Comparative Ecophysiological Analyses of Melaleuca Irbyana and Melaleuca Bracteata – a Narrowly Versus Widely Distributed Congeneric Species

COMPARATIVE ECOPHYSIOLOGICAL ANALYSES OF MELALEUCA IRBYANA AND MELALEUCA BRACTEATA – A NARROWLY VERSUS WIDELY DISTRIBUTED CONGENERIC SPECIES

Thita Soonthornvipat

Submitted in fulfilment of the requirements of the degree of

Doctor of Philosophy

School of Earth, Environmental and Biological Sciences

Science and Engineering Faculty

Queensland University of Technology

Brisbane, Australia

2018

Keywords

Adaptive ability, basal area, biodiversity, canopy, competitive ability, critically endangered species, critically threatened species, crucial abiotic factors, diameter at breast height, dominant, ecological community, ecosystems, edaphic factors, ecological strategies, ecophysiology, extinction, fitness traits, germination characteristics, geographical range, growth performance, habitat fragmentation, habitat’ s specificity, interspecific variation, intraspecific variation, Kaplan-Meier estimate, leaf area index, life history traits, Melaleuca bracteata, Melaleuca irbyana, monoculture, non-dominant, nutrient acquisition, phenotypic plasticity, photosynthetic active radiation, photosynthetic rate, physiological performance, physiology, plant herbivores, Principal Component Analysis, reciprocal seedlings, regeneration programs, regional management plans, relative growth rate, reproductive capability, resin bag, resource acquisition, resource availability, resource use efficiency, restoration, restricted distribution, revegetation programs, seed ecology, seed germination, seedling survival, shoot elongation, soil conditions, stem density, survival traits, tree density, widely distributed, widespread distribution.

Abstract

Melaleuca irbyana R. T. Baker, is a small to medium size tree that forms a unique habitat, in that the forests in which it is found are dense monocultures located mainly in the the South-East Queensland region. Melaleuca irbyana forests were originally restricted in their distribution, but are now listed federally as critically endangered under the Environment Protection and Biodiversity Conservation ( EPBC) Act 1999. Despite considerable conservation efforts to protect remaining M. irbyana forests, it remains under threat of extinction due to increased land clearing for urban expansion, coal seam gas exploration, and the indirect effects of urbanisation, e. g. , eutrophication. The genus Melaleuca is made-up of 290 species. Commonly distributed species include Melaleuca quinquenervia with a distribution ranging from Cape York in Queensland to Botany Bay in New South Wales. Under the EPBC Act 1999, 11 species of Melaleuca are considered endangered within Australia, including M. irbyana. To conserve the remaining M. irbyana populations, there is an urgent need to better understand how this species grows, including reproductive and life history traits, nutrient cycling, and other habitat requirements. In my doctoral research, I compared how M. irbyana and the common and often co-occurring M. bracteata grow across life history stages, from seed germination, seedling survival, and growth, to changes in height, diameter at breast height (DBH), and shoot elongation in adult populations.

To understand the reproductive and life history traits of both of these Melaleuca species, I measured seed germination success rates under different controlled environmental conditions in growth cabinets, as well as germination success rates in the field, across four growing seasons. I hypothesised that lower reproductive capabilities may contribute to M. irbyana’ s original narrow distribution and therefore rare status. Melaleuca irbyana displayed germination characteristics that were restricted to a narrower range of temperatures than those of M. bracteata. Melaleuca irbyana showed a lower germination success rate in response to cooler temperatures and lower light conditions when compared to M. bracteata, which displayed high germination rate at all temperatures (15, 25 and 30°C) and photoperiod regimes (0, 10, 12 and 15 hours). Melaleuca irbyana’s germination appears to occur under a narrower range of temperatures than M. bracteata, but overall at higher

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temperatures (e. g. , 30°C), M. irbyana had a high germination success rate than originally expected. I measured seedling survival and growth rates in a reciprocal seedling experiment grown under the canopies of four mature M. irbyana or M. bracteata forests. Overall, I found that M. irbyana showed a lower seedling survival and growth rate than M. bracteata, whether growing under a canopy of M. irbyana or M. bracteata. However, M. irbyana had better survival and growth rates when seedlings were grown under M. bracteata canopies. While, M. bracteata seedlings survived better and had more enhanced growth rates than M. irbyana under both canopies, their survival and growth rates were lower under M. irbyana canopies. Reduction in seedling survival and growth rates under M. irbyana mature trees suggests that these endangered habitats may inhibit recruitment, possibly due to dense canopies, litter, or specific soil conditions (such as high alluvial clay levels in the soil). Adult tree densities in M. irbyana forests may have negative effects on seedlings’ survival and growth because of reduced light availability. Low survival rates and slower growth rates of seedlings in natural environments of M. irbyana may contribute to explaining its original restricted distribution. Ecophysiological traits such as aspects of rate of shoot growth, plant height and tree DBH of mature trees were measured to produce useful information on the performance of adult M. irbyana versus M. bracteata, as possible indicators of the competitive ability of these species. Melaleuca irbyana presented high growth performance in the adult stage, refuting our hypothesis that rare species would present lower performances at every stage of their life cycle. Measurements, although recorded cover three growing seasons, were taken during drought periods, which provides some evidence that M. irbyana may access groundwater, which may explain its higher growth rates during this period than M. bracteata, which is not thought to be groundwater dependent. I compared the soil properties and nutrients levels in remnant mature forests of M. irbyana and M. bracteata in terms of different habitat conditions. Soil nutrient analyses showed similar levels of nutrients in soils collected from both M. irbyana and M. bracteata forests. I found, however, that M. irbyana sites showed higher inorganic nitrogen levels, and that M. bracteata forests had higher levels of sand in the soil, which suggests that M. irbyana forests are more nutrient-rich that M. bracteata. I also found higher levels of some metals such as aluminium in M. irbyana soils, which can be toxic to plants at high concentrations.

The next steps with these analyses would be to focus on understanding how the two species acquire nutrients from the soil. Understanding resource acquisition and use in these tree species would involve comparing nutrient levels in plant tissue (e.g., leaves, young shoots, roots, and old wood) in M. irbyana and M. bracteata possibly under controlled conditions in the glasshouse. My research findings will assist with the management of remaining populations of M. irbyana by providing much needed information on basic survival and growth characteristics to assist with future revegetation projects. My research results can also contribute more broadly to understanding the traits and environmental conditions that limit some plant species’ range, while enabling the widespread distribution of other species. In this research, I found that M. irbyana had more specialised germination requirements than M. bracteata under controlled conditions in a growth cabinet, but under field conditions, both species showed low germination successalthough M. irbyana in every test had a lower germination success rate. These findings suggest that when growing seedlings for restoration projects, M. irbyana will germinate successfully under warmer conditions where light is available, but direct seeding projects are not likely to be a viable option for restoration of either species, and that natural recruitment under canopies of either species may be limited. I also found that seedling survival and growth rates of M. irbyana are higher in areas with higher light availability, which indicates that the most suitable site for restoration of this species is not under dense canopies, but in open areas or areas with sparse tree cover. A key result of comparing the growth and habitat-specific conditions of remnant populations of M. irbyana and M. bracteata is that mature populations of M. irbyana had a higher growth rate in terms of height and diameter at breast height (DBH) than M. bracteata. This finding suggests that once M. irbyana reaches the adult stage of its life cycle it possibly has fewer limitations on its growth, explaining why and how it can form dense monocultural plantations. I also found some differences in soil characteristics between M. irbyana and M. bracteata sites, which may explain recruitment of M. irbyana at some localities and not others. These differences in soil may also be explained by M. irbyana adult monocultures altering the environment to suit their own successful growth and development, which may hinder the recruitment of other species in its canopy. Collectively, my findings show that it is probable that M. irbyana originally had a limited distribution because of specialised requirements in the early stages of its life cycle, but adult populations of M.

irbyana appear highly successful in their growth characteristics even during drought periods. This leads to important recommendations for conserving existing adult populations and also points to key habitat characteristics needed for revegetation programs to expand distribution. A key recommendation that stems from my research is that studies aimed at comparing congeneric species that are common versus restricted distribution should focus on comparing species across all stages of their life cycle from germination to adult populations, in order to capture important differences and shifts in potential competitive advantages.

Table of Contents Abstract ...... iii List of Figures ...... x List of Tables ...... xvii List of Abbreviations ...... xxi Statement of Original Authorship ...... xxii Acknowledgements ...... xxiii Chapter 1 ...... 1 General Introduction ...... 1 1.1 Introduction ...... 2 1.2 Thesis structure ...... 5 Chapter 2 ...... 7 Literature Review ...... 7 2.1 Background ...... 8 2.2 Definitions of rarity ...... 9 2.3 Causes of rarity ...... 12 2.3.1 Endogenous or intrinsic causes of rarity ...... 13 2.3.2 Exogenous or extrinsic causes of rarity...... 23 2.3.2.1 Land alteration...... 23 2.4 Approaches to conservation and management of rare and endangered plants ...... 25 2.5 Description, distribution and habitats of Melaleuca species ...... 26 2.5.1 Taxonomy...... 26 2.5.2 Morphology ...... 27 2.5.3 Reproductive traits ...... 28 2.6 Characteristics of the study species: Melaleuca irbyana and Melaleuca bracteata .. 30 2.7 Conclusion ...... 33 Chapter 3 ...... 34 Comparing in vitro and in situ germination attributes between a rare and a common Melaleuca species ...... 34 3.1. Introduction ...... 35 3.2 Materials and methods ...... 39 3.2.1 Seed germination ...... 39

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3.2.2 Soil sampling and analyses ...... 44 3.3 Results ...... 47 3.3.1. In vitro germination experiment ...... 47 3.3.2 In situ germination trial ...... 53 3.4 Discussion ...... 57 3.4.1 In vitro germination experiment ...... 57 3.4.2 In situ germination trial ...... 58 3.5 Conclusion ...... 61 Chapter 4 ...... 62 Understanding differences in seedling microhabitat conditions using a reciprocal planting experiment ...... 62 4. 1. Introduction ...... 63 4.2 Materials and Methods ...... 67 4.2.1 Field sites...... 67 4.2.2 Reciprocal seedlings ...... 71 4.3 Results ...... 73 4.3.1 Light availability and seedling survival ...... 73 4.4 Discussion ...... 82 4.4.1 Light availability and seedling survival ...... 82 4.5 Conclusion ...... 85 Chapter 5 ...... 87 Effect of exogenous traits on physiological characteristics of remnant mature trees . 87 5.1 Introduction ...... 88 5.2. Materials and methods ...... 90 5.2.1 Field sites...... 90 5.2.2 Growth measurements ...... 94 5.2.3. Soil sampling ...... 95 5.2.4 Soil characterisation preparation ...... 96 5.2.5 Soil nutrient analyses ...... 97 5.3 Results ...... 103 5.3.1. Remnant mature tree performances...... 103 5.3.2 Habitats’ nutrient availability ...... 110

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5.3.2 Nitrogen mineralisation and nitrification ...... 122 5.4 Discussion ...... 129 5.4.1 Plant growth performance ...... 129 5.4.2 Habitat quality, nutrient availability and soil pH ...... 130 5.5 Conclusions ...... 131 Chapter 6: General discussion and recommendations ...... 133 6.1 Purpose of the study ...... 133 6.2 Key outcomes and limitations of the experimental chapters ...... 133 6.3 Recommendations for conservation and restoration of Melaleuca irbyana ...... 135 6.4 Future research directions ...... 136 Bibliography ...... 138 Appendix 1 ...... 166 Appendix 2 Conference Abstracts ...... 172

List of Figures

Chapter Two

Figure 2. 1 The life cycle of a hypothetical plant population model (After Schemske et al., 1994, p. 590)...... 17 Figure 2.2 A conceptual framework showing the relationship and impact of endogenous and exogenous processes in landscape modification and their influence on species traits and behaviour (adapted from Fischer and Lindenmayer, 2007, p. 268)...... 24 Figure 2.3 Pictures of Melaleuca irbyana: (A) dense thickets of mature trees; (B) leaves and flowers; (C) fruits/capsules containing seeds. (Photograph taken by Thita Soonthornvipat, 2014)...... 31 Figure 2. 4 Pictures of Melaleuca bracteata: (A) mature trees; (B) leaves and flowers ( Photograph taken by Thita Soonthornvipat, 2016) ; ( C) fruits/ capsules containing seeds (Tanetahi, 2010)...... 31 Figure 2. 5 Distribution map based on Australian herbarium records for (A) Melaleuca irbyana (red dots), (B) Melaleuca bracteata (red dots). Maps were generated using the online tool the Atlas of Living Australia (map created on 27/7/2016)...... 33 Chapter Three

Figure 3. 1 Monthly mean maximum and minimum temperature at Henderson Reserve site during 2013-2014. Values are from Australian Bureau of Meteorology, 2014...... 40 Figure 3. 2 Comparison of seed size between Melaleuca irbyana and Melaleuca bracteata...... 41 Figure 3. 3 In situ seed germination experimental set- up at Henderson Reserve site, Jimboomba (A and B). Each experiment was divided into 16 sub-plots of 1 m × 1 m (C and D). Seeds mixed with sand were sown in summer (C) and winter (D)...... 42 Figure 3. 4 Kaplan-Meier estimates of germination functions for the first germination seeds in growth chamber between Melaleuca irbyana and Melaleuca bracteata

under 15°C and different photoperiod conditions (A-D). Orange lines indicate the first instance of germination in each of the conditions...... 50 Figure 3. 5 Kaplan-Meier estimates of germination functions for the first germination seeds in growth chamber between Melaleuca irbyana and Melaleuca bracteata under 25°C and different photoperiod conditions (E-H). Orange lines indicate the first instance of germination in each of the conditions...... 51 Figure 3. 6 Kaplan-Meier estimates of germination functions for the first germination seeds in growth chamber between Melaleuca irbyana and Melaleuca bracteata under 30°C and different photoperiod conditions (I-L). Orange lines indicate the first instance of germination in each of the conditions...... 52 Figure 3.7 Comparison estimate of mean for soil between Melaleuca irbyana (MI) and Melaleuca bracteata (MB) microhabitat (A) Moisture content (%); and (B) Organic matter (%)...... 53 Figure 3.8 Seed germination at Henderson Reserve site, Jimboomba. (A) Seedlings of Melaleuca bracteata (yellow circles) under M. bracteata overstorey with the litter treatment (B) Seedlings of Melaleuca irbyana (red circles) under M. irbyana overstorey without the litter treatment; (C) Seedlings of M. irbyana (red circles) under M. bracteata overstorey without the litter treatment; (D) Seedlings of M. irbyana (red circles) under M. irbyana overstorey with the litter treatment...... 55 Figure 3. 9 Kaplan- Meier estimates of seed germination functions for the first germination seeds in field conditions ( Henderson Reserve site) between Melaleuca irbyana ( MI) and Melaleuca bracteata ( MB) under different understoreys of remnant mature trees. Orange lines indicate the first instance of germination in each of the conditions...... 56 Chapter Four

Figure 4. 1 Aerial view of Melaleuca irbyana and Melaleuca bracteata field sites (red circle); (A) Henderson Reserve, Jimboomba (B) Victoria Park (C) Bottlebrush Park (D) Moffatt Park (E) Waterford West Park (Google Earth, 10/1/2017). 68 Figure 4. 2 Monthly mean maximum temperatures at Jimboomba, Logan City in five research sites. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia)...... 69

Figure 4. 3 Monthly mean maximum temperatures at Logan City, Jimboomba in five research sites. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia)...... 69 Figure 4.4 Mean monthly rainfall (mm) at Jimboomba, Logan City in five research sites. Values are presented in three years of study during 2014- 2016 ( Bureau of Meteorology of Australia)...... 70 Figure 4.5 Mean monthly photoperiod (hours) in Brisbane. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia)...... 70 Figure 4. 6 Pictures of a Melaleuca irbyana seedling (A) and a Melaleuca bracteata seedling (B) recently planted for reciprocal experiment...... 71 Figure 4. 7 Higher-order fixed effects from LMEM terms where the response variables are light availability of photosynthetically active radiations (PAR), and how it varies with year of observation (2014-2016) in each habitat of remnant mature trees between Melaleuca irbyana (MI) and Melaleuca bracteata (MB). Error bars represent ± standard error...... 74 Figure 4. 8 Higher-order fixed effects from LMEM terms where the response variables are light availability of leaf area index (LAI) and how it varies with year of observation ( 2014- 2016) in each habitat of remnant mature trees between Melaleuca irbyana (MI) and Melaleuca bracteata (MB). Error bars represent ± standard error...... 76 Figure 4. 9 Kelplan-Meier estimate of the survival rate of seedlings between Melaleuca irbyana ( MI) and Melaleuca bracteata ( MB) overstorey of both remnant mature trees (12 months, 2015-2016) of experiment in five sites. Orange line indicates the first time of seedling death occurring in each of the conditions tested...... 78 Figure 4. 10 Comparison of total seedling survival after 12 months of experiment when grown under canopies of Melaleuca irbyana (MI) and Melaleuca bracteata (MB) trees...... 79 Figure 4. 11 Comparison of seedling height when grown under canopies of Melaleuca irbyana ( MI) and Melaleuca bracteata ( MB) trees after 12 months of experiment...... 81

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Chapter Five

Figure 5. 1 Aerial view of Melaleuca irbyana and Melaleuca bracteata field sites (red circles); (A) Henderson Reserve, Jimboomba (B) Victoria Park (C) Bottlebrush Park (D) Moffatt Park (E) Waterford West Park (Google Earth, 10/1/2017). 91 Figure 5. 2 Monthly mean minimum temperatures at Jimboomba, Logan City in five research sites over the study period of 2014 to 2016 (Bureau of Meteorology of Australia)...... 92 Figure 5. 3 Monthly mean maximum temperatures at Logan City, Jimboomba in five research sites over the study period of 2014 to 2016 (Bureau of Meteorology of Australia)...... 92 Figure 5.4 Mean monthly rainfall (mm) at Jimboomba, Logan City in five research sites over the study period of 2014 to 2016 (Bureau of Meteorology of Australia)...... 93 Figure 5.5 Mean monthly photoperiod (hours) in Brisbane over the study period of 2014 to 2016 (Bureau of Meteorology of Australia)...... 93 Figure 5. 6 Resin bag preparation; (A) Resin bags at soil surface (0 cm depth) were covered with litter, (B) Resin bags at 10 cm depth were covered with soil, (C) Resin bags at 20 cm depth were covered with soil (D) Resin bags were tagged with labels tied to pegs to facilitate identification and retrieval of bag...... 100 Figure 5. 7 Soil, litter and resin bag preparations and equipment for available nutrients analyses: (A) Soil samples, (B) Mixing of soil suspension (1 part soil and 5 parts water) on rotary suspension mixer for soil pH measurement, (C) Mixing resin bag suspension with 1 M KCl solution on rotary suspension mixer, (D) LECO® machine for total carbon and total nitrogen measurements (E) Litter samples, (F) Example of resin bags before commencement of experiment, (G) Thermo ScientificTM ( GALLERYTM Automated Photometric Analyzer, - + Ammonia High method) for NO3 and NH4 , ( H) X- ray fluorescence spectrometer, (I) Claisse® The Ox® automated fusion machine and (J) XRF fused glass discs...... 101 Figure 5. 8 Higher-order fixed effects from LMEM terms where the response variables are height and how it varies with year of observation (2014-2016) in each

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habitat of remnant mature trees between Melaleuca irbyana ( MI) and Melaleuca bracteata (MB). Error bars represent ± standard error...... 104 Figure 5. 9 Higher-order fixed effects from LMEM terms where the response variables are DBH and how DBH varied depending on the year of measurement (2014- 2016) in Melaleuca irbyana ( MI) and Melaleuca bracteata ( MB) adult populations. Error bars represent ± standard error...... 105 Figure 5.10 Higher-order fixed effects from LMEM terms where the response variables are basal area (BA) and how BA varied depending on the year of measurement (2014-2016) in Melaleuca irbyana (MI) and Melaleuca bracteata (MB) adult populations. Error bars represent ± standard error...... 106 Figure 5. 11 Higher-order fixed effects from LMEM terms where the response variable is stem density per hectare and how stem density varied depending on the year of measurement ( 2014- 2016) in Melaleuca irbyana ( MI) and Melaleuca bracteata (MB) adult populations. Error bars represent ± standard error. ... 107 Figure 5. 12 Higher-order fixed effects from LMEM terms where the response variable is shoot elongation and how shoot elongation varied depending on the year of measurement ( 2014- 2016) in Melaleuca irbyana ( MI) and Melaleuca bracteata (MB). Error bars represent ± standard error...... 108 Figure 5.13 Comparison of nutrients available in soil (14) and litter (3) in remnant mature habitats between Melaleuca irbyana (MI) and Melaleuca bracteata (MB). The asterisk (*) indicates that there was a significant difference at P <0.05 and “. ” at P < 0.1, whereas no signs were shown as not significant...... 112 Figure 5. 14 Comparison of nutrient elements found in Melaleuca irbyana (MI) and Melaleuca bracteata (MB) habitats, which are significantly (or marginally) different. (A) Aluminium; Al (%) (B) Potassium; K (%) (C) Magnesium; Mg (%) (D) Sodium; Na (%) (E) Manganese; Mn (%) and (F) Silicon; (Si) (%)...... 113 Figure 5.15 Comparison of % moisture content (A) and % organic matter (B) in soils between Melaleuca irbyana (MI) and Melaleuca bracteata (MB) habitats in 28 plots of five experiment sites...... 114 Figure 5. 16 Histogram of the Principal Components Analysis illustrating the principal components variances of the nutrient characteristics measured from 28 plots

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between Melaleuca irbyana habitats and Melaleuca bracteata habitats in five sites...... 118 Figure 5.17 Graphical ordination of the five sites of the results of Principal Components Analysis (PCA) for soil nutrient availability across all 28 plots. Both nutrient availability scores and quadrant scores of the first two ordination axes are plotted. Each colour dot (M. bracteata) and triangle (M. irbyana) represents nutrient availability in the soil conditions for each species The x-axis represents the scores for the first principal component, the y-axis the scores for the second principal components; in the axis label, the percentage of variance explanation is given in parentheses. Component variables are represented by arrows that indicate the proportion of the original variance explained by the first two principal components. Directions of the arrows indicate the relative loadings on the first and second principal components. The orientation of arrows indicates the direction in ordination space where the soil variables change most rapidly, and in which they have maximum correlation with the ordination configuration, whereas the length of the arrows indicates the rate of change...... 119 Figure 5.18 The contribution histogram of variable PC 1 (29.1%) illustrates the variance of all the principal components which run along the X axis of previously shown PCA Figure 5. 17. They are primarily driven by Si, Fe, P and Mn elements in the soil measured from 28 plots in five sites...... 120 Figure 5.19 The contribution of variable PC 2 (18.1 %) illustrates the variance of all the principal components which run along the Y axis of previously shown PCA Figure 5.17. They are primarily driven by K, Na, and soil total carbon elements in the soil measured from 28 plots from five sites...... 121 + Figure 5. 20 Soil nitrogen measured as NH 4 including differences in depth of buried resin bags (0, 10, and 20 cm) between M. irbyana (MI) and M. bracteata (MB) remnant mature trees...... 125 - Figure 5.21 Soil nitrogen measured as NO3 including differences in depth of buried resin bags (0, 10, and 20 cm) between M. irbyana (MI) and M. bracteata (MB) remnant mature trees...... 125

Figure 5. 22 Graphical ordination of five sites, 28 plots of the result of Principal Component Analysis (PCA) based on inorganic nitrogen is generated in the soil by in situ ion exchange using resin bags between M. bracteata and M. irbyana remnant mature trees; 95% confidence ellipses are shown for each species. Both inorganic nitrogen uptake scores and quadrant scores of the first two ordination axes are plotted. Each coloured dot (M. bracteata) and triangle + - (M. irbyana) represents nitrogen rate (NH4 and NO3 respectively) for each species. The scores in the axis label the percentage of variance, and explanation is given in parentheses. The original variables are represented by arrows that indicate the proportion of the original variance. The direction of the arrows indicates the relative loadings on the first and second principal components. The orientation of arrows indicates the direction in ordination space in which the inorganic nitrogen availability changes most rapidly and in which they have maximum correlation with the ordination configuration, whereas the length of the arrows indicates the rate of change...... 128

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List of Tables Chapter Two

Table 2. 1 Definitions of rarity as defined by Rabinowitz, 1981 ...... 10 Table 2.2 An updated scheme of pattern of rarity adding the notion of habitat occupancy (adapted from Rey Benayas et al., 1999, p.2)...... 11 Table 2. 3 Pattern of rarity for vascular plants as defined by Fiedler and Ahouse (1992) adapted from (Leverington, 2011, p.12)...... 12 Table 2. 4 Comparison in morphology and distribution between Melaleuca irbyana (narrowly distributed species) and Melaleuca bracteata (widespread species)...... 32 Chapter Three

Table 3.1 Monthly Mean Daily photoperiod (hours) in Brisbane ...... 40 Table 3.2 Monthly mean minimum temperatures (degree Celsius) in Brisbane ...... 43 Table 3.3 Monthly mean maximum temperatures (degree Celsius) in Brisbane ...... 43 Table 3.4 Monthly Rainfall (millimetres) in Brisbane ...... 43 Table 3.5 Monthly Mean Daily photoperiod (hours) in Brisbane ...... 43 Table 3. 6 Result of ANOVA comparing total germination success between Melaleuca irbyana and Melaleuca bracteata, temperatures ( 15, 25, 30°C) and photoperiods (10, 12, 15 hours)...... 47 Table 3.7 Results of Tukey’s HSD test performed on an ANOVA of germination data for Melaleuca irbyana and Melaleuca bracteata...... 48 Table 3. 8 Effect of temperature and light regimes on the time ( day) to germination initiation in Melaleuca irbyana and Melaleuca bracteata...... 49 Table 3.9 The ANOVA result of abiotic factors (climatic conditions related to season) on total germination between Melaleuca irbyana and Melaleuca bracteata seeds under canopies of Melaleuca irbyana and Melaleuca bracteata remnant mature trees with an error structure of plots nested within sites...... 54 Chapter Four

Table 4.1 Experimental design for reciprocal seedlings...... 72 Table 4. 2 Result from a Wald F-test of a linear mixed effect model with the response variable of photosynthetic active radiation ( PAR) conducted to assess the

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significance of the fixed effects of habitats (Melaleuca irbyana and Melaleuca bracteata canopies) and year...... 74 Table 4. 3 Result from a Wald F-test of a linear mixed effect model with the response variable of leaf area index (LAI) conducted to assess the significance of the fixed effects of habitats ( Melaleuca irbyana and Melaleuca bracteata canopies) and year...... 75 Table 4. 4 The ANOVA result of the effect of Melaleuca irbyana and Melaleuca bracteata canopies to seedling survival rate between M. irbyana and M. bracteata seedlings and month with an error structure...... 77 Table 4. 5 The ANOVA result of light availability under Melaleuca irbyana and Melaleuca bracteata canopies to vegetative growth rate ( height) between Melaleuca irbyana and Melaleuca bracteata seedling at the end of experiment (12 months) with an error structure...... 80 Table 4.6 Result of Tukey multiple comparison of means test performed on an ANOVA of seedling growth rate (height) under canopies of both mature remnant trees. The symbols within brackets define habitat overstorey of each remnant mature tree symbols without brackets define seedlings species...... 80 Chapter Five

Table 5. 1 Results from Wald F-tests of a linear mixed effect model with the response variables of height, DBH, BA, number of stems per hectare, and shoot elongation, conducted to assess the significance of the fixed effects of nutrient availability between Melaleuca irbyana and Melaleuca bracteata remnant mature trees and year...... 109 Table 5.2 Results from an ANOVA used to assess differences in nutrient concentrations between Melaleuca irbyana and Melaleuca bracteata habitats in five sites at Logan City region with an error structure of plots nested within site...... 111 Table 5. 3 Principal component loadings of the data set, eigenvalues and their contribution to the correlations, showing only the first five components. ... 116 Table 5. 4 The table shows eigenvalues extracted from the correlation matrix of the selected soil properties and present variations and cumulative percentage of variance explained by each principal component axis (PC) for the entire dataset

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between Melaleuca irbyana and Melaleuca bracteata habitats in Logan City region...... 117 Table 5.5 ANOVA results using an error structure of plots nested within sites, comparing + - levels of ammonium (NH 4) and nitrate (NO3 ) in soils between Melaleuca irbyana and Melaleuca bracteata sites at different depths (0, 10 and 20 cm) in in situ ion exchange resin bags...... 123 Table 5. 6 Result of Tukey multiple comparison of means test of ANOVA results using an error structure of plots nested within sites comparing levels of ammonium + - (NH 4) and nitrate(NO3 ) in soil between Melaleuca irbyana and Melaleuca bracteata sites at different depths (0, 10 and 20 cm) in in situ ion exchange resin bags...... 124 Table 5. 7 Principal component loadings of the data set, eigenvalues and their contribution to the correlations, showing the three components...... 126 Table 5.8 Summary statistics for principal components analysis of nitrogen generated in soil of mature remnant trees between Melaleuca irbyana and Melaleuca bracteata in Jimboomba, Logan City region. The table shows eigenvalues, present variations and cumulative percentages of variance explained by each principal component axis (PC) for the entire dataset...... 127

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List of Appendices

Appendix 1 Summary of research on seed germination and seedling growth depending on whether they are rare or common...... 166 Appendix 2 Conference abstract…………………………………………………….. 172

List of Abbreviations

Abbreviations Descriptions ANOVA Analysis of variance PCA Principal Component Analysis PAR Photosynthetically active radiation, mol photon m-2s-1 LAI Leaf area index, m2/m2 DBH Diameter at breast height BA Basal area pH Potential of hydrogen

- NO3 Nitrate

+ NH4 Ammonium LMEMs Linear mixed effects models ML Maximum likelihood numDF Degrees of freedom numerator denDF Degrees of freedom denominator

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Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

QUT Verified Signature Signature:

Date: March 2018

xxii Acknowledgements

First and foremost, I would like to express my sincere appreciation and profound gratitude to my Principal Supervisor, Associate Professor Jennifer Firn, for her invaluable guidance, wise counsel, constructive comments and unfailing encouragement throughout my study at Queensland University of Technology (QUT).

