Terrestrial Habitat Requirements of a Suite of Anuran Species Inhabiting a Semi- Arid Region of South East Queensland

TERRESTRIAL HABITAT REQUIREMENTS OF A SUITE OF ANURAN SPECIES INHABITING A SEMI- ARID REGION OF SOUTH EAST QUEENSLAND

Joanne Chambers B. App.Sc (Hons) Queensland University of Technology

A thesis submitted in part fulfilment of the requirements for the degree of Doctor of Philosophy at Queensland University of Technology School of Natural Resource Sciences

2008

Key words:

Amphibian, anuran, terrestrial habitat, habitat choice, burrowing frog, evaporative water loss, ground cover, soil pH, Barakula State Forest, frog conservation.

ABSTRACT

Hypothesised causes of the observed world-wide decline of amphibian populations are varied and in some cases contentious. Insufficient information relating to the autecology of many amphibian species can cause erroneous speculations regarding critical habitat requirements and hence management programs designed to enhance population viability are often unsuccessful. Most amphibians display a bi-phasic life history that involves occupation of an aquatic breeding habitat and terrestrial habitats that are used for foraging, and shelter from predation and environmental stress. However, the focus of most amphibian research is centred on the breeding habitat, with limited research being conducted into the terrestrial habitat requirements of most amphibian species.

Barakula State Forest is a large continuous area of open woodland situated in the semi-arid region of Queensland. The forest supports 21 species of endemic anurans, many of which use ephemeral waterbodies for breeding. This area is, therefore, an ideal location to test the relative importance of terrestrial habitat on the distribution of a suite of frogs that display different morphological and physiological characteristics.

On the landscape scale, the attributes of the terrestrial environment at three survey areas within Barakula were similar. However, at the patch scale, ground truthing showed there were considerable variations in vegetation and ground cover attributes within and between each survey site. Measured properties of the soil also tended to vary within and between sites. Soil texture ranged from sandy to heavy clay, soil pH ranged from 3.9 to 6.4 and soil moisture varied considerably.

Agar models, used for testing evaporative moisture loss at different microhabitats, retained significantly higher levels of moisture when positioned in the buried

microhabitat during summer, but in winter, models that were placed under leaf litter retained higher levels of moisture. Variations in levels of moisture loss at the five different microhabitats were evident within and between the survey sites.

Despite a prolonged drought, 1844 native frogs representing 17 species were pitfall trapped. Members from the family Myobatrachidae comprised 94% of these captures, and burrowing species accounted for 75% of total captures. Species were not randomly distributed within or between the survey sites. Vegetation attributes and soil properties played a significant role in influencing the catch rates and traplines that supported similar vegetation and soil attributes also tended to catch similar species. Capture rates of six of the seven burrowing species were significantly influenced by soil properties.

When given a choice of four different microhabitats created in enclosures, individuals from five species showed varying responses to habitat choice during night time activity. During daylight all species tended to avoid bare areas and burrowing species tended to burrow under some form of cover. Pseudophryne bibronii metamorphs showed a significant avoidance to soils with high pH. The number of Limnodynastes ornatus metamorphs was significantly and positively correlated with moisture levels surrounding a breeding area. Limnodynastes ornatus metamorphs tended to avoid areas that did not support some form of cover.

Embryos from the terrestrial egg laying P. bibronii translocated to sites with varying levels of soil pH, suffered increased mortality where the soil pH was >4.8. In the laboratory, embryonic survival was not significantly different between the four pH treatments. There was a significant influence of fungal infection on survival rates and ranked fungal infection was significantly different between the four pH treatments.

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The terrestrial environment at the three survey sites has provided sufficient protection from environmental elements to allow a large diversity of anurans to persist for long periods without access to permanent water. Management must consider the importance of the non-breeding habitat when defining buffer zones, restoration programs and conservation strategies to ensure that the complete set of ecological requirements for frog species are provided.

Table of Contents

KEY WORDS: ...... I ABSTRACT...... II STATEMENT OF ORIGINALITY ...... VIII ACKNOWLEDGMENTS ...... IX CHAPTER 1: GENERAL INTRODUCTION...... 1 1.0 INTRODUCTION ...... 1 1.2 PROJECT AIMS ...... 9 1.3 SUMMARY OF THESIS ...... 11 CHAPTER 2: TERRESTRIAL HABITAT ASSESSMENT ...... 13 2.0 INTRODUCTION ...... 13 2.1 METHODS...... 15 2.1.1 STUDY AREA ...... 15 2.1.2 TERRESTRIAL HABITAT ASSESSMENT ...... 26 2.2 DATA ANALYSIS...... 28 2.2.1 HABITAT ASSESSMENT ...... 28 2.3 RESULTS ...... 29 2.3.1 HABITAT ASSESSMENT...... 29 2.3.2 SOIL PROPERTIES...... 36 2.4 DISCUSSION ...... 38 CHAPTER 3:...... 41 EVAPORATIVE WATER LOSS IN DIFFERENT MICROHABITATS...... 41 3.0 INTRODUCTION ...... 41 3.1 METHODS...... 44 3.1.1 MODELS ...... 44 3.1.2 MICROHABITATS ...... 45 3.1.3 SAMPLING PERIODS...... 46 3.2 RESULTS ...... 47 3.2.1 WINTER SAMPLING PERIOD...... 49 3.2.2 SUMMER SAMPLING PERIOD ...... 50 3.2.3 ASSOCIATIONS BETWEEN MOISTURE LOSS IN MODELS AND HABITAT VARIABLES ...... 52 3.3 DISCUSSION ...... 54 CHAPTER 4:...... 60 FROG CENSUS AND TERRESTRIAL HABITAT ASSOCIATIONS ...... 60 4.0 INTRODUCTION ...... 60 4.1 METHODS...... 63 4.1.1 STUDY STIES...... 63 4.1.2 FROG CENSUS ...... 64 4.1.3 DATA ANALYSIS...... 65 4.2 RESULTS ...... 66 4.2.1 SPECIES COMPOSITION ...... 66 4.2.2 SPECIES COMPOSITION AND HABITAT ASSOCIATIONS ...... 73 4.2.3 INDIVIDUAL SPECIES AND HABITAT ASSOCIATIONS...... 78 4.2.3.1 General Observations...... 78 4.2.3.2 Habitat Associations ...... 79 4.3 DISCUSSION ...... 86 CHAPTER 5:...... 92 HABITAT CHOICE TRIALS...... 92 5.0 INTRODUCTION ...... 92 5.1 METHODS...... 96 5.1.1 HABITAT CHOICE TRIALS ...... 96 5.1.2 PSEUDOPHRYNE BIBRONII CHOICE TRIALS ...... 99

5.1.3 METAMORPH MICROHABITAT USE...... 100 5.2 RESULTS ...... 101 5.2.1 HABITAT CHOICE TRIALS ...... 101 5.2.2 PSEUDOPHRYNE BIBRONII CHOICE TRIALS...... 104 5.2.3 METAMORPH CHOICE TRIALS...... 105 5.3 DISCUSSION ...... 109 CHAPTER 6:...... 115 SOIL PH AND HATCHING SUCCESS IN ...... 115 PSEUDOPHRYNE BIBRONII ...... 115 6.1 INTRODUCTION ...... 115 6.2 METHODS...... 118 6.2.1 BROAD-SCALE ASSOCIATIONS...... 118 6.2.2 MEASUREMENT OF SOIL PH...... 118 6.2.3 EMBRYO MORTALITY - FIELD EXPERIMENTS...... 119 6.2.3.1 Study 1...... 120 6.2.3.2 Study 2...... 120 6.2.3.3 Study 3...... 121 6.2.4 EMBRYO MORTALITY – LABORATORY EXPERIMENT ...... 122 6.2.5 DATA ANALYSIS...... 123 6.3 RESULTS ...... 124 6.3.1 BROAD-SCALE ASSOCIATIONS WITH SOIL PH ...... 124 6.3.2 EGG MORTALITY – FIELD EXPERIMENTS...... 126 6.3.2.1 Study 1...... 126 6.3.2.2 Study 2:...... 126 6.3.2.3 Study 3:...... 126 6.3.3 LABORATORY STUDY ...... 128 6.4 DISCUSSION ...... 129 CHAPTER 7:...... 134 OVERVIEW AND GENERAL DISCUSSION ...... 134 7.1 OVERVIEW ...... 134 7.2 GENERAL DISCUSSION ...... 136 BIBLIOGRAPHY...... 142

LIST OF FIGURES

Figure 2.1. Map showing the position of the study area within Barakula State Forest in eastern Australia (a) and a satellite photo of Barakula State Forest showing the continuous vegetation cover in the 265,000 ha area (b)…...... 16

Figure 2.2. Regional Ecosystem Mapping for Barakula State Forest showing the three survey sites used to identify terrestrial habitat associations in frogs...... 20

Figure 2.3. Photo showing typical ephemeral breeding site for frogs at Barakula State Forest………………….……………………………………...…..….21

Figure 2.4. Aerial photograph showing the position of the survey sites used to study terrestrial habitat associations in frogs within Barakula State Forest …………………………………………………………………….....22

Figure 2.5. Photo showing areas within the survey sites at Barakula State Forest, where ground cover was dominated by grasses…………………….….23

Figure 2.6. Photo showing dense cover of leaf litter that occurred in areas within Barakula State Forest that supported a dense growth of Acacia……..23

Figure 2.7. In areas within Barakula State Forest where a dense growth of Callitris sp. occurred, the ground layer contained large amounts of downed timber………………………………………………………………………..24 Figure 2.8. Photo showing ground layer of sedges that occurred in some low-lying areas of the three survey sites within Barakula State Forest….……...24

Figure 2.9. Histograms showing variations in percentage ground cover variables measured at the 36 sampling points within the three survey sites at Barakula State Forest.………….....………………...... 30

Figure 2.10. Histograms showing variations in percentage ground cover variables measured at the 36 sampling points within the three survey sites at Barakula State Forest……………………………………………………...31

Figure 2.11. Multi Dimensional Plot showing similarity of the three survey sites within Barakula State Forest in relation to the measured vegetation attributes…………………………………………………………………….32

Figure 2.12. MDS plot showing similarity of the three survey sites in relation to measured soil properties collected from each sampling point at the three sites within Barakula State Forest ………………….………….....35

Figure 3.1. Photo showing the three different sized frog-shaped agar moulds used to test evaporative moisture loss under different microhabitats within the three survey sites at Barakula State Forest……………….………..44

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Figure 3.2. Mean (+/- 2SE) percent moisture lost from agar models placed in different microhabitats in summer and winter. Sample sizes are shown along x axis. ………………….…………………………………………….47

Figure 3.3. Mean (+/- 2SE) percent moisture lost from agar models placed in different microhabitats at the three study sites during winter...... 48

Figure 3.4. Means (+/- SD) showing moisture loss in agar models positioned at the five different microhabitats within Barakula State Forest during summer……………………………………………..……………………….50

Figure 4.1 Species accumulation curve showing the number of frog species captured in pitfall traps at the three survey sites within Barakula State Forest, during each sampling day..……………………………………....67

Figure 4.2. MDS plot showing similarity in species assemblages captured at each pitfall trapline at the three survey sites within Barakula State Forest...68

Figure 4.3. Scatter plot showing relationship between the number of adult frogs captured in pitfall traps at each survey site and the distance (m) from the nearest breeding pond where captures were recorded……….…..74

Figure 4.4. Mean (+/- 95%CI) capture rates of P. bibronii and mean soil pH (+/- 95%CI) at the three survey sites within Barakula State Forest..…...…82

Figure 5.1. Photo showing the enclosures used to test microhabitat choice in different frog species captured in pitfall traps at Barakula State Forest...... 94

Figure 5.2. Photo showing the four different microhabitats (vegetation, debris, leaf litter and bare) created in each enclosure to test microhabitat choice in different frog species…………………………………..…..……………...95

Figure 5.3. Pseudophryne bibronii metamorph used to test soil pH preferences in experimental chambers……………………………………………….…101

Figure 5.4. Mean (+/- 2SE) number of observed L. ornatus metamorphs per m2 and moisture levels (mean rank) in relation to distance from edge of the water body.…………………………………...... …………………….…...102

Figure 5.5. Scatter plot showing the relationship between the presence of Limnodynastes ornatus metamorphs in quadrats with varying levels of ground cover …………………………………………………………...... 103

Figure 5.6. Mean (+/- SD) showing the relationship between the average number of metamorphs/m2 observed in the habitat manipulation trials, and moisture levels in relation to distance from the water body ………....105

Figure 5.7. Mean (+/- 2SE) number of Limnodynastes ornatus metamorphs per m2 in relation to bare (1), normal cover (2) and extra cover (3) habitat categories and three moisture categories.……………...…………...... 106

Figure 6.1. Photo showing typical Pseudophryne bibronii nest where eggs are laid in direct contact with the soil…………………………………...………..114

Figure 6.2. Artificial nest sites used for translocated Pseudophryne bibronii eggs to test if soil pH influenced hatching success of embryos place in artificial nest sites within Barakula State Forest……………………………...…116

Figure 6.3. Mean (+/- 2SE) soil pH at 10 sites within Barakula State Forest where Pseudophryne bibronii are present, and 10 sites where they are absent. …………………………………………………….………….…..121

Figure 6.4. Scatterplot showing the relationship between soil pH and embryonic survival of Pseudophryne bibronii in artificial nests used in Study 3 conducted at Barakula State Forest …………………………..…....…124

Figure 6.5. Photo showing unidentified fungi on eggs in a P. bibronii nest observed at Barakula...... 125

LIST OF TABLES

Table 2.1. Mean monthly rainfall for Barakula State Forest (mm) and total monthly rainfall recorded during the study period of February 2001 – January 2004………………………...……………………………………..17

Table 2.2. Anuran species previously recorded from within Barakula State Forest and a brief description of their terrestrial and breeding habitat requirements…………………………………………………………..19

Table 2.4. Mean, minimum and maximum levels of habitat variables measured at each sampling point within the three survey sites used to assess terrestrial habitat associations in frogs within Barakula State Forest...29

Table 2.5. ANOVA results for the measured habitat variables at each sampling site that were significantly different between the three survey sites used to assess terrestrial habitat associations in frogs within Barakula State Forest……………………………………………………….………….…....33

Table 2.6. The number of sampling points at each survey site within Barakula State Forest that fell into the different ground cover categories. Also shown are the mean, and minimum and maximum percent coverage for each group of sampling points………………………….…….……...…..34

Table 2.7. The number of sampling points at each survey site within Barakula State Forest that occurred in each dominant vegetation category …..34

Table 2.8. Mean, minimum and maximum values for the measured soil properties at each of the three survey sites used to assess terrestrial habitat associations in frogs at Barakula State Forest, and overall, mean for sites combined….……………………………………………………….....35

Table 3.1. Minimum and maximum percentage moisture loss in the agar models used to simulate evaporative moisture loss in frogs, during winter and summer sampling periods in the five microhabitats at the three survey sites within Barakula State Forest……………………..………..……….47

Table 4.1. Frog species and total numbers of frogs captured in pitfall traps at the three survey sites at Barakula State Forest between February 2001 and January 2004……………………………………………..……….…..66

Table 4.2. Shows mean capture rates per survey site at Barakula State Forest and ANOVA results for site differences in total frog captures, species richness, total burrowing species and individual species per trapline/100 days…………………………………………………………...68 Table 4.3. Results of Mantel Test comparing similarity between frog capture rates and measured vegetation and soil attributes at each sampling point within Barakula State Forest. Significant correlations are in bold print…………………………………………………………………………..71

Table 4.4. Forward stepwise multiple regression results for frog species composition and habitat attributes within and between survey sites at Barakula State Forest …………………………..……………….….....….73

Table 6.1. Mean and standard deviation percent survival of Pseudophryne bibronii embryos exposed to different soil pH levels in three separate field trials conducted at Barakula State Forest, to test if soil pH influenced hatching success………………………..………………………..………123 Table 6.2. Range, Mean and standard deviation of soil pH recorded at artificial nest sites used in Study 3 conducted at Barakula State Forest, to experimentally test the influence of soil pH on hatching success of Pseudophryne bibronii embryos……………………………………..….123

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STATEMENT OF ORIGINALITY

I certify that this thesis does not incorporate without acknowledgement, any material previously submitted for a degree or diploma in any University, and that, to the best of my knowledge and belief, does not contain any material published or written by other persons, except where due reference is made in the text.

Joanne Chambers

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ACKNOWLEDGMENTS

Completion of a work of this extent would not have been possible without the assistance and support of certain persons. I would like to take this opportunity to thank the following people who contributed so much towards making the dream of achieving the honourable status of holding a PhD Degree, a possibility.

The person who deserves the most credit is my supervisor, Dr Ian Williamson. Ian’s humour and enthusiasm have kept me going, especially when continuing drought conditions caused me to make considerable and frequent changes to the direction the study was heading. His encouragement throughout this long journey has expediated the completion of this work, and his constructive criticism of the many drafts of this thesis has helped to produce a comprehensive report of the study.

Ian’s dedication to his family and work are an inspiration to me and all other students who have the pleasure of getting to know him.

Thanks are also due to my co-supervisor, the late Dr John Wilson. His vision was an inspiration, and although his specialty was rodents, he happily spent many hours walking through the bush at Barakula to gain a better understanding of how frogs operate. It was he who suggested that different microhabitats might provide greater protection against evaporative water loss, but thankfully I came up with a better methodology to achieve this (he wanted to use incontinence pads). I miss you John, and hope you would have been proud of this thesis.

Special thanks go to the wonderful guys who volunteered to assist with digging in

309 pitfall traps, Doug Harding, Pete Prentis and Dave Elmouttie. In particular, Dave has become my closest and dearest friend and has spent many a cold, wet night at

Barakula, and many a night at the local pubs, giving me the enthusiasm and

encouragement to keep going. Thanks Dave, for your good humour, you are a true friend.

I would like to thank Gary and the staff at Barakula Forestry Office for making my time there such a memorable occasion in my life. Thanks also to the staff at QUT, especially Kathy, Sean, Mark, Eilish, Janet and Tricia, who helped with field gear and processing the never-ending paperwork associated with university life.

Thanks are also due to my employer, Biodiversity Assessment and Management Pty

Ltd, for gracefully allowing me the time off work to complete this thesis. The support and encouragement of Glen Ingram, Adrian Caneris, Paulette Jones and Chris

Schell is highly valued.

Special thanks go to my family for their undying support and patience. My Mum, in particular, has provided unwavering support and her trust in my judgement throughout life gave me the motivation to commence this journey. I also thank my beautiful children, Shallon and Robert, for sacrificing so much when I left full-time employment to become a full-time student. It was difficult at times, and I really appreciate your support. In addition, I thank Shallon for encouraging me to share my love of nature with her beautiful son, Tyson.

x Introduction Chapter 1

Chapter 1: GENERAL INTRODUCTION

1.0 INTRODUCTION

A decline in amphibian populations has been observed in many parts of the world during the past two decades (Beebee et al. 1990; Crump et al. 1992; Alford and

Richards 1997; Osborne et al. 1999). The reports of amphibian declines were so recurrent and widespread that a Global Amphibian Assessment task force, consisting of almost 600 scientists in 60 countries was established in 2004 to determine the scale of the current extinction crisis in amphibians (IUCN 2006). The current (2006) results from this review show that almost one third of the world’s amphibians are listed as threatened. When compared to the number of mammal species (1 in 4) and bird species (1 in 8) listed as threatened (IUCN 2006), it appears that amphibians are suffering population declines at a greater rate than other vertebrate taxa.

Australia supports only one Order of Amphibians, namely Anurans (frogs) of which five families are represented: Hylidae (Tree Frogs), Myobatrachidae (Southern

Frogs), Microhylidae (Narrow-mouthed Frogs), Ranidae (True Frogs) and Bufonidae

(True Toads) (Barker et al. 1995). Many of these species are threatened with extinction, with 14 species listed as critically endangered on the World Conservation

Union Red List of Threatened Species (IUCN 2006). Three Australian species are listed as extinct and nine species are listed as “data deficient” which means we do not have enough information about their distribution or ecology to make an informed assessment of their status. The remaining 52 species are listed as Endangered,

Vulnerable, Threatened or Near Threatened.

Joanne Chambers Page 1 Introduction Chapter 1

Widespread reports of amphibian declines are cause for alarm as amphibians are considered by many to be indicator species (Pearson and Cassola 1992). The biphasic life history of most amphibians means they are exposed to both aquatic and terrestrial environments, and because they have highly permeable skins they may be more sensitive to a variety of environmental contaminants (Bradford et al.

1993; Blaustein et al. 1994a).

Because of their diversity and abundance, amphibians are among the most important vertebrate components of wetland ecosystems. The large potential size of both adult and larval amphibian populations has important implications for both predators and prey, and for patterns of energy transfer (Scale 1980). Through their contribution to trophic dynamics in a variety of communities, a world-wide decline in amphibians could have an important impact on other organisms (Blaustein et al.

1994b). The loss of amphibians from an area could have significant detrimental effects on the ecosystem dynamics, by altering the composition and order of predator/prey associations.

Although amphibian populations have been the subject of many experimental and monitoring studies, the autecology of most amphibians is poorly understood (Alford and Richards 1999), therefore, assigning causes to declines and losses has been difficult in many cases. Numerous hypotheses concerning possible causes of declines have been advanced: UV-B radiation (Blaustein et al 1994b), introduced species (Kiesecker et al. 2001), pollution (Sparling et al. 2001), disease (Daszak et al. 2001), and habitat loss/alteration (Pearman 1997). However, little information has been presented or published that allows many of these hypotheses to be tested.

Separation of natural from anthropogenic causes requires considerable observational and experimental evidence, which is rarely available because research is usually governed by time and monetary constraints. Amphibian behaviour varies seasonally with weather and internal rhythms of the animal (Alford

Joanne Chambers Page 2 Introduction Chapter 1 and Richards 1999). The habitats that amphibians occupy also vary seasonally, so it can be expected that amphibian population sizes may vary considerably between survey times. Long-term studies of some species indicate that natural populations regularly display significant fluctuations in population size (Pechman and Wilbur

1994; Alford and Richards 1999), therefore, it may be necessary to census populations frequently over a considerable time span (at least five years) to ensure accurate knowledge of population dynamics. To date, there have been very few long-term field studies conducted on amphibian populations, which makes it difficult to assess the population status of many amphibian species.

In spite of the difficulty in assigning causes to population declines, one causal factor is recognised as having a significant effect on not only amphibians, but also on all biodiversity: habitat loss and range contraction due to habitat fragmentation (Bibby

1995; Ehrlich 1995; Thomas and Morris 1995). In order to understand the role of habitat loss it is important to understand how habitat alteration impacts a threatened taxon.

Theory predicts there is a relationship between habitat selection by animals and improved fitness, with ideal habitats being those that provide high reproduction and growth Begon et al. (1996). Identifying the characteristics of the habitat that provide increased fitness for an animal is often compounded by the fact that animals may utilise different habitats on a spatial and temporal basis, depending on age, sex, reproduction status, and resource availability (Buskirk and Ostfeld 1998). Habitat selection occurs through a hierarchical spatial scaling process, ranging from the geographic range, the home range, specific sites within the home range and finally to how an animal utilises resources within these specific sites (Hutto 1985). The relationships between wildlife and habitats are often distinctly different at different scales (Hall et al. 1997), and without knowledge of utilisation at the landscape- and

Joanne Chambers Page 3 Introduction Chapter 1 patch-scale, possible differential habitat usage may not be adequately identified

(Martin and McComb 2003).

Amphibians typically display a complex biphasic life-cycle that involves both an aquatic and terrestrial stage. Most anuran larvae feed, grow and develop until metamorphosis in an aquatic environment, after which they migrate as juveniles to terrestrial habitats. Most species remain in the terrestrial environment until they reach reproductive maturity when they return to the breeding pond. The larval period for anurans typically lasts weeks or at the most months, but adults can live for a number or years, therefore most anurans spend the majority of their life cycle in terrestrial habitats.

Despite this fact, the focus of most research into amphibian habitat requirements has been on the aquatic phase of the amphibian life cycle. A significant amount of research has identified the preferred physical and chemical characteristics of aquatic habitats for many amphibian species (Kiesecker and Skelly 2000; Gillespie

2002; Glos et al. 2003), whilst others have studied the effects of changes in environmental parameters and amphibian declines (Blaustein et al. 1994b; Sparling et al. 2001) as well as inter- and intraspecific competition within the aquatic environment (Azevedo-Ramos et al. 1999; Orizaola and Braha 2006).

To date, there has been limited research conducted into the type and amount of terrestrial habitat required by different species in order to maintain viable populations. The presence of suitable water bodies is essential for egg and larval life history stages. However, persistence of populations is not possible without nearby areas of suitable terrestrial habitat (Semlitsch et al. 1996). Even in common species, the potential for decline due to the alteration of terrestrial habitats needs much further investigation (Dodd and Cade 1998). Without a complete knowledge of all

Joanne Chambers Page 4 Introduction Chapter 1 ecological requirements it will be difficult to successfully manage amphibian populations.

For amphibian species that do not have access to permanent water, such as flowing streams, lakes or dams, they require three distinct habitats: a breeding pond used by adults during spawning time, and tadpoles for development, terrestrial habitat for foraging and protection from exposure and predation, and suitable habitat for hibernation or aestivation during prolonged dry, hot or cold periods (Pope et al.

2000). The ability to survive until the next breeding opportunity is critical to the persistence of a population, therefore, the characteristics of the terrestrial environment may play an important role in determining the distribution and abundance of amphibian species across the landscape.

Many amphibians spend the majority of their life cycle away from water bodies and only return to “ponds” during breeding seasons (sometimes only 2 – 3 days per year). For instance, Semlitsch and Bodie (1998) found that adult salamanders spent between 86% - 99% of their annual cycle in terrestrial habitats. Studies in Australia

(Williams and Hero 2001), Britain (Denton et al. 1997), Canada (Dupuis et al. 1995),

Amazonian Ecuador (Pearman 1997) and USA (Delis et al. 1996) show that frog

species richness is related to terrestrial habitat heterogeneity. Increased structural

diversity, through its potential to provide sites that may act as refuges, is associated

with increased species richness in all of these studies.

