fire

ephemerals generally exhibit similar germination and dormancy patterns.

The second experimental chapter, ‘Dormancy release in Australian fire

ephemeral seeds during burial increases germination response to smoke water or heat’,

has been published in Seed Science Research. It has been inferred that seeds of fire

ephemerals persist in the soil. In this chapter, the proportion of seeds of eight fire

ephemerals that can persist in the soil for twelve months without germinating or

decaying is assessed. Fresh or laboratory-stored fire ephemeral seeds often produce low

germination in response to fire-related cues such as heat and smoke, even though these

species germinate after fire under natural conditions. Also, seeds may undergo changes

in dormancy states during burial. Thus, for each species, the response to fire-related

cues such as heat and smoke water are examined after six (spring) and twelve (autumn)

months of burial.

The third experimental chapter, ‘The changing window of conditions that

promotes germination of two fire ephemerals, Actinotus leucocephalus (Apiaceae) and

Tersonia cyathiflora (Gyrostemonaceae)’ has been published in Annals of Botany. In

the previous chapter both species produced a higher response to smoke water following

12 months of burial and exhumation in autumn, than after six months of burial and

exhumation in spring. In this chapter, germination of seeds in smoke water is examined

over a longer period of burial to determine whether changing response to smoke water

is a result of dormancy cycling or an increase in receptivity to smoke water with time.

The range of conditions at which autumn exhumed seeds can germinate is compared

with that of laboratory-stored seeds of a similar age. Temperature and seed moisture

storage trials are also undertaken to try to determine what is driving dormancy changes

in the soil and in an attempt to mimic dormancy release in the laboratory.

Following these experimental chapters, a brief General Discussion will

highlight the main findings of this study including the dormancy classification of each

species, means of overcoming dormancy, and species requirements for germination

4

Chapter 1

stimulants. The implications of these findings for conservation, horticulture and land

rehabilitation are discussed, followed by suggestions for future research.

5

Chapter 1

6

Chapter 1

7

LITERATURE REVIEW

Fire, Mediterranean Ecosystems and Vegetation Fire, in addition to temperature and rainfall, plays a major role in shaping the world�s

vegetation (Bond et al. 2005). Fire-prone vegetation types include grasslands, savannas,

boreal forests and Mediterranean shrublands (Archibold 1995; Bond et al. 2005). The

five areas of the world with Mediterranean ecosystems are found in the Mediterranean

Basin, the Western Cape Province of South Africa, California, Central Chile and

southern and south-western Australia (Dallman 1998). A range of vegetation types

occur within each of these areas but the main ones associated with fire are the

sclerophyllous shrublands, referred to in each of these areas as the maquis and garrigue,

fynbos, chaparral, matorral, and kwongan and mallee, respectively (Cowling et al. 1996;

Dallman 1998). Climatically, these areas are typified by hot, dry summers and mild, wet

winters (Dallman 1998). Vegetation in these Mediterranean regions is susceptible to

fire, because drought coincides with the hottest time of the year, and sufficient rain falls

during winter for vegetation growth and litter accumulation (Dallman 1998).

All five Mediterranean regions feature in the world�s 25 most biodiverse

conservation hotspots ranked according to species endemism and degree of threat

(Myers et al. 2000). Due to geographic disparity and high species endemism, there is

little species overlap between these regions (Ornduff 1996). Four regions are fire prone.

However, fire is not considered to be a historically selective agent in Chile (Cowling et

al. 2004), because natural fires are less frequent and widespread due to the absence of

summer lightning and fire-spreading winds (Mooney 1977; Montenegro et al. 2004).

Differences may have also arisen in the flora of the four fire-prone

Mediterranean regions as a result of differences in topography, soil nutrient status,

severity of summer drought, and reliability and predictability of rainfall. High

mountains are found in California and the Mediterranean Basin, South Africa is less

mountainous, and south-western Australia is an ancient plateau (Beard 1990; Dallman

1998). Soil nutrient levels are generally lower in Australia and South Africa than in the

northern hemisphere Mediterranean regions (Dallman 1998). Summer drought is more

severe in California than the other three regions, although average annual rainfall is

similar across all regions (Cowling et al. 2004). Winter rainfall in the southern-

hemisphere Mediterranean regions is more reliable and occurs in many small rainfall

events, compared with the northern hemisphere regions, where rainfall occurs in less

predictable large events (Cowling et al. 2004).

Chapter 1

8

Despite the differences among these regions, the Mediterranean climate and

occurrence of fire has resulted in the convergence of vegetative traits (Cowling and

Witkowski 1994; Ornduff 1996; Keeley and Bond 1997; Pignatti et al. 2002). It is

thought that the flora in these regions evolved in response to conditions that prevailed

prior to onset of Mediterranean climates such as drought (Gill 1975; Bell et al. 1984),

rather than fire, but the species present today have persisted in the prevailing fire-prone

environment. These plants can be classified according to their life history response to

fire (Pate and Hopper 1993). Seeders are killed by fire, but regenerate from seeds,

whereas resprouters survive the fire and regrow from epicormic buds or lignotubers.

Seeders may store their seeds in fruit structures in the plant canopy

(serotinous/bradysporous) or in the soil seedbank. Geophytes are generally unaffected

by fire, because they grow during winter and persist over summer as under-ground

storage organs. Regular ephemerals are seeders that generally complete their life cycle

over winter when fires are unlikely to occur, and fire ephemerals only germinate after

fire and generally complete their life cycle before a subsequent fire (Pate and Hopper

1993). In the following section, the traits of fire ephemerals will be examined in more

detail.

Fire Ephemerals

Fire ephemerals are a functional group of plants that have an obligate requirement for

fire to germinate, grow rapidly, often reach reproductive maturity early, produce

copious amounts of seeds and often die before a subsequent fire (Hunter et al. 1998).

These species only form a transient component of the above-ground flora and remain in

the soil seedbank for long periods (Egerton-Warburton 1998). The two main categories

of fire ephemerals are monocarpic fire ephemerals, which flower and produce seeds

once and are also known as fire annuals, and polycarpic fire ephemerals, which are also

referred to as fire perennials and flower and produce seeds usually for three to four, but

occasionally for up to ten years or longer (George 1982; Pate et al. 1985). Other terms

that have been used to describe species with similar characteristics include fire

followers (Keeley and Pizzorno 1986; Naveh 1994), annual pyrophytes (Milberg 1994),

fire weeds (Pate et al. 1985; Bell et al. 1993), fire recruiters (Brits et al. 1993; Egerton-

Warburton 1998), fire annuals and fire endemics (Thanos and Rundel 1995), pyrophytes

(Naveh 1994) and pyrophyte endemics (Keeley et al. 1985), fire-type plants (Quick

1959) and fire-chasers (Preston et al. 2004).

Chapter 1

9

Monocarpic and polycarpic fire ephemerals are found in a number of families in

the chaparral (California), kwongan (south-western Australia) and fynbos (Cape of

South Africa; le Maitre and Midgley 1992; Table 1). Some families, such as the

Apiaceae, are represented by fire ephemeral species in all three regions, whereas the

Gyrostemonaceae is endemic to Australia. Although fire ephemerals are often found in

Mediterranean climate regions (Bell et al. 1984; Keeley et al. 1985; Pate et al. 1985; le

Maitre and Midgley 1992), Australian fire ephemerals are not restricted to southern and

south-western Australia (George 1982; Hunter et al. 1998). A number of Australian

species have been cited as fire ephemerals or possessing fire ephemeral characteristics

(Appendix). Yet fire ephemerals constitute a smaller proportion of the flora in Australia

and South Africa than in California (Bell et al. 1984; Delfs et al. 1987; le Maitre and

Midgley 1992; Ornduff 1996). This may be linked to differences between these regions

described above, such as less severe summer drought in south-western Australia and

South Africa than in California, resulting in lower selection for short-lived species that

complete most of their life cycle during the wet season (le Maitre and Midgley 1992).

Lower soil nutrients in Australia and South Africa than California may also select

against annuals (le Maitre and Midgley 1992; Beard et al. 2000).

Fire ephemerals are highly adapted to the post-fire environment. Australian fire

ephemerals exhibit highly plastic growth responses to resource availability, and

seedlings transplanted into unburnt areas usually exhibit nutrient deficiencies and die

(Pate et al. 1985; Pate and Hopper 1993). Similarly, plants of the Californian fire

ephemeral, Nicotiana attenuata, exhibit resource related growth plasticity, producing

twelve times more seeds when grown in burnt soil than unburnt soil (Preston and

Baldwin 1999). Two Australian fire ephemerals, Tersonia cyathiflora and Gyrostemon

ramulosus, produce extensive fibrous root systems which may enhance their ability to

exploit the transient availability of resources after fire (Pate et al. 1985).

Chapter 1

10

Table 1. Families with monocarpic (M or m) or polycarpic (P or p) fire-ephemeral taxa

in the chaparral, kwongan and fynbos. Capital letters indicate that fire ephemerals are

widespread in the family, whereas lower-case letters denote that they are less important.

Some families do not contain fire ephemerals (n). Other families include species, which

are fire ephemerals, but the extent of the trait is undetermined (f) and in other families

the presence or absence of fire ephemerals is unknown (u). Based on le Maitre and

Midgley (1992).

Vegetation Type and Region Family Chaparral

California USA

Kwongan Western Australia

Australia

Fynbos Cape

South Africa Aizoaceae - M Mp Apiaceae Mp M P Asteraceae MP M MP Boraginaceae M f n Cistaceae P u u Crassulaceae M u P Euphorbiaceae f M M Fabaceae MP n P Geraniaceae f f P Goodeniaceae u P u Gyrostemonaceae n-family not

present in this region

P n-family not present in this

region Hydrophyllaceae Mp u f Liliaceae n M n Lobeliaceae f M mP Loganiaceae f M n Malvaceae f P P Mesembryanthemaceae f u mp Onagraceae M U f Poaceae f M mp Polemoniaceae M u u Polygalaceae u n P Polygonaceae Mp n f Portulaceae M M m Santalaceae u n P Scrophulariaceae MP f MP Solanaceae P P p* *most Solanum species are long-lived

Chapter 1

11

Fire Regimes in Australia

Plants are adapted to particular fire regimes as opposed to fire per se (Gill 1975). Fire

frequency, intensity and season are all interrelated components of the fire regime (Gill

1975; Bond and van Wilgen 1996). The exact frequency of fires in Australia in the past

is debatable and would vary both spatially and temporally. Fires occur in Australian

heathlands every 1-50 years (Keith et al. 2002). Bell et al. (1984) suggest that the

�natural� fire interval of the kwongan/northern sandplain is between 25 and 50 years,

whereas van der Moezel et al. (1987) propose a natural fire frequency of 8-15 years.

Based on the optimum fire interval required for Banksia hookeriana recruitment,

Enright et al. (1996) inferred that fires in the past occurred at intervals of 15-18 years.

Despite some variations in the estimates of past fire frequency, Bell et al. (1984), van

der Moezel et al. (1987) and Enright et al. (1996) all agree that in recent years fires

have occurred more frequently (5-11 years) due to human intervention. In contrast,

Lamont et al. (2003) suggest that fire frequency was higher in the past than in recent

years. By removing the leaf blades and examining the banding on Xanthorrhoea stems,

Lamont et al. (2003) predict that the average fire interval between 1859 and 1919 on the

kwongan was approximately five years and that since 1940 the average fire interval has

increased to ten years. Irrespective of whether fire frequency has increased or decreased

in recent years, Cowling et al. (2004) estimate that the fire interval in south-western

Australia is presently 3-20 years. This is a shorter interval than that experienced in the

three other major fire prone regions of the world; the south-western cape of South

Africa (5-40 years), the Californian region of the United States (30-90 years) and the

Mediterranean Basin (>50 years; Bond and van Wilgen 1996; Cowling et al. 2004).

Frequent fires may lead to a decline in the number of fire sensitive species if

plants are killed before seeds are produced (Auld and Scott 2004). This does not

generally affect fire ephemerals because these short-lived species mainly germinate in

the rainy season following fire and reach reproductive maturity early, thus completing

their life cycle before the likely event of a subsequent fire (Bell et al. 1984; Pate and

Hopper 1993). However, if fire ephemerals require a number of years of soil burial

before optimal germination, frequent fires may influence the recruitment of these

species. Conversely intervals between fires that exceed seed longevity could be

detrimental to the persistence of fire ephemerals at a site (Holmes and Newton 2004).

Australian monocarpic fire ephemerals usually dominate after frequent mild burns

whereas polycarpic fire ephemerals are observed after less frequent hotter fires (Pate

and Hopper 1993). This may suggest that monocarpic species have shorter-lived seeds

Chapter 1

12

than polycarpic species, or that monocarpic seeds are less heat tolerant. Although heat

from a fire may kill seeds, some species require exposure to certain temperatures to

promote germination (Shea et al. 1979; Auld and O'Connell 1991). It is unknown

whether heat is an important factor in the germination of Australian fire ephemerals.

Fire intensity is influenced by fire frequency because longer durations between fires

results in greater fuel build up (Dallman 1998). Season of burn can also influence fire

intensity because it affects the dryness of fuel and hence its flammability (Dallman

1998).

In Australia, fire seasons are largely influenced by rainfall and temperature, with

most fires in south-western Australia occurring in summer and autumn (Figure 1;

Lindesay 2003). The season of fire can influence germination. Seedling recruitment in

bushland north of Perth is much higher following an autumn burn than a spring burn

(Hobbs and Atkins 1990). Likewise, germination and establishment of plants in south-

western Australia is higher following smoke treatment of sites in autumn than either

winter or spring (Roche et al. 1998). Thus fire frequency, intensity and seasonality are

all factors that may affect the occurrence and distribution of fire ephemerals.

Figure 1. Season when fires are most likely to occur in different parts of Australia

(Australian Bureau of Meteorology, 2005).

Chapter 1

13

The Problem:

Australian Fire Ephemerals Are Difficult To Germinate Under natural conditions, fire ephemerals primarily germinate after fire (Bell et al.

1984). The basis for fire-stimulated germination has not been investigated in many

Australian fire ephemerals. For those Australian fire ephemerals that have been

examined, attempts to germinate these species ex situ has proven difficult (Dixon et al.

1995; Rossetto et al. 1995; Fuss et al. 1996; Parsons 1997; Hunter 1998; Tieu et al.

1999;2001a). The discovery that smoke water could stimulate seed germination (de

Lange and Boucher 1990) has resulted in the germination of many previously difficult-

to-germinate Australian species (Dixon et al. 1995). However, some species, including

many fire ephemerals, still produce low or variable germination in response to smoke

(Dixon et al. 1995; Rossetto et al. 1995; Tieu et al. 1999;2001a). Heat is the other main

cue associated with fire (Hunter 1998). Even combining heat and smoke cues has not

promoted germination of some of these species, such as Tersonia cyathiflora (Tieu et al.

2002). The low response of these species to smoke and heat is surprising considering

that these species are cued to germinate by fire in their natural environment (Hunter

1998).

Here we will examine the germination requirements of the following Australian

fire ephemerals representing seven genera across five families: Actinotus leucocephalus

Benth. (Apiaceae), Codonocarpus cotinifolius (Desf.) F.Muell., Gyrostemon racemiger

H.Walter, Gyrostemon ramulosus Desf., Tersonia cyathiflora (Fenzl) J.W.Green

(Gyrostemonaceae), Alyogyne hakeifolia (Giord.) Alef., Alyogyne huegelii (Endl.)

Fryxell (Malvaceae), Austrostipa compressa (R.Br.) S.W.L.Jacobs & J.Everett,

Austrostipa macalpinei (Reader) S.W.L.Jacobs & J.Everett (Poaceae), and Grevillea

scapigera A.S.George (Proteaceae). All of these species occur within the Mediterranean

climate region of south-western Australia (Figure 2), although many are not restricted to

this area (Figure 4). This south-west botanical region (Figure 3) is biologically very

diverse, containing approximately 5710 plant species, of which about 79% are endemic

to Western Australia (Beard et al. 2000).

Some Fabaceae species also germinate after fire and are short-lived (le Maitre

and Midgley 1992; Vigilante and Bowman 2004). However, Acacias and other species

in the Fabaceae will not be considered in this investigation. Extensive work has already

been undertaken on the germination requirements of Fabaceae seeds, many of which are

impermeable to water but become able to imbibe water and germinate following

Chapter 1

14

scarification or a heat shock (Clemens et al. 1977; Shea et al. 1979; Auld 1986a; Auld

and O'Connell 1991; Morrison et al. 1998). There are also some other differences

between many of the fire ephemerals that will be investigated and the Fabaceae. For

example, fire ephemerals often obtain their nutrients via dense networks of shallow

fibrous roots (Pate et al. 1985) and in contrast to the Fabaceae, do not form mycorrhizal

associations or fix nitrogen (Pate and Dixon 1996).

Figure 2. Western Australian bioclimates based on seasonality and amount of rainfall

(Beard 1990).

Chapter 1

15

Figure 3. The three main botanical provinces of Western Australia (Northern, Eremaean

and South-West) and regions within them (Beard 1990).

Chapter 1

16

Why Examine the Germination Requirements of these

Australian Fire Ephemerals? Fire ephemerals have been largely overlooked in Australia because plants only appear

for short periods of time after fire and constitute a small proportion of the flora (Bell et

al. 1984; Delfs et al. 1987; le Maitre and Midgley 1992). Many fire ephemerals are rare

or threatened or closely related to rare species. A number of the species under

investigation are congeneric to species that are listed as Rare or Threatened Australian

Plants (Briggs and Leigh 1996). Examining unthreatened species may provide insights

into the germination requirements of related rare species. For rare species, seed

availability is often limited and a wide range of germination or dormancy breaking

treatments cannot be tested. Thus an understanding of the germination requirements of

related seeds with similar life histories may direct research towards successful methods

of germinating rare species and thus minimise seed wastage and assist in plant

conservation.

Some fire ephemerals are widespread across Western Australia eg Gyrostemon,

whereas others are restricted to the northern sandplain. Many of these species occur on

mining leases. For example, Tersonia cyathiflora is present at Iluka (near Eneabba),

Gyrostemon tepperi was collected at Nifty Copper Mine (East Pilbara; Matthew Barrett

pers. comm. 2001), and Austrostipa compressa seeds for this project were collected at

Rocla (Perth). In post-mining rehabilitation areas, seed dormancy can result in the loss

of key families and a reduction in plant biomass by up to 60% (Adkins et al. 2002a). In

addition, fire ephemerals may be overlooked in pre-mining vegetation surveys because

they are only transitory components of the above-ground flora after fire. Nevertheless

their inclusion in rehabilitation programs is essential for maintaining landscape function

and biodiversity. Fire ephemerals are early successional species (Bell et al. 1984) and

potentially useful as cover crops in land rehabilitation programs. Factors which render

these species suitable as cover crops include their ability to grow in bare soil, their rapid

growth rates, dense root networks which bind the soil thus minimising erosion, and a

short life span which allows other species to establish under protected conditions (Pate

et al. 1985). The presence of these fast growing species would also reduce the potential

for nutrient loss following a fire (Christensen 1994). However, understanding the

germination requirements of these species would be necessary to be able to use these

species as cover crops.

Chapter 1

17

A number of Western Australian polycarpic fire ephemerals are also being

investigated for use in the production of paper, pulp and reconstituted wood panels.

These species have rapid growth rates and low density wood, which are desirable

attributes for source material. However, for any commercial development of these

species to be feasible, reliable methods of germination are required (Olsen et al. 2003).

Chapter 1

18

(a) Actinotus leucocephalus

(b) Alyogyne hakeifolia

(c) Alyogyne huegelii

(d)Austrostipa compressa

(e) Austrostipa macalpinei

(f) Grevillea scapigera

(g) Codonocarpus cotinifolius

(h) Gyrostemon racemiger

(i) Gyrostemon ramulosus

(j) Tersonia cyathiflora

Figure 4. Western Australian distribution of the ten

fire ephemerals that will be examined in this thesis.

Each red dot represents a herbarium specimen

(Western Australian Herbarium 1998-).

Chapter 1

19

Description of the Study Species

Below is a brief description of each of the ten fire ephemerals that are examined,

including reasons why their germination requirements should be examined and the

previous work that has been undertaken on them and their closely-related species.

Actinotus leucocephalus (Apiaceae)

The Apiaceae family has a cosmopolitan distribution with approximately 3000 species

in 300 genera worldwide (Marchant et al. 1987). There are 15 species in the Actinotus

genus, 14 of which are endemic to Australia (Powell 1992). This genus is characterised

by an umbel of flowers contracted into a head surrounded by woolly involucral bracts

(Figure 5a; Marchant et al. 1987; Lee and Goodwin 1994). In fact, �actino� means ray or

star-shaped, referring to the arrangement of the bracts (Stearn 1992). Another feature of

species in this genus are dry, dorsally-compressed one-seeded fruit called mericarps,

which have a persistent calyx (Figure 5b; Marchant et al. 1987; Lee and Goodwin

1994). Mericarps of Apiaceae species also usually have five main longitudinal ribs.

0.3 cm

b

Figure 5. Actinotus leucocephalus a) head ofumbels surrounded by white woolly bracts, b)mericarps with a persistent calyx, and c) plantgrowing to approximately 45 cm tall.

a c

Chapter 1

20

Actinotus leucocephalus is an annual herb that grows to approximately 45 cm in

height (Figure 5c) and the white flower heads (including the bracts) are approximately

1.5 to 4.5 cm in diameter (Figure 5a; Marchant et al. 1987). Accordingly, leucocephalus

comes from �leuco� meaning white and �cephalus�, which means head (Stearn 1992).

This species is found on the northern sandplain of Western Australia and is distributed

from Geraldton to south of Perth. Some plants have also been collected north of Albany

(Figures 3, 4a).

Monocarpic fire ephemerals are found within the Apiaceae family in Australia

(Bell et al. 1984; Pate and Dixon 1996) and California (le Maitre and Midgley 1992).

Actinotus leucocephalus is a monocarpic fire ephemeral because it is an annual that

grows prolifically after fire (Blombery 1965; Elliot and Jones 1982; Keighery 1982;

Offord and Tyler 1996).

Actinotus leucocephalus has horticultural potential as a rockery, bedding display

or pot plant species, but this potential has been restricted by difficulties in germinating

seeds (Fuss et al. 1996; Offord and Tyler 1996). Examining the germination behaviour

of Actinotus leucocephalus may assist in understanding how to propagate this species as

well as other closely related species with horticultural potential such as Actinotus

helianthi, A. schwarzii and A. forsythia (Offord and Tyler 1996). An understanding of

the germination requirements of Actinotus leucocephalus may also provide insights into

the requirements of related rare species. Four Australian Actinotus genera are listed as

rare or threatened Australian plants, with three (A. paddisonii, A. rhomboideus and A.

whicherae) being poorly known and the fourth, A. schwarzii, listed as vulnerable

(Briggs and Leigh 1996). Actinotus schwarzii only occurs in limited numbers in a small

number of populations, and it has been recommended that research be undertaken to

overcome the difficulties in propagating this species (Leach 1992).

Previous studies have shown that Actinotus leucocephalus seeds require either

heat or smoke to germinate (Tieu et al. 1999;2001a). However, less than 20% of A.

leucocephalus seeds germinated in response to smoke water or the optimum heat

treatments (40°C for 90 days or 100°C for 3 h; Tieu et al. 1999;2001a), and in some

studies none of these seeds responded to aerosol smoke (Roche et al. 1997). Maximum

germination was slightly higher (approximately 30%) when heat (50°C for 10 days) and

smoke treatments were combined (Tieu et al. 2001a). Further research is required to

determine how to increase germination further.

Germination of a related species from eastern Australia, Actinotus helianthi, is

notoriously variable (von Richter and Offord 1998). In a similar manner to A.

Chapter 1

21

leucocephalus, A. helianthi seeds are believed to persist in the soil seedbank for many

years and germinate prolifically after fire (Lee and Goodwin 1994; Offord and Tyler

1996). Germination of fresh A. helianthi seeds is generally low (<30%) and may

increase following a period of after-ripening (Lee and Goodwin 1994; von Richter and

Offord 1998). Smoke treatments have also enhanced germination (Roche et al. 1997;

von Richter and Offord 1998).

Alyogyne hakeifolia and Alyogyne huegelii (Malvaceae)

The Malvaceae is another family with a worldwide distribution, being found in all but

very cold regions (Mitchell and Norris 1990). Worldwide there are approximately 2000

species in 85 genera, and in Australia there are about 160 species in 24 genera (Mitchell

and Norris 1990). Alyogyne is an Australian endemic genus with four species: A.

cuneiformis, A. hakeifolia, A. huegelii and A. pinoniana, and a number of varieties

(Marchant et al. 1987; Paczkowska and Chapman 2000; Pfeil and Craven 2004).

However, the group is presently undergoing revision and many more species and

varieties are in the process of becoming formally recognised (Western Australian

Herbarium 1998-).

Alyogyne hakeifolia and A. huegelii are fast-growing shrubs reaching up to 3 m

in height (Elliot and Jones 1982; Keena 2002). Both species produce large flowers in a

range of colour forms (Figure 6a-d). Alyogyne hakeifolia flowers can be yellow, pink or

mauve and often have red centres whereas A. huegelii flowers can be white, pink,

yellow, lilac, mauve or deep purple (Keena 2002). Alyogyne species have an undivided

style, which differentiate them from the closely-related Hibiscus species which have a

divided style (Marchant et al. 1987; Keena 2002). Accordingly, Alyogyne is derived

from �alytos�, meaning undivided (Keena 2002) and �gyno� meaning female organs

(Stearn 1992). The seeds of Alyogyne and Hibiscus also differ; generally Alyogyne seeds

have more endosperm and smaller and simpler embryos (Fryxell 1968). The seeds

(Figure 6e,f) of both Alyogyne species are released from dehiscent capsules (Figure

6g,h; Marchant et al. 1987; Keena 2002). Alyogyne huegelii plants are usually covered

in stellate hairs and the leaves are variable in form but often broad and sometimes lobed

(Figure 6a,c,h; Marchant et al. 1987). In contrast, the leaves of A. hakeifolia are linear

to terete and glabrous (Figure 6c,g; Elliot and Jones 1982). The specific epithet

�hakeifolia� refers to a similarity of the leaves to those of the genus Hakea (Stearn 1992;

Sharr 1996).

Chapter 1

22

a d

f g

h

e

1 cm

Figure 6. Flowers (a-d), seeds (e,f) and dehiscent capsules (g,h) of Alyogyne hakeifolia (b,d,e and g) and Alyogyne huegelii (a,c,f and h).

b

c

Chapter 1

23

Alyogyne hakeifolia occurs across most of the south-west botanical province

except in the south-western corner of the state which has relatively high rainfall and a

moderate Mediterranean climate (Figures 2, 3, 4b; Beard 1983). Alyogyne huegelii is

primarily found within the south-west botanical province of Western Australia within

the mallee and heath vegetation types on the northern sandplain stretching from Perth to

just north of Geraldton and southern sandplain from Albany to Esperance (Figures 3, 4c;

Beard 1990). The distribution of both species extend into South Australia (Keena 2002).

A number of polycarpic fire ephemerals from the Malvaceae are found in the

South African Cape and Australia (le Maitre and Midgley 1992). Alyogyne hakeifolia

and Alyogyne huegelii have frequently been cited as polycarpic fire ephemerals (Pate et

al. 1985; Weston 1985; Pate and Hopper 1993; Pate and Dixon 1996). Alyogyne

hakeifolia has also been referred to as a fire ephemeral by Brown and Hopkins (1984),

Hopkins (1985), Bell et al. (1993) and Yates et al. (2003). Alyogyne hakeifolia and A.

huegelii plants regenerate from seeds after fire and die within six or seven years (Brown

and Hopkins 1984; Pate et al. 1985). These seeds are thought to persist in the soil

seedbank for decades between fires because these conspicuous species are not observed

except in the immediate post-fire years (Weston 1985). Elliot and Jones (1982) and

Keena (2002) have noted that Alyogyne seeds retain their viability for a number of

years.

It is important to understand how to germinate seeds of Alyogyne and related

species because Hibiscus cravenii, known as Alyogyne cravenii until a recent taxonomic

revision (Pfeil and Craven 2004), is listed as poorly known. This means that Hibiscus

cravenii may be rare or threatened but further information is required to confirm this

(Briggs and Leigh 1996). An understanding of how to germinate Alyogyne species is

also important for horticultural propagation. Alyogyne species are valued in ornamental

horticulture because they have numerous, large showy flowers and the plants grow

rapidly and do not have prickles (Elliot and Jones 1982; Keena 2002). Cultivars of these

species are already grown in Europe, the United States, Australia and New Zealand

(Keena 2002). One Alyogyne hakeifolia cultivar grown in Australia is �Melissa Anne�

(Figure 6b). In addition to ornamental horticulture, Alyogyne hakeifolia and A. huegelii

have been identified as potential future wood sources for paper and panel board

construction. For any future commercial production of these species as crops it is

essential that their germination requirements are understood (Olsen et al. 2003).

Some Alyogyne seeds are reportedly easy to germinate, with germination being

enhanced by treatments that fracture the impermeable seed coats such as nicking (Keena

Chapter 1

24

2002). In this thesis this claim will be investigated. If validated, a comparison with

seeds of other fire ephemerals may provide insights into means of germinating these

other species.

Austrostipa compressa and Austrostipa macalpinei (Poaceae)

The Poaceae is another family with a cosmopolitan distribution and it is one of the

largest plant families with approximately 700 genera and 10 000 species worldwide

(McCusker 2002). There are 62 Austrostipa species endemic to Australia (Jacobs and

Everett 1996). These species were formerly included in the genus Stipa but the

Australian species have recently been transferred into a separate genus, Austrostipa

(Jacobs and Everett 1996). Austro comes from the term �australis� meaning southern

(Stearn 1992). Species in this genus are commonly known as Speargrasses because they

have a pointed callus (Osborn et al. 1931; U. Bell 1999). Austrostipa compressa and A.

macalpinei are annual tufted grasses that grow to 70 cm and 90 cm tall respectively

(Vickery et al. 1986). However, the actual size of plants may vary according to the

availability of nutrients (Pate et al. 1985; Roche et al. 1998). Austrostipa compressa and

A. macalpinei are placed in the subgenus Longiaristatae, because of their long awns

(Jacobs and Everett 1996). In both species this awn is bent twice (Figure 7). The column

of the awn is twisted (Jacobs and Everett 1996) and can assist in seed burial as it

unwinds (Ghermandi 1995; Smith et al. 1999). These species can be differentiated on

the basis of the ligule; in A. compressa it is glabrous, whereas in A. macalpinei it is

tomentose (Blackall and Grieve 1981). Austrostipa compressa is primarily distributed

along the south-western Australian coast from Geraldton to Albany in sandy areas

(Figure 4d; Vickery et al. 1986). Austrostipa macalpinei has a wider distribution from

western Victoria to southern South Australia and also in Western Australia from Perth

to Geraldton and also in pockets near Albany and Esperance (Figure 4e; Vickery et al.

1986). The specific epithet macalpinei is not in reference to an alpine habitat. Instead it

alludes to the plant pathologist, Daniel McAlpine (Sharr 1996).

Chapter 1

25

A number of Austrostipa species have been classified as fire ephemerals (Bell et

al. 1984; Pate et al. 1985) including Austrostipa compressa (Roche et al. 1998; Rokich

et al. 2000) and A. macalpinei (Gill 1993). Austrostipa compressa and A. macalpinei

have also been described as primarily, (Bennett 1988; U. Bell 1999) or only,

germinating after fire (Specht et al. 1958; Baird 1984). Austrostipa compressa may also

germinate after track-grading or other soil disturbances (U. Bell 1999; Rokich et al.

2000). U. Bell (1999) suggests that A. compressa can form a persistent seedbank as over

100 viable seeds per metre squared were recorded at Yule Brook Reserve, Perth, over 45

years after a fire (Smith et al. 1999). However, this is based on the assumption that

these plants only germinate after fire which may not be entirely true (Bennett 1988;

Rokich et al. 2000). Nevertheless it does suggest that this species has the potential to

form persistent seedbanks, which is a characteristic of fire ephemerals (Parker and Kelly

1989).

Researching the germination and dormancy requirements of Austrostipa

compressa and A. macalpinei is important because most work on growing Australian

native grasses has focused on eastern Australian species (U. Bell 1999). Austrostipa

species have also been identified as potential native fodder grasses (Osborn et al. 1931)

and as suitable for land rehabilitation (Hagon 1976; U. Bell 1999), and thus an

understanding of how to germinate these species will facilitate their utilisation.

Austrostipa compressa and A. macalpinei are not rare or threatened Australian plants

but 13 other Stipa species (now Austrostipa) are listed by Briggs and Leigh (1996). One

species is endangered, two are vulnerable, six are rare and four are poorly known.

Hence knowledge about the propagation of Austrostipa compressa and A. macalpinei

may be transferable to these other species.

