From Molecules to Bird Communities: a Mistletoe Story

Home , Acacia acuminata, Acanthiza, Amyema, Amyema miquelii, Amyema preissii, Amyema quandang

FROM MOLECULES TO BIRD COMMUNITIES:

A MISTLETOE STORY

This thesis was submitted in fulfilment of the requirements for the degree of Doctor of

Philosophy (Veterinary Studies), Murdoch University, by:

Kathryn Renee Napier

BSc., BSc. (Hons)

School of Veterinary and Life Sciences

Murdoch University

Murdoch, Western Australia

June 2013

DECLARATION

I declare that this thesis is my own account of my research and contains as its main content of work, which has not previously been submitted for a degree at any tertiary education institution.

Kathryn Renee Napier

ABSTRACT

Worldwide, mistletoes act as a keystone resource, providing food (nectar, fruit and foliage) and structural (nesting sites) resources to hundreds of fauna species. 75 species of ‘showy’ Loranthaceae mistletoes are native to Australia, and are found in wooded habitats throughout the mainland. The mistletoebird (Dicaeum hirundinaceum) is considered the primary disperser of mistletoe fruit in south-west Western Australia

(WA). Other ‘generalist’ species, including several honeyeater species and the silvereye (Zosterops lateralis), also regularly consume and disperse mistletoe fruits.

This thesis takes a broad, eco-physiological approach to investigate the interactions between two Australian loranthaceous mistletoes species (Amyema miquelii and A. preissii), their host plants and their avian consumers. This was achieved through a combination of intensive field surveys and sampling at five sites where mistletoe was extremely abundant, and laboratory experiments assessing various aspects of avian digestive physiology of three frugivorous bird species; the mistletoebird (specialised frugivore), silvereye (generalist frugivore) and singing honeyeater (Lichenostomus virescens; generalist nectarivore).

A stable isotope approach was used to investigate the parasitic relationship between A. miquelii and A. preissii and their eucalypt or acacia hosts (respectively).

Results demonstrate that these mistletoes regulate their water use in relation to the supply of nitrogen available from the host.

Next, the importance of mistletoe to bird communities in south-west WA was investigated through surveys and the use of stable isotopes. The presence of fruiting

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(but not flowering) mistletoe was associated with significant changes in bird community structure. Mistletoebirds were more likely to be recorded during months when ripe mistletoe fruit was present, and overall bird species richness was higher for these survey months. The contribution of mistletoe fruit to the diet of mistletoebirds ranged from

33% to 55%, demonstrating that despite mistletoe fruit being low in nitrogen, it is an important source of nutrients. Fruiting mistletoes therefore provide important food resources to bird communities in south-west WA.

Various aspects of avian digestive physiology were compared for three species that include mistletoe fruit in varying degrees to their diet. Mistletoebirds, silvereyes and singing honeyeaters demonstrated similar patterns of sugar preferences with similarly high (>97.5%) apparent assimilation efficiencies (AE*) for sucrose, glucose and fructose and lower AE* for the pentose monosaccharide xylose (56-78%), yet demonstrated differences in their absorption of dietary sugars. Mistletoebirds, in contrast to the other two species, did not vary bioavailability (f) with diet concentration, and appear to absorb xylose through both mediated and paracellular mechanisms. This may be a result of the short, specialised intestinal tract of mistletoebirds, which facilitates faster transit rates of mistletoe fruit compared to silvereyes and honeyeaters.

This thesis presents new insight into the parasitic relationship between mistletoes and hosts, and the relationship between mistletoes and its avian consumers.

Mistletoebirds differ from other opportunistic mistletoe feeders in their ability to process large numbers of mistletoe fruit quickly, while obtaining sufficient nutrients such as nitrogen and carbohydrates from these fruits.

ACKNOWLEDGMENTS

Firstly, I’d like to thank my supervisors – Patricia Fleming, Todd McWhorter and

Carlos Martínez del Rio. I don’t think I could express just how much I have enjoyed working with you over the last 6+ years. Trish, you have taught me more than I would have ever thought possible – your brilliant advice, encouragement, and unbelievable dedication to your students are such an inspiration. I would not be at this stage in my research career nor have experienced such fantastic opportunities without your constant encouragement, support, patience and friendship, and I will always be grateful.

Todd, I really appreciate the encouragement, support and advice given over the long distances between Perth, Laramie and Adelaide. Your patience, kind words and knowledge freely given have helped me immensely over the years. I have dearly missed working with you in person, and hope that I may rejoin you someday in the lab.

Carlos, you gave me the opportunity of a lifetime by accepting me into your lab group at the University of Wyoming. I thank you and Martha for the support and help you gave in those first few weeks at 2184 m! Laramie and Wyoming now have a special place in my heart, and the Medicine Bow Mountains are one of my favourite places on this earth.

My deepest gratitude is also given to my sources of funding and awards, without which this research would not have been possible: the 2010 Western Australian

Fulbright Scholarship and Gregory Schwartz Enrichment Grant (Australian-American

Fulbright Association), the Holsworth Wildlife Research Endowment, the Stuart Leslie

Bird Research Award and travel grant (Birdlife Australia and Birdlife WA), and the

Jean Gilmore Postgraduate research bursary (Federation of University Women, SA).

The financial support given by Murdoch University is also very much appreciated.

Sincere thanks are also given to the staff at the Araluen Country Club and

Resort, Dr Manda Page and Jo Kuiper from the Australian Wildlife Conservancy, and

Peter Monger and family in York for allowing access to their field sites. I would also like to thank reviewers whose comments greatly improved previous versions of the manuscripts in Chapters 3, 5 and 6.

I would also like to thank Suzanne Mather for her assistance with bird surveys

(and your willingness to get up at 4 am!), the use of your ladder, and for worrying if I would fall off the ladder. You’re an inspiration.

I would also like to thank the following people for their assistance with various aspects of my research: Tony Start (Western Australian Herbarium, Department of

Environment and Conservation) for his mistletoe knowledge and comments on a draft version of Chapter 3; Simon Cherriman, for climbing trees and tagging them for me at

Paruna; Joao Paulo Coimbra (University of Western Australia) for the provision of avian intestinal tissues; Clare Auckland and John McCooke (Murdoch University) for their assistance with intestinal enzyme assays; Bill Bateman for demonstrating his sensational grasshopper catching skills and assisting with mist-netting; Shannon

Dundas, Tracey Moore and Penny Nice (Murdoch University) for their assistance with field work; the staff at the Murdoch University Animal House, particularly Derek

Mead-Hunter, for their assistance in keeping birds in captivity; David Perry (University of Wyoming Macromolecular Analysis Core) for HPLC analysis; and the staff at the

University of Wyoming Stable Isotopes Facility for being so very patient with me while

we processed hundreds of samples. Special thanks must also go to Susan Nicolson

(University of Pretoria) for her advice and collaboration.

To my fellow research students in the dungeon and bat cave offices, past and present, I thank you for your friendship and support. To Penny, Tracey, Narelle, Wil,

Heather, Gill, Shannon, Ivan, Chelsea, Bryony, Renata, John, Kelly, Tegan (honorary dungeon member), Buddy and Rocket (dungeon mascots) – my time at Murdoch was a truly enjoyable experience thanks to you wonderful, fantastic people (and animals). To

Jon and Brenna in Laramie, I thank you for your friendship and advice, and for not laughing at me (too much) during my first bear encounter. I look forward to future collaborations!

Last, but not least, I would like to thank my family and friends for their constant support and love: to my parents, Jeff and Clarice Napier, Sandy Chaney, siblings David and Alison Napier and future brother in-law Eddie Terry, friends Brett and Lois

Andrijich and Liz Snyder-Campion, and my husband Simon Aplin. Simon, your love, our adventures, and your constant support of my academic endeavours enrich my life in countless ways. I thank you from the bottom of my heart.

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DISCLAIMER

This PhD thesis consists of chapters that have been prepared as stand-alone manuscripts. These manuscripts have either been accepted for publication (Chapters 3 and 5), submitted for consideration of publication (Chapter 6), or are being prepared for future submission (Chapters 2 and 4). As a consequence, there may be some repetition between chapters. To reduce unnecessary replication of references between chapters, the references are compiled together in Chapter 8.

To maintain consistency in formatting throughout the thesis, the chapters may differ slightly from the future published manuscripts.

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PUBLICATIONS ARISING FROM THIS THESIS

Napier, K. R., McWhorter, T. J., Nicolson, S. W., and Fleming, P. A. (2013). Sugar

preferences of avian nectarivores are correlated with intestinal sucrase activity.

Physiological and Biochemical Zoology 86, 499–514.

Napier, K. R., Mather, S. H., McWhorter, T. J. and Fleming, P. A. (2013). Do bird

species richness and community structure vary with mistletoe flowering and

fruiting in Western Australia? Emu In press.

OTHER PUBLICATIONS ARISING FROM THE PERIOD OF CANDIDATURE

Purchase, C., Napier, K. R., Nicolson, S. W., McWhorter, T. J. and Fleming, P. A.

(2013). Gastrointestinal and renal responses to variable water intake in

whitebellied sunbirds and New Holland honeyeaters. Journal of Experimental

Biology 216, 1537–1545.

Napier, K. R., McWhorter, T. J. and Fleming, P. A. (2012). A comparison of

pharmacokinetic methods for in vivo studies of nonmediated glucose absorption.

Physiological and Biochemical Zoology 85, 200–208.

Napier, K. R., McWhorter, T. J. and Fleming, P. A. (2008). Mechanism and rate of

glucose absorption differ between an Australian honeyeater (Meliphagidae) and

a lorikeet (Loriidae). Journal of Experimental Biology 211, 3544–3553.

Napier, K. R., Purchase, C., McWhorter, T. J., Nicolson, S. W. and Fleming, P. A.

(2008). The sweet life: diet sugar concentration influences paracellular glucose

absorption. Biology Letters 4, 530–533.

Fleming, P. A., Xie, S., Napier, K., McWhorter, T. J. and Nicolson, S. W. (2008).

Nectar concentration affects sugar preferences in two Australian honeyeaters and

a lorikeet. Functional Ecology 18, 223–232.

TABLE OF CONTENTS

DECLARATION ...... II

ABSTRACT ...... III

ACKNOWLEDGMENTS ...... V

DISCLAIMER ...... VIII

PUBLICATIONS ARISING FROM THIS THESIS ...... IX

OTHER PUBLICATIONS ARISING FROM THE PERIOD OF CANDIDATURE ...... IX

TABLE OF CONTENTS ...... XI

LIST OF FIGURES ...... XVI

LIST OF TABLES ...... XVIII

1 GENERAL INTRODUCTION ...... 1

1.1 MISTLETOE-HOST INTERACTIONS ...... 2

1.2 MISTLETOE-ANIMAL INTERACTIONS ...... 5

1.2.1 Provision of resources ...... 5

1.2.2 Pollination and dispersal ...... 7

1.3 PHYSIOLOGICAL ADAPTATIONS TO FRUGIVORY ...... 9

1.4 STUDY AIM ...... 13

1.5 MISTLETOE STUDY SPECIES ...... 13

1.6 AVIAN STUDY SPECIES ...... 18

1.6.1 Mistletoebird (Dicaeum hirundinaceum) ...... 18

1.6.2 Silvereye (Zosterops lateralis) ...... 19

1.6.3 Singing honeyeater (Lichenostomus virescens) ...... 19

2 CARBON AND NITROGEN STABLE ISOTOPES OF AUSTRALIAN MISTLETOES AND THEIR HOSTS...... 22

STATEMENT OF AUTHOR CONTRIBUTION ...... 23

2.1 ABSTRACT ...... 24

2.2 INTRODUCTION ...... 25

2.3 METHODS ...... 27

2.3.1 Field sites and sampling ...... 27

2.3.2 General statistics ...... 30

2.4 RESULTS ...... 32

2.4.1 Carbon ...... 32

2.4.2 Nitrogen ...... 32

2.4.3 Relationships between mistletoes and hosts ...... 36

2.5 DISCUSSION ...... 39

2.5.1 δ13C of mistletoes and hosts: water relations and effects of aridity ...... 39

2.5.2 Relationships with nitrogen ...... 40

2.5.3 Conclusions ...... 41

3 DO BIRD SPECIES RICHNESS AND COMMUNITY STRUCTURE VARY WITH MISTLETOE FLOWERING AND FRUITING IN WESTERN AUSTRALIA? ...... 42

STATEMENT OF AUTHOR CONTRIBUTION ...... 43

3.1 ABSTRACT ...... 44

3.2 INTRODUCTION ...... 45

3.3 METHODS ...... 49

3.3.1 Study sites ...... 49

3.3.2 Fruit and flower phenology ...... 50

3.3.3 Bird species ...... 52

3.3.4 Statistical analysis ...... 52

3.4 RESULTS ...... 53

3.4.1 Presence of mistletoe flowers ...... 54

3.4.2 Presence of ripe mistletoe fruit ...... 55

3.5 DISCUSSION ...... 60

3.5.1 Reliability of mistletoe resources ...... 60

3.5.2 Abundance of mistletoe ...... 61

3.5.3 Degree of consumer specificity ...... 62

3.5.4 Temporal redundancy of mistletoe ...... 64

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3.5.5 Resources (functions) not otherwise present ...... 65

3.5.6 Conclusions ...... 66

4 MISTLETOEBIRDS VARY THEIR DIETARY INTAKE OF ARTHROPODS DEPENDING ON TIME OF YEAR ...... 69

STATEMENT OF AUTHOR CONTRIBUTION ...... 70

4.1 ABSTRACT ...... 71

4.2 INTRODUCTION ...... 72

4.3 METHODS ...... 74

4.3.1 Study sites ...... 74

4.3.2 Breath, blood and feather samples ...... 76

4.3.3 Diet sources ...... 78

4.3.4 Stable isotopes analysis ...... 78

4.3.5 Isotope mixing model...... 79

4.3.6 General statistics:...... 80

4.4 RESULTS ...... 83

4.4.1 Diet sources ...... 83

4.4.2 Mistletoebird tissues ...... 84

4.4.3 Isotope mixing model...... 87

4.4.4 Comparison with other bird species ...... 87

4.5 DISCUSSION ...... 92

4.5.1 Detecting diet switching of mistletoebirds ...... 92

4.5.2 Contribution of fruit vs arthropods ...... 94

4.5.3 Comparison with other bird species ...... 95

4.5.4 Arthropods and mistletoe fruit are both important sources of dietary nitrogen ...... 96

5 SUGAR PREFERENCES OF AVIAN NECTARIVORES ARE CORRELATED WITH INTESTINAL SUCRASE ACTIVITY ...... 98

STATEMENT OF AUTHOR CONTRIBUTION ...... 99

5.1 ABSTRACT ...... 100

5.2 INTRODUCTION ...... 102

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5.3 MATERIALS AND METHODS ...... 108

5.3.1 Birds and their maintenance ...... 108

5.3.2 Apparent assimilation efficiency (AE*) ...... 109

5.3.3 Sugar preference trials ...... 111

5.3.4 Intestinal enzymes...... 113

5.3.5 Foraging data and Australian nectar composition ...... 117

5.3.6 General statistical analysis ...... 118

5.4 RESULTS ...... 120

5.4.1 Apparent assimilation efficiency (AE*) ...... 120

5.4.2 Sugar preferences ...... 120

5.4.3 Intestinal enzymes...... 123

5.4.4 Foraging data and Australian nectar composition ...... 126

5.5 DISCUSSION ...... 132

5.5.1 Are there differences in apparent assimilation efficiency between sugar types? ...... 133

5.5.2 Can we explain hexose preferences on dilute diets? ...... 134

5.5.3 Can we explain sucrose preference on concentrated diets? ...... 135

5.5.4 Do laboratory results reflect foraging preferences in the wild? ...... 136

5.5.5 Conclusions ...... 137

6 MISTLETOEBIRDS AND XYLOSE: AUSTRALIAN FRUGIVORES DIFFER IN THEIR HANDLING OF DIETARY SUGARS ...... 139

STATEMENT OF AUTHOR CONTRIBUTION ...... 140

6.1 ABSTRACT ...... 141

6.2 INTRODUCTION ...... 142

6.3 MATERIALS AND METHODS ...... 147

6.3.1 Study species ...... 147

6.3.2 Apparent assimilation efficiency (AE*):...... 149

6.3.3 Pharmacokinetic experiments ...... 151

6.3.4 Gut passage times (GPT) ...... 152

6.3.5 Statistical analysis ...... 153

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6.4 RESULTS ...... 153

6.4.1 Apparent assimilation efficiency (AE*) ...... 153

6.4.2 Pharmacokinetic experiments ...... 156

6.4.3 Gut passage times (GPT) ...... 160

6.5 DISCUSSION ...... 160

6.5.1 Apparent assimilation efficiency (AE*) ...... 161

6.5.2 Mechanism of sugar absorption ...... 162

6.5.3 Metabolism of xylose ...... 165

6.5.4 Conclusions ...... 166

7 GENERAL CONCLUSIONS ...... 167

7.1 MISTLETOE-HOST INTERACTIONS ...... 167

7.2 MISTLETOE-ANIMAL INTERACTIONS ...... 168

7.3 PHYSIOLOGICAL ADAPTATIONS TO FRUGIVORY ...... 170

7.4 FUTURE DIRECTIONS ...... 172

7.5 CONCLUSIONS ...... 174

8 REFERENCES ...... 176

LIST OF FIGURES

Figure 1.1 The location of five study sites in south-west Western Australia...... 16

Figure 1.2 Two Loranthaceae mistletoe species common in south-west Western Australia: the box (or stalked) mistletoe Amyema miquelii and wireleaf mistletoe Amyema preissii...... 17

Figure 1.3 Digestive adaptations associated with consuming a mistletoe fruit diet were investigated for three focal frugivorous bird species...... 21

Figure 2.1 Stable isotope values and percentages of carbon and nitrogen for mistletoe, infected and uninfected host samples on acacia (black symbols) or eucalyptus (white symbols) hosts...... 35

Figure 2.2 Correlations between mistletoe and host a) δ13C (‰), b) carbon content (%), c) δ15N (‰) and d) nitrogen content (%) ...... 38

Figure 3.1 Flowering and fruiting phenology of the mistletoes Amyema preissii (left hand panel) and Amyema miquelii (right hand panel) from February 2010 to January 2011...... 58

Figure 3.2 The number of bird species recorded at each site in the presence (1) or absence (0) of open flowers (a) and ripe fruit (c), and the number of nectarivorous bird species recorded at each site in the presence (1) and absence (0) of open flowers (b)...... 59

Figure 4.1 Median, 25th and 75th quartile values of δ15N (‰) signatures of arthropods sampled at three sites in south-west Western Australia...... 82

Figure 4.2 δ13C (a) and δ15N (b) values (‰) of breath, whole blood and feathers for mistletoebirds at three sites in south-west Western Australia...... 86

Figure 5.1 Evolutionary relationships...... 116

Figure 5.2 Concentration-dependent total sugar intake...... 121

Figure 5.3 Concentration-dependent sugar preferences...... 122

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Figure 5.4 Gut nominal surface area and intestinal enzymes...... 125

Figure 5.5 Feeding observations for New Holland honeyeaters, red wattlebirds, singing honeyeaters and silvereyes in Western Australia (Brown et al. 1997) and rainbow lorikeets in Western Australia and the Queensland-New South Wales border region (Cannon 1984; Brown et al. 1997)...... 127

Figure 5.6 Average nectar composition from 16 Australian plant genera (mean fructose, glucose, sucrose)...... 129

Figure 6.1 Apparent assimilation efficiency (AE*) of D-xylose (solid) and D-glucose (lines)...... 155

Figure 6.2 Bioavailability (f, %) of radiolabelled L-glucose (white) and D-xylose (grey) at two diet concentrations (0.25 and 1 mol·L-1, solid and lines respectively)...... 159

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LIST OF TABLES

Table 2.1 The characteristics of the five field sites in south-west Western Australia...... 29

Table 2.2 Leaf carbon and nitrogen content (%), δ13C and δ15N values (‰) of mistletoe, host and uninfected host plants...... 31

Table 2.3 δ13C and δ15N values (‰) and carbon and nitrogen content (%) for all samples (mistletoe, infected and uninfected hosts) were analysed as dependent variables in separate mixed-model ANOVAs...... 34

Table 3.1 The characteristics of the five sites surveyed in south-west Western Australia...... 51

Table 3.2 Bird species recorded in 53 surveys over 5 sites in south-west Western Australia from February 2010 to January 2011...... 68

Table 4.1 The characteristics of the three field sites in south-west Western Australia...... 75

Table 4.2 Stable isotope values δ13C and δ15N (‰) and carbon and nitrogen concentrations (%) for mistletoebird tissues (breath, blood and feathers) and diet sources (arthropods and mistletoe fruit)...... 85

Table 5.1 Summary of sugar type preferences, apparent assimilation efficiency (AE*) and digestive capacity...... 107

Table 5.2 Details of birds euthanased for digestive enzymes...... 119

Table 5.3 Summary of maltase activity...... 130

Table 5.4 Nectar composition of 16 Australian plant genera...... 131

Table 6.1 Relative sugar composition (% dry mass±SEM) of mistletoe fruit viscin...... 146

Table 6.2 Parameters used to determine bioavailability (f) of radiolabelled L-glucose and D-xylose in mistletoebirds, silvereyes and singing honeyeaters at two diet concentrations (0.25 and 1 mol·L-1 hexose solutions)...... 158

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1 GENERAL INTRODUCTION

Examining the interactions of plants and animals addresses some of the most fundamental and challenging questions in ecology (Sutherland et al. 2013). The interactions between mistletoes, their hosts, and animals are particularly interesting due to the significant influence of mistletoes on the ecological communities they inhabit.

Mistletoes are a diverse group of shrubby, aerial, flowering plants with over

1,500 species found over a wide range of habitats and across all continents with the exception of Antarctica (Kuijt 1969; Calder 1983; Watson 2001). Their growth form of obligate hemiparasitism exhibits a unique strategy by combining the parasitism of a host species with their own ability to photosynthesise (Těšitel et al. 2010). There are five families within the order Santalales, with Loranthaceae and Viscaceae comprising the majority (> 98%) of mistletoe species (Nickrent 2002; Mathiasen et al. 2008). Most mistletoes rely on birds or insects for pollination and fruit dispersal, which leads to a broad range of mistletoe-animal interactions, yet the closest interactions mistletoes form would be their hemiparasitic relationships with their hosts, in order to obtain water and nutrients (Miller et al. 2003).

This thesis includes five experimental chapters, investigating the relationships between mistletoes, their hosts, and the avian community. These studies use a variety of experimental methods to examine these relationships from a molecular to community level. Firstly, I investigated the parasitic relationships between mistletoes and their hosts (Chapter 2). Secondly, I investigated the importance of mistletoe as a food resource to bird communities, and the contribution of mistletoe fruit to the diet of

mistletoebirds (Chapters 3 and 4). Thirdly, I examined the digestive adaptations associated with consuming a mistletoe fruit diet for three focal avian species (Chapters

5 and 6). This introductory chapter therefore focuses on three aspects; the interactions between mistletoes and their hosts, the interactions between mistletoes and animals, and the physiological adaptations associated with frugivory.

1.1 MISTLETOE-HOST INTERACTIONS

One general characteristic of parasites is the deleterious relationship they have with their hosts. The infection of commercially-valuable conifers with the dwarf mistletoe

(Arceuthobium), and subsequent reductions in growth, fruiting and seed production

(Mathiasen et al. 2008), is an example of a classic parasite-host interaction. However, the relationship between mistletoe and host may switch from parasitism to mutualism by the introduction of a third species, such as an avian disperser. For example, juniper hosts infected with mistletoe attracted significantly more avian seed dispersers compared to uninfected host trees, which resulted in higher recruitment of juniper seedlings in stands where mistletoe was present (van Ommeren and Whitham 2002).

While some mistletoe species, like the dwarf mistletoe, can have deleterious effects on their hosts, most mistletoes actually play a key role in ecosystems. As mistletoes depend solely on their hosts for their water and the majority of their nutritional requirements, it is not in the best interest of the mistletoe to impact on the mortality of its host. While infection by mistletoes can negatively impact their hosts in some cases, mature, healthy trees in general do not suffer major ill effects, aside from the loss of the branch distal to a mistletoe infestation (Tennakoon and Pate 1996). The host safeguards the mistletoe by providing a buffer against large-scale fluctuations in

the availability of nutrients and resources that may limit the growth and distribution of other plants (Ehleringer and Marshall 1995). Mistletoe plants have a low annual survival rate for a variety of reasons. Susceptibility to fire (Rowe 1983) and frost

(Smith and Wass 1979), and a high light requirement for successful germination, establishment, and maturation, has lead to a narrow microsite tolerance in many mistletoe species (Lamont 1982; Knutson 1983; Lamont 1983a; Sargent 1995; Yan and

Reid 1995; Hawksworth and Wiens 1996; Polhill and Wiens 1998). Mistletoes are also generally more sensitive to water shortages in comparison to their hosts, leading to increased mistletoe mortality during periods of drought (Watson 2011).

Loranthaceous mistletoes are dependent on their hosts for water and mineral nutrients (Ehleringer et al. 1985; Marshall and Ehleringer 1990), and obtain these by parasitising the xylem of their hosts (Calder 1983; Marshall et al. 1994). Despite the lack of a connection to the phloem, xylem-tapping mistletoes can also obtain varying amounts of carbon from the host photosynthate (Hull and Leonard 1964; Marshall and

Ehleringer 1990; Dörr 1997; Govier et al. 1967). The haustorium (a vascular connection which attaches the mistletoe to the stem of a host plant) facilitates the one- way flow of water and dissolved nutrients from the host xylem stream (Lamont 1983b;

Ehleringer and Marshall 1995), although the mechanisms of this movement are still not completely understood. Transpiration rates of mistletoes are generally higher then those of their hosts, and this is likely achieved by mistletoes keeping their stomata open, even under dry conditions or at night, when other plants typically close their stomata to minimise water loss (Schulze et al. 1984; Ehleringer and Marshall 1995). These high transpiration rates also cause reduced xylem water potentials in the host branches, which acts to reduce the net photosynthetic rate of the host (Knutson 1983; Stewart and

Press 1990; Mathiasen et al. 2008). Mistletoes accumulate high concentrations of xylem-transport cations in their tissues, particularly phosphorous and potassium

(Lamont 1983b), which further facilitates the absorption of water and solutes by promoting lower xylem water potential in mistletoe tissue compared with their hosts

(Knutson 1983; Mathiasen et al. 2008). The xylem hydraulic conductivity of host branches distal to a mistletoe infection is likely to be decreased due to transpiration by the mistletoe, causing the death of the end of the host branch while the mistletoe remains living and attached to the host (Tennakoon and Pate 1996).

Parasitism of a host by mistletoes therefore inevitably effects water, nitrogen and carbon assimilation of host plants (Wang et al. 2007). Studies of stable isotopes of carbon and nitrogen have been instrumental in the development of the knowledge of mistletoe biology. Conventionally expressed as a ratio of the heavy to the light isotope

(δ) in units per mil (‰), the studies of carbon (13C to 12C) and nitrogen (15N to 14N) stable isotope ratios have facilitated our understanding of how mistletoes acquire carbon and nitrogen from their hosts. The δ13C of plant material also gives an indication of the long term water use efficiency of the plant, due to the strong correlation between carbon isotope discrimination and the ratio between carbon assimilation and transpiration (i.e. water use efficiency) (Farquhar et al. 1982). This relationship results from the discrimination against 13C in favour of 12C during the diffusion of carbon dioxide into plant leaves and during the fixation of carbon by ribulose-1,5-biphosphate carboxylase in the mesophyll (Toft et al. 1989).

More positive values of δ13C (i.e. 13C-enriched) are therefore found in more water-efficient plants. In arid areas, the δ13C values for mistletoes are generally more

negative than those of their hosts, and the greatest differences in δ13C between mistletoes and hosts were on hosts with low nitrogen contents (Ehleringer et al. 1985).

This suggests that a greater differential in water use efficiency is required to extract nitrogen from nitrogen-poor hosts. I therefore used δ13C and δ15N to examine the parasitic relationships of five unique mistletoe-host pairs from five sites in south-west

Western Australia (WA) in terms of the water use efficiency of mistletoes, their hosts and uninfected host plants.

1.2 MISTLETOE-ANIMAL INTERACTIONS

1.2.1 Provision of resources

While some mistletoe species can have deleterious effects on their hosts, most mistletoes actually play a key role in ecosystems (Watson 2001; Mathiasen et al. 2008).

Mistletoes offer reliable food and shelter resources for hundreds of fauna species, providing resources and ecosystem services out of proportion to their abundance and contribution to biomass (Davidson et al. 1989b; Watson 2001; Watson 2002; Mathiasen et al. 2008). Mistletoes may therefore be considered a ‘keystone species’ (discussed in further detail in Chapter 3). For example, mistletoe plants provide a broad range of nutritional resources to both obligate and partially-dependent animal species, and create a habitat that is used for nesting and roosting by a wide variety of species (Watson

2001; Mathiasen et al. 2008).

Mistletoes are used as a food source by a wide range of animals, with species from 66 families of birds and 30 families of mammals recorded feeding on mistletoe nectar, fruit and leaves worldwide (reviewed by Watson 2001). A wide range of bird

and insect species are known to pollinate mistletoes; many of these birds and insects tend not to be dependent on mistletoe nectar as a primary food source, while there are few specialised mistletoe fruit dispersers (Davidar 1983; Davidar 1985; Reid 1986;

Watson 2001). Bats are also known to visit mistletoe flowers, but their role as a potential pollinator has not yet been confirmed (Mathiasen et al. 2007). In addition to flowers and fruit, mistletoe leaves also provide an extremely nutritious food source to folivorous animals such as possums, deer and gorillas, and mistletoe clumps are often used as a foraging substrate by insectivorous species (Watson 2001).

A wide range of species have been recorded nesting in mistletoes, including mammals, marsupials, and birds (see extensive reviews by Watson 2001; Cooney et al.

2006). In Australia, 66% of all arboreal-nesting bird species (217 species from 29 bird families) have been reported nesting in mistletoe; the dense clumps provide structural support for nests, a more attractive microclimate, and also a ‘hiding’ spot for the nests of smaller passerines, with lower predation rates demonstrated for nests in mistletoe compared to eucalypts (Cooney et al. 2006; Cooney and Watson 2008).

Mistletoes have recently been described as the Robin Hood of woodlands, by taking nutrients from their hosts to redistribute to those in need, a more favourable association then being compared to Dracula, taking nutrients from an unfortunate host

(Press 1998). While mistletoes provide abundant food and nesting resources, mistletoe leaf litter (which is shed year-round) also returns valuable nutrients such as potassium, phosphorous, and nitrogen back to the host and surrounding plant community (March and Watson 2010). The high concentration of minerals and water present in mistletoe leaves, coupled with the high rates of mistletoe leaf turn over, act to increase leaf litter

mass and the availability of mineral nutrients, and improve soil fertility by slowing the overall rate of decomposition and providing a steady supply of nutrients (March and

Watson 2007; March and Watson 2010; Watson 2011).

1.2.2 Pollination and dispersal

With the exception of the insect pollinated root-parasitic Western Australian Christmas tree, Nuytsia floribunda, and Atkinsonia, Atkinsonia ligustrina, the majority of

Australian loranthaceous mistletoes are pollinated by birds (Hawkeswood 1981; Watson

2011). These ornithophilous mistletoes tend to display brightly-coloured, odourless flowers, with abundant, sugar-rich nectar (up to 60% total sugar content, primarily glucose and fructose sugars, Reid 1986; Stiles and Freeman 1993; Baker et al. 1998).

Although they are not entirely dependent on mistletoe nectar as a primary food source, a wide range of bird species pollinate these ornithophilous mistletoes, including spinebills, wattlebirds, and singing, spiny-cheeked and New Holland honeyeaters

(Meliphagidae; Reid 1986; Watson 2001; Watson 2011).

Although the seeds of several lineages of mistletoe (e.g. Viscaceae) are rather spectacularly self-dispersed by hydrostatic explosion (Hawksworth and Geils 1996), mistletoes in general are bird dispersed. Mistletoe fruits contain chlorophyllus seeds which are enclosed in a thick pericarp. This pericarp is made up of epicarp, mesocarp and endocarp layers (Salle 1983). The thick mesocarp is composed of hairy cells, spherical gel, and a mucilaginous substance known as viscin (Salle 1983; Azuma et al.

2000). Viscin tissue occupies a significant proportion of the mature fruit, and is composed of cellulose in a mixture of both acidic and neutral polysaccharides (Salle

1983; Gedalovich et al. 1988; Azuma et al. 2000). The seeds voided by the bird are still surrounded by a layer of viscin, which is unique to parasitic plants (Richardson and

Wooller 1988; Azuma et al. 2000), and functions in the dissemination of mistletoe by birds. As the mistletoe seeds can adhere to the cloaca of the bird, wiping by the bird may be required to remove the seeds and thus adhere them to host branch, where they remain ‘glued’ prior to germination (Azuma and Sakamoto 2003).