Dr Firn assisted me with every need that arose in my PhD journey. I have learned so much from her and have grown as a person through my many interactions with her. I have learnt scientific thinking and feel that I have become more analytical and disciplined under her guidance. Regarding my scholarship, I sincerely thank Dr Firn for her persistence in securing the supervisor scholarship for the last four months of my PhD candidature.

I extend my sincere appreciation and heartfelt gratitude to my Associate Supervisor, Professor Acram Taji, who has been like my mother and an angel in my life, always coming up with a miracle and supporting me in every step of my studies and, saving me whenever I faced difficulties.

There are no words that can sufficiently express my gratitude towards my two wonderful supervisors. They have changed me forever and will remain my inspirational role models in my academic life.

I am profoundly indebted to Anna Markula from the Logan City Council, Brisbane, Australia for providing funding support for my research. I gratefully acknowledge the financial support provided by Chiang Mai Rajabphat University in Thailand and the tuition fee scholarship from QUT.

I greatly appreciate Dr Joshua Comrade Buru’s guidance and kind support in reading and editing a draft of my thesis. I am thankful to Karyn, Sophie and Christian from Academic Language and Learning Service, QUT, for their kindness in editing and providing useful advice for my thesis draft. My professional Editor, Dr Christina Houen of Perfect Words Editing, has provided copyediting and proofreading services according to the University- endorsed national policy guidelines.

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I wish to extend my gratitude to the following people for their help in the field: Jack, Eleanor, Sahnice, Elysia, Samantha, Morgan, Victoria, Jacob and Lawson. Special thanks to the EEBS, CARF, and QUT vehicle staff and technicians: Anne-Marie, Amy, Karina, Aarshi, Shane, Rachael, John, Scott for their advice and support during field and lab work.

I would like to express my thanks to Gustavo H. Puma Tejada for his support, encouragement, and patience in teaching me computer techniques. I am grateful to all my friends from EEBS and other schools/ faculties at QUT: Joshua, Purnika, Peraj, Tharanga, Karma, Sarah, Noors, Kuan and Naimul, for their encouragement, advice and support during the course of my studies and stay in Brisbane.

I dedicate this thesis to the memory of my biological mother, Mrs Ahmporn Soonthornvipat, who passed away when I was young, and to my step-mother, Mrs Wanpen Soonthornvipat, who passed away during my PhD studies. I would like to express my sincere thanks to my brother and sister for their support, advice and encouragement and for taking care of our father during the period of my PhD studies in Australia.

Finally, my heartfelt thanks go to my beloved father, Mr Sahmruay Soonthornvipat, for his caring wishes, for staying beside me in every situation during my life, and for being an unlimited source of love, support, good advice and encouragement throughout my life, and especially while undertaking my PhD so far away from my country.

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Chapter 1 General Introduction

Photos showing an example of old growth Melaleuca irbyana forest at Henderson Reserve site, which is one of the largest remaining and contiguous examples of this threatened ecological community in the early period of the study (2014). Photograph taken by Thita Soonthornvipat.

1.1 Introduction

Biodiversity is being lost at unprecedented rates globally, due to increased land clearance, introduction of exotic species, eutrophication and other anthropogenic pressures such as climate change (Millennium Ecosystem Assessment, 2005). These drivers of the loss of biodiversity have led to an increasing number of studies investigating the role that biodiversity plays in terms of ecosystem function and services, and how remnant populations can be restored or managed to develop increased resilience in the face of further human- induced perturbations (Lyons & Schwartz, 2001).

Worldwide, there are a large number of plant species that are naturally rare, and others that, overtime, have become rare because of altered ecological interactions affecting species distributions. For a species to be categorised as rare, it normally has a limited geographical distribution, and/or specialised habitat needs that are infrequently found in the landscape ( Buist et al. , 2002; Rabinowitz, 1981) . These specialised habitat conditions include nutrients, light and water availability (Pate & Hopper, 1994; Rabinowitz, 1981). In some cases, the rare species may not have specialised habitat requirements, but may instead show a low competitive ability when growing across a broad range of abiotic conditions and consequently may be confined to a narrow range of specialised habitat requirements, also called a realised niche (Dobson et al., 2006; Matesanz et al., 2009).

Rare species play an important role in maintaining overall ecosystem functionality, and as such a number of studies have been conducted to help understand the causes of rarity (Gaston, 1994b; 1996; Kunin & Gaston, 2012; Kunin & Shmida, 1997). Some plants become rare because of low phenotypic variability ( Phenotypic plasticity) , and therefore have specific habitat requirements (Hermant et al., 2013), or have less competitive physiological characteristics such as low growth rates, low reproductive fitness and low rate of seedlings survival (Pohlman et al., 2005; Richards, A. et al., 2003). Gaston (1994a) listed a number of causes for rarity and suggested that breeding systems that favour selfing, low reproductive investment, low amounts of genetic variation, low population densities, and chaotic population dynamics, are amongst the key drivers of rarity.

The focus of this thesis is Melaleuca irbyana R.T. Baker (swamp tea-tree), a small to medium sized tree listed federally as critically endangered under the Environment

Protection and Biodiversity Conservation Act 1999. Despite considerable conservation efforts to protect the integrity of the remaining populations of M. irbyana, which are largely located in the south- west peri- urban fringe of South East Queensland in Australia, this species remains under the threat of extinction due to increased land clearing for urban expansion, coal seam gas exploration, and common indirect effects of urbanisation, e. g., eutrophication. Prior to European settlement, M. irbyana had a limited distribution when compared to other widespread and common species within the same genus such as Melaleuca bracteata F. Muell. To improve the management of the existing M. irbyana populations, there is a need to understand how this species grows, including reproductive and life history traits, nutrient availability, and other habitat requirements.

The physiological and ecological traits of M. irbyana are not well known. It has been proposed that M. irbyana forests are groundwater dependent ecosystems, which are defined as ecosystems requiring access to groundwater on a permanent or intermittent basis to meet all or some of their water requirements (Bertrand et al., 2012; Kløve et al., 2011; Murray et al. , 2006). The main objective of this project was to develop a better understanding of the ecological, and physiological traits of the critically endangered species M. irbyana, as compared to the more commonly distributed and often co-occurring species, M. bracteata, to help improve the management of the existing M. irbyana populations.

Comparative analyses of seed germination under different controlled environmental conditions in the laboratory, as well as seed germination and seedling development in the field sites during four seasons for both M. irbyana and M. bracteata, were undertaken to shed light on the reproductive and life history traits of these species, because lower reproductive capability is an indicator of why species become rare. Measurements of physiological traits such as aspects of photosynthetic capability, rate of shoot growth, plant height and tree diameter at breast height of mature trees, seedling growth and development under the mature canopy of each species, and their reciprocal performance, were taken to produce useful information on the competitive abilities of M. irbyana versus M. bracteata; the point of the comparison was that lack of competitive ability in species may lead them to becoming rare. Habitat traits such as soil types and soil nutrients provide information on what is available in the environments where these two species grow, and help to understand whether M. irbyana has become a threatened species because there are fewer common

resources, and/ or whether there is a narrower range of resources available to it compared with M. bracteata. On the other hand, the same resources in the environment may be available to both M. irbyana and M. bracteata, but rare plants’ poor competitive abilities may make them less able to meet their needs from the environment.

The findings of this thesis will assist with the management of remaining populations of M. irbyana and improve the effectiveness for future M. irbyana revegetation projects as well as any related species. This research will also contribute more broadly to understanding the traits and environmental conditions that limit some plant species’ range, while fostering the widespread distribution of other species. Further, the information collected in this project will be useful for developing more detailed and effective regional management plans to protect this critically threatened species and its endangered ecosystems, and will help advance our theoretical understanding of the ecological and physiological traits of narrowly distributed species versus those which are more common and widely distributed. The three key questions that form the basis of this thesis are as follow:

1. What are the main ecological differences in the habitats between M. irbyana and M. bracteata that could explain the reasons for narrow distribution of M. irbyana?

2. Do limitations in reproductive capabilities and physiological traits in M. irbyana lead to its becoming rare?

3. What are the differences in the habitat of M. irbyana and M. bracteata, especially in terms of soil and nutrient conditions?

This research project is defined by two main aims and five respective objectives, as follows:

Aim 1: Compare the germination and early growth characteristic of M. irbyana (the endangered species) and M. bracteata (common and co-occurring species) seedlings to better inform restoration projects.

Objective 1: Compare the time to and success rates for germination of M. irbyana and M. bracteata as affected by temperature, photoperiod and light availability in in vitro and in situ conditions.

Objective 2: Compare the growth and survival of M. irbyana and M. bracteata when grown in the understory of mature forests of these species.

Aim 2: Compare the growth and abiotic conditions of mature populations of M. irbyana ( the endangered species) and co- occurring populations of M. bracteata ( the common species).

Objective 1: Compare the basal area, stem density and canopy structure of mature thickets of M. irbyana and M. bracteata forests.

Objective 2: Compare the rate of growth of stem and new leaf production of mature individuals of M. irbyana and M. bracteata.

Objective 3: Compare the soil properties and nutrient levels in mature forests of M. irbyana and M. bracteata.

1.2 Thesis structure

As described above, the overall aim of my research was to investigate the ecophysiology and reproductive characteristics of M. irbyana (the narrowly distributed and threatened species) and compare these characteristics with a common and often co-occurring congener species, M. bracteata. The thesis consists of six chapters, below:

Chapter 1, the introduction, sets the context of the study including the main aims and objectives of the thesis.

Chapter 2, the literature review, provides a critical analysis of the literature relevant to the thesis, focusing on causes of rarity and covering endogenous or intrinsic characteristics of the species, exogenous or extrinsic traits, as well as human induced factors which result in a species becoming rare. The taxonomy, morphology and reproductive biology of the study genus, Melaleuca, are described. Finally, detailed characteristics of the rare M. irbyana and its common congener M. bracteata are described.

Chapter 3 sheds light on reproductive and life history traits of M. irbyana and M. bracteata, because lower reproductive capability is indicative of why species become rare. Seed germination experiments conducted under different controlled environmental conditions four seasons in the laboratory (in vitro), as well as seed germination in situ for both M. irbyana and M. bracteata provide some answers to the narrow distribution of M. irbyana.

Chapter 4 explains measurements of seedling development in the field during the four seasons, as well as seedling growth and development under the mature canopy of each species. Measurable aspects of photosynthetic capabilities such as photosynthetically active radiation (PAR), leaf area index (LAI), survival rate, and their reciprocal performance, provide useful information on the competitive abilities of M. irbyana versus M. bracteata, because lack of competitive ability in species may lead to them becoming rare.

Chapter 5 looks at growth traits of shoot growth, plant height, and tree diameter at breast height (DBH) of mature trees. Habitat traits such as soil type and soil nutrients provide information on what is available in the environments where M. irbyana and M. bracteata are growing. Such information unravels if M. irbyana has become a threatened species, because there are fewer common resources and/ or a narrower range of resources available to it compared with M. bracteata; alternatively, the same amount of resources may be available to both M. irbyana and M. bracteata in the environment, but the specific requirement of M. irbyana requires highly fertile soil of nutrients and water ( resource acquisition) from the environment, especially in the early stage of the life cycle, has resulted in this plant’s rare status.

This thesis closes with Chapter 6, which provides a general discussion on the ecophysiological and reproductive traits of the study plant, M. irbyana, and puts forward strategies for the management of remaining populations and revegetation projects for this plant in Logan City in Queensland, Australia. Generalisations are drawn for the application of these strategies to other related species in other environments, the key outcomes from all chapters are synthesised, and the overall study findings discussed. The practical implications and limitations of the study, as well as future research arising from this work, are discussed in this chapter.

Chapter 2 Literature Review

Photos showing an example of old growth Melaleuca irbyana forest at Henderson Reserve site, which is one of the largest remaining and contiguous examples of this threatened ecological community, in the third year of the study (2016). Photograph taken by Thita Soonthornvipat.

2.1 Background

In this literature review, I discuss the following topics: definition of rarity, endogenous and exogenous causes of rarity, approaches to conservation, and management of M. irbyana populations. I will conclude by summarising the known ecological, physiological and morphological traits of the Melaleuca genera, and more specifically of M. irbyana and M. bracteata.

Rapid expansion of cities in Australia has modified the natural landscape, and has fragmented habitats, resulting in a significant loss of biodiversity (Spies et al. , 1988). In Australia, 92%, (nearly 16,000) of vascular plants are endemic; 7% of these are considered threatened with extinction, and are listed in Australia’ s EPBC Act 1999. This is approximately 14% of the world’s threatened vascular plants (Chapman, 2009). The high proportion of endemic species is indicative of the length of time Australia has been separated from other continents and its long-term geological stability (Braithwaite, 1990; Groves, 1994). The highest number of threatened species and ecosystems occur in most cleared areas of southern and eastern Australia ( National Land and Water Resources Audit, 2002) . Furthermore, habitat fragmentation and segregation contribute to the risk of extinction in natural populations, due to inbreeding and loss of genetic variation (Frankham, 2005; Young et al., 2000). Australia is not unique in the world in terms of these pressures.

Landscape modification, habitat destruction and fragmentation are globally the major drivers of loss of species (Fazey et al. , 2005; Fischer & Lindenmayer, 2007; Haila, 2002; Millennium Ecosystem Assessment, 2005). Many factors are involved in determining species’ abundance or rarity, and many questions remain unanswered in the search to understand the survival or extinction of a threatened species (Gaston, 1994b; Schemske et al., 1994). Understanding the biological status and characterising the biotic interactions and habitat requirements of a species are important first steps towards developing management plans to conserve threatened species. This is the approach taken in this research, because many management plans for threatened species lack key information on the characteristics of these species, and therefore can only provide generic recommendations on best practice.

Studies to explore the causes of rarity largely have focussed on genetics (Baskauf et al. , 1994; Dodd & Helenurm, 2002; Shapcott, 2007) , life history, and demographic

characteristics of restricted species (Byers & Meagher, 1997). Previous studies include the comparative ecophysiology of Graptophyllum species in Australia (Le Buu Thach, 2007) and geographic range size, seedling ecophysiology and phenotypic plasticity in Australian Acacia species (Pohlman, 2005). Species may be restricted to a given environment because they lack appropriate physiological traits to establish, reproduce, compete and defend themselves in other habitats. Therefore the focus of this thesis is to investigate the ecological and physiological characteristics of endangered Melaleuca irbyana with an often co- occurring and common congener species, Melaleuca bracteata, to help understand their potential impact on species’ rarity. Understanding causes of plant rarity including endogenous and exogenous factors is important in helping us with approaches to restoration and management of M. irbyana and other rare species, discussed in detail below.

2.2 Definitions of rarity

While there is no one universally accepted definition of rarity, in general and at its simplest level, species are considered rare if the total population of the species is made up of few individuals, or is restricted to a narrow geographic range, or both. Some rare species occur sparsely over a broad area. Others have many individuals, but these are reserved to just a few locations. A third kind of rare species are those with both few individuals and a narrow geographic range. These are considered the rarest of the species (Gaston, 1994b; Main, 1982).

Seven types of rarity in plant species were described by Rabinowitz (1981) based on three important characteristics: 1) extent of geographical range, 2) local population size and 3) habitat specificity (Table 2.1). Based on Rabinowitz (1981) and Rabinowitz et al. (1986) species are considered rare if they have a restricted or narrow range of distribution, if they occur only in one or a few specific habitats, and/ or if the size of the population is always small. Gaston (1996) supported these concepts, finding evidence that abundance and large spatial distributions are generally positively correlated. Within taxonomic groups, locally abundant species tend to be widespread, and locally rare species tend to have restricted distribution. For example, Glycine latrobeane (clover glycine) is considered a rare species because its populations are low in density, even though it grows over a large geographic

range in 17 conservation reserves in Australia, and it can be found in diverse habitats from open grassland to woodlands (Cropper, 1993).

Table 2. 1 Definitions of rarity as defined by Rabinowitz, 1981

Geographic Habitat Local population Rarity types Category range specificity size Large Wide Large, dominant Locally abundant in several Common somewhere habitats over a large range of (not rare) geographic area Large Wide Small, non-dominant Constantly sparse in several Sparse everywhere habitats over a large range of geographic area Large Narrow Large, dominant Locally abundant in a Predictable somewhere specific habitat over a large range of geographic area Large Narrow Small, non-dominant Constantly sparse in a Predictable everywhere specific habitats over a large range of geographic area Small Wide Large, dominant Locally abundant in several Unlikely somewhere habitats over a small geographic area Small Wide Small, non-dominant Constantly sparse and Unlikely everywhere geographically restricted in several habitats Small Narrow Large, dominant Locally abundant in a Endemics somewhere specific habitat over a small geographic area Small Narrow Small, non-dominant Constantly sparse in a Endemics everywhere specific habitat over a small geographically restricted Adapted from (Leverington, 2011, p.11; Williams et al., 2014, p.5).

More recently, Rey Benayas et al. (1999) expanded on Rabinowitz’s scheme for classifying species as common or rare by adding a fourth criterion, the ability of a species to occupy a larger or smaller fraction of its potential suitable habitats (habitat occupancy), a criterion which is independent of the three categories put forward by Rabinowitz (1981) (Table 2.2).

Table 2. 2 An updated scheme of pattern of rarity adding the notion of habitat occupancy (adapted from Rey Benayas et al., 1999, p.2).

Geographic range Wide Narrow Habitat specificity Broad Restricted Broad Restricted Abundance Large Small Large Small Large Small Large Small Habitat occupancy high Common Widespread Endemic Locally Non- Endemic Indicator Common existent indicator Habitat occupancy low Highly Sparse Locally Potentially Endangered dispersed endangered endangered

The state of rare species is influenced by the rate of change in a given population over time (temporal scale) and types of fluctuation experienced by that population’s natural changes or human induced changes (Harper, 1981). Common definitions of species status which include time scale and changes in population size are:

• Species that have always been rare (stable but naturally restricted) • Species that were common, but are now rare (relics, threatened) • Species that were rare and are now common (newly evolving) • Species that have always been common (common) Fiedler, P.L and Ahouse (1992) proposed a framework to include and relate multiple criteria with multiple mechanisms to describe and explain patterns of rarity. The two criteria they used are spatial/ geographic distribution (narrow or wide) and temporal persistence (short or long), resulting in four categories-species that have existed for a long time over wide or narrow ranges, and those that have existed for only a short time over wide or narrow ranges (Table 2.3).

Table 2.3 Pattern of rarity for vascular plants as defined by Fiedler and Ahouse (1992) adapted from (Leverington, 2011, p.12).

Temporal Persistence Short Long Spatial Wide Short/Wide Long/Wide Distribution Narrow Short/Narrow Long/Narrow

Although rare plants can be classified by their patterns of distribution and site level abundance, as argued by Rabinowitz (1981), Rabinowitz et al. (1986) and Fiedler, P.L and Ahouse ( 1992) , these broad patterns of population level measures do not identify the mechanisms that explain these patterns. For example, is a low abundance at the site level (Nathan & Muller-Landau, 2000), and a limited distribution at a landscape level explained by a species’ inability to germinate unless experiencing very specialised conditions or is this rarity instead explained by anthropogenic disturbances (Murray et al., 2002).

2.3 Causes of rarity

The main causes and consequences of rarity fall within three categories: low genetic variation, life history traits, and ecology (Walck et al., 2001). In the context of life history, rare species can be considered as both ancient relics of previously widely dispersed species, or a new species that has not yet dispersed (Cropper, 1993). Secondly, low genetic variation has been discussed as a cause of rare species, with a low level of genetic variation compared with common species (Gitzendanner & Soltis, 2000). However, the results are conflicting, because rare species can have either low or high genetic variation compared with common species (Godefroid et al., 2011; Stebbins Jr, 2013). Finally, distribution of plant species can be limited because of ecological traits, including the dispersal factors, competitive ability with other species in natural environment, or restricted due to abiotic (Garnier & Poorter, 2007; Hölzel & Otte, 2004; Leeson & Kirkpatrick, 2004; Parmesan, 2006). Unravelling the causes of rarity is complex, because the causes are often multidimensional. Physiological studies on abiotic interactions such as light, water and nutrient relationships between rare species and widespread congeneric species may help unravel causes of rarity and provide some answers as to why some species naturally occur

in small, restricted population, while others are widely distributed and are common (Richards, A. et al. , 2003). Richards, A. et al. (2003) investigated how physiological characteristics ( e. g. , assimilation rates, water use) vary depending on environmental characteristics ( light, water and nutrients) between three restricted and widespread congeneric North Queensland species. They found that causes of rarity were unique to each species. Under high light conditions, Gardenia ovularis, a widespread species, was found to have a greater photosynthetic rate than Gardenia actinocarpa, which is a restricted species. Xanthostemon formosus, a restricted species, was found to have a low photosynthetic capacity (low electron transport rate and CO2 fixation rate), higher transpiration rate and high concentration of foliar manganese when compared with Xanthostemon chrysanthus, a widespread species, because X. chrysanthus is more adaptive and able to cope better with variability in the environment. Additionally, restricted Archidendron kanisii had higher electron transport rates, larger dissipative capacity for exclusion of excess light, and an efficient system for using nitrogen in the process of photosynthesis when compared with Archidendron whitei, a widespread species (Richards, A. et al., 2003). Thus, understanding the physiological traits and their interactions with species’ genetic and ecological attributes is important in developing conservation plans for species at risk of extinction. Rarity may be explained by endogenous (intrinsic or natural) or biological traits such as age, stage of reproductive development, genetics, and germination, including levels of plant regulators, for example hormones and reactive oxygen species ( Munné- Bosch & Alegre, 2004). Or it can be described by exogenous (extrinsic) causes defined by harmful human activities that have reduced the distribution or abundance of species independent of their biological requirements, such as light, soil, water, human effects, habitat fragmentation, climate change, etc. These causes are described in the following sections ( Fischer & Lindenmayer, 2007).

2.3.1 Endogenous or intrinsic causes of rarity

There are a number of inherent biological and ecological characteristics ( endogenous) that have been found to be associated with rare species, and these characteristics are often used to classify species into categories of rarity for regulatory or conservation management purposes (Dawson et al., 2012).

Intrinsic factors result in changes in biology of the species due to biotic interactions with their habitat. They include:

1. Low phenotypic variability and low competitive physiological characteristics (Cordell et al., 1998). 2. Low reproductive fitness including seed dispersal, seed germination and seedling survival ( Kunin & Shmida, 1997; Osunkoya & Swanborough, 2001; Timmerman-Erskine & Boyd, 1999; Yates & Ladd, 2005).

2.3.1.1 Species traits

Species traits that are considered endogenous causes of rarity include factors that can affect population vital rates (e. g. , growth rates, survival rates, reproductive traits, etc.). Species with “ slow” life histories such as slow growth and slow reproduction after disturbance may be predisposed to extinction risks (Brunet & Von Oheimb, 1998; Dupré & Ehrlén, 2002; Grashof‐Bokdam, 1997; Turnbull et al., 2000; White, 1979).

Low phenotypic variability and low competitive physiological characteristics

Phenotypic plasticity refers to the capacity of a single genotype to exhibit different characteristics or phenotypes in response to differences and/or changes in the environment (Sultan, 1995; 2001; Sultan, S. E., 2000). This is fundamental to the way in which organisms cope with environmental variations. Phenotypic plasticity can manifest as changes in physiology, morphology, biochemistry, behaviour or life history (phenology). Phenotypic plasticity can be passive, anticipatory, and instantaneous, responding to environmental conditions, especially stress, by adapting in phenotypic variation such as increased number of leaves, and reduced leaf size following environmental variation (Richards, C. L. et al., 2006; Richards, C. L. et al., 2005).

Morphological traits such as root characteristics have been suggested as predictors of species distributions, whether narrow or widespread (Poot & Lambers, 2003). Poot and Lambers (2003) studied root morphology of seven South-Western Australian floras. When comparing rare and common Hakea spp. (Proteaceae) they found that, the rare species survived well under low phosphorus soils due to inherent differences in their root-system architecture, such as differences in initiation of lateral and cluster root primordia.

Additionally, they proposed that highly specialised, habitat-specific morphological and physiological adaptations could explain success in the rare species adaptation to a given habitat. Conversely, species were not able to survive in the surrounding habitats whose soils were characterised by higher phosphorus availability due to reduced phenotypic plasticity in their traits. Therefore, traits essential to thriving in their own specialised habitat, reduced their fitness to survive in other habitats (Poot & Lambers, 2003).

It has been hypothesised that rare plant species have traits that make them more efficient at resource conservation than at resource acquisition ( Reich et al. , 1999) . For example, plants in high- resource ecosystems succeed through high rates of resources acquisition, while species adapted to low- resource ecosystems largely display traits associated with resource conservation, such as slow growth, high tissue longevity, and resource-use efficiency (Funk, 2013; Imhoff, 2010). Thus, some rare species are limited to resource poor habitats because they cannot compete with species that are resource acquisition specialists when they are grown in nutrients rich habitats (Funk, 2013). High nutrient levels increase plant productivity and aboveground biomass (Hautier et al., 2009). Therefore, compared with rare plants, common species may have higher survival rates when they are under competition. As competition intensifies with increased resources such as nutrients, the difference in survival rates becomes more accentuated than when plants are under competition with lower nutrient availability in the soil. However, there is a dearth of information on experiments involving a large number of plant species, that compare competition and nutrient responses of rare plants species with those of common plant species (Lloyd et al., 2002). In recent years Dawson et al. (2012) undertook research on competitive responses between 23 native plant species (9 common and 14 rare) and 18 introduced species (8 common, 10 rare). Introduced common species were found to have higher biomass than native and rare species. Common species’ total biomass levels were higher in response to nutrient addition than the rare species, regardless of species origin, whether they were native or non-native. Furthermore, they found that common species marginally coped better with competition than did the rare species (Dawson et al., 2012).

Most studies comparing rare and common or widespread species have demonstrated that widespread species have the ability to survive a greater range of environments, than do endangered or rare species, due to their higher phenotypic plasticity and ecological

versatility (Bevill & Louda, 1999; Brown, J. et al., 2003; Maliakal-Witt et al., 2005; Novotný & Basset, 2000; Powell et al., 2011; Simon & Du Vall Hay, 2003; Siqueira et al., 2012). In terms of studies comparing rare and common species in the environment, most research provides information that relies on physiological characteristics such as the growth rate, plant survival rate and the fitness of plants under natural conditions (Brown, J. H. , 1984; Gibson, 2012; Lavergne et al., 2004; Lloyd et al., 2002).

Linder (1995) assessed the distribution patterns of 16 species of the Southern African terrestrial orchid from the genus Herschelia and classified them into the Rabinowitz (1981) rarity categories. A strong correlation between habitats’ age, the relative age of the species, and the degree of rarity was found. Further, Linder (1995) concluded that the degree of rarity was correlated with habitat types and with phylogenetic history (life history). A recent study by Siqueira et al. (2012) comparing widespread and narrowly distributed species from different regions of Brazil found that certain traits such as dispersal, survival, and recruitment stages of plants are critical in their distribution. For example, occurrence and distribution of Acacia ausfeldii and Acacia williamsonii were negatively affected by more than 150 years of human impact as a result of mining, timber harvesting, agricultural activities and land clearance causing habitat fragmentation and population reduction in these species (Brown, J. et al., 2003).