Figure 7. Caryopsis of a) Austrostipa compressa and b) Austrostipa macalpinei showing doubly bent awn and twisted column.

a b1 cm

Chapter 1

26

Some previous germination studies have been undertaken on Austrostipa

compressa seeds (Dixon et al. 1995; Roche et al. 1998; Smith et al. 1999) but not, as far

as it is known, on Austrostipa macalpinei seeds. Germination of Austrostipa compressa

is generally higher under a 12 h daily light regime than in continuous darkness and is

also influenced by wavelength (red light producing more germination than far red light

but the R:FR ratios were not recorded; Smith et al. 1999). Similarly, Stipa nitida seeds

germinated better in light than darkness (Osborn et al. 1931) but Austrostipa scabra

subsp. scabra germination was unaffected by light conditions (Clarke et al. 2000).

Maximum germination was low for Stipa nitida (12.5%; Osborn et al. 1931) and

Austrostipa scabra subsp. scabra seeds (approximately 30%; Clarke et al. 2000) but

high levels of germination (>80%) were recorded for Austrostipa compressa (Smith et

al. 1999). Germination trials at Kings Park on Austrostipa compressa were unable to

replicate the levels of ex situ germination reported by Smith et al. (1999; K.W. Dixon

pers. comm. 2001).

Smoke water enhanced germination of 16 month old Austrostipa compressa

seeds that had been stored in the laboratory, but aerosol smoke (90 min application) had

no effect on the germination of seeds stored in the soil for between 14 months and more

than 45 years (Smith et al. 1999). Conversely, Dixon et al. (1995) found that aerosol

smoke promoted germination of seeds in situ. Variable response to smoke water has

also been found in other Austrostipa species. For example, germination of Stipa scabra

subsp. scabra was unaffected by smoke water in one study (Clarke et al. 2000) but was

suppressed relative to the control in another (Read and Bellairs 1999).

Bennett (1988) suggested that the heat from fires may cue Austrostipa

compressa seeds to germinate. However, heating Austrostipa compressa seeds for

between 10 and 60 min at 70-90ºC suppressed germination (Smith et al. 1999).

Similarly an 80ºC for 15 min heat treatment suppressed germination of Austrostipa

scabra subsp. scabra compared to unheated controls (Clarke et al. 2000). Neither study

investigated whether heat suppressed germination because it imposed secondary

dormancy or killed seeds.

Chapter 1

27

Grevillea scapigera (Proteaceae)

The Proteaceae is a predominantly Southern Hemisphere family and the majority of

species are found in Australia and South Africa (Flora of Australia 1995). In total there

are approximately 79 genera and 1700 species in the family, of which about 46 genera

and 1100 species are found in Australia (Flora of Australia 1995). There are 362 species

of Grevillea, most (355) of which are endemic to Australia (Makinson 2000). Grevillea

scapigera is a prostrate shrub that grows to approximately 2 m in diameter (Makinson

2000). The leaves are deeply divided (Figure 8a) and the green-white flowers (Figure

8b) are borne in globular heads (4 cm diameter) on emergent scapes to 0.4 m in height

(Brown et al. 1998; Makinson 2000). It is this from this distinctive peduncle or scape

that the species has been given the specific epithet, scapigera (Olde and Marriott 1995).

There are three main types of grevillea seeds; those with a membranous wing, those

with revolute margins and those with an inner flat surface and an outer convex mottled

surface (Olde and Marriott 1995). Grevillea scapigera seeds fall into the third category

(Figure 8c).

a b

1 cm

Figure 8. Grevillea scapigera a) plant showing divided leaves b) flower head and c)seeds. The flat surface of the seeds is shown on the left and the convex, mottled side onthe right.

c

b

Chapter 1

28

This species occurs on sandplains in the Corrigin region, which is located at the

drier margin of the south-west botanical zone (Figures 2, 3, 4f; George 1974). These

shrubs are short-lived (≤9 years) and require disturbance for seedling establishment

(Olde and Marriott 1995; Rossetto 1995; Rossetto et al. 1995; Brown et al. 1998).

Approximately 77% of seeds buried in situ did not germinate and retained their viability

for at least three years (Rossetto 1995). Thus this species appears to display fire

ephemeral characteristics even though, as far as it is known, no species in the

Proteaceae have been formally classified as fire ephemerals (Table 1; le Maitre and

Midgley 1992).

There are a number of reasons why research is required into the germination and

dormancy requirements of this species. Grevillea scapigera is a critically endangered

species that has a geographic range of less than 100 km (Briggs and Leigh 1996; Brown

et al. 1998). In 1994 only four populations of Grevillea scapigera were known, with the

largest population totalling only 16 plants (Rossetto et al. 1995). This species is

reportedly difficult to grow from seed (Rossetto 1995; Rossetto et al. 1995). Another

175 Grevillea taxa are listed as rare or threatened (Briggs and Leigh 1996) and insights

gained about the dormancy and germination of this species may be applicable to other

rare grevilleas. In addition, understanding the germination requirements of this species

may be beneficial to the horticultural industry. This species has potential as a rockery

plant, ground cover or pot plant because of its unique head of lightly scented flowers,

blue-grey foliage and trailing habit (Wrigley and Fagg 1989; Olde and Marriott 1995).

Although Grevillea scapigera has been described as difficult to grow from seed

(Rossetto 1995; Rossetto et al. 1995), some seeds have been induced to germinate by

scarification (Vallee et al. 2004). In another trial approximately 55% of Grevillea

scapigera seeds germinated over 154 days following a combination of scarification,

smoke water and gibberellic acid treatments (Cochrane et al. 2002). However, it is not

known which factors contributed to germination. In disturbance experiments (aerosol

smoking, burning, cultivating and mulching) at translocation field sites, germination

only occurred in the aerosol smoked site, but it is difficult to conclude whether seeds are

smoke responsive as seedling numbers totalled only six over two years (Vallee et al.

2004). However, 50 ± 25% of Grevillea scapigera seeds germinated following

glasshouse soil storage and smoke treatment (Roche et al. 1997). Numerous

germination studies have been undertaken on other Grevillea species with many

responding to heat, smoke or scarification treatments, or a combination of these

Chapter 1

29

(Edwards and Whelan 1995; Roche et al. 1997; Kenny 2000; Morris 2000; Morris et al.

2000; Pickup et al. 2003). A period of burial also enhanced germination in some of

these species (Roche et al. 1997; Pickup et al. 2003).

Gyrostemon racemiger, Gyrostemon ramulosus, Codonocarpus cotinifolius and

Tersonia cyathiflora (Gyrostemonaceae)

The Gyrostemonaceae is an Australian endemic family containing five genera:

Codonocarpus, Gyrostemon, Tersonia, Cypselocarpus and Walteranthus (George 1982;

Paczkowska and Chapman 2000). Many species in this family are characterised by red,

brown or orange stems (Figure 9a,i; George 1982). Species in this family are also

typified by separate male and female flowers. Male flowers have one or more whorls of

stamens on a disc (Figure 9c) and female flowers have a single whorl of tepals

(Carlquist 1978). The seeds are reniform and red to brown with a prominent aril (Figure

9d-g). The seed surface is ribbed and each rib is more finely striated. Internally the

embryo is curved and endosperm is present (George 1982; Tobe and Raven 1991). Most

species in this family germinate after fire or disturbance and are primarily distributed in

arid areas (George 1982). Species in the Gyrostemonaceae have frequently been

described as fire ephemerals (Bell et al. 1984; Pate et al. 1985; Delfs et al. 1987; Pate

and Hopper 1993; Pate and Dixon 1996; Yates et al. 2003).

Codonocarpus cotinifolius is a pyramidal shrub or tree that can grow to 10 m

tall. The name �Codonocarpus� comes from �codon� meaning bell and �carpus� meaning

fruit, in reference to the bell-shaped fruit (Stearn 1992; Sharr 1996). The specific epithet

cotinifolius refers to the similarity of the leaves to those of the genus Cotinus (Figure

9h; Sharr 1996). This species is monoecious (separate male and female flowers on the

same plant). This species has a widespread distribution in dry areas (Figure 4g),

occurring in all states of Australia except for Tasmania and the Australian Capital

Territory (Carlquist 1978; George 1982).

There are twelve Gyrostemon species, all of which are shrubs or small trees

(George 1982). Gyrostemon is from �gyrus� meaning ring and �stemon� meaning

stamens in reference to the arrangement of the stamens (Stearn 1992). These species are

dioecious (male and female flowers on separate plants) and are found in all states of

Australia (George 1982). The two species that will be investigated here are Gyrostemon

racemiger (Figure 9a-c) and Gyrostemon ramulosus. These two species can be

distinguished from one another by the arrangement of the flowers (George 1982). The

Chapter 1

30

dc

ba

f

e

g

i h

j

kl

1 cm

Figure 9. Gyrostemon racemiger a) plants growing en masse 1.5 years after fire to over 2 m tall, b) male flowers, and c) dry carpels releasingseeds. Seeds of d) Codonocarpus cotinifolius, e) Gyrostemon ramulosus, f) Tersonia cyathiflora and g) Gyrostemon racemiger. Codonocarpus cotinifolius seedling h) showing leaf form. Tersonia cyathiflora i) female sprawling plant, j) mature indehiscent fruit along stem, k) inside of fruitand l) flower.

Chapter 1

31

specific epithet, �racemiger�, means bearing a raceme and hence Gyrostemon racemiger

flowers are arranged in racemes (Figure 9b,c). In contrast, �ramulosus� means having

many small branches and Gyrostemon ramulosus plants have many solitary flowers

(Stearn 1992; Sharr 1996). Gyrostemon ramulosus occurs widely, from the west coast

of Australia, across central western Australia to southern Northern Territory and South

Australia, with some populations also in south-western Queensland (Figure 4i; George

1982). In contrast, Gyrostemon racemiger is a less widely distributed species on a

continental scale but is widespread in south-western Australia, particularly in a band

from Geraldton to Salmon Gums (Figure 4h; Carlquist 1978; George 1982).

Tersonia is a monotypic dioecious genus (George 1982). It differs from the other

Gyrostemonaceae genera examined above in that female plants are usually, although not

always, prostrate (Figure 9i), and the fruit is hard and indehiscent (Figure 9j,k; George

1982). Tersonia is from tersomai meaning �to be dried up� with reference to the dry

fruit (Sharr 1996), and �cyathiflora� means cup-shaped flowers (Figure 9l; Stearn 1992).

The genus is endemic to south-western Western Australia and occurs near the coast

from Bunbury to Kalbarri (Figure 4j; George 1982).

The germination of Gyrostemonaceae species should be examined for a range of

reasons. A number of species congeneric to those that will be studied are rare or poorly

known (Briggs and Leigh 1996). Codonocarpus pyramidalis is classified as vulnerable,

and Gyrostemon brownii, G. ditrigynus, G. prostratus, G. sessilis and G. thesioides are

poorly known (George 1982; Briggs and Leigh 1996). Another species, Gyrostemon

reticulatus, was thought to be extinct (Briggs and Leigh 1996) until it was rediscovered

in 1990 near Geraldton (Chant 2001; Stack and English 2002). The research in this

thesis has been listed as an action in the Recovery Plan for this species (Stack and

English 2002). In addition, Codonocarpus cotinifolius, Gyrostemon racemiger and

Gyrostemon ramulosus are among the species being investigated for their potential use

in paper and panel board production, and so an understanding of how to germinate these

species will assist in developing these species as a crop (Olsen et al. 2003).

Codonocarpus cotinifolius also has potential as an ornamental plant, but its use has been

restricted by the difficulties in germinating seeds (Kenneally et al. 1996).

At present, many species in the Gyrostemonaceae are regarded as difficult to

germinate (Fox 1985; Dixon et al. 1995; Stack and English 2002). Tersonia cyathiflora

seeds did not respond to heat and smoke cues or scarification and smoke (Tieu et al.

2002). Gyrostemon ramulosus and Codonocarpus cotinifolius seeds placed in the soil

and watered were not induced to germinate but a small percentage (<15%) of viable

Chapter 1

32

seeds germinated following smoke treatment of punnets (Dixon et al. 1995). Another

Codonocarpus species, C. pyramidalis failed to germinate in water but 45% of seeds

germinated in response to gibberellic acid (Loveys and Jusaitis 1994).

Requirements for Seeds to Germinate A seed is a plant dispersal unit containing an embryo and its food source (Bewley

1997). This �packaged living plant� is often in a state of arrested growth. For seeds to

germinate they must be viable and have adequate water and suitable temperature.

Sometimes germination does not proceed due to a block to germination within the seed,

termed dormancy. Non-dormant seeds may also require specific germination stimulants

such as light or nitrate (Vleeshouwers et al. 1995).

Viability

A variety of techniques have been developed to assess seed viability including

germination tests, tetrazolium chloride (Lakon 1949; Moore 1973; International Seed

Testing Association 2005), cut tests (Roche et al. 1997; Tieu et al. 1999; Turner et al.

2005) and embryo excision (Meney and Dixon 1988; Paynter and Dixon 1990). The

most widespread technique presently used, apart from germinating seed, is the

tetrazolium chloride test. This test involves soaking seeds in a 1% solution of 2,3,5-

triphenyl-tetrazolium chloride in the dark, as the solution is light sensitive (Lakon 1949;

Moore 1973; International Seed Testing Association 2005). Living cells reduce the

colourless tetrazolium to triphenyl-formazan, which is red and non-diffusable, and thus

stains viable tissue red (International Seed Testing Association 2005). Seeds are

dissected before soaking to facilitate entry of the solution into the seeds (Lakon 1949).

The duration of soaking required varies between species (Lakon 1949; International

Seed Testing Association 2005). Maize requires only three to four hours at room

temperature (Lakon 1949), whereas many other species require longer periods (Ooi et

al. 2004; International Seed Testing Association 2005). For example, some Leucopogon

species require seven days at 24ºC for staining to occur (Ooi et al. 2004). Generally,

seeds are not soaked for more than 24 hours to prevent the growth of microorganisms,

which may cause staining even if seeds are not viable (Lakon 1949). Elevating the

temperature can hasten the staining reaction (Lakon 1949) and the recommended

temperature for staining seeds is 25-30°C (Moore 1973; International Seed Testing

Association 2005).

Chapter 1

33

There are some limitations with using the tetrazolium chloride test to assess seed

viability. The pattern of staining is the primary determination of seed viability (Moore

1973). Some commonsense interpretation of staining patterns can be used and essential

structures for seedling development, such as meristematic tissue, must be stained

(International Seed Testing Association 2005). Protocols for assessing staining patterns

are lacking for many Australian species, which may make it difficult to interpret the

wide range of patterns that can result (Gravina and Bellairs 2000). Ooi et al. (2004)

suggest that the variation in staining intensity of Leucopogon seeds is related to the rate

of respiration, which is possibly linked to dormancy. Some viable seeds do not stain at

all when dormant (Mohamed et al. 1998; Cohn 2002), and this may result in an

underestimation of viability. In addition, the tetrazolium chloride test is more time

consuming than alternative methods. Nevertheless, despite the problems, this test

appears to have widespread acceptance.

Although the tetrazolium chloride test is standard it may be appropriate to

undertake multiple viability tests to confirm results and to determine whether simpler

tests can be used as less time-consuming surrogates. The cut test is a simple rapid

method of estimating viability (Ooi et al. 2004; International Seed Testing Association

2005). Seeds are dissected, and scored as potentially viable if the contents are full,

white, firm and possess an embryo. This technique has been used in a large number of

studies, particularly with Australian species including those by Roche et al. (1997), Tieu

et al. (1999) and Turner et al. (2005). The cut and tetrazolium chloride tests produced

similar estimates of seed viability in Leucopogon (Ooi et al. 2004). Here all filled seeds

were viable which might be expected in fresh seeds. However, with duration of

laboratory-storage seeds may lose viability but remain filled, thus overestimating seed

viability. For example, difficulty in distinguishing between live and dead seed tissue in

some species was noted by Paynter and Dixon (1990). Although the cut test may

overestimate viability, it is simple, quick and clearly identifies what proportion of the

seed lot is not potentially germinable.

Apart from tetrazolium chloride, a number of other stains have been used to

assess seed viability including fluorescein diacetate (Pritchard 1985; Sukhvibul and

Considine 1994; Noland and Mohammed 1997), indigo carmine (Booth and Hendry

1993) and Evans blue (Batty et al. 2001). Following soaking in fluorescein diacetate,

cells with an intact plasmolemma, which indicates viability, fluoresce under ultraviolet

light. This technique is mainly used for assessing the viability of single cells or simple

organisms such as orchid seeds (Pritchard 1985). Relatively few studies have utilised

Chapter 1

34

fluorescein diacetate in viability tests of larger seeds, although Sukhvibul and Considine

(1994) and Noland and Mohammed (1997) have applied it successfully. A problem with

this technique is that some seeds autofluoresce, that is, fluoresce without staining (Baker

and Mock 1994). Another stain that can be used to test seed viability, indigo carmine, is

oxidised to a colourless compound by living tissue, but dead tissue stains red (Booth

and Hendry 1993). Seeds can also be stained with Evans blue, which accumulates in

dead cells (Baker and Mock 1994). As for fluoroscein diacetate, Evans blue is primarily

used on single cells or orchid seeds (Batty et al. 2001).

Another method of assessing seed viability, particularly for species that are

difficult to germinate and do not produce meaningful tetrazolium staining patterns, is in

vitro culture of extracted embryos (Meney et al. 1994). In vitro culture, also referred to

as excised-embryo tests, entails aseptically extracting embryos and placing them on agar

containing half-strength Murashige and Skoog (1962) salts, and vitamins, sugars and

hormones to induce germination (Meney and Dixon 1988). This technique has also been

used to verify tetrazolium chloride staining patterns (Paynter and Dixon 1990). Dormant

embryos of some cereals will grow in water alone after excision (Cohn 1996). In fact,

viable embryos of most species with non-deep or intermediate physiological dormancy

will produce normal germination when excised (J. Baskin and Baskin 2004). Growth

indicates the presence of living tissue, but this is a very time-consuming technique as

the excision of small, intact and delicate embryos is difficult and laborious.

Basic Conditions Essential For Germination

Most seeds require appropriate temperatures, water and oxygen to germinate (Bradbeer

1988). Temperature and soil water availability are two of the main factors that influence

the timing of seed germination in the field (Ross 1976). Often seeds germinate at

temperatures that coincide with optimal conditions in their natural range for growth and

survival (Bell and Bellairs 1992). In south-western Western Australia most germination

occurs during the wet winter season (D. Bell 1999). Four of the species that will be

examined in this thesis were collected from the northern sandplain region of south-

western Australia (Figure 3), and other species from this area have had optimum

germination at incubation temperatures between 15 and 20ºC, which coincide with

temperatures during the wetter months (Bellairs and Bell 1990).

Species may germinate effectively at constant temperatures equivalent to the

average of fluctuating field temperatures (Ross 1976). Germination of Stipa nitida

(Poaceae) seeds is similar irrespective of whether seeds are held under alternating

Chapter 1

35

temperatures or the average constant temperature (Osborn et al. 1931). In other species

higher levels of germination occur under alternating temperatures (Harty and McDonald

1972; Thompson 1974).

Germination Stimulants

Viable seeds may still not germinate at appropriate temperatures when supplied with

water and oxygen due to requirements for germination stimulants or because dormancy

is present. Vleeshouwers et al. (1995) proposed that dormancy-breaking and

germination stimulation are distinct processes. Non-dormant seeds may require

germination stimulants such as light or nitrate before germination can proceed

(Vleeshouwers et al. 1995). Fluctuating temperatures may also operate as germination

stimulants (Vleeshouwers et al. 1995) because they are an absolute requirement for the

germination of some species and promote higher levels of germination as dormancy is

alleviated (Benech-Arnold and Sánchez 1995). The actual mechanism of germination

stimulants is not fully understood but it has been proposed that receptors in seeds, which

can set in train a sequence of steps that lead to germination when activated, may become

more available for binding as dormancy is alleviated (Derkx and Karssen 1993;

Vleeshouwers et al. 1995). For germination to proceed non-dormant seeds may not

require any germination stimulants (J. Baskin and Baskin 1994), one germination

stimulant, such as light (Baskin and Baskin 1990), or multiple stimulants such as light

and nitrate (Derkx and Karssen 1993).

Benech-Arnold et al. (2000) use slightly different but equally valid terminology

to Vleeshouwers et al. (1995) to describe germination stimulants. Requirements for

light, nitrate and alternating temperatures are regarded as ultimate constraints to

germination rather than germination stimulants. However, the terminology of

Vleeshouwers et al. (1995) will be adopted here because it is increasingly gaining

acceptance and it may provide a clearer explanation of the separate roles of burial and

fire-cues in this study.

Dormancy

If viable seeds do not germinate when provided with suitable temperature and moisture

conditions, and requirements for germination stimulants are satisfied, seeds may be

dormant. In this thesis dormancy will be considered a characteristic of seeds and not

their environment (Thompson et al. 2003). Dormancy is an internal block to

germination (Vleeshouwers et al. 1995). Thus dormancy should not be equated with the

Chapter 1

36

absence of germination resulting from inappropriate external conditions (Vleeshouwers

et al. 1995). A feature of the most common class of dormancy, physiological dormancy

(Baskin and Baskin 2003), is that seeds are not simply dormant or non-dormant. Rather,

the degree of physiological dormancy is continuous and influences the range of

conditions at which seeds can germinate (Vleeshouwers et al. 1995).

Dormancy Classification

An understanding of the type of dormancy present in seeds may provide insights

into ways to alleviate that dormancy and vice versa. According to Hobson (1981), who

worked on tuber dormancy, there may be as many dormancy classifications as there are

practitioners working in the field. However the dormancy classification scheme for

seeds that appears to be most widely accepted at present is that proposed by J. Baskin

and Baskin (2004; Table 2) which is a modification of the work of Nikolaeva

(1969;1977). This system is comprised of five dormancy classes based on seed

permeability to water, embryo morphology and physiological response of whole seeds

to temperature (J. Baskin and Baskin 2004). Seeds with undifferentiated embryos form

another specialised type of morphological dormancy (C. Baskin and Baskin 2004) and

will not be examined here because they are not representative of any of the fire

ephemerals examined in this thesis. The J. Baskin and Baskin (2004) classification

scheme differs from that of Nikolaeva's (1969;1977) in the exclusion of mechanical

dormancy (now considered to be an aspect of physiological dormancy) and chemical

dormancy. Here chemical dormancy refers to chemicals in the seed covering layers that

inhibit germination as opposed to chemicals involved in metabolic pathways within

seeds that are involved in the imposition and release of dormancy in seeds with

physiological dormancy. J. Baskin and Baskin (2004) argue that the presence of

germination inhibitors in seed covering structures does not prove that these play a role

in seed dormancy under natural conditions. Nikolaeva (2004) has recently supported the

changes proposed by J. Baskin and Baskin (2004).

Chapter 1

37

Table 2. Main distinguishing characteristics of non-dormant seeds and the five

dormancy classes according to Baskin and Baskin (2003), C. Baskin and Baskin (2004),

and J. Baskin and Baskin (2004).

Dormancy class Seed permeable to water

Underdeveloped embryo (as opposed to developed)

Seeds germinate within 30 days at appropriate temperatures (water-impermeable seeds scarified)

Proportion of species with each of these dormancy types (%)

Non-dormant ! ! 30.4

Physiological (PD) ! 45.1

Physical (PY) ! 14.5

Combinational

(PY + PD)

0.5

Morphological (MD) ! ! ! 1.5

Morphophysiological (MPD)

! ! 8.1

Physical dormancy is presently known to occur in 15 families (Baskin and

Baskin 1998; Baskin et al. 2000). Of these, only the Malvaceae is represented amongst

the fire ephemerals that will be examined in this thesis. Even if physical dormancy is

found within a family, it is not necessarily present in all species within that family

(Baskin and Baskin 1998), and the proportion of seeds with physical dormancy may

also vary between seedlots (Rolston 1978). Seeds with physical dormancy

characteristically do not imbibe water when soaked (Table 2; Baskin and Baskin 1998).

However, following scarification these seeds rapidly imbibe water and can double in

weight within 24 hours (Baskin and Baskin 1974;1997; Morris 2000; Baskin et al.

2004; Turner et al. 2005). Except for seeds with combinational dormancy, seeds with

physiological dormancy do not require scarification for water to be imbibed. Following

soaking, these seeds usually increase in weight by approximately 15 to 40%, and

occasionally up to 70%. Pre-scarifying water permeable seeds does not alter patterns of

water uptake (Morris 2000; Hidayati et al. 2000;2001).

Physiological and morphophysiological dormancy can be subdivided into levels

(intensity of dormancy) and types (physiological patterns; J. Baskin and Baskin 2004).

Morphophysiological dormancy can be subdivided into eight levels on the basis of

warm or cold stratification requirements, timing of embryo growth and response to

gibberellic acid (C. Baskin and Baskin 2004; J. Baskin and Baskin 2004). Physiological

dormancy can be divided into non-deep (most common), intermediate and deep

dormancy. Seeds with intermediate and deep dormancy require cold stratification before

they will germinate whereas seeds with non-deep physiological may require either

Chapter 1

38

warm or cold stratification to alleviate dormancy. These levels are also distinguished by

the response of seeds to gibberellic acid, whether or not scarification promotes

germination and whether isolated embryos can germinate normally (J. Baskin and

Baskin 2004). Five different types of non-deep physiological dormancy have been

identified based on the changing range of temperatures at which seeds germinate during

dormancy release (J. Baskin and Baskin 2004). Seeds with type 1 dormancy initially

germinate at low temperatures and become able to germinate at increasingly higher

temperatures as dormancy is alleviated. Seeds with type 2 dormancy initially germinate

at high temperatures and as dormancy is relieved also become able to germinate at

progressively lower temperatures. The remaining three types are less common. Seeds

with type 3 dormancy can initially germinate at intermediate temperatures and

incrementally germinate at both higher and lower temperatures as dormancy is

alleviated. Seeds with type 1, 2 or 3 dormancy have the potential to exhibit dormancy

cycling. These three types of dormancy were also described by Vegis (1964). Seeds

with types 4 and 5 dormancy do not exhibit a gradual widening in the temperatures at

which seeds germinate. Rather once dormancy is released, seeds with type 4 dormancy

germinate at high temperatures and seeds with type 5 dormancy germinate at low

temperatures. As well as variations in the width of temperatures at which seeds will

germinate, the responsiveness of seeds to germination stimulants is altered by changes

in the level of seed dormancy (Hilhorst 1998). Thus physiological seed dormancy is not

static and the conditions suitable for germination become more or less specific

according to prior exposure of seeds to environmental conditions (Vegis 1964; Baskin

and Baskin 1985). Seeds in this continuum between dormant and non-dormant states are

defined as conditionally dormant (Vegis 1964; Baskin and Baskin 1985).

Primary and secondary dormancy are temporal classifications of physiological

dormancy (Hilhorst 1995). Seeds acquire primary dormancy during maturation drying

before they are shed from the mother plant (Hilhorst 1995; J. Baskin and Baskin 2004).

After primary dormancy is alleviated, secondary dormancy may be imposed if

environmental conditions are not conducive to germination (Vleeshouwers et al. 1995).

Secondary dormancy may be imposed and alleviated seasonally over many years of

burial, in a process termed dormancy cycling (Hilhorst 1995; Vleeshouwers et al. 1995;

Hilhorst 1998). Generally only seeds that exhibit primary dormancy can enter secondary

dormancy (J. Baskin and Baskin 2004). There is no conclusive evidence to determine

whether primary and secondary dormancy are distinct physiological processes (Hilhorst

1998). However it has been suggested that they are different because the alleviation of

Chapter 1

39

primary dormancy and induction of secondary dormancy may occur concurrently

(Totterdell and Roberts 1979; Probert et al. 1985) and in some species the response to

gibberellic acid differs during alleviation of primary versus secondary dormancy (Derkx

and Karssen 1993; Vleeshouwers et al. 1995).

Many other dormancy classification schemes have been proposed and some of

these overlap in certain aspects and represent a development of ideas over time. For

example as early as 1916, Crocker outlined some of the dormancy characteristics upon

which the Nikolaeva (1969;1977) and J. Baskin and Baskin (2004) schemes are based

including rudimentary embryos, barriers to water uptake and embryo dormancy. Other

schemes include those by Amen (1968) who emphasises the role of hormones in

dormancy regulation, Atwater (1980) who links different seed morphological

characteristics with germination requirements, and Lang et al. (1987) who attempt to

define a dormancy system applicable to all plant growth structures. In addition, seeds

can be classified according to morphology. Martin (1946) classified seeds according to

the size, position and morphology of the embryo. Although morphology is an effective

means of classifying seeds, more information about possible seed dormancy

characteristics can be gained when both morphological and physiological traits are

considered (Nikolaeva 2004).

Although this thesis follows the dormancy classification system of J. Baskin and

Baskin (2004), Harper�s (1977) seed dormancy scheme will be examined briefly as it is

widely used despite recent criticism (Vleeshouwers et al. 1995; Thompson et al. 2003;

J. Baskin and Baskin 2004). Harper (1977) defined three types of seed dormancy;

innate, induced and enforced. Innate and induced dormancy are similar to primary and

secondary dormancy respectively, as defined above (J. Baskin and Baskin 2004).

However, Harper�s (1977) definition of induced dormancy is much narrower than that

of secondary dormancy (Baskin and Baskin 1985). Induced dormancy refers to seeds

that have not previously experienced innate dormancy and thus this system does not

accommodate dormancy cycling and is too restrictive to define the range of dormancy

behaviour present in seeds (J. Baskin and Baskin 2004). The validity of the third type of

dormancy, enforced dormancy, has also been questioned (Vleeshouwers et al. 1995;

Thompson et al. 2003). Enforced dormancy is applied to seeds that do not germinate or

possess either innate or induced dormancy. Germination in these seeds is prevented by

the lack of appropriate environmental conditions such as adequate water or suitable

temperatures (Harper 1977). However as pointed out by Thompson et al. (2003), this

would include seeds that persist in the soil, yet these seeds are not necessarily dormant.

Chapter 1

40

Similarly Vleeshouwers et al. (1995) criticised this classification system because it does

not accommodate the definition of dormancy as an internal characteristic of the seed,

masking differences between dormancy breaking and germination. Thompson et al.

(2003) notes that despite the paper by Vleeshouwers et al. (1995), the less useful system

of Harper (1977) is still being followed. This is partially because Harper�s (1977)

terminology is embedded in the literature (Richards and Beardsell 1987; Bradbeer

1988). In this thesis the terminology of Vleeshouwers et al. (1995) will be adopted.

Possible Methods of Inducing Fire Ephemerals to Germinate Possible means of inducing fire ephemerals to germinate at a single point in time will

now be considered. The emphasis here will be on methods to stimulate or artificially

induce germination as opposed to alleviating physiological dormancy. These can be

grouped into natural and artificial means of inducing germination. Natural means of

inducing germination include fire-related cues such as heat pulses and smoke and

germination stimulants such as light and nitrate. Artificial means of inducing

germination include external applications of hormones and seed scarification. Although

these treatments will not necessarily unravel the requirements for these seeds to

germinate in their natural environment, they can provide insights into the kinds of

dormancy present (J. Baskin and Baskin 2004) and may provide avenues for producing

seedlings, which is particularly important for rare species and where other methods of

germinating these seeds is unknown.

Fire-Related Cues

The stimulatory effect of fire on the germination of many species is well established

(Levyns 1935; Went et al. 1952; Keeley et al. 1985; Auld and O'Connell 1991). Two of

the main components of fire that promote germination are heat (Warcup 1980) and

smoke (de Lange and Boucher 1990). Heat and smoke can influence germination either

separately or in combination (Keith 1997; Gilmour et al. 2000; Kenny 2000; Morris

2000; Tieu et al. 2001a; Wills and Read 2002; Thomas et al. 2003; Clarke and French

2005). Other products from fire include ash and charred wood. Exudates from charred

wood have promoted germination of species primarily from California, but also from

South Africa (Keeley et al. 1985; Brown 1993a; Keeley and Bond 1997). However the

germination of other fire-prone fynbos species has not been enhanced by charred wood

(Kilian and Cowling 1992), nor has the germination of most Australian species tested

Chapter 1

41

(Bell et al. 1987; Kenny 2000; Enright and Kintrup 2001). Similarly, ash has been less

effective than heat or smoke in promoting germination across a range of Australian

species (Enright et al. 1997) and aqueous extracts of ash did not stimulate the

germination of Nicotiana attenuata (Solanaceae), a Californian postfire annual

(Baldwin et al. 1994). Fire may also influence light and nutrient availability

(Humphreys and Craig 1981) and thus these factors will be considered although they are

not solely related to fire.

Heat

Heat can alleviate both physical and physiological dormancy. In seeds with physical

dormancy (water-impermeable) species, heat can crack the seedcoat, allowing water to

enter the seed and germination to proceed (Stone and Juhren 1951). For example, dry

heat (70°C for 1h) effectively overcame physical dormancy in Phylica ericoides

(Rhamnaceae; Kilian and Cowling 1992). Heat also alleviates physiological dormancy

in some species, such as Anigozanthos manglesii (Haemodoraceae; Tieu et al. 2001a).

The mechanism of how heat enhances the germination of water permeable seeds is still

not fully understood, although a number of explanations have been proposed. The most

likely is that heat causes changes in dormancy via alterations to seed membranes

(Hallett and Bewley 2002). This possibility is supported by the importance of

temperatures in driving changes in physiological seed dormancy (Vleeshouwers et al.

