ECOLOGICAL IMPACT of MYRTLE RUST (Austropuccinia Psidii) in a WET SCLEROPHYLL FOREST

ECOLOGICAL IMPACT OF MYRTLE RUST (Austropuccinia psidii) IN A WET SCLEROPHYLL FOREST

Santiago Diaz Torres Bachelor of Biological Sciences

Submitted in fulfilment of the requirements for the degree of

Master of Philosophy

School of Biological, and Environmental Sciences

Faculty of Science and Engineering

Queensland University of Technology

2021

Keywords

Acmena smithii, Archirhodomyrtus beckleri, Austropuccinia psidii, canopy gap fraction, Decaspermum humile, ecological communities, ecological impact, ecological populations, ecological succession, forest structure, Gossia hillii, invasive species, Myrtaceae, myrtle rust, Rhodamnia maideniana, pathogen spillover, plant-pathogenic fungi, plant–soil feedback, species composition, specific leaf area, light availability, soil nutrients, South East Queensland, Tallebudgera Valley, vegetation monitoring program, wet sclerophyll forest

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Abstract

Myrtle rust is an infection caused by Austropuccinia psidii, a fungal pathogen that threatens the persistence of plant species within the Myrtaceae family. Myrtle rust was first detected in Australia in April 2010 on the central coast of New South Wales. Myrtle rust originates from South America and has now also spread to other regions where Myrtaceae species are native, including New Caledonia, South Africa, Indonesia, Singapore and New Zealand. The Myrtaceae family is the largest plant family in Australia with over 2250 species and 70 genera. The species of this family make up a large proportion of iconic Australian forests such as dry and wet sclerophyll forests. The importance of studying the ecological impacts of myrtle rust in wild populations has been highlighted in multiple studies in Australia. Understanding its effects in the field will help in the prioritisation of species and environments at risk and understand how plant communities will change over time in response to myrtle rust infection.

Previous studies of wet sclerophyll forests have found high infection rates of myrtle rust on susceptible Myrtaceae species growing in the midstory and understory canopies of the forests. However, given that the arrival of myrtle rust in Australia is relatively recent, the impact of myrtle rust on populations over time and on plant community composition and key resources, such as light availability, is equivocal. In this study, I evaluated the impact of myrtle rust on a wet sclerophyll forest in the Tallebudgera Valley in South East Queensland and compared recent survey results with previous survey results, recorded over multiple time periods since 2016. I examined how increasing infection rates of myrtle rust has affected the survival of five species of Myrtaceae: Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana. I also studied how tree mortality in the midstory of the forest canopy changes the resource availability and plant community composition in the understory.

Archirhodomyrtus beckleri and Decaspermum humile are tree species that make up the overstory of the canopy of the forest. Acmena smithii, Gossia hillii and Rhodamnia maideniana can grow to shape the midstory of the forest but are shrub species that are more likely to be found in the understory. I regard these are five Myrtaceae species as key species for the forest because they create a complex setting for the community and ecosystem dynamics. Having these five key species a fundamental role in the forest by altering light

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 3 dynamics in the forest and bringing nourishment to animal species through edible fruits that are consumed by birds and mammals.

Populations of the five Myrtaceae species that make up the structure of the forest decreased over the four years of recorded infection. I used Kaplan–Meier plots to estimate the probability of survival of the five most abundant Myrtaceae species, based on data collected from three time periods over four years. I then used Markov models to predict their probability of survival for the next three years. Over the next three years, predictions from this research suggest that populations of Archirhodomyrtus beckleri and Decaspermum humile may become locally extinct (extirpation), Gossia hillii has a low probability of survival as well, but Acmena smithii and Rhodamnia maideniana have higher probabilities of survival.

I used specific leaf area (SLA) as a proxy measure of the physiological adaptations of populations to the local environment that could predict susceptibility to myrtle rust. This is because leaves are usually the common structure associated with infection by A. psidii. I measured SLA in the five Myrtaceae key species described above. I did not find a significant correlation between high SLA in species with higher mortality rates. Although the overall trend was promising, further research should be conducted in different environments and with more species and samples to understand whether SLA is a good predictor of susceptibility to myrtle rust.

I hypothesised that the high mortality rates found at these sites in the mid-canopy and understory would result in more light reaching the forest floor and greater soil nutrient levels. I measured canopy gap fraction as a surrogate measure of light availability for the forest floor and soil nutrients associated with plant decomposition in regions of the forest with and without mortality. I found a significant variance between canopy gap fractions but no significant difference in soil nutrients. I surveyed the understory species composition of the forest. I found that 11 non-native species, seven of them regarded as invasive because of high impacts and/or spread, were present on the forest floor (e.g. Ageratina adenophora and Ochna serrulata). However, I did not find a significant correlation between quadrats that showed higher mortality because of myrtle rust and the cover of invasive species in the understory.

In this thesis, I aimed to understand the impact of myrtle rust over four years of recorded infection on five prominent Myrtaceae species in a wet sclerophyll forest and the

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 4 subsequent effects that increasing mortality caused by myrtle rust might have on the plant community composition of the ground cover. I found that myrtle rust affects the composition of the forest over time by killing prominent species in the mid- and understory and this mortality could impact on longer-term community assembly by changing light availability reaching the forest floor. However, I did not record a significant shift in plant composition on the forest floor and soil nutrient conditions, which may be explained by the short period (four years) of recorded infections. Most studies research the impacts of invasive species and in particular invasive pathogens after 10 to 30 years of infection. This study represents an important interim examination of how invasive pathogens impact in the early stages of arrival and an opportunity to understand how resultant processes begin to shift. The significant differences found in the canopy gaps because of mortality in the mid- and understory suggest that changes in composition and other resource availability may become more pronounced over time. Annual follow-up studies are highly recommended to document the impact on populations not just after 10 years but every couple of years so that timelines of impact can be better understood and the timing of management interventions optimised.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 5

Table of Contents

Keywords ………………………………………………………………………….….………1 Abstract …………………………………………………………………………….….……...2 Table of Contents ……………………………………………………………………..………5 List of Figures ……………………………………………………………………….…….….8 List of Tables ……………………………………………………………………….…….….11 Statement of Original Authorship ……………………………………………….…………..12 Acknowledgements ………………………………………………………………….……....13 Chapter 1: Introduction …………………………………………………………….…….. 14 1.1 Background ………………………………………………………………….………14 1.2 Literature review …………………………………………………………………….15 1.2.1 Value of the Myrtaceae family in Australia …………………………………………16 1.2.2 Wet sclerophyll Forest and the Tallebudgera Valley ………………………………. 17 1.2.3 Ecological value of five focal Myrtaceae species of the Tallebudgera Valley wet sclerophyll forest …………………………………………………………………… 19 1.2.4 A. psidii life cycle and susceptibility categorisation ……………………………….. 23 1.2.5 Plant traits and A. psidii ………………………………………………...………….. 24 1.2.6 Canopy structure and light dynamics ………………………………………………. 25 1.2.7 Impact of Austropuccinia psidii on keys species of a wet sclerophyll forest…….….26 1.2.8 Impact of mortality on light availability and community assembly of wet sclerophyll forests…………………………………………………...………..……. 28 1.3 Research scope ……………………………………………………………..………. 30 1.4 Data chapter outline …………………………………………………………....…….30 Chapter 2: Population impact: Impact of Austropuccinia psidii on keys species of a wet sclerophyll forest over time ……..…………………………………………………………32 2.1 Aims and objectives …………………………………………………………..……. 32 2.2 Research design ……………………………………………………….……………. 33 2.2.1 Assessing mortality (percentage of dead trees) in focal Myrtaceae populations …... 33 2.2.2 Specific leaf area measurements and susceptibility classification …………...….…..34 2.3 Analysis ……………………………………………………………………..……….35 2.3.1 Assessing mortality (percentage of dead trees) in focal Myrtaceae populations overtime ……………………………………………………………………….……. 35

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2.3.2 Predicting probability of survival for focal Myrtaceae species …………………….. 36 2.3.3 Specific Leaf Area ………………………………………………………………..… 38 2.4 Results ……………………………………………………………………...……… 38 2.4.1 How populations of five focal Myrtaceae species, Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana, have been impacted by A. psidii over four years of infection? ……………………………..…. 39 2.4.2 Based on the survival rates of focal Myrtaceae species; what are the predicted survival probability of focal populations in the forest? ………………………………………40 2.4.3 Does Specific Leaf Area (SLA) correlate with mortality rates (as a surrogate measure of populations susceptibility) for focal Myrtaceae species in the forest? Show variance between different susceptibility ranking categories? ……………………………..... 43 2.5 Discussion………………………………………………………………..…...………45 2.5.1 Impact of A. psidii for Myrtaceae focal species over four years of infection and their predicted probability of survival ………………………………………….…………46 2.5.2 SLA correlation with mortality of focal species in the forest, and as a surrogate measure of susceptibility……………………………………………….…………… 49 2.6 Conclusion ……………………………………..…………………………………….50 Chapter 3: Austropuccinia psidii mortality impacts on light availability and foster conditions for changes in the community assembly of Wet Sclerophyll forests ………..52 3.1 Aims and objectives ……………………………………………………...………….52 3.2 Research design ………………………………………………………..…………….52 3.2.1 Field assessment …………………………………………….………...…………….52

3.2.2 Light measurements ………………………………….……………...………………53

3.2.3 Soil Sampling ……………………………………………………………….……….54

3.2.4 Seedling composition ………………………………….……….……………………55

3.3 Analysis …………………………………………………………….………….…….55 3.3.1 Soil nutrients ………………………………………………………….……………..55

3.3.2 Ground cover species richness ……………………………...……………………….55

3.3.3 Seedling and Ground cover abundance …………………………………...……….. 56

3.4 Results ……………………………………………………………………………….56

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3.4.1 Does canopy gap fraction (as a surrogate measure of light) and ground cover differ, in regions of the forest that present mortality due to A. psidii? ……………..…………56

3.4.2 Does nutrients in the soil change in regions of a forest with mortality due to A. psidii? ……………………………………………………………………………….………58 3.4.3 Is seedling composition in the forest affected as a direct or indirect consequence of A. psidii? ………………………………………………….…………………………… 59

3.5 Discussion ……………………………………………………………….……………..64 3.5.1 Implications of the increment in the availability of light in the ground cover …….. 64

3.5.2 Soil nutrient and mortality in the forest due to A. psidii …………………………….66

3.5.3 Seedling composition and invasive species richness ………………………………..66

3.6 Conclusion …………………………………………...………………………………68

Chapter 4: Discussion and Conclusions …………………………………………………..69 4.1 Discussion ……………………………………………………………….……….….69 4.1.1 A. psidii impact on high abundant Myrtaceae species over time ………….……..… 69 4.1.2 SLA as a tool to monitor populations susceptibility to A. psidii …….….………...…71 4.1.3 Light dynamics and ground cover ………………………………………………….. 72 4.2 Limitations …………………………………………….…………………………….73 4.3 Future research …………………………………………………..…………………..75 4.4 Conclusions ……………………………………...…………………………………. 76 Bibliography ………………………………..……………………………………………….79 Appendices ……………………………..……………………………………………………94

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List of Figures

Figure 1.1 History of Austropuccinia psidii invasion events and biotypes around the world. EU/SJ, collections from Eucalyptus spp. and Syzygium jambos from Brazil, Paraguay, and Uruguay; S-Africa, South Africa. Reprinted from Carnegie and Pegg, 2018 with copyright permission contained in Appendix A.

Figure 1.2 Regional ecosystems and biodiversity status of the Ryans Rd site, Tallebudgera Valley, South East Queensland. Transects locations are indicated in in orange rectangles. The area that is still used for cattle purposes is shown as endangered (in purple) for the biodiversity status and under no concern at present the area corresponding to the wet sclerophyll forest (in green). Retrieved from Queensland Globe 15 May 2020 from https://qldglobe.information.qld.gov.au/.

Figure 1.3 Distribution in Australia of the five most abundant Myrtaceae species found in the wet sclerophyll forest of the Tallebudgera Valley and of A. psidii. The distribution of Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana was retrieved from Atlas of Living Australia (2020) from https://www.ala.org.au/ and the distribution of A. psidii from Makinson (2018).

Figure 1.4 Percentage of live and dead trees within different forest layers in a wet sclerophyll forest, Ryans Road, Tallebudgera Valley, Queensland. Data used to construct the plot were data shared and collected in 2016 by Dr Geoff Pegg and colleagues from the Queensland Department of Agriculture, Fisheries and Forestry. The data presented are based on a modification of the information presented in Pegg et al. (2017) to include the total number of species per canopy strata.

Figure 2.1 Markov chain diagram for Acmena smithii based on the transitional matrix for the species. Arrows indicate the direction of the change and numbers the probability of staying or changing state.

Figure 2.2 Intercept (which represents Acmena smithii) and slope estimates from the LME model comparing morality (arcsine square-root transformed) between the five focal

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 9

species with a random-effects structure of the transect nested in time. Longer error lines indicate estimates of confidence intervals and bolded error lines the standard error.

Figure 2.3 Probability of survival of 13 local Myrtaceae species in the wet sclerophyll forest in the Tallebudgera Valley after four years of continuous infection by A. psidii. Surveys were conducted at the site in 2016 (year 1), 2017 (year 2), and 2019 (year 4). Shaded areas show 95% confidence intervals around these estimates.

Figure 2.4 Probability of survival for five of the most abundant focal Myrtaceae species after continuous infection by A. psidii. Surveys were conducted at the site in 2016 (year 1), 2017 (year 2), and 2019 (year 4). Shaded areas show 95% confidence intervals around these estimates.

Figure 2.5 Specific leaf area variance and mortality in the highly affected species Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii. Each dot represents the average SLA and mortality (percentage of dead trees) for the species in 2019.

Figure 2.6 Specific leaf area variance for the focal species Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia maideniana in a wet sclerophyll forest at the Ryans road site, Tallebudgera Valley, Queensland. Different letters indicate statistically significant differences between species SLA based on Dunn’s post hoc test.

Figure 2.7 Dead trunk of Decaspermum humile on the ground in one of the transects at the study site in Tallebudgera Valley, Queensland. Figure 3.1 a) Zenith division for hemispherical photography of the canopy; b) Zenith restriction angle for the area of the quadrat. : Zenith restriction angle, : angle of view, d: quadrat size, : Horizontal projection.