Plant ecophysiology is an experimental science that aims to describe the physiological mechanisms that underline ecological observations (Lambers et al. , 1998). Species rarity could be explained by ecophysiological traits, especially if rare species are found to have lower environmental tolerance than their common congeners (Brown, J. H., 1984) . Two theories are thought to explain plant growth and survival, Shelford’ s and Liebig’ s law. Shelford’ s law of tolerance proposes that the range of environmental conditions that are suitable for a species will determine its distribution (Krebs, 1994). The second law, Liebig’s law of the minimum, is concerned with plant growth and nutrition. This law indicates that “plant growth will be limited by the nutrient in shortest supply even when other nutrients are abundant, giving rise to hyperbolic growth response curves to individual nutrients” (Austin, 2007, p.5).

Many abiotic factors influence where plant species live, including light availability, temperature, moisture, the physical properties of soil, soil pH, soil nutrient availability, and

so on (Akula & Ravishankar, 2011). Species can become restricted to a narrow climatic range because they lack physiological traits to recruit, reproduce, compete and protect themselves from the environment of adverse habitats (Thuiller et al., 2005). Plants that can regulate their physiology and cope with a wide-range of environments are often thought of as resilient plants, and illustrate how physiological characteristics may be linked to the conservation status of plants (Pohlman et al. , 2005). Thus, measuring the environmental conditions that impact on a species’ germination, growth rate, and reproductive characteristics, may assist with understanding why some species are rare while others are common in terms of their populations size and distribution (Begon et al., 2006).

Low reproductive fitness

Ecological and evolutionary processes that operate at all stages of the plant life cycle affect plant population dynamics. Reproductive processes such as pollination, seed production, seed dispersal, seed size, seed germination and seedling establishment influence species distribution, and hence their ability to colonise a range of new environments (Ashman et al., 2004; Grime, 1977; 2006).

Figure 2. 1 The life cycle of a hypothetical plant population model (After Schemske et al., 1994, p. 590).

The life cycle presented in Figure 2.1 above shows five life history stages generally used to represent a plant’s life cycle, starting from seeds, seedlings, juveniles, sub-adults, and adults and then cycling back to seeds. This life cycle summarises most possible transitions within and between stages for a hypothetical plant population. The arrows indicate the probability of individuals growing to another stage (increase in size), survival (remaining in the same stage as previous year), or reproduction (Schemske et al., 1994).

Comparing reproductive success based on pollination biology between five native Cirsium spp. (C. fontinale var. fontinale, C. andrewsii, C. brevistylum. C. occidentale, and C. quercetorum) and an invasive congener, Cirsium vulgare, Powell et al. (2011) found that the invasive C. vulgare produced more flower heads per plant than most native species, and that C. vulgare attracted more pollinators than its congeners. They found that, unlike the rare species (C. fontinale and C. andrewsii), C. vulgare did not require pollinator visits for a high rate of seed production. The remaining native species set fewer seeds than C. vulgare without a pollinator. However, differences in insect visitation and autonomous self- pollination did not lead to differences in pollination rates across species or between populations. Based on their results, the researchers suggested that factors other than pollination biology may explain the difference in reproductive success of these species (Powell et al., 2011). Osunkoya and Swanborough ( 2001) compared reproductive patterns of seed germination and ecophysiological traits between an Australian rare rainforest plant Gardinia actinocarpa and its common congener G. ovularis (Rubiaceae). Gardinia actinocarpa was found to have a longer reproductive period (nine months) with lower fecundity than G. ovularis. Gardinia ovularis showed a shorter reproductive duration ( four months) and produced more fruit ( 35. 76 fruits per female tree) . However, under low levels of photosynthetically active radiation (PAR), the pattern and percentages of seed germination of both species were similar (70-90%). Although at 37% PAR (open forest) G. actinocarpa had lower seed germination and lost seed viability faster than G. ovularis, the rate of its seedlings’ growth was not inferior to G. ovularis at 37% PAR or at 2.5% PAR levels (closed forests). Osunkoya and Swanborough (2001) suggested that the rarity in G. actinocarpa may be a result of limited fecundity, reduced seed dispersal ability and lack of soil seed- banks, rather than to inferior vegetative and ecophysiological characteristics. Thus, the

regeneration of plant communities depends on the seed being in an appropriate physiological state in order to germinate (Murdoch & Ellis, 2000).

Seed germination

The important factor for seed germination is conditions of ambient temperature, especially during the period of soil water availability (Mijani et al., 2013). The interaction between the effects of temperature and light conditions may also substantially impact germination and increase the survival rate and establishment at the seedling stage (Baskin, C. C. & Baskin, 1998). Plants which have faster seed germination rates are able to establish root systems rapidly and tap into the unexploited areas to extract soil moisture in deeper soil profiles and further afield (temporal and spatial adjustment) (Schenk, 2006). For example, Schütz et al. (2002), investigating seed germination, seedling survival and seedling growth of four Eucalyptus species on two major soil types in south-western Australia, found that seed size and speed of germination in Eucalyptus species play an important role in their rapid root growth and establishment, especially in water deficient soils, enabling roots to reach the water deep in the soil profile (Schütz et al., 2002).

Seedling growth and survival

Seedling growth is important in the distribution of plant species (Daws et al., 2005; Standish et al. , 2007). Numerous adaptations in plant species may increase the ability of seedlings to cope with variations in climate such as drought, when soil water availability becomes limited. Some species are better able to survive in water deficient environments (Bartlett et al., 2012; Jump & Penuelas, 2005). Trees are long-lived plants, but as seedlings they arguably at their most vulnerable to incidents of stress or injury due to drought, frost, poorly drained soils and herbivory (Buell et al. , 1971; Gill & Marks, 1991; Inouye et al., 1994; Myster & McCarthy, 1989) . Studies conducted in natural and anthropogenic grasslands and savannahs have identified water stress as an important cause of woody seedlings’ mortality (Beckage & Clark, 2003; Canham et al., 1999; Engelbrecht et al., 2005; Green et al. , 2010; Liu et al. , 2011; Moles & Westoby, 2004; Navarro & Guitian, 2003; Padilla & Pugnaire, 2007; Queenborough et al., 2007; Villar-Salvador et al., 2012; Walters, M. B. & Reich, 2000). Furthermore, shade from tree canopies may either promote or inhibit the survival of woody seedlings depending on soil water availability (Holmgren et al., 1997).

Zutter et al. (1986) argued that at dry sites, shade would enhance the survival rate of woody seedlings by reducing water stress, while in wet sites, shade would inhibit seedlings by increasing competition for light. Climate variability can also affect the survival rate of seedlings (Engelbrecht et al., 2005). Davis et al. (1999) found that long dry seasons and wet periods increased the survival, growth and photosynthetic rate of oak seedlings. The survival rate increased constantly when increasing water. However, seedling performance in dry and wet summers varied depending on light regimes and the competitive capabilities of the seedlings (Davis et al., 1999; Montes-Hernández & López-Barrera, 2013).

Walters, M. B. and Reich (2000) investigated the relationship between seed size and growth rates of ten shade tolerant North American trees ( Populus tremuloides, Betula papyrifera, Betula alleghaniennsis, Acer saccharum, Larix laricina, Pinus banksiana, Pinus resinosa, Pinus mariana, and Abies balsamea) and their contributions to seedlings’ survival with time and with variations in light and resource availability below ground. They found that the relative growth rate (RGR) and survival rate of seedlings after germination increased with light across all species. Whilst RGR and survival of seedlings also increased with nitrogen supply, this increase occurred at the two highest light levels, and only for the shade- intolerant, broad-leaved Populus and Betula species. Their results showed that seedling survival was positively related to RGR for all species. Moreover, they found that the relationship of seedlings’ survival and RGR differed for each species, depending on their seed mass. Generally, at any given RGR, large-seeded, shade-tolerant species had higher survival than small-seeded, shade-intolerant species. Furthermore, they showed that across species, in most light and nitrogen treatments there was a positive correlation between seed mass and seedling survival, but not with RGR (Walters, M. B. & Reich, 2000).

Walters, M. B. and Reich ( 2000) found the availability of light, nitrogen and differences in species characteristics were important predictors of the growth rates of seedlings. In young seedlings growing in deeply shaded microsites, nitrogen supply does not seem to be important, and only shade-tolerant species survive, due to their large seed size and physiological traits other than RGR. In moderate shade, RGR and survival rate are higher in all species except in small-seeded and broad-leaved species. Intolerant species have low survival and RGR at low nitrogen supply. Overall these findings provide considerable information for understanding the generic silviculture of tree species, as they

suggest that broad-leaved shade-intolerant species compete more effectively in moderate shade on richer soils than on poorer soils (Walters, M. B. & Reich, 2000).

Nutrients may reduce seedlings’ survival. For example, in conditions of nitrogen enrichment and low water availability, Quercus rubra and Quercus prinus seedlings showed the lowest physiological performance (Kleiner et al. , 1992). Beckage and Clark (2003) examined how heterogeneity in understorey cover, mineral nutrients, and moisture, and their interactions with canopy gaps, contribute to the coexistence of three common and co- occurring tree species by studying seedling survival and growth: Acer rubrum (red maple), Liriodendron tulipifera (yellow poplar) and Quercus rubra (red oak) planted in one of five understorey treatments (removal of understory vegetation, trenched, trenched plus removal of understorey vegetation, fertilisation of 10g Agriform planting tablets consisting of NPK [ 20-10-5 plus microelements] , and a control without trenches and removal of understory vegetation) . They found canopy gaps and understorey conditions had large effects on survival rates. Differences in seedling growth and survival rates across modified microenvironments may contribute to the coexistence of some species, especially in high- resourced microenvironments (Beckage & Clark, 2003). Thus, the survival rate of seedlings depend on many factors such as tolerance to drought, high capacity to reach nutrients in deep soil and having high ability to compete for resources in natural habitat, especially under harsh conditions and limited resources. The works of researchers on seed germination and seedling growth are summarised in Appendix 1.

2.3.1.2 Ecosystem characteristics

Ecosystem characteristics can impact survival, growth rates and population size of species. The capacity of an ecosystem largely depends on its nutrient cycling characteristics, which are driven by edaphic and climatic condition ( Luo et al. , 2010; Zechmeister- Boltenstern et al., 2015).

Edaphic characteristics

Many rare plant species are found on relatively infertile soils made-up of geological substrates such as sandstone, limestone, granite, gypsums or serpentine (de Lange & Norton, 2004; Leeson & Kirkpatrick, 2004; Mattner et al., 2002; McIntyre & Martin, 2001). John et

al. (2007) found that the spatial distribution of 36-51% of tree species in Colombia showed strong association with soil nutrient availability. These results indicate that belowground resource availability plays an important role in tree communities, and provides a basis for investigation of resource competition in between plant species. Plants obtain nutrients necessary for their growth and development from soil. The growth of terrestrial plants requires the uptake of organic nutrients from the soil as a result of annual nutrient cycling in soil, which is closely associated with decomposition (Galloway et al., 2004).

Eutrophication is widely believed to be the reason for the decline of some rare plant species, especially in terrestrial ecosystems. Numerous researchers have shown that there is a relationship between increasing nitrogen availability and decreasing plant species richness (Moore et al. , 1989; Stevens et al. , 2004; Wheeler & Shaw, 1991). Villar-Salvador et al. (2012) found that there is positive relationship between plant procedures, seedling size, and soil nutrient concentrations in seedling survival and growth rates in Mediterranean-climate plantations. Physiological processes such as nitrogen remobilisation, carbohydrate storage, and plant hydraulic conductance, are crucial in the survival of seedlings.

Water and light conditions

Water availability plays an important role in seedling recruitment, especially in natural habitats (James & Svejcar, 2010; Lauenroth et al., 1994; Noumi et al., 2010). Low soil moisture is the primary cause of seedling mortality. Seedling survival and vegetative growth are improved by prolonged root water absorption and efficient water transport (Folk & Crossnickle, 1997; Grossnickle, 2005; 2012; Hernández et al., 2010). Drought can inhibit photosynthesis in plants (Hájek et al. , 2009). Some plant species cannot cope with harsh conditions and, as a consequence of having low survival rates in natural habitats, finally become rare (Flores & Jurado, 2003). In some cases seedlings are established by animal dispersal, such as birds and herbivores. This enables seeds to establish under the canopies of mature plants, benefitting from the less stressful microclimate, and providing protection against moisture stress (Ellner & Shmida, 1981; Gibson, A. et al., 1998).

2.3.2 Exogenous or extrinsic causes of rarity

Extrinsic causes of rarity are human induced and can lead to a species being threatened with extinction. These include:

1. Conversion and fragmentation of natural habitat to cater for increasing human population. 2. Biotic stresses as a result of introduction of invasive non-native species.

Habitat loss and habitat degradation may rank as the most important extrinsic factors leading to increased rarity (Sutton & Morgan, 2009). All the above mentioned factors are described further in this section.

2.3.2.1 Land alteration

Agriculture and urbanisation result in habitat fragmentation and habitat loss for many species (Kerr & Deguise, 2004; Luck et al. , 2004). Landscape modification and habitat fragmentation have become major research topics in conservation biology (Fazey et al., 2005) . Fischer and Lindenmayer ( 2007) developed a conceptual framework to help understand the relationship and impact of processes altering landscape (Figure 2.2).

Figure 2.2 A conceptual framework showing the relationship and impact of endogenous and exogenous processes in landscape modification and their influence on species traits and behaviour (adapted from Fischer and Lindenmayer, 2007, p. 268).

Habitat modification and fragmentation

Fragmentation occurs when habitat is progressively sub- divided into smaller, geometrically altered and more isolated fragments as a result of both natural and human (anthropogenic) causes (McGarigal & McComb, 1995; Watson, D., 2002). Humans have the highest impact on the rate of fragmentation, with indexes strongly correlated with the proportion of habitat loss in landscape (Fahrig, 2003). Some species may not be able to disperse naturally without a suitable range of habitats if fragmentation creates barriers to plant distribution ( Hamrick et al. , 1991; Lenz et al. , 2011; Primack & Miao, 1992) . Fragmented areas have been shown to have lower diversity and abundance of flowering plants, with species receiving fewer pollinators causing low seed set (Cunningham, 2000; Jennersten, 1988; Menz et al. , 2011; Steffan-Dewenter & Tscharntke, 1999; Vanbergen,

2013). Fragmentation may also affect the coevolution of species, affecting, for example, plant and pollinators interactions (Dupré & Ehrlén, 2002; Jennersten, 1988).

2.4 Approaches to conservation and management of rare and endangered plants

Schemske et al. ( 1994) suggested a “ top- down” ( broad overview or strategic perspective) approach to development of recovery plans for rare and endangered plant species. They proposed that the following issues need to be addressed before developing recovery plans:

1. Understanding the biological status of the species— assembling the necessary demographic information (life cycle) to determine if the numbers of individuals and populations of a species are increasing, decreasing, or are stable (For example a life cycle population model as per Figure 2.1). 2. Narrowing down on the life history stages that have the greatest effect on population growth and species persistence. This will help focus efforts on those aspects of the species biology that constitute the greatest threat to survival. 3. Knowing the biological causes of variation in those life history stages that have a major demographic impact.

At the species level, assessments of conservation status are generally performed using the guidelines provided by International Union for Conservation of Nature (IUCN, 2001). The IUCN Red List combines the following characteristics into an endangerment index: 1) population size, 2) growth rate, 3) population fluctuation, 4) habitat fragmentation, and 5) range size.

Only one of these five needs to be met for a species to be included in the IUCN Red List, but all need to be considered. Based on these guidelines in Australia, Commonwealth and State legislation uses estimates of potential threats and potential for extinction as criteria for examining the conservation status of species (IUCN, 2011).

Comparative studies are one of the main tools to contribute to research and find answers to why some species have narrow distributions and become rare, while others remain common and widespread. Comparative studies provide a way to gather significant information on key similarities and differences between closely related species that differ in

abundance and distribution (Bevill & Louda, 1999; Elith & Burgman, 2002; Godefroid et al. , 2011) . Furthermore, comparison of congeneric species can minimise confounding effects of disparate phylogenetic histories ( Gitzendanner & Soltis, 2000) , providing information on the key similarities and differences between closely related species that differ in terms of abundance and distribution (Bevill & Louda, 1999). According to Baskin, J. M. and Baskin (1986), such studies have the potential to identify the mechanisms that explain a rare species’ limited distribution. The benefit of a multiple-species approach is that in general, patterns of rarity can be elucidated from a broad spectrum of life forms, life cycles and taxonomic categories (Walck et al., 2001). Therefore, comparative ecophysiological analyses of closely related species appear to be a good choice for a study to understand the specialised requirements of rare species, which is the focus of this thesis.

2.5 Description, distribution and habitats of Melaleuca species

The genus Melaleuca L. comprises of 300 species, and is the third largest angiosperm genus in Australia after Acacia and Eucalyptus (Craven, L. & Lepschi, 1999; Craven, L. A. , 2009; Edwards et al., 2010). The genus Melaleuca L. goes by numerous common names such as bottlebrush trees, broad- leaved paperbarks, cajuputi, niaouli, honey myrtles (broombush), tea-trees, paper-barks, and punk trees. The name Melaleuca is derived from the Greek, melas, meaning black or dark, and leucon meaning white, presumably referring to the white branches and black trunk of the first named species, M. leucadendra, the trunks of which are often blackened by fire. The common name for this genus, paperbark, refers to the distinctive papery bark of the trunks that can be peeled off in sheets, particularly prominent on the larger trees (Southwell & Lowe, 2003).

2.5.1 Taxonomy

The genus Melaleuca is a member of the Myrtaceae or myrtle family. There are approximately 130 genera and 3,000 species recorded in this family (Serbesoff-King, 2003; Watson, L. & Dallwitz, 1992) . Family Myrtaceae consists of trees and shrubs, usually evergreen, mostly with simple leaves, commonly opposite leaves, and rarely found with alternate leaves (Long, R. W. & Lakela, 1971; Mabberley, 2008). This family is noted for its spicy, aromatic scent because of the variety of essential oils present in the oil glands on the leaves (Gentry, 1993; Tomlinson, 1980; Zomlefer, 1989). Members of the Myrtaceae

family occur in temperate, sub-tropical, and tropical regions, mostly in Australia and tropical America (Watson, L. & Dallwitz, 1992). Specifically, the Myrtaceae family is divided into two subfamilies: Leptospermoideae and Myrtoideae (Mabberley, 2008). Leptospermoideae is mostly found in the southern or eastern hemisphere, with the main centre of distribution being Australia. They have dry woody fruits ( capsules) . Plants from the subfamily Myrtoidea are distributed in Australia, tropical America and the South Pacific, and some have fleshy fruits (Tomlinson, 1980; Wunderlin, 1982).

The genus Melaleuca L. is taxonomically grouped in the subfamily of Myrtoidea which overall has a wide geographic distribution, characterised by a diverse range of environmental conditions. In nature, Melaleuca spp. are found along watercourses or along the edges of swamps. They can be found growing in open forests, woodlands or shrublands, and are popular for gardens and landscaping both in Australia and overseas (Barlow, 1987). Melaleuca spp. are found in all states and territories in Australia (Craven, L. & Lepschi, 1999; Craven, L. A. , 2009; Edwards et al., 2010), with Western Australia having the most diverse collection of about 80-90 species. However, several species occur in areas near Indonesia and Papua New Guinea. For example, Melaleuca cajuputi is thought to have expanded from Australia northwards, especially from Northern Queensland, the Northern Territory and North-western Australia, to the Asian mainland, where it was most likely introduced for cultivation, but also occurs naturally (Barlow, 1987; 1988; Barlow & Cowley, 1988; Blake, 1968; Byrnes, 1986; Cheel, 1924; Cowley et al., 1990).

2.5.2 Morphology

Plants in the genus Melaleuca are evergreen, mostly small to medium size shrubs, but a few species can reach heights of up to 30 meters. The bark of Melaleuca spp. is typically thick, spongy, a whitish-colour when young, exfoliating in cinnamon-coloured, papery layers that can easily be peeled off from the stem of mature trees. Bark consists about 15-20% of the stem volume. The leaves are mostly four to 12 mm long, simple, narrowly elliptic to lanceolate-elliptic, with the principal veins parallel; they have very short petioles arranged in five spiral rows (Barlow, 1987; Barlow & Cowley, 1988; Quinn et al., 1989).

Leaf blades are densely pubescent when young, becoming glabrous with age. They are dull green in colour on both surfaces, with reddish oil glands containing essential oils

which have been used for millennia by indigenous people in Australia for medicinal purposes. At present a thriving tea tree oil industry exists in Australia (Barlow, 1987; Barlow & Cowley, 1988; Rayamajhi et al. , 2002; Serbesoff-King, 2003; Van et al. , 2002). The showy parts of the flowers of Melaleuca are the stamens, the petals being small and inconspicuous. The stamens are often brightly coloured, with red, pink, mauve, purple and yellow being common ( Barlow & Cowley, 1988) . The Melaleuca “ flower” is an inflorescence formed by a cluster of small flowers. In Australia, peak flowering for most species is spring (September to November). The flower clusters may occur terminally at the ends of branches or in short spikes along the branches. Each flower has five sepals approximately 2 mm long and obtuse, and five petals about 3 to 4 mm long, white, and obovate to orbicular (Barlow & Cowley, 1988; Serbesoff-King, 2003). Following flowering, three-celled woody seed capsules develop, with each capsule containing numerous small reddish seeds. The capsules usually remain tightly closed unless stimulated to open by fire or by the death of the plant (Bodle et al. , 1994; Chiang & Wang, 2007; Godfrey, 1981; Langeland, 2008; Long, R. W. & Lakela, 1971; Turner et al., 1997; Woodall, 1981). Based on the observations of Van et al. (2000, 2002) the diversity in morphological traits including those of leaves and flowers as well as the reproductive traits such as fruit setting in the Melaleuca genus may contribute to some species within this genus exhibiting widespread distribution while others become narrow endemic species (Craven, L. A. , 2009).

2.5.3 Reproductive traits

As mentioned above, the peak flowering season for most Melaleuca species in Australia is spring (September to November) (Barlow & Cowley, 1988; Van et al., 2002). The flower clusters may occur terminally at the ends of branches or in short spikes along the branches. Melaleuca flowers are radially symmetrical, stalkless, and perfect, with a cup shaped floral tube (hypanthium). The ovary is inferior and three celled, with few to many ovules per cell. The top of the ovary is convex and almost always hairy with a central depression around the style. Styles are simple, terminal and threadlike, with very small stigma. The five sepals that are connected to the floral tube are small and usually fall off in fruit. The five petals are free and overlapping, short, clawed or narrowed at the base, broad near the top. The 15 or so stamens are joined together (united) into groups, with each group joining the floral tube as a unit. Each Melaleuca flower contains five of these groups or

“staminal bundles”. The anthers are versatile, not erect, with two parallel cells, and release pollen through a lengthwise slit (Barlow, 1987; Barlow & Cowley, 1988; Cowley et al., 1990; Craven, L. A. , 2009). Melaleuca flowers are mostly insect-pollinated with a wide range of flower colours. Half of all species have filaments of white through cream to yellow or green, while the others have pink, red or mauve filaments (Brophy et al., 2013). The fruit consists of a three-loculed cup-shaped capsule within a usually woody to subwoody fruiting hypanthium, with no stalk. Capsules are small and not larger than 0. 5 cm long x 0. 5 cm in diameter (Serbesoff-King, 2003; Van et al., 2000; Van et al., 2002).

Melaleuca fruits are dehiscent and usually many seeded. The seed has a membranous or rarely coriaceous testa containing an embryo but no endosperm. The numerous small seeds ( mean length of 1 mm) are released through slits at the top ( midway between partitions) . Seeds are contained within capsules and are retained in the canopy upon maturity. Capsules’ dehiscence and seed release are triggered by capsules desiccation. Natural seed release is triggered by mechanical injury, fire, frost, and ageing (Brophy et al., 2013). Typically in the Melaleuca genera, as in several other genera of the family Myrtaceae, the fine particles that dehisce from the fruit are a mixture of viable seed and unfertilised ovules commonly referred to as ‘chaff’ (Barlow, 1987; 1988; Cowley et al., 1990; Craven, L. & Lepschi, 1999; Craven, L. A. , 2009). Hybridisation occurs widely across the genus and examples noted in both the field and the herbarium have been listed by Craven, Lyn A (2006). In all, over 20 examples are known. It is expected that, as comprehensive DNA studies are undertaken on species complexes within the genus, more will become known as to the extent of past and more recent hybridisation events (Edwards et al., 2010).

The majority of Melaleuca species are diploid with 2n=22 (Rye, 1979). Polyploidy appears to be relatively infrequent in this genus, with only a few recorded instances of aneuploidy (2n + 2 =24), triploidy (3n = 33), tetraploidy (4n = 44) and hexaploidy (6n = 66) (Brophy et al., 2013; Rye, 1979) which have been linked with hybridisation and apomixes. Apomictic species may have populations that are diploid, triploid or tetraploid (Dickinson et al., 2007).

About 160 species of Melaleuca species produce only morphologically bisexual (hermaphroditic) flowers (e.g., M. quinquenervia and M. viminalis). About 90 species are

always andromonoecious, that is, there are male and hermaphroditic inflorescences on the one plant (e.g., M. gibbosa and M. uncinata). About 30 species include some plants that are hermaphroditic and others that are andromonoecious (e. g. , M. hamulosa and M. incana) (Brophy et al., 2013). One species, M. cornucopiae, is particularly interesting as some plants are monoecious (with both male and female inflorescence on the same plant) and others are gynoecious (with female inflorescence only). Generally broad-leaved paper bark group are hermaphroditic; all of the broombrush group are andromonoecious (Baskorowati, L. et al., 2010).

2.6 Characteristics of the study species: Melaleuca irbyana and Melaleuca bracteata

Melaleuca irbyana is a small to medium size tree, (8-12 m high), with black papery bark and tiny leaves ( 4- 5 mm long and 1- 1. 5 mm wide) ( Barlow & Cowley, 1988) . Branchlets are shortly puberulous or glabrous, growing mostly under the shade of eucalypt trees. Melaleuca bracteata is a large shrub to a medium tree usually up to 15 m tall, with small, narrow and hairy leaves, (3-12 mm long) (Byrnes, 1986). Flowers of M. irbyana are white-cream, 6-12 or 15 triads and 10-25 mm long with 8-12 stamens per bundle (Carrick & Chorney, 1979). In M. bracteata, flowers are small bottlebrushes up to 20 mm long and occur near the end of the twigs. The fruit and seed set in M. irbyana and M. bracteata are similar. Their fruits are woody, small, cup-shaped capsules. In M. irbyana the capsules are 3-5 mm long, 3.5-4 mm wide. Flowering in M. irbyana occurs in spring and summer (Barlow & Cowley, 1988). In M. bracteata the capsules are 2-3 mm long and 2. 5-3 mm wide appearing on branches. Generally M. bracteata can flower for the whole year, but more in spring and summer (Barlow & Cowley, 1988; Van et al., 2002) (Figures 2. 3-2. 4; Table 2.4).

C A B

Figure 2.3 Pictures of Melaleuca irbyana: (A) dense thickets of mature trees; (B) leaves and flowers; ( C) fruits/ capsules containing seeds. ( Photograph taken by Thita Soonthornvipat, 2014).

A B C

Figure 2. 4 Pictures of Melaleuca bracteata: ( A) mature trees; ( B) leaves and flowers (Photograph taken by Thita Soonthornvipat, 2016); (C) fruits/ capsules containing seeds (Tanetahi, 2010).

Melaleuca irbyana is generally found growing on poor draining clay soil, and M. bracteata is generally found growing close to waterholes and along watercourses where heavy-textured deep clay soil persists. The distribution of M. irbyana is in North-eastern New South Wales and South-eastern Queensland; while, M. bracteata is distributed through Eastern Australia from central New South Wales to Cape York Peninsula, Western Queensland, Central Australia and in the Pilbara and Kimberley of Western Australia (Barlow, 1988; Byrnes, 1986) (Figure 2. 5). Recent mapping activities found only 998 hectares of M. irbyana forest remaining, which is about 8% of its pre-European distribution ( Accad & Neil, 2006) . Melaleuca irbyana populations’ mono- dominant stands, often referred to as “thickets”, grow in poorly draining clay soils known as alluvial or cracking clays (Barlow, 1988).

Table 2. 4 Comparison in morphology and distribution between Melaleuca irbyana (narrowly distributed species) and Melaleuca bracteata (widespread species).