1995). However it has not been established whether a heat pulse operates in the same

way as heating seeds for longer durations at lower intensities. An alternative mechanism

by which heat pulses may induce germination is by scarifying outer layers of seeds

through desiccation, thus increasing oxygen uptake (Brits et al. 1993).

Soil temperatures, even without the passage of fire, may heat seeds sufficiently

to alleviate dormancy (Warcup 1980; Steadman et al. 2003). For example in exposed

sites in the chaparral, temperatures 6 mm below the soil surface can reach 60ºC for

several hours a day during summer (Stone and Juhren 1951) and on the soil surface on

sites directly exposed to the sun, temperatures may reach 70 to 80ºC (Keeley and

Fotheringham 2000). In the south-western botanical province of Western Australia,

surface soil temperatures frequently exceed 60 to 70°C (Hnatiuk and Hopkins 1981),

and the mean temperature maxima in the top 5 mm of soil can exceed 65°C for several

months of the year (Mott 1972).

Chapter 1

42

Smoke

The stimulatory effect of smoke on germination was first reported by de Lange

and Boucher (1990) who found that smoke, applied either as aerosol smoke or in smoke

water, promoted the germination of Audouinia capitata seeds (Bruniaceae). However,

subsistence farmers in South Africa traditionally stored maize in huts over fireplaces,

suggesting that the benefits of smoking seeds might not be such a recent discovery

(Modi 2002). Since 1990, the effect of smoke on the germination of a large number of

species has been tested from the following Mediterranean climate regions; the Cape

region of South Africa (Brown 1993b; Brown et al. 1993;1994; Brown and Botha

2004), California (Keeley and Fotheringham 1998a) and south-western Australia (Dixon

et al. 1995; Roche et al. 1997; Tieu et al. 1999).

Although investigations into the response to smoke have focused on species

from fire-prone areas with Mediterranean climates (Brown 1993b; Dixon et al. 1995;

Keeley and Fotheringham 1998a), species from other fire-prone environments have also

exhibited enhanced germination in smoke water. For example, Themeda triandra

(Poaceae) seeds collected from montane grassland in Natal (Baxter et al. 1994),

Hypericum gramineum (Clusiaceae) and Wahlenbergia gracilis (Campanulaceae) and

six native grass species from sclerophyll forest communities in New South Wales (Read

et al. 2000), and seven eastern Australian Grevillea species (Proteaceae; Morris 2000)

all exhibit enhanced germination in smoke water and are from areas without a

Mediterranean climate.

It is still unresolved whether smoke stimulated germination is primarily found in

species from fire-prone habitats or whether it evokes a more general germination

response. Three papers that have frequently been cited to suggest that smoke may

enhance germination of species from non fire-prone areas are those by Drewes et al.

(1995), Pierce et al. (1995) and Thomas and van Staden (1995). In two of the species

examined, lettuce (Lactuca sativa, Asteraceae; Drewes et al. 1995) and celery (Apium

graveolens, Apiaceae; Thomas and van Staden 1995), smoke effectively replaces a light

requirement as opposed to an obligate germination requirement for smoke, and in the

celery cultivar, only when gibberellic acid was also applied. Generalisations about the

germination response to smoke of species from non fire-prone areas should also be

tentative given that the taxa examined are cultivars that have undergone selection and

breeding outside their natural environment. Nevertheless, the paper by Pierce et al.

(1995) does appear to suggest that smoke responsive germination within the

Mesembryanthemaceae is not confined to species from fire-prone areas, assuming that

Chapter 1

43

the non fire-prone karoo species evolved in fire-free habitats. Species examined in all

three papers are from families that also occur within fire-prone areas and exhibit smoke

stimulated germination (Brown 1993b; Roche et al. 1997; Brown et al. 2003; Brown

and Botha 2004). Before it can be concluded whether smoke responsive germination is a

general trait rather than a fire-related cue, and whether it is a family-related trait, a

larger number of species need to be examined.

Within fire prone areas, such as South Africa, seeders and species with soil-

stored seeds are more likely to respond to smoke water than resprouters and serotinous

species (Brown et al. 2003). Thus Australian fire ephemerals, which are seeders with

soil-stored seedbanks, may germinate in smoke water. Furthermore, fire ephemerals

generally germinate after fire, suggesting that fire-related cues are involved in their

germination, and the germination of some fynbos and chaparral fire ephemerals are

enhanced by smoke (Keeley and Fotheringham 1998a; van Staden et al. 2000).

Nevertheless, some fire-following species have not germinated in response to

smoke water as expected. Some species do not germinate at all in response to smoke

water, such as the eastern Australian fire ephemeral, Monotaxis macrophylla

(Euphorbiaceae; Hunter 1998) and some other species exhibit a small increase in

germination following smoke treatment but retain a large proportion of non-germinating

viable seeds (Brown et al. 1994; Roche et al. 1997), including species in the fire

ephemeral genus Erepsia (Mesembryanthemaceae; Pierce et al. 1995). Consequently,

the function of smoke as an ecological cue has been questioned (Pierce et al. 1995).

Thus the cues required to germinate fire-following species that produce low germination

in response to smoke, including some fire ephemerals, need to be established to help

determine whether smoke plays an ecological role as a germination cue. Perhaps some

of these species require additional cues before the full response to smoke can be

expressed (Brown et al. 1993;1994).

Variation in germination response to smoke within a species is another aspect of

smoke stimulated germination that requires further investigation. In a number of

species, different seedlots of the same species have produced different responses to

smoke (Dixon et al. 1995; Morris et al. 2000; Tieu et al. 2001b). For some species this

variation can be explained by provenance. For example, more Anigozanthos manglesii

(Haemodoraceae) seeds germinate in response to smoke with an increase in latitude

(Tieu et al. 2001b). Other interrelated factors that may contribute to variation in

germination response to smoke within a species include seed age, quality, storage and

dormancy status (Dixon et al. 1995; Morris et al. 2000). In addition, the concentration

Chapter 1

44

of smoke water or duration of smoke exposure can influence the germination response.

In many species, high concentrations of smoke water inhibit germination or produce

less germination than lower concentrations (Dixon et al. 1995; Drewes et al. 1995; van

Staden et al. 1995a; Jäger et al. 1996a,b; Doherty and Cohn 2000). This is possibly

because a number of compounds in smoke inhibit germination (Baldwin et al. 1994).

Thus it may be necessary to test a range of smoke dilutions to determine whether a

species is smoke responsive. It also highlights the importance of using consistent smoke

water dilutions for comparisons.

The mechanism by which smoke water stimulates germination is still unknown

(van Staden et al. 1995b; Gardner et al. 2001; Modi 2002), which is not surprising

given that the process of germination itself has been studied for many years yet is still

not fully understood. Possible mechanisms by which smoke water may stimulate the

germination process include altering membrane permeability and influencing the

response of receptors to hormones (van Staden et al. 1995b), which are similar to the

possible mechanisms suggested for germination stimulants such as light and nitrate.

However, unlike light and nitrate, smoke has not been described explicitly as a

germination stimulant (Vleeshouwers et al. 1995). Alternative mechanisms by which

smoke may influence dormancy of water-permeable seeds are via external scarification

of seedcoats and/or increased permeability of the sub-testa cuticle (Egerton-Warburton

1998). Although permeability and scarification mechanisms may operate in some

species, smoke has enhanced germination of seeds of other species which have had their

seedcoats removed, indicating that smoke does not only influence germination via the

seedcoat (Morris et al. 2000).

There are thousands of chemicals present in smoke water (Adriansz et al. 2000).

Baldwin et al. (1994) investigated 71 compounds that were identified in fractions of

smoke water that stimulated germination. Research into the action of the germination

promoting component in smoke would have been confounded by the responses from

unrelated chemicals (van Staden et al. 1995b). However the recent isolation of a

butenolide as a germination stimulating agent in smoke water (Flematti et al. 2004), will

probably assist future investigations into the action of smoke water (van Staden et al.

2004).

The Search for the Active Chemical in Smoke

This thesis is not specifically concerned with the search for the chemical(s) in smoke

that stimulates germination. However, as smoke is an important fire-related germination

Chapter 1

45

cue, and in recent years a significant amount of smoke-related research has focused on

discovering the chemical(s) in smoke that promote germination, some of the

investigations that have led to the discovery of a butenolide as a germination enhancing

agent in smoke water will be outlined.

A large number of chemicals are present in smoke and the material burnt can

influence the presence and relative quantities of different chemicals (Guillen and

Manzanos 2002). Baldwin et al. (1994) identified 71 chemicals in fractions of smoke

water that promoted germination and Guillen and Manzanos (2002) detected 215

compounds in smoke derived from burning Quercus sawdust. The smoke generated

from a range of plant species that have been burnt stimulates seed germination (Baxter

et al. 1995; Jäger et al. 1996a). Perhaps the first suggestion that chemicals in smoke

may stimulate seed germination was the investigation by van de Venter and Esterhuizen

(1988) into the effect of ethylene and ammonia on seed germination. Although ethylene

is a known component of smoke (McElroy 1960) and stimulates germination in some

species (van de Venter and Esterhuizen 1988; Sutcliffe and Whitehead 1995; Ne�eman

et al. 1999), it is unlikely to be the ubiquitous smoke chemical that stimulates the

germination of many species because ethylene does not stimulate the germination of

specific smoke responsive species, such as Audouinia capitata (Bruniaceae; de Lange

and Boucher 1990) and Themeda triandra (Poaceae; Baxter et al. 1994). Furthermore,

germination of these species was stimulated by smoke water but the smoke water is

thought to contain only negligible concentrations of ethylene (de Lange and Boucher

1990).

Octanoic acid has a similar effect as smoke on Cyclopia intermedia (Fabaceae)

germination and it was hypothesised that the acid increased seed sensitivity to ethylene

(Sutcliffe and Whitehead 1995). This suggested that octanoic acid and ethylene together

may have been the active constituents in smoke. Although ethylene and octanoic acid

also enhance seed germination in lettuce, another smoke responsive species, the

concentration of smoke water required for octanoic acid to be at levels that stimulate

germination is inhibitory (Jäger et al. 1996b). Fractionated components of smoke most

stimulatory to lettuce seeds do not correspond to the presence of either of these

chemicals, and Jäger et al. (1996b) concluded that neither octanoic acid nor ethylene are

the active chemicals in smoke.

Different nitrogen compounds have also been considered as the basis for smoke

stimulated germination. Nitrate stimulated the germination of two smoke-responsive fire

annuals, Emmenanthe penduliflora and Phacelia grandiflora (both in the

Chapter 1

46

Hydrophyllaceae), which led to the suggestion that nitrogenous compounds may induce

post-fire germination (Thanos and Rundel 1995). Although these species are also

stimulated by smoke, Keeley and Fotheringham (1998b) conclude that nitrate is not the

stimulatory agent in smoke because germination levels are correlated with the

concentration of H+ rather than the concentration of dissociated NO3 ions. Also, smoke

water stimulates >90% of Emmenanthe penduliflora seeds to germinate when buffered

at a pH of 5 or 6 whereas nitrate produces <50% germination at the same pH (Keeley

and Fotheringham 1998b). Furthermore, Lloyd (2001) detected very low concentrations

of nitrogen in smoke water and found that nutrient application to field sites does not

invoke the germination response of smoke, indicating that nutrients in smoke water are

not responsible for smoke stimulated germination. Likewise, levels of nitrite in smoke

water are too low to account for the germination response of Oryza sativa to smoke

water (Doherty and Cohn 2000). Nitrogen oxides were also proposed as the germination

stimulant in smoke because they are present in smoke (McElroy 1960) and produce

similar high levels of Emmenanthe penduliflora germination as smoke (Keeley and

Fotheringham 1997). However, the stimulating chemical in smoke water is water

soluble (de Lange and Boucher 1990) and although smoke water stimulates germination

of Emmenanthe pendiflora and Nicotiana attenuata, the end product of NOx in water,

nitrite, was not detected. This suggests that nitrogen oxides are not the germination

stimulant in smoke water (Preston et al. 2004). Also, Preston et al. (2004) found that

nitrogen oxides at concentrations up to 42 µM do not stimulate germination in these

species. Light and van Staden (2003) suggest that nitrogen oxides are not the only

germination stimulating agents in smoke water because when nitric oxide scavenging

chemicals are added to smoke water, the stimulatory effect of smoke is not suppressed.

Furthermore, the germination stimulating chemical in smoke can be produced by the

combustion of pure cellulose (Jäger et al. 1996a). This indicates that the active chemical

is comprised of C, H and O only, indicating that no nitrogen compounds are likely to be

involved in smoke-mediated germination stimulation.

Adriansz et al. (2000) suggested that 1,8-cineole is a germination cue present in

smoke water. This chemical is less effective than smoke water at stimulating

germination and so Adriansz et al. (2000) concluded that there may be a number of

germination enhancing chemicals in smoke. Flematti et al. (2001) repeated this

experiment but concluded that 1,8-cineole was not responsible for the promotion of

germination by smoke water because it did not promote germination.

Chapter 1

47

Subsequently Flematti et al. (2004) identified 3-methyl-2H-furo[2,3-c]pyran-2-

one, which is a butenolide, as a chemical in smoke produced from cellulose combustion

that promotes germination. This was confirmed by using the synthesised butenolide to

stimulate germination of lettuce and other smoke-responsive species from fire-prone

regions. van Staden et al. (2004) identified the same chemical as a component of plant-

derived smoke that stimulated germination, providing further confidence that at least

one of the ubiquitous germination promoting chemicals in smoke water has been

identified.

Germination Stimulants

Light

Light is regarded as a germination stimulant, that is, a factor that may be required for

germination, but one that does not alter the dormancy state of the seed itself

(Vleeshouwers et al. 1995). Seeds of some species require light for germination, such as

Aristida contorta (Poaceae; Mott 1972), some are inhibited by light, including Spinifex

hirsutus (Poaceae; Harty and McDonald 1972) and Trachyandra divaricata

(Asphodelaceae; Bell et al. 1999), while others are unaffected by light conditions, such

as Emmenanthe penduliflora (Hydrophyllaceae; Keeley and Fotheringham 1998b). A

light requirement, which is often characteristic of small seeds, might prevent seeds

germinating at depths where they will have insufficient reserves to reach the soil

surface. Conversely, the requirement for darkness might be an adaptation to ensure that

seeds germinate below the soil surface where moisture availability is higher (D. Bell

1999).

Light exposure can vary in intensity, spectral composition and duration (Pons

2000). Germination of light sensitive seeds is promoted by exposure to wavelengths in

the red region of the visible spectrum and inhibited by exposure to wavelengths from

the far red, and to a lesser extent the violet/blue/green region of the spectrum (Flint and

McAlister 1935). The red to far red ratio (R:FR) can enable seeds to detect whether they

are buried, or shaded by other plants. At midday this ratio is approximately 1.05-1.25 in

unfiltered sunlight and 0.05 to 1.15 under a plant canopy (Neff et al. 2000). In many

germination studies the light conditions are not stipulated. Others just stipulate the light

source (Harty and McDonald 1972).

In seeds light is perceived by phytochrome photoreceptors, which are located in

the embryo (Casal and Sánchez 1998; Pons 2000). The presence of these photoreceptors

was first deduced by Borthwick et al. in 1952. There are two main types, phytochrome

Chapter 1

48

A and phytochrome B (Casal and Sánchez 1998; Pons 2000). These phytochromes

occur in two interchangeable forms; the red-absorbing form and the far-red absorbing

form. Shifts between these forms rarely occur in dry seeds (Bartley and Frankland 1984;

Casal and Sánchez 1998).

Nitrate

The use of nitrate is recommended for germinating a large number of species

(International Seed Testing Association 2005). This chemical is regarded as a

germination stimulant because it enables the germination of non-dormant seeds

(Vleeshouwers et al. 1995). Accordingly, germination in response to nitrate increases as

seeds after-ripen (Adkins et al. 1984).

Potassium nitrate may stimulate the germination of fire ephemerals as it has

stimulated germination in many species that are classified as disturbance opportunists

(Pons 1989; Plummer et al. 2001; Cruz et al. 2003) and fire followers (Baldwin et al.

1994; Thanos and Rundel 1995). For many of these species, around 10 mM of

potassium nitrate stimulates germination. In natural systems soil nitrate levels increase

after fire (Christensen 1973; Stock and Lewis 1986). Bell et al. (1984) also observed

fire ephemerals growing in unburnt nutrient enriched areas and suggested that these

species might germinate in response to nutrients. The growth of fire ephemerals is

highly responsive to nutrients (Pate et al. 1985) and thus nitrate may provide a signal to

ensure that these plants germinate when conditions are optimal. Thus the influence of

nitrate on the germination of a suite of Western Australian fire ephemerals will be

examined.

Gibberellic Acid

Gibberellic acid is a plant hormone that is involved in the regulation of seed dormancy

and germination (J. Baskin and Baskin 2004). Unlike nitrate or smoke, gibberellic acid

is not a naturally occurring external germination cue that seeds perceive. Rather seeds

either produce or become more receptive to gibberellic acid during dormancy-release.

Thus exogenous applications of gibberellic acid effectively bypass the normal sequence

of events involved in dormancy release in many seeds (Bewley 1997). Gibberellic acid-

induced germination is characteristic of species with non-deep physiological dormancy

or non-deep simple morphological dormancy (Nikolaeva 2001).

Chapter 1

49

Ethylene

Ethylene plays a role in the development and growth of plants (Klassen and Bugbee

2004). Ethylene enhances seed germination between concentrations of 0.1 and 200 µL

L-1 (Corbineau and Côme 1995). The optimum ethylene concentration varies between

species and is influenced by the length of time that seeds are imbibed prior to ethylene

contact.

Ethylene is produced in many seeds during germination. There is some dispute

as to whether it is a requirement or a product of the germination process (Matilla 2000).

In many species exogenous ethylene appears to act as a germination stimulant as

opposed to a dormancy-breaking agent because most seeds respond to this treatment

after dormancy has been alleviated (Abeles and Lonski 1969; Adkins and Ross 1981).

Under natural conditions, ethylene may influence seed germination as it is present in the

natural environment. Microorganisms (Arshad and Frankenberger 1991) and plant roots

(Klassen and Bugbee 2004) release ethylene into the soil, and ethylene is a component

of smoke (McElroy 1960) and it is released by wet ash (Ne'eman et al. 1999).

Applied ethylene stimulates germination of a large number of species, including

some that germinate after fire such as Erica hebecalyx (Ericaceae; van de Venter and

Esterhuizen 1988), Cyclopia intermedia (Fabaceae; Sutcliffe and Whitehead 1995) and

Rhus coriaria (Anacardiaceae; Ne'eman et al. 1999). For germination to proceed, the

later two species required physical dormancy to be overcome by heat, as well as

ethylene to overcome embryo dormancy. Non fire-prone species that respond to smoke,

such as lettuce, also respond to ethylene (Jäger et al. 1996b). However, ethylene has

been eliminated as the ubiquitous compound in smoke that stimulates germination

because other smoke responsive species do not germinate when ethylene is applied (de

Lange and Boucher 1990; Baxter et al. 1994). Likewise, other fire-followers, such as

Emmenanthe penduliflora (Hydrophyllaceae), do not respond to ethylene (Keeley and

Fotheringham 1997). Usually the response of seeds to ethylene is tested during

imbibition or once imbibition has commenced (Abeles and Lonski 1969; Sutcliffe and

Whitehead 1995; Jäger et al. 1996b; Ne'eman et al. 1999). Emmenanthe penduliflora

seeds were exposed to ethylene when they were dry and this may be why they were not

influenced by this treatment. However, some species, such as Erica hebecalyx respond

to ethylene whether seeds are dry or imbibed (van de Venter and Esterhuizen 1988).

Exposure of seeds to ethylene may enhance the germination of Australian fire

ephemerals since these species normally germinate after fire, ethylene is produced in

Chapter 1

50

smoke and some other fire-following species are stimulated to germinate by this

chemical.

Polyvinylpyrlidone (PVP)

Phenols might be present in the long-lived seeds of fire ephemerals, as seed persistence

in soil has been positively correlated with ortho-dihydroxyphenol concentration

(Hendry et al. 1994). There are many different types of phenols and they have a number

of functions in seeds such as conferring antimicrobial properties, herbivory defence and

being oxidised to increase seedcoat impermeability (Marbach and Mayer 1974; Scalbert

1991). In Nyctanthes arbor-tristis (Oleaceae), the addition of an antioxidant,

polyvinylpyrlidone (PVP), correlates with a reduction in phenol leaching and an

increase in germination (Bhattacharyya et al. 1999). Bhattacharyya et al. (1999)

suggested that PVP enhances germination by increasing oxygen availability to the

embryo through binding to phenols and thus preventing the phenols from scavenging

oxygen.

The presence of phenols in seeds does not imply that they play a role in

dormancy. Cynoglossum officinale (Boraginaceae) seeds have high levels of phenols but

these levels do not change with treatments that induce germination (Qi et al. 1993).

However, Qi et al. (1993) concluded that dormancy was due to a lack of oxygen, and

this may have been related to changes in phenolic composition. Fire ephemerals are

inferred to remain in the soil seedbank for long periods of time between fires and if

phenols are involved with longevity they may have a role in dormancy and germination

of these species.

Seed Scarification

A variety of techniques can be used to scarify seeds such as manual scarification, or

soaking seeds in strong acids or bases. Sulphuric acid is a well established laboratory

technique to overcome impermeability in seeds (Rolston 1978), although its

effectiveness is not restricted to water-impermeable seeds (Koller and Negbi 1959).

Potassium hydroxide treatment of seeds is less common but it has also been used

effectively to increase seed germination (Hou and Simpson 1994; Sukhvibul and

Considine 1994; Gao et al. 1998). Scarification by potassium hydroxide and sulphuric

acid is not identical. Hydroxides saponify lipids and undergo basic reactions with

inhibitors, whereas acids are corrosive (Sukhvibul and Considine 1994).

Chapter 1

51

Scarification has increased germination in species from some of the same

families as the fire ephemerals under examination. For example, manual scarification

has enhanced germination in four eastern Australian Grevillea species from the

Proteaceae (Morris 2000) and Malva parviflora (Sumner and Cobb 1967) and Iliamna

corei from the Malvaceae (Baskin and Baskin 1997). Treatment of seeds with sulphuric

acid increases germination of Rhus ovata (Anacardiaceae; Stone and Juhren 1951) and

Iliamna corei (Baskin and Baskin 1997) and Oryzopsis miliacea (Poaceae; Koller and

Negbi 1959). Potassium hydroxide treatment of seeds increases germination in Avena

fatua (Poaceae; Hou and Simpson 1994) and Angelica atropurpurea (Apiaceae; Gao et

al. 1998). In addition, high levels of germination have been induced by manual

scarification or sulphuric acid treatment in fire ephemerals from California (Keeley and

Fotheringham 1998a,b).

These scarification techniques can increase germination in both water-

impermeable (Stone and Juhren 1951; Das and Saha 1999) and water permeable seeds

(Koller and Negbi 1959; Hou and Simpson 1994; Keeley and Fotheringham 1998a,b;

Morris 2000). In water-impermeable seeds (those with physical dormancy), scarification

allows seeds to imbibe water, enabling germination (Stone and Juhren 1951; Das and

Saha 1999). A range of suggestions have been proposed as to how scarification

promotes germination in water permeable seeds. These include; increases in seed

permeability to water (Hou and Simpson 1994), solutes (Keeley and Fotheringham

1998b) or oxygen (Mott 1974), removal of germination inhibitors (Koller and Negbi

1959) and reduced mechanical restraint to the embryo by covering layers (Adkins et al.

2002b; J. Baskin and Baskin 2004). Generally water permeable seeds that germinate in

response to scarification treatments have non-deep physiological dormancy (J. Baskin

and Baskin 2004).

In the soil it is unlikely that seeds will be exposed to strong acids or bases.

However coat-imposed dormancy may be relieved under natural conditions by

saprophytic fungi, acids encountered during animal digestion, temperature fluctuations

and other aspects of weathering, and caustic potash after fires (Adkins et al. 2002b).

Hydrogen Peroxide

Hydrogen peroxide is another chemical that may enhance germination through

scarification (Chien and Lin 1994; Keeley and Fotheringham 1998a). Alternative

mechanisms of enhancing germination by hydrogen peroxide include reacting with

chemical inhibitors in seeds (Ogawa and Iwabuchi 2001), increasing oxygen availability

Chapter 1

52

(Mott 1974; Brits 1986) and possibly influencing seed metabolism (Bewley and Black

1994; Fontaine et al. 1994).

Hydrogen peroxide has enhanced seed germination in many species including a

number from the Poaceae (Roberts 1964; Mott 1974) and Proteaceae families (Brits

1986). Some of the fire ephemerals that will be examined in this thesis are from these

families and hence they may respond to similar germination treatments. Hydrogen

peroxide also enhances germination of two Californian fire ephemerals and so this

technique may be effective for germinating Australian fire ephemerals (Keeley and

Fotheringham 1998a,b). In a number of these studies, germination is promoted

following soaking of the seeds in hydrogen peroxide solutions for 24 to 48 hours (Mott

1974; Brits 1986, Fontaine et al. 1994, Keeley and Fotheringham 1998a,b).

Seedbank Ecology May Provide Insights Into Germination

Requirements The previous section has focused on possible methods of germinating fire ephemerals at

a point in time with an emphasis on post-fire cues. Another feature of the life history of

fire ephemerals, that may influence subsequent germination, is their incorporation into

the soil seedbank. Seeds that germinate after fire, are either stored in the plant canopy

(serotinous/bradysporous) and released primarily after fire, or shed after maturity and

stored in the soil seedbank (Bell et al. 1993). Here we will focus on the latter

characteristic, because all of the fire ephemerals examined in this thesis have soil-stored

seeds. Some of the characteristics of fire ephemeral seedbank ecology will be examined,

such as seed persistence, and compared with those of other plant functional groups.

Following this, methods of studying seed persistence will be explored. Finally, the

potential influence of burial on dormancy and germination will be examined, as well as

the components of burial that may drive these changes.

Seedbank Longevity

Based on longevity, seedbanks can be classified as either transient or persistent

(Thompson and Grime 1979). Seeds in transient seedbanks generally survive for less

than one year, either germinating, being predated or dying within that time (Thompson

and Grime 1979). These seedbanks generally form when seeds are dispersed at a time of

year inappropriate for germination (Thompson 2000). Thus this type of seedbank

primarily exists to shift germination from an unfavourable season to a more favourable

Chapter 1

53

one for seedling establishment (Thompson 2000). Seeds in transient seedbanks are

generally large (>0.5 mg), and germinate under both light and dark conditions

(Thompson et al. 1993). Seeds forming persistent seedbanks can remain viable in the

soil for over a year, are often small, have certain requirements for germination such as

light, and frequently germinate after soil disturbance (Thompson and Grime 1979).

Generalisations about seed size and persistence may not always apply due to the

influence of habitat, with larger seeds also forming persistent seedbanks in frequently

disturbed and fire-prone areas (Thompson et al. 1993). Indeed, in a number of

Australian studies there was no consistent relationship between seed mass and longevity

(Lunt 1995; Leishman and Westoby 1998; Moles et al. 2003). Similarly, a study on seed

persistence in the fire-prone South Africa fynbos found no correlation between seed

mass and persistence (Holmes and Newton 2004). Recently, Walck et al. (2005) have

suggested that transient and persistent seedbanks should be differentiated on the basis of

whether seeds persist until the second germination season. This provides an ecological

rather than an arbitrary time frame for this classification and accommodates the many

species in temperate areas that disperse seeds and germinate in distinct seasons, often

with the second germination season occurring about one-and-a-half years after

dispersal.

Dormancy plays a role in many transient seedbanks where seed dispersal and

germination occur in different seasons (Thompson 2000). Other transient seedbanks,

especially those that are briefly formed when seeds are dispersed shortly before

germination, generally do not possess any dormancy mechanisms (Parker and Kelly

1989). Likewise, persistent seedbanks may or may not possess dormancy. In these

seedbanks the basis for persistence is often a requirement for a germination stimulant,

rather than the presence of dormancy (Thompson 2000). The main exception to this is

seeds with physical dormancy. These seeds may persist in the soil for many years until

dormancy, imposed by the hard impermeable seedcoat, is overcome (Portlock et al.

1990; Baskin and Baskin 1997; Thompson 2000).

Seed persistence may be correlated with environmental variability and/or plant

life histories. In environments where there is variability between years in rainfall,

nutrient availability, competition from other plants or other factors that may influence

plant establishment and fitness, seed persistence enables seeds to remain in the soil until

suitable conditions cue germination (Brown and Venable 1986; Thompson 2000). For

species that require fire to germinate, persistent seedbanks are particularly important for

the continuation of a species at a site if plants live for shorter durations than the average

Chapter 1

54

fire-interval, and seed dispersal is limited (Bullock 1989; Parker and Kelly 1989; Auld

et al. 2000; Thompson 2000; Holmes and Newton 2004).

Fire ephemerals are inferred to persist in the seedbank for long durations

because plants are short-lived and seeds primarily germinate after fire (Figure 10a,b;

Weston 1985; Parsons 1997). For example, Alyogyne hakeifolia and A. huegelii plants

usually live for less than 6 years, but seeds of these species germinated after fire in an

area that had possibly not been burnt for 170 years (Weston 1985). This implies the

existence of a persistent seedbank. Few studies have tested the seed persistence of fire

ephemerals directly. Smith et al. (1999) indirectly showed that Austrostipa compressa

seeds could form persistent seedbanks by counting seeds in areas of soil with different

fire histories. Seed density declined with time from a fire but even after a 45 year

interval there were still 119 Austrostipa compressa seeds per square metre. This study

was based on the assumption that seeds were only produced in the immediate post-fire

years and that seeds were not transported in from other areas.

Monocarpic fire ephemerals generally live for less than a year whereas

polycarpic fire ephemerals live for longer, often for at least three to nine years (Figure

10a,b; Pate et al. 1985). Both monocarpic and polycarpic fire ephemerals produce large

numbers of seeds during their short lives (Pate and Dixon 1996; Hunter et al. 1998).

Monocarpic fire ephemerals produce seeds over a shorter time period than polycarpic

fire ephemerals and allocate a larger proportion of their biomass to seed production

(Pate et al. 1985). According to Pate et al. (1985), polycarpic fire ephemerals usually

reach reproductive maturity in their second season. This has been observed in Tersonia

cyathiflora. However some polycarpic fire ephemerals, such as Gyrostemon racemiger,

Alyogyne hakeifolia, Alyogyne huegelii and Grevillea scapigera have been observed

flowering and fruiting in their first year (Baker pers obs.). Pate and Hopper (1993) noted

that monocarpic fire ephemerals are more common after frequent mild burns whereas

polycarpic fire ephemerals tend to be associated with less frequent, severe fires. Thus

polycarpic fire ephemerals may persist in the soil seedbank for longer durations than

monocarpic fire ephemerals (Pate and Hopper 1993). Generally these species only

germinate and contribute to the seedbank during the immediate postfire period, and

hence seed numbers decline during the interfire period as seeds die (Figure 10a,b).

However, some fire ephemerals, particularly monocarpic species, occasionally

germinate in the interfire period (Pate and Hopper 1993), which may supplement

seedbanks.

Chapter 1

55

Seeders are killed by fire and regenerate from seed, whereas resprouters usually

survive fire and sprout from epicormic buds (Figure 10c,d; Bell et al. 1993).

Consequently, seeders have a greater reliance on a seedbank for re-establishment after

fire and thus usually produce more seeds than resprouters (Bell et al. 1993) and are

more likely to have persistent seedbanks (Parker and Kelly 1989). Fire ephemerals have

been described as �extreme versions� of seeders (Bell et al. 1984), and thus form one

end of the continuum of adult longevity, seed production and seed persistence. Shorter

lived seeders are more likely to form persistent seedbanks such as Acacia suaveolens

(Fabaceae) which produces most seeds in a few fruiting seasons following fire (Auld

1986b) than some longer lived seeders including a range of fynbos species (Musil 1991)

and Persoonia pinifolia (Proteaceae; Auld et al. 2000) which exhibit turnover in the

seedbank as shorter-lived seeds die and new seeds are added. In fact, there is a

continuum between the functional groups. In general, the shorter the life span of adult

plants, the greater the amount of resources allocated to seed production and the greater

the selection pressure for persistent seedbanks (Rees 1994).

Chapter 1

56

Figure 10. The relative abundance of plants and seeds over time following a fire in four

groups of species classified according to their life history and response to fire: a)

monocarpic fire ephemeral, b) polycarpic fire ephemeral, c) seeder and d) resprouter.

The filled region represents plant numbers whereas the area of diagonal lines depicts the

number of viable seeds in the seedbank. Based on Bond and van Wilgen (1996).

