Seasonal physiological responses in the Cape Rockjumper

(Chaetops frenatus): a Fynbos endemic bird shows limited capacity to deal with

temperature extremes.

By

Krista Natasha Oswald

Submitted in fulfilment of the requirements for the degree of

Masters of Science in the

Faculty of Science at

Nelson Mandela Metropolitan University

Port Elizabeth

Date of submission: August 5th 2016

Supervisor: Dr. Ben Smit Co-Supervisor: Dr. Alan T K Lee Krista Oswald Dissertation Introduction

Preface

The experimental work described in this dissertation was carried out at Blue Hill Nature Reserve, Western Cape, South Africa, between July 2015 and January 2016. The study was conducted under the supervision of Dr. Ben Smit, and co-supervision of Dr. Alan Lee.

This dissertation contains original work by the author and none of its contents have been submitted in any other form for any other degree or diploma to any other institution.

The Research Ethics Community (Animal) at Nelson Mandela Metropolitan University approved all procedures related to the use of animals in this work under document A15-SCI-ZOO-007.

……………………………………….

Krista N Oswald

Port Elizabeth

8 November 2016

i Krista Oswald Dissertation Introduction

Table of Contents

Table of Contents ………………………………………………...………………………...…. ii

Acknowledgments ………………………………………………..……………………...…… iv

Summary ………………………………………………………………..….…………...……… vi

List of Abbreviations …………..………………………………………………...………….viii

Chapter 1: General Introduction …………..………………………...………...………….. 1 Physiological mechanisms ………………………...……………..…...... 1

Mechanisms for coping with cold climates ...... 2

Mechanisms for coping with Tair above Tb ……………….………………...………….... 4

Cape Rockjumper: a Fynbos-endemic as a model species ……………………....…….. 5

References ………….…………………………………………………...…...……...…….. 7

Chapter 2: Seasonal cold tolerance responses in an alpine-restricted bird, the Cape Rockjumper (Chaetops frenatus): maximum thermogenic capacity increases despite maintenance metabolic decreases in winter Abstract ………………………………………………………………………….………. 13

Introduction ………………………………………………………………….....……….. 14

Methods …………………………………………………………………..……………… 18

ii Krista Oswald Dissertation Introduction

Results …………………………………………………………………...…………...…... 25

Discussion ……………………………………………………………..….………...……. 36

References ………………………………………………………………….……………. 44

Appendix ………………………………………………………………….....…………... 50

Chapter 3: Seasonal physiological responses show low heat tolerance thresholds in the Cape Rockjumper (Chaetops frenatus) Abstract …………………………………………………………….….……..………..… 52 Introduction ………………………………………………..…….…………………..….. 53

Methods ……………………………………………………..….………….…………..… 57

Results ……………………………………………………..….…………………..……... 61

Discussion ………………………………………………………………………….……. 74

References ………………………………………………...……………………..………. 82

Chapter 4: General Conclusion …………………………………...…...…………...…….. 88

References ……………………………………………………...……………….……….. 90

iii Krista Oswald Dissertation Introduction

Acknowledgments

I thank Nelson Mandela Metropolitan University and the NRF of South Africa for providing funding for this study. I would also like to thank the Lee family, the owners of Blue Hill Nature

Reserve, where the fieldwork for this study was conducted. I am grateful to them, not only for allowing the study to occur, but also for providing accommodation (for myself and my assistants) at the cost of a song and a dance.

For dealing with my flowery, verbose, and adverb-heavy writing, and attempting to turn it into something succinct and resembling of scientific writing, I would like to thank Dr. Ben Smit. Ben, you forced me to remove at least half of my semi-colons, of which I am so proud, and replace them with short and to-the-point sentences (I hope). You took an ornery student and painstakingly taught her about physiology, dealing with frantic phone calls that, although initially occurring on a daily basis, were hopefully far less often as the season continued. I would like to thank Ben sincerely for his attention to detail, and for encouraging me in every endeavour, even when the tasks and deadlines seemed insurmountable. My co-supervisor, Dr. Alan Lee was present throughout the fieldwork, and although he intends on remaining proudly ignorant of the physiology, taught me more about rockjumpers in my first year than most people will ever learn.

He made me feel at home on the family farm, allowing me free access to the entire 2000 ha of rockjumper-friendly terrain, and was always up for (and generally the instigator of) early morning capture sessions. I am sincerely grateful for everything you two taught me, and look forward to our future undertakings.

iv Krista Oswald Dissertation Introduction

I would also like to thank my field assistants, who spent many long, long, long, hours out trekking in territory that only a bird or klipspringer would comfortably navigate. Namely, my winter field assistants: Audrey Miller (sparrow-weaver), Alacia Welch (rogue), and Gavin Emmons (rock kestrel), who were so surprisingly capable that Alan (bald eagle) felt comfortable having us go out into the field without him (sure praise, indeed!), and my summer assistants: Audrey Miller (she strikes again!), Jenny Tartini (blue jay), and Christina Ebneter (scrub hare). I would also like to thank my colleagues Nick Pattinson (ocelot) and Cuen Muller (red-finned minnow), for helping me wrap up my winter session. From honey badger to all of you — thank you!

Lastly, but arguably most importantly, I would like to thank my husband, Matthew Dunn (northern cardinal). You’ve been by my side for the past 8 years, and encouraged me to pursue my dreams, even to the point of your own (and my) sadness when we spend so much time on different sides of the ocean. I wouldn’t be here if it wasn’t for you, and I appreciate you every day—even the days when I make you come out into the field with me to take notes and try to not get sunburned. I am also grateful to my mom, who insists she is proud of me, despite my not pursuing a career in public relations.

v Krista Oswald Dissertation Chapter 1: General Introduction

Summary

The Fynbos biome in south-western South Africa is a global biodiversity hotspot vulnerable to climate change. Of the six Fynbos-endemic passerines, Cape Rockjumpers (hereafter Rockjumpers;

Chaetops frenatus) are most vulnerable to increases in temperature, with population declines correlated with warming, and low physiological heat thresholds. Rockjumper’s preferred mountain habitat is predicted to decrease as they lack opportunity to move to cooler regions as temperatures warm. As Rockjumpers currently occupy the coldest regions of the Fynbos, I hypothesized their thermal physiology would show cold adaptation at the expense of lowered ability to cope with higher temperatures. I aimed to determine the seasonal 1) maintenance metabolism and cold tolerance, and 2) thermoregulatory responses to high temperatures of Rockjumpers. I measured seasonal maintenance metabolic rate, thermal conductance, and maximum thermogenic capacity. I also measured seasonal resting metabolic rate, evaporative water loss, evaporative cooling efficiency, and body temperature at high air temperatures.

In winter, Rockjumpers had higher maximum thermogenic capacity, lower maintenance metabolic rate, and lower thermal conductance. Lower maintenance metabolic rates (and thus, lower metabolic heat production) combined with the decreased thermal conductance, confers substantial energy savings in winter. The increased winter maximum thermogenic capacity of Rockjumpers was expected, although the mean seasonal values fell below those expected for a ~ 50 g bird using a global data set, suggesting Rockjumpers are not especially cold tolerant. I further show that in summer Rockjumpers had higher elevations in resting metabolic rates, evaporative water loss, and body temperature, denoting higher rates of heat production and lower heat thresholds in summer compared to winter. My results suggest that Rockjumpers are best suited for relatively mild

vi Krista Oswald Dissertation Chapter 1: General Introduction

temperatures. While I found further support for a physiological basis for declining Rockjumper populations, further studies on other mechanisms Rockjumpers may possess to cope with climate warming (e.g. behavioural adjustments) are needed in order to truly understand their vulnerability to climate change.

Key Words: Cape Rockjumper, cold tolerance, heat thresholds, climate change, Fynbos, seasonal acclimatization, basal metabolic rate, summit metabolism, evaporative water loss

vii Krista Oswald Dissertation Chapter 1: General Introduction

List of Abbreviations and Symbols

EHL - evaporative heat loss EWL - evaporative water loss BMR – maintenance metabolic rate C – thermal conductance

CBMR – thermal conductance at BMR

[CO2] - concentration of CO2 EHL/MHP – evaporative cooling efficiency LM – linear model LME – linear mixed-effects model

Mb - body mass

ME – metabolic expansibility, Msum/BMR MHP - metabolic heat production MR – metabolic rate

Msum – maximum thermogenic capacity

[O2] - concentration of O2 RMR - resting metabolic rate

RQ – repiratory quotient, [CO2]/[O2]

Tair - air temperature

Tb - body temperature

Tcl – cold limit temperature

TEWL – Tair of EWL inflection

Tlc – lower critical limit of thermoneutrality TNZ – thermoneutral zone

VCO2 - rate of CO2 emission

VO2 - rate of O2 consumption

viii Krista Oswald Dissertation Chapter 1: General Introduction

Chapter 1: General Introduction

Mediterranean-type biomes are considered a global conservation priority due to their vulnerability to anthropogenic threats and climate warming (Lee and Barnard 2015). Of all terrestrial biomes,

Mediterranean biomes are projected to have the largest proportional loss of biodiversity by the year

2100 (Klausmeyer and Shaw 2009; Sala et al. 2000; Underwood et al. 2009). The south-western tip of South Africa contains the Cape Floristic Kingdom, a Mediterranean-type biome also referred to as the Fynbos biome (hereafter Fynbos). Even though most Fynbos habitat loss to date has occurred from conversion to agriculture and urbanisation in lowland regions (Gillson et al. 2013), increasing temperatures linked to climate change may become the leading cause of biodiversity loss over the next few decades (Kruger and Sekele 2013). While some species may already possess the ability to adjust both their physiology and behaviour to existing temperature fluctuations

(Dupoué et al. 2015), they may also need the ability to adjust to the more extreme or short-term temperature fluctuations expected to occur with climate warming. Especially for those species that cannot relocate to more favourable habitats, adaptive responses will be required for them to persist under increasing temperatures through genetic changes (i.e. genetic adaptation across generations), and physiological or behavioural plasticity (i.e. acclimatization by individuals to temperature changes occurring within days or weeks; Bellard et al. 2012; Boyles et al. 2011; Chevin et al. 2010;

Hofmann and Todgham 2010; Sinervo et al. 2010; Underwood et al. 2009).

Physiological Mechanisms

While ectotherms derive their heat primarily from the environment (i.e. behavioural mechanisms), endotherms derive their heat primarily from metabolism (i.e. physiological mechanisms; Weather and van Riper III 1982; Huey et al. 2012; Tattersall et al. 2012; Nilson et al. 2016). The

1 Krista Oswald Dissertation Chapter 1: General Introduction

endothermic strategy for thermoregulation evolved by mammals and birds involves using metabolism to maintain body temperature (Tb) in air temperatures (Tair) outside their thermoneutral zone (TNZ; the range of Tair in which an organism experiences the lowest resting metabolic rates

(RMRs) with correspondingly lowest minimum metabolic heat production (MHP)). Endothermic maintenance of Tb is generally accomplished by adjusting metabolic heat production and rate of heat loss. Birds provide an excellent group for studying physiological mechanisms in endotherms as they have relatively high metabolic rates and body temperatures, and so require more energy than mammals. In general, birds at Tair below Tb maintain Tb by shivering and increasing their

MHP (Tattersall et al. 2012), while birds at Tair above Tb maintain Tb by panting or gular fluttering to increase respiratory evaporative water loss, or by increasing cutaneous evaporative water loss, increasing their evaporative heat loss (EHL; Wolf and Walsberg 1996; Wolf 2000). These mechanisms are costly, as increasing MHP consumes energy, and increasing EHL requires increased water demands. One principal method for examining how birds will adjust their physiology to different temperatures is through seasonal acclimatization studies (Van de Ven et al.

2013), which can show phenotypically flexible responses to temperatures above and below TNZ.

Phenotypic flexibility is the temporary and reversible adjustment that can be used to alter behaviour, morphology, or physiology (Piersma and Drent 2003).

Mechanisms for coping with cold climate

Although it seems intuitive that birds inhabiting cooler environments would have an increased maintenance metabolic rate (BMR), which would aid in maintaining Tb in colder Tair by increasing

MHP, this is not always the case. Increasing MHP is energetically demanding, and may require energy normally allocated to other critical functions such as growth and reproduction (Hofmann and Todgham 2010), and the direction of seasonal change in subtropical avian BMR has been

2 Krista Oswald Dissertation Chapter 1: General Introduction

equivocal. While temperate species tend to increase BMR in winter (Cooper 2002; Cooper and

Swanson 1994; Liknes and Swanson 1996; Swanson and Liknes 2006), subtropical species often lower their BMR in winter, although there is considerably more variation at lower latitudes

(McKechnie et al. 2015). Whereas some subtropical species typically decrease their BMR in winter

(Smit and McKechnie 2010; Van de Ven et al. 2013), others maintain a seasonally stable BMR

(Lindsay et al. 2009a, b; Noakes and McKechnie unpublished data) and still others increase their

BMR in winter (Moldonado et al. 2009; Smit and McKechnie 2010). The ambiguity of BMR seasonal change may be because increasing BMR in winter is costly and requires greater energy demands to elevate MHP; adjusting insulation (i.e. lowering thermal conductance), may allow birds may be able to lower energy requirements by decreasing BMR in winter (Wu et al. 2015).

In colder Tair birds generally show a higher maximum thermogenic capacity (Msum; the maximum metabolic rate by an animal at rest). The positive relationship between Msum and cold tolerance in birds suggests that Msum may be an important, and possibly prerequisite, indicator of cold acclimatization (McKechnie et al. 2015; Swanson 2010; Swanson and Garland Jr 2009). The consequence of occupying cold climates, associated with scarce food availability, is a reduction in energy required to raise Tb via metabolic heat production (Tattersall et al. 2012). In winter, the increased energy requirements of an elevated BMR require high rates of energy consumption, leading to a mismatch in energy supply and demand as winter often has decreased access to food and fewer daylight hours for foraging (Lima 1987, 1988),. Maximum thermogenic capacity was shown to be strongly influenced by both short-term temperature fluctuations (Swanson and

Olmstead 1999) and site-specific minimum temperatures (Petit and Vézina 2014), suggesting in cold Tair increasing Msum may be an alternative to increasing BMR.

3 Krista Oswald Dissertation Chapter 1: General Introduction

Mechanisms for Coping With Tair above Tb

At Tair above TNZ, increasing evaporative cooling mechanisms (e.g. gaping or panting) has been well documented as a physiological method to help avoid hyperthermia in birds (Battley et al.