Figure 3.2 Box and whisker quadrat showing canopy gap fraction (%) in quadrats with and without mortality. The average canopy gap fraction was 67% in regions of the forest with mortality caused by A. psidii, and 53% in regions without mortality A higher percentage means a larger gap in the canopy, which implies more light (Wilcoxon rank-sum test, W = 7201.5, p < 1.204  10–13).

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Figure 3.3 Estimated percentage of ground cover (%) in quadrats with and without mortality. Ground cover did not differ significantly between quadrats with and without A. psidii infection (Wilcoxon rank-sum test, W = 13,442, p > 0.1).

Figure 3.4 Variance in soil element composition (%) for major elements needed for plant growth (potassium, nitrogen, phosphorus and sulphur) in quadrats with and without mortality caused by A. psidii. Soil element composition did not differ significantly between quadrats with and without mortality (Kruskal–Wallis ranks test, p > 0.05).

Figure 3.5 Non-metric multidimensional scaling model (nMDS) for all species in the four transects. There was a significant difference for richness of species between transects

(F196 = 12.3, p < 0.001; Figure 3.5A) but no difference between quadrats with and

without mortality (F196 = 1.28, p > 0.1; Figure 3.5B).

Figure 3.6 Relative abundance of Myrtaceae seedlings after four years of continuous infection by A. psidii in 2016 (n = 98) and 2019 (n = 81) within a wet sclerophyll forest at Ryans Road, Tallebudgera Valley, Queensland.

Figure 3.7 The cumulative ground cover (%) of non-native species over 400 quadrats in a wet sclerophyll forest, South East Queensland. The dotted line represents quadrats with mortality caused by A. psidii and sustained line without mortality.

Figure 3.8 Total cumulative ground cover (%) over 400 quadrats in a wet sclerophyll forest, South East Queensland. The dotted line represents quadrats with mortality caused by A. psidii and sustained line without mortality.

Figure 3.9 Percentage of ground cover of native, non-native species and invasive species in quadrats with mortality (3.3% invasive, 6.6 % native and 8.1% non-native) and without (16.4% invasive, 29.2% native and 26.4% non-native) mortality caused by A. psidii.

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List of Tables

Table 1.1 Description and conservation status of the most abundant Myrtaceae species in the wet sclerophyll forest of the Tallebudgera Valley. Information based on the Atlas of Living Australia (2020).

Table 2.1 Comparison between Kaplan–Meier probabilities of survival and Markov chain probabilities of survival for Decaspermum humile and Gossia hillii for the second survey year (2017).

Table 2.2 Mortality of Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana over four years period ( 2016-2019), recorded in three surveys (2016, 2017 and 2019).

Table 2.3 Probability of survival of Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana for the next three years(2020-2021).

Table 3.1 Species in the regenerating area of the forest, under one meter in height.

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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.

Signature: QUT Verified Signature

Date: 04/01/2021.

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Acknowledgments

I acknowledge the traditional owners of the land: The Daggan Balum (Five Rivers) people where my research took place and the Yugara and Turrbal people where the QUT stands. I pay my respects to their Elders, lores and customs.

I would like to thank my supervisory team for their guidance and encouragement through this process: Professor Jennifer Firn, Dr Geoff Pegg and Dr Ramona Maggini. I also thank the Plant Biosecurity Science Foundation for funding my research through a grant awarded in 2019. Special thanks to Dr Jarrah Wills for his kindness and invaluable contribution to the project; to Douglas and Lyndria Cook, who kindly allowed me to work on their property; and to my laboratory team Simone-Louise Yasui, Erica Porter and Febriana Wayana for their encouragement and support.

I give a special acknowledgement to all of the people who volunteered to participate in this project’s fieldwork: Stephane Gomes Batista, Maria Teresa Carvalho, Salvatore Bonanno, Grechel Alejandra Taucare and Erica Heaton. I could not have collected any of my data without all your wonderful help. Finally, I would like to thank my family for their continued support and especially my wife Claudia, who was by my side during the whole process; without her, its completion would have been impossible.

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Chapter 1: Introduction

1.1 BACKGROUND

Austropuccinia psidii (G. Winter) Beenken is a fungal pathogen that threatens the persistence of plant species within the Myrtaceae family, which as a plant family dominates 11 of the 13 major vegetation types in Australia (Pegg et al., 2017; Specht et al., 1981). A. psidii was first detected in Australia in the state of New South Wales in 2010 and, since its arrival, has spread into a wide range of native ecosystems in Australia, such as wet sclerophyll, subtropical and tropical rainforests (Carnegie et al., 2015). Widescale detections of A. psidii have been made in New South Wales, Queensland (including offshore islands such as K’gari) (Wild Preservation Society of QLD, 2019) and the Northern Territory. Early detections in Victoria and Tasmania are restricted to mainly urban and peri-urban environments (CRC, 2018; Westaway, 2016).

The Myrtaceae family is the largest plant family in Australia, with over 70 genera and 2250 species (Makinson, 2018). Species of this family have shaped iconic Australian ecosystems such as sclerophyll forests, where they are the dominant plant species (Peters & Butler, 2014). Given its apparent ease of proliferation, Australia’s Threatened Species Committee considers A. psidii as a serious threat to biodiversity (Morin et al. 2012), and over 392 species in Australia have been identified as being susceptible to infection (Soewarto et al., 2019). To date, there are 182 confirmed species infected with A. psidii based on field observations; however, only 19% of the species within the Myrtaceae family have been assessed for infection susceptibility in Australia (Berthon et al., 2019).

The consequence of the loss of Myrtaceae species in these forests is unknown, and the current research focuses primarily on the impact of A. psidii infection at a species level. Therefore, the aim of my master’s research was to assess the ecological impact that A. psidii has had on a subtropical lowland wet sclerophyll forest of a South East Queensland hinterland valley where repeated A. psidii infections over time have resulted in the death of some of the Myrtaceae species at the site and caused significant declines in species abundance. Rainforest pioneer species, including Archirhodomyrtus beckleri (F. Muell.) A.J.

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Scott, Decaspermum humile (G. Don) A.J. Scott, Gossia hillii (Benth.) Snow & Guymer, and Rhodamnia maideniana C.T. White (Pegg et al., 2017) dominate the regenerating understory and midstory. The overstory is dominated by Eucalyptus grandis and Lophostemon confertus and scattered within site are mature rainforest Myrtaceae including Syzygium corynanthum, Syzygium oleosum and the threatened species Syzygium hodgkinsoniae and Syzygium moorei.

1.2 LITERATURE REVIEW

A. psidii has at least four biotypes (Carnegie and Pegg, 2018), which are now considered pandemic diseases in the United States, Costa Rica, Jamaica, Puerto Rico, Mexico, China, Australia, New Caledonia, Indonesia, New Zealand and Colombia (Berthon et al. 2018; Carnegie and Pegg, 2018; Steward et al., 2018). A. psidii is also been detected in 16 other countries (Brazil, Paraguay, Uruguay, Ecuador, Cuba, Dominican Republic, Venezuela, Trinidad and Tobago, Argentina, Dominica, Guatemala, El Salvador, Panama, Japan, South Africa and Singapore). A. psidii was first described (as Puccinia psidii) in Brazil in 1884 and has become a cosmopolitan fungal pathogenic species that is present in four continents and 27 countries. It is more prevalent in tropical and subtropical regions, as shown in Figure 1.1.

A. psidii has infected more than 520 Myrtaceae species globally (Berthon et al., 2019) and affects multiple hosts. It is called several names around the globe such as guava rust, eucalyptus rust, ohi’a rust and myrtle rust among others. A. psidii causes extensive damage to commercial plantations of Eucalyptus species, such as Metrosideros polymorpha Gaudich., Pimenta dioica and other commercial species around the globe (Carnegie and Pegg, 2018). This pathogen also infects multiples species in natural forests, such as Rhodamnia rubescens and Rhodomyrtus psidioides in Australian rainforests (Fernandez-Winzer et al., 2020).

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1.2.1 Value of the Myrtaceae family in Australia

To understand the risk A. psidii poses to Australian forests, it is important to understand the ecological, social and economic value of tree species in the Myrtaceae family. Species within this family produce edible fruits (Mitra et al., 2012), which are a source of nourishment for many animal species including birds and marsupials, such as Trichoglossus moluccanus and Trichosurus vulpecula among other species. For example, the rainforest bird, the green catbird, is a local endemic species of South East Queensland and is known to eat the berries of the rose myrtle, Archirhodomyrtus beckleri (Floyd, 1989).

Tree species within the Myrtaceae family provide an important framework for the overstory, midstory and understory strata in wet sclerophyll forest. The depletion of the

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 17 canopy could have a direct impact on the understory species assemblage, as the loss of complexity because of increase mortality by A. psidii can shift the light dynamics in the forest by reducing populations that act a natural filter of light in the forest, as previous studies on the effects of selective logging have shown (Döbert et al. 2017; Friday et al., 2008). This loss of trees and shrubs leads to an overall reduction in net primary productivity in these ecosystems (Ruitta et al. 2018). With time, seedlings in the understory will grow to be part of the mid- and overstory strata of the forest, and a shift of the understory will result in a shift in the canopy of the forest over time. Other species may benefit from the new conditions and proliferate. For example, studies suggest that invasive weeds such as Lantana camara could be recruited under such higher light conditions (Carnegie and Pegg, 2018), which may result in a gradual shift in the composition of the forest with a possible shift in structure, function and biodiversity.

From a social perspective, plants within the Myrtaceae family have a social and cultural value for Aboriginal communities in Australia. Aboriginal people use Callistemon citrus (Curtis) Skeels, which is native to Australia, as food and medicine in the treatment of bacterial, fungal, parasitic and viral diseases (Radulović et al. 2015). Callistemon citrus has not been assessed as a susceptible species yet, but species within the Callistemon genus are depicted as susceptible (Berthon et al. 2019). The Aboriginal Dharawal people also use different Eucalyptus spp. that have anti-inflammatory properties to treat inflammatory conditions such as asthma, arthritis and rheumatism, among others (Akhtar et al. 2016).

Finally, from an economic perspective, A. psidii has had a significant impact on the lemon myrtle and nursery industries that contribute significantly to the Australian economy (Plant Health Australia, 2012). These examples of the importance of tree species within the Myrtaceae family provide some insight into the magnitude of the problem A. psidii may cause over both the short and long term, such as the potential loss of many species from this family and changes in plant community structures. Aspects that provide the necessary context to understand the scope of my research is described below.

1.2.2 Wet sclerophyll forest and the Tallebudgera Valley

Wet sclerophyll forests are one of the most iconic and unique Australian biomes, and the canopy layers of this forest type are dominated by Myrtaceae tree species (Krishnan et al. 2019). This makes the structure, species composition and likely ecosystem functioning of wet

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 18 sclerophyll forests highly vulnerable to A. psidii (Carnegie & Pegg, 2018). The characteristic features of wet sclerophyll forests include a canopy of 10–30 meters, canopy cover of 50– 80% and an understory comprising rainforest plant species, grassy cover with a shrub layer or a combination of both, and soils rich in carbon as a consequence of an accumulation of biomass and wood debris (Peters & Butler, 2014).

Wet sclerophyll forests are typically found in areas of high precipitation of >1200 mm of rain per year (Geoscience Australia, 2007), such as South East Queensland, Tasmania and a few locations in South West and North Western Australia. Wet sclerophyll forests have a narrow range and represent only 4% of the forest in Australia (Department of the Environment and Energy, 2017). Despite their small range, wet sclerophyll forests provide a key habitat for multiple species of marsupials (Wormington & Lamb, 1999) and >100 species of residential and non-residential birds (Chapman et al. 2010); including endemic species such as Lichenostomus frenatus (Floyd 1989), the bridled honeyeater. Wet sclerophyll forests normally form an ecotone between dry sclerophyll forests and rainforests in undisturbed conditions, but without periodical fire disturbances, wet sclerophyll forests can transition to rainforest (Krishnan et al. 2019).

The Tallebudgera valley is located in South East Queensland and is mainly dominated by wet sclerophyll forest patches in a rural residential landscape. Pegg et al. (2017) have described the study area as a subtropical wet sclerophyll forest with an understory dominated by Myrtaceae species. The study site has been allowed to regenerate naturally after being logged and cleared for cattle grazing purposes (Pegg et al. 2017). The area that remains is shown in Figure 1.2 as endangered (in purple) for its biodiversity status and under no concern at present the area corresponding to the wet sclerophyll forest (in green).

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1.2.3 Ecological value of five focal Myrtaceae species of the Tallebudgera Valley wet sclerophyll forest

Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana are the most common mid- and understory Myrtaceae species in the wet sclerophyll forest at the study site (Pegg et al., 2017). Archirhodomyrtus beckleri and Decaspermum humile are tree species that shape the canopy of the forest. Acmena smithii, Gossia hillii and Rhodamnia maideniana can grow to shape the canopy of the forest but are shrub species that are more likely to be found in the understory. All five species are categorised as ‘least concern’ under the Queensland conservation status, as shown in Table 1.1.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 20

Acmena smithii is widely distributed on the east coast of Australia. Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii share similar distribution patterns and are distributed along the coast of Queensland and north and central New South Wales. Populations of Rhodamnia maideniana are restricted to the Queensland–New South Wales border. All species distributions overlap with the distribution of A. psidii in Australia, as shown in Figure 1.3.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 21

Table 1.1 Description and conservation status of the most abundant Myrtaceae species in the wet sclerophyll forest of the Tallebudgera valley. Information based on the Atlas of Living Australia (2020).

Species Description Conservation status Acmena smithii Acmena smithii is a Least concern shrub species, under the commonly known as Queensland lilly pilly, and produces conservation edible fruits that are status source of nourishment for mammals and bird species.

Archirhodomyrtus Archirhodomyrtus Least concern beckleri beckleri is a rainforest under the tree species, commonly Queensland known as rose myrtle conservation and native to Australia status east region.