The physiological characteristics/constraints displayed by different frog species

dictate their broad-scale distribution and their habitat choice at a finer scale. Their

occurrence in a given area is mainly determined by the need to minimise deleterious

effects caused by environmental influences (e.g. desiccation). Most amphibians are

highly susceptible to desiccation and low humidity can limit their activity (Tracy

1976, Moore and Moore 1980). Anuran species can be broadly grouped into three

Joanne Chambers Page 5 Introduction Chapter 1

assemblages depending on their associations with the breeding pond, which often

influences their requirements for prevention of desiccation. These broad groupings

are:

I. Stream-dwelling species - Stream-dwelling species usually have unrestricted

access to water, either directly by submersion in streams, or indirectly through

water spray from fast flowing streams (Hodgkison and Hero 2001). These

species minimise the risk of desiccation by remaining close to flowing water,

therefore, they may not need to utilise large amounts of terrestrial habitat to

search for appropriate retreat sites;

II. Permanent water species – Species that are associated with permanent water

bodies such as lakes, dams or other human-made water storage areas, are

most often habitat “generalists” and are usually common species that do not

possess any specific morphological/behavioural adaptation for the prevention of

desiccation (Vos and Chardon 1998). These species can usually be found

sheltering under debris within close proximity to the water body; and

III. Temporary/ephemeral water species- Species that only breed in temporary or

ephemeral water bodies need to be capable of finding refuge/retreat sites that

will allow them to survive long periods without access to water for rehydration

(Richter et al. 2001). For these species, suitable retreat sites may often be long

distances from the water body and microhabitats within the terrestrial habitat

may play an important role in influencing their distribution.

An examination of the degree to which habitat characteristics were correlated with

population demographic and genetic characteristics in the common toad (Bufo bufo),

showed the relative importance of terrestrial and aquatic environments varied in

relation to different life history stages and seasonal activities (Scribner et al. 2001).

Genetic characteristics were related to both aquatic variables within the breeding

Joanne Chambers Page 6 Introduction Chapter 1 ponds and to landscape variables, while toad presence and population size were related exclusively to the landscape and terrestrial variables. Terrestrial variables measured immediately adjacent to the pond appeared to have little predictive value for this species of toad.

The common approach for wetland conservation is to retain a terrestrial buffer zone.

However, lack of knowledge with regard to terrestrial habitat requirements for many species of amphibians means that the minimum buffer zone width (for example 10 -

50 m in Queensland, Australia, depending on stream order, and 30.8 m in USA) is often inadequate to encompass the movement of most species. To understand variation in the structure of amphibian assemblages Mitchell et al. (1997) conducted

a two year study of several montane forest stands, in different stages of regrowth.

The study showed that juvenile and adult amphibians utilised habitat up to several

hundred meters from their breeding locations and the authors suggested the

importance of maintaining suitable terrestrial habitat around aquatic breeding sites

to ensure persistence of amphibian populations. An analysis of studies concerned

with terrestrial requirements of pool-breeding amphibians showed that juveniles and

adults were found on average between 69 and 125 m from the edge of aquatic

habitats (Semlitsch 1998). A buffer zone width of 164 m was recommended, to

ensure 95% of Ambystoma salamander populations’ annual requirements were

encompassed within that zone (Semlitsch 1998).

Characteristics of the terrestrial environment surrounding breeding ponds may affect

metamorph and adult survival via their effects on food and other resources, such as

refuge and hibernation/aestivation sites (Scribner et al. 2001). While poor choice of

breeding habitat may result in a lack of breeding success in one season, the

improper choice of hibernation/aestivation habitat may be fatal (Hazell et al. 2001),

therefore, maintenance of amphibian biodiversity and abundance depends on the

Joanne Chambers Page 7 Introduction Chapter 1 protection and management of both aquatic breeding sites and the surrounding terrestrial habitat.

Until more research is directed towards gaining information on terrestrial habitat requirements, we may not be in the position to provide adequate management for anuran populations. For example, a recovery program for natterjack toad (Bufo calamita) in Britain highlighted the need to take into consideration all aspects of a species’ ecological requirements to ensure conservation plans are successful

(Denton et al. 1997). Extensive autecological studies identified aquatic and terrestrial habitat features of critical importance to natterjacks and 26 ponds were created in an effort to enhance population sizes. However, at more than half the sites where pools had been created, there was no evidence of a significant change in toad abundance and insufficient management of terrestrial habitat has been cited as the reason these ponds were not colonised. To ensure we have all the relevant information needed to successfully manage amphibian populations, it is critical that we improve our understanding of terrestrial habitat requirements at each life-history stage and threats that reduce the quality or quantity of terrestrial habitat around wetlands should be identified.

In Australia there is a paucity of studies that have identified specific terrestrial habitat requirements of anurans. Those that have recognised the importance of this environment for persistence of populations have focused their research on stream- dwelling species (McDonald, 1992, Parris, 1999), with little or no research into terrestrial habitat requirements of ephemeral breeders or burrowing species. In addition few studies have focused on Australian species and their mechanisms to cope with climatically unpredictable environments. This is despite the fact that one third of Australian species are morphologically equipped to burrow so they can avoid desiccation and freezing and many Australian species are explosive breeders that may need to survive long periods without coming into contact with standing water.

Joanne Chambers Page 8 Introduction Chapter 1

The semi-arid and arid regions of Queensland experience intermittent rainfall patterns that are usually governed by summer monsoonal rains occurring in northern Australia. Seasonal droughts are common in most of inland Australia

(Linacre and Hobbs 1977). Terrestrial retreat sites for frogs in these areas may be particularly important as individuals often need to withstand extreme environmental conditions in appropriate microclimates for long periods (Tracey 1976, Pough et al.

1983).

The occurrence of rapid declines in amphibians from unknown causes leaves biologists initially with very few options for action. This is even more difficult when base-line data on population distribution and abundance, or even the most basic demographic data have not been obtained (Mahony et al. 1999). The debate as to

whether the population declines observed in amphibians represent temporary

fluctuations (Blaustein et al. 1994; Pechmann and Wilbur 1994) or are real

catastrophic declines (IUCN 2006) will continue until more long-term population

monitoring is conducted and an understanding of species demography is attained.

Both intensive field studies at a few sites and extensive multi-site monitoring will be

needed to identify most of the habitat variables important to anurans. Knowledge of

broad habitat associations is critical if we hope to reverse declines in amphibian

populations (Knutson et al. 1999). It is inconceivable to think that we can

successfully manage anuran populations if we do not possess the knowledge of how

much, or what type, of terrestrial habitat different species utilise away from the

breeding habitat.

1.2 PROJECT AIMS

There is a lack of knowledge about the role terrestrial habitats have in determining species abundance. This is particularly true for species that utilise temporary-water for breeding. While restoration of potential breeding sites is imperative, it is possible

Joanne Chambers Page 9 Introduction Chapter 1 that conservation effort could be wasted if we ignore the importance of terrestrial habitat surrounding breeding sites. In particular we need information on the preferred microhabitat and the amount of terrestrial habitat different species utilise to complete their life cycle. If we are serious about reversing the population declines of any species we must gain an understanding of their entire ecological requirements.

The present study has been designed to fill in the knowledge gaps that currently exist in Australia regarding terrestrial habitat requirements for a number of endemic frog species. For the purpose of this study, terrestrial habitat utilisation is restricted to the fine-scale usage within the animals’ home range. This project will focus on frogs that inhabit open woodlands in a semi-arid region of Queensland. For these species, the microhabitats within the terrestrial environment are likely to be especially important for providing suitable refuge sites during long dry periods. This study will provide information where frogs are found within the terrestrial environment in an open woodland area, and on whether different frog species favoured or avoided particular types of terrestrial habitat.

The proposed study area contains 21 species, ~ 10% of Australian anuran species

(Barker et al. 1995), and these species display significant interspecies variation in body size, therefore, it is expected that different species will have distinct terrestrial habitat requirements relating to morphology and behaviour.

To date, there have been no other reported studies of this kind in Australia, therefore, the study will provide important ecological data on the habitat requirements of a number of endemic frog species. The study has been designed to:

• Quantify the terrestrial microhabitat characteristics within a large area of open

woodland, to identify the level of heterogeneity that exits at a fine-scale;

Joanne Chambers Page 10 Introduction Chapter 1

• Identify the terrestrial microhabitat components that provide efficient protection

from desiccation; the variable considered most important for habitat choice in

frogs that do not have access to permanent water;

• Examine the diversity and distribution of anuran species occupying open

woodland habitats in a semi-arid region of South-east Queensland;

• Identify whether different frog species utilise the quantified terrestrial

microhabitats at varying degrees; and

• Qualify the reasons why particular terrestrial habitat preferences prevail.

1.3 SUMMARY OF THESIS

This thesis consists of seven chapters, three of which are experimental that were designed to investigate how terrestrial microhabitats influence evaporative water loss, refuge site selection and embryonic survival. In Chapter 1 I have reviewed the literature on amphibian declines and amphibian terrestrial habitat requirements.

Chapter 2 will quantify the terrestrial microhabitat attributes within three study sites, each occupying approximately 6.5 ha. I have used agar moulds, as replicates for live frogs, in Chapter 3 to determine which microhabitats provide the greatest protection against evaporative water loss. These two chapters were designed to provide a background to the study area and to allow identification of terrestrial microhabitat variations that are predicted to be utilised advantageously by different frog species.

In Chapter 4 the distribution and activity patterns of the anuran assemblage within the study area was investigated. This is followed by an assessment of associations between capture rates and the measured terrestrial microhabitats as defined in

Chapter 2.

Joanne Chambers Page 11 Introduction Chapter 1

Newly emerged metamorphs and adult frogs were used in Chapter 5 to experimentally test the observed microhabitat associations from Chapter 4 and to investigate terrestrial habitat choice. In Chapter 6 I investigate how one of the measured habitat attributes influences the hatching success of Pseudophryne bibronii embryos (a version of this Chapter has been published (Chambers et al.

2006)). In the final chapter (Chapter 7) I provide an overview of the results and a general discussion within the context of current knowledge of anuran terrestrial habitat requirements.

Joanne Chambers Page 12 Terrestrial Habitat Assessment Chapter 2

Chapter 2: TERRESTRIAL HABITAT ASSESSMENT

2.0 INTRODUCTION

The broad distribution of a species is limited by its tolerance limits to environmental factors such as altitude, temperature, rainfall, the physical structure of the habitat and the availability of resources, such as nesting and shelter sites and food availability. Broad scale studies can identify the environmental parameters that most likely predict the geographic distribution of a species. However, within that broad range, other biotic or abiotic conditions may produce further spatial structuring of a species distribution and many species display a patchy distribution within their known range. This patchy distribution is often a result of habitat heterogeneity that is influenced by topography, aspect, hydrological regimes, or geology. For example

Beckett and Webster (1971) showed that the relative level of variation in soil parameters within one square meter was often half or more of the total range of variation within the entire agricultural field. The small-scale heterogeneity, which is often present in an area could influence the distribution of plant and animal species, with the presence of one species often influencing the presence of others (Ceballos

et al. 1999, Brose 2001).

Habitat diversity or habitat complexity represents spatial levels of structural

complexity of vegetation, plant species composition, connectivity and heterogeneity

and is recognised as having an important influence on biodiversity (Lindenmayer et

al. 2000). Research on birds (Mac Nally et al. 2000), invertebrates (Siemann et al.

1998), reptiles (Fox and Fox 2000), small mammals (Kotler and Brown 1988), and amphibians (deMaynadier and Hunter 1999; Halverson et al. 2003; Semlitsch and

Bodie 2003), has shown that habitat structural complexity, provided mainly by

plants, is often strongly correlated with animal species diversity.

Joanne Chambers Page 13 Terrestrial Habitat Assessment Chapter 2

This association may be explained by the recognition that plants provide different microhabitats or substrates that offer a large variety of resources and conditions that can be utilised by different animal species. Variations in understorey and overstorey structure and floristics within a habitat lead to stratification of the environment (e.g, change in light penetration, humidity, etc.) and hence stratification in faunal communities ( number of ground dwellers, arboreal dwellers).

Variation in microhabitat conditions may promote species diversity by enabling coexistence of different plant species, which in turn can promote increased animal species diversity. Biologically generated patterns, such as stands of Acacia that host nitrogen-fixing bacteria, create spatially heterogeneous environments by modifying the chemical, physical and biological properties of soils (Franklin et al. 2000), which may then influence the distribution of other species of plants and animals, therefore, within the broad range of a species it is often a suite of microhabitat attributes that will determine whether a species is present or absent from a site.

Assemblages of pond-breeding frogs are usually spatially aggregated due to their dependence on water for breeding (Johnson and Semlitsch 2003). However, different species within that assemblage may require varying amounts and types of terrestrial habitat to ensure persistence at the pond across multiple breeding seasons (Williams and Hero 2001). It is often necessary to look at microhabitat conditions of breeding habitat and complimentary terrestrial habitat, rather than landscape parameters to gain an understanding of which features facilitate or inhibit the local distribution of a species (Pope et al. 2000). For example, Parris and

McCarthy (1999) showed that understorey vegetation composition surrounding streams, was a strong environmental predictor for the presence of many frog species in the forest streams in southeast Queensland, whereas broad forest type showed no significant influence on frog assemblages in that area. The availability and use of suitable terrestrial microhabitat is important because

Joanne Chambers Page 14 Terrestrial Habitat Assessment Chapter 2

incorrect choice of terrestrial habitat may lead to a reduction in fitness caused by increased risk of predation (Azevedo-Ramos et al. 1999; Bronmark and Edenhamn

1993; Kats et al. 1992), or desiccation (Cohen and Alford 1996; Schwarzkopf and

Alford 1996), or reduced foraging success (Scribner et al. 2001).

Soil moisture appears to be a major source of water for some amphibian species

(Shoemaker and Nagy 1977). Many of the species inhabiting semi-arid regions

burrow to avoid predation during daylight, desiccation during summer, or freezing

during winter, causing these species to be in direct contact with the substrate for

long periods. Therefore, properties of soil may influence where frogs choose to

burrow.

Many of the frog species in semi-arid and arid habitats do not have access to

permanent water, and it is hypothesised that variations within the terrestrial habitat

will play a major role in influencing their distribution.

The aim of this chapter is to describe the heterogeneity of the terrestrial habitat

surrounding ephemeral frog breeding areas in a semi-arid region of Queensland.

Variation in habitat attributes that are considered most likely to influence frog

distribution, were quantified at three sites to assess levels of habitat heterogeneity

between and within each site. The focus was placed on ground cover attributes and

soil properties, as these factors are most likely to be important for ground-dwelling

and burrowing frog species in this area (Tyler 1994).

2.1 METHODS

2.1.1 STUDY AREA

In order to examine the role that terrestrial microhabitats play in influencing the

distribution of frogs, the study area needed to display the following characteristics:

Joanne Chambers Page 15 Terrestrial Habitat Assessment Chapter 2

• An extensive area of habitat to allow the placement of three survey sites to

ensure sufficient replication could be established. These survey sites would

need to be separated by at least 2 – 3 km to ensure independence of data

collection.

• A continuous area of habitat that was not impacted by fragmentation or large

and/or frequent disturbances. This was deemed important because it is

necessary to minimise any external factors that may influence the behaviour and

composition of assemblages of frogs within an area.

• There should be variation in vegetation and ground layer (i.e. potential

microhabitat variation) within survey sites, but the broad vegetation composition,

i.e., Regional Ecosystem, and topography, would need to be similar across all

survey sites to ensure similar variability between sites.

• The study area would need to be in a relatively dry region and the survey sites

would need to be positioned away from permanent water sources, such as

creeks and damp drainage lines, to ensure the terrestrial habitat was the

dominant feature likely to influence the distribution and movement patterns of

frogs.

This research was conducted within the Barakula State Forest, a large area of continuous forest (265,000 ha) situated approximately 350 km west of Brisbane,

Queensland (Figure 2.1). The region is described as semi-arid (Dalal et al. 2003) and has an average annual rainfall of 660 mm, the majority of which falls in late summer. The area has a sub-tropical climate, with hot moist summers and cold dry winters (Halford 1995). Annual temperatures range from average winter minimum of

4oC to an average summer maximum of 34oC.

Joanne Chambers Page 16 Terrestrial Habitat Assessment Chapter 2

Figure 2.1. The position of Barakula State Forest in eastern Australia (a) and a satellite photo of Barakula State Forest showing the continuous vegetation cover in the 265,000 ha area (b).

During the current study winter temperatures often fell below 0oC and summer temperatures regularly exceeded 40oC. Average rainfall for the 64 years prior to this

Joanne Chambers Page 17 Terrestrial Habitat Assessment Chapter 2

study is presented in Table 2.1, together with the recorded rainfall during the current study period. It can be seen that rainfall in the first two years of the study was considerably less than the average for the preceding 60 years, but rainfall was slightly higher than average in 2003. However, on closer inspection of the data, it can be seen that during the study period (January 2001 to February 2004) summer rainfall was lower than the average, particularly in the first two years of the study.

Lack of summer rainfall had the greatest influence on the captures of frogs, as most species reduce their activity once average night time temperatures begin to fall. Also during the study period summer storms that usually fill the ephemeral breeding sites, were infrequent and when they did occur follow-up rain rarely eventuated, which meant that the duration of peak frog activity was usually only two-three days.

Table 2.1. Mean monthly rainfall for Barakula State Forest (mm) and total monthly rainfall recorded during the study period of February 2001 – January 2004. Note: summer rainfall includes December from the previous year. Season Month Av. 64years 2001 2002 2003 2004 Summer December 95.1 34 61 84 164 Summer January 91.9 18.2 37 47 52.2 Summer February 89.1 36.0 66.2 94.6 32.8 Autumn March 54.6 114.3 57 134 Autumn April 38.3 53.2 0.6 106 Autumn May 39.0 10.4 23.4 14.2 Winter June 34.4 0.4 78.2 39.4 Winter July 35.6 33.6 2 13.6 Winter August 29.5 8.2 56.6 66.8 Spring September 29.5 28.2 2.7 3 Spring October 64.1 58 45 83.8 Spring November 73.4 145.8 7.2 9 Total Rainfall 674.5 540.5 437.1 695.8

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Within the study area there is limited variation in the topography, with only a few minor hills occurring throughout the forest. Thirteen different, sometimes overlapping soil types have been identified within Barakula State Forest (Maher

1996). These soils are described as deep loamy soils over a sandy clay subsoil, that are infertile and poorly drained (DEH 2006). The forest is dominated by tall

Eucalyptus-Callitris woodlands, and mixed woodland forest. The mid and lower stratum consists of numerous Acacia sp., Allocasuarina sp. and Callitris glaucophyla and C. endlicheri. The ground layer consists of grasses, sedges and numerous herbaceous plants.

Despite the relatively low rainfall and fairly extreme temperature range, the forest supports 21 species of endemic frogs (Mason 1997) and the introduced cane toad

(Table 2.2).

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Table 2.2. Anuran species previously recorded from within Barakula State Forest and a brief description of their terrestrial and breeding habitat requirements. (Nomenclature follows Ingram et al. (2002). Habitats as noted in Barker et al. (1995) and Anstis (2002)). Scientific Name Common Name Habit Terrestrial Habitat Breeding Habitat Bufo marinus Cane Toad Terrestrial Occurs in a wide range of Ephemeral and habitats permanent water Crinia Beeping Froglet Terrestrial Usually associated with Ephemeral and parinsignifera grassy areas near temporary water temporary or semi- permanent water Cyclorana Greenstripe Frog Burrowing Woodlands, cleared lands Ephemeral and alboguttata and drier forested areas permanent water C. brevipes Superb Collared Burrowing Open grassland and lightly ephemeral Frog forested areas C. novaehollandiae Eastern Burrowing Usually associated with ephemeral Snapping Frog black soil plains and flooded plains C. platycephala Water-holding Burrowing Grasslands, temporary ephemeral Frog swamps, claypans C. verrucosus Rough Collared Burrowing Open grasslands ephemeral Frog Limnodynastes Barking Frog Terrestrial Usually in association with Ephemeral and fletcheri rivers and lakes permanent water L. ornatus Ornate Burrowing Burrowing Occurs in a variety of All types Frog habitats L. salmini Salmon-striped Burrowing Restricted to semi-arid Ephemeral and Frog regions, usually in permanent water association with dams and flooded grasslands L. tasmaniensis Spotted Terrestrial Widespread usually in Ephemeral and Grassfrog association with grass-lined permanent water streams and flooded paddocks L. terraereginae Scarlet-sided Burrowing Prefers permanent water Ephemeral and Pobblebonk where there is adequate permanent water vegetation cover Litoria caerulea Common Green Arboreal Widespread. Usually breeds Ephemeral and Treefrog in temporary water permanent water L. fallax Eastern Arboreal Common species usually All types Sedgefrog associated with coastal areas L. latopalmata Broad-palmed Terrestrial Associated with temporary Ephemeral and Rocketfrog or permanent waters near permanent water open forest L. peronii Emerald-spotted Arboreal Associated with a variety of Ephemeral and Treefrog habitats including highly permanent water disturbed areas. L. rubella Ruddy Treefrog Terrestrial Inhabits a variety of regions Ephemeral and from coastal to desert areas permanent water L. verreauxii Whistling Arboreal Associated with swamps, Ephemeral and Treefrog lagoons and creeks in permanent water woodland, forest, farmland and heathland Neobatrachus Meowing Frog Burrowing Inhabits woodlands, Ephemeral water sudelli grasslands and shrubland

Notaden bennetti Holy Cross Frog Burrowing Mainly associated with black Ephemeral water soil flood plains which are often topped with red sand Pseudophryne Bibron’s Toadlet Terrestrial Once common in forest, Ephemeral water bibronii heath and cleared lands, now less frequently observed Uperoleia rugosa Chubby Gungan Burrowing Grassy plains usually in Ephemeral water association with temporary and semi-permanent water bodies

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Within Barakula State Forest there is considerable variation in vegetation communities, and this variation is expected to have an influence on the structure and composition of the ground layer.

Three survey sites were selected on the basis of having similar levels of variation in vegetation and therefore, variation in ground layer, but also having similar types of vegetation composition represented at each site (as defined by Regional Ecosystem

Mapping (EPA 2007) (Figure 2.2).

Figure 2.2. A section from the Regional Ecosystem Mapping for Barakula State Forest showing the location of the three survey sites (red squares) in relation to the regional ecosystem types.

Sites also contained a frog breeding area (both ephemeral in Sites 2 and 3, or temporary ponds and a simple earth dam without emergent vegetation or vegetation around the margins in Site 1). Sites were all approximately 350 m above sea level and there was minimal topographic variation between the three sites (Site 1 = 346m

Joanne Chambers Page 21 Terrestrial Habitat Assessment Chapter 2

ASL, Site 2 = 347m ASL, Site 3 = 357m ASL). Sites were separated by at least 5 km from each other and were positioned within areas of continuous forest (Figure 2.3).

The frog breeding areas were not the dominant feature of the sites (Figure 2.4).

Figure 2.3. Aerial photograph showing the position of the survey sites used to study terrestrial habitat associations in frogs within Barakula State Forest.

Joanne Chambers Page 22 Terrestrial Habitat Assessment Chapter 2

Figure 2.4. A typical ephemeral frog breeding site following a rainfall event within Barakula State Forest.

Four broad vegetation types were represented at each site (Figures 2.5 – 2.8).

Where tall eucalypts dominated a section of a study site, the ground layer contained a covering of grasses (Figure 2.5), but where Acacias were the dominant plant species the ground had a thick layer of leaf litter (Figure 2.6). Callitris sp. often grew in very dense stands which caused overcrowding and ultimately death to many trees. In sections within a study area where this occurred, the ground was covered in logs and branches of varying sizes (Figure 2.7). In some of the lower-lying sections of the survey sites the ground layer was dominated by a covering of sedges

(Figure 2.8).

Joanne Chambers Page 23 Terrestrial Habitat Assessment Chapter 2

Figure 2.5. Photo showing areas within the survey sites at Barakula State Forest, where ground cover was dominated by grasses.

Figure 2.6. Photo showing dense cover of leaf litter that occurred in areas within Barakula State Forest that supported a dense growth of Acacia.

Joanne Chambers Page 24 Terrestrial Habitat Assessment Chapter 2

Figure 2.7. In areas within Barakula State Forest where a dense growth of Callitris sp. occurred, the ground layer contained large amounts of downed timber.

Figure 2.8. Photo showing ground layer of sedges that occurred in some low-lying areas of the three survey sites within Barakula State Forest.

Joanne Chambers Page 25 Terrestrial Habitat Assessment Chapter 2

2.1.2 TERRESTRIAL HABITAT ASSESSMENT

2.1.2.1 Ground Cover and Vegetation Structure

Habitat structure and complexity was quantified during March 2002 in a 250 m x 250 m area at each survey site. Thirty-six sampling points were positioned 50 m apart in a 6 x 6 grid at each survey site. Sampling points corresponded to the centre of 10 m drift fence arrays used to sample frogs (see Chapter 4). At each sampling point a

25m tape was laid out along a randomly generated compass bearing and then in the reverse direction of the compass bearing. At 2 m intervals along this transect, the form of structure (bare, grass, shrub, sedge, leaf litter, debris) the tape touched was recorded. If the tape touched leaf litter, the depth of litter was also recorded. This procedure was repeated using a different randomly selected compass bearing, giving 52 recordings of ground cover per sampling point. These data were pooled and converted to give estimates of percent coverage of each variable within 25 m of the sampling points.