2003; Huey et al. 2012; Wolf 2000). Increasing evaporative cooling often necessitates a trade-off between hyperthermia and dehydration due to high rates of evaporative water loss (EWL) that increase with increasing Tair (Boyles et al. 2011; Dupoué et al. 2015; McKechnie and Wolf 2004;

Smit et al. 2013; Tattersall et al. 2012; Whitfield et al. 2015; Wolf 2000; Wolf and Walsberg

1996). For example, a study by Wolf and Walsberg (1996) on Desert Verdins (Auriparus flaviceps) showed modest water loss through both respiratory and cutaneous components between

30 and 36 °C (6.47 mg g-1 hr-1), with rates increasing significantly between 38 and 50 °C (176.93 mg g-1 hr-1). Elevated EWL demands are especially costly in arid or semi-arid areas where water is scarce and summers are warm and dry (du Plessis et al. 2012), with strong selection for adjustments that minimize energy expenditure or water loss (Williams and Tieleman 2005).

Overall water requirements are predicted to be much higher in future decades compared with current values (McKechnie and Wolf 2009). Under high Tair we may then expect arid-zone species to depend on mechanisms other than EWL, such as a higher normothermic Tb (average resting body temperature) or facultative hyperthermia, which can reduce EWL demands (Smit et al. 2013).

However, Williams and Tieleman (1999) compared water loss and hyperthermia among different sizes of bird (~ 10 g, ~100 g, and ~ 1000 g), and found larger birds had significantly increased water loss when hyperthermic compared to normothermic after five hours of hyperthermia.

While it is important to note that the link between short-term metabolic responses (i.e. physiological acclimatization) and long-term evolutionary responses (i.e. genetic adaptation) to climate change is as yet unclear, seasonal studies remain an important tool in determining how

4 Krista Oswald Dissertation Chapter 1: General Introduction

species may handle temperature variation (Mckechnie and Swanson 2010). Additionally, high- altitude species that experience generally low annual temperatures may have abandoned the ability to acclimatize physiologically to high summer temperatures in favour of increasing their cold tolerance (Cooper 2002; Mugaas and King 1981; Weathers and van Riper III 1982). For example, cool climate birds such as Black-billed Magpies (Pica pica; Mugaas and King 1981) and Mountain

Chickadees (Poecile gambeli; Cooper 2002) have relatively high BMR as they occupy habitats where Tair is often below their TNZ, and therefore are limited in distribution to regions with cooler temperatures. Indeed, high altitude communities are most at risk from climate change due to their dependency on cooler mountain slopes (Hijmans and Graham 2006; Hill et al. 2011; Parmesan

2006; Simmons et al. 2004; Visinoni et al. 2015), as well as the possibility of lower altitude species expanding upwards as environments become more suitable (Sinervo et al. 2010).

Cape Rockjumper: a Fynbos-endemic as a model species

Of the six Fynbos-endemic passerine species, the Cape Rockjumper (Rockjumper; Chaetops frenatus) may be the most adversely affected by increasing temperature (Lee and Barnard 2015;

Simmons et al. 2004), and so serves as an excellent study species to examine thermal physiology thresholds. Although currently not listed as threatened by the IUCN, in 2004 they were ranked among the top three Fynbos specialists most susceptible to climate change in South Africa

(Simmons et al. 2004). In addition, Rockjumpers were recently upgraded to locally “near threatened” by BirdLife South Africa (BirdLife 2015) due to their relatively small climatic space and low population density (Lee and Barnard 2015), position as a global outlier in terms of heat thresholds, and evidence of population decline linked to increases in temperature (Milne et al.

2015). Moreover, the Rockjumper’s range is likely over-estimated as they are restricted to high- altitude rocky Fynbos in the Cape Fold Mountains (Holmes et al. 2002; Lee and Barnard 2015) and

5 Krista Oswald Dissertation Chapter 1: General Introduction

therefore do not have any opportunity of moving to cooler areas either further south or at higher altitudes (Huntley and Barnard 2012; Parmesan 2006; Simmons et al. 2004; Walther et al. 2002).

A study of pollen-bearing sedimentary fossils from the Cederberg mountain range (a Rockjumper habitat stronghold) showed evidence of surprising climatic stability, relevant on an evolutionary timescale (Cowling and Lombard 2002; Cowling et al. 2015; Meadows and Sugden 1993).

Historic climate stability and a lack of extreme seasonal variation may suggest Rockjumpers have not adapted significant physiological flexibility to accommodate for predicted seasonal temperature extremes, as currently Rockjumper occurrence is limited by both mean annual temperature and the mean temperature of the warmest quarter of the year (Lee and Barnard 2015). Data on physiological flexibility will be a useful model for predicting the effects of increased temperatures on similarly range-restricted species in order to predict their future ranges and the future distribution of biodiversity (Hill et al. 2011). This is especially important in currently declining populations such as the Rockjumper, which has experienced reduction in both relative abundance and range size over the past two decades (Milne et al. 2015).

This dissertation contains a general introduction and conclusion, as well as two stand-alone data chapters written in manuscript style. Because of this, there is inevitably some overlap between the two data chapters.

In Chapter 2, I determine seasonal cold tolerance in wild-living Rockjumpers. I did this by recording Tb and resting metabolic rates (RMRs) over a range of Tair to determine the TNZ, as well as BMR, and Msum.

6 Krista Oswald Dissertation Chapter 1: General Introduction

In Chapter 3, I determine seasonal heat thresholds by collecting evaporative cooling capacity data in wild-living Rockjumpers. I did this by collecting Tb, RMR, and EWL data over a range of increasing Tair. I then converted EWL to evaporative heat loss (EHL) and RMR to MHP to determine evaporative cooling efficiency (EHL/MHP).

In Chapter 4, I discuss my general conclusions and proposed areas for future research.

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Weathers, W.W., and C. van Riper III. 1982. Temperature regulation in two endangered Hawaiian honeycreepers: the Palila (Psittirostra bailleui) and the Laysan Finch (Psittirostra cantans). Auk: 667-674.

Whitfield, M.C., B. Smit, A.E. McKechnie and B.O. Wolf. 2015. Avian thermoregulation in the heat: scaling of heat tolerance and evaporative cooling capacity in three southern African arid-zone

11 Krista Oswald Dissertation Chapter 1: General Introduction passerines. J Exp Biol 218: 1705-1714.

Williams, J. B., and B. I. Tieleman. 2000. Flexibility in basal metabolic rate and evaporative water loss among hoopoe larks exposed to different environmental temperatures. J Exp Biol 203: 3153- 3159.

Williams, J.B., and B.I. Tieleman. 2005. Physiological adaptation in desert birds. Bioscience 55: 416-425.

Wolf, B. 2000. Global warming and avian occupancy of hot deserts; a physiological and behavioral perspective. Rev Chil Hist Nat 73: 395-400.

Wolf, B., and G. Walsberg. 1996. Respiratory and cutaneous evaporative water loss at high environmental temperatures in a small bird. J Exp Biol 199: 451-457.

Wu, M. X., L. M. Zhou, L. D. Zhao, Z. J. Zhao, W. H. Zheng and J. S. Liu. 2015. Seasonal variation in body mass, body temperature and thermogenesis in the Hwamei, Garrulax canorus. Comp Biochem Physiol A Mol Integr Physiol 179: 113-119.

12 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Chapter 2: Seasonal cold tolerance responses in an alpine-restricted bird, the Cape Rockjumper (Chaetops frenatus): maximum thermogenic capacity increases despite maintenance metabolic decreases in winter.

Abstract

For many birds, maintaining body temperature at air temperatures below their thermoneutral zone is energetically costly. Seasonal physiological flexibility studies have shown that while some species increase their maintenance metabolic rate (BMR) in winter, some decrease BMR.

However, increasing maximum thermogenic capacity (Msum) in winter has been correlated with improved cold tolerance. I examined seasonal cold responses in the Cape Rockjumper (“Rockjumper”; Chaetops frenatus), a range-restricted Fynbos-endemic bird. I hypothesised that, given their high-altitude habitat preference, Rockjumpers were physiologically specialised for cooler temperatures. I collected body mass (Mb), BMR, Msum, and thermal conductance at BMR

(CBMR) in wild-living Rockjumpers during winter and summer (n = 13 winter, 5 females and 8 males; n = 12 summer, 6 females and 6 males). I found Rockjumpers had decreased BMR and

CBMR, and increased Msum and Mb, in winter compared to summer. My findings of BMR, CBMR, and

Mb indicate Rockjumpers may conserve energy in winter using increased insulation, while increased Msum in winter suggests Rockjumpers are able to produce more metabolic heat during cold fronts. When added to a global data set, Rockjumper Msum was below average, indicating Rockjumpers are not especially cold adapted, but may be well-suited to relatively mild temperatures in the Fynbos biome, and so vulnerable to climate warming.

13 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Introduction

Endotherms such as birds and mammals need to allocate a large component of their maintenance energy budget toward heat production at cold temperatures. In particular, birds are an ideal taxon for studying mechanisms involved in maintaining normothermic body temperature (Tb) in cooler temperatures due to their relatively high Tb compared to mammals. While within the thermoneutral zone (TNZ; the range of temperatures with minimum metabolic heat production) body temperature can be maintained using only heat produced from their maintenance metabolic rate (BMR), below

TNZ maintaining Tb may require an increase in metabolic heat production (MHP; Swanson 1991;

Swanson and Weinacht 1997; Angiletta et al. 2010, Smit and McKechnie 2010, Boyles et al. 2011).

Increased MHP may be a pre-requisite for species exposed to generally low annual temperatures, such as high-altitude species (Cooper 2002; Mugaas and King 1981; Weathers and van Riper III

1982). For example, alpine birds such as Mountain Chickadees (Poecile gambeli) and Juniper

Titmouses (Baelophus griseus; Cooper 2002), and non-alpine—albeit cool-climate inhabiting—

Black-billed Magpies (Pica pica; Mugaas and King 1981), all have a relatively high BMR.

However, for some species increasing BMR to maintain Tb at low Tair may not be an option, as the physiological mechanisms required often divert energy allocation away from other critical functions such as growth and reproduction (Hofmann and Todgham 2010). In these cases, species inhabiting cooler environments may use other mechanisms (e.g. increased insulation and lower thermal conductance) to conserve metabolically produced heat while maintaining Tb in cooler weather (Marsh and Dawson 1989; Williams and Tieleman 2000; Wu et al. 2015).

Cold regions often have scarce food ability for birds, which may require birds inhabiting regions or niches that experience colder temperatures to show less energy demanding mechanisms than increasing MHP to maintain Tb (Tattersall et al. 2012). If a bird is sufficiently insulated, and can

14 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

maintain a stable Tb with a relatively low BMR, they may be able to conserve energy reserves for colder events. Increased insulation allows for decreased food requirements and environmental heat loss, and is supported as a mechanism for maintaining Tb in colder Tair by the negative correlation found between BMR and thermal conductance (Marschall and Prinzinger 1991). For example, increased insulation due to increased feather mass, resulting in lower thermal conductance, was suggested as why Superb Fairy-wrens (Malurus cyaneus) decreased metabolic rates in winter as compared to summer (Lill et al. 2006). Alternatively, it may be that in environments with less variation in Tair among seasons, a change in BMR is not necessary or optimal compared to an increased maximum thermogenic capacity (Msum; the maximum rate of metabolic thermogenesis by a bird at rest). The relationship between Msum and cold tolerance in birds suggests that Msum may be an important (and possibly prerequisite) indicator of cold adaptation (McKechnie 2015; Swanson

2010; Swanson and Garland Jr 2009; Swanson and Liknes 2006).

As Msum and BMR are not fixed within individuals, they may be adjusted depending on environmental cues (Minnaar et al. 2014). Metabolic expansibility (ME; the ratio of Msum to BMR) has been suggested as an indicator of individual or species physiological flexibility (Minnaar et al.

2014; Swanson and Garland Jr 2009; Van de Ven et al. 2013a). For those species that regulate a wide range of Tb on a short-term basis, such as inhabiting region with wide ranges of Tair or lowering Tb during torpor, a large ME value may be expected from a decreased BMR, and an increased Msum used as an aid in re-warming (Minnaar et al. 2014).

Currently, our understanding of seasonal physiological variation is still biased toward high latitude species, with less data from subtropical species (McKechnie 2008; Smit and McKechnie 2010; Van de Ven et al. 2013b). Emerging patterns from subtropical regions are showing a more diverse range of species-specific, or even population-specific, avian responses in BMR and Msum. While multiple

15 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

subtropical species typically decrease their BMR in winter (McKechnie et al. 2015; Smit and

McKechnie 2010), a few species such as Southern Red Bishops (Euplectes orix) showed site- specificity in regards to whether BMR increased or decreased in winter (Van de Ven et al. 2013a).

Among temperate species such as the Black-capped Chickadee (Parus atricapillus), seasonal difference in the directional change of BMR also showed site-specificity; while no populations decreased BMR in winter, some populations showed no seasonal change in BMR (Chaplin 1976;

Rising and Hudson 1974), and others showed increased BMR in winter compared to summer

(Cooper and Swanson 1994; Dawson et al. 1983a; Sharbaugh 2001). The equivocal or contradictory results between regions and species highlight the deficiency of information regarding the relationship between BMR, Msum, and cold tolerance. As most studies have occurred in either cold-temperate northern species, or warm-subtropical southern species, more data from species inhabiting cold regions at low latitudes (i.e. < 40 °) would improve our understanding of why this variation exists.

In the present study I aim to study cold tolerance in an endemic species of the Cape Fold

Mountains within the Fynbos biome (“the Fynbos”) of South Africa. The Fynbos is a

Mediterranean biome consisting of a relatively narrow range of annual temperature variation with cool wet winters and warm dry summers (Cowling et al. 1996). As a region, the Fynbos provides an interesting model habitat; it is at a subtropical latitude with precipitation occurring mainly during the Austral winter, and primary productivity is often centered around the coldest times of the year (Cowling 1992). Although long term climate data from the upper peaks of the Cape Fold

Mountains are limited, it has been suggested that the Fynbos has historically been extremely mild and stable in its Tair variation (Cowling and Lombard 2002; Cowling et al. 2004).

16 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

The Cape Rockjumper (Chaetops frenatus; “Rockjumper”) serves as a highly suitable study species for examining seasonal physiological flexibility. Rockjumpers’ preferred habitat is the high- altitude regions of the Cape Fold Mountains (Lee and Barnard 2015b) that have generally mild annual Tair, with cool productive winters and warm dry summers (Cowling and Lombard 2002;

Péron and Altwegg 2015). Despite generally mild annual Tair, Fynbos endemics occur mainly in regions that are on average cooler than the mean annual Fynbos temperatures (Huntley and Barnard

2012; Milne et al. 2015). This may partially explain why a previous study found Rockjumpers were a global outlier in terms of their low heat tolerance thresholds (Milne et al. 2015). Rockjumpers are a useful study species because they are a lower latitude subtropical species in a cooler climate, and so may be the link between past subtropical and temperate studies.