Decaspermum Decaspermum humile is Least concern humile a tree species under the commonly known as Queensland currant myrtle and conservation produces fruits. status

Gossia hillii Gossia hillii is a small Least concern tree or shrub species under the that produces edible Queensland fruits that are used as conservation source of nourishment status for birds and mammal species. Rhodamnia Rhodamnia maideniana Least concern maideniana is a rare rainforest under the species, commonly Queensland known as smooth scrub conservation turpentine. status

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 22

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 23

1.2.4 Austropuccinia psidii life cycle and susceptibility categorisation

A. psidii is regarded as an autoecious plant pathogen (Makinson, 2018), which means it completes its life cycle on one host (McTaggart et al. 2018). Autoecious rust pathogens can produce infectious urediniospores on one host, which permit completion of faster life cycles and an extensive spread to new geographic areas and hosts (Stewart et al. 2018). Urediniospores are spores produced in the uredium, a reproductive structure of the fungi, and can spread the infection within the host by forming hyphae structures to find new entry points into the plant (Dyakov, 2007). However, A. psidii infection most commonly occurs through the leaf cuticle (Yong et al. 2019) and not through the stomata, as with other fungal pathogens.

Urediniospores are the dominant spore stage for A. psidii, although teliospores and basidiospores are also present and play a role in host infection (Glen et al. 2007; McTaggart et al. 2018). Teliospores are also produced in the uredium but often under adverse conditions. They are located in the host epidermis and have more condensed cells than the urediniospores, a thicker wall and no vacuole (Dyakov, 2007). The third kind of spores are the basidiospores, which are formed in the basidium and contain two haploid cells.

Spread by wind, urediniospores develop optimally in winter at temperatures between 15 °C and 20°C, teliospores between 21°C and 25°C, and basidiospores at 21°C (MacTaggart et al. 2018). However, in a study of four susceptible species (Metrosideros excelsa, Lophomyrtus bullata, Lophomyrtus obcordata and Syzygium jambos) in Australia and New Zealand, Beresford et al. (2020) showed that spore development was optimal between 22°C and 28°C. In field observations, Pegg et al. (2014) and Carnegie et al. (2015) observed that urediniospores were associated all year with disease incidence and were generally lower in the drier times of the year, between September and January. Teliospores were more common in the cooler autumn months and on affected host species.

If the conditions are appropriate for the development of the telia reproductive structure, such as low rainfall, A. psidii does not need to invest energy in the formation of a uredinium (MacTaggart et al. 2018). This implies that different structures for fungal

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 24 reproduction are formed depending on the environmental conditions. Moreover, as the ideal temperature of spore production ranges between 22 °C and 28 °C, phenological events such as the change in season could trigger times of infection (Beresford et al., 2020).

Methods have been developed to categorise the levels of infection by A. psidii, such as susceptibility scoring systems. These systems classify infection rates of tree species according to three different levels of susceptibility to A. psidii (Pegg et al., 2012; Sandhu and Park, 2013; Yong et al. 2019,): resistant, tolerant and susceptible. Species-level susceptibility depends on the plant response to the rust by visible signs of infection, such as pustules, fleck and necrosis. To develop a scale to measure the response to rust of different species to A. psidii, Sandhu and Park (2013) worked originally with two species Eucalyptus globulus (Labill) and Eucalyptus obliqua (L’Herit.) to assess the pathways of infection, germination of spores and colonisation of the different phenotypes.

There are several approaches to categorise susceptibility to A. psidii. Sandhu and Park used a wheat rust-rating model. Previous model used to categorise susceptibility in the wet sclerophyll forest in the Tallebudgera Valley was a rust-screening assessment method. The wheat rust-rating model comprises a scale system to categorise the host response according to the visible signs of infection (Draz et al., 2015; Sandhu and Park, 2013). However, this method does not consider that differences in susceptibility to A. psidii could be due to genotypic variation between and within-host species (Makinson, 2018). Rust-screening methods assess susceptibility based on quantitative analysis of polymorphisms in genes that evaluate markers linked to resistance (Junghans et al., 2003).

1.2.5 Plant traits and Austropuccinia psidii

Plant communities respond to changes in environmental conditions by altering species composition and by adjusting their physiology. Such a response can be captured by the measurement of specific functional traits (Dwyer et al., 2014). Leaf traits are considered to be one way to categorise investment strategies (Wright et al., 2004).

Some plant traits, such as the presence of cuticle waxes on leaves, have been found to contribute to the defence of Eucalyptus species to A. psidii (Dos Santos et al. 2018). Cuticle waxes on leaves are solvent-soluble lipids that are involved in cuticle permeability and non-

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 25 host resistance, which is a plant defence mechanism (Yeats et al. 2013). However, cuticle waxes are difficult and costly to measure. To date, no studies have investigated the potential use of soft traits of leaves (i.e. ones that are easy to measure and can be accessed in global datasets such as TRY; Kattge et al., 2020) as predictors of host susceptibility or whether commonly used leaf traits such as specific leaf area (SLA, or the leaf area divided by leaf dry weight) may be a more rapid measure of possible susceptibility to A. psidii infection.

SLA is a relative measure of how fast (higher SLA) or slow (lower SLA) plants grow (Wright et al.2004; Wills et al., 2018). SLA has been found to predict growing characteristics of species. Understanding how traits differ between and within species with different levels of susceptibility may provide a tool to predict the impact of disease and to help prioritise species for conservation. SLA has been described as a surrogate measure for plant energy investment strategies in terms of how plant invests in the production of its overall photosynthetic area and the amount of dry weight tissue it invest for protection against pathogens and herbivores (Westoby et al., 2002; Wright et al., 2005). SLA has been found to vary depending on abiotic conditions such as soil nutrient availability (Lavorel & Garnier, 2002) and temperature (Kumar et al., 2012). SLA might then be a proxy measurement of the physiological adaptations of populations to the local environment that could be associated with host susceptibility and therefore could present a tool for in-situ evaluation of populations at risk.

1.2.6 Canopy structure and light dynamics

The forest canopy structure is a complex aspect of a forest in which environmental factors set the parameters for the species ensemble of the forest layers through intra- and interspecific competition (Ligot et al., 2014). At the same time, the forest canopy structure can modify environmental conditions inside the forest, such as temperature (Brüllhardt et al., 2020) and soil composition (Majasalmi & Rautaiainen, 2020) by affecting light dynamics in the forest and plant–soil feedback mechanisms, respectively. Therefore, analysing the forest canopy structure is key to understanding aspects of the forest that are inherently spatial and that impact on forest composition, function and, ultimately, on the condition of the ecosystem.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 26

The conventional vertical forest structure is divided into layers denoted as over-, mid- and understory (Leiterer et al., 2015). The overstory makes up the canopy or roof of the forest, and the mid- and understory are usually described as a subcanopy structure. The vertical forest structure plays an important role as a natural filter of light and other environmental conditions, such as rain and wind (Baldocchi et al., 2002).

Light availability in the forest floor is highly variable (Tateno and Takeda, 2003) and depends on the forest structure, topography and climate. Light availability may act as an estimator of disturbance in a forest because openings in the canopy correlate positively with the arrival of early colonist species whose growth characteristics are favoured by highlight conditions and, in some cases, invasive non-native plants (Théry, 2001; Goodale et al., 2012; Seidler, 2017; Rees, 2018; Döbert et al., 2017).

1.2.7 Impact of Austropuccinia psidii on keys species of a wet sclerophyll forest

Since it was initially reported in New South Wales in 2010 (Carnegie et al. 2010) A. psidii has spread across Australia into a wide variety of ecosystems, such are rainforest (Fernandez-Winzer et al., 2020) coastal swamp woodlands (Fernandez-Winzer et al., 2018) and wet sclerophyll forests (Pegg et al., 2017). It has been reported in Queensland (Pegg et al., 2014), New South Wales (Carnegie et al., 2010; Carnegie et al., 2016; Pegg et al., 2014), Northern Territory (Westaway, 2016), Victoria and Tasmania (CRC, 2018). To date, 378 species of 50 genera have been reported as host species for A. psidii (Soewarto et al., 2019). However, the full impact of A. psidii on plant community and species populations is unknown because only a few wild populations have been assessed in detail, including Rhodomyrtus psidioides (Carnegie et al., 2016, Fensham et al., 2020; Fernandez-Winzer et al., 2020) and Rhodamnia rubescens (Carnegie et al., 2016, Fernandez-Winzer et al., 2020). Despite these studies, the longer-term impacts on plant communities and ecosystem function remain equivocal.

The wet sclerophyll forest in the Tallebudgera Valley has been the subject of studies that aim to understand the impact of A. psidii on Myrtaceae populations since 2014 (Carnegie et al., 2015). From 2016, long-term study transects were established by the Queensland Department of Agriculture and Fisheries to track the impact of increasing infection and mortality on ecosystem functions in the forest. When the transects were established in 2016,

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 27

Myrtaceae species were estimated as comprising 36% of forest regeneration, 64% of the understory and 94% of the midstory, and the mortality rates were 44.5% in the midstory, 18.1% in the understory and 2.2% in the regeneration strata (Pegg et al., 2017) (Figure 1.4). Species such as Archirhodomyrtus beckleri were highly abundant in the forest and accounted for 28% of Myrtaceae species reported in 2016, although they were described as a highly susceptible species in the study site (Pegg et al., 2017). However, populations of Archirhodomyrtus beckleri have been assessed in North Queensland (Makinson, 2018) and across New South Wales (Manea et al., 2019). These findings suggest that susceptibility of Myrtaceae species to A. psidii may differ between regions. Similar results have been found for Melaleuca quinquenervia (Pegg et al., 2018).

Infection severity is variable between, and in some cases within, species, but can also depend on the ecological conditions where a species is growing. Therefore, finding a set of plant traits that could be used as a predictor of susceptibility would be valuable. A. psidii

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 28 infects juvenile growth, including young leaves, shoots and stems. Infection also occurs on flower buds, flowers, and immature and mature fruit (Pegg et al., 2014). Leaves are one structure that is commonly associated with infection (McTaggart et al., 2018; Pegg et al. 2012; Sandu & Park 2013; Pegg et al. 2012; Yong et al., 2019).

Experiments in natural systems present a unique opportunity to assess the impact of A. psidii within a region. These allow for a better understanding of the influence of local conditions on disease development and impact, and how the characteristics of the different plant populations might influence disease development and impact of A. psidii in both the short and long term. Understanding the diversity of a species and its influence on disease susceptibility will provide essential information to the long-term survival of these species and broader ecological impacts of A. psidii. Understanding of these patterns, coupled with improved knowledge of the ecological role these species play, will be essential for the development of impact management strategies and establishment of long-term field studies that provide information about the host response to infection over time.

1.2.8 Impact of mortality on light availability and community assembly of a wet sclerophyll forests

Natural and anthropogenic disturbances are essential factors that can explain community assembly in ecosystems (Johnson et al., 2007). Such disturbances are usually a consequence of an event that leads to a process of community change in which species replace each other until a stable community is reached (Johnson & Miyanishi, 1991). Some of these events have been studied widely, such as the effects of selective logging on forest function and species composition (Brocardo et al., 2018; Friday et al., 2008; Imai et al., 2019). However, for some events such as the arrival of an exotic plant pathogen, more time is needed to build up infection rates and impact to be noticeable on native ecosystems (Burdon et al., 2006).

Plant pathogens can influence plant communities through two mechanisms: plant–soil feedbacks and pathogen spillover (Chen & Nan, 2015). In plant–soil feedback, pathogens directly affect the availability of nutrients in the soil by infecting plants and increasing mortality, and therefore plant litter input, or indirectly by altering soil properties (Veen et al., 2019). Van der Sande (2018) found that changes in the elemental composition of the soil,

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 29 specifically in nitrogen and phosphorus, limit the number and type of species in a forest. The pathogen spillover phenomenon is based on the presence of multiple host species for a plant pathogen in an ecosystem where less-affected species act as a reservoir of the pathogen (Cobb et al., 2010). The availability of multiple host species gives continuity to the pathogen in the environment and ensures that multiple hosts are available for the long-term survival of the pathogen, even though the initial species may no longer appear to be susceptible to infection (Chen & Nan, 2015; Power et al., 2004).

Plant pathogens can control key processes, such as community assembly and nutrient cycling, through their impact on the survivability of some species over others (Burdon et al., 2006). Community assembly theory assumes that species that co-occur in an ecosystem do not assemble in a location randomly but as the product of abiotic (nonliving) filters and biotic (living) interactions that allow for some species to live together in a location and others to not (Götzenberger et al., 2012). Plant pathogens can impact the community assembly by altering species composition and the proportion of adult to juvenile plant species present at a site (Dobson & Crowley, 1994; Roy, 1993). There are multiple examples of how introduced pathogenic fungus in North America, such as Cronartium ribicola (Smidt & Puettmann, 1998), Fusarium carinatum (Burdon et al., 2006), Cryphonectria parasitica (Rigling et al.,2018) and Phytophthora ramorum (Grünwald et al., 2019) have had an impact on plant community composition and forest structure by decreasing populations of native species that shaped the forest canopy by killing adult trees, which then created larger and more frequent openings in forests (Burdon et al., 2006). Decreasing number of individuals for susceptible species in the forest and increasing competence in understory species by increasing light availability in the forest floor, leading to changes on the community assembly over time.

In an Australian context, a pathogenic fungus has had a devastating effect on forest canopy in the past. Phytophthora cinnamomic, was reported in Western Australia in 1920. Initially, this fungus decreased populations of Eucalyptus marginate, which structured the canopy of jarrah forest and were highly susceptible to the pathogen (Dell et al., 2005). This was recognised as a key threatening process to biodiversity in Australia after 43 years of spread, during which time it spread to affect 2500 species of the ~5700 species described for Western Australia (Hardham, 2005), or~44% of the species in the region. Highly impacting ecosystems such as banksia woodlands and jarrah forest (Hill et al., 1994). It is known that Phytophthora cinnamomic has caused permanent damage to the ecosystems by dramatically

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 30 modifying the structure and composition of native ecosystems (Wills, 1993) and reducing primary productivity and functionality (Department of the Environment, 2014) through habitat loss for flora and fauna, which have been irreversible (Department of the Environment, 2020). This pathogen is also considered to be of major environmental significance because of its destruction of vegetation in World Heritage areas and National Heritage places (Department of the Environment, 2020).

The impacts of the mortality of Myrtaceae species on forest light availability, seedling recruitment, and soil nutrients have not yet been measured. Given that the forest canopy creates important microenvironments inside the forest for species on the forest floor, including newly recruited seedlings, the loss of canopy layers because of A. psidii infection will likely impact heavily on the future functioning and composition of forests. Understanding the effects of early invasion of A. psidii and the rate of impact will be important for designing appropriate management strategies (Peters & Butler, 2014).