The nutritional composition of the fleshy fruit pulp varies among species, but most mistletoe fruits contain a high proportion of carbohydrates, lipids and protein

(Walsberg 1975; Godschalk 1983a; Herrera 1987; McPherson 1987; Witmer 1996;

López de Buen and Ornelas 2001; Barea 2008). While a small number of highly specialised avian species tend to be associated with the dispersal of mistletoe (e.g.

Phainopepla nitrens in North America, Walsberg 1975; Dicaeum species in southern

Asia and Australia, Davidar 1983; Reid 1986), generalist frugivores also contribute to the dispersal of mistletoe fruit (e.g. Colius indicus in South Africa, Godschalk 1983b;

Acanthagenys rufogularis in Australia, Reid 1989). Mistletoe specialists exhibit specific anatomical adaptations to a mistletoe diet, which results in extremely rapid gut passage times of mistletoe seeds compared to dietary generalists (Murphy et al. 1993).

Dietary generalists may therefore be particularly important in the establishment of new mistletoe populations (Mathiasen et al. 2008), due to the fast gut transit time of mistletoe specialists often precluding long dispersal distances. Several studies have demonstrated that the ‘seed rain’ of specialist mistletoebirds (Dicaeum hirundinaceum) is limited, as they tend to visit trees that are already infected with mistletoe, thereby maintaining only existing populations of mistletoe (Ward and Paton 2007; Rawsthorne et al. 2012). The establishment of new populations of mistletoes is likely to be carried

out by non-specialists such as the spiny-cheeked honeyeater (Aca. rufogularis), which has been reported to visit both infected and uninfected stands within the (longer) time taken to defecate mistletoe seeds (Rawsthorne et al. 2011). Migrating generalists, such as silvereyes (Zosterops lateralis), have also been speculated to be dispersers of mistletoe to islands, through the deposition of mistletoe seeds stuck to legs or feathers

(Watson 2011).

Several recent studies have examined how mistletoes may influence changes in

Australian bird communities (see studies by Turner 1991; Watson 2002; Watson et al.

2011; Watson and Herring 2012), but the interactions between mistletoes and birds in

Western Australia (WA) have not been studied in great detail. I therefore examined the importance of mistletoe as a food resource to bird communities in south-west WA through the use of field surveys and stable isotopes analysis. This was achieved by conducting bird and mistletoe flowering and fruiting phenology surveys approximately every five weeks from February 2010 and January 2011 (Chapter 3). Stable isotopes of carbon and nitrogen measured in plant and animal tissue samples were subsequently used to investigate the relative importance of mistletoe fruit and insects as a source of dietary protein to mistletoebirds at three of the five study sites (Chapter 4).

1.3 PHYSIOLOGICAL ADAPTATIONS TO FRUGIVORY

Fruit pulp and nectar is the ‘reward’ offered to dispersers and pollinators; however sugary fruits and nectar are characterised by a high water and carbohydrate content, generally containing low levels of lipids and protein (Herrera 1987; Nicolson 1998).

Specialist frugivores and nectarivores must therefore assimilate large amounts of sugars and process large volumes of preformed water, while conserving proteins and salts

(Yokota et al. 1985; Beuchat et al. 1990; Murphy et al. 1993; Witmer and van Soest

1998; McWhorter and Martinez del Rio 1999; Levey and Martinez Del Rio 2001;

McWhorter et al. 2003; Lotz and Martínez del Rio 2004). The reliance on a predominantly frugivorous diet is associated with several specialised adaptations to frugivory that allows these birds to meet their relatively high energy demands.

Firstly, specialised frugivores typically demonstrate a wide and short alimentary tract (Richardson and Wooller 1988; Witmer 1998). In mistletoebirds, this allows for the rapid processing of large numbers of mistletoe fruit, with mistletoebirds demonstrating shorter gut retention times in comparison to non-specialised frugivores

(discussed in Chapter 6). To rely on mistletoe fruit as a main dietary source of carbohydrates and nitrogen, these birds must be able to efficiently utilise these nutrients.

Most frugivorous birds, like specialised nectarivores, demonstrate high assimilation efficiencies of the sugars found in fruit (Witmer 1999; Lotz and Schondube 2006).

The fruit pulp of fruits consumed by birds in generally rich in the monosaccharides glucose and fructose (collectively termed hexoses), while the disaccharide sucrose averages only 8% of total sugars in passerine-consumed fruits

(Baker et al. 1998). While glucose and fructose are quite easily absorbed by both passive (i.e. paracellular) and mediated (active) mechanisms, sucrose must be hydrolysed to its hexose components by the intestinal enzyme sucrase-isomaltase

(hereafter referred to as sucrase) before it can be absorbed (Nicolson and Fleming

2003b). The ability to assimilate sucrose varies widely between different bird species due to the presence or absence of sucrase; frugivores in the Sturnidae-Muscicapoidea lineage that lack this enzyme are unable to tolerate sucrose (Martínez del Rio and

Stevens 1989; Brugger et al. 1993; Sabat and Gonzalez 2003; Gatica et al. 2006; Brown et al. 2012). Most frugivores and nectarivores are able to efficiently assimilate both sucrose and hexoses (Lotz and Schondube 2006; Fleming et al. 2008; Napier et al.

2008a). However, some opportunistically frugivorous birds such as the yellow-rumped warbler (Dendroica coronata), which does express sucrase but has high rates of food passage and intake, appear to be constrained by the time taken in the hydrolysis of sucrose (Afik and Karasov 1995). This may explain the presence of hexoses as the dominant sugars in bird consumed fruits.

The significant reliance on paracellular absorption for uptake of glucose and small water-soluble nutrients in small birds and bats is thought to compensate for a reduction in intestinal absorptive surface area (Caviedes-Vidal et al. 2007). These animals face strong selection pressures to digest food rapidly in order to reduce digesta mass carried during flight, and this may be accomplished by the rapid absorption of glucose via the paracellular pathway. Paracellular absorption provides a non-saturable absorptive process that matches glucose absorption capacity to acute changes in dietary nutrient concentration (Afik et al. 1997; Ferraris 2001); therefore the relative contribution of paracellular to total glucose absorption increases with an increase in diet concentration, presumably due to increased digesta retention time in the intestine

(McWhorter et al. 2006; Napier et al. 2008b). Some frugivorous birds and bats (as well as specialised avian nectarivores), are known to rely extensively on the paracellular pathway for the absorption of glucose (Karasov and Cork 1994; McWhorter et al. 2006;

Tracy et al. 2007; Napier et al. 2008b; McWhorter et al. 2010), although similar tests have not been carried out for specialist frugivores. The mediated absorption of glucose has been examined in one specialised frugivore, the cedar waxwing (Bombycilla

cedrorum), which demonstrates three times the mediated transport of glucose in comparison to the American robin (Turdus migratorius), a seasonal frugivore (Karasov and Levey 1990). It has been suggested that this allows cedar waxwings to maintain efficient absorption of sugars in spite of relatively high intake and processing rates of fruit (Witmer and van Soest 1998).

Secondly, specialised frugivores have relatively low protein requirements in comparison to dietary generalists (Witmer 1998). Fruits typically contain low amounts of protein, and, while frugivory is a common dietary strategy, exclusive frugivory is considered rare (Morton 1973). For most avian frugivores, fruits are a non-exclusive food resource that is supplemented with animal protein (such as insects) and nectar.

Even specialist frugivores (such as mistletoebirds) exhibit diet switching and consume insects during periods when protein requirements are particularly high (e.g. during moult and female reproduction) (Higgins et al. 2006). Protein is a vital dietary component for birds, as it is the principle structural and catalytic component of all tissues and birds lack the enzymes necessary for the synthesis of nine of the 20 amino acids required (Klasing 1998). Consequently, birds must consume dietary protein to meet their essential amino acid requirements and to supply nitrogen for the synthesis of non-essential amino acids (Klasing 1998).

Thirdly, a large proportion of fruit mass is taken up by indigestible seeds (30–

50% dry fruit mass, see Levey and Grajal 1991 and references therein) which increases the energy expenditure of flight and can also decrease the capacity to fill the gut with more food due to their bulk. The ability to efficiently process these seeds is therefore of significant importance to frugivorous birds. Some frugivores may regurgitate seeds or

discard seeds before ingesting the fruit pulp; while several mistletoe dispersers regurgitate mistletoe seeds, such as the streak-necked flycatcher (Mionectes striaticollis)

(Montaño-Centellas 2013) and Australian Raven (Corvus coronoides) (Watson 2011),

Australian mistletoe specialists defecate mistletoe seeds. Frugivorous birds that avoid regurgitating seeds have demonstrated the ability to separate fruit pulp from seeds in the intestine, and defecate seeds quicker than fruit pulp (Levey and Grajal 1991). This rapid passage of seeds increases capacity for fruit consumption, and therefore assimilation of nutrients from fruit pulp (Levey and Grajal 1991).

I investigated various aspects of digestive physiology in three focal avian frugivorous species, including the apparent assimilation efficiency of sucrose and hexoses, sugar preferences and the capacity to hydrolyse sucrose (Chapter 5). The mechanisms of absorption of glucose and xylose (a pentose monosaccharide that is abundant in some species of mistletoe fruit) were examined for the three focal avian species (Chapter 6).

1.4 STUDY AIM

This thesis takes a multi-faceted approach in the investigation of the interactions between two Australian loranthaceous mistletoes species (Amyema miquelii and A. preissii), their hosts, and their avian consumers in south-west Western Australia.

1.5 MISTLETOE STUDY SPECIES

Two endemic Australian Loranthaceae mistletoe species common in south-west WA were the focal mistletoe species for this thesis. Field work was conducted at five study

sites in south-west WA (Fig. 1.1) – detailed site descriptions are included in Chapters 2 and 3 (Tables 2.1 and 3.1).

The box (or stalked) mistletoe, A. miquelii, (Lehm. ex Miq.) Tiegh., is found in open forest and woodlands dominated by Eucalyptus species. It is dependent on eucalypts as a host, although it occasionally parasitises Acacia species (Western

Australian Herbarium 1998–; Watson 2011). Amyema miquelii is the most widespread

Australian mistletoe (Western Australian Herbarium 1998–; Watson 2011). It is often found in extremely high abundances and is a popular nesting site for a wide range of bird species (Watson 2011). It has red flowers, grouped in threes on individual stalks, and smooth fruits that are green when unripe and yellow upon ripening (Fig. 1.2a, b).

This mistletoe is highly distinguishable across the landscape due to the yellow-bronzed colour of its leaves when exposed to sunlight. Three study sites contained A. miquelii parasitising Corymbia calophylla (Forrestfield and York 2) and Eucalyptus accedens

(Paruna).

The wireleaf mistletoe, A. preissii (Miq.) Tieghem, is found in open forest, woodland and semi-arid woodlands, and is primarily dependent on Acacia species although it has been recorded growing on other host species (Western Australian

Herbarium 1998–; Watson 2011). It has a wide spread inland distribution across the mainland of the continent with scattered distributions extending to the coast of WA

(Western Australian Herbarium 1998–; Watson 2011). Amyema preissii has abundant red flowers, borne in groups of three, with pale pink fruits (Fig. 1.2c, d). It is also a popular foraging site for a range of insectivorous bird species (Watson 2011). It is the only mistletoe with slight, needle shaped leaves, and despite its common name, its

foliage is succulent and rather delicate (Watson 2011). Two study sites contained A. preissii parasitising Ac. baileyana, Ac. podalyriifolia (Araluen) and Ac. acuminata

(York 1).

Figure 1.1 The location of five study sites in south-west Western Australia.

Perth is indicated by a star for reference.

Figure 1.2 Two Loranthaceae mistletoe species common in south-west Western Australia: the box (or stalked) mistletoe Amyema miquelii and wireleaf mistletoe Amyema preissii. a) scenescent flowers and yellow leaves of A. miquelii, with the mistletoe moth (Comocrus behri); b) the yellow ripe fruits of A. miquelii; c) open flowers and needle like leaves of A. preissii and d) ripening pink fruits of A. preissii.

1.6 AVIAN STUDY SPECIES

1.6.1 Mistletoebird (Dicaeum hirundinaceum)

The mistletoebird (body mass mb ~8 g) is the only Australian species of the family

Dicaeidae, a group of passerine birds found mainly in southern Asia and New Guinea

(Richardson and Wooller 1988). The interaction between the mistletoebird and mistletoe is considered the most specific of all seed dispersal systems in Australia

(Serventy 1973). The mistletoebird is able to grasp and swallow mistletoe fruits (after removing the exocarp by squeezing the fruit with its beak) that are relatively large in comparison to its small body size (Liddy 1983; Richardson and Wooller 1988).

Mistletoebirds also include nectar and insects in their diet (Reid 1990). The mistletoebird displays a short, specialised alimentary tract that is typical of specialised frugivores, which allows the rapid processing of the large number of mistletoe fruits that are ingested (Richardson and Wooller 1988). The mistletoebird is locally nomadic, and its presence tends to correspond with the availability of fruiting mistletoe (Higgins et al. 2006; Rawsthorne et al. 2012). The distribution of the mistletoebird ranges throughout mainland Australia to several Indonesian islands in the Arafura Sea (Higgins et al. 2006). The sexes are dichromatic, with males (Fig. 1.3a) displaying a glossy bluish-black head, wings and upperparts with a bright red throat, chest and undertail, and a white belly with a central dark streak. Females are grey above with a grey streak on a white belly, with a paler red undertail. Immature birds resemble paler females, but have a bright orange beak and gape (Fig. 1.3b).

1.6.2 Silvereye (Zosterops lateralis)

The silvereye (mb~11 g), is a small generalist frugivore that feeds on fruit, nectar and insects (Wilkinson 1931; Thomas 1980; Richardson and Wooller 1986). The silvereye does not have a specialised intestinal tract, but in line with its ingestion of insects, has a more muscular gizzard than specialist nectarivores (Richardson and Wooller 1986).

The silvereye has been recorded feeding on both fruits and flowers of A. preissii and A. miquelii (see Reid 1986 and references therein), and defecating mistletoe seeds resulting in successful establishment when transferred to suitable host branches (Keast 1958).

The silvereye is widespread throughout eastern, southern and western Australia, New

Zealand and many outlying islands, and is rather complex in its geographical variation – nine subspecies are recognised in the region (Australia, New Zealand, Antarctica and surrounding islands; Higgins et al. 2006). Zosterops lateralis chloronotus is recognised in western South Australia and WA, where it is usually considered resident through most of its range in WA, but is a seasonal visitor or nomadic in low rainfall areas of the

WA wheatbelt (Higgins et al. 2006). Plumage is usually indistinguishable between the sexes, with Z. l. chloronotus (Fig. 1.3c) displaying a conspicuous ring of white feathers around the eye, yellowish olive upperparts, with the head, back rump and uppertail coverts duller and paler, and grey belly and undertail (Higgins et al. 2006).

1.6.3 Singing honeyeater (Lichenostomus virescens)

The singing honeyeater (mb ~20–35 g) is widely distributed throughout mainland

Australia, and is generally present in open shrublands and low woodlands that are dominated by acacias. It is considered resident or sedentary throughout its range, with

four subspecies recognised (Higgins et al. 2001). The nominant L. v. virescens is recognised in south-west and southern Australia (Higgins et al. 2001). There is no sexual dimorphism in plumage. Birds have a distinctive black streak through the eye bordered with a yellow streak below the eye, a white throat and white to grey underbody with brown to grey streaks (Fig. 1.3d; Higgins et al. 2001). The singing honeyeater is a considered a nectarivore that also ingests a relatively high proportion of insects and fruit in its diet (Collins and Morellini 1979; Richardson and Wooller 1986). Like silvereyes, singing honeyeaters also have a more muscular gizzard than specialist nectarivores

(Richardson and Wooller 1986). Singing honeyeaters have been recorded feeding on the flowers and fruit of several mistletoe species, including A. miquelii and A. preissii, though its role as a potential disseminating agent of mistletoe has not been investigated

(Reid 1986).

a) b)

c) d)

Figure 1.3 Digestive adaptations associated with consuming a mistletoe fruit diet were investigated for three focal frugivorous bird species. a) male mistletoebird, b) juvenile mistletoebird, c) silvereye, and d) singing honeyeater.

2 CARBON AND NITROGEN STABLE ISOTOPES OF AUSTRALIAN

MISTLETOES AND THEIR HOSTS

Kathryn R. Napier1,2, Todd J. McWhorter3, Carlos Martínez del Rio2, Patricia A. Fleming1

1 School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150,

AUSTRALIA

2 Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA

3 School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, SA

5371, AUSTRALIA

STATEMENT OF AUTHOR CONTRIBUTION

K.R. Napier: Initiated the research, performed the leaf sampling and prepared samples for stable isotopes analysis, entered and analysed the data, and wrote the manuscript.

T.J. McWhorter: Assisted with the design and initiation of the research and provided editorial comments to versions of the manuscript from draft to final versions.

C.M. del Rio: Provided training and assistance in stable isotopes analysis, including preparation of samples and statistical analysis, and provided editorial comments to final versions of the manuscript.

P.A. Fleming: Assisted with the design and initiation of the research, advised and assisted K.R. Napier with statistical analyses, and provided editorial comments to versions of the manuscript from draft to final versions.

2.1 ABSTRACT

Australia has 75 species of native loranthaceaous mistletoes. To date, very few data have been published on the δ13C values of Australian mistletoes, while mistletoe δ15N has not been investigated in Australian mistletoes to date. This paper presents δ13C,

δ15N and nitrogen and carbon content (%) data on two loranthaceous mistletoes

(Amyema preissii (Miq.) Tieghem and A. miquelii (Lehm. ex Miq.) Tiegh.) on five unique mistletoe-host pairs from five sites in south-west Western Australia. We also investigated the effects of nitrogen-fixing hosts (Acacia species) vs. non nitrogen-fixing hosts (Eucalyptus species), aridity and season on these data. Australian loranthaceous mistletoes demonstrate a relationship with δ13C and δ15N that is consistent with those found in previous studies. Unlike other mistletoes studied to date, proportionally more nitrogen was taken up by mistletoes parasitising nitrogen-fixing (and therefore nitrogen rich) acacia hosts, compared to mistletoes on nitrogen-poor eucalypts. Australian mistletoes regulate their water use in relation to the supply of nitrogen available from the host.

2.2 INTRODUCTION

Loranthaceous mistletoes are predominantly arboreal hemiparasites, which parasitise the xylem stream of their hosts (Calder 1983; Marshall et al. 1994) via the haustorium

(a vascular connection which attaches to a host plant, Lamont 1983b; Ehleringer and

Marshall 1995). Xylem-tapping mistletoes are dependent on their hosts for water

(Ehleringer et al. 1985) and dissolved nutrients, including nitrogen (Marshall and

Ehleringer 1990; Schulze et al. 1991; Marshall et al. 1994; Wang et al. 2007), but do not rely extensively on organic solutes from the host phloem (Hull and Leonard 1964).

Studies of carbon (δ13C) and nitrogen (δ15N) stable isotope ratios have aided and greatly developed our understanding of how mistletoes obtain nutrients like carbon and nitrogen from their hosts. While hemiparasitic plants obtain virtually all water and mineral nutrients from their host plants, their reliance on their hosts for carbon varies widely, partly due to the photosynthetic capacity of the mistletoe (Press 1995).

High transpiration rates of mistletoes, as well as high resistances in the hydrolytic pathway between host and mistletoe (especially at the haustorial interface,

Davidson and Pate 1992), are the principal mechanisms maintaining a water potential gradient by which water and dissolved solutes flow towards the parasite (Davidson et al. 1989a). A strong correlation between carbon isotope discrimination and the ratio between carbon assimilation and transpiration (i.e. water use efficiency, Farquhar et al.

1982) first led to the generalisation that more positive (i.e. 13C-enriched) values of δ13C are found in more water-efficient plants. Ehleringer et al. (1985) reported that δ13C values for mistletoes were generally more negative than those of their hosts in samples taken from arid or semi-arid regions of Europe, Africa, North America and Australia, suggesting mistletoes are more profligate in their water use (Bannister and Strong

2001). In more mesic environments, however, where water is not limiting, both mistletoe and host may transpire freely and gain an adequate supply of water and xylem-transported nutrients (Panvini and Eickmeier 1993; Bannister and Strong 2001), leading to smaller differences in δ13C between mistletoes and hosts.

Ehleringer et al. (1985) also demonstrated that differences in δ13C between mistletoe and host were least for pairs where the host has high nitrogen content. Large differences in δ13C between mistletoes and hosts appear to be a consequence of high leaf transpiration rates in mistletoes, necessary for the parasites to extract sufficient nitrogen from the xylem of a nitrogen-poor host. Small differences in δ13C are associated with lower water conductance rates that are adequate to extract sufficient amounts of nitrogen from a nitrogen-rich host. Therefore, nitrogen content (Ehleringer et al. 1986; Bannister 1989) and δ15N (Schulze et al. 1991; Wang et al. 2007;

Tennakoon et al. 2011) of mistletoes and hosts are usually strongly correlated.

The differences in δ13C may also be related to the degree of heterophic carbon gain (i.e. the proportion of carbon in mistletoes obtained from its host) by mistletoes.

Smaller differences in δ13C are expected if mistletoes obtained higher amounts of carbon from their hosts (Bannister and Strong 2001). Heterotrophic carbon gain ranges from 5 to ~80% worldwide (see review by Bell and Adams 55 2011), but Australian mistletoes studied to date appear to source only 5 to 21% of their carbon from the host

(Marshall et al. 1994).

Australia has 75 species of native loranthaceaous mistletoes which are found in wooded habitats throughout the mainland (Barlow 1984; Barlow 1992; Watson 2011).

Very few data have been published on the δ13C values of Australian Loranthaceae

mistletoes to date (Marshall et al. 1994; Miller et al. 2003). This paper presents δ13C and δ15N stable isotope ratios and nitrogen and carbon content (%) data on six mistletoe-host pairs and uninfected host plants from five sites in south-west Western

Australia (WA). We investigated the differences between δ13C, δ15N, nitrogen and carbon content (%) between mistletoes and their hosts (where hosts are either nitrogen- fixing acacias or non-nitrogen-fixing eucalypts), and determine the effects of aridity and season on these data.

2.3 METHODS

2.3.1 Field sites and sampling

The study was conducted at five sites (100 x 200 m) in south-west WA where mistletoe was extremely abundant (see Table 2.1 for site details). Two sites contained Amyema preissii (Miq.) Tieghem parasitising acacia hosts, while three contained A. miquelii

(Lehm. ex Miq.) Tiegh. parasitising eucalypt hosts. Mistletoes and their host plants as well as uninfected host plants from the same sites (nearby plants that had no visible signs of previous or current mistletoe infestation) were sampled at each location in May

(autumn) and October (spring) 2010 and March (summer) 2011. A total of 212 mistletoe-host pairs and 85 uninfected host plants were sampled; all had similar exposure in the canopy. Host leaves were collected proximally to a mistletoe infestation (Bannister and Strong 2001). Approximately 15-20 fully-expanded leaves from each plant were collected and combined for isotopic analysis.

Leaf samples were dried at 60 °C for 72 hours, then ground to a fine powder and homogenised. Prepared samples were analysed at the University of Wyoming Stable

Isotope Facility, USA. Total carbon and nitrogen content (%) and δ13C and δ15N compositions (‰) were determined using a Finnigan Delta Plus XP continuous flow inlet Stable Isotope Ratio Mass Spectrometer online with a Costech EA 1108 Elemental analyser or CE Elantech 2500 CHN Elemental analyser. δ13C values are expressed with respect to Vienna Pee Dee Belemnite (V-PDB) and δ15N values with respect to atmospheric molecular nitrogen.

Table 2.1 The characteristics of the five field sites in south-west Western Australia.

Rainfall range is shown for ‘winter’ (May to September inclusive). Weather data obtained for 1981–2010 (Bureau of Meteorology 2011) from the nearest meteorological station to the study areas: Araluen and Forrestfield – Gosnells City (32°02′S, 115°58′E); Paruna – Pearce RAAF (31°40′S, 116°1′E); York 1 and York 2 – York (31°53′S, 116°46′E).

Site Mistletoe species Host species Host Aridity Site description Days with Temp. max. system scale >35°C, min <2°C; Avg. annual rainfall (winter rainfall range) Nitrogen fixing hosts Araluen 1 Araluen Country Club, Amyema preissii Acacia baileyana Acacia Mesic A heavily watered suburban garden 35, 1 day Araluen 2 Roleystone, WA A. preissii Ac.podalyriifolia Acacia Mesic with both non-native and native plants 795 mm (83–165 mm) (32°08′S, 116°05′E) present York 1 Private farming A. preissii Ac. acuminata Acacia Semi-arid A fragmented, semi-arid acacia 45, 47 days property, York, WA woodland subject to grazing 401 mm (44–71 mm) (31°51′S, 116°44′E) Non–fixing hosts Paruna Paruna Sanctuary, A. miquelii Eucalyptus accedens Eucalyptus Intermediate Australian Wildlife Conservancy, 31, 3 days Avon Valley, WA pristine warm temperate eucalypt 669 mm (70–133 mm) (31°41′S, 116°7′E) woodland Forrestfield Forrestfield, WA A. miquelii Corymbia calophylla Eucalyptus Mesic Roadside suburban reserve in a 35, 1 day (32°0′S, 116°1′E) fragmented low eucalypt woodland 795 mm (83–165 mm)

York 2 Private farming A. miquelii C. calophylla Eucalyptus Semi-arid A fragmented, semi–arid eucalypt 45, 47 days property, York, WA woodland, subject to grazing 401 mm (44–71 mm) (31°50′S, 116°44′E)

2.3.2 General statistics

δ13C, δ15N, C and N content (%) and Δδ13C values for all leaf samples were analysed as dependent variables in separate mixed-model (MM) ANOVAs with plant sample

(mistletoe, host or uninfected host leaves), host taxon (acacia or eucalypt), and degree of aridity (i.e. mesic, intermediate and semi-arid) as categorical factors, and season

(autumn, spring, and summer) as a random factor (see Table 2.2). Linear regression was used to assess the relationships between mistletoe and host δ15N/ δ13C, nitrogen/carbon content (%), and differences in mistletoe and host δ13C values (Δδ13C,

‰). Data are reported as mean ± 1 SD throughout. Statistical analyses were performed with Statistica (StatSoft Inc 2007). Statistical significance was accepted for α<0.05.

Table 2.2 Leaf carbon and nitrogen content (%), δ13C and δ15N values (‰) of mistletoe, host and uninfected host plants.

Data are averaged over three collection periods (May (Autumn) and October (Spring) 2010, March (Summer) 2012), with the apparent heterotrophy of mistletoes calculated from equation 2.1 and n referring to number of mistletoe and host pairs. Data are presented as mean ± 1 SD.

%C %N δ13C (‰) δ15N (‰) Site Aridity Mistletoe Host Mistletoe Host Mistletoe Host Mistletoe Host Uninfected Mistletoe Host Uninfected n host host Nitrogen-fixing hosts

Araluen 1 Mesic Amyema preissii Acacia baileyana 44.06±1.95 52.31±4.38 3.93±0.84 2.41±0.38 -30.84±0.99 -29.78±1.72 -31.14±1.05 -0.55±1.15 -0.24±0.97 -0.62±0.51 36

Araluen 2 Mesic A. preissii Ac. podalyriifolia 44.16±2.20 53.05±1.49 3.49±0.80 2.22±0.33 -30.37±0.98 -28.89±1.39 -29.21±0.55 -0.69±0.83 -0.57±1.17 -0.78±0.68 50

York 1 Arid A. preissii Ac. acuminata 46.86±2.06 51.42±1.23 2.88±0.56 2.45±0.20 -29.17±0.98 -27.72±0.77 -27.63±0.95 1.52±0.61 1.17±0.70 1.36±0.93 37

Non-fixing hosts

York 2 Arid A. miquelii Corymbia calophylla 50.75±1.11 49.49±0.86 1.04±0.19 1.09±0.11 -29.15±0.90 -26.55±0.98 -27.39±1.22 3.20±1.19 1.79±1.02 1.47±0.95 24

Forrestfield Semi-arid A. miquelii C. calophylla 51.19±1.34 51.25±1.22 0.80±0.20 1.06±0.15 -30.40±0.78 -28.27±0.57 -28.76±0.84 -3.44±0.57 -3.64±0.28 -3.42±4.53 39

Paruna Semi-arid A. miquelii Eucalyptus accedens 50.15±4.21 51.79±1.57 0.65±0.16 0.84±0.07 -31.20±0.60 -28.27±0.87 -28.82±0.59 -1.82±1.50 -1.47±0.90 -2.03±0.79 26

2.4 RESULTS

δ13C, δ15N, C and N content (%) values of mistletoes, infected hosts and uninfected hosts are summarised in Table 2.2.

2.4.1 Carbon

δ13C values (‰) and carbon content (%) of leaves differed significantly between mistletoes, hosts, and uninfected host plants (Table 2.3), with host plants and uninfected hosts significantly enriched in δ13C compared to mistletoe leaves (Fig. 2.1a) and mistletoes having a lower carbon content than the host plants (Fig. 2.1c). Host genus had a significant effect on δ13C values and carbon content (%) (Table 2.3) of plant samples (i.e. mistletoe and infected and uninfected host leaves), with eucalypt infected hosts and uninfected host plants enriched in δ13C compared to samples taken from acacia plants (infected host and uninfected hosts, Fig. 2.1a), and mistletoes on eucalypt hosts demonstrating a higher percentage of carbon compared to mistletoes on acacia hosts (Fig. 2.1c). Samples (including mistletoe and infected and uninfected host leaves) collected from semi-arid sites were enriched in comparison to mesic and intermediate sites, but degree of aridity had no effect on carbon % (Table 2.3). δ13C and carbon % values did not differ significantly with season (Table 2.3).

2.4.2 Nitrogen

δ15N and nitrogen content (%) differed significantly between mistletoes, infected and uninfected host plants (Table 2.3), with samples taken from acacia plants (mistletoe and infected and uninfected hosts) significantly enriched in δ15N and with higher nitrogen

contents compared to samples taken from eucalypt plants (mistletoe and infected and uninfected hosts) (Figs. 2.1b, d). Nitrogen content from mistletoes on acacia hosts was higher compared to infected and uninfected acacia hosts (Fig. 2.1d). Degree of aridity had a significant effect on δ15N values and nitrogen content (%) (Table 2.3), with samples from semi-arid sites enriched in δ15N and with higher N content by comparison with mesic and intermediate sites. Season also had a significant effect on δ15N values and nitrogen content (%) (Table 2.3), with δ15N values more enriched in autumn samples compared to spring and summer samples, while nitrogen content was highest in spring and lowest in summer.

Categorical factors included plant sample, host genus and degree of aridity. Season was set as a random factor. Significant variables are indicated in bold.

C (%) N (%) δ13C (‰) δ15N (‰)

Plant sample (mistletoe, host, uninfected host) F2,502=127, P<0.001 F2,503=103, P<0.001 F2,503=170, P<0.001 F2,503=6.70, P=0.001

Host genus (acacia or eucalypt) F1,271=35.9, P<0.001 F1,46=1030, P<0.001 F1,185=21.1, P<0.001 F1,51=82.7, P<0.001

Degree of aridity (mesic, intermediate and semi-arid) F2,268=0.23, P=0.794 F1,45=6.58, P=0.003 F2,181=107, P<0.001 F2,50=211, P<0.001

Season (autumn, spring, summer) F2,501=1.78, P=0.170 F2,501=6.85, P=0.001 F2,501=2.55, P=0.079 F2,501=6.38, P=0.002

-26 4 a) Acacia b) Eucalypt -27 2

-28 0

-29

C(‰)

N (‰) N

13 15 -2 -30

-4 -31

-32 -6 mistletoe host uninfected host mistletoe host uninfected host

60 c) 5 d) 58

56 4

52 3

C (%) C (%) N 48 2

44 1

40 0 mistletoe host uninfected host mistletoe host uninfected host

Figure 2.1 Stable isotope values and percentages of carbon and nitrogen for mistletoe, infected and uninfected host samples on acacia (black symbols) or eucalyptus (white symbols) hosts. a) δ13C values (‰), b) δ15N values (‰), c) C content (%), and d) N content (%). Data are presented as mean ± 1 SD.

2.4.3 Relationships between mistletoes and hosts

The δ13C values of mistletoes are significantly correlated with those of their hosts

2 (linear regression: F1,210=91.9; P<0.001, R =0.304; Fig. 2.2a), even though there is considerable variation around the regression line. Mistletoes were generally more depleted in δ13C than their hosts (Fig. 2.1a, Table 2.2), but for 18 individual samples

(out of 212), mistletoes were more positive than their hosts (Fig. 2.2a). The relationship between mistletoe and host carbon content (linear regression: F1,207=32.9; P<0.001,

R2=0.137) significantly deviates from a 1:1 ratio (95% confidence interval of the slope:

-0.48 to -0.99; Fig. 2.2b), indicating that mistletoes do not obtain all of their carbon from their hosts. Differences in δ13C between mistletoes and hosts were significantly affected by host taxon (F1,190=54.7; P<0.001) and degree of aridity (F2,189=3.50;

P=0.031), but not season (F2,205=1.16; P=0.317); differences were greater on nitrogen- poor eucalypt hosts compared to nitrogen-rich acacia hosts (Fig. 2.2e), and were greater at mesic and semi-arid sites compared to intermediate sites.