The microhabitat conditions that may play a role in influencing frog distribution can also be affected by the amount of light penetration reaching the ground layer. The structure and density of vegetation can influence ground temperatures and moisture regimes. To ascertain variation in habitat structure, all plants in height classes of 0-1 m, 1-3 m and >3 m were recorded in two 50 m x 2 m belt transects that followed the original transects. The number of logs <10cm and >10cm in diameter were also counted in this transect. The variables measured for terrestrial habitat assessment are listed in Table 2.3.

Prior to measuring the habitat attributes, each sampling point was subjectively designated to a broad ground cover classification and dominant vegetation category based on the observed nature of the habitat surrounding the sampling point. These being:

Joanne Chambers Page 26 Terrestrial Habitat Assessment Chapter 2

Ground cover categories Dominant Vegetation Categories

1 = logs 1 = Acacia 2 = grass 2 = Cypress 3 = leaf litter 3 = Eucalypt 4 = sedge 4 = Leucopodum 5 = Mixed

Table 2.3. Summary of variables used to measure terrestrial habitat structure at the 36 sampling points within the three survey sites used for the study of terrestrial habitat associations in frogs within Barakula State Forest. Variables marked with * were converted to number per 50m2. Ground Cover Attributes Structure Bare ground (%) Number of plants in 0-1 m height class * Grass cover (%) Number of plants in 1-3 m height class * Sedge cover (%) Number of plants in >3 m height class * Herbaceous plant cover (%) Leaf litter cover (%) Leaf litter depth (cm) Debris cover (sticks or logs) (%) Number of logs < 10cm in diameter * Number of logs > 10cm in diameter *

2.1.2.2 Soil Properties

To ascertain the variation in soil properties within a site, approximately 50 g soil samples were collected from areas within 5 m of each sampling point. As it is not known to what depth different frog species burrow, soil samples were collected from three depths considered appropriate for comparisons of soil properties that may influence retreat site choice for burrowing species. A 10 cm diameter auger was used to extract soil from 15 cm, 30cm and 45 cm depths at each sampling point.

Each individual sample was labelled and sealed in a snap-lock plastic bag and transported to the university laboratory for analysis of moisture content, physical structure and pH.

Moisture content was measured by filling a small aluminium container with soil and accurately weighing the sample before and after drying in a 60oC oven for 48 hours.

Joanne Chambers Page 27 Terrestrial Habitat Assessment Chapter 2

The difference in weight is classified as “moisture received” (Rowel 1994) and allows comparisons to be made between moisture content of soils at different sampling points at the time of sampling.

Soil texture is reflected in the mechanical analysis of soil in terms of clay and sand

(Murphy 1991). Soil samples were classified into six texture groups with increasing proportions of fine particles as follows:

1 = sand 4 = light clay

2 = sandy loams 5 = heavy clay

3 = loams

The stoniness of samples was recorded using a ranking system, from no stones (0) to very stony (6).

To ascertain if pH varied between soils at different sampling points the methodology recommended by Rowel (1994) was utilised. This procedure uses 10g of air-dried soil to which 25ml of deionised water is added. The solute was placed in a screw top plastic jar and agitated for 15 minutes, after which time the pH of the solute was recorded, using a glass electrode probe.

2.2 DATA ANALYSIS

2.2.1 HABITAT ASSESSMENT

All measured variables were pooled for each sampling point to give a portrayal of the habitat structure within a 25m radius of the sampling point. Ground cover attributes were converted to percent coverage and arcsine transformed before analysis and counts (logs and trees) were log10 transformed. The 12 variables were used to construct a site-by-site similarity matrix using the Bray-Curtis index (Greig-

Smith 1983). Multi Dimension Scaling (MDS) produces a graphical representation of the similarities of sites based on the ground cover and structure: more similar sites

Joanne Chambers Page 28 Terrestrial Habitat Assessment Chapter 2

are closer together in ordination space and have a stress value >0.15, whilst more dissimilar ones further apart, with a lower stress value.

Analysis of Variance (ANOVA) was performed to allow identification of specific variables that differed within and between sites.

2.3 RESULTS

2.3.1 HABITAT ASSESSMENT

There was considerable variation in vegetation and ground cover attributes within

and between the three sites (Table 2.4 and Figure 2.9 and 2.10). For example,

percent coverage of bare and grass ranged from 0 at some sampling points to 69%

and percent cover of leaf litter ranged from 1 to 86%. The number of small logs at a

sampling point ranged from 8 to 83 and the number of trees in the 1 – 3m height

class ranged from 0 to 78 at a sampling point at Site 3. The variance in habitat

attributes was higher within a site than between sites (Table 2.4).

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Table 2.4. Mean, minimum and maximum percentage cover of habitat variables measured at each sampling point within the three survey sites used to assess terrestrial habitat associations in frogs within Barakula State Forest. Densities of logs and trees are in numbers per 200m2. Measured Site 1 Site 2 Site 3 Overall, Variable mean (min-max) mean (min-max) mean (min-max) mean Bare soil % 12.4 (0-52) 13.9 (0-69) 19.4 (0-52) 15.3 Grass % 16.5 (0-69) 23 (2-42) 7.95 (0-32) 15.7 Shrub % 1.9 (0– 1) 1.8 (0-5) 3.74 (0-17) 2.5 Sedge % 7.2 (0-19) 5.6 (0-25) 9.14 (0-36) 7.3 Leaf % 50.9 (15-77) 38.5 (1-63) 46.4 (9-86) 45.3 Debris % 11.05 (0-19) 16.4 (7-36) 12.6 (0-32) 13.3 Leaf depth 1.18 (0.5-2) 1.01 (0.5-2) 1.24 (0.6-9) 1.15 cm Logs <10cm 42 .2 (8-67) 40.4 (9-83) 28.35 (8-50) 36.91 Logs >10cm 9.95 (4-25) 9.2 (2-24) 7.8 (2-13) 8.97 Trees <1m 4.04 (1-13) 5.06 (0-15) 4.71 (1-28) 4.6 Trees 1-3m 8.73 (0-50) 2.98 (0-6) 12.21 (1-78) 8.01 Trees >3m 8.93 (3-23) 7.79 (1-20) 7.43 (2-19) 8.1

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25 Mean = 15.3317 30 Std. Dev. = 13.68195 Mean = 15.7374 N = 109 Std. Dev. = 12.61367 N = 109

25 20

15 Frequency Frequency 10

5 5

0 0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 bare grass

Mean = 2.5051 40 Std. Dev. = 2.99025 50 Mean = 7.3571 N = 109 Std. Dev. = 7.70546 N = 109

40 30

20 Frequency Frequency 20

10 10

0 0 0.00 5.00 10.00 15.00 20.00 0.00 10.00 20.00 30.00 40.00 shrub sedge

20 Mean = 45.342 25 Mean = 13.3712 Std. Dev. = 15.53144 Std. Dev. = 7.06038 N = 109 N = 109

20 15

10 Frequency Frequency 10

5 5

0 0 0.00 20.00 40.00 60.00 80.00 100.00 0.00 10.00 20.00 30.00 40.00 leaf debris

Figure 2.9. Histograms showing variations in percentage ground cover variables measured at the 36 sampling points within the three survey sites at Barakula State Forest.

Joanne Chambers Page 31 Terrestrial Habitat Assessment Chapter 2

Mean = 36.9169 80 20 Mean = 1.1607 Std. Dev. = 15.50796 Std. Dev. = 0.88308 N = 109 N = 109

60 15

40 10 Frequency Frequency

20 5

0 0 0.00 2.00 4.00 6.00 8.00 10.00 0.00 20.00 40.00 60.00 80.00 100.00 leaf depth (cm) log <10cm wide

Mean = 8.9799 25 50 Mean = 4.6089 Std. Dev. = 4.31123 Std. Dev. = 3.84016 N = 109 N = 109

20 40

15 30 Frequency Frequency 10 20

5 10

0 0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 logs >10cm wide treed <1m high

Mean = 8.0189 70 20 Mean = 8.1055 Std. Dev. = 12.45582 Std. Dev. = 4.22409 N = 109 N = 109

15 50

Frequency 30 Frequency

20 5

0 0 0.00 20.00 40.00 60.00 80.00 0.00 5.00 10.00 15.00 20.00 25.00 trees 1-3m high trees >3m high

Figure 2.10. Histograms showing variations in vegetation structural attributes measured at the 36 sampling points within the three survey sites at Barakula State Forest.

Joanne Chambers Page 32 Terrestrial Habitat Assessment Chapter 2

Despite the broad range in values for many of the measured habitat variables, the

MDS plot conducted on a Bray Curtis similarity matrix shows that the three survey sites are similar in vegetation structure and composition (Figure 2.11). Most sampling points are grouped closely together and the relatively low stress value of

0.18 indicates that the three survey sites are somewhat similar as defined by the measured habitat variables, although some variation is evident between the three sites.

Figure 2.11. Multi Dimensional Plot showing similarity of the three survey sites within Barakula State Forest in relation to the measured vegetation attributes.

One way ANOVAs conducted on the habitat variables showed that there were significant differences in six of the measured variables between the three survey sites (Table 2.5), with all but one of the differences occurring in ground cover attributes.

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Table 2.5. ANOVA results for the measured habitat variables at each sampling site that were significantly different between the three survey sites used to assess terrestrial habitat associations in frogs within Barakula State Forest. (Bold = significant p value). See Table 2.4 for means. Habitat Variable F df P value Tukey Post Hoc Results Grass 23.12 2 <0.001 Sites 1-2 p = 0.33 Sites 1-3 p<0.001 Sites 2-3 p<0.001

Shrub 3.45 2 0.035 Sites 1-2 p = 0.888 Sites 1-3 p = 0.117 Sites 2-3 p = 0.040

Leaf 6.22 2 0.003 Sites 1-2 p = 0.002 Sites 1-3 p = 0.468 Sites 2-3 p = 0.059

Debris 5.49 2 0.005 Sites 1-2 p = 0.007 Sites 1-3 p = 0.854 Sites 2-3 p = 0.030

Logs <10 cm 9.51 2 <0.001 Sites 1-2 p = 0.740 Sites 1-3 p<0.001 Sites 2-3 p = 0.003

Trees 1-3 m 0.71 2 <0.001 Sites 1-2 p = 0.001 Sites 1-3 p = 0.990 Sites 2-3 p<0.001

Examination of the grouped ground cover variable within each survey site showed that sites had varying numbers of sampling points within each ground cover category (see page 25; logs, grass, leaf litter, sedge) (Table 2.6), but overall, the three sites contained similar numbers of sampling points supporting the predetermined ground cover groupings. However, Site 2 had considerably more sampling points situated in grassy areas and fewer sampling points dominated by leaf litter coverage.

The number of sampling points within the dominant vegetation categories (see page

25) also varied between sites (Table 2.7), with Site 1 supporting less Acacia sites but more Cypress sites, Site 2 contained more sampling points within Acacia

Joanne Chambers Page 34 Terrestrial Habitat Assessment Chapter 2

dominated areas and Site 3 had more areas supporting the densely-growing

Leucopodum sp. and less Eucalyptus sites.

Table 2.6. The number of sampling points at each survey site within Barakula State Forest that fell into four different ground cover categories. Also shown are the mean, and minimum and maximum percent coverage for each group of sampling points. Ground Cover Site 1 Site 2 Site 3 Logs No. of sampling points 8 7 8 Min - Max (3.1-4.2) (3.6-4.3) (1.4-2.7) Site Mean 3.7 3.6 3.2 Grass No. of sampling points 9 16 8 Min - Max (22-56%) (16-41%) (20-34%) Site Mean 24% 28% 12% Leaf No. of sampling points 14 8 13 Min - Max (37-55%) (35-53%) (41-68%) Site Mean 45% 38% 43% Sedge No. of sampling points 5 5 8 Min - Max (14-25%) (20-30%) (18-48%) Site Mean 14% 12% 15%

Table 2.7. The number of sampling points at each survey site within Barakula State Forest that occurred in each dominant vegetation category. No. of sampling points Dominant vegetation Site 1 Site 2 Site 3 Acacia 3 16 14 Cypress 23 9 15 Eucalypt 8 4 1 Leucopodum 1 0 5 Mixed* 1 7 2 *Mixed = Eucalypt, Cypress and Acacia.

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2.3.2 SOIL PROPERTIES

There was some variation in the measured soil properties between the three depths and between the three survey sites (Table 2.8). Percent moisture content across the three survey sites ranged from almost totally dry (4%) to relatively moist (26%).

Moisture tended to increase with depth. The ranked stoniness of the soil ranged from no stones (0) to soils containing large amounts of stones (6) and the amount of stones increased with depth. Texture ranged from sandy to heavy clay (rank 1-6) with soils tending towards higher clay content with depth. Soil pH ranged from very acidic (3.9) to almost neutral (6.4) and except for Site 2, pH tended to become less acidic with depth.

Table 2.8. Mean, minimum and maximum values for the measured soil properties at each of the three survey sites used to assess terrestrial habitat associations in frogs at Barakula State Forest, and overall, mean for sites combined. Site Site 1 mean (min-max) Site 2 mean (min-max) Site 3 mean (min-max) Depth Depth Depth Depth Depth Depth Depth Depth Depth Depth 1 2 3 1 2 3 1 2 3

Moisture 13.5% 14.9% 16.0% 13.1% 15.2% 16.7% 13.2% 14.7% 17.0% (4-21) (4-26) (6-28) (8-18) (11-25) (8-23) (10-17) (10-26) (11-26) Overall, mean 13.3% 14.6% 16.6%

Stoniness 1.3 2.0 2.5 0.9 1.2 1.7 0.9 2.0 2.9 (0-3) (0-4) (0-6) (0-3) (0-5) (0-6) (0-3) (0-6) (0-6) Overall, mean 1.1 1.7 2.4

Texture 2.9 3.5 3.9 3.0 3.5 3.8 2.8 3.3 3.7 (2-5) (2-6) (2-6) (2-5) (2-6) (2-6) (2-4) (2-6) (1-6) Overall, mean 2.9 3.4 3.8

5.2 5.2 5.3 4.8 4.6 4.7 5.1 5.3 5.6 pH (4.1- (4.3- (4.6- (4.3- (3.9- (4.1- (3.9-6.1) (4.7- (5.1- 6.4) 6.1) 6.2) 5.9) 5.5) 5.7) 5.9) 6.2) Overall, mean 5.0 5.0 5.2

Joanne Chambers Page 36 Terrestrial Habitat Assessment Chapter 2

An MDS plot conducted on a Bray Curtis similarity matrix indicated there was slight variation in measured soil properties between the three survey sites (Figure 2.12).

Most sampling points are grouped closely together and the relatively low stress value of 0.19 indicates that the three survey sites are similar as defined by the measured habitat variables.

Stress: 0.19 Site

Figure 2.12. MDS plot showing similarity of the three survey sites in relation to measured soil properties collected from each sampling point at the three sites within Barakula State Forest.

One way ANOVA showed that the three sites were similar in mean moisture content, and mean texture (Mean moisture F2,104 = 0.047, p = 0.954; Mean texture F2,105

=0.53, p = 0.56). However, there were differences in mean stoniness and mean pH

(Mean stoniness F2,105 = 4.77, p = 0.01; Mean pH F2,104 = 32.03, p<0.001). Tukey

Post hoc tests showed that in both stoniness and pH, Site 2 was significantly

different from the other two sites (stoniness vs Site 1 p = 0.026, vs Site 3 p = 0.021;

pH vs Site 1 p <0.001, vs Site 3 p<0.001), with Site 2 having significantly lower

amounts of stones and significantly lower pH (Table 2.9).

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A Mantel Test, comparing Bray Curtis similarity matrices for soil properties and vegetation variables, was conducted to ascertain whether sites that were more similar in soil properties were also more similar in vegetation attributes. Results showed there was no similarity between the two matrices (Correlation = 0.095, p>0,05), suggesting that soil properties did not play a strong role in influencing the measured vegetation attributes at the scale at which these factors were examined.

2.4 DISCUSSION

The results of the terrestrial habitat assessment show that the three survey sites are similar in terms of most ground cover, structural attributes, and soil properties. All three sites, with the exception of Site 2 in the grass category, contained a comparatively similar number of sampling points that were dominated by the broad ground cover attributes.

On a regional scale, vegetation communities are influenced by a combination of geology, landform and soils (Sattler and Williams 1999). On a smaller scale even slight variations in aspect, topography, hydrology (i.e., water table depth) and soil properties can influence the structure and floristics within a vegetation community

(Gough et al. 2000) and productivity of the ecosystem can influence tree death and leaf litter accumulation (Aarssen 2001). The floristics within the survey sites were not quantified as part of the habitat assessment, because the aim of this study was to identify variation in habitat attributes that were considered likely to influence frog utilisation, rather than provide a description of the vegetation communities within a site.

All three survey sites had similar range of ground cover and soil attributes. The recorded differences in the measured habitat attributes were much greater within a survey site in comparison to between survey sites. These variations often occurred from one sampling point to another, with, for example, the ground cover at one

Joanne Chambers Page 38 Terrestrial Habitat Assessment Chapter 2

sampling point being dominated by grass and the next by a dense covering of debris and logs. These changes were expected to be caused by the soil properties.

However, this was not the case and it is likely that other features of the landscape that were not quantified, such as underlying geology, aspect, chemical properties of the soil, soil evaporation rate, etc. may have influenced the observed variation in habitat attributes.

The observed spatial difference in the recorded terrestrial habitat attributes may play an important role in influencing the activity of frog species at Barakula, although unmeasured biotic effects, such as predation and competition may also contribute to factors that influence frog activity. Considerable variation exists in the morphology of frogs, which suggests that species may exhibit differential success in their movements through a terrestrial environment. For example, some small frogs move from one area to another by crawling along the ground, rather than jumping. For these species, areas that contain large amounts of debris may restrict their movement and it is expected these species may avoid traversing these areas.

Burrowing species may avoid areas that are covered by grasses, because these frogs may find difficulty in burrowing through a dense fibrous root system that is associated with most grasses. It is also predicted that burrowing species would avoid soils that contained large amounts of stones, as these soils may be difficult to burrow in. Species that are classified as ‘generalists’ are most likely to be found throughout all areas within a survey site, whereas habitat ‘specialists’ may be more restricted to certain areas that are governed by their ecological or morphological characteristics.

The positioning of the three survey sites at Barakula was determined to minimise as much as possible, the number of abiotic variables (i.e other than microhabitat variables) that could play a role in influencing the distribution of individuals of the

Joanne Chambers Page 39 Terrestrial Habitat Assessment Chapter 2

different frog species. All sites were located directly adjacent to or are dissected by an unsealed forestry road, therefore, all sites should be subject to the same disturbances usually related to roadways (fragmentation, edge effects, increased mortality), although traffic along these roads is very infrequent. The lack of topographic variation between the sites also means that the disturbance regime and topography should not unduly influence any observed differences in frog distribution across the three survey sites.

Hibernating and aestivating animals need to maintain vital physiological processes during long periods of torpor or inanition (Bayomy et al. 2002). Prevention of dehydration is probably the most critical process exhibited by these animals. Frogs are able to manage water loss by physiological means (cocoon formation) or by selecting appropriate microhabitats. Many of the species inhabiting Barakula State

Forest burrow to avoid predation during daylight, desiccation during summer, or freezing during winter, causing these species to come into direct contact with the substrate for long periods, therefore, moisture holding potential, texture and pH of the substrates may influence where frogs choose to burrow.

In the next Chapter experimental tests were conducted on the measured habitat variables and soil properties within each sampling point at the three study sites to ascertain if these characteristics of the terrestrial environment offered differential protection against water loss.

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Chapter 3:

EVAPORATIVE WATER LOSS IN DIFFERENT MICROHABITATS

3.0 INTRODUCTION

Dry environmental conditions inhibit the activities of most amphibians (Shoemaker and Nagy 1977) because, in most instances, their permeable skin is not capable of preventing evaporative water loss (EWL) (Shoemaker et al. 1972, Blaylock et al.

1976). The rate of water loss through evaporation from the skin of an animal is

essentially the difference in the concentration of water vapour within the animal

(below the skin) and in the free air beyond the adhering boundary layer of air next to

the animal surface (Spotila and Berman 1976). In the absence of solar radiation,

exposed surface area and movement are the major factors determining the

magnitude of EWL (Heatwole et al. 1969).The rate of EWL has been shown to be

correlated with species (Lillywhite 1975), temperature and wind speed (Cohen

1975), skin resistance (Spotila and Berman 1976) and habitat selection

(Schwarzkopf and Alford 1996). For the majority of species, control of EWL is

primarily related to the use of moist microhabitats, and to reduction of the skin area

being exposed to the desiccating environment (Shoemaker et al. 1992).

Different species of anurans display morphological, physiological or behavioural

modifications to minimise EWL. Arboreal species in general are exposed to a lesser

availability of shelter and free water and to stronger air movement (Yorio and Bently

1977) and may therefore, be at a greater risk of desiccation than terrestrial species

(Amey and Grigg 1995). However, research has shown that many arboreal species

lose water across the skin at a lower rate than terrestrial species (Toledo and Jared

1993). These species have been termed ‘water-proof’ frogs (Withers et al. 1984).

These species do not actively avoid the hot dry conditions of their environment;

Joanne Chambers Page 41 Evaporative Water Loss Chapter 3

rather they have continuous layers of lipids in the dorsal skin that form a barrier to water loss (Amey and Grigg 1995).

Species of toads are able to tolerate large amounts of water loss (< 50% of body weight) and can reabsorb water from the bladder (Shoemaker and Nagy 1977). The

Cane Toad, Bufo marinus, has an additional adaptation to reduce water loss: a reduction in preferred temperature in arid regions, which lowers the driving gradient for EWL, thereby conserving water (Malvin and Wood 1991).

The formation of a protective cocoon during aestivation is an adaptation displayed by a variety of Australian frogs (Lee and Mercer 1967). The cocoon consists of a series of compact layers of keratinised epidermal cells (van Beurden 1982) that completely envelopes the frog except for the external nares (Withers 1998). The cocoon markedly reduces EWL (Davies and Withers 1994) and provides equivalent or better ‘water-proofing’ than that shown by arboreal frogs (Withers 1998).

Metabolic depression is a common phenomenon in a number of vertebrate and invertebrate animals, in response to cold, anoxia, starvation or water deprivation

(Withers 1993). During enforced aestivation of two Australian anuran genera, it was shown that metabolic rate declined rapidly in Neobatrachus and Cyclorana with metabolic depression being measured at 70% (Withers 1993). A 60-80% depression in metabolic demand extends the duration of aestivation by 3-5 fold, and the significance of such an extension in the duration of aestivation is important for the survival of species inhabiting regions that suffer from prolonged hot or cold dry spells (Etheridge 1990).

Behavioural responses to changes in moisture availability occur in many amphibian species (Huey and Slatkin 1976, Jaeger 1980). Amphibians usually respond to dry conditions by limiting their activities to microhabitats that retain moisture. However, some species can remain active during dry conditions by changing their posture. In

Joanne Chambers Page 42 Evaporative Water Loss Chapter 3

a study of a Puerto Rican frog, Pough et al. (1983) showed that Eleutherodactylus

coqui were able to minimise moisture loss during dry nights by adopting a water-

conserving posture previously described for other species by Heatwole et al. (1969).

Anuran species that inhabit moist tropical regions may not rely on refuge shelters

that provide protection against moisture loss as much as those species that occur in

more arid regions, because high humidity will decrease EWL (Pough et al. 1983,

Sinch 1984). The need for refuge sites that provide protection from desiccation may

also be minimal for stream-dwelling species, as these species will be able to

rehydrate when necessary by either submerging in the stream or by positioning

themselves in areas that receive spray from flowing streams.

Anuran species that occur in the semi-arid and arid zones of the world typically

reduce the risk of desiccation by becoming inactive between periods of rainfall

(Withers 1993). During hibernation and aestivation, animals need to maintain vital

physiological processes and water is considered the most vital substance to ensure

their survival during long dormancy periods (Bayomy et al. 2002). The presence of

terrestrial habitats that provide suitable refuge sites for protection from

environmental extremes, are critical for the persistence of populations in these semi-

arid and arid areas (Stewart and Pugh 1983).

It is considered unethical to place undue stress on experimental vertebrates, a

consideration that whilst necessary and desired, limits the scope for measuring EWL

in anurans. However, in a study of the role of skin in controlling water loss in

amphibians and reptiles, Spotila and Berman (1976) used agar models as well as

live animals to compare moisture loss. Results of this experiment showed that

evaporation rates in the live animals were similar to the physical duplicates of the

agar models. This methodology was successfully adopted by Schwarzkopf and

Alford (1996) to measure desiccation and shelter-site use in Cane Toads.

Joanne Chambers Page 43 Evaporative Water Loss Chapter 3

Results from the previous chapter indicate that there was significant variation in soil and ground cover properties within and between study sites in Barakula State

Forest. This variation is likely to provide differences in the effectiveness of sites for reducing evaporative water loss. By using agar models as replicates for frogs, it is possible to gain an understanding of variation in the protection that might be provided at different sites, and how this might compare to variation in EWL provided by different types of microhabitats.

The relatively harsh environmental conditions prevailing in Barakula State Forest mean that species confronted with long dry periods will need to select appropriate refuge sites to prevent desiccation. In this chapter, agar models have been used to investigate if different microhabitats at the three study sites at Barakula offer differential protection against EWL, a factor that maybe related to terrestrial microhabitat utilisation by the frog assemblage within Barakula State Forest.

3.1 METHODS

3.1.1 MODELS

Agar models of frogs were created using ‘plaster of Paris’ moulds of preserved frogs of three different sizes. The size of moulds used was determined by the average sizes of the suite of frogs inhabiting Barakula State Forest (i.e., large = 65 mm snout to vent length (SVL), medium = 45 mm, small = 25 mm) (Figure 3.1). The moulds were not created to represent a particular species; rather they were used to give a generalised indication of water loss in different sized frogs. The strength/composition of agar used for the models followed the recipe used by

Schwarzkopf and Alford (1996), being 10 g of agar mixed with 100 ml of boiling water.