1.3 RESEARCH SCOPE

A. psidii constitutes a significant threat to the persistence of susceptible Myrtaceae species in multiple ecosystems (Berthon et al., 2019). Through my Master’s thesis, I aimed to understand the impact of A. psidii infection over four years on the five most abundant Myrtaceae species in a wet sclerophyll forest: Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia maideniana. A further aim was to investigate the subsequent effects of A. psidii infection on the community composition and ground cover. The extend of the impact of A. psidii is discussed in the next two chapters along with the impact of A. psidii on populations and the effects on forest communities.

1.4 DATA CHAPTER OUTLINE

In the work described in Chapter 2, I re-surveyed the long-term observational experiment described above, section 1.2.7, in the Tallebudgera Valley to understand how Myrtaceae populations have been affected over four years of sustained infection by A. psidii. The aim was to use these data to predict the probability of survival of five focal Myrtaceae species

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 31

(the most dominant at the site) and to investigate whether an easy-to-measure and widely available plant trait, specific leaf area, could be used as a general predictor of susceptibility to A. psidii. In this chapter, I address the following three questions.

1. How have populations of five locally abundant Myrtaceae species, namely Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia Maideniana, been impacted by A. psidii over four years of sustained infection?

2. Based on the survival rates of the five focal Myrtaceae species, what are their predicted survival probabilities of focal populations in the forest?

3. Does SLA correlate with mortality rates for the focal Myrtaceae species and provide a surrogate measure of the level of infection susceptibility for these host species? Does SLA exhibit variance between different susceptibility ranking categories?

In the work described in Chapter 3, I aimed to provide evidence of the ecological impact of A. psidii on the plant community in the same wet sclerophyll site at Tallebudgera Valley. In this chapter, I specifically address the following three questions.

1. Do canopy gap fraction (as a surrogate measure of light) and ground cover differ between regions of the forest with and without mortality caused by A. psidii?

2. Do soil nutrients differ in regions that exhibit mortality in the midstory and understory due to A. psidii?

3. Are seedling composition and ground cover in the forest affected as direct and/or indirect consequences of A. psidii?

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 32

Chapter 2 - Population impact: Impact of Austropuccinia psidii on keys species of a wet sclerophyll forest over time

2.1 AIMS AND OBJECTIVES

Through this chapter of my master thesis, I aimed to contribute to the understanding of the long-term impact of A. psidii on the population of five Myrtaceae species in the Tallebudgera Valley. I studied populations of Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana, which I regard as key species because they are highly abundant in the sampled area. These species shape the midstory and understory of the forest and have a fundamental role in the forest by mediating light dynamics in the forest. Bringing nourishment to animal species through the provision of edible fruits that are consumed by birds and mammals (Floyd, 1989) and are a source of nectar and pollen for pollinator species (Fensham et al., 2020). I also studied SLA as a proxy measure of the plant energy investment strategies that respond to specific abiotic characteristics of populations in their local environment that may be linked the susceptibility to A. psidii.

In this study, I aimed to answer the following key questions.

1. How have populations of five locally abundant Myrtaceae species, Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia Maideniana, been impacted by A. psidii over four years of sustained infection?

2. Based on the survival rates of the five focal Myrtaceae species, what are their predicted survival probabilities of focal populations in the forest?

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 33

2.2 RESEARCH DESIGN

The study site is a private property located in the Tallebudgera Valley, South East Queensland (coordinates: 28°127.28S, 153°21.66 E). This site is part of a long-term surveillance project that aims to evaluate the impact of A. psidii on the region (Pegg et al., 2017). The impact of A. psidii on 13 Myrtaceae species has been surveyed on four transects measuring 2 m wide and 50 m long (Pegg et al., 2017). From 2016, long term study transects were established to track the impacts of increasing infection and mortality on ecosystem functions in the forest. When the transects were established in 2016, Myrtaceae species composed 36% of forest regeneration, 64% of the understory and 94% of the mid-story, and showed a mortality rate of 44.5% in the midstory, 18.1% of the understory and 2.2% of the regeneration strata (Pegg et al., 2017). Data recorded for the work described in this chapter were collected between September to December of 2019.

2.2.1 Assessing mortality (percentage of dead trees) in focal Myrtaceae populations

In this study, I conducted the third measurement in 2019 using similar methods to those of Pegg et al. (2017). In the same manner to Pegg et al. (2017), I recorded mortality of Myrtaceae species over four transects and the position within the canopy; as Ove- , Mid- , Under- story or regeneration. However, Pegg et al. (2017) included all Myrtaceae species, while this study emphasised in the five most abundant Myrtaceae species in the forest. Pegg et al. (2017) also recorded percentage of dead branches and dieback, measures that were not included in this study and instead I include SLA as a proxy measure of the physiological adaptations of populations to the local environment that could predict susceptibility to A. psidii.

Along the four transects, I recorded 13 Myrtaceae species. Five of these species were the most abundant, containing more than 30 individuals as a representative sample for statistical inference. These five abundant Myrtaceae species became the focal species of the study: Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana. The number of dead trees in different layers of the forest was recorded; trees without foliage were recorded as dead.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 34

Tree height was measured with a laser range finder (Nikon Forestry Pro II) or with a measuring tape for trees under a meter of height. Their position in the canopy was classified according to height as follows: overstory for trees over 15 m; midstory for trees between 4 m and less than 15 m; and understory for trees between 1 m and less than 4 m; and regeneration for trees under 1 m. Newly collected data were compared with previous data collected in the same way by Pegg et al. (2017).

2.2.2 Specific leaf area measurements and susceptibility classification

Leaf area measurements were performed using a CI-203 CID Bio-Science leaf area meter on 50 leaves collected from the field site for each of the five focal Myrtaceae species; 10 leaves were collected from each tree and five trees per species. Leaves were collected from both infected and uninfected trees. Fully expanded leaves were collected as stated in Pérez-Harguindeguy et al. (2013). However, given the nature of the infection, some of the leaves were not in perfect shape, but efforts were made to collect leaves that showed as little infection as possible. However, SLA has been used to compare across ecosystems and capturing these differences is important for its use in this study.

Each leaf was weighed after drying at 65°C for 48 hours to calculate SLA (Pérez- Harguindeguy et al., 2013). To compare SLA between species and according to the different levels (categories) of susceptibility, SLA values for the species ranked in Pegg et al. (2018) and species depicted as threatened or near-threatened in the Australian jurisdiction (Makinson, 2018) were retrieved from the TRY Plant Trait Database (https://www.try- db.org/TryWeb/Home.php - Kattge et al., 2020). All species were classified according to five categories: 1, resistant; 2, relatively tolerant; 3, moderately susceptible; 4, highly susceptible; and 5, extremely susceptible, ass were the categories used in previous studies in populations at the study site by Pegg et al. (2017).

Only species for which it was possible to obtain SLA measurements, either from field collection or through TRY Database, were included in this study: Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Eucalyptus grandis, Gossia hillii, Lophostemon confertus, Rhodamnia maideniana and Syzygium oleosum (all from Pegg et al., 2017); and Baeckea linifolia, Eucalyptus globulus, Eucalyptus pauciflora, Eucalyptus

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 35 radiata, Eucalyptus viminalis and Syzygium paniculatum (all from Berthon et al., 2019). Species in both of the studies were chosen as the studies use a similar approach to categorize susceptibility.

2.3 ANALYSIS

Data analyses were conducted using R statistical computing package version 3.6.1 (R Development Core Team, 2013), through the R studio platform, version 1.2.5001.

2.3.1 Assessment of mortality (percentage of dead trees) in focal Myrtaceae populations over time

Mortality was defined as the percentage of dead trees and was compared between populations of the five focal species over three time periods, four years of recorded infection and between canopy layers. To meet the assumptions of normality, mortality data were arcsine square-root transformed and analysed using a linear mixed-effects (LME) model. LME models use fixed and random effects to quantify variation between units, which is different from fixed-value methods that quantify the exact values of a predictor variable. I used the package ‘lmerTest’ in R to compare differences in the morality of species through time. The model was of the following form:

푀표푟푡푎푙푖푡푦 ~ 푆푝푒푐푖푒푠 + (1|푇푖푚푒/푡푟푎푛푠푒푐푡)

In this model, the random effects were the transects nested in time, with time representing the three survey time periods, and the fixed effects were the five focal species. To compare the variance between species, I then ran an analysis of variance (ANOVA) on the LME model through the Kenward–Roger method for denominator degrees of freedom and F statistics. Data recorded over three surveys (2016, 2017 and 2019) over four years (2016 to 2019) were merged into a file that contained the information about the four transects.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 36

2.3.2 Predicting the probability of survival for focal Myrtaceae species

Kaplan–Meier probability of survival

Kaplan–Meier survival estimates are used to model survival in biomedical sciences and ecology (Crawley et al.; 2002; Rich et al., 2010). The Kaplan-Meier survival estimates is the simplest method for calculating survival depending on time and because the probability of surviving is calculated using many small intervals missing time periods or uneven data can be accounted for in the analyses (Goel, Khanna and Kishore 2010). I used this model to assess the probability of survival of focal species and created two models, one for the 13 local Myrtaceae species present in the transect and a second for the five focal Myrtaceae species. The Kaplan–Meier analysis was conducted using the R packages ‘survival’, and ‘survmine’ and I used individual deaths caused by A. psidii infection as the event. All of the results using this model are shown with predicted 95% confidence intervals.

Markov chain

Markov models predict time series probabilities based on transitional matrices. Markov chain models are stochastics models using to model random systems and assumed that distribution of the variable depends only on the distribution of the previous state (Seneta, 2006). Based on the information on the probability of survival given by the Kaplan–Meier analysis, I created a Markov chain model to predict the probability of survival for the five focal species for 2020 to 2022. The R package ‘markovchain’ was used to predict the survival rate for the five focal Myrtaceae species. The initial state matrix is the proportion of individuals in each state at time zero. I created a model with two stages, living and dead, and used the probabilities estimated using the Kaplan–Meier analyses to set the probabilities of transition between states, as illustrated in Figure 2.1.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 37

To check the accuracy of the model, I compared the model estimate to the known survival rates for Decaspermum humile and Gossia hillii, from year 1 (2016) to year 2 (2017). These two species were chosen because they are the only species that showed mortality from the first year of the surveillance surveys. Once the accuracy of the model was confirmed, as shown in Table 2.1, the model was used to predict the probability of survival for the five focal Myrtaceae species in the forest from 2020 to 2022.

Table 2.1 Comparison between Kaplan–Meier probabilities of survival and Markov chain probabilities of survival for Decaspermum humile and Gossia hillii for the second year (2017).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 38

Kaplan– Markov Species Meier chain Decaspermum humile 0.48 0.47 Gossia hillii 0.72 0.73

2.3.3 Specific Leaf Area

SLA was calculated by taking the leaf area and dividing it by the leaf dry mass (Pérez- Harguindeguy et al. 2013) for Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia maideniana. I tested the significance of correlations between SLA and mortality (percentage of dead trees) using a linear regression.

Kruskal–Wallis

Kruskal–Wallis is a test on ranks for non-parametric data that is used to determine the variance between two or more groups ("Kruskal-Wallis H Test | Procedure for Statistical analysis 2020). As SLA data was non-normal distributed, I used Kruskal–Wallis to compare the variance between SLA in the five focal species (Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia maideniana). Once it was established a significant difference between the five species, I ran post hoc test called Dunn’s, to investigate specific variance between groups.

A second Kruskal–Wallis test was performed to evidence the difference in SLA between different categories of susceptibility presented in A. psidii, presented in Berthon et al., 2019 and Pegg et al., 2017. SLA for the next species was retrieved from the TRY plant database: Baeckea linifolia, Eucalyptus globulus, Eucalyptus pauciflora, Eucalyptus radiata, Eucalyptus viminalis, and Syzygium paniculatum.

2.4 RESULTS

2.4.1 How have populations of five locally abundant Myrtaceae species, Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana, been impacted by A. psidii over four years of sustained infection?

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 39

Mortality (dead trees 0 to 1.0) of Myrtaceae focal species differed significantly over the three survey periods over four years of A. psidii infection (ANOVA of the LME model fit: F4,124 = 23.2, p < 0.0001). Acmena smithii (the intercept) had similar morality rates to Rhodamnia maideniana. Decaspermum humile and Archirhodomyrtus beckleri had the highest mortality rates followed by Gossia hillii (Figure 2.2). LME models were based in the mortality recorded in three different surveys over a four years period (Table 2.2)

Figure 2.2 Intercept (which represents Acmena smithii) and slope estimates from the LME model comparing morality (arcsine square-root transformed) between the five focal species with a random-effects structure of the transect nested in time. Longer error lines indicate estimates of confidence intervals and bolded error lines the standard error.

Species 2016 2017 2019 Acmena smithii 0% 0% 5% Archirhodomyrtus beckleri 16% 32% 88% Decaspermum humile 38% 53% 83% Gossia hillii 10% 27% 34% Rhodamnia maideniana 0% 0% 20%

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 40

2.4.2 Based on the survival rates of five focal Myrtaceae species; what are the predicted survival probabilities of focal populations in the forest?

Based on the Kaplan–Meier model, the overall probability of survival for the 13 local Myrtaceae species surveyed in the transects was estimated at 96% in the first year (2016), 80% in the second year (2017) and 45% in the fourth year (2019) (Figure 2.3).

The Kaplan–Meier model for the five focal Myrtaceae species showed a high probability of survival for Acmena smithii (90%) after four years of being monitored at the site), followed by Rhodamnia maideniana (72%), Gossia hillii (32%) and, with a very low probability of survival, Decaspermum humile (9%) and Archirhodomyrtus beckleri (6%) (Figure 2.4).

Figure 2.3 Probability of survival of 13 local Myrtaceae species in the wet sclerophyll forest in the Tallebudgera Valley after four years of continuous infection by A. psidii. Surveys were

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 41 conducted at the site in 2016 (year 1), 2017 (year 2), and 2019 (year 4). Shaded areas show 95% confidence intervals around these estimates.

Figure 2.4 Probability of survival for five of the most abundant focal Myrtaceae species after continuous infection by A. psidii. Surveys were conducted at the site in 2016 (year 1), 2017

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 42

(year 2), and 2019 (year 4). Shaded areas show 95% confidence intervals around these estimates.