Characteristics Melaleuca irbyana (narrow range Melaleuca bracteata (common species) (Barlow & Cowley, 1988) species) (Byrnes, 1986) Habit Shrub or tree 8-12 m tall, under canopy Shrub or tree usually up to 15 m tall of eucalypt trees. Leaves Leaves ovate 4-5 mm long, 1-1.5 mm Small, narrow 3-12 mm long, 2.5-3 wide, acuminate with 1-3 longitudinal mm wide, hairy venation visible Flowers White-cream, inflorescence 6-12 or 15 White-cream, small bottlebrushes up triads, 10-25 mm long, stamens 8-12 per to 20 mm long, near the end of twigs bundle Fruits Woody, small, cup-shape capsules 3-5 Woody, small, cup-shaped capsule 2- mm long, 3.5-4 mm wide 3 mm long and 2.5-3 mm wide on branch Seed 200-300 very small seeds in each capsule 200-300 very small seeds in each capsule Habitat Poorly draining clay soils as alluvial or Around waterholes and along cracking clays watercourses often where heavy- textured deep clay soil persists Distribution North-eastern New South Wales and Eastern Australia from central New South-eastern Queensland (Figure 2.5, South Wales to Cape York Peninsula, A) Western Queensland, Central Australia and in Pilbara and Kimberly (Figure 2.5, B) Flowering time Winter to Spring Generally whole year but more in winter, spring and summer

A B

Figure 2. 5 Distribution map based on Australian herbarium records for (A) Melaleuca irbyana (red dots), (B) Melaleuca bracteata (red dots). Maps were generated using the online tool the Atlas of Living Australia (map created on 27/7/2016).

2.7 Conclusion

The literature review identifies knowledge gaps relating to rare plants that this study aims to address. In particular, little has been studied on M. irbyana despite its rarity. There is a dearth of information on why M. irbyana has restricted distribution compared with co- occurring and common congener M. bracteata. Although there have been some studies on Melaleuca spp. comparing rare with common and contiguous species in terms of seed biology, physiological and ecological characteristics, most of these studies did not factor in influences of local habitats on the life cycles of seed germination, seedling establishment, nutrient acquisition and use in natural conditions (Baskorowati et al., 2010; Edwards et al., 2010; Geary et al., 1981; Gomes & Kozlowski, 1980; Hamilton-Brown et al., 2009; Hewitt et al., 2014; Hewitt et al., 2015; Martins et al., 2013; Thai et al., 2005). Thus, this study will quantify and compare the dynamics of seed biology, seedling growth and survival rate, including mature tree traits associated with resource acquisition and performance, for both M. irbyana and M. bracteata. I expect to find that M. bracteata exhibits superior characteristics to M. irbyana in terms of better fitness and survival under environmental stress, and more efficient nutrient uptake, resulting in it spreading more widely. My research findings are aimed at assisting conservation and management efforts for remaining populations of M. irbyana, and have the potential to improve the effectiveness of management efforts to restore populations of M. irbyana.

Chapter 3 Comparing in vitro and in situ germination attributes between a rare and a common Melaleuca species

The figure shows remnant mature trees of both Melaleuca species co-occurring at Henderson Reserve site, Jimboomba: (A) Mature trees of Melaleuca irbyana; (B) Mature trees of Melaleuca bracteata.

3.1. Introduction

Seed germination is the most important stage in the life cycle of a plant, as it enables a plant to initiate its growth ( Cerabolini et al. , 2004; Donohue et al. , 2010; Fenner & Thompson, 2005) . The amount of time it takes for a seed to germinate ( germination initiation) and the number of seeds that successfully germinate over time, define germination success rates as important drivers of the early stages of plant population dynamics, and have been found to be predictors of species success over other stages in their life cycle (Ramírez- Padilla & Valverde, 2005; Soltani et al., 2002). Studies have found that some threatened or rare plant species will only germinate under a narrow range of environmental conditions (Murray et al., 2002). More common species are often, but not always, found to germinate under a wide range of environmental conditions, facilitating opportunities for these species to grow under a wider range of habitat conditions, and thereby increasing their chance of having more common distributions (Baker, 1974; Lockwood et al., 2005).

A species is considered rare when its distribution is restricted and/ or occupying specific habitats (Ramírez-Padilla & Valverde, 2005). Species with small populations both in size and range and those displaying low reproductive capacities are highly vulnerable to extinction (Krebs, 1994). Because dispersal and propagule pressure are key characteristics defining population growth and spread, a comparative understanding of the reproductive biology and seed germination requirements may provide important clues as to why some plants species have restricted or widespread distribution ( Ramírez- Padilla & Valverde, 2005).

Maintaining viable populations that are able to continue to efficient reproduction and have increased genetic diversity is important for species survival, especially for those species that are threatened with extinction (Gaston & Fuller, 2008; Yates & Broadhurst, 2002). Poor reproductive success may be explained by low flower production, specialised pollination requirements, low seed set, and overall poor recruitment success, which may be explained by dispersal, germination and seedling establishment (Hobbs & Yates, 2003). Small plant populations are at risk of failure at the reproductive stage and further demographic decline, even without noticeable external threats to their population persistence (Gaston & Kunin, 1997). Thus, fragmentation and low numbers of small populations are

likely to have bottleneck populations and may lead to reduced genetic diversity and decreased gene flow between populations (Westergaard et al., 2011).

Comparing seed germination rate between rare and common plant species that co- occur is a key point to consider when developing conservation plans for rare species. Several studies on seed germination between rare and common plant species have shown that the critically endangered plant species frequently exhibit low germination rates and slow seedling emergence in comparison to the common species. For instance, a study of seed size under different water, temperature, and light regimes between common and endangered Melaleuca spp. of rare M. deanei, and the three common congeners of M. styphelioides, M. thymifolia, and M. nodosa, found that M. deanei showed the lowest capability to germinate in all experiments of temperature, fire, light, and shade regimes ( Hewitt et al. , 2015) . Likewise, Ranieri et al. (2012) tested seed germination of three species in the Gesneriaceae family (tribe Sinningieae) which share the same habitat zone in rocky fields. They compared seed germination rate between restricted Sinningia rupicola and two common Paliavana sericiflora and Sinningia allagophylla under controlled light but varied temperature (10- 40°C), and found that at 15-30°C S. rupicola had a lower germination rate (45%) than both P. sericiflora (80%) and S. allagophylla (90%). Another study on the effect of fire (heat and smoke) between two rare (A. ausfeldii and A. williamsonii) and three common Acacia spp. (A. pycnantha, A. genistifolia and A. paradoxa) (Brown, J. et al. , 2003) found a low percentage of germination and a slow germination rate in the Acacia species. Similarly, a study of light treatments under natural habitats between narrow endemic Petrocoptis species (P. grandiflora and P. viscosa) (Navarro & Guitian, 2003) found a low percentage of germination and a slow germination rate in these narrow endemic species. Additionally, Prober (1992) studied five temperature regimes in a controlled environment of restricted Eucalyptus paliformis and common E. fraxinoides, and found that the restricted species had a low germination rate. Moreover, Prober (1992) also found that low temperatures reduced the rate of seed germination in both common and restricted species. Similarly, the study between rare mariposa lilies (Calochortus obispoensis, C. tiburonensis, and C. pulchellus) and common lilies (C. albus) on seed germination in natural habitats found that the rare Calochortus species showed low levels of seedling establishment compared to the common species (Fiedler, Peggy Lee, 1987). Based on the above reviews, it appears that critically

endangered species have frequently exhibited a low capacity for seedling establishment in contrast with co-occurring congeneric species that are more common.

Soil moisture or soil water holding capacity ( field capacity) is one of the most important factors in the early stages of seed germination and seedling growth (Ahmad et al., 2009). A drought environment negatively influences seed germination and can severely affect the early stages of seedling growth (Guan et al. , 2013). For instance, comparative studies in seed germination between five restricted Acacia species (A. cincinnata, A. elata, A. fulva, A. trachyphloia, and A. silvestris) with five common Acacia species ( A. melanoxylon, A. irrorata, A. implexa, A. dealbata, and A. mearnsii) in Eastern Australia, found that all seed germination occurred at 20°C where adequate soil moisture was present (Pohlman et al., 2005). However, they found that common Acacia species responded better to soil water availability than the restricted Acacia species (Pohlman et al., 2005). Another study conducted under field conditions with the tree species Acacia suaveolens found that seedling emergence and survival were affected by soil water stress, with the highest seedling mortality occurring under low soil water potential (Auld, 1987). Therefore, understanding soil water availability is an important consideration for understanding seed germination and seedling survival of a rare plant in its conservation and management (Rühl et al., 2015).

Soil organic matter can act to retain water making it more available for plants and potentially increasing nutrient availability (Thornton et al., 2015). Organic matter in the soil can also provide beneficial services such as decreasing water-runoff, ameliorating oxygen levels by increasing aeration, and providing better soil structure (Diacono & Montemurro, 2010). Soil organic matter can then be used as an indicator of soil structure that is more conducive to seed germination and plant growth (Herridge, 2011).

Temperature and light are also important cues for seed germination, particularly when adequate soil moisture is present (Ghaderi et al. , 2008; Mijani et al. , 2013; Tilki & Çiçek, 2005) . Temperature is an important factor in controlling germination in natural environments (Probert, 2000). However, temperature and light requirements for germination vary from species to species (Phartyal et al., 2003). For instance, Geissler and Gzik (2010) stated that fluctuating daily temperatures, set at an incubator of 12/12 hours in temperature regimes (22/ 15°C and 22/ 10°C), did not have an effect on germination of Juncus atratus and Gratiola officinalis even in a suitable microsite. Temperature changes may influence

processes which control seed germinability after absorption of water. Temperature affects germination by breaking the seed coat and increasing membrane permeability, the membrane-bound activities, and the cytosolic enzymatic activities which break down the stored food resources (Jalilian et al. , 2005; Zhu et al. , 2006). On the other hand, high fluctuation in temperatures as a result of climate change may lead to decreased germination success rates in some plant species (Luna et al., 2012).

Due to the limited number of studies on the ecology of M. irbyana, little is known about the optimal germination requirements for this species. The purpose of this study was to investigate whether the influence of temperature, light, and soil moisture on seed germination could potentially explain the restricted distribution of M. irbyana as compared to the more common and co-occurring relative, M. bracteata. The study sought to compare the time needed to germinate and germination success rates between M. irbyana and M. bracteata under controlled conditions, and under more natural conditions in the field. Specifically, the study addressed the following questions: 1) Do endangered M. irbyana and common M. bracteata show specific germination response patterns to temperature and photoperiod regimes? The hypothesis is that endangered M. irbyana will show lower germination percentages and slower germination rates than M. bracteata with respect to temperature (low/high) and photoperiod (short/long). 2) Do endangered M. irbyana and common M. bracteata show specific response patterns of germination when they are in the natural environment? The hypothesis is that M. irbyana seeds will germinate optimally in situ under the tree canopy of mature remnant M. irbyana habitats, and M. bracteata will be able to germinate over a wider range of habitats.

3.2 Materials and methods

3.2.1 Seed germination

3.2.1.1 In vitro germination experiment

The main objective of in vitro ( within the laboratory) germination trials was to compare the time to initial germination and germination success rate in M. irbyana and M. bracteata under different temperature and photoperiod regimes. The temperatures (15°C, 25°C and 30°C) (Figure 3.1) and photoperiods (10, 12 and 15 hours) (Table 3.1) were used to mimic seasonal changes occurring in the natural environment ( Jimboomba site, Queensland, Australia) where these plants occur (Bureau of Meteorology, 2014). A no light (total darkness) treatment was included to determine if the photoperiod and consequently light availability had an effect on seed germination of M. irbyana and M. bracteata. Seeds of both species were collected from the Logan region, Brisbane, Australia in 2014. Seeds were stored at room temperature in a cool and dry place in bags inside a desiccator ( containing silica gel) to ensure no loss of viability before the commencement of germination trials. Melaleuca irbyana seeds weigh approximately 0. 0045 g per 100 seeds, while M. bracteata seeds weigh approximately 0.0031 per 100 seeds (Figure 3.2). For each treatment, 3 replicates of 100 seeds of M. irbyana and M. bracteata were placed in 9 cm Petri dishes on filter paper (Whatman® number 1) moistened using deionised water. The Petri dishes were then randomly positioned within growth chambers (Adaptis by Conviron, series CMP 6010) set to the appropriate temperatures and photoperiods as indicated above. The Petri dishes were checked daily and additional deionised water was added to Petri dishes when required to maintain a constant moisture level during the experiment. Seeds were considered to have germinated when there was emergence of a radicle (Baskin, C. C. & Baskin, 1998). The number of days until radicle emergence (radicle length >3mm) were recorded daily for four weeks. Germinated seeds were counted and removed from the Petri dishes daily.

C) 0 20

Temperature Temperature ( 10

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

Mean maximun temperature Mean minimun temperature

Figure 3. 1 Monthly mean maximum and minimum temperature at Henderson Reserve site during 2013-2014. Values are from Australian Bureau of Meteorology, 2014.

Table 3.1 Monthly Mean Daily photoperiod (hours) in Brisbane

Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Annual 2013 13.5 13.4 12 11.2 10.7 10.3 10.4 10.9 11.8 12.6 13.3 13.5 11.97 2014 13.4 12.8 12 11.3 10.5 10.3 10.3 11 11.7 12.5 13.3 13.5 11.88 * Source: Bureau of Meteorology of Australia

Figure 3.2 Comparison of seed size between Melaleuca irbyana and Melaleuca bracteata. Melaleuca irbyana seeds weigh approximately 0. 0045 g per 100 seeds while Melaleuca bracteata seeds weigh approximately 0.0031 g per 100 seeds.

3.2.1.2 In situ germination trial a

This experiment was set up in September 2015 at Henderson Reserve, located in Jimboomba, South-east Queensland, and run until December 2015 (spring/summer seasons in Australia). During this period, the average minimum temperature was 16°C, while the average maximum temperature was 28°C (Tables 3.2-3.3; Figure 3.1). The average rainfall was 54 mm (Table 3.4) and photoperiod ranged from 12-14 hours on average (Table 3.5) (Bureau of Meteorology, 2016). Four plots of 1 m × 1 m were established under M. bracteata thickets. The experimental design was a factorial of the two plant species × four blocks × with and without litter, totalling 16 sub-plots. The litter largely consisted of leaves and twigs of plants in the area including Eucalyptus trees. In eight sub-plots 20 seeds of M. irbyana were planted, and in the remaining eight sub-plots 20 seeds of M. bracteata were planted. For each species, four sub- plots were covered with litter and four sub- plots remained uncovered ( no litter) . The number of seedlings that emerged from each sub- plot was recorded weekly for 14 weeks (Figure 3.3, A - D).

Figure 3. 3 In situ seed germination experimental set- up at Henderson Reserve site, Jimboomba (A and B). Each experiment was divided into 16 sub-plots of 1 m × 1 m (C and D). Seeds mixed with sand were sown in summer (C) and winter (D).

To understand the effects of seasonality on germination in situ, the aforementioned experiment was also repeated again in the autumn/ winter season from April 2016 to September 2016. During this period, the average minimum temperature was 11°C, and the average maximum temperature was 24°C (Tables 3.2-3.3; Figure 3.1). The monthly rainfall and photoperiod in spring/ summer was 58 mm and 13 hours respectively, whereas in autumn/winter rainfall was 36 mm and photoperiod was 11 hours (Tables 3.4-3.5) (Bureau of Meteorology, 2016). The duration of data collection in this experiment was longer (26 weeks) than for the spring/ summer experiment, as seeds took longer to germinate than in the spring/ summer germination in situ. Experimental plots were established in different areas during the spring/ summer experiments versus autumn/ winter trials. However, the characteristics of these plots were similar to those used for the spring/ summer in situ seed germination (Figure 3.3).

Table 3.2 Monthly mean minimum temperatures (degree Celsius) in Brisbane

Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Annual 2015 21.6 20.3 20.3 15.9 13 11.2 8.8 10.5 11.3 14.1 16.7 18.6 15.2 2016 19.6 21.0 20.0 17.5 13.4 11.9 11.0 10.7 14.1 14.6 18 19 15.9 * Source: Bureau of Meteorology of Australia

Table 3.3 Monthly mean maximum temperatures (degree Celsius) in Brisbane

Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Annual 2015 30.2 28.4 29.6 26.0 24.3 21.3 20.6 23.3 24.3 26.8 29.1 29.0 26.1 2016 29.9 31.3 29.4 28.1 26.8 21.6 22.4 22.4 24.9 27 29.3 30 24.68 * Source: Bureau of Meteorology of Australia

Table 3.4 Monthly Rainfall (millimetres) in Brisbane

Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Annual 2015 155.5 243 126.9 199 250 53.7 19 23 30.2 52.3 88.3 61 108.56 2016 74.7 26.3 79.1 13 15.5 18.9 19 40.8 54.5 68 53 145 64.83 * Source: Bureau of Meteorology of Australia

Table 3.5 Monthly Mean Daily photoperiod (hours) in Brisbane

Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Annual 2015 13.4 12.8 12.1 11.3 10.5 10.2 10.3 10.9 11.7 12.5 13.2 13.5 11.88 2016 13.4 12.8 11.9 11.2 10.5 10.3 10.4 11.33 12.5 12.5 13.2 13.5 11.96 * Source: Bureau of Meteorology of Australia

3.2.2 Soil sampling and analyses

Soil sampling was undertaken around both M. irbyana and M. bracteata remnant mature trees at Henderson Reserve site in Logan City in order to compare microsite soil conditions between remnant forests dominated by both species. In each plot, soil samples were collected from a depth of between 0-15 cm using a soil auger sampler. Soil samples were refrigerated at 4°C and analysed for different chemical properties that are described below.

3.2.2.1 Soil organic matter

Soil organic matter and moisture content were measured using Loss on Ignition methods. Loss on Ignition ( LOI) quantifies the organic matter content (%OM) of soil samples. Soil samples were dried at 40°C for 24-48 hours. The dried samples were ground with Cr-steel Ring Mill Pulveriser (ROCKLABS®) to produce homogenous samples for laboratory analyses. Ring mill cups were washed and dried between samples to avoid sample cross contamination. Ceramic crucibles were weighed and recorded, then samples were weighed at approximately 5 g in the crucible and dried in the thermostatically controlled oven set at 105°C for 24 hours. After cooling in a desiccator, samples were reweighed, and then placed in a muffle furnace (Thermolyne® Small Benchtop) at 375°C for at least 16 hours. The dried samples were removed from the furnace, cooled in the desiccator and reweighed. The percentage of organic matter was calculated using the following equation:

푝푟푒 푖푔푛푖푡푖표푛 푤푒푖푔ℎ푡 (푔) − 푝표푠푡 푖푔푛푖푡푖표푛 푤푒푖푔ℎ푡 (푔) %푂푀 = × 100 푝푟푒 푖푔푛푖푡푖표푛 푤푒푖푔ℎ푡 (푔)

3.2.2.2 Soil moisture

Soil moisture content ( MC) was measured following a standard protocol (Department of Sustainable Natural Resource, 1990). Approximately 5 g of sieved soil was placed in the ceramic crucible. The samples were dried in the controlled temperature oven set at 105°C for 16 hours. The dried samples were removed and cooled in the desiccator and reweighed. The moisture content was calculated using the formula below:

(푊2 − 푊3) 푀퐶% = × 100 (푊3 − 푊1)

where, W1 = Weight of crucible (g) W2 = Weight of moist soil + crucible (g) W3 = Weight of dried soil + crucible (g)

3.2.1.3 Data analyses

Time- to- event analysis is most useful in analysing seed germination data with standard statistical models, including Kaplan- Meier estimators ( McNair et al. , 2012) . Germination rates were calculated using Kaplan-Meier survivor function estimators, where germination probability and ANOVA were generated to investigate differences in germination using the R statistical program on RStudio, and R version 3. 3. 0 functions ‘survfit’ and ‘survreg’ in the R survival package. In general, this package have two variables at any one time: the number of deaths, d(ti), and the number at risk, r(ti) (i.e. those that have not yet died: the survivors). The Kaplan-Meier survivor function model is depicted below (Crawley, 2014, p. 876).

푟(푡푖) − 푑(푡푖) 푆̂퐾푀 = ∏ 푟(푡푖) 푡푖<푡

This model takes into consideration delays in the onset of seed germination as well as the mixture of germinable and non-germinable seeds. For the seed germination analysis, the data from both in vitro (controlled growth chamber) and in situ (field condition) were analysed with the RStudio and R program (R development Core Team 2013); R: language and environment for statistical computing. R.F.S Computing Vienna, Austria, version 3.3.0 functions ‘survfit’ and ‘survreg’ from R ‘survival’ package (Crawley, 2014; Therneau, 2016) (http://www.R-project.org/) (https: //cran.rproject.org/web/packages/survival/survival.pdf). The package survival requires the status variable to be expressed in binary terms: 0 (not germinated) and 1 (germinated); thus, seedlings which had a growth were considered to

have a status of one: they survived, while those with growth of zero remained not germinated. The temperature and photoperiod regimes were included as fixed effects in Analysis of Variance (ANOVA) models testing germination indices. A Tukey’s Honest Significant Test (HSD) was performed to assess the germination differences between the temperature and photoperiod treatments. One of the limitations to the in vitro experiments described here is that the day and night temperatures were kept constant at 15°C, 25°C and 30°C, which are different from differential temperatures that seeds experience when germinating in situ. Furthermore, due to limitations of the equipment used, the light intensity within the growth chambers for all treatments (except no light treatments) was maintained at a constant 500 µmoles m-2 s-1.

In situ germination trials were analysed using the same analysis as for in vitro trials, but field data was analysed using an ANOVA with an error structure, to compare seed germination success between two species. The nested analyses in the S-plus concept of an error structure of plots nested within sites were used (Crawley, 2014).

Soil characteristics were analysed using an ANOVA with an error structure to test differences between two species in the same conditions. Models were developed with an error structure of plots nested within sites as representative of the experimental design used to collect the soils.

3.3 Results

3.3.1. In vitro germination experiment

3.3.1.1 Effect of temperatures and photoperiods on seed germination

The germination success of M. irbyana and M. bracteata varied significantly depending on temperature and photoperiod. Melaleuca irbyana showed a significantly lower total seed germination rate than M. bracteata (F1, 2136 = 1588.82, P < 0.001), and this trend remained consistent across temperatures (F2, 2136 = 571. 35, P < 0. 001) and photoperiod duration (F3, 2136 = 57. 82, P < 0. 001) (Table 3. 6). Melaleuca bracteata showed high germination success under all temperatures (15, 25, 30°C) and photoperiods (10, 12, 15 hours). However, M. irbyana only showed high germination success under the warmer temperatures 25°C (52%) and 30°C (71%) respectively (Figures 3.4-3.6). For all possible combinations of temperature and photoperiod, the Tukey HSD post hoc test indicates significant differences in germination response between M. irbyana and M. bracteata (Table 3.7).

Table 3. 6 Result of ANOVA comparing total germination success between Melaleuca irbyana and Melaleuca bracteata, temperatures (15, 25, 30°C) and photoperiods (10, 12, 15 hours).

Variables Df Sum of squares F-value P-value

Temperature 2 661146 571.35 <2e-16 ***

Photoperiod 3 100355 57.82 <2e-16 ***

Species 1 919256 1588.82 <2e-16 ***

Temperature × Photoperiod 6 64196 18.49 <2e-16 ***

Temperature × Species 2 144647 125.00 <2e-16 ***

Photoperiod × Species 3 390041 22.49 2.46e-14 ***

Temperature × Photoperiod × Species 6 24836 22.49 1.44e-07 *** Residuals 2136 1235845 7.15 Significant codes: ***P < 0.001

Table 3. 7 Results of Tukey’s HSD test performed on an ANOVA of germination data for Melaleuca irbyana and Melaleuca bracteata.

Treatment M. irbyana × M. bracteata P- value Temperature / photoperiod 15°C /dark < 0.05 25°C / dark < 0.05 30°C / dark < 0.05 15°C / 10 hours < 0.05 25°C / 10 hours < 0.05 30°C / 10 hours < 0.05 15°C / 12 hours < 0.05 25°C / 12 hours < 0.05 30°C / 12 hours < 0.05 15°C / 15 hours < 0.05 25°C / 15 hours < 0.05 30°C / 15 hours < 0.05

3.3.1.2 Time to germination initiation

The amount of time needed to initiate germination differed significantly between M. irbyana and M. bracteata, with M. bracteata showing the fastest time to initial germination. At 15°C under all photoperiods, M. bracteata seeds started germination earlier (approximately five days) than M. irbyana (~15 days) (Figure 3.4; A-D). However, the results showed that at a photoperiod of 15 hours, M. bracteata took only one day for seed to initiate germination under 15°C (Table 3. 8). Additionally, at 25 and 30°C, M. bracteata seeds germinated at approximately two days and one day respectively; while M. irbyana took approximately five days at 25°C and four days at 30°C (Table 3. 8). Measurement at all temperatures (15, 25 and 30°C) and 12 and 15 hours of light showed that M. irbyana seeds germinated earlier than at 10 hours photoperiod or total darkness (Figure 3. 5; E-H and Figure 3.6; I-L). At 25°C and 30°C M. irbyana seeds germinated approximately five days earlier, as did M. bracteata, under all light conditions (Figures 3.5-3.6). However, M. bracteata’ germination rate was significantly higher than for M. irbyana at both 25°C and 30°C. The germination of M. irbyana seems inhibited by a decrease in temperature and the most suitable temperature was found to be 30°C.

Table 3.8 Effect of temperature and light regimes on the time (day) to germination initiation in Melaleuca irbyana and Melaleuca bracteata.

Treatment Time (days) to germination initiation (Temperature/photoperiod) M. irbyana M. bracteata 15°C / dark 19 6 15°C / 10 hours 18 6 15°C / 12 hours 17 6 15°C / 15 hours 12 1 Mean response 16.5 4.75 25°C / dark 4 2 25°C / 10 hours 5 2 25°C / 12 hours 5 2 25°C / 15 hours 6 2 Mean response 5 2 30°C/ dark 3 1 30°C / 10 hours 4 1 30°C / 12 hours 4 1 30°C / 15 hours 4 1 Mean response 3.75 1

Figure 3.4 Kaplan-Meier estimates of germination functions for the first germination seeds in growth chamber between Melaleuca irbyana and Melaleuca bracteata under 15°C and different photoperiod conditions ( A- D). Orange lines indicate the first instance of germination in each of the conditions.

Figure 3.5 Kaplan-Meier estimates of germination functions for the first germination seeds in growth chamber between Melaleuca irbyana and Melaleuca bracteata under 25°C and different photoperiod conditions ( E- H) . Orange lines indicate the first instance of germination in each of the conditions.

Figure 3.6 Kaplan-Meier estimates of germination functions for the first germination seeds in growth chamber between Melaleuca irbyana and Melaleuca bracteata under 30°C and different photoperiod conditions (I-L). Orange lines indicate the first instance of germination in each of the conditions.

3.3.2 In situ germination trial

3.3.2.1 Spring/summer season (September – December 2015)

Total germination rate

Overall, soil moisture contents in M. irbyana and M. bracteata understoreys did not differ significantly being estimated at 21% and 22 % moisture respectively (F1, 6 = 2.02; P = 0. 20; Figure 3. 7; A). Similarly, no significant difference was found in organic matter between M. irbyana and M. bracteata sites (8.76% and 7.95% respectively) (F1, 6 = 0.18; P = 0.68; Figure 3.7; B).

Figure 3. 7 Comparison estimate of mean for soil between Melaleuca irbyana (MI) and Melaleuca bracteata (MB) microhabitat (A) Moisture content (%); and (B) Organic matter (%).

Seed germination rates in situ were not found to differ significantly between M. irbyana and M. bracteata sites (F1,1175 = 0. 31, P = 0. 58; Table 3. 9). For both species, germination in the field started after six weeks under M. bracteata overstoreys that were covered with litter. The percentage of M. bracteata seedlings that emerged was estimated at 0.15% (2 of 1280); while the percentage of M. irbyana seedlings that emerged was estimated at 0.08% (1 of 1280) (Figure 3.8; D). After week 6, no further germination was recorded until week 10 for both species. In week 10, for the sub-plots in M. irbyana microsite that were not covered with litter, the total percentage of M. bracteata seedlings that emerged was

estimated at 0. 23% ( 3 of 1280) , whereas the percentage of M. irbyana seedlings that emerged was estimated at 0.31% (4 of 1280) (Figure 3.8; A). In sub-plots of M. irbyana microsite covered with litter and sub-plots of M. bracteata microsite without litter cover, both species had the same average percentage of seedlings emerged, at 0. 23% (3 of 1280) ( Figure 3. 9; B- C). The results showed that no seedling survival was recorded after emergence until end of the experiment.

Table 3. 9 The ANOVA result of abiotic factors (climatic conditions related to season) on total germination between Melaleuca irbyana and Melaleuca bracteata seeds under canopies of Melaleuca irbyana and Melaleuca bracteata remnant mature trees with an error structure of plots nested within sites.