To persist, seeds must remain viable but not germinate. Different attributes have

been proposed as protection against pathogen attack. Defence chemicals, such as

glucosinolates are found in sixteen plant families, including the Gyrostemonaceae

(Bottomley and White 1950; Kjaer and Malver 1979; Fahey et al. 2001). Many species

in this family are fire ephemerals (George 1982; le Maitre and Midgley 1992; Pate and

Dixon 1996), four of which will be examined in this thesis. These chemicals have

fungicidal and bacteriocidal properties (Fahey et al. 2001), which may protect seeds

from attack in the soil. Products of some glucosinolates also inhibit the germination of

certain weed species (Angelini et al. 1998) and suppress germination of wheat (Bialy et

al. 1990).

a) Monocarpic Fire Ephemeral (Fire Annual)

Time since fire

Num

ber o

f pla

nts

(fille

d) o

r see

ds (s

tripe

d) b) Polycarpic Fire Ephemeral (Fire Perennial)

Time since fire

d) Resprouter

Time since fire

c) Seeder

Time since fire

Num

ber o

f pla

nts

(fille

d) o

r see

ds (s

tripe

d)Time of fire Time of fire

Time of fire Time of fire

Chapter 1

57

Methods of Assessing Seed Persistence

There are different methods of assessing seed longevity. For orthodox seeds, longevity

under artificial storage conditions may differ from longevity in the field (Thompson

2000; Walters et al. 2005). The Apiaceae family has relatively short-lived propagules

under laboratory storage conditions (Walters et al. 2005). However, viable fruit of two

species in this family, Conium maculatum and Daucus carota, persist in the soil for

more than five years (Thompson et al. 1993). Generally, seed longevity increases as

temperature and moisture are reduced (Roberts 1973). However if seeds are fully

hydrated (above approximately 15 - 28% seed moisture content, depending on whether

seeds are oily or non-oily), longevity is also enhanced (Roberts and Ellis 1989) because

sufficient water is available for seeds to metabolise and repair damaged cellular

components (Villiers 1974). Seeds in the soil are exposed to a wide range of relative

humidities and temperatures. Therefore, to estimate the longevity of soil seedbanks, it is

best to examine seeds in the soil.

Three different methods of assessing soil seedbank longevity include

germinating seeds from soil samples, manually extracting seeds from the soil and

testing viability, and burying seeds and exhuming them later (Auld 1986b; Kilian and

Cowling 1992). Germinating seeds from soil samples may underestimate persistence of

dormant seeds (Thompson et al. 2003) and physical extraction of seeds can be time-

consuming (Auld 1986b).

Many seed persistence studies have entailed burying seeds in the soil in nylon

mesh bags and exhuming them after set intervals of time (Auld 1986b; Lunt 1995; Auld

et al. 2000; Tieu et al. 2001c; Holmes and Newton 2004; Yates and Ladd 2005).

Preferably, experiments should be designed so that seeds do not require reburial after

exhumation as exposure to light may stimulate germination of seeds that may have

otherwise persisted (Holmes and Newton 2004). Seedbank longevity studies are most

relevant when seeds are buried in their naturally occurring soil type (Csontos and Tamás

2003). Soil is often placed in the mesh bags with the seeds so that conditions are as

close as possible to those in the surrounding soil. It also dilutes the concentration of

seeds and thus reduces the likelihood that seeds will be attacked by saprophytic fungi

(van Mourik et al. 2005). van Mourik et al. (2005) argue that burying seeds in mesh

bags may overestimate seed decline due to the increased likelihood of fungal attack with

highly concentrated seeds. However others argue that this method may have the

opposite effect (Thompson et al. 1993; Lunt 1995). Seeds are usually deposited on the

soil surface and become incorporated into the soil in a variety of ways such as falling

Chapter 1

58

into soil cracks or burial by ants. Burying seeds may increase the likelihood of seeds

persisting in the soil (Thompson et al. 1993) by, for example, preventing germination of

light requiring seeds (Wesson and Wareing 1967). In six species placed in mesh bags on

the soil surface and at 3 cm depth in south-eastern Australia, half of the species

persisted to higher levels when buried, whereas the remaining species exhibited similar

levels of persistence regardless of seed placement (Lunt 1995). Burying seeds in mesh

bags may also lead to overestimates of seed persistence by excluding predators (Lunt

1995). Nevertheless the advantage of burying seeds in mesh bags is that known numbers

of seeds can be monitored (van Mourik et al. 2005).

Depth of Seed Burial

To ensure the ecological relevance of burial studies, seeds should be placed at depths

approximating natural soil storage. Most seeds in Australian soil seedbanks are located

near the soil surface. In the jarrah forest of south-western Western Australia, the

majority (93%) of seeds in the top 10 cm of the soil profile occur in the surface 2 cm of

soil (Tacey and Glossop 1980). Similarly 83% of seeds of the fire ephemeral,

Austrostipa compressa, were found at 0-5 cm depth (Smith et al. 1999) and Grevillea

scapigera seeds were only found in the surface 3 cm of 10 cm deep soil cores (Rossetto

1995). Furthermore, seeds of Grevillea rivularis, which germinate after disturbances

such as fire, were most numerous in the surface 2 cm of soil (Pickup et al. 2003).

Consequently, in burial studies undertaken in Australia, where the topsoil is relatively

shallow, seeds are generally buried at shallower depths than in European and North

American studies. For example, in south-western Australia, Tieu et al. (2001c) buried

seeds in the field at 2 cm depth and in south-eastern Australia, Lunt (1995) buried seeds

3 cm below the surface. In contrast, in northern America, C. Baskin and Baskin (1994)

and J. Baskin and Baskin (1994) buried seeds in pots 7 cm below the surface to

approximate conditions at 10 cm depth in the field, and in Europe, Bouwmeester and

Karssen (1992) and Derkx and Karssen (1993) buried seeds at 10 cm depth in pots

buried in the field.

The persistence of seeds in the soil can be modelled using an exponential decay

curve (Roberts 1972; Auld 1986b; Auld et al. 2000; Holmes and Newton 2004).

Seedbank half-life can be estimated using the equation: y=ae-bt, where y is the

proportion of filled seeds remaining, t is time in years, and a and b are constants.

However in a two year burial study, Auld et al. (2000) found that it was a poor predictor

of seed longevity for seeds from a fire-prone area of eastern Australia.

Chapter 1

59

Influence of a Period of Burial on Seed Dormancy and Germination

Fire ephemerals are inferred to persist in the soil seedbank for long periods of time.

Hence burial potentially influences the dormancy state of these seeds. Some species that

typically appear after disturbances such as fire, have seeds that germinate more readily

after a period of soil burial, including Passerina paleacea (Thymelaeaceae; Kilian and

Cowling 1992), Thryptomene calycina (Myrtaceae; Beardsell et al. 1993) and Grevillea

rivularis (Proteaceae; Pickup et al. 2003) and 11 out of 110 difficult to germinate

Western Australian species (Roche et al. 1997). Other species that usually germinate

after fire become more responsive to smoke following a period of burial, including

Verticordia fimbrilepis (Myrtaceae) from south-western Australia (Yates and Ladd

2005), Audouinia capitata (Bruniaceae) from South Africa (de Lange and Boucher

1993b), and Dendromecon rigida (Papaveraceae), Dicentra chrysantha and Trichostema

lanatum (Lamiaceae) from California (Keeley and Fotheringham 1998a). Additionally,

7 of the 110 south-western Australian species examined in a glasshouse burial trial by

Roche et al. (1997) required both burial and smoke to germinate, but germination levels

were generally low (<15%).

A number of reasons have been suggested to explain higher germination

following a period of burial. Dormancy in water impermeable seeds may be overcome

during burial as a result of heat from the passage of fire (Keeley 1987; Bradstock and

Auld 1995; Baskin and Baskin 1997), or otherwise high (Warcup 1980; Auld and

Bradstock 1996) and/or fluctuating soil temperatures (Quinlivan 1971; van Staden et al.

1994). Microbial action and abrasion by soil particles are also frequently cited as natural

agents that break physical dormancy eg Rolston (1978), but Baskin and Baskin (2000)

argue that there is little evidence to support these claims. Enhanced germination of

water permeable seeds following burial may be a result of increased permeability of

embryo covering layers (Tieu and Egerton-Warburton 2000) and/or changes in the level

of seed dormancy arising from the influence of temperature and possibly also moisture

(Roberts and Neilson 1982; Benech-Arnold et al. 2000).

Increased germination of water permeable seeds following burial may arise from

alterations in the permeability of embryo-covering layers

Seed scarification leads to germination in many species with water permeable seeds

(Keeley and Fotheringham 1998a; Pickup et al. 2003; J. Baskin and Baskin 2004).

Consequently it has been suggested that seedcoat weathering during burial may play a

key role in the germination of some species (Pickup et al. 2003). Enhanced germination

Chapter 1

60

following burial was at least partially attributed to natural scarification and weathering

of the seed covering layers in Audouinia capitata (Bruniaceae), Thryptomene calycina

(Myrtaceae), Leucopogon conostephioides (Epacridaceae) and Grevillea rivularis

(Proteaceae; Beardsell et al. 1993; de Lange and Boucher 1993a; Pickup et al. 2003).

Higher germination of Audouinia capitata and Leucopogon conostephioides seeds in

response to smoke following burial was correlated with increased weathering of the

water-permeable indehiscent fruit (de Lange and Boucher 1993a; Tieu and Egerton-

Warburton 2000). de Lange and Boucher (1993a) inferred that a period of burial and

fruit weathering enhanced Audouinia capitata germination by reducing mechanical

resistance to the embryo whereas Tieu and Egerton-Warburton (2000) suggested that

increased germination in Leucopogon conostephioides was related to greater solute

permeability. Egerton-Warburton (1998) proposed that the embryo coverings of water

permeable seeds may allow the exchange of water and gases but impede the exit of

inhibitors thus enforcing dormancy. Alternatively, improved permeability may allow the

chemical in smoke that promotes germination to reach sites in the seed where it can be

effective. However, not all of the species examined by Tieu and Egerton-Warburton

(2000), particularly those without woody seed coverings, exhibited correlations between

enhanced permeability and germination. Similarly, the germination response of

Nicotiana attenuata (Solanaceae) to smoke was not altered by scarification (Baldwin et

al. 1994), suggesting that changes in permeability are not a requirement for smoke

receptiveness in all species. J. Baskin and Baskin (2004) argue that there is no

conclusive evidence that weakening of the embryo covering layers reduces the level of

dormancy in water permeable seeds, which suggests that burial may alter seed

dormancy in some other way.

Potential dormancy changes in water permeable seeds during burial

Some species that produce higher germination following burial may require a period of

after-ripening, rather than weathering. Although Anigozanthos manglesii

(Haemodoraceae) seeds became more permeable and germinated more readily as

duration of burial increases, shelf-stored seeds also produced higher germination with

time, without the burial-induced permeability changes (Tieu et al. 2001c). This

highlights the importance of comparing the germination of seeds stored in the laboratory

with buried seeds to separate the effects of after-ripening and other possible influences

of burial.

Chapter 1

61

The effect of a period of after-ripening may also explain the changes in smoke

response observed in a number of species. For example, in some populations of the

Californian fire annual, Emmenanthe penduliflora (Hydrophyllaceae), seeds did not

respond to charred wood immediately after collection but germinated readily when this

treatment was applied to the same seed lot two years later (Keeley et al. 1985).

Likewise, Roche et al. (1997) found that some species produced an enhanced response

to smoke water as seeds aged.

Thus during burial, physiological dormancy may be alleviated. If seeds do not

germinate or die, they may either remain non-dormant or conditionally dormant, or shift

to a more dormant state (J. Baskin and Baskin 2004). Seeds are considered to undergo

dormancy cycling if the state of dormancy increases and decreases in concert with the

seasons. For example, many winter annuals germinate in autumn and produce seeds in

spring (Baskin and Baskin 1985; Nikolaeva 2001). These species require a period of

warm temperatures (warm stratification or after-ripening) before they germinate and are

often found in hot climates. Autumn germination prevents exposure of young seedlings

to harsh dry summer conditions. Conversely summer annuals germinate in spring and

produce seeds in autumn, and require a period of cold temperatures (cold stratification)

before they germinate. These species are often found in regions with very cold winters.

By requiring a period of cold stratification, germination is delayed until the end of

winter when temperatures are less severe for seedling establishment. Dormancy cycling

is found in perennials as well as annuals (Meyer and Kitchen 1992; Nikolaeva 2001).

Although dormancy cycling has been recognised for a long time (Courtney 1968), most

studies have been restricted to North America (Baskin and Baskin 1990; C. Baskin and

Baskin 1994) and Europe (Roberts and Neilson 1982; Bouwmeester and Karssen 1992).

Seeds most likely to exhibit dormancy cycling occur where there are predictable and

distinct favourable and unfavourable seasons for seedling establishment (Baskin et al.

1993). Thus dormancy cycling is potentially a characteristic of species in the

Mediterranean climate region of south-western Australia.

Annual rhythms in seed germinability have also been reported in a few species

that have been stored under dry laboratory conditions including Mesembryanthemum

nodiflorum (Aizoaceae; Gutterman and Gendler 2005), Stylidium crossocephalum

(Stylidiaceae) and Conostylis neocymosa (Haemodoraceae; Tieu et al. 2001c). Although

the changing responses of these seeds has been attributed to internal biological clocks

that ensure germination in appropriate seasons, perhaps these seeds sensed slight shifts

in laboratory temperatures and dormancy was alleviated or imposed accordingly.

Chapter 1

62

Dunbabin and Cocks (1999) claimed that Arctotheca calendula (Asteraceae) seeds

exhibit dormancy cycling under constant laboratory conditions. However the multiple

germination peaks within each year following laboratory storage do not correspond with

annual dormancy cycling in the field.

Generally, dormancy cycling is considered to be driven by temperature

(Vleeshouwers et al. 1995). Soil moisture may modulate the influence of temperature

(Hilhorst 1998; Benech-Arnold et al. 2000). To date, the influence of seed moisture

content has largely been examined with regard to its effect on subsequent rates of

germination. Wetting and drying seeds, known as priming, increases the rate of

germination in many species when seeds are subsequently imbibed (Berrie and Drennan

1971; Heydecker et al. 1973; Bradford 1986; Schrauf et al. 1995; Nascimento 2003).

More rapid germination is possibly a result of osmotic adjustment (when seeds are

primed with osmotic solutions rather than pure water) which induces metabolic

processes involving enzyme activity (Berrie and Drennan 1971; Bradford 1986). Less is

known about the influence of moisture on dormancy. Dormancy release in some species

is accelerated by storing seeds at higher seed moisture contents (up to approximately

18% dry weight; Baskin and Baskin 1979; Leopold et al. 1988; Steadman et al. 2003).

Similarly, Adkins and Ross (1981) and Probert et al. (1985) attributed faster after-

ripening in seeds stored in the soil than in the laboratory to the wetter soil environment.

However, Bouwmeester and Karssen (1992; 1993) found that moisture content was not

important in regulating dormancy changes. Most studies have either held seeds at

constant moisture conditions or in uncontrolled soil environments where conclusions are

difficult to draw. According to Kigel (1995), very little is known of the influence of

moisture on seed dormancy. More recently, the effect of periodic summer rainfall in

Mediterranean climates has been simulated under controlled conditions, where it was

suggested that the rate of dormancy release may be accelerated in dry seeds following a

period of imbibition (Gallagher et al. 2004). Thus the effect of wet/dry cycles on seed

dormancy requires further investigation.

Chapter 1

63

Aims of This Thesis As this literature review has shown, little is understood regarding the germination

requirements of many Australian fire ephemerals. Many of these species have not been

studied previously, and of those that have, germination has either been difficult or

inconsistent. The primary aim of this thesis is to determine how to germinate these

species. This will involve an investigation into seed viability, and the possibility of

dormancy and/or a requirement for one or more germination stimulants. Possible

treatments that may induce germination in the laboratory will be tested. An endeavour

will also be made to understand the ecology of these species; what cues these seeds to

germinate in nature. Thus the following chapters will aim to unravel the germination

ecophysiology of this functional group of south-western Australian fire ephemerals.

Chapter 1

64

Chapter 2

CHAPTER 2

Seed dormancy and germination responses of nine Australian fire ephemerals

Abstract

Fire ephemerals are short-lived plants with seeds that persist in the soil and germinate

after a fire or physical soil disturbance. Ex situ germination of many Australian fire

ephemerals has previously been difficult. Dormancy was present in most of the nine fire

ephemerals examined. Alyogyne hakeifolia (Giord.) Alef. and Alyogyne huegelii (Endl.)

Fryxell (Malvaceae) seeds had physical and possibly also physiological dormancy,

Actinotus leucocephalus Benth. (Apiaceae) seeds had morphophysiological dormancy,

Austrostipa compressa (R.Br.) S.W.L.Jacobs & J.Everett and Austrostipa macalpinei

(Reader) S.W.L.Jacobs & J.Everett (Poaceae) seeds were either non-dormant or

possessed physiological dormancy, and seeds of all remaining species possessed

physiological dormancy. A proportion of the Alyogyne hakeifolia, Alyogyne huegelii,

Austrostipa compressa and Austrostipa macalpinei seed populations were non-dormant

because some seeds could germinate at the various incubation temperatures without

further treatment. At 20ºC, artificial methods of inducing germination such as manual or

acid scarification were among the optimal treatments for Austrostipa compressa,

Austrostipa macalpinei, Alyogyne huegelii, Actinotus leucocephalus and Grevillea

scapigera A.S.George (Proteaceae), and gibberellic acid induced maximum germination

of Tersonia cyathiflora (Fenzl) J.W.Green (Gyrostemonaceae) seeds. Heat (70ºC for 1

h) and smoke water was one of the most effective treatments for germinating Actinotus

leucocephalus and Codonocarpus cotinifolius (Desf.) F.Muell. (Gyrostemonaceae)

seeds. Germination of Grevillea scapigera, Codonocarpus cotinifolius, Gyrostemon

racemiger H.Walter (Gyrostemonaceae) and Tersonia cyathiflora did not exceed 40%

and may require other treatments to overcome dormancy. Although the nine fire

ephemerals examined require fire to germinate under natural conditions, a range of

germination responses and dormancy types was observed.

67

Chapter 2

Introduction

The three Mediterranean-climate regions of the world where fire-stimulated germination

is most pronounced are south-western Australia, the California chaparral and the south-

western Cape of South Africa (Cowling et al. 2004). Fire ephemerals are a functional

group of species in each of these regions that predominantly germinate after fire, are

short-lived and persist between fires as seeds in the soil seedbank (Bell et al. 1984; Pate

et al. 1985; le Maitre and Midgley 1992; Thanos and Rundel 1995). Fire ephemerals can

be either monocarpic (fire annuals), flowering and producing seeds once, or polycarpic

(fire perennials), flowering and producing seeds over three to four and occasionally up

to ten years (Pate et al. 1985; Pate and Dixon 1996). Fire ephemerals form a larger

proportion of the flora in California than either the South African Cape or south-western

Western Australia and hence the germination requirements of Californian species have

been more extensively studied (Keeley 1987; le Maitre and Midgley 1992; Thanos and

Rundel 1995; Keeley and Fotheringham 1998a).

For those Australian fire ephemerals that have been examined, the germination

of fresh or ex situ stored seeds has been difficult, with many viable seeds failing to

germinate under a range of conditions (Dixon et al. 1995; Rossetto et al. 1995; Tieu et

al. 1999). Germination of fresh or ex situ stored seeds is important because many fire

ephemerals and closely related species are rare (Briggs and Leigh 1996; Pate and Dixon

1996) and fire ephemerals have potential as cover crops in land rehabilitation (Baker et

al. 2005). In addition, the inclusion of fire ephemerals in revegetation programs is

important for maintaining biodiversity. Thus the germination requirements of this

ecologically important class of plants in Australia require further investigation.

Seeds may require particular temperature and/or light regimes for germination to

proceed. Optimum germination temperatures usually correspond with seasonal

conditions favourable for the growth and survival of seedlings (Bellairs and Bell 1990).

Many other treatments have also been employed to stimulate the germination of seeds.

These include applying oxidisers, such as hydrogen peroxide (Ogawa and Iwabuchi

2001), which has stimulated the germination of other fire ephemeral species (Keeley

and Fotheringham 1998a) or chelating agents, like polyvinylpyrolidone, to seeds

(Bhattacharyya et al. 1999). Scarification is another germination pre-treatment which

may be applied manually or by soaking seeds in strong acids or strong bases (Sukhvibul

and Considine 1994; Keeley and Fotheringham 1998a). Under natural conditions, fire-

related cue(s) appear to play an important role in the germination of fire ephemerals

from the soil seedbank, as highly synchronous germination events occur in the growing

68

Chapter 2

season following a fire (Pate and Dixon 1996). Heat and smoke are two of the main

components of fire that are known to promote germination, and these can operate

separately or in combination (Kenny 2000; Morris 2000; Tieu et al. 2001a; Thomas et

al. 2003). Germination has also been promoted by ethylene, which is a component of

smoke (van de Venter and Esterhuizen 1988) and is released from wet ash (Ne'eman et

al. 1999), and nitrate (Thanos and Rundel 1995), which becomes more abundant in the

soil following fire (Christensen 1973). Gibberellic acid is a plant hormone that plays a

role in releasing seed dormancy in many species (Bewley 1997), including the fire

ephemeral Codonocarpus pyramidalis (F.Muell.) F.Muell. (Loveys and Jusaitis 1994).

In attempting to release seed dormancy, a useful approach is to firstly classify

seeds into their respective dormancy classes. Recently J. Baskin and Baskin (2004) have

proposed a revised system of a seed dormancy classification scheme based on the work

of Nikolaeva (1977). This scheme defines five core classes of seed dormancy based on

attributes such as water permeability of seeds and embryo development. The most

common type of dormancy, physiological dormancy, can be further subdivided on the

basis of cold stratification requirements and whether seeds germinate in response to

gibberellic acid or scarification, or whether excised embryos produce normal seedlings.

This study investigates the germination requirements of the following nine fire

ephemerals: Austrostipa compressa, Austrostipa macalpinei (Poaceae), Alyogyne

hakeifolia, Alyogyne huegelii (Malvaceae), Actinotus leucocephalus (Apiaceae),

Codonocarpus cotinifolius, Gyrostemon racemiger and Tersonia cyathiflora

(Gyrostemonaceae) and Grevillea scapigera (Proteaceae; Bell et al. 1984; Pate et al.

1985; Rossetto et al. 1995; Pate and Dixon 1996). Austrostipa compressa, Austrostipa

macalpinei and Actinotus leucocephalus are monocarpic fire ephemerals and the

remaining species are polycarpic fire ephemerals. All of these species occur within the

south-west botanical province of Western Australia which has a Mediterranean climate,

although not all species are restricted to this area. The aims of this research were to

classify the dormancy types of these species according to the system of J. Baskin and

Baskin (2004), which will provide a better understanding of the conditions required to

alleviate dormancy, and to identify optimum methods for ex situ germination of fresh

and laboratory stored seeds through examining the effects of various incubation

temperatures, light conditions, chemical stimulants, scarification, and heat and smoke

treatments.

69

Chapter 2

Materials and methods

Seed source

Seed collection and provenance data is outlined in Table 1. Voucher specimens of

Actinotus leucocephalus, Austrostipa compressa, Austrostipa macalpinei and

Gyrostemon racemiger were lodged at the Western Australian Herbarium (PERTH).

After collection, seeds were air-dried and stored in sealed containers at 15ºC until use in

germination tests, except Alyogyne hakeifolia, Alyogyne huegelii and Codonocarpus

cotinifolius seeds which were stored at 4°C until February 2002 and then transferred to

15ºC. Experiments were undertaken on the mericarps of Actinotus leucocephalus

(hereafter referred to as ‘seeds’) and the seeds of all other species.

Table 1. Date and provenance of seed collection for each of the species studied

Species Family Collection Date

Source

Austrostipa compressa (R.Br.) S.W.L.Jacobs & J.Everett

Poaceae 4-10/12/2001 Unburnt rehabilitation site (re-spread topsoil) Rocla, Armadale Road, Perth, WA 32º07.706’S, 115º53.343’E

Austrostipa macalpinei (Reader) S.W.L.Jacobs & J.Everett

Poaceae 25/11/2001 Burnt site 40 km N of Gin Gin, WA 31º01.231’S, 115º43.297’E

Alyogyne hakeifolia (Giord.) Alef.

Malvaceae 12/1999 Salmon Gums, WA

Alyogyne huegelii (Endl.) Fryxell

Malvaceae 12/1999 Near Esperance, WA

Actinotus leucocephalus Benth.

Apiaceae 12/2001 Burnt site 40 km N Gin Gin, WA, 31º01.231’S, 115º43.297’E

Grevillea scapigera A.S.George

Proteaceae 12/2001 Translocated population at Corrigin, WA

Codonocarpus cotinifolius (Desf.) F.Muell.

Gyrostemonaceae 1/11/97 Hyden, WA

Gyrostemon racemiger H.Walter

Gyrostemonaceae 21/12/01 Burnt site 55 km N Gin Gin, WA 30º54.384’S, 115º38.417’E

Tersonia cyathiflora (Fenzl) J.W.Green

Gyrostemonaceae 6/1/02 Burnt site at Beekeepers Reserve, N of Eneabba, WA 29º32.315’S, 115º03.415’E

Viability tests, seed type and embryo growth

For the cut test, three replicates of 50 intact seeds of each species were dissected and the

proportion of seeds that were filled, contained an embryo and appeared healthy, were

noted. Seeds were classified according to the morphological types of Martin (1946) and

embryos were described as either developed or underdeveloped (Baskin and Baskin

1998; J. Baskin and Baskin 2004).

For species that produced <90% germination in all trials, a tetrazolium chloride

test was conducted to determine seed viability. Three replicates of 50 seeds of each of

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Chapter 2

the Alyogyne species (nicked first to facilitate water uptake) and three replicates of 10

seeds for remaining species were imbibed in distilled water for 24 h at 25°C. Seeds

were dissected longitudinally and placed in 1% w/v 2,3,5-triphenyl tetrazolium chloride

for 24 h at 30°C in the dark. Seeds were then classified as viable (most of the embryo

stained including the radicle), possibly viable (patchy or faint staining of embryo) or

dead (embryo either stained very faintly or unstained).

Five excised Codonocarpus cotinifolius and Tersonia cyathiflora embryos were

placed on filter paper moistened with distilled water to test whether they could grow

normally without additional hormones. Petri dishes were sealed with Parafilm,

incubated at 20°C under a 12 h light, 12 h dark regime and assessed for evidence of

embryo growth after three weeks.

Imbibition test

For each species, four replicates of 10 seeds were incubated in distilled water at

20°C and weighed 2-hourly during the initial 12 h of imbibition and then 4-hourly for a

further 12 h to establish whether seeds had imbibed water. Before weighing, seeds were

blotted with paper towels to remove excess moisture. Increase in seed weight over time

was calculated. Measurements were repeated on an individual seed basis for the two

Alyogyne species.

Germination protocol

Germination tests were performed between April 2002 and April 2003. To prevent

fungal contamination, seeds were shaken for 30 s in a 2% w/v NaOCl solution

containing two drops of a non-ionic surfactant (Plus 50, Ciba-Geigy, Sydney) per 250

mL, unless noted otherwise. Seeds in this solution were held under vacuum for 5 min,

returned to normal air pressure for 5 min and placed under vacuum for a further 5 min.

Seeds were then transferred to a laminar flow, washed three times with sterile distilled

water, soaked in water for 10 min and then rinsed again.

Four replicates of 25 seeds were used in each treatment. Seeds were placed on

two pieces of filter paper (Whatman No. 1) over three 4 cm2 pieces of absorbent sponge

in 90 mm diameter plastic Petri dishes. These were moistened with 10 mL of sterile

distilled water containing 0.15% v/v of the fungicide Previcur (Aventis, Melbourne,

active ingredient 600 g L-1 propamocarb) unless noted otherwise. All Petri dishes were

sealed with Parafilm and placed in transparent plastic bags. Light was provided by cool

white fluorescent tubes (50 µmol m-2s-1 PAR; R:FR = 9:1). Seeds were monitored

71

Chapter 2

weekly for 84 days and germination was scored when the radicle emerged >2 mm.

Seeds that displayed cotyledon but not radicle emergence were noted separately.

Light and incubation temperature

To determine whether light was required for germination at 20°C, four replicate dishes

of each species were exposed to the incubator light/dark regime and four dishes were

wrapped in aluminium foil to exclude light. Germination was assessed after 84 days.

To test the effect of incubation temperature on germination, seeds were

incubated at 5°C, 10°C, 15°C, 20°C, and 25/15°C (alternating 12 h/ 12 h regime). Light,

on a 12 h light, 12 h dark cycle, was provided by cool white fluorescent tubes (30-50

µmol m-2s-1; R:FR 9:1) for the constant temperatures, and fluorescent and incandescent

lights for the 25/15°C treatment (50 µmol m-2s-1; R:FR=1.1:1). Codonocarpus

cotinifolius and Gyrostemon racemiger were also incubated at 30°C. After 84 days at

5°C, seeds were transferred to 20°C for a further 84 days to test the effect of cold

stratification.

The two Austrostipa species were also incubated at 10°C (constant) with 12 h of

light daily provided by fluorescent and incandescent lights (R:FR =1.1:1), and at

25/15°C with 12 h of light during the warm phase provided by fluorescent lights only

(R:FR = 9:1) to test whether light quality influenced germination at these temperatures.

A R:FR ratio of 1.1:1 is similar to that in unfiltered sunlight whereas a R:FR ratio of 9:1

is typical of most standard germination incubators but does not occur in nature.

Chemical, scarification, smoke water and heat treatments

Seed germination response to a range of chemical, scarification, smoke water and heat

treatments was tested at 20ºC under a 12 h light/12 h dark regime. Control seeds were

incubated in 10 mL of sterile distilled water and Previcur (0.15% v/v), which had a pH

of 5.6. For the hydrogen peroxide (H2O2) treatment, seeds were presoaked in a 1% v/v

solution for 24 h at 20°C. For the polyvinylpyrolidone (PVP) treatment, dried surface

sterilised seeds were placed in 20 mL of 2% w/v PVP, shaken for 30 s and soaked in the

solution for a further 30 min. Following these treatments, seeds were washed five times

in distilled water.

Three scarification treatments were tested: acidic, basic and manual. Seeds were

soaked in concentrated sulphuric acid (H2SO4) for 5 min or 5 M potassium hydroxide

(KOH) for 10 min and then washed five times in sterile distilled water. For manual

scarification the palea and lemma were peeled from the Austrostipa seeds and the

72

Chapter 2

pericarp was removed from Actinotus leucocephalus mericarps. For the remaining

species a 1 mm2 portion of the seedcoat was removed from the side of seeds to prevent

damage to the radicle.

For the ethylene (C2H4) treatment, seeds were imbibed at 20°C for 24 h then

exposed to 200 μL L-1 C2H4 for a further 24 h. This C2H4 concentration was used

because concentrations of 0.1 to 200 μL L-1 are known to stimulate germination and

higher concentrations generally lead to higher germination (Corbineau and Côme 1995).

For the gibberellic acid (GA3), seeds were incubated in 30 μM GA3, which is an

effective concentration for the germination of other species (Plummer and Bell 1995).

For the potassium nitrate (KNO3) treatment seeds were incubated in 10 mM KNO3,

which had a pH of 5.9. This concentration of nitrate was optimal for Californian fire

ephemerals and approximates the increase in soil nitrate after fire (Thanos and Rundel

1995).

Smoke water (Seed Starter, Kings Park and Botanic Garden, Perth) was prepared

according to Tieu et al. (1999) except that oaten hay was burnt in place of native

vegetation. Seeds were incubated in smoke water (pH=4.0), which was filtered (0.2 µm)

and diluted with distilled water to 1:10 v/v.

The impact of heating seeds at different temperatures and for different durations

was also examined. Seeds were wrapped in aluminium foil and placed in an oven at

70ºC for 10 min, 70ºC for 1 h, 70ºC for 5 h or 100ºC for 1 h. For the 5 h treatment seeds

were alternately heated for 1 h and cooled at 20ºC for 1 h five times. After heating all

seeds were placed at 20°C for 12 h prior to incubation. Seeds were heated in aluminium

foil rather than paper envelopes to ensure that smoke-related gases were not produced

during heating because heating plant material may produce the stimulatory compound(s)

found in smoke (Jäger et al. 1996a). For the combined heat and smoke water treatments,

heat treatments were repeated (apart from 70°C for 10 min) and seeds were incubated in

smoke water instead of water.

Influence of heating seeds at 100ºC for 1 h on seed viability and Alyogyne permeability

The impact of the 1 h heat treatment at 100ºC on seed viability was determined using

the tetrazolium chloride test. The test was performed as described above in July 2004 on

three replicates of 15 non-heated and heat-treated seeds of each species. The influence

of the 100ºC treatment on Alyogyne seed permeability was tested by placing heated and

non-heated seeds in water for 24 h to determine whether they imbibed water.

73

Chapter 2

Smoke water dilution

Actinotus leucocephalus and Tersonia cyathiflora seeds were incubated

continuously at each of the following smoke water dilutions: 0.1, 1, 5, 10, 15, 20 and

30% v/v. Seeds were also imbibed in 30% v/v Seed Starter for 24 h at 20ºC, rinsed three

times and transferred to fresh Petri dishes for incubation in distilled water alone.