Prediction model

Survival predictions for five focal Myrtaceae species using Markov chain models showed that Acmena smithii and Rhodamnia maideniana are the most likely to survive over the next three years. Despite showing some early signs of infection, Acmena smithii had the highest probability of survival of 78% in 2020 to 66% in 2022. The respective probabilities were 37% to 19% for Rhodamnia maideniana and 7% to 1% for Gossia hillii. Based on the recorded trends over four years, the probability of survival for Archirhodomyrtus beckleri and Decaspermum humile is 0 in the next three years (Table 2.3).

Table 2.3 Probability of survival of Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana for the next three years.

Species 2020 2021 2022 Acmena smithii 0.78 0.72 0.66 Archirhodomyrtus beckleri 0 0 0 Decaspermum humile 0 0 0 Gossia hillii 0.07 0.02 0.01 Rhodamnia maideniana 0.37 0.27 0.19

2.4.3 Does SLA correlate with mortality rates for the focal Myrtaceae species and provide a surrogate measure of the level of infection susceptibility for these host species? Does SLA exhibit variance between different susceptibility ranking categories?

Correlational analysis of mortality (percentage dead trees) as a function of SLA in five focal tree species produced a coefficient of determination of 0.58, which was not significant (p > 0.1). There was an apparent tendency toward high SLA in species with the highest mortality, i.e. Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii. A second tendency was also noted as species that showed a higher variability of SLA at the study site showed the highest mortality (Figure 2.5). The variance between SLA for the five

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 43

focal species was significant based in the Kruskal–Wallis test by ranks (2 = 164.4, df = 4, p < 2.2  10–16; Figure 2.6).

Figure 2.5 Correlation between SLA and mortality in the highly affected species Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii. Each dot represents the mean SLA and mortality (percentage of dead trees) for the species in 2019.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 44

Figure 2.6 SLA variance for the focal species Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii, and Rhodamnia maideniana in a wet sclerophyll forest at the Ryans road site, Tallebudgera Valley, Queensland. Different letters indicate differences between species SLA based on Dunn’s post hoc test.

SLA values from the TRY database were used to examine this trend for a wide range of species categorised based on the observed levels of A. psidii susceptibility (Berthon et al.,

2019; Pegg et al., 2017). The SLA variance showed no significant difference (Kruskal–Wallis test by ranks, 2 = 5.83, df = 4, p > 0.1). Baeckea linifolia, Gossia hillii, Lophostemon confertus, Rhodamnia maideniana and Syzygium paniculatum show little variation in SLA as there were just one or two values of SLA for these species in the database. Species with few measurements or categorized in multiple categories could be creating a bias in this analysis make it difficult to find evidence if there is a significant difference in the variation in SLA between categories in the susceptibility ranking.

2.5 DISCUSSION

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 45

From 2016 to 2019, the Myrtaceae species, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana, were highly impacted by A. psidii. Archirhodomyrtus beckleri and Decaspermum humile populations have decreased critically by 72% and 45%, respectively, and are predicted to potentially experience a local extinction within the next three years at the sites. On the other hand, Acmena smithii is showing some infection (Geoff et al., 2017), but has had a low mortality of 5% to date. However, 2019 was the first year in which this species’ mortality was recorded; if the rate continues, the model predicts that mortality will increase to 44% by 2022.

2.5.1 Impact of A. psidii on the five Myrtaceae focal species over four years of infection and their predicted probability of survival

Populations of Archirhodomyrtus beckleri have different levels of susceptibility in different regions. In Northern Queensland, populations are reported to be relatively tolerant to A. psidii (Makinson, 2018) compared with South East Queensland, where populations are highly susceptible (Pegg et al., 2018). Similarly, populations in New South Wales have different levels of susceptibility between regions and notable difference in morbidity and mortality (Manea et al., 2019). Archirhodomyrtus beckleri was once one of the most abundant Myrtaceae species in the mid- and understory canopy of the forest at the study site of Tallebudgera (Pegg et al., 2017). However, since 2017, it has experienced a dramatic decrease in its survival at the field site as a result of A. psidii infection. The population is nearly extinct in the area sampled and has a Kaplan–Meier probability of survival of 6% for 2019 and a predicted probability be zero from 2020.

The condition of the Myrtaceae populations at the study site was assessed during the period between September to November 2019, a very dry period for the region and for Australia in general. In February 2020, after rains, basal shoots were found in several trees that were recorded previously as dead. However, the data were not updated for mortality as fieldwork was interrupted by the 2020 COVID-19 pandemic. A. psidii has been recorded in the past to have a high infection rate of basal shoots in susceptible species such as Rhodomyrtus psidioides (Fensham et al, 2020), whose basal shoots become heavily infected and do not survive for long. Therefore, Archirhodomyrtus beckleri basal shoots may show similar infection rates and mortality, although follow-up studies are needed to verify whether this is a common trend in species infected by A. psidii.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 46

Gossia hillii mortality has increased over the last four years, and the predicted Kaplan–Meier probability of survival indicates a 32% probability of survival for populations in 2019 and a predicted decrease in the next three years to only 1% in 2022. Gossia hillii is showing that the impact of repeated A. psidii infection has increased over time, as shown in Table 2.2. This species is considered to be high to extremely susceptible and has been reported as severely impacted in populations across its natural range at different life stages, from seedlings to mature trees (Makinson et al., 2018). Gossia hillii and Archirhodomyrtus beckleri are common plant species found in the understories of wet sclerophyll forests that are transitioning to rainforests.

Decaspermum humile mortality has increased gradually over the last four years. To date, there is a 9% probability of survival estimated with Kaplan–Meier analyses and a predicted probability close to zero for 2020. At the initial assessment, populations of Decaspermum humile were shown to be highly susceptible to A. psidii and were listed as one of the most impacted species at the site in 2016 (Pegg et al., 2017). During the survey of 2019, dead trunks of Decaspermum humile were found on the ground in several regions of the forest (Figure 2.8). Dissimilarities in susceptibility between regions were also observed for Decaspermum humile. Northern populations of Decaspermum humile have shown minor to severe infection rates by A. psidii (Makinson, 2018).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 47

Figure 2.7 Dead trunk of Decaspermum humile on the ground in one of the transects at the study site in Tallebudgera Valley, Queensland.

Rhodamnia maideniana was also initially classified as extremely susceptible and of high concern in the first study at this site in 2016 (Pegg et al., 2017). Populations of Rhodamnia maideniana are restricted to northern New South Wales and South East Queensland, a region depicted as highly suitable for A. psidii (Pegg et al., 2017). Rhodamnia maideniana initially showed severe dieback of all the trees assessed between 2016 and 2017 but with no mortality. Populations of Rhodamnia maideniana assessed in 2019 demonstrated mortality in different canopy layers, particularly in the regenerating strata of the forest (midstory 6%, understory 3%, regenerating 33%). Despite being highly susceptible to A. psidii, this species shows resilience to the effects of infection in the field, especially in the adult stage. Model predictions suggest a 37% probability of survival for Rhodamnia maideniana over the site for 2020 and a 19% probability of survival for 2022.

Acmena smithii has demonstrated relative tolerance to moderate susceptibility to A. psidii infection (Pegg et al. 2017) and had the lowest mortality of all the focal species

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 48 recorded at the study site. Survival models in this study estimate a 90% probability of survival for the species in 2019 and the highest predicted probability of survival over the next three years, 2020 (78%), 2021 (72%) and 2022 (66%).

A. psidii has remained active at the site over the last six years since it was first reported in 2014 (Carnegie et al., 2015). A probable explanation for the subsistence of A. psidii in the study site is that species that are less affected, such as Acmena smithii and to some extent Rhodamnia maideniana, may act as a reservoir for the pathogen. Species that are highly tolerant of specific pathogens can be regarded as pathogen reservoirs because these species harbour the pathogen but do not show the same level of mortality, which allows the pathogen to build its own population and spread from less susceptible individuals (Chen & Nan, 2015). Seemingly, a coherent explanation given the observations of Pegg et al. (2014) and Carnegie et al. (2015) who had observed spores throughout the year and not only during short periods of time as might be expected when waves of infections are driven for other factors such as in insect vectored pathogens (Eigenbrode et al., 2018).

2.5.2 Correlation between SLA and mortality of focal species in the forest and SLA as a surrogate measure of susceptibility

Plant communities respond to changes in the environment by altering species composition and, in some cases, the environmental conditions favour the establishment and survival of species with different traits; in this way, the environment and biotic interactions filter the traits of surviving populations (Funk et al., 2008). Such a response can be captured by the measurement of key functional traits and their comparisons between and within species across environmental gradients or in response to different disturbances (Dwyer et al., 2014). Leaf traits are considered to be a way to understand investment strategies as part of the physiological response of species to the environment (Wright et al.2004). SLA is a relatively easy trait to measure and has been recorded in worldwide trait databases such as TRY (Kattage et al., 2011). SLA is one of the key leaf traits described as a surrogate measure of plant energy investment strategies between photosynthetic area and protection (Wright et al., 2005; Westoby et al., 2002).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 49

Results from the ANOVA to compare SLA between focal Myrtaceae species showed higher values for extremely affected species Archirhodomyrtus beckleri and Decaspermum humile and lower values for Acmena smithii, Gossia hillii, and Rhodamnia maideniana. Low- SLA species are depicted as species with low net photosynthesis rate and low growing rates but with longer life span, whereas high SLA species have fast-growing rates and shorter life spans (Dwyer et al., 2014; Wright & Westoby, 2000). This suggests that there may be a relationship between species investment strategies, as a proxy measure of physiological adaptation of the species to the local environment, and the susceptibility of populations within a region.

When accessing the recorded SLA values for a wider range of Myrtaceae species from the TRY database (Kattage et al., 2011), I did not find a significant correlation between variance in SLA and predicted susceptibility to A. psidii. Species susceptibility differ between regions, which means that there could be a bias in the analysis that includes the same record of mortality for different susceptibility categories. Therefore, studies that assess the variance between local populations where the susceptibility is known and agreed on for populations in the region may show a stronger correlation between SLA and susceptibility.

Most of the time, SLA is considered a responsive variable from the impact of a pathogen on a plant species (Call et al., 2017; Fernandez-Winzer et al., 2020). For A. psidii, SLA was previously studied in inoculated plants to examine the effect of A. psidii on leaf area available for photosynthesis (Fernandez-Winzer et al., 2020). This thesis is the first study to investigate whether SLA can be used as a surrogate measurement of physiological processes that could confer resistance to A. psidii. However, the changes in SLA complex because changes in SLA estimates could be a product of the leaf phenology response to A. psidii. Carnegie et al. (2015) reported that repeated infection of Rhodamnia rubescens by A. psidii affects their leaf phenotypic characteristics by producing leaf distortion. Further studies are necessary to observe whether similar results would be obtained with other species and to determine whether SLA is accurate as a predictive tool of the susceptibility of populations to A. psidii infection and the response to repeated A. psidii infection over time

2.6 CONCLUSION

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 50

The five focal Myrtaceae species survey in this study have shown an increase in mortality over time, but this increase has been more pronounced in some species. Archirhodomyrtus beckleri had a mortality rate of 16% in 2016 and 88% in 2019, whereas Acmena smithii mortality rate was 0% in 2016 and 5% in 2019, as shown in Table 2.2 . The data presented here indicate the importance of tracking the impact of infections over time because A. psidii infection is relatively new (<10 years) to the study site, and there has already been a significant decrease in the populations of Myrtaceae species. Species that initially were categorised as tolerant to the infection (e.g., Acmena smithii), but that with time their populations have been increasingly infected and starting to show some mortality.

The seemingly dead trunks of Archirhodomyrtus beckleri showed some basal shoots after a period of rain. This is an example of how some species respond to climatic events, such as rain, which is an important factor to consider when assessing the impact of A. psidii within a region. Being a fungal pathogen, A. psidii is also affected by site moisture levels and temperatures (Talley et al., 2002). Therefore, further studies are recommended to assess the effects phenological events play on the impact of A. psidii infection and understanding the role that weather plays in the plant–pathogen interaction in different climatic regions and under different phenological conditions. Studies of foreign strains of A. psidii indicate climatic suitability through tropical and subtropical regions where moisture conditions and moderate temperatures are more consistent between seasons (Kriticos et al., 2013). However, the role that phenological events, such as change of seasons, play in determining the impact of A. psidii infection on populations in their natural systems remains equivocal.

Leaf traits such as SLA can be used as surrogate measurements of physiological processes that manifest in morphological features and reflect products of the interaction between plants and their environments. SLA is relatively easy to measure leaf trait and can be used as a relative measure of growth strategy (Garnier et al., 2004). In this study, I found some evidence of a potential relationship, but more data are needed. SLA may prove useful because it also shows an intraspecific variation (Dwyer et al., 2014) and therefore, might capture the site-level differences in susceptibility also found with mortality.

I did not find a significant correlation between SLA and mortality rates. Also, I did not find a correlation between mortality and SLA values sourced from the TRY database (Kattage et al., 2011). This may be because SLA can be highly variable depending on the environmental conditions in which a species grows (Albert et al., 2010) and so too can the

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 51 susceptibility of a species to A. psidii (Makinson, 2018). Further research should be conducted in different environments where SLA can be measured to capture its variation and where species can be categorised at the site level for susceptibility to A. psidii.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 52

Chapter 3: Impact of Austropuccinia psidii mortality on light availability and fostering conditions and changes in the community assembly of wet sclerophyll forests

3.1 AIM AND OBJECTIVES

In the work described in this chapter of my thesis, I aimed to provide evidence of the impact of A. psidii on the plant community following the increasing mortality of Myrtaceae species that structure the mid- and understory of the wet sclerophyll forest. I focused specifically on ground cover composition, seedling recruitment and environmental conditions such as light and soil nutrients. I hypothesised that the high mortality rates found at these sites in the mid-canopy and understory would result in more light reaching the forest floor and higher soil nutrients. These changes could then provide suitable conditions for the arrival of early colonising plant species and potentially more invasive non-native plant species, which depend on light availability in the forest floor (Burdon et al., 2006), and changes in soil nutrients (Krishna & Mohan, 2017). In particular, this study aimed to answer the following key questions:

1. Do canopy gap fraction (as a surrogate measure of light) and ground cover differ between regions of the forest that with and without mortality caused by A. psidii?