Both δ15N and nitrogen content (%) of mistletoe leaves are highly correlated

15 with those of their hosts. The relationship between mistletoe and host δ N (F1,210=862;

P<0.001, R2=0.804) was not significantly different from 1 (95% confidence interval of the slope: 0.96 to 1.10; Fig. 2.2c). The relationship between mistletoe and host nitrogen

2 content (F1,210=612.65; P<0.001, R =0.745) deviates from a 1:1 ratio (95% confidence interval of the slope: 1.59 to 1.87; Fig. 2.2d). Mistletoes on nitrogen-rich acacia hosts tend to have higher leaf nitrogen concentrations than their hosts (F1,121=4.25; P=0.041,

R2=0.03; relationship deviates from a 1:1 ratio with the 95% confidence interval of the slope from 0.019 to 0.923; Fig. 2.2d), compared to mistletoes on nitrogen-poor eucalypt

2 hosts (F1,87=45.3; P<0.001, R =0.342; 95% confidence interval of the slope: 0.613 to

1.13; Fig. 2.2d; slope differs significantly from mistletoes on acacia hosts F2,208=9.1;

P<0.001 ). The differences between mistletoe and host δ13C (Δδ13C) were greatest when host leaf nitrogen content was low (linear regression: F1,4=24.45; P=0.008,

R2=0.859; Fig 2.2e).

-22 a) 58 b) 56 Col 21 vs Col 23 -24 Col 19 vs Col 21 54 Col 21 vs Col 23: -1.0440 Col 21 vs Col 23: -1.4880 -26 Col 19 vs Col 21: 2.4100 Col 19 vs Col 21: 2.2200 52 Col 21 vs Col 23: -1.4260 50 -28 Col 19 vs Col 21: 2.4500 Col 21 vs Col 23: -2.6020 Col 19 vs Col 21: 1.0900 48 Col 21 vs Col 23: -2.1320 -30 Col 19 vs Col 21: 1.0600 Col 21 vs Col 23: -2.2940

C mistletoe 46

13 Col 19 vs Col 21: 0.8400 -32 Plot 1 Regr C % Mistletoe 44 42 -34 40 2 2 F1,210=91.91; P<0.001, R =0.304 F1,207=32.86; P<0.001, R =0.137 -36 38 -36 -34 -32 -30 -28 -26 -24 -22 38 40 42 44 46 48 50 52 54 56 58 13C host Host C % 6 c) 6 d)

4 5

2 F1,121=4.25, P=0.04, R =0.03 2 4

N (‰)

15 0 3 Araluen 1 (A, mesic) Araluen 2 (A, mesic) Forrestfield (E, semi-arid) -2 2 Paruna (E, semi-arid) Mistletoe N (%) Mistletoe York 1 (A, arid) Mistletoe Mistletoe York 2 (E, arid) 2 -4 1 F1,87=45.3, P<0.001, R =0.745 F =862, P<0.001, R2=0.804 2 1,210 F1,210=612, P<0.001, R =0.342 -6 0 -6 -4 -2 0 2 4 6 0 1 2 3 4 5 6 Host 15N (‰) Host N (%) 1 e)

-1

-2

misttletoe-host, ‰ misttletoe-host,

13 -3

2 F1,4=24.5, P=0.008, R =0.859 -4 0.5 1.0 1.5 2.0 2.5 3.0 Host N (%)

Figure 2.2 Correlations between mistletoe and host a) δ13C (‰), b) carbon content (%), c) δ15N (‰) and d) nitrogen content (%)

Dashed lines represent a 1:1 correspondence between mistletoe and host nitrogen. e) relationship between average differences in mistletoe and host δ13 values (Δδ13C, ‰) with standard deviations as error bars. Grey symbols indicate Amyema miquelii on nitrogen-poor eucalypt hosts (E), white symbols indicate A. preissii on nitrogen-rich acacia hosts (A).

2.5 DISCUSSION

We found significant effects of plant sample (mistletoe, uninfected and infected hosts), host taxon (eucalypt, acacia), aridity and season on δ13C and δ15N values and on carbon and nitrogen content. These patterns reflect water and nitrogen relations in these plants which reveal some differences in the ecology of Australian mistletoes compared with other mistletoes previously described. The differences between mistletoe and host δ13C were greatest at semi-arid, and surprisingly also at mesic sites on nitrogen-poor eucalypt hosts, indicating lower water use economy by mistletoes in both of these situations.

2.5.1 δ13C of mistletoes and hosts: water relations and effects of aridity

The range of δ13C values reported in this study are consistent with previously published results, with values of approximately -28 to -30‰ reported for Australian mistletoes and approximately -26‰ for their hosts (Marshall et al. 1994; Miller et al. 2003). As dry conditions reduce stomatal conductance, which leads to reduced discrimination against

δ13C, water-stressed plants in more arid environments (such as York in this study) tend to have more positive δ13C values than more mesic environments (such as Araluen and

Forrestfield) (Farquhar et al. 1989; Stewart et al. 1995). Agricultural land-use practices, which alter the exposure of plants to sunlight, may also influence plant δ13C values, resulting in more enriched δ13C values compared to forested areas due to water use efficiency mechanisms and the removal of the canopy effect which tends to lower the δ13C values of plants (Hobson 2007).

Given that mistletoes need to maintain a water potential gradient in order to receive water and nutrients from their hosts, host water status has been hypothesised to

be an important determinant of mistletoe growth (Miller et al. 2003). However, like

Miller et al. (2003), we found no differences between δ13C leaf values between infected and uninfected hosts, suggesting that infection with mistletoe is not leading to increased water stress on these hosts. Average mistletoe δ13C values were lower than their hosts at all sites, supporting the relationship observed between mistletoe and host δ13C observed in semi-arid and arid locations world wide (Ehleringer et al. 1985; Schulze et al. 1991; Marshall et al. 1994). The significant differences between mistletoe and host

δ13C values appear to be related to both nitrogen availability (between nitrogen-rich acacia and nitrogen-poor eucalypt hosts, Fig. 2.2e), and differences in water availability between semi-arid and intermediate sites.

2.5.2 Relationships with nitrogen

Like other mistletoe species studied to date (Schulze et al. 1991; Bannister and Strong

2001; Wang et al. 2007; Tennakoon et al. 2011), the two species of Australian loranthaceous mistletoes in this study also acquire nitrogen from their hosts, confirmed by strong correlations between mistletoe and host leaf δ15N values (Fig 2.3c). Nitrogen contents of mistletoes and their hosts are usually also strongly correlated, with a slope that deviates from a one-to-one relationship. Previous studies report lower nitrogen concentrations in mistletoes compared to nitrogen-rich hosts, but higher nitrogen concentrations in mistletoes compared to nitrogen-poor hosts (Ehleringer et al. 1986;

Bannister 1989; Schulze et al. 1991; Bannister and Strong 2001). However, this was not the case in the present study, with mistletoes having lower concentrations of nitrogen in comparison to their nitrogen-poor eucalypt hosts, but more nitrogen than their hosts for mistletoes parasitising nitrogen-rich acacia hosts (Fig. 2.2d).

The relationship between the differences in mistletoe and host δ13C and host leaf nitrogen content is similar to those predicted and found by Ehleringer et al. (1985) and

Schulze et al. (1991) - the difference in δ13C is least when host nitrogen content is high

(i.e. acacia hosts, Fig. 2.2c). These differences in water use efficiency between mistletoe and host are therefore related to the nutritional quality of the host - the most negative differences in δ13C suggest that a greater differential in water use efficiency is required to extract nitrogen from nitrogen-poor eucalypt hosts.

2.5.3 Conclusions

Two species of Australian loranthaceous mistletoes demonstrate a relationship with

δ13C and δ15N that is consistent with those found in previous studies. Unlike other mistletoes studied to date, proportionally more nitrogen was taken up by mistletoes on nitrogen-rich acacia hosts, compared to mistletoes on nitrogen-poor eucalypt hosts. A greater differential in water use efficiency is therefore required for mistletoes on nitrogen-poor eucalypt hosts to extract nitrogen, resulting in more negative differences in δ13C in comparison to mistletoes on acacia hosts. Differences in water use efficiency between Australian mistletoes and hosts are therefore related to the nutritional quality of the host, with Australian mistletoes regulating their water use in relation to the supply of nitrogen available from the host.

3 DO BIRD SPECIES RICHNESS AND COMMUNITY STRUCTURE VARY

WITH MISTLETOE FLOWERING AND FRUITING IN WESTERN

AUSTRALIA?

Kathryn R. Napier1, Suzanne H. Mather2, Todd J. McWhorter3, Patricia A. Fleming1

1 School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, AUSTRALIA

2 3 Hardy Road, Nedlands, WA 6009, AUSTRALIA

3 School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, SA 5371, AUSTRALIA

This chapter was accepted for publication in the journal Emu on March 25, 2013

(published online early, http://dx.doi.org/10.1071/MU12098).

STATEMENT OF AUTHOR CONTRIBUTION

K.R. Napier: Initiated the research, performed the mistletoe phenology and bird surveys, collected and entered data, analysed the data, and wrote the manuscript.

S.H. Mather: Provided training and assistance in the conduction of bird surveys, and provided editorial comments to a draft version of the manuscript.

T.J. McWhorter: Assisted with the design and initiation of the research and provided editorial comments to versions of the manuscript from draft to publication.

3.1 ABSTRACT

Worldwide, mistletoes act as a keystone resource, providing food (nectar, fruit and foliage) and structural (nesting sites) resources to hundreds of fauna species. In

Australia, loranthaceous mistletoes depend on birds for pollination and dispersal, and provide important nectar and fruit resources to a large number of nectarivorous and frugivorous bird species. We investigated whether bird species richness and community structure varies with flowering and fruiting of two common mistletoe species (family

Loranthaceae, Amyema preissii and A. miquelii) through monthly surveys for one year at five sites in south-west Western Australia. Flowering and fruiting periods were distinct and differed both amongst sites and between mistletoe species. Nectar and ripe fruit were available for up to 5 and 6–7 months (A. miquelii and A. preissii respectively) at individual sites, but were available every month of the year across all sites. The presence of fruiting, but not flowering, mistletoe was associated with changes in bird community structure. Mistletoebirds (Dicaeum hirundinaceum) were significantly more likely to be recorded during months when ripe mistletoe fruit was present and the overall bird species richness was higher for these survey months. Mistletoes provide important resources, but further investigation is required to assess keystone species status in south-west WA.

3.2 INTRODUCTION

Mistletoes are a polyphyletic group of shrubby, aerial hemiparasitic flowering plants with over 1,500 species found over a wide range of habitats and across all continents with the exception of Antarctica (Kuijt 1969; Calder 1983; Watson 2001). A diversity of ‘showy’ mistletoes (family Loranthaceae) is native to Australia, with 75 species currently recognised (Barlow 1984; Barlow 1992; Watson 2011). Watson et al. (2001) first proposed that mistletoes act as a keystone resource (sensu Power et al. 1996) in forests and woodlands worldwide, due to the pervasive effects they have on these habitats through the provision of nutritional and nesting resources (confirmed by

Watson and Herring 2012). Peres (2000) identified four criteria used to define a keystone plant: reliability and abundance of resources, degree of consumer specificity and temporal redundancy. Kotliar (2000) further proposed that keystone species should perform functions not otherwise carried out. In this study, we address whether two species of loranthaceous mistletoes in south-west Western Australia (WA) meet the keystone criteria established by these authors.

In Australia, mistletoe nectars and fruits are consumed by at least 50 bird species, including several honeyeater species (Keast 1958; Reid 1987; Turner 1991;

Brown et al. 1997). In addition to providing a nutritious food source, many mistletoe species also have extended flowering and fruiting phenologies that minimise competition with other plant species, but is also important for sustaining populations of their avian pollinators and dispersers. Australian mistletoes continually draw upon their host’s water and nutrient resources and consequently are able to flower and set fruit during dry seasons when little other nectar or fruit is available in the landscape (Paton

and Ford 1977; Reid 1986; Watson 2001). In addition to flowering and fruiting at different times to most other plants, discontinuous ripening (both within a species and amongst species within communities) extends flowering and fruiting periods (Reid

1986; Hawksworth and Wiens 1996; Watson 2001). For example, most temperate southern Australian mistletoes flower during summer, contrasting with largely spring and autumn flowering for the majority of ornithophilous plant species present in these areas (Reid 1986; Watson 2011). In more arid areas, where mistletoes are a critical food source to nectarivorous species, some mistletoe species flower during winter, others during summer, while other species exhibit all year round flowering (Reid 1986;

Watson 2011).

Mistletoes also provide the most reliable (and sometimes the only) source of fruit for their avian dispersers. Further, their semi-succulent leaves have a high concentration of nutrients and are consumed by herbivores (Watson 2001). Their dense, multibranched structure means that many mistletoe species offer important nest and foraging sites for animals in an otherwise open canopy (see review by Watson 2001).

In Australia, bird species from more than 60 families (across 16 orders) and several mammalian families nest in mistletoe, and 66% (of 330 species) of Australian arboreal nesting bird species have been recorded using mistletoes as nest sites (Cooney et al.

2006). Many insectivorous species (e.g. thornbills, whistlers) also use mistletoe clumps as a foraging substrate, as mistletoe often has abundant and distinctive insect assemblages (Turner 1991; Watson 2001; Start 2011; Watson 2011); however Burns et al. (2011) found no difference in insect assemblages. Mistletoes therefore reliably provide important food and shelter resources for hundreds of fauna species, providing resources and ecosystem services out of proportion to their abundance and contribution

to biomass (Davidson et al. 1989b; Watson 2001; Watson 2002; Mathiasen et al. 2008;

Watson and Herring 2012).

Loranthaceous mistletoes provide birds with important nectar and fruit resources

(Kuijt 1969; Calder 1983; Davidar 1985; Reid 1986; Ladley et al. 1997; Robertson et al. 1999). With two exceptions, the root-parasitic Western Australian Christmas tree,

Nuytsia floribunda, and Atkinsonia, Atkinsonia ligustrina (Hawkeswood 1981; Watson

2011), all Australian loranthaceous mistletoes are pollinated by birds. These mistletoes tend to display brightly-coloured, odourless flowers, with abundant, sugar-rich nectar

(up to 60% total sugar content, primarily glucose and fructose sugars, Reid 1986; Stiles and Freeman 1993; Baker et al. 1998), characteristics typically associated with ornithophilous pollination. Although they are not dependent on mistletoe nectar as a primary food source, a wide range of bird species pollinate these mistletoes, including several species of honeyeaters (Reid 1986; Watson 2001; Watson 2011). The degree of consumer specificity between mistletoes and their pollinators has led several authors to suggest that long term negative consequences for both interacting organisms and perhaps indeed the entire ecosystem may ensue if this balance were disrupted (Reid et al. 1995; Watson 2001).

Birds are also responsible for seed dispersal for nearly all Australian loranthaceous mistletoes (with the exception of N. floribunda, which is wind dispersed

(Watson 2011)). Mature mistletoe fruits are often brightly coloured, fairly large and sweet and act as a food source for obligate and opportunistic bird species worldwide

(see review by Watson 2001). The composition of the fleshy fruit pulp surrounded by a layer of viscin varies among species, but most mistletoe fruits contain a high proportion

of carbohydrates, lipids and protein (López de Buen and Ornelas 2001; Watson 2001;

Barea 2008).

In addition to supporting generalist feeders, Australian mistletoes also support two mistletoe fruit specialists. The mistletoebird, Dicaeum hirundinaceum, is found throughout mainland Australia (Keast 1958; Blakers et al. 1984) and is locally nomadic, its presence corresponding with the availability of fruiting mistletoe (Rawsthorne et al.

2012). The rare painted honeyeater, Grantiella picta, found across the eastern inland side of the continent (Reid 1986), is considered the original (Australian) ‘mistletoe bird’ as the mistletoebird did not colonise Australia until possibly as recently as the Holocene

(~12,000 YBP, Reid 1991). The diversification and radiation of mistletoes across

Australia has therefore depended upon species such as the painted honeyeater and its ancestors (Watson 2011).

Despite their importance as a food resource, there have been few studies examining how mistletoe may influence changes in Australian bird communities (see studies by Turner 1991; Watson 2002; Watson et al. 2011; Watson and Herring 2012).

Additionally, we have extremely limited information on mistletoe fruiting and flowering phenology in south-west WA. The aim of this study was to investigate if bird species richness and community structure varies with flowering and fruiting of two

Loranthaceae mistletoe species common in south-west WA, the wireleaf mistletoe

(Amyema preissii) and box mistletoe (A. miquelii). This study examined how flowering and fruiting phenology compared with year-round bird community surveys. We predicted that: 1) there would be a greater number of bird species present (i.e. greater species richness) at our study sites when mistletoe flowers or fruit were available, and

2) bird community structure at our study sites would reflect variability in available resources, and 3) mistletoebirds would only be present in an area when ripe mistletoe fruit was available. We then discuss the findings of this study in relation to the keystone criteria identified by Peres (2000) and Kotliar (2000).

3.3 METHODS

3.3.1 Study sites

The study was conducted at five sites (100 x 200 m) in south-west WA where mistletoe was extremely abundant, from February 2010 to January 2011 (Table 3.1). Two sites contained A. preissii parasitising Acacia spp. hosts, whilst three contained A. miquelii parasitising Eucalyptus and Corymbia hosts. All sites were dominated by mistletoe with few other sources of fruit or nectar present (Table 3.1), with the exception of

Araluen. Araluen is surrounded by a dense urban matrix, including flowering and fruiting plants in extensive adjacent gardens; and while Forrestfield was also surrounded by suburban matrix, the area is more rural with larger property sizes and more native vegetation (Table 3.1). One of the sites (York 2) contained both species of mistletoe, however A. preissii was present in extremely low densities on 2 individual trees only.

In south-west WA, flowering and fruiting records suggested that both mistletoe species have narrow flowering and fruiting periods (A.N. Start, Western Australian Herbarium,

WA Department of Environment and Conservation, pers. comm.). Flowering (January to March) and fruiting (April to July) periods of A. preissii differ from those of A. miquelii in south-west WA, which has flowering records from March to April and fruiting records from June, November and December (A.N. Start, pers. comm.).

South-west WA experiences hot summers and cool wet winters. York (in the highly fragmented agricultural wheatbelt of WA) is situated further inland than the other sites, and experiences a more extreme temperature range and lower rainfall compared to the other sites (Table 3.1).

3.3.2 Fruit and flower phenology

The flowering and fruiting phenologies of A. preissii and A. miquelii were measured by counts of flowers (categorised as ‘bud’, ‘open’ or ‘senescent’) and fruit (‘immature’,

‘unripe’, ‘ripe’ or ‘bare’) at approximately 5 week intervals. At each site, 30 cm sections of tagged branches (measured proximally from the tip of the branch) of up to

21 randomly-selected mistletoe plants were monitored for each survey period, as per

Barea and Watson (2007). These branches ranged in height from 0.5–15m high. Each tagged branch was monitored every 5 weeks, with counts made of flowers (buds through senescence) and fruit (unripe through to stalks where fruit had been removed

(‘bare’)). As the total number of flowers and fruits varied between branches, an index of relative fruit and flower abundance was calculated (expressed as the proportion of fruit and flowers per 30 cm of branch, summing to 1) which were averaged across all mistletoe plants for each site/host. Mistletoes surveyed included A. preissii parasitising

Acacia baileyana (n=21), Ac. podalyriifolia (n=21) and Ac. acuminata (n=21), and A. miquelii parasitising Eucalyptus accedens (n=14) and Corymbia calophylla (n=16, n=18; Table 3.1).

Table 3.1 The characteristics of the five sites surveyed in south-west Western Australia.

The range of all mistletoe plant heights are presented underneath species names, with heights of tagged mistletoe plants underlined. Dates exclude the month of July for all sites. Rainfall range is shown for ‘winter’ (May to September inclusive). Weather data obtained for 1981-2010 (Bureau of Meteorology 2011) from the nearest meteorological station to the study areas: Araluen and Forrestfield – Gosnells City (32°02′S, 115°58′E); Paruna – Pearce RAAF (31°40′S, 116°1′E); York 1 and York 2 – York (31°53′S, 116°46′E).

Site Total time recorded: Mistletoe Host species Site description Vegetation Days with Temp. max. >35°C, min dates species <2°C; Avg. annual rainfall (winter rainfall range) 1 ‘Araluen’ Araluen Country 11 months: Amyema preissii, Acacia baileyana A heavily watered suburban Nerium oleander, Corymbia calophylla, 35, 1 day Club, Roleystone, Feb 2010–Jan 2011 0.5–2m Ac. podalyriifolia garden with both non–native Eucalyptus wandoo, E. forestiana, as well 795 mm (83–165 mm) WA (non WA natives) and native plants present as various Eucalyptus, Grevillea and (32°08′S, 116°05′E) Callistemon species and one species of Rubus also present 2 ‘York 1’ Private farming 11 months: A. preissii Ac. acuminata A fragmented, semi arid acacia Dominated by Ac. acuminata 45, 47 days property, York, WA Feb 2010–Jan 2011 0.5–2–5m woodland subject to grazing 401 mm (44–71 mm) (31°51′S, 116°44′E) 3 ‘Paruna’ Paruna Sanctuary, 11 months: A. miquelii E. accedens Australian Wildlife E. accedens and E. wandoo, with scattered 31, 3 days Avon Valley, WA Feb 2010–Jan 2011 10–15–25m E. wandoo Conservancy, pristine warm C. calophylla 669 mm (70–133 mm) (31°41′S, 116°7′E) C. calophylla temperate eucalypt woodland 4 ‘Forrestfield’ Forrestfield, WA 11 months: A. miquelii C. calophylla Roadside suburban reserve in Dominated by C. calophylla with scattered 35, 1 day (32°0′S, 116°1′E) Feb 2010–Jan 2011 2–4–20m E. wandoo a fragmented low eucalypt E. wandoo, with Hovea pungens, Goodenia 795 mm (83–165 mm) woodland fasciculata and Banksia spp present 5 ‘York 2’ Private farming 9 months: A. miquelii C. calophylla A fragmented, semi-arid Dominated by C. calophylla and E. wandoo. 45, 47 days property, York, WA April 2010–Jan 2011 2–4–20m E. wandoo eucalypt woodland, subject to Gastrolobium and Banksia spp. also 401 mm (44–71 mm) (31°50′S, 116°44′E) grazing present, as well as Ac. acuminata (parasitised with very low densities of Am. preissii).

3.3.3 Bird species

Monthly surveys of bird species presence/absence at each site were carried out using a standardised search method (Watson 2003). We did not attempt to estimate bird species abundance due to different detectabilities of species. At least three 20-minute surveys were conducted back-to-back, with the stopping rule (after three surveys being reached) that the number of species seen in a single sampling period of 20 minutes was less than or equal to the number of species seen in two previous subsequent sampling periods

(Watson 2003; Watson 2004b). Surveys commenced within 90 minutes after sunrise

(0503 to 0718).

3.3.4 Statistical analysis

Differences in fruiting and flowering periods were compared among sites (and host species in the case of Araluen) and month by two-way ANOVA, with Tukey-Kramer post hoc tests for unequal sample sizes as required. Proportions of ripe fruit and open flowers were arcsine squareroot transformed prior to analysis to meet requirements of parametric statistics.

1) Bird species richness (total number of bird species recorded) was compared among sites and for months when flowers or fruit were present (‘open’ and ‘ripe’) or absent (‘bud’, ‘senescent’ and ‘immature’, ‘unripe’, ‘bare’) by two-way ANOVA, with

Tukey-Kramer post hoc tests for unequal sample sizes as required.

2) Bird community structure: Bird data, classified as presence (1) or absence (0) for each bird species for each monthly survey, were analysed by multidimensional

scaling (MDS) in the program PAST 2.08b (Hammer et al. 2001). Bird community structure was then compared via two-way analysis of similarity (ANOSIM, Bray-Curtis similarity matrices; PAST 2.08b), using site (1–5) and either flowering (comparing months with or without ‘open’ flowers) or fruiting (comparing months with or without

‘ripe’ fruit available) as independent factors. The ANOSIM test statistic (R) contrasts the differences among groups with variation within groups, a large positive R (up to 1) signifying dissimilarity between groups; significance is calculated via permutation

(Clarke 1993). Similarity Percentage (SIMPER) was then used to assess which individual species were primarily responsible for observed differences (Clarke 1993) and then subsequently for feeding guilds (frugivore, nectarivore, insectivore, granivore and omnivore, see Table 3.2).

3) Presence/absence of bird species: To examine the relationship of bird species and mistletoe flower presence, contingency tables were constructed for each month of the presence/absence of individual bird species compared to the presence/absence of mistletoe flowers or fruit. These contingency tables were analysed for significance via

Fisher’s exact probability using STATISTICA (StatSoft Inc 2007), followed by a

Bonferroni correction.

Statistical significance was set to α<0.05. Results are presented as mean ± 1 SD.

3.4 RESULTS

41 bird species were recorded over 53 surveys at the five sites, including 2 frugivorous

(mistletoebird and silvereye) and 6 nectarivorous species (brown, New Holland, singing and white-cheeked honeyeaters, red wattlebird and western spinebill, Table 3.2).

3.4.1 Presence of mistletoe flowers

Flowers (and therefore nectar) of both mistletoe species were available for 2 to 3 months at each site, spanning the Australian summer and autumn months (from

December to February; A. preissii, Fig. 3.1a, c, e; from December to May; A. miquelii,

Fig. 3.1b, d, f), with peak abundances occurring at different times at each site (Fig. 3.1).

A significant site by month interaction (F48,1014=59.66, P<0.001) showed that flowering periods differed among sites and between mistletoe species. We note, however, that differences between hosts at the same site did not affect flowering or fruiting phenology

(A. preissii was monitored on hosts Ac. baileyana and Ac. podalyriifolia at Araluen, see

Fig. 3.1c, e).

1) Bird species richness: There were no significant differences in bird species richness (i.e. number of species recorded) between months when mistletoe was flowering or not (flowers present: 11.1±4.1, n=13 monthly surveys; flowers absent:

9.8±4.0 bird species, n=40 surveys; F1,43<0.01, P=0.975), or differences among sites

(F4,43=0.57, P=0.686). However, a significant interaction term (site x presence of flowers: F4,43=6.49, P<0.001, Fig. 3.2a), showed that Araluen had significantly higher bird species richness compared to all other sites (with the exception of York 1) when flowers were not present (outside the summer months, Tukey-Kramer post hoc tests).

There were also no significant differences in bird species richness when the presence of nectarivorous species only (n=6 species) was compared for months when mistletoe was flowering vs months when flowers were absent (flowers present: 1.9±1.8, n=13 monthly surveys; flowers absent: 1.7±1.3 bird species, n=40 surveys; F1,43=1.11, P=0.297).

There was a significant difference among sites (F4,43=20.71, P<0.001), and a significant

interaction term (site x presence of flowers: F4,43=2.80, P=0.037, Fig. 3.2b) demonstrated that even when flowers were not present, Araluen had significantly more nectarivorous species compared to all other sites (with the exception of York 2, Tukey-

Kramer post hoc tests).

2) Bird community structure: Bird community structure also varied among sites

(two-way ANOSIM; site: R=0.75, P<0.001), but the presence of flowering mistletoe

(comparing months with or without ‘open’ flowers) did not have a significant effect on bird community structure (flowering: R=0.08, P=0.208).

3) Presence/absence of bird species: The grey butcherbird (Cracticus torquatus) was the only bird species that was more likely to be recorded during months when mistletoe was flowering (Fisher’s exact test: P=0.042), while the western gerygone was less likely to be recorded during these months (P=0.042). However, as these differences were not significant after Bonferroni correction, these results will not be discussed further. During surveys, four nectarivorous honeyeater species (brown, New

Holland, singing, and white-cheeked honeyeaters) were directly observed feeding on flowers of both mistletoe species.

3.4.2 Presence of ripe mistletoe fruit

Ripe fruit appeared some 2–7 months after flowering, and was available for 2 to 5 months, with peak abundances occurring in winter (June and August: A. preissii, Fig.

3.1a, c, e) and spring/summer (November and January: A. miquelii, Fig. 3.1b, d, f). A significant interaction term (site x month: F48,1014=26.66, P<0.001) demonstrated that fruiting periods differed among sites and between mistletoe species.

1) Bird species richness: There were significantly more bird species present (i.e. greater species richness) when surveys were carried out when ripe mistletoe fruit was present (fruit present: 12.06±4.27, n=16 monthly surveys; fruit absent: 9.24±3.65, n=37 surveys; F1,43=7.94, P=0.007). There was also a significant difference among sites

(F4,43=13.60, P<0.001) and a significant interaction term (site x month: F4,43=3.77,

P=0.010; Fig. 3.2c) with the two sites surrounded by urban matrix showing signficiantly higher (Araluen) or lower (Forrestfield) bird species richness (Fig. 3.2c).

2) Bird community structure: Bird community structure was significantly different for months when ripe mistletoe fruit was present, compared with months when fruit was absent or unripe (two-way ANOSIM; fruiting: R=0.28, P=0.002), with significant differences also present among sites (site: R=0.88, P<0.001). The mistletoebird made the greatest contribution of all species (n=41) to the distinction between bird community structure for fruiting and non-fruiting months (6.3%,

SIMPER). When analysed for the five feeding guilds (SIMPER, standardised by the number of species within each guild), the two frugivore species contributed an average of 32.7% to the distinction, compared with 25.2% for six nectarivore species, 17.6% for five omnivorous species, 14.9% for seven granivore species and 9.6% for 21 insectivore species.

3) Presence/absence of bird species: The mistletoebird was the only species that was significantly more likely to be recorded during months when ripe mistletoe fruit was present (Fisher’s exact test: P<0.001, significant after Bonferroni correction), although mistletoebirds were also recorded at three sites (York 1, Paruna, York 2) during months when no ripe mistletoe fruits were recorded (2, 1 and 5 months for these

sites, respectively). The mistletoebird and a parrot, the Australian ringneck, Barnardius zonarius, were the only bird species directly observed consuming mistletoe fruit during our surveys (the Australian ringneck feeding on A. preissii; mistletoebirds feeding on both mistletoe species). Mistletoebirds were frequently observed probing the green unripe fruits of A. miquelii and pale pink fruits of A. preissii and then either rejecting (A. miquelii and A. preissii) or occasionally ingesting the unripe fruit (A. preissii only).

Many other species were observed perching within mistletoe clumps, including the yellow-rumped thornbill, red-capped robin, rufous whistler, grey fantail, varied sittella, weebill, striated pardalote, western spinebill and silvereye.

b) Paruna: A. miquelii on E. accedens a) York 1: A. preissii on Ac. acuminata Fl. bud 1.0 1.0 Fl. open Fl. senescent Fr. immature 0.8 0.8 Fr. unripe Fr. ripe Bare

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0

c) Araluen: A. preissii on Ac. baileyana d) Forrestfield: A. miquelii on C. calophylla 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

Proportion per 30cm branch per Proportion

0.2 0.2

0.0 0.0

e) Araluen:A. preissii on Ac. podalyrifolia f) York 2: A. miquelii on C. calophylla 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0 Feb Mar Apr May June Aug Sept Oct Nov Dec Jan Feb Mar Apr May June Aug Sept Oct Nov Dec Jan

Figure 3.1 Flowering and fruiting phenology of the mistletoes Amyema preissii (left hand panel) and Amyema miquelii (right hand panel) from February 2010 to January 2011.

A. preissii: two locations on 3 host species; York 1: Acacia acuminata (a) n=21; Araluen: Ac. baileyana (c) n=21, Ac. podalyrifolia (e) n=21. A. miquelii: three locations on 2 host species; Paruna: Eucalyptus accedens (b) n=14; Forrestfield: Corymbia calophylla (d) n=16; York 2: C. calophylla (e) n=18. Values are expressed as the average proportion of flowers and fruits per 30cm of branch. Lines denote flowers, solids denote fruits. Course lines denote flowers in ‘bud’, crossed lines denote ‘open’ and fine lines denote ‘senescent’. Light grey denotes ‘immature’ fruits, grey denote ‘unripe’, black denote ‘ripe’ and white denote ‘bare’. Arrows indicate peak abundances of flowers (grey) and fruit (black).