Joanne Chambers Page 44 Evaporative Water Loss Chapter 3

Prior to placement in the field, each agar model was weighed to nearest 0.01g and placed in a labelled individual air-tight plastic zip-lock bag, which was then placed in eskys for transportation from the laboratory at QUT to the study sites.

3.1.2 MICROHABITATS

To measure EWL in various refuge sites, agar models were positioned in five

different microhabitats at every sampling point at the three study sites. Four of the

microhabitats were chosen on the basis of potentially providing refuge sites for

ground-dwelling frogs in Barakula State Forest (under logs, leaf litter, vegetation,

and buried). The vegetation microhabitat consisted of clumps of grass or sedges,

but where these were not available models were placed under low shrubs. The

buried microhabitat was chosen because of the prevalence of burrowing species

within the study area. The fifth microhabitat, open, was used to give an indication of

the rate of moisture loss if a frog did not find a suitable refuge site.

Joanne Chambers Page 45 Evaporative Water Loss Chapter 3

At each sampling point a model of the three different size classes was placed in the designated microhabitat within 3 m of the centre of the habitat assessment sampling points (see Section 2.1.2, Chapter 2). The three models used for the buried habitat were placed within 1 m of the sampling point at a depth of 25 cm, and models used for the open habitat were placed immediately adjacent to the centre sampling point in an area that had been cleared of all ground cover. At all habitats care was taken to position the three models so they were not touching each other, a factor that may influence water loss in the models.

Models were left for five days at each site, after which they were collected and any adhering debris was carefully removed with a fine paint brush. The models were then placed in the original zip-lock bags, transported back to QUT in an esky and reweighed. The difference in weight was regarded as moisture lost during exposure in the different microhabitats and is expressed as percentage moisture loss of the original model. Percentage moisture loss was arcsine transformed before analysis to minimise the risk of heteroscedasticity of the data (Sokal and Rohlf 1995).

3.1.3 SAMPLING PERIODS

To ascertain if there was temporal variation in protection against desiccation at the different microhabitats the experiments were conducted during winter and summer.

The winter experiment commenced on the 24th July 2002. Mean temperatures for that period at Barakula were maximum 20.1oC and minimum 0.1oC. The last rainfall that had been recorded at Barakula was 20.8 mm on the 17th June. The summer experiment commenced on the 16th February 2003. Mean maximum and minimum temperatures for that period were 30.9oC and 20.9oC respectively. The previous rainfall event was 48 mm on the 5th February 2003. No rainfall occurred during the sampling periods.

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Following the winter experiment, it was found that EWL for the three different sized models was equivalent at all treatments when the surface to volume ratio (surface area/volume) was incorporated into the measurements, therefore, moisture loss for the three models was combined to give a mean moisture loss for each treatment at every trapline (expressed as a percentage of moisture lost for medium sized models).

Because size of models made no difference to rate of EWL, only medium sized models were used during the summer sampling period.

3.2 RESULTS

At two treatments during the winter and nine treatments during summer, models could not be located. In most instances these were the open treatments, although two disappeared from the leaf treatments and one from a vegetation treatment. It is probable that these models were removed by animals. These missing treatments were omitted from analysis.

Moisture loss varied considerably across treatments and sampling positions from little to no moisture loss, to almost complete dehydration (98%) (Table 3.1). There was also a clear difference in moisture loss between the winter and summer sampling periods (Figure 3.2, Table 3.1). Average summer moisture loss for the different microhabitats ranged from 27% to 98% compared with winter averages from 0% to 75%, and average moisture loss was noticeably higher in all treatments during summer. This result is expected because of the very dry conditions that prevail during most summer days at Barakula.

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120

100

Summer 20 % Moisture Lost (Mean +- 2 SE)

0 Winter N = 109 109109 109 109 109 109 109 109 109 buried leaf logs open veg

Microhabitat Type

Figure 3.2. Mean (+/- 2SE) percent moisture lost from agar models placed in different microhabitats in summer and winter. Sample sizes are shown along x axis.

Table 3.1: Minimum and maximum percentage moisture loss in the agar models used to experimentally simulate evaporative moisture loss in frogs, during winter and summer sampling periods in the five microhabitats at the three survey sites within Barakula State Forest. Microhabitat Site Min % loss Max % loss Min % loss Max % loss winter winter summer summer Buried 1 12 50 32 94 Buried 2 16 52 27 69 Buried 3 4 41 44 96 Leaf litter 1 0 33 77 96 Leaf litter 2 -2 25 74 96 Leaf litter 3 -1 24 74 97 Logs 1 5 50 92 96 Logs 2 9 38 80 97 Logs 3 0 71 82 98 Open 1 32 64 95 97 Open 2 19 75 85 97 Open 3 9 60 96 97 Vegetation 1 1 57 89 97 Vegetation 2 12 47 80 97 Vegetation 3 -1 55 84 97

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Because of the observed differences in moisture loss across the two sampling periods, the data were analysed separately for winter and summer.

3.2.1 WINTER SAMPLING PERIOD

Moisture loss over the five days ranged from 0% in some leaf microhabitats and under some logs, to 76% in the open (Table 3.1). Some models also showed slight increases in weight (1-2%) suggesting that they absorbed some moisture from heavy dew, or that small amounts of undetected debris remained on the models when re-weighed. In either case it suggests minimal moisture loss.

A Two-way ANOVA conducted on arcsine transformed data indicated clear differences in mean weight lost in microhabitats between the three study sites (F2,527

= 39.6, p<0.001) (Figure 3.3). Tukey post hoc tests showed that moisture loss at

Site 3 was significantly different to the other two sites (p <0.001). Moisture loss between the five microhabitats at all study sites was also significantly different (F4,527

= 117.1, p=<0.001). Rates of water loss in the leaf microhabitat were significantly lower than all other treatments (Tukey p<0.001). Log and vegetation treatments, which recorded similar rates of evaporative moisture loss in all sites (Tukey p=0.99) were also lower than buried and open treatments (Tukey, p<0.001), and buried was lower than open (Tukey p<0.001) (Figure 3.3). The magnitude of the difference in moisture loss between microhabitats was larger than differences between sites

(Figure 3.3) and there was no interaction between microhabitats and site (F8,527 =

0.73, p = 0.662).

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Site 1.00 30 2.00 3.00

20 % Moisture Loss (Mean +- 2 SE) Loss % Moisture

buried leaf logs open veg Microhabitat

Figure 3.3. Mean (+/- 2SE) percent moisture lost from agar models placed in different microhabitats at the three study sites during winter.

3.2.2 SUMMER SAMPLING PERIOD

During the summer trial period average moisture loss ranged from 27% in the buried microhabitat at site 2, to around 97% in at least one of each of the microhabitats at each of the three survey sites (Table 3.1). Average moisture loss for the different microhabitats at each site is shown in Figure 3.4. There was a clear trend for models in buried microhabitats to lose much less moisture than all other treatments.

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100

Site 70 1.00 2.00 3.00

60 % Moisture Loss (Mean +-2SE) Moisture %

buried leaf logs open veg Microhabitat

Figure 3.4. Means (+/- SD) showing moisture loss in agar models positioned at the five different microhabitats within Barakula State Forest during summer.

A two-way ANOVA on the arcsine transformed data showed a significant interaction between site and treatment (F8,519 = 5.99, p<0.001). Because of this interaction, one-

way ANOVAs were performed on moisture loss within sites to identify differences

between treatments. At all three sites there were significant differences in moisture

loss between the treatments (Site 1, F4,169 =361.8, p<0.001; Site 2, F4,170 = 368.3, p<0.001; Site 3, F4,180 =178.8, p<0.001).

Tukey post hoc tests indicated that average moisture loss was significantly lower in the buried microhabitat at all sites (p<0.001). Moisture loss was also lower in leaf litter compared with vegetation, logs and open microhabitats (sites 1 and 2, p<0.001, site 3 p = 0.031 leaf v logs, p = 0.026 leaf v vegetation, leaf v open p<0.001).

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During both sampling periods moisture loss between the buried and leaf microhabitats was always significantly different to the other treatments, with the buried microhabitats providing the greatest protection against EWL in summer

(Figure 3.3), but during winter models in the leaf microhabitats lost less moisture

(Figure 3.2).

3.2.3 ASSOCIATIONS BETWEEN MOISTURE LOSS IN MODELS AND HABITAT VARIABLES

The data indicate considerable variation in moisture loss in the different microhabitats within each study site, and generally smaller differences within a treatment between sites. Site to site differences may be caused by overall, differences in surrounding cover. To examine if the type of ground cover or dominant tree cover influenced the moisture loss rates at a sampling point within a study area, one-way ANOVAs were performed on the moisture loss data in the two sampling periods in relation to the broad ground layer and the dominant tree cover categories at each sampling point (see Section 2.1.2 Chapter 2).

The type of ground cover had a significant influence on moisture loss in only one microhabitat; the winter log treatment (Table 3.2). Moisture loss in areas dominated by sedges (33.8%) tended to be higher than for other ground cover types (leaf =

27.6%, grass = 26.4%, debris = 25.4%) (SNK Test, p<0.05). Similarly, moisture loss varied significantly between the different dominant vegetation types for only two microhabitats; models placed under logs in summer (mixed vegetation [73.7%] lower than eucalypts [78.7%]), and in vegetation in winter (cypress [27.3%] lower than eucalypt [30.1%]) (Table 3.2).

Overall, the dominant vegetation or the ground layer type did not play a significant role in influencing moisture loss in the different microhabitats. Therefore, rates of moisture loss in the models were also examined in a stepwise multiple regression against four vegetation cover variables at each sampling point (% bare ground,

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density of trees <1m, density of trees 1-3m, and density of trees >3m). For models that were buried, an additional variable (soil moisture at 25cm - as recorded earlier in the study – see Chapter 2) was added to the multiple regression. There was a clear trend for the rate of moisture loss in the non-buried models to be affected by the density of trees 1-3m high (Table 3.3). Sampling points with high densities of these trees had slightly lower rates of moisture loss. Buried models were not influenced by relative levels of soil moisture.

Table 3.2. ANOVA results conducted on moisture loss in agar models used to simulate evaporative water loss in frogs, and ground layer type and dominant tree species at each sampling point within Barakula State Forest. Dominant vegetation Ground layer type F df p F df p Summer leaf 1.67 4,102 0.162 0.69 3,103 0.561 log 2.97 4,104 0.023 0.72 3,105 0.542 open 0.37 4,98 0.828 0.80 3,99 0.498 vegetation 2.17 4,103 0.077 0.91 3,104 0.441 buried 0. 18 4,102 0.951 0.15 3,103 0.929 Winter leaf 1.98 4,102 0.102 0.46 3,103 0.710 log 0.37 4,104 8.28 3.93 3,105 0.011 open 3.59 4,103 0.009 0. 70 3,104 0.553 vegetation 1.51 4,104 0.204 0.30 3,105 0.822 buried 0.46 4,104 0.767 0.92 3,105 0.433

Table 3.3. Results of multiple regression analysis examining relationship between moisture loss in agar models used to simulate evaporative moisture loss in frogs, and vegetation variables at each sampling point within Barakula State Forest. Season Microhabitat Variable Slope r2 F df p Summer buried tree 1-3 3.9 0.052 5.76 1,105 0.001 %bare 0.24 0.088 5.02 2,104 0.008 ground leaf nil log nil open nil vegetation nil Winter buried nil leaf trees 1-3m -2.81 0.045 5.06 1,107 0.026 log trees 1-3m -3.26 0.043 4.78 1,107 0.031 open trees 1-3m -4.89 0.092 10.86 1,107 0.001 vegetation trees 1-3m -0.14 0.091 10.76 1,107 0.001

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3.3 DISCUSSION

Microhabitats within the terrestrial environment in Barakula State Forest provide varying degrees of protection against EWL. This variation in protection occurs both spatially and temporally within the three study areas. Significant differences in moisture loss were observed between the three sites in both trial periods, despite results from the previous chapter showing that the three sites were similar in habitat structure and complexity. This suggests that some other environmental factor not quantified in this study, may be influencing the capacity for the microhabitats to provide protection against moisture loss. However, the difference in moisture loss between the five treatments was clearly stronger than differences between sites, suggesting that the quality of microhabitats within a site may be more important for the survival of frogs than the overall, site attributes.

It was expected that the results of this current study would show a correlation between moisture loss in the buried models and the previously measured relative moisture content of the soil (refer Section 2.3.2, Chapter 2), with areas where the soil had a higher moisture content giving greater protection against EWL, as suggested in Cohen and Alford (1996). However, the results did not support this prediction, and failure to do so could to be due to the fact that the models were not buried in the exact spot where the soil samples were obtained, therefore, the moisture level of the soil at the point of sampling may have been different to that at the point of burying the models. A suitably designed study using experimental plots containing soils with different properties (e.g, Cohen and Alford 1996) would be needed to elucidate this argument.

Most of the frog species within Barakula State Forest, with the exception of

Pseudophryne bibronii, breed over a period of several months during the summer, if

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suitable rainfall occurs. During this time adult frogs may move to and from the terrestrial habitat surrounding the breeding site on numerous occasions, but need to find adequate refuge sites during the daytime when, for most species, breeding activity ceases. Most of the frogs at Barakula are explosive breeders and they respond rapidly to favourable weather conditions, therefore, protection against EWL is probably not the most critical issue facing these frogs during the breeding season, because of the presence of moisture in the environment. However, at the completion of the breeding season, when the environment starts to dry out, suitable refuge sites must be found to ensure survival until the next breeding opportunity. In addition, newly emerged metamorphs move away from the breeding site during this period and their movement may be at much greater distances than shown by adults

(Breden 1987). Throughout the dispersal stage appropriate refuge sites must be selected to prevent desiccation.

The results of this study showed that EWL within the microhabitats varied considerably between the summer and winter sampling periods. These results suggest that microhabitat utilisation by the frogs at Barakula may vary temporally.

Seasonal shifts in habitat preference in frogs can be initiated by age, reproductive activity, foraging, or when adverse environmental conditions prevail (Lamoureux and

Madison 1999). For frogs that breed in ephemeral ponds, the hibernation sites, breeding sites, and foraging areas may be temporally and spatially separated and individuals would need to move between these sites in seasonal cycles (Sinsch

1990; Richter et al. 2001). Studies have shown that species change preferred

shelters during different seasons (Schwarzkopf and Alford 1996, Regosin et al.

2003). Seasonal changes in movement and behaviour of cane toads were

associated with seasonal variation in moisture levels in the environment (Seebacher

and Alford 1999). A change in effectiveness in microhabitats providing protection

between summer and winter was observed in Schwarzkopf and Alford (1996) and

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was likely to be due to the lower environmental temperatures experienced during the winter period and hence lower evaporative water loss during this time.

Approximately 35% of Australia’s native frogs can burrow (Tyler 1994), an adaptation that enables species to inhabit areas that become arid for long periods

(Sanders and Davies 1984, Shoemaker et al. 1992). Other species shelter down cracks in the soil or seek shelter under some form of structure, such as logs, shedding bark, leaf litter etc. This study has demonstrated by using agar models, that frogs that burrow into the soil sustain the least amount of EWL during the summer period. Burrowing species are able to survive underground during long dry periods because water from the moist soil is absorbed through capillary action from the soil particle matrix to the surface of the animal (Hillyard 1976, Brekke et al.

1991). The models used were buried 25cm beneath the surface. There is no published literature that provides the depth attained by burrowing species at

Barakula, so this depth was used to give an indication only of how soil properties at this depth provide protection from moisture loss. The results of this experiment effectively demonstrate that burrowing species are afforded greater protection against desiccation, than would be experienced by non-burrowing species.

During winter at Barakula, the results showed that sheltering under leaf litter is likely to provide the greatest protection against moisture loss, rather than buried. This change in effectiveness in microhabitats providing protection between summer and winter is likely due to the lower environmental temperatures experienced during the winter period and hence lower evaporative water loss during this time. It is expected that soil moisture would have decreased considerably since the end of the summer wet season which would explain why EWL in buried habitats was higher in winter.

Different pressures may apply to refuge site use during summer and winter in

Barakula, with prevention of desiccation being the overriding influence during

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summer when temperatures can be extremely high. However, during the colder months of winter, active species would need to utilise microhabitats that not only provide protection against EWL, but also allow absorption of warmth during daylight hours. Endotherms are capable of lowering metabolic heat production as a means of reducing water loss (Withers 1993). In ectotherms, such as frogs, reducing heat gain from the environment has a similar effect on water economy and is especially important during warmer months (Malvin and Wood 1991). However, during cooler months active species need to acquire sufficient warmth from the environment to provide energy for daily activities. The results of this study show that sheltering under leaf litter during winter at Barakula may provide the means for active individuals to maintain their body temperature and hence provide them with the energy required for foraging and breeding activity during this period, whilst at the same time minimising EWL. Further research using techniques where individuals could be followed would be needed to identify if seasonal changes in microhabitat use are employed by species inhabiting Barakula State Forest.

Ambient temperature and air humidity are other factors that may influence water loss in amphibians (Malvin and Wood 1991). The environmental temperature plays a

direct role in influencing metabolic rate and hence water loss (Withers 1992). The

study on Cane Toads (Schwarzkopf and Alford 1996) showed that this species used

burrows more frequently during the hot dry season in comparison to shelter use

under wetter conditions. In temperate environments protection from low

temperatures has been shown to influence refuge site choice in frogs (Carey 1978,

Sinsch 1984). Temperatures at the different microhabitats were not recorded during

this study and further research is needed to clarify if the physical structures of the

habitat, the temperature within the habitat or both are responsible for the observed

moisture loss rates.

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The physiological constraints of frogs may play a significant role in influencing their choice of refuge sites. The results from the agar mould experiments show that selection of appropriate refuge sites, particularly during summer months, may be of critical importance to the survival of frogs at Barakula. In particular, species that are capable of burrowing have the greatest opportunity to prevent desiccation during dry spells. Refuges used above the soil surface may lead to significant moisture loss if used for extended periods, therefore, non-burrowing species must find suitable refuge during those hot dry periods if they are to persist in Barakula State Forest.

Potential microhabitats for non-burrowing species in Barakula include cracks in the soil, unused burrows of other animals, hollows under trees, inside or under logs, buried in leaf litter or under dense vegetation such as clumps of grass or sedges.

These microhabitats may be more plentiful within some areas thus providing sufficient refuge sites for individuals, but in other areas they may be scarce and individuals may need to move greater distances to find appropriate refuge sites.

The results from this chapter relate to moisture loss from passive models, and the behaviour of frogs will obviously vary from these models. Nevertheless, the data clearly demonstrate that there is significant variation in microhabitats in their suitability as refuge sites. The previous chapter (Chap. 2) also indicated that there is significant variation in the amount, quality and spatial distribution of these microhabitats at the different sampling points. In the next chapter I will compare the results of three years of frog census with the measured terrestrial habitat variables to identify if there is any relationship between distribution/activity of a species and any of the measured habitat attributes. Due to the unpredictable and often minimal rainfall events that occur in semi-arid regions of Australia, frog activity in these regions is often episodic, causing irregular utilisation of the terrestrial habitat. This factor makes it difficult to identify accurately which particular habitat variable/s are utilised more frequently. However, censusing over a medium-term study should

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produce some evidence of repeated usage/avoidance of different areas within a survey site, thus allowing predictions to be made regarding which habitat variable/s are important for the persistence of different frog species.

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CHAPTER 4:

FROG CENSUS AND TERRESTRIAL HABITAT ASSOCIATIONS

4.0 INTRODUCTION

The physiological characteristics of different frog species will dictate both their broad-scale distribution and their habitat choice at a finer scale. The occurrence of a species in a given area is determined by the need for food, reproduction, predator avoidance and to minimise deleterious effects caused by environmental influences

(e.g, desiccation). Most amphibians are highly susceptible to desiccation, and low humidity can limit their activity (Tracy 1976, Moore and Moore 1980) and also plays a role in influencing the distribution of species. In environments where water is scarce or highly intermittent, amphibian species require specific behavioural, physiological or morphological characteristics to enable persistence.

Based on their associations with the breeding pond, which often reflects their requirements for prevention of desiccation, anuran species can be broadly grouped into three assemblages (as discussed in Chapter 1). Species within each assemblage often possess similar physiological characteristics; Stream-dwelling species that can generally obtain hydration readily and permanent water species are often habitat ‘generalists’ that have permanent access to water for hydration. In contrast, species in less mesic environments that use temporary/ephemeral ponds, need to find refuge sites that prevent desiccation during dry spells.

To understand variation in the structure of amphibian assemblages Mitchell et al.

(1997) conducted a 2 year study of several montane forest stands in different stages of regrowth. The study showed that juvenile and adult amphibians utilised terrestrial habitat up to several hundred meters from their breeding locations and suggested the importance of maintaining suitable terrestrial habitat around aquatic breeding

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sites to ensure persistence of amphibian populations. An analysis of studies concerned with terrestrial requirements of amphibians (Semlitsch, 2002) showed that juveniles and adults were found on average between 69 and 125 m from the edge of aquatic habitats. The common approach for wetland conservation is to retain a terrestrial buffer zone. However, lack of knowledge with regard to terrestrial habitat requirements for many species of amphibians means that the minimum buffer zone width (for example 10 – 100m in Queensland, depending on stream order, and 30.8 m in USA) is often inadequate to encompass the movement of most species. A buffer zone width of 164 m was recommended by Semlitsch and Bodie

(2003) to ensure 95% of amphibian populations’ annual requirements were encompassed within that zone.

The environment at Barakula State Forest, Queensland, represents a very different range of habitats for amphibians relative to the more mesic environments examined in other studies of terrestrial habitat use (e.g., Parris and McCarthy 1999, Wilkins and Peterson 2000, Parris 2004). In this semi-arid region, anurans are utilising habitats comprising either temporary or small breeding pools set amongst large areas of dry open woodland, often at considerable distances from permanent water sources. The type of terrestrial microhabitats occupied by anurans is likely to be of extreme importance in this environment.

The aim of this chapter is to establish if the frog species inhabiting Barakula State

Forest show any preference/avoidance to the microhabitat variables described in

Chapter 2 and tested in Chapter 3. To assess habitat utilisation of frogs, three frequently used methodologies were considered for their appropriateness on frogs at

Barakula:

1. Radio/spool tracking: This methodology is used for studying

movements of a single individual as it allows precise identification of habitat

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usage. However, this method is restricted by the body shape of individual

frogs, with belts regularly slipping off (e.g., Watson et al. 2003) and by the

low numbers that can be tracked (Miaud and Sanuy 2005). For this study I

was interested in identifying terrestrial habitat associations for an

assemblage of species, many of which are burrowing species. Attachment of

a transmitter or cotton spool would affect their burrowing ability and

therefore, their normal movement patterns, use of refuge and survival.

2. Pit-fall trapping: This method has been identified as an effective

way to assess the distribution of small vertebrates (Heyer et al. 1994). This

method allows collection of data for multiple individuals from a suite of

species, over long periods. Capture rate in pitfalls is related to the number of

animals in the target area and their activity levels, with more captures being

recorded where densities and/or activity levels are highest. Data from the

capture records can be used to assess if more species/individuals are active

in areas that support specific habitat attributes. However, if individuals are

not marked (see 3 below), the data is likely to contain recaptures which could

confound the results of the study, as species may just be moving through the

area to get to another site. Alternatively, multiple captures of the same

species within a particular trapline could indicate a preference for a particular

microhabitat. Nevertheless, over time, cumulative captures of a species

would be expected to be greater in areas that support the preferred

microhabitat conditions. That is, although there will be some variability,

overall, if a frog species spends more time in a particular area it should have

a higher catch rate in that area.

3. Toe-clipping: Research suggests that toe clipping of frogs can be

detrimental to survival rate (Williamson and Bull 1996, McCarthy and Parris

2004). Ricter and Sergel (2002) showed that recapture rates on toe clipped

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frogs were low in the first two years, with no recaptures occurring after the

second year. This research was conducted over a three-year period and

repeated captures of the same individuals were expected, therefore, at the

risk of obtaining limited data, I would potentially be subjecting individuals to

reduced chance of capture and possible increased mortality rates.

Marking of individuals is also labour intensive and time consuming, thus

greatly increasing exposure of trapped individuals to the risk of desiccation

and predation by ants. Estimating population size or abundance was not the

primary aim of this research, therefore, marking of individuals was not

deemed necessary.

The aim of this research was to examine terrestrial habitat associations for an assemblage of frogs. By using pitfall traps and positioning traps and drift fence arrays in a heterogeneous landscape for a three year trapping period, it was believed there would be sufficient data to allow identification of any obvious habitat preference/avoidance of microhabitat types for different species. From these baseline data, hypotheses regarding terrestrial habitat preferences can then be developed and tested experimentally.

4.1 METHODS

4.1.1 STUDY STIES

Pitfall trapping was established at the three survey sites used for the habitat assessment (Chap 2). Site 1 was situated in an area adjacent to an unsealed forestry road that was used infrequently. The area contained a small semi- permanent dam and an ephemeral pool in a natural depression. Traplines were established on the northern side of the roadway and ran in a north-south, west-east grid. Site 2 was situated 6 km from Site 1 on the same dirt road. This site contained two ephemeral pools in claypans that were situated on opposite sides of the road

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and separated by approximately 100m. The traplines were positioned in the same direction as in Site 1 but 21 traplines were positioned on the northern side of the road and 15 were on the southern side. Site 3 was positioned approximately 10 km to the south-west of Site 1, on another unsealed road with slightly higher use than the roadway at Sites 1 and 2. Site 3 contained a large ephemeral claypan on the eastern side of the roadway. Traplines were positioned in a west-east, north-south direction at this site. Twenty-two traplines were established on the eastern side of the roadway and 15 were on the western side. An additional trapline was established at Site 3 near the claypan because the size and shape of the ephemeral water body (approximately 60 x 50 m) did not allow symmetrical arrangement of traps.