Predictor variables Df Sum of squares F-value P-value Species 1 0.05 0.31 0.58 NS Week 1 0.31 2.14 0.14 NS Overstorey with litter treatment 3 0.54 1.24 0.29 NS NS Species × Week 1 0.31 0.00 0.93 Species × Overstorey 3 0.00 1.37 0.25 NS Week × Overstorey 3 0.21 0.49 0.69 NS NS Species × Week × Overstorey 3 0.31 0.70 0.55 Residuals 1775 259.69 S Significant codes: NS (non-significant)

Figure 3. 8 Seed germination at Henderson Reserve site, Jimboomba. ( A) Seedlings of Melaleuca bracteata (yellow circles) under M. bracteata overstorey with the litter treatment (B) Seedlings of Melaleuca irbyana (red circles) under M. irbyana overstorey without the litter treatment; (C) Seedlings of M. irbyana (red circles) under M. bracteata overstorey without the litter treatment; (D) Seedlings of M. irbyana (red circles) under M. irbyana overstorey with the litter treatment.

(A) (B)

Figure 3.9 Kaplan-Meier estimates of seed germination functions for the first germination seeds in field conditions (Henderson Reserve site) between Melaleuca irbyana (MI) and Melaleuca bracteata (MB) under different understoreys of remnant mature trees. Orange lines indicate the first instance of germination in each of the conditions.

3.3.2.2 Autumn/winter season (April - September 2016) No M. irbyana and M. bracteata seedlings were recorded during this experimental period.

3.4 Discussion

3.4.1 In vitro germination experiment

Total germination rate

In line with our hypothesis, M. irbyana showed a lower germination success rate than its more commonly distributed congener M. bracteata under all conditions investigated in this study. However, at high temperatures (25-30°C) we found M. irbyana presenting relatively high total seed germination rate, especially when seeds were exposed to long photoperiods (12 and 15 hours). In response to a decline in temperature, there was a stronger decrease in germination performance of M. irbyana than of M. bracteata. Low germination performance by M. irbyana could potentially result in lower recruitment in the field. Temperature and photoperiod were also found to have an interacting significant effect on the germination initiation for M. irbyana, suggesting that this rare and critically endangered species has likely a narrower germination niche than M. bracteata. The relationship between temperature and light conditions may also substantially impact on germination, including increasing seedling survival rate and seedling establishment through provision of synergistic environmental resources ( Bakker, 2001) . Thus, optimal germination conditions for M. irbyana appear to occur under warm temperatures with longer photoperiods. Previous studies have found that declines in populations exhibited by endangered species were related to low germination rates or specific germination requirements (Hölzel & Otte, 2004; Schütz, 2000; Schütz et al., 2002). This is also consistent with Hewitt et al. (2015), who studied one rare (Melaleuca deanei) and three common (Melaleuca styphelioides, M. thymifolia and M. nodosa) Melaleuca species of the Sydney region. They found that the rare M. deanei had specific germination requirements, which resulted in its being rare (Hewitt et al. , 2015). Thus, slow germination rates in rare Melaleuca species may result in their limited germination niche, whereas common Melaleuca species that are able to germinate faster tend to have enhanced capacity for colonisation success and hence to have greater distribution capacity. As a common species, M. bracteata presented higher capability of germination (germination rate and less time to germination initiation) that could potentially facilitate its establishment even at low temperatures and short periods of light. It appears that this species

is therefore able to persist in unfavourable conditions. This is corroborated in several studies of germination traits comparing common and endangered plant species, that found common species may respond more flexibly to abiotic stress than endangered plant species, even in changing conditions related to climate change ( Rühl et al. , 2015) . A narrower set of germination requirements may contribute, then, to the narrow distribution or the rarity of M. irbyana species in Queensland. This result is consistent with another study comparing the effects of constant temperature (5, 10, 15, 20 and 25°C) and two alternating temperatures with 8 hours of light (25/10 and 30/15°C) on seed germination in Central-eastern Sardinia ( Italy) ; the authors found that common ( Centranthus ruber) species were capable of germination over a wider range of temperature and photoperiods than endangered plant species (Centranthus amazonum) (Mattana et al., 2010).

These results show that temperature and photoperiod influence M. irbyana germination performance, as our results of time to germination initiation showed that at room temperature (25°C), M. irbyana had a faster germination speed (~5 days) than at 15°C (~17 days). Ambient temperature has been identified previously as a cue for initiation of germination, especially during the periods when water availability was not limited (Mijani et al. , 2013). Most Melaleuca species in Australia germinate within the range of 20-35°C, usually taking approximately 5-40 days to initiate germination (Turnbull, J. & Doran, 1987). Results of our study showed that germination in our focal species appears to fall within these general parameters, with both M. irbyana and M. bracteata exhibiting high germination rates above 20°C. Hewitt et al. (2015) compared the germination rates of M. nodosa, M. styphelioides and M. thymifolia, and found that a high percentage of seed germination (60- 80%) occurred at 20 to 35°C.

3.4.2 In situ germination trial

Total germination rate

In general, the total germination rate and establishment of M. irbyana in the field was similar to M. bracteata, with very low germination rates during the spring/ summer season and no germination found during the autumn/ winter season in the years this study was conducted. Additionally, germination and seedling emergence for M. irbyana and M. bracteata were unaffected by four microhabitats of litter conditions, with low numbers of

seedling emergence occurring under canopy for both species. Light availability in the understorey is likely to be one of the limiting factors that explain the low germination rates found. This finding concurs with germination and light condition experiments conducted by Hewitt et al. (2015). Their study compared seed germination between rare Melaleuca deanei with three common M. thymifolia, M. thymifolia and M. styphelioides. They found light played an important role in the seed germination of M. styphelioides, with 95% of final germination occurring under light conditions (low, medium or high), but only 10% of seeds germinating when placed in the dark (Hewitt et al., 2015).

Studies conducted with other Melaleuca spp. showed that low soil water potential reduced both germination rates and success (Bradford, 1995). In this study, there was no significant difference in soil water content and organic matter under canopy of both species. However, the weather conditions over the time period of this study could also be a key factor in contributing to low germination rates in the field for both study species. The field sites received an unseasonably low amount of rainfall during the experiment periods: September to December, 2015 ( ~58 mm) and April to September, 2016 ( ~27 mm) ( Bureau of Meteorology of Australia, 2016). These unusually low rainfall levels during the study in the field experiment may have contributed to lack of seed germination and survival of both species from the first season of experiment, and no result for seed germination in the second season of experiment. Similarly, Raabová et al. (2007) demonstrated that germination percentage of rare Aster amellus was very low (0.7-2%) in the field due to low soil moisture content. Low germination rates in the field experiment could have been because of insufficient soil moisture content, implying that microhabitats were inappropriate for seeds to germinate. Similar results have also been demonstrated by Liu et al. (2011), who found that limited sources of water stops seedling establishment.

Soil type might be another factor contributing to low germination rate in the field. In some plots in the Henderson Reserve site, where field experiments were carried out, generally the soil was characterised as sand-clay loam and heavy clay loam (Offord et al., 2004). Clay soils usually have high water retention capacity, which can lead to water logging during high rainfall periods, resulting in growing conditions that can impact negatively on seed germination (Liu et al., 2011). Studies of germination rates of Camelia nitidissima in sandy soil and clay soil types have shown that this species has a higher germination rate in

sandy soil than in clay soil (Yang et al., 2008). Domènech and Vilà (2008) also found that seeds of Cortaderia selloana had high emergence rates when located in sandy soil and decreased emergence when sites contained a high level of clay. Thus, soil type might be the cause of a low rate of seed germination in some plots.

The difference in temperature in the natural habitats could be another factor resulting in no germination in the autumn/ winter season. According to temperature data from the Bureau of Meteorology in Brisbane (2016), temperatures were above 30°C in summer and below 12°C in winter. Results of our growth chamber experiments showed a low germination rate at 15°C for M. irbyana. The results are consistent with another study on Melaleuca spp. which found that Melaleuca thymifolia germination reduced when temperature dropped below 20°C, with major reductions in germination in winter (Hewitt et al. , 2015). Çiçek and Tilki (2007) who studied three Ulmus species in Turkey, found that temperatures had a significant effect on the germination percentages of some Ulmus spp., particularly Ulmus mino and Ulmus glabra, which produced the lowest germination at 20 °C. On the other hand, high temperatures, may not influence seed germination of Melaleuca spp., which is consistent with the results of Vickers (2004), who found that heat treatments had no effect on M. irbyana germination. Thus, the variation of seed germination in the field cannot be attributed to temperature and light regimes only, but may also be correlated with habitat conditions, particularly water availability (Baskin, C. & Baskin, 2014).

Seed dispersal by insects such as ants, bugs and beetles, or wind, may also be responsible for seed relocation from the area of study, since Melaleuca seeds of the study species are very tiny (Beardsell et al. , 1993; Zomlefer, 1994). To reduce the likelihood of this happening, I mixed seeds with fine sand to prevent seed dispersal, allowing for more accurate observation. Evidence of seed being relocated by ants and insects and so on is in line with observations of Donohue et al. (2010) , who attributed the missing seedling emergence and spread of seeds (Gillia tricolor) outside areas of observation to the small seed size of their reviewed study species.

3.5 Conclusion

Overall, the results of this study have shown that the endangered M. irbyana species showed a lower germination rate under both controlled conditions of growth cabinets than did the common co-occurring M. bracteata, although germination rates for both species were overall much lower in the field. Melaleuca irbyana’s germination success appears to be more sensitive to cooler temperatures and lower light availability than M. bracteata. This study presents the first detailed analysis of the germination characteristics of M. irbyana, providing key information needed to develop more effective prescriptions for propagation this species.

Chapter 4 Understanding differences in seedling microhabitat conditions using a reciprocal planting experiment

Reciprocal planting experimental site: under canopies of Melaleuca irbyana (first) and under canopies of Melaleuca bracteata (second) at Henderson Reserve, Logan City, Brisbane, Queensland.

4. 1. Introduction

Melaleuca irbyana was listed as a critically endangered ecological community under the Environment Protection and Biodiversity Conservation Act 1999 (EPBC Act), due to its very restricted distribution and vulnerability to ongoing threats. Most of the forest has been cleared or modified by anthropogenic activities, feral animals, or invaded by weeds. Recent records show that less than 30% of the original forest remains in Australia (Earless et al., 2002). In spite of its biological and ecological importance, little is known about M. irbyana’s physiological and ecological characteristics. Microclimates influence a wide range of important ecological processes such as seedling establisment, plant growth and soil nutrient (Bonan, 2015). Seedling establishment is an essential stage for tree regeneration (Herrera & Garcia, 2010; Hewitt et al., 2015; Kaye & Brandt, 2005). To date there have been no studies published that investigated the physiological and ecological characteristices of M. irbyana and the common co-occurring M. bracteata. A better understanding of the conditions best suited for M. irbyana seedlings to survive and grow will assist with restoration efforts. Limitations in adaptive abilities for reproduction, dispersal, competition, niche size, recruitment, and the environmental conditions, influence the extent of species distributions (Pulliam, 2000). Thus, understanding the causes for reduced ecological breadth (such as the range of habitats that species successfully occupy and are able to grow and reproduce in) and including species distribution may help describe factors that can be targeted to impact positively on the growth and survival rate of endangered plant species (Matesanz et al., 2009).

There are several models that explain the existence of narrowly distributed species. The refuge model states that endemic species are generally stress-tolerant and do not display specific adaptations to habitats where they occur, but are limited to stressful habitats, where competition from other species is reduced; in this respect species may be rare in terms of distribution but also have stress tolerance (Gankin & Major, 1964; Meyer et al. , 1992). In this hypothesis, narrowly endemic species should show different stress-tolerance traits to commonly distributed species (Poorter, 1999). The narrowly distributed species are usually associated with low shoot and root ratio, low height, low photosynthetic rate, or low specific leaf area (Grime, 1977; 2006; Lavergne et al., 2004). Another model, the specialist model, suggests that rare species are specifically adapted to the habitats where they occur (Meyer,

1986). Under this model, rare species show optimal growth characteristics for surviving under specific habitats, but lack the phenotypic plasticity to grow and prosper under a wide range of habitat conditions (Caley & Munday, 2003). In this context, common species would cope with a wider range of environments, in contrast to specialist species (Richards, C. L. et al. , 2005; Sultan, S. E. , 2000) . The physiological performance of related species or congeneric species, along with their potential to affect plant survival, vegetative growth, and fitness under natural conditions, is a topic that has been rarely studied (Pohlman et al., 2005; Richards, A. et al. , 2003). Plant reciprocal transplanting is generally used as a tool for restoration; plant materials are transferred to a new location or to extinct local sites (Vallee, 2004). This is especially important for the purpose of increasing population numbers in the juvenile stage, particularly in the critically endangered or threatened plant species (Millsom, 2002). Transplanting is also an approach used to determine the critical factors that limit germination and seedling establishment, including growth and survival rate (Moir et al., 2012; Volis & Blecher, 2010) . Thus, understanding the physiological and ecological characteristics in the field of reciprocal transplanting may shed some light on the appropriate habitats for M. irbyanan, for restoration purposes.

Understanding the survival and growth rate of rare and endangered plant species grown in understoreys of local forests can help explain their limited distribution (Winkler et al. , 2005) . Microhabitats play an important role in plant species establishment and restoration (Landero & Valiente-Banuet, 2010). Light is an essential resource for plant growth and development, and in a forest it is the canopy that determines the quantity of light reaching the understorey (Stan & Daniels, 2010). Light availability is a key factor in seedling performance, particularly in terms of the phenotypic plasticity traits, which tend to be low in shade-tolerant species in the natural environment (Valladares & Niinemets, 2008). The distribution of optical properties ( the interpretation of the spectrum such as reflection, transmission and absorption) of the plant canopy may also be considered as part of the canopy structure (Serbin et al., 2011).

The structure of a forest’s canopy can be described by the position, orientation, size and shape of the trees and vines that make up the top layer of the forest (Ross, 1981). Canopy characteristics, such as the size of gaps between neighbouring trees and the number of layers in a canopy, impact plant productivity and germination success rates including the life form

and species type recruited into the understorey (Ryu et al. , 2010). Tree species can be classified as shade tolerant or intolerant (Niinemets, 2010). Shade intolerant tree species such as Tsuga heterophylla and Abies amabillis are unable to grow under low light conditions, but thrive under high light, while others tree species like Thuja plicata are shade tolerant (Stan & Daniels, 2010). Shade tolerant species can adapt their phenotype to cope with shaded environments (Gommers et al., 2013), for example, by increased leaf size, high longevity, and elongation, including high relative growth rate (Valladares & Niinemets, 2008). Evidence suggests that the seedlings of some species perform better under unshaded treatment, such as a rare Melaleuca deanei (Hewitt et al., 2015) and Melaleuca triumphalis (Crase et al., 2006). However, some shade tolerant plant species such as common Melaleuca nodosa and Melaleuca thymifolia would perform better under low and medium shade conditions ( Hewitt et al. , 2015) . The density of surrounding trees may also create a microhabitat where there is competition for light, water and nutrients (de Chantal et al., 2003). Generally, if species are co-occurring with closely related or congeneric species, it may reduce their survival rate, due to increasing competitive interactions for the same essential and often limited resources ( Matesanz et al. , 2009; Prinzing, 2001) . Thus, understanding light availability and how it impacts on seedling survival and growth rate may provide information on seedling establishment and performance under forest canopies.

Seedling recruitment is one factor used to determine the level of survival and growth in critically endangered or threatened plant species, for the purposes of regeneration programs. Recruitment limitation is a major cause of rarity and generally occurs when some species fail to regenerate (Wright, 2002). For instance, Ågren and Schemske (2012) studied the adaptation of reciprocal transplants of Arabidopsis thaliana between two locations, Italy and Sweden, and found that A. thaliana exhibited strong evidence that adaptive differentiation ( ability to adapt to new biotic and abiotic conditions) in tolerance to temperature, especially freezing, contributed to fitness variation and survival rate between the two sites. Webb and Peart (1999) observed seedling survival of 149 species established in Gunung Palung, Indonesia for 19 months, and found that the survival rate had declined with the increase of seedling density (1 m2 quadrats) and of adult basal area (surrounding 0.16-ha). Therefore, seedling recruitment may provide insights that help us understand the adaptive ability in each plant species, especially endangered plant species. This information may be useful in developing restoration and conservation programs for these plants.

Seedling performance is affected by various abiotic factors, but generally light, temperature, water, and nutrient availability are paramount (Bejarano et al. , 2010; Sack, 2004). The relationship between specific environmental factors and differential seedling performance has not been clearly identified (Matesanz et al., 2009). Reciprocal experiments could provide important information concerning the range of local adaptation and highlight the factors responsible for adaptive differentiation in plants (Hereford, 2009). Identification of environmental factors could be used to inform restoration and revegetation programs, or to help achieve higher rates of success for restoration endeavours (Boyd & Davies, 2012). Several studies have been carried out on the effects of canopy habitats on the ecology of low lying vegetation (Maestre et al. , 2003). For example, a study of the performance of Stipa tencissima seedlings under a Pinus halepensis canopy found a negative effect on S. tencissima performance; S. tencissima seedlings had slow growth rate due to the reduction of light (Navarro-Cano et al. , 2009). Generally, a low-light level reduces plant growth (Quero et al. , 2008; Sánchez‐Gómez et al. , 2006), and increases the biomass proportion allocated to leaves and stems at the expense of roots ( Ruiz- Robleto & Villar, 2005) . Therefore, it is important to identify the appropriate environmental conditions in order to establish effective M. irbyana restoration practices. The purpose of this study was to examine abiotic factors, focusing particularly on light availability in the understorey of remnant mature trees, and the effect of natural conditions (e.g., soil nutrients and soil moisture, temperature, rainfall, and photoperiod) on seedling recruitments. The study sought to compare the seedlings’ performance, comparing vegetative growth and survival capability between the common M. irbyana and co-occurring M. bracteata in local habitats. In general, this study predicted that seedling performances of both species would be higher in microhabitats with shade and shrub covers. This study focuses on the abiotic factors such as light availability in each microhabitat, and how they affect seedling survival and growth rate. The study addressed the following questions: (1) Do local habitats have a greater effect on the survival and growth of seedlings in study species? (2) Do M. irbyana seedlings have the capacity to survive and grow under the same resource conditions as M. bracteata? Here, I hypothesise that M. irbyana will present low vegetative growth and survival rates compared to M. bracteata at all local habitats. (3) Do M. irbyana seedlings require specific habitats for their growth and survival when they are

under the canopy of mature remnant trees? The hypothesis is that M. irbyana seedlings will show higher performance under M. irbyana canopies than M. bracteata canopies.

4.2 Materials and Methods

4.2.1 Field sites

The research reported in this chapter was conducted within the Logan City region, Queensland, Australia, at five sites (Henderson Reserve, Victoria Park, Bottlebrush Park, Moffatt Park and Waterford West) within the Jimboomba and Waterford West regions of Logan. Field sites are generally located on alluvial soils derived from sedimentary rocks. The climate of this area was characterised by an average rainfall of 292 mm, an average temperature minimum of 7.4°C, an average temperature maximum of 29.8°C in 2014 when the sites were first established. Experiments were conducted during September 2014 to October 2016. Figures 4. 2, 4. 3, 4. 4 and 4. 5 summarise minimum and maximum temperatures, average monthly rain-fall and photoperiods (Bureau of Meteorology, 2016) in Waterford station (27. 68°S, 153. 19°E). Regulations of vegetation management and plants fall under the Logan Planning Scheme 2006, which includes the broad Queensland regional ecosystem type of Eucalyptus crebra (Ironbark) and Eucalyptus tereticornis (Forest red gum), where both study species, M. irbyana (the endangered species) and M. bracteata (the common species), are found within the understorey. There are remnant populations of both M. irbyana and M. bracteata where thickets are present in forest reserves in Henderson Reserve and Moffatt Park; while at Victoria Park and Waterford West only M. irbyana is present and at Bottlebrush Park only M. bracteata (Figure 4.1: A-E).

C) 0 15

10 Temperature Temperature ( 5

0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2014 21.1 20.8 19.7 17.3 14.5 11.3 7.4 11 11.8 15.3 19.9 20.6 2015 21.6 20.3 20.3 15.9 13 11.2 8.8 10.5 11.3 14.1 16.7 18.6 2016 19.6 21 20 17.5 13.4 11.9 11 10.7 14.1 14.6 18 19

Figure 4. 2 Monthly mean maximum temperatures at Jimboomba, Logan City in five research sites. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia).

25 C) 0 20

10 Temperature Temperature ( 5

0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2014 29.9 29.7 28.8 28.1 24.4 23.1 22.3 22.2 24.6 27.6 29.8 29.8 2015 30.2 28.4 29.6 26 24.3 21.3 20.6 23.3 24.3 26.8 29.1 29 2016 29.9 31.3 29.4 28.1 26.8 21.6 22.4 22.4 24.9 27 29.3 30

Figure 4. 3 Monthly mean maximum temperatures at Logan City, Jimboomba in five research sites. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia).

300

250

200

150

Rainfall(mm) 100

0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2014 116.8 22.5 122.9 13 40 29.4 9 134 35.5 2.8 39.9 161.8 2015 155.5 243.4 126.9 199.4 249.8 53.7 19.2 23.2 30.2 52.3 88.3 61 2016 74.7 26.3 79.1 13.1 15.5 189 19 40.8 54.5 68 53 145

Figure 4. 4 Mean monthly rainfall (mm) at Jimboomba, Logan City in five research sites. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia).

Mean monthly photoperiod 16 14 12 10 8 6

Photoperiod(hours) 4 2 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2014 13.4 12.8 12 11.3 10.5 10.3 10.3 11 11.7 12.5 13.3 13.5 2015 13.4 12.8 12.1 11.3 10.5 10.2 10.3 10.9 11.7 12.5 13.2 13.5 2016 13.4 12.8 11.9 11.2 10.5 10.3 10.4 11.33 12.5 12.5 13.2 13.5

Figure 4.5 Mean monthly photoperiod (hours) in Brisbane. Values are presented in three years of study during 2014-2016 (Bureau of Meteorology of Australia).

4.2.2 Reciprocal seedlings

4.2.2.1 Plant materials

The one-year old seedlings of M. irbyana and M. bracteata were obtained from Paten Park Native Nursery at the Gap in Queensland, Australia. The total number of seedlings used in the reciprocal seedling development experiments was 720, including those needing to be replaced due to drought and a high rate of mortality during the first year at Henderson Reserve site.

4.2.2.2 Method of reciprocal seedlings

Initially, seedlings were planted in spring (September) of 2014 in the Henderson Reserve in 10 m × 10 m plots, each with 10 seedlings of M. irbyana and 10 seedlings of M. bracteata in the understorey of mature trees of both species. In spring 2015, the planting was done in the plots of the same size and same number and age of seedlings at Victoria Park, Bottlebrush Park, Moffatt Park and Waterford West with a total of 28 plots (Table 4.1). On a monthly basis over one year (spring 2015 to spring 2016) the plants were checked and the height of seedlings and their survival rates were recorded (Figure 4.6: A-B).

Figure 4.6 Pictures of a Melaleuca irbyana seedling (A) and a Melaleuca bracteata seedling (B) recently planted for reciprocal experiment

Table 4.1 Experimental design for reciprocal seedlings.

Site name Plots Number in each plot Total number of seedlings 1. Henderson site • 4 plots of mature M. irbyana • 10 seedlings M. irbyana • 160 2. Moffatt Park • 4 plots of mature M. bracteata • 10 seedlings M. bracteata • 160 3. Victoria Park • 4 plots of mature M. irbyana • 10 seedlings M. irbyana • 80 4. Waterford West • 10 seedlings M. bracteata • 80 5. Bottlebrush Park • 4 plots of mature M. bracteata • 10 seedlings M. irbyana • 80 • 10 seedlings M. bracteata

4.2.2.3 Growth measurements

Melaleuca irbyana and M. bracteata seedling growth and survival rates were measured every month from September 2015 to October 2016. Leaf area index (LAI) and photosynthetically active radiation (PAR), which is the spectrum of light that a plant requires to perform photosynthesis, were measured during the growing season of the preliminary planting seedling in 2014, and measurements continued at the same period of the growing season of retransplanted seedlings in 2015 and again in 2016. The proportion of canopy in each plot was measured using a Plant Canopy Image (CID Bio-Science) which estimates PAR and LAI. Canopy measurements were taken in all the 28 plots at the five experimental sites ( Henderson Reserve, Victoria Park, Bottlebrush Park, Moffatt Park and Waterford West). Canopy analyses were completed in five locations for each plot. The PAR and LAI measurements were taken using Plant Canopy Image (CID Bio-Science) from the point marked in between the pegs and the centre of each plot.

4.2.2.4 Data analysis

Seedling growth and survival rate depending on habitat of both M. irbyana and M. bracteata seedlings were analysed using an ANOVA with an error structure to compare seedling survival and growth rate. A Tukey’s Honest Significant Test (HSD) was performed to assess the survival and growth rate differences between each habitat. The survival rates were calculated using the Kaplan-Meier estimator (McNair et al., 2012). Survival rates were calculated to estimate survival probability with the R statistical program (R development Core Team 2013) on RStudio and R version 3.3.0 functions ‘survfit’ and ‘survreg’ in the R

survival package (Crawley, 2014; Therneau, 2016); the package survival which requires the status variable to be expressed in binary terms: 0 (non-deaths) and 1 (deaths).Thus, seedlings which died were considered to have a status of one, while those with growth of zero were deemed to survive. The data was log transformed to normalise errors and homogenise variance. This study has a balanced design which used F-statistic to assess the significance of fixed effects as explanations of variation in the response variables (Pinheiro et al., 2009); (http://www.R-project.org/(https://cran.rproject.org/web/packages/survival/survial.pdf). To assess the relationship between canopy structure and abiotic conditions of LAI ( proxy for light availability) and PAR, a mixed effects general linear model was used (LMEMs), estimated with maximum likelihood (ML). The structure of plots nested within the sites were treated as random effects, and the canopy of each remnant mature tree as a fixed effect. All LMEMs were fitted using the package “nlme”, R version 3.3.0) (Pinheiro et al., 2016). Wald F-tests were then used to assess the significance of the interactions of the fixed effects in the models.

4.3 Results

4.3.1 Light availability and seedling survival

Overall, M. irbyana and M. bracteata canopies differed significantly in the amount of light that was able to be filtered through to the understoreys. Photosynthetically active radiation between both species’ canopies was significantly different (F1,420 = 35. 05, P <

0.001). The year of plant growth also had a significant effect on PAR (F1,420 = 26.01, P < 0. 001). Based on the interaction test and the interaction plot, it appeared that the effect of the year on PAR depended on species’ canopies (F1,420 = 29. 33, P < 0. 001; Table 4. 2). When comparing each year, the result showed that M. bracteata canopies had a higher rate of PAR than M. irbyana canopies during the three years of the study. In each year during the growing season, PAR under M. bracteata showed a constant increase every year. On the contrary, PAR measured on M. irbyana canopy decreased dramatically every year until the end of the experiment (Figure 4.7).

Table 4. 2 Result from a Wald F- test of a linear mixed effect model with the response variable of photosynthetic active radiation (PAR) conducted to assess the significance of the fixed effects of habitats (Melaleuca irbyana and Melaleuca bracteata canopies) and year.

Variables numDf denDf F-value P-value (PAR) Species 1 22 35.05 <.0001 *** Year 2 368 26.01 <.0001 *** Species × Year 2 368 29.33 <.0001 *** Residual 420 Significant codes: *** P < 0.001

Figure 4.7 Higher-order fixed effects from LMEM terms where the response variables are light availability of photosynthetically active radiations (PAR), and how it varies with year of observation (2014-2016) in each habitat of remnant mature trees between Melaleuca irbyana (MI) and Melaleuca bracteata (MB). Error bars represent ± standard error.

The leaf area index (LAI) was also significantly different between species (F1,420 = 4.61, P < 0.05). Interaction effects indicate that the LAI depended on both species and which the year the measurements were taken (F1,420 = 5.00, P < 0.01). However, on its own, the year did not have a significant effect on LAI (F1,420 = 0.07, P = 0.93; Table 4.3). Melaleuca bracteata canopy showed a slight decrease of LAI every year. Conversely, LAI of M. irbyana canopy increased slowly over the year of experiment (Figure 4.8).

Table 4. 3 Result from a Wald F- test of a linear mixed effect model with the response variable of leaf area index (LAI) conducted to assess the significance of the fixed effects of habitats (Melaleuca irbyana and Melaleuca bracteata canopies) and year.

Variables numDf denDf F-value P-value (LAI) Species 1 22 4.61 0.04 * Year 2 368 0.07 0.93 NS Species × Year 2 368 5.00 0.00 ** Residual 420 Significant codes: ** P < 0.01, * P < 0.05 and NS (non-significant)

Figure 4.8 Higher-order fixed effects from LMEM terms where the response variables are light availability of leaf area index (LAI) and how it varies with year of observation (2014- 2016) in each habitat of remnant mature trees between Melaleuca irbyana ( MI) and Melaleuca bracteata (MB). Error bars represent ± standard error.