18 a) All bird species

10 Araluen (n=8, 3) 8 York 1 (n=8, 3) Paruna (n=8, 3) 6 Forrestfield (n=9, 2) 4 York 2 (n=7, 2)

2 0 1 Open flower absence (0) or presence (1)

6 b) Nectarivorous species

3 Araluen (n=8, 3) Number of speciesof Number 2 York 1 (n=8, 3) Paruna (n=8, 3) 1 Forrestfield (n=9, 2) York 2 (n=7, 2) 0

0 1 Open flower absence (0) or presence (1)

18 c) All bird species

10 Araluen (n=7, 4) 8 York 1 (n=8, 3) Paruna (n=9, 2) 6 Forrestfield (n=7, 4) York 2 (n=6, 3) 4

2 0 1 Ripe fruit absence (0) or presence (1)

Numbers of surveys/months contributing to each data point for absent and present, respectively, are shown in parentheses. Values are expressed as mean ± 1 SD.

3.5 DISCUSSION

In this study, we recorded 1) greater bird species richness and 2) altered bird community structure in the presence of fruiting mistletoe, with 3) a higher chance of sighting mistletoebirds for months when ripe fruit was present. Can mistletoes in south-west

WA therefore be described as a keystone resource? We discuss the findings of this study and literature records in relation to the keystone criterion identified in the introduction.

3.5.1 Reliability of mistletoe resources

The wire leaf mistletoe A. preissii and box mistletoe A. miquelii are important resources for bird communities in south-west WA due to the wide distribution of these plants as well as their extended flowering and fruiting times. A. preissii and A. miquelii each had flowers or fruit available for half the year (A. preissii 6–7 months and A. miquelii 5 months) and across sites timing of flowering and fruiting was offset so that resources were available over all months of the year. We predicted that there would be a greater number of bird species present at our study sites when mistletoe flowers or fruit were available, and that bird community structure would reflect available resources. The presence of ripe mistletoe fruit was correlated with significantly higher bird species richness and altered community structure. Importantly, the patterns of fruit presence did not coincide across our field sites (maximum 100 km apart), and yet the presence of bird species was associated with site-specific timing of fruit presence. Therefore, while we are not able to entirely discount ecosystem-wide effects such as bird detectability

(Field et al. 2002), spring and winter migration (see review on partial migration by

Chan 2001) and rainfall driven surges attracting locally nomadic species (as well as

promoting mistletoe recruiting and fruiting, Reid 1987; Yan and Reid 1995), the findings of the present study are consistent with those of previous studies that have positively linked increased species richness to mistletoe density (Turner 1991; Bennetts et al. 1996), and manipulative studies comparing the avifauna of two adjacent woodland remnants where one site had been manually cleared of mistletoe (Watson 2002; Watson and Herring 2012). Reid (1986) reported that mistletoe is one of the few reliable sources of fruit in eucalypt forests of south-eastern Australia, and while we only recorded fruiting phenology over one year, anecdotal observations (K. Napier, pers. obs.) suggest that mistletoe fruit is predictably available at each site from year to year, and is therefore extremely reliable. Mistletoes in south-west WA appear to fulfil the criterion of resource reliability, as they appear to be predictably available every year to sustain consumers such as the mistletoebird.

3.5.2 Abundance of mistletoe

Crude measures of mistletoe abundance at each site in the present study indicated that mistletoe is super-abundant in the 2 ha search areas at each, but resource patch density

(sensu Peres 2000) was not measured in the present study. With this caveat, mistletoe appears to be super-abundant at the sites presented in this study, typical of fragmented landscapes in south-western WA, and fulfilling the criterion of high abundancy (sensu

Peres 2000). However, the attribute of resource abundance as a criterion for keystone species status is seen as secondary to the redundancy, reliability and specificity of a given resource (Peres 2000).

3.5.3 Degree of consumer specificity

Resources may range from being extremely generalised, if they are consumed by at least half of the species in a bird community, to extremely specialised, if they are consumed by 5% or less of the species (Peres 2000). Mistletoes promote biodiversity by providing plentiful resources such as nutrient-rich fruit, nectar and leaf litter, as well as sheltered nesting sites and foliar arthropods (Watson 2001; Cooney et al. 2006; Watson et al.

2011). While the influence of these resources on the structure of the avian community can be difficult to elucidate (Watson et al. 2011), a number of studies have documented the link between the presence of aerial mistletoes and greater avian species richness

(e.g. Turner 1991; Bennetts et al. 1996; Watson 2002; Watson and Herring 2012). The present study clearly indicates temporal effects in bird species presence correlated with the availability of ripe mistletoe fruit. We had predicted that frugivorous birds would be more likely to be present when ripe mistletoe fruit was available. Not surprisingly, presence of the only mistletoe fruit specialist found in WA, the mistletoebird, was associated with the presence of fruiting mistletoe, and mistletoebirds were observed feeding on both species of mistletoe fruit. Australian ringneck parrots are opportunistic feeders (Higgins 1999) and were observed ingesting A. preissii fruits and have been previously recorded (Forde 1986) feeding on the fruits of A. quandang and Lysiana exocarpi (both species are found in WA, but are generally restricted to the southern edge of the Great Victoria Desert and the Nullarbor Plain; Western Australian

Herbarium 1998–; Watson 2011). Several other bird species (nectarivorous, granivorous and insectivorous) that are known to regularly consume or opportunistically supplement their diets with mistletoe fruit, including the singing honeyeater, red wattlebird, silvereye, and yellow-rumped thornbill (see Reid 1986 and references

therein), were recorded in the present study, although none were sighted feeding on the fruit. Although these individual species did not show significant patterns in presence/absence on their own, the overall pattern was a significant increase in species richness and changes in the structure of the bird community for months with mistletoe fruit present compared with months when fruit was absent.

Many nectarivorous, as well as insectivorous and generalist species feed on the nectar of Australian mistletoes (see Watson 2001). In the present study, although four nectarivorous honeyeater species were observed feeding on the nectar of both mistletoe species, we did not find that the presence of these species at our study sites was linked with the presence of mistletoe flowers. Many insectivorous species were also observed perching in mistletoe clumps, which may reflect enhanced foraging opportunities presented by the abundance of insects associated with mistletoes (Turner 1991; Burns et al. 2011; Watson 2011; Watson et al. 2011).

Findings from the literature therefore suggest support for the criterion of consumer specificity proposed by Peres (2000), although in the present study, only the presence of a single species (the mistletoebird) was positively correlated with fruiting mistletoe; mistletoe, in this study, would therefore be classified as an extremely specialised resource, and fails to meet this criterion as it is not consumed by a large proportion of the bird assemblage with which they coexist (i.e. extremely generalised resource). This criterion requires further investigation.

3.5.4 Temporal redundancy of mistletoe

Under the keystone criterion of temporal redundancy density (sensu Peres 2000), a resource may be considered entirely indispensable if it is available during periods of overall resource scarcity. Mistletoes in the fragmented landscapes assessed in this study may then be considered a ‘low redundancy’ resource as they were often the only source of fleshy fruit and nectar available (with the exception of Araluen, where blackberry

Rubus sp. was present, see Table 3.1), and would therefore be considered entirely indispensable. Temporal and spatial fluctuations within and among habitats due to both individual movements and population processes occur in most bird communities

(Malizia 2001). The distribution and abundance of food resources, in particular, influences the movements of many birds (Levey 1988). The foraging efficiency of nectarivores and frugivores is also affected by the temporal pattern of flower and fruit availability: if nectar and fruits are temporally and spatially predictable, animals may retain this information and visit plants with available resources without random, undirected searching (Wright 2005). Unlike A. quandang (surveyed at Middleback

Station, South Australia, Reid 1990), which exhibits continuous ripe fruit production due to an overlap in successive annual fruit crops, the fruiting and flowering within A. preissii and A. miquelii at each site was fairly distinct. However, across all five sites examined in the present study, nectar and/or ripe fruit was available for every month of the year due to staggered flowering and fruiting of these two species, as well as geographic variation in timing. While the study by Reid (1990) showed that A. quandang was able to sustain permanent populations of mistletoebirds due to the continuous fruit availability, it appears that the distinct, but staggered fruiting phenology of A. preissii and A. miquelii in south-west WA (and South Australia, Yan

1993) supports more locally-nomadic movements of mistletoebirds. We note that mistletoebirds were present at three sites during months when no ripe mistletoe fruits were recorded, and that this may be attributed to slight differences in fruiting and flowering phenology that were not captured by our survey methods (e.g. ripe fruit present on mistletoe plants that were not monitored during surveys), or mistletoebirds feeding on the unripe fruits of A. preissii. With the caveat that fruiting and flowering phenology was recorded for only one year, mistletoe appears to be a temporally reliable source of fruit and nectar (see also Yan 1993). Through the provision of fruit and nectar resources, these mistletoes act to sustain nomadic populations of mistletoebirds and assist to sustain permanent populations of nectarivorous birds in the local area throughout the year.

3.5.5 Resources (functions) not otherwise present

About 18 million ha or 86.5% of the agricultural region of Western Australia has been cleared, and in the wheatbelt, the percentage is estimated to be 93% (DEP 1997). For example, 22 districts in the wheatbelt have less than 10% native vegetation cover

(Shepherd et al. 2001), and the few patches of remaining native vegetation exist in fragmented and isolated patches. Similar environmental disturbances have occurred in agricultural landscapes across Australia. The quality of food resources in these fragmented landscapes is an important consideration in terms of sustenance for fauna species. For example, Norton et al. (1995) predicted that in extremely fragmented habitats (such as the heavily cleared wheatbelt area), mistletoes would eventually become extinct due to regional declines in key avian pollinators and dispersers

(Saunders 1993), as has been recorded in the wheatbelt area north of the town of

Kellerberrin (Norton et al. 1995). Loranthaceous mistletoes naturally have a patchy distribution across the landscape, due to both the patterns of bird dispersal (Reid and

Lange 1988; Reid et al. 1995) and narrow microsite tolerances coupled with host specificity (Knutson 1983; Yan and Reid 1995). The distribution of Australian mistletoes in fragmented habitats is likely dependent on mistletoe distribution prior to fragmentation and the impact of fragmentation on the avian pollinators and dispersers

(Norton et al. 1995). Mistletoes have become more abundant in fragmented habitats in southeast Australia (Reid et al. 1994; Watson 2001), and tend to be either super- abundant or absent in fragmented areas of southwest WA (Norton et al. 1995). The presence of these plants (and the resources they provide) in fragmented, otherwise resource-poor habitats, may therefore counteract the detrimental effects caused by habitat fragmentation (Kelly et al. 2000; Watson 2002) and may further support their recognition as important bird resources. Mistletoes therefore play a unique role in the fragmented landscapes examined in the present study in their provision of vital food resources to mistletoebirds.

3.5.6 Conclusions

Our findings of increased species richness and changes in the structure of the bird community demonstrate that A. preissii and A. miquelii may provide important food and shelter resources for bird species in fragmented south-west Western Australian woodlands. However, while mistletoes produce highly reliable, low redundancy fruit resources that play a unique role in fragmented landscapes in south-west WA, we failed to find evidence that mistletoe nectar and fruit are consumed by a wide range of bird species. Instead, the only bird species reliant on these food resources is the specialist

mistletoebird. The potential for the keystone status of mistletoes in south-west WA requires further investigation, with comprehensive experimental ‘mistletoe removal’ tests such as those performed by Watson (2002) and Watson and Herring (2012) recommended.

Table 3.2 Bird species recorded in 53 surveys over 5 sites in south-west Western Australia from February 2010 to January 2011.

Surveys were conducted approximately every 5 weeks from February 2010 to January 2011 (11 surveys in total for Araluen, York 1, Paruna and Forrestfield; 9 surveys in total for York 2 from April 2010 to January 2011). The total number of observations was n=511. F refers to frugivore, I (insectivore), N (nectarivore), G (granivore), and O (omnivore). Nomenclature follows Christidis and Boles (2008).

Number of surveys present

Mistletoe present at site: Amyema preissii Amyema miquelii

Feeding Araluen York 1 Paruna Forrestfield York 2 Scientific name: Common name: guild: Columbidae Phaps chalcoptera Common bronzewing G 11 2 Ocyphaps lophotes Crested pigeon G 1 Cacatuidae Calyptorhynchus banksii Red-tailed black-cockatoo G 1 3 Calyptorhynchus latirostris Carnaby's black-cockatoo G 3 Eolophus roseicapillus Galah G 7 1 3 5 Psittacidae Barnardius zonarius Australian ringneck G 11 7 9 11 8 Purpureicephalus spurious Red-capped parrot G 3 6 Cuculidae Cacomantis pallidus Palid cuckoo I 1 Cacomantis flabelliformis Fan-tailed cuckoo I 1 Climacteridae Climacteris rufa Rufous treecreeper I 8 Maluridae Malurus splendens Splendid fairy-wren I 8 1 2 Acanthizidae Smicrornis brevirostris Weebill I 11 7 2 Gerygone fusca Western gerygone I 10 5 8 7 Acanthiza chrysorrhoa Yellow-rumped thornbill I 11 9 2 Acanthiza apicalis Inland thornbill I 7 Pardalotidae Pardalotus striatus Striated pardalote I 8 9 7 Meliphagidae Acanthorhynchus superciliosus Western spinebill N 6 6 Lichenostomus virescens Singing honeyeater N 4 9 Anthochaera carunculata Red wattlebird N 11 1 2 Lichmera indistincta Brown honeyeater N 4 4 7 8 8 Phylidonyris novaehollandiae New Holland honeyeater N 11 1 Phylidonyris niger White-cheeked honeyeater N 8 5 Pomatostomidae Pomatostomus superciliosus White-browed babbler O 4 Neosittidae Daphoenositta chrysoptera Varied sittella I 4 Campephagidae Coracina novaehollandiae Black-faced Cuckoo-shrike I 1 2 1 Pachycephalidae Pachycephala rufiventris Rufous whistler I 3 3 2 3 3 Colluricincla harmonica Grey shrike-thrush O 4 6 1 Artamidae Artamus cinereus Black-faced Woodswallow I 2 Cracticus torquatus Grey butcherbird O 2 2 Cracticus tibicen Australian magpie I 10 3 2 Strepera versicolor Grey currawong O 2 Rhipiduridae Rhipidura albiscapa Grey fantail I 5 9 9 3 Rhipidura leucophrys Willie wagtail I 8 9 Corvidae Corvus coronoides Australian raven O 6 4 7 1 4 Monarchidae Grallina cyanoleuca Magpie-lark I 2 Petroicidae Petroica boodang Scarlet robin I 3 2 Petroica goodenovii Red-capped robin I 9 5 Timaliidae Zosterops lateralis Silvereye F 5 1 5 5 Hirundinidae Petrochelidon nigricans Tree martin I 2 5 Nectariniidae

4 MISTLETOEBIRDS VARY THEIR DIETARY INTAKE OF

ARTHROPODS DEPENDING ON TIME OF YEAR

Kathryn R. Napier1,2, Todd J. McWhorter3, Carlos Martínez del Rio2, Patricia A. Fleming1

1 School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150,

AUSTRALIA

2 Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA

3 School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, SA

5371, AUSTRALIA

STATEMENT OF AUTHOR CONTRIBUTION

K.R. Napier: Initiated the research, mist-netted and sampled mistletoebirds and sampled dietary sources, prepared samples for stable isotopes analysis, entered and analysed the data, and wrote the manuscript.

T.J. McWhorter: Assisted with the design and initiation of the research and provided editorial comments to versions of the manuscript from draft to final versions.

C.M. del Rio: Provided training and assistance in the conduction of stable isotopes analysis, including preparation of samples and statistical analysis, and provided editorial comments to final versions of the manuscript.

P.A. Fleming: Assisted with the design and initiation of the research, assisted

K.R. Napier with mist-netting and bird sampling, advised and assisted K.R. Napier with statistical analyses, and provided editorial comments to versions of the manuscript from draft to final versions.

4.1 ABSTRACT

Mistletoebirds (Dicaeum hirundinaceum, family Dicaeidae) inhabit a wide variety of wooded and forested habitats across Australia. Movements of mistletoebirds are poorly described, but they appear to be locally nomadic and follow fruiting mistletoe around the landscape. Mistletoebirds are the primary dispersers of mistletoe seeds and are mistletoe fruit specialists, with a specialised alimentary system adapted for the ingestion and quick passage of mistletoe fruits. They are also known to ingest other fruits, nectar, and invertebrates. We used stable isotopes of carbon (δ13C) in breath, blood and feathers and nitrogen (δ15N) in blood and feathers from mistletoebirds to detect potential changes in diet through time and to investigate the proportional contribution of arthropods vs. fruit to their diet. Mistletoebirds were mist-netted and sampled and diet sources (i.e. arthropods and mistletoe fruit) sampled at three sites in south-west Western

Australia (WA) when ripe mistletoe fruit was available at each site. Sampling occurred during the austral autumn, winter and summer months. We found that mistletoebirds appear to change their diet over time, as indicated by the significant differences in δ13C values of breath and feathers (tissues that have significantly different turnover rates) from birds at two of the three sites. We also found that the contribution of arthropods to the diet of mistletoebirds varies depending on the time of year, or between sites. The contribution of arthropods to the diet of mistletoebirds ranged from 45% to 67%, and could be associated with increased protein requirements during breeding and moulting or differences in availability of food sources between the sites occupied at these times.

4.2 INTRODUCTION

Mistletoebirds (Dicaeum hirundinaceum, family Dicaeidae) inhabit a wide variety of wooded and forested habitats across Australia. Movements of mistletoebirds are poorly described, and vary with descriptions ranging from largely resident with some local dispersion to dispersive over large distances, but are usually associated with the presence of fruiting mistletoe (Higgins et al. 2006). Of 190 recoveries, 98.9% of banded birds were recovered <10 km from their original banding location (Higgins et al. 2006). In south-west Western Australia (WA), mistletoebirds are generally only present in an area when ripe mistletoe fruit is available (Napier et al. 2013). These birds are the primary dispersers of mistletoe seeds (Higgins et al. 2006) and are mistletoe specialists, with a specialised alimentary system adapted for the ingestion and quick passage of mistletoe fruits (Richardson and Wooller 1988), but are also known to ingest other fruits, nectar, and invertebrates (Reid 1990; Higgins et al. 2006).

Exclusive frugivory in birds is fairly rare, as frugivorous birds may face nutritional limitations due to the low protein content in most fruits (Morton 1973).

Many frugivorous birds supplement their diet with insects as a means to source adequate levels of dietary protein, which is essential to meet amino acid requirements

(Klasing 1998). Observational studies of foraging behaviour, food ingested and analysis of faeces have established that mistletoebirds primarily feed on mistletoe fruit, but supplement their diet with insects and spiders during the breeding season and when fruit is scarce (Higgins et al. 2006). These observational studies, however, are unable to determine the nutritional importance of these different dietary components, as they do not measure the assimilation of nutrients into the tissues of consumers (Barea and

Herrera 2009). Stable isotopes analysis allows the quantification of the origin of

assimilated nutrients (Hobson 1999), which gives an estimation of their dietary importance (Herrera et al. 2001a; Barea and Herrera 2009), making it a powerful tool in the reconstruction of diets and estimation of trophic position.

Barea and Herrera (2009) recently used a stable isotope approach to determine the extent to which mistletoebirds and painted honeyeaters (Grantiella picta, the only other mistletoe fruit specialist in Australia) in south–east Australia obtain assimilated nitrogen from mistletoe fruit versus arthropods, and reported that both mistletoebirds and painted honeyeaters obtain approximately half of their nitrogen from mistletoe fruit

(mean proportional contribution of mistletoe fruit±SE, mistletoebirds: 0.5±0.14; painted honeyeaters: 0.4±0.04). Herrera et al. (2009) also used a similar stable isotope approach and estimated that a tropical mistletoe fruit specialist, the yellow-throated euphonia (Euphonia hirundinacea), obtained most of their assimilated nitrogen from various fruits (76-100%), which increased with increasing fruit availability at different times of the year.

We followed a similar, yet slightly more comprehensive approach to Barea and

Herrera (2009) and Herrera et al. (2009) to determine if we could detect changes in the diets of mistletoebirds over time by sampling birds at three locations in south-west WA at different times of the year (i.e. when peak abundances of mistletoe fruit were available at each site), and by also sampling several tissues from each individual bird.

We used stable isotopes of carbon (δ13C) in breath, blood and feathers and nitrogen

(δ15N) in blood and feathers from mistletoebirds to firstly detect potential changes in diet and to provide an estimate of the timeframe of these dietary changes, and secondly, investigate the proportional contribution of arthropods vs. fruit to the diet of

mistletoebirds. We also present δ13C and δ15N values for other bird species captured and sampled along with mistletoebirds, to provide a comparison with birds from other feeding guilds.

4.3 METHODS

4.3.1 Study sites

The study was conducted at three sites in south-west WA where mistletoe was abundant

(see Table 4.1 for details). Two sites contained Amyema preissii (family Loranthaceae) parasitising acacia hosts, while the other site contained A. miquelii parasitising a eucalypt host.

Table 4.1 The characteristics of the three field sites in south-west Western Australia.

A, J, M, F, U indicate adult, juvenile, male, female and unknown sex. Fruiting and flowering phenology surveys determined when ripe mistletoe fruit was available, with the average proportion of ripe fruit available per 30 cm branch presented in parentheses (Napier et al. 2013). Rainfall range is shown for ‘winter’ (May to September inclusive). Weather data were obtained for 1981-2010 and for the months of sampling (Bureau of Meteorology 2011) from the nearest meteorological station to the study areas: Araluen– Gosnells City (32°02′S, 115°58′E); York 1 and York 2 –York (31°53′S, 116°46′E).

Site Number of Mistletoe Host species Site description Sample period Ripe mistletoe Days with Temp. max. >35°C, min <2°C birds sampled species (proportion of fruit fruit available Avg. annual rainfall (winter rainfall range) available) Total rainfall during sampling; avg. max. Temp

York 1 Private farming A (M=2; F=1) Amyema Acacia A fragmented, semi arid acacia April 2010 Apr–June 2010 45, 47 days property, York, WA J (U=2) preissii acuminata woodland subject to grazing (0.38) 401 mm (44–71 mm) (31°51′S, 116°44′E) 2.6 mm; 27°C Araluen Araluen Country Club, A (M=1; F=1) A. preissii A heavily watered suburban June 2010 May–Sept 2010 35, 1 day Roleystone, WA Ac. baileyana garden with both non-native (0.37) 795 mm (83–165 mm) (32°08′S, 116°05′E) Ac. podalyriifolia and native plants present (0.13) 60.5 mm; 19.7°C York 2 Private farming A (M=3) A. miquelii Corymbia A fragmented, semi-arid Dec 2010–Jan Nov 2010–Jan 45, 47 days property, York, WA J (M=1) calophylla eucalypt woodland, subject to 2011 2011 401 mm (44–71 mm) (31°50′S, 116°44′E) grazing (0.21–0.38) Dec: 20.5 mm; 31.5°C, Jan: 28.8 mm; 35°C

4.3.2 Breath, blood and feather samples

11 individual mistletoebirds (8 adults: 6 males, 2 females; and 3 juveniles: 1 male, 2 undetermined sex), were captured by mist-nets placed in front of fruiting mistletoe from

March 2010 to January 2011 – the sampling period was determined by the availability of ripe mistletoe fruit, and therefore differed at each site (see Table 4.1). One individual was re-captured >2 weeks after initial sampling at the same site and was re-sampled; data were therefore averaged for this individual. At least 300 trap hours were conducted at each site. Three other avian species were also captured at at least two of the sites: the nectarivorous brown honeyeater (Lichmera indistincta, n=10), the frugivorous silvereye

(Zosterops lateralis, n=26) and insectivorous yellow-rumped thornbill (Acanthiza chrysorrhoa, n=25). None of these species are sexually dimorphic in their plumage, so sex was not determined.

We selected three tissues (breath, whole blood and feathers) that can be sampled both quickly and non-destructively, and that also have significantly different turnover rates so that we were able to detect potential changes in diet over time. Tissues that are metabolically active reflect the average diet of an individual, and vary according to the turnover rate of the tissue. δ13C obtained from breath samples reflects the diet consumed several hours since the last meal, with a half-life of approximately 4.4 hours in small birds (Podlesak et al. 2005). The half-life of carbon and nitrogen in whole blood in small birds is approximately 4–6 and 11 days respectively (Hobson and

Bairlein 2003; Pearson et al. 2003). Feathers, in contrast, are metabolically inert and therefore their isotopic composition reflects the diet consumed during feather formation

(Chamberlain et al. 1997).

Upon capture, birds were held in a calico bag and carried to a central sampling station. Birds were then weighed and banded. Breath samples were collected as described by Carleton et al. (2006). Birds were placed into a 100 ml cylindrical tube, which was fitted with two one-way stopcock valves. The tube was gently flushed with

~500 ml of CO2-free air over a period of 30 s. The tube was then sealed, and exhaled

CO2 accumulated for two min. A 30ml air sample was extracted and injected into pre- evacuated gastight vials (Exetainer, Labco Ltd, Buckinghamshire, UK) until a positive pressure was achieved. Approximately 50 µl of whole blood was collected from the brachial vein with heparinised capillary tubes by venepuncture. Blood samples were dried in sample vials in the field initially, and then upon return to the laboratory, at

60°C for 72 h. Samples were then ground to a powder, homogenised and stored at 4°C until stable isotopes analysis. Body feathers (chest) were obtained from the calico holding bag or during handling. Feathers were cleaned in 90% EtOH, finely cut, and stored in paper envelopes until stable isotopes analysis. It was noted that mistletoe seeds (free from arthropods) were excreted in the calico bags of 7 of 12 mistletoebird captures. Birds were then released at the site of capture.

All field work and experimental procedures were approved by the Murdoch

University Animal Ethics Committee (approval R2175/08), Western Australian

Department of Environment and Conservation (SW012746 and SF0073330) and the

Australian Bird and Bat Banding Scheme (2699).

4.3.3 Diet sources

Collections of >100 ripe mistletoe fruit from at least 20 mistletoe plants were collected at each study site, placed on ice and stored at -20°C. Samples were prepared for stable isotopes analysis by manually removing and discarding the exocarp from frozen fruits, which were then freeze dried to facilitate removal of the fruit pulp from the seed.

Individual fruits, minus the seed, were then dried at 60°C for 72 h and stored at 4°C until stable isotopes analysis. Stable isotope data were analysed for a sub sample of 27 fruit samples from Araluen, 29 from York 1 and 36 from York 2.

Diurnal arthropods were collected from the ground, mistletoe branches and air during the same sampling period. After capture, arthropods were initially stored in 70%

EtOH prior to drying at 60°C for 72 h, then ground to a fine powder, homogenised and stored at 4°C until stable isotopes analysis. Arthropods represented 9 orders, with the number of morphospecies within each order in brackets. Predatory arthropods (York 1 and York 2 only) included: Arachnida (1), Odonata (2), Diptera (5), and Hymenoptra

(7); while non-predatory arthropods (all sites) included: Coleoptera (4), Diptera (15),

Hemiptera (3), Hymenoptra (9), Lepidoptera (1), Mantodea (1), and Orthoptera (10), with 11 samples from Araluen, 24 from York 1 and 23 from York 2.

4.3.4 Stable isotopes analysis

Prepared samples were transported to the University of Wyoming Stable Isotope

Facility, USA. Total carbon and nitrogen content (%) and δ13C and δ15N compositions

(‰) were determined using a Finnigan Delta Plus XP continuous flow inlet Stable

Isotope Ratio Mass Spectrometer (IRMS) online with a Costech EA 1108 Elemental

analyser or CE Elantech 2500 CHN Elemental analyser. δ13C values are expressed with respect to V-PDB and δ15N values with respect to atmospheric molecular nitrogen.

Breath samples were analysed using a Thermo Finnigan Gasbench II online gas preparation and introduction system online with an IRMS as described above.

4.3.5 Isotope mixing model

Isotope mixing models were carried out on whole blood values only, as while we were able to apply average discrimination values for whole blood, we were unable to find similar published discrimination values for breath. Due to uncertainty of the location of mistletoebirds when they moulted, the contribution of various diet sources to the stable isotope values of feathers also could not be estimated using mixing models.

Isotope values (δ15N, δ13C) were compared by 2-way ANOVA with diet source

(all-arthropods or fruit) and location (site) as factors and Tukey HSD post hoc tests for unequal sample sizes as required. Significant differences in δ13C and δ15N were detected between sites for fruit but not for arthropods (see results). Each site was therefore analysed by separate isotope mixing models. We classified arthropods into two groups based on their trophic level (i.e. predatory and non-predatory arthropods, see

Fig. 4.1a, b, c) for York 1 and York 2. This resulted in three diet sources (fruit, predatory and non-predatory arthropods) for the two York sites while Araluen only contained two diet sources (fruit and non-predatory arthropods) as no predatory arthropods were sampled at this site. Contributions of non-predatory and predatory arthropods were combined for statistical analysis to allow comparison across the three sites.

As it is critical to consider the concentration of elements when attempting to reconstruct the diet of animals through the use of isotopic signatures (Phillips and Koch

2002), a Bayesian isotopic mixing model, using both carbon and nitrogen isotopes, was used to determine the contribution of fruit and arthropods to the diet of mistletoebirds in the package Stable Isotope Analysis in R (SIAR) (Parnell et al. 2010). SIAR uses a

Markov Chain Monte Carlo simulation to estimate probability distributions of different dietary sources to a mixture (i.e. whole bird blood) while accounting for the observed variability in isotopic signatures, dietary isotopic fractionation and elemental concentration. The solo command in SIAR was used with individual mistletoebirds as single data points, with the resulting values for each individual compared between sites via MANOVA. As we did not measure the tissue specific discrimination factors for mistletoebirds, we followed the approach of Herrera et al (2006); the diet source values of arthropods and fruit were corrected using the average whole blood-diet carbon and nitrogen discrimination factors for two insectarivores (that also eat fruit) studied to date

(yellow-rumped warblers and garden warblers) which were fed under controlled conditions on predominately fruit (δ13C: 1.41±3.68‰; δ15N: 1.98±0.39‰) or insect diets (δ13C: 2.35±0.21‰; δ15N: 2.6±0.14‰) (Hobson and Bairlein 2003; Pearson et al.

2003).

4.3.6 General statistics:

Stable isotope values of mistletoebird tissues were analysed via mixed-model (MM)

ANOVAs with individual ID as a random factor and tissue and site as fixed factors, followed by one-way post hoc ANOVAs with tissue or site as fixed factors. Stable isotope values of breath and blood were compared in separate MM-ANOVAs with

species and site as fixed factors, followed by one-way post hoc ANOVAs with species or site as fixed factors. Data are presented as mean ± 1 SD throughout, with significance accepted for α<0.05.

14 a) York 1 14 b) Araluen 14 c) York 2

12 12 12

10 10 10

8 8 8

N (‰)N

15 6 6 6

4 4 4

2 2 2

0 0 0

Diptera (4) Diptera (1) Diptera

Diptera (10) Diptera

Mantodea (1) Mantodea

Hemiptera (3) Hemiptera

Coleoptera (2) Coleoptera

Orthoptera (4)

Orthoptera (6)

Coleoptera (2) Coleoptera

Lepidoptera (1) Lepidoptera

Diptera pred (1) pred Diptera

Diptera pred (4) pred Diptera

Hymenoptera (3) Hymenoptera

Hymenoptera (1) Hymenoptera

Hymenoptera (5) Hymenoptera

Odonata pred (2) pred Odonata

Arachnida pred (1) pred Arachnida

Hymenoptera pred (6) pred Hymenoptera Hymenoptera pred (1) pred Hymenoptera

Figure 4.1 Median, 25th and 75th quartile values of δ15N (‰) signatures of arthropods sampled at three sites in south-west Western Australia. a) York 1, b) Araluen and c) York 2 with number of samples in parentheses. Pred indicates predatory arthropods.

4.4 RESULTS

δ15N and δ13C values and C and N concentrations for mistletoebird tissues and diet sources are summarised in Table 4.2.

4.4.1 Diet sources

Significant differences in both δ13C and δ15N were detected between sites for fruit and

13 15 all-arthropods (two-way ANOVA; site: δ C: F2,131=7.16, P=0.001; δ N: F2,131=10.93,

P<0.001; Table 4.2). Mistletoe fruit were significantly depleted in δ13C and δ15N compared to all-arthropods; mistletoe fruit was significantly depleted in δ13C compared to the arthropod samples (all-arthropods) (2-way ANOVA; diet source: F1,131=354.9,

P<0.001; Table 4.2). Fruit δ15N values averaged from 0.53 to 4.08‰ and were also significantly depleted compared to the arthropod samples (all-arthropod), which ranged from 6.62 to 7.41‰ (2-way ANOVA: diet source; F1,131=195.7, P<0.001). Mistletoe fruit from York 1 was significantly enriched in both δ13C and δ15N compared to fruit

13 from Araluen and York 2 (two-way ANOVA; diet source x site: δ C: F2,131=22.16,

15 p<0.001; δ N: F2,131=9.78, P<0.001).

Arthropods (across all orders) contained a significantly higher proportion of nitrogen (averaging 11.63±1.56%) compared to mistletoe fruit (1.37±1.87%) (2-way

ANOVA; diet source: F1,129=1088, P<0.001). Fruit contained a significantly higher proportion of carbon (50.83±11.32) compared to arthropods (48.84±3.40%) (2-way

ANOVA; diet source: F1,129=6.78, P=0.010), with a significant interaction term (2-way

ANOVA; diet source x site: F2,129=19.62, P<0.001) demonstrating that fruit from York

1 had a significantly higher proportion of carbon compared to all other arthropod and fruit values (Table 4.2).