2. Do soil nutrients differ in regions that exhibit mortality in the midstory and understory due to A. psidii? 3. Are seedling composition and ground cover in the forest affected as direct and/or indirect consequences of A. psidii?

3.2 RESEARCH DESIGN

3.2.1 Field assessment

The community impact of A. psidii was recorded in four transects of 2 m  50 m in the wet sclerophyll forest at the Ryans Road site, Tallebudgera Valley. Each metre of the transect was divided into two 1 m2 quadrats for a total of 100 quadrats per transect and 400 quadrats in total, quadrats with mortality due to A. psidii were mark as present and without

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 53 mortality as absent. Giving an area in the forest with and without mortality. Trees without foliage were recorded as dead. It was assumed that dead was a consequence of A. psidii, as there is no other pathogen in the region associated to mortality events in populations of Myrtaceae (Pegg et al., 2017), and species evaluated were previous recorded infected in the region (Carnegie et al., 2015; Pegg et al., 2017). Canopy gap per quadrat was measured as a surrogate measure of light conditions in the forest.

The transects were established by Pegg et al. (2017) in 2016 as a long-term study site to record the impact of A. psidii in the Myrtaceae populations because they provide a representative sample of the condition of wet sclerophyll forest in the region. One-meter quadrats were used to record canopy cover estimates, seedling composition and ground cover (%), and soil nutrients of the forest in regions of the forest with and without mortality as a result of A. psidii infection. This was the first time that this information was recorded in the transects because previous surveys focused on the state of the Myrtaceae population in 2016, 2017 and 2019. Data recorded for the work described in this chapter were collected in December 2019 to March 2020.

The percentages of non-native and native species were estimated in the 1 m2 quadrats and soil was sampled for the elemental analysis to identify changes in soil nutrients. Mortality in the quadrats was recorded as a binary variable: yes, if there were any dead trees or shrubs in the canopy, and no, if absent.

3.2.2 Light measurements

The canopy gap fraction was recorded in each of the 1 m2 quadrats in the four transects using a CI-110 CID Bio-Science plant canopy imager. To ensure that similar measurements were recorded under similar conditions, these were taken between 10 am and 2 pm under cloudy conditions, following the methodology of Gibson (2015). Other possible light measurements include photosynthetically active radiation, which is dependent on the intensity of the light. However, canopy gap fraction was selected as a more stable measurement and is defined as the percentage of light passing through the canopy without encountering foliage or other plant elements (Negrón Juárez et al., 2009).

The area of measurement in the canopy was restricted to the area corresponding to each quadrat. Having a zenith angle of 10°, based in the equation shown below and following the zenith division method for hemispherical photography (Walter et al., 2010), as shown in Figure 3.1.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 54

d(α) 휃 = 훽

휃: 푍푒푛푖푡ℎ 푟푒푠푡푟푖푐푡푖표푛 푎푛푔푙푒, 훼: 푎푛푔푙푒 표푓 푣푖푒푤, 푑: 푝푙표푡 푠푖푧푒 , 훽: 퐻표푟푖푧표푛푡푎푙 푝푟표푗푒푐푡푖표푛

Figure 3.1 a) Zenith division for hemispherical photography of the canopy; b) Zenith restriction angle for the area of the quadrat. : Zenith restriction angle, : angle of view, d: quadrat size, : Horizontal projection.

3.2.3 Soil sampling

Soil nitrogen, phosphorus, potassium and sulphur were compared between quadrats with and without mortality because these elements are usually found in plant litter decomposition (Krishna & Mohan, 2017) and therefore may show differences depending on the tree species found at local areas at a site. Soil samples corresponding to the horizon A (10 cm of depth) were taking; quadrats were numerated from 1 to 400 , as shown in Appendix B, and 30 quadrats were randomly selected through single random sampling for soil sample collection (11 samples were taken from along transect 1, five along transect 2 , five along transect 3 and nine along transect 4). Soil samples were preserved in a cold room at 4° C and later a representative soil sample was taken from the existing soil sample of each quadrats using the quartering method (Washington State University, 2018) to be dried in the laboratory

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 55

at –50C using a freeze-drying methodology (Neil et al., 2002). Each sample was ground in a vibrating disk mill at 220–240 V and 50–60 Hz for 3 minutes.

To compare the percentages of nitrogen in the soil samples, 1 g of soil per sample was analysed using LECO combustion analysis (Wang & Anderson, 1998). To measure traces of other elements, the soil samples were subjected to X-ray fluorescence analysis (Cerato and Miller, 2013).

3.2.4 Seedling composition

A 1 m2 Polyvinyl chloride quadrat was used to record the species composition for each 1 m2 of the transect. Two 1 m2 quadrats were recorded for each metre of the transect and named as a right or left quadrat. Only species <1 m of height were included, and the percentage of cover for each species was recorded using the Daubenmire technique ( Daubenmire, 1959)

3.3 ANALYSIS

The data were analysed using R statistical computing package version 3.6.1 (R Development Core Team, 2013) through the R studio platform, version 1.2.5001.

Gap canopy fraction was compared between quadrats with and without mortality. I used the Wilcoxon rank-sum non-parametric test (Nelson, 1963) as the data were non- normally distributed and I was aiming to find differences between a variable with two groups: regions of the forest with (yes) and without (no) mortality due to A. psidii.

3.3.1 Soil nutrients

To compare soil percent nitrogen, phosphorus, potassium and sulphur between areas with and without mortality, I used the Wilcoxon rank-sum test for each element. As the data was not normally distributed a non-parametric analysis was a appropriate to compare the variance between the two groups: regions of the forest with (yes) and without (no) mortality.

3.3.2 Ground cover species richness

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 56

I analysed seedling composition using data recorded for the 400 quadrats in the four transects. The community-level species richness of seedlings in the understory was analysed using non-metric multidimensional scaling (nMDS), in which the species were recorded as present or absent and categorised as native, non-native and invasive according to Queensland Government species list for the Tallebudgera Valley (Queensland Government, 2020). Each category was compared against two factors, morality caused by A. psidii and identity of the transect surveyed. To elucidate the ground cover richness and its relationship with mortality in the canopy or site-specific characteristics of the transects, I performed nMDS analysis using the r package ‘vegan’, using a Bray-Curtis ordination matrix and permutational multivariate ANOVA to compare species dispersion over the two factors (mortality and transects), and set on 999 permutations. The species were set as a binomial variable: species absent (0) or present (1).

3.3.3 Seedling and ground cover abundance

The abundance of seedlings of Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana, which were considered as focal Myrtaceae species, was recorded and compared with that from previous records to identify changes in seedling composition over time (Pegg et al., 2017). Previous proportional data recorded in 2016 were updated with the number of seedlings for the five focal species in the sampled area for 2019. Plant species ground cover (%) of native and non-native species was recorded in each quadrat for plant species <1 m in height. Ferns were considered relevant as an environmental indicator of moisture but were treated as grouped because differences within species in the group were of low relevance for the purpose of this study.

I created a cumulative graph based on the ground cover per species estimates to visualise the relative abundance of non-native species in quadrats with and without mortality caused by A. psidii. The proportions of ground cover of native, non-native and invasive species were compared using the Kruskal–Wallis test by ranks because the data were not normally distributed and were compared between one variable with three levels. When a difference between ground cover was significant, Dunn’s post hoc test was used to identify the difference between the three groups.

3.4 RESULTS

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 57

3.4.1 Do canopy gap fraction (as a surrogate measure of light) and ground cover differ between regions of the forest that with and without mortality caused by A. psidii?

Canopy gap fraction differed significantly between quadrats with and without mortality due to A. psidii. The median canopy gap fraction was >60% in quadrats with mortality and <60% in quadrats without mortality (W = 7201.5, p < 1.204  10–13, Figure 3.2). Ground cover estimates did not differ in quadrats with and without mortality caused by A. psidii (W = 13442, p > 0.1) Figure 3.3).

Figure 3.2 Box and whisker plot showing canopy gap fraction (%) in quadrats with and without mortality. The average canopy gap fraction was 67% in regions of the forest with mortality caused by A. psidii and 53% in regions without mortality A higher percentage means a larger gap in the canopy, which implies more light shining through the larger gaps (Wilcoxon rank-sum test, W = 7201.5, p < 1.204  10–13).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 58

3.4.2 Do soil nutrients differ in regions that exhibit mortality in the midstory and understory due to A. psidii?

The composition of major elements required for plant growth in soil samples showed no significant difference between quadrats with and without mortality caused by A. psidii (all Wilcoxon rank-sum tests :p > 0.05) (Figure 3.4).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 59

3.4.3 Are seedling composition and ground cover in the forest affected as direct and/or indirect consequences of A. psidii?

Seedlings recorded in the understorey of the forest included 24 species, 10 tree species, 8 shrub species and 6 herbaceous species, plus a plant group Polypodiopsida (ferns). From the 24 species, 11 were non-native and 13 species plus Polypodiopsida were considered as native species, as shown in Table 3.1.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 60

Table 3.1 Species in the regenerating area of the forest, under 1 m in height.

Species Category Life form Invasive Acmena smithii Native Tree Aphananthe philippinensis Native Tree Archirhodomyrtus beckleri Native Tree Beilschmiedia elliptica Native Shrub Decaspermum humile Native Tree Ficus coronata Native Tree Flindersia australis Native Tree Gossia hillii Native Shrub Guioa semiglauca Native Tree Hibbertia dentata Native Herb Jagera pseudorhus Native Tree Macaranga tanarius Native Tree G Polypodiopsida Native Shrub/Herb Rhodamnia maideniana Native Shrub Ageratina adenophora Non-native Shrub * Ageratina riparia Non-native Herb * Ageratum houstonianum Non-native Herb * Ardisia crenata Non-native Shrub Cinnamomum camphora Non-native Tree Paspalum mandiocanum Non-native Herb * Erigeron canadensis Non-native Herb * Lantana camara Non-native Shrub * Ochna serrulata Non-native Shrub * Psychotria sp. Non-native Shrub Setaria palmifolia Non-native Herb Note: Species were categorised as native or non-native to Australian ecosystems. Polypodiopsida is the plant group (G) corresponding to ferns. Invasive species are shown with a star under the invasive column (Queensland Government, 2020).

For the model that included all species under 1 m height, no significant difference

(F196 = 1.28, p >0.1) was found between quadrats with and without mortality. However, a significant difference was found in plant seedling communities between the four transects

(F196 = 12.3, p < 0.001). Transect 4 had the highest dispersion distance; the differences in species composition are shown in Figure 3.5A.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 61

Figure 3.5 Non-metric multidimensional scaling model (nMDS) for all species in the four transects. There was a significant difference for richness of species between transects (F196 = 12.3, p < 0.001; Figure 3.5A) but no significant difference was found between quadrats with and without mortality (F196 = 1.28, p > 0.1; Figure 3.5B).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 62

The relative abundance of Myrtaceae seedlings after four years of infection by A. psidii increased by 14% for A. smithii and 1% for G. hillii and decreased by 8% for A. beckleri, 4% for D. humile and 2% R. maideniana between 2016 (n = 98) and 2019 (n = 81) (Figure 3.6).

Cumulative quadrats of native and non-native species showed higher overall ground cover for quadrats without mortality than those with mortality caused by A. psidii. The findings are shown in Figure 3.7 for only non-native species and in Figure 3.8 for all species surveyed in the ground cover.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 63

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 64

Overall variance between ground cover under native, non-native and invasive species showed significant differences based on a Kruskal-Wallis test by ranks (X2 =41.2, df = 2 p<1.11 x 10-09). A post-hoc test, Dunn’s test, show significant difference between native and invasive ground cover (p<9.91 x 10-06), non-native and invasive ground cover (p<9.91 x 10- 09) and no difference between native and non-native ground cover (p>0.1), as seen in Figure 3.9.

29.5

28.5

27.5

Ground cover (%) 26.5

25.5

25 native non native

3.5 DISCUSSION

3.5.1 Implications of the increment in the availability of light in the ground cover

Overall, I found that the canopy gap fraction was higher in regions where trees in the mid- and understory of the forest had been killed because of A. psidii infection. These results suggest that the quadrats where mortality occurred are experiencing increasing amounts of light in the understory. Several studies have reported on the ecological impact of fungal pathogens on the canopy structure by decreasing species in the overstory of the forest (Burdon et al., 2006; Dillon et al., 2019). Burdon et al. (2006) noted that a potential chain of events may ensue that causes changes to local environments. This includes the increasing

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 65 abundance of recruits (Blanco et al., 2015) or the arrival of light specialist species, which are often early colonising species (Théry, 2001). Over time the composition of these plant species changes, driven by pathogen spillover, which then impacts the soil microbial communities and soil nutrient availability.

Pathogen spillover will affect the interspecific competition by disabling the competition of the affected species by impairing their ability to compete through redirecting the way plants invest resources in the need to re-grow affected structures or simply by the extirpation of a species in the ecosystem (Chen & Nan, 2015). Newly recruited plant species may eventually play a role in changing abiotic conditions in the forest by changing the soil composition through plant–microbe feedback mechanisms (Bennett et al., 2019).

Increased light reaching the ground in the forest will contribute to the establishment of plant species that are competitively favoured by light (Brocardo et al., 2018). The identity of the seedlings recorded here provides evidence of the recruitment of early successional native species such as Macaranga tanarius, a rainforest early colonising species. This may also recruit non-native species that could be detrimental for the persistence of the forest, such as Ochna serrulata and Ardisia crenata; which are both considered as invasive species in South East Queensland (Queensland Government, 2020).

Usually, plants in low-light conditions grow slower than in environments with greater light available (Tateno & Takeda, 2003). However, plant species respond differently to light conditions in the forest. Invasive species have been found to be more competitive than native species under high-light conditions, particularly when light is available as a consequence of a disturbance in an environment (Pattison et al., 1998). Seven invasive species were recorded in the understory. Species such as Lantana camara and Ochna serrulata were present in one of the transects but were not linked to mortality events caused by A. psidii, but instead to the heterogeneity of the forest microenvironments. Invasive plant species are known for their ability to compete for resources (Pattison et al., 1998; Perry, 2006), and under the right conditions could lead to a decrease in plant diversity of the forest (Gallardo et al., 2019). Therefore, with more time (over four years of A. psidii infection) the number of invasive plants could start to increase in the understory with the increased light conditions and create patches in the forest with a different composition and function. As most invasive species recorded in this are mostly herbaceous or scrub-like in habit, and it seems unlikely that this

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 66 species will replace the forest structure and more likely that they could lead to changes in the community assembly.