Statistical analysis

A one-way ANOVA was employed to compare cumulative germination after 84 days at

the different incubation temperatures for each of the species and to compare the effect

of treatment on germination levels for each of the species. Treatments that produced

zero germination in all four replicates were excluded to satisfy the ANOVA assumption

of equal variances, and remaining cut test adjusted percentage data were arcsine square

root transformed prior to analysis. Fisher’s protected LSD test was performed as a post-

hoc test if the treatments had a significant effect on germination. Species that produced

greater germination when the heat (70ºC for 1 h) and smoke water treatments were

combined than when applied separately were analysed using a two-way ANOVA to

examine whether the treatments interacted. A t-test was employed to compare light and

dark regimes, viability of heated and unheated seeds and the proportion of Alyogyne

seeds permeable to water before and after a 100ºC heat treatment. Differences were

regarded as statistically significant if P<0.05. All statistical analyses were undertaken

with Genstat version 6 (VSN International, Oxford, UK).

Results

Viability tests, seed type and embryo growth

A high proportion (≥88%) of seeds were filled in all species except Gyrostemon

racemiger (Table 2). For this species approximately half of the seeds were filled. High

viability of Austrostipa compressa and Austrostipa macalpinei seeds was inferred since

germination of these species exceeded 90% (Figure 4). Seed viability of Alyogyne

hakeifolia, Actinotus leucocephalus, Grevillea scapigera and Codonocarpus cotinifolius

was also high (>70%) according to tetrazolium chloride staining (Table 2).

74

Chapter 2

Table 2. Percentage (mean ± se) of filled seeds, and percentage of filled seeds that were viable, possibly viable or dead according to the tetrazolium chloride test

Species Filled seeds (%)

Viable (%)

Possibly viable (%)

Dead (%)

Austrostipa compressa 98 ± 1.0 - - - Austrostipa macalpinei 98 ± 0.0 - - - Alyogyne hakeifolia 89 ± 1.5 72 ± 3.5 10 ± 1.2 18 ± 2.3 Alyogyne huegelii 92 ± 3.0 28 ± 1.5 43 ± 3.6 29 ± 2.4 Actinotus leucocephalus 88 ± 0.0 90 ± 5.8 0 10 ± 5.8 Grevillea scapigera 97 ± 0.6 87 ± 3.3 0 13 ± 3.3 Codonocarpus cotinifolius 96 ± 0.6 73 ± 11.8 10 ± 5.8 17 ± 9.0 Gyrostemon racemiger 53 ± 2.1 37 ± 3.3 33 ± 13.3 30 ± 10.0 Tersonia cyathiflora 90 ± 0.0 60 ± 15.3 20 ± 10.0 20 ± 5.8

Morphological dormancy is only found in seeds with either rudimentary or

linear embryos (Baskin and Baskin 1998). According to the seed types of Martin

(1946), based on embryo morphology, size and position, the two Austrostipa species

had lateral seeds, the two Alyogyne species had folded seeds, Grevillea scapigera had

investing seeds, the three Gyrostemonaceae species had linear seeds and Actinotus

leucocephalus had rudimentary seeds. Embryos of all species were fully developed

except for Actinotus leucocephalus, which had differentiated but underdeveloped

embryos indicating that this was the only species with morphological dormancy (Baskin

and Baskin 1998). Two of the five Codonocarpus cotinifolius embryos placed on filter

paper grew normally (both the radicle and cotyledons extended). Although all Tersonia

cyathiflora embryos produced growth, four of the five grew abnormally with extended

cotyledons but no radicle development.

Seed imbibition

Seeds of all species increased in weight by 20-60% after 24 h of soaking in distilled

water, indicating that they were permeable to water (data not presented). Large

variability between replicates was noted for seeds of the two Alyogyne species.

Measurements on individual seeds revealed that approximately half of the Alyogyne

hakeifolia (56 ± 4.6%) and Alyogyne huegelii (48 ± 1.2%) seeds imbibed within 24 h.

Remaining Alyogyne seeds were impermeable to water.

75

Chapter 2

Light and incubation temperature

All germination results have been adjusted to express germination as a percentage of

filled seeds. Both Austrostipa species produced greater germination at 20ºC when seeds

were incubated at the 12 h light/ 12 h dark regime than continuous darkness, whereas

germination levels were similar for Alyogyne hakeifolia and Alyogyne huegelii in the

light and dark (Figure 1). The five remaining species produced <3% germination under

both light r

egimes at 20ºC.

Austro

stipa

compre

ssa

Austro

stipa

mac

alpine

i

Alyogy

ne ha

keifo

lia

Alyogy

ne hu

egeli

i

Actino

tus le

ucoc

epha

lus

Greville

a sca

pigera

Codon

ocarp

us co

tinifo

lius

Gyroste

mon ra

cemige

r

Terson

ia cy

athiflo

ra

Ger

min

atio

n (%

)

0

10

20

30

40

50

a

aa

a

a

b

b

a aa

aa

Figure 1. Comparison of the effect of a 12 h diurnal light/dark regime (open bars) and continuous darkness (filled bars) on germination of the nine fire ephemeral species. Germination is expressed as a percentage of filled seeds (mean ± se) after imbibition for 84 days at 20°C. Different letters above the bars indicate significant differences (P<0.05) in germination between the light regimes for each species.

Germination of both Austrostipa species exceeded 70% after 84 days at optimal

temperatures of 10 and 15ºC for Austrostipa compressa, and 5, 10 and 15ºC for

Austrostipa macalpinei, although germination of A. macalpinei was initially delayed at

5ºC (Figure 2a,b). In both Austrostipa species germination was negligible (≤3%) at

15/25°C, despite 37% and 20% of A. compressa and A. macalpinei seeds, respectively,

germinating at the mean constant temperature of 20°C (Figure 2a,b).

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Chapter 2

For both Alyogyne species, cumulative germination after 84 days was similar

across the incubation temperatures tested, except that A. huegelii germination was lower

at 5°C (Figure 2c,d). In both species, germination was delayed at 5°C compared to the

other temperatures. At 15/25ºC, A. huegelii produced more rapid germination than at the

other temperatures tested. Actinotus leucocephalus, Grevillea scapigera and the three

Gyrostemonaceae species produced <3% germination in all temperature treatments.

0

20

40

60

80

100

Cum

ulat

ive

germ

inat

ion

(%)

0

20

40

60

80

100

Time (days)

0 20 40 60 80

Cum

ulat

ive

germ

inat

ion

(%)

0

10

20

30

40

50

Time (days)

0 20 40 60 800

10

20

30

40

50

(a) Austrostipa compressa (b) Austrostipa macalpinei

(c) Alyogyne hakeifolia (d) Alyogyne huegelii

a

a

bb

a

aaa

a

aaa

b

a

a

aa

b

Figure 2. Cumulative germination at 5ºC ( ), 10ºC ( ), 15ºC ( ), 20ºC ( ) and 15/25ºC ( ) for the four species (a) Austrostipa compressa, (b) Austrostipa macalpinei, (c) Alyogyne hakeifolia and (d) Alyogyne huegelii that produced >3% germination at one or more temperatures. Germination is expressed as a percentage of filled seeds (mean ± se) after imbibition for 84 days under a 12 h diurnal light/dark regime. Different letters indicate significant differences (P<0.05) between final germination after 84 days at the different incubation temperatures at which seeds germinated.

Light quality influenced germination of Austrostipa macalpinei at 10°C (Figure

3b). Austrostipa compressa germination was high at 10°C at R:FR ratios of 1.1:1 and

9:1 (Figure 3a), whereas A. macalpinei germination at the same temperature was

77

Chapter 2

enhanced only by the higher R:FR ratio (Figure 3b). Germination was negligible (≤1%)

for both Austrostipa species at 15/25°C irrespective of light quality.

0 20 40 60 80

Cum

ulat

ive

germ

inat

ion

(%)

0

20

40

60

80

100

Time (days)

0 20 40 60 80

(a) Austrostipa compressa (b) Austrostipa macalpinei

Time (days) Figure 3. Effect of red to far-red ratio of 1.1:1 (open symbols) v. 9:1 (filled symbols) during the light phase of a 12 h light/dark diurnal cycle at 10ºC ( , ) and 15/25ºC ( , ) on (a) Austrostipa compressa and (b) Austrostipa macalpinei. Germination is expressed as the percentage of filled seeds (mean ± se) over 84 days at 10°C and 15/25°C.

Chemical, scarification, smoke water and heat treatments

For all species cumulative germination after 42 days of incubation is presented (Figure

4) as few additional seeds germinated between days 42 and 84, unless noted. For

Austrostipa compressa and A. macalpinei, sulphuric acid and manual scarification were

the two most effective treatments for stimulating germination at 20ºC (Figure 4a,b). A

third scarifying treatment, potassium hydroxide, did not increase germination above the

control in either species. Hydrogen peroxide and polyvinlypyrolidone stimulated

germination of both Austrostipa species and gibberellic acid enhanced germination of A.

compressa. Heat treatments of 70ºC for 10 minutes and 1 h enhanced germination of A.

macalpinei but had no effect on A. compressa. Germination of neither species was

enhanced by the addition of smoke water.

Approximately one-third of Alyogyne seeds germinated at 20ºC without further

treatment (Figure 4c,d). For Alyogyne hakeifolia, none of the treatments significantly

altered germination levels after 42 days of incubation (Figure 4c). By contrast, the

sulphuric acid, polyvinylpyrolidone, and combined 70ºC for 1 h and smoke water

treatments all enhanced Alyogyne huegelii germination (Figure 4d). Germination of this

species was suppressed by the potassium hydroxide, ethylene and 100ºC heat

treatments. Heating the seeds for 1 h at 100°C increased the number of water permeable

Alyogyne hakeifolia (from 58 ± 6 to 100 ± 0%, F1,4=140, P<0.001) and A. huegelii

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Chapter 2

(from 62 ± 11% to 96 ± 2%, F1,4=11, P=0.028) seeds. According to the tetrazolium

chloride test, heating seeds for 1 h at 100ºC killed all A. huegelii seeds, but the decline

in viability of A. hakeifolia seeds following heating (56 ± 15% to 33 ± 12%) was not

significant (F1,4=1.45, P=0.294).

0 20 40 60 80 100

0 20 40 60 80 100

0 20 40 60 80 100

100°C 1 h + sw100°C 1 h

70°C 1 h x 5 + sw70°C 1 h + sw

70°C 1 h x 570°C 1 h

70°C 10 minsmoke water (sw)potassium nitrate

gibberellic acidethylene

manual scarificationpotassium hydroxide

sulphuric acidpolyvinylpyrolidonehydrogen peroxide

control

0 20 40 60 80 100

100°C 1 h + sw100°C 1 h

70°C 1 h x 5 + sw70°C 1 h + sw

70°C 1 h x 570°C 1 h

70°C 10 minsmoke water (sw)potassium nitrate

gibberellic acidethylene

manual scarificationpotassium hydroxide

sulphuric acidpolyvinylpyrolidonehydrogen peroxide

control

abcabcd

def

f

gcghgh

bchbch

bc

abc

bch

aa

a

bc

d

abe

ce

ef

f

d

d

a

bc

0 20 40 60 80 100

(a) Austrostipa compressa (b) Austrostipa macalpinei (c) Alyogyne hakeifolia

(d) Alyogyne huegelii

acdecdef

efef

gcdeh

gbcdh

fab

defhcdehdefh

iefh

iaabc

abcd

ab

cdce

fag

h

ab

b

gibe

j

dji

dj

gi

gi

be

(e) Actinotus leucocephalus

e

0 20 40 60 80 100

(f) Grevillea scapigera

0 20 40 60 80 1000 20 40 60 80 100

100°C 1 h + sw100°C 1 h

70°C 1 h x 5 + sw70°C 1 h + sw

70°C 1 h x 570°C 1 h

70°C 10 minsmoke water (sw)potassium nitrate

gibberellic acidethylene

manual scarificationpotassium hydroxide

sulphuric acidpolyvinylpyrolidonehydrogen peroxide

controlaa

abc

cab

de

a

cde

bc

(g) Codonocarpus cotinifolius

a

(i) Tersonia cyathiflora

Germination (%)0 20 40 60 80 100

(h) Gyrostemon racemiger

a

bb

b

a

a

a

a

b

aa

aa

a

a

aa

aa

aa

a

aa

aa

a

a

a

Figure 4. Germination in response to a range of chemical, scarification, smoke water and heat treatments in (a) Austrostipa compressa, (b) Austrostipa macalpinei, (c) Alyogyne hakeifolia, (d) Alyogyne huegelii, (e) Actinotus leucocephalus, (f) Grevillea scapigera, (g) Codonocarpus cotinifolius, (h) Gyrostemon racemiger and (i) Tersonia cyathiflora. Germination is expressed as a percentage of filled seeds (mean ± se) after 42 days of imbibition under a 12 h diurnal light/dark regime at 20°C. Different letters beside the bars indicate significant differences (P<0.05) between germination for each species in response to the treatments.

79

Chapter 2

The most effective treatments for enhancing Actinotus leucocephalus

germination were manual scarification, and heating the seeds for 1 to 5 h at 70ºC before

incubation in smoke water (Figure 4e). The heat (70ºC for 1 h) and smoke water

treatments did not produce a significant interaction when combined (F1,12=0.33,

P=0.577). Heating seeds for 1 to 5 h at 70ºC without subsequent incubation in smoke

water also enhanced germination, with 5 h of heating producing more germination than

1 h. Smoke water and gibberellic acid each produced similar levels of germination as

heating seeds to 70ºC for 1 h. Sulphuric acid, potassium nitrate and a combination of

100ºC heating for 1 h with smoke water incubation all increased Actinotus

leucocephalus germination above the control, but germination levels were low (5-15%).

The 1 h 100ºC heat treatment reduced seed viability, according to the tetrazolium

chloride test, from 96 ± 4% to 33 ± 20% (F1,4=10.89, P=0.030).

Manual scarification was the only treatment that induced >1% of Grevillea

scapigera seeds to germinate (Figure 4f). Germination of Grevillea scapigera following

manual scarification was 14 ± 3% after 42 days (Figure 4f) but increased to 49 ± 7%

after 84 days. According to the tetrazolium chloride test, all Grevillea scapigera seeds

were killed by a 100ºC for 1 h treatment.

Eleven treatments induced some Codonocarpus cotinifolius germination (Figure

4g). After 42 days, maximum germination was attained after smoke water treatment,

either with or without a 70ºC for 1 h heat treatment. No interaction was detected

between the heat (70ºC for 1 h) and smoke water treatments after 42 days of incubation

(F1,12=0.06, P=0.811). The only treatment to germinate beyond 42 days was the

combined heat and smoke water treatment, where germination reached 54 ± 3% after 84

days. After 84 days an interaction was detected between the heat and smoke water

treatments (F1,12=6.01, P=0.031) and germination was increased synergistically. At least

10% of seeds also germinated following the gibberellic acid treatment and after five

cycles of heating to 70ºC for 1 h before incubation in smoke water. Codonocarpus

cotinifolius was the only species that exhibited enhanced germination following the

potassium hydroxide treatment. Germination of this species was also enhanced by

sulphuric acid. For Gyrostemon racemiger, only the sulphuric acid, gibberellic acid and

combined 100ºC for 1 h and smoke water treatments elicited germination, albeit at low

levels (<4%; Figure 4h). The tetrazolium chloride test indicated that the 100ºC for 1 h

treatment killed all Gyrostemon racemiger and Codonocarpus cotinifolius seeds. For

Tersonia cyathiflora, the optimum germination treatment was gibberellic acid (Figure

4e). Less than 4% of T. cyathiflora seeds germinated in the sulphuric acid, smoke water

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Chapter 2

and combined 70ºC and smoke water treatments, and remaining treatments produced no

germination. Although the 100ºC treatment did not produce any Tersonia cyathiflora

germination and this treatment caused a reduction in seed viability from 96 ± 4%

(F1,4=14.97, P=0.018), 53 ± 10% of seeds remained viable after heating. Although

manual scarification did not lead to any of the Gyrostemonaceae species germinating

normally, 44 ± 5, 22 ± 4 and 10 ± 2% of Codonocarpus cotinifolius, Gyrostemon

racemiger and Tersonia cyathiflora seeds, respectively, exhibited cotyledon protrusion

without radicle emergence.

Smoke water dilution

Germination of Actinotus leucocephalus in smoke water was dose dependent with

maximum germination occurring when seeds were imbibed in smoke water dilutions of

1 to 20% (Figure 5). Placing the seeds in 30% smoke water for 24 h before transfer to

distilled water resulted in similar levels of germination as continuous incubation in 30%

smoke water (56 ± 9% v. 51 ± 4% respectively). Germination of Tersonia cyathiflora

seeds did not exceed 3% at any of the smoke water dilutions tested.

Smoke water dilution (%, log scale)

0.1 1 5 10 20 30

Ger

min

atio

n (%

)

0

20

40

60

80

100

0

a

b

cde

dee de

cde

c

Figure 5. Germination of Actinotus leucocephalus (filled symbols) and Tersonia cyathiflora (open symbols) seeds incubated at a range of smoke water dilutions for 35 days at 20°C. Germination (mean ± se) is expressed as a percentage of filled seeds. Different letters above the symbols indicate significant differences (P<0.05) between germination at the different smoke water dilutions.

81

Chapter 2

Discussion

This study provides the first analysis of seed dormancy patterns in the ecologically

significant group of plants in Australia known as fire ephemerals. Surprisingly, whereas

all the study species respond to fire in their natural habitat, this unifying relationship

was not evident in the diverse responses of seeds to germination treatments. Grevillea

scapigera, Codonocarpus cotinifolius, Gyrostemon racemiger, and Tersonia cyathiflora

had fully developed embryos yet did not germinate readily, and hence had physiological

dormancy (J. Baskin and Baskin 2004). These species are likely to possess non-deep

physiological dormancy as by definition, intermediate and deep physiological dormancy

are overcome by cold stratification (J. Baskin and Baskin 2004) and these species were

not induced to germinate by this treatment nor do they receive long periods of cold

temperatures in their natural habitat. Furthermore non-deep physiological dormancy is

the prevalent form of physiological dormancy in Mediterranean climate regions

(Nikolaeva 2004). However all four species did not germinate in response to gibberellic

acid or scarification, or produce normal seedlings following embryo excision, typical

characteristics of seeds with non-deep physiological dormancy (J. Baskin and Baskin

2004). However there is much variation within seeds with non-deep physiological

dormancy (Nikolaeva 2004), suggesting that as additional species are investigated, this

level of physiological dormancy may require revision.

Another species with physiological dormancy was Actinotus leucocephalus since

untreated water-permeable seeds did not germinate within four weeks at a range of

incubation temperatures (J. Baskin and Baskin 2004). However, in contrast to the other

species examined, the embryos of this species were rudimentary meaning that

morphophysiological dormancy was present.

The two Austrostipa species were either non-dormant or possessed non-deep

physiological dormancy because high germination could be obtained at specific

temperature and light regimes. These species germinated readily at winter temperatures

that coincide with high soil moisture availability in their natural Mediterranean climate

habitat, which suggests that they would germinate readily in the field. This appears to

conflict with the notion that these species usually form persistent seedbanks and require

fire to germinate (D. Bell 1999; Smith et al. 1999). However germination of these

species is prevented in the field because the hygroscopic awn buries the seeds

(Ghermandi 1995), thus placing the seeds in darkness and inhibiting germination.

Furthermore, the red to far-red ratios that produced most Austrostipa macalpinei

germination are unlikely to be encountered in nature.

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Chapter 2

An important characteristic of seeds with non-deep physiological dormancy is

that they may after-ripen in dry storage (Schütz et al. 2002; J. Baskin and Baskin 2004).

In this study, seeds were stored for variable periods at 15°C following air drying prior to

germination testing. It is therefore likely that the physiological status of the seeds, and

hence their response to some of the germination stimulating treatments such as smoke

and nitrate, will have changed as a result of after-ripening since the time of collection.

Thus whilst the results may be indicative of species behaviour, any ecological

interpretation can only be tentative.

Approximately half of the Alyogyne seeds had physical dormancy as they were

impermeable to water and approximately one-third were non-dormant as they

germinated across a range of incubation temperatures. Acid scarification (H2SO4)

increased germination of Alyogyne huegelii seeds, and this has been reported for many

other species with impermeable seeds that germinate after fire, such as Rhus ovata

Wats. (Stone and Juhren 1951). For the two Alyogyne species none of the scarification

treatments resulted in germination of all viable and possibly viable seeds. Either the

scarification treatments damaged some of the seeds, their potential viability was

overestimated by the tetrazolium chloride test or seeds had combinational dormancy

(physiological as well as physical dormancy) as found in another Malvaceae species,

Malva parviflora L. (Sumner and Cobb 1967).

Scarification treatments also induced germination of seeds with physiological or

morphophysiological dormancy. At supra-optimal incubation temperatures both manual

and acid scarification produced high levels of Austrostipa compressa and A. macalpinei

germination. Manual scarification was also one of the optimal germination treatments

for Actinotus leucocephalus and Grevillea scapigera, although acid scarification was

less effective at promoting germination in each of these species. Scarification has

previously been found to promote germination in a number of Poaceae (Adkins et al.

2002b) and Proteaceae (Morris 2000) species. However, Tieu et al. (1999) found that

removing the pericarps from Actinotus leucocephalus (Apiaceae) fruit did not promote

germination in the absence of supplemental GA3 and zeatin, or smoke water. The

difference with the present study may have arisen due to different light conditions

during seed incubation (continuous darkness in the study by Tieu et al. (1999) compared

to an alternating light/dark regime in the present study). Another seed scarifying

treatment, KOH (Sukhvibul and Considine 1994), did not stimulate germination of any

of the above species, although it did promote germination of Codonocarpus cotinifolius.

The different impacts of the acidic and basic scarifying treatments may have arisen

83

Chapter 2

because H2SO4 weakens the seed coat by corrosion, whereas KOH may saponify lipids

in the seed coat or undergo basic rather than acidic reactions with inhibitors (Sukhvibul

and Considine 1994).

Scarification of permeable seeds could induce germination by increasing gas

permeability, mitigating the effects of germination inhibitors, reducing mechanical

restriction on embryo expansion (Adkins et al. 2002b), or by inducing wound responses

such as ethylene production (Cranston et al. 1996). Ethylene did not stimulate the

germination of any of these species and so it is unlikely that ethylene production was

the mechanism by which scarification promoted germination. Polyvinylpyrolidone,

which possibly promotes germination by binding to oxygen scavengers in seeds and

allowing greater oxygen supply to the embryo (Bhattacharyya et al. 1999), and H2O2,

which may promote germination by oxidising inhibitors (Ogawa and Iwabuchi 2001),

also stimulated germination in the two Austrostipa species. Thus, perhaps scarification

enhances germination in these two species by mitigating the effects of oxygen

scavengers and/or other seed-based inhibitors.

Some of the other chemical treatments, including ethylene and nitrate, which are

products of fire, were less effective at promoting germination. Ethylene, which is

produced in smoke (van de Venter and Esterhuizen 1988) and released from ash

(Ne'eman et al. 1999), did not promote germination of any of the fire ephemerals

examined, and although nitrate levels increase in the soil after fire (Christensen 1973),

nitrate only stimulated Actinotus leucocephalus to germinate. Although ethylene or

nitrate stimulate the germination of many species from fire-prone areas, many other

species from these areas are not responsive to these treatments (van de Venter and

Esterhuizen 1988; Thanos and Rundel 1995; Keeley and Fotheringham 1998a).

Germination of Actinotus leucocephalus in response to nitrate supports the suggestion

that some fire ephemerals can germinate in unburnt, nutrient enriched areas (Bell et al.

1984). The general low response to ethylene and nitrate may indicate that most of these

species are unresponsive to these treatments or that the concentrations tested were not

optimal.

Another fire-related treatment, smoke water, stimulated germination of Actinotus

leucocephalus and Codonocarpus cotinifolius. High smoke water concentrations (30%)

were less effective at promoting Actinotus leucocephalus germination than lower

concentrations (1-20%) and similar inhibitory effects of high concentrations have been

found by others using Seed Starter (Tieu et al. 1999) and other smoke-based

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Chapter 2

preparations (Brown 1993b). This is possibly because compounds exist in plant-derived

smoke that are toxic to germination at higher concentrations (Baldwin et al. 1994).

Smoke water or aerosol smoke produced less than 25% germination in

laboratory-stored Codonocarpus cotinifolius seeds in the present study and in the study

by Dixon et al. (1995). However heat (70°C for 1 h), despite promoting only low levels

of germination when seeds were incubated in water, produced a synergistic effect when

seeds were incubated in smoke water, as observed in Grevillea buxifolia (Sm.) R.Br.

(Kenny 2000) and Kunzea capitata (Sm.) Heynh. (Thomas et al. 2003). This suggests

that the heat rendered the seeds more smoke-responsive. In contrast, the combined

treatments had an additive effect on Actinotus leucocephalus germination, as observed

in Grevillea sericea (Sm.) R.Br. (Kenny 2000), Gahnia sieberiana Kunth and Kunzea

ambigua (Sm.) Druce (Thomas et al. 2003). For these species, although germination

levels resulting from the heat and smoke treatments were additive, it is unknown

whether the two treatments operated separately or interacted when applied in

combination.

Heat treatments that enable germination vary considerably between species. A 3

h heat pulse at 100°C stimulated approximately 20% of Actinotus leucocephalus seeds

to germinate (Tieu et al. 2001a), while heating Salvia apiana Jepson seeds at 70°C for 1

h enhanced germination of this fire-following species (Keeley 1987). In contrast to Tieu

et al. (2001a), treatments at 100°C failed to enhance germination of any species

including Actinotus leucocephalus. Germination suppression in many species following

heating for 1 h at 100°C was due to seed mortality. The 100°C heat treatment overcame

water-impermeability in both Alyogyne species but did not enhance germination. For

Alyogyne huegelii this can be attributed to a concurrent decline in seed viability.

Perhaps heating seeds in aluminium foil in the present study, in place of paper

envelopes, as in the study by Tieu et al. (2001a) contributed to seed death, as the

aluminium foil may have restricted moisture loss, and seed deterioration is enhanced

when high temperatures are combined with elevated moisture contents (Merritt et al.

2003). Heating seeds to 70ºC for 1 h promoted germination in three species with water-

permeable seeds: Austrostipa macalpinei, Actinotus leucocephalus and Codonocarpus

cotinifolius. The mechanism by which a heat pulse induces germination of

physiologically dormant seeds is still unknown but it is possibly related to changes

occurring at the membrane level (Hallett and Bewley 2002).

In conclusion, scarification treatments were one of the most effective methods

for germinating five of the nine fire ephemerals examined, and optimum germination of

85

Chapter 2

another species was induced by gibberellic acid. Although these treatments are unlikely

to induce germination in situ, they effectively overcame dormancy and increased

germination in these species. Heat and smoke treatments, which were most effective for

inducing germination in a further two species, are possibly a greater reflection of agents

that stimulate germination in the soil. Scarification, gibberellic acid, and heat and smoke

treatments could thus be employed to propagate fire ephemeral species for land

rehabilitation or horticulture programs and would perhaps also be effective in

propagating congeneric rare species. Although these treatments stimulated the

germination of many seeds, some seeds with non-deep physiological dormancy

remained dormant. During ex situ storage these seeds may not have received the

environmental cues required to overcome dormancy and allow germination in response

to the treatments tested. Further investigation is required into the conditions in the soil

environment that may influence the subsequent germination of these species.

86

Chapter 3

CHAPTER 3

Dormancy release in Australian fire ephemeral seeds during burial increases

germination response to smoke water or heat

Abstract

Fire ephemerals are short-lived plants that primarily germinate after fire. Fresh and

laboratory-stored seeds are difficult to germinate ex situ, even in response to fire-related

cues such as heat and smoke. Seeds of eight Australian fire ephemeral species were

buried in unburnt and recently burnt sites of natural bushland during autumn. Seeds

were exhumed after 6 and 12 months and incubated in water and smoke water, either

with or without a 70ºC for 1 h heat treatment. Generally germination did not increase

after 6 months of burial but after 12 months of burial germination was enhanced in

seven of the eight species. Actinotus leucocephalus produced higher germination

following 12 months of burial without any further treatment, and smoke water and heat

further improved germination. The four Gyrostemonaceae species, Codonocarpus

cotinifolius, Gyrostemon racemiger, Gyrostemon ramulosus and Tersonia cyathiflora,

only germinated in the presence of smoke water and their germination was enhanced by

burial. Burial improved germination in response to a heat treatment in Grevillea

scapigera and Alyogyne huegelii seeds, but did not enhance Alyogyne hakeifolia

germination. During concurrent dry laboratory storage of seeds at 15ºC, only Actinotus

leucocephalus produced increased germination in response to smoke water and heat

over time. In summary, soil burial can alter the dormancy status of a number of

Australian fire ephemeral seeds, rendering them more responsive to germination cues

such as smoke water and heat. The requirement for a period of burial before seeds

become responsive to smoke and/or heat would ensure that seeds persist in the soil until

a subsequent fire when there is an increase in nutrients available for growth and reduced

competition from other plants.

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Chapter 3

Introduction

Fire ephemerals are a functional group of plants that are found in many fire-prone

regions of the world such as California, South Africa and Australia (le Maitre and

Midgley 1992). These species form transient components of the above-ground flora;

usually only germinating after fire, growing rapidly, flowering, producing seed and

dying before a subsequent fire (Bell et al. 1984; Hunter 1998). Monocarpic fire

ephemerals flower and fruit once and usually live for less than one year whereas

polycarpic fire ephemerals flower and fruit for more than one season and may live for

up to 10 years (Pate et al. 1985). After the plants die, these species exist only as seeds in

the soil seedbank.

Australian fire ephemerals germinate from the soil seedbank in large numbers

after fire in natural bushland (Bell et al. 1984). However, germination of fresh or

laboratory-stored seeds is difficult. Many viable fire ephemeral seeds, that are fresh or

laboratory-stored, fail to germinate following incubation at a range of temperatures or

treatment with fire-related cues such as heat, smoke, nitrate and ethylene (Chapter 2;

Parsons 1997; Hunter 1998). Even setting fire to hay over fresh or laboratory-stored fire

ephemeral seeds placed in the soil does not induce germination (Baker unpublished

results). It has been inferred that under natural conditions, seeds of fire ephemerals

persist in the soil seedbank for long periods between fires (Pate and Hopper 1993;

Egerton-Warburton 1998). Thus low germination of laboratory-stored seeds compared

to the profuse emergence of these species from the soil seedbank after bushfires

suggests that some form of pre-fire conditioning is required.

A period of soil burial alters the conditions necessary for germination in a

number of species. Some species produce higher germination after a period of burial

(Kilian and Cowling 1992; Pickup et al. 2003) and others change in their sensitivity to

germination stimulants such as light, nitrate (Wesson and Wareing 1967; Derkx and

Karssen 1993) and smoke (Doherty and Cohn 2000). Germination of fire-following

species has also been enhanced by a period of burial. In South Africa, more seeds from

Audouinia capitata fruits germinated in response to smoke after a period of burial than

when freshly collected (de Lange and Boucher 1993b). In California, three fire-

following species which did not germinate when initially treated with heat, smoke,

nitrate and ethylene, germinated in response to smoke following a year of burial (Keeley

and Fotheringham 1998a). Likewise, a number of Western Australian species produced

higher seedling emergence in response to aerosol smoke following a period of burial

(Roche et al. 1997; Tieu et al. 2001c). Many of these studies did not separate the effects

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Chapter 3

of ageing from burial. However Tieu et al. (2001c) concurrently examined the effects of

laboratory storage and found that some species required burial whereas others just

required a period of dry storage. Roche et al. (1997) investigated the effect of storing

seeds in soil punnets in a glasshouse whereas Tieu et al. (2001c) buried seeds in

bushland to better reflect natural conditions of soil burial. Both studies measured

changes in germination response as seedling emergence from soil punnets. However

seedling emergence may have underestimated actual germination.

Here we investigated whether the germination of eight fire ephemerals from the

south-western botanical province of Western Australia was influenced by six or twelve

months of soil burial in natural bushland in unburnt and recently burnt soil. It was

hypothesised that recently burnt soil would be more conducive to overcoming dormancy

than unburnt soil. The requirement for further treatments such as smoke and heat

following burial was also examined. To separate any effects of burial and ageing,

germination tests were also undertaken on seeds concurrently stored under laboratory

conditions. In contrast to Roche et al. (1997) and Tieu et al. (2001c) these tests were

undertaken in Petri dishes rather than soil punnets to measure germination rather than

emergence. A glasshouse trial was also established to determine whether glasshouse soil

storage mimicked burial in the field. The effect of soil burial under natural conditions

has not been examined previously in any of these species. A better understanding of the

requirements for dormancy breaking and germination stimulation in these species will

ensure greater efficiency in the use of these seeds in land rehabilitation and conservation

programs.