The relationship between PAR and LAI results under M. irbyana canopies was correlated to M. irbyana and M. bracteata seedling performances. Significant differences were detected in survival rates between M. bracteata and M. irbyana seedlings under both canopies (Figure 4.9). After one year since restart of the reciprocal experiment (September 2015-October, 2016), only 24% (68/280) of M. irbyana seedlings had survived. On the other hand, 40% (112/280) of M. bracteata seedling survived at the end of the experiment. Our results showed that seedling survival rates of both species were significantly different from each other (F1,5632 = 180.35, P < 0.001). There were also significant effects of the month

(F1,5632 = 15.01, P < 0.001) and overstorey (F1,5632 = 801.82, P < 0.001) on seedling survival rate. However, based on the interaction test and the interaction plot, indications are that there was no month and species interaction to influence seedling survival rates (F1,5632 = 0.61, P

= 0.82). Likewise, there was no significant interaction of species and overstorey (F1,5632 =

3.60, P = 0.80), or interaction between species × month × overstorey (F1,5632 = 0.24, P = 0.99). However, overstorey had a marginally significant relationship with month on survival rates (F1,5632 = 0. 24, P = 0. 99; Table 4. 4). Monthly mortality declined with increasing seedling age in both species (Figure 4.9).

Table 4.4 The ANOVA result of the effect of Melaleuca irbyana and Melaleuca bracteata canopies to seedling survival rate between M. irbyana and M. bracteata seedlings and month with an error structure.

Predictor Variables Df Sum of squares F-value P-value

Species 1 37.0 180.35 <2e-16 *** Month 11 33.9 15.01 <2e-16 *** Overstorey 1 164.5 801.82 <2e-16 *** Species × Month 11 1.4 0.61 0.82 NS

Species × Overstorey 1 0.0 0.06 0.80 NS Month × Overstorey 11 8.1 3.60 4.41e-05 *** Species × Month × Overstorey 11 0.5 0.24 0.99 NS Residuals 5632

Significant codes: ***P < 0.001 and NS (non-significant)

Overall, when comparing seedling survival under each canopy, the results showed that M. bracteata seedlings had the highest survival rate (9%) under both canopies of M. irbyana and M. bracteata. This was followed by M. irbyana seedlings (6%) under M. bracteata canopy, and M. bracteata seedlings under M. irbyana canopy (5%). Melaleuca irbyana seedlings planted under M. irbyana canopy had the lowest survival rate ( 2% ) (Figures 4.9-4.10).

Figure 4. 9 Kelplan-Meier estimate of the survival rate of seedlings between Melaleuca irbyana (MI) and Melaleuca bracteata (MB) overstorey of both remnant mature trees (12 months, 2015- 2016) of experiment in five sites. Orange line indicates the first time of seedling death occurring in each of the conditions tested.

Figure 4. 10 Comparison of total seedling survival after 12 months of experiment when grown under canopies of Melaleuca irbyana (MI) and Melaleuca bracteata (MB) trees.

Seedling growth

The results indicated that there was no significant difference in seedling growth between species (F1,556 = 2.33, P = 0.13). However, there were significant differences in seedling growth rate between the species when considering the canopies that they were grown under (F1,556 = 43.38, P < 0.001) (Table 4.5). Vegetative growth rate of both species depending on overstorey type, with M. irbyana and M. bracteata seedlings having high growth rates under M. bracteata canopy (F1,556 = 4.51, P < 0.05; Table 4.5). A post hoc Tukey HSD test showed that there was significant variation in seedling growth rate between most overstoreys of remnant mature tree. However, the difference between M. irbyana seedlings growing under M. irbyana canopies and M. bracteata seedlings growing under M. irbyana canopies were not significantly different (Table 4.6).

Table 4.5 The ANOVA result of light availability under Melaleuca irbyana and Melaleuca bracteata canopies to vegetative growth rate ( height) between Melaleuca irbyana and Melaleuca bracteata seedling at the end of experiment (12 months) with an error structure.

Variables Df Sum of squares F-value P-value Species 1 1113 2.33 0.13NS Overstorey 1 23051 48.38 9.9e-12 *** Species × Overstorey 1 2152 4.51 0.034 * Residuals 556 Significant codes: ***P < 0.001, * P < 0.05 and NS (non-significant)

Table 4.6 Result of Tukey multiple comparison of means test performed on an ANOVA of seedling growth rate (height) under canopies of both mature remnant trees. The symbols within brackets define habitat overstorey of each remnant mature tree symbols without brackets define seedlings species.

Treatment P-value M. irbyana (MI) × M. bracteata (MB) MI : (MB) × MB : (MB) < 0.05 MB : (MI) × MB : (MB) < 0.05 MI : (MI) × MB : (MB) < 0.05 MB : (MI) × MI : (MB) < 0.05 MI : (MI) × MI : (MB) < 0.05 MI : (MI) × MB : (MI) 0.99

The results also showed that under M. bracteata canopies, M. bracteata seedlings had the highest vegetative growth rate (6 cm) from the initiation of reciprocal seedlings until the end of the experiment (12 months) under both canopies. The height of M. irbyana seedlings reduced when grown under both M. bracteata and M. irbyana canopies (-3 and - 20 cm, respectively). A similar decline in height also occurred with M. bracteata seedlings under M. irbyana canopies (-22 cm) (Figure 4.11). The main cause of reduction in seedling height was herbivores foraging (e.g., wild grey kangaroos, feral goats and hares). Overall, the results showed that seedling performance was better (higher seedling survival rates and

greater height increase) for seedlings growing under M. bracteata canopies compared to M. irbyana canopies (Figures 4.9- 4.11).

Figure 4. 11 Comparison of seedling height when grown under canopies of Melaleuca irbyana (MI) and Melaleuca bracteata (MB) trees after 12 months of experiment.

4.4 Discussion

4.4.1 Light availability and seedling survival

The microhabitats created by M. bracteata canopies differed from the microhabitats created by M. irbyana canopies because LAI values were lower in M. bracteata forests, which led to higher levels of PAR availability for seedlings growing in the understorey. This then suggests that M. bracteata forests provided higher levels of the light in a range that plants need for carbon assimilation, leading to better survival and higher growth rates. The distribution of PAR from solar irradiance can affect transpiration and, developmental processes of seedling growth underneath the canopy (Choudhury, 2001). The M. bracteata forest showed sparse canopies (usually 1 or 2 trees in each 10 m × 10 m plot) which created large light gaps in this habitat, resulting in more light availability; whereas M. irbyana forests had mature trees, hence denser canopies (approximately 20-45 trees in each 10 m × 10 m plot) which blocked most light, resulting in low light availability to the forest floor. From the findings, it is evident that M. irbyana forests had higher LAI than M. bracteata forests. The differences in high LAI and low PAR observed mean that M. irbyana forests had clumped distribution of mature trees and fewer gaps in the canopy. Tree canopies block part of the solar radiation, which results in reduction of light levels and complicated irradiance patterns at the forest floor (Katul et al., 2011). Dense canopies with high LAI can block up to 95% of visible light from reaching low lying vegetation ( Bonan, 2015) . Monitoring the changes of LAI (which influences PAR) is important for the assessment of growth, vigour and light transmission through the canopy of plants (De Bei et al. , 2016; Dokoozlian & Kliewer, 1995). These changes are important in controlling the interactions between terrestrial environments and spatial variables due to land use and local climate change (Hardwick et al., 2015). There was a strong relationship between LAI (which affects PAR) and microhabitat, indicating that LAI and PAR are crucial in differentiating the forest habitats of the two species. These findings indicate that light levels impacted less on M. bracteata seedling performance, while M. irbyana had a better performance when seedlings were grown under M. bracteata canopies. Canopies with high LAI absorb most of the sunlight, thus the amount of sunlight reaching the ground level will be similar whether on cloudy or sunny days (Hardwick et al., 2015). Melaleuca irbyana canopies provide low light, which affects early

seedling establishment in M. irbyana. In contrast to these results, some argue that seedlings may show high survival rates when they occur beneath mature trees due to shading (high LAI canopies), because soil moisture retention is high due to low evaporation from soil surface (Rich et al., 2008). A study of a critically endangered species (Quercus insignis) by Montes-Hernández and López-Barrera (2013) found that Q. insignis had high survival rates and increased height when growing under shaded environments than in direct sunlight areas. Thus, in some species, light variations, which may indirectly affect soil moisture content could be a crucial determinant of young seedling survival under remnant habitat, and also for revegetation (Iszkulo, 2010).

Generally, seedlings that are able to obtain a larger size quickly are considered to have a better survival rate than smaller seedlings (Close et al., 2010). Large seedlings have greater ability to establish themselves, survive and cope with stressful conditions than small seedlings (Tsakaldimi et al., 2013). Seedling size may be related to differences of mortality rates because of competition for resources when plants coexist (Close et al. , 2010; Gilbert et al. , 2001). Large seedlings with higher height may be more able to reach the resources such as light and soil nutrients than small seedlings (Metz et al. , 2010). In this study, I observed that seedlings of M. bracteata had a better chance of survival and were generally larger in size than M. irbyana seedlings of the same age (one year). Another study of seedling mortality on 163 species in Yasuní National Park, Ecuador, reported that survival in some plant species was dependent on seedling size and density of adult trees (Metz et al., 2010). The results are consistent with the hypothesis that larger seedlings have a higher survival rate (Jakobsson & Eriksson, 2000). Wendelberger et al. (2008), also observed that seedling size is an important consideration for survival rate after transplanting in the wild.

The high survival rate of M. bracteata seedlings even during drought conditions suggests that M. bracteata seedlings could be more drought tolerant than M. irbyana seedlings. Soil moisture availability plays an important function for seedling establishment and survival, especially in the early stages after transplanting (Wendelberger et al., 2008). The low survival rate of seedlings in both species at the start of the experiment may be because our reciprocal experiment was set up when there was an unprecedented low rainfall for several months (~40-60 mm). Drought incidence during early stages of the reciprocal seedling experiment (2014-2015) at five sites has been identified as a major reason for

seedling mortality, although seedlings were watered weekly at the beginning to help their establishment. The low survival rate at the early stages of the reciprocal seedling experiment can be attributed to physiological drought caused by high temperatures (Iszkulo, 2010). During physiological drought, the transport of water from the soil is stopped or strongly reduced; this leads to leaf desiccation and ultimately causes plant death (Iszkulo, 2010). Dry summers can be expected to influence seedling recruitment negatively ( Mitchell et al. , 2008), but dry summers and drought conditions are common in the natural areas where M. irbyana occur. Cabin et al. (2002) studied the effect of microsite, water, and weeding on seedlings of six endangered shrubs native to Hawaii, and concluded that lack of water is the major cause of low survival and slow growth rate for both native and invasive species. Thus, water deficiency could be a major cause of both M. irbyana and M. bracteata seedlings’ mortality in this experiment. Intense drought appeared to surpass the acclimation potential of M. bracteata seedlings, but not for M. irbyana seedlings under the same conditions.

Overall, growth rates were higher under M. bracteata canopies, suggesting that the increased light availability for seedlings was not only beneficial for survival but also for growth. This pattern is consistent with the hypothesis that microhabitats play an important role in vegetative growth (Rolhauser et al. , 2011). If microhabitats have moderate shade conditions along with an intermediate level of light, seedlings can have a higher growth rate when compare with full sunlight areas (Montes-Hernández & López-Barrera, 2013).

The findings provided some evidence for a congruent suite of functional traits associated with rarity in M. irbyana, since this species showed traits of low survival and growth rate. We first hypothesised that M. irbyana would present lower growth rates than M. bracteata seedlings. We found M. bracteata had greater height than M. irbyana seedlings under both M. bracteata and M. irbyana canopies, agreeing with a stress-tolerant strategy (individual survival via maintenance of metabolic performance in variable and unproductive niches) (Lavergne et al., 2004; Matesanz et al., 2009). In this context, Lavergne et al. (2004) studied 20 congeneric species in the Mediterranean region and found that rare species were shorter than widespread species. The slow growth rate of M. irbyana seedlings indicates that this species may be unable to adapt and perform well under shaded conditions (M. irbyana microhabitat) when compared to M. irbyana seedlings under M. bracteata canopy. On the other hand, M. bracteata seedlings showed high performance (high survival and growth

rate) in either low or high light conditions, which may define a shade- tolerant species. Matesanz et al. (2009) , studied seedling performance between Thymus loscosii, a rare endemic of semiarid Spain, and the widespread Thymus vulgaris. The rare T. loscosii had lower performance (height and biomass) than widespread species due to low competitive ability when located with congeneric species. Ramsay and Fotherby (2007) observed that the population size of rare species declined and changed in spatial patterns because of their inability to compete for resources with co-occurring species. Vegetative growth rate has a positive relationship with seedling survival rate (Rodríguez-Calcerrada et al., 2010). In this study, the results showed that M. bracteata seedlings had a better performance (survival and growth rate) than M. irbyana seedlings under both canopies. The high performance of M. bracteata seedlings could be because M. bracteata seedlings are more shade tolerant than M. irbyana seedlings. We found the growth rate results showed some seedlings of both species reduced in height when calculated at the end of experiment. We assumed that herbivores might have been responsible for this observation. This is consistent with Moles and Westoby’s study (2004b), which observed that herbivory is the major cause of seedling mortality in natural habitats. Predation of M. irbyana and M. bracteata seedlings is presumably carried out by grey kangaroos, feral goats and hares, although direct evidence is lacking. Wahungu et al. (2011) stated that the seedling stage of the plant life cycle is generally the most vulnerable to herbivores, due to small size and total reliance on nutrient reserves stored within their cotyledons.

4.5 Conclusion

Overall, M. bracteata seedlings were better able to survive and increase in height than M. irbyana under canopies of both species. Seedling performance of both species was reduced under a canopy of M. irbyana, suggesting that these endangered habitats inhibit recruitment, possibly because of dense canopies, litter, or specific soil conditions. Low survival and slower vegetative growth rates of M. irbyana may mean its colonisation capacity requires specific conditions in a natural environment, and possibly explain its original rare distribution. The results also suggest that M. irbyana may be a habitat specialist, providing evidences for congruence traits associated with rarity in M. irbyana, such as low survival rate and slow growth under natural severe weather. Similarly, Rabinowitz (1981) stated that the most frequent type of rare species are habitat specialist plants.

The results demonstrate that dense canopies of mature trees in M. irbyana forests tend to have a negative effect on seedling survival and growth rate for restoration of M. irbyana seedlings. Generally, in light- limited environments, the survival rate of shade- tolerant species is higher than that of less shade-tolerant species (Erefur et al. , 2011; Kobe & Coates, 1997). These findings suggest that variations in light availability in each forest could be a crucial consideration for young seedling survival in regeneration programs ( Iszkulo, 2010; Montes- Hernández & López- Barrera, 2013) . Thus, in this context, the adaptive response to performance and distribution between narrowly and widely distributed species suggests that narrowly distributed species might be unable to perform well, that is, might have low survival and slow growth rates, and be unable to maintain high performance when resource are limited and they co-occure with congeneric species (Fridley et al., 2007; Scheiner, 2002) . Matching a suitable habitat to species can improve success rate by providing safe habitats for seedling survival. I recommend that future research focus on investigating interactions between abiotic and biotic factors (Matías & Jump, 2012; Meier et al. , 2010) and their effect on the physiological characteristics of M. irbyana, that is, photosynthesis, biomass and specific leaf area. Such a focus may bring us closer to a full understanding of the cause of limitations in the distribution of M. irbyana.

Chapter 5 Effect of exogenous traits on physiological characteristics of remnant mature trees

Mature remnant (A) Melaleuca irbyana the endangered species and (B) Melaleuca bracteata a common species often growing at the same sites but in distinct communities. (C) Melaleuca irbyana seedlings 1 year old and (D) Melaleuca bracteata seedlings 1 year old. Nutrient analyses were conducted at QUT’s Central Analytical Research Facility (CARF) laboratory to measure various soil properties. (E)GalleryTM Plus Automated Photometric Analyser, (F) X-ray fluorescence spectrometer, (G) LECO® machine, (H) Carbolite Furnace, (I) Claisse® The Ox® automated fusion machine and (J) XRF fused glass discs.

5.1 Introduction

Abiotic conditions such as soil nutrients, water, and light, are essential for plant growth and survival, and the minimum amount required by a species can affect competitive interactions in plant communities (Kobe, 1996; Tilman, 2004). Studies of the effect of soil characteristics on growth traits and plant performance may then explain why some species have restricted distribution while others do not. Studies of rare or threatened plant species and how they respond to abiotic environmental conditions have been informative, although a lot still remains to be learnt (Siqueira et al., 2012). In the late twentieth century, more than 1,700 flora and ecological communities were reported to be either threatened or at the brink of extinction in Australia (Baillie et al., 2004). Causes of species extinction are varied, but environmental deterioration and habitat fragmentation are two of the most significant threats (Fischer & Lindenmayer, 2007). Habitat fragmentation due to land clearing and conversion of habitat patches to other land-uses can result in the isolation of species and, a reduction in gene flow (Vranckx et al., 2012). Reduction of gene flow is considered one of the most important causes of the decrease in number of plant species and increase in extinction rates (Heinken & Weber, 2013; Kuussaari et al., 2009; Ouborg et al., 2006). Plant communities worldwide are now highly fragmented and as a result many species are under threat of extinction (Myers et al., 2000).

The ecological niche breadth hypothesis describes the relationship between species abundance and the habitat, and has been used to explain differences in distribution between common and rare species (Brown, J. H. , 1984; Gaston & Kunin, 1997). According to the niche breadth model, rare species occupy a relatively smaller area of habitat than common species because rare species have restricted environmental tolerance (Brown, J. H. , 1984). Thus, in habitats where environmental conditions are favourable for a specialist species, such species may be abundant in those areas, but rare across the landscape (Brown, James H et al., 1995; Siqueira et al. , 2012). In contrast, common species may be widespread because the habitats they occupy are widespread, whereas rare species might be restricted in distribution because their habitats are also rare (Lennon et al., 2011; Thompson et al., 1998). Therefore, when environmental factors vary in space, generalist species would be able to occupy more sites and reach greater abundances levels than the specialist species (Brown, J.H., 1984).

Nutrient availability at a site affects plant growth and survival, and can either promote or restrict the establishment and survival of some species over others (Baskauf & Eickmeier, 1994; Greulich et al. , 2000; Walters, M. & Field, 1987). A study comparing competitive interactions between common and rare species found that common species tended to be more competitive than rare species where nutrient availability was high; whereas rare species tended to be more competitive than common species where nutrient availability was low (Dawson et al., 2012). However, Siqueira et al. (2012) found that the distribution and population size of common species were mostly influenced by environmental conditions, and rare species most influenced by fewer opportunities to disperse and colonise new sites due to dispersal limitations. Rare species may be affected by interacting factors that are complex to unravel, such as abiotic covariance, whereas common species may be affected mainly by conspicuous environmental variables (Lennon et al. , 2011) . The variation of habitat factors, especially nutrition availability, is often correlated to plant physiological performance (growth rate) and plant distribution (John et al., 2007; Russo et al., 2005). Therefore, investigating the performance of adult plants might improve our understanding of plant distributions.

Understanding the relationship between species distribution and environmental conditions (e. g. , rainfall, light, temperature and soil level) may inform development of biodiversity conservation and management strategies, especially in the context of rare and/or endangered species (Lennon et al., 2011). Plant performance and yield are generally used to predict the development of forest stands (Sharma & Parton, 2007). Diameter at breast height (DBH) and total tree height are indicators of plant growth and yield development, and these traits depend on soil nutrient availability (Lilles & Astrup, 2011). Tree performance traits such as height, DBH, BA, stem density, and shoot elongation, are important, as they directly relate to resource acquisition and use, and they are indicators of how trees convert resources into productivity, also known as resources- use efficiency. For example, many endemic and/ or narrowly distributed plant species have a lower height than widespread species (Lavergne et al., 2004; Matesanz et al., 2009; Medrano et al., 2006), which may provide a broad indication of lower performance, as taller plants are better able to compete for light resources to enhance photosynthesis than are shorter plants (Falster & Westoby, 2003; Ryan & Yoder, 1997).

The purpose of this chapter is to investigate the effect of soil chemical properties on the growth rates of remnant mature trees in different sites with adult populations of M. irbyana and adult populations of M. bracteata. The study compares the soil chemical concentrations in each habitat and whether nutrients correlate with growth characteristics of M. irbyana and M. bracteata populations. Based on the narrow distribution of M. irbyana remnant mature trees, it was hypothesised that it would show a lower level of growth (height, DBH, BA, stem density and shoot elongation) than M. bracteata over the three measurement periods (September 2014, 2015 and 2016). This study specifically addresses the following questions: (1) Do the growth rates of M. irbyana and M. bracteata differ over time? (2) Does variation in soil characteristics correlate with growth rates of M. irbyana and M. bracteata populations? and (3) Do growth rates and soil characteristics have the potential to explain the causes of narrow distribution of M. irbyana?

5.2. Materials and methods

5.2.1 Field sites Research on only remnant adult populations of both M. irbyana and M. bracteata were conducted at five sites in the Logan City Council region of Queensland Australia: 1) Henderson Reserve, 2) Victoria Park, 3) Bottlebrush Park, 4) Moffatt Park and 5) Waterford West. Field sites were the same as described in Chapter 4; the reciprocal seedling experiment (Figure 5. 1) and weather conditions were also the same as the environmental conditions reported in Chapter 4, as the studies were conducted during the same time period, but are shown again in this chapter (minimum and maximum temperatures, average rainfall and photoperiod summarised in Figures 5.2-5.5) (Bureau of Meteorology, 2016) in Jimboomba station ( 27. 68°S, 153. 19°E) . Remnant mature populations of both M. irbyana and M. bracteata were present in forest reserves in Henderson Reserve and Moffatt Park; while at Victoria Park and Waterford West, only M. irbyana populations were present, and at Bottlebrush Park, only M. bracteata populations.

C) 0 15

10 Temperature Temperature ( 5

Figure 5. 2 Monthly mean minimum temperatures at Jimboomba, Logan City in five research sites over the study period of 2014 to 2016 (Bureau of Meteorology of Australia).

25 C) 0 20

10 Temperature Temperature ( 5

Figure 5. 3 Monthly mean maximum temperatures at Logan City, Jimboomba in five research sites over the study period of 2014 to 2016 (Bureau of Meteorology of Australia).

300

250

200

150

Rainfall(mm) 100

Figure 5. 4 Mean monthly rainfall (mm) at Jimboomba, Logan City in five research sites over the study period of 2014 to 2016 (Bureau of Meteorology of Australia).

Mean monthly photoperiod 16 14 12 10 8 6

Figure 5.5 Mean monthly photoperiod (hours) in Brisbane over the study period of 2014 to 2016 (Bureau of Meteorology of Australia).

5.2.2 Growth measurements

All individual remnant trees of M. irbyana and M. bracteata were counted in 28 plots of 10 m × 10 m established at Henderson Reserve, Victoria Park, Bottlebrush Park, Moffatt Park and Waterford West. To compare the growth rate of the study plants their diameter at breast height (DBH), and tree height (Ht) were measured. Diameter at breast height was used to measure stand basal area, which in the equation below is denoted by the symbol G, and represents the sum of basal areas of all living trees in a stand. As individual tree basal area is related to tree volume, biomass, crown parameters, etc. , so the stand basal area is related to stand volume, biomass, etc. To calculate G, the following equation was used where DBH is the diameter in cms of all trees in a known area (a in ha) (Long, J. N., 1985):

휋 ∑ 푑푏ℎ2 ∑ 푑푏ℎ2 퐺 = ∗ = 0.0000785398 ∗ 40000 푎 푎

Heights of M. irbyana and M. bracteata plants were measured using a laser Rangefinder ( Nikon Forestry Pro Laser) . The total tree volume in cubic meters was calculated from DBH and the total tree height (Ht) using the equation below. Assuming all individual trees had the same shape, an estimate of the total tree volume can be made using the following equation, where Ht is the total tree height and TBA is tree basal area (Hahn, 1984).

DBH ( )2 x 3.142 x Ht TBA x Ht Tree volume (m3) = 200 or 3 3

Shoot elongation in mature trees of M. irbyana and M. bracteata was measured once every year over three years (September 2014, 2015 and 2016) by marking 20 new young shoots with bird bands of different colours. Bird bands acted as the marker points on each shoot and the elongations from these points were then measured using a measuring tape.

5.2.3. Soil sampling

In each of the 10 m × 10 m plot, three soil samples were collected from a depth of 0 to15 cm using a soil auger. Sampling in the upper layer of soil in depths less than 20 cm is a common protocol used in ecology to study soil fertility for measurement of nitrogen (N), phosphorous (P), potassium (K), calcium (Ca) and carbon (C) (Wigley et al., 2013). Soil samples were then placed inside zip lock bags and each bag labelled with the location. The soil samples were stored in a refrigerator set at 4°C until they were analysed in the laboratory. Each soil sample was handled separately and any equipment used was thoroughly cleaned each time during the analyses of the different soil characteristics.

5.2.3.1 Soil organic matter

Soil organic matter and moisture content were measured using Loss on Ignition methods. Loss on Ignition (LOI) quantifies the organic matter (OM) content (%OM) of the soil sample. Soil samples were dried at 40°C for 24-48 hours. The dried samples were ground with Cr- steel Ring Mill Pulveriser (ROCKLABS®) to produce homogenous samples for laboratory analyses. Ring mill cups were washed and dried between samples to avoid sample cross contamination. Ceramic crucibles were weighed and the weights recorded, then ≈ 5 g of the soil samples were weighed in crucibles and dried in the thermostatically controlled oven at 105°C for 24 hours. After cooling in a desiccator, samples were reweighed, and then placed in a muffle furnace (Thermolyne® Small Benchtop) at 375°C for at least 16 hours. The dried samples were removed from the furnace, cooled in the desiccator and reweighed. The percentage of OM was calculated using the following equation:

5.2.3.2 Soil moisture

Soil moisture content (MC) was measured following a standard protocol developed by the Department of Sustainable Natural Resource (1990). Approximate weights of 5 g of sieved soil (moist soil) were placed in the ceramic crucible. The samples were dried in the

controlled temperature oven set at 105°C for 16 hours. The dried samples were removed and cooled in the desiccator and reweighed. The MC was calculated using the formula below:

(푊2 − 푊3) 푀퐶% = × 100 (푊3 − 푊1)

where, W1 = Weight of crucible (g) W2 = Weight of moist soil + crucible (g) W3 = Weight of dried soil + crucible (g

5.2.4 Soil characterisation preparation

5.2.4.1 Soil air-drying

Prior to any analyses of the soil nutrient characteristics, soil samples had to be air- dried. There are a number of ways to air-dry samples; for example, within heat controlled ovens or desiccators, or by exposing soil to the air in a large temperature controlled room. Soil samples were spread in shallow trays and air-dried at approximately 40°C, for 24-48 hours or until a constant mass was achieved. The air-drying temperature of around 40°C is recommended because temperatures above 65°C could activate carbon oxidation. Each soil sample used in this study was spread on an aluminium foil tray and air-dried in an oven set at 40°C for 24 hours or until the soil was dried. Once air-drying was completed, any visible organic debris in the soil, including leaves, stalks and roots were removed.

5.2.4.2 Crushing, sieving and splitting

Aggregates within the soil were broken up and crushed using pestle and mortars. Soil was placed into sturdy plastic bags to minimise spillage during manual crushing. Each soil sample was sieved to separate the soil particles to fractions of 2 mm or less. The sieve was gently shaken to allow the soil to pass through. If soil aggregates were present in the ≥ 2 mm fractions after first sieving, the crushing and sieving steps were repeated.

The <2 mm fractions of soil from each core or composite sample were mixed thoroughly to produce a homogenous sample. Thorough mixing is important to ensure that the sub-samples are homogenous and representative of each sample to be used for laboratory analyses. If samples are not mixed thoroughly, the variance in soil carbon between sub-

samples is likely to be large. Such a large variation would make it difficult to detect statistically significant changes in soil organic carbon stocks over time. A range of methods has been used to create representative sub- samples. For soil analyses described here, a Rotary Micro Riffler (Quantachrome Instruments) was used to obtain representative sub- samples. Sub-samples were then grinded by Cr- steel Ring Mill Pulveriser (ROCKLABS®) to produce homogenous samples for laboratory analysis.