3.5.2 Soil nutrient and mortality in the forest due to A. psidii

Soil nutrients such as carbon, nitrogen, and phosphorus are dependent on the biomass accumulation (Krishnan et al., 2017) and soil feedback mechanisms (Chen & Nan, 2015). Studies have shown that increased mortality in the forest could lead to changes in soil nutrients that, at the same time, foster conditions for the arrival of early colonising plant species or invasive non-native plants (Burdon et al., 2006). In this study, I found no difference in soil nutrients between parts of the forest with and without evidence of mortality caused by A. psidii in the canopy.

3.5.3 Seedling composition and invasive species richness

The seedling composition in the forest showed a higher percentage of native species, as shown in Figure 3.9. Native seedlings were still dominant in the forest in regions with and without mortality. However, there was a clear impact of A. psidii infection on the seedling recruitment of Myrtaceae species in the forest. Five of the 13 native species, excluding ferns, under 1 m were Myrtaceae, and though field observation most of the Myrtaceae seedlings showed infection symptoms such as pustules, spores or necrosis. It is unknown how much of this will persist in the forest over time. Mortality in some species seems to be restricted to seedlings, which may reduce the recruitment of this species in the forest; Acmena smithii mortality was present only in seedlings under 1 m in height and in Rhodamnia maideniana 70% of the mortality reported for in 2019 correspond to seedlings under the same height.

The decrease of species richness in a forest canopy usually leads to two outcomes. The first is that the remnant species and/or species that had a redundant role in the forest will occupy niches in the forest. Functional redundancy in ecology is the phenomena by which multiple species can share similar roles in the functionality of an ecosystem (Rosenfeld, 2002). In a study of subtropical grasslands in Brazil, Pillar et al. (2013) found that species with a redundant role enhance community resilience and stability. In that study, the authors aimed to understand the role of functional redundancy in ensuring ecosystems processes after perturbations as a result of temporary or permanent local species extinction. Transferring such a scenario to the wet sclerophyll forest in the Tallebudgera Valley may mean that the species composition could change in the forest but without affecting the structure of an

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 67 already established native ecosystem. In the wet sclerophyll forest context, species such as Macaranga tanarius, Guioa semiglauca, and Jagera pseudorhus could structure the makeup of the canopy over time.

A second scenario is described by Burdon et al. (2006) who reported changes in the community dynamics in a forest following the reduction of forest canopy because of infection by a pathogenic fungus called Cronartium ribicola, where an increase in the availability led to a change in the community assembly over time. Despite a tangible reduction of the focal Myrtaceae population seedlings in the forest I studied, 2016 (n = 98) and 2019 (n =81), and an increase of light in the forest floor, (Figure 3.2), there is a low abundance of invasive plant species in ground cover, being 16.4% of the ground cover reported including quadrats with and without mortality in the canopy, as shown in Figure 3.9.

A probable explanation of the low abundance of invasive species in ground cover is that native species composition in the understory of the forest could be intercepting significant light that otherwise would be available for invasive non-native species. Such an effect has been documented in a forest in Hawaii in a study that evaluated the impact of selective logging on native and invasive species abundance. Areas of the forest without understory native fern species exhibited an increase in light from 10.1% to 42.5% and, consequently, an increase in invasive grasses and tree seedlings (Friday et al., 2008).

Another explanation for such phenomenon is shade tolerance. How trees grow up into different parts of the canopy is usually influenced by two factors, its genetic pre-disposition to height, the amount of nutrients, water and light available and early in its development its ability to tolerate light conditions that are not optimal for its height (Grebner et al., 2013). Shade tolerance is defined as the ability of a plant species to survive under low-light environments (Ward & Stephen, 1993). Shade tolerance can vary between species (Hugh et al., 2003) and within species (Ntawuhiganayo et al., 2020), and shade-tolerant physiology generally increases with the decreasing of canopy openness (Hugh et al., 2003). This suggests that even if the species on the forest floor are accustomed to low levels of light, this could change after being exposed to new levels of light. Cumulative graphs are used in ecology to study different components (e.g. richness, abundance) of the diversity of an environment by showing the relationships between the components and ranked categorical data (Matthews & Whittaker, 2014; Thompson & Withers, 2003). The cumulative ground cover data (Figures 3.7 and 3.8) show more ground cover in regions of the forest without mortality events in the

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 68 non-native and in all species recorded. Through this study, I have linked the impact in the canopy of the forest with a higher amount of light that reaches the forest floor. Therefore, a possible explanation of greater ground cover in areas of the forest without mortality caused by A. psidii may be that some species are accustomed to low-light conditions.

Topography is another factor that could explain the composition of species in the ground cover. Topography reflects the arrangements of the physical features of the land and influences light and soil nutrient availability (Tateno & Takeda, 2003). Plant species richness in the ground cover was not found to differ between patches with and without mortality caused by A. psidii. However, there was significant difference in the richness between the transects sampled. Most of the invasive species were recorded in transects 3 and 4, and transect 4 showed the highest number of invasive species richness. Transect 4 was the only transect with a natural opening of the canopy caused by what seemed to be the presence of an ephemeral stream and a flat region on one site of the stream where two of the most abundant invasive species were found: Paspalum mandiocanum was 9.2% and Ageratina Adenophora was 4.1% of the total ground cover. This result may indicate that the presence of these species is more related to topographic features of the transects than to the impact of A. psidii. However, the increasing canopy gap percentage measured because of mortality from A. psidii infection may exacerbate the presence of invasive species in the forest over time by changing the environmental conditions suitable for their proliferation (Brocardo et al., 2018).

3.6 CONCLUSION

In conclusion, I found that A. psidii is impacting the plant community in the wet sclerophyll forest by changing seedling composition of the five most Myrtaceae species in the forest and increasing the canopy gap size. However, I did not record a significant shift in ground cover and soil nutrient conditions, which may reflect that A. psidii infection and impact have been recorded for only four years. Other studies of forests affected by pathogens (e.g. Ophiostoma ulmi) have found that up to 10–30 years may be needed to start to see the change in community assembly (Boddy, 2016). Despite the short time of monitoring in this forest, significant differences were found in the canopy gaps because of the mortality in the mid- and understory, which suggest that changes in the composition and other resource availability may become more pronounced over time. Follow-up studies in 10 years are highly recommended to document further and record the ecological impacts of A. psidii.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 69

Chapter 4: Discussion and conclusion

4.1 DISCUSSION

Wet sclerophyll forests in South East Queensland are composed largely of tree and shrub species in the Myrtaceae family. Plant species from this prominent family make up the upper-, mid- and understory canopies. A. psidii infections have decreased populations of Myrtaceae species at the study site and in forests along the eastern coast of Australia (Carnegie et al., 2015; Pegg et al., 2017). The data collected in this study show that infection rates of different species changes over time. Some species showed few signs of infection initially (Pegg et al., 2017), but after four years of infection have started to show more signs of infection and even mortality. I also found that the mortality experienced in the midstory and understory has increased canopy gap size, which with increased incidental light could over the longer term change the species composition in the understory and ground cover. However, I did not find significant evidence of species compositional changes in the understory after four years of A. psidii infection. This increased canopy gap size and likely resultant increase in light reaching the forest floor could lead to changes in species composition and nutrient cycling in the future as the forest responds to the loss of complex layers in the canopy.

4.1.1 A. psidii impact on highly abundant Myrtaceae species over time

Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana were surveyed at this study site over three time periods and over four years of A. psidii infection. Overall, the populations have decreased over time as a result of A. psidii persistent infection. According to the prediction models described in Chapter 2 and Table 2.2, Archirhodomyrtus beckleri, Decaspermum humile, and Gossia hillii populations had a lower probability of survival and are likely to become locally extinct by extirpation in the next three years. Populations of Acmena smithii and Rhodamnia maideniana showed a lower mortality rate when infected with A. psidii. Nevertheless, mortality rates were 4.8% for Acmena smithii and 20% for Rhodamnia maideniana over the last four years.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 70

Mortality events in Acmena smithii were present only in seedlings under 1 m in height, and 70% of the mortality found for Rhodamnia maideniana corresponded to seedlings with the same characteristics. It appears that A. psidii could be decreasing the recruitment of seedlings of these two species at the study site. Thompson et al. (2010) studied the effects of a plant pathogenic fungi, Diplodia mutila, on tree recruitment in a lowland tropical forest in Peru and found that it impacted the understory of the forest by reducing the seedling population with predicted community changes in the forest over time. Therefore, reducing the seedling recruitment of native species in the forest could have a detrimental effect in the forest structure and function over time. As adult tree populations that structure the forest canopy and understory are decreasing and there is high mortality in recruits, decreasing the number of native species in the forest will likely impact on animal species (Floyd, 1989).

Other examples of how pathogenic fungus have impacted the structure of forests are Cryphonectria parasitica in chestnuts (Rigling et al.,2018) and Ophiostoma ulmi in elms (Boddy, 2016). These two pathogenic fungal species have had a significant effect on forest canopies in North America and Europe, respectively. Cryphonectria parasitica was first reported in North America in the early 1900s and resulted in a high mortality rate of plant species in the genus Castanea (family Fagaceae), chestnut trees, where most of the populations exhibited a decrease in the canopy density and an increase in seedling mortality (Burke, 2011). Similarly, Ophiostoma ulmi reduced the populations of Ulmus spp. and Zelkova carpinifolia, elms trees, in North America by 40% and in Europe by 10% (Boddy, 2016).

In Australia, an introduced pathogenic fungus, Phytophthora cinnamomi, was first reported in 1920 and 43 years after was recognised as a key threatening process for Australian biodiversity as it spread to about 44% of the species in Western Australia (Hardham, 2005) and caused permanent damage to the ecosystems by dramatically modifying their structure and functionality through habitat lost for flora and fauna in jarrah forests and banksia woodlands ( Hill et al., 1994; Wills, 1993), destruction of vegetation in World Heritage areas and National Heritage places that, to date, have been irreversible (Department of the Environment, 2020).

The impact of A. psidii in the natural systems has been the focus of a few studies in Australia that assessed populations in a rainforest (Fernandez-Winzer et al., 2020) coastal

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 71 swamp woodlands (Fernandez-Winzer et al., 2018) and wet sclerophyll forests (Pegg et al., 2017). However, the arrival of A. psidii in Australia is relatively recent, in the past 10 years, and it is unknown what effect this pathogen will have on Australian ecosystems over time. In the wet sclerophyll forest in the Tallebudgera Valley, A. psidii has impacted the canopy of the forest, where species such as Archirhodomyrtus beckleri, Decaspermum humile, and Gossia hillii have been shown to be extremely susceptible to A. psidii over time. All Myrtaceae species included in this study are listed as least concern under the Queensland Government conservation status. However, based on the predictions of this study Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii are likely to become locally extinct over the next three years.

4.1.2 SLA as a tool for monitoring populations susceptibility to A. psidii

SLA is the leaf area divided by the amount of dry tissue and has been found to be a surrogate measure for plant energy investment strategies between photosynthetic area and protection against herbivores (Westoby et al., 2002; Wright et al., 2005). Species generally with a higher SLA adopt what is described as a more ‘disposable’ strategy in which the investment of dry matter per leaf is lower than in species with a lower SLA, and overall this investment is in faster growth rates and less in herbivore and pathogenic defence mechanisms (Dwyer et al., 2014). I tested whether SLA could be used as a surrogate measure for mortality rates of different species at a site level because of the infection pathway of A. psidii being prominently through the leaves. I did not find a significant correlation between SLA and mortality rates.

Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii showed higher SLA and higher levels of mortality, as shown in Figure 2.5 in Chapter 2. Overall, I did not have a large enough sample size and therefore suggested a more extensive study should be performed to include more species and sites to explore if there is a trend between high SLA and mortality. Susceptibility to A. psidii can vary within a species, such is the case of Melaleuca quinquenervia (Pegg et al., 2018), and this could be the product of phenotypical changes within a species than confer resistance (Yong et al.2019). Such a response can be captured by the measurement of specific functional traits (Dwyer et al., 2014).

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 72

Intraspecific plant trait variation has been shown to differ over an elevational gradient. For example, Xu et al. (2020) found a decrease in SLA with an increase in elevation in Pittosporopsis kerrii, an abundant rainforest species in China. Similar results for SLA were reported in Pinus sylvestris in sclerophyll woodlands by Laforest-Lapointe et al. in 2014. Elevation gradient affects nutrient availability, temperature and soil moisture (Grebner et al., 2013; Burdick et al., 2001). Consequently, changes in environmental features that affect plant traits could be a surrogate measure of plant physiological adaptations to their environment that could be related to the degree of susceptibility of populations to A. psidii.

In addition to intraspecific plant trait variations, there was a second interesting trend in SLA of the populations sampled in this study. As shown in Figure 2.5 in Chapter 2, the most affected species Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii, showed a high variation in SLA between organisms within their species in leaves sampled at the study site. By contrast, less affected species such as Acmena smithii and Rhodamnia maideniana showed a lower variance in SLA values. Intraspecific variation in SLA has been positively linked to the fertility of a habitat; that is, species in soils with higher nitrogen levels show high SLA values (He et al., 2018). However, in this study, there was no difference between soil nitrogen content in regions of the forest with and without mortality. It is only possible at this point to speculate that other environmental conditions, such as soil moisture, may be related to SLA variability within these species.

In summary, the relationship between SLA and species mortality was not statistically significant, and therefore the relation between SLA and susceptibility to A. psidii is still equivocal. High variability in SLA was observed in the more affected species in the sampled area, but more research is needed to elucidate if high SLA could be a proxy measurement of populations at risk.

4.1.3 Light dynamics and ground cover

Canopy cover in wet sclerophyll forests is estimated at 50% to 80% cover under normal conditions (Peters & Butler, 2014). I recorded an average canopy gap fraction of 67% in the regions of the forest with mortality caused by A. psidii and 53% in regions without mortality. This equates to an average canopy cover of 37% in areas with and 47% in areas

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 73 without mortality in the forest. Both measurements are lower than the canopy cover normally recorded in a wet sclerophyll forest and suggest a more significant impact for canopy gap in regions of the forest that experience mortality events caused by A. psidii (Chapter 3). Therefore, more light is reaching the forest floor in areas of the forest canopy with mortality caused by A. psidii. Despite the greater light levels in the forest floor because of A. psidii, the impact on the forest canopy of the did not show significant differences between the ground cover in regions with and without mortality.