4.4.2 Mistletoebird tissues

Feathers were significantly enriched in δ13C compared to blood, with breath showing

13 the most depleted δ C values (2-way MM-ANOVA; tissue: F2,15=25.80, P<0.001).

Significant site (F2,8=5.03, P=0.038) and ID (F9,12=3.16, P=0.033) effects were recorded. For York 1, the δ13C values of the three mistletoebird tissues did not vary significantly (one-way post hoc ANOVA; tissue: F2,12=0.1.23, P=0.326), but for both

Araluen (F2,3=60.19, P=0.004) and York 2 (F2,9=16.26, P=0.001), feathers were significantly enriched compared to breath (Fig. 4.2a). There was a significant site

13 difference in breath δ C (one-way post hoc ANOVA; site: F2,8=23.10, P<0.001) and

13 blood δ C (F2,9=11.63, P=0.003) values, with samples from York 1 significantly enriched in comparison to York 2 and Araluen (Fig. 4.2a). There were no site

13 differences for feather δ C values (F2,7=0.05, P=0.950).

Feathers and blood did not differ in their δ15N values (2-way MM-ANOVA; tissue: F1,10=0.24, P=0.636, see Fig. 4.2b). There were no ID (F9,5=4.22, P=0.064) or site (F2,8=0.33, P=0.730) differences.

Mistletoe fruit: Amyema preissii (Araluen and York 1) and A. miquelii (York 2). Data are presented as mean ± 1 SD with number of samples in parentheses.

York 1 Araluen York 2

δ13C (‰) δ15N (‰) C% N% δ13C (‰) δ15N (‰) C% N% δ13C (‰) δ15N (‰) C% N%

Breath -23.29±0.65 (5) -27.31±0.40 (2) -25.82±1.00 (4)

Whole blood -22.56±0.84 (6) 6.88±0.56 (6) 44.89±1.73 (6) 13.65±0.57 (6) -24.68±0.71 (2) 4.78±0.35 (2) 45.72±0.73 (2) 13.31±0.88 (2) -24.58±0.54 (4) 5.21±1.67 (4) 45.39±0.35 (4) 13.56±0.32 (4)

Feathers -22.14±5.49 (4) 5.49±1.29 (4) 43.89±0.66 (4) 13.79±0.29 (4) -22.07±7.18 (2) 7.18±0.57 (2) 41.56±4.70 (2) 13.09(2)1.52 (2) -22.39±6.18 (4) 6.18±1.10 (4) 43.84±1.18 (4) 13.57±0.31 (4)

All-arthropods -24.01±1.85 (24) 7.05±2.60 (24) 48.46±3.61 (24) 11.62±1.71 (24) -22.95±0.40 (11) 7.41±0.19 (11) 48.77±1.02 (11) 11.28±0.33 (11) -23.33±1.32 (23) 6.62±2.55 (23) 48.51±2.03 (23) 11.81±1.77 (23)

-Non-predatory -23.81±2.13 (16) 5.80±2.14 (16) 48.73±4.35 (16) 11.88±1.90 (16) -23.64±1.30 (16) 5.24±1.51 (16) 49.45±2.37 (16) 11.25±1.85 (16)

-Predatory -24.42±1.09 (8) 9.54±1.31 (8) 48.94±1.34 (8) 11.09±1.17 (8) -22.64±1.21 (7) 9.75±1.31 (7) 49.62±1.04 (7) 13.10±0.44 (7)

Mistletoe fruit -26.44±1.47 (29) 4.08±1.83 (29) 49.70±11.79 (29) 1.12±0.27 (29) -28.11±1.02 (27) 0.82±1.81 (27) 47.20±7.13 (27) 2.17±3.00 (27) -28.62±1.03 (36) 0.53±1.30 (36) 54.47±12.63 (36) 0.89±0.25 (36)

-18 a) 8 b) a* a* a* all-arthropods (A) a* -20 a* 7 a* all-arthropods (Y 1) all-arthropods (Y 2) a* 6 a* a* -22 a* a* all-arthropods (A) 5 all-arthropods (Y 2) b**b** -24 all-arthropods (Y 1) fruit (Y 1)

N (‰) N C (‰) C 4 b**

13 3 -26 Araluen fruit (Y 1) c** York 1 2 York 2 -28 fruit (A) fruit (Y 2) 1 fruit (A) fruit (Y 2) -30 0 breath blood feather blood feather

Figure 4.2 δ13C (a) and δ15N (b) values (‰) of breath, whole blood and feathers for mistletoebirds at three sites in south-west Western Australia.

Sites are represented by: York 1 (Y 1, white triangles), Araluen (A, black circles) and York 2 (Y 2, black squares). Data are presented as mean ± 1 SD. Horizontal lines represent δ13C and δ15N values for diet sources: mistletoe fruit (solid lines) and arthropods (dashed lines). Diet sources have not been corrected by diet-tissue discrimination factors. Letters indicate significant differences between sites while asterisks denote differences between tissues obtained from one-way ANOVAs with Tukey HSD post hoc tests for unequal sample sizes.

4.4.3 Isotope mixing model

δ13C and δ15N values for mistletoebird whole blood and diet sources are presented in

Fig. 4.3a. There were significant differences in the dietary contribution of arthropods and fruit between sites (MANOVA: site; F2,8=64.37, P<0.001, Fig. 4.4a). Post hoc tests revealed that fruit made a significantly greater dietary contribution to mistletoebird diets at Araluen compared with the other two sites (Araluen: 0.55±0.03; York 1: 0.33±0.01;

York 2: 0.38±0.04) while arthropods made a significantly greater dietary contribution at

York 1 and York 2 (Araluen: 0.45±0.03; York 1: 0.67±0.01; York 2: 0.62±0.04).

At the York sites, we collected non-predatory as well as predatory arthropods, with their respective dietary contributions as follows; York 1: 0.51±0.03 and 0.16±0.04 and York 2: 0.56±0.01 and 0.07±0.04 Variation amongst individuals (i.e. adults vs juveniles, males vs females) could not be assessed via statistical methods due to small sample sizes, but are presented graphically (Fig. 4.4b).

4.4.4 Comparison with other bird species

δ13C values for breath and δ13C and δ15N values for whole blood and feathers from brown honeyeaters, silvereyes and yellow-rumped thornbills are presented in Table 4.3, with average δ13C and δ15N values for whole blood and diet sources presented in Fig.

4.3b. Breath δ13C values were depleted at Araluen compared to the two semi-arid sites at York (2-way MM-ANOVA; site: F2,52=12.58, P<0.001). A significant species effect was also recorded (F3,52=5.18, P=0.003), with the insectivorous yellow-rumped thornbill with the most enriched breath δ13C values while the nectarivorous brown honeyeater had the most depleted. At Araluen, the breath δ13C values of the yellow-

rumped thornbill were significantly enriched compared to the frugivorous silvereye

(one-way post hoc ANOVA: F3,26=4.50, P<0.011), but species differences were not evident at either York site (York 1: F3,17=1.79, P=0.190; York 2: F2,9=2.79, P=0.114).

Whole blood δ13C values were depleted at Araluen, in comparison to both York

1 and 2, with the most enriched δ13C values from York 1 (2-way MM-ANOVA; site:

F3,53=11.62, P<0.001). A significant effect of species (F3,53=11.62, P<0.001) and an interaction between site and species were also recorded (F4,53=12.39, P<0.001). At

Araluen and York 2, the whole blood δ13C values of the yellow-rumped thornbill were significantly enriched in comparison to silvereyes (Araluen only) and mistletoebirds

(one-way post hoc ANOVA; Araluen: F3,23=18.16, P<0.001; York 2: F1,10=43.96,

P<0.001), while species differences were not evident at York 1 (F3,20=2.47, P=0.091).

Whole blood δ15N values were more depleted at Araluen in comparison to the two York sites (2-way MM-ANOVA; site: F2,53=9.75, P<0.001). A significant effect of species was also evident (F3,53=12.03, P<0.001), with yellow-rumped thornbills having the most enriched δ15N values and brown honeyeaters the most depleted. A significant interaction between site and species were also recorded (F4,53=4.50, P=0.003). At

Araluen and York 2, the δ15N values of yellow-rumped thornbills were significantly enriched compared to silvereyes (Araluen only) and mistletoebirds (one-way post hoc

ANOVA; Araluen: F3,23=18.16, P<0.001; York 2: F1,10=42.56, P<0.001). Species differences were not evident at York 1 (F3,20=3.87, P=0.025) when post hoc analyses were applied.

14 a)

10 Blood- York 2 Blood- Araluen 8 Blood- York 1 Fruit- York 2 Fruit- Araluen N (‰) N 6 Fruit- York 1

15 All-arthropods- York 1 All-arthropods- Araluen 4 All-arthropods- York 2 Feathers- York 1 2 Feathers- Araluen Feathers- York 2

-30 -28 -26 -24 -22 -20 -18 13C (‰) 14 b) arthropod signature

12 Brown honeyeater- York 1 Brown honeyeater- Araluen Mistletoebird- York 2 10 Mistletoebird- Araluen Mistletoebird- York 1 Silvereye- York 1 8 Silvereye- Araluen Yellow-rumped thornbill- York 2

N (‰) N Yellow-rumped thornbil- Araluen 15 6 Yellow-rumped thornbill- York 1 Fruit- York 1 Fruit- Araluen 4 Fruit- York 2 All-arthropods- York 2 All-arthropods- Araluen 2 All-arthropods- York 1

0 -30 -28 -26 -24 -22 -20 -18 13C (‰)

Figure 4.3 δ13C and δ15N values (‰) of a) whole blood and feathers from individual mistletoebirds and b) average whole blood values for mistletoebirds, silvereyes, brown honeyeaters and yellow- rumped thornbills at three sites in south-west Western Australia. a) Blood samples are represented by circles and feathers by diamonds at the sites York 1 (black), Araluen (grey) and York 2 (white). δ13C and δ15N values for diet sources are represented by inverted triangles (mistletoe fruit) and squares (arthropods). b) Mistletoebirds are represented by inverted triangles, silvereyes by squares, brown honeyeaters by circles, and yellow-rumped thornbills by diamonds at the sites York 1 (black), Araluen (grey) and York 2 (white). δ13C and δ15N values for diet sources are represented by triangles (mistletoe fruit) and hexagons (arthropods). Diet sources have not been corrected by tissue-diet discrimination factors. Data are presented as mean ± 1 SD.

0.8 a) York 1 (n=5) Araluen (n=2) York 2 (n=4)

a a

0.6 *

** 0.4 **

Proportional contribution Proportional 0.2

Mistletoe fruit Non-predatory arthropods Predatory arthropods 0.0 Jan Feb Mar April May June Jul Aug Sept Oct Nov Dec

moult moult

breeding (eggs)

0.7 b)

0.6

0.5

0.4

0.3 A,F A,M A,M J,U J,U A,F A,M A,M J,M A,M A,M

0.2

Proportional contribution of fruit contribution of Proportional 0.1

0.0 York 1 Araluen York 2

Figure 4.4 Proportional contribution of fruit and insects to the whole blood of mistletoebirds a) and proportional contribution of mistletoe fruit to individual mistletoe birds b) obtained from a dual-isotope (δ13C, δ15N) Bayesian isotope mixing model (SIAR). n= number of mistletoebirds sampled for whole blood at each site. Letters and asterisks indicate significant differences between sites for all-insects and fruit respectively, obtained from MANOVA with Tukey HSD post hoc tests for unequal sample sizes. Breeding and moulting information for Western Australia obtained from Higgins et al. (2006). A indicates adult, J juvenile, F female, M male, U unknown sex. Arthropod contribution = 1- mistletoe fruit contribution.

Table 4.3: Stable isotope values for breath and whole blood for four avian species. Data are presented as mean ± 1 SD with number of samples in parentheses.

York 1 Araluen York 2 δ13C (‰) δ15N (‰) δ13C (‰) δ15N (‰) δ13C (‰) δ15N (‰) Brown honeyeater Breath -24.27±1.46 (2) -27.50±1.00 (7) -25.82 (1) Whole blood -22.25± 0.91(2) 3.92±1.97 (2) -23.84±0.33 (6) 6.01±1.13 (6) Feathers -21.76±1.04 (2) 6.20±0.36 (2) -21.68±0.57 (7) 6.48±0.61 (7) -20.64 (1) 7.71 (1) Silvereye Breath -24.22± 0.99 (9) -27.36±1.24 (10) Whole blood -22.13±0.43 (11) 8.11±2.42 (11) -24.61±0.15 (11) 5.86±0.83 (11)

Feathers -21.48±0.51 (9) 10.03±2.60 (9) -22.69±1.52 (15) 5.37±0.49 (15) Yellow-rumped thornbill Breath -24.53± 0.76 (5) -24.52±3.04 (11) -24.26±1.18 (7)

Whole blood -22.92±0.23 (5) 8.56±0.41 (5) -23.19±0.67 (8) 7.11±0.16 (8) -22.97-±0.32 (8) 9.06±0.37 (8)

Feathers -21.44± 0.60 5) 9.57±0.58 (5) -18.28±1.52 (12) 8.47±0.49 (12) -22.44±1.72 (8) 10.02±1.15 (8)

4.5 DISCUSSION

We found that mistletoebirds appear to change their diet over time, as indicated by the significant differences in δ13C values of breath and feathers. We also found that the contribution of arthropods to the diet of mistletoebirds varies depending on the time of year or between sites, which could be associated with increased protein requirements during breeding and moulting or differences in availability of food sources between the sites occupied at these times. For example, our sampling of arthropods at Araluen during winter was likely to be hampered by low ambient temperatures (less than 20°C on average), which may also influence arthropod availability for the birds.

4.5.1 Detecting diet switching of mistletoebirds

We measured carbon and nitrogen stable isotopes in three tissues with different isotopic turn-over rates to determine if we could detect changes in the diet of mistletoebirds over time. For small birds, diet-tissue discrimination values for carbon and nitrogen isotopes are generally enriched by 1-3.5‰ relative to diet, with tissue isotope values influenced by the signature of the diet fed to the animal, the signature of macronutrients within the diet, as well as the nutrient composition of the tissue (Hobson and Bairlein 2003;

Pearson et al. 2003; Podlesak et al. 2005). Following the approach of Podlesak et al.

(2005), we interpret the significantly different δ13C values of the various tissues (>3‰) as indication that mistletoebirds had switched between diets that were isotopically distinct. For example, the significant differences between feathers, which provide an estimate of the past diet of mistletoebirds, and breath, which has a very short half-life of

~4.5 hours, indicates a recent change in diet at two out of the three sites (Podlesak et al.

2005).

The δ13C and δ15N values of mistletoebirds captured at York 1 (sampled in autumn) were <1.5‰ different across tissues, suggesting that these birds had been ingesting food with relatively consistent C and N signatures for an extended period of time. The δ15N values of mistletoebirds sampled at York 2 (sampled in summer) and

Araluen (sampled in winter) were <1.5‰ different across tissues, suggesting that these birds had also been ingesting food with relatively consistent N signatures for an extended period of time, however C signatures varied.

The δ13C values of breath, blood and feathers from mistletoebirds captured at

York 2 and Araluen were >2‰ different, with the greatest differences evident between breath and feathers (>3‰), suggesting that these birds had recently changed their diet to a carbon signature that closely resembles mistletoe fruit (Fig. 4.2a). In birds and bats,

δ13C values from breath generally reflect immediate oxidation of ingested food, rather than endogenous energy stores (Welch et al. 2006; Voigt et al. 2008). We mist-netted birds in front of fruiting mistletoe plants, and noted that 58% of birds excreted mistletoe seeds while held in calico bags, with no visible evidence of arthropods present in excreta. This, combined with the δ13C values obtained from breath samples, strongly indicates that mistletoebirds were ingesting a high proportion of mistletoe fruit in the hours preceding capture. Conversely, the δ13C values of mistletoebird feathers strongly resembles that of arthropods (Fig. 4.2a), indicating that these birds were supplementing their fruit diet with arthropods during their moult, a period when dietary protein requirements are higher (Klasing 1998) .

4.5.2 Contribution of fruit vs arthropods

We were able to estimate the proportional contribution of mistletoe fruit and arthropods to the diet of mistletoebirds by sampling whole bird blood, arthropods and mistletoe fruit from the area where birds were mist-netted. Bird and plant surveys indicated that mistletoebirds were sighted in the study sites during months when ripe mistletoe fruit was available (Napier et al. 2013), so we were confident that we managed to obtain an accurate representation of the dietary sources available to mistletoebirds at each location.

Mistletoebirds varied the contribution of arthropods to their diet, ranging from

45% to 67% (Fig. 4.4a), depending on the time of year. Mistletoebirds are known to ingest insects, primarily during the breeding season, or when fruit is scare (Higgins et al. 2006). As mistletoe fruit was abundant at each of the sites during the sampling period (with proportions of ripe mistletoe fruit available ranging from 0.13 to 0.38 per

30 cm of branch, Table 4.1), it is not likely that a lack of fruit availability was the cause of this variation. Frugivorous birds are also known to supplement their fruit diet with insects during moulting periods (Klasing 1998). As mistletoebirds moult from summer to autumn (Higgins et al. 2006), we may expect arthropods to contribute a higher proportion to the diet of mistletoebirds at these times. Indeed, we see that mistletoebirds obtained significantly greater contributions of arthropods to their diet at

York 1 and York 2, which were sampled in April (autumn) and December/January

(summer) and presumably during their moulting period which is associated with an increased demand for dietary nitrogen. Araluen was sampled during June (winter), presumably post moulting. The breeding period of mistletoebirds in WA is poorly

described, with unspecified breeding records from the months of July to January (with eggs recorded in September and October) and from March to May. We did mist-net juvenile mistletoebirds at two sites, York 1 (2 out of 5 birds) and York 2 (1 out of 4 birds), with juveniles of undetermined sex sampled at York 1 and a single first immature male (as defined by Higgins et al. 2006) mist-netted at York 2 (Fig. 4.4b).

Therefore, it is likely that a combination of breeding and moulting contributed to the increase in arthropods in the diet of mistletoebirds at York 1 and York 2, compared to

Araluen.

We did not see the level of intrapopulation variation described by Barea and

Herrera (2009), rather, differences were evident between populations of mistletoebirds

(Figs. 4a, b). We could not test for sex- (or age-) based dietary differences although none were evident in our data, as also noted by Barea and Herrera (2009).

4.5.3 Comparison with other bird species

As we did not determine the tissue specific discrimination factors for mistletoebirds for use in the isotope mixing model, these estimates were obtained through the application of average discrimination values from two studies that fed two species of warblers fruit and insect based diets (Hobson and Bairlein 2003; Pearson et al. 2003), following the approach of Herrera et al. (2005; 2006; 2009) and Barea and Herrera (2009). Yellow- rumped and garden warblers belong to two diverse families of insectivorous birds

(Parulidae and Sylvidae respectively), while mistletoebirds are specialised frugivores belonging to the family Dicaeidae. To date, there have been no studies on tissue to diet discrimination factors in specialist frugivorous birds (see Hahn et al. 2012). As

discrimination factors have been shown to vary between taxonomic groupings (Caut et al. 2009), the extrapolation of these average discrimination factors is problematic, and our conclusions based upon the diet mixing model must be treated with some caution.

We therefore also compared the average values of δ13C and δ15N with birds from other taxonomic and feeding guilds, captured and sampled at the same time as mistletoebirds. As insectivores feed at higher trophic levels than frugivores, we expect that the tissues of yellow-rumped thornbills would have higher δ15N values compared to mistletoebirds (Gagnon and Hobson 2009). The average increase of 3.4 in δ15N for each increase in trophic level (Kelly 2000; Post 2002) allows the detection of diet shifts, such as insectivory vs. frugivory (Herrera et al. 2001b; Herrera et al. 2003).

For birds sampled at Araluen and York 2, yellow-rumped thornbills had more enriched δ15N and δ13C values for whole blood compared to mistletoebirds also sampled at these sites (Fig 4.3b). The δ15N and δ13C values for mistletoebirds and yellow- rumped thornbills at York 1 were similar, lending support to the findings of the isotope mixing model that mistletoebirds were supplementing their diets with a higher proportion of arthropods at this site.

4.5.4 Arthropods and mistletoe fruit are both important sources of dietary

nitrogen

Mistletoebirds did not depend on mistletoe fruit as a sole food source; they incorporate arthropods in their diet to varying degrees. However, despite its low nitrogen content

(Table 4.2), mistletoe fruit makes a significant contribution to the assimilated nitrogen of mistletoebirds. The high reliance on mistletoe fruit as a source of protein by these

specialised frugivores is assisted by their high intake rates of mistletoe fruit, as well as specialised intestinal morphology (Richardson and Wooller 1988) that results in extremely rapid transit times of mistletoe fruit compared to other non-specialised frugivores (Murphy et al. 1993; Barea 2008; Napier et al. 2013).

Stable isotope techniques are an extremely useful tool in the reconstruction of animal diets. This study is the first to investigate the changes in diets of mistletoebirds over time, and demonstrates the increased contribution of arthropods to the diet of mistletoebirds during breeding or moulting.

5 SUGAR PREFERENCES OF AVIAN NECTARIVORES ARE

CORRELATED WITH INTESTINAL SUCRASE ACTIVITY

Kathryn R. Napier1, Todd J. McWhorter1,2, Susan W. Nicolson3 and Patricia A.

Fleming1

1 School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150,

AUSTRALIA

2 School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, SA

5371, AUSTRALIA

3 Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, SOUTH

AFRICA

This chapter has been published in the journal Physiological and Biochemical Zoology

(86(5):499–514, DOI: 10.1086/672013).

STATEMENT OF AUTHOR CONTRIBUTION

K.R. Napier: Initiated the compilation of the research, mist-netted and kept birds in captivity, performed all laboratory experiments on mistletoebirds, silvereyes and singing honeyeaters and collected, entered and analysed the data. Also performed sugar preference experiments on the New Holland honeyeaters detailed in Fleming et al.

(2008). Collected intestinal tissue and performed enzyme assays on five species (as detailed in Table 5.2, years 2010–2011), and wrote the manuscript.

T.J. McWhorter: Collected intestinal tissue and performed enzyme assays on four species (as detailed in Table 5.2, years 2006–2007) and provided access to unpublished data as cited throughout. Assisted with the design and initiation of the research, advised and assisted K.R. Napier with enzyme assay and apparent assimilation efficiency procedures and statistical analyses, and provided editorial comments to versions of the manuscript from draft to publication.

S.W. Nicolson: Provided data on nectar composition as detailed in Figure 5.6 and

Table 5.4 and provided editorial comments to versions of the manuscript from draft to publication.

P.A. Fleming: Assisted with the design and initiation of the research, assisted

K.R. Napier with mist-netting, advised and assisted K.R. Napier with statistical analyses, and provided editorial comments to versions of the manuscript from draft to publication.

5.1 ABSTRACT

Nectar-feeding birds generally demonstrate preference for hexose solutions at low sugar concentrations, switching to sucrose/no preference at higher concentrations. Species vary in the concentration at which the switch from hexose preference occurs; this could reflect physiological constraints that would also influence nectar selection when foraging. We recorded concentration-dependent sugar type preferences in three opportunistic/generalist Australian nectarivorous species: the mistletoebird (Dicaeum hirundinaceum), silvereye (Zosterops lateralis) and singing honeyeater (Lichenostomus virescens). All three preferred hexoses up to sugar concentrations of 0.25 mol·L-1 and switched to sucrose/no preference for higher concentrations. Using these and literature records, we investigated physiological mechanisms that may explain the concentration- dependence of sugar type preferences and compared diet preference data with foraging records. We measured sucrase activity in silvereyes and singing honeyeaters as well as three specialised nectarivorous species; red wattlebirds (Anthochaera carunculata),

New Holland honeyeaters (Phylidonyris novaehollandiae) and rainbow lorikeets

(Trichoglossus haematodus) for comparison with published concentration-dependent sugar preference data. Sucrase activity varied between these species (P=0.003). The minimum diet concentration at which birds show no sugar preference was significantly correlated with sucrase activity for the eleven species analysed (P=0.005). Birds with the lowest sucrase activity showed hexose preference at higher diet concentrations and birds with the greatest sucrase activity either showed no hexose preference or hexose preference on only the most dilute diets. Foraging data compiled from the literature also support the laboratory analyses, e.g. rainbow lorikeets (preference for hexose over a wide range of diet concentrations, low sucrase activity) also feed primarily on hexose

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nectars in the wild. Intestinal sucrase activity is likely to contribute to diet selectivity in nectarivorous bird species.

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5.2 INTRODUCTION

Nectar and fruit are an important carbohydrate-rich food source for many bird species.

The disaccharide sucrose and its monosaccharide components glucose and fructose (i.e. hexoses, which are similar in chemical structure and in energy content per unit mass) are among the most common carbohydrates in nectar and fruit (Levey and Martinez Del

Rio 2001). The composition and concentration of sugars in nectar and fruit pulp varies widely amongst plant species (Whiting 1970; Pyke and Waser 1981; Baker and Baker

1982b; Baker and Baker 1983; Baker et al. 1998; Nicolson and Van Wyk 1998;

Nicolson 2002; Wilson and Downs 2012). Fruit pulp tends to be hexose-dominant, with sucrose content averaging only 8% of total sugars in fruits consumed by passerines

(Martínez del Rio et al. 1992; Baker et al. 1998). Nectar may be sucrose-dominant, hexose-dominant, or contain a mixture of both sucrose and hexoses (Nicolson and

Fleming 2003b; Johnson and Nicolson 2008). Many nectarivorous and frugivorous bird species exhibit distinct preferences for these sugars (see review by Lotz and Schondube

2006), although past studies were commonly conducted using a single sugar concentration and so the role that energy density may play in sugar selection is not clear. These past studies also used a wide variety of experimental methodologies, which can make comparing results among different studies difficult (Brown et al.

2008).

The potential physiological mechanisms underlying the sugar preferences of birds and the extent to which the sugar composition of natural nectars reflects selection by birds have long been debated. Dramatic differences in the composition of sugars in nectar were first reported by Baker and Baker (1982b; 1983). While these differences

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in plant nectar sugar composition were first thought to reflect selective pressures from their hummingbird (sucrose-dominant nectars) or passerine (hexose-dominant nectars) pollinators (Martínez del Rio 1990; Martínez del Rio et al. 1992), subsequent studies on the digestive enzymes of various avian lineages have shown that both hummingbirds and nectar-specialist passerines are capable of efficient digestion and assimilation of sucrose (see review by Lotz and Schondube 2006). The intestinal enzyme sucrase- isomaltase is responsible for the hydrolysis of sucrose into its monosaccharide hexose components. Most specialist and occasional nectarivores and frugivores are able to efficiently assimilate both sucrose and hexoses (Lotz and Schondube 2006; Fleming et al. 2008; Napier et al. 2008a), with the exception of frugivores in the Sturnidae-

Muscicapoidea lineage that lack sucrase (Martínez del Rio and Stevens 1989; Brugger et al. 1993; Sabat and Gonzalez 2003; Gatica et al. 2006; Brown et al. 2012). Some occasional nectarivores, however, exhibit lower apparent assimilation efficiencies for both sucrose and hexoses (Brown et al. 2010b; Brown et al. 2010a) and some occasionally nectarivorous and frugivorous passerines exhibit sucrose assimilation efficiency that is significantly lower than that for hexoses (Lane 1991; Odendaal et al.

2010). These patterns are consistent with findings presented by Johnson and Nicolson

(2008), who demonstrated that nectars of plants pollinated by specialist nectarivorous passerines are strongly convergent with those of plants pollinated by hummingbirds.

Specifically, plants pollinated by specialist avian nectarivores tend to have small volumes of concentrated, sucrose-dominant nectars, while those pollinated by generalists tend to have large volumes of dilute, hexose-dominant nectars.

One important finding of recent studies is that sugar type preference varies with sugar concentration. Nectarivorous birds tested using a range of concentrations of

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‘equicaloric’ (Fleming et al. 2004) sucrose or hexose diets generally demonstrate preference for hexose solutions at low sugar concentrations (i.e. energy densities), with a switch to sucrose or no preference at higher concentrations. This has been demonstrated in specialist nectarivores including sunbirds, hummingbirds, honeyeaters and lorikeets (Schondube and Martinez del Rio 2003; Fleming et al. 2004; Lotz and

Schondube 2006; Fleming et al. 2008; Brown et al. 2010c), and occasional nectarivores such as the speckled mousebird (Colius striatus) and village weaver (Ploceus cucullatus) (Brown et al. 2010a; Odendaal et al. 2010, see Table 5.1). Although these species demonstrate a similar pattern in sugar preferences, they differ in the concentration at which the switch from hexose preference to no preference occurs.

Most specialist nectarivores prefer hexoses at extremely dilute diets only, e.g. the red wattlebird (Anthochaera carunculata), New Holland honeyeater (Phylidonyris novaehollandiae), white-bellied sunbird (Cinnyris talatala), Malachite sunbird

(Nectarina famosa), magnificent hummingbird (Eugenes fulgens) and cinnamon-bellied flowerpiercer (Diglossa baritula) (Table 5.1). Some opportunistic nectar feeders (the speckled mousebird and village weaver) prefer hexoses up to slightly higher concentrations than these specialised nectarivores, yet the dark-capped bulbul

(Pycnonotus tricolor, a nectar generalist), and rainbow lorikeet (Trichoglossus haematodus, a nectar specialist) prefer hexoses at much higher sugar concentrations

(Table 5.1). Brown and colleagues suggested that these findings help to explain the dichotomy reported by Johnson and Nicolson (2008); however, aside from the work by

Brown et al. (2010a; 2010b) and Odendaal et al. (2010), little comparative data on sugar preferences in generalist nectar feeders has been available to date. Compared with

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nectarivores, we know far less about the concentration-dependence of sugar preferences of opportunistic or generalist avian frugivores.

Compensatory feeding, where birds increase volumetric intake rate as food energy density decreases, allows birds to deal with variations in nectar concentration

(Martínez del Rio et al. 2001; Nicolson and Fleming 2003a). Lotz and Schondube

(2006) and Fleming et al. (2008) have hypothesised that the concentration-dependence of sugar preferences in nectarivorous birds may be attributed to varying levels of sucrase activity and the need for constant energy assimilation (i.e. compensatory feeding). Birds that exhibit a lower capacity to hydrolyse sucrose are more likely to show preference for hexoses over sucrose solutions on dilute diets in this scenario, because digesta transit rates will be faster and substrate concentration for the sucrase enzyme will be lower, limiting the hydrolysis rate (McWhorter and Martinez del Rio

2000). In this study, we have tested this prediction with new and available published data. We investigated sugar preferences and apparent assimilation efficiency in three opportunistic/generalist Australian nectarivorous species: the mistletoebird (Dicaeum hirundinaceum), silvereye (Zosterops lateralis) and singing honeyeater (Lichenostomus virescens). We also analysed the activity of the intestinal enzymes sucrase-isomaltase

(EC 3.2.1.48, hereafter ‘sucrase’) and maltase-glucoamylase (EC 3.2.1.20, hereafter

‘maltase’) in silvereyes and singing honeyeaters, as well as three specialised nectarivorous species (New Holland honeyeaters, rainbow lorikeets and red wattlebirds) for comparison with published sugar preference data for these species (Fleming et al.

2008). Finally, we compiled foraging data for these species and Australian nectar sugar compositions, where available, from the literature. We predicted that:

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1) specialised nectarivorous species would exhibit greater apparent assimilation efficiencies for both hexoses and sucrose than generalist nectarivores;

2) the degree of preference for hexose over sucrose solutions would be correlated with variation in the capacity to hydrolyse sucrose; and

3) specialist nectarivores should preferentially forage on sucrose-rich nectars compared with generalist species.

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Table 5.1 Summary of sugar type preferences, apparent assimilation efficiency (AE*) and digestive capacity.

- indicates not measured or not available. Diet: Specialist nectarivore (SN, bold), generalist nectarivore (GN), frugivore (Fr). References: 1this study; 2Fleming et al. (2008); 3Napier et al. (2008a); 4Fleming et al. (2004); 5Schondube and Martínez del Rio (2003); 6Schondube and Martínez del Rio (2004); 7McWhorter et al. (unpublished), 8Köhler et al. (2010), 9Brown et al. (2010a), 10Brown et al. (2010b), 11Brown et al. (2010c), 12Odendaal et al. (2010), 13Wilson and Downs (2011), 14Downs (1997) 15Brown et al. (2012) 16Martínez del Rio (1990) 17Martínez del Rio (1990), 18McWhorter and Martínez del Rio (2000), 19Bizaare et al.(2012). Sugar preferences: dark grey: hexose (H) or glucose (G),

light grey: sucrose (S), ns: no significant preference. Total activity: nd: not detectable. Vmax: *we were advised by the authors that this published value is incorrect, and should be 9.16±1.1. This correct value was used in the analyses detailed in Figure 5.4. Km and pH optima: Kinetic parameters obtained using at least n=1 tissue homogenate (proximal intestinal section): † two data sets for birds caught in 2010–2011 (n=1) or 2006–2007 (n=1).