5.2.5 Soil nutrient analyses

Soil characteristics such as the composition of clay, sand, and silt, and nutrient availability, for example total nitrogen, total carbon, and other elements such as phosphorous, potassium, calcium and manganese, were measured in soil samples taken from under mature thickets of M. irbyana and M. bracteata. These measurements are described below in sections 5.2.5.1 to 5.2.5.5. These soil analyses were completed using the Central Analytical and Research Facilities (CARF) at Queensland University of Technology (QUT).

5.2.5.1. Soil pH measurements

The pH of soil samples was measured using a pH meter (TPS smart CHEM-LAB- Cond/pH). The pH meter was calibrated according to the manufacturer’s instructions, using buffer solutions at pH 4.0 and 7.0. The soil suspensions were prepared by mixing 10 grams of air-dried soil samples with 50 mL deionised water in a plastic bottle and then shaken by a rotary suspension mixer (RATEK Instrument) for 1 hour at 50 rpm. Each soil suspension was then placed on a magnetic plate with a magnetic stirrer and was constantly stirred while the pH electrode was lowered into the soil solution. The pH value was recorded once equilibrium was reached (Rayment & Higginson, 1992). The pH was read three times for each soil sample. The pH meter electrode was thoroughly rinsed with deionised water between each measurement.

5.2.5.2. Soil total carbon and total nitrogen measurements

The soil samples were prepared with the same method as described for soil pH measurements. Total carbon and total nitrogen were determined by weighing approximately 1 g of each soil sample. The samples were run in a LECO® (TruMac® Series Macro) against the standards (MOD ~ 1 g 3 crucibles, clay ~ 0.75 g 2 crucibles and Gy soil ~ 1 g 2 crucibles).

The fundamental of the LECO® process is combustion of soil samples under 1450°C and in the presence of oxygen. During this process carbon is oxidised to produce CO2. Incorporating gas fusion under a flowing stream of helium, LECO measures combustion gases through infrared absorption and thermal conductivity. During this process, oxygen and carbon combine to form CO2, and nitrogen is released as N2 (Figure 5. 7; D) (Case et al., 2012; McDowell et al., 2012).

5.2.5.3. Soil major elements measurements

X-ray fluorescence (XRF) was used to measure key elements for plant nutrition in the soil. X-ray fluorescence is widely used for chemical analysis, particularly in materials such as metals, glass or glass fibres, soil, rocks, ceramics, etc. X-ray fluorescence analysis involves the emission of the characteristic fluorescent X-rays of the atoms of reference and analysis substances that have been excited by the discrete spectrum and the “bremsspectrum” of an X-ray tube. The radiation emitted by the sample is monochromatised and split up by the analyser crystals in the spectrometer so that the intensities of individual spectral lines and/ or regions can actually be measured ( wavelength dispersive X- ray spectrometry or WDX) (Figure 5.7; H).

Soil samples collected from all 28 sites were crushed using a Cr-steel Ring Mill Pulveriser (ROCKLABS®). Major elements were acquired using the X-ray Fluorescence (XRF) technique. Major elements were measured on 40 mm diameter fused glass discs. Approximately 1. 15 g of samples was fused for 25 minutes at 1050°C with approximately 8.85 g of 50:50 mix lithium metaborate-lithium tetraborate flux containing 0.5 wt% LiBr as a wetting agent. Discs were prepared using a Claisse® The Ox® automated fusion machine.

A PANAlytical Axios wavelength dispersive (WD) X-ray fluorescence spectrometer equipped with a 1kW Rh tube was used for all XRF analyses. The instrument is equipped with PX1, PE002, LiF220 and LiF220, analysing crystals, scintillation, duplex, and P10 flow proportional counters, brass and Al tube filters, and 700 µm and 300 µm collimators. Major elements were acquired using PANAlytical’s WROXI application, and calibration standard procedures modified slightly to suit in-house requirements. Loss-On-Ignition data derived from a separate analysis procedure were incorporated for each sample for matrix correction purpose. X-ray fluorescence major elements data are reported as oxide up to 21 elements,

but here the focus is on key plant nutrients including Fe, Mn, Ca, K, S, P, Si, Al, Mg, and Na. Calibration procedures were validated using combination of USGS, Mintek, BCS and BAS certified reference materials ( CRMs) , and in- house well- characterised rock samples (Figure 5.7; H-J) (Wang, D. et al., 2015).

5.2.5.4 Analysis of litter samples

Dry leaf litter was collected in the autumn of 2014 in the study sites of Henderson Reserve, Victoria Park, Bottlebrush Park, Moffatt Park and Waterford West, in Jimboomba within Logan City from 1 m × 1 m areas in the understorey of mature trees of both M. irbyana and M. bracteata species in 28 plots, each with three replicates. The fresh weights of litter samples were recorded before samples were oven dried at 70°C for 48 hours, and then dry weights were recorded. The litter samples were ground using a Cr-steel Ring Mill Pulveriser (ROCKLABS®) with a 2 mm mesh screen and analysed for total carbon and total nitrogen content using LECO® (TruMac® Series Macro) as described above (Section 5.3.5.2, Figure: 5.7; E).

5.2.5.5 Nutrient availability in mature forests

Soil nitrogen availability rate was assessed in situ using Ion Exchange Resin (IER) bags (Erskine et al. 1998). Resin bags were made from nylon net. The nylon net material was cut and assembled into 10 cm × 10 cm bags. Each bag was filled with 5 g fresh weight of mixed bed resin (Dowex MR-3, Sigma, St. Louis, MO, USA). Five bags were buried vertically at different depths within each of the 28 plots in the five study sites: two at a depth of 20 cm, two at a depth of 10 cm, and one at the ground level amongst the leaf litter. The purpose of using resin bags on the top 20 cm of the soil is to capture incoming ions, which originate above the soil core (Hart & Firestone, 1989).

The bags were left in situ for 35 days (April to June 2016) (Figure 5.6; A-D). During this time frame, 27. 5 mm of rain fell, and an additional 177 mm of rain fell over just three days before the collection of the resin bags. After removing the bags from the ground they were stored in separate plastic bags, labelled, and refrigerated at 4°C until all laboratory analyses were completed. The extractions of compounds consisted of placing resin bags in 20 mL of 1 M KCl solution on rotary suspension mixers (RATEK Instrument) at 60 rpm for

30 min. The eluent was decanted and filtered using Whatman® No 1 filter paper. The above steps were repeated five times to ensure thorough extraction from each of IER bags. The bulked eluent for each IER bag was analysed for Total Oxidised Nitrogen (TON) using Thermo ScientificTM ( GALLERYTM Automated Photometric Analyzer, Ammonia High - method) (Figure 5. 7; F-G). The TON was analysed for nitrate (NO3 ), and ammonium + (NH4 ) (Binkley, 1984; Livesley et al., 2011).

Figure 5.6 Resin bag preparation; (A) Resin bags at soil surface (0 cm depth) were covered with litter, (B) Resin bags at 10 cm depth were covered with soil, (C) Resin bags at 20 cm depth were covered with soil (D) Resin bags were tagged with labels tied to pegs to facilitate identification and retrieval of bag.

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Figure 5.7 Soil, litter and resin bag preparations and equipment for available nutrients analyses: (A) Soil samples, (B) Mixing of soil suspension (1 part soil and 5 parts water) on rotary suspension mixer for soil pH measurement, (C) Mixing resin bag suspension with 1 M KCl solution on rotary suspension mixer, (D) LECO® machine for total carbon and total nitrogen measurements (E) Litter samples, (F) Example of resin bags before commencement of experiment, (G) Thermo ScientificTM (GALLERYTM Automated - + Photometric Analyzer, Ammonia High method) for NO3 and NH4 , (H) X-ray fluorescence spectrometer, (I) Claisse® The Ox® automated fusion machine and (J) XRF fused glass discs.

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5.2.5.6 Data analyses

Soil characteristics were statistically compared between adult populations of M. irbyana and M. bracteata using an ANOVA with an error structure. The nested analyses of an error structure of plots nested within sites was used to re-partition variations in line with the sampling design of this study (Crawley, 2014). The different parameters of height, DBH, basal area, stem density, total tree volume and canopy structure of mature thickets of M. irbyana and M. bracteata forests were compared using linear mixed effects models ( LMEMs) and estimated with maximum likelihood (ML) in R statistical program. All LMEMs were fitted using the package “nlme”, R version 3.3.0 (Pinheiro et al., 2016). Random effects were plots nested within sites and growth traits of each forest as a fixed effect in the models (Pinheiro et al. , 2016). Wald F- tests were used to assess the significance of the interactions of the fixed effects in the models. Principal Component Analysis

Principal Component Analyses (PCA) were used to compare the variation in soil characteristics between mature thickets of M. irbyana and M. bracteata forests, as well as litter variation under a non-hypothesis scenario (Wildi, 2010). Commonly, PCA is used to ascertain whether there are any obvious subdivisions. Importantly for the assessment of soil characteristics between two species co-occurring habitats, PCA was used to identify which soil characteristics are particularly variable or constant in each habitat of the two study species. Principal Component Analysis ( PCA) was run using the R statistical computing packages: devtools, factoextra, FactoMineR, ggplot2, rgl, ggbiplot, git2r, lattice and permute http://agrocampus-rennes.fr/math/ (Husson et al., 2007; Lê et al., 2008).

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5.3 Results

5.3.1. Remnant mature tree performances

The research was conducted within the Jimboomba and Waterford West regions of Logan City, Queensland, Australia at five sites. The climate of this area is characterised by an average rainfall of 292 mm (2014-2016). However, during the study period (2014-2016) there was unprecedented low rainfall for several months (~40-60 mm). Drought incidence during early stages of the study in 2014 and 2015 at five sites has been identified as a major reason for low soil moisture content. Soil moisture availability is an important factor for plant growth (Wendelberger et al., 2008).

Overall, height did not differ significantly between M. irbyana and M. bracteata remnant mature trees during the three years of study (F2,2233 = 3.27, P = 0.07). However, when comparing the effect of year to the height of mature remnant trees, significant differences were found (F2,2233 = 113.34, P < 0.001; Table 5.1). The effect of year on plant height depended on the species of remnant mature trees (F2, 2233 = 7.62, P < 0.001; Table 5.1), as indicated by an interaction between plot and sites. While, the height of trees in both M. irbyana and M. bracteata increased every year, the increase was more pronounced in M. irbyana (Figure 5.8).

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Figure 5.8 Higher-order fixed effects from LMEM terms where the response variables are height and how it varies with year of observation (2014-2016) in each habitat of remnant mature trees between Melaleuca irbyana (MI) and Melaleuca bracteata (MB). Error bars represent ± standard error.

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A significant difference was found in DBH between adult populations of M. irbyana and M. bracteata (F1,2233 = 28. 79, P < 0. 001), with DBH increasing over time for both species (F2,2233 = 23.21, P < 0.001; Table 5.1), but the increase of M. irbyana was higher than for M. bracteata forests. Melaleuca irbyana showed overall a higher mean DBH than for M. bracteata in each of the three years of measurement. However, year on year, these differences were not significant (F2,2233 = 1.18, P = 0.30; Figure 5.9).

Figure 5.9 Higher-order fixed effects from LMEM terms where the response variables are DBH and how DBH varied depending on the year of measurement ( 2014- 2016) in Melaleuca irbyana ( MI) and Melaleuca bracteata ( MB) adult populations. Error bars represent ± standard error.

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No significant differences were observed in basal area (BA) between the two species

(F2,2233 = 0.35, P = 0.55), and year (F2, 2233 = 1.06, P = 0.34). However, BA did differ significantly depending on the year of measurement (F2,2233 = 24.25, P < 0.001) (Table 5.1), (Figure 5.10).

Figure 5.10 Higher-order fixed effects from LMEM terms where the response variables are basal area (BA) and how BA varied depending on the year of measurement (2014-2016) in Melaleuca irbyana ( MI) and Melaleuca bracteata ( MB) adult populations. Error bars represent ± standard error.

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Stem density per hectare or species abundance did not differ significantly between

M. irbyana and M. bracteata species (F1,1375 = 1.68, P = 0.21) (Table 5.1). Our results showed that species abundance had slightly declined in both M. irbyana and M. bracteata species during the three years of study. The effect of years showed that the decline in tree numbers in M. irbyana is less than for M. bracteata (F1,1375 = 6.05, P < 0.01). During, the first and the second year of observation M. irbyana had a similar number of stems, but the numbers reduced in the third year (Figure 5.11).

Figure 5. 11 Higher-order fixed effects from LMEM terms where the response variable is stem density per hectare and how stem density varied depending on the year of measurement (2014-2016) in Melaleuca irbyana (MI) and Melaleuca bracteata (MB) adult populations. Error bars represent ± standard error.

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No significant differences were observed with respect to shoot elongation in M. irbyana and M. bracteata remnant mature trees (F2, 1568 = 0.38, P = 0.54; Table 5.1). The effect of year on shoot elongation depended on species of remnant mature trees (F2,1568 = 416.98, P < 0.001; Table 5.1), as indicated by an interaction between plots and sites (Figure 5.12).

Figure 5. 12 Higher-order fixed effects from LMEM terms where the response variable is shoot elongation and how shoot elongation varied depending on the year of measurement (2014-2016) in Melaleuca irbyana (MI) and Melaleuca bracteata (MB). Error bars represent ± standard error.

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Independent variables numDf denDf F-value P-value (Height) Species 1 2233 3.27 0.07 NS Year 2 2233 113.34 < .0001 *** Species × Year 2 2233 7.62 0.0005 ***

(DBH) Species 1 2233 28.79 <.0001 *** Year 2 2233 23.21 <.0001 *** Species × Year 2 2233 1.18 0.30 NS

(Basal area) Species 1 2233 0.35 0.55 NS Year 2 2233 24.25 <.0001 *** Species ×Year 2 2233 1.06 0.34 NS

(Stem/ha) Species 1 22 1.68 0.21 NS Year 2 48 6.05 0.0045 ** Species × Year 2 48 1.40 0.26 NS

(Shoot elongation) Species 1 22 0.38 0.54 NS Year 2 1568 416.98 0.001 *** Species × Year 2 1568 2.03 0.13 NS

Significant codes: *** P < 0.001, ** P < 0.01, * P < 0.05 and NS (non-significant)

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5.3.2 Habitats’ nutrient availability

Major and minor element availability

This section presents the results of soil analyses that compared 14 major and minor nutrients and three characteristics of litter samples between M. irbyana and M. bracteata habitats. There were no significant differences in elements that are considered macronutrients for plants between habitats (Table 5.2). Soils, however, showed marginally significant differences in aluminium (Al) content between the two habitats (F1, 27 = 9.80, P < 0.05), with M. irbyana habitats showing slightly higher levels than M. bracteata habitats.

A similar, trend was found for magnesium (Mg) (F1, 27 = 20.97, P < 0.05) and manganese

(Mn) (F1, 27 = 0.03, P = 0.07). There were also marginally significant differences between habitats in potassium (K) (F1, 27 = 8.39, P < 0.05), sodium (Na) (F1, 27 = 1.95, P < 0.05), and significant differences in silicon (Si) (F1, 27 = 6.21, P < 0.1; Figures 5.13 - 5.14) (Table 5. 2), but in the case of these elements M. bracteata soils showed higher levels than M. irbyana habitats. Overall, soil moisture contents in M. irbyana and M. bracteata habitats did not differ significantly, being estimated at 4% and 3% moisture respectively (F1, 26 = 2.17; P = 0.15; Figure 5.15; A). In contrast, organic matter differed significantly between M. irbyana and M. bracteata habitats, with M. irbyana soil showing higher levels of organic matter than M. bracteata habitats (10% and 7% respectively) (F1, 26 = 7.75; P < 0.01; Figure 5.15; B).

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Table 5. 2 Results from an ANOVA used to assess differences in nutrient concentrations between Melaleuca irbyana and Melaleuca bracteata habitats in five sites at Logan City region with an error structure of plots nested within site.

Variables Df Sum of squares F-value P-value Soil total carbon 27 4.76 0.01 0.90 NS

Soil total nitrogen 27 0.01 0.00 0.98 NS

Soil carbon and nitrogen ratio (C/N) 27 29.49 0.03 0.87 NS

Litter total carbon 27 160.75 0.36 0.58 NS

Litter total nitrogen 27 0.26 0.17 0.70 NS

Litter carbon and nitrogen ratio (C/N) 27 1098.40 0.10 0.76 NS

pH 27 0.24 0.91 0.39 NS

Al 27 8.85 9.80 0.04 *

Ca 27 0.05 1.46 0.29 NS

Fe 27 14.33 0.95 0.38 NS

K 27 0.02 8.39 0.04 *

Mg 27 0.04 20.97 0.01 *

Mn 27 0.01 0.03 0.07(.)

Na 27 0.07 1.95 0.02*

P 27 0.01 0.47 0.53 NS

S 27 0.09 0.60 0.50 NS

Si 27 33.84 6.21 0.06 (.)

Significant codes: * P < 0.05, “.” < 0.1 and NS (non-significant)

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Figure 5.13 Comparison of nutrients available in soil (14) and litter (3) in remnant mature habitats between Melaleuca irbyana (MI) and Melaleuca bracteata (MB). The asterisk (*) indicates that there was a significant difference at P <0.05 and “. ” at P < 0.1, whereas no signs were shown as not significant.

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Figure 5. 14 Comparison of nutrient elements found in Melaleuca irbyana ( MI) and Melaleuca bracteata (MB) habitats, which are significantly (or marginally) different. (A) Aluminium; Al (%) (B) Potassium; K (%) (C) Magnesium; Mg (%) (D) Sodium; Na (%) (E) Manganese; Mn (%) and (F) Silicon; (Si) (%).

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Figure 5. 15 Comparison of % moisture content (A) and % organic matter (B) in soils between Melaleuca irbyana (MI) and Melaleuca bracteata (MB) habitats in 28 plots of five experiment sites.

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5.3.2.1 Principal Component Analysis of soil properties

Nutrient availability

Principal Component Analysis (PCA) results, which were used to summarise the main variations between M. irbyana and M. bracteata habitats based on 17 characteristics of nutrient levels, are shown in Tables 5.3 and 5.4. The first four major axes explain 73.09% of the variation in soil availability data (Figures 5.16-5.19). The first axis (with 29.1% of explanatory power) was primarily driven by levels of silicon (Si), iron (Fe), phosphorous (P), and manganese (Mn) in the soil. The second axis, with 18.07% explanatory power, was driven by potassium (K) and sodium (Na). The third and the fourth axes (with 13. 7 and 12.21% explanatory power, respectively) were driven by soil total nitrogen (TN), litter C/N ratio, and litter total nitrogen (TN) on axis three (Table 5.3). The structure of PCA ordination revealed that the two habitats had shared some traits of nutrient levels and had similar levels of macro and micro-nutrients. Correlation between the first PC (axis one) and the measured soil properties are depicted in Figure 5.17. However, four elements appear to differ between the two habitats, especially along axes one ( Na and K elements) and two ( Fe and Si elements), but there is a considerable amount of dispersion between soil samples collected from M. irbyana and M. bracteata (Figure 5.17). Histograms showing the variance of all the principal components nutrients available from 28 plots at five sites from the maximum to the minimum levels run along the X axis or the first axis (PC1) (Figure 5. 18) and the second histograms run along from the maximum to the minimum levels of nutrients available of Y axis or the second axis (PC2) (Figure 5.19).

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Table 5.3 Principal component loadings of the data set, eigenvalues and their contribution to the correlations, showing only the first five components.

Nutrient characteristics PC1 PC2 PC3 PC4 PC5 Total nitrogen (soil) 0.13 0.15 0.58 0.02 0.06 Total carbon (soil) 0.07 0.44 0.34 0.03 0.05 C/N ratio (soil) 0.11 0.34 0.12 1E-04 0.02 pH 0.11 0.02 0.25 0.05 0.34 Total nitrogen (litter) 0.03 0.18 0.00 0.54 0.01 Total carbon (litter) 0.01 0.01 0.30 0.28 0.01 C/N ratio (litter) 0.05 0.09 0.09 0.75 0.00 Fe 0.71 0.04 0.12 5E-04 0.00 Mn 0.47 9E-04 0.20 0.03 0.03 Ca 0.48 0.02 0.02 6E-04 0.37 K 0.02 0.70 0.04 0.12 0.04 S 0.42 0.08 0.12 0.00 0.05 P 0.70 0.15 0.01 0.00 0.03 Si 0.80 0.00 0.04 0.03 0.07 Al 0.26 0.21 0.05 0.06 0.27 Mg 0.56 0.02 0.01 0.07 0.15 Na 0.02 0.63 0.03 0.10 0.15 Importance of components Eigen values 4.95 3.07 2.33 2.08 1.64 Proportion explained 29.10 18.07 13.70 12.21 9.70 Cumulative proportion 29.11 47.18 60.88 73.09 82.75

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Table 5.4 The table shows eigenvalues extracted from the correlation matrix of the selected soil properties and present variations and cumulative percentage of variance explained by each principal component axis (PC) for the entire dataset between Melaleuca irbyana and Melaleuca bracteata habitats in Logan City region.

PC Eigenvalues % Variance Cumulative % 1 4.948324 29.108 29.108 2 3.071883 18.070 47.178 3 2.328845 13.699 60.877 4 2.07593 12.211 73.088 5 1.643346 9.667 82.755 6 0.868394 5.108 87.863 7 0.818966 4.817 92.681 8 0.560233 3.295 95.976 9 0.252392 1.485 97.461 10 0.191236 1.125 98.586 11 0.096934 0.570 99.156 12 0.081572 0.480 99.636 13 0.031708 0.187 99.822 14 0.015985 0.094 99.916 15 0.009313 0.055 99.971 16 0.004061 0.024 99.995 17 0.000876 0.005 100

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Figure 5. 16 Histogram of the Principal Components Analysis illustrating the principal components variances of the nutrient characteristics measured from 28 plots between Melaleuca irbyana habitats and Melaleuca bracteata habitats in five sites.

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Figure 5. 17 Graphical ordination of the five sites of the results of Principal Components Analysis (PCA) for soil nutrient availability across all 28 plots. Both nutrient availability scores and quadrant scores of the first two ordination axes are plotted. Each colour dot (M. bracteata) and triangle (M. irbyana) represents nutrient availability in the soil conditions for each species The x-axis represents the scores for the first principal component, the y- axis the scores for the second principal components; in the axis label, the percentage of variance explanation is given in parentheses. Component variables are represented by arrows that indicate the proportion of the original variance explained by the first two principal components. Directions of the arrows indicate the relative loadings on the first and second principal components. The orientation of arrows indicates the direction in ordination space where the soil variables change most rapidly, and in which they have maximum correlation with the ordination configuration, whereas the length of the arrows indicates the rate of change.

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Figure 5.18 The contribution histogram of variable PC 1 (29.1%) illustrates the variance of all the principal components which run along the X axis of previously shown PCA Figure 5.17. They are primarily driven by Si, Fe, P and Mn elements in the soil measured from 28 plots in five sites.

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Figure 5. 19 The contribution of variable PC 2 (18. 1 %) illustrates the variance of all the principal components which run along the Y axis of previously shown PCA Figure 5. 17. They are primarily driven by K, Na, and soil total carbon elements in the soil measured from 28 plots from five sites.

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5.3.2 Nitrogen mineralisation and nitrification

5.3.2.1 In situ ion-exchange using resin bags

Overall, in the top 20 cm of the soil, nitrogen availability, especially ammonium + (NH4 ), differed significantly between M. irbyana and M. bracteata (F1,134 = 4.80, P < 0.05) (Table 5. 5 and Figure 5. 20), which contrasts with the results found for total soil nitrogen stocks in the soil, as they did not differ significantly. The results show that M. irbyana soils + - contained only a narrow range of both ammonium (NH4 ) and nitrate (NO3 ). Melaleuca + bracteata soils had a higher availability of NH4 (Figures 5.20-5.21), except at 0 cm or soil - surface. In contrast, there was no significant difference in NO3 between soils collected from sites dominated by both species (F1,134 = 0.26, P = 0.61). A post hoc Tukey test showed that + - there was significant variation in the NH4 and NO3 availability at different soil depths (Table 5.6).

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Table 5.5 ANOVA results using an error structure of plots nested within sites, comparing + - levels of ammonium (NH 4) and nitrate (NO3 ) in soils between Melaleuca irbyana and Melaleuca bracteata sites at different depths (0, 10 and 20 cm) in in situ ion exchange resin bags.

Variables Df Sum of squares F-value P-value

+ NH 4 Species 1 9.05 4.80 0.03* Depth 2 17.15 4.55 0.01* Species × Depth 2 6.32 1.68 0.19NS Residuals 134

- NO 3 Species 1 60 0.26 0.61NS Depth 2 5084 11.15 3.32e-05*** Species ×Depth 2 68 0.15 0.86NS Residuals 134 Significant codes: *** P < 0.001, * P < 0.05 and NS (non-significant)

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Table 5. 6 Result of Tukey multiple comparison of means test of ANOVA results using an + error structure of plots nested within sites comparing levels of ammonium ( NH 4) and - nitrate(NO3 ) in soil between Melaleuca irbyana and Melaleuca bracteata sites at different depths (0, 10 and 20 cm) in in situ ion exchange resin bags.

+ Treatment (NH 4) P-value M. irbyana (MI) × M. bracteata (MB) MB : 10 - MB : 0 < 0.05 MB : 10 - MI : 0 < 0.05 MI : 20 - MB : 10 < 0.05

- Treatment (NO 3) P-value MB : 10 - MB : 0 < 0.05 MI : 10 - MB : 0 < 0.05 MB : 10 - MI : 0 < 0.05 MI: 10 - MI : 0 < 0.05 MI : 20 - MI : 10 < 0.05

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+ Figure 5. 20 Soil nitrogen measured as NH 4 including differences in depth of buried resin bags (0, 10, and 20 cm) between M. irbyana (MI) and M. bracteata (MB) remnant mature trees.

- Figure 5. 21 Soil nitrogen measured as NO3 including differences in depth of buried resin bags (0, 10, and 20 cm) between M. irbyana (MI) and M. bracteata (MB) remnant mature trees.

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5.3.2.2 Principal component analysis of nitrogen uptake

Nitrification related to nitrogen mineralisation

The graphical representations of PCA were used to summarise the variation between M. irbyana and M. bracteata remnant mature trees’ availability of inorganic nitrogen from resin bags at three depths (0, 10 and 20 cm). The two major axes explain 100% of the variation in inorganic nitrogen data (Figure 5.22). The first axis was primarily driven by + - variations in NH4 and NO3 (with 69% of explanation power; Tables 5.7-5.8). The results + - showed the relation between NH4 and NO3 are equal (with the same length of arrow, Figure 5. 22) in terms of value and found M. bracteata had wider variance of data in axis one (PC1= 69%), while M. irbyana had a narrower range of variance of data in axis two (PC2 = 31%). The structure of PCA ordination revealed that the two habitats contain similar levels of inorganic nitrogen.

Table 5.7 Principal component loadings of the data set, eigenvalues and their contribution to the correlations, showing the three components.

Nutrient characteristics PC1 PC2 + NH4 0.8307909 -0.5565846 - NO3 0.8307909 0.5565846 Importance of components Eigen values 1.1749 0.7871 Proportion explained 0.6902 0.3098 Cumulative proportion 0.6902 1.0000

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Table 5.8 Summary statistics for principal components analysis of nitrogen generated in soil of mature remnant trees between Melaleuca irbyana and Melaleuca bracteata in Jimboomba, Logan City region. The table shows eigenvalues, present variations and cumulative percentages of variance explained by each principal component axis (PC) for the entire dataset.

PC Eigenvalues % Variance Cumulative % 1 1.3804271 69.02135 69.02135 2 0.6195729 30.97865 100.00

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Figure 5.22 Graphical ordination of five sites, 28 plots of the result of Principal Component Analysis (PCA) based on inorganic nitrogen is generated in the soil by in situ ion exchange using resin bags between M. bracteata and M. irbyana remnant mature trees; 95% confidence ellipses are shown for each species. Both inorganic nitrogen uptake scores and quadrant scores of the first two ordination axes are plotted. Each coloured dot (M. bracteata) + - and triangle (M. irbyana) represents nitrogen rate (NH4 and NO3 respectively) for each species. The scores in the axis label the percentage of variance, and explanation is given in parentheses. The original variables are represented by arrows that indicate the proportion of the original variance. The direction of the arrows indicates the relative loadings on the first and second principal components. The orientation of arrows indicates the direction in ordination space in which the inorganic nitrogen availability changes most rapidly and in which they have maximum correlation with the ordination configuration, whereas the length of the arrows indicates the rate of change.