There are several explanations on why there are no significant differences between the ground cover in regions with and without mortality. Species in the regeneration strata of the forest, both native and non-native, could become accustomed to lower levels of light as regions with morality in the forest had less ground cover. However, Hugh et al. (2003) and Ntawuhiganayo et al. (2020) suggested that the physiological mechanisms of the response to lower light levels in a forest can change because shade tolerance can vary both between and within species in response to changes in the canopy openness and under an increase in light availability. This implies that regions of the forest where the canopy was recently opened and therefore received more light, may experience a change in the ground cover composition over time. Another possible explanation is the role of the site topography because parts of the forest have natural openings and these present with a greater abundance of native and non- native species. These areas exhibited no mortality events and may therefore have contributed to equalising the ground cover in regions with and without mortality in the analysis.

Number of individual trees in the understory has decreased over time: by 8% for Archirhodomyrtus beckleri, 4% for Decaspermum humile and 2% for Rhodamnia maideniana between 2016. Of the 25 species recorded in the forest floor, 11 are non-native, and seven were categorised as invasive. There was no difference recorded in soil composition between regions of the forest with and without mortality. However, the increase in light in the understory is considered as a fostering condition for light-specialist species (Brocardo et al., 2018), such as some of the invasive species that are already present in the study site (e.g. Lantana camara, Paspalum mandiocanum and Ageratina Adenophora. Follow-up studies are recommended in 10 years to examine whether A. psidii has had further impact on the forest function and composition.

4.2 LIMITATIONS

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 74

The study presents some limitations, particularly around the time of surveying. The first part of this study was conducted between September and December 2019, which when Pegg et al. (2014) and Carnegie et al. (2015) estimated that the disease incident is lowest because it is a generally dry period in south east Queensland. The year 2019 was the second driest year on record for Queensland (Bureau of Meteorology, 2020). After a period of rain in 2020, basal shoots of Archirhodomyrtus beckleri appeared. During that period, I was collecting the data for the third chapter of this thesis. However, once the COVID-19 pandemic restrictions were in place, it was not possible for me to record the impact of A. psidii infection on the basal shoots and the further effect of A. psidii infection on the focal species populations, nor to update the mortality records for Archirhodomyrtus beckleri.

There are also several limitations presented with the tests for accuracy between the Kaplan-Meier survival estimated and Markov chains models. First, there is a lack of uniformity in the sampling periods of the mortality data, as there is no data available for 2018. This data gap means it is not possible to compare the prediction of the Markov models for the mortality from 2017 to 2018 Kaplan-Meier probability of survival or predict 2019 mortality, as there is no data from the previous year. This means I could only use one accuracy test that is presented in table 2.1. Further records of the mortality in the next years are necessary to evaluate more thoroughly accuracy of the method. Second, there are factors that could affect the reliability of how mortality was recorded in this study, trees without foliage were recorded as dead. As the 2019 survey was completed during a dry season for the region and after rain basal shoots were detected. Indicating a clear bias in the way mortality was recorded and therefore affecting Kaplan-Meier estimates and Markov chain predictions; this is however a well-known issue when recording tree mortality and further highlights the importance of repeated sampling, as conducting in this study.

As for SLA, I did not collect sufficient data to establish whether there is a correlation between the susceptibility of species to A. psidii and SLA. However, there was an indication of a trend in which high SLA species seemed to be more susceptible to mortality caused by A. psidii infection. I aimed to compare SLA according to the categories of susceptibility. However, there are different approaches to the categorisation of A. psidii susceptibility (Draz et al., 2015; Junghans et al., 2003; Sandhu and Park, 2013) and differences in the impact of populations between different regions (Carnegie and Pegg, 2018; Makinson 2018; Pegg et al.,

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 75

2018).. Also, some species had few SLA values in the database. These make it difficult to investigate the relationship between the susceptibility of a species and SLA.

The survey design of the impact that A. psidii has on the community impact has several limitations. As it is mention through chapter 3 of the thesis 2019 was the first survey for understory impact. Therefore, there is no previous data of how the understory community, light and soil nutrients have changed over time. Such information is necessary to estimates with precision how the plant community has changed over time and predict the impact that A. psidii will have in the plant community of the wet sclerophyll forest at the Tallebudgera Valley.

A. psidii infection is relatively new at the study site, less than 10 years, and the results showing no significant difference within regions of the forest with and without mortality may change with time.

4.3 FUTURE RESEARCH

All Myrtaceae species surveyed in this study are categorised as low concern under the Queensland Government conservation status. However, through the work presented in this thesis, these species are threatened by A. psidii at the study site. I recommend that surveys at more site are conducted to accurately identify the susceptibility of Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii in Queensland and NSW. Further assessment is necessary to investigate the extent of the impact of A. psidii infection on other Myrtaceae species that are present in the forest overstory, such as Eucalyptus grandis and Lophostemon confertus (Pegg et al., 2017), because as infection rates increase in the mid- and understory the overstory species may begin to show signs of susceptibility. Evidence I present here after just four years of infection, shows how species susceptibility can increase over time.

Further research on susceptible and resistant species, particularly those species whose susceptibility varies between regions, is recommended to clarify whether SLA is a predictor of the susceptibility of populations within a region to A. psidii. I recommend that direct SLA measurements are used in future studies of regional assessments for populations of the five focal species Acmena smithii, Archirhodomyrtus beckleri, Decaspermum humile, Gossia

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 76 hillii, and Rhodamnia maideniana. This information could be recorded for different environments to elaborate on the trends found in this study.

Follow-up studies that record the impact of A. psidii on ground cover composition, soil nutrients and recruitment in the forest are necessary to identify the long-term impacts of A. psidii on the community ensemble of the wet sclerophyll forest in the Tallebudgera Valley.

4.4 CONCLUSION

Several studies in Australia have shown that A. psidii threatens the persistence of susceptible Myrtaceae species and is bringing some species to the border of extinction (Fensham et al., 2020). Nevertheless, the impact of A. psidii on natural systems over time remains equivocal. In the research presented in this thesis, I have shown that the impact of A. psidii on populations of Myrtaceae species increased over the four years of monitoring in a wet sclerophyll forest in South East Queensland, where species that structure and have a function in the forest by providing roof and nourishment are decreasing and some are likely to be locally extinct. I recommend a survey of the state of the highly susceptible species Archirhodomyrtus beckleri, Decaspermum humile and Gossia hillii be undertaken to update their conservation status in Queensland and in general for Australia.

Overall, the evaluation of populations in this study site showed evidence of an increase in mortality over time. This trend was more dramatic for some species; for example, the mortality of Archirhodomyrtus beckleri was 16% in 2016 and 88% in 2019, whereas that of Acmena smithii was of 0% in 2016 and 4.8% in 2019. The increased mortality of species that initially showed a low or zero mortality is important for the conservation of these species in the Tallebudgera Valley area because this may imply that populations that were categorised initially in low risk could be endangered over time. Moreover, as mortality changes from year to year, the susceptibility status may not be a conserved trait but, instead, susceptibility to A. psidii may vary depending on local environmental conditions.

I also examined the effects of A. psidii in the community composition and ground cover. The canopy gap fraction differed between regions of the forest with and without mortality, which suggests that increased light reaches the understory of the forest as a result of A. psidii. However, the species composition and soil nutrients composition did not differ

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 77 between regions of the forest in regions with and without mortality. Two factors might explain this phenomenon: the topography (there was a difference between species richness between the transects) and time (infection has been recorded for only four years). Species may become accustomed to low levels of light irradiation in the forest. However, it is known that time and increasing levels of light change shade tolerance physiology (Hugh et al., 2003).

Across the transects, 11 non-native species were recorded in the understory, seven of which are declared as invasive species. Invasive species have been found to be more competitive than native species under high-light conditions, particularly when light is available as a consequence of high levels of disturbance in an environment (Pattison et al., 1998). No invasive species were linked to mortality events. However, changes in light are described as fostering conditions for the spread of invasive species (Döbert et al. 2017). Therefore, the increase in light may lead to more colonisation events over time.

I also recorded an impact of A. psidii on the recruitment of native Myrtaceae species in the forest. Mortality events in Acmena smithii were observed only in seedlings under 1 m in height, and most of the mortality recorded for Rhodamnia maideniana corresponded to seedlings with the same characteristics. The impact on seedling recruitment after changes in the canopy of the forest was previously recorded in Phytophthora cinnamomic (Cahill et al., 2008).

The impact of Phytophthora cinnamomic in Western Australia is an example of how an introduced fungal pathogen can spread over multiples ecosystems, and its impact increase gradually (over 40 years; Hardham, 2005). This can modify the structure and composition of the forest in the region and have an impact on primary productivity, ecological function and habitat loss of flora and fauna that so far are irreversible (Department of the Environment, 2020). A. psidii was first reported in April 2010, a little over 10 years ago, and since then has spread to over 180 native species in their natural systems (Berthon et al., 2019), native ecosystems (coastal swamp woodlands; Fernandez-Winzer et al., 2018), rainforests (Fernandez-Winzer et al., 2020) and sclerophyll forests (Pegg et al., 2017). A. psidii has being linked with the imminent extinction of native Myrtaceae species (Fensham et al., 2020) and is predicted to cause the local extinction of Archirhodomyrtus beckleri, Decaspermum humile, and Gossia hillii in the sclerophyll forests in the Tallebudgera Valley. An interesting question

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 78 is whether the impact of A. psidii in Australian ecosystems will be similar to that of Phytophthora cinnamomic in Western Australia.

The research findings in this thesis will help to understand further the impacts on populations of Myrtaceae in the wet sclerophyll forest and begin to capture the subsequent impacts on the understory and ground cover of the forest. This information will be useful for monitoring the impact of A. psidii in populations at risk in the Tallebudgera Valley, South East Queensland, and contributing to the understanding of the long-term impact of A. psidii in wet sclerophyll forests.

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 79

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Appendixes

Appendix A- Copyright licence Carnegie, A. J., & Pegg, G. S. (2018).

Queensland University of Technology

22/09/2020

Santiago Diaz Torres - [email protected]

Dear Dr. Angus Carnegie and Geoff Pegg,

Request for Copyright Licence

My name is Santiago Diaz Torres. I am completing a Mater thesis at Queensland University of Technology. I am seeking your permission, in the form of a non-exclusive licence, to include your works in the online digital version of my thesis. The thesis will be available for anyone to access online via the Australian Digital Theses database and our institutional repository QUT ePrints/ https://eprints.qut.edu.au/ . Your works will be fully and correctly referenced.

My thesis includes the following copyright material:

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 95

Austropuccinia psidii invasion events and biotypes of each location, history of the events of myrtle rust invasion around the world. EU/SK abbreviation is used for the collection of Eucalyptus spp. and Syzygium spp. from Brazil, Paraguay, and South Africa. Reprinted from Carnegie and Pegg, 2018

From the following works for which I believe you hold the copyright: Carnegie, A. J., & Pegg, G. S. (2018). Lessons from the Incursion of Myrtle Rust in Australia. Annu Rev Phytopathol, 56, 457-478. doi:10.1146/annurev-phyto-080516-035256 Please sign below if you agree and return to me a copy. If you do not agree, or do not hold the copyright in the above works would you please notify me of this in writing.

I Angus Carnegie agree to grant you a non-exclusive licence for an indefinite period to include the above materials for which I am the copyright owner, into your online digital thesis. I understand that this thesis will be made available on the internet for online public access via the Australian Digital Theses database and your institutional repository.

Date: 22/9/2020

Signature (Copyright Owner)

Yours Sincerely,

Santiago Diaz Torres

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 96

Appendix B – Soil random sampling

Transect Transect Transect Transect Transect Transect Transect Transect Metres 3 3 4 4 1 1 2 2 R L R L R L R L 1 1 2 101 102 201 202 301 302 2 3 4 103 104 203 204 303 304 3 5 6 105 106 205 206 305 306 4 7 8 107 108 207 208 307 308 5 9 10 109 110 209 210 309 310 6 11 12 111 112 211 212 311 312 7 13 14 113 114 213 214 313 314 8 15 16 115 116 215 216 315 316 9 17 18 117 118 217 218 317 318 10 19 20 119 120 219 220 319 320 11 21 22 121 122 221 222 321 322 12 23 24 123 124 223 224 323 324 13 25 26 125 126 225 226 325 326 14 27 28 127 128 227 228 327 328 15 29 30 129 130 229 230 329 330 16 31 32 131 132 231 232 331 332 17 33 34 133 134 233 234 333 334 18 35 36 135 136 235 236 335 336 19 37 38 137 138 237 238 337 338 20 39 40 139 140 239 240 339 340 21 41 42 141 142 241 242 341 342 22 43 44 143 144 243 244 343 344 23 45 46 145 146 245 246 345 346 24 47 48 147 148 247 248 347 348 25 49 50 149 150 249 250 349 350 26 51 52 151 152 251 252 351 352 27 53 54 153 154 253 254 353 354 28 55 56 155 156 255 256 355 356 29 57 58 157 158 257 258 357 358 30 59 60 159 160 259 260 359 360 31 61 62 161 162 261 262 361 362 32 63 64 163 164 263 264 363 364 33 65 66 165 166 265 266 365 366 34 67 68 167 168 267 268 367 368 35 69 70 169 170 269 270 369 370 36 71 72 171 172 271 272 371 372 37 73 74 173 174 273 274 373 374 38 75 76 175 176 275 276 375 376 39 77 78 177 178 277 278 377 378 40 79 80 179 180 279 280 379 380 41 81 82 181 182 281 282 381 382 42 83 84 183 184 283 284 383 384 43 85 86 185 186 285 286 385 386 44 87 88 187 188 287 288 387 388

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest 97

45 89 90 189 190 289 290 389 390 46 91 92 191 192 291 292 391 392 47 93 94 193 194 293 294 393 394 48 95 96 195 196 295 296 395 396 49 97 98 197 198 297 298 397 398 50 99 100 199 200 299 300 399 400

Ecological impact of myrtle rust (Austropuccinia psidii) in a wet sclerophyll forest