Materials and methods

The germination responses of eight Western Australian fire ephemeral species from the

Apiaceae, Gyrostemonaceae, Malvaceae and Proteaceae families were examined. All

species were polycarpic fire ephemerals except for Actinotus leucocephalus, which is a

monocarpic fire ephemeral. Seeds were either collected during summer 2001/02 or

purchased from commercial seed suppliers (Table 1). Prior to use all seeds were

cleaned, air dried and stored in sealed containers at 15ºC. Seeds obtained from

Nindethana Seed Service had been stored at 4ºC prior to purchase in February 2002.

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Chapter 3

Table 1. Seed collection date and provenance for the species studied. Species Family Collection Date Source Actinotus leucocephalus Benth.

Apiaceae 12/2001 Collected from burnt site 40 km N Gin Gin, WA, 31º01.231’S, 115º43.297’E

Alyogyne hakeifolia (Giord.) Alef.

Malvaceae 12/1999 Purchased from Nindethana Collection from Salmon Gums, WA

Alyogyne huegelii (Endl.) Fryxell

Malvaceae 12/1999 Purchased from Nindethana Collection from near Esperance, WA

Grevillea scapigera A.S.George

Proteaceae 12/2001 Received from Kings Park, WA Collection from a translocated population at Corrigin, WA

Codonocarpus cotinifolius (Desf.) F.Muell.

Gyrostemonaceae 1/11/97 Purchased from Nindethana Collection from Hyden, WA

Gyrostemon racemiger H.Walter

Gyrostemonaceae 21/12/01 Collected from burnt site 55 km N Gin Gin, WA 30º54.384’S, 115º38.417’E

Gyrostemon ramulosus Desf.

Gyrostemonaceae Pre-1989 Purchased from Nindethana Collection from Carnarvon, WA

Tersonia cyathiflora (Fenzl) J.W.Green

Gyrostemonaceae 6/1/02 Collected from burnt site Beekeepers Reserve, North of Eneabba, WA, 29º32.315’S, 115º03.415’E

Field burial trial

A burial trial was undertaken in an area of Banksia attenuata woodland on Karrakatta

sands in the Spearwood dune system (McArthur 1959) at The University of Western

Australia’s Shenton Park Field Station, Perth. Part of the bushland had been burnt in

January 2002. Rainfall and temperature data for the duration of the trial were obtained

from the nearby Department of Agriculture Floreat Park weather station (Fig. 1). Mean

monthly minimum and maximum temperatures were calculated from daily minimum

and maximum temperatures.

Seeds were buried in nylon mesh bags in March 2002 (autumn) before the onset

of the rainy season (Fig. 1) at four random sites within each of the burnt and unburnt

areas of bushland. Seeds were buried in both burnt and unburnt sites to determine

whether post-fire soil conditions were more conducive to alleviating dormancy. The

finely woven mesh bags allowed the transfer of water and solutes but contained the

seeds. For each species, 130 seeds that appeared healthy and intact were placed in each

bag together with an approximately equal volume of fine white sand. At each of the

eight sites, the surface 2 cm of soil was removed from a 2 m2 area, and then the

underlying 3 cm of soil was removed separately. Ten free draining trays were positioned

in the hole and the lower 3 cm of soil placed in the trays. A seed bag of each species

was placed in each tray and covered with the surface 2 cm of soil. Bird netting was

stretched over each site to reduce seed predation.

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Chapter 3

Jan 02Feb 02

Mar 02Apr 02

May 02Jun 02

Jul 02Aug 02

Sep 02Oct 02

Nov 02Dec 02

Jan 03Feb 03

Mar 03

Air

tem

pera

ture

(ºC

)

0

5

10

15

20

25

30

35

Rai

nfal

l (m

m)

0

20

40

60

80

100

120

140

160

180Fire Seeds buried Spring retrieval Autumn retrieval

Figure 1. Mean monthly minimum (dashed line) and maximum (solid line) air temperature (ºC) and monthly rainfall (mm, bars) between January 2002 and March 2003.

Half of the trays at each of the four plots within the burnt and unburnt sites were

exhumed six months after burial in late September 2002 (spring) as air temperatures

started to rise and rainfall decreased, and the remainder exhumed one year after burial in

March 2003 (autumn) at the end of the summer dry season. At each exhumation the

number of seedlings that had germinated in the plots were noted. The moisture content

(% fresh weight) was determined for the seeds retrieved from burial in spring, at the end

of the winter wet season. Exhumed seeds were sealed in plastic bags and within 24 h of

collection, a subsample of each species (10-80 seeds depending on seed size) from each

of the plots within each of the burnt and unburnt sites was weighed, placed in an oven

for 17 h at 105°C, and reweighed (International Seed Testing Association 1999). Seeds

exhumed in spring were allowed to air dry in the laboratory for one week prior to

germination testing. For all exhumed seeds, damaged, flat or germinated seeds were

counted and discarded. For each of the eight plots (four replicates in each of the burnt

and unburnt sites), 25 healthy seeds of each species were assigned to control, smoke

water, heat and combined heat and smoke water treatments. Seeds assigned to either the

heat, or heat and smoke water treatments were wrapped in aluminium foil and placed in

an oven at 70ºC for 1 h and then cooled at 20ºC for 24 h. To minimise fungal

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Chapter 3

contamination, all seeds were surface sterilised prior to incubation by shaking for 30 s

in 2% v/v NaOCl containing a non-ionic surfactant (one drop per 125 mL of Plus 50,

Ciba-Geigy, Sydney). Seeds in NaOCl were then placed under vacuum for 5 min,

returned to normal air pressure for 5 min and placed under vacuum again for a further 5

min to remove air pockets around the seeds. Seeds were then transferred to a laminar

flow, rinsed three times in sterile distilled water, and soaked in sterile distilled water for

10 min before a further rinsing. Seeds assigned to smoke water treatments were air dried

in the laminar flow for 8-12 h before transfer to Petri dishes to ensure imbibition of the

smoke water.

Seeds were placed on two pieces of Whatman No. 1 filter paper over three

pieces of 4 cm2 absorbent sponge in sterile plastic Petri dishes (90 mm diameter). Petri

dishes were moistened with either 10 mL of sterile distilled water or a 1:10 dilution of

smoke water. Both solutions contained 0.15% v/v Previcur fungicide (Aventis,

Melbourne, active ingredient 600 g L-1 propamocarb). Smoke water was prepared by

filtering (0.2 μM) Seed Starter (Kings Park and Botanic Garden, Perth) produced

according to Tieu et al. (1999) except that hay was burnt instead of native vegetation.

Aliquots of Seed Starter were frozen until required to minimise any possible changes

over time. Petri dishes were sealed with Parafilm to retain moisture. Seeds were

incubated under a daily 12 h light, 12 h dark regime at a constant 20ºC. Light was

provided by cool white fluorescent tubes (50 μmol m-2 s-1 PAR). Seeds were monitored

weekly for five weeks and seeds removed as they germinated. Seeds were considered to

have germinated once the radicle emerged >2 mm. Remaining seeds were dissected to

determine whether they were filled. Results are presented as a percentage of filled

seeds.

The same germination tests (water, smoke water, heat, heat and smoke water)

were undertaken on seeds stored at 15ºC in the laboratory at the time seeds were buried

(autumn 2002) and when seeds were exhumed for the second time (autumn 2003) to

determine whether any differences in germination of buried seeds were due to the

storage environment or age. At the time of the first seed exhumation (spring 2002) all

four germination tests were undertaken on laboratory-stored Actinotus leucocephalus

and Codonocarpus cotinifolius seeds and remaining species were germination tested in

water only.

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Chapter 3

Shadehouse burial trial

To determine whether field conditions could be replicated under nursery conditions, the

effect of soil storage of seeds in a shadehouse was also investigated. At the end of

autumn 2002, laboratory-stored seeds of all eight species were placed in seedling

punnets (13 x 8 x 4.5 cm deep) containing a pasteurised mix of sand, composted

sawdust and peat (1:1:1) as per Roche et al. (1997) and Tieu et al. (2001c). Seeds were

covered with a fine layer of sand, watered and placed in a tin shed. Smoke produced by

hay burnt in a metal drum was fan-forced through a pipe into the shed. Control punnets

were set up in a similar way but not smoked. Punnets were transferred to a shadehouse

at The University of Western Australia, Perth and watered daily until spring 2002. Over

summer the punnets remained dry except during a few showers (Fig. 1). In autumn 2003

the smoked punnets were re-smoked following the same procedure. Watering to all

punnets then recommenced and continued until spring 2003. Seedling emergence was

monitored weekly during periods of watering.

Statistics

One-way ANOVAs were undertaken to determine whether the percentage of filled

seeds changed over time when seeds were buried in each of the burnt and unburnt sites,

and to compare germination within each species over time under controlled laboratory

conditions and after burial in the burnt and unburnt plots. Where germination was

absent from all four replicates, the treatment was excluded from analysis to satisfy the

ANOVA assumption of equal variances. T-tests were used to compare the percentage of

filled seeds and germination levels after 6 and 12 months between the burnt and unburnt

sites. A two-way ANOVA was undertaken to determine whether seed moisture content

was similar between the burnt and unburnt plots. Percentage data were arcsine square-

root transformed prior to analysis. Fisher’s protected LSD test was used as a post-hoc

test. Treatments were regarded as significantly different if P<0.05. Statistical analyses

were performed in Genstat version 6 (VSN International, Oxford, UK).

Results

Field burial trial

During 12 months of soil burial many of the species experienced a decline in the

number of filled seeds, calculated as the proportion of seeds filled out of the percentage

of seeds retrieved from burial that were undamaged and firm (Fig. 2). Four species,

Alyogyne hakeifolia, Alyogyne huegelii, Codonocarpus cotinifolius and Gyrostemon

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Chapter 3

ramulosus, exhibited large declines (44-84%) in the proportion of filled seeds during the

first six months of burial (Fig. 2B, C, E, G), which coincided with the wet winter season

(Fig. 1). Although the two Alyogyne species displayed similar declines in filled seeds

between the burnt and unburnt sites, greater declines in the proportion of filled seeds

occurred in the burnt sites for Codonocarpus cotinifolius and Gyrostemon ramulosus.

Gyrostemon racemiger and Tersonia cyathiflora, which are in the same family as

Codonocarpus cotinifolius and Gyrostemon ramulosus, did not experience any

reduction in the proportion of filled seeds during 12 months of burial (Fig. 2F, H). The

remaining species, Actinotus leucocephalus and Grevillea scapigera, exhibited a small

decline (18-33%) in filled seeds over the 12 months of burial (Fig. 2A, D). In spring,

some seedlings, mainly Actinotus leucocephalus, Alyogyne hakeifolia and Alyogyne

huegelii, were observed in the burnt plots, representing <0.6% of seeds buried at that

site. No seedlings of the study species were observed in either the unburnt plots or the

surrounding area. No seedlings were observed in autumn in either the burnt or unburnt

plots.

A Actinotus leucocephalus

Fille

d se

eds

(%)

0

20

40

60

80

100B Alyogyne hakeifolia C Alyogyne huegelii

E Codonocarpus cotinifolius

0 6 120

20

40

60

80

100

D Grevillea scapigera

F Gyrostemon racemiger

0 6 12

G Gyrostemon ramulosus

Duration of burial (months)

0 6 12

H Tersonia cyathiflora

0 6 12

Figure 2. Filled seed (mean ± se) at the time of burial and after 6 and 12 months of burial in burnt and unburnt soil for each species. (A) Actinotus leucocephalus; (B) Alyogyne hakeifolia; (C) Alyogyne huegelii; (D) Grevillea scapigera; (E) Codonocarpus cotinifolius; (F) Gyrostemon racemiger; (G) Gyrostemon ramulosus; and (H) Tersonia cyathiflora.

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Prior to burial, germination of many species was low (Figs 3, 4). Only Alyogyne

hakeifolia and Alyogyne huegelii seeds germinated in water, but germination of neither

species exceeded 30% (Fig. 3E, I). Smoke water led to some germination of pre-buried

Actinotus leucocephalus and Codonocarpus cotinifolius seeds but germination levels

were still below 40% (Figs 3B, 4B). Heat enhanced germination of Actinotus

leucocephalus and Alyogyne huegelii resulting in 27% and 45% germination

respectively (Fig. 3C, K). Combining the heat and smoke water treatments produced

higher germination than either treatment individually for Actinotus leucocephalus (78%

germination, Fig. 3D) and Codonocarpus cotinifolius (31% germination, Fig. 4D). Four

species, Grevillea scapigera, Tersonia cyathiflora, Gyrostemon racemiger and

Gyrostemon ramulosus produced negligible germination prior to burial irrespective of

incubation in water or smoke water, or whether they were heat treated (Figs 3M-P, 4E-

P).

Germination was influenced by the duration of burial. Six months of burial

during the winter wet season failed to enhance germination of most species, and actually

suppressed germination of those species that had germinated before burial (Figs 3, 4).

For example, prior to burial Actinotus leucocephalus produced up to 78% germination

in the heat and smoke water treatments, but after six months of burial neither heat nor

smoke water induced any germination in this species (Fig. 3B-D). In contrast, Grevillea

scapigera and Tersonia cyathiflora, which did not germinate prior to burial, produced

low levels of germination (<15%) after burial for six months; Grevillea scapigera

responded to heat and Tersonia cyathiflora responded to smoke water (Figs 3O, 4N).

Following a further 6 months of burial over the hot, dry summer (Fig. 1),

germination of most species was enhanced above pre-burial levels (Figs 3, 4). Actinotus

leucocephalus was the only species that produced higher germination in water after 12

months of burial (Fig. 3A). Most other species required either smoke water and/or heat

treatments following burial to increase germination above pre-burial levels. Actinotus

leucocephalus germination was also enhanced further by the smoke water and heat

treatments (Fig. 3A-D). Heat treatments produced higher germination in the two

Alyogyne species after one year of burial compared to non-heat treated seeds (Fig. 3E-

L). All four Gyrostemonaceae species exhibited an increase in germination following

burial for 12 months, but only when incubated in smoke water (Fig. 4). The only species

that produced lower germination after 12 months of burial than before burial were

unheated Alyogyne hakeifolia seeds and Alyogyne huegelii seeds exhumed from the

burnt plot (Fig. 3E, F, I, J).

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water

0

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smoke water heat heat and smoke water

0

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Ger

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n (%

)

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0 6 120

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Duration of burial or laboratory storage (months)

0 6 12 0 6 12 0 6 12

A B C D

E F G H

I J K L

M N O P

Actinotus leucocephalus

Alyogyne hakeifolia

Alyogyne huegelii

Grevillea scapigera

Figure 3. Germination (mean ± se) as a percentage of filled seeds for (A-D) Actinotus leucocephalus; (E-H) Alyogyne hakeifolia; (I-L) Alyogyne huegelii; (M-P) and Grevillea scapigera when treated with water (A, E, I, M), smoke water (B, F, J, N), heat (C, G, K, O) and heat and smoke water (D, H, L, P). Seeds were stored in the laboratory at a constant 15ºC ( ), or buried in burnt ( ) or unburnt ( ) soil for 6 and 12 months.

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water

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smoke water heat heat and smoke water

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Duration of burial or laboratory storage (months)0 6 12

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0 6 12 0 6 12 0 6 12

A B C D

E F G H

I J K L

M N O P

Codonocarpus cotinifolius

Gyrostemon racemiger

Gyrostemon ramulosus

Tersonia cyathiflora

Figure 4. Germination (mean ± se) as a percentage of filled seeds for the four Gyrostemonaceae species. (A-D) Codonocarpus cotinifolius; (E-H) Gyrostemon racemiger; (I-L) Gyrostemon ramulosus; and (M-P) Tersonia cyathiflora when treated with water (A, E, I, M), smoke water (B, F, J, N), heat (C, G, K, O) and heat and smoke water (D, H, L, P). Seeds were stored in the laboratory at a constant 15ºC ( ), or buried in burnt ( ) or unburnt ( ) soil for 6 and 12 months.

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For both Alyogyne species, any filled seeds remaining at the conclusion of the

germination tests, regardless of treatment, were impermeable to water.

Generally, germination was either similar between seeds exhumed from the

burnt and unburnt sites, or higher in the latter. For example, germination of Tersonia

cyathiflora was similar following burial in the burnt and unburnt sites (Fig. 4M-P),

while Codonocarpus cotinifolius seeds produced higher germination in smoke water

after burial in the unburnt site than the burnt site (Fig. 4B). In Actinotus leucocephalus,

germination in water was higher after seeds were buried for 12 months in the unburnt

site than the burnt site (Fig. 3A), but after a heat treatment this difference was

diminished (Fig. 3A, C). Similarly, smoke water enhanced more Gyrostemon racemiger

seeds to germinate after 12 months of burial in the unburnt site than the burnt site, but

heating the seeds prior to smoke water treatment resulted in similar levels of

germination between the two sites (Fig. 4F, H). The moisture content of seeds exhumed

in spring varied between species (8-27%) but was consistently higher in those seeds

exhumed from the burnt site than the unburnt site (Fig. 5).

Actino

tus le

ucoc

epha

lusAlyo

gyne

hake

ifolia

Alyogy

ne hu

egeli

iGre

villea

scap

igera

Codon

ocar

pus c

otinif

olius

Gyroste

mon ra

cemige

r

Gyroste

mon ra

mulosu

sTe

rsonia

cyath

iflora

Seed

moi

stur

e co

nten

t (%

fres

h w

eigh

t)

0

5

10

15

20

25

30

Figure 5. Seed moisture content (% fresh weight, mean ± se) of each species following exhumation in spring 2002 from the burnt (filled bars) and unburnt (open bars) sites, and after air dry storage in the laboratory at 15ºC (bars with diagonal lines).

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Chapter 3

Germination of seeds stored in a controlled temperature room at 15ºC exhibited

different patterns to those buried in the field (Figs 3, 4). Laboratory-stored seeds did not

exhibit the increase in germination noted for many of the seeds after 12 months of

burial, nor suppression of seed germination observed after six months of burial. In fact

germination levels of most laboratory-stored seeds remained unchanged throughout the

year. The only species to exhibit a change in germination response during the year were

Actinotus leucocephalus and Alyogyne huegelii. Germination of Actinotus

leucocephalus increased over time when seeds were incubated in smoke water or heated

(Fig. 3B, C), whereas Alyogyne huegelii germination declined when heated and

incubated in smoke water (Fig. 3L).

0

10

20

30

40

50 A Control

B Aerosol smoke

Actinotus le

ucoce

phalus

Alyogyn

e hakeifo

lia

Alyogyn

e huegelii

Codonocarpus c

otinifo

lius

Grevillea sc

apigera

Gyroste

mon race

miger

Gyroste

mon ramulosu

s

Tersonia cy

athiflora

Cum

ulat

ive

germ

inat

ion

(%)

0

10

20

30

40

50

Figure 6. Germination (mean ± se) of the eight species when placed in soil (A) and (B) given an aerosol smoke treatment. Aerosol smoke treatments were performed in autumn 2002 and autumn 2003. Filled bars indicate germination during the first winter season and the open bars represent germination over the second winter in the soil. Germination is expressed as a percentage of filled seeds buried at the start of the experiment.

Shadehouse burial trial

Fresh or laboratory-stored seeds placed in soil and either aerosol-smoked for an hour or

left unsmoked generally produced low levels of emergence (≤5%; Fig. 6). However,

moderate numbers (25-35%) of Alyogyne hakeifolia and Alyogyne huegelii seedlings

emerged in the first year, with no difference in emergence between the smoked and

unsmoked punnets. Emergence of unsmoked Actinotus leucocephalus, Grevillea

scapigera, Gyrostemon racemiger and Gyrostemon ramulosus seedlings was negligible

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(≤1%), and only slightly higher (3-5%) when aerosol-smoked. Some Actinotus

leucocephalus and Codonocarpus cotinifolius seeds emerged after an aerosol-smoke

treatment in the second year, but only to low levels (7 and 4% respectively).

Discussion

Seeds of fire ephemerals are typically exposed to a period of soil burial because these

species predominantly germinate after fire, release their seeds into the soil seedbank and

adult plants usually die before a subsequent fire (Pate et al. 1985). Burial influenced the

subsequent germination of the eight species examined. Germination of five of the six

species with water-permeable seeds and physiological dormancy (Chapter 2) was

enhanced in smoke water by 12 months of burial. Thus, in these species, physiological

dormancy was alleviated during burial, allowing them to respond to germination

stimulants such as smoke (Vleeshouwers et al. 1995).

Fire ephemerals characteristically germinate after fire, grow rapidly and reach

reproductive maturity quickly (Bell et al. 1984; Pate et al. 1985; Hunter 1998).

Consequently, seeds may be dispersed into an environment with some residual smoke

cues (Roche et al. 1998) but without the other conditions optimal for growth of these

highly plastic fire ephemerals, such as the flush of nutrients released post-fire and

reduced competition from established plants (Bell et al. 1984; Pate et al. 1985). A

requirement for a period of seed burial before seeds become responsive to smoke would

ensure that seeds persist in the soil until a subsequent fire rather than germinate in a

suboptimal environment. These findings also highlight that other species from fire-

prone regions which produce negligible or low germination in response to smoke water

when freshly collected or after laboratory storage (Pierce et al. 1995; Hunter 1998) may

not necessarily lack a smoke response once dormancy is alleviated.

The only monocarpic species examined, Actinotus leucocephalus, differed from

the remaining species, which were polycarpic fire ephemerals, in that it after-ripened

during twelve months of laboratory storage and produced high levels of germination

after burial without any further heat or smoke cues. Pate and Hopper (1993) suggest that

monocarpic fire ephemerals have shorter-lived seedbanks than polycarpic fire

ephemerals, and perhaps the characteristics displayed by Actinotus leucocephalus may

indicate greater ease of dormancy release and the ability to germinate in the absence of

fire. These features would increase the likelihood of species survival if the fire interval

exceeded seed longevity. However more monocarpic fire ephemerals need to be

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examined before general conclusions about monocarpic versus polycarpic fire

ephemerals can be drawn.

The duration and/or seasonality of burial influenced germination. Six months of

winter burial did not enhance germination in any of the species, and actually suppressed

germination of some species that germinated prior to burial, such as Actinotus

leucocephalus. However, following subsequent summer burial, germination of all but

one species was higher than after six months of burial. This may suggest the presence of

dormancy cycling in response to the seasons and dormancy cycling would confirm the

presence of non-deep physiological dormancy in many of these species (Chapter 2; J.

Baskin and Baskin 2004). Dormancy cycling is exhibited by numerous species (Auld et

al. 2000; Derkx and Karssen 1993; Benech-Arnold et al. 2000; Tieu et al. 2001c) and

may be present in other species with soil-stored seeds in south-western Australia, as

seedling emergence is higher after field sites are smoked in autumn, than in spring

(Roche et al. 1998). Similarly, seed germination from soil-stored Audouinia capitata

fruit in South Africa, which also has a Mediterranean climate, varies in response to

smoke according to season (de Lange and Boucher 1993b). Few seedlings that

germinate in spring survive the subsequent summer drought (Roche et al. 1998),

highlighting possible evolutionary selection for seeds that germinate in autumn in

environments with winter dominant rainfall.

Alternatively, observed germination may have been lower after six months of

winter burial if seeds had already germinated while buried. For example, as some

Codonocarpus cotinifolius seeds were smoke responsive prior to burial, they may have

germinated in the burnt site when smoke chemicals leached through the soil with the

onset of winter rain. Indeed, fewer filled Codonocarpus cotinifolius seeds were

retrieved from the burnt site than the unburnt site and seeds from the burnt site exhibited

a reduced response to smoke water. For Alyogyne hakeifolia and Alyogyne huegelii,

which have physical and possibly also physiological dormancy (Chapter 2), the

proportion of filled seeds declined during the first six months of burial and all remaining

filled seeds were impermeable. This suggests that the permeable fraction of the seed lots

(approximately 50%; Chapter 2) either germinated or decayed, and winter conditions

did not alleviate physical dormancy in the remaining seeds.

Seed persistence in the soil is important for species such as fire ephemerals that

require disturbance for recruitment and have plants that live for shorter periods than the

average disturbance interval (Auld et al. 2000; Holmes and Newton 2004). As in other

species in the hard-seeded Fabaceae, Geraniaceae and Malvaceae families, the

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impermeable fraction of the Alyogyne hakeifolia and Alyogyne huegelii seedlots are

likely to persist in the soil for many years (Halloin 1983; Holmes and Newton 2004).

However seed persistence in the soil is not limited to species with impermeable seed

coats (Thompson et al. 2003). Gyrostemon racemiger and Tersonia cyathiflora

exhibited no reduction in the proportion of filled seeds during 12 months of burial,

indicating that seeds of these species could persist in the soil for many years. However

Gyrostemon ramulosus and Codonocarpus cotinifolius experienced large reductions in

the proportion of filled seeds during 12 months of burial, possibly because these seeds

were older when buried.

When germination of exhumed seeds differed between the burnt and unburnt

sites it was generally higher following burial in the latter contrary to expectations. In the

process of burying seeds the ground cover was removed from both sites. However there

was greater regrowth on the burnt site, primarily of the weed Ehrharta calycina, which

may have shaded the soil and reduced temperatures. Seed moisture content was higher

in the burnt site than the unburnt site at the end of winter suggesting that moisture levels

differed between the two sites. Hence temperature and soil moisture may have differed

between the sites, which would have influenced the depth of physiological seed

dormancy (Vleeshouwers et al. 1995; Benech-Arnold et al. 2000).

Germination following burial in the field and incubation in the laboratory was

generally higher than emergence after soil-storage in the shadehouse. Emergence in the

shadehouse was similar to that obtained by Roche et al. (1997) for species that required

a period of burial before seeds responded to smoke. Lower germination in the

shadehouse may have resulted from different conditions between punnet and field soil

storage. Differences in soil temperatures and moisture contents between the shadehouse

and field would be anticipated due to differences in the amount and timing of water

application (daily sprinkler versus rain), and differences in solar radiation, atmospheric

humidity and soil type (Businger 1966; Bachelard 1985). Alternatively, seeds in the

shadehouse may have germinated but seedlings did not emerge above the soil surface

and went undetected. Regardless, field burial and germination under controlled

conditions provided a greater estimate of the proportion of seeds that were responsive to

smoke.

Germination of three of the species was not greatly enhanced by burial or post-

burial treatment. Alyogyne hakeifolia germination was not enhanced by burial and

increases in Alyogyne huegelii and Grevillea scapigera germination were less than 20%.

Although the 70ºC for 1 h heat treatment induced some germination of Alyogyne

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huegelii seeds after 12 months of burial, ungerminated seeds retained physical

dormancy. Heating seeds to 100ºC for 1 h overcomes physical dormancy in these

Alyogyne species but reduces viability (Chapter 2), suggesting that an intermediate heat

treatment may be more appropriate, such as 100ºC for 5 minutes, which promotes the

germination of a number of chaparral shrubs (Keeley 1987). Grevillea scapigera

germination was variable, with low germination resulting from heat treatments after

field burial or aerosol smoke after shadehouse storage. A higher response to aerosol

smoke was observed by Roche et al. (1997) but there was large variability between

replicates. This variability may suggest that this species requires specific soil

temperature and moisture requirements for dormancy release during burial, which might

reflect the narrow distribution of this species (Rossetto et al. 1995). Burial of Grevillea

scapigera seeds in the Corrigin region where this species occurs (drier, hotter and with

lower and less seasonal rainfall than Perth; Australian Bureau of Meteorology 1975)

might result in higher subsequent germination in response to heat and smoke water than

after burial in Perth.

In summary, the germination of a number of Australian fire ephemerals can be

enhanced by smoke water or heat treatments following twelve months of soil burial in

natural bushland and exhumation in autumn. Six months of burial and exhumation in

spring either did not enhance germination or suppressed germination levels indicating

that seeds were undergoing seasonal dormancy cycling or required a longer duration of

burial to enhance germination. Previously many of these species were considered

difficult to germinate. Improved understanding of the requirement for burial prior to

smoke exposure in many of these seeds will ensure more effective and efficient use of

seeds which has potential cost benefits for horticultural and land rehabilitation

industries, and conservation benefits for rare species. These findings highlight that

conditions of seed storage can alter seed dormancy and hence influence their response

to stimulants such as smoke water. Further investigations are required to confirm

whether temperature and soil moisture are the factors altering the dormancy states of

these seeds during burial and enabling these seeds to respond to fire-related cues.

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Chapter 4

CHAPTER 4

The changing window of conditions that promotes germination of two fire

ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora

(Gyrostemonaceae)

Abstract

Following a period of burial, more Actinotus leucocephalus (Apiaceae) and Tersonia

cyathiflora (Gyrostemonaceae) seeds germinate in smoke water. The main aim of this

study is to determine whether these fire ephemeral seeds exhibit annual dormancy

cycling during burial. This study also aims to determine the effect of dormancy

alleviation on the range of light and temperature conditions at which seeds germinate,

and possible factors driving changes in seed dormancy during burial. Seeds were

collected in summer, buried in soil in mesh bags in autumn and exhumed every 6

months for 24 months. Germination of exhumed and laboratory-stored (15ºC) seeds was

assessed at 20ºC in water or smoke water. Germination response to light or dark

conditions, incubation temperature (10, 15, 20, 25, 30ºC), nitrate and gibberellic acid

were also examined following burial or laboratory-storage for 24 months. In the

laboratory seeds were also stored at various temperatures (5, 15, 37 and 20/50ºC) for 1,

2 and 3 months followed by germination testing in water or smoke water. Both species

exhibited dormancy cycling during soil burial, producing low levels of germination in

response to smoke water when exhumed in spring and high levels of germination in

autumn. In autumn, seeds germinated in both light and dark and at a broader range of

temperatures than laboratory-stored seeds, and some Actinotus leucocephalus seeds also

germinated in water alone. Dormancy release of Actinotus leucocephalus was slow

during dry storage at 15ºC and more rapid at higher temperatures (37 and 20/50ºC);

weekly wet/dry cycles further accelerated the rate of dormancy release. Cold

stratification (5ºC) induced secondary dormancy. In contrast no Tersonia cyathiflora

seeds germinated following any of the laboratory storage treatments. Temperature and

moisture influence dormancy cycling in Actinotus leucocephalus seeds. These factors

alone did not simulate dormancy cycling of Tersonia cyathiflora seeds under the

conditions tested.

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Introduction

Fire ephemerals are species that primarily germinate after fire and normally live for 3

months to 4 years (Bell et al. 1984). It is widely inferred that their seeds persist in the

soil for many years between fires, although these species may occasionally germinate in

areas of soil disturbance enriched with nutrients (Bell et al. 1984; Pate and Dixon 1996).

A number of Australian fire ephemeral species possess non-deep physiological

dormancy and consequently germination of freshly-collected seeds can be difficult,

even in response to fire-related cues such as heat and smoke (Chapter 2). However, in

many fire-following species, germination in response to smoke is enhanced by a period

of burial (Chapter 3; Roche et al. 1997; Keeley and Fotheringham 1998a; Tieu et al.

2001c).

Different explanations have been proposed for enhanced germination in

response to smoke following burial. Increased permeability of the seed coat or

breakdown of seed covering structures may allow the entry and movement of chemicals

in smoke (eg butenolides) to sites in seeds where they stimulate germination (de Lange

and Boucher 1993b; Tieu and Egerton-Warburton 2000; Flematti et al. 2004).

Alternatively, it may be the temperature and moisture conditions in the soil that enhance

the response of seeds to smoke following a period of burial (Keeley and Fotheringham

1998a). Buried seeds with non-deep physiological dormancy characteristically cycle

seasonally in their degree of dormancy (J. Baskin and Baskin 2004). As dormancy is

alleviated, the range of conditions such as temperature and light, or responsiveness to

germination stimulants suitable for germination widen (Vegis 1964; Derkx and Karssen

1993). Likewise, as dormancy is re-imposed there is a narrowing of the window of

conditions at which seeds can germinate. Although fire ephemerals produce higher

germination in response to smoke cues after a period of burial (Chapter 3; Keeley and

Fotheringham 1998a), it has not been established whether these species cycle seasonally

in their response to smoke water.

At present temperature is considered to be the main factor that regulates

dormancy levels in seeds with physiological dormancy (Vleeshouwers et al. 1995) and

there is debate as to whether seed moisture content also plays a role. Bouwmeester and

Karssen (1992;1993) concluded that soil moisture had no effect on dormancy cycling in

Sisymbrium officinale (Brassicaceae) or Polygonum persicaria (Polygonaceae) seeds.

However, small increases in seed moisture or short periods of imbibition have been

shown to accelerate dormancy release during after-ripening of Oryza sativa and Lolium

rigidum (Poaceae; Leopold et al. 1988; Steadman et al. 2003; Gallagher et al. 2004).

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For species that exhibit dormancy loss at low temperatures, seeds must be imbibed for

dormancy alleviation to occur, as in Polygonum aviculare (Polygonaceae; Kruk and

Benech-Arnold 1998).

Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora

(Gyrostemonaceae) are both Australian fire ephemerals with soil-stored seeds (Bell et

al. 1984). Both occur within the Mediterranean climate region of south-western

Australia and under natural conditions primarily germinate in the first wet season after

fire. Actinotus leucocephalus seeds have morphophysiological dormancy whereas

Tersonia cyathiflora seeds have physiological dormancy only (Chapter 2). Germination

of both species in response to smoke water is enhanced when seeds are buried in

autumn and exhumed 12 months later (Chapter 3). We aim to determine whether

germination in response to smoke water increases with duration of seed burial or cycles

annually in these species, and whether seeds can persist in the soil for at least 24

months. In addition to the change in response to smoke, we will examine whether the

breadth of conditions suitable for germination, such as incubation temperatures and light

regimes, are influenced by a period of burial. We investigate whether enhanced

germination response to smoke water following exhumation in autumn is a result of

summer burial only or whether winter followed by summer burial is required. We also

aim to determine what environmental components of burial influence the changes in

dormancy in these species, such as temperature, seed moisture and physical weathering.

These investigations will provide a better understanding of the germination

ecophysiology of these two Australian fire ephemerals.

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Chapter 4

Materials and methods

Seed source

Freshly-matured Actinotus leucocephalus (Apiaceae) seeds (technically mericarps but

herein referred to as seeds) were collected in Dec. 2001 from plants in a Banksia

woodland site (40 km north of Gin Gin, WA, 31°01.231’S, 115°43.297’E) burnt the

previous summer. Tersonia cyathiflora (Gyrostemonaceae) seeds were collected from

plants at Beekeepers Reserve (north of Eneabba, WA, 29°32.315’S, 115°03.415’E) in

Jan. 2002 from a site burnt during a wildfire in Jan. 2000. Seeds were air-dried and

stored in sealed containers at 15°C prior to use. To determine the moisture content of

seeds (% fresh weight) during storage, four replicates of fifty seeds were weighed and

then placed at 103°C for 17 h before reweighing (International Seed Testing

Association 1999).

Experiment 1 Seed burial and retrieval over 24 months

The purpose of the first experiment was to determine whether seed germination was

influenced by a period of burial and whether buried seeds could persist in the soil

without germinating for at least 24 months. For each species, 130 seeds were placed in

nylon bags with an approximately equal volume of white sand to seeds. The nylon

material allowed the transfer of water and solutes between the contents of the bags and

the soil environment. Seeds were buried in Mar. 2002 (autumn) in an area of Banksia

woodland on Spearwood dunes (31°56.98’S, 115°47.83’E) at The University of

Western Australia Shenton Park Field Station (Perth), part of which was burnt in Jan.

2002. Randomly distributed plots were established within burnt (four plots) and

unburnt (four plots) areas with similar aspect (westerly) and slope (slight). Ten bags of

each species were buried 2 cm below the soil surface in seedling trays at each of the

eight plots. The trays enabled easy removal of bags at each exhumation date without

disturbing other buried seeds. Seeds were exhumed during daylight hours in Oct.

(spring) 2002 and 2003 and Mar. (autumn) 2003 and 2004.

After seeds were exhumed, damaged and flat seeds were counted and discarded.

Remaining seeds were assigned to one of four treatments; water, smoke water, heat

followed by incubation in water, or heat followed by incubation in smoke water. All

treatments consisted of four replicates of 25 seeds, and each of the four plots in the

burnt and unburnt sites formed the replicates. For the heat treatment, seeds were

wrapped in aluminium foil and placed in a 70ºC oven for 1 h and then transferred to

20ºC for 24 h before further treatment. To surface sterilise seeds, and hence minimise

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fungal contamination, all seeds were shaken for 30 s in 2% v/v NaOCl containing a non-

ionic surfactant (one drop per 125 mL of Plus 50, Ciba-Geigy, Sydney) and placed

under vacuum (5 min on, 5 min off, 5 min on) to remove air bubbles and ensure contact

of the solution with the seed surface. Seeds were transferred to a laminar flow cabinet,

rinsed three times with sterile distilled water, soaked for 10 min and rinsed again. Prior

to incubation in smoke water, seeds were air-dried in the laminar flow cabinet for 8-12 h

to ensure that smoke water was imbibed.

Seeds were placed in sterile plastic Petri dishes (90 mm diameter) on two pieces

of Whatman No. 1 filter paper. Three pieces of 4 cm2 sponge were placed under the

filter papers to absorb excess moisture. Petri dishes were moistened with either 10 mL

of sterile distilled water or a 1:10 dilution of smoke water (Seed Starter, Kings Park and

Botanic Garden, Perth), both containing 0.15% v/v Previcur fungicide (Aventis,

Melbourne, active ingredient 600 g L-1 propamocarb), and sealed with Parafilm. Smoke

water aliquots were frozen and only thawed when required, to minimise any possible

changes over time. After thawing, the smoke water was filtered (0.2 μM) and diluted.

Seeds were incubated at 20ºC, and exposed to 12 h of light daily from cool white

fluorescent tubes (50 μmol m-2 s-1 PAR). For all germination experiments, seeds were

monitored every 7 days for 5 weeks and removed as they germinated (radicle emerged

>2 mm). Seeds not germinating within 5 weeks were dissected and the number of filled

seeds noted. Water, smoke water, heat or heat and smoke water treatments were also

undertaken on laboratory-stored seeds (15ºC) at the time seeds were buried and to

coincide with each seed exhumation.

Breadth of conditions suitable for germination

Assignment of seeds from the burnt and unburnt sites to the different treatments was

based on seed availability. After 24 months of burial (Mar. 2004), surface sterilised

seeds exhumed from the unburnt site were incubated at a range of constant temperatures

(10, 15, 20, 25 and 30ºC) in water or smoke water. At all temperatures, seeds were

exposed to 12 h of light daily. Seeds exhumed from the unburnt site were also placed in

water or smoke water, wrapped in aluminium foil to exclude light and incubated at

20ºC. Dark incubated seeds were only monitored at the end of the five week period to

avoid exposing seeds to light during the trial.

Seeds exhumed from the burnt site in Mar. 2004 were surface sterilised and

incubated at 20ºC in 10 mL of one of the following solutions; 30 μM gibberellic acid

(GA3), 10 mM KNO3, smoke water plus 30 μM GA3, and 10 mM KNO3 plus 30 μM

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GA3. The GA3 (Sigma-Aldrich, St Louis, USA) was dissolved in 4 mL L-1 ethanol, and

all solutions contained 0.15% v/v Previcur. Additional seeds were soaked in

concentrated H2SO4 for 1 min, rinsed five times in sterile distilled water in the laminar

flow cabinet and incubated in water or smoke water. Seeds treated with H2SO4 were not

surface sterilised. All treatments were undertaken concurrently on seeds stored in the

laboratory at 15ºC.

Influence of burial on seed morphology

To determine whether burial altered the surface of seeds, seeds stored in the laboratory

or buried in the unburnt site for 24 months (exhumed Mar. 2004) were examined and

imaged using an Electroscan E3 environmental scanning electron microscope (ESEM).

The influence of surface sterilization on buried and laboratory-stored seeds was also

examined because this was a standard procedure before germination tests. The impact of

soaking buried T. cyathiflora seeds in concentrated H2SO4 for 1 min was also

investigated to tie in with pertinent impacts on germination. Images of A. leucocephalus

were captured with the Electroscan E3 ESEM operating at 30 kV, using both

backscatter and secondary detectors. To detect finer surface detail, all T. cyathiflora

images were captured at 15 kV using a secondary detector only (since secondary

electrons originate from surface features).

Experiment 2 Seed burial and retrieval over summer

Laboratory-stored seeds were buried in the unburnt site on 5 Nov. 2003 (late spring) to

determine whether enhanced germination in autumn (Experiment 1) arose from winter

followed by summer burial or summer burial only. Nylon mesh bags were prepared as

before (this time 150 Actinotus leucocephalus and 115 Tersonia cyathiflora seeds per

bag). An equal number of seeds were also placed in sealed foil packets to separate the

effects of temperature (foil packets) from combined temperature and moisture shifts

(nylon mesh bags). The moisture content of seeds at the start of the experiment was

determined using three replicates of 50 seeds of each species as outlined in Experiment

1. For each species, three nylon and two foil bags were buried in seedling trays adjacent

to each of the four unburnt plots. Dataloggers (T-Tec 501-1, Temperature Technology,

Henley Beach, South Australia) were buried (2 cm deep) in the seedling trays at two of

the four unburnt plots, and the temperature recorded at hourly intervals for 25 days from

the date of seed burial. Seeds were exhumed in Mar. 2004 and the germination of firm

and undamaged seeds was tested in water or smoke water, with and without a 70ºC for 1

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h heat treatment, as for seeds buried in Mar. 2002. In addition, non-surface-sterilised

seeds from the foil packets and nylon bags were soaked in H2SO4 for 1 min, rinsed with

distilled water five times and then germination tested in water or smoke water. All

germination results were adjusted according to the number of filled seeds determined at

the end of the trial using a cut test. However, germination results for the H2SO4

treatments were adjusted for the proportion of seeds filled according to the other

treatments, as this treatment may have damaged seeds.

A tetrazolium chloride test was used to estimate the viability of seeds of each

species prior to and following burial in nylon mesh and foil bags as it was suspected that

seeds may have died in the foil bags but appeared viable because they were filled. For

each of the four sites, three replicates of 25 intact and firm seeds were imbibed in

distilled water for 24 h at 25°C. A small portion of the T. cyathiflora seed coat and A.

leucocephalus pericarp and seed coat were then removed from each seed to facilitate

uptake of the stain. Seeds were placed in a 1% solution of 2,3,5-triphenyl tetrazolium

chloride for 24 h at 30°C in the dark (International Seed Testing Association 1999). The

embryo in each seed was then examined microscopically and those that appeared

healthy (firm and without necrotic tissue) and were stained red were scored as viable.

Experiment 3 Temperature simulation of seasons under controlled conditions

Laboratory-based investigations were undertaken to determine whether temperature was

the primary factor that drove dormancy cycling in the soil, and whether seed moisture

content also played a role. In Aug. 2003 laboratory-stored seeds were placed in paper

bags and stored at either 20/50°C (alternating regime with 12 h at each temperature) or

37°C (constant) to simulate summer conditions in the soil. Seeds were removed after 1,

2 and 3 months of storage, surface sterilised and germination tested in water or smoke

water at 20°C with 12 h of light daily. To examine the effect of cold stratification

(winter conditions) surface sterilised seeds were incubated at 5°C in Petri dishes

moistened with 10 mL water and wrapped in aluminium foil to exclude light. After 1, 2

and 3 months, seeds of each species were transferred to fresh Petri dishes, containing

either water or smoke water, and incubated at 20°C with 12 h of light daily for a further

5 weeks. In an additional treatment, seeds were cold stratified for 2 months, air-dried for

24 hours at 20°C and placed in paper envelopes at 20/50°C for 1, 2 and 3 months (and 6

months for A. leucocephalus only). Seeds were again surface sterilised and incubated in

either water or smoke water at 20°C as above.

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Chapter 4

Statistical analysis

One- or two-way ANOVAs were performed to determine whether treatments influenced

germination, whether the proportion of filled seeds remained unchanged over time

during burial in each of the burnt and unburnt sites and whether burial over summer in

nylon and foil influenced seed viability across the four unburnt sites. When germination

was absent from all four replicates, the treatment was excluded from analysis to satisfy

the ANOVA assumption of equal variances. Prior to analysis, data were arcsine square-

root transformed. Fisher’s protected LSD test was used as a post-hoc test. Treatments

were regarded as significantly different if P<0.05. Statistical analyses were performed

in Genstat version 6 (VSN International, Oxford, UK).

A linear regression was applied to the germination of laboratory-stored A.

leucocephalus seeds in response to smoke water, heat, and heat plus smoke water, to

determine whether there were any changes in germination response to each of these

treatments over time. Germination of seeds following different durations of a range of

laboratory temperature and moisture regimes were also tested against linear regressions

to examine if these regimes altered germination over time. All regression analyses were

performed in SigmaPlot version 8 (Systat Software, Richmond, CA, USA).

Results

Experiment 1 Seed burial and retrieval over 24 months

The number of A. leucocephalus and T. cyathiflora seeds that germinated

following exhumation from burial cycled annually (Fig. 1). Prior to burial, no T.

cyathiflora seeds germinated at 20ºC, even in response to smoke water (Fig. 1E-H). In

contrast, some A. leucocephalus seeds germinated when treated with smoke water

(35%) or heat (29%) or both (73%) before burial (Fig. 1B-D). After 6 months of winter

burial, germination in response to these treatments was very low (<5%) in A.

leucocephalus and low (0-15%) in T. cyathiflora (Fig. 1). Following subsequent

summer burial all A. leucocephalus seeds and approximately 60% of T. cyathiflora

seeds germinated in smoke water (Fig. 1B,F). Seeds of A. leucocephalus exhumed from

the unburnt site after summer also germinated in water (84%, Fig. 1A) but smoke water

was an absolute requirement for germination of T. cyathiflora (Fig. 1E-H). For both

species, lower germination after winter burial and higher germination after summer

burial was repeated in the subsequent year. Actinotus leucocephalus seeds exhumed in

autumn produced maximum germination (100%) in smoke water after 12 months of

burial whereas germination of T. cyathiflora seeds buried in the unburnt site was

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Chapter 4

enhanced by a longer duration of burial, with higher germination in smoke water after

24 months of burial than after 12 months (79% vs 65%; F1,68.60, P=0.026).

0 6 12 18 240 6 12 18 24

Actinotus leucocephalus

0 6 12 18 24

Ger

min

atio

n (%

)

0

20

40

60

80

100

Tersonia cyathiflora

Water Smoke water Heat Heat and smoke water

Duration of burial or laboratory storage (months)

0 6 12 18 24

Ger

min

atio

n (%

)

0

20

40

60

80

100

A B C D

E F G H

Figure 1. Germination (mean ± se) of Actinotus leucocephalus (A-D) and Tersonia cyathiflora (E-H) seeds following 6, 12, 18 and 24 months of burial in nylon mesh bags in recently burnt ( ) or unburnt soil ( ), or laboratory storage at 15ºC ( ). Germination was assessed by incubating seeds at 20ºC in water (A,C,E,G) or smoke water (B,D,F,H) for 35 days without preheating (A,B,E,F) or following pretreatment at 70ºC for 1 h (C,D,G,H).

Actinotus leucocephalus seeds stored in the laboratory at 15ºC (5.7 ± 0.3%

moisture content) exhibited an increase in germination response to smoke water (35-

75%) and a 70ºC heat treatment (29-52%) applied separately (Fig. 1B,C). Germination

in response to smoke water increased linearly over time (slope=1.97, R2=0.82,

F1,18=82.4, P<0.0001) and at a slower rate in response to heat (slope=0.40, R2=0.49,

F1,18=17.4, P=0.0006). In contrast, over the same time period, A. leucocephalus

germination in water remained below 10% (Fig. 1A). The combined heat and smoke

water treatment produced approximately 80% at the start of the trial and did not increase

further over the 24 month storage period (F1,182.2, P=0.15, Fig. 1D). Tersonia

cyathiflora seeds stored in the laboratory at 15ºC (5.0 ± 0.1% moisture content) did not

germinate at 20ºC, irrespective of germination treatment (Fig. 1E-H).

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During 24 months of burial the proportion of filled seeds declined in both

species, particularly during the first winter of burial (Fig. 2). Despite seed fill declining

over time, over 65% of A. leucocephalus (Fig. 2A) and over 75% of T. cyathiflora seeds

(Fig. 2B) remained filled after 24 months of burial.

When seeds were dissected to determine the proportion that were filled it was

observed that embryo growth in A. leucocephalus, which has underdeveloped embryos

at seed dispersal, had not visibly commenced during winter or summer burial.

B Tersonia cyathiflora

0 6 12 18 24

A Actinotus leucocephalus

Duration of burial (months)

0 6 12 18 24

Fille

d se

eds

(%)

0

20

40

60

80

100 a

b bc c bca

bc c c

ab

b b ba

bc bc

b

Figure 2. Number of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds filled (mean ± se) after burial in nylon mesh bags for 6, 12, 18 and 24 months in unburnt ( ) and recently burnt ( ) soil. Different letters indicate significant differences in the proportion of filled seeds over time. Letters above and below the symbols refer to seeds buried in the unburnt and burnt sites respectively.

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Breadth of conditions suitable for germination

For both species, seeds exhumed from burial in autumn germinated at a broader range

of temperatures than laboratory-stored seeds (Fig. 3). Germination of A. leucocephalus

was >85% following soil burial and incubation in smoke water at 15 to 25ºC (Fig. 3A).

Buried seeds incubated in water alone produced maximum germination at a similar

temperature range (15-25ºC) but germination levels were lower (50-75%) than for seeds

incubated in smoke water (87-99%; F1,1865.39, P<0.001). Laboratory-stored seeds

incubated in smoke water produced similar optimal levels of germination as buried

seeds incubated in water, however the optimum incubation temperature range was

narrower (15-20ºC). Germination of laboratory-stored seeds in water did not exceed 3%

across the range of temperatures tested. Tersonia cyathiflora seeds only germinated

following burial and incubation in smoke water (Fig. 3B). High numbers of T.

cyathiflora seeds germinated (70-80%) across a broad range of temperatures (15-30ºC)

following exhumation from the unburnt site in autumn and incubation in smoke water.

Tersonia cyathiflora

10 15 20 25 300

20

40

60

80

100

Actinotus leucocephalus

Incubation temperature (ºC)

10 15 20 25 30

Ger

min

atio

n (%

)

0

20

40

60

80

100

a

b b

c

d

b b

b

c

aa

b

b

c

a

bb

bb

A B

a

Figure 3. Germination (mean ± se) of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds at a range of incubation temperatures (10, 15, 20, 25, 30ºC) following 24 months of burial in nylon mesh bags in unburnt soil ( , ) or laboratory-storage ( , ) at 15ºC. Seeds were incubated in water (open symbols) or smoke water (closed symbols) for 35 days. Letters indicate significant differences in germination across incubation temperatures for each treatment.

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Chapter 4

Following 24 months of laboratory-storage, A. leucocephalus seeds produced

higher germination in smoke water under an alternating light/dark regime (75%) than in

constant darkness (19%; Fig. 4A). Conversely, the germination of A. leucocephalus

seeds buried in the soil for the same duration of time was not promoted by light (Fig.

4A,B). Germination of T. cyathiflora seeds was unaffected by light environment

irrespective of laboratory or soil storage (Fig. 4C,D).

Tersonia cyathiflora smoke water

laboratory unburnt soil

Ger

min

atio

n (%

)

Actinotus leucocephalussmoke water

Actinotus leucocephalus water

Ger

min

atio

n (%

)

0

20

40

60

80

100

Tersonia cyathiflora water

Storage

laboratory unburnt soil

Ger

min

atio

n (%

)

0

20

40

60

80

100

A

C

D

D

B

*

Figure 4. Germination (mean ± se) of Actinotus leucocephalus (A,B) and Tersonia cyathiflora (C,D) seeds under an alternating light/dark regime (12 h light/12 h dark, open bars) or continuous darkness (filled bars) for 35 days. Seeds were incubated in water (A,C) or smoke water (B,D) at 20ºC. Prior to testing seeds had been stored in the laboratory (15ºC) or buried in nylon mesh bags in unburnt soil for 24 months. Stars (*) indicate significant differences in germination between light regimes for each treatment.

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Following treatment with a range of chemicals, A. leucocephalus seeds exhumed

from the soil in autumn or stored in the laboratory produced maximum germination in

response to smoke water (Fig. 5A). Gibberellic acid, nitrate and sulphuric acid also

stimulated germination of laboratory-stored A. leucocephalus seeds, but of these

chemicals, only nitrate stimulated the germination of soil-stored seeds. Smoke water

was the only chemical treatment that produced higher germination of A. leucocephalus

seeds after exhumation from burial in autumn than after laboratory-storage.

Likewise, following a range of chemical treatments, germination of T.

cyathiflora seeds was only higher following soil-storage than laboratory-storage in the

presence of smoke water (Fig. 5B). Gibberellic acid induced low levels of germination

in both laboratory and soil-stored seeds but neither nitrate nor sulphuric acid alone

induced any germination. However, pre-treating seeds with sulphuric acid enhanced

germination response to smoke water in both laboratory- and soil-stored seeds.

water sw

sw & GA3 GA3

GA3 & KNO3 KNO3

H2SO4

H2SO4 & sw

Actinotus leucocephalus Tersonia cyathiflora

water sw

sw & GA3 GA3

GA3 & KNO3 KNO3

H2SO4

H2SO4 & sw

Ger

min

atio

n (%

)

0

20

40

60

80

100 BA

aab

b

c

b

c

c

b

d

ab

cd

d

cd

a

b

ab

a

b

ab

b

ab

c

ab

cb

d* *

* *

*

**

*

Figure 5. Germination (mean ± se) of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds following 24 months of burial in nylon mesh bags in burnt bushland (filled bars) or laboratory-storage (15ºC; open bars) when incubated at 20ºC for 35 days in water, smoke water (sw), sw plus 30 μM GA3, 30 μM GA3, 30 μM GA3 plus 10 mM KNO3, or 10 mM KNO3. Additional seeds were soaked in H2SO4 for 1 min prior to incubation in water or smoke water. Letters indicate differences in germination across treatments for each storage regime (laboratory or burial). Stars (*) indicate significant differences in germination between the storage regimes for each treatment.

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Influence of burial on seed morphology

A period of burial resulted in morphological changes to the outer layers of A.

leucocephalus (Fig. 6) and T. cyathiflora seeds (Fig. 7). Most notably, in A.

leucocephalus, hairs remained intact in laboratory-stored seeds (Fig. 6A,B) but were

mostly lost during burial (Fig. 6C,D). In addition to the removal of hairs, a period of

burial resulted in the lifting of a membrane over the base of the hairs (Fig. 6D). When

seeds were surface sterilised prior to germination testing laboratory-stored seeds were

unaltered but the outer layer of the pericarp became completely detached from buried

seeds (irrespective of whether they were exhumed in autumn or spring), exposing an

underlying pitted layer of the pericarp (Fig. 6E,F). Laboratory-stored T. cyathiflora

seeds had a fine surface texture (Fig. 7A,B) that was weathered by burial (Fig. 7C,D).

The sulphuric acid treatment, which was associated with an increase in germination

response to smoke water in both laboratory- and soil-stored T. cyathiflora seeds (Fig.

5B), fractured the seedcoat of both laboratory- and soil-stored seeds (Fig. 7E,F),

exposing the outer layer of the endosperm.

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Chapter 4

A B

C D

FE

50 µm

50 µm

50 µm500 µm

500 µm

500 µm

Figure 6. Environmental Scanning Electron Microscope images of the surface of Actinotus leucocephalus seeds following laboratory-storage (A,B), burial in the unburnt site for 24 months (C,D), and surface sterilization following burial in the unburnt site for 18 months (E,F). For each pair of images the whole dispersal units is shown (A,C,E) and then the mid-section of the seeds is captured at higher magnification (images B,D and F respectively).

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Chapter 4

A B

DC

FE

50 µm500 µm

500 µm

500 µm 20 µm

20 µm

Figure 7. Environmental Scanning Electron Microscope images of the surface of Tersonia cyathiflora seeds following laboratory-storage (A,B), burial in the unburnt site for 24 months (C,D), and burial in the unburnt site for 24 months and soaking in H2SO4 for 1 min (E,F). For each pair of images the whole dispersal units is shown (A,C,E) and then the mid-section of the seeds is captured at higher magnification (B,D and F respectively).

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Experiment 2 Seed burial and retrieval over summer

Germination was generally higher following burial over summer in nylon mesh bags

than in foil packets (Fig. 8). Germination of A. leucocephalus seeds exhumed in autumn

following burial in nylon mesh bags for 4 months over summer in the unburnt site (Fig.

8A) was similar to that of seeds buried for 24 months at the same site (Fig. 1A-D).

Sulphuric acid suppressed the smoke water response of A. leucocephalus seeds buried in

the burnt site for 24 months (Fig. 5A), but did not suppress the germination response to

smoke water of seeds buried in nylon mesh bags over summer only (Fig. 8A).

Actinotus leucocephalus

germ

water sw 70ºC70ºC & sw

H2SO4

H2SO4 & sw

Ger

min

atio

n (%

)

0

20

40

60

80

100A

*

*

*

*

*

Tersonia cyathiflora

water sw 70ºC70ºC & sw

H2SO4

H2SO4 & sw

B

**

Figure 8. Germination (mean ± se) of Actinotus leucocephalus (A) and Tersonia cyathiflora (B) seeds following burial in an unburnt area for 4 months over summer in nylon mesh bags (open bars) or sealed foil packets (filled bars). Seeds were incubated at 20ºC for 35 days in water or smoke water (sw), with or without a 1 h 70ºC heat or 1 min H2SO4 pretreatment. Stars (*) indicate significant differences between germination of seeds stored in nylon mesh bags versus foil packets for each treatment

The moisture content of A. leucocephalus seeds when sealed in the foil packets

was 6.3 ± 0.1 %. Following summer burial in the foil packets, 91 ± 5.8% of filled A.

leucocephalus seeds from the most shaded plot (plot 3) were viable, but viability was

<2% at the other three plots (plots 1, 2 and 4). In contrast, viability of A. leucocephalus

seeds buried over summer in nylon mesh bags exceeded 93% at all four unburnt plots.

The average soil temperatures at plots 1 and 3 during the first 25 days of burial, 2 cm

beneath the soil surface, were 28.1ºC and 22.0ºC respectively. At plot 1, temperatures

ranged from 12.5 to 64.3ºC, and exceeded 50ºC on 18 of the 25 days. At plot 3

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Chapter 4

temperature fluctuations were lower with minimum and maximum temperatures of 14.2

and 33.3ºC.

The moisture content of T. cyathiflora seeds sealed in foil packets was 5.9 ±

0.1%. After summer burial, T. cyathiflora seed viability did not differ significantly

between nylon mesh bags and foil packets (F1,3=0.00, P=0.969), and was higher in

seeds exhumed from plot 3 (85%) than from the three other plots (52 to 68%;

F3,16=16.06, P<0.001).

Germination of T. cyathiflora seeds in smoke water, as a proportion of filled

seeds, was higher after burial for 24 months in nylon mesh bags than after a single

summer (Figs 1F and 8B). For seeds buried over summer only, germination response to

smoke water was higher after burial in nylon mesh bags than in foil packets (Fig. 8B).

Following sulphuric acid treatment, the germination response to smoke water of seeds

buried in foil packets was enhanced by smoke water (F3,12=6.56, P=0.007), whereas the

germination of seeds buried in nylon mesh was unchanged (F2,9=0.39, P=0.685).

Experiment 3 Temperature simulation of seasons under controlled conditions

At the commencement of this experiment, A. leucocephalus seeds were already partially

after-ripened following storage at 15ºC for 20 months, producing 60% germination in

smoke water (Fig. 9A-D). Storage at alternating 20/50ºC temperatures for 3 months led

to no detectable change in germination in smoke water (Fig. 9A; F1,14=0.4, P=0.52).

However, after 6 months of storage at 20/50ºC, germination increased to 81 ± 6%

(F1,6=10.08, P=0.019). During 3 months of dry storage at 37ºC, Actinotus leucocephalus

germination in smoke water increased linearly to 83% (Fig. 9B; F1,14=13.6, P=0.0024).

Subjecting seeds to weekly wet/dry cycles increased germination in smoke water to

90% after 1 month of after-ripening at 37ºC (Fig. 9C).

Conversely, cold stratification (5ºC) induced dormancy, suppressing subsequent

germination of A. leucocephalus. Following 1 month of stratification, no A.

leucocephalus seeds germinated in smoke water at 20ºC and germination remained

negligible (≤1%) following 2 and 3 months of stratification (Fig. 9D). Transferring

seeds that were cold stratified for 2 months to warm dry storage 20/50ºC resulted in a

gradual increase in germination response to smoke with duration of storage (Fig. 9E).

In contrast to A. leucocephalus, germination of T. cyathiflora seeds was

negligible (<1%) following all laboratory temperature/moisture regimes tested when

subsequently imbibed in water or smoke water at 20ºC.

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Chapter 4

D

0 1 2 3

Ger

min

atio

n (%

)

0

20

40

60

80

100E

Duration of storage (months)

0 1 2 3

A

0 1 2 3

Ger

min

atio

n (%

)

0

20

40

60

80

100B

0 1 2 3

C

0 1 2 3

Figure 9. Germination (mean ± se) of Actinotus leucocephalus in water ( ) and smoke water ( ) at 20ºC following laboratory storage of seeds for 1, 2 and 3 months at air-dry storage at 20/50ºC (A), air-dry storage at 37ºC (B), a weekly regime of air-dry storage at 37ºC for 5 days, imbibition of seeds on wet absorbent sponge for 1 day at 20ºC followed by one day of air-drying at 20ºC (C), 5ºC stratification (D), or air-dry storage at 20/50ºC after 2 months of 5ºC stratification (E).

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Discussion

Both Actinotus leucocephalus and Tersonia cyathiflora seeds exhibited annual

dormancy cycling, with a widening of conditions suitable for germination occurring

after summer burial and a narrowing after winter burial. According to the dormancy

types of J. Baskin and Baskin (2004), T. cyathiflora possessed non-deep physiological

dormancy and A. leucocephalus had morphophysiological dormancy (Chapter 2).

Dormancy release in A. leucocephalus occurred following warm stratification, and

observations indicated that embryo growth only proceeded after physiological

dormancy was overcome, suggesting that A. leucocephalus seeds had non-deep simple

morphophysiological dormancy (J. Baskin and Baskin 2004). This kind of dormancy

was first described for another Apiaceae species, Chaerophyllum tainturieri (Baskin and

Baskin 1990). The germination phenology of seeds with non-deep morphophysiological

dormancy is generally similar to winter annuals with non-deep physiological dormancy

in terms of dormancy release occurring during summer and dormancy induction in

winter, with the primary difference being the presence of underdeveloped embryos in

the former and fully developed embryos in the latter (J. Baskin and Baskin 1994).

Actinotus leucocephalus and T. cyathiflora are fire ephemerals and thus

primarily germinate after fire (Bell et al. 1984). In south-western Australia most fires

occur in summer and autumn (Lindesay 2003). Seeds of both species germinated to

higher levels in response to smoke water when exhumed in autumn than in spring.

Smoke is an important component of fire and a germination stimulant in smoke, a

butenolide (Flematti et al. 2004), becomes available to seeds when smoke deposits on

the soil surface are leached through the soil profile with the onset of rain in autumn.

Germination stimulation by smoke water ensures these seedlings benefit from the post-

fire environment when there is a flush of nutrient availability and reduced competition

from other plants (Bell et al. 1984).

In addition to enhanced response to a germination stimulant, in the form of

smoke water, seeds exhumed from burial in autumn could germinate at a broader range

of temperatures than seeds stored dry in the laboratory at 15ºC. Buried A. leucocephalus

seeds incubated in either water or smoke water produced >50% germination across a

wider range of temperatures than laboratory-stored seeds of the same age. A similar

widening of temperatures appropriate for germination occurs in many other species as

dormancy is released (Vegis 1964; Bouwmeester and Karssen 1992; Schütz et al. 2002).

Furthermore, when dormancy was alleviated the requirement for specific light

conditions was overcome, as has been found in some other species (Vegis 1964; C

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Baskin and Baskin 1994). Laboratory-stored Actinotus leucocephalus seeds exhibited a

strong light-stimulated germination response while dormancy release associated with

soil burial removed any influence of light environment on germination. As seeds were

retrieved during hot dry environmental conditions and seeds must be hydrated to

respond to a red light stimulus (Bartley and Frankland 1984), exposure of seeds to light

during exhumation is unlikely to have influenced the germination of seeds subsequently

incubated in the dark. Another winter annual, Corydalis flavula, germinates in both light

and darkness at autumn temperatures and consequently lacks the potential to form large

persistent seedbanks (J Baskin and Baskin 1994). The additional requirement for a

smoke stimulant enables A. leucocephalus and T. cyathiflora to form persistent

seedbanks.

Previously it has been inferred that seeds of fire ephemerals persist in the soil

seedbank for long periods between fires (Bell et al. 1984; Pate and Dixon 1996). Here,

after 24 months of burial in Perth bushland over 65% of A. leucocephalus seeds and

over 75% of T. cyathiflora seeds persisted as filled seeds without germinating. Seed

persistence is particularly important for the continuation of species that require fire to

germinate and have a life span shorter than the average fire interval (Holmes and

Newton 2004). On the Northern Sandplain in south-western Australia, where both these

species occur, estimates of natural fire intervals vary (as opposed to human managed

fire frequency). van der Moezel et al. (1987) suggest that fires occur naturally every 8-

15 years whereas Bell et al. (1984) estimate longer intervals of 25-50 years. Both

estimates are substantially longer than the life spans (<1-4 years) of A. leucocephalus

and T. cyathiflora plants. Actinotus leucocephalus may be able to germinate in the inter-

fire period because autumn exhumed seeds germinated in response to water, however T.

cyathiflora required a smoke cue to germinate suggesting that germination may be

restricted to the post-fire environment. Interestingly the conditions in the unburnt soil

compared to the burnt soil were more conducive to dormancy release in A.

leucocephalus, allowing germination even in the absence of smoke water. This may be

an ecological adaptation to ensure some germination and hence persistence of this

species at a site in the absence of fire, especially as Pate and Hopper (1993) suggest that

the seed longevity of monocarpic fire ephemerals, such as A. leucocephalus, is shorter

than that of polycarpic fire ephemerals, such as T. cyathiflora.