Sugar type preferences Apparent assimilation efficiency (AE*) Digestive capacity (sucrase) Diet Concentration (Sucrose Gut nominal Body Equivalents, mol·L-1) surface area Total activity Vmax pH Bird species Diet mass (g) Ref 0.075 0.1 0.25 0.5 0.75 1 2 Sucrose Glucose Fructose n (cm2) (µmol·min-1) (μmol∙min-1) Km (mmol) optima Rainbow lorikeet (Trichoglossus haematodus) SN 137±14 1,2,3 H H H H H ns S >98 99.7±0.1 - 7 31.6±7.1 25.8±12.1 42.8±21.7 21.7, 16.7† 5, 5† New Holland honeyeater (Phylidonyris novaehollandiae) SN 20.5±3.4 1,2 H H ns ns ns ns ns >99 - - 9 6.0±1.7 12.6±7.9 25.3±15.3 39.0, 25.0† 6, 6† Red wattlebird (Anthochaera carunculata) SN 105±3 1,2 H ns ns ns ns S S >99 99.8±0.1 - 8 15.3±4.2 41.7±23.6 77.9±37.0 42.9, 20.2† 5.5, 6† White-bellied sunbird (Cinnyris talatala) SN 9.0±1.4 4,7,8 - H ns ns S ns - 99.8±0.05 99.7±0.2 99.7±0.1 4 3.3±0.5 8.3±2.2 12.7±3.1 15.4±4.5 5.5 Broad-tailed hummingbird (Selasphorus platycercus) SN 3.3±0.1 4,6,7,18 - - ns ns ns ns - 95.0±0.02 - - 3 2.1±0.4 4.8±1.6 Silvereye (Zosterops lateralis) Fr, GN 9.0±0.4 1 H H H ns S S S 98.7±0.3 99.9±0.1 97.7±0.5 4 6.2±1.1 4.1±1.5 7.5±2.8 22.9 5 Singing honeyeater (Lichenostomus virescens) GN 28.9±4.1 1 H H H ns ns ns ns 99.6±0.2 99.9±0.1 99.3±0.3 7 8.3±1.7 13.6±8.6 23.7±14.5 24.9, 20.0† 5.5, 6† Mistletoebird (Dicaeum hirundinaceum) Fr, GN ~8 1 H H H ns ns ns ns 98.4±1.4 99.8±0.2 99.3±0.4 - - - - - 0.146 0.584 1.168 Magnificent hummingbird (Eugenes fulgens) SN 7.1±0.2 5,6 H ns S 99 99 99 3 3.5±0.5 21.4±4.2 * - - - Cinnamon-bellied flowerpiercer (Diglossa baritula) SN 8.1±0.2 5,6 H ns S 99 99 99 4 3.7±0.2 3.3±0.6 10.2±1.9 59.5 6 0.146 0.73 1.022 Broad-billed hummingbird (Cynanthus latirostris) SN 2.9±0.2 16,17 ns ns ns 99±2.4 97±4.9 98±2.4 3 1.7 5.6±0.9 - - - 0.146 0.29 0.438 0.584 0.73 'Hexoses' Malachite sunbird (Nectarina famosa ) SN ~16 11,14 H ns ns ns S >99 >99 Village weaver (Ploceus cucullatus) GN 36.7±2.8 12 H H ns ns ns ~90-94 ~96–98 Speckled mousebird (Colius striatus) GN ~47 9 H H ns ns S ~84-87 ~89–93 Dark-capped bulbul (Pycnonotus tricolor) GN ~37 10 H H H H H ~65-85 ~75–95 Red-winged starling (Onychognathus morio) GN ~126 15,19 H H H H 107ns 0 ~64–73 nd 0.193 0.643 Knysna tauraco (Tauraco corythaix) Fr ~260 13 S(vs G) ns Purple-crested tauraco (Tauraco porphyreolophus) Fr ~250 13 ns ns

5.3 MATERIALS AND METHODS

5.3.1 Birds and their maintenance

The mistletoebird is a specialised frugivore that feeds primarily on mistletoe fruit

(Richardson and Wooller 1988), but also includes nectar and insects in its diet (Reid

1990). The silvereye is a generalist, feeding on fruit, nectar and insects (Wilkinson

1931; Thomas 1980; Richardson and Wooller 1986). The singing honeyeater is a nectarivore that also ingests a relatively high proportion of insects (Collins and

Morellini 1979; Richardson and Wooller 1986); both singing honeyeaters and silvereyes have more muscular gizzards than specialised nectarivores due to their ingestion of insects (Richardson and Wooller 1986), therefore we have classified these species as generalist nectarivores (Table 5.1).

Singing honeyeaters (n=8) and silvereyes (n=8) were captured on the grounds of

Murdoch University, Perth, Western Australia (WA; 32°04′S, 115°50′E) by mist-netting in May 2009 and January 2010, respectively. There is no measure for sexual dimorphism in plumage for either species, so their sex could not be determined.

Mistletoebirds (four male and two female) were captured on private property at York,

WA (31°50′S, 116°44′E), in December 2010, January and March 2011. All birds were acclimated to captive conditions for at least two weeks before the commencement of experimental trials.

Birds were housed in individual outdoor aviaries (116 x 160 x 210 cm), but were confined to smaller cages (47 x 54 x 41 cm) placed within each aviary for the experiments. During the period of captivity, all three species were fed a maintenance

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diet of Wombaroo® nectarivore mix (Wombaroo Food Products, South Australia), which contains sucrose as the main sugar type, supplemented with additional sucrose or equal parts of glucose and fructose for a total sugar content of c. 25% w/w dry matter.

Birds fed through a small hole (c. 1-1.5 mm diameter) from plastic, stoppered syringes hung on the sides of the cage. The frugivorous silvereyes and mistletoebirds were also fed a variety of fleshy fruits (e.g. mistletoe fruit, watermelon, grapes, apricots) daily.

Martínez del Rio (1990) reported that measured sugar preferences in hummingbirds were not correlated with the sugar type of their maintenance diet. All animal care procedures and experimental protocols adhered to Murdoch University Animal Ethics

Committee regulations (R1137/05 and R2175/08). Birds were collected under permits issued by the Western Australian Department of Environment and Conservation (DEC).

5.3.2 Apparent assimilation efficiency (AE*)

Singing honeyeaters (n=8), silvereyes (n=8) and mistletoebirds (3 male, 2 female) fed ad libitum from sucrose and hexose solutions at three concentrations (0.25, 0.5, 1 mol·L-

1) for 24 h. Each bird fed from each sugar solution at each diet concentration, with sugar type and concentration randomised. Trials commenced within 30 min after sunrise (0500 to 0716 WST). Maintenance diet was removed one hour before sunrise to ensure all previously ingested food (i.e. from the previous day) was voided before trials commenced. Trays were placed under experimental cages to collect excreta, and liquid paraffin was placed in containers directly beneath feeders to collect any diet spilt. Food intake was recorded over 24 h by weighing feeders (0.01 g). Excreta produced over 24 h were allowed to evaporate and were then reconstituted and collected with a known volume of distilled H2O and stored at -20 °C until analysis.

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Glucose assays: Two replicates of each excreta sample (100 µl) were incubated at room temperature (~21 °C) for 15 min with 500 µl of hexokinase-glucose-6- phosphate dehydrogenase enzymatic assay reagent (G3293, Sigma Aldrich).

Absorbance was then measured at 340 nm relative to distilled water by spectrophotometry (UV mini 1240, Shimadzu Scientific Instruments, Balcatta, WA,

Australia). Standard ‘reagent blanks’ were included for each excreta sample.

Fructose assays: Two replicates of each excreta sample (45 µl) were incubated at room temperature (~21 °C) for 15 min with 650 µl of hexokinase-glucose-6- phosphate dehydrogenase enzymatic assay reagent (G3293, Sigma Aldrich) and 5 µl phosphoglucose isomerase from baker’s yeast (F2668, Sigma Aldrich). Absorbance was then measured at 340 nm relative to distilled water by spectrophotometry. Standard

‘reagent blanks’ were included for each excreta sample.

Sucrose assays: Two replicates of each excreta sample (25 µl) were incubated at room temperature (~21 °C) for 10 min with 25 µl invertase from baker’s yeast sucrose assay reagent (S1299, Sigma Aldrich). 650 µl of hexokinase-glucose-6- phosphate dehydrogenase enzymatic assay reagent (G3293, Sigma Aldrich) was then added, and samples incubated for a further 15 min. Absorbance was then measured at

340 nm relative to distilled water by spectrophotometry. Standard ‘reagent blanks’ were included for each excreta sample.

Apparent assimilation efficiency (AE*) was estimated separately for sucrose, glucose and fructose as:

AE* = (sugarin-sugarout) / (sugarin) (eq. 5.1)

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-1 where sugarin (g) is calculated as the concentration (g·L ) of sugar in the ingested diet multiplied by the volume of solution ingested (L), and sugarout (g) is the sugar concentration (g·L-1) in the total volume of excreta plus rinse water (L).

AE* data were arcsine square root transformed (Zar 1999) before analysis.

Differences in AE between sugar type, sugar concentration, species and total sugar intake were assessed by ANCOVA with total sugar intake as a covariate and Tukey-

Kramer post hoc tests for unequal sample sizes as required. Additional data for sucrose

AE* (excluding species from the sub-family Muscicapoidea) were obtained from

Fleming et al. (2008) and differences between specialist (n=21 species) and generalist

(n=13 species) nectarivores assessed by Mann-Whitney U test.

5.3.3 Sugar preference trials

Singing honeyeaters (n=8), silvereyes (n=8) and mistletoebirds (four males) participated in sugar preference trials which, following the methodology of Fleming et al (2008) for consistency, lasted for 6 h, commencing within 30 min of sunrise (0535 to 0705 WST).

Sugar preferences were examined by comparing the intake of seven paired concentrations of sucrose and energetically equivalent hexose (1:1 glucose:fructose) solutions: 0.075, 0.1, 0.25, 0.5, 0.75, 1 and 2 mol·L-1 Sucrose Equivalents (SE). Hexose diets were equicaloric with, but had approximately twice the osmolality of sucrose solutions (Fleming et al. 2008). Birds were simultaneously presented with pairs of feeders containing sucrose and hexose concentrations of the same SE molarity in random order. To account for potential sources of side bias (Jackson et al. 1998a;

Jackson et al. 1998b), the start position of each feeder was random, with the positions of

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the feeders switched half way though each trial. Each concentration was also tested on each bird twice, with the starting position of the feeders reversed on the second trial.

Liquid paraffin was placed in containers directly below feeders to collect any diet spilt.

Sugar intake was determined by weighing the feeders before and after trials (0.01 g) and calculating the mass of sugar ingested by taking into account the density of each diet.

Trials were conducted approximately every second day, with at least one day of rest and maintenance diet between trials. Trials were repeated for a third time in the instance of low diet intake (a few individuals did not drink when first offered the lowest concentration of 0·075 mol·L-1 SE, but increased intake during subsequent trials). The average intake over all trials for each diet was used to calculate a sugar preference index, with hexose intake expressed as a proportion of total sugar intake (H/(H+S), where a value of 0.5 indicates no preference whilst a value close to 1 indicates a strong hexose preference).

Average food intake (g sugar in 6 h of each trial) was analysed via one-way

ANOVA for each species, with diet sugar concentration as the independent factor and

Tukey’s Honest Significant Differences (HSD) post-hoc tests as required. Preference data were arcsine square root transformed (Zar 1999) before analysis by one-way

ANOVA for each species, with diet sugar concentration as the independent factor and

Tukey’s HSD tests as required. Differences in preferences between species and diet concentrations were assessed via two-way ANOVA with Tukey-Kramer post hoc tests for unequal sample sizes as required. For each species, sugar preference at each concentration was analysed by one-sample t-tests (Sokal and Rohlf 1995) comparing the arcsine-transformed square root of preference indices against 0.5 (no preference).

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5.3.4 Intestinal enzymes

Study species and dissection: Red wattlebirds (n=3), singing honeyeaters (n=7), silvereyes(n=4), New Holland honeyeaters (n=9) and rainbow lorikeets (n=7) were captured by mist- or cannon-netting near Perth, Western Australia, between 2007 and

2011 (see Table 5.2 for details). We did not have access to mistletoebirds for this part of the study. Birds were not fasted prior to euthanasia. Birds were euthanised via

Isoflurane overdose or a 1:1 sodium pentobarbital:distilled H2O solution injected into the heart. Sex was determined by examination of reproductive organs upon dissection.

The intestines were removed from stomach to cloaca within 10 min of euthanasia, dissected length-wise, cut into three sections (proximal, medial and distal) and measured (length and width to calculate nominal surface area, cm2). The intestinal sections were then rinsed in 0.75 mol·L-1 NaCl, blotted, and weighed (0.001 g). Each section was then frozen in liquid nitrogen and stored at -80 °C until enzyme activity analysis (<12 months after euthanasia). All animal care procedures and experimental protocols adhered to Murdoch University Animal Ethics Committee regulations

(R1137/05). Birds were collected under permits issued by DEC. Some tissues were kindly provided by Joao Coimbra (The University of Western Australia Animal Ethics

Committee RA/3/100/927 and DEC permit SF007556).

Disaccharidase assays: Intestinal samples were thawed at room temperature

(21±2 °C) and homogenised (Heidolph ‘DIAX 600’, Heidolph Instruments, Schwabach,

Germany) in 0.3 mol·L-1 mannitol in 0.001 mol·L-1 HEPES/KOH pH 7.5 buffer (99 to

128 mg intestine·mL-1 of homogenate). Aliquots of homogenates were immediately diluted in 0.3 mol·L-1 mannitol in 1.0 mmol·L-1 HEPES/KOH pH 7.5 buffer (1:40 for

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sucrase, 1:300 for maltase) and frozen in liquid nitrogen and stored at -80 °C until disaccharidase (sucrase and maltase) assays were performed.

Disaccharidase activity was measured according to Dahlqvist (1984) as modified by Martínez del Rio et al. (1995). Diluted intestinal homogenates (30 µL) were incubated with 30 µL of 0.056 mol·L-1 sugar substrate (maltose or sucrose) solutions in

0.1 mol·L-1 maleate NaOH pH 6.5 buffer at 40 °C for 20 min. 400 µL of a stop/develop reagent was then added, and samples were vortexed and incubated at 40 °C for a further

30 min. Stop/develop reagent was made by dissolving one bottle of Glucose oxidase/peroxidase reagent (G3660, Sigma Aldrich) in 19 mL 0.5 mol·L-1 phosphate

-1 buffer (NaH2PO4/Na2HPO4) pH 7.0 plus 19 mL 1 mol·L Tris/HCl pH 7.0, plus 2 mL

O-dianisidine solution (2.5 mg O-dianisidine dihydrochloride [D3252, Sigma Aldrich] per mL dH2O). Lastly, 400 µL 12NH2SO4 was added and the absorbance read at 540 nm. Maltase and sucrase activity (µmol·min-1) was measured for each section of intestine and summed together to calculate ‘total activity’ for each individual. Total enzyme activity for each individual bird was then adjusted to optimal pH, and then standardised for nominal gut surface area (µmol·min-1·cm2).

Differences in standardised sucrase activity between the five species were assessed by one-way ANOVA followed by Tukey-Kramer post hoc tests for unequal sample sizes. Least squares linear regression was also used, with data averaged for species to assess relationships between log body mass (mb) and log gut nominal surface area and log total sucrase and maltase activity.

As maltose may be hydrolysed by both sucrase and maltase (Alpers 1987;

Martinez del Rio 1990), the activity of both disaccharidases were measured. The slope

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of the relationship between sucrase and maltase indicates the amount of maltase activity relative to sucrase activity and the y-intercept provides an estimate of maltase activity that occurs in the absence of sucrase (Martinez del Rio 1990). The relationship between standardised sucrase and maltase activities was therefore examined using least squares linear regression.

Least squares multiple linear regression was also used to assess the relationship between hexose preference (scored as the minimum diet concentration at which birds show no sugar preference) and standardised sucrase activity with data averaged for all individuals for eleven species (Table 5.1). Studies that have used only a few diet concentrations may not yield accurate information in this regard, but the use of the minimum no-preference concentration is a conservative estimate of sugar type preference. Minimum no-preference concentration values also allowed inclusion of species that do not exhibit hexose preference, e.g. broad-billed (Cynanthus latirostris) and broad-tailed hummingbirds (Selasphorus platycercus). The red-winged starling

(Onychognathus morio), like other starlings, lacks the intestinal enzyme sucrase and therefore has non-detectable levels of sucrase activity (Bizaare et al. 2012). We then included the red-winged starling in the analyses with a sucrase activity value of 0.

Phylogenetic analyses: As phylogenetic relationships may confound the inferences of allometric analyses (Garland et al. 1992; Garland and Adolph 1994;

Rezende and Diniz-Filho 2012), these conclusions were corroborated using phylogenetically-independent contrasts. Felsenstein’s (1985) independent contrasts method was used in the computer program PDAP (Garland et al. 1992; Garland et al.

1993; Garland et al. 1999; Garland and Ives 2000) running through Mesquite (Version

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2.75) (Midford et al. 2009). Phylogenetically-independent contrasts (PIC) of dependent and independent variables were calculated and standardised utilising the branch length transformation (Garland et al. 1992). Evolutionary relationships (Fig. 5.1) were determined using the phylogenetic tree of Ericson et al. (2006) as a ‘backbone’ with sets of pseudo-prosterior samples of the dated phenologies built by Jetz et al. (2012) subsampled and then pruned for our full set of species. Regressions were fitted to standardised PIC values, forcing the data through the origin (Garland et al. 1992).

Cynanthus latirostris

Eugenes fulgens

Selasphorus platycercus

Trichoglossus haematodus

Lichenostomus virescens

Anthochaera carunculata

Phylidonyris novaehollandiae

Zosterops lateralis

Onychognathus morio

Cinnyris talatala

Diglossa baritula

Figure 5.1 Evolutionary relationships.

Phylogenetically independent contrast values were calculated using the evolutionary phylogenetic tree of Ericson et al. (2006) as a backbone, with sets of pseudo-posterior samples of the dated phenologies built by Jetz et al. (2012) subsampled and then pruned for our full set of species, through the website birdtree.org.

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5.3.5 Foraging data and Australian nectar composition

Foraging data for silvereyes, singing and New Holland honeyeaters, red wattlebirds and rainbow lorikeets were compiled from the Western Australian Pollination Database

(Brown et al. 1997). As the foraging records for rainbow lorikeets in Western Australia

(Brown et al. 1997) were rather limited (due to their recent introduction in the 1960s and subsequent establishment as a pest species in Perth, WA), detailed foraging records for rainbow lorikeets were also compiled from the Queensland-New South Wales border region (Cannon 1984). Nectar compositions of Australian plants were compiled from published and unpublished sources (Baker and Baker 1982b; Paton 1982;

Gottsberger et al. 1984; McFarland 1985; Nicolson 1994; Davis 1997; Baker et al.

1998; Nicolson and Van Wyk 1998; Hölscher et al. 2008; Morrant et al. 2010; S.W.

Nicolson and B.-E. Van Wyk, pers. comm.). The ratio of hexoses to sucrose was calculated as H/(H+S), and nectars classed as hexose-dominant (>0.8), hexose-rich

(0.6–0.8), mixed (0.4–0.6), sucrose-rich (0.2–0.4) or sucrose-dominant (<0.2). Ratios were adapted from Baker and Baker (1982b), with new classifications developed for this study. To examine the relationship between nectar type and foraging preference of the five species, a contingency table was constructed for foraging data for each species and the five nectar classifications (excluding plants for which we lack information on the nectar composition – classified as ‘unknown’) and analysed for significance by

Pearson’s χ2 square analysis (with Bonferroni correction applied).

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5.3.6 General statistical analysis

Data are reported as mean ± 1 SD throughout, with n referring to the number of animals.

Statistical analyses were performed with Statistica (StatSoft Inc 2007) and SPSS (SPSS

Inc, Chicago, IL, USA). Statistical significance was accepted for α<0.05.

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Table 5.2 Details of birds euthanased for digestive enzymes.

*Nedlands – grounds of The University of Western Australia (UWA); Murdoch – grounds of Murdoch University; Shenton Park – grounds of the UWA Shenton Park Field Station; Roleystone – grounds of the Araluen Country Club; Bentley – grounds of Curtin University (bird flew into window and died ~1 h later); Wattle Grove – bird obtained from Wattle Grove Veterinary Clinic after an unknown period in captivity; Perth Airport – grounds of Perth Domestic Airport as part of a Department of Conservation

(DEC) culling program. +Birds were euthanased via 1:1 sodium pentobarbital:distilled H2O solution injected into the heart or by Isoflurane overdose.

Period Species Year of Method of Sex of Location of capture* capture capture (M, F) captivity Method of euthanasia+

Red wattlebird Nedlands, WA (31°58′S, 115°49′E) 2010 Mist-netting (3, 0) < 72 h Sodium pentobarbital (Anthochaera carunculata) Murdoch, WA (32°04′S, 115°50′E) 2007 Mist-netting (4, 1) 5 mo Isoflurane

Singing honeyeater Shenton Park, WA (31°57′S, 115°47′E) 2010 Mist-netting (2, 1) < 7 h Sodium pentobarbital (Lichenostomus virescens) Murdoch, WA (32°04′S, 115°50′E) 2007 Mist-netting (4, 0) 5 mo Isoflurane

New Holland honeyeater Roleystone, WA (32°08′S, 116°05′E) 2011 Mist-netting (3, 1) < 7 h Sodium pentobarbital (Phylidonyris novaehollandiae) Murdoch, WA (32°04′S, 115°50′E) 2007 Mist-netting (5, 0) 14 mo Isoflurane

Silvereye Roleystone, WA (32°08′S, 116°05′E) 2011 Mist-netting (3, 1) < 7 h Sodium pentobarbital (Zosterops lateralis)

Rainbow lorikeet Bentley, WA (32°0′S, 115°53′E) 2011 Unknown (1, 0) < 1 h Natural death (Trichoglossus haematodus) Wattle Grove, (32°0′S, 115°59′E) 2011 Unknown (1, 0) Unknown Sodium pentobarbital

Perth Airport, WA (31°55′S, 115°57′E) 2006 Cannon-netting (5, 0) >12 mo Isoflurane

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5.4 RESULTS

5.4.1 Apparent assimilation efficiency (AE*)

Silvereyes, singing honeyeaters and mistletoebirds displayed high assimilation efficiencies for all three sugar types (>97.5%, Table 5.1). AE* was not different between sugar concentrations (F1,191 =2.56, P=0.111) but varied with sugar intake (F1,191

=10.20, P=0.002), where AE* increased as sugar intake decreased. AE* varied between species (F2,191=7.19, P=0.001), being greatest for singing honeyeaters and least for silvereyes overall. AE* also varied between sugars (F2,191 =65.54, P<0.001), being greatest for glucose and least for fructose. The significant sugar type by species interaction (F4,191=11.54, P<0.001) demonstrated that silvereyes assimilated less fructose than mistletoebirds and singing honeyeaters, and singing honeyeaters assimilated more sucrose than mistletoebirds and silvereyes.

AE* for sucrose differed significantly between generalist (86.24±16.21%, n=13) and specialist nectarivores (98.09±1.25%, n=21) (U=64, Z=2.62, P=0.0093).

Comparable data for hexoses were not available.

5.4.2 Sugar preferences

Silvereyes, singing honeyeaters and mistletoebirds all failed to consume sufficient volumes to maintain energy balance on the most dilute diets, with significantly lower intakes of sugar compared with the more concentrated diets. Compensatory feeding

(identified here as diet concentrations where sugar intake was not significantly different from the most concentrated diets) was observed for singing honeyeaters for diets ≥0.25

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mol·L-1, but only for diets ≥0.5 mol·L-1 in mistletoebirds and silvereyes (Fig. 5.2).

Sugar preferences were influenced by sugar concentration (F6,279=36.17, P<0.001), with all three species showing significant preferences for hexose solutions at low sugar concentrations (Fig. 5.3). Sugar preferences differed significantly between the species

(F2,279=4.460, P=0.012), with silvereyes displaying significant preferences for sucrose diets at the higher concentrations (i.e. ≥0.75 mol·L-1; Fig. 5.3).

(n=8)

2 (n=8) (n=4)

Diet sugar intake (g) sugar intake Diet 1

0 0.0 0.5 1.0 1.5 2.0 2.5

-1 Diet concentration (mol·L )

Figure 5.2 Concentration-dependent total sugar intake.

Mistletoebirds (circle), silvereyes (triangle) and singing honeyeaters (square) were offered paired sucrose and hexose (fructose + glucose) solutions of varying concentrations: 0·075, 0·1, 0·25, 0·5, 0·75, 1 and 2 mol·L-1 sucrose equivalents (SE). Diets where birds did not achieve energy balance (statistically lower intake than the maximal sugar intake) are indicated with increasingly lighter shaded symbols (one-way ANOVA and Tukey’s HSD test). n refers to number of individuals.

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a) Mistletoebirds (n=4) b) Silvereyes (n=8) c) Singing honeyeaters (n=8) *a *a 1.0 1.0 1.0 *a *a *a *b,c 0.8 0.8 0.8 c,d c,d Hexose b b c,d b d 0.6 0.6 b 0.6 *a b *b *b *a,b *a *b 0.4 0.4 0.4

Sugar preference Sugar

0.2 0.2 0.2

Sucrose

0.0 0.0 0.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0

Diet concentration mol·L-1

Figure 5.3 Concentration-dependent sugar preferences. a) mistletoebirds (circle), b) silvereyes (triangle) and c) singing honeyeaters (square) were offered paired sucrose and hexose (fructose + glucose) solutions of varying concentrations: 0·075, 0·1, 0·25, 0·5, 0·75, 1 and 2 mol·L-1 sucrose equivalents (SE). Diets where birds did not achieve energy balance (statistically lower intake than the maximal sugar intake) are indicated with increasingly lighter shaded symbols. Letters indicate diets that are statistically different from each other (one-way ANOVA with Tukey’s HSD test). Asterisks indicates concentrations where there was a signiﬁcant preference for either hexose or sucrose diets (one-sample t-test). n refers to number of individuals. 122

5.4.3 Intestinal enzymes

Body mass, gut nominal surface area, total sucrase activity and kinetic parameters for rainbow lorikeets, silvereyes, singing and New Holland honeyeaters and red wattlebirds are summarised in Table 5.1, with data for additional species reported from the literature. Total maltase activity and associated kinetic parameters are detailed in Table

5.3. Sucrase and maltase activity, as a function of substrate concentration, followed

Michaelis-Menten kinetics. Sucrase and maltase activities were highest in proximal sections of the intestine and decreased distally (data not shown). There were species differences in standardised sucrase activity (one-way ANOVA: F5,26=4.87, P=0.003); rainbow lorikeets and silvereyes had significantly lower sucrase activity than red wattlebirds (post hoc: P=0.012, P=0.032). When comparing data averaged for each species, gut nominal surface area increased with body mass (F1,9=120.88, P<0.001,

2 PIC R =0.94; Fig. 5.4a). This result was confirmed by PIC analysis of log10body mass

PIC 2 and log10gut nominal surface area (F1,8=62.53, P<0.001, R =0.887). Total sucrase activity was also significantly correlated with body mass (F1,9=12.46, P=0.008,

2 2 R =0.61, Fig. 5.4b; confirmed by PIC analysis: F1,8=15.08, P=0.006, R =0.653). Total maltase activity showed a borderline correlation with body mass, which was not upheld

2 by PIC analysis (F1,8=5.97, P=0.04, R =0.43, Fig. 5.4c; PIC: F1,8=0.432, P=0.532,

R2=0.051). Standardised maltase activity was not significantly correlated with

2 standardised sucrase activity (F1,9=0.18, P=0.686, R =0.02; Fig. 5.4d), which was

2 confirmed by PIC analysis (F1,8=4.113, P=0.082, R =0.340).

For eleven species tested, hexose preference (the minimum no-preference concentration) was significantly correlated with standardised sucrase activity

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2 (F1,10=13.44, P=0.005, R =0.60; Fig. 5.4e). Phylogenetically-corrected analysis

2 confirmed this result (F1,9=18.0, P=0.003, R =0.667). Birds with the lowest standardised sucrase activity showed hexose preference at more concentrated diets (i.e. greater minimum no-preference concentration), and birds with the greatest standardised sucrase activity either showed no hexose preference (e.g. hummingbirds) or hexose preference on only the most dilute diets.

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1.6 a) 1.6 b)

]

1.4 ] -1 1.4 1.2

1.0 1.2

0.8 Cinnamon-bellied flowerpiercer 1.0 Magnificent hummingbird 0.6 New Holland honeyeater Rainbow lorikeet Red wattlebird 0.8 0.4 Silvereye Singing honeyeater White-bellied sunbird 0.2 0.6

Broad-billed hummingbird µmol·min activity, [sucrase log

log [gut nominal surface area, cm area, surface nominal [gut log Broad-tailed hummingbird 0.0 0.4 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 log [body mass, g] log [body mass, g]

2.5 18 d) c) Red wattlebird

] ) Singing honeyeater

2 -1 2.0 16

·cm

-1 14 1.5

12 1.0 Silvereye

10 White-bellied sunbird

0.5 Broad-tailed hummingbird 8 Cinnamon-bellied flowerpiercer 0.0 6 Magnificent hummingbird

-0.5 Broad-billed hummingbird log [maltase activity, µmol·min activity, [maltase log 4

maltase activity (µmol·min activity maltase New Holland honeyeater Rainbow lorikeet -1.0 2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0 1 2 3 4 5 -1 2 log [body mass, g] sucrase activity (µmol·min ·cm ) 1.2 e)

Broad-tailed hummingbird 1.0 Broad-billed hummingbird Cinnamon-bellied flowerpiercer Magnificent hummingbird 0.8 New Holland honeyeater Rainbow lorikeet Red wattlebird Red-winged starling 0.6 Silvereye Singing honeyeater White-bellied sunbird 0.4

0.2

0.0 Minimum no-preference concentration (mol) concentration no-preference Minimum 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 -1 2 sucrase activity (µmol·min ·cm )

Figure 5.4 Gut nominal surface area and intestinal enzymes.

Relationships between body mass and a) gut nominal surface area; b) total sucrase activity and c) total maltase activity. d) Relationship between maltase and sucrase activity (both standardised by gut nominal surface area). e) Relationship between degree of hexose preference (i.e. minimum no-preference concentration) and standardised sucrase activity. Data are averaged for each species. White symbols denote generalist nectarivores, grey symbols denote specialised nectarivores. See Table 5.1 for details of references, numbers of individuals and diet categories.

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5.4.4 Foraging data and Australian nectar composition

Foraging data (Cannon 1984; Brown et al. 1997) are summarised in Fig. 5.5. Foraging records indicate that all of the focal species have a diverse diet, including multiple plant taxa in their diets (Fig. 5.5a). Nectar composition was available for 16 Australian genera (Fig. 5.6, Table 5.4). There was a significant association between bird species

2 and nectar type (χ 16=532.77, P<0.001; Fig. 5.5b), with rainbow lorikeets avoiding sucrose and mixed nectars in favour of hexose-rich nectars and the three honeyeater species avoiding hexose nectars in favour of sucrose-dominant, -rich and mixed nectars.

The foraging preferences of silvereyes were not very clear, which may reflect few foraging records (n=44) for this species.

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1.0 a) Other Tristania Schefflera Macaranga 0.8 Paraserianthes Beaufortia Acacia Lambertia 0.6 Calothamnus Grevillea Banksia Melaleuca 0.4 Eucalyptus Adenanthos Callistemon Amyema 0.2 Anigozanthos

Proportion of foraging observations Corymbia Hakea

0.0

1.0 b) *P unknown sucrose dominant ***P sucrose rich 0.8 mixed ***A ***A *A hexose rich *A hexose dominant

0.6 ***P

0.4 ***P ***P

***A ***A ***P ***P 0.2

Proportion of foraging observations ***P

0.0

(1174, 30) Silvereye (59, 23)

Red wattlebird (264, 19) Rainbow lorikeet Singing honeyeater (198, 25) New Holland honeyeater (433, 31)

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a) Feeding observations grouped by plant genus. White lines indicate hexose dominant and rich nectars, solid light grey indicates mixed sugars, dark grey lines indicates sucrose dominant and rich nectars. Dots indicate unknown sugar composition, solid white indicates other genera comprising <2% of feeding observations (including Agonis, Adansonia, Astroloma, Billardiera, Blancoa, Bombax, Bossiaea, Braxychiton, Brachysema, Chasmanthe, Chorilaena, Cosmelia, Crotalaria, Darwinia, Diplolaena, Eremophilia, Erythina, Gastrolobium, Hardenbergia, Hybanthus, Jacksonia, Jansonia, Kunzea, Leptosema, Leptospermum, Loranthus, Lysiana, Macropidia, Microcorys, Muiriantha, Nematolepis, Nicotiana, Nutysia, Pimelea, Pittosporum, Psoralea, Regelia, Temletonia, and Xanthorrea). In parentheses: total number of foraging observations, number of plant genera. b) Feeding observations grouped by nectar composition; see text for definitions of nectar categories. Asterisks denote significant preference (P) or avoidance (A) of nectar categories as determined by χ2 analysis with Bonferroni correction applied (P<0.05*, P<0.01**, P<0.001***).