4.1.2 FROG CENSUS

Traplines were centred on the sampling points used for the vegetation and soil analysis. Hence, thirty-six traplines were positioned 50 m apart in a 6 x 6 trapline grid surrounding the main water body at each of the three sampling sites. Traplines consisted of a shade cloth drift fence and three pit traps. Drift fences were 10 m long by 45 cm high, supported by steel rods attached every 1.5 m and buried to a depth of 10 cm to prevent escape of animals under the fence. Traps consisting of 11 L plastic straight-sided buckets were buried in the ground so the rim of each was flush with ground level. Three traps were positioned along each trap line (middle and each end). Drift fences were extended through the centre of each trap to a point approximately 40 cm from both ends of trap lines. Three 5 mm holes were drilled in the bottom of each bucket to prevent them rising due to water-table pressure. A piece of PVC pipe (15 cm x 4.5 cm diameter) containing a water-saturated wad of cotton wool was placed in the bottom of each bucket to provide moisture and refuge for animals. In addition a 6cm cube of saturated foam rubber was also placed in the bottom of each bucket to provide additional moisture and shelter.

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Many anurans breed opportunistically following adequate rainfall (Tyler, 1989). Non- breeding activity (e.g, foraging) is also likely to be associated with moisture, therefore, trapping events corresponded with adequate rainfall events. Traps were checked between 0445 and 1030 hours daily and again between1600 and 1900 hours. Captured animals were identified (Barker et al. 1995), counted, and age- classed (adult or juvenile), then released immediately at point of capture in suitable refuge at least 10 m to the side of traplines. When traps were not in use a 30 cm x

10 cm piece of plastic gutter guard, which simulated a “ladder”, was placed in each bucket before lids were replaced. This ladder offered a means of escape for any animals inadvertently caught if feral horses or other large animals trampled lids.

Trapping commenced on 12th June 2001 and continued sporadically, dependent on

rainfall events, until 4th March 2004. An official weather station is situated near the

Barakula Forestry Office and daily rainfall is reported to the Bureau of Meteorology

(BOM). The BOM web site (www.bom.gov.au) was accessed daily and whenever possible, trapping occurred within one day of sufficient rainfall (>20 mm) being recorded at Barakula.

During the survey period there were a total of 11 separate trapping events; 2 in

2001, 2 in 2002, 5 in 2003 and 2 in 2004. These trapping sessions ranged in duration from 3 to 10 consecutive days, but the majority were of 3 day duration because frog activity declined rapidly within the first three days of rainfall. During the survey period 27 trapping days occurred in summer, 9 in winter, 5 in autumn and 3 in spring.

4.1.3 DATA ANALYSIS

Approximately two – three hours were spent clearing traps at each survey site. To avoid exposing animals to detrimental factors over an extended period (e.g, desiccation or predation), traplines at two sites only were opened during very hot

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conditions, therefore, all captures were converted to captures per 100 days to allow for dissimilarity of trapping days between sites. For all analyses individuals were grouped by adult, juvenile, total species, species richness and guilds (i.e. burrowing and non-burrowing species).

Analyses of variance (ANOVAs) were performed to ascertain if there were differences between capture rates of different species within and between the three survey sites.

Forward Stepwise Multiple Regression analysis was performed on capture data and terrestrial habitat attributes. This allowed identification of any relationships between the catch rate of a species and particular site characteristics. To include the possibility that catch rate at a trapline was influenced by distance from areas where free-standing water occurred, a distance category was added to the analysis.

Distance was calculated to the nearest 5 m, by measuring how far each trapline was away from the closest point to where ponds and ephemeral pools occurred. .

Similarity matrices, using the Bray-Curtis similarity measure were constructed for habitat attributes (Chap. 2) and frog captures and measures of the similarity/dissimilarity between species composition and habitat attributes were analysed using the MANTEL Test in PopTools (Hood 2006).

4.2 RESULTS

4.2.1 SPECIES COMPOSITION

The region surrounding the study area was “drought declared” in February, 2001 by the Queensland Department of Primary Industries (www.LongPaddock.qld.gov.au).

This drought status continued for the region over the entire study period. Lack of sufficient rainfall meant that trapping events were significantly lower than initially anticipated: Site 1 = 27 days; Site 2 = 38 days; Site 3 = 44 days.

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Despite the dry conditions, a total of 1844 native frogs, representing two families, eight genera and seventeen species were captured over the study period (Table

4.1). The introduced species, Bufo marinus was also caught from one site in low numbers. Of the 21 species previously recorded from Barakula (see Table 2.2,

Chap. 2) Cyclorana platycephala, C. novaehollandiae, Litoria peronii and Litoria

fallax were never captured and none of these four species was heard calling from

any of the survey sites over the study period.

Members from the family Myobatrachidae made up the majority of captures (97%).

This result was expected as most species from the family Hylidae, except for the

Cycloranas, are morphologically equipped to either climb or jump from pitfall traps.

Captures of adult Cyclorana verrucosus and Litoria caerulea occurred only when

rain had fallen during the evening, causing the buckets to fill with water, thus making

it difficult for these individuals to escape from the buckets. Uperoleia rugosa,

species from the genera Limnodynastes and Neobatrachus sudelli comprised the

majority (83.6%) of captures (688, 578 and 273 respectively), while burrowing

species from the families Myobatrachidae and Hylidae accounted for 75% of total

captures. Adult frogs accounted for 72% of all captures. For the purpose of this

chapter individuals were classed as juveniles if they were smaller than the average

‘snout to vent length’ (SVL) of adults. In most instances, no distinction was made

between juveniles and recently metamorphosed individuals.

Fourteen species were recorded at Sites 1 and 2, and 17 species were recorded

from Site 3 (Table 4.1). Most species were located at all three survey sites, with the

exception of Limnodynastes fletcheri and Litoria caerulea, which were only recorded

from Site 3, Cyclorana alboguttata and Litoria verreauxii, were not recorded from

Site 2, and Cyclorana verrucosus was not recorded from Site 1. These species

tended to have lower catch rates than species caught at all sites.

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Figure 4.1 shows species accumulation curves for the three survey sites. These data suggest that the number of species captured at each site had plateaued, although there were small jumps in the curves associated with additional sampling periods at different times of the year. It is possible that additional sampling may have produced more species, especially at Sites 1 and 2.

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Table 4.1. Frog species and total numbers of frogs captured in pitfall traps at the three survey sites at Barakula State Forest between February 2001 and January 2004. Numbers in italics relate to captures per 100 days (rounded up to nearest whole number). * refers to burrowing species. Family/Species Site 1 Site 2 Site 3 Total Total adult juv. adult juv. adult juv. adult juv. Bufo marinus 0 0 3 1 0 0 3 1 Myobatrachidae Uperoleia rugosa* 133 6 284 8 238 19 655 33 493 22 747 21 541 43 1781 86 Limnodynastes 53 22 45 7 109 36 207 65 tasmaniensis 196 81 118 18 248 82 562 181 Limnodynastes 17 50 11 13 6 9 34 72 terraereginae* 63 185 29 34 14 20 106 239 Limnodynastes ornatus* 1 16 20 19 5 89 26 124 4 59 53 50 11 202 68 311 Limnodynastes salmini* 1 0 1 0 26 7 28 7 4 3 59 16 66 16 Limnodynastes fletcheri 0 0 0 0 5 10 5 10 11 23 11 23 Neobatrachus sudelli* 17 2 104 20 96 34 217 56 63 7 274 53 39 77 376 137 Pseudophryne bibronii 5 0 53 3 3 1 61 4 19 139 8 5 2 163 10 Crinia parinsignifera 4 3 25 2 11 0 40 5 15 11 66 5 25 106 16 Notaden bennetti* 3 3 0 1 17 30 20 34 11 11 3 39 68 50 82 Hylidae Cyclorana brevipes* 2 18 1 3 0 43 3 64 7 67 3 8 98 10 173 Cyclorana verrucosus* 0 0 1 0 1 3 2 3 3 2 7 5 7 Cyclorana alboguttata* 1 0 0 0 2 10 3 10 4 5 23 9 23 Litoria verreauxii 8 1 0 0 2 4 10 5 30 4 5 9 35 13 Litoria latopalmata 2 12 1 0 1 4 4 16 7 44 3 2 9 12 53 Litoria rubella 5 3 2 0 3 3 10 6 19 11 5 7 7 31 18 Litoria caerulea 0 0 0 0 1 0 1 0 2 2 Total Captures per site 252 136 551 77 526 302 1329 515 933 504 1442 203 1196 686

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SITE 1 SITE 2 SITE 3

6 Cumultive number of species

0 0 5 10 15 20 25 30 35 40 45 50 Number of sample days

Figure 4.1. Species accumulation curve showing the cumulative number of frog species captured in pitfall traps at the three survey sites within Barakula State Forest, with each additional sampling day.

The MDS plot (Figure 4.2) shows the general relationship between species assemblages caught at each trapline and survey site. The plot indicates some similarity between traplines at different survey sites, but shows a general trend for the three sites to have slightly different frog assemblages. The plot suggests that

Sites 1 and 2 were more similar to each other than to Site 3.

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Figure 4.2. MDS plot showing similarity in species assemblages captured at each pitfall trapline at the three survey sites within Barakula State Forest.

Capture rates for some species were relatively low; therefore, analysis of variance

(ANOVA) was only conducted on species that recorded > 30 captures (10 species), and on total adults and total juveniles. The total number of species (species richness) and the number of burrowing species were also examined in relation to microhabitat variation.

A series of one way ANOVAs show that most species and guilds were not caught at the same rates at the three survey sites, although total captures of adult frogs was similar across all sites (Table 4.2). Mean species richness per site (mean per trapline per 100 days, adjusted for sampling effort) differed significantly between sites, with each trapline at Site 3 catching an average of 6.6 species, compared to

4.3 and 4.6 at Sites 1 and 2 respectively (Table 4.2). Site 3 also recorded higher richness of burrowing species (Table 4.2). Site 2 had significantly lower captures of

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juveniles. Differences in mean capture rate per trapline at the three sites for eight of the ten species were also evident (Table 4.2) (see also 3.3.1).

Table 4.2. Shows mean capture rates per survey site at Barakula State Forest and ANOVA results for site differences in total frog captures, species richness, total burrowing species and individual species per trapline/100 days (figures in bold indicate significant differences). Species Site 1 Site 2 Site 3 Tukey Post ANOVA values capture rate capture rate capture rate Hoc Results Species richness F =15.91; p <0.001 4.3 4.6 6.6 Site 3 >1,2 Burrowing species richness F =23.81; p <0.001 2.9 2.9 4.8 Site 3 >1,2 Total Burrowing species 18.0 31.0 30.97 No difference Total Adults F =2.2, p=0.11 25.9 40.1 32.3 No difference Total Juveniles F =12.1, p<0.001 14.0 5. 6 18.5 Site 2 <1,3 Crinia parinsignifera Adult F =1.42, p=0.246 0.41 1.83 0.68 No difference Juvenile F = 0.69, p=0.503 0.31 0.15 0.00 No difference Cyclorana brevipes Adult F 1.29, p=0.281 0.2 0.1 0.0 No difference Juvenile F 5.96, p<0.00 1.8 0.2 2.6 Site 2 <1,3 Limnodynastes ornatus Adult F= 8.85, p<0.001 0.1 1.5 0.5 Site 2 >1,3 Juvenile F= 6.42 p=0.00 1.6 1.4 5.5 Site 3 >1,2 Limnodynastes. tasmaniensis Adult F 3.48, p=0.034 5. 5 3.2 6.7 Site 2 <1,3 Juvenile F 7.58, p=0.001 2.3 0.5 2.2 Site 2 <1,3 Limnodynastes terraereginae Adult F 3.52, p=0.033 1.7 0.8 0.3 Site 1 > 3 Juvenile F =27.92, p<0.001 5.1 0.9 0.5 Site 1 >3 Limnodynastes salmini Adult F=14.42, p<0.001 0.1 0.1 1.6 Site 3 >1,2 Juvenile F =6.03, p<0.00 0.0 0.0 0.4 Site 3 >1,2 Neobatrachus sudelli Adult F=9.69, p<0.001 1.7 7.6 5.9 Site 1 <2,3 Juvenile F= 9.83, p<0.00 0.2 1.5 2.1 Site 1 <2,3 Notaden bennetti Adult F8.38, p<0.01 0.3 0.0 1.0 Site 3 >1,2 Juvenile F 15.01, p<0.0 0.3 0.1 1.8 Site 3 > 1,2 Pseudophryne bibronii Adult F= 27.66, p<0.001 0.5 3.9 0.2 Site 2>1,3 Juvenile F=2.03, p=0.13 0.0 0.2 0.1 No difference Uperolea rugosa Adult F =1.4, p=0.25 13.7 20.8 14.6 No difference Juveniles F = 1.2, p=0.318 0.6 0.6 1.2 No difference

These analyses show that total captures of juveniles varied considerably between sites with Site 3 showing significantly higher capture rates than Sites 1 and 2 and

Site 1 recorded significantly more juveniles than Site 2. On inspection of the data

(Table 4.2) it can be seen that most of the variation in abundance is caused by the

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increased number of captures of just three species (C. brevipes, L. ornatus and L.

terraereginae).

4.2.2 SPECIES COMPOSITION AND HABITAT ASSOCIATIONS

To test if sampling sites that were similar in frog species assemblages were also

similar in measured soil and vegetation habitat variables, Mantel tests were

performed on Bray-Curtis similarity matrices for total burrowing species, total adults

and total juveniles. Results showed that for the combined data for all sites and the

data set for Site 2, the assemblage of burrowing species were similar where

traplines had similar vegetation (Table 4.3). There was a significant positive

correlation between burrowing species composition and soil properties for all sites

combined and at Sites 1 and 2 (Table 4.3), indicating that traplines which had similar

soil properties also caught similar numbers of burrowing species. The species

composition at a site (adults of all species) was positively associated with vegetation

at Sites 2 and soil properties at all sites combined and at Sites 1 and 2 (Table 4.3).

In general, composition of juvenile captures was similar at sites that had similar

vegetation and were positively correlated to soil properties at Site 3.

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Table 4.3. Results of Mantel Test comparing similarity between frog capture rates and measured vegetation and soil attributes at each sampling point within Barakula State Forest. Significant correlations are in bold print. Comparison Variable All Site 1 Site 2 Site 3 sites Burrowing Species Vegetation r value 0.159 0.143 0.158 0.003 p value <0.001 0.105 0.026 0.496 Soil r value 0.126 0.276 0.205 0.111 p value 0.015 0.014 0.006 0.078 Adult Captures (all species) Vegetation r value 0.012 0.128 0.168 -0.033 p value 0.372 0.097 0.031 0.339 soil r value 0.137 0.319 0.186 0.077 p value 0.001 <0.001 0.020 0.181 Juvenile Captures (all species) vegetation r value 0.071 0.124 0.037 -0.033 p value 0.047 0.104 0.307 0.332 soil r value -0.012 -0.102 0.011 0.189 p value 0.378 0.144 0.400 <0.001

Multiple regression analyses (Table 4.4) were conducted on species richness, burrowing species, total adult and total juvenile captures to test if specific habitat attributes around traplines influenced frog distribution across the three survey sites.

Where there were significant differences in capture rates between the three sites

(Table 4.2: species richness, burrowing species and total juveniles), these multiple regression analyses were repeated on captures within similar sites to test whether site to site abundance might be influencing regression results.

Results showed that species richness was lower around traplines that had a high covering of grass, but higher in areas that had a higher percentage of bare ground

(Table 4.4). Species richness was significantly higher at Site 3 than the other two sites (Table 4.2) and Site 3 had less grassy areas and more bare areas than Sites 1 and 2 (see Table 2.4 Chap 2). Regression analysis was repeated on Sites 1 and 2 combined and results showed that at Sites 1 and 2 capture rates were higher in

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areas that supported more sedges and had higher moisture content at the depth 1

(Table 4.4).

Captures of burrowing species were lower in areas with high amounts of grass and where soil pH was higher at depth 1 (Table4.4). Higher burrowing species richness was also associated with areas where the soil texture at depth 1 was sandier and with areas that had higher soil moisture content at depth 2, higher soil pH at depth 3 and more stones at depth 3 (Table 4.4). Because burrowing species richness was also highest at Site 3, Sites 1 and 2 were examined separately from Site 3 in further regression analyses. Results show a weak association between captures and sedges at Sites 1 and 2 (Table 4.4). At Site 3 higher captures were recorded where the soil contained more stones at depth 3.

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Table 4.4. Forward stepwise multiple regression results for frog species composition and habitat attributes within and between survey sites at Barakula State Forest. Species/guild Variable Slope R2 F p Species Richness grass -0.06 0.121 14.33 <0.001 All sites distance -0.01 0.206 13.37 <0.001 stones 3 0.327 0.270 12.58 <0.001 Sites 1 and 2 only distance -0.01 0.121 9.36 0.003 sedge 0.05 0.10 6.90 0.002 moisture 2 0.12 0.220 6.20 0.001 Site 3 stones 3 0.65 0.224 9.81 0.004 Burrowing Species grass -0.05 0.134 16.13 <0.001 Richness stones 3 0.25 0.201 12.92 <0.001 All sites pH 1 -0.76 0.244 10.95 <0.001 pH 3 0.97 0.300 10.80 <0.001 texture 1 -0.42 0.336 10.13 <0.001 moisture 2 0.08 0.363 9.42 <0.001 Site 1 and 2 only sedge 0.04 0.073 5.38 0.023 Site 3 stones 3 0.463 0.202 8.63 0.006 Total Burrowing Species distance -0.16 0.181 22.95 <0.001 moist 2 1.41 0.221 14.61 <0.001 Total Adult distance -0.21 0.193 26.05 <0.001 tree <1m 9.91 0.221 15.91 <0.001 Total Juvenile bare 0.37 0.100 12.62 0.001 distance -0.05 0.150 10.24 <0.001 grass -0.25 0.192 9.32 <0.001 texture1 -3.57 0.235 9.05 <0.001 tree>3m -6.05 0.268 8.470 <0.001 tree 1-3m 3.27 0.303 8.63 <0.001 Site 2 nil

Total captures of burrowing species was negatively associated with distance and more burrowers were captured at sites where the soil moisture at depth 2 (15-30 cm) was higher.

Multiple Regression analysis showed that more adults were captured closer to water

(-ve distance) and/or in areas that supported a higher number of small trees (trees

<1m). As all trapping events corresponded to rainfall events, it is likely that some of the adults captured were moving towards or away from the water body in response to reproductive cues, and therefore, had a higher chance of being captured in pitfalls

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positioned nearest the water. Of the 109 traplines throughout the three survey sites,

56 (51%) were located at distances <120 m from ponds or ephemeral pools and 53

(49%) were located >120m from water, therefore, if individuals were caught at random, the probability of frogs being captured in traplines closer to water (i.e.,

<120m) was roughly similar to that for frogs being captured at greater distances from water. However, 67% of adults were captured in traplines located <120m from the nearest pond or ephemeral pool, but a plot of total adult captures and distance from the nearest water (Figure 4.3) shows that adults were also regularly captured at distances > 150 m from the water, particularly at Sites 1 and 3.

200 site 1 2 3

150

100 Total Adult Captures Adult Total

0 50 100 150 200 250 Distance from Breeding Pond (m)

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4.2.3 INDIVIDUAL SPECIES AND HABITAT ASSOCIATIONS

4.2.3.1 General Observations

Of the seventeen species captured during this study, all but one, Lit. caerulea, (of which only one individual was caught), were caught as both adults and juveniles, indicating that despite the drought conditions, recruitment events had either occurred just prior to the commencement of the study and/or were occurring throughout the study period.

There was significant variation in capture rates between the three survey sites in all but three species (U. rugosa, C. parinsignifera and Cyclorana brevipes) (Table 4.2), and where variation in capture rates occurred, site to site differences were not uniform. For example, Site 2 recorded significantly more captures of adult L. ornatus and P. bibronii than the other two sites, but significantly fewer captures of L. tasmaniensis (Table 4.2). Site 3 recorded higher numbers of Notaden bennetti but fewer captures of L. terraereginae (Table 4.2).

Capture rates of adults for three species, U. rugosa, L. tasmaniensis and N. sudelli, were generally higher than for other species across the three survey sites, although

Site 1 recorded lower captures of L. tasmaniensis and N. sudelli in comparison to the other two sites.

Captures of juveniles, however, was sporadic and generally coincided with recent metamorphosis of individuals. In most cases, juveniles were caught in traplines closest to the breeding pond in high numbers on the first day of trapping, but numbers decreased rapidly over the next two days, and juveniles tended to be caught further from the water over the successive days. For example on the 13th

June 2001 41 juvenile L. ornatus were captured at Site 3, of which 33 (80%) were in traplines located less than 75 m away from the breeding pond. On the 14th June only

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6 juvenile L. ornatus were captured and most of these were at least 100 m from the pond. Similarly, on the 11th January, 2004, 17 juvenile L. brevipes were caught at

Site 3, the majority (12) of which were in traplines that were located less than 90 m

away from the water. By the 13th January, 2004, only 3 juvenile L. brevipes were caught in two traplines and these traplines were 120 and 160 m from the water.

The rapid decrease in captures of juveniles over a short period of time, and the trend of captures occurring furthest from the breeding pond as time progressed, suggests that recently metamorphosed individuals were migrating away from the pond in a random manner, rather than utilising habitats in the terrestrial environment immediately adjacent to the waterbody. For this reason, captures of juveniles will not be analysed further, as habitat associations are most likely to be influenced by movement patterns (e.g., Sinsch 1990), rather than daily use of the terrestrial environment.

4.2.3.2 Habitat Associations

Multiple Regression analysis was performed on adult captures of the ten species that recorded >25 captures across the three survey sites and whenever captures were >20 within a site. Within site analysis was performed to test if low or high catch rates at a site were driving overall, regression results. Results showed significant influences of many measured habitat variables on capture rates in all species except for C. brevipes (Table 4.5).

Of the measured vegetation variables, grass, debris and logs in both sizes were the most influential ground cover variables, whilst trees in the height class > 3m in height also tended to influence the activity patterns in one of the species (Table 4.5).

Measured soil properties taken at the three depths, played a role in influencing activity patterns in five of the six burrowing species (Table 4.5). Four species were

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influenced by distance to the breeding pond, with this group tending to be caught more regularly within a short distance to the water (Table 4.6).

Table 4.5. Regression analysis results showing the negative and positive relationships between frog species, distance from pitfall traplines and some attributes of the terrestrial environment within Barakula State Forest. Species variable slope R2 F p U. rugosa* distance -0.14 0.188 24.1 <0.001 moisture 2 1.16 0.228 15.21 <0.001 Neobatrachus sudelli* leaf -0.16 0.057 6.25 0.014 stones 2 -0.97 0.096 5.44 0.006 distance -0.02 0.105 5.08 0.003 Sites 2 and 3 log <10cm 14.20 0.091 7.05 0.010 distance -0.28 0.157 6.41 0.003 pH3 4.04 0.239 7.10 <0.001 stones 1 -1.84 0.288 6.78 <0.001 Site 1 leaf depth -7.27 0.329 15.70 <0.001 L. tasmaniensis grass -0.35 0.192 24.64 <0.001 tree >3m -3.84 0.284 20.46 <0.001 debris -0.14 0.374 20.35 <0.001 distance -0.02 0.405 17.19 <0.001 Sites 1 and 3 log <10cm -6.46 0.193 16.28 <0.001 tree >3m -5.20 0.304 14.61 <0.001 debris -0.36 0.389 14.04 <0.001 grass -0.14 0.435 12.53 <0.001 tree<1m 3.68 0.500 12.82 <0.001 Site 2 sedge -0.30 0.241 10.81 0.002 L. ornatus* pH 2 -1.28 0.149 18.20 <0.001 bare 0.03 0.203 13.12 <0.001 L. terraereginae* moisture 1 2.10 0.050 6.26 0.021 texture 1 -0.70 0.102 6.14 0.004 texture 3 0.56 0.170 5.67 <0.001 texture 2 -0.84 0.226 7.397 <0.001 P. bibronii pH 1 -1.55 0.062 6.89 0.01 tree1-3m -0.92 0.127 7.42 0.001 Notaden bennetti* grass -0.04 0.1202 11.86 0.001 shrub 0.05 0.142 6.54 <0.001 C. parinsignifera bare 0.11 0.102 11.86 0.001 distance -0.01 0.142 8.54 <0.001 Site 2 log<10cm -6.64 0.27 12.56 0.001 L. salmini* grass -0.05 0.126 14.97 <0.001 stones 1 -0.47 0.194 12.38 <0.001 texture 2 -0.31 0.207 10.11 <0.001 Site 3 stones 1 -0.94 0.136 5.35 0.027 moist 1 -0.45 0.288 6.67 0.004

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Table 4.6. Minimum, maximum and mean distances (m) of traplines from the breeding pond where species were captured, including the proportion of captures that were in traplines located >120m from closest breeding pond (N = number of traplines each species were caught at). Species N Min Max Mean SD Proportion >120m U. rugosa 100 5 250 107.25 61.07 26% N. sudelli 74 5 250 116.9 62.77 41% L. tasmaniensis 74 5 225 106.28 61.5 45% L. ornatus 17 5 250 107.06 67.55 61% L. terraereginae 24 20 210 100 59.2 35% P. bibronii 35 15 250 98.28 58.03 34% N. bennetti 17 20 210 117.65 71.15 40% C. parinsignifera 16 15 180 83.44 60.68 11% L. salmini 18 30 210 134.17 53.09 68%

A summary of the multiple regression results for individual species is provided below. Information relating to each species habits is based on available literature and personal observations.

Uperoleia rugosa

Uperoleia rugosa is one of the smallest (18-32 mm SVL) burrowing species. This was the most frequently captured species, with 655 adult captures but only 33 juveniles recorded during the survey period. There were no significant differences in capture rates between the three sites. U. rugosa would face a higher risk of desiccation in comparison to larger frogs, so for this reason it is not unexpected to capture more adults closest to the water (Table 4.4 –ve to distance). Table 4.6 shows that only 26% of all captures were recorded at distances >120 m from the closest waterbody.