Buried seeds of both these fire ephemerals varied seasonally in their response to

germination stimulants, such as smoke water. Actinotus leucocephalus seeds buried in

nylon mesh over summer only, germinated to similar levels in autumn as seeds buried

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for 24 months, suggesting that summer conditions only are required to alleviate

dormancy. Accordingly, after-ripening of dry seeds at warm temperatures (15, 37 and

20/50ºC) enabled dormancy release of A. leucocephalus seeds, as in other

Mediterranean climate species (Schütz et al. 2002; Steadman et al. 2003). Furthermore,

seeds induced into secondary dormancy by cold stratification were also released from

dormancy by warm dry after-ripening. Actinotus leucocephalus occurs in south-western

Australia which has a Mediterranean climate, with hot, dry summers and cool, wet

winters. Dormancy alleviation over summer and germination in autumn increases the

likelihood that seeds will germinate when there is sufficient moisture available for

seedling survival. Dormancy release of Actinotus leucocephalus seeds occurred more

rapidly at warm temperatures when seeds were wetted and dried than when stored dry.

Wetting and drying is usually associated with accelerating germination rates but has

also alleviated dormancy (Lush et al. 1981; Gallagher et al. 2004). The acceleration of

dormancy release in A. leucocephalus seeds by wetting and drying has practical

applications for the ex situ germination of this species. Ecologically, the transition from

the dry to wet seasons is characterised by sporadic rain events during otherwise warm

dry periods. Thus, accelerated dormancy release associated with wetting and drying

would increase the likelihood that dormancy has been alleviated by the onset of the wet

season. Few A. leucocephalus seeds germinated at high temperatures (30ºC), indicating

that germination would be unlikely in most sites following short periods of unseasonal

summer rain.

In contrast to A. leucocephalus, laboratory-storage at warm temperatures did not

simulate burial-induced dormancy release in T. cyathiflora seeds. This was unexpected

because after-ripening occurs in many other seeds with non-deep physiological

dormancy (J. Baskin and Baskin 2004). Fewer T. cyathiflora seeds germinated in smoke

water after summer burial only than after 24 months of burial, suggesting that

something other than, or in addition to, warm temperatures are involved in dormancy

release in this species. In addition to temperature, T. cyathiflora seeds may require some

form of seed coat weathering because sulphuric acid treatment enhanced germination in

response to smoke water. Scarification alone does not stimulate germination of T.

cyathiflora seeds in water in contrast to many other species with non-deep physiological

dormancy (J. Baskin and Baskin 2004). Scarification may alter germination response to

smoke water by altering embryo-covering layers other than, or in addition to, the seed

coat. Many water-permeable seeds may be impermeable to certain solutes due to the

presence of a membrane in the inner layer of the seed coat adjacent to the endosperm

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(Taylor et al. 1997). An increase in permeability of the embryo covering layers may be

required for movement of the smoke chemical(s) to site(s) that stimulate germination

(Tieu and Egerton-Warburton 2000). However J. Baskin and Baskin (2004), argue that

there is little evidence for the modification of embryo covering layers being a natural

germination requirement in water permeable seeds. Indeed, the seed coats of both

species exhibited alterations in morphology following burial but changes in A.

leucocephalus dormancy status were attributable to storage temperatures that did not

alter the seed coat. In addition, the presence of dormancy cycling, wherein dormancy is

alternately alleviated and re-imposed suggests that weathering alone, which is a non-

reversible process, is not wholly responsible for changes in germination response to

smoke water. Thus further work is required to conclusively determine whether

breakdown of the embryo covering layers plays a key role in enabling germination in T.

cyathiflora under natural conditions and whether there are any other factors involved.

These fire ephemerals have similar ecological niches, primarily germinating

after fire, having a short life span and persisting in the soil seedbank until a subsequent

fire. Both species exhibited dormancy cycling, with dormancy being alleviated by

autumn and reimposed by spring, which would ensure that seeds germinate when

moisture is most likely to be available for seedling establishment. Despite the

similarities in the ecophysiology of these species, some differences emerged. Smoke

water was the only environmental stimulant tested that enabled T. cyathiflora to

germinate when exhumed in autumn. In contrast, some A. leucocephalus seeds could

germinate in water alone, suggesting that T. cyathiflora has a greater requirement for

fire to germinate than A. leucocephalus. Temperatures alone drove dormancy cycling in

A. leucocephalus but none of the temperature regimes enabled any T. cyathiflora seeds

to germinate, even in smoke water, suggesting that other factors are required before

germination in this species can proceed.

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GENERAL DISCUSSION

One of the primary aims of this thesis was to determine how to germinate a group of

south-western Australian fire ephemerals. These species can be grouped according to

family: Actinotus leucocephalus (Apiaceae), Alyogyne hakeifolia and Alyogyne huegelii

(Malvaceae), Austrostipa compressa and Austrostipa macalpinei (Poaceae), Grevillea

scapigera (Proteaceae) and Codonocarpus cotinifolius, Gyrostemon racemiger,

Gyrostemon ramulosus and Tersonia cyathiflora (Gyrostemonaceae). Previous

germination studies on many of these species were either non-existent or indicated that

germination was difficult or variable under test conditions. Here, the main insights

gained into the germination and dormancy behaviour of these species will be discussed,

as well as their implications for conservation, horticulture, and land rehabilitation and

revegetation. This will be followed by a discussion of potential future research

directions arising from the findings of this thesis.

Main Insights

The first step in determining the germination requirements of seeds is to examine their

response to a range of incubation temperatures and light conditions, as seeds may

require no further treatment to germinate (Chapter 2). Three main responses emerged;

high levels of germination at particular temperature and light regimes (the two

Austrostipa species), a proportion of the seed lot germinating (the two Alyogyne

species), and no germination under any of the conditions tested (Actinotus

leucocephalus, Grevillea scapigera and the Gyrostemonaceae species). The relative

ease at which the two Austrostipa species germinated at 10 and 15°C was unexpected,

because these temperatures coincide with wet winter conditions in the range of these

species which would allow germination in the absence of fire. However, fire ephemerals

are thought to form persistent seedbanks, and primarily only germinate after fire. These

assumptions may still stand, because most Austrostipa seeds required light to germinate,

and under natural conditions these seeds are usually placed in darkness by the

hygroscopic awn, thus preventing germination. Furthermore, the red-to-far red (R:FR)

ratio of light that promoted most germination of Austrostipa macalpinei seeds is typical

of standard germination cabinets, whereas R:FR ratios more typical of sunlight

produced lower germination.

Many of the fire ephemerals examined have been observed growing in profuse

numbers after bushfires in native ecosystems, suggesting that one or more factors

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136

associated with fire may cue their germination. Generally, fire-related germination cues

such as heat, smoke, ethylene and nitrate did not induce high levels of germination in

fresh or laboratory-stored seeds (Chapter 2). However, for example, maximum

germination of Actinotus leucocephalus and Codonocarpus cotinifolius seeds was

induced by combining heat and smoke water treatments (Table 1). Overall, poor

germination in response to fire-related cues was not due to low seed viability. Thus, low

response to fire-related cues, as well as a range of germination temperatures and light

regimes suggested the presence of seed dormancy in some of the species. The class of

seed dormancy was primarily determined on the basis of germination response to a

range of temperatures, embryo development and permeability of seeds to water (J.

Baskin and Baskin 2004). Interestingly, all fire ephemerals examined did not possess

the same class of dormancy (Table 1). Those species with physiological dormancy were

further classified as having non-deep physiological dormancy based on the fact that

seeds with either of the other two levels of physiological dormancy by definition require

cold stratification for dormancy release which is not experienced in the environments in

which these species occur. The presence of non-deep physiological dormancy in a

number of these species was confirmed by germination in response to gibberellic acid

and scarification; characteristics of seeds with this level of physiological dormancy (J.

Baskin and Baskin 2004). Nevertheless not all species with suspected non-deep

physiological dormancy responded to both these treatments, as discussed below.

This is the first time that the dormancy type of these species had been assessed

according to the J. Baskin and Baskin (2004) classification scheme. The presence of

physiological dormancy in many of these species indicated that either warm or cold

stratification was required to alleviate dormancy (Baskin and Baskin 1998; Nikolaeva

2001). Due to the possible requirement for a period of dormancy alleviation, and

because a period of soil burial had increased germination response to smoke in some

other species (de Lange and Boucher 1993b; Roche et al. 1997; Keeley and

Fotheringham 1998a; Tieu et al. 2001c), seeds were buried in autumn and retrieved six

(spring) and twelve months later (autumn; Chapter 3). Generally, germination of species

that had responded to smoke water prior to burial was suppressed when seeds were

exhumed six months later. However after twelve months of burial, germination in seven

of the eight species tested was enhanced in either smoke water and/or following a heat

treatment (70°C for 1 h) to pre-burial levels or higher.

Chapter 5

137

Table 1. Dormancy classification and optimal germination treatment for 10 fire ephemeral species at the time of initial testing Species Family Class of

dormancy Level/Type Optimum Germination at

Time of Initial Testing* Optimum Germination Following Burial*

Actinotus leucocephalus

Apiaceae Morphophysiological Non-deep simple

70°C 1h + smoke water (80%) 1yr burial (autumn), unburnt soil 70°C 1h + smoke water (100%)

Alyogyne hakeifolia

Malvaceae Physical (+ possibly Physiological)

Non-deep Sulphuric acid (41%) (not significantly higher than control)

Not significantly enhanced by burial

Alyogyne huegelii

Malvaceae Physical (+ possibly Physiological)

Non-deep Sulphuric acid (61%) 1yr burial (autumn), unburnt soil 70°C 1h (66%)

Austrostipa compressa

Poaceae Non-dormant or Physiological

Non-deep Sulphuric acid (91%) Not tested

Austrostipa macalpinei

Poaceae Non-dormant or Physiological

Non-deep Manual scarification (99%) Not tested

Grevillea scapigera

Proteaceae Physiological Non-deep Manual scarification (14%) 1yr burial (autumn), unburnt soil 70°C 1h (19%)

Codonocarpus cotinifolius

Gyrostemonaceae Physiological Non-deep 70°C 1h + smoke water (34%) 1yr burial (autumn), unburnt soil 70°C 1h + smoke water (74%)

Gyrostemon racemiger

Gyrostemonaceae Physiological Non-deep Sulphuric acid (4%) 1yr burial (autumn), unburnt soil 70°C 1h + smoke water (63%)

Gyrostemon ramulosus

Gyrostemonaceae Physiological Non-deep Sulphuric acid (6%)** 1yr burial (autumn), unburnt soil 70°C 1h + smoke water (35%)

Tersonia cyathiflora

Gyrostemonaceae Physiological Non-deep Gibberellic acid (29%) 2 yr burial (autumn), unburnt soil 70°C 1h + smoke water (87%)

*Germination of seedlings was at 20°C over 6 weeks (initial testing) or 5 weeks (after burial) in the light **Data excluded from Chapter 2 to simplify paper

Chapter 5

Germination of all species was higher after twelve months of burial than after

six months. This suggested that either duration of burial or season of retrieval

influenced germination. Examination of two of the species, Actinotus leucocephalus and

Tersonia cyathiflora, over a second year of burial confirmed the presence of seasonal

dormancy cycling. Ecologically, a major role of dormancy cycling is to prevent seeds

germinating during unseasonal periods of favourable conditions too short to ensure

seedling establishment (Hilhorst 1998). Species examined in this thesis were either

restricted to the Mediterranean region of south-western Australia or seed was collected

from within this range of the species. Dormancy cycling, with dormancy alleviation

over summer, would ensure that germination occurred in autumn, at the start of the wet

season. This would increase the likelihood of seedling establishment during winter

when most rainfall occurs in Mediterranean regions, before the onset of summer

drought.

Although these species undergo annual dormancy cycling, most only germinate

when given additional smoke and/or heat cues, which restricts germination to the post-

fire environment. Thus, smoke water is operating as a germination stimulant, according

to the terminology of Vleeshouwers et al. (1995). In species such as Tersonia

cyathiflora, which have an absolute requirement for a germination stimulant, in this

instance smoke, it is easier to distinguish between dormancy breaking and germination

stimulation, than in species that can produce some germination without a stimulant

(Hilhorst 1997). Thus this thesis is one of the first instances where smoke water has

been demonstrated to operate as germination stimulant and described as such.

Fire ephemerals characteristically germinate in the wet season after fire, and

then mature rapidly. Seeds are likely to be dispersed into an environment with some

residual smoke cues, but without the other advantages of the post-fire environment,

such as reduced competition from other plants and the post-fire flush of nutrients. A

requirement for a period of burial before these species become responsive to fire-related

cues, such as smoke, could thus prevent seeds from germinating in a suboptimal

environment.

Much of the Australian literature on seed germination to date has focused on

breaking dormancy at a single point in time. This is appropriate for species with

physical dormancy only; however physiological dormancy is not static and can change

in response to environmental conditions. Dormancy cycling in these Australian fire

ephemerals highlights that the state of physiological dormancy can change over time.

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Thus, testing germination at one point in time may not detect the potential of a species

to respond to a germination stimulant, such as smoke.

Dormancy cycling of Actinotus leucocephalus was shown to be driven by

temperature. Under natural conditions in the soil, seeds would be exposed to wet/dry

cycles. Here wetting and drying cycles indeed accelerated the rate of dormancy release

at warm temperatures. In contrast, in Tersonia cyathiflora, which also exhibited

dormancy release during burial and has a similar distribution as Actinotus

leucocephalus, did not germinate following any of the same simulated dormancy-

release treatments. The importance of seed moisture in temperature-driven dormancy

release requires further investigation. Other factors, in addition to temperature and

moisture, appear to be required for dormancy release in Tersonia cyathiflora.

Fire ephemerals are inferred to persist for long periods in the soil seedbank, and

the species examined in this study persisted for at least a year in the soil. Two species

were examined over two years of burial, Actinotus leucocephalus and Tersonia

cyathiflora, and over 60% of these seeds persisted for this duration. The greatest decline

in filled seeds occurred during the first six months of burial, and only minimal

reductions occurred thereafter, suggesting that seeds are at least short-term persistent

and are probably long-term persistent (Thompson 1993). The two Alyogyne species also

exhibited an initial decline in seed fill, leaving only the impermeable fraction of the

seed lot. These impermeable seeds may persist, as in many other hard-seeded species

(Holmes and Newton 2004).

Dormancy did not appear to be alleviated under concurrent dry storage at 15ºC,

nor was dormancy imposed. Thus, over a year at 15ºC, none of the eight species

included in the burial trial changed in their responsiveness to smoke water or a 70ºC

heat treatment during laboratory storage. In other species with physiological dormancy,

such as Sisymbrium officinale (Brassicaceae), there are no detectable signs of dormancy

alleviation during ten years of dry storage (Hilhorst 1997). In this study the one

exception was Actinotus leucocephalus, the only monocarpic species included in this

trial, which underwent dormancy alleviation during storage. None of these species

exhibited dormancy cycling under constant conditions.

Over time, seeds within a cohort did not always produce a consistent response to

treatments such as smoke. Whilst there are genetic similarities within species,

provenance, environment and maternal effects influence phenotype, and subsequent

handling and storage influence the way seeds respond to germination treatments. In this

thesis the influence of the post-dispersal seed environment, such as temperature and

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moisture, and its duration, on the physiological state of mature seeds was highlighted.

This may partly explain the loss of a smoke requirement in Syncarpha vestita

(Asteraceae) over time (Boucher and Meets 2004), the different responses to smoke in

Grevillea wilsonii (Proteaceae) reported by Morris et al. (2000) and Dixon et al. (1995),

and the conflicting germination results reported for the in situ germination response to

aerosol smoke of Austrostipa compressa (Poaceae; Dixon et al. 1995; Smith et al.

1999).

Implications for Germinating Seeds of Fire Ephemerals for Conservation,

Horticulture and Land Rehabilitation Purposes

Fire ephemerals are an important component of the Australian flora and many are rare

or threatened. Grevillea scapigera is an endangered species and most of the other fire

ephemerals examined are closely related to species classified as rare or threatened

(Briggs and Leigh 1996). It is possible that treatments that stimulate the germination of

these species could also be effective in propagating closely related rare species,

especially those with similar life histories.

Several species have commercial potential as ornamentals or for wood products.

Understanding their seed propagation may assist in the commercial development of

these species. This would include development of Actinotus leucocephalus and

Codonocarpus cotinifolius as ornamentals, which has been previously hindered by

difficulties in germinating seeds (Kenneally et al. 1996; Offord and Tyler 1996). It

could also assist in the development of Codonocarpus cotinifolius, Gyrostemon

racemiger and Gyrostemon ramulosus as a crop species for panel board and paper

production (Olsen et al. 2003). These findings could also be trialed on horticulturally

important congeneric species that have demonstrated difficult and variable germination

such as Actinotus helianthi.

The distribution of fire ephemerals coincides with a number of minesites. These

species have traits that are desirable for species used in land rehabilitation, such as rapid

growth rates, high biomass production and soil binding characteristics (Baker et al.

2005). For mining and other land rehabilitation industries, understanding the

germination ecology of seeds is also important to ensure the most effective and efficient

use of seeds. Spreading seeds of fire ephemerals onto an area to be rehabilitated without

alleviating dormancy is unlikely to be effective because one of the aims of seeding on

rehabilitation sites is to develop a quick ground cover, and fire ephemerals may require

a year of burial before they will respond to smoke. Scarification treatments such as

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sulphuric acid effectively increased germination (Table 1). However, scarifying seeds

before they are broadcast into the soil is generally inadvisable because seeds are more

likely to dry out or be attacked by fungi in the soil without the protection of intact seed

coats (Halloin 1983; Lamont and Milberg 1997). To ensure dormancy is released prior

to seed broadcast, seeds could be placed under outdoor conditions in containers of soil

(for easy retrieval) either over summer or from one autumn to the next. Then these seeds

could be smoke-treated along with other seeds from the seeding mix prior to sowing.

Seeds of some fire ephemerals, such as Actinotus leucocephalus, simply require warm

temperatures for dormancy release. This could be during ex situ storage at elevated

temperatures or during summer following earlier broadcast. Fire ephemerals primarily

germinate in burnt sites where there is little competition from other plants, and hence it

is inadvisable to broadcast them where other species have already established. In

addition, fertiliser application may be required when fire ephemeral seeds are broadcast

on unburnt rehabilitation sites to simulate the post-fire flush of nutrients.

For long term landscape functioning, untreated fire ephemeral seeds could be

broadcast in rehabilitated areas, especially if the site is likely to be burnt within a few

years. Although the seeds are unlikely to germinate directly after seeding, they may

germinate after a future fire, which would increase the biodiversity and resilience of a

rehabilitated area.

Future Research

Despite the insights gained into the dormancy and germination attributes of these fire

ephemerals, not all of the questions about these species have been answered. For

example, in some species such as Grevillea scapigera, burial and heat treatments

enhanced germination but many viable seeds still did not germinate. The classification

of physiological dormancy, as well as physical dormancy in the Alyogyne species, was

only tentative, and further research is required to confirm this. This study has also

demonstrated that many fire ephemerals can persist in the soil for at least one to two

years, but due to time constraints of the project longer durations of burial were not

examined. Thus longer-term studies are required because it has been inferred that these

seeds persist in the soil for considerable periods but no direct tests have been

undertaken (Weston 1985; Pate and Dixon 1996). Dormancy cycling in Actinotus

leucocephalus during burial was found to be driven by temperature and modulated by

wetting/drying, whereas further work is required to ascertain what factors drive

dormancy cycling in Tersonia cyathiflora.

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In addition, the research undertaken in this thesis has raised further questions.

For example, variable germination responses to gibberellic acid and scarification in

seeds with non-deep physiological dormancy suggest that this level of dormancy may

require further subdivision. The germination and dormancy patterns obtained were

based on fire ephemerals collected from the south-western Mediterranean region of

Western Australia, which raises questions of whether fire ephemerals either in the same

species or from different taxa from non-Mediterranean regions of Australia would

exhibit similar patterns. In addition, the discovery that germination responsiveness to

smoke water may vary in accordance with degree of physiological dormancy calls into

question some of the conclusions drawn regarding the smoke-responsiveness of certain

species.

Suggestions for further research:

1. Grevillea scapigera has a very narrow distribution near Corrigin (Rossetto et

al. 1995; Briggs and Leigh 1996) which is drier and has less seasonal rainfall

than Perth (Australian Bureau of Meteorology 1975). Seeds were buried in

Perth which might not have provided the optimal soil conditions required to

alleviate dormancy. A burial trial should be established in its natural range.

Ideally trials on all species should be conducted within their local

provenance. Seeds were only exhumed twice per year and shorter intervals

may be worth examining to ensure that optimum responsiveness is detected.

2. There was some indication that Alyogyne species exhibited physiological

dormancy but further tests would be required to confirm this. Scarification

did not induce germination of all apparently viable seeds. These tests could

be repeated and additional methods such as embryo rescue should be used to

confirm viability if necessary. Scarification should be undertaken following

short intervals of storage at warm temperatures. This would indicate changes

in physiological dormancy and confirm its presence. Seeds that germinate

immediately following scarification have physical dormancy only. If viable

seeds do not germinate when physical dormancy has been alleviated, it

indicates that physiological dormancy is also present. Physiological

dormancy can be confirmed by changes in response to scarification

following periods of warm or cold storage.

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3. Longer burial trials need to be established. Fire ephemerals are inferred to

persist in the soil seedbank for very long periods however no long term

studies have been undertaken on these species. Accelerated ageing

techniques could be undertaken to indicate the possible longevity of seeds ex

situ but the only way of establishing the longevity of seeds in situ is by

burial.

4. Dormancy cycling was shown to be driven by storage temperature in

Actinotus leucocephalus but not Tersonia cyathiflora. It has been

hypothesised that primary and secondary dormancy may be different

processes (Probert et al. 1985; Hilhorst 1998). Perhaps temperatures drive

dormancy cycling in Tersonia cyathiflora once primary dormancy has been

alleviated. After 12 months of burial from autumn one year to autumn the

next, dormancy is alleviated in Tersonia cyathiflora seeds. Dormancy is then

re-imposed (secondary dormancy) by the following spring. To determine

whether secondary dormancy can be alleviated by warm temperatures alone,

seeds could be exhumed from burial in spring (buried for at least 18

months), placed at elevated temperature storage regimes in the laboratory

(37°C and 20/50°C) and germination tested in smoke water after varying

periods of time. Increased permeability of the embryo covering layers during

burial may also be required to enable Tersonia cyathiflora seeds to

germinate in response to smoke water and this should be investigated.

5. In general, germination of seeds with non-deep physiological dormancy is

promoted by both gibberellic acid and scarification (J. Baskin and Baskin

2004) and many of the species responded to these treatments. However the

Gyrostemonaceae species produced abnormal seedlings following

scarification which is characteristic of species with deep physiological

dormancy. Perhaps abnormal seedlings were produced as a result of embryo

damage during scarification. Tersonia cyathiflora (Gyrostemonaceae)

produced normal germination in response to gibberellic acid (GA3). Further

work is required to determine whether other species, such as Gyrostemon

racemiger (Gyrostemonaceae) and Grevillea scapigera (Proteaceae) could

germinate with normal seedlings with application of GA3 at other

concentrations or other gibberellic acids. Most research into the levels of

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physiological dormancy has been undertaken on northern hemisphere

species. This level of physiological dormancy is highly variable with some

species requiring cold temperatures, and others, warm temperatures, for

dormancy release (Nikolaeva 2001). Although a number of studies have

been undertaken on physiological dormancy release by warm temperatures

(Mott 1972; Baskin and Baskin 1986; Schütz et al. 2002), most research has

focused on the influence of low temperatures (Probert 2000). Further

research is required into non-deep physiological dormancy release by warm

temperatures and the patterns of seed responses to different treatments such

as gibberellic acid to determine whether additional subdivision or revision of

this level is necessary.

6. This study focused on the dormancy and germination of fire ephemerals in

the south-western region of Western Australia where dormancy cycling is

advantageous to ensure germination at the start of the wet season and not

during the hot dry season. However a number of the fire ephemerals studied

are not confined to the south-western region of Australia. For example, the

distribution of Codonocarpus cotinifolius and Gyrostemon ramulosus extend

into the more arid regions of the continent (George 1982; Paczkowska and

Chapman 2000) where rainfall is less predictable (Australian Bureau of

Meteorology 1975). Other species exhibit variable dormancy states in

different ecological habitats, which are considered to be the result of

different selection pressures (Allen and Meyer 1998; Dunbabin and Cocks

1999). Dormancy cycling may still operate in more arid regions (Baskin et

al. 1993). However where the environmental conditions suitable for seedling

establishment were less seasonally predictable, conditional dormancy and

non-dormant cycles were exhibited rather than dormant, non-dormant cycles

(Baskin et al. 1993). The responses identified in Table 1 need to be explored

in other provenances of these species and other fire ephemerals from across

Australia (Appendix).

7. The germination responsiveness of species may vary according to their state

of physiological dormancy. Failure of viable seeds to germinate at any given

time does not necessarily mean that they are incapable of responding to a

specific germination stimulant. The likelihood that seeds will germinate may

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change given pre-treatments such as soil burial or a period of warm or cold

stratification to alleviate dormancy. Tests could be undertaken on those

species with water permeable seeds that normally germinate after fire, but

have not produced the expected response to smoke including some of the

species examined by Brown et al. (1994), Pierce et al. (1995) and Hunter

(1998).

Conclusion

This thesis provides new knowledge of the germination eco-physiology of Australian

fire ephemerals. It provides dormancy alleviating and germination stimulating

treatments for several prominent south-western Western Australian fire ephemerals.

These species are from five families, encompassing monocotyledons and dicotyledons,

monocarpic and polycarpic plants, rare and common flora, and species with wide and

narrow distributions. Although these species represented a number of dormancy types, a

unifying factor was the presence of non-deep physiological dormancy. For ex situ

germination many of these species responded to either scarification and/or gibberellic

acid. During soil burial these species exhibit annual dormancy cycling with reduced

dormancy in autumn and greater levels in spring. Seeds respond to smoke when

dormancy is alleviated in autumn. Temperature and moisture are important factors

driving dormancy cycling in the soil.

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146

References

153

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Appendix

APPENDIX

Table 1. Fire ephemerals in Australia Species Family Reference Carpobrotus modestus S.T.Blake Aizoaceae Parsons 1997 Laxmannia sessiliflora Decne. Anthericaceae Roche et al. 1998 Sowerbaea multicaulis E.Pritz. Anthericaceae Rossetto 1995 Actinotus superbus O.H.Sarg. Apiaceae Elliot and Jones 1982 Trachymene anisocarpa (Turcz.) B.L.Burtt Apiaceae Pate et al. 1985 Trachymene cyanopetala (F.Muell.) Benth. Apiaceae Pate et al. 1985 Trachymene pilosa Sm. Apiaceae Pate et al. 1985 Bulbine semibarbata (R.Br.) Haw. Asphodelaceae Bell et al. 1984 Uldinia ceratocarpa (W.Fitzg.) N.T.Burb. = Trachymene ceratocarpa (W.Fitzg.) Keighery & Rye

Apiaceae Bell et al. 1984

Athrixia asteroides (Turcz.) C.A.Gardner = Asteridea asteroides (Turcz.) Kroner

Asteraceae Pate et al. 1985

Argentipallium obtusifolium (Sonder) Paul G.Wilson

Asteraceae Gill 1993

Calomeria amaranthoides Vent. Asteraceae Gill 1993 Ixodia achillaeoides R.Br. Asteraceae Specht 1981; Gill 1993 Senecio linearifolius A.Rich. Asteraceae Ashton 1981 Senecio quadridentatus Labill. Asteraceae Ashton 1981; Specht 1981; Gill

1993 Senecio vagus F.Muell. Asteraceae Ashton 1981 Senecio velleioides A.Cunn. ex DC. Asteraceae Ashton 1981 Leptorhynchos gatesii (H.B.Will.) J.H.Willis Asteraceae Gill 1993 Podotheca gnaphalioides Graham Asteraceae Pate et al. 1985 Waitzia paniculata (Steetz) Benth. = Pterochaeta paniculata Steetz

Asteraceae Pate et al. 1985

Waitzia suaveolens (Benth.) Druce Asteraceae Pate et al. 1985 Cardamine dictyosperma Hook. = Rorippa dictyosperma (Hook.) L.A.S.Johnson

Brassicaceae Ashton 1981

Monotaxis macrophylla Benth. Euphorbiaceae Hunter and Clarke 1997; Hunter 1998; Shelly and Lewer 2002

Poranthera microphylla Brongn. Euphorbiaceae Bell et al. 1984 Acacia gonocarpa F.Muell. Fabaceae Vigilante and Bowman 2004 Acacia stigmatophylla Cunn. Ex Benth. Fabaceae Vigilante and Bowman 2004 Goodenia laevis Benth. Goodeniaceae Hopkins 1985 Scaevola aemula R.Br. Goodeniaceae Weston 1985; Gill 1993 Scaevola phlebopetala F.Muell. Goodeniaceae Pate et al. 1985 Codonocarpus cotinifolius (Desf.) F.Muell. Gyrostemonaceae Bell et al. 1984; Fox 1985; Pate et

al. 1985; Bell et al. 1993; Pate and Dixon 1996; Fletcher 1997; Yates et al. 2003

Gyrostemon australasicus (Moq.) Heimerl Gyrostemonaceae Gill 1993 Gyrostemon ramulosus Desf. Gyrostemonaceae Bell et al. 1984; Hopkins 1985;

Pate et al. 1985; Bell et al. 1993; Pate and Hopper 1993; Pate and Dixon 1996

Gyrostemon reticulatus A.S.George Gyrostemonaceae Stack and English 2002 Gyrostemon subnudus (Nees) Baill. Gyrostemonaceae Pate et al. 1985; Yates et al. 2003 Tersonia brevipes Moq. = Tersonia cyathiflora (Fenzl) J.W.Green

Gyrostemonaceae Bell et al. 1984; Pate et al. 1985; Delfs et al. 1987; Bell et al. 1993; Pate and Hopper 1993; Pate and Dixon 1996

149

Appendix

Species Family Reference Anigozanthos humilis subsp. chrysanthus Hopper

Haemodoraceae Pate and Hopper 1993

Anigozanthos viridis Endl. subsp. terraspectans Hopper

Haemodoraceae Hopper 1993

Anigozanthos kalbarriensis Hopper Haemodoraceae Hopper 1993

Anigozanthos onycis A.S.George Haemodoraceae Hopper 1993

Haloragis odontocarpa F.Muell. Haloragaceae Gill 1993 Isotoma hypocrateriformis (R.Br.) Druce Lobeliaceae Pate et al. 1985 Alyogyne hakeifolia (Giord.) Alef. Malvaceae Brown and Hopkins 1984;

Hopkins 1985; Pate et al. 1985; Weston 1985; Bell et al. 1993; Pate and Hopper 1993; Pate and Dixon 1996; Yates et al. 2003

Alyogyne huegelii (Endl.) Fryxell Malvaceae Pate et al. 1985; Weston 1985; Bell et al. 1993; Pate and Hopper 1993; Pate and Dixon 1996

Commersonia rosea S.A.J. Bell & L.M. Copel. Malvaceae Bell and Copeland 2004 Villarsia parnassifolia (Labill.) R.Br. Menyanthaceae Weston 1985 Macarthuria apetala Harv. Molluginaceae Pate et al. 1985 Danthonia induta Vickery Poaceae Wark et al. 1987; Gill 1993 Dryopoa dives (F.Muell.) Vickery Poaceae Ashton 1981 Stipa compressa R.Br. = Austrostipa compressa (R.Br.) S.W.L.Jacobs & J.Everett.

Poaceae Roche et al. 1998

Stipa elegantissima Labill. = Austrostipa elegantissima (Labill.) S.W.L.Jacobs & J.Everett

Poaceae Pate et al. 1985

Stipa macalpinei Reader = Austrostipa macalpinei (Reader) S.W.L.Jacobs & J.Everett

Poaceae Specht et al. 1958; Specht 1981;

Gill 1993 Muehlenbeckia costata K.L.Wilson & R.Makinson

Polygonaceae Hunter et al. 1998

Calandrinia corrigioloides Benth. Portulacaceae Pate et al. 1985 Grevillea refracta R.Br. Proteaceae Vigilante and Bowman 2004 Distichostemon hispidulus (Endl.) Baillon Sapindaceae Vigilante and Bowman 2004 Anthocercis littorea Labill. Solanaceae Pate et al. 1985 Solanum aviculare G.Forst. Solanaceae Ashton 1981 Solanum simile F.Muell. Solanaceae Weston 1985 Rulingia procumbens Maiden & Betche Sterculiaceae Shelly and Lewer 2002; Bell and

Copeland 2004 Pimelea brevifolia R.Br. Thymelaeaceae Hopkins 1985 Pimelea nervosa Meisn. = Pimelea angustifolia R.Br.

Thymelaeaceae Hopkins 1985

Triumfetta bradshawii F.Muell. Tiliaceae Vigilante and Bowman 2004

150