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Stenocarpus (1) Hakea (4) % fructose % glucose Corymbia (1) % sucrose Anigozanthos (1) Amyema (1) Telopea (1) Callistemon (1) Erythrina (1) Adenanthos (3) Eucalyptus (18) Melaleuca (6) Banksia (23) Grevillea (25) Calothamnus (1) Macadamia (1) Lambertia (3)

0 20 40 60 80 100 Nectar compostion (% fructose, glucose, sucrose)

Figure 5.6 Average nectar composition from 16 Australian plant genera (mean fructose, glucose, sucrose).

In parentheses: number of species sampled for each genus. Fructose is represented by white bars, glucose by grey and sucrose by black. Hexose-dominant nectars include: Sternocarpus, Hakea, Corymbia, Anigozanthos, Amyema, Telopea, Callistemon, Erythrina and Adenanthos; hexose-rich nectars: Eucalyptus and Melaleuca; mixed nectars: Banksia (including former Dryandra species); sucrose-rich nectars: Grevillea and Calothamnus; sucrose-dominant nectars: Lambertia and Macadamia.

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Table 5.3 Summary of maltase activity.

Data are presented as mean ± 1 SD. - indicates not tested or not available. References: 1Data was obtained from this study; 6Schondube and Martínez del Rio (2004); 7McWhorter et al. (unpublished), 17Martínez del Rio (1990). Km and pH optima: Kinetic parameters obtained using at least n=1 tissue homogenate (proximal intestinal section): † two data sets for birds caught in 2010–2011 (n=1) or 2006–2007 (n=1).

Digestive capacity (maltase) V ref Total activity (µmol·min-1) max Km (mM) pH optima (μmol∙min-1) Rainbow lorikeet, Trichoglossus haematodus (n=7) 1 174±89.7 207.4±103.2 4.5, 5.8 5, 6 Silvereye, Zosterops lateralis (n=4) 1 50.7±20.3 60.5±24.2 5.4 6.5 Singing honeyeater, Lichenostomus virescens (n=7) 1 91.3±47.9 100.6±51.3 3.9, 2.6 5.5, 4.5 New Holland honeyeater, Phylidonyris novaehollandiae (n=9) 1 40.6+19.0 50.5±22.0 12.5, 4.3 4.5, 5 Red wattlebird, Anthochaera carunculata (n=8) 1 213.9±119 258.7±130.2 12.6, 4.3 5, 5.5 Broad-tailed hummingbird, Selaphorus platycercus (n=2) 6 7.7±1.4 - - - Magnificent hummingbird, Eugenes fulgens (n=3) 6 17.0±3.3 - - - Cinnamon-bellied flowerpiercer, Diglossa baritula (n=4) 6 30.1±4.0 33.2±4.4 2.8 5.5 White-bellied sunbird, Cinnyris talatala (n=4) 7 41.0±7.9 44.3±8.5 2.2 5 Broad-billed hummingbird, Cynanthus latirostris (n=3) 17 14.0±2.3 - - -

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Table 5.4 Nectar composition of 16 Australian plant genera.

Data are presented as mean ± 1 SD, n=total number of species.

Family Genus Fructose (%) Glucose (%) Sucrose (%) n Reference Fabaceae Erythrina 46 51 3 1 (Baker and Baker 1982a) Haemodoraceae Anigozanthos 45 55 0 1 (Hölscher et al. 2008) Loranthaceae Amyema 59 40 1 1 (Paton 1982) Myrtaceae Callistemon 48 50 2 1 S.W. Nicolson and B.-E Van Wyk, unpublished Calothamnus 12 8 80 1 S.W. Nicolson and B.-E Van Wyk, unpublished Corymbia 52 48 0 1 (Nicolson 1994) Eucalyptus 41.6±15.3 31.40±12.15 26.91±21.68 18 (Nicolson 1994; Davis 1997; Baker et al. 1998; Morrant et al. 2010; S.W. Nicolson and B.-E Van Wyk, unpublished) Melaleuca 41.5±7.5 31.76±12.95 26.70±19.56 6 (Morrant et al. 2010; S.W. Nicolson and B.-E Van Wyk, unpublished) Proteaceae Adenanthos 46.0±3.0 46.7±5.1 7.3±8.1 3 (Nicolson and Van Wyk 1998) Banksia 23.6±21.0 24.1±21.0 52.2±41.7 23 (McFarland 1985; Nicolson and Van Wyk 1998) Grevillea 8.5±16.9 12.5±24.6 82.7±33.7 25 (Gottsberger et al. 1984; Nicolson and Van Wyk 1998) Hakea 49.4±1.3 50.6±1.3 0 4 (Nicolson and Van Wyk 1998) Lambertia 1.3±0.6 1.0±1.0 97.7±1.5 3 (Nicolson and Van Wyk 1998) Macadamia 4 4 92 1 (Nicolson and Van Wyk 1998) Stenocarpus 51.7±3.1 48.3±3.1 0 1 (Nicolson and Van Wyk 1998) Telopea 49.0±0.0 49.3±1.5 1.7±1.5 1 (Nicolson and Van Wyk 1998)

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5.5 DISCUSSION

We investigated physiological mechanisms that may explain the concentration- dependence of sugar type preferences using data obtained from laboratory trials and literature records, and compared diet preference data with foraging records.

Supporting our first prediction, we found that specialised nectarivorous species exhibited greater apparent assimilation efficiencies for sucrose than generalist nectarivores when comparing broadly, using data available from this study (Table 5.1) and the literature (Fleming et al. 2008). Not enough information for glucose and fructose assimilation was available for generalist nectarivores (n=3) so we were unable to make this broader comparison for hexoses. However, the Australian generalist nectarivore species studied exhibited high apparent assimilation efficiencies (AE*) for sucrose, glucose and fructose (all >97.5%) comparable with specialist nectarivores.

These results suggest that these Australian generalist nectarivores should be as capable of feeding on both sucrose- and hexose-rich nectars as specialist nectarivores.

In terms of our second prediction, both specialist and generalist nectarivores demonstrated concentration-dependent sugar preferences. The degree of preference for hexose over sucrose solutions on dilute diets (assessed as the minimum no-preference concentration) was negatively correlated with the capacity to hydrolyse sucrose. For example, rainbow lorikeets, a specialist nectarivore, had one of the lowest sucrase activity levels and correspondingly preferred hexose diets over a broad range of diet concentrations. Hummingbirds, with the greatest sucrase activity levels, showed no preference for hexose over sucrose.

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Our third prediction was that diet preferences would match foraging records.

While some specialist nectarivores (e.g. New Holland honeyeaters) preferentially foraged on sucrose nectars over hexose nectars in the wild, others (e.g. rainbow lorikeets) preferred hexose-rich nectars to mixed and sucrose-rich and sucrose-dominant nectars. These data therefore do not support a simplistic differentiation in diet preferences between specialist and generalist nectarivores and indicate that the digestive physiology of each species is more closely correlated with its diet preferences

(measured in the laboratory or foraging records in the field) than broad classifications have led us to expect.

5.5.1 Are there differences in apparent assimilation efficiency between sugar

types?

Although all three Australian generalist nectarivore species assessed show high apparent assimilation efficiencies of sucrose and hexoses, there were some differences between these sugar types. AE* was greatest for glucose and least for fructose, and varied by species. Greater AE* for glucose over sucrose has been noted in studies of other species (Table 5.1) and may reflect the direct assimilation of glucose, but the need for hydrolysis of sucrose before its constituent monosaccharides can be assimilated.

Fructose absorption (by GLUT5 transporters) appears to be more concentration- dependent than the absorption of D-glucose (Holdsworth and Dawson 1964; Rand et al.

1993), therefore the lower AE* for fructose may reflect the availability of GLUT transporters and reliance on facilitated diffusion (rather than secondary active transport via SGLT1 transporter proteins as for glucose).

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Our data revealed a clear distinction between specialist and generalist nectarivores in terms of their AE*for sucrose. Specialist nectarivores uniformly have high AE* for sucrose, while many generalist nectarivores have lower AE* which could reflect lower sucrase activity. We could only compare AE* between generalist and specialist nectarivores for sucrose, due to lack of available data for the other sugars.

However, because AE* values for Australian generalist and specialist nectarivores feeding on all three sugars are >97.5%, these differences are not likely to be functionally significant or impact the sugar preferences or foraging choices of these birds.

5.5.2 Can we explain hexose preferences on dilute diets?

We examined whether hexose preference on dilute diets could be influenced by the amount of intestinal sucrase activity. Across eleven bird species, hexose preference

(minimum no-preference concentration) was significantly negatively correlated with sucrase activity (Fig. 5.4e). Birds with lesser capacities to digest sucrose show a significant preference for hexose solutions at higher sugar concentrations. For example, the rainbow lorikeet assessed in this study does not have the same sucrose digestive capacity shown by other specialist nectarivores, with only one third the sucrase activity of the red wattlebird, a similar-sized honeyeater (Fig. 5.4d, e). Rainbow lorikeets prefered hexose solutions up to 0.75 mol·L-1. By contrast, birds with greater capacities to digest sucrose showed hexose preference on only the most dilute diets or no preference over the range of concentrations tested. We included data for two hummingbird species which show no sugar type preference at room temperature for the minimum diet concentrations they have been tested on (0.146 and 0.25 mol·L-1 diets,

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respectively). When tested at lower diet concentrations (0.1 mol·L-1), the broad-tailed hummingbird resorted to torpor rather than feeding on the dilute solutions (Fleming et al. 2004). Challenging them with colder ambient temperatures (i.e. increasing their metabolic demands; Fleming et al. 2004) may be the only way to test for evidence of a hexose preference in hummingbirds. These data, together, demonstrates that preference for hexose at low diet concentrations reflects the digestive capacity of bird species.

Most specialist nectarivores prefer hexoses at only the most dilute diet concentrations tested, while many species of generalist nectarivores (e.g. the dark- capped bulbul and red-winged starling) show hexose preference across a greater range of diet concentrations (Table 5.1). However, the rainbow lorikeet (a specialist nectarivore) shows significant hexose preference for more concentrated diets than other specialist nectarivores. Furthermore, the simplistic categorisation of honeyeater species as specialist or generalist, in itself, may also be problematic. These data therefore do not support a simplistic distinction between specialist and generalist nectarivores across all avian lineages. Compared to specialist avian nectarivores, we know far less about the concentration-dependence of sugar preferences of avian frugivores (see Table 5.1).

5.5.3 Can we explain sucrose preference on concentrated diets?

A number of species have now been shown to switch to preference for sucrose solutions at high diet concentrations (Table 5.1). Significant sucrose preference has been somewhat puzzling, since these solutions have similar energetic value compared with the hexose equivalents, and sucrose solutions require sucrose hydrolysis before assimilation. As sucrose-dominant nectars tend to be more concentrated than

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predominantly hexose nectars (Nicolson 1998), birds may prefer sucrose at high concentrations and hexose at low diet concentrations as this reflects the pattern found in natural floral nectars (Lotz and Schondube 2006).

It has also been suggested that the preference for sucrose on high sugar concentrations could reflect taste preferences. By human tastes, fructose is 1.3x sweeter than sucrose while glucose has only 0.7x the sweetness of sucrose (Harborne 1993).

Birds may also show discrimination in sugar tastes. A recent study demonstrated that the broad-billed hummingbird perceives glucose, fructose and sucrose differently and is able to detect fructose at ~30% lower concentrations than sucrose or ~20% lower than glucose, indicating that fructose has a more intense flavour for this hummingbird

(Medina-Tapia et al. 2012). These authors suggested that hummingbirds were selecting sugar solutions in relation to their relative sweetness, and that gustatory thresholds may play an important role in determining sugar selection at least for more dilute diets

(Medina-Tapia et al. 2012). The role of taste in sugar type preference for concentrated diets remains to be tested.

5.5.4 Do laboratory results reflect foraging preferences in the wild?

The three honeyeater species examined (New Holland and singing honeyeaters and red wattlebirds) feed preferentially on sucrose nectars, avoiding hexose nectars; these foraging data reflect their preferences for hexoses only on very dilute sugar concentrations when tested in the laboratory. By contrast, rainbow lorikeets feed predominantly on hexose nectars, avoiding sucrose nectars; again, these data reflect preferences of these birds for hexoses at much higher sugar concentrations under

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laboratory conditions. We have few foraging data for silvereyes to date, therefore it is difficult to make any conclusions about their foraging preferences.

We have been limited by several constraints in our comparison between laboratory sugar type preferences and foraging choices in the wild. Firstly, there are very few data available on nectar sugars of Australian plants. While foraging observations are identified to plant species, the nectar composition data for these same plant species are often unavailable. We therefore present nectar composition data for plant genera rather than species (even so, we still lack data on nectar composition data for plant genera accounting for an average of 15% of foraging records for the five bird species examined for this measure). Secondly, these bird species also forage widely at plant species outside of Western Australia (we have not found comparative data of foraging observations for the rest of the country). Finally, where nectar data are available for multiple species of a plant genus, averaging values for sugar composition obscures the fact that some genera (notably Banksia and Grevillea), show a dichotomy in nectar composition, with some species having sucrose nectars and other species hexose nectars (Nicolson and Van Wyk 1998). Many species included in this data set

(e.g. Grevillea spp.; Table 5.4) may be not be primarily bird-pollinated, although birds may visit their flowers on an opportunistic basis.

5.5.5 Conclusions

In the Americas and Africa, nectar-feeding birds are relatively easily categorised as specialised (hummingbirds and sunbirds, respectively) or generalist (all other bird taxa) due to distinctions between bird lineages. However, there are ~180 species of

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Australasian honeyeaters (Family Meliphagidae) which exhibit a range of diets, from predominantly nectar through to predominantly insect diets. This makes a simplistic dichotomy between specialised and generalist/opportunistic nectarivores difficult for

Australian honeyeaters.

We have identified that sucrase activity is likely to be a key digestive constraint directly influencing the concentration-dependence of sugar type preferences shown in birds. To our knowledge, this is the first study to compare sugar preferences assessed in the laboratory with both aspects of digestive physiology and wild foraging observations.

We suggest that further comparative work on generalist and specialist nectarivores, particularly in larger birds such as lorikeets, takes a similarly multi-faceted approach by incorporating avian ecology and behaviour with digestive physiology.

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6 MISTLETOEBIRDS AND XYLOSE: AUSTRALIAN FRUGIVORES

DIFFER IN THEIR HANDLING OF DIETARY SUGARS

Kathryn R. Napier1, Patricia A. Fleming1 and Todd J. McWhorter2

1 School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150,

AUSTRALIA

2 School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, SA 5371,

AUSTRALIA

This chapter has been submitted to the journal Physiological and Biochemical Zoology for consideration of publication.

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STATEMENT OF AUTHOR CONTRIBUTION

K.R. Napier: Initiated the research, mist-netted and kept birds in captivity, performed all laboratory experiments on mistletoebirds, silvereyes and singing honeyeaters, collected, entered and analysed the data and wrote the manuscript.

P.A. Fleming: Assisted with the design and initiation of the research, assisted

K.R. Napier with mist-netting and paracellular absorption experiments, advised and assisted K.R. Napier with statistical analyses, and provided editorial comments to versions of the manuscript from draft to publication.

T.J. McWhorter: Assisted with the design and initiation of the research, advised

K.R. Napier with apparent assimilation efficiency, paracellular absorption, and HPLC experimental methodologies, procedures and statistical analyses, and provided editorial comments to versions of the manuscript from draft to publication.

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6.1 ABSTRACT

Carbohydrate-rich mistletoe fruits are consumed by a wide range of avian species.

Small birds absorb a large portion of water-soluble nutrients, such as glucose, via the paracellular pathway. D-xylose, a pentose monosaccharide, is abundant in some nectars and mistletoe fruits consumed by birds, and it has been suggested that it is most likely absorbed via the paracellular pathway in birds. We measured apparent assimilation efficiency (AE*) and bioavailability (f) for D-xylose and D- and L-glucose in three frugivorous Australian bird species. Mistletoebirds, silvereyes and singing honeyeaters showed significantly lower AE* for D-xylose than for D-glucose. Across two diet sugar concentrations, silvereyes and singing honeyeaters significantly increased f of both L-glucose (a metabolically inert isomer of D-glucose commonly used to quantify paracellular uptake) and D-xylose on the more concentrated diet, probably due to increased gut processing time. By contrast, mistletoebirds (mistletoe fruit specialists) did not vary f of either sugar with diet concentration. Mistletoebirds also showed higher f for D-xylose than L-glucose and eliminated D-xylose more slowly than silvereyes and singing honeyeaters, demonstrating differences in the handling of dietary xylose between these species. Our results suggest that D-xylose may be absorbed by both mediated and non-mediated mechanisms in mistletoebirds.

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6.2 INTRODUCTION

Mistletoe fruit is used by a wide range of bird species, with at least 30 bird species recorded feeding on Australian mistletoe fruits (Reid 1986; Reid 1989; Barker and

Vestjens 1990; Higgins et al. 2001). The composition of the fleshy fruit pulp varies among species, but most mistletoe fruits are high in carbohydrates, lipids and protein

(Walsberg 1975; Herrera 1987; McPherson 1987; Witmer 1996; López de Buen and

Ornelas 2001; Barea 2008), with 24–74% of the dry mass comprising of soluble carbohydrates (Godschalk 1983a; Lamont 1983b; Restrepo 1987; Snow and Snow

1988; Gedalovich-Shedletzky et al. 1989). The sugar composition varies between species, with fructose, rhamnose, mannose, glucose, arabinose, galactose and xylose present in varying amounts in four Santalaceae and Loranthaceae species studied to date

(Table 6.1, Gedalovich-Shedletzky et al. 1989; Azuma et al. 2000).

Xylose is a pentose monosaccharide that has been reported as one of the most abundant sugars in the fruit of several species of the North American mistletoe families

Santalaceae and Loranthaceae and is present in one species of Australian Loranthaceae mistletoe, Amyema preissii (see Table 6.1). Xylose is also abundant in nectar sugar of two genera of Proteaceae, comprising up to 39% of the total nectar sugar by weight in

Protea and Faurea nectars examined (Nicolson and Van Wyk 1998). The high concentration of xylose in these mistletoe fruits and floral nectars suggests that this sugar could be an important dietary component for nectarivores and frugivores.

However, few vertebrates are able to tolerate high concentrations of xylose in food, and it is unclear whether many vertebrates are able to directly metabolise it (Lotz and

Nicolson 1996; Franke et al. 1998; Jackson et al. 1998a; Jackson et al. 1998b). Xylose

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appears to be absorbed to some degree in nectar feeding birds, indicated by reasonably high apparent assimilation efficiencies (AE*) in two southern African species: the Cape sugarbird (Promerops cafer, AE* ~53%) and the Cape white-eye (Zosterops pallidus,

AE* ~61%) (Franke et al. 1998; Jackson et al. 1998a; Jackson et al. 1998b), although assimilation of sucrose and hexoses including glucose and fructose are still much higher with AE* close to 100% in most nectarivores and frugivores tested to date (Lotz and

Schondube 2006; Fleming et al. 2008; Napier et al. 2008a). Some birds have been shown to exhibit osmotic diarrhoea (resulting from unabsorbed xylose) after ingesting pure xylose solutions (Lotz and Nicolson 1996; Jackson et al. 1998b; Jackson and

Nicolson 2002). While nectar feeding bird species tested to date have shown avoidance of pure xylose solutions (Lotz and Nicolson 1996), a southern African mammal pollinator, the Namaqua rock mouse, Aethomys namaquensis, freely consumed xylose solutions with an AE* of 97% (Johnson et al. 1999). Xylose only has nutritional value, however, if it is able to be absorbed and metabolised. The mechanisms of absorption of this sugar are therefore of direct interest in interpreting how animals deal with the presence of xylose in nectar and fruit.

There are two routes for sugar absorption at the intestine, via carrier-mediated transport through the intestinal epithelial cells themselves (the transcellular route), or diffusion between these cells (the paracellular route). Small birds and bats with reduced intestinal absorptive surface area, as an adaptation to flight (Caviedes-Vidal et al. 2007), absorb a large portion of their water-soluble nutrients (e.g. glucose) via the paracellular pathway (reviewed by McWhorter 2005). Paracellular absorption provides a non- saturable absorptive mechanism that matches absorption capacity to acute changes in dietary nutrient concentration (Afik et al. 1997; Ferraris 2001). The relative

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contribution of paracellular to total glucose absorption in nectarivorous birds increases with an increase in diet sugar concentration, an effect which may be largely due to increased digesta retention time of more energy dense nectars in the intestine

(McWhorter et al. 2006; Napier et al. 2008b). Frugivorous birds and bats also rely extensively on the paracellular pathway for the absorption of glucose (Tracy et al. 2007;

McWhorter et al. 2010), but the effects of diet energy density and thus digesta transit or residence times have not been assessed in frugivorous species to date.

The mechanism of absorption of xylose appears to vary amongst species, and probably includes both paracellular and carrier-mediated components. There is evidence that intestinal absorption of xylose in chickens, hamsters, rats, frogs, rabbits and cows occurs at least in part by active carrier-mediated transport; it requires energy expenditure and it is Na+-dependent and inhibited by phlorizin (Salem et al. 1965;

Alvarado 1966; Lassen and Csaky 1966; Heyman et al. 1980; Scharrer and Grenacher

2000). However in humans, xylose appears to be absorbed only via passive (i.e. paracellular) diffusion (Ohkohchi et al. 1986; Fine et al. 1993). It has been suggested that xylose is likely to be absorbed via the paracellular pathway in nectarivorous birds

(Jackson and Nicolson 2002), although no direct uptake measurements have been done.

We investigated the AE* of D-xylose (assumed to be absorbed by paracellular mechanisms) and D-glucose (absorbed by both paracellular and transcellular pathways) in three bird species: a mistletoe fruit specialist, a generalist frugivore and a generalist nectarivore that also takes mistletoe fruit. We also explored the mechanism(s) of absorption by assessing the bioavailability of radiolabelled L-glucose (an isomer of D- glucose that is not metabolised and is absorbed only by the paracellular pathway) and

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D-xylose. We predicted, firstly, that all three study species would exhibit high AE* for

D-glucose but lower AE* for D-xylose (as shown in previous studies, e.g. Lotz and

Nicolson 1996; Franke et al. 1998; Jackson et al. 1998b). Because L-glucose is only absorbed via the paracellular pathway (Chang and Karasov 2004), a high bioavailability of L-glucose is indicative of significant paracellular uptake of D-glucose. Therefore, we further predicted that if D-xylose is also absorbed solely by the paracellular pathway, both L-glucose and D-xylose bioavailability would increase with diet sugar concentration.

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Table 6.1 Relative sugar composition (% dry mass±SEM) of mistletoe fruit viscin.

Viscin is the mucilaginous tissue that surrounds the endosperm and embryo of mistletoe fruit. Tr indicates trace. References include 1Azuma et al. (2000), 2Gedalovich-Shedletzky et al. (1989) and 3Napier et al. (unpublished data). * refers to fruits obtained from plants grown in a greenhouse. Sugar composition for Amyema species (Napier et al., unpublished data) were obtained from HPLC and GC-MS, with + indicating presence, as determined by GC-MS. Sucrose, glucose and fructose were the only three sugars detected with

confidence by HPLC.

Total Sugar

Xylose

Sucrose Glucose

Fructose

Mannose

Raffinose

Galactose Arabinose Family Species Harvest region (% dry mass) Rhamnose Ref

Santalaceae Viscum album France 61.6 1.1 0.9 4.0 12.5 10.9 10.4 1

Phoradendron californicum USA 51±2 14.4 Tr 2.3 36.1 15.6 19.4 11.1 2

Arceuthobium americanum Canada 55±4 6.8 Tr 5.7 39.5 20.3 24.4 3.4 2

Loranthaceae Phthirusa pyrifolia Canada* 24±1 43.5 4.4 32.7 5.4 11.8 2.2 2

Amyema preissii Western Australia +7.4 +34.4 +58.2 + + + 3

Amyema miquelii Western Australia 5.0 38.3 56.7 3

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6.3 MATERIALS AND METHODS

6.3.1 Study species

Our study species were three Australian bird species that are known to ingest mistletoe fruit (Chaffer 1966; Paton and Ford 1977; Bernhardt 1984; Forde 1986; Reid 1986).

The mistletoebird (Dicaeum hirundinaceum) is a specialised frugivore that feeds primarily on mistletoe fruit (Richardson and Wooller 1988). The short, specialised alimentary tract of the mistletoebird is typical of specialised frugivores; mistletoebirds possess smaller and less muscular gizzards than insectivorous birds of similar sizes, and the gizzard, proventriculus and duodenum are all in the same plane, which allows the more direct and rapid processing of the large number of mistletoe fruit that are ingested

(Richardson and Wooller 1988).

The silvereye (Zosterops lateralis) is a generalist frugivore (Wilkinson 1931;

Thomas 1980; Richardson and Wooller 1986) and the singing honeyeater

(Lichenostomus virescens) is considered a generalist nectarivore (Collins and Morellini

1979; Richardson and Wooller 1986). The digestive tract of honeyeaters is less specialised then that of the mistletoebird (Richardson and Wooller 1988). Silvereyes and singing honeyeaters show no distinct morphological adaptations for frugivory, with intestine lengths expected for nectarivorous birds of their size, and more muscular gizzards that fall between those of specialist frugivores and specialist insectivores

(Richardson and Wooller 1986; Stanley and Lill 2002). The junction of the gizzard and duodenum is also on a separate plane from that of the proventriculus and gizzard in these species (Richardson and Wooller 1988).

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The transit time of mistletoe seeds is shorter in specialist mistletoe feeders such as the mistletoebird and painted honeyeater (Grantiella picta) than in more generalist frugivores and honeyeaters. For example, the mean gut passage time of Amyema quandong mistletoe seeds by the spiny-cheeked honeyeater (Acanthagenys rufogularis), an opportunistic mistletoe fruit feeder, in Australia, is 40.57±0.60 (SE) min, nearly three times longer than for the mistletoebird at 13.67±0.48 (SE) min (Murphy et al. 1993) and nearly twice as long as painted honeyeaters at 24.43±1.27 (SE) min (Barea 2008).

Keast (1958) also reported longer voiding times in silvereyes feeding on A. miquelii and

A. qaudichaudii fruits (30 to 80 min) compared to mistletoebirds (25 to 60 min). Seed transit times for silvereyes feeding on Coprosma quadrifida and Rhagodia parabolica fruits (slightly smaller than A. preissii fruit) average 18 and 31.5 min, respectively

(French 1996; Stanley and Lill 2002).

Silvereyes (body mass; mb 9.0±0.4 g) and singing honeyeaters (mb 28.9±4.1 g) were captured on the grounds of Murdoch University, Perth, Western Australia (WA;

32°04′S, 115°50′E) by mist-netting in May 2009 and January 2010, respectively. There is no measure of sexual dimorphism by plumage in either species and we did not conduct genetic sexing, so gender is not known for these species. Mistletoebirds (mb

8.7±0.6 g) were captured on a private property at York, WA (31°50′S, 116°44′E), in

December 2010 and January and March 2011. All birds were acclimatised to outdoor captive conditions (see below) for at least two weeks before the commencement of experimental trials. All three species were fed a maintenance diet of Wombaroo ® nectarivore mix (Wombaroo Food Products, Glen Osmond, South Australia), which contains sucrose as the main sugar type, supplemented with additional sucrose or equal parts of glucose and fructose for a total sugar content of ~25% w/w dry matter.

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Silvereyes and mistletoebirds were also fed a variety of fleshy fruits daily (e.g. mistletoe fruit, watermelon, grapes, apricots).

Birds were housed in individual outdoor aviaries (116 x 160 x 210 cm), and confined to individual cages (47 x 54 x 41 cm) within the aviary for apparent assimilation efficiency (AE*) and gut processing time (GPT) experiments. During pharmacokinetic experiments, birds were housed individually in opaque plastic cages

(42 x 54 x 50 cm) with an automatic lighting regime (12:12 h), and a one way mirror to minimise disturbance during sample collection. Excreta were collected from waxed paper which was rolled through slots in the bottom of the cage, allowing samples to be collected immediately upon defecation.

All animal care and experimental procedures were approved by the Murdoch

University Animal Ethics Committee (approval R2175/08).

6.3.2 Apparent assimilation efficiency (AE*):

Seven silvereyes, 8 singing honeyeaters and 5 mistletoebirds (3 male, 2 female) fed ad libitum from D-glucose:D-xylose (4:1) solutions at three concentrations (0.25, 0.5, 1 mol·L-1 total sugar) for 24 h, through inverted, stoppered syringes. Each bird fed from each solution with sugar concentration randomised and trials commencing within 30 min after sunrise (0500 to 0716 WST). Maintenance diet was removed 1 h before sunrise to ensure all ingested food was voided before trials commenced. Trays were placed under experimental cages to collect excreta and small containers of liquid paraffin were placed directly below feeders to collect any diet spilt. Food intake was recorded over 24 h by weighing feeders (0.01 g). Dried excreta were reconstituted with

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a recorded amount of distilled rinse water. AE* was estimated separately for glucose and xylose as:

AE* = (sugarin-sugarout) / (sugarin) (eq. 6.1)

-1 where sugarin (mg) is the concentration (mg·ml ) of sugar in the ingested diet multiplied by the volume of solution ingested (ml), and sugarout (mg) is the sugar concentration

(mg·ml-1) in the total volume of excreta plus rinse water (ml).

NSW, Australia). Standard ‘reagent blanks’ were included for each excreta sample.

Absorbance was then measured at 340 nm relative to distilled water by spectrophotometry (UV mini 1240, Shimadzu Scientific Instruments, Balcatta, WA,

Australia).

Xylose assays: Two replicates of each excreta sample (50 µl) and two standard

‘reagent blank’ samples were incubated at room temperature (~21 °C) for 10 min with

+ 200 µl of TEA/MgCl2 buffer solution, 200 µl of NAD /ATP solution and 10 µl of hexokinase suspension (D-xylose assay kit, Megazyme International, Wicklow, Ireland) in order to remove any D-glucose present in the sample. Absorbance (A1) was then measured at 340 nm relative to distilled water by spectrophotometry. The xylose detection reaction was then initiated by the addition of 10 µl of β-xylose dehydrogenase/xylose mutarotase solution and incubated for 10 min. Absorbance (A2) was then measured at 340 nm relative to distilled water by spectrophotometry, with the absorbance difference calculated (A2-A1).

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6.3.3 Pharmacokinetic experiments

Bioavailability of L-glucose and D-xylose was measured using [14C] and [3H] radiolabelled L-glucose (180.16 g mol-1) and D-xylose (150.13 g mol-1), administered orally and by intramuscular (IM) injection to 8 silvereyes, 8 singing honeyeaters and 4 mistletoebirds (2 male, 2 female) as described below. Oral and IM trials were performed in separate experiments. To vary food intake rate, birds received two diet sugar concentrations (0.25 and 1 mol·L-1 hexose solutions) in separate feeding experiments. Both the order of trials and treatments given were randomly assigned, and followed published protocol (McWhorter et al. 2006; Napier et al. 2008b).

Bioavailability (f) was calculated as:

F = (P·S·Kel) / I (eq. 6.2) where P is the steady-state feeding concentration of radiolabelled sugars in plasma

(d.p.m.·mg-1 of plasma); S is the probe distribution space of radiolabelled sugars in plasma (mg of plasma); Kel is the elimination rate constant for the removal of radiolabelled sugars from plasma and its excretion in urine (min-1); and I is the ingestion rate of radiolabelled sugars (d.p.m.·min-1) (Karasov and Cork 1994; McWhorter et al.

2006; Napier et al. 2008b).