Uperoleia rugosa were also captured more frequently in areas that supported higher soil moisture at depth 2 (Table 4.5). The depth to which U. rugosa burrow is

unknown, but because of its small size it is expected this species would not burrow

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below 30cm (depth 2) and it could be expected that increased soil moisture 15-30cm below the surface would help prevent evaporative water loss for this species.

Neobatrachus sudelli

Neobatrachus sudelli is a relatively small burrowing species (38-49 mm SVL). This was the second most frequently captured species, with 217 adults and 56 juveniles being recorded across the three sites. Capture rates for both adults and juveniles were similar between Sites 2 and 3, but were significantly lower at Site 1 (Table 4.2).

Significantly more captures occurred in areas with low leaf cover. N. sudelli also tended to avoid areas that had a higher stone content in soil at depth 2 and was caught more frequently closest to the breeding pond, although 46% of all captures were recorded at distances > 120 m from the nearest waterbody (Table 4.6).

Because capture rates at Sites 2 and 3 were similar, regression analysis were repeated on the combined Site 2 and 3 data. Results show that N. sudelli was more frequently caught at sites that had a higher covering of small logs (logs <10cm) and where the pH at depth 3 was less acidic, but tended to avoid areas with increased amounts of stones at depth 2.

Limnodynastes tasmaniensis

This is a medium sized (31-47 mm SVL) very common species that is distributed over much of south-east Australia (Barker et al. 1995). Of the 207 captures of adults, over half (109) were recorded from Site 3. The results of the regression analysis for this species showed that habitat associations were generally influenced by ground cover attributes (grass and debris), with this species tending to avoid areas that had a high covering of grass and more debris. L. tasmaniensis were caught more frequently closest to the breeding pond (Table 4.6), but 45% of all captures of this species were recorded at distances >120 m from the breeding pond.

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Capture rates for this species were lower in areas that were dominated by trees in the 1-3 m height class.

When regression analysis was conducted on the two sites that had the lower capture rates (Sites 1 and 3), capture rates at these two sites were also negatively associated with logs <10cm and positively associated with trees <1m (Table 4.5). At site 2, where more captures of this species were recorded (Table 4.2), capture rates were lower in areas that supported more sedges.

Limnodynastes ornatus

Limnodynastes ornatus is described as a versatile, medium sized (29-42cm SVL)

common species (Barker et al. 1995, Anstis 2002) which is known to breed almost

anywhere there is standing water, which includes roadside ditches. Of the 150

captures recorded for this species, almost half (124) were of juveniles or

metamorphs (Table 4.1), and adults were rarely captured at Sites 1 or 3 (Table 4.2).

Regression analysis showed that this species was more frequently caught where the

soil pH was more acidic at depth 2 (15-30cm) and in bare areas (Table 4.5).

Limnodynastes terraereginae

Limnodynastes terraereginae is a fairly large (55-79 mm SVL) burrowing species

that is distributed along the entire Queensland coastline and into north central New

South Wales. 106 captures of this species were recorded, with the majority (72)

being juveniles. Site 1 recorded significantly more adults and juveniles than Site3,

but catch rates at these sites were similar to those at Site 2 (Table 4.2). Catch rates

of juveniles were higher than for adults at all three sites (Table 4.1).

The regression results indicate that movement activity for this large frog is

influenced strongly by the moisture and texture of the soil, rather than vegetative

cover. In particular, most captures of this species occurred in areas where the soil

contained less clay; except at depth 3. Burrowing through heavy clay may be

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difficult, but clay at the third depth would retain moisture for a longer period and thus provide greater protection against evaporative water loss.

Pseudophryne bibronii

Pseudophryne bibronii is a relatively small (22 – 32 mm SVL) ground-dwelling frog that is usually associated with areas that support damp soil and sufficient cover

(Anstis 2002). A total of 65 P. bibronii was captured during the study period, with the majority (61) of these being adults. Juveniles were not captured at Site 1 and the only juvenile caught at Site 3 was not a recent metamorph, suggesting that recruitment for this species might not have occurred at Sites 1 and 3 during the survey period or in recent times prior to the commencement of trapping. Catch rates for this species varied significantly between sites (Table 4.2) with the majority of captures (53) occurring at Site 2 (Table 4.1). Site 2 had significantly lower soil pH than the other two sites and multiple regression analysis, including all sites, indicated that catch rates were negatively related to soil pH, and the density of trees in the 1-3 m height class (Table 4.5).

Within Site 2, multiple regression analysis did not reveal any association between catch rates and vegetation or soil. However, this site had more areas containing highly acidic soil (Figure 4.4), with pH ranging from 4.3 to 5.9 (mean = 4.7), whereas at Sites 1 and 3 the range of soil pH values was greater (4.1 to 6.4 (mean = 5.2) and

4.0 to 6.1 (mean = 5.3) respectively). The relatively low range in variations of soil pH between the traplines at Site 2 may make it harder to detect a relationship between pH and catch rate at traplines for this species.

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Figure 4.4. Mean (+/- 95%CI) capture rates of P. bibronii and mean soil pH (+/- 95%CI) at the three survey sites within Barakula State Forest.

Notaden bennetti

Notaden bennetti is a medium-sized (42- 68 mm SVL) burrowing frog that spends

most of its life deep underground (Barker et al. 1995). A total of 54 captures was

recorded for this species, with the majority of these (34) being juveniles. Site 3

recorded significantly higher capture rates for both adult and juveniles (Table 4.2).

Site 1 recorded similar numbers of both adults and juveniles (3) and only one

juvenile was recorded from Site 2.

Like other burrowing species in this study, N. bennetti tended to avoid heavily

grassed areas, and was more commonly captured in areas that supported a higher

coverage of shrubs. Site 3 had significantly more captures of this species, and Site 3

also tended to have more shrub cover.

Crinia parinsignifera

This species is a very small (18-23 mm SVL) terrestrial frog that is usually found beneath debris at the edge of swamps or ponds (Barker et al. 1995). A total of 45 captures of this species was recorded across the three sites, with only 5 of these being juveniles. Although more of this species tended to be caught at Site 2, there

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were no significant differences in capture rates between the three sites (Table 4.2).

Higher catch rates of adults were associated with traplines in areas with low ground cover closer to water bodies (-ve distance) (Table 4.5). Only 11% of all captures for this species occurred at distances >120 m from the closest waterbody (Table 4.6).

Limnodynastes salmini

Limnodynastes salmini is a large (61-76 mm SVL) burrowing species. A total of 28 adult captures and 7 juvenile captures was recorded for this species. Adult catch rates were significantly higher at site 3 (Table 4.2), and all juveniles were captured at this site. Capture rates appeared to be influenced by soil properties, with this species avoiding areas that contained higher amounts of clay soil and higher levels of stones. Both of these characteristics of the soil are expected to influence the burrowing behaviour of this species. Catch rates were also negatively related to the amount of grass around a trapline. Within Site 3, where the majority of individuals of this species were captured, the stoniness and moisture of the upper levels of the soil tended to influence where this species was more active. There were no significant associations between capture rates and habitat attributes on the combined data for

Sites 1 and 2 (Table 4.5).

4.3 DISCUSSION

Despite the persistent drought conditions, a relatively high number of frog captures

(1844) was recorded during the survey period. These captures represented a diverse range of species that exhibit very different morphological, physiological and ecological traits. In particular, 828 captures, representing 17 species, ranging from the very small ground-dwelling C. parinsignifera to the large and robust burrowing species like L. terraereginae and L. salmini, were recorded from Site 3. The total trapping area of Site 3 was approximately 6.25 ha, and this relatively small patch of semi-arid woodland supported 17 frog species. Survey Sites 1 and 2 were of similar

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size to that of Site 3, and relatively high species richness (14 species) was recorded at these two sites. It is possible that more species would have been recorded at these two sites if trapping events were uniform across all sites.

It is not surprising, given the fairly harsh environmental conditions that prevail at

Barakula, and the fact that the closest permanent water (a tributary of Cutthroat

Creek) is situated approximately 7 km to the north-east, that over half of the species captured during this survey (9) are burrowing species. Cramp et al. (1998) found 17

of the 21 native species in a broadscale survey of 132 sites in Barakula State Forest

and adjacent farm land. For their study they used egg and larval sampling, hand

capture and aural surveys around breeding ponds, and 13 of the species recorded

in their study were common to this project. The ephemeral pool at Site 3 was part of

their survey and they recorded nine species at this site (Cramp unpublished data).

Although amphibian species diversity is usually found to be higher in studies

covering larger areas with more mesic conditions (e.g., 25 anuran species in an east

coast closed forest; Parris 2004), other studies have shown that small isolated

patches of wetlands, and/or temporary ponds also support high amphibian species

diversity (Dodd 1992; Dodd and Cade 1998 Gibbons et al. 2006). These three

studies recorded similar numbers of species to that found during this study (15, 16

and 17 respectively), although it should be noted that two of these studies (Dodd

1992 and Dodd and Cade 1998) were not restricted to anurans, and were conducted

in wetter environments. As many species in the present research were recorded in

the furthest traplines, it is likely that the frog assemblages at Barakula are utilising

greater areas of terrestrial habitat in the adjacent woodlands than the study

encompassed (i.e., >6.5ha at each survey site).

In general, the terrestrial habitat associations found in this study matched what is

known about the ecology of each species, and the fine-scale terrestrial habitat

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characteristics within Barakula State Forest have some effect on the movement patterns and/or composition of frog assemblages. Captures of burrowing species and species richness were consistently higher in areas that had lower grass cover.

Most grasses tend to form a thick cover of stolons and a dense network of fibrous roots and it has been shown that frogs find it difficult to burrow through grasses to gain refuge in the soil below (Jansen et al. 2001). Other patterns of associations with measured vegetation attributes were less obvious, and it is likely that more detailed and/or experimental studies (e.g., Faccio 2003; Van Sluys et al. 2007) are needed before more definitive habitat associations can be identified.

To my knowledge, this is the first Australian study that has systematically studied the influence of soil properties on the distribution of burrowing species, despite the fact that one third of Australian anuran species are burrowers. Although the reasons why and how species burrow have received a fair amount of interest and study

(Hudson and Franklin, 2002; Lemckert and Brassil 2003; Cartlegde et al. 2006), the role that the physical and chemical characteristics of soil plays in influencing the distribution of burrowing species has received very limited attention.

The results of the Mantel tests and regression analyses convincingly showed that capture rates of burrowing species was strongly related to the soil properties at a trapline. Where traplines supported similar soil properties they also tended to support similar numbers and diversity of burrowing frogs, although this association was not evident at Site 3. This result is interesting, in that four of the nine burrowing species were either never caught or were caught in significantly lower numbers at

Sites 1 and 2 than at Site 3. This result suggests that a combination of vegetation and soil properties are responsible for influencing an increase in diversity and abundance of burrowing species at Site 3.

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Overall, the soil properties within Barakula State Forest played a significant role in influencing the distribution of six of the seven burrowing species examined. The texture of the soil, amount of stones present and moisture content were the contributing factors to the observed associations. Burrowing species appear to employ two different strategies to avoid desiccation during prolonged dry periods

(Shoemaker 1988), with these strategies being driven by soil type (Booth 2006). In areas that support sandy soils, frogs dig deeper into the soil over time as the soil dries (Ruibal et al. 1969), whereas in areas that support soils with high clay content, frogs tend to form burrows (10-30 cm below the surface) where they stay until the next heavy rainfall event (Booth 2006). It is expected that the different burrowing species at Barakula are also employing similar strategies, with two of the three

Limnodynastes species L. terraereginae and L. salmini) avoiding areas with higher clay content in the top 30 cm that would tend to be harder to burrow through, due to the adhesive nature of clay particles (Booth 2006). At the time of writing, there appears to be no documented evidence pertaining to these two Limnodynastes

species forming burrows or cocoons, although L. spenceri is known to form cocoons

(Lee and Mercer 1967). It is however, predicted that the observed associations with soil texture in these two species is driven by the second strategy employed by other species to avoid desiccation (Shoemaker et al. 1969). The observed avoidance of soils containing higher amounts of stones by Neobatrachus sudelli and Lim. salmini

is predicted to be the result of stony soils being difficult to burrow through (Jansen et

al. 2001).

Results from the Chapter 3 showed that frogs that burrowed at Barakula should

sustain the least amount of evaporative water loss, in comparison to sheltering

under some form of vegetative cover. High humidity is known to influence

evaporative water loss and it is expected that soil moisture would be higher and

would remain for longer periods in areas that have a more dense covering of some

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form. Additional studies are needed before more decisive predictions can be made regarding the influence soil properties play in the distribution of burrowing frog species. Although it is difficult to mark burrowing frogs in a manner that would allow observations of where they burrowed to be made, Lemkert and Brassil (2003) were successful in implanting radio-transmitters into a large burrowing frog. However, although they were able to locate where the frogs had burrowed, they did not record fine-scale attributes of the vegetation or soil properties at the burrowing site.

Of the three non-burrowing species, two (L. tasmaniensis and C. parinsignifera) were caught more frequently in traplines positioned closest to the breeding pond.

However, inspection of the data (Table 4.6) shows a considerable number of L. tasmaniensis were regularly caught in traplines >120 m from the breeding pond, particularly at Sites 1 and 3, although very few captures of C. parinsignifera were recorded in traplines >120 m. It is likely that the high capture rates of L. tasmaniensis (26) at one trapline in Site 1 was driving the regression result, and in fact this species regularly moves considerable distances from the breeding pond.

Because of the greater surface to volume ratio, small species, such as C. parinsignifera, face a particularly high risk of desiccation, which probably explains why this species was more commonly captured nearest water, or areas where temporary pools form. Traplines closest to the water tended to have more bare areas and it is expected that this species would tend to find shelter in cracks that appear in the clay soil surrounding the waterbodies.

The association between captures of P. bibronii and soil pH is difficult to explain, as this species does not burrow and it was unexpected to find an attribute of the soil influencing the distribution of this species. Soil pH has been shown to play a role in influencing the distribution of some amphibian species (Wyman 1988), with species generally avoiding acidic soils. However, in this study, total burrowing species, species richness and the capture rates of two species, L. ornatus and P. bibronii,

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tended to be higher in areas where the upper layer of the soil tended to be more acidic. The reasons why this suite of frogs tended to be different in their associations with soil pH in comparison to other studies which suggest that amphibians avoid acidic areas, is interesting, and it is possible that other synergistic relationships exist, such as flora species composition and hence leaf litter accumulation influences the soil pH and frog species distribution.

The environment in semi-arid regions of Australia, such as Barakula State Forest, can be particularly harsh for all organisms. Long, hot, dry periods are a regular occurrence, as are cold, dry periods. As this is the first intense research that has attempted to identify which terrestrial microhabitat attributes influence the distribution of a suite of frogs living in a semi-arid region, reference to previous studies that would allow comparisons of results is very limited.

Overall, the results of the habitat associations conducted during this study suggest that the morphology/physiology of the different species influences where they are more found, with even small variations in microhabitat attributes playing a role in the observed distribution patterns. However, because the correlations identified in this study do not imply causality, and because individuals were not marked during this study, the role these habitat attributes play in influencing refuge site selection in different species is unclear. In the next two chapters I experimentally test some of the observed relationships by manipulating attributes of the terrestrial environment to test the response of individuals and/or species to changes in microhabitat conditions.

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Chapter 5:

HABITAT CHOICE TRIALS

5.0 INTRODUCTION

The study of how and why organisms select particular habitats is regarded as central to ecology (Huey 1991) and the interactions between organisms and their environment determine their distribution and abundance (Krebs 1972). Habitat choice reflects the animal’s need to utilise the environment advantageously to ensure survival, with peculiar biologies of different species matching the features of the environments in which they live (Begon et al. 1996b).

Habitat choice is often driven by physiologically mediated interactions between organisms and their physical environments (Huey 1991). This may be particularly evident in ecototherms as these are more sensitive to temperature than endotherms and the thermal consequences of their habitat selection may be relatively conspicuous (Porter and Gates 1969). Research on reptiles has shown that habitat selection can influence the potential energy gain of an ectotherm and influences the energetic costs associated with thermoregulatory movements (Huey and Slatkin

1976).

Habitat choice in the aquatic environment has been widely studied in frogs. Factors that influence breeding habitat choice or oviposition site choice are varied and include interspecific tadpole predation (Petranka et al. 1994), physical characteristics of the water body (Laurila 1998, Hagman and Shine 2006), and the presence of fish (Orizaola and Braha 2006). However, fewer studies have examined the underlying reasons for within site variations in amphibian distributions.

Adult amphibians are carnivorous and urinary excretion of ammonia or urea requires amounts of water vastly in excess of inputs from the diet and oxidative metabolism

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(Shoemaker et al. 1975). Also water moves readily by osmosis across the skin of amphibians. When amphibians are maintained in fresh water this results in a very large net water influx (Shoemaker and Nagy 1977), and they may incur substantial water deficits during periods spent out of the water. In many anuran species loss of body water elicits an increase in the hydraulic conductivity of the skin, which enables the animal to rehydrate very rapidly either in water or from a wet surface

(Shoemaker and Nagy 1977). These physiological constraints may have a compelling influence on the habitat selection in amphibians, with choice being driven by the need to maintain a sustainable level of body water.

The daily movement of frogs is governed by their needs to find food, shelter and mates (Woolbright 1985). During these movements, the regulation of body moisture and thermoregulation may dictate which areas different species utilise in search of their required resources. For example, fossorial frogs have the ability to absorb more water from dryer soil than species that do not normally burrow (Walker and

Whitford 1970) which may explain why burrowing frogs persist so well in arid regions.

Small sized frogs that have a larger surface to volume ratio lose more water per unit of surface than larger frogs (Seymour and Lee 1974), therefore, small frogs may be more likely to limit their movements to areas providing greater protection from desiccation (i.e., moist substrates and shaded areas), than larger frogs. Cricket frogs (Acris crepitans) are a very small frog, with males averaging only 25mm. A laboratory examination of their microhabitat preferences (Smith et al. 2003) showed that when given a choice of wet or dry substrates 87% of the experimental frogs chose to move to the moist substrate.

The survival and movement of individuals through the terrestrial environment is the critical process that ensures successful dispersal and recolonisation among

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metapopulations (Semlitsch 2002) and dispersal has been shown to have a stabilising effect on local populations (Ruxton et al. 1997). Research shows that juvenile dispersal from the natal breeding pond is the major process that facilitates colonisation of new breeding ponds (Breden 1987; Berven and Grudzien 1990).

Juvenile dispersal rate has been shown to be extensive (up to 62% annually) and can occur over long distances (>5 km) (Funk et al. 2005). Quantitative models of amphibian populations have demonstrated that reduction in juvenile or adult survival was more likely to lead to population declines than the same magnitude of reduction in embryo survival. Ultimately the attributes of terrestrial habitat can influence the distribution, demographic and ecological performance of frog species (Huey 1991).

Predation is considered to be the major selective pressure for various physiological and behavioural strategies (Woodbury 1986). However, it is possible that more than one selective pressure is guiding the observed behavioural outcomes. For example, substrate choice in amphibians is known to occur for physiological reasons independent of predation risk (Tracey 1989) and the grouping of newly metamorphosed anurans has been shown to be related to desiccation avoidance rather than an antipredator behaviour (Heinen 1993a). Substrate choice in toads was also shown to be independent of the presence of a predator (Heinen 1993b).

The need to broaden our knowledge of terrestrial habitat preferences in amphibians should drive researchers into conducting more quantitative studies, as these can provide a more accurate assessment of habitat preferences. However, quantitative studies are often labour-intensive. For this reason, experimental trials investigating habitat use are useful. Those studies that have used amphibian species in experimental trials have focused on the larvae (Heyer et al. 1975, Smith 1983,

Stredl and Collins 1992, Marsh et al. 1999), oviposition site (Hopey and Petranka

1994, Johnson and Semlitsch 2003, Kiesecker and Skelly 2000) and migration distances for adults (Streatfeild 1999, Richter et al. 2001, Faccio 2003).

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The results from the previous chapters indicate that frogs inhabiting a semi-arid region tend to avoid particular microhabitats during nocturnal movements and results from the agar models suggest that different shelter sites provide vastly different levels of protection against desiccation. Shelter site choice may be particularly important for this suite of frogs because they often have to withstand extreme environmental conditions. The aim of this chapter is to investigate if different species actively avoid or seek out particular microhabitats during their nightly activity and if refuge site choice of different species is influenced by the type of microhabitat they are presented with.

The ecological studies of particular animals not only focuses on the relationship between organisms and their environment, but also on how these relationships are taking place (Schmid 1965). One approach to answering the latter question is to fabricate controlled conditions and study the organisms responses within those conditions. As discussed previously, marking of individuals to assess habitat utilisation was not considered for the current study. However, indications of habitat utilisation can be made if appropriate enclosures that simulate natural conditions are created (Hairston, 1989, Abamsky et al. 1990, Everett and Ruiz 1993). To this end I

fabricated ten enclosures to test habitat preferences in five frog species. A short

laboratory trial was also conducted to assess microhabitat preference in an

additional species. Availability (i.e., which species were active at the time of trials)

limited species selected, though all species used showed non-random distribution in

relation to pitfall trap captures and therefore, may utilise specific microhabitat types

more frequently.

In addition to the habitat choice experiments, juvenile movements away from the

natal breeding ponds were opportunistically observed at Barakula. An experimental

manipulation of the surrounding terrestrial habitat was also conducted to gain an

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insight into habitat utilisation for a group of newly metamorphosed Limnodynastes ornatus individuals.

5.1 METHODS

5.1.1 HABITAT CHOICE TRIALS

To test whether different frog species showed any preference or avoidance of particular microhabitats, it was necessary to create escape-proof enclosures that would allow observations of the movements of the frogs during the evenings. To accomplish this, ten 1.5 m x 1 m x 1 m high escape-proof enclosures were constructed using clear plastic sheeting attached to metal frames, positioned in the yard of the forestry house at Barakula (Figure 5.1).

Two elements of habitat use were examined: refuge site choice, and microhabitat use while active. Where sample sizes were large enough, refuge site choice was examined using Chi square goodness of fit tests, or binomial tests for lower sample sizes. However, analysis of data on microhabitat use while active was treated descriptively because data included repeated observations on the same individuals

(4 x observations on each individual taken at 1 hour intervals) and independence of observations could not be assumed, and because some observations were missing.

Missing observations were more likely to occur when frogs were using one of the three non-bare microhabitats, therefore, estimates of the use of non-bare microhabitats were likely to be underestimated.

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Figure 5.1. Photo showing the enclosures used to test microhabitat choice in different frog species captured in pitfall traps at Barakula State Forest.

Each enclosure contained a 10 cm layer of soil obtained from near one of the study

sites. In each quarter of the enclosures different microhabitats were created using

plants, leaf litter and debris, in the form of bark and sticks, obtained from one of the

study sites (Figure 5.2). The fourth quarter was bare and contained no cover. For

the first four nights shrubs were used as the plant material and the remaining five

nights clumps of grasses were used. Microhabitats were chosen on the basis of

those available at sampling points (Chap. 2) and perceived habitat preferences from

pit-fall trapping data (Chap. 4). Microhabitats were allocated randomly within

enclosures. The trials commenced on the 3rd February, 2004 and ran for nine days.

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Figure 5.2. Photo showing the four different microhabitats (vegetation, debris, leaf litter and bare) created in each enclosure to test microhabitat choice in different frog species.

To ensure that variation in soil moisture did not influence site selection, a watering can was used to apply equal amounts of rain water to each microhabitat within the enclosures before each trial. At the completion of each trial, the soil was raked in an effort to mask any chemical traces that the previous occupant may have left that could inadvertently influence the movement patterns of frogs. The soil was then smoothed over to allow identification of where individuals had burrowed during the evening, as evident by disturbance to the soil.

Frogs used for the trials were captured from the pitfall traps the previous evening and were kept in 11 L buckets that contained sufficient cover and moisture. Prior to placement in the trials, frogs were fed with termites obtained from a nearby nest.

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At dusk (~6.30pm) on each evening one individual was placed in the centre of each enclosure. At hourly intervals until approximately 10.30pm each enclosure was inspected and the microhabitat that each frog was located in was recorded. To minimise disturbance during these inspection times a hand-held torch, covered with red cellophane paper, was used for illumination. If a frog could not be observed at the specified hour, no attempt was made to search the habitats, as this form of disturbance may have biased results, therefore, some observations were recorded as ‘missing’.

At 6.00am the following morning the enclosures were thoroughly searched and the habitat in which the individual had sheltered was recorded. At this time frogs were removed from the enclosures, placed back in holding enclosures and released near point of capture that evening.

5.1.2 PSEUDOPHRYNE BIBRONII CHOICE TRIALS

This experiment was conducted in the laboratory to explore the hypothesis that individual P. bibronii could discriminate between differences in soil pH.

Approximately 0.4m3 of soil with pH of 4.6 was collected from one site in Barakula

State Forest. Half of this soil was treated with garden lime to raise the pH to 5.6. A

15 cm layer of each soil type (pH 4.6 or 5.6) was placed in each half of a 50 x 50 x

30 cm container with a 14.5 cm high plastic divider that separated the soil types, but allowed free surface access to both sides of the container. Two small depressions were created in each half and leaf litter was placed over the depressions. Four replicate containers and twenty laboratory-reared P. bibronii metamorphs were used for these trials. Each morning the side of the container where the individual had sought refuge was noted. To mask any affect left by the previous occupant, the soil was disturbed and new depressions created before replacement of metamorphs on the next evening.

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5.1.3 METAMORPH MICROHABITAT USE

On the 4th March, 2004 numerous Limnodynastes ornatus metamorphs were observed at the edge of a drainage line situated in open woodland, approximately

12 km north of Site 3. This water body was approximately 1.5 m in width and ~ 50 meters long and oriented in an east-west direction. To determine whether these metamorphs were showing any preference or avoidance of microhabitats, a series of observations on the distribution of these metamorphs was made, followed by a short manipulation experiment.