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5.4 Discussion

5.4.1 Plant growth performance

Despite being limited in distribution, M. irbyana adult populations showed higher levels of growth in terms of increment in height, DBH, BA, and shoot elongation than M. bracteata adult populations. These results are consistent with Richards, A. et al. (2003) who found that restricted Archidendron kanisii presented higher maximum electron transport rate, greater dissipative capacity for transferring excess light, and enhanced nitrogen in photosynthetic systems than widespread A. whitei species. Lavergne et al. (2004) compared biological and ecological characteristics of 20 congeneric narrow endemic plant species with their widespread congeners in the French Mediterranean region. They found that rare endemic species did not show differences in traits that related to resource acquisition (specific leaf area, leaf nitrogen content, photosynthetic rate) or resource conservation (leaf dry matter content) (Lavergne et al., 2004). Thus, the causes of rarity may be unique in each species and each stage of the plant’s life cycle.

Soil characteristics at M. irbyana-dominated sites were higher in organic matter, Al, Mg and Mn, than at M. bracteata-dominated sites. The maximum tree height is an important feature of forest vegetation, due to its direct connection to the local demographic, standing biomass and resources used (Kempes et al., 2011). Baltzer et al. (2007) demonstrated that widespread species showed more conservative patterns of growth and responsiveness to variations in local edaphic conditions when compared with restricted distribution species. The higher levels of nutritional elements in M. irbyana forests may contribute to the higher growth performance measured in adult populations than for M. bracteata.

The findings that M. irbyana presented higher growth rate than M. bracteata at the mature stage even during the drought periods may due to several factors. First, M. irbyana may not have more limited habitat requirements than M. bracteata at mature stages in its life-cycle. Secondly, M. irbyana may perform well at the same level of resources as M. bracteata, but may still respond negatively to changes in the specific habitat or suitable habitats, due to habitat fragmentation due to human activities. Thirdly, some evidence showed that M. irbyana may be groundwater dependent, because it showed a higher growth rate than M. bracteata when there was unprecedented low rainfall during the time these

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measurements were taken. Finally, the finding from Chapter 3 (seed germination) was that M. irbyana had a slower rate of seed germination and overall success rate of germination than M. bracteata. The slower rate might affect seedling recruitment and survival in the early stages of this species’ life cycle; a trend that was also found at the seedling stage in Chapter 4 (reciprocal seedlings) where M. irbyana seedlings showed lower survival rates and slower growth rate than M. bracteata.

These lower germination and survival rates and slower growth in the early stages of M. irbyana’s growth might limit the distribution, as adult populations are performing well. Matesanz et al. (2009) compared the functional ecology between a rare Thymus loscosii with widespread T. vulgaris in semiarid Spain, and found that T. loscosii is not a habitat - specialist species but behaved as a refuge endemic (competition between two species when they co-occur). The same study also stated that T. loscosii had restricted competitive ability, which is associated with vegetative growth traits (Matesanz et al., 2009). These results are also consistent with Osunkoya and Swanborough (2001), who found that rare Gardenia actinocarpa ( Puttock) has a surprisingly superior physiological performance and competitive ability than widespread G. ovularis F. M. Bailey, by presenting greater plasticity in the seedling stage. This study also suggested that the rarity of G. actinocarpa might be because of low fecundity, lack of seed dispersal ability and soil seed banks, rather than inferior vegetative growth rate and ecophysiological traits ( Osunkoya & Swanborough, 2001).

5.4.2 Habitat quality, nutrient availability and soil pH

The soil data showed that the major and minor elements of soil chemistry were similar in both M. irbyana and M. bracteata forests. Similarly, Richards, A. et al. (2003) found that soil nutrient availability in the habitats of restricted and widely distributed species did not differ significantly, suggesting that species grow under comparable soil nutrient conditions. However, M. irbyana forests had marginally higher concentrations of soil Al, Mg, and Mn elements than M. bracteata forests. In contrast, M. bracteata had significantly higher concentrations of soil K, NA and Si than were found in M. irbyana forests. Melaleuca bracteata soils are higher in sand and salts than M. irbyana soils and inorganic nitrogen availability was lower than in M. bracteata soils. Soils with high sand contents are known

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to be lower in nutrient availability because of reduced cation exchange capacities (Weil et al. , 2016), which may further explain M. bracteata’s lower growth performance than M. irbyana, whose adult populations tend to grow on higher nutrient soils with lower amounts of sand. Sandy soils have larger particles and consequently larger pores, which increase aeration and more easily drain water (Rawls et al., 1982). Harms and Noble (1996) stated that M. irbyana generally occurred in clay type soils. The clay soil type, however, is richer in nutrient availability (cabon and nitrogen) and has higher water retention capacity (Lee et al. , 2002). Our results showed that M. irbyana forests had higher organic matter than M. bracteata forests. The high clay content in soil is positive correlated to the high amount of organic matter (Saxton & Rawls, 2006). Soil organic matter maintains soil structure by increasing the cation exchange capacity and improving water retention (Havlin et al., 2005). As a consequence, soil organic matter can be positively correlated with an increasing growth rate of plants (Kononova, 2013). Specific plant species may have considerable effects on the quality and quantity of soil organic matter (Ehrenfeld et al., 2005). The results for nitrogen availability from the resin bags experiment revealed that the + - rate of production of NH4 and NO3 levels in the soil of M. bracteata forests was higher + - than in M. irbyana forests at all depths (5, 10 and 20 cm). Maximum NH4 and NO3 levels were found at depths of 10 cm. Kinney and Lindroth ( 1997) found that high nitrate availability in soil increased the relative growth rate in some plant species (91% of aspen growth). The relationship between growth rate and net nitrification potential may explain why M. irbyana in the mature stage is well adapted to soil conditions and grows well under poor soil and low ammonium nutrition.

5.5 Conclusions

This is the first observational study of the performance of remnant mature M. irbyana (narrowly distributed species) compared with the common congeneric M. bracteata trees (widely distributed species). Our results showed that M. irbyana had a higher tree growth rate in mature stages compared with M. bracteata, which was contrary to our hypothesis that rare species would present lower performance in every stage in terms of physiological and ecological characteristics. This demonstrates that the naturally dense monoculture stands

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that M. irbyana populations form may be beneficial for growth rates. However, causes of species’ narrow distribution seem to be unique in each species (Richards, A. et al., 2003). According to the Environment Protection and Biodiversity Conservation (EPBC) Act 1999, M. irbyana species is thought to require access to groundwater on a permanent or intermittent basis to meet all or some of their water requirements. Information about groundwater may provide useful knowledge for how M. irbyana maintain their communities at the mature stage. It should be noted that data on performance (height, DBH, BA, stem density, and shoot elongation) were obtained from a short period of observation (2014-2016). Future investigations on the effect of environmental conditions on growth rates of M. irbyana and the common co-occurring M. bracteata should take place over longer periods of time. Future studies may also focus on physiological factors such as photosynthesis rate, specific leaf area, relative growth rate (RGR), leaf biomass, and leaf nitrogen assimilation. Furthermore, growth rate studies should include monthly monitoring of shoot elongation for every season, as recommended from previous research (Rossi et al., 2009; Seo et al., 2010; Wang, Y. et al., 2012).

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Chapter 6: General discussion and recommendations

6.1 Purpose of the study

The main purpose of this study was to compare the performance of the critically endangered M. irbyana and the common and often co-occurring M. bracteata species during the whole life cycle of these plants. Specifically, the study aimed to determine the optimal conditions for seed germination, seedling survival and growth rate, and the growth of remnant mature trees. My findings also contributes more broadly to understandings of the growth traits and environmental conditions that limit some plant species’ range, while fostering the widespread distribution of other related species. The overarching research question addressed in this study is: “Why does M. irbyana have a narrower distribution than the co-occurring M. bracteata?” More specifically, the following three key questions were addressed by this thesis:

• What are the main ecological differences between M. irbyana and M. bracteata habitats that might explain the reasons for the narrow distribution of M. irbyana? • Do limitations in the reproductive capabilities and physiological traits of M. irbyana lead to its becoming rare? • What are the differences between the habitats of M. irbyana and M. bracteata especially in terms of soil and nutrient characteristics?

In the following section, I summarise the findings of this study in regards to each of these questions. I also include a general discussion of the research findings and make recommendations for M. irbyana restoration and management of existing remnant populations. I conclude this chapter by making suggestions and recommendations for future research.

6.2 Key outcomes and limitations of the experimental chapters

In Chapter three, I compared the time to and success rates of germination between M. irbyana and M. bracteata seeds as affected by temperature, photoperiod and light availability under in vitro and in situ conditions. The results show that the endangered M.

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irbyana had a lower germination success rate and germination was also slower under controlled conditions in growth cabinets and in the field, compared to the common co- occurring M. bracteata. Melaleuca irbyana germination success appears to be particularly sensitive to cooler temperatures and light availability, compared to M. bracteata. Furthermore, M. irbyana’ s seed germination occurred under a narrower range of temperatures, with higher germination success rate under higher temperature (e. g. , 30°C, 71%), than lower temperatures (e.g., 15°C, 3% and 25°C, 52%). Contrary to the findings under controlled conditions, in the field germination study, both species displayed low germination success rates, although again M. bracteata seeds overall germinated faster and more often. A limitation of the findings from the field germination study was that the experiment occurred during a growing season of unusually low rainfall, which may explain the low germination success of both species. In Chapter four, I compared survival and growth rate of both M. irbyana and M. bracteata seedlings that were planted in the understory of remnant mature populations of both species. I found that M. bracteata seedlings had a higher survival rate and growth rate (as measured using apical height) than M. irbyana seedlings under canopies of both species. Seedling survival and growth rates of both M. irbyana and M. bracteata were reduced under the canopy of M. irbyana, suggesting that these endangered habitats may inhibit recruitment, possibly because of dense canopies, litter or specific soil conditions. Furthermore, the low survival and slower vegetative growth rates of M. irbyana may mean its colonisation capacity requires specific conditions in the natural environment; for example, increased light availability. There findings suggest then that at the early stages of growth, M. irbyana may be a habitat specialist, and that restoration of M. irbyana is best not conducted under dense canopies, but better in open areas or sites with sparse tree cover. A limitation of this reciprocal planting study was that due to severe weather events the rate of seedling mortality at the beginning of the study, especially during summer, was very high during the time when the seedling experiment was first being established. Additionally, during this experiment, some plots, such as those at the Henderson Reserve site and Bottlebrush, were flooded, making it difficult access the experimental sites to observe the seedlings’ performance. In Chapter five, I compared the growth and habitat-specific conditions of remnant mature populations of M. irbyana and M. bracteata. I found that at the adult stage, M.

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irbyana presented a higher growth rate in terms of height and DBH when compared to M. bracteata. This finding suggests that once M. irbyana reaches maturity, there may be less of an impediment to its growth, resulting in dense foliage typical of the natural monocultures of M. irbyana. This study also found differences in soil characteristics in M. irbyana and M. bracteata habitats. Soil characteristics may explain why the adult M. irbyana monocultures require their own specific habitat for successful growth and development. Overall, M. irbyana seems to have a limited distribution because of specific habitat requirements at the early stages of its life cycle; however, adult populations of M. irbyana appear highly successful in growth during severe weather or drought periods, which provides some evidence of the suspected groundwater dependency of this species. This suggests that habitat characteristics are the main factors to consider for the conservation and extension of existing M. irbyana remnant mature populations. These findings will assist management and improve the effectiveness of restoration programs for M. irbyana. While this chapter provided insights into the kind of nutrients available in the soil environments of M. irbyana and M. bracteata, to have a better understanding of the impact of these nutrients on M. irbyana’ s and M. bracteata’ s growth and development, further research is required into the topic of nutrient acquisition. How much is taken up by these plants?

6.3 Recommendations for conservation and restoration of Melaleuca irbyana

Habitat modification and fragmentation has resulted in the loss of biodiversity worldwide ( Hanski, 2015) . Restricted distribution of some species may be due to their inability to establish and compete with other species. Thus, it is imperative for conservation programs to be based on an understanding of optimal habitat conditions for species like M. irbyana that are threatened with extinction, including biotic and abiotic factors. The findings of my research provide key insights into the probable causes of rarity for M. irbyana, with a hierarchal ranking of those causes (Tables 2.1-2.3) (Fiedler, P.L & Ahouse, 1992; Rabinowitz, 1981; Rey Benayas et al. , 1999) . Melaleuca irbyana has a naturally limited distribution largely centred in south-eastern Queensland. Land clearing and habitat fragmentation from urban expansion into the M. irbyana region have reduced its occurrence and the size of populations; populations are becoming more fragmented, with an even narrower distribution than the species originally had. Rabinowitz (1981) described three characteristics of species’ rarity: geographical range (small vs large); local population

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size ( large, dominant vs small, non- dominant) ; and habitat’ s specificity ( wide versus narrow) . Melaleuca irbyana originally displayed a small geographic range and a large population size, which categorises it as an endemic rare species. The findings of in vitro and in situ experiments show lower germination success rates and lower seedling survival and growth in contrast to M. bracteata. These findings suggest that the early stages of recruitment should be the key focus for M. irbyana conservation programs, as recruitment will be key for ensuring the viability of remaining populations (Lynch & Drury, 2006). Australia is an old continent, and soil nutrients have played an important role in the analysis of the ecophysiology of Australian plants (Stewart & Schmidt, 1999). Soil type, soil structure, and drainage, may also be causes of reduced species distribution, which hamper seedlings’ establishment (Beadle, 1954; Honnay et al. , 1999). The findings show that M. irbyana and M. bracteata forests are likely to acquire nutrients from soil in different ways for their growth at the adult stage. More research is needed on this topic, as I measured nutrient levels in the soil and not nutrient uptake characteristics of M. irbyana and M. bracteata.

6.4 Future research directions

The research I present here led to the identification of a number of important future research directions to increase our understanding and knowledge of the ecophysiology of remaining populations M. irbyana for future revegetation projects.

1. Habitat fragmentation can influence pollination and eventual seed dispersal. Therefore, future research directions could focus on understanding relationships between species in geographically isolated areas and their pollinators, as well as potential seed dispersal limitations.

2. Only a few habitat factors, such as soil type, soil moisture, nutrient availability, light and temperature regimes, were investigated in the present studies in the Jimboomba region. Expanding these studies to more habitats and focusing on the interactions between plants and their environment as well as their neighbouring environments, which can affect seedling establishment, may provide further useful information concerning growth requirements of M. irbyana.

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3. The study of reciprocal seedlings provided indications of seedling growth and survival rates of M. irbyana and the benefits of increased light availability for both species studied. Other physiological factors such as measurements of the rate of photosynthesis, regulative growth rate ( RGR) affecting biomass, and specific leaf area should also be investigated. Such detailed information would help improve our understanding of the constraints to seedling growth and survival, providing further explanations for the rarity of M. irbyana.

4. Melaleuca irbyana forests are presumed to be groundwater dependent ecosystems. Such ecosystems require access to groundwater on a permanent or intermittent basis to meet all or some of their water requirements. Further investigation is required to establish the relationship between groundwater and the occurrence of M. irbyana. If it is found that M. irbyana relies on groundwater, it is then of paramount importance to protect aquifer recharge and place restrictions on extractions and development. Additionally, it needs to be determined how urban expansion in the Logan region, one of the fastest growing councils in south- east Queensland, and the associated reduction in catchment permeability, may cause changes to water table levels, amount of run-off, sedimentation, water flow and soil compaction, including how these may in turn impact M. irbyana.

5. Undertaking detailed molecular studies will help better understand M. irbyana’ s population genetics, providing useful information about phylogenetic relationships between remnant populations and levels of genetic diversity remaining (Burrough, 2016).

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Zutter, B. , Glover, G. , & Gjerstad, D. (1986). Effects of herbaceous weed control using herbicides on a young loblolly pine plantation. Forest Science, 32(4), 882-899.

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Appendix 1 Summary of research on seed germination and seedling growth depending on whether they are rare or common.

Species Traits Factors Findings Reference Rare Melaleuca deanei and Seed size Water potential 1. Melaleuca deanei had significantly larger seeds and also (Hewitt et al., the three common congeners Temperature and fire cues slower germinate in all experiments than the common 2015) of Melaleuca styphelioides, Light and shade levels species Melaleuca thymifolia, 2. Melaleuca deanei required specific habitats for Melaleuca nodosa species of germination such as require light for seedlings the Sydney region establishment, but in medium and high-light reducing seedlings survival 3. Melaleuca deanei had similar range of temperature and water potential requirement for germination as the three common species 4. Melaleuca deanei had a few seedlings recruitment in the field 5. Germination in all four species was not responded with heat and smoke 6. Melaleuca deanei may require all fire release from canopy, high-light after post-fire and rainfall all together for seedlings survival 150 relative vulnerability Seed mass, Soil types 1. Viability of fresh seeds is the cause of seed germination (Moles et al., species in Sturt National Seed viability and increase rate of seedling growth compare with 2003) Park, north-west New South diaspore mass while buried in soil for 1 year Wales, Australia 2. Species that have big seed mass had higher survival rate than small-seeded species 3. Buried seeds had better survival rate than seeds on soil surface Two rare Acacia species (A. Seed size, Fire (high temperature) effect 1. Fire stimulated seed germination in all species (Brown, J. et al., ausfeldii and A. Seed viability 2. Significantly lower rate of germination in small-seeded 2003) williamsonii) and three 3. Seed viability increased seed germination rate common Acacia species: A. pycnantha, A. genistifolia and A. paradoxa) co- occurring in south-east Australia

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Species Traits Factors Findings Reference

Eucalyptus of E. Seed size Soil types, 1. The large-seeded species germinated faster than small- (Schütz et al., macrocarpa, E. tetragona, Soil moisture, seeded 2002) E. loxophleba and E. wando Temperature (low 100C and 2. High rates of seedling mortality were found for all in South-Western Australia high 280C) species in the beginning after planting even in high soil moisture content 3. Germination rate in darkness in loamy soils was lower than in sandy soils Narrow endemic Petrocoptis Seed germination, Cold treatment, 1. Both species produce large number of seeds (Navarro & species P. grandiflora and Seedling survival, Light treatment, 2. The specific microhabitat of cracks and crevices of Guitian, 2003) P. viscosa Seed production, Natural habitat limestone rockfaces significantly increased seed (Caryophyllaceae) from Seed weight germination and seedling survival northwest lberian Peninsula 3. Seeds stored in darkness had higher germination rate than those in light (12:12 h photoperiod) 4. Exposure to a short cold period had no significant effect on germination in both species 5. High seed weight positively affected seed germination and seedling survival 6. In natural habitat, less than 10 % of germinated seeds survived by the end of experiment ( 30days) Eremosparton songoricum Seed germination, Burial depth, 1. Seed germination occurred at burial depth of maximum (Liu et al., 2011) (Fabaceae) a rare species in Seedlings survival Sand dune types, 6 cm Gurbantunggut Desert, Soil water content, 2. The deeper the burial the lower percentage of seed China Distribution pattern emergence 3. Seedlings which emerged from seed on the surface of sandy soil had higher survival success than seedlings that emerged from buried seeds 4. Limited sources of water stops seedlings establishment 5. Sowing seeds at the right time of the year and suitable place can help endangered species restoration

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Species Traits Factors Findings Reference Rare Gardenia actinocarpa Reproductive, Moisture regime, 1. Moisture had no significant effect on seed germination (Osunkoya & (Rubiaceae) and common traits Light regime in both species Swanborough, co-occurring G. ovularis Ecophysiological 2. High-light inhibited germination rate of both species 2001) north Queensland, Australia traits 3. Rare G. actinocarpa has long reproductive period (9 months) while common G. ovularis has shorter (4 months) 4. Rare G. actinocarpa produces 1.77 fruits per female trees while common G. ovularis produced more fruits (35.76 fruit per female tree) Common Mimosa claussenii Seed germination, Burial in natural condition 1. No correlation exists between seed germination and (Simon & Du Vall and three rare species of M. Seed production seed density in rare species Hay, 2003) decorticans and M. 2. Common M. claussenii occurred in dried and rocky heringeri, and M. environments; while, two rare species occurred in wetter setosissima in Central Brazil habitats 3. Rare species had high level of fruit set, more viable seeds per plant 4. Germination rate was low in all species 5. Germination occurred in the first month but none of the seedlings survived 5 pairs of geographically Seed germination, Soil water 1. Seeds germination occurred at 20˚C where adequate soil (Pohlman et al., Acacia species, restricted Seedling growth, moisture was present. 2005) species (A. cincinnata, A. rate 2. No significant difference in growth rate and biomass elata, A. fulva, A. Morphology, was observed in any of the species trachyphloia and A. physiology 3. Widespread species had better adaptive morphology silvestris) compare with than restricted species widespread species (A. 4. Widespread species had significantly higher melanoxylon, A. irrorata, photosynthetic capacity than narrowly distributed species and A. implexa, A. dealbata 5. Both species had adaptation to cope with soil moisture and A.mearnsii) in Eastern Australia Eucalyptus paliformis Seed germination, Five temperature regimes in 1. Germination of E. paliformis was inhibited at (Prober, 1992) restricted species and E. Seedling survival controlled environment temperature below 11/210C fraxinoides common species cabinets 2. E. fraxinoides germinated at 17/27˚C and later at in Wadbilliga Plateau of 14/24˚C south-eastern NSW

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Species Traits Factors Findings Reference 3. Interaction between temperature and seed collection place significantly increased rate of germination in both species 4. Low temperature reduced rate of seed germination in both species Narrowly endemic Seed germination, Temperature regimes 1. Heat treatment enhanced rate of seed germination and (Baskin, Jerry M Echinacea tennesseensis, Morphological Stratification methods seedlings survival in E. tennesseensis and E. angustifolia et al., 1997) Iliamna corei, Solidago trails, 2. Fire can reduce seed dormancy in I. corei and I. shorti) compared with Physiological rivularis widespread congener (E. traits, 3. S. shorti and S. altissima need cold stratification for angustifolia, I. rivularis, S. Cytological traits seed germinate altissima in America 4. S. shorti and S. altissima germinated during spring season 5. No differences were found in ecological, morphological, physiological, cytological, genetic or life history characteristics in Echinacea species but differences in their geographical distribution 6. Seed bank of widespread E. angustifolia and E. angustifolia persisted well and is a better competitor than narrow Echinacea tennesseensis and Solidago shorti Rare mariposa lilies Seed germination Natural field study 1. C. obispoensis and C. tiburonensis produced smaller (Fiedler, Peggy (Calochortus obispoensis C. Seed production bulbs, fewer flowers and less fruit set than common Lee, 1987) tiburonensis, and C. Seedling survival species pulchellus) and common (C. 2. C. obispoensis and C. tiburonensis had low seed albus) in Santa Lucia survival and seedlings establishment Mountains of central coastal 3. C. obispoensis , C. tiburonensis and C. pulchellus had California low adult mortality and slow growth 4. C. albus and C. pulchellus shared the same reproductive pattern producing high amount of seeds Two rare Hakea species Biomass Greenhouse condition 1. Rare Hakea species had root biomass and total root (Poot & Lambers, (Proteaceae) H. oldfieldii Root morphology length greater than widespread congeners 2003) and H. tuberculata with co- 2. Rare Hakea species roots grew deeper and had large occurring mass in 10 cm and 40 cm-deep pots than common species

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Species Traits Factors Findings Reference five widespread congeners 3. Rare Hakea had considerably greater total root length species (H. ceratophylla, H. than widespread congeners when compare in the same varia) and H. linearis plant mass occurring on winter-wet or nonwetland habitats on deeper soil (H. lissocarpha H. cyclocarpa) in mediterranean-climate south-western Australia Two rare Hakea Seedling survival Different habitats 1. Rare Hakea had horizontal uniform of root distribution (Poot & Lambers, (Proteaceae) H. oldfieldii Adaptive ability than common species 2008) and H.tuberculata compare 2. In reciprocal transplant traits, rare Hakea species had with four congers commonly higher seedling survival rate in their own habitat than other occurring (H. ceratophylla habitats Smith, H. varia) nearby 3. No relationship between habitat and adaptive abilities in winter-wet or nonwetland terms of root characteristics such as total root length, habitats on deeper soil (H. average root diameter, specific root length or root mass lissocarpha, H. cyclocarpa) ratio were observed in Mediterranean-climate south-western Australia Common wetland species Seed germination Habitat sizes, 1. Carex species had 35% less biomass, 30% fewer tillers, (Hooftman et al., (Carex davalliana and Seedling growth Degree of habitat isolation and 45% fewer flowering tillers than plants in large areas 2003) Succisa pratensis) in 18 Reproductive 2. Succisa species from small isolated habitat had 19% habitat islands, Switzerland capacity more biomass, 14% more flower heads, and 35% more flowers per flower head than plants from large areas 3. Succisa species from small isolated habitats yielded 32% more rosettes than plants in small connected islands 4. Reciprocal transplant of Succisa produced 7% more rosettes than large ones 5.There are no effects of small habitat size and isolation on germination in both species Narrowly distributed Seed germination, Cold treatment (cold/not- 1. The specific microhabitat of cracks and crevices of (Navarro & Petrocoptis species P. Seedling survival cold), limestone rockfaces significantly increased seed Guitian, 2003) grandiflora endemic species Light treatment (light/dark), germination and seedling survival

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Species Traits Factors Findings Reference and P. viscosa Natural condition / 2. Seed weight positively influenced seed germination and (Caryophyllaceae) in microhabitat seedling survival northwest lberian Peninsula 3. Seed in darkness showed higher germination percentage than 12:12 h photoperiod 4. The short period of cold period had no significant effect on either species 5. Seedlings in non-rockface soil microhabitat suffered more from herbivory or interspecific competition than seedlings in crevices in the rockface 3531 of individual seedlings Seedling survival, Community compensatory 1. The survival of seedlings was positively correlated with (Queenborough et in both rare and common Seedling growth, trend (CCT) seedlings’ height al., 2007) species which coexistent Seedling 2. More abundant of species had higher seedling mortality from the family recruitment (25-ha scale) Myristicaceae in highly 3. Seedling survival was inversely related to the relative diverse neotropical rain basal area of trees forest, Ecuador 4. Neighbourhood effects on seedling survival varies with tree species abundance

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Appendix 2 Conference Abstracts Appendix A: Conference Abstracts Conference Conference: Ecological Society of Australia Annual conference; 28 November to 2 December 2016, Fremantle, Western Australia

Title: Do habitat characteristics reduce growth and survival capacities of remnant mature endangered Melaleuca irbyana?

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Conference Abstracts 2 Conference: Society for conservation Biology 4th Oceania Congress; 5-8 July 2016, Brisbane, Australia

Title: Growth and survival capacity of endangered Melaleuca irbyana seedlings is more limited than a widespread congener

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Conference Abstracts 3 Conference: Ecological Society of Australia Annual conference; 29 November to 3 December 2015, Adelaide, South Australia

Title: Germination capacity of endangered Melaleuca irbyana is more restrictive than a widespread congener

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Conference Abstracts 4 Conference: International Student Conference on Conservation Science; 19-25 January 2015, Brisbane, Australia

Title: Comparing the ecological and physiological traits of the critical endangered Melaleuca irbyana to the more commonly occurring Melaleuca bracteata

Thita Soonthornvipat, Jennifer Firn, and Acram Taji

Melaleuca irbyana (swamp tea-tree) is a small to medium tree that is listed federally as critically endangered under the Environment Protection and Biodiversity Conservation Act 1999 and is protected under the Beaudesert Shire Planning scheme 2007 as an overlay in the Nature Conservation Overlay. Melaleuca irbyana’s distribution co-occurs peri-urban fringe of South East Queensland and therefore it is under threat from increased clearing and common effects of urbanisation such as eutrophication including coal- seam gas production. Recent mapping activities found only 998 hectares of M. irbyana forest in its original form, which is just 8.1% of its pre-European distribution. Not only its habitat under threat but M. irbyana is also a species with a limited original distribution; therefore it may have a set of reproductive, water-use and nutrient-use traits that explains its limited distribution and if understood could aid its recovery. The purpose of this research is quantify a number of key ecological and physiological traits of M. irbyana (adult and seedlings) and compare these characteristics to the more common Melaleuca bracteata that overlaps in its distribution and range with M. irbyana. The early results have shown that the critically endangered M. irbyana has a significantly lower germination success rate and seedling height development under a range of different temperatures and light conditions than the more commonly distributed M. bracteata. Overall, the traits this research measuring will assist with both the more effective management of existing populations of M. irbyana, and improved efficiency for planting programs by allowing us to pinpoint the traits that are likely limiting survival and expansion of M. irbyana.

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Appendix B: Awards and Grants 1. Earth, Environmental and Biological Science (QUT) Early Career Travel Grant-The grant was used to cover costs while attending Ecological Society of Australia Annual conference at Adelaide, 2015-valued at AUD 1,500.00.

2. Earth, Environmental and Biological Science (QUT) Early Career Travel Grant-The grant was used to cover costs while attending Ecological Society of Australia Annual conference at Fremantle, Western Australia, 2016-valued at AUD 1,800.00.

Appendix C: Membership of Professional Societies

1. Ecological Society of Australia (ESA).

2. Society for Conservation Biology (SCB).

3. British Ecological Society (BES).

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