For IM administration, each bird was injected into the pectoralis muscle with

~40 μl of a solution of 175 mmol·L-1 NaCl containing 500 KBq [14C]-L-glucose per mg

3 mb, or 2,220 KBq [ H]-D-xylose per mg mb. The osmolality of the IM injection solution

-1 was controlled at approximately 350mmol·kg , so that the solution was isosmotic with avian blood (Goldstein and Skadhauge 2000). After IM administration, excreta were

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collected continuously for ~2 h, followed by collection of a small blood sample from the brachial vein. The parameters for the mono- and bi-exponential models were derived for each individual by non-linear curve fitting of the concentration of radiolabelled sugars in excreta after IM administration versus time, by use of the

Marquardt-Levenberg algorithm (Marquardt 1963). For oral administration, birds fed in separate trials from a hexose solution containing radiolabelled sugars ad libitum for ~3 h (3H-D-xylose: 120 KBq·ml-1 and 200 KBq·ml-1; 14C-L-glucose: 60 KBq·ml-1 and 90

KBq·ml-1 for 0.25 and 1 mol·L-1 hexose diets respectively). One small blood sample was collected 3 h after introduction of the radiolabelled diet; 4 silvereyes, 8 singing honeyeaters and 4 mistletoebirds (3 male, 1 female) reached steady-state with regard to radiolabel ingestion and excretion by 90 min, after returning to steady-state feeding within 30 min after introduction of the radiolabelled diet (data not shown). The final analyses were conducted on these individuals only, as four silvereyes and one mistletoebird did not return to steady-state feeding despite repeated attempts at oral trials, violating this assumption (Napier et al. 2012).

6.3.4 Gut passage times (GPT)

Eight silvereyes, 8 singing honeyeaters and 5 mistletoebirds (3 male, 2 female) were offered fresh, ripe mistletoe fruits on branches, either A. miquelii (singing honeyeaters) or A. preissii (mistletoebirds and silvereyes), depending on time of year and seasonal availability (the bird species were not held simultaneously due to space and logistic constraints). Birds were then observed via video camera, and the GPT of mistletoe fruits was determined by recording the time of ingestion and defecation of mistletoe fruits and calculating the difference; it was assumed that fruits were defecated in the

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order they were ingested (Karasov and Levey 1990; Murphy et al. 1993; Witmer 1994).

Trials commenced within 1 h after sunrise (0500 to 0716 WST) and lasted for up to 3 h, until all ingested seeds had been defecated. Maintenance diet was removed 1 h before sunrise to increase appetite for the mistletoe fruit (Murphy et al. 1993). GPT was calculated for each fruit defecated during the feeding trial by viewing of the video footage, with up to two trials per bird conducted on separate days.

6.3.5 Statistical analysis

Proportional AE* data were arcsine square root transformed (Zar 1999) before analysis.

Differences in AE*, total sugar intake and pharmacokinetic parameters between sugar type, sugar concentration and species were assessed by 3-way RM-ANOVA and 2-way

RM-ANOVA with sugar concentration and sugar type as repeated measures with

Tukey-Kramer post hoc tests for unequal sample sizes as required. Data are reported as mean ± 1 SD throughout. Statistical analyses were performed with Statistica (StatSoft

Inc 2007) and SPSS (SPSS, Inc, Chicago, IL, USA). Statistical significance was accepted for α<0.05.

6.4 RESULTS

6.4.1 Apparent assimilation efficiency (AE*)

All three species modulated their sugar intake with changes in diet concentration (i.e. exhibited compensatory feeding), ingesting the same mass of sugar irrespective of diet concentration (3-way RM-ANOVA; sugar concentration: F4,68=0.605, P=0.660). A separate analysis examining AE* revealed a significant three-way interaction term

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(sugar concentration x sugar type x species; F4,34=3.1, P=0.029) which indicated that the three bird species were handling the sugars differently. Glucose AE* was higher than xylose AE* (sugar type: F1,17=733.8, P<0.001). Glucose AE* was extremely high

(averaged over all concentrations: mistletoebirds: 99.77±0.39%; silvereye:

99.85±0.14%; singing honeyeater: 99.85±0.14%) and did not differ with species or diet concentration (Fig. 6.1; post hoc analyses shown). Xylose AE* ranged from

56.08±8.10 to 78.3±4.07% (Fig. 6.1). Diet concentration did not have a significant effect on xylose AE* for silvereyes, but singing honeyeaters assimilated significantly

-1 -1 less xylose on the 1 mol·L diet compared to the 0.25 mol·L diet, and mistletoebirds assimilated significantly less xylose on the 0.25 mol·L-1 diet compared to the 0.5 mol·L-1 diet (Fig. 6.1).

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a a a a a a a a a 100 b

b,c b,c b,c,d 80 b,c,d b,c,d b,c,d c,d

d 60

40 0.25 mol·L-1 D-xylose 0.5 mol·L-1 D-xylose 1 mol·L-1 D-xylose 20 0.25 mol·L-1 D-glucose 0.5 mol·L-1 D-glucose

Apparent assimilation efficiency (AE*, %) efficiency Apparent assimilation 1 mol·L-1 D-glucose 0 Mistletoebird Silvereye Singing honeyeater

Figure 6.1 Apparent assimilation efficiency (AE*) of D-xylose (solid) and D-glucose (lines).

Birds were fed solutions containing 80% D-glucose and 20% D-xylose at three total sugar concentrations (0.25 (white), 0.5 (light grey) and 1 mol·L-1 (grey)) for mistletoebirds, silvereyes and singing honeyeaters. Values are presented as mean ± 1SD; mistletoebirds (n=5), silvereyes (n=7), singing honeyeaters (n=8). Superscripts refer to results of Tukey-Kramer post hoc tests for unequal sample sizes (3-way RM- ANOVA).

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6.4.2 Pharmacokinetic experiments

Pharmacokinetic data were available for all singing honeyeaters (n=8) tested, but only for four out of the original eight silvereyes and four of the original five mistletoebirds, due to silvereyes and one mistletoebird failing to recommence steady-state feeding within a sufficient timeframe. These individuals were therefore excluded from pharmacokinetic analyses due to the violation of steady-state feeding conditions (Napier et al. 2012). Over the three hour trial period, birds drank approximately three times the

-1 volume of the dilute diet (0.25 mol·L ) compared to the more concentrated diet (Table

6.2). The mean steady-state concentration (P) of L-glucose and D-xylose in plasma was relatively high in all species, indicating significant absorption of both compounds

(Table 6.2). The elimination of both L-glucose and D-xylose after IM injection did not fit a bi-exponential model significantly better than a mono-exponential model for all individual birds of all species (glucose: F<3.29, P>0.1; xylose: F<4.49, P>0.06), indicating single compartment kinetics. Xylose was eliminated faster than glucose in singing honeyeaters, but the opposite was the case for mistletoebird (2-way RM-

ANOVA: Kel; sugar type x species; F2,13=240, P<0.001, Table 6.2). Probe distribution space (S) did not differ between glucose and xylose for mistletoebirds and silvereyes but did for singing honeyeaters (Table 6.2).

In terms of bioavailability (f) of the two sugars, there was a significant species by sugar type interaction (3-way RM-ANOVA; sugar type x species: F2,13=7.65,

P=0.006) indicating the three species handled glucose and xylose differently; we therefore analysed the data for each species separately by two-way RM-ANOVA (sugar and diet concentration as the repeated-measures factors). Mistletoebirds did not vary

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either glucose or xylose f with diet concentration (concentration: F1,3=0.006, P=0.941), while the other two species did (silvereyes concentration: F1,3=97.69, P=0.002; singing honeyeaters concentration: F1,7=33.38, P=0.001; see Fig. 6.2). Furthermore, mistletoebirds showed higher f for xylose compared with glucose, while the opposite pattern was observed for singing honeyeaters (no significant effect for silvereyes, see

Fig. 6.2).

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Values are presented as mean ± 1 SD. Feeding rate: mistletoebirds and singing honeyeaters were presented both L-glucose and D-xylose in a single solution with double-labelled isotopes while silvereyes were fed L-glucose and D-xylose in separate feeding trials. Superscripts refer to significant differences obtained from 2-way RM-ANOVA (between -1 species: Kel and S; within species: f) and Tukey post hoc tests for unequal sample sizes. †calculated only once for each bird while feeding on 0.25 mol·L hexose solution.

Mistletoebirds (n=4) Silvereyes (n=4) Singing honeyeaters (n=8) L-glucose D-xylose L-glucose D-xylose L-glucose D-xylose Feeding rate (mg·min-1) 0.25: 48.44±8.14 0.25: 48.44±8.14 0.25: 26.99±4.95 0.25: 26.42±9.55 0.25: 65.16±32.97 0.25: 65.16±32.97 1: 16.08±6.93 1: 16.08±6.93 1: 5.72±2.66 1: 9.611±3.61 1: 22.28±5.78 1: 22.28±5.78 Intake rate, I (d.p.m.·min-1) 0.25: 76215±12811 0.25: 86764±14583 0.25: 67002±16493 0.25: 99876±52152 0.25: 597098±30308 0.25: 61976±30509 1: 43132±40343 1: 63568±27376 1: 16289±6503 1: 25860±8544 1: 218258±59577 1: 31294±9709 Steady-state plasma, P 0.25: 282±156 0.25: 832±156 0.25: 707±160 0.25: 1310±465 0.25: 2335±667 0.25: 157±56 (d.p.m.·mg-1 plasma) 1: 101.89±56 1: 658±256 1: 500±193 1: 653±291 1: 1668±456 1: 142±63

Elimination constant, Kel (min-1) † 0.044±0.005a 0.014±0.003b,c 0.021±0.009 b,c 0.025±0.009 b,c 0.014±0.009 c 0.022±0.005 b

Probe distribution space, S 2100±1062b 4015±1772a,b 1456±590 b 1631±1117 b 6268±2243a 3631±2033 b (mg plasma) † Bioavailability, f (%) 0.25: 32.88±19.72 b 0.25: 51.98±23.46 a 0.25: 29.70±12.69 d 0.25: 53.31±16.24 b,d 0.25: 36.53±13.35 b 0.25: 25.06±17.13 d 1: 32.92±27.84 b 1: 54.15±14.75 a 1: 81.81±17.23 a,b 1: 89.44±12.07 a 1: 65.22±21.93 a 1: 36.86±14.89 b

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a a,b 100 a

80 a a b,d b

, %) ,

f 60 b b b

d d 40

Bioavailability ( Bioavailability

0.25 mol·L-1 L-glucose 1 mol·L-1 L-glucose 20 0.25 mol·L-1 D-xylose 1 mol·L-1 D-xylose

0 Mistletoebirds Silvereyes Singing honeyeaters

Figure 6.2 Bioavailability (f, %) of radiolabelled L-glucose (white) and D-xylose (grey) at two diet concentrations (0.25 and 1 mol·L-1, solid and lines respectively).

Values are presented as mean ± 1 SD; mistletoebirds (n=4), silvereyes (n=4), singing honeyeaters (n=8). Superscripts refer to significant differences between diets within species (2-way RM-ANOVA with Tukey-Kramer post hoc tests for unequal sample sizes).

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6.4.3 Gut passage times (GPT)

Mistletoebirds were the only species to ingest mistletoe fruits whole, after removing the exocarp. None of the 8 singing honeyeaters ingested mistletoe fruit over their two 3- hour trials (i.e. 6 h observations for each individual bird). One of 8 silvereyes ingested small amounts of A. preissii fruit pulp (2 out of 14 available fruits; in one trial) by prising open the exocarp of the fruit using its beak in a pliers-like motion to open a small hole into a wider split that it could feed from, but did not consume any seeds.

When the seed was removed from the exocarp, the bird stuck it to the mistletoe branch and continued to eat the fruit pulp.

The average GPT of A. preissii ingested by mistletoebirds was 16.45±7.21 min, and ranged from 2.0 to 40.0 min (n=177, total number of trials n=8). The number of fruits presented to individual birds ranged from 22 to 32, with 84–100% ingested by birds and 0–16% of fruits manipulated, but dropped before consumption. The average feeding period (71.99±16.73 min) ranged from 49.0 to 98.0 min, while the total trial period (last defecation-first feeding) averaged 87.95±18.89 min (ranging from 54.0 to

109.0 min). The average number of fruits in the birds intestine over the total trial period was 5.49±2.49 (range from 1–12). The average number of seeds defecated over the total trial period was 3.08±1.03 (range from 1–5).

6.5 DISCUSSION

Xylose was only first reported as a nectar sugar in southern African Proteaceae by Van

Wyk and Nicolson in 1995, but we know very little about its prevalence and presence in other plant nectars across the globe. Xylose is also evident in the fruits of some

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mistletoe species (Table 6.1 and references therein). Xylose may therefore be more prevalent in nectar and fruit diets for birds than we currently are aware of, and birds that have evolved with a high-xylose diet are likely to have developed mechanisms of handling this sugar. Mistletoe birds clearly process xylose differently from the other two frugivores tested in this study. While there was no clear pattern between the species in terms of the AE* of xylose, the three species handled the absorption of glucose and xylose differently: silvereyes and singing honeyeaters showed significantly greater bioavailability for both sugars on the more concentrated diet, but mistletoebirds did not vary f with diet sugar concentration. Furthermore, mistletoebirds showed higher f for D-xylose compared with L-glucose; while the opposite pattern was observed for the singing honeyeaters (no significant effect for silvereyes). We discuss these findings firstly in terms of the assimilation of these sugars, followed by their mechanisms of absorption.

6.5.1 Apparent assimilation efficiency (AE*)

All three species assimilated significantly less D-xylose than D-glucose. As mistletoebirds consume mistletoe fruit (previous studies reveal several species of mistletoe contain large quantities of xylose sugar, Table 6.1 and references therein), these birds should have been better able to deal with xylose than the other two bird species. However we did not find this, as mistletoebirds did not demonstrate higher D- xylose AE* values then the other two species.

All three species exhibited compensatory feeding, that is, they were able to adjust their intake rate so they ingested a similar total mass of sugar over the range of

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sugar concentrations. Sugar concentration did not affect D-xylose AE* in silvereyes, but varying effects of concentration were demonstrated in mistletoebirds and singing honeyeaters, with no clear discernable pattern. The AE* experiments integrate digestive function over a longer time period in comparison to the pharmacokinetic measures of bioavailability, and so may be more sensitive to variation in feeding rate and frequency than the latter. It is also possible that varying amounts of microbial degradation of xylose in the excreta collection pans may contribute to this variation, but the absolute causes of the varying effects of diet concentration between bird species on the D-xylose AE* values are unclear.

We also found slightly higher AE* of D-xylose in our study species (56–78%) compared to other bird species studied to date (53–61%) (Franke et al. 1998; Jackson et al. 1998b), but much lower than the Namaqua rock mouse (~97%) (Johnson et al.

1999), suggesting that, like other bird species studied, xylose is not absorbed efficiently by these Australian birds.

6.5.2 Mechanism of sugar absorption

Silvereyes and singing honeyeaters showed greater bioavailability of both L-glucose and D-xylose on the more concentrated diet, where slower gut passage time would promote increased absorption via the paracellular route (likely due to increased contact time of digesta with absorptive surfaces), as has been previously described in three specialised nectarivores to date (McWhorter et al. 2006; Napier et al. 2008b). On the other hand, mistletoebirds did not vary f with diet concentration for either sugar. This could suggest that absorption of these compounds known to be absorbed by non-

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mediated (i.e. paracellular) mechanisms is somehow decoupled from digesta retention time in this species. Although we were unable to directly compare the transit rates of mistletoe fruit between our study species, mistletoebirds have previously demonstrated much faster transit rates of mistletoe fruit through their highly specialised intestinal tracts compared to silvereyes and honeyeaters (Keast 1958; Murphy et al. 1993; French

1996; Stanley and Lill 2002; Barea 2008). We are not aware of any measurements of digesta retention time in mistletoebirds feeding on liquid diets, but it is highly likely that mistletoebirds, like many other species (Lopez-Calleja et al. 1997; Levey and Martinez del Rio 1999; McWhorter and Lopez-Calleja 2000; McWhorter et al. 2006; Wilson and

Downs 2011), do vary retention time with energy density. A high capacity for mediated glucose uptake (not quantified in this study) might mean that mistletoebirds can meet their energy needs without an increased reliance on paracellular uptake. Indeed, the significantly lower f of L-glucose for mistletoebirds in comparison to silvereyes and singing honeyeaters suggests a decreased reliance on the paracellular pathway in mistletoebirds. One other specialised frugivore, the cedar waxwing, has three times the active transport of glucose compared to the American robin (Karasov and Levey 1990).

This allows cedar waxwings to maintain a high and efficient absorption of sugars, even at relatively high intake and processing rates of fruit (Witmer and van Soest 1998). If mistletoebirds have relatively high capacity for mediated carbohydrate uptake, digesta retention time may vary less with energy density than in other species studied, providing a potential mechanism to explain the apparent decoupling of f and diet sugar concentration in this species.

The paracellular space discriminates according to molecular size, similar to a sieve (Friedman 1987; Chediack et al. 2003). Therefore, if D-xylose and L-glucose

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were both absorbed via the paracellular pathway, we would expect higher f values for

D-xylose based on its lower molecular mass (Chediack et al. 2003). Due to these differences in molecular mass between D-xylose and L-glucose and the fact that diffusion in water declines with molecular weight (MW0.5), the bioavailability values of

D-xylose can be corrected by a decrease of 8.7% [(180.160.5-150.130.5)/180.160.5)*100] to assess the effects of molecule size on absorption. Mistletoebirds were the only species to demonstrate a significantly higher f for D-xylose than L-glucose, and this difference is not solely accounted for by differences in molecular mass (i.e. average D- xylose f values are >8.7% higher than L-glucose, Table 6.2). This suggests that xylose could possibly be absorbed by both mediated and paracellular routes in these birds- possibly in the same manner as D-glucose (i.e. both paracellular and carrier-mediated active transport). Further work is required to determine if xylose is actively transported by membrane proteins in these birds.

Evidence also suggests that in some other animals, D-xylose is actively transported along with D-glucose, although the affinity of the transporter for D-xylose is much lower than that of D-glucose (Salomon et al. 1961; Bihler et al. 1962; Csaky and

Lassen 1964; Alvarado 1966; Ohkohchi and Himukai 1984). In chickens, Savory

(1992) reported D-xylose was absorbed slower than D-glucose and D-galactose, but faster than D-arabinose and D-mannose, which concurs with results previously reported in chicks (Wagh and Waibel 1967), rats (Kohn et al. 1965) and humans (Wood and

Cahill 1963); suggesting that the absorption rates of these sugars may depend on their relative contributions of active and passive (transcellular and paracellular) transfer mechanisms (Savory 1992).

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6.5.3 Metabolism of xylose

Xylose is only beneficial as a source of energy to these birds if it is able to be metabolised, either by gastrointestinal microbes or directly by bird tissues (discussed in depth by Jackson and Nicolson 2002). Animals such as the Namaqua rock mouse, through its caecal fermentation chamber in its gut, are able to ferment xylose to convert it into a source of energy (Johnson et al. 2006). The caecal enlargement demonstrated in chicks fed xylose suggests a similar process of bacterial fermentation (Schutte 1990;

Schutte et al. 1992). While frugivorous and nectarivorous birds like mistletoebirds and honeyeaters tend to have rudimentary or small caeca (Richardson and Wooller 1986;

Richardson and Wooller 1988), the presence of intestinal microbes (that may be able to metabolise xylose), has not been studied in these birds. Xylose is also able to be catabolised by certain mammalian tissues, with mammalian cells demonstrated to be able to survive with xylose or xylitol as their sole energy source (Demetrakopoulos and

Amos 1978), but to date, this has not been investigated in birds.

The presence of xylose in nectar and fruit remains puzzling due to the aversion shown by birds and rodent pollinators (Jackson and Nicolson 2002). Although the

Namaqua rock mouse is able to efficiently absorb and metabolise xylose, it is the least preferred sugar in preference tests (Johnson et al. 1999). A co-evolutionary explanation for xylose as an attractant for pollinators and dispersers therefore remains contentious

(Jackson and Nicolson 2002), and the study of the potential catabolism of xylose by intestinal microbes or systemic catabolism in birds certainly warrants further investigation.

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6.5.4 Conclusions

Mistletoebirds showed higher bioavailability for xylose compared with glucose; exactly the opposite pattern that was observed for singing honeyeaters (data for silvereyes were not statistically significant). This implies that mistletoebirds may be absorbing D- xylose by both mediated and non-mediated mechanisms. Mistletoebirds also eliminated xylose more slowly than silvereyes and singing honeyeaters, suggesting that xylose may have been incorporated into cells or used in biochemical pathways in mistletoebirds; it might also reveal a delay due to xylose passing through the gut enterocytes on its way to the circulation.

The observation that mistletoebirds did not vary f with diet sugar concentration, unlike silvereyes and singing honeyeaters, is also very intriguing – while possible explanations may include the decoupling of retention time and absorption of compounds absorbed by non-mediated mechanisms, a decreased reliance on the paracellular pathway, relatively less modulation of digesta retention time with diet energy density, or the presence of intestinal microbes in this species, we did not measure digesta mean retention time, mediated carbohydrate uptake or quantify the intestinal microbiome of birds in the present study.

These data build upon our understanding of the handling of sugars in frugivorous Australian birds, with new insight in particular to a specialised frugivore.

While mistletoebirds do not assimilate more xylose than the more generalist frugivores assessed in this study, they may absorb xylose differently. These three species therefore reveal differences in how they handle dietary sugars.

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7 GENERAL CONCLUSIONS

Australian mistletoes are a fascinating example of biological diversity, and are considered a keystone species due to the key role they play in ecosystems. There is an extremely diverse community of birds, ants, butterflies and mammals that live around mistletoes and their hosts, with mistletoe associations ranging from the highly specific relationships between mistletoes and their hosts and the specialised dispersers of mistletoe fruit, to the more general relationships between opportunistic feeders of mistletoe fruit, nectar and leaves.

The main aim of this thesis was to use a multi-faceted approach in the investigation of the interactions between two mistletoe species, Amyema miquelii and A. preissii, their hosts, and their avian consumers in the south-west of Western Australia

(WA). This was achieved through intensive field surveys and sampling, followed by a series of laboratory studies assessing various aspects of avian digestive physiology. By using a variety of experimental and analytical techniques, including field surveys, stable isotopes analysis, multivariate analysis and pharmacokinetic modelling, this thesis has provided new insight into the relationships between mistletoes and their hosts, and the food resources mistletoes provide to a mistletoe specialist, the mistletoebird.

7.1 MISTLETOE-HOST INTERACTIONS

The mistletoe story presented in this thesis began with a study of the parasitic and host relationship that determines how Australian mistletoes obtain their water and nutrients

(Chapter 2). Unlike other mistletoe species studied to date, A. miquelii obtained little

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nitrogen from its nitrogen-poor eucalypt hosts, while A. preissii obtained more nitrogen from its nitrogen-fixing (and therefore nitrogen-rich) acacia hosts. This study also demonstrated that a greater differential in water use efficiency was required for mistletoes on nitrogen-poor eucalypt hosts to extract nitrogen, resulting in more negative differences in δ13C in comparison to mistletoes on acacia hosts. The investigation of the degree of heterotrophy in Australian mistletoes would also be a valuable contribution to this area of research. The future investigation of proportional heterotrophy requires a back-to-basics approach, however; greenhouse-based pot- culture experimental methodologies that measure gas exchanges and biomass and control for water availability, as well as investigations into the assumptions such as estimated carboxylation factors and confounding effects of identical carbon isotope discrimination, are recommended (Bannister and Strong 2001; Tennakoon et al. 2011).

7.2 MISTLETOE-ANIMAL INTERACTIONS

This thesis then transitioned into the investigation of the importance of mistletoe as a food resource to bird communities in south-west WA, with particular focus on the mistletoebird. The presence of fruiting mistletoe was associated with significant changes in bird community structure, and mistletoebirds were more likely to be recorded during months when ripe mistletoe fruit was present (Chapter 3). The use of stable isotopes allowed the estimation of the proportional contribution of mistletoe fruit to the diet of mistletoebirds, which ranged from 33% to 55% across the three sites/sampling times tested (Chapter 4). The low nitrogen content of mistletoe fruit

(0.89–2.17%) is significantly less then that of arthropods (11.09–13.10%).

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Mistletoebirds therefore supplement their diet with arthropods, and appear to increase the dietary contribution of arthropods during breeding and moulting periods.

One limitation of the study of the dietary contributions of arthropods and mistletoe fruit to mistletoebirds detailed in Chapter 4 is the limited sample sizes of both dietary sources and mistletoebird tissues, particularly at the Araluen site. Mistletoebirds proved difficult to mist-net in large numbers, with only two individuals mist-netted and sampled at Araluen. I was unable to attempt mist-netting and sampling at two of the five original study sites due to the extreme height of mistletoe in the canopy (Paruna), and the overlapping availability of ripe fruit between Paruna and York 2 meant that field work could only be conducted at one site (York 2). Cold weather conditions also contributed to the decreased diversity of arthropods sampled at Araluen. Nevertheless, this study is the first attempt to estimate the diets of mistletoebirds at more than one site and at different times throughout the year through the use of dual isotopes (δ13C and

δ15N) and multiple avian tissues, and also takes into account the variation in dietary sources and differences in the elemental concentration of isotopically distinct dietary sources.

The nitrogen content of mistletoe fruit sampled in this study from York 1 (A. preissii, 1.12±0.27%) and York 2 (A. miquelii, 0.89±0.25%) is similar to other bird dispersed fruits (0.90±0.57%, Levey et al. 2000), and also to another Australian

Amyema species (A. quandang, 0.7%, Barea 2008). The higher nitrogen content of A. preissii fruits sampled from Araluen (2.17±3.00%) is intriguing; while A. preissii, parasitising nitrogen-rich acacia hosts, exhibit higher foliar nitrogen contents compared to A. miquelii (parasitising nitrogen-poor eucalypt hosts, Chapter 2), there is no clear

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pattern in fruit nitrogen content. Considerable variation in the nutritional content of fruit, due to environmental and genetic factors, has previously been demonstrated within species (Herrera 1987; Obeso and Herrera 1994; Izhaki et al. 2002), but further research is required to determine if the differences demonstrated within A. preissii in this study are due to local abiotic factors.

The specialisation to a diet consisting of high carbohydrate, low protein fruits and nectar is associated with relatively low nitrogen requirements (Witmer 1998;

Schondube and Martinez del Rio 2004; Tsahar et al. 2006). This, combined with high intake rates of mistletoe fruit and specialised intestinal morphology, has been hypothesised to allow mistletoebirds to rely on mistletoe fruit as a dietary source of protein (Barea and Herrera 2009). It would have been particularly valuable to have quantified the nitrogen requirements of mistletoebirds (i.e. minimal nitrogen requirements, MNR), which would be beneficial for future research (see ‘future directions’). If mistletoebirds maintain the general pattern observed in other frugivores

(and specialised nectarivores) of having lower than expected nitrogen requirements compared with generalist-feeding bird species, the physiological mechanisms which they use to be able to survive with such low nitrogen requirements would also be of great interest.

7.3 PHYSIOLOGICAL ADAPTATIONS TO FRUGIVORY

This mistletoe story concluded with the study of various digestive adaptations associated with consuming a fruit diet in three focal avian species that consume fruit to various degrees in their diet. Like the majority of bird-consumed fruits, fruit pulp from mistletoe species studied to date contains low amounts of, or no, sucrose (Chapter 6).

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For the mistletoe species analysed to date, the sugar composition of mistletoe fruit pulp varies, with glucose and xylose the most prevalent sugars. The two Australian mistletoe species studied in this thesis appear to differ from published studies of other mistletoe species due to the high levels of fructose present in mistletoe fruit pulp (with the caveat that the HPLC analysis performed was not sensitive enough to detect prevalence or concentrations of sugars other than sucrose and hexoses). Mistletoebirds demonstrated comparable values for the assimilation of sucrose and hexoses to both the more generalist feeders in this study (silvereyes and singing honeyeaters) and other specialised nectarivores studied to date, with apparent assimilation efficiency (AE*) values >98% for sucrose, glucose and fructose. While intestinal enzyme data were not available for mistletoebirds due to ethical reasons, the study detailed in Chapter 5 is the first to demonstrate that intestinal sucrase activity is likely to contribute to diet selectivity in frugivorous and nectarivorous bird species. Foraging data available for nectarivorous species supported the laboratory analyses, with the rainbow lorikeet, which demonstrated preferences for hexose over a wide range of diet concentrations in laboratory preference trials, was also demonstrated to feed primarily on hexose nectars in the wild. Mistletoebirds, unlike silvereyes, did not demonstrate a significant preference for sucrose solutions at high diet concentrations, which may be influenced by the low sucrose concentrations in mistletoe fruit.

The mistletoebird is currently the only specialised frugivore that has been examined for concentration-dependent sugar preferences over a wide range of dietary concentrations. More conclusive research into the sugar composition of Australian mistletoe fruit is required to confirm that mistletoe fruits in general contain low amounts of sucrose, as well as the assessment of sucrase activity.

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Mistletoebirds demonstrated similar D-xylose AE* values to silvereyes and singing honeyeaters (Chapter 6). Both silvereyes and singing honeyeaters showed significantly increased bioavailability (f) of L-glucose and D-xylose on a more concentrated diet, where slower gut passage time would promote increased absorption via the paracellular route. By contrast, mistletoebirds did not vary f with diet concentration and mistletoebirds appear to absorb xylose through both mediated and paracellular mechanisms. This lower dependence on paracellular sugar absorption

(compared with the other two species tested in this thesis as well as various nectarivorous bird species tested to date) is likely due to the short intestinal tract of mistletoebirds, specialised for processing fruit. Mistletoebirds may rely more on the mediated transport of glucose in comparison to silvereyes and singing honeyeaters

(whose relatively slower gut passage times would promote increased absorption via the paracellular route).

7.4 FUTURE DIRECTIONS

How frugivorous and specialist nectarivores are able to survive with such low nitrogen requirements remains unknown. While it has been suggested that these birds may have lower rates of endogenous protein turnover and high nitrogen recycling capacity

(Witmer 1998; Pryor et al. 2001), yet this has not been confirmed to date. Birds excrete urate in their urine in spherical concretions, which also contain protein and inorganic ions (Casotti and Braun 1997; Casotti and Braun 2004). Tsahar et al. (2005), upon reporting that protein concentration was lower in excreta than in ureteral urine in two frugivorous bird species, hypothesised that some of the protein associated with urate- containing spheres is digested in the lower intestine and recovered. This corresponds

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with the findings of high activities of aminopeptidase-N (which is often used as a proxy measure for the ability to digest protein, Maroux et al. 1973; Kania et al. 1977;

Martínez del Rio et al. 1995) in the terminal portion of the intestine of cedar waxwings

(Witmer and Martínez del Rio 2001). McWhorter et al. (unpublished data) also demonstrated that aminopeptidase activity was highest for medial and distal portions of the intestine for honeyeaters, sunbirds and a lorikeet. McWhorter et al. (unpublished data) also reported the curious finding that nectarivorous and non-nectarivorous passerines have similar total aminopeptidase activity, despite the lower than expected nitrogen requirements of small nectarivores. Future research into the nitrogen requirements of frugivores such as mistletoebirds and silvereyes is therefore of great interest.

While singing honeyeaters and silvereyes have been recorded feeding on the fruits of the two focal mistletoe species in this thesis in the literature, neither species ingested mistletoe fruit whilst in captivity, nor were recorded feeding on mistletoe fruit in the wild during surveys. Stable isotopes analysis has the advantage of allowing the evaluation of the origin of assimilated nutrients – in this thesis, this approach demonstrated that mistletoebirds supplement their diets with arthropods, despite the lack of arthropod remains evident in the faeces of captured birds. During the field sampling of mistletoebirds, I also sampled other bird species, including silvereyes and singing honeyeaters (captured in mist-nets in front of fruiting mistletoe), with the aim to compare differences in dietary contributions between species; however several limitations resulted in the exclusion of this data from this thesis. Limitations included the low capture numbers of some species, and the unsuccessful stable isotopes analysis of other available dietary sources, such as nectar and sap.

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Stable isotopes analysis has also been used in the study of other keystone species in ecosystems around the world. For example, Symes et al. (2011) used carbon stable isotopes (δ13C) to investigate the importance of Aloe marlothii nectar to opportunistic avian nectarivores in South Africa. Wolf and Martínez del Rio (2003) also used δ13C and δD to demonstrate how the nectar and fruit resources provided by saguaro

(Carnegiea gigantea) provide for a large proportion of the diets of many insectivorous, granivorous and frugivorous avian species in the Sonoran Desert. This thesis has demonstrated that stable isotopes analysis can be extremely useful in the investigation of the nutritional ecology of mistletoebirds, and with a more complete isotopic sample of available dietary sources, this approach can be utilised to investigate the nutritional importance of mistletoe fruit and nectars to a wide range of bird species.

While this thesis makes some progress, the mechanisms of xylose absorption in birds are still unclear. It appears that mistletoebirds may absorb D-xylose by both mediated and paracellular mechanisms of absorption; further research to confirm the mechanisms of absorption and potential catabolism of xylose by intestinal microbes or systemic catabolism in birds would be advantageous.

7.5 CONCLUSIONS

This thesis presents new insight into the parasitic relationship between mistletoes and hosts, and the relationship between mistletoes and its avian consumers. This thesis presents the first data on mistletoe fruiting and flowering phenology and the influence of mistletoe on bird communities in south-west WA, and demonstrates differences in the mechanisms of sugar absorption by a mistletoe specialist, the mistletoebird. This

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thesis also provides conclusive evidence that intestinal sucrase activity is likely to contribute to diet selectivity in frugivorous and nectarivorous bird species.

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