Observations: On March 4, a total of 31 1 x 1 m quadrats were placed across three different distance categories from the edge of the water (<5m, 5-10m, 10-15m) on both the northern and southern side of the waterbody. Each quadrat was thoroughly searched and the number of metamorphs counted. Moisture level was ranked on a scale from 1 (dry) to 5 (wet), and the amount of ground cover was estimated by eye to the nearest 10%. Quadrats were positioned haphazardly within each distance category, with the aim of getting a range of ground cover values within each zone.

Habitat Manipulation: In this trial, the ground cover on the southern side of the waterbody was manipulated to give the following three habitat types:

1. Bare – all leaves, vegetation and debris were removed

2. Untouched – normal covering of leaf litter and debris

3. Additional Cover - more leaf litter and debris were added

Each habitat type was created in a 1 m x 10 m belt transect and treatments were replicated four times to give a total of 12 transects.

The moisture content of the substrate was ranked by visual observation and by touch, from 1 being very dry soil to 5 which was very wet soil. Each transect was

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subdivided into 5 x 2 m lengths (quadrats) and the distance from the waters edge was categorised from 1 to 5, with 1 being within the first 2 m, and five being within the last 2 m of the transect (8-10 m from the water).

Observations were made over five days. Each transect was walked and the number of metamorphs in each 2 m section were recorded along with the moisture of the substrate in that section. Whilst traversing the transects due care was taken to detect any individuals moving between quadrats in response to my presence. When this occurred the original quadrat where the individual was first observed was recorded.

5.2 RESULTS

5.2.1 HABITAT CHOICE TRIALS

Twenty (20) Limnodynastes terraereginae, 20 Limnodynastes ornatus, 16

Neobatrachus sudelli, 20 Cyclorana brevipes and 10 Limnodynastes tasmaniensis were used for the choice trials. Results for each species are provided separately.

Limnodynastes tasmaniensis

About 20% of observations of L. tasmaniensis night-time activity were missing but

the distribution of observations across microhabitats suggests that individuals did

not show any tendency to avoid or favour particular microhabitats (Table 5.1).

However, observations of diurnal shelter site choice suggested a preference for the

debris over the other three treatments (binomial test – debris versus leaf + grass +

bare; p <0.001).

Limnodynastes terraereginae

Less than 10% of observations of active L. terraereginae were recorded in the bare

microhabitat (Table 5.1). Although more than 40% of observations were missing, it

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is unlikely that these involved individuals on the bare areas. The data suggest that L. terraereginae avoid bare areas while active. Data for diurnal shelter site choice also suggest that L. terraereginae avoid bare microhabitats, although the data were not significant at the 0.05 level (χ2 = 6.8, p=0.079).

Limnodynastes ornatus

Limnodynastes ornatus showed no tendency to avoid or favour particular microhabitats during night-time observations (Table 5.1). One quarter of the observations was missing, but individuals were found in all microhabitat types. All 20

L. ornatus individuals tested burrowed for diurnal shelter and there was a significant trend for this species to avoid bare and grass microhabitats (χ2 = 21.2, p<0.001).

Neobatrachus sudelli

This species also showed no tendency to avoid or favour any microhabitat during night-time activity, although only 12.5% of observations were on the bare microhabitat (Table 5.1). N. sudelli was the only species that sought shelter in the bare habitat, though there was still a trend to avoid burrowing in bare microhabitats

(1 of 16 frogs tested). The distribution of shelter site choice was not significant at the

0.05 level (Binomial test – bare versus debris + leaf + shrub; p=0.053).

Cyclorana brevipes

Cyclorana brevipes showed no preference for any microhabitat in the night-time activity observations (Table 5.1). However, the shelter site data showed a significant trend for this species to favour debris microhabitats (70% of observations) (χ2 =

26.4, p<0.001).

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Table 5.1. Results of habitat selection trials conducted in enclosures at Barakula State Forest, showing the number and percentage of frogs recorded in each manipulated microhabitat. Species Trial Debris leaf grass bare missing TOTAL L. tasmaniensis Activity No 8 5 5 13 9 40 % 20 12.5 12.5 32.5 22.5 Retreat No 8 1 1 0 10 % 80 10 10 0 L. terraereginae Activity No 20 6 13 7 34 80 % 25 7.5 16.25 8.75 42.5 Retreat No 7 7 6 0 20 % 35 35 30 0 0 L. ornatus Activity No 8 19 10 23 20 80 % 10 23.75 12.5 28.75 25 Retreat No 6 13 1 0 20 % 30 65 5 0 N. sudelli Activity No 11 15 17 8 13 64 % 17.2 23.4 26.6 12.5 20.3 Retreat No 5 5 5 1 16 % 31.25 31.25 31.25 6.25 C. brevipes Activity No 19 22 11 14 14 80 % 23.8 27.5 13.8 17.5 17.5 Retreat No 14 0 6 0 20 % 70 0 30 0

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5.2.2 PSEUDOPHRYNE BIBRONII CHOICE TRIALS

Of 20 juvenile P. bibronii individuals tested, significantly more (17) settled on the low pH soil (χ2 = 9.8; P = 0.002). Most (15) were located in the created depressions

(Figure 5.3) under the cover provided, suggesting that movement was instigated by a need to seek shelter.

Figure 5.3. Pseudophryne bibronii metamorph used to test soil pH preferences in experimental chambers.

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5.2.3 METAMORPH CHOICE TRIALS

Observations

A total of 141 L. ornatus metamorphs was counted across the 31 quadrats, with

numbers ranging from 0 to 20 per quadrat (mean = 4.46/m2, SD = 4.11). Mean

numbers tended to decline with distance from the edge of the water body (F2,28

=5.94, p = 0.007) (Figure 5.1). Median rank moisture level also declined with distance from the water body (Kruskal-Wallis H = 20.3, df = 2, p<0.001) (Figure 5.4).

Ground cover ranged from 5 to 90% (mean = 30.0%, SD = 22.9%), but did not vary with distance from the edge of the water body (Kruskal-Wallis H = 0.47, df = 2, p =

0.790).

12 Metamorphs Moisture

6 Mean +- 2 SE Mean +- 2

1 2 3 Distance category

Figure 5.4. Mean (+/- 2SE) number of observed L. ornatus metamorphs per m2 and moisture levels (mean rank) in relation to distance from edge of the water body.

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Because there were no quadrats in some moisture categories (e.g, all driest ranked quadrats were furthest from the water and most wet ranked quadrats were close to the water), and because the distance covered was only 15 m, distance was not considered in the following analysis. There was a positive and significant correlation between the number of metamorphs per quadrat and quadrat moisture levels

(Spearman’s r = 0.705, p<0.001, n = 31). Given that numbers increased with moisture level, the effect of ground cover on numbers per quadrat was examined with a partial correlation (correcting for moisture) (Daniel, 1995). There was a positive and significant partial correlation between these variables (Spearman’s partial r = 0.442, p = 0.014), suggesting larger numbers of metamorphs occurred in quadrats with higher levels of cover (Figure 5.5).

moisture 20 1 2 3 4 5

10 metamorphs

0 20 40 60 80 100 Ground cover (%)

Figure 5.5: Scatter plot showing the relationship between the number of Limnodynastes ornatus metamorphs and % ground cover in quadrats with varying levels of moisture.

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Habitat Manipulation

In the manipulation trial a total of 312 L. ornatus metamorphs was counted over the

five days of observation (mean = 0.52/m2, sd = 0.67), which was lower than the

mean number counted in the initial observations five days earlier (mean = 4.46 / m2,

sd = 4.11). This difference represents dispersal and mortality over the study period.

Totals counted on each day ranged from 47 on day 4, to 82 on day 5 (mean = 62.4,

sd = 14.7), indicating that there were different numbers of metamporphs using the

area around the water body on the different days, therefore, for the following

analyses, numbers were averaged across each quadrat over the five days, and the

median moisture index for each quadrat over the five days was used.

Mean numbers per quadrat also showed a decline with distance from the pond edge

in the manipulation study (F4,55 = 11.25 p=<0.001) (Figure 5.6). Moisture and

distance were also related in these observations (Kruskal Wallis H = 52.46, df = 4, p

< 0.001) (Figure 5.6). Given its relationship with moisture, and the likelihood that

metamorphs are likely to move freely over the area studied, distance was omitted

from further analyses. Also, because not all moisture categories were represented in

each habitat, the moisture categories were further condensed by combining ranks 3,

4 and 5.

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Average metamorphs 5 Median Moisture level

2 Mean +- 2 SE

1 2 3 4 5 Distance categories

Figure 5.6. Mean (+/- 2SE) number of metamorphs/m2 and moisture levels in relation to distance from the water body for the habitat manipulation trials.

A two way ANOVA examining the effects of moisture (3 levels) and habitat (cover) indicated there was a positive and significant effect of moisture (F2,51=25.16, p<0.001) but no effect of habitat (F2,51=0.936, p<0.399) although Figure 5.7 suggests there is a trend towards metamorphs showing preference to microhabitats with cover, rather than bare.

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Moisture category 3 1 2 3

1 Average metamorphs (Mean +- 2 SE) 0

1 2 3 Habitat category

Figure 5.7. Mean (+/- 2SE) number of Limnodynastes ornatus metamorphs per m2 in relation to bare (1), normal cover (2) and extra cover (3) habitat categories and three moisture categories.

5.3 DISCUSSION

Of the limited number of studies that have experimentally tested terrestrial habitat preferences in amphibians by providing choices of different microhabitats (Heinen

1993b, Hamer et al. 2003, Rittenhouse et al. 2004) only Heinen (1993b) provided test individuals with a choice between bare and covered substrates. To my knowledge this is the first study that has experimentally tested activity levels of different anuran species in microhabitats that provide varying levels of cover.

Of the 86 frogs tested in the microhabitat choice trials, all but one sought shelter under some form of cover during daylight hours. A single Neobatrachus sudelli was found in the bare habitat in the morning, but this individual had burrowed. Burrowing species comprised 88% (76 individuals) of frogs used in this experiment, with 10 of these being juveniles, 20 metamorphs and 46 adults. The only adult frogs that were

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not buried in the mornings were all Limnodynastes terraereginae. This species has been observed sheltering under cover, rather than burrowing on numerous occasions (personal observations) when the soil moisture is relatively high, so it is expected that changes in environment cues may prompt this species to burrow, rather than burrowing on a daily basis. An increase in vegetation cover has been shown to produce lower soil temperatures and higher soil moisture (Friedl and Davis

1994, Raich and Tufekciogul 2000), therefore, frogs that burrow into soil beneath some form of vegetation of cover would have lower desiccation rates than those that burrow into unprotected soil.

Of the 10 juveniles used in this experiment, only two had burrowed, but none of the metamorphs burrowed. Physiological limitations associated with small body size may force recently metamorphosed individuals to congregate in places and be active at times that are different to adults (Taigen and Pough 1981). There have been limited studies on changes in habitat preferences between adult and juvenile amphibians (Stewart 1985, Freeland and Kerin 1991, Beard et al. 2003) with no studies comparing burrowing behaviour between adult and juvenile frogs. It is therefore unclear if the observed lack of burrowing in juvenile frogs in comparison to adult frogs is a behaviour normally occurring in the natural environment.

The habitat choice trials were conducted primarily to quantitatively validate the habitat associations observed in the pitfall trapping (Chapter 4). Three of the four species used for the trials, had shown associations with one or more of the quantified habitat variables discussed in Chapter 4. Brief descriptions of the behaviour and habitat preferences for the four species are provided below, together with a summary of the results of the habitat choice trials.

Limnodynastes tasmaniensis is a medium sized terrestrial frog that is regarded as a habitat generalist (Schell 2001). It is a widespread species that is typically found in

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marshy country in the vicinity of grass-lined streams and ponds or flooded paddocks. During the day it shelters under logs, stones and debris (Barker et al.

1995). Results of the pitfall trapping showed a negative association with debris, grass and trees >3m, suggesting that this species readily uses areas that are devoid of vegetative cover. Evening activity during the choice trials occurred across all microhabitats, but more often in the bare habitat. Also 8 of the 10 individuals trialled sought shelter under debris in the mornings and avoided bare and grass microhabitats.

Limnodynastes terraereginae is a large burrowing species that is associated with dams, flooded areas and ditches in forests, woodland, cleared land and farmland.

The associations observed in the pitfall trapping indicate that soil properties, rather than vegetative cover play a greater role in influencing where this species tends to be more active. Soil properties were not considered in the choice trials. However, night time activity occurred more often in the debris and shrub habitats, and all individuals avoided the bare area during the day and chose to shelter under some form of cover. This result suggests that although no associations with vegetation attributes were observed in Chapter 4, vegetation may play a role in the relationship between the distribution of this species and soil properties.

Limnodynastes ornatus is a medium sized burrowing species that occurs in a variety of habitats often some distance away from permanent water (Barker et al. 1995).

Results from Chapter 4 showed significant and positive correlation with bare areas.

During the choice trials this species was regularly observed in the bare areas during the evening, but sought shelter under debris during the day. These results only partially confirm the results from the field.

Cyclorana brevipes is a medium sized burrowing frog that inhabits dry forest and grasslands, where it burrows underground for most of the year (Barker et al. 1995).

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No significant habitat associations were observed during the pitfall trapping, which could be a result of the low capture rates. The results of the choice trials for this species also suggest that this species shows no preference for specific habitat characteristics during night time activity, but tends to prefer to shelter under debris.

Neobatrachus sudelli is a medium sized burrowing frog that is associated with open grasslands and lightly wooded areas (Barker et al. 1995). Results from the pitfall data suggest that this species avoids areas that have a high cover of leaf litter.

However, in the choice trials this species was found significantly more often sheltering in the leaf during the day.

The inconsistencies in the choice trials and field data may be due to a number of factors:

A single day trial may not be long enough to allow the individuals to become

acclimatised to their new environment, and their movements may have been

driven by a need to escape, rather than reflect the normal movement patterns

they would display when foraging or moving to or from the breeding pond;

The enclosures may not have adequately reflected the natural environment; or

There may have been insufficient numbers of individuals of each species

trialled to give a true reflection of habitat utilisation.

Nevertheless, the data do indicate that adult and juvenile frogs distinguish between microhabitat types even when burrowing.

Results from Chapter 4 indicated that the presence of Pseudophryne bibronii was negatively associated with high soil pH. In the choice trials metamorphs of this species consistently chose more acidic soils for shelter. Post-metamorphic dispersal by juveniles from the natal nesting site has been shown to be common in anurans

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(Breden 1987; Pope et al. 2000; Sjogren 1991). The migration of individuals to appropriate sites creates the metapopulation structure observed in many amphibian communities, and reduces the potential for genetic differentiation in large populations (Blaustein 1994). The choice trials showed that juveniles discriminate between differences in soil pH and may therefore, avoid areas with unsuitable soil pH. Thirteen different, sometimes overlapping, soil types have been identified within

Barakula State Forest, which typically display pH levels ranging from 4.4 to 6.8

(Maher 1996). The data suggest that these different soil types influence the distribution of P. bibronii and support the results from the trapping data in Chapter 4.

Dispersal of newly metamorphosed frogs away from the natal pond can place the individuals under increased pressure from predation or loss of body moisture and recently metamorphosed frogs may exhibit patterns of activity and habitat selection that differ from those of adults of the same species (Freeland and Kerin 1991). The observations of the L. ornatus metamorphs suggest that for this species the need to maintain a moist epidermis was stronger than the need to avoid predation, as individuals did not show a preference for quadrats that contained some form of cover.

During the three year period of this study, cane toad metamorphs were observed during daylight hours at the edge of permanent water bodies in Barakula State

Forest on numerous occasions. L. ornatus species were the only endemic species exhibiting this same behaviour, with all other species metamorphosing and dispersing away from the water during the evening. Research has found that metamorphs of cane toads congregate in large numbers near the waters edge, are heliophilic (Van Beurden 1980) and activity in recently metamorphosed individuals is promoted by warm temperatures, moist substrates and conditions that allow for evaporative cooling (Freeland and Kerin 1991). I have been unable to locate any published literature that suggests that L. ornatus metamorphs are also heliophilic

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and avoid nocturnal activity, at least in the first five days of emergence from the aquatic environment.

In this chapter, I have shown that all species tested in the habitat choice trials showed some level of association with microhabitats that provided cover for day time sheltering sites. It is uncertain if this cover is necessary for protection from desiccation or from predation and it would involve more labour-intensive experimental studies to determine the precise cause for the observed habitat associations. However, the association between P. bibronii and soil pH is not likely to be due to predator avoidance, and the soil properties would not play a role in reducing EVL because P. bibronii is not a burrowing species. In the next chapter I will investigate why the distribution of P. bibronii could be influenced by soil pH.

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CHAPTER 6: SOIL PH AND HATCHING SUCCESS IN PSEUDOPHRYNE BIBRONII

6.1 INTRODUCTION

Many species display a patchy distribution within their broad geographic range and it is often necessary to look at microhabitat conditions rather than landscape parameters, to gain an understanding of the features that facilitate or inhibit local distributions (Parris and McCarthy 1999; Pope et al. 2000). Before initiating an ecological investigation at the microhabitat scale, it is important to identify those attributes most likely to affect a species distribution. Soil properties are one environmental attribute that influences broad and fine scale distributions. For example, factors such as soil type, texture, moisture level, chemical composition

and pH have been shown to play a significant role in influencing the spatial structure

of plants (Bigelow and Canham 2002), invertebrates (Antvogel and Bonn 2001),

mammals (Claridge and Barry 2000) and herpetofauna (Woinarski et al. 1999). If any aspect of the life cycle of the target species is reliant on properties of the substrate for food resources, shelter sites or breeding sites, examination of this characteristic of the habitat should be included in the study design.

Results from Chapter 4 indicated that capture rates of the Brown Toadlet,

Pseudophryne bibronii, were related to the pH of the soil with higher capture rates associated with low soil pH. Habitat choice trials in Chapter 5 showed that P. bibronii metamorphs chose to shelter in soil with low pH. Previous studies have found that low soil pH can have a negative impact on the distribution of terrestrial amphibians

(Wyman and Hawksley-Lescault 1987, Wyman 1988), but it is not clear why soil pH influences the distribution of P. bibronii. One possibility is related to the terrestrial egg laying habit of P. bibronii, in that soil pH might influence hatching success (see below). Although this is not an attribute associated with adult or juvenile utilisation of

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terrestrial habitats, it is important to investigate whether this might be the reason for the association between a terrestrial habitat attribute and the distribution of individuals of this species.

P. bibronii is a relatively small (SVL 23-33mm), terrestrial frog distributed through south-eastern Queensland, eastern New South Wales, Victoria and south-eastern

South Australia. Like most other species of the genus, P. bibronii is a terrestrial egg layer. The breeding season is typically autumn (March/May) when males initiate breeding by calling from nest sites. These are usually small depressions located under leaf litter or logs, or at the base of clumps of vegetation. Males also call from burrows/tunnels constructed by other animals (e.g., spiders). Females attracted by the mating call will deposit between 60-160 large eggs (2-2.5mm diameter) (Tyler

1994) in direct contact with the underlying soil (Figure 6.1). Embryos develop within the capsule to Gosner stage 27 (Woodruff 1976), then embryonic diapause is initiated until sufficient rainfall inundates the eggs and hatching commences. In many areas of the distribution of this species, rainfall is highly unpredictable and eggs may need to remain viable within the nests in intimate contact with the soil for long periods (White 1993). Appropriate nest site choice is therefore, critical for preventing desiccation and predation of eggs.

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Figure 6.1. Photo showing typical Pseudophryne bibronii nest where eggs are laid in direct contact with the soil.

Previous studies on terrestrial egg laying frogs have shown that moisture content of nests influences reproductive success (Mitchell 2001) and embryonic mortality

(Woodruff 1976, Mitchell 2002b). Given the observed relationship between P. bibronii abundance and soil pH at the three study sites, soil pH might be influencing

P. bibronii distribution via an effect on survival of embryos that remain in contact with soil for extended periods. In this part of the study, I was interested in identifying whether soil pH might play a role in influencing the distribution and persistence of P.

bibronii populations via an influence on embryo survival. The aims of this chapter

were:

(i) to determine whether the pattern of P. bibronii distribution at the three study

sites was evident at a broader scale by examining soil pH at other breeding

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sites within Barakula State Forest where P. bibronii was known to occur,

and at potential breeding sites where P. bibronii was absent; and

(ii) to investigate whether pH levels within the terrestrial nest affect embryonic

survival.

6.2 METHODS

6.2.1 BROAD-SCALE ASSOCIATIONS

Previous studies in Barakula Sate Forest examined presence and absence of anuran species at 85 separate breeding sites (Simon Cramp, unpublished data).

Prior to the P. bibronii breeding season in 2003, 20 of these sites were selected; 10 where P. bibronii had previously been recorded as present, and 10 where P. bibronii had not been found. At each site soil samples were taken from positions typically used by P. bibronii as nest sites (e.g., depressions in low lying areas) and pH was determined. A subset of these sites was used for field experiments (Study 3 below), and at each visit any signs of P. bibronii presence were noted (calling males, presence of eggs).

6.2.2 MEASUREMENT OF SOIL PH

For all experiments, soil pH was measured using a calibrated glass electrode.

Following the methodology of Rowel (1994), 10 g of soil was added to 25 ml of demineralised water and the solution was agitated for 15 minutes prior to measuring pH. Soil types within the study area are typically acidic, ranging from pH 4.4 to 6.8

(Maher 1996). For this study, soils were classed as “low pH” for pH 4.8 or lower, and

“high pH” when pH was 4.9 or greater.

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6.2.3 EMBRYO MORTALITY - FIELD EXPERIMENTS

A series of field experiments were conducted to investigate the effect of soil pH on embryonic survival. In April 2003, thirty P. bibronii nests were assessed for position, moisture level, and amount of cover, to determine the normal environmental conditions to which embryos are exposed. Artificial nest sites used in the following experiments were constructed to match these conditions. These artificial nests were created to protect P. bibronii eggs that had been translocated from original nest sites.

Artificial nests were constructed using 120 mm lengths of 80 mm diameter flexible down-pipe screwed ~20 mm into the substrate (Figure 6.2). Eggs were collected within one to eight days after being laid and placed in a depression formed in the centre of the soil enclosed by the pipe. Eggs were covered with leaf litter found adjacent to the new nest site. After four weeks, eggs were removed from nests and placed in separate plastic containers, inundated with pond water for 48 hours, after which time hatched and unhatched embryos were counted. Unhatched eggs were considered viable if movement was detected under a hand-lens.

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Figure 6.2. Artificial nest sites used for translocated Pseudophryne. bibronii eggs to test if soil pH influenced hatching success of embryos place in artificial nest sites within Barakula State Forest.

6.2.3.1 Study 1

Twenty P. bibronii nests were located at one breeding site (pH ≈ 4.2) in May 2003.

Eggs were removed from each nest using a sterilised stainless steel spatula, placed on moistened sterilised gauze in separate petri dishes, counted and staged (Gosner

1960). Each clutch of eggs was divided into approximately three equal portions. One portion was returned to the original nest site, one portion was placed in an artificial nest next to the original nest, and one portion was transferred to an artificial nest at a breeding site approximately 15 km away that contained soil of high pH (pH ≈ 4.9).

Every possible effort was taken to ensure handling time was similar for each portion of eggs.

6.2.3.2 Study 2

The above experiment was repeated in July 2003 using ten nests from a different breeding site (pH ≈ 4.2). For this trial, eggs were separated into three portions; one

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set returned to original nest site, one set moved to artificial nests at a high pH site ~

5 km away (pH ≈ 4.9), and the third set was translocated to artificial nests at another breeding site with low pH ~ 12 km away (pH ≈ 4.2).

6.2.3.3 Study 3

Differences in hatching success for treatments in Studies 1 and 2 could indicate differences in the effect of soil pH, or they may be due to unique site effects because each pH treatment was restricted to a single site. A third study was conducted in May 2004 following the same procedures as Studies 1 and 2, but using five source sites and ten translocation sites. A combination of four treatments used in Studies 1 and 2 were applied in this experiment: a portion of eggs returned to nest, a portion to an artificial nest near original nest, a portion to an artificial nest at a site with high pH, and one portion of eggs taken to an artificial nest at a site with low pH. Note that each block or replicate comprised three sites; a source site for eggs and two translocation sites (low pH and high pH). Ten sites were used in total (5 high pH sites and 5 low pH sites), with each low pH site being a source site for one replicate and a recipient site for another). Distances between source and translocation sites ranged from ~ 3 – 18 km. These treatments were applied to five nests located at each of five different source sites, giving 20 observations per source site. Average values of the five replicates of each treatment from each source site (4 treatments x 5 sites) were used for comparisons between treatments

(randomised complete block ANOVA blocked by source site).

Soil pH at each artificial nest was recorded in study 3 to enable direct comparisons of the effect of soil pH on egg survival. Following the initiation of this experiment the study area experienced very cold dry conditions, that may have increased rates of soil moisture loss, so each artificial nest was inspected after two weeks, and 100mls of deionised water was added to the substrate adjacent to the eggs. All nests were left undisturbed for a further two weeks to ensure eggs reached Gosner stage 27.

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Eggs from the original nests were checked for signs of mortality and viable eggs were enumerated by visual inspection. However, hatching of these eggs was not induced as studies 1 and 2 had shown that there were no effects of the artificial nest environment, therefore, survival at nest was not included in statistical comparisons of survival.

Ideally, the treatments used in Study 3 should have been replicated using eggs from nests located at breeding sites containing high soil pH. However, although males occasionally were heard calling from sites with high soil pH during the two years of this study, inspection of these calling sites failed to find any eggs, suggesting low reproductive rates in these areas.

6.2.4 EMBRYO MORTALITY – LABORATORY EXPERIMENT

Any effect of soil pH on embryonic survival in field conditions may be a direct effect of soil pH or an effect of some other factor interacting with soil pH (e.g., some inhibitory agent whose impact varies with pH). To determine the role of pH, a laboratory trial was conducted using sterilised soil of four pH levels. Approximately

0.4m3 of soil with a pH of 4.3 was collected from around one of the breeding ponds and returned to the laboratory. Small quantities of garden lime were added to different amounts of this soil to raise the pH to 4.6, 5.0 and 5.3 and a quantity was left un-manipulated to give four pH treatments. Twenty replicates of water-saturated soil of each pH level were placed in 250ml screw-top jars. A depression was created in the centre of the soil in each jar, and then the soil and containers were sterilised by autoclaving under high pressure and temperature for twenty minutes. Four additional containers of soil were also autoclaved to determine whether the above procedure affected moisture or pH levels. No effect was detected.

Sterilised containers were transported to a breeding pond in the study area for addition of P. bibronii eggs. Twenty new nests were located, and using the handling

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techniques described above, twenty eggs from each nest were randomly assigned to each container of different pH (20 replicates of four pH levels). Containers were then returned to the laboratory and kept at 150C in a temperature-controlled cabinet for four weeks. At the completion of the trials, containers were inspected for signs of egg mortality and all embryos were submerged in containers filled with pond water.

After forty-eight hours, hatching success and egg survival was enumerated. Given that it is difficult to sterilise terrestrially laid frog eggs, the eggs used for laboratory treatments were not sterilised and therefore, might have had fungal or bacterial spores adhering to the capsules. Before immersion in water, the level of fungal infection in each container was scored on a point ranked scale (0 = no infection, 5 = high infection).

6.2.5 DATA ANALYSIS

Comparison of mean soil pH at field sites was made using a Student’s t test. Field and laboratory egg mortality trials were analysed with random blocks ANOVA

(blocked by nest for field studies 1 and 2 and lab study, blocked by source breeding site for field study 3) using arc sin transformed percent survival values. The blocked design allowed for any differences between nests (maternal effects on hatching success, differences in clutch sizes and developmental stages) to be accounted for in comparisons between treatments. A Pearson correlation was used to examine the relationship between soil pH and survival for field study 3. In the laboratory study, relationships between ranked fungal infection levels and survival were investigated with a Spearman rank correlation, and differences in ranked fungal infection levels and soil pH were tested with a Friedman nonparametric two-way

ANOVA.

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6.3 RESULTS

6.3.1 BROAD-SCALE ASSOCIATIONS WITH SOIL PH

Average soil pH at sites where P. bibronii had been recorded as present (mean =

4.80; SD = 0.21; n = 10) was significantly lower than pH at sites where P. bibronii had been recorded as absent (mean = 5.15; SD = 0.17; n = 10)(t18 = 3.61; p =

0.019). Subsequent visits to 10 of these 20 sites indicated that P. bibronii were present at low pH sites (calling males and/or eggs) (5 of 5 sites examined), and generally absent from all high pH sites examined (5 sites) (Figure 6.3). Some males were heard calling at one high pH site, but males were few in number compared with low pH sites, and searches failed to find any signs of eggs. The pH of water bodies at sites with high and low soil pH were similar (high – 2 sites: pH 6.3 and 6.7; low – 2 sites: pH 5.8 and 6.5), suggesting that soil pH did not have a large influence on pH of water bodies across the range of soil pH values examined in this study.

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5.6

5.4

5.2

5.0

4.8

Mean Soil pH ( +- 2 SE) 2 +- ( pH Soil Mean 4.6

4.4

4.2

Absent Present DistributionDistribution of Pseudophryne of P. bibronii bibronii

Figure 6.3. Mean (+/- 2SE) soil pH at 10 sites within Barakula State Forest where Pseudophryne bibronii are present, and 10 sites where they are absent.

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6.3.2 EGG MORTALITY – FIELD EXPERIMENTS

6.3.2.1 Study 1

Mean number of eggs per nest used for this trial was 130 (SD = 23, n = 20) and number of eggs per treatment ranged from 30 to 52. Development stages ranged from 2 to 25. One artificial nest was disturbed by animals, so this replicate was omitted from analysis. There was a significant effect of treatment on egg survival

(F2,36 = 10.49, P<0.001; Table 6.1). There was no significant difference in hatching success between eggs from the original nest and those moved to artificial nests adjacent to original nests (Tukey post hoc, P = 0.873), indicating that environments created within artificial nests did not adversely affect egg survival. However, there was a significant effect of soil pH, with survival significantly lower at the high soil pH site than at the low pH source site (Tukey post hoc, P = 0.001).

6.3.2.2 Study 2:

Mean nest size for these trials was 90 eggs (SD = 31, n =10), and numbers used in each replicate ranged from 14 to 72. Developmental stages ranged from Gosner stage 13 to 23. Egg survival varied significantly between treatments (F2, 18 = 12.05, P

< 0.001, Table 6.1) with survival significantly lower in the high soil pH treatment than both the low pH treatment (Tukey post hoc, P = 0.016) and nest (Tukey post hoc, P

< 0.001). There was no difference in survival between the nest and the low pH treatment (Tukey post hoc, P = 0.214).

6.3.2.3 Study 3:

Mean number of eggs per nest used in this experiment was 256 (SD = 104.7, n =

25), suggesting that nests contained clutches from more than one female. Numbers of eggs per replicate ranged from 27 to 182. Development stages were not recorded. Survival rates varied considerably between treatments (F2, 8 = 17.6, P =

0.001) (Table 6.1), but were consistently lower at the high soil pH sites compared to

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both the low pH sites and the nest sites (Tukey post hoc, P < 0.005). pH values at artificial nest sites ranged from 4.0 to 4.7 at low pH sites and from 4.8 to 5.5 at high pH sites (Table 6.2). There was a significant negative correlation between soil pH at the artificial nests and survival (r2 = 0.4.7, P < 0.001, n = 75) (Figure 6.4).

Table 6.1. Mean (+/- SD) percent survival of Pseudophryne bibronii embryos exposed to different soil pH levels in three separate field trials conducted at Barakula State Forest, to test if soil pH influenced hatching success. Study % egg survival % egg survival % egg survival % egg at nest near nest at low soil pH survival at high soil pH Study 1 N = 19 87 ± 23 82 ± 27 N/A 48 ± 33

Study 2 N = 10 81 ± 37 N/A 73 ± 29 52 ± 20

Study 3 N = 25 70 ± 22 63 ± 28 71 ± 21 21 ± 24

Table 6.2. Range, Mean and standard deviation of soil pH recorded at artificial nest sites used in Study 3 conducted at Barakula State Forest, to experimentally test the influence of soil pH on hatching success of Pseudophryne bibronii embryos. Translocation Minimum Maximum Mean Standard Site pH pH Deviation Original nest site 4.0 4.6 4.3 0.15

Low pH site 4.0 4.7 4.3 0.20

High pH site 4.8 5.5 5.1 0.18

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100.00

80.00

60.00

40.00 % Embyronic Survival

20.00

R Sq Linear = 0.467

0.00

4.00 4.25 4.50 4.75 5.00 5.25 5.50 Soil pH

Figure 6.4. Scatterplot showing the relationship between soil pH and embryonic survival of Pseudophryne bibronii in artificial nests used in Study 3 conducted at Barakula State Forest.

6.3.3 LABORATORY STUDY

In the laboratory study using sterilised soil of varying pH, there was no difference in survival rates in relation to pH (F3, 57 = 1.61, p = 0.19). Survival at low pH was similar to field levels, while survival at high pH was higher than for high soil pH sites in the field (mean % ± SD: pH 4.3 = 59 ± 24; pH 4.6 = 62 ± 23; pH 5.0 = 73 ± 18; pH 5.3 =

64 ± 24). Although soil was sterilised, the majority of replicates (79 of 80) showed some level of fungal infestation, indicating that the fungus was present on the eggs at the low pH sites where breeding occurs and were transported with the eggs to the laboratory. Figure 6.3 shows fungal infection of P. bibronii eggs in a nest at a high pH site, and although the species of fungi was not identified, it appeared similar to that found on the eggs in the laboratory. There was a significant negative relationship between survival and rank infection levels (Spearman rank; N = 80, r = -

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0.28, P = 0.012). There was a significant difference in rank fungal infection levels between pH treatments (Friedman χ2 = 12.39, P = 0.006, df = 3), with pH treatment

4.6, having lower fungal infection levels than other treatments (mean ranks; pH 4.3

= 2.6, pH 4.6 = 1.7, pH 5.0 = 2.8, pH 5.3 = 2.9).

Figure 6.5. Photo showing unidentified fungi on eggs in a P. bibronii nest observed at Barakula.

6.4 DISCUSSION

It is readily accepted that the broad distribution of a species is dictated by its tolerance limits to environmental factors such as temperature and moisture.

However, within that broad geographical range, a patchy distribution arises because there may be other biotic or abiotic conditions that further restrict the spatial and temporal use of habitats. This patchy distribution is particularly evident for amphibians that require both aquatic and terrestrial habitats to complete their life cycle.

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This study provides strong evidence that, within its broad geographical distribution, the local distribution of Pseudophryne bibronii may be influenced by soil pH rather than conditions of the breeding pond. The pH data from sites selected from previous studies of P. bibronii in Barakula State Forest (Cramp unpublished data) suggested that P. bibronii was more likely to be located at sites with low soil pH. The influence of pH of water in breeding ponds on amphibians has been well documented (Pough

1976; Pierce 1985; Ferraro and Bergin 1993; Glos et al. 2003). Studies show that acidic conditions cause mortality and abnormalities at the embryonic stage (Freda and Dunson 1985; Beebee 1986) and delayed development and abnormalities at the larval stage (Beebee et al. 1990; Dunson et al. 1992) with disruption of ionic regulation cited as the major cause of reduced fitness. There is also evidence to suggest that considerable intraspecific variation to acid stress in amphibians exists

(Pierce and Wooten 1992; Picker et al. 1993; Glos et al. 2003). However, few studies have investigated the role that soil pH plays on the distribution patterns of amphibians (Mushinsky and Brodie, 1975; Wyman and Hawksley-Lescault 1987;

Wyman 1988). These studies indicate that highly acidic conditions (<3.8) are not tolerated by most amphibian species. The present study suggests that P. bibronii shows an association with acidic soil environments in Barakula State Forest. Future studies of local distribution patterns of P. bibronii should be conducted at other sites within its range to ascertain if all populations of this species display a similar association with acidic soils. Other Pseudophryne species may also favour breeding sites with low soil pH. For example, Pseudophryne corroboree is confined to high alpine country where they breed in burrows under sphagnum moss. Soils in sphagnum bogs are also acidic (Clarke and Martin 1999). Further work could also examine the possibility of a preference for acidic conditions in this genus.

It is not clear from this study how pH affects survival. One possibility is that pH effects on mortality occur because pH influences proliferation of fungi (Figure 6.3).

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Both field and laboratory studies have shown that soil pH influences the distribution and abundance of microfungi, with colonisation and sporulation increasing with higher pH (Coughlan et al. 2000; Grishkan et al. 2003). Fungal infestation of frog eggs has also been shown to be associated with embryonic mortality (Kiesecker and

Blaustein 1997; Warkentin et al. 2001), with the jelly capsules of amphibian eggs

constituting suitable substrate for the growth and maturation of fungi (Woodruff

1977). Fungal infection is either saprovoric, where infestation only occurs when

fungal hyphae penetrate capsules of dead embryos while healthy eggs resist

infection (Woodruff 1976), or fungi are pernicious parasites that attack healthy eggs

(Villa 1979; Williamson and Bull 1994; Warkentin et al. 2001). Although I lack

unambiguous field data that demonstrates that fungi were responsible for the

differential mortality between low and high pH sites, two elements of the

experiments suggest parasitic fungi are responsible for embryonic mortality. First, all

embryos used in our manipulations appeared viable when collected, and handling

techniques were standardised, therefore, if manipulations damaged any eggs, then

all nests had the same probability of receiving a dead embryo that would have

initiated saprovoric fungal infestation. Second, the laboratory trials suggested that

variation in pH alone did not result in variation in survival. The field and laboratory

results, plus the observation that fungi were associated with all egg batches

collected in the field from low pH sites and brought to the laboratory on sterile soil,

support the argument the fungi is capable of killing embryos, but proliferation in the

field is more likely when soil pH is above 5.

The difference between laboratory and field experiments may be caused by complex

interactions between microorganisms within soil. The techniques used for sterilising

the soil for this experiment were sufficient to destroy microfungi and other micro-

organisms (Coventry et al. 2001), therefore it is possible that autoclaving the soil

caused the destruction of other microbial organisms that may naturally inhibit

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sporulation and proliferation of fungi, causing the observed fungal infestations in nearly all replicates. Because eggs could not be sterilised, fungal spores were retained on eggs and fungi were able to proliferate in an environment relatively free of the inhibiting micro-organisms. pH is also known to influence the distribution of bacteria (Glenn and Dilworth 1990; Frostegard et al. 1993) and it has been shown that the change in distribution of bacterium influences the distribution of fungi

(Toyota et al. 1996; Arao 1999) because bacterium show antibiosis against some fungal species (Arao 1999). A complex microbial interaction may explain the relatively uniform survival in the laboratory and the variation in survival observed in our field trials, with soil pH displaying a synergistic influence on microbial organisms.

Thirteen different, sometimes overlapping, soil types have been identified within

Barakula State Forest, which typically display pH levels ranging from 4.4 to 6.8

(Maher 1996). Data from this research suggest that these different soil types play a significant role in determining the distribution of P. bibronii, but it does so via an effect on embryos of the species. That is, soil pH is affecting distribution via its effect on reproductive ecology rather that via an influence on the microhabitat selection of adults and juveniles. However, it is also possible that soil pH does play a role in determining microhabitat selection, as the choice trials (Chapter 5) showed that juvenile P. bibronii discriminate between differences in soil pH and may therefore, avoid areas with unsuitable soil pH. Post-metamorphic dispersal by juveniles from the natal nesting site has been shown to be common in anurans (Breden 1987;

Sjogren 1991; Pope et al. 2000). However, it is not possible from this study to determine the relative importance of habitat choice by post-metamorphic individuals, or differential mortality at egg laying sites, in influencing the distribution of P. bibronii. Nevertheless, the data illustrate that terrestrial habitat attributes are playing a role in influencing the distribution of P. bibronii, and that an understanding of naturally occurring terrestrial microhabitat variation that results in a patchy

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distribution of amphibians within a local area may have important implications for the management of amphibian populations.

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Chapter 7:

OVERVIEW AND GENERAL DISCUSSION

7.1 OVERVIEW

The word-wide decline in amphibian populations will most likely continue and more species may become extinct before the true meaning/causes of declines are fully understood. Significantly more research is required that focuses on gaining fundamental information relating to the complete set of habitat requirements for a species, coupled with more rigorous studies on the factors that affect population dynamics (Alford and Richards 1999). This knowledge is necessary so conservation managers can instigate appropriate programs aimed at preventing further declines

(Chapter 1).

Given the lack of information on terrestrial habitat requirements for many amphibian species, this study was designed to fill some of the knowledge gaps for a suite of

Australian frog species. The study sites were selected because they were not dominated by aquatic habitats and were situated in a semi-arid open woodland that was relatively free from human disturbance. These criteria for site selection were deemed necessary to ensure the habitat attributes of the terrestrial environment were the overriding force facilitating the persistence of the frog community, rather than the breeding habitats.

The results of the terrestrial habitat assessment (Chapter 2) show the highly heterogeneous nature of the habitats within Barakula State Forests. Although the regional ecosystem mapping suggests that the vegetation communities within all three sampling sites are similar, the fine scale ground truthing has shown that substantial variation exists within each site in important habitat attributes, such as ground cover and soil properties. For larger or arboreal vertebrate species, these

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fine-scale variations may not have a substantial effect on their activity patterns or distribution, but for smaller, ground-dwelling species, and in particular, species that rely on the terrestrial habitat for protection against the harsh elements of the environment, even small changes in habitat attributes might play a significant role in influencing their distribution and activity patterns (Palmer and Dixon 1990; Atauri and Lucio 2001). Of particular interest was the extensive variation in measured soil properties over such a relatively small area. The use of agar models in Chapter 3 has shown that the terrestrial microhabitats within Barakula State Forest offer differing degrees of protection against evaporative water loss, a factor that plays an important role in ensuring anuran survival in areas that do not provide access to permanent water.

The focus of this research was to identify the value of terrestrial habitat to a suite of anuran species that inhabit a xeric environment. One of the study sites (Site 3), which contains only an ephemeral breeding site that was dry during the majority of the study period, and is situated at least 4 km from the nearest permanent waterbody, supports 17 species of anurans. Results of this study provide evidence that the vegetation and soil microhabitats are playing a significant role in influencing the distribution and activity patterns in a number of frog species (Chapter 4) and that species select terrestrial habitats that suit their ecological requirements to ensure persistence. In particular, it was shown that the physical and chemical composition of soil influenced where burrowing species were most likely to be captured. Results also showed that many species utilise more terrestrial habitat than is generally perceived, with 68% of captures for one species (L. salmini) occurring at distances

>120 m from the nearest waterbody.

Manipulative experiments (Chapter 5) highlighted the importance of terrestrial habitat when 99% of individuals tested sought shelter under some form of habitat structure during daylight hours. Shelter provides protection from predation (Everett

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and Ruiz 1993), but is also necessary in minimising evaporative moisture loss, particularly in burrowing species, as increased cover provides lower soil temperatures and retains higher soil moisture (Raich and Tufekciogul 2000). The result from Chapter 4 showing the distribution of the non-burrowing frog, P. bibronii, was significantly influenced by soil pH was interesting, and without further investigation the role soil pH plays in influencing the observed distribution would not have been identified (Chapter 6).

7.2 GENERAL DISCUSSION

This study was designed to identify terrestrial habitat utilisation by anuran species at the fine-scale level. It was shown that the vegetation and soil attributes within the three study sites exhibit a significant amount of variation over a relatively small area, and the frogs at Barakula State Forest were utilising the different microhabitats at varying degrees. These results would not have been evident if the study had only looked at terrestrial habitat selection on a larger scale, and they provide strong evidence for the need for further studies aimed at identifying habitat preferences on the finer scale to ensure we have complete knowledge of species habitat requirements. For example, in Queensland, Australia, the Environmental Protection

Agency (EPA) uses broad-scale habitat requirements to identify and map ‘Essential

Habitat’ for different animal species and any development in these habitats is strictly governed (EPA 2007). However, ground-truthing of these essential habitats often reveals that key components necessary for the persistence of the species in the area are missing (BAAM 2006).

The major finding of this research is that despite prolonged drought conditions and no access to permanent water, the frogs at Barakula State Forest are able to persist in these harsh conditions. Reproductive failure was noted on numerous occasions, when dead tadpoles were observed in breeding areas that had dried up. It is

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therefore, obvious that the non-breeding habitats at Barakula provide the required microhabitat attributes to support a diverse range of anuran species. These habitats are being utilised advantageously by different species depending on their morphological and physiological characteristics. Whilst it is accepted that a breeding area is necessary for the long-term persistence for these anuran species, the quality and type of breeding area may not be as important for their fitness as the quality of the surrounding terrestrial habitats.

Anuran populations that utilise ephemeral waterbodies are dynamic and complex, particularly when these waterbodies occur in areas that receive low and often unpredictable rainfall. It is therefore, often difficult to ascertain whether populations are in decline, without long-term studies (Alford and Richards 1999). Throughout the study period, the three survey sites were visited during the evenings on most trapping events to allow aural identification of which species were attempting to breed. On most occasions, the species captured the following morning did not represent the species that were heard calling the previous night, indicating that not all species bred opportunistically. In addition, not all species were captured at every trapping event. For example, of the 11 separate trapping events, 54 Notaden bennetti were captured during only four of these events, with half of these captures

(27) occurring during the final trapping event in January, 2004, and the species was only recorded calling during this last event. If the terrestrial habitat surrounding the breeding areas had not been sampled repeatedly over a lengthy period, it is likely this species would have been missed and the conclusion may have been drawn that the species was absent from the areas surveyed. Relying on surveys conducted solely on what is happening at the breeding habitat is not always a consistent or appropriate method to ascertain population distribution or abundance.

It is often difficult to pinpoint specific microhabitat attributes that influence species distributions, therefore, the recognition of the importance of these attributes in

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facilitating persistence of a population is often lacking. This lack of knowledge is evident in anuran species, particularly those that inhabit xeric environments and/or utilise ephemeral water bodies. This research has shown that different species display significant variation in habitat associations depending on their ecological needs. This indicates that it is not always appropriate to make generalisations about habitat requirements for anuran populations (Lemckert 2004).

The information that has been presented in this research will provide a beneficial tool to organisations that make decisions on habitat management for anurans at two levels. First, this project has demonstrated the importance of terrestrial habitat requirements for anurans. Restoration of degraded wetlands is recognised as an important process in promoting amphibian population persistence (Richter et al.

2001), and without complete knowledge of both the breeding and non-breeding habitat requirements, restoration efforts may be unsuccessful in providing ideal habitat conditions for a species (Semlitsch 2002). Although there has been an increased awareness of the importance of terrestrial habitats for the persistence of amphibian populations (e.g, Maud and Sanuy 2003; Regosin et al. 2005), significant gaps in the knowledge of terrestrial habitat preferences for many species, particularly Australian anurans, still exist. The lack of critical baseline ecological data on non-stream-dwelling species makes successful protection and management of these species and their habitats almost unattainable.

The present research adds to this growing body of knowledge about the importance of terrestrial habitat for an assemblage of anurans occupying a relatively dry open woodland environment. It also provides a useful starting point for further studies of terrestrial habitat requirements of particular species, and some possible management considerations for some assemblages. For example if a project is aimed at restoring habitat for a burrowing species, the results from this present study suggest that aspects of ground cover and soil are extremely important.

Joanne Chambers Page 138 Overview and General Discussion Chapter 7

The second area that this research provides useful information for management, lies in the approach that might best be used to conserve some anuran assemblages. A current approach to habitat management that incorporates terrestrial habitat into the management program, tends to focus on the breeding habitat, and then works outwards from there to preserve some terrestrial habitat that surrounds the breeding site. For example, there are increasing numbers of studies investigating the nature and size of terrestrial buffer zones for anurans (Richter et al. 2001; Bulger et al.

2003; Wilson and Dorcas 2003). Buffers or buffer zones are generally described as

a fixed width boundary around wetlands and other breeding sites such as streams

(Semlitsch and Bodie 2003). The availability of this surrounding terrestrial habitat is

important as studies have shown that migration away from the breeding pond is not

always predictable (Fellers and Kleeman 2007), and can influence the sex ratio with

distance from the breeding pond (Regosin et al. 2003). Distances moved from the

breeding pond also vary considerably within and between species (Richter et al.

2001; Miaud and Sanuy 2005). Whilst buffer widths of 20 – 30 m may be appropriate

to accommodate habitat requirements for stream-dwelling species that move

relatively short distances from the breeding area (e.g. Streatfeild 1999), species that

inhabit open woodland and other relatively dry environments utilise significantly

more terrestrial habitat that needs protection to ensure population persistence

(Richter et al. 2001; Porej et al. 2004).

Clearly it would be difficult to set an arbitrary buffer width that encompasses the

entire area that is being utilised by species, without first conducting more research

into the importance of the non-breeding habitat in influencing the distributions and

persistence of a species. However, what the present study suggests is that for some

anuran assemblages, the issue of determining a suitable buffer zone can be ignored

because the breeding site might not be the most important habitat to consider first.

For assemblages occupying woodland habitat, it may be more appropriate to focus

Joanne Chambers Page 139 Overview and General Discussion Chapter 7

on preserving terrestrial habitat, and as long as that habitat contains some ephemeral pools for breeding, significant populations of anurans might persist.

Given that many anuran species (including the species inhabiting Barakula) utilise artificial breeding sites (pools in quarries, dams etc), it may also be possible to add breeding habitats to existing woodland habitat. Conservation of amphibian populations in open woodland environments may be achieved in conjunction with conservation programs directed at other small ground-dwelling vertebrates (lizards and small mammals).

For the most part, current management strategies for anurans have focused on restoring aquatic habitats (QPWS 2001; Hines 2002) and very strict Local, State and/or National government regulations are now in place to prevent further degradation of significant wetlands and waterways. While this might be important in some habitats, the present study suggests that for species not occupying areas that support permanent breeding habitats, maintenance of the non-breeding terrestrial habitat should be the overriding factor that influences decisions regarding management / conservation of anuran species.

Pseudophryne bibronii is a case in point. The species is currently listed as ‘Near

Threatened’ (IUCN 2006), because this species is in significant decline due to widespread habitat loss throughout much of its range (Gillespie et al. 2004). This species, like others that were studied at Barakula, would not be afforded protection if management used buffer zone criteria as the basis for delineating protection areas.

The frogs studied at Barakula occupy woodland habitats that are not associated with any notable waterbody; rather, many of the ephemeral breeding areas within

Barakula State Forest are scattered, often small in area, for most part totally dry, and therefore, not considered as important habitats requiring preservation for conservation of anurans. To successfully manage frog populations that occupy areas in open woodlands, managers would need to recognise the importance of the

Joanne Chambers Page 140 Overview and General Discussion Chapter 7

terrestrial habitats (i.e. non-breeding habitats) and apply the appropriate management practices to this habitat, rather than focusing entirely on the breeding habitat.

The fact that such a large diversity of frogs were captured during the present study, despite the low rainfall, and hence reduced breeding opportunities, indicates that the terrestrial habitat is the most critical feature of the environment in allowing persistence of these frog assemblages at Barakula. Significantly more research into the terrestrial habitat requirements of anurans, especially on species with specialised habitat needs, should be conducted to ensure successful management decisions regarding the future of amphibian populations are made.

Joanne Chambers Page 141 Bibliography

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