In this study I determine seasonal responses in maintenance metabolic rate (BMR) and cold tolerance (Msum) in the Rockjumper. I hypothesised the high altitude habitat preference of the

Rockjumper led to a physiological specialisation for colder climates, similar to patterns observed in

Mountain Chickadees and Black-billed Magpies. I predicted that physiological responses of the

Rockjumper would be centred on improved cold tolerance, shown by elevated BMR and Msum, and lowered thermal conductance and cold limit temperature (Tcl; the Tair at which Msum is reached) in winter compared to summer.

17 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Methods

Study Site and Bird Capture

This study took place between July 2015 and February 2016 at Blue Hill Nature Reserve (BHNR;

33.59 S; 23.41 E; 2 230 ha; 1 000 – 1 530 m, above sea level) in Western Cape Province, South

Africa. BHNR has vegetation consisting mainly of Fynbos, with mixed thicket in the lower valleys.

The specific territories occupied by Rockjumpers ranged from ~1 100 – 1 500 m above sea level, and consisted solely of Fynbos. BHNR is home to an estimated 30 Rockjumper family groups

(A.T.K. Lee, personal observations), each comprising two to five individuals of varying sex and age composition, generally with a single breeding pair. Rainfall and temperature (Tair) data were collected every 30 minutes using an on-site weather station (Vantage Vue, Davis Instruments

Corp., California USA). The mean annual rainfall in 2015 was 496.2 mm, while average daily Tair from two weeks before the first bird was captured until the day the last bird was captured was 8.71

°C ± 6.00 in winter (July 24th to August 31st 2015) while in summer it was 20.54 °C ± 5.94

st st (January 1 to January 31 2016. Minima and maxima Tair experienced over the study period were

-2.6 °C to 27.5 °C in winter, and 6.9 °C to 35.4 °C in summer.

I collected physiological data during the winter months of July and August 2015 as well as the summer month of January 2016. Data were collected on wild-living adult Rockjumpers captured using mist-nets and spring traps baited with mealworms. After capture, birds were held in cloth bags and then transported to a field laboratory station set up at BHNR. Physiological measurements were obtained on the day of capture (day one) as well as the day after capture (day two).

Physiological data collected comprised body temperature (Tb), and oxygen consumption (VO2) and

carbon dioxide production (VCO2) to determine maintenance metabolic rate (BMR) and maximum thermogenic capacity (Msum). BMR and Msum are standard metabolic measurements, and are known

18 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

indicators of a species cold tolerance (Swanson 1991, 1993, 2001; Swanson and Liknes 2006;

Mckechnie and Swanson 2010; Smit and McKechnie 2010; Swanson and Bozinovic 2011).

During July and August 2015 (subsequently “winter”) I captured 17 individuals comprised of nine males and eight females. To obtain a reliable estimate of BMR metabolic rate must be measured within the thermoneutral zone (TNZ). I estimated the TNZ by subjecting the first five individuals to five Tair between 15 and 30 °C for a minimum of two hours at each Tair, and performed a visual inspection of the metabolic rate profiles to look for elevations in resting metabolic rate (RMR) indicative of lower critical temperature (Tlc) of thermoneutrality. I obtained BMR estimates from

13 individuals (eight males and five females), and Msum from 12 individuals (eight males and four females) during winter. Maintenance metabolic rate and Msum were also collected from four additional individuals (one male and three females) who showed clear physical signs of breeding

(i.e. well-developed brood patches; henceforth referred to as breeders). Rockjumpers breed anytime between August and January (Holmes et al. 2002).

During January 2016 (subsequently “summer”) I captured 17 individuals comprised of six males, six females, and five immature. I followed the same procedure described above to ensure BMR measurements were obtained within TNZ, by again subjecting the first five individuals to Tair between 15 and 30 °C for a minimum of two hours at each Tair. I obtained BMR and Msum estimates from 12 additional individuals (six males and six females) during summer. The majority of data represent different individuals between seasons, although four individuals were re-captured (two males and two females). Between experimental stages birds were held in cloth-covered birdcages

(length x width x height: 50 x 30 x 50 cm) in a dark and quiet room to rest, with mealworms provided ad libitum. Each bird was not held for longer than 48 h, after which they were released at

19 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

the site of capture.

Body Temperature Measurements

Individual birds were injected with a small, temperature-sensitive, Passive Integrated Transponder

(PIT) tag intra-peritoneally (Ratnayake et al. 2014) to measure Tb throughout experimentation. PIT tags transmit large quantities of accurate and reliable Tb data to a PIT-tag reader, with Tb then uploaded into an Excel spreadsheet. PIT tags provide Tb information while minimising handler effects, with no significant alteration of individual condition (Gerson et al. 2014; Ratnayake et al.

2014), and no significant negative effects in wild bird populations (Ratnayake et al. 2014).

Metabolic Measurements

Body mass (Mb) was measured to within 0.1 g before and after each experimental procedure and all birds maintaining mass of < 5 % capture Mb. Birds were placed individually in a 4-L chamber constructed from airtight plastic (Lock & Lock, Mumbai, India) fitted with a wire-mesh platform raised 15 cm to ensure normal perching posture. A thin layer of mineral oil was placed at the base of the chamber to cover any faeces, ensuring faecal water did not factor into evaporative water loss

(EWL) measurements. Chamber temperature (Tair) was measured using a thermistor probe (model

TC100, Sable Systems, Las Vegas, NV, USA) inserted one cm into bird chambers through a small hole in the lid.

Metabolic rates (MRs) were measured indirectly as oxygen consumption (VO2) and carbon dioxide

-1 emission (VCO2) in mL min using a portable open-flow respirometry system. For all measurements, I controlled flow rate of either atmospheric air for RMR and BMR measurements, or Helox (21 % Oxygen, balance Helium; Afrox, Johannesburg, South Africa) for Msum

20 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

measurements. Air was pushed through bird chambers using FMA-series mass flow controllers

(Omega, Bridgeport, NJ USA) calibrated using a 1-L soap bubble metre (Baker and Pouchot 1983).

-1 Flow rates of between 1.5 – 3 L min were selected to ensure [O2] to the chamber remained within

0.5% of incurrent [O2]. Subsampled air was then pulled from the bird chamber through an O2 and

CO2 analyser (Foxbox-C Field Gas Analysis System, Sable Systems, Las Vegas, Nevada USA).

The Foxbox included a subsampling pump, and allowed for digital outputs to be recorded using

Expedata Data Acquisition and Analysis Software (Sable Systems, Las Vegas, Nevada USA).

For RMR and BMR measurements, I placed the respirometry chambers in a custom-made environmental chamber consisting of a100-L cooler box lined with copper tubing through which temperature-controlled water was pumped by a circulating water bath (FRB22D, Lasec, Cape

Town, South Africa; Smit and McKechnie 2010). A small fan was placed inside the environmental chamber to ensure a uniform distribution of air temperature. An infrared light source and closed circuit security camera with live video feed placed within the environmental chamber allowed for continuous monitoring of the bird. A BioMark PIT tag reader was placed next to the chamber to monitor and record Tb every minute (Gerson et al. 2014; Whitfield et al. 2015).

For Msum bird measurements I placed respirometry chamber in a 47 L Portable ARB Fridge Freezer

(ARB 4x4 Accessories, Melbourne AUS) with Tair controlled using the digital display. Birds were constantly monitored by placing a small red light source and a GoPro Hero 3+ Silver Edition

(GoPro Inc. USA) within the ARB chamber with constant streaming using the “preview” option on the GoPro App for smartphones (GoPro Inc., USA). A BioMark PIT-tag reader was placed near the

ARB chamber to check Tb when hypothermia was suspected due to a steady maximum VCO2 reading on the monitor (see Msum Protocol below).

21 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

After all experimental runs birds were weighed and returned to holding cages and provided with mealworms ad libitum.

Maintenance Metabolic Rate Protocol

Food was withheld from birds from 16:00 h (UTC + 2 h) to ensure birds were post-absorptive during measurements (Smit and McKechnie 2010; Van de Ven et al. 2013b). At 19:00 h (UTC + 2 h) in winter and 20:00 h (UTC + 2 h) in summer birds were weighed and placed in individual chambers that were then placed within the cooler box.

I established TNZ by subjecting five post-absorptive birds overnight during their rest-phase to a series of Tair (~ 15, 20, 25, 30 °C from 19:00 h to 7:00 h in winter; ~ 18, 23, 28, and 33 °C from

20:00 h to 6:00 h in summer). Each bird spent between 2 and 2.5 h at each Tair overnight in ascending order for all five birds during winter and for the first three birds during summer. The last two birds in summer were done at ~ 28 and 33, and then ~18 and 23 °C as the Tair of the cooler box could not be brought below 23 °C, leaving the ~ 18 and 23 °C measurements to be done in the

ARB. A baseline was recorded for a minimum 15 min at the beginning and end of each Tair interval. Birds did not spend more than 12 h consecutively in the respirometry chamber. BMR was measured by placing individual birds at Tair = 26 - 28 °C for at least 2 h, with at least 15 min of baseline data recorded at the beginning and end of each run. Although a clear Tlc for thermoneutrality was not found (see Appendix: Figure 9), I am confident that BMR measurements at Tair = 26 and 28 °C were within TNZ and thus representative of thermoneutrality as average

TNZ for birds is between 24 and 35 °C (Araújo et al. 2013).

22 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Thermal conductance (C) was calculated using the equation C = MR / Tb – Tair using MR values measured during BMR, and representing thermal conductance of birds at BMR (CBMR). Although minimum thermal conductance (Cmin) would have been the preferred measurement, Cmin is measured below TNZ, which was not clearly identified. While CBMR may not have been indicative of Cmin, due to my inability to identify Tlc, it is still useful for seasonal comparisons.

Maximum Thermogenic Capacity Protocol

Msum was measured as the maximum cold-induced VCO2 in a Helox atmosphere. Msum was measured by subjecting an individual bird to decreasing temperatures with Helox gas during their active phase (between 8:00 h and 16:00 h UTC + 2 h). The use of Helox is well-documented in studies of Msum in birds (Bozinovic and Rosenmann 1989; Swanson 2010; Swanson et al. 1996) as it allows for maximum rates of heat loss to be achieved at moderate Tair, and therefore does not carry the risk of frost bite or tissue damage.

After weighing birds and recording initial Tb, I placed birds in chambers and subjected to normal atmospheric air for a minimum of 5 min, allowing birds to become used to chambers at ~ 15 °C. I then switched the air source to Helox and recorded a 5 min baseline. After setting the ARB digital

display to 10 °C I continuously monitored VCO2 output until maximum VCO2 was reached for a

minimum 3 min (i.e. did not increase with further decrease in Tair). When VCO2 levels reached a plateau, the ARB was opened briefly to take a Tb reading; if Tb remained above 35 °C the run was continued, but if Tb was below 35 °C, Msum was considered as having been reached and the bird was removed from the chamber. A 5 min baseline measurement was also performed at the end of each run. After each run birds were placed near a heat source until Tb rose above 37 °C after which

I returned them to their holding cage with mealworms provided ad libitum. Experimental runs took

23 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

a maximum of 60 min as the maximum VCO2 always occurred within this timeframe.

Data Analyses

Expedata data files were corrected for VO2 drift in baselines using the relevant algorithms in

Warthog LabAnalyst X (www.warthog.ucr.edu). Data are presented as means ± SD.

I could not identify a clear Tlc during TNZ runs, suggesting that Rockjumpers have a wide range of

thermoneutrality. BMR was determined as the lowest continuous average VO2 over ten minutes

(Liknes et al. 2002; Petit and Vezina 2014; Paruk et al. 2015), which always occurred at least 1 h after birds were placed in the chamber, supporting the opinion that birds were at rest. Whole- animal BMR values were calculated in Watts (W) using a respiratory exchange rate (RQ) of 0.71 for BMR (mean winter RQ = 0.72 ± 0.06, mean summer RQ = 0.71 ± 0.02) with a Joule conversion

-1 factor of 19.8 J mL O2. Msum measurements were taken as the maximum VO2 over a 60 sec interval during the run, and the Tair for this interval was recorded as Tcl. Whole-animal Msum values were calculated in W using an RQ of 0.85 (mean winter RQ = 0.82 ± 0.07, mean summer RQ =

-1 0.80 ± 0.07) with a Joule conversion factor of 20.79 J mL O2. Total metabolic rates were used, as studies have suggested they are more informative than mass-specific rates when conducting seasonal comparison (Cooper 2002; Swanson 1991).

All analyses were conducted in R version 3.1.2 (The R Foundation for Statistical Computing,

2014). Data were analyzed for normality (Shapiro-Wilk test) and homogeneity (Levene’s test) prior to analyses. I report statistical significance for p values < 0.05. I used analyses of variance

(ANOVA) to determine which predictor variables (sex, season, Mb) determined best model fit (i.e. lowest AIC), with linear models then fitted to response variables (BMR, CBMR, Msum, Tcl, and ME).

24 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Only those predictor variables with significant outputs were used in the final models. Seasonal comparisons were done using only adult, non-breeding individuals. Additional analyses were done on the aforementioned factors with the addition of an “age” factor (0 = adult, 1 = immature, 2 = breeder) that included immature individuals and breeders.

Animal ethics clearance (A15-SCI-ZOO-007) was obtained from the Research Ethics Committee

(Animal) at Nelson Mandela Metropolitan University, and a bird capture permit (0037-AAA041-

00060) was issued by Cape Nature, Western Cape, South Africa.

Results

Body Mass and Condition

Mean capture mass of adult birds was significantly different between seasons, with summer birds having significantly less mass compared to winter birds (summer = 49.9 ± 4.9 g, winter = 55.3 ±

4.4 g; F1,25 = 27.63; p < 0.01; Table 1).

25 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Table 1 Capture mass, total basal metabolic rate (BMR), maximum thermogenic capacity (Msum), metabolic expansibility (ME), thermal conductance at BMR (CBMR), Tair at Msum (Tcl), and sample sizes for wild-living Cape Rockjumpers (Chaetops frenatus) captured at Blue Hill Nature Reserve, South Africa. Data were collected in July and August 2015 (winter) and January 2016 (summer) and are reported as means ± SD.

Capture BMR Msum ME (Msum/ CBMR Season Body Tcl (°C) (Watts) (Watts) BMR) (W/ °C-1) Mass (g)

Winter* 55.3±3.0v .64±0.1v 2.73±0.3iv 4.38±0.7iv 3.50±0.6iv -1.38±4.0iv

Winter** 56.8±6.7ii .67±0.1ii 2.49±0.5ii 3.61±1.2i 3.56±0.7i 5.27 ± 3.8ii

Summer* 50.6±5.5v .88±0.3iv 2.21±0.5iv 2.71±1.3iv 6.13±1.7iv 2.94±4.8iv

Summer*** 48.7±1.1iii 1.04±0.4 1.81±0.3iii 1.87±0.6iii 7.39±3.0iii 7.94±4.9iii i n = 3; ii n = 4; iii n = 5; iv n = 12; v n = 13. *non-reproductive adult; ** breeder (adult with brood patch); *** immature

There was no significant difference in mass between the sexes within summer (males = 52.4 ± 5.2, females = 47.3 ± 3.2; F1,10 = 4.10; p = 0.07), however females had significantly lower mass than males in winter (males = 57.7 ± 3.3, females = 52.1 ± 3.5; F1,14 = 10.77; p < 0.01). I then determined how mass (g) changed as a factor of tarsus length (mm; Fig 1). Based on the tarsus- corrected residuals of body mass, winter birds were in significantly better condition than summer birds (F1,30 = 39.57; p < 0.01). Within season, sex was not a significant factor on residuals in summer (F1,10 = 1.27; p = 0.29) or in winter (F1,11 = 3.81; p = 0.08).

26 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

70 summer

65 winter

60

55 body mass (g) 50

45

40 32 33 34 35 36 37 38 39 40 41 tarsus length (mm)

Figure 1 Body mass (g) and tarsus length (mm) measurements from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 12) and winter (n = 17) at Blue Hill Nature Reserve, South Africa. The trendline represents a significant relationship (p < 0.05). Mean winter residuals = 3.4 ± 2.7, mean summer residuals = -2.8 ± 3.4.

Maintenance Metabolic Rate

Metabolic rates are presented in Watts (W). Total BMR was significantly higher in summer

compared to winter (summer = 0.93 ± 0.30 W, winter = 0.64 ± 0.09 W; F1,23 = 10.06; p < 0.01; Fig

2). Similarly, mass-specific BMR remained significantly higher in summer compared to winter

-1 -1 -1 -1 (summer = 0.93 ± 0.30 J g hr , winter = 0.64 ± 0.09 J g hr ; F1,23 = 15.98; p < 0.01).

27 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 2 Basal metabolic rate (W) data collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 12) and winter (n = 13) at Blue Hill Nature Reserve, South Africa. Boxplots show 1st quartile, median, and 3rd quartile, whiskers are standard deviation.

Among sex and age groups, total BMR was not significantly different among males, females, and immature in summer (males = 0.87 ± 0.28 W, females = 0.89 ± 0.27 W, immature = 1.04 ± 0.38 W;

F2,25 = 0.22; p = 0.80), nor among males, females, and breeders in winter (males = 0.65 ± 0.11 W, females = 0.61 ± 0.05 W, breeders = 0.67 ± 0.08 W; F2,13 = 0.70; p = 0.51; Fig 3).

28 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 3 Basal metabolic rate (W) data collected from wild-living Cape Rockjumper (Chaetops frenatus) in summer (males n = 6, females n = 6, immature n = 5) and winter (males n = 8; females n = 5; adults with brood patches [breeders] n = 4), from Blue Hill Nature Reserve, South Africa. Boxplots show 1st quartile, median, and 3rd quartile, whiskers are standard deviation.

Body temperature (Tb) of individuals during BMR measurements were significantly higher in summer compared to winter (summer = 38.78 ± 0.35 °C, winter = 38.31 ± 0.38 °C; F1,22 = 4.34; p <

0.05). Among sex and age groups, Tb during BMR did not differ significantly for males, females, and immature in summer (males = 38.5 ± 0.37 °C, females = 39.0 ± 0.87 °C, 38.8 ± 0.58 °C; F2,14 =

0.90; p = 0.43), but was significantly higher in breeders compared to both males and females in winter (males = 38.3 ± 0.42 °C, 38.4 ± 0.4 °C, breeders = 39.1 ± 0.4 °C; F2,12 = 5.66; p < 0.05; Fig

4).

29 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 4 Body temperature (°C) during respiromtery runs at thermoneutrality in wild-living Cape Rockjumper (Chaetops frenatus) during summer (males n = 6, females n = 6, immature n = 5) and winter (males n = 8; females n = 4; adults with brood patches [breeders] n = 3), from Blue Hill Nature Reserve, South Africa. Boxplots show 1st quartile, median, and 3rd quartile, whiskers are standard deviation.

Thermal conductance at BMR (CBMR) was significantly higher in summer compared to winter

-1 -1 (summer = 6.13 ± 1.69 W °C , winter = 3.50 ± 0.60 W °C ; F1,22 = 25.83; p < 0.01; Table 1). The

Tb of one female in winter was excluded from the analyses of thermal conductance at BMR (CBMR) as her PIT tag could not be read, leaving CBMR sample size as eight males and four females in winter, and six males and six females in summer. Among sex and age groups, there were no significant differences in CBMR among males, females, and immature in summer (males = 5.63 ±

-1 -1 -1 1.47 W °C , females = 6.63 ± 1.88 W °C , immature = 7.39 ± 3.00 W °C ; F2,14 = 0.93; p = 0.42), or among males, females, and breeders in winter (males = 3.46 ± 0.66 W °C-1, females = 3.58 ±

-1 -1 0.52 W °C , breeders = 3.56 ± 0.66 W °C ; F2,12 = 0.07; p = 0.94).

30 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Maximum Thermogenic Capacity

I did not obtain Msum from one female, as I was not able to ensure absence of hypothermia due to

PIT tag failure (see above). Total Msum was significantly lower in summer compared to winter

(summer = 2.09 ± 0.46 W, winter = 2.67 ± 0.33 W; F1,22 = 10.69; p < 0.01; Fig 5). Mass-specific

Msum, metabolic rate remained significantly lower in summer compared to winter (summer = 2.09 ±

-1 -1 -1 -1 0.46 J g hr , winter = 2.67 ± 0.33 J g hr ; F1,22 = 5.05; p < 0.05). Seasonally, Tcl (°C) was significantly higher in summer compared to winter (summer = 2.94 ± 4.81 °C, winter = -1.38 ±

4.00 °C; F1,22 = 5.72; p < 0.05; Fig 5).

Figure 5 Seasonal variation in summit metabolism (Msum) and temperature of cold limit (Tcl) collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 12) st and winter (n = 12) from Blue Hill Nature Reserve, South Africa. Boxplots show Msum 1 quartile, median, mean (×’s), outliers (filled circles), 3rd quartile, and SD. Filled squares with whiskers show mean and SD at which Cape Rockjumpers reached Tcl.

31 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

In summer, total Msum among sex and age groups was significantly higher for males than both females and immature (males = 2.46 ± 0.28 W, females = 1.95 ± 0.53 W, immature = 1.81 ± 0.25

W; F2,14 = 4.65; p < 0.05), with no significant difference between females and immature (F1,9 =

0.29; p = 0.61; Fig 6). In winter, total Msum among sex and age groups did not differ significantly for males, females, or breeders in winter (males = 2.82 ± 0.19 W, females = 2.55 ± 0.33 W, breeders = 2.39 ± 0.55 W; F2,13 = 2.65; p = 0.11; Fig 6). In summer, among sex and age groups males had significantly lower Tcl than both females and immature (males = 0.03 ± 3.65 °C, females

= 5.85 ± 4.15 °C, immature = 7.94 ± 4.89 °C; F2,14 = 5.35; p < 0.05), with no significant difference between females and immature (F1,9 = 0.59; p = 0.46). In winter, among sex and age groups males also has significantly lower Tcl as compared to females and breeders (males = -2.35 ± 3.55 °C, females = 0.58 ± 4.64 °C, breeders 5.27 ± 3.83 °C; F2,13 = 6.65; p < 0.05), with no significant difference between females and breeders (F1,6 = 3.68; p = 0.10).

32 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 6 Summit metabolism (W) data collected from wild-living Cape Rockjumper (Chaetops frenatus) in winter (males n = 8; females n = 4; adults with brood patches [breeders] n = 3) and in summer (males n = 6, females n = 6, immature n = 5), from Blue Hill Nature Reserve, South Africa. Boxplots show 1st quartile, median, and 3rd quartile, whiskers are standard deviation.

Metabolic Expansibility

Metabolic expansibility was significantly lower in summer compared to winter (summer = 2.46 ±

1.21, winter = 4.23 ± 0.83; F1,22 = 14.77; p < 0.01; Fig 7).

33 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 7 Metabolic expansibility (ratio of BMR to Msum) from summer (n = 12) and winter (n = 11) collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured from Blue Hill Nature Reserve, South Africa. Boxplots show 1st quartile, median, and 3rd quartile, whiskers are standard deviation.

Among sex and age groups, ME was not significantly different between males, females, and immature in summer (males = 3.18 ± 1.73, females = 2.23 ± 0.63, immature = 1.87 ± 0.61; F2,14 =

1.97; p = 0.18), or between males, females, or breeders in winter (males = 4.50 ± 0.66, females =

4.15 ± 0.84, breeders = 3.61 ± 1.16; F2,12 = 1.39; p = 0.29; Fig 8).

34 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 8 Metabolic expansibility (ratio of BMR to Msum) data collected from wild-living Cape Rockjumper (Chaetops frenatus) in winter (males n = 8; females n = 4; adults with brood patches [breeders] n = 3) and in summer (males n = 6, females n = 6, immature n = 5), from Blue Hill Nature Reserve, South Africa. Boxplots show 1st quartile, median, and 3rd quartile, whiskers are standard deviation.

35 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Discussion

My findings on seasonal metabolic adjustments in the Rockjumper showed mixed support for my hypotheses. While Msum was indeed higher, I did not expect BMR to be lower in winter compared to summer. My findings included winter birds being in better body condition, having a lower BMR and lower CBMR, and having a higher Msum at a lower Tcl, all of which may be prerequisites aimed at conserving energy in winter. A lower BMR makes sense if energy resources are limited in winter

(Smit et al. 2010), and when combined with a lower CBMR indicates Rockjumpers are able to conserve more metabolically produced heat in winter compared to summer.

Body Mass and Condition

The current study found Rockjumpers in significantly better body condition in winter than in summer (Fig 1). Mass varied with sex in winter only, however body condition was not significantly different between the sexes in either season. Further, Mb was not a significant factor in regards to seasonal comparisons of BMR, Msum, CBMR, or Tcl. Seasonal changes in Mb have resulted in equivocal results in other studies; whereas some species showed no seasonal change (Cooper and

Swanson 1994; Dawson et al. 1983b; Noakes et al. 2016; Smit and McKechnie 2010; Thompson et al. 2015), some had a winter decrease in Mb (Lill et al. 2006), and others had a winter increase in

Mb (Chamane and Downs 2009; Liknes and Swanson 1996, 2011; Van de Ven et al. 2013a; Vezina et al. 2006; Wu et al. 2015).

Increased Mb in winter has been correlated with organ and muscle mass increase, which is itself correlated with an upregulation in metabolic activity such as increased BMR and Msum (Williams and Tieleman 2000; Liknes and Swanson 2011). However, the decreased winter BMR of

Rockjumpers suggests other mechanisms drive the increase in Mb, such as higher food availability

36 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

and winter fattening. While I did not quantify insect abundance during my study, arthropod abundance in the Fynbos peaks in spring (September/October), although correlated more with warmer temperatures than with season (Lee and Barnard 2015). The milder climate experienced at

BHNR as compared to the western sections of Rockjumper habitat (e.g. Cederberg mountains) may mean Rockjumpers at my study site had more insect availability than those further west, but again, this is speculative. If the milder climate at BHNR meant Rockjumpers were able to obtain enough food in winter to store fat (i.e. energy reserves), this could then be used to aid in general insulation

(Marsh and Dawson 1989).

It also may be that Rockjumpers do not see a winter increase in Mb so much as a summer decrease.

Rockjumpers are spring/summer breeders (Holmes et al. 2002) and adults likely forage solely for themselves during the winter, as opposed to the spring and early summer (i.e. November and

December) when individuals often are foraging for one or more immature individual (K.N.

Oswald, personal observations). While neither fat score nor feather density were recorded seasonally throughout the study, thus verifying Mb difference was indeed seasonal and not due to sample populations, I am certain the difference was due to changes within individuals, as the four recaptured individuals lost 2.7, 7.8, 8.4, and 9.1 g (4.6 – 15.5 % Mb) respectively between winter and summer (Fig 1). These values are similar to the 10 % average summer decrease in Mb recorded in the Hwamei (Garrolax canorus; Wu et al. 2015).

Maintenance Metabolic Rate

The lower BMR in winter compared to summer along with reduced body condition in summer indicate greater summer energy demands, possibly due to breeding depleting fat reserves, or decreased energy availability as mentioned above. An increased BMR in summer may also be the

37 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

result of more food availability in summer (contrary to what was speculated above), allowing rockjumpers to spend energy on breeding in spring/summer. I also found that CBMR was significantly lower in winter compared to summer, similar to early findings of Dawson and Carey

(1976), with lower conductance indicative of increased insulation (Lill et al. 2006; Swanson 1993;

Thompson et al. 2015; Wu et al. 2015). Conversely, a previous study by Wu et al. (2015) on the

Hwamei found higher thermal conductance in winter than summer. However, these authors also found a higher BMR in winter compared to summer, and so their study, and the current study, corroborate the correlation between BMR and C found in multiple bird species by Marschall and

Prinzinger (1991). The lower winter CBMR in the present study supports my idea that Rockjumpers reduce winter thermoregulatory costs with improved insulation instead of increased metabolic heat.

Though, as mentioned in the methods, my estimate of CBMR does not necessarily represent minimum thermal conductance, which is normally measured below TNZ, this difference is unlikely to change the outcome of the seasonal patterns I observed.

Considering the sexually dimorphic nature of Rockjumpers, it was surprising that Mb and sex were not influential in the top weighted models for predicting BMR, especially as a previous study showed that “sex” was highly influential in the best models for the monomorphic Cape White-eye

(Zosterops virens; Thompson et al. 2015). However, sex differences in the Cape White-eye were attributed to female-specific reproductive activity (Thompson et al. 2015). In contrast, both male and female Rockjumpers share in parental care (including the development of a brood patch in the male breeder), and it is possible that reproductive metabolic changes occur equally for both sexes.

My data suggests the lower BMR in winter for Rockjumpers follows the pattern of other subtropical birds, and not the pattern of Northern hemisphere temperate birds as I had

38 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

hypothesized. However, I then fitted my results into a previous data set that collated seasonal BMR and Msum data from subtropical and temperate bird species (McKechnie et al. 2015). I was surprised to find that when placed within the global set, the winter BMR of Rockjumpers seemed to fit with expected mass-transformed values (Fig 10B), while the summer BMR of Rockjumpers was above the expected values (Fig 10A).

In contrast to my expectations, these results indicate that Rockjumper BMR is unlikely to be associated specifically with cold adaption, and they perhaps require less physiological flexibility because they inhabit the relatively mild Tair experienced annually in the Fynbos. It is also possible that Rockjumpers do not require a higher BMR in the winter to maintain Tb, corroborating the findings in subtropical species or other species where winters are not extremely cold (Smit and

McKechnie 2010; Thompson et al. 2015). The seasonal Tair at my field site did not drop below - 2.6

°C, even in the coldest months of the year. By comparison, seasonal studies at temperate sites often report mean daily Tair well below zero, for example, Black-capped chickadees often experience Tair

< -20 °C (Petit and Vézina 2014). Moreover, winter rainfall in Fynbos regions results in peak insect abundance during October (Procheş and Cowling 2006), and although foliage invertebrate abundance show little seasonal variation, aerial invertebrate abundance peaks in late spring and summer (Pryke and Samways 2008). Decreased food availability in winter may result in

Rockjumpers conserving energy by reducing their general metabolic requirements and instead rely on a lower thermal conductance combined with increased insulation.

The lower winter BMR of Rockjumpers may also be due to temperatures during the study period not dropping low enough to require a general winter upregulation, with a low BMR reflective of the need to conserve energy for cold fronts. This brings into question why Rockjumpers increase

39 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

their BMR in summer. One suggestion is that increased metabolic rate is associated with the breeding activities of Rockjumpers during summer, such as increased thyroid activity or increased organ mass (Vezina et al. 2006; Wingfield and Farner 1993). However, I can only speculate that breeding activities influenced summer energy demands in Rockjumpers. My finding that

Rockjumpers which began showing signs of breeding in winter (i.e. breeders) had significantly higher BMR and Tb compared to non-breeding conspecifics, and similar to Rockjumpers caught during summer, provides further support that breeding activities increase maintenance metabolic demands.

Maximum Thermogenic Capacity

Overall I found a significantly higher Msum in winter compared to summer, which aligns with past studies that have fairly unequivocally shown winter Msum increases (see Introduction). It is unclear at this stage why in summer males would have significantly higher Msum than females –– when there was no significant difference in Mb between sexes –– while in winter males and females had no significant difference in Msum –– when there was a significant difference in Mb between sexes.

Again, after fitting the data into the global data set from McKechnie et al. (2015), I found both seasons were below average predicted Msum values expected for their Mb (Fig 10C, D). I found this surprising, as Msum is driven by thermogenic requirements in winter, and many of the species with mass-specific Msum values greater than those of Rockjumpers are from warmer environments (i.e. latitude greater than 38 degrees; McKechnie et al. 2015). This seems to contradict Swanson and

Garland (2009) who suggested that a higher Msum indicates cold adaptation.

This may be due to a lack of extreme cold temperatures at my study site, as Camacho et al. (2015) suggest extreme temperatures, and not mean temperatures, are often better descriptors of a species’

40 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

thermal environment. For comparison, an unpublished data set recorded seasonal Msum in White- browed Sparrow-weavers (~42 g) at a desert site near Askham, SA as 3.93 W in winter (Noakes,

McKechnie, unpublished data). The higher Msum of sparrow-weavers compared to the mean winter

Msum value for rockjumpers of 2.67 W may be due to differences in site minimum temperatures.

The mean and minimum Tair for July 2013 at Twee Rivieren (near Askham), were 1 °C and -5 °C respectively (wunderground.com), which is lower than those at my field site (8.7 °C and -2.6 °C respectively). Alternately, Araújo et al. (2013) suggest it is solely extreme maximum temperatures that determine thermal limits, with Twee Rivieren reaching an annual mean and maximum of 39 °C and 43 °C respectively (wunderground.com), which is considerably higher than those at my field site (20.5 °C and 35.4 °C).

Given the higher winter Msum of Rockjumpers, it remains likely that Rockjumpers can substantially increase their metabolic heat production to maintain Tb during the coldest temperatures experienced. Although Minnaar et al. (2014) found no seasonal change in the Msum of Wahlberg’s

Epauletted Fruit Bats (Epomorphus wahlbergi), my finding of higher Msum in winter compared to summer aligns with predictions for birds in cold climates (Dawson et al. 1983b; McKechnie et al.

2015; Smit and McKechnie 2010; Swanson and Garland Jr 2009; Swanson and Liknes 2006), as well as numerous other empirical studies (Arens and Cooper 2005; Cooper 2002; Cooper and

Swanson 1994; Liknes et al. 2002; Petit and Vézina 2014; Van de Ven et al. 2013a; Wu et al.

2015). It seems possible that while the average Tair of my study site may not be low enough to require an upregulation in BMR, a higher Msum in winter was nevertheless necessary to cope with the lower temperatures experienced in winter. Rockjumpers also had a significantly lower Tcl in winter compared to summer, which combined with a higher Msum emphasizes that they have increased ability to endure colder Tair in winter.

41 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Metabolic Expansibility

Metabolic expansibility was significantly greater in winter than in summer, which was expected given the seasonal changes found in both BMR and Msum. Although interpreting ME can be difficult, it seems intuitive that a greater ME means greater physiological flexibility. In the case of the Rockjumper, it may be that a greater ME is more important than BMR and Msum separately, as it indicates a greater capacity to increase maximum metabolic rates despite lower maintenance metabolic demands. I did not observe any obvious negative metabolic effects associated with reduced BMR in the Rockjumpers during winter; there was no compromise in thermogenic capacity (Msum was still higher in winter), nor a decrease in average Rockjumper Tb.

Few studies have tried to directly interpret ME, although Cooper and Swanson (1994) suggested a larger ME was indicative of a greater ability to elevate metabolism and thus a greater cold tolerance. Minnaar et al. (2014) attempted to relate ME to use of hibernation or torpor, and in their case found that a wider ME in winter was correlated with re-warming in fruit bats (from a seasonal decrease in BMR and unchanged Msum). In contrast, Van de Ven (2015) made no attempt to explain why they found a wider ME in summer. Rockjumper ME varied between 2.45 ± 1.21 (n = 12 in summer) and 4.22 ± 0.83 (n = 11 in winter). These values are slightly below expected values for endotherms (4 to 8 for birds and mammals; Hinds et al. 1993). Typical values for temperate bird species range from 6.8 to 7.2 (Swanson 1993). ME also seems to be an equivocal indicator of tolerance, as some species had larger ME in warmer Tair or in summer (Arens and Cooper 2005;

Van de Ven et al. 2013a), while others followed the same pattern of the Rockjumpers with a larger

ME in winter (Cooper and Swanson 1994).

42 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Limitations

My study presented a number of constraints that limited my interpretation of my findings. Firstly, I could not detect a clear lower limit of TNZ in either winter or summer. My TNZ test runs did not show a clear increase in RMR at either end of the thermal range examined, indicating the range of

Tair tested (~ 15, 20, 25, and 30 °C) was not wide enough (Fig 11). It is therefore possible that applying a wider range of Tair would determine TNZ. Another limitation was the technical constraint in recording Tb continuously during Msum experiments, as the PIT tag reader would not fit into the ARB alongside the respirometry chamber. As a result, I had to temporarily interrupt

Msum runs in order to check birds were not hypothermic. Although the cold air released from the chamber while checking did increase run time of Msum experiments, I did not observe a noticeable difference in the O2/CO2 trace obtained.

Conclusions

My findings provided inconclusive support for my hypothesis that low heat thresholds previously shown in Rockjumpers (Milne et al. 2015) are the result of an adaptation to cold. The greater Mb of winter birds was likely related to winter fattening and increased insulation as mechanisms to help lower thermal conductance. In all likelihood, Rockjumpers do not require increased BMR to maintain Tb in winter, despite experiencing lower temperatures than in summer. My findings also indicate that BMR remains an equivocal factor when trying to infer cold or warm adaptation. As suggested by Araújo et al. (2013), physiological responses (i.e. thermal limits) in birds were only explained by the maximum Tair. The conclusions drawn in my study may still be a case of site- or temporal-specific results, as all of the birds measured were from a single site in the Eastern Cape province; many studies have shown large differences in BMR and Msum across the species geographical range.

43 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Future studies could involve a repetition of the experiment across the geographical range to see if the difference in rainfall and temperature results in similar patterns found in my study. I would also suggest Rockjumper RMR be collected over a broader range of temperatures, perhaps as wide a range as 5 to 35 °C, to properly identify TNZ. Additionally, collecting data on fat scores and feather weight as proxies for insulation, following similar procedure to Lill et al. (2006), in summer compared to winter and breeding compared to non-breeding would allow for better interpretation of the above results. Finally, for range-restricted species with populations decreases associated with warming temperatures, such as the Rockjumper, identifying current thermal niches aid in determining future range changes under predicted climatic change.

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Tattersall, G.J., B.J. Sinclair, P.C. Withers, P.A. Fields, F. Seebacher, C.E. Cooper and S.K. Maloney. 2012. Coping with thermal challenges: physiological adaptations to environmental temperatures. Comprehensive Physioliology.

Thompson, L.J., M. Brown and C.T. Downs. 2015. Seasonal metabolic variation over two years in an Afrotropical passerine bird. J Therm Biol 52: 58-66.

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Van de Ven, T.M., N. Mzilikazi and A.E. McKechnie. 2013b. Seasonal metabolic variation in two populations of an Afrotropical euplectid bird. Physiol Biochem Zool 86: 19-26.

48 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Vezina, F., K. Jalvingh, A. Dekinga and T. Piersma. 2006. Acclimation to different thermal conditions in a northerly wintering shorebird is driven by body mass-related changes in organ size. J Exp Biol 209: 3141-3154.

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Wu, M.-X., L.-M. Zhou, L.-D. Zhao, Z.-J. Zhao, W.-H. Zheng and J.-S. Liu. 2015. Seasonal variation in body mass, body temperature and thermogenesis in the Hwamei, Garrulax canorus. Comp Biochem Physiol A Molec Integr Physiol 179: 113-119.

49 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Appendix

Figure 9 Resting metabolic rate (W) over a range of air temperatures (15 to 30 °C) from wild- living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 5) and winter (n = 5) at Blue Hill Nature Reserve, South Africa.

50 Krista Oswald Dissertation Chapter 2: Seasonal Responses to Cold

Figure 10 Scaling of Cape rockjumoer (Chaetops frenatus) body mass (Mb) and A - maintenance metabolic rate (BMR) during summer, B - maintenance metabolic rate (BMR) during winter, C - maximum thermogenic capacity (Msum) during summer, D – maximum thermogenic capacity

(Msum) during winter, all overlaid on temperate and subtropical seasonal data from McKechnie et al. 2015. Trendlines are shown for subtropical (solid) and temperate (dashed) data from McKechnie et al. 2015.

51 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Chapter 3: Physiological responses show low heat tolerance thresholds in the Cape Rockjumper (Chaetops frenatus).

Abstract

Recent studies have shown that a Fynbos endemic, the Cape Rockjumper (Rockjumper; Chaetops frenatus), is at risk from increases in temperature, with greatest population declines observed in regions that experienced warming over the last two decades. Moreover, is was shown previously that Rockjumpers have low air temperature thresholds for evaporative cooling, suggesting a direct link between high thermoregulatory costs during hot temperatures and population declines. In this study I determine if Rockjumpers show seasonal flexibility in thermoregulatory costs at high temperatures. I collected data on resting metabolic rate (RMR), evaporative water loss (EWL) and body temperature (Tb) at high temperatures in a wild population of Rockjumpers during winter and summer (n = 11 winter, 4 females and 7 males; n = 11 summer, 6 females and 5 males). I found no evidence that Rockjumpers reduce the physiological costs of thermoregulation in summer compared to winter. During summer, Rockjumpers showed elevated RMR (at whole-animal and mass-specific level), and EWL, compared to winter. This study provides further evidence of a physiological basis for the Rockjumpers' cool climate niche and population declines reported in parts of the Fynbos region that experienced significant warming. Rockjumper habitat will become both hotter and drier, and a better understanding of their physiological and behavioural mechanisms in dealing with high temperatures is urgently needed.

52 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Introduction

Endotherms maintaining body temperature (Tb) in air temperatures (Tair) above Tb are presented with physiological challenges in avoiding hyperthermia. Conductance, convection, and radiation can no longer be relied upon to help dissipate heat, with reliance instead on the physiological mechanism of evaporative cooling (Tieleman et al. 2002). A reliance on evaporative cooling (i.e. panting, sweating, or gular fluttering), involves trade-offs that can affect behaviour, reproduction, or body condition (Cunningham et al. 2013; du Plessis et al. 2012; Gardner et al. 2016; Smit et al.

2013; Tieleman et al. 2002; Williams and Tieleman 2005; Wolf and Walsberg 1996). As heat wave duration and frequency increase mostly due to anthropogenic climate change (Field 2012) these affects are likely to increase. Understanding the functional value of species’ thermal physiological variation on a regional and temporal scale is directly relevant to testing future predictive climate- envelope models (Boyles et al. 2011; Dawson et al. 2011). A recent review suggested studies focus on how species cope with extreme temperature events, and not average temperatures, in predictive modeling (Camacho et al. 2015). Such studies include examining the thermal physiology of individuals over a range of increasing Tair (Noakes et al. 2016; Tieleman et al. 2002; Whitfield et al.

2015).

At high Tair, we may expect species at rest to reduce metabolic heat production (MHP) with a lower resting metabolic rate (RMR; the lowest metabolic rate by an organism at any given temperature;

Swanson 1991, Chamane and Downs 2009, Swanson 2010). However, increases in evaporative water loss (EWL) are well documented as the only physiological mechanism of heat dissipation among avian taxa when Tair exceeds normothermic Tb (Battley et al. 2003; du Plessis et al. 2012;

Huey et al. 2012; Wolf 2000). Dependence on EWL necessitates a trade-off between hyperthermia and dehydration as EWL rates increase with increasing Tair (Boyles et al. 2011; Dupoué et al. 2015;

53 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

McKechnie and Wolf 2004; Smit et al. 2013; Tattersall et al. 2012; Whitfield et al. 2015; Wolf

2000; Wolf and Walsberg 1996). It follows that EWL demands are especially costly in arid and semi-arid areas where water is scarce and summers are warm and dry (Chamane and Downs 2009; du Plessis et al. 2012; Smit and McKechnie 2015; Williams and Tieleman 2000). As average global temperatures increase with climate warming, overall water requirements are predicted to be much higher in future decades, resulting in a mismatch between supply and demand (McKechnie and Wolf 2009).

Determining phenotypic flexibility, for example by examining seasonal variation, is a useful tool for understanding how species will respond to predicted future climate variation (Chamane and

Downs 2009; Hofmann and Todgham 2010; Smit and McKechnie 2015; Tieleman et al. 2002).

Seasonal physiological flexibility can not only give us an idea of species’ reaction norm to seasonal weather changes, but may also be indicative of a species capacity to respond to changing temperature extremes (Valladares et al. 2014). Phenotypic flexibility in response to high summer

Tair includes seasonal changes in evaporative cooling capacity (i.e. the ability to maintain Tb over a gradient of Tair) and evaporative cooling efficiency, defined as the ratio of evaporative heat loss

(EHL –– calculated from EWL) to MHP. While some studies have examined inter- and intra- specific evaporative cooling capacity (Caldwell et al. 2015; Kelly et al. 2012; McKechnie and Wolf

2004; Milne et al. 2015; Whitfield et al. 2015; Noakes et al. 2016), investigations of intra-specific seasonal evaporative cooling capacity remain relatively rare. While a recent study on three populations of white-browed sparrow-weavers (Plocepasser mahali) unsurprisingly found all three populations increased EHL/MHP as Tair increased, they also found that not only was EHL/MHP higher in summer compared to winter, but in summer birds had reduced rates of Tb increase

(Noakes et al. 2016). These authors suggest that the summer reduction in EWL and Tb, although

54 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat accompanied by no clear pattern in RMR, indicated the ability of sparrow-weavers to enhance their evaporative cooling capacity at higher Tair to conserve water, a pattern that may be true for most arid or semi-arid species.

The south-western section of South Africa contains the Cape Floristic Region (hereafter referred to as “Fynbos”), one of only six floristic kingdoms, containing some of the highest levels of plant endemism (Péron and Altwegg 2014). The IPCC identified Mediterranean biomes as threatened by desertification under relatively mild climate warming, with the potential for species range shifts

(WDPA Consortium 2004). Furthermore, the six Fynbos-endemic passerines have been labelled as among the most at risk from climate change (Foden et al. 2013). Of the six Fynbos-endemic passerine species, the Cape Rockjumper (Chaetops frenatus; from here on “Rockjumper”) may be the most adversely affected by increased temperature (Lee and Barnard 2015a), and so serves as an excellent study species to examine thermal physiological thresholds. Currently not listed on the

IUCN Red List of Threatened Species, in 2004 Rockjumpers were ranked among the top three

Fynbos endemics most susceptible to climate change in South Africa (Simmons et al. 2004).

Recently, Rockjumpers have been upgraded to “near threatened” by BirdLife South Africa (Taylor et al. 2015) due to their relatively small climatic space and low population density (Lee and

Barnard 2015a); this was further corroborated by a recent study linking low thermal thresholds and population decreases to increases in temperature (Milne et al. 2015). Moreover, Rockjumper range is likely over-estimated as it is range-restricted to high altitude rocky habitat islands of the south- west corner of South Africa’s Cape Fold Mountains (Holmes et al. 2002; Lee and Barnard 2015a).

In the current study I examined seasonal adjustments in Rockjumper evaporative cooling capacity in Rockjumpers by looking at Tb, EWL, RMR, and EHL/MHP. I hypothesized that, similar to

55 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat semi-arid subtropical species, I would find increased evaporative cooling capacity in summer as compared to winter; specifically, I expected to find more gradual increases in RMR, EWL, and Tb with increasing Tair, and a higher ratio of EHL/MHP. The Fynbos environment has historically maintained an unprecedented level of environmental stability (Cowling et al. 2004; Jansson and

Dynesius 2002), with mild historic climatic changes arguably resulting in Fynbos endemics avoiding the need for flexible physiology (Huntley et al. 2016). In Chapter 2, I found no evidence that Rockjumpers are unusually cold tolerant for their mass, with scaled mass-specific values of their summit metabolism (a suggested correlative of cold tolerance) falling below scaled values of other temperate and subtropical species (McKechnie et al. 2015). Because of this, my alternative hypothesis is a historically stable environment has lead the Rockjumper to have no variation in evaporative cooling capacity between seasons, shown by no seasonal differences in RMR, EWL,

Tb, and EHL/MHP.

As a high altitude-preferring species, Rockjumpers face future challenges for coping with climate warming, and will serve as an excellent indicator of how similarly habitat-constrained and range- restricted species may cope. High altitude communities are among those most at risk from climate change due to their dependency on cooler mountain slopes (Hijmans and Graham 2006; Hill et al.

2011; Parmesan 2006; Simmons et al. 2004; Visinoni et al. 2015), competition from the upward expansion of lower altitude species (Sinervo et al. 2010), and an inability to move to cooler areas either further south or at higher altitudes (Huntley and Barnard 2012; Parmesan 2006; Simmons et al. 2004; Walther et al. 2002).

56 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Methods

Study Site and Species

This study took place between July 2015 and February 2016 at Blue Hill Nature Reserve (BHNR; see Chapter 2). Rainfall and temperature (Tair) data were collected every 30 minutes using an on- site weather station (Vantage Vue, Davis Instruments Corp., California USA). The mean annual rainfall in 2015 was 496.2 mm, while average daily Tair from two weeks before the first bird was captured until the day the last bird was captured was 8.71 °C ± 6.00 in winter (July 24th to August

31st 2015) while in summer it was 20.54 °C ± 5.94 (January 1st to January 31st 2016. Minima and maxima Tair experienced over the study period were -2.6 °C to 27.5 °C in winter, and 6.9 °C to 35.4

°C in summer.

During July and August 2015 (subsequently “winter”) I captured 17 individuals comprised of nine males and eight females. During January 2016 (subsequently “summer”) I collected heat data

(RMR, EWL, and Tb) from 17 individuals (six males, six females, and five immature). Four individuals (one male and three females) were excluded from experimentation as they showed clear physical signs of breeding (i.e. brood patches). Heat data was not collected from any of the same individuals between seasons, as the four individuals re-captured in summer were those excluded from winter experiments due to signs of breeding (see Chapter 2).

Body Temperature Measurements

Individual birds were injected with a small, temperature-sensitive, Passive Integrated Transponder

(PIT) tag inter-peritoneally (Ratnayake et al. 2014) to measure Tb throughout experimentation. PIT tags provide Tb information while minimising handler effects, with no significant alteration of individual condition (Gerson et al. 2014; Ratnayake et al. 2014), and no significant negative effects

57 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat in wild bird populations (Ratnayake et al. 2014).

Metabolic Measurements

Body mass (Mb) was measured to within 0.1 g before and after each experimental procedure and all birds maintaining mass of < 5 % capture Mb. Birds were placed individually in a 4-L chamber constructed from airtight plastic (Lock & Lock, Mumbai, India) fitted with a wire-mesh platform raised 15 cm to ensure normal perching posture. A thin layer of mineral oil was placed at the base of the chamber to cover any faeces, ensuring faecal water did not factor into evaporative water loss

(EWL) measurements. Chamber temperature (Tair) was measured using a thermistor probe (model

TC100, Sable Systems, Las Vegas, NV, USA) inserted one cm into bird chambers through a small hole in the lid.

Metabolic rates (MRs) were measured indirectly as oxygen consumption (VO2) and carbon dioxide

-1 emission (VCO2) in mL min using a portable open-flow respirometry system. For all measurements, I controlled flow rate of atmospheric air through bird chambers using FMA-series mass flow controllers (Omega, Bridgeport, NJ USA) calibrated using a 1-L soap bubble metre

-1 (Baker and Pouchot 1983). Flow rates of between 1.5 – 3 L min were selected to ensure [O2] to the chamber remained within 0.5% of incurrent [O2]. Subsampled air was then pulled from the bird chamber through an O2 and CO2 analyser (Foxbox-C Field Gas Analysis System, Sable Systems,

Las Vegas, Nevada USA). The Foxbox included a subsampling pump, and allowed for digital outputs to be recorded using Expedata Data Acquisition and Analysis Software (Sable Systems,

Las Vegas, Nevada USA).

58 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

I placed the respirometry chambers in a custom-made environmental chamber consisting of a100-L cooler box lined with copper tubing through which temperature-controlled water was pumped by a circulating water bath (FRB22D, Lasec, Cape Town, South Africa; Smit and McKechnie 2010). A small fan was placed inside the environmental chamber to ensure a uniform distribution of air temperature. An infrared light source and closed circuit security camera with live video feed placed within the environmental chamber allowed for continuous monitoring of the bird. A BioMark PIT tag reader was placed next to the chamber to monitor and record Tb every minute (Gerson et al.

2014; Whitfield et al. 2015).

Heat Protocol

Heat tolerance measures were taken during the active phase of birds on either day one or day two.

Birds were subjected to a ramped series of Tair (30, 33, 36, 39, 42 °C) for a minimum of 15 minutes each. Baseline was recorded for a minimum of 5 minutes at the beginning of each run, as well as between each Tair and again at the end of the run. Birds were held at the final test temperature of 42

°C for up to 20 minutes to allow a better assessment of RMR, EWL, and Tb regulation at Tair close to Tb (Whitfield et al. 2015). I recorded Tair at which birds began panting (Tpanting).

After all experimental runs birds were weighed and returned to holding cages and provided with mealworms ad libitum.

Statistical Analysis

Two individuals were removed from analyses from either Mb decrease > 5 % for one (n = one male in summer) or prolonged agitation at Tair > 39 °C (n = one female in winter), leaving me with sample sizes of 11 in both summer (five males, six females) and winter (seven males, four

59 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat females). Only adult individuals were used for seasonal comparisons. The five immature individuals (all collected in summer) were then compared to adults collected in summer. Values are presented as mean ± standard deviation (SD).

Expedata data files were corrected for VO2 drift in baselines using the relevant algorithms in

Warthog LabAnalyst X (www.warthog.ucr.edu). All measurements were taken from the minimum

VO2 over a 60 second interval during the last five minutes at each Tair to ensure birds had the highest likelihood of being at RMR. Total metabolic rates were used, as studies have suggested they are more informative than mass-specific values when doing seasonal comparison (Cooper

2002; Swanson 1991). RMR values were calculated using VO2 and a respiratory quotient of 0.76

(winter average = 0.76 ± 0.01, summer average = 0.76 ± 0.10) with a Joule conversion of 20.1 J

-1 mL O2. Evaporative heat loss in Watts (W) was calculated from EWL using latent heat of vaporisation 2.4 J mg-1, and evaporative cooling efficiency was calculated as the ratio of EHL to

MHP.

All analyses were carried out in R version 3.1.2 (The R Foundation for Statistical Computing,

2014). I report statistical significance for p values < 0.05. Data assumptions were checked visually

(i.e. plotting residuals), for normality (Shapiro-Wilk test), and for homogeneity (Levene’s test; car package). For all factors, significance did not vary with total vs. mass-specific values, so total values are reported.

Model selection was determined by lowest AIC values, with segmented models and linear mixed- effects models (LMEs) then fitted to data to determine significant inflection points (segmented and lme packages), with. I initially ran models using a full array of potential predictor variables (Mb, sex, Tair, and season). I then ran analyses on each potential response variable (RMR, EWL, Tb,

60 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

EHL/MHP, and Tpanting). Sex and Mb were removed as there were no significant interactions (all p

> 0.05), and their removal in subsequent analyses either did not affect or improved model fit (i.e. decrease or no change in AIC value). Linear mixed-effects models were fitted to response variables that included repeated measurements of birds at multiple temperatures (RMR, EWL, Tb, and EHL/MHP), with individual included as a random effect. A linear model was fitted to Tpanting data as panting data included a single data point from each individual both seasons.

Seasonal comparisons were done using only adult individuals. Additional analyses were done on the aforementioned factors with the addition of an additional “age” factor (0 = adult, 1 = immature) using summer data (no immature individuals were present in winter).

Animal ethics clearance was secured from Nelson Mandela Metropolitan University (A15-SCI-

ZOO-007) and a bird capture permit (0037-AAA041-00060) was issued by Cape Nature, Western

Cape, South Africa.

Results

Body Condition

Mean capture mass of adult birds was significantly different between seasons, with summer birds being significantly lighter than winter birds (summer = 51.1 ± 5.3 g, winter = 55.4 ± 3.3 g; F1,20 =

5.29; p < 0.05). Males were significantly heavier than females in winter (males = 57.0 ± 2.3 g, females = 52.6 ± 3.2 g; t1,9 = 2.59; p = 0.03), however there was no significant difference in summer (males = 54.2 ± 5.6 g, females = 48.5 ± 3.5 g; t1,9 = 4.26; p = 0.07). I then determined how mass (g) changed as a result of tarsus length (mm; Fig 1). Based on the tarsus-corrected residuals of body mass, winter birds were in significantly better condition than summer birds (F1,20 = 11.74;

61 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

p < 0.01). Within seasons, sex was not a significant factor on residuals in either winter (F1,9 = 3.47, p = 0.10) or in summer (F1,9 = 2.29; p = 0.16).

65 summer

60 winter

55

50

45 body mass (g)

40

35 33 34 35 36 37 38 39 40

tarsus length (mm)

Figure 1 Body mass (g) and tarsus length (mm) measurements from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 11) and winter (n = 11) at Blue Hill Nature Reserve, South Africa. The trendline represents a significant relationship (p < 0.05). Mean winter residuals = 2.3 ± 2.7, summer residuals = -2.3 ± 3.5.

Resting Metabolic Rate

Total RMR (W) increased significantly with increasing Tair (mean slope ± SE = 0.03 ± 0.01; t1,81 =

5.52; p < 0.01), and was significantly higher in summer compared to winter (mean slope ± SE =

0.69 ± 0.28; t1,81 = 2.49; p < 0.05; Fig 2). The rate of RMR increase was significantly higher in summer compared to winter (mean slope ± SE = -0.02 ± 0.01; t1,81 = -2.73; p < 0.01; Fig 2).

62 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

2.5 summer

winter 2

1.5

1

0.5 resting metabolic rate (W) summer winter y = 0.03x - 0.10 y = 0.01x + 0.59 0 28 30 32 34 36 38 40 42 44

air temperature (°C)

Figure 2 Resting metabolic rate (W) data collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 11) and winter (n = 11) from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C).

Evaporative Water Loss

-1 Evaporative water loss (EWL; mg hr ) increased significantly with increasing Tair (mean slope ±

SE = 103.68 ± 9.47; t1,41 = 10.94; p < 0.01), and was significantly higher in summer compared to winter (mean slope ± SE = 1784.27 ± 496.55; t1,41 = 3.59; p < 0.01; Fig 3). The estimated EWL breakpoint was ~ 36 °C in both summer and winter (summer = 36.0 °C, winter = 36.1 °C); above this Tair the rate of EWL increase in summer was significantly higher in summer compared to winter (mean slope ± SE = -51.23 ± 12.66; t1,41 = -4.05; p < 0.01; Fig 3).

63 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

2000 summer ) -1 1600 winter

summer y = 103.7x - 3284.2 1200

800

400 winter y = 52.5x - 1500.0

evaporative water loss (mg hr 0 28 30 32 34 36 38 40 42 44 air temperature (°C)

Figure 3 Evaporative water loss (mg hr-1) data collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 11) and winter (n = 11) from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C).

The Tair at which birds began panting (Tpanting) was similar to the EWL inflection point in summer

(35.83 ± 1.19 °C), but was slightly lower than the EWL inflection point in winter (34.32 ± 1.43

°C). Panting temperature was significantly different between seasons, with birds in summer panting at higher Tair than those in winter (t1,91 = -3.59; p = 0.17; Fig 4).

64 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

43

41 × mean

) 39

° C outlier ( 37

panting 35 T

33

31

29 summer winter

Figure 4 Panting air temperature (Tpanting; °C) data from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 11) and winter (n = 11) from Blue Hill Nature Reserve, South Africa, and subjected to a range of air temperatures (29 – 43 °C).

Body Temperature

Body temperature (Tb; °C) increased significantly as Tair increased (mean slope ± SE = 0.20 ± 0.02; t1,82 = 12.36; p < 0.01), and was significantly lower in summer compared to winter (mean slope ±

SE = 2.32 ± 0.79; t1,82 = 2.93; p < 0.05; Fig 5). The rate of Tb increase was higher in summer compared to winter (mean slope ± SE = -0.06 ± 0.02; t1,82 = -2.67; p < 0.01; Fig 5).

65 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

45 summer

44 winter C)

° (

43 summer y = 0.20x + 34.0

42

41

40 winter body temperature y = 0.15x + 36.3

39 28 30 32 34 36 38 40 42 44 air temperature (°C)

Figure 5 Body temperature (°C) data collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 11) and winter (n = 11) from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C).

Evaporative Cooling Efficiency

Evaporative cooling efficiency (EHL/MHP) increased significantly with increasing Tair (mean slope ± SE = 0.03 ± 0.00; t1,83 = 14.07; p < 0.01) but was not significantly different between seasons (mean slope ± SE = 0.10 ± 0.12; t1,83 = 0.89; p < 0.01; Fig 6). There was no significant difference in the rate at which evaporative cooling efficiency increased as a result of Tair in summer compared to winter (mean slope ± SE = -0.01 ± 0.00; t1,82 = 0.81; p = 0.10; Fig 6).

66 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

1 summer winter

0.75 summer y = 0.02x - 0.427

0.5

0.25 winter y = 0.02x - 0.525 evaporative cooling efMiciency 0 28 30 32 34 36 38 40 42 44 air temperature (°C)

Figure 6 Evaporative cooling efficiency (evaporative heat loss/metabolic heat production) data collected from wild-living Cape Rockjumpers (Chaetops frenatus) captured in summer (n = 11) and winter (n = 11) from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C).

Immature and Adult Comparison

The Mb of immature individuals was not significantly lower than adult males (immature = 48.5 ±

1.5 g, males = 54.2 ± 5.6 g; t1,8 = 1.66; p = 0.14), or from adult females (females = 48.5 ± 3.5 g; t1,9

= 0.52; p = 0.75). Immature RMR (W) increased significantly with increasing Tair (mean slope ±

SE = 0.10 ± 0.04; t1,16 = 2.32; p < 0.05) and also increased at significantly greater rate compared to adult (mean slope ± SE = -0.07 ± 0.03; t1,54 = -2.41; p < 0.05; Fig 7).

67 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

5 immature y = 0.10x - 1.64

4

3

2

1 adult

resting metabolic rate (W) y = 0.03x - 0.10 0 28 30 32 34 36 38 40 42 44

air temperature (°C)

Figure 7 Resting metabolic rate (W) data of immature wild-living Cape Rockjumpers (Chaetops frenatus; n = 5) captured in summer 2015 from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C) with symbols displayed for immatures only, and lines of best fit shown for immature (black) and adult (grey dashed).

-1 Evaporative water loss (mg hr ) increased significantly with increasing Tair (mean slope ± SE =

67.24 ± 22.59; t1,16 = 2.98; p < 0.01) but did not increase at significantly greater rate compared to adult (mean slope ± SE = -12.88 ± 18.88; t1,54 = -0.68; p = 0.50; Fig 8).

68 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

2500 )

-1 immature y = 126.9x - 3712.5 2000

1500

1000

500 adult y = 103.7x - 3284.2

evaporative water loss (mg hr 0 28 30 32 34 36 38 40 42 44 air temperature (°C)

Figure 8 Evaporative water loss (mg hr-1) data of immature wild-living Cape Rockjumpers (Chaetops frenatus; n = 5) captured in summer 2015 from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C) with lines of best fit shown for immature (black) and adult (grey dashed).

Body temperature (°C) for immature individuals did not increase significantly with increasing Tair

(mean slope ± SE = 0.09 ± 0.07; t1,16 = 1.19; p= 0.25), with no significant difference in the rate of increase compared to adult (mean slope ± SE = 0.10 ± 0.06; t1,54 = 1.75; p = 0.08; Fig 9).

69 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

44 immature y = 0.09x + 38.6

C ) 43 °

42

41

40

body temperature ( adult y = 0.20x + 34.0

39 28 30 32 34 36 38 40 42 44 air temperature (°C)

Figure 9 Body temperature (°C) data of immature wild-living Cape Rockjumpers (Chaetops frenatus; n = 5) captured in summer 2015 from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C) with lines of best fit shown for immature (black) and adult (grey dashed).

Immature evaporative cooling efficiency (EHL/MHP) differed significantly at increasing Tair (mean slope ± SE = 0.01 ± 0.00; t1,16 = 2.81; p < 0.05), and increased at a significantly lower rate compared to adult (mean slope ± SE = 0.02 ± 0.01; t1,54 = 3.01; p < 0.01; Fig 10).

70 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

1

0.9 immature 0.8 y = 0.01x + 0.08

0.7

0.6

0.5

0.4

0.3

0.2 adult 0.1 y = 0.02x - 0.427

evaporative cooling efMiciency 0 28 30 32 34 36 38 40 42 44 air temperature (°C)

Figure 10 Evaporative cooling efficiency data of immature wild-living Cape Rockjumpers (Chaetops frenatus; n = 5) captured in summer 2015 from Blue Hill Nature Reserve, South Africa, over a range of air temperatures (29 – 43 °C) with lines of best fit shown for immature (black) and adults (grey).

Four immature individuals, as well as one adult male in summer and one adult female in winter, were removed from the chamber before reaching Tair = 42 °C, as they showed signs of stress (i.e.

Tb rising above 43.9 °C; see Methods). Table 1 shows comparative data for response variables

(RMR, EWL, Tb, and EHL/MHP) by season and age group at 30 and 39 °C. Air temperatures of 30 and 39 °C were chosen to include all individuals.

71 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Table 1 Body temperature (Tb), resting metabolic rate (RMR), evaporative water loss (EWL), and evaporative cooling efficiency (evaporative heat loss [EHL] / metabolic heat production [MHP]) at 30 and 39 °C from adult and immature wild-living Cape Rockjumpers (Chaetops frenatus) captured during summer 2016 and winter 2015 at Blue Hill Nature Reserve, South Africa.

Summer Winter Immatureii Adulti Adulti

RMR (W) at Tair ≈ 30 °C 1.59 ± 0.46 1.04 ± 0.38 0.85 ± 0.15

RMR (W) at Tair ≈ 39 °C 2.40 ± 1.36 1.17 ± 0.51 0.87 ± 0.13

-1 EWL (mg hr ) at Tair ≈ 30 °C 0.50 ± 0.14 0.32 ± 0.16 0.16 ± 0.06

-1 EWL (mg hr ) at Tair ≈ 39 °C 0.76 ± 0.34 0.53 ± 0.23 0.52 ± 0.23

Tb (°C) at Tair ≈ 30 °C 40.78 ± 0.59 40.44 ± 0.56 40.73 ± 0.48

Tb (°C) at Tair ≈ 39 °C 41.58 ± 0.83 41.95 ± 0.71 41.75 ± 0.50

EHL/MHP at Tair ≈ 30 °C 0.32 ± 0.05 0.31 ± 0.08 0.19 ± 0.04

EHL/MHP at Tair ≈ 39 °C 0.35 ± 0.11 0.47 ± 0.11 0.35 ± 0.04 I n = 11; ii n = 5

Table 2 shows summary statistics (t-value, F-value, sample size (n), and degrees of freedom (df)) for each response variables (RMR, EWL, Tb, and EHL/MHP) with each predictor variable (Tair, season, interaction of Tair and season, sex, and Mb).

72 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

response variedaWhenvariablesignificantly (T temperature air include variables Predictor [MHP]). production (T temperature body (EWL), loss water evaporative (29 temperatures air of range a over Africa, South Reserve, ( Rockjumpers Cape of variables response ( results Statistical 2 Table F - value, value, P - value, degrees of freedom [df] and sample size [ size sample and [df] freedom of degrees value, Chaet

with a with predictor variable, the a ops frenatus ops b , n eaoaie fiiny eaoaie et os EL / eaoi heat metabolic / [EHL] loss heat (evaporative efficiency evaporative and ), atrd uigsme 21 n wne 05a Bu Hl Nature Hill Blue at 2015 winter and 2016 summer during captured ) –

43 air ), season, the T the season, ), ° C ). Response variables include resting metabolic rate (RMR), (RMR), rate metabolic resting include variables Response ). P - value is presented in isvalue bold. air

× n season interaction, body mass (M mass body interaction, season ]) from linear from ])

mixed

- effects

models fitted to to fitted models b ), and sex. and ),

73 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Discussion

Contrary to my hypothesis and alternative hypothesis, greater rates of increase in resting metabolic rate (RMR), and evaporative water loss (EWL) as a function of air temperature (Tair) in summer suggest Rockjumpers have less evaporative cooling capacity (i.e. are more sensitive to high temperatures) in summer compared to winter. Although higher evaporative cooling efficiency

(evaporative heat loss (EHL) / metabolic heat production (MHP)) ratios in summer compared to winter indicate Rockjumpers do increase their evaporative cooling efficiency, the concomitant elevation in RMR and EWL in summer (up to 14.5 % increase in RMR and up to 36.2 % increase in EWL compared to winter) suggests higher energy demands high Tair in summer compared to winter. Rockjumpers also show greater increases of body temperature (Tb) as a function of Tair in summer, suggesting the increase in evaporative cooling efficiency is not enough to maintain Tb at high Tair. In contrast, white-browed sparrow-weavers from a desert site have adjustments centred on reduced EWL and RMR in mid-summer (Noakes et al. 2016). My data suggest that energy and water demands were substantially lower during winter in Rockjumpers. In addition, the breeding period of Rockjumpers, which extended into summer during my study (August through December, personal observations) may increase the energetic demands of reproductive organs, although I can only speculate this as a reason for the metabolic elevation observed in summer.

Body Condition

As discussed in the previous chapter, Rockjumpers were in significantly better condition in winter compared to summer, likely due to a combination of environmental factors and physical changes

(see Chapter 2). I initially expected that insect productivity would be increased in summer (Lee and

Barnard 2015b) with excess food availability allowing individuals to show an increase in metabolic rates, but this would generally be accompanied by an increase (or at least maintaining) of Mb.

74 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

These findings suggest Rockjumpers were not in energy balance during summer—their high summer energy demands exceeding energy supply. To date I am not aware of any attempts to quantify seasonal insect productivity in the Fynbos. I did not quantify insect abundance during either season, but I found it unexpected that Rockjumpers showed such a strong reduction in body condition during the summer season.

Resting Metabolic Rate

The higher RMR in summer compared to winter was unexpected, as I expected a lower RMR in summer to maintain a lower Tb to reduce overall MHP if they were acclimatized to summer heat

(Tieleman and Williams 2000; Williams and Tieleman 2005). However, if greater insulation in winter allows Rockjumpers to restrict the loss of metabolically produced heat (see Chapter 2:

Discussion), decreased insulation in summer may allow Rockjumpers to more readily lose heat to the environment and offset higher MHP. Although temperatures above 40 °C (~ Tb as discussed below) are rare at BHNR, the high summer metabolic rate of Rockjumpers could limit activity at

Tair approaching Tb. In fact, behavioural data on Rockjumpers has shown that at Tair > 25 °C

Rockjumpers show a strong preference for shaded microsites (K. N. Oswald, S. J. Cunningham, A.

T. K. Lee, P. Barnard, and B. Smit, unpublished data). The above shade-seeking patterns, taken together with finding that Rockjumpers have elevated metabolic heat production rates in summer, strongly suggest Rockjumpers may be at risk of excessive heat loading at relatively mild Tair during summer (Tattersall et al. 2012; Weathers and van Riper III 1982).

A higher summer RMR compared to winter may indicate Rockjumpers experience a change in energy demands, as lower metabolic rates are correlated with lower energy requirements (Williams and Tieleman 2005). As was discussed previously in relation to maintenance metabolic rate (see

75 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Chapter 2: Discussion), higher metabolic rates in summer may be due to breeding, which requires increased energy demands from changes in organ size/metabolic intensity (Vezina et al. 2006), and increased parental energy expenditure (Swanson 2010).

Evaporative Water Loss

The surprising increase in summer RMR was accompanied by elevated summer EWL rates; although Rockjumpers increased their MHP with a higher RMR in summer compared to winter, they also increased their heat dissipation through higher EWL. In general, most studies examining

EWL, both seasonal and non-seasonal, have been on desert species, and it is possible that non- desert birds do not follow the same patterns. Blue Hill Nature Reserve (BHNR) has low annual rainfall of 397 ± 98 mm (see Chapter 2: Methods) placing BHNR closer to a mesic climate (275 to

900 mm annual rainfall) as opposed to an arid climate (100 to 275 mm annual rainfall; Allsopp,

2014).

While arid-zone species decrease their EWL in summer as a mechanism to conserve water,

Rockjumpers may not require similar water conservation mechanisms; indeed, the two mesic populations of sparrow-weavers in Noakes et al. (2016) found similar EWL decreases in winter compared to summer. The results of Noakes et al. (2016) corroborated earlier EWL patterns observed by Tieleman et al. (2002) in comparing two mesic lark species (Skylarks, Alauda arvensis, and Woodlarks, Lullula arborea) to two desert lark species (Hoopoe Larks, Alaemon alaudipes, and Dunn’s Larks, Eramalauda dunni). Tieleman et al. (2002) suggested the increased

EWL patterns identified in mesic species was a response to higher metabolic heat production, both of which were identified in summer acclimatized Rockjumpers during this study.

76 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

A previous comparative study on heat thresholds in Fynbos-endemics and non-endemics, also from

BHNR, found Rockjumpers had an EWL inflection point (TEWL) at Tair = 31.2 °C (Milne et al.

2015), as opposed to TEWL at Tair = 36 °C in summer and Tair = 35.9 °C in winter measured for

Rockjumpers in my study. I suspected that BHNR experienced warmer Tair during my study, resulting in Rockjumpers being acclimatized to warmer Tair; however the average Tair at BHNR was 15.0 °C between 23 September and 29 November 2013 for Milne et al. (2015), which is warmer than BHNR during the winter portion of my study and colder than BHNR during the summer portion of my study (see Methods). The change in TEWL may be explained by differing methodology; I started my heat experiments at a Tair of 30 °C, which may not have been low enough to get a true inflection point of TEWL, whereas the experimental procedure of Milne et al.

(2015) began at 24 °C, lending their TEWL estimate greater statistical power. However, I also found

Rockjumpers began panting at a higher Tair than those in the previous study (34.2 °C in winter and

35.8 °C in summer compared to 33.6 °C in Milne et al. 2015).

Birds in the wild have been recorded as panting in general at Tair above 30 °C from increased activity and thus MHP (Gardner et al. 2016). It is possible MHP levels of Rockjumpers in my study differed from those in Milne et al. (2015), potentially from more demanding breeding activity. The

Rockjumpers captured for thermal testing during October to November 2013 from Milne et al.

(2015) consisted of six males (1 with a developing brood patch) and four females (one with well- defined brood patch, two with developing brood patch, and one with a re-growing brood patch; source: A.T.K. Lee, personal observations). Although this does not tell us what stage of breeding birds were, it is likely their Rockuumpers were still in early stages, whereas the birds captured during summer for my study all had fledged (or nearly fledged) young requiring greater provisioning by parents.

77 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Body Temperature

As with both RMR and EWL, Rockjumper Tb did not follow the expected pattern of a lower increase in Tb at increasing Tair as seen in most subtropical species. Body temperature values for

Rockjumpers at Tair = 30 °C are likely representative of Tb at Tair within TNZ, and thus representative of normothermic Tb. While Rockjumpers had a lower Tb in summer compared to winter at Tair = 30 °C (summer average = 40.44 °C; winter average = 40.73 °C; Table 1), they had a steeper increase in Tb at increasing Tair in summer compared to winter (Fig. 5). It has been suggested that allowing Tb to rise above normothermic levels is a mechanism for storing metabolically produced heat when the risks of dehydration from EWL are too high (i.e. facultative hyperthermia), again mostly in desert birds (Smit et al. 2013; Tieleman and Williams 1999; Wolf

2000). However, facultative hyperthermia involves a corresponding decrease in EWL, which did not occur in Rockjumpers, and so the greater increase of Tb at increasing Tair in summer was not facultative hyperthermia.

This may have to do with the generally mild temperatures of the Fynbos (Milne et al. 2015), as

birds that do not often experience Tair above Tb may not have the mechanisms to cope with those

Tair, such as was the case with the milder climate inhabiting population in Noakes et al. (2016).

What is interesting to note is that Tb of birds in summer was greater than Tb of birds in winter at Tair

> 39.3 °C (near the likely normothermic Tb values mentioned above), which was the average Tb =

39.3 °C for Rockjumpers at Tair = 30 °C from Milne et al. (2015). This lends further support that in

summer Rockjumpers were thermally stressed at Tair above Tb compared to winter. It is important to note the difference in Tb may be from differences in methodology, as Milne et al. (2015) measured Tb using cloacal thermocouples as opposed to my use of intra-peritoneal PIT tags.

78 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Evaporative Cooling Efficiency

While past studies suggested increases in RMR and EWL in summer compared to winter were indicative of a lower evaporative cooling capacity (see Introduction), the Rockjumpers’ increase in evaporative cooling efficiency as air temperatures were increased suggests they compensated for an elevated metabolic heat production by also elevating their evaporative water loss. However, average EHL/MHP ratios at 42 °C for Rockjumpers (summer = 0.52, winter = 0.43) were still much lower than the mean ratios recorded in three sparrow-weaver populations (summer = 1.32, winter = 1.04; Noakes et al. 2016). A low evaporative cooling efficiency of Rockjumpers suggests

they may not possess adequate mechanisms for maintaining Tb at Tair > 42 °C. As well, although

Rockjumpers did show a tendency toward having greater efficiency in summer compared to winter, the difference was not statistically significant.

Immature vs. Adult

Immature Rockjumpers had increasing RMR, EWL, and EHL/MHP at increasing Tair, showing similar patterns to adult Rockjumpers. However, while immature Rockjumper EWL and Tb did not increase at different rates compared to adults, immature Rockjumper RMR increased at a greater rate than adults. I hesitate to draw any conclusion on the differences between immature and adult

Rockjumpers due to small sample sizes and a large amount of variance. However, it seems plausible to speculate the variance in thermoregulatory responses to heat of the immature

Rockjumpers compared to adults indicates a lack of well-established metabolic machinery; this may be expected as immature individuals were still developing (the immature individuals in this study were less than three months old), and may not have fully developed heat dissipation

mechanisms. Additionally, a statistical comparison of the Tair at which immature individuals began panting compared to adults could not be accomplished as immature individuals all began panting

79 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

immediately after entering the chamber at Tair = 30 °C.

The even lower heat threshold of immature individuals compared with adults suggests a few explanations for the decreasing population of Rockjumpers observed in warmer areas (Milne et al.

2015), on which I can speculate. Firstly, I speculate that part of the population decline is due to survival rates of immature individuals. While during my study I did not witness any casualties of either immature or adult individuals (birds identified using a unique combination of colour rings;

K. N. Oswald unpublished data), the environmental Tair at BHNR was rarely as warm as those experienced by birds in our experimental conditions (although operative Tair may be very high).

Secondly, although there is currently no information on Rockjumper dispersal patterns, if immature birds disperse, their increased heat sensitivity may be a limiting factor. With climate change likely to increase temperatures in the already warm valleys separating the Cape Fold Mountain ranges,

Rockjumpers may find themselves unable to disperse using their normal patterns. Both of these speculations are avenues for future study in determining the cause of population declines for

Rockjumpers.

Limitations

I identified two limitations in my study. Firstly, although I managed to obtain a larger sample size than initially anticipated, a larger sample size would have lent more strength to my statistical analyses, especially in regards to immature individuals. Although my sample of adult birds had sufficiently low variation, the large variation among immature individuals detracted from any statistical conclusions in comparing immature to adult Rockjumpers. Secondly, the metabolic measurements I have obtained may have been less to represent steady-state values than in Milne et

al. (2015), as the latter study had Rockjumpers at each Tair for up to 30 min (up to four hours total)

80 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

mine were kept at each Tair a maximum of 20 min (up to two-and-a-half hours total). While the values in Milne et al. (2015) may have been a better indication of birds at rest, I am able to say that constant monitoring gave me confidence that birds had reached steady-state RMR and EWL traces

as a function of Tair, and so this difference is likely negligible.

Conclusions

Overall, Rockjumpers do seem to respond seasonally to warmer temperatures in summer, but to the best of my knowledge not to the same scope as reported in other species. While evaporative cooling efficiency was elevated in summer, indicating greater evaporative cooling capacity in summer, the corresponding elevation of RMR and EWL seem costly in terms of water and energy demands in an environment where the summer period is the hottest and driest. However, seasonal heat response data on birds remains scarce, and Rockjumpers occupy cooler regions than all other published studies. To the best of my knowledge, seasonal heat threshold studies on the White- browed Sparrow-weaver(Noakes et al. 2016).

I found very little evidence that Rockjumpers possess similar heat tolerance typical to other subtropical birds, which in combination with previously found lack of evidence for extreme cold adaptation (Chapter 2), provides strong evidence of a thermal niche specific to moderate climate characteristic of the Fynbos biome. Considering the declining Rockjumper population associated with increased temperature, I suspected that the population decrease can be partially explained by the low heat thresholds of immature Rockjumpers if it decreases their survival rate or if they are the main avenue of dispersal. Moreover, my findings that EWL is high in summer further

corroborates findings from Milne et al. (2015) that elevated water demands in Rockjumpers at Tair

> 30 °C could play an important role in their sensitivity to increasing temperatures.

81 Krista Oswald Dissertation Chapter 3: Seasonal Responses to Heat

Although, as was suggested in Milne et al. (2015), declining populations of Rockjumpers may be due to physiological constraints combined with geographical barriers, I suggest a few avenues of study to further determine possible causes. Firstly, I suggest an examination of the temperature dependency of behavioural thermoregulation mechanisms by obtaining Rockjumper behaviour data over a range of temperatures. In general, organisms can cope with temperature extremes through phenotypic adjustments of both physiological and behavioural mechanisms (Chevin et al. 2010;

Valladares et al. 2014), and so Rockjumpers may adjust their behaviour instead of adjusting their

physiology in warmer Tair, and use behavioural mechanisms such as huddling to stay warm in colder Tair. Furthermore, to truly understand if climate change and its accompanying warmer Tair are affecting population declines in Rockjumpers, I suggest a genetic analysis of current Rockjumper gene-flow and diversity among the various Cape Fold mountain ranges. Rockjumper populations may have becoming genetically isolated due hot and dry valleys that separate the ranges of the

Cape Fold Mountains. After analysing Rockjumper population genetics, and identifying any potential genetic bottlenecks, the results can be overlaid on predicted climatic data for the Fynbos to help identify both Rockjumper range changes and dispersal barriers. This in turn will identify corridors that remain for dispersal, with these given conservation priority.

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87 Krista Oswald Dissertation Chapter 4: General Conclusion

Chapter 4: General Conclusion

Determining a species’ thermal niche has important implications for determining future ranges under climate warming; this is especially true for those species that have shown declining populations in areas of their habitat experiencing warming temperatures. In general, the most common technique used to predict species’ distributional responses to a changing environment is climate envelope modeling (Pearson and Dawson 2003; Hijmans and Graham 2006; Williams et al.

2008; Buckley and Kingsolver 2012) often referred to as climatic—or bio-climatic—envelope models (CEMs or BCEMs). While these BCEMs have often been based solely on correlations built on environmental factors and species absence/presence, there is an increasing amount of interest in BCEMs incorporating phenotypic flexibility to improve prediction models (Chevin et al.

2010; Dawson et al. 2011; Hijmans and Graham 2006). Indeed, Hijmans and Graham (2006) suggest physiological flexibility in particular will be necessary for future efficacy of BCEMs. The aim of my study was to determine whether Cape Rockjumpers (hereafter “Rockjumpers”) possess seasonal physiological flexibility, and so may possess physiological mechanisms to deal with predicted increases in temperatures. In particular, I hoped to find evidence as to why their populations are declining in areas experiencing the most warming.

By statistically testing physiological responses at both ends of the thermal environment, I found that Rockjumpers are not especially tolerant toward temperatures of either cool or warm extremes.

In testing cold acclimatization I found Rockjumpers had lower maintenance metabolic rates

(BMR), lower thermal conductance at BMR (CBMR), and higher maximum thermogenic capacity

(Msum), providing mixed support for my hypothesis regarding their cold acclimatization in winter.

Instead of regulating body temperature (Tb) in winter by increasing BMR and thus increasing

88 Krista Oswald Dissertation Chapter 4: General Conclusion

metabolically produced heat, I suggest that Rockjumpers maintain Tb by lowering BMR and increasing insulation to conserve what heat is produced. A higher Msum in winter compared to summer was expected from previous seasonal studies (Cooper and Swanson 1994; Dawson 1958;

Dawson et al. 1983; Liknes et al. 2002; McKechnie et al. 2015; O'Connor 1995). However, mean

Rockjumper Msum values for both seasons fell below the expected values for a ~ 50 g bird using a global data set (McKechnie et al. 2015), suggesting Rockjumpers are not especially cold tolerant.

I also found unexpected results in testing Rockjumper heat thresholds. I found that Rockjumpers had greater values, as well as greater rates of increase, in their resting metabolic rates (RMR), evaporative water loss (EWL), and Tb, all of which suggest lower evaporative cooling capacity in summer compared to winter. However, I did find that Rockjumpers had higher values for evaporative cooling efficiency in summer compared to winter, and so may be able to compensate for their elevated summer RMR. My data suggest that energy and water demands for Rockjumpers were substantially lower during winter. Additionally, the initiation of breeding during winter for some of the Rockjumpers used in this study (n = 4), and the presence of recently fledged young during summer (personal observations), may have provided unanticipated affects and results.

While my study provides support for a physiological basis for Rockjumper population declines in areas that experienced the most warming, physiological flexibility is not the only mechanism for determining how a species can cope with a changing environment. I suggest that future studies involve a more thorough examination of other mechanisms the Rockjumper possesses to cope with climate warming in order to more properly assess its vulnerability. For example, behavioural flexibility can also be used to adjust to short-term environmental changes (Dupoué et al. 2015).

89 Krista Oswald Dissertation Chapter 4: General Conclusion

As a range-restricted species with no ability to move to higher altitude or latitude under climate warming, an understanding on current geographic barriers to dispersal may give a clearer understanding of future potential barriers. I suggest a genetic study be done to determine current genetic diversity among Rockjumper sub-populations and to determine barriers to population dispersal. Physiological flexibility, behavioural flexibility, phylogeography, and landscape genetics, have all been suggested as methods for improving BCEMs and conservation planning

(Bernardo 2014; Scoble and Lowe 2010; Valladares et al. 2014).

While the Rockjumper is a single species model, I suggest it is an excellent study species for these future endeavours for three main reasons. Firstly, as was stated in the Chapter 1, it is a range- restricted species with population declines linked to climate warming (Milne et al. 2015).

Secondly, data on Rockjumper physiological flexibility for both warm and cool temperatures have already been collected by Milne et al. (2015), as well as in the current study. Thirdly, as a range- restricted species, thermal limitations for the Rockjumper may be a useful proxy for understanding such limitations for other range-restricted species unable to move to more thermally favourable habitats as temperatures increase. Collecting phenotypic flexibility data at different temperatures, and using that data to create BCEMs predicting future distributions, may help isolate which mechanisms species’ will rely on most for coping with climate warming.

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