METABOLIC PERFORMANCE AND DISTRIBUTION IN BLACK- CAPPED (POECILE ATRICAPILLUS ) AND CAROLINA CHICKADEES (P. CAROLINENSIS )

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Jennifer R. Olson

*****

The Ohio State University 2009

Dissertation Committee:

Professor Joseph B. Williams, Advisor Approved By

Professor H. Lisle Gibbs Advisor Professor William M. Masters Evolution, Ecology, and Organismal Biology Graduate Program

ABSTRACT

In endotherms, metabolic performance is associated with a wide array of ecological traits, including species distribution. Researchers have previously suggested that the northern boundaries of North American passerines are limited by their ability to sustain high metabolic rates required for thermoregulation. Black-capped chickadees

(Poecile atricapillus , BC) are year-round residents in most of Canada and the northern half of the United States, whereas Carolina chickadees ( Poecile carolinensis , CA) are found exclusively in the southeastern United States. These species hybridize along a narrow contact zone, which has been moving northward at a rate of about 1.6 km per decade, coincident with warming temperatures in Ohio. The location of the chickadee hybrid zone in Ohio closely matches air temperature isotherms, further suggesting that metabolic rate may correlate with distribution in these species.

We tested the hypothesis that distribution patterns of chickadees are linked with their rate of metabolism. For populations of BC and CA chickadees, we measured basal

(BMR) and cold-induced peak metabolic rates (PMR) from areas that differ in winter temperatures and supplemented this information with data from other studies. Our findings suggest a general relationship between colder temperatures and higher metabolic rates, though this trend was not robust among all locations.

ii Within Ohio, hybrids had a significantly higher mass-corrected BMR than either parental species. We suggest that the mtDNA-nDNA mismatch of hybrids may produce less efficient mitochondrial protein complexes, which in turn affects the efficiency of

ATP production, thereby increasing rate of oxygen consumption to meet ATP demands.

In chickadees, heat production during cold exposure is primarily achieved through shivering thermogenesis, an energetically expensive process that requires high rates of

ATP production. Thus, we predicted that northern populations would exhibit higher PMR compared to southern populations, facilitated by an elevation in enzyme activity of oxidative pathways. To explore this relationship, we measured in vitro activity of three

pectoral muscle metabolic enzymes that catalyze non-equilibrium, highly exergonic

reactions: citrate synthase (CS), phosphofructokinase (PK), and L-3-hydroxyacyl-CoA-

dehydrogenase (HOAD).

While we did not find any differences in mass- or protein-corrected enzyme

activities between the two species, we did observe a higher level of total muscle CS

activity in BC chickadees. In addition, PMR in BC chickadees was significantly

correlated with pectoralis mass and total muscle enzyme activity of CS, PFK, and

HOAD. We concluded that among BC chickadees, higher metabolic rates were primarily

a result of greater pectoralis mass, rather than an increase in tissue-specific metabolic

enzyme activity.

iii

This dissertation is dedicated to my loving and supportive Family.

iv

ACKNOWLEDGMENTS

I would like to thank the faculty and staff at Ohio State University Department of

Evolution, Ecology, and Organismal Biology for their continued support, especially Dr.

Joe Williams for his endless advice, inspiration, and sense of humor. I would also like to thank the additional members of my committee, Dr. Lisle Gibbs and Dr. Mitch Masters, for their guidance throughout my project.

This project would not have been possible without the cooperation of multiple landowners in Ohio and Tennessee, especially Steve Humrichauser and Judy Varner, who allowed me to capture birds on their properties. I would also like to acknowledge Sheldon

Cooper for his assistance trapping Wisconsin birds, as well as Dave Swanson for supplying data on South Dakota birds, and Sue Chaplin for going beyond the call of duty to find data that was covered in dust on New York chickadees. I would also like to acknowledge Dr. Mike Braun, Chris Huddleston, and Sarah Kingston at the Smithsonian

Institution for their assistance and advice in my genetic identification of chickadees. In addition, Dr. Marianne Jurkowitz provided indispensible assistance designing and executing the enzyme assays, with additional help from the lab of Dr. Douglas Pfeiffer.

Many thanks to both the Williams and Grubb Lab groups for their continued advice and comments on earlier drafts of these manuscripts, especially Dr. Thomas

Grubb for his role in initially developing this project and for his early guidance.

v Finally, I would like to thank my entire family for their continued love and support. Special thanks go to my parents, Len and Diane, and to Clint and Kyle for their selflessness and support.

This study was funded in part by NSF IBN 0212587 to Dr. Joe Williams.

vi

VITA

April 6, 1980……………..…………… Born – Chicago, IL.

2002…………………………………… B.S. Biology. Illinois Wesleyan University.

2002 – present…………………...…… . Teaching and Research Associate The Ohio State University.

PUBLICATIONS

1. Olson, J. R. and Grubb, T. C. Jr. (2007). Winter adaptations in chickadees and

titmice and the added effect of habitat fragmentation. In: Otter, K. A. ed. Ecology and

behavior of chickadees and titmice: an integrative approach. Oxford University Press,

London.

2. Desrochers, A., Otter, K. A., Bélisle, M. and Olson, J. R. (2007) Landscape

ecology, behavior, and conservation issues. In: Otter, K. A. ed. Ecology and behavior of

chickadees and titmice: an integrative approach. Oxford University Press, London.

3. Juliano, S. A., Olson, J. R., Tillman, E. G., & Hatle, J. D. (2004) Plasticity and

canalization of insect reproduction: testing alternative models of life history transitions.

Ecology 85 : 2986-2996.

vii FIELDS OF STUDY

Major Field: Evolution, Ecology, and Organismal Biology

viii

TABLE OF CONTENTS

Page Abstract……………………………………………………………………………... ii

Dedication………………………………………………………………………...... iv

Acknowledgements………………………………………………………………… v

Vita…………………………………………………………………………………. vii

List of Tables……………………………………………………………………….. xi

List of Figures……………………………………………………………………… xii

Chapters:

Winter adaptations in chickadees and titmice and the added effect of habitat 1. 1 fragmentation…………………………………………………………………... 1.1. Introduction………………………………………………………………... 1 1.2 Physiological adaptations to winter conditions…………………………….. 2 1.2.1 Nocturnal hypothermia and seasonal metabolic adjustments……… 2 1.2.2 Thermogenesis by shivering……………………………………….. 8 1.3 Behavioral modifications and ecological adaptations to winter conditions... 10 1.3.1 Over-wintering in heterospecific flocks……………………………. 10 1.3.2 Foraging behavior and food caching……………………………….. 12 1.3.3 Cavity roosting……………………………………………………... 15 1.4 Responses to habitat fragmentation………………………………………... 16 1.4.1 Species richness and identity………………………………………. 17 1.4.2 Woodlot edge effect………………………………………………... 17 1.5. Effects of fragmentation on wintering permanent resident birds………….. 18 1.5.1 Survivorship………………………………………………………... 18

ix 1.5.2 Effects of wind and temperature on isolated woodlots…………….. 22 1.5.3 Woodlot connectivity and inter-patch movement…………………. 24 List of References………………………………………………………………. 28 Metabolic Performance and Distribution in Black-capped and Carolina 2. 34 Chickadees……………………………………………………………………… 2.1 Abstract……………………………………………………………………. 35 2.2 Introduction………………………………………………………………... 36 2.3 Material and Methods……………………………………………………... 41 2.4 Results……………………………………………………………………... 47 2.5 Discussion…………………………………………………………………. 59 List of References………………………………………………………………. 65 Metabolic Performance and Aerobic Enzyme Activity in Black-capped and 3. 78 Carolina Chickadees 3.1 Abstract……………………………………………………………………. 78 3.2 Introduction………………………………………………………………... 79 3.3 Materials and Methods……………………………………………………. 83 3.4 Results……………………………………………………………………... 89 3.5 Discussion…………………………………………………………………. 101 List of References………………………………………………………………. 107 Bibliography………………………………………………………………………… 119

x

LIST OF TABLES

Table Page 2.1 Mean metabolic rate by location………………………………………... 49

3.1 Results of ANOVA and ANCOVA comparing total body mass and pectoral mass specific rates across species and populations………………………………………………….………….... 91

3.2 Results of ANOVA comparing whole-organism and mass-specific metabolic rates across species and populations…………………………. 95

3.3 Results of ANOVA and ANCOVA comparing metabolic enzyme activity across species and populations…………………………………. 98

xi

LIST OF FIGURES

Figure Page 1.1 Relationship of body temperature to ambient temperate for willow tits during different seasons with different L:D cycles: summer (crosses), late autumn or early spring (open circles), and midwinter (filled circles). Each point represents the minimum value recorded on a single night. Curves are fitted by hand. (From: Reinertsen and Haftorn 1983).. 6

1.2 Daily body temperatures of summer and winter acclimatized mountain chickadees and juniper titmice. Each point represents 1 individual. Lines represent a quadratic fit of the data. (From: Cooper and Gessaman 2005)…………………………………………………….. 7

1.3 Shivering amplitude (mV/sec) of six black-capped chickadees exposed for at least two hours to ambient temperatures of 2oC, 10oC, 15 oC, 20 oC, and 27 oC. One data point represents the mean amplitude of 10 shivering bursts. (From: Chaplin 1976)………………………………… 9

1.4 Wind-dependent (A) and temperature-dependent (B) foraging curves for black-capped chickadees and tufted titmice wintering in a deciduous woodlot. Each “sawtooth” consists of a slope representing the average rate of individual movement, and a flat segment denoting the time spent on an average stop. The diagonal common to all sawteeth indicates the average foraging speed over one minute. (From: Grubb 1978)………… 14

xii 1.5 Relationship between annual probability of survival, woodlot area, and the presence of supplemental food for Carolina chickadees and tufted titmice. (From: Doherty and Grubb 2002)……………………………… 19

1.6 Linear regressions for the distance of Carolina chickadees and tufted titmice from the windward edges of woodlots against the wind speed, blocked by temperature range (a-d). (From: Dolby and Grubb 1999)….. 23

2.1 Position of the chickadee hybrid zone in relation to mean minimum January temperatures. We approximated zone position from North American Breeding Bird Survey data (http://www.mbr- pwrc.usgs.gov/bbs/bbs.html), Tanner 1952, Brewer 1963, Rising 1968, Robbins et al. 1986, Grubb et al. 1994, and Sattler and Braun 2000. Isotherms are based on the mean minimum daily temperature in January as depicted in U.S. Geological Survey (1970)………………………….. 39

2.2 Genetic identity of Ohio birds along a transect of Ashland County, OH (n=174). Distance zero on the x-axis is the northern most woodlot sampled, located at 41°6'N. Distance forty on the x-axis is equivalent to forty km south of this woodlot. Birds assigned a probability of being BC >75% are labeled BC (filled circles), 25-75% are HY (crosses), and <25% are CA (open circles). The genetic identities of samples from Wisconsin (WI, n=10) and Tennessee (TN, n=10) are also included for reference, as well as samples from extreme northern and southern Ohio (G = Geauga County, 41°22'N, n=6; L = Lawrence County, 38°43'N, n=18). The additional Ohio samples are included for genetic reference only, and are not incorporated in metabolic or statistical analyses…………………..…………………………………………….... 48

xiii 2.3 The relationship of BMR (A) and PMR (B) to mass in BC (filled circles) and CA (open circles). Linear regression lines are plotted solid for BC and dashed for CA ( p<0.001 for both). Sample sizes: BC BMR n=71, CA BMR n=24, BC PMR n=77, CA PMR n=22………………… 51

2.4 Mean BMR (A) and PMR (B) versus mean minimum January temperature for all locations included in this study. Metabolic rate is presented as least square means controlling for mass. Linear regressions were calculated by combining data for both species ( p<0.001 for both BMR and PMR)………………………………………………………… 52

2.5 Mean BMR (A) and PMR (B) for CA and BC chickadees. Metabolic rate is presented as least square means controlling for mass. Different letters identify significant differences in mass-corrected metabolic rate between populations. Sample sizes can be found in Table 2.1. Abbreviations used: AK=Alaska, WI=Wisconsin, SD=South Dakota,

NY=New York, OH M=Ohio, Munzinger (1974) data, OH BC =Ohio

black-capped chickadee data from this study, OH CA =Ohio Carolina chickadee data from this study, and TN=Tennessee…………………… 54

2.6 Mean BMR (A) and PMR (B) for each Ohio species. Metabolic rate is presented as least square means controlling for mass, and different letters identify significant differences in mass-correct metabolic rate between populations. Sample sizes can be found in Table 2.1.

Abbreviations used: OH BC =Ohio black-capped chickadee, OH CA =Ohio Carolina chickadee, and OH =Ohio hybrid chickadee.……………….. HY 56

xiv 2.7 Mean Tcl for each chickadee population. Metabolic rate is presented as least square means controlling for mass, and different letters identify significant differences in mass-correct metabolic rate between

populations. Sample sizes are as follows: WI n=11, OH BC n=18, OH CA n=10, TN n=13………………………………………………………….. 57

3.1 Mean total body mass (A - species, B - populations) and pectoralis mass (C - species, D - populations) for chickadee species and

populations. Sample sizes: BC n=22, CA n=16, WI n=11, OH BC n=11,

OH CA n=5, TN n=5. Pairs designated with a ( ∗) are significantly different (P<0.05)……………………………………………………….. 90

3.2 The relationship of BMR (A) and PMR (B) to mass in BC (filled circles) and CA (open circles). Trend lines are plotted solid for BC and dashed for CA. Sample sizes: BC BMR n=22, CA BMR n=9, BC PMR n=20, CA PMR n=9…………………………………………………….. 93

3.3 Mean whole-organism and mass-specific BMR (A) and PMR (B) for CA and BC chickadees. Sample sizes: BC BMR n=22, CA BMR n=9, BC PMR n=20, CA PMR n=9. Pairs designated with a ( ∗) are significantly different ( P<0.05)…………………………………………. 94

3.4 The relationship of BMR (A) and logPMR (B) to pectoralis mass in BC (filled circles) and CA (open circles). Trend lines are plotted solid for BC and dashed for CA. Sample sizes: BC BMR n=22, CA BMR n=9, BC PMR n=20, CA PMR n=9……………………………………. 96

xv 3.5 Scatterplots of total pectoralis enzyme activity for CS (A), HOAD (B), and PFK (C) versus logPMR for BC (filled circles) and CA (open circles). Trend lines are plotted solid for BC and dashed for CA. Correlation coefficients and significance levels are presented next to each plot. Sample sizes: BC n=20, CA n=9…………………………….. 99 3.6 Scatterplots of total pectoralis enzyme activity for CS (A), HOAD (B),

and PFK (C) versus logPMR for WI (filled triangles) and OH BC (open

triangles). Trend lines are plotted solid for WI and dashed for OH BC . Correlation coefficients and significance levels are presented next to each plot. Sample sizes: WI n=9, OH n=11………………………….. BC 101

xvi

CHAPTER 1

WINTER ADAPTATIONS IN CHICKADEES AND TITMICE AND THE ADDED EFFECT OF HABITAT FRAGMENTATION 1

Jennifer R. Olson and Thomas C. Grubb, Jr.

Department of Evolution, Ecology, and Organismal Biology, The Ohio State University

1.1. Introduction

Many North American Paridae have ranges that expose them to temperate winter

seasons. Chickadees and titmice possess several physiological and behavioral adaptations

that allow them to manage the reduced food supply and high thermoregulatory demands

of this environment. Yet, increased habitat fragmentation posed by agricultural and urban

expansion also presents a further stress to these populations. Furthermore, the interaction

between responses to winter and responses to fragmentation is assumed to be a major

factor constraining populations that are presented with both of these tasks. With this

1Chapter one of this dissertation has been previously published. Oxford University Press has granted permission in order to reprint the chapter within the dissertation.

1 chapter, we review research on physiological adaptations to reduced temperatures, and then address how microclimate change induced by habitat fragmentation can affect the response potential of over-wintering birds.

1.2. Physiological adaptations to winter conditions

Over-wintering in north-temperate regions can be energetically challenging for small passerines that must endure cold temperatures, reduced food supply and a shorter foraging time. For example, during harsh winters at the northern edge of their range, black-capped chickadees are exposed to normal low temperatures of -30 oC and as few as

5 hours of daylight (Sharbaugh 2001).

Relative to their body size, chickadees and titmice possess a large surface area

over which heat can be lost. Most mature adults are in the 10-20g range and are therefore

challenged by a high surface-to-volume ratio. Such birds have an internal body

temperature of approximately 40 oC, so exposure to extreme winter environments (-50 oC) can result in a 90 oC temperature gradient in less than 2 cm, between ambient conditions

and body core (Sharbaugh 2001). In order to survive in conditions of high

thermoregulatory demand, chickadees and titmice have developed several physiological

adaptations, including the use of nocturnal hypothermia, and shivering thermogenesis.

1.2.1 Nocturnal hypothermia and seasonal metabolic adjustments

Maintaining an appropriate body temperature is metabolically demanding and

birds can partially overcome this challenge through the use of nocturnal hypothermia.

Nocturnal hypothermia has been observed in several families of Order Passeriformes,

2 including Parids (Reinertsen 1996). This moderate reduction in body temperature is not as extreme as the torpor observed in hummingbirds (Wolf and Hainsworth 1972) and poor-wills (Jaeger 1949). Several species of European (Grossman and West 1977) and

North American Parids exhibit moderate nocturnal hypothermia in response to low ambient temperatures (Chaplin 1976), and the exact nocturnal energy savings to a hypothermic bird depend on several factors, including ambient temperature, body temperature, body mass, and the duration of hypothermia.

In order to survive overnight in winter, birds must achieve a thermal balance where the metabolic heat production equals the heat lost. In low ambient temperatures, such a balance is accomplished by either increasing consumption of energy stores or decreasing the core body temperature. Reinertsen and Haftorn (1983) suggested that a change in posture or erection of the feathers is an initial response to colder temperatures, followed by a reduction in body temperature until a thermal balance is achieved. It is at this body temperature that the bird will remain throughout the night.

Entering a hypothermic state can result in substantial energetic savings throughout the night, perhaps allowing for survival otherwise not possible. For example, lowering the body temperature from 39 oC to 33 oC in an ambient temperature of -30 oC will result in

a 10% reduction in oxygen consumption. For an 11.5g bird, this yields an overnight

energy savings of approximately 925 calories (Reinertsen and Haftorn 1983, 1986).

Another study showed that reducing nocturnal body temperature by only 8 oC will provide a chickadee with 72 additional minutes of fasting endurance, an energy savings that could mean the difference between overnight survival and death (Sharbaugh 2001).

3 The proximate factor that induces a hypothermic state is unknown, although the degree of hypothermia is correlated with body mass and fat reserves. Reinertsen (1996) suggests that hormones or plasma metabolites contribute to a sequence of events transforming information on energy reserves into the appropriate thermal response.

There is some lack of consistency in the results of studies on Parid hypothermia, which might be attributed to several factors, including different techniques of body temperature measurement and the period of time for which the birds were held captive prior to testing.

Grossman and West (1977) observed an average decrease in body temperature of only

3oC (to 38.5 oC) for winter-acclimatized black-capped chickadees in Fairbanks, Alaska

(65 oN) when the birds were measured at ambient temperatures of 27 oC to -50 oC.

However, birds of the same species in New York have shown body temperatures

approximately 10 oC below normal when exposed to ambient temperatures of 0 0C, which resulted in a 32-45% lower oxygen consumption than predicted for homeotherms of a similar size (Chaplin 1976). Nocturnal body temperatures of European great tits (Steen

1958) and willow tits (Reinertsen and Haftorn 1983) showed similar patterns to New

York chickadees and were 6-8oC lower than their diurnal temperatures. In addition, the stable hypothermic body temperature of willow tits was strongly correlated with ambient temperature.

North American and Eurasian Parids apparently differ in their arousal from a

hypothermic state. Chaplin (1976) found that black-capped chickadees captured in New

York and kept at overnight ambient temperature of 0-5oC were still hypothermic in the

early morning, but could regain normal body temperatures with 1-2 minutes of

4 intermittent flying. This does not conform to the data from European species that returned to normal body temperatures in the morning even if kept in dark chambers (Chaplin

1976).

Wind intensity can also affect a bird’s use of hypothermia. Mayer et al. (1982) found that Carolina chickadees would reduce their body temperatures to 35 oC at 5 oC

ambient temperatures in the absence of wind. Body temperatures were reduced further (to

30 oC) when birds were exposed to -10 oC temperatures and a wind speed of 10 km/h, and this decrease in body temperature corresponded to a 33% energy savings when compared to the metabolic requirements for a bird maintaining a 40 oC body temperature under

those same conditions.

Seasonal variation in hypothermic states is also present in North American

chickadee populations. For black-capped chickadees collected in South Dakota (42 oN), a

larger percentage of winter than summer birds became hypothermic in cold stress tests

(Cooper and Swanson 1994). Similar trends were also evident in willow tits from Norway

(63 oN); birds acclimatized to winter conditions showed a much larger decrease in body

temperature than summer-acclimatized birds (Reinertsen and Haftorn 1983) (Figure 1.1).

In contrast, measurements from mountain chickadees and juniper titmice captured in

Utah (41 o52’N) showed that both species achieved considerable nocturnal hypothermia throughout the year (Cooper and Gessaman 2005). Depth of hypothermia also did not vary seasonally for either species (Figure 1.2), and the authors suggest that the region’s high climatic variability may be the source of this indifference. Both species reduced their body temperatures by 3-11 oC, which resulted in nocturnal energy savings of up to

28% in titmice and up to 50% in chickadees. Similar results were seen in Alaskan

5

Figure 1.1. Relationship of body temperature to ambient temperate for willow tits during different seasons with different L:D cycles: summer (crosses), late autumn or early spring

(open circles), and midwinter (filled circles). Each point represents the minimum value recorded on a single night. Curves are fitted by hand. (From: Reinertsen and Haftorn

1983.)

6

Figure 1.2. Daily body temperatures of summer and winter acclimatized mountain chickadees and juniper titmice. Each point represents 1 individual. Lines represent a quadratic fit of the data. (From: Cooper and Gessaman 2005.) 7 black-capped chickadees which also utilize nocturnal hypothermia in both winter and summer months (Sharbaugh 2001). This could be due, in part, to the relatively cold and short summers experienced at this latitude—conditions favoring individuals that maintain their cold acclimatization year-round.

Several studies also provide evidence that winter-acclimatized birds are more cold tolerant than summer birds. Standard metabolic rates of winter chickadees were 18% greater than those of summer birds, and winter birds also showed a significant increase

(36%) in maximal thermogenic capacity, another likely adaptation to winter conditions

(Cooper and Swanson 1994). These differences in physiology occurred despite the fact that there was no seasonal variation in body mass or visible fat (Cooper 2002).

In contrast, the standard metabolic rates of Alaskan black-capped chickadees did not differ between seasons (Sharbaugh 2001).

1.2.2 Thermogenesis by shivering

In addition to lowering nocturnal body temperatures, chickadees also respond to cold ambient temperatures with shivering thermogenesis, facilitated primarily by the pectoral muscles. Shivering as a means of heat production has been documented in several avian species, including pigeons (Columba livia , Hohtola 1982), house finches

(Carpodacus mexicanus , Marsh et al. 1984), and Japanese quail (Coturnix coturnix japonica , Hohtola and Stevens 1986), and the inverse relationship between shivering magnitude and ambient temperatures is well-established.

Chaplin (1976) observed an increase in both frequency of shivering bursts and mean duration of bursts at lower ambient temperatures in winter-acclimatized

8

Figure 1.3. Shivering amplitude (mV/sec) of six black-capped chickadees exposed for at least two hours to ambient temperatures of 2 oC, 10 oC, 15 oC, 20 oC, and 27 oC. One data

point represents the mean amplitude of 10 shivering bursts. (From: Chaplin 1976.)

9 black-capped chickadees. When these measurements were combined, there was a seven- fold increase in total shivering time as temperature decreased from 27 oC to 2 oC..

Electromyographic measurements of the pectoralis muscle also showed an increase in shivering amplitude with decreasing temperature, corresponding to a dramatic increase in heat production (Figure 1.3). The fact that during their normal daily activities chickadees spend so little of their time in continuous flight supports the hypothesis that the pectoral muscles have a vital role as organs of heat production. Cooper (2002) found that pectoral muscle mass of wintering mountain chickadees and juniper titmice was 33% and 18% greater, respectively, than during the summer. This seasonal variation could help to satisfy the increased need for shivering thermogenesis experienced by wintering birds.

1.3. Behavioral modifications and ecological adaptations to winter conditions

Permanent-resident woodland birds, such as chickadees and titmice, possess several behavioral modifications that may reduce energetic costs under conditions of high thermoregulatory demand. This can be accomplished through adjustments in their foraging and roosting locations in order to minimize exposure to harmful winds and temperatures. However, as members of heterospecific flocks, these birds must do so while minimizing interspecific competition for food and roosting sites.

1.3.1 Over-wintering in heterospecific flocks

Most North American Parids over-winter as members of mixed-species flocks.

Within these flocks, chickadees and titmice serve functionally as nuclear species, facilitating the flocks’ formation and movement. Additional, satellite species, such as

10 woodpeckers and nuthatches, maintain a more passive role as flock followers (Morse

1970; Austin and Smith 1972; Buskirk 1976; Hutto 1994; Dolby and Grubb 1999b;

Chapter 15 1). Removing chickadees and titmice from small woodlots resulted in satellite

species occurring together less often, further supporting the nuclear species’ role in flock

cohesion (Dolby and Grubb 1999b).

Although this multi-species social arrangement can provide individuals with both antipredator defense and increased foraging efficiency (Morse 1970; Berner and Grubb

1985), the resulting interspecific competition can also have a negative effect. For example, Carolina chickadees and tufted titmice forage together during the non-breeding season, with the titmice the more socially dominant species. When titmice were removed from a woodlot, the more subordinate chickadees responded by increasing feather growth rate (an index of an energetically demanding process) and expanding their foraging niche

(Cimprich and Grubb 1994). This suggests the presence of interspecific competition between the species for local food resources. This response has also been observed in

Palearctic Parids. When varied tits were removed from flocks containing the smaller great tits, allowing the great tits to expand their niche and forage at greater heights

(Jablonski and Lee 2002). A similar change in foraging location was also observed in socially subordinate coal tits when dominant crested tits and willow tits were removed from Swedish forests (Alatalo et. al 1985). Coal tits shifted their foraging sites in trees to

1Citations of other chapters refer to the original publication.

11 include the inner regions previously occupied by the more dominant species, suggesting the presence of either interference or exploitation competition. In a subsequent study

(Alatalo et. al 1987), the smaller species were removed, causing the dominant species to expand their foraging regions, suggesting that in forest tits, competition for limited resources can occur even without interference from dominants.

In Cimprich and Grubb’s experiment, the removal of titmice from a woodlot may have also reduced competition for roosting sites, providing the remaining chickadees with more protection from harsh winter conditions. Although competition for roosting sites is common among great tits and blue tits (Dhondt et al. 1991), the refusal of chickadees to use artificial roost boxes in winter suggests that roosting sites in this system may not be a limiting resource.

1.3.2 Foraging behavior and food caching

Inclement winter weather can have a deleterious effect on the foraging of chickadees and titmice by reducing the variety of locations a bird can exploit while keeping thermal stress at a minimum. In a study examining the effect of several climatic factors on the foraging rates of black-capped chickadees and tufted titmice in New Jersey,

Grubb (1975) found that temperature and wind speed significantly affected the choice of substrate. In cold and windy weather, birds adjusted their foraging habits to minimize exposure to these elements. As wind speed increased, both chickadees and titmice reduced their foraging height and spent more time foraging in shrub-type vegetation, both of which would offer more protection from the wind.

12 In addition to altering their foraging location, black-capped chickadees and tufted titmice reduced foraging rates as ambient temperature decreased and wind speed increased (Grubb 1978). For example, in conditions of high wind (3.1-4.0 m/s), tufted titmice spent 11.5% more time stationary and traveled only 15.3% as rapidly as in low winds (0.1-1.0 m/s). An increase in temperature from -10 oC to 20 oC quadrupled the foraging speed of chickadees. The difference in foraging rate can be partially explained by a change in foraging substrate, and it may not be the weather conditions themselves that dictate the speed at which an individual feeds. In higher winds, both chickadees and titmice spent more time foraging on the ground where their movements were dominated by small, short “hops.” The change in location was then more directly responsible for modifying the foraging technique to a method that was not as rapid (Figure 1.4).

For birds with access to supplementary food at bird feeders, as temperatures decrease, time spent participating in energetically demanding activities, such as foraging and flight, decreases and more time is spent at rest. During cold weather, black-capped chickadees spent as much as 70% of the day at their roosting site, and 10% of the time not spent at the roost was spent resting (Kessel 1976). Foraging ranges also decrease in size with temperature, presumably to minimize time spent away from the roost. Black- capped chickadee populations in Alaska tend to become more concentrated at higher elevations due to the presence of thermal inversions. For example, temperatures can be

25 oC warmer at 1000 m than in lowland areas (Hotly 1973).

Many Parids collect and store food for consumption at a later time. Although time from caching to food retrieval varies (see Chapter 2), birds do accumulate caches during late autumn in preparation for the colder months when nutritional resources are more

13

Figure 1.4. Wind-dependent (A) and temperature-dependent (B) foraging curves for black-capped chickadees and tufted titmice wintering in a deciduous woodlot. Each

“sawtooth” consists of a slope representing the average rate of individual movement, and a flat segment denoting the time spent on an average stop. The diagonal common to all sawteeth indicates the average foraging speed over one minute. (From: Grubb 1978.)

14 difficult to find and foraging time is limited due to shorter, colder days. Caching of food resources has many benefits to birds that over-winter in harsh climates. The presence of a predictable food source decreases a wintering bird’s risk of starvation while allowing it to maintain a lower body mass, thereby reducing the risk of predation. In addition, caching birds are able to spend less time foraging, minimizing their exposure to low temperatures and high winds (Pravosudov and Grubb 1998). Further discussion of food caching in

Parids can be found in Chapters 2 and 3.

1.3.3 Cavity roosting

Chickadees and titmice utilize both natural and artificial cavities as nocturnal roost sites (Bent 1947). The microclimate of a roosting cavity can minimize thermoregulatory stress by providing shelter from wind and precipitation, and by reducing radiative heat loss. North American and Eurasian Parids roost in a variety of locations, but most roosting sites can be found in holes and recesses of dead or rotting trees (Bent 1947; Pitts 1976; Perrins 1979). Most roosts observed by Pitts (1976) had only a single side opening, but others had multiple openings and did not offer any better protection than dense vegetation. Willow tits and Siberian tits will also roost in holes in the snow (Smith 1991), and this behavior has also been witnessed in black-capped chickadees (C. C. St. Clair, pers. comm.).

Communal roosting is not employed by chickadees or titmice, however, several different chickadees may use the same roost over several nights (Pitts 1976).

Occasionally, these roosts were also occupied by other species, such as tufted titmice.

15 Small cavities provide a relatively stable microclimate for overnight roosting, especially in metabolically challenging winter conditions. Mayer et al. (1982) found that by roosting in a cavity, a bird’s net radiant heat loss could be reduced 60 to 100 percent, and convective heat loss was essentially zero due to the lack of any wind inside the roost site.

In fact, wind protection may be the primary benefit conveyed by cavity roosts. Cooper

(1999) showed that when air temperatures inside a cavity were 4.3-5.6 oC warmer than open sites for chickadees (1.7-6.3 oC warmer for titmice), the standard operative temperature ( Tes )—which incorporates solar and thermal radiation, ambient temperature, and windspeed with a species' resistance to heat loss—was much higher: 4.5-14.8 oC

warmer than open sites for chickadees and 3.2-23.0 oC warmer for titmice. This dramatic

increase in Tes translates into significant overnight energy savings. For example, in

Cooper’s study, the wind reduction experienced by winter cavity roosting led to nocturnal

energy savings of 37.6% for chickadees and 25.1% for titmice, which increased fasting

endurance by 7.3 and 5.7 hours, respectively. Fasting endurance can become especially

important when foraging is hindered by inclement weather.

1.4. Responses to habitat fragmentation

The ranges of many North American Parids are at least partially coincident with

agricultural and urban habitat modifications, which often results in a fragmented or

patchy distribution of the birds (see also Chapter 15). Habitat fragmentation in avian

populations has received much attention, especially in light of recent conservation

concerns. The negative effects of habitat fragmentation on avian communities are well

documented and include reduced food supply, higher nest predation, and greater risk

16 from predators of adults. In addition, smaller woodlots may contain lower-quality individuals. These consequences are of concern regarding persistence of chickadee and titmice populations.

1.4.1 Species richness and density

Several studies have shown species richness and within-species density to be strongly associated with woodlot size; in general, woodlots with greater area will harbor more species and more individuals. In studies of permanent-resident birds in northern

Ohio, including Carolina chickadees and tufted titmice, Doherty and Grubb (2000) found that woodlot area was the most important predictor of species presence and density.

Smaller woodlots tend to have lower food supplies, fewer refuges from predation, and greater thermoregulatory costs due to less protection from harsh winter temperature and wind, all of which presumably contribute to the observed trends in avian presence.

Researchers (Telleria and Santos 1995) working in winter landscapes have also detected highly significant positive relationships between woodland fragment size and both species richness and density in Eurasian Parid species, including great tits, crested tits, and coal tits, as well as several other woodland passerines. However, blue tits failed to show an increase in density with woodlot size.

1.4.2 Woodlot edge effect

Forest fragmentation also contributes to an increase in edge habitat. Some avian species will nest along woodland edges in addition to the interior, though with different degrees of success. For example, the nests of Carolina chickadees (in addition to other

17 Poecile spp.) are often usurped by migratory house wrens (Troglodytes aedon ). Due

presumably to the house wrens’ preference for edge habitat, chickadee nesting success is

significantly greater in woodland interiors (Doherty and Grubb 2002).

1.5. Effects of fragmentation on wintering permanent resident birds

1.5.1 Survivorship

In addition to being associated with species presence and density, it stands to

reason that woodlot size also affects the annual survivorship of permanent-resident birds.

Combining previously mentioned constraints of a fragmented landscape (e.g. lower food

supplies, higher predation rates, etc.) with the increased thermoregulatory demands of

winter may reduce survivorship in such woodlots. Temperature may magnify any effects

of fragmentation alone because sufficient energy and nutritional resources may become

even more difficult to find on cold, short winter days (Grubb and Pravosudov 1994).

Although studies have shown temperature to be positively correlated with great tit

survivorship in Holland and Finland (see citations in Doherty and Grubb 2000), its effect

on black-capped chickadee populations was minimal (Loery and Nichols 1985; Loery et

al. 1987).

Doherty and Grubb (2002) were the first to explore the relationship of survival in

a fragmented landscape to factors influencing winter severity, including the presence or

absence of supplemental food. Their study was executed in a highly-fragmented

agricultural county of Ohio, with ~10% forested area (Steiger et al. 1979) and two

riparian corridors. Carolina chickadee numbers responded more strongly to woodlot size

and supplemental food than the other species tested. Results showed that the annual

18

Figure 1.5. Relationship between annual probability of survival, woodlot area, and the presence of supplemental food for Carolina chickadees and tufted titmice. (From:

Doherty and Grubb 2002.)

19 probability of survival was positively associated with woodlot area in chickadees, however, a similar association was not apparent in tufted titmice (Figure 1.5). In addition, the presence of supplemental food also increased survivorship, most notably for the chickadees, a result that is consistent with observations from gray jays ( Perisoreus canadensis , Waite 1990) and European nuthatches ( Sitta europaea , (Nilsson et al. 1993).

Due to their subordinate status within a heterospecific flock, chickadees are often forced to forage in suboptimal areas (Pierce and Grubb 1981). The possible foraging locations in a smaller woodland patch are, therefore, even further reduced, putting an even greater strain on this species to consume the necessary resources for over-night survival. Chickadees are also less likely than other flock species to cross gaps between woodlots in search of more optimal foraging conditions (Cimprich and Grubb 1994;

Chapter 15). Logically, increasing a woodlot’s size or providing supplemental food would help to counteract these disadvantages.

Woodlot size and supplemental food not only influence the annual survivorship of

Carolina chickadees in a fragmented landscape, but also the nutritional condition of those that do survive (Doherty and Grubb 2003). Food consumption has a direct effect on the fitness of winter birds, and the health of individuals can be compromised without making them victims of over-night mortality.

Through the use of ptilochronology, Doherty and Grubb (2003) were able to evaluate the nutritional condition of permanent-resident birds in winter and ascertain whether food availability was having a direct effect on survivorship, as opposed to other factors associated with a small woodlot size, such as increased predation (Nupe and

20 Swihart 1998). Using the width of growth bars on induced feathers as a surrogate for overall nutritional health, the researchers compared birds from both large and small woodlots, with and without supplemental food.

Daily feather growth of Carolina chickadees was positively correlated with the

woodlot area x food interaction, such that birds from large, supplemented woodlots had

the widest growth bars. Similar results were not observed in other species tested—tufted

titmice, white-breasted nuthatches ( Sitta carolinensis ), and downy woodpeckers

(Picoides pubescens ) — all of which maintain distinctly different foraging niches.

Previous studies on these species did record benefits from food supplementation (Grubb

1989; Grubb and Cimprich 1990), however, that work was conducted in a landscape of

more continuous woodland. The additional food in this study may not have been enough

to counteract the harm from such a small woodlot.

Snow cover can also impact the survivorship of winter-resident birds. In

fragmented woodlots, tufted titmice showed higher survivorship in years with less snow

cover (Doherty and Grubb 2000). Similarly, when examining feathers for evidence of

nutritional condition, the researchers found that titmice had significantly wider growth

bars in years with less snow cover (Doherty and Grubb 2003). Relationships between

snow cover and either survivorship or nutritional condition were not seen in any

chickadees, woodpeckers, or nuthatches. Tufted titmice spend more time foraging on the

ground than the other three species (Rybcynski 1977), so snow cover may not have had

as strong an impact on birds that concentrate their foraging on other substrates. Reduced

snow cover due to the effects of global warming may be one mechanism promoting the

northward range expansion of tufted titmice (Harrap and Quinn 1996).

21 1.5.2 Effects of wind and temperature on isolated woodlots

The effects of wind and temperature below the thermoneutral zone become much more pronounced in isolated woodlots, reducing the suitability of such patches for permanent resident birds. Edge effects become much more pronounced. In the most extreme conditions, such effects could potentially penetrate deep enough into a woodlot to render it completely uninhabitable (Dolby and Grubb 1999a).

Dolby and Grubb (1999a) examined windchill effects specifically on small (mean area = 5.3 ha) isolated wooded patches in rural Ohio, each containing one mixed-species flock. As observed in previous studies (Grubb 1975, 1978), both wind speed and temperature influenced the vertical foraging location of the birds within a woodlot. As wind speed increased, both chickadees and titmice were found increasingly lower in the canopy. The increased wind did not elicit the same response from downy woodpeckers or white-breasted nuthatches. Chickadees and titmice tend to forage on smaller-diameter substrates and may not be able to shelter themselves from the wind as easily as woodpeckers and nuthatches which are more likely to respond by foraging on the leeward side of a tree or by moving to substrates of larger diameter (Grubb 1975, 1977).

In addition to adjustments in foraging height, birds observed in these small fragments also modified their foraging location with respect to the woodlot’s edge. The investigators found a significant correlation between a wind/temperature interaction term and the distance of birds from the woodlot’s edge. The relationship with wind speed was most pronounced at the lowest temperatures observed (Figure 1.6). A resident flock often

22

Figure 1.6. Linear regressions for the distance of Carolina chickadees and tufted titmice from the windward edges of woodlots against the wind speed, blocked by temperature range (a-d). (From: Dolby and Grubb 1999.)

23 adjusted their location in the woodlot based not only on wind speed, but also on its direction. 62% of the time, the researchers observed a majority of the flock on the leeward side of the woodlot, presumably there to minimize exposure to the wind.

These observations provide evidence for an additional challenge to the flock. If severe conditions persist and the flock remains constricted to one section of a woodlot, the availability of food resources may potentially become an exigent issue, further reducing the suitable habitat. In addition, returning to windward edges for the purpose of cache retrieval may become difficult, if not averted altogether. Other permanent resident species may not be as distressed by low temperatures and high winds as the smaller chickadees, but woodpeckers and nuthatches were also present on the leeward side of woodlots. One explanation for this behavior is that they followed chickadees and titmice to that location to maintain the anti-predation and foraging benefits of social flocking

(Dolby and Grubb 1999a).

The available foraging substrate may also affect flock distribution within a woodlot (Telleria and Santos 1995). Patch edges that contain dense vegetation may provide the same wind protection as the woodlot’s interior or the leeward side. Likewise, a lack of appropriate foraging substrate on the leeward side of the woodlot may reduce a species’ presence even when conditions may warrant wind protection.

1.5.3 Woodlot connectivity and inter-patch movement

In a fragmented landscape, the degree of patch connectivity can affect winter movement among wooded habitat patches (see also Chapter 15). Also, if adverse winter conditions result in individual mortality and, in the most severe cases, local patch

24 extinctions, the reoccupation of a woodlot may be directly related to its connectivity within the landscape. The presence of travel corridors, such as fencerows, may help to facilitate inter-patch movements, especially during times of inopportune weather conditions when protection from wind and precipitation, as well as the usual protection from aerial predators, would be more advantageous.

The impact of winter weather conditions on movements among fragmented woodlands has been demonstrated by Groom and Grubb (2006), who simulated local patch extinctions by removing Carolina chickadees from selected woodlots in an agricultural landscape, and then monitoring re-colonization events. All 25 woodlots selected for the study were eventually recolonized, and birds moved sooner into those patches that contained more wooded area and were connected via fencerows to another woodlot. If, as previous studies have suggested (Grubb 1975, 1977, 1978; Dolby and

Grubb 1999b,1999a), abiotic factors, such as wind, can reduce the amount of suitable habitat in a woodland patch, occupants may be more likely to disperse into and remain in larger patches. During the winter, a larger patch will offer greater protection from the wind effects experienced at a woodlot edge. Mated pairs will also benefit by remaining in a larger woodlot during the breeding season. As discussed earlier, nesting in a larger patch provides an opportunity for the pair to maximize their distance from the woodlot’s edge, minimizing the risk of nest usurpation by house wrens.

In addition, Groom and Grubb (2006) discovered that chickadees were more likely to reoccupy a woodlot during periods of relatively benign wind chill. During conditions of high thermoregulatory demand, minimizing exposure to deleterious abiotic factors, such as wind, is critical to survival. Inter-patch dispersal may, therefore, be

25 further regulated by the current microclimatic conditions, in addition to degree of woodlot isolation. Inclement weather may not only restrict the current foraging habitat, but could also prevent individuals from seeking more suitable habitat.

For small birds, the task of surviving temperate winter seasons can be physiologically demanding, and the additional stress created by a fragmented habitat can further reduce a population’s annual survivorship and the nutritional condition of individuals. Many chickadees and titmice have large ranges that expose them to a variety of habitats, seasonal intensities, and microclimates. While several studies have documented energy-saving physiological adaptations and behavioral modifications to better manage increased thermoregulatory demands, most studies have been limited to a single species or to multiple species in the same area. The intraspecific variation of these responses across a broad geographical range has received less attention. Comparisons of this sort, along with a better understanding of the physiological chain of events that induce these responses, will provide insights into how a population may respond in environments that differ in thermal and spatial structure. The development and use of innovative techniques, such as ptilochronology, and improvements to current technology, such as radio tracking and thermal sensors, will continue to provide additional information as well.

Habitat fragmentation is likely to increase due to agricultural and urban expansion. Global climate change in also underway, though the magnitude and consequences at the regional scale are still hotly debated. In both matters, an effect on

26 avian communities is likely to be observed. Combining a complete knowledge of ecological and physiological constraints will facilitate more effective conservation and management of over-wintering Parid species.

27

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33

CHAPTER 2

METABOLIC PERFORMANCE AND DISTRIBUTION IN BLACK-CAPPED AND CAROLINA CHICKADEES

Jennifer R. Olson 1, Sheldon J. Cooper 2, David L. Swanson 3, Michael J. Braun 4, and

Joseph B. Williams 1

1Department of Evolution, Ecology and Organismal Biology, Ohio State University, 318

W 12 th Ave., Columbus, OH 43210, USA

2Department of Biology & Microbiology, University of Wisconsin-Oshkosh, 800 Algoma

Blvd., Oshkosh, WI 54901-8640

3Department of Biology, University of South Dakota, 414 E. Clark Street

Vermillion, SD 57069, USA

4Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian

Institution, 4210 Silver Hill Road, Suitland, Maryland 20746, USA

and

Behavior, Ecology, Evolution, and Systematics Program, University of Maryland,

College Park, MD 20742, USA

34

2.1 ABSTRACT

In endotherms, metabolic performance is associated with a wide array of ecological traits, including species distribution. Researchers have previously suggested that the northern boundaries of North American passerines are limited by their ability to sustain high metabolic rates required for thermoregulation. Black-capped chickadees

(Poecile atricapillus , BC) are year-round residents in most of Canada and the northern half of the United States, whereas Carolina chickadees ( Poecile carolinensis , CA) are found exclusively in the southeastern United States. These species hybridize along a narrow contact zone, which has been moving northward at a rate of about 1.6 km per decade, coincident with warming temperatures in Ohio. The location of the chickadee hybrid zone in Ohio closely matches air temperature isotherms, further suggesting that metabolic rate may correlate with distribution in these species.

We tested the hypothesis that distribution patterns of chickadees are linked with their rate of metabolism. For populations of BC and CA chickadees, we measured basal

(BMR) and cold-induced peak metabolic rates (PMR) from areas that differ in winter temperatures and supplemented this information with data from other studies. Although our findings suggest a general relationship between colder air temperatures and higher metabolic rate among chickadee populations, this trend was not robust among all species or locations.

35 Within Ohio, hybrids had a significantly higher mass-corrected BMR than either parental species. We suggest that the mtDNA-nDNA mismatch of hybrids may produce less efficient mitochondrial protein complexes, which in turn affects the efficiency of

ATP production, thereby increasing rate of oxygen consumption to meet ATP demands.

2.2 INTRODUCTION

Metabolic performance is a fundamental physiological attribute that correlates

with many aspects of a species’ ecology, including its distribution, survival, behavior,

and life history (Aschoff and Pohl 1970, Weathers 1979, White et al. 2007, Wiersma et

al. 2007a). Two indices are commonly used to assess metabolic performance: basal

metabolic rate (BMR), the minimum metabolic rate of a quiescent, postabsorptive animal,

in its thermal neutral zone and rest phase (McNab 1997), and peak metabolic rate (PMR),

the maximum rate of energy expenditure, often achieved by exposing birds to cold in

metabolic chambers (Wiersma et al. 2007b). Cold-induced PMR defines the maximum

heat generating capacity of an endotherm and correlates positively with endurance to cold

(Swanson 2001). Because BMR makes up 25-40% of a bird’s field metabolic rate (Bryant

1997) and often correlates with field metabolism (White & Seymour 2004; Daan et al .

1991), it has been used as a proxy for energy expenditure in the wild. Unlike BMR,

which is largely a function of central organs (e.g. brain, heart, kidneys and gut; Rolfe &

Brown 1997), heat generation in birds during cold exposure relies on shivering by

skeletal muscles (Bennett 1991, Weibel et al . 2004). Although within species

comparisons of BMR and PMR often show a positive correlation (Hinds and Rice-

36 Warner 1992, Dutenhoffer and Swanson 1996, Liknes and Swanson 1996, Rezende et al.

2002, Wiersma et al. 2007b), some studies have shown that these two variables are unrelated (Koteja 1987; Vézina et al . 2006).

The impact of metabolic performance on the distribution patterns of birds remains

controversial (Root 1988b, Castro 1989, Root 1989, Repasky 1991, Canterbury 2002). In

North America, there is evidence that the northern limits of some bird species are

correlated with metabolic performance. Root (1988b) found that the northern boundary of

60% of birds wintering in North America coincided with isotherms of minimum daily

January temperature, and concluded that winter distributions are limited to areas where

the birds do not need to raise their metabolic rate more than 2.5 times BMR. Some

investigators have challenged Root’s analyses and conclusions, rendering the generality

of her predictions uncertain (Castro 1989, Repasky 1991, Canterbury 2002).

Variation in metabolic performance across populations of the same species,

especially those with a large geographic range that transects multiple thermal

environments has not been addressed (Root 1988a, Swanson in press ). Such studies could

provide insights into the role of metabolism in defining distributional limits of species

and insights into how the local environment impacts levels of adjustment of metabolic

rate. One might surmise that individuals of the same species inhabiting generally warmer

areas will have a lower BMR and PMR than birds that inhabit colder climes, the local

adjustment hypothesis. Hence, birds of the same species that experience different winter

isotherms might be expected to show corresponding adjustments to BMR and/or PMR.

37 North American chickadees (Family: Paridae) provide a unique system for exploring possible relationships between metabolic performance and distribution. Black- capped chickadees ( Poecile atricapillus , BC) are year-round residents in most of Canada and the northern half of the United States, whereas Carolina chickadees ( Poecile carolinensis , CA ) are found exclusively in the southeastern United States. The ranges of these two species meet in Kansas eastward to New Jersey where they form a narrow band of hybrids (Ohio: Bronson et al. 2005, Pennsylvania: Reudink et al. 2007, Missouri:

Sawaya 1990, Virginia and West Virginia: Sattler and Braun 2000). The northern limit of

CA chickadees is associated with an isotherm at -6.7 oC (mean minimum January temperature, Repasky 1991) and BC chickadees live as far north as the -26.5 oC isotherm,

(Department of Energy 1974, Smith 1993).

Variation in thermal environments suggests that ambient temperature ( Ta) is

associated with the distribution of BC and CA chickadees (Figure 2.1). Unlike a number

of other hybrid zones, the contact zone between BC and CA chickadees includes

southward extensions of BC chickadees into upper elevations of the Appalachian

Mountains, as well as extensions into Illinois, Missouri, and Kansas; each interdigitation

southward occurs in a region with colder winter climate than other regions at similar

latitudes (US Geological Survey 1970). In addition, as Tas have warmed, populations of

CA and BC chickadees have moved northward. Over the last half century, the mean daily

January temperature in Ohio has warmed by approximately 4 oC (NOAA 2008).

Coincidentally, the contact zone between BC and CA chickadees in Ohio has been moving northward at a rate of approximately 1.6 km per decade (Wheaton 1882,

Trautman 1940, Grubb et al. 1994, Bronson et al. 2003a, Bronson et al. 2003b).

38

Figure 2.1. Position of the chickadee hybrid zone in relation to mean minimum January temperatures. We approximated zone position from North American Breeding Bird

Survey data (http://www.mbr-pwrc.usgs.gov/bbs/bbs.html), Tanner 1952, Brewer 1963,

Rising 1968, Robbins et al. 1986, Grubb et al. 1994, and Sattler and Braun 2000.

Isotherms are based on the mean minimum daily temperature in January as depicted in

U.S. Geological Survey (1970).

39 We examined metabolic performance of five populations of BC chickadees, from

Alaska, South Dakota, Wisconsin, Ohio, and New York, and two populations of CA chickadees, from Ohio and Tennessee, to test the hypothesis that distribution is related to metabolic performance. In cold, temperate climates, winter-acclimatized birds increase their BMR, PMR, and cold tolerance relative to summer-acclimatized birds, in response to the increased energetic demands of thermoregulation (Dawson and Marsh 1989,

Swanson 1990, Cooper and Swanson 1994). Using strong inference (Platt 1964), we envisioned several non-mutually exclusive hypotheses that could describe the relationship between BMR and PMR with latitude in these species. If chickadees optimize their metabolism for the local environment, birds living at higher latitudes should have a higher BMR and PMR compared with conspecifics at lower latitudes.

Alternatively, chickadees may not exhibit any latitudinal variation in metabolism.

Hybridization between species of chickadees could impact metabolic performance of offspring, which in turn may affect the position and northward movement of the hybrid zone. Oxidative phosphorylation and oxygen consumption of endotherms are largely a function of their mitochondria (Weibel and Hoppeler 2005, Pon and Schon 2007), and protein complexes within the mitochondria consist of polypeptides encoded by both nuclear and mitochondrial DNA. Because the mtDNA is maternally-inherited, hybrid chickadees will have polypeptides derived from both the BC and CA genomes. If mitochondrial genes have diverged in tandem with mitochondrial proteins encoded by the nuclear genome, one might predict that ATP production of hybrids would be less efficient than that parent species, perhaps as a result of increased proton leak or less efficient metabolic enzyme activity (Ellison and Burton 2008, Dobzhansky 1936). Assuming that

40 muscle mass is not affected by hybridization, we hypothesized that hybrids would have higher BMR than either parental population due to greater oxygen consumption needed to overcome inefficiencies in ATP production. Assuming that maximum oxygen consumption is limited by the rate of ATP synthesis, we also predicted that hybrids would have a lower PMR than either parental population if hybrids do not compensate for this inefficiency through other physiological means (e.g. higher muscle mitochondrial density or increased activity of metabolic enzymes).

2.3 MATERIAL AND METHODS

Capture of Birds

We trapped 181 chickadees (BC, CA, and hybrids) in Ashland County, Ohio

(40 o51’N, 82 o10’W) during three winters from December 2003 through February 2006.

Traps were located in 26 privately owned woodlots along a 40 km north-south x 5 km

east-west transect that encompassed the hybrid zone between chickadee species as

determined by Bronson et al. (2003a). In addition, we captured 6 BC in Chagrin Falls,

OH (41 o22’N, 81 o22’W), 12 BC in Oshkosh, WI (44 o01’N, 88 o33’W) and 15 CA in

Paris, TN (36 o18’N, 88 o19’W) using mist nets during Feb 2006. Birds from Chagrin

Falls, OH were not used for metabolic measurements and were incorporated for genetic

analyses only. D. Swanson measured BMR and PMR of BC in South Dakota (n=47) and

S. Chaplin provided data for BMR for New York BC ( n=10) (Chaplin 1974).

41 We housed birds in 30 cm x 25.5 cm x 35.5 cm wire mesh cages and provided them with sunflower seeds, mealworms, and water ad libitum . Cages were kept in a university-

approved animal housing room at ambient indoor temperatures with lights turned on

during daylight hours (9L:15D). We made all metabolic measurements within a week of

capture. Experiments were approved by the ILACUC committee of Ohio State University

(Protocol 2004A0065) .

Species identification

Morphological similarity between BC, CA, and hybrids, along with high levels of backcrossing, make species recognition difficult in regions where BC and CA potentially coexist (Sattler and Braun 2000). Based on amplified fragment length polymorphism

(AFLP) analysis (Vos et al. 1995), with modifications proposed by Kingston and Rosel

(2004), we designated birds as BC, CA, or hybrid. At the time of capture, we punctured the bird’s brachial vein using a 25-gauge needle, collected blood in a heparinized capillary tube, placed the sample in lysis buffer (100 mM Tris at pH 8.0, 100 mM EDTA,

10 mM NaCl, 0.5% SDS; Longmire et al. 1988), and stored it at 4 oC. We extracted DNA from samples using the methods of Bronson et al. (2003). We digested extracted DNA from blood samples with restriction enzymes Eco RI and Taq I, followed by two rounds of

PCR (preselective and selective) to both amplify fragments and fluorescently label them for detection. Fragments were sized using GeneMapper v3.7 software (Applied

Biosystems, Foster City, CA) and we assigned individuals to populations using

STRUCTURE v2.1 (Pritchard et al. 2000). In addition to the birds above, we also analyzed extracted DNA from 18 CA chickadees previously captured in Lawrence

42 County, OH (38 o43’N, 82 o34’W, Sattler and Braun 2000) for inclusion as an additional parental population. We analyzed 245 loci from 218 individuals using 100,000 estimation steps after 50,000 burn-in steps, and each value of K (the number of clusters) 1 to 5. We achieved the most logical population structure using a K of 2, and we classified birds as hybrids if they were assigned a 25-75% probability of belonging to either parental species. We also used restriction fragment length polymorphism to determine mitochondrial haplotypes of chickadees from Ohio, Wisconsin, and Tennessee using methods described in Reudink et al (2006).

Measurements of Basal Metabolic Rate

Birds collected in Wisconsin, Ohio, and Tennessee were transported to the lab for measurements of metabolism. For measurements of BMR, we removed food from birds 2 hrs prior to measurements to assure that they were postabsorptive. Then, at 20:00 hrs, we placed birds in one liter stainless-steel chambers lined with dark brown Teflon to reduce reflective radiation and minimize adherence of water molecules to the chamber (Porter

1969). Each chamber contained a wire mesh platform and a plastic perch above a layer of mineral oil to capture feces, thus eliminating them as a source of water vapor. Rubber gaskets rendered the lids airtight and excluded light. We placed four chambers into a temperature-controlled cabinet maintained at 32 oC, a temperature within the thermal neutral zone of these species (Rising and Hudson 1974), using a Peltier device (Sable

Systems Pelt 4). To monitor Ta within chambers, we inserted 30-gauge copper-constantan thermocouples into each chamber. Compressed air was directed through a column of

Drierite to remove water, then through mass flow controllers (Tylan/Mykrolis, Chaska,

43 MN) that we had previously calibrated using a glass bubble meter (500 mL-Levy 1964), and then into the chamber. An automated system of solenoids sequentially sampled the outflow air from each chamber. Excurrent air passed through a Dew Prime II hygrometer to measure dewpoint (EdgeTech, Marlborough, Massachusetts), and then through tubes containing silica gel, ascarite, and silica gel to remove water and CO 2, before progressing

through an Ametek S3AII oxygen analyzer to measure fractional concentration of

oxygen. We directed outputs from the oxygen analyzer, dew point hygrometer, and

thermocouples to a CR23X Micrologger® (Campbell Scientific, Logan, Utah) with a one

minute sampling rate. Data from each chamber were recorded for 12 minutes before the

solenoid system switched to the next chamber; we continued measurements throughout

the night. Mass (±0.1g) and cloacal temperature (±0.1 oC) of each bird was recorded

before and after each trial. We averaged oxygen consumption over 10 minute intervals

during the sampling period and selected the lowest constant value for BMR. We

calculated rate of oxygen consumption, V& , using equation 4 from Hill (1972). O2

Measurements of Peak Metabolic Rate

One to two days after measurement of BMR we measured PMR between 08:00 and

18:00 hrs. Two hours before measurements, we removed food to assure that birds were

post-absorptive. We measured PMR using a system similar to that for BMR, except that

the temperature of the chamber was controlled by a mixture of antifreeze circulating in

copper coils that surrounded the chamber, the entire assembly insulated by Styrofoam.

The temperature of the antifreeze was controlled by a refrigerated water bath (NESLAB

RTE-140, Pittsburgh, PA). Incurrent air was a mixture of 79% helium-21% oxygen 44 (heliox) (Rosenmann and Morrison 1974). We set the initial water bath temperature at

32 oC, and birds were allowed a minimum of 30 min to adjust to the chamber before we

o began decreasing Ta by about 1.0 C per min. Measurements terminated when oxygen consumption no longer increased with a decrease in chamber temperature (Swanson et al.

1996). We recorded bird mass (±0.1g) and cloacal temperature (±0.1 oC) before and after

each trial.

For PMR, we calculated instantaneous rates of oxygen consumption according to the equations of Bartholomew et al. 1981 and averaged values over 10 minute intervals. We took the highest 10 minute mean over the entire test period as PMR. We defined cold tolerance as the temperature at the cold limit (Tcl , Saarela et al. 1995), which was the

temperature at which a bird achieved peak metabolic rate during cold trials, and used Tcl

as a measure of thermal endurance.

Data from the literature - BMR and PMR

Oxygen consumption of South Dakota and New York chickadees was measured

as described in Swanson et al. (1996) and Chaplin (1974), respectively. We obtained

BMR measurements from two additional studies on chickadee metabolism. We averaged

the eight data points from Fig. 1 of Grossman and West (1977) that represented

measurements of BMR on BC chickadees from Fairbanks, AK, made in the birds’

thermoneutral zone. We also obtained approximations of BMR for Ohio BC (Ashtabula

Co., OH) and CA chickadees (Hamilton Co., OH) from Table 4 of Munzinger (1974).

Because these values represent average nocturnal oxygen consumption from 24 hour

45 trials that were conducted at a lower temperature than our measurements (25 vs 32 oC), these data are included in figures for comparison, but were not used in any statistical analyses.

Statistical Analyses

We analyzed data from our measurements both within and across species to separate

the influence of thermal environment and genetic identity on metabolic rate. In order to

determine how metabolism varied within species, we compared whole-organism

metabolic rates across the five populations of BC chickadees (Alaska, Wisconsin, South

Dakota, New York, and Ohio) and across the two populations of CA chickadees (Ohio

and Tennessee) using an analysis of variance (ANOVA). Hybrids were not included in

these analyses. We then evaluated the whole-organism metabolic rates of chickadees

across the hybrid zone in Ohio.

In order to control for the effect of body size on metabolic rate (King and Farner

1961, Lasiewski and Dawson 1967, Weibel and Hoppeler 2005), we used an analysis of covariance (ANCOVA) to examine metabolic rate across populations, using population or species as a fixed factor and mass as a covariate (see Packard and Boardman 1988,

1999, Hayes 2001). Data presented in figures are least square means ± SE when controlling for mass. As with whole-organism metabolism, we compared both BC and

CA chickadees across populations, and BC, CA, and hybrid chickadees across the OH hybrid zone. When applicable, we employed Sidak post hoc comparisons to identify

46 significant differences between populations. We performed statistical analyses using

SPSS 15.0 and all mean values are reported ± 1 S.E. P<0.05 was chosen as the lowest acceptable level of significance.

2.4 RESULTS

Identification of individuals to species

In 1994, Bronson sampled chickadees along a north-south transect in Ohio across the putative boundary between BC and CA chickadees (Bronson et al. 2003a). Using five enzyme-probe markers, these authors identified a band of hybrids approximately 15 km wide that ran from 40 o46’3” N to 40 o55’46” N, as judged by visual inspection of their graphs. We extended our sampling region both north and south of that used by Bronson et al. (2003a), and using AFLP analysis, classified all birds that we measured as BC, CA, or hybrid. The genetic identities of our samples are displayed in Figure 2.2.

Body mass and metabolic rate

Body mass of CA chickadees in Tennessee did not differ significantly from conspecifics in Ohio (Table 2.1; P<0.08). However, among BC chickadees, there was a significant effect of location on body mass ( F4,101=29.0, P<0.001), and post hoc analyses indicated that BC chickadees in South Dakota weighed more than those from Wisconsin,

New York, or Ohio ( P <0.008 for all comparisons), but not more than BC chickadees

47

Figure 2.2. Genetic identity of Ohio birds along a transect of Ashland County, OH

(n=174). Distance zero on the x-axis is the northern most woodlot sampled, located at

41°6'N. Distance forty on the x-axis is equivalent to forty km south of this woodlot. Birds assigned a probability of being BC >75% are labeled BC (filled circles), 25-75% are HY

(crosses), and <25% are CA (open circles). The genetic identities of samples from

Wisconsin (WI, n=10) and Tennessee (TN, n=10) are also included for reference, as well as samples from extreme northern and southern Ohio (G = Geauga County, 41°22'N, n=6; L = Lawrence County, 38°43'N, n=18). The additional Ohio samples are included for genetic reference only, and are not incorporated in metabolic analyses.

48

Table 2.1. Mean metabolic data by location.

T Species and Location Latitude min Mass (g) b n BMR (W) n PMR (W) n T (oC) n Citation (oC) a Mass BMR PMR cl Tcl BCCH: Alaska 64°54'N -27.7 12.6 ± 0.08 8 0.43 ± 0.02 8 Grossman & West 1977 Wisconsin 44°00'N -11.3 11.1 ± 0.24 13 0.27 ± 0.01 13 1.5 ± 0.06 11 -2.3 ± 1.8 11 this study South Dakota 42°47'N -10.6 12.9 ± 0.15 47 0.32 ± 0.01 31 2.1 ± 0.03 47 this study New York 42°41'N -7.9 11.8 ± 0.25 10 0.30 ± 0.01 10 Chaplin 1974 Ohio (Ashtabula Co.) 41°52'N -7.9 11.1 0.26 ± .02 4 Munsinger 1974 Ohio (Ashland Co.) 40°51'N -6.9 10.8 ± 0.17 28 0.25 ± 0.006 27 1.5 ± 0.05 20 6.6 ± 1.6 18 this study Hybrid: Ohio (Ashland Co.) 40°51'N -6.9 10.3 ± 0.48 7 0.26 ± 0.01 7 1.6 ± 0.05 4 2.9 ± 1.2 3 this study

4 CACH: 9

Ohio (Ashland Co.) 40°51'N -6.9 9.5 ± 0.18 11 0.21 ± 0.01 9 1.3 ± 0.05 10 8.1 ± 1.9 10 this study Ohio (Hamilton Co.) 39°30'N -4.7 9.9 0.24 ± 0.01 4 Munsinger 1974 Tennessee 36°18'N -2.4 9.4 ± 0.14 16 0.21 ± 0.01 15 1.3 ± 0.04 13 5.6 ± 1.2 13 this study

a Tmin is the minimum daily temperature in January averaged over 10 years (1997-2006). bMass and metabolic measurement are reported ± 1 S.E.

49 from Alaska ( P >0.9). Within Ohio, mass was significantly different between groups

(F2,43 =7.9, P =0.001): BC chickadees weighed the most, CA the least, and hybrid intermediate. Post hoc tests revealed that hybrid mass did not differ significantly from either parental species ( P >0.05 for both).

Within each species, basal metabolic rate increased with mass (Fig. 2.3). For CA chickadees, the relationship between BMR and mass was described as log BMR (W) = -

1.42 + 0.77 log mass (g) ( R2=0.22, P =0.02), whereas for BC chickadees, it was log BMR

2 (W) = -1.98 + 1.35 log mass (g) ( R =0.45, P< 0.001). Slopes and intercepts of equations

for BMR did not differ significantly between species.

For CA chickadees, log PMR (W) = -1.19 + 1.34 log mass (g) ( R2=0.33, P=0.004) and for BC chickadees, log PMR (W) = -0.99 + 1.12 log mass (g) ( R2=0.52, P<0.001).

Slopes and intercepts of PMR equations did not differ significantly between species.

Ta and metabolic rate across locations

With data pooled for BC and CA, we found that metabolism was generally related to mean minimum January temperature 1997-2006 (whole-organism BMR: R2 = 0.61, P<

0.001; mass-corrected BMR: R2 = 0.48, P<0.001; whole-organism PMR: R2 = 0.42,

P<0.001; mass-corrected PMR: R2 = -0.38, P<0.001; Fig. 2.4). The relationships between metabolic rate, both whole-organism and mass-specific, and temperature are as follows:

o o BMR (W) = 0.191 - 0.009 Ta ( C); BMR (W/g) = 0.0199 - 0.0005 Ta ( C); PMR (W) =

o o 0.942 – 0.092 Ta ( C); PMR (W/g) = 0.1238 -0.0029 Ta ( C).

50

Figure 2.3. The relationship of BMR (A) and PMR (B) to mass in BC (filled circles) and

CA (open circles). Linear regression lines are plotted solid for BC and dashed for CA

(P<0.001 for both). Sample sizes: BC BMR n=71, CA BMR n=24, BC PMR n=77, CA

PMR n=22.

51

Figure 2.4. Mean BMR (A) and PMR (B) versus mean minimum January temperature for all locations included in this study. Metabolic rate is presented as least square means controlling for mass. Linear regressions were calculated by combining data for both species ( P<0.001 for both BMR and PMR).

52 Comparison of BMR and PMR across species

Mean BMR of all BC was significantly greater than the mean BMR of all CA chickadees; F1,111=37.7, P<0.001). In an analysis of covariance, there was no significant difference in the BMR of BC or CA chickadees after including mass as a covariate.

BC chickadees had a mean PMR of 1.86 ± 0.04 W, and CA chickadees 1.29 ± 0.03 W.

Whole-organism PMR was significantly higher in BC chickadees than in CA chickadees

(F1,99=53.6, P<0.001), but this difference disappeared when we included mass as a covariate.

Comparison of BMR and PMR within species

We evaluated BMR within species by comparing populations of the same species from different locations (Fig. 2.5a). For CA chickadees, BMR in Tennessee birds did not differ significantly from those in Ohio. We obtained the same results when we included mass as a covariate (see Table 2.1 for values). In contrast, there were significant differences for whole-organism BMR among BC chickadee populations (F4,84 =40.8,

P<0.001), although more northerly populations did not consistently have higher BMR.

Alaskan BC had a significantly higher BMR than BC from any other location (P<0.001 for all comparisons). South Dakota BC had a higher BMR than both Wisconsin and Ohio

(P<0.001 for both comparisons). Location was also significantly related to BMR in BC after including mass as a covariate ( F4,83 =23.6, P<0.001). Again, post hoc tests revealed that Alaskan birds had a significantly higher mass-corrected BMR than birds from any other location ( P<0.001 for all comparisons). Among the remaining groups, New York

53

Figure 2.5. Mean BMR (A) and PMR (B) for CA and BC chickadees. Metabolic rate is

presented as least square means controlling for mass. Different letters identify significant

differences (P<0.05) in mass-corrected metabolic rate between populations. Sample sizes

can be found in Table 2.1. Abbreviations used: AK=Alaska, WI=Wisconsin, SD=South

Dakota, NY=New York, OH M=Ohio, Munzinger (1974) data, OH BC =Ohio black-capped chickadee data from this study, OH CA =Ohio Carolina chickadee data from this study, and

TN=Tennessee. 54 and Wisconsin BC chickadees did not differ significantly from any other location, and the

South Dakota BC chickadees had a significantly higher BMR than Ohio birds ( P=0.009).

Whole-organism and mass-corrected PMR did not differ between CA chickadees in Ohio and Tennessee (Fig. 2.5b). In contrast to CA chickadees, there was a significant effect of location on whole-organism PMR of BC chickadees ( F2,75 =59.3, P<0.001);

South Dakota birds had a significantly higher PMR than both Wisconsin and Ohio

chickadees ( P<0.001 for both comparisons). Mass-corrected PMR of BC chickadees was

also significantly different among populations ( F2,74 =17.1, P<0.001), and again South

Dakota birds had a higher PMR than both Wisconsin and Ohio birds ( P<0.001 for both comparisons). There was no significant difference between Wisconsin and Ohio BC chickadees in either whole-organism or mass-corrected PMR.

Metabolic rate and cold tolerance across the hybrid zone

BC, CA, and hybrid chickadees from across the Ohio hybrid zone, and therefore a

similar environment, had significantly different BMR (Fig. 2.6a; F2,40=7.6, P=0.002).

Carolina chickadees had significantly lower BMR than hybrid (P=0.01) and BC chickadees ( P=0.002), and there was no significant difference between BC and hybrid

chickadees. There was also a significant difference in BMR among the Ohio BC, CA, and

hybrid chickadees after including mass as a covariate ( F2,39 =6.1, P=0.005). Hybrids had a significantly higher mass-corrected BMR than either CA ( P=0.01) or BC ( P=0.01) chickadees, which did not differ significantly from each other.

55

Figure 2.6. Mean BMR (A) and PMR (B) for each Ohio species. Metabolic rate is

presented as least square means controlling for mass, and different letters identify

significant differences (P<0.05) in mass-correct metabolic rate between populations.

Sample sizes can be found in Table 2.1. Abbreviations used: OH BC = Ohio black-capped chickadee, OH CA =Ohio Carolina chickadee, and OH HY = Ohio hybrid chickadee.

56

Figure 2.7. Mean Tcl for each chickadee population. Metabolic rate is presented as least square means controlling for mass, and different letters identify significant differences

(P<0.05) in mass-correct metabolic rate between populations. Sample sizes are as follows: WI n=11, OH BC n=18, OH CA n=10, TN n=13.

57 Across the Ohio hybrid zone, we found that PMR was significantly related to genetic identity (Fig. 2.6b; F2,31 =6.0, P=0.006). Peak metabolic rate of CA chickadees was significantly lower than PMR of both BC ( P=0.02) and hybrid chickadees ( p=0.02),

which did not differ significantly from each other. We did not find a significant

difference in PMR between any of the three OH groups (CA, BC, hybrid) after

controlling for mass.

Cold tolerance

The average Tcl of BC chickadees, combining data for birds from Wisconsin and

Ohio, did not differ significantly from the average Tcl of CA chickadees from Ohio and

Tennessee (Fig. 2.7; P=0.07). We did find a significant effect of location on Tcl within

each species ( F3,48 =6.9, P=0.001). The cold tolerance of Wisconsin chickadees was significantly higher than that of Ohio BC, Ohio CA, or Tennessee CA (P<0.01 for all comparisons), which did not differ significantly from each other. There was no significant difference between the average Tcl of BC, CA, or hybrid chickadees across the

Ohio hybrid zone.

Mitochondrial Haplotypes

For statistical analyses, we used AFLP to identify hybrids based on STRUCTURE scores. However, we did identify three BC and one CA that possessed mtDNA of the other species, and these birds presumably represent some level of backcross hybrid. To ensure the accuracy of species identification in our analyses, we removed these four

58 individuals and repeated all statistical tests with the same results. Although these chickadees did not meet our a priori definition of a hybrid, we also repeated statistical tests with them classified as such. These tests rendered the comparison of BMR in Ohio birds insignificant, after including mass as a covariate ( F2,39 =1.1, P=0.3). All other results

remained the same.

2.5 DISCUSSION

If chickadees maintain the minimum metabolic machinery necessary to survive ambient winter temperatures, their BMR and PMR should be associated with temperature isotherms. This “machinery-matching” strategy is consistent with the seasonal metabolic variation found in permanent resident, winter passerines. In a previous study, winter BC chickadees had a standard metabolic rate that was 18% higher than summer birds

(Cooper and Swanson 1994). In addition to seasonal variation, there is also evidence of significant winter-to-winter variation in both BMR and PMR of BC chickadees, which are negatively correlated with the mean minimum temperature of that season (Swanson and Olmstead 1999). This suggests that chickadees use seasonally variable, temperature- associated metabolic limits in order to facilitate winter survival. The presence of lower metabolic rates during both the summer and less severe winters implies some physiological cost to maintaining machinery that would result in increased metabolism year-round, or during times when a lower level would suffice, although the physiological mechanisms controlling this variation remains unknown (Vézina et al. 2007, Swanson in

59 press ). Thus, we predicted that metabolic rates would be negatively correlated with temperature across our sampling sites: northern birds would have higher metabolic rates than conspecifics at lower latitudes.

Our data did support the notion that metabolism of chickadees is associated with winter temperatures, at least within BC, but the relationships were complex. Although we saw a general correlation between colder temperatures and higher metabolic rates (Fig.

3), this trend was not robust among all locations. For example, although Wisconsin was the coldest location sampled in the lower forty-eight states, the BMR of Wisconsin BC chickadees did not differ significantly from that of Ohio BC chickadees (the southern- most BC chickadee population), even before correcting for body mass. The mean minimum daily January temperatures of Wisconsin and Ohio differ by only 4.4 oC, a difference that is less than the mean daily temperature span at either location (WI =

8.6 oC, OH = 8.1 oC, NOAA 2008). If chickadees in Ohio tolerate average daily temperature changes of 8.1 oC, exposure to an environment that is, on average, only 4.4 oC colder may not present a significant thermal challenge requiring a change in metabolic rate. However, other factors that we have not measured, such as wind and solar radiation, also influence heat flux in these small birds.

South Dakota BC chickadees had a significantly higher mass and PMR than any other BC chickadee population in the continental United States, even though the temperature profile of South Dakota is similar to that of Wisconsin. Researchers have consistently recorded masses around 13g for this South Dakota population (Cooper and

Swanson 1994, Dutenhoffer and Swanson 1996, Swanson and Liknes 2006). However, even after correcting for mass, these birds still had higher PMR than any other population

60 in this study. Although possibly influenced by slight differences in methodology, such as rate of cooling during PMR tests (see Swanson 1990, Cooper and Swanson 1994), these data suggest that South Dakota chickadees have means other than increasing mass to elevate metabolic rate, such as elevating oxidative enzyme activity (Liknes 2005). It also suggests that this population may be exposed to environmental factors, other than temperature, that require an elevated metabolism. Southeastern South Dakota has the lowest forest cover, approximately 4% (Castonguay 1982), among all of our sampling sites, and the effects of both wind and temperatures below the thermoneutral zone become much more pronounced in isolated woodlots. Wind exposure can elevate metabolic rate in small birds (Bakken and Lee 1992, Dolby and Grubb 1999, Bakken et al. 2002). The exceedingly fragmented woodland landscape of South Dakota may amplify the effect of wind and explain in part why these birds had a higher metabolic rate than conspecifics at similar latitudes.

We saw little variation in mass-corrected BMR of chickadees across a wide range of latitudes (Fig. 2.5), suggesting that, with the exception of Alaskan BC, chickadees increase their body mass in response to lower Ta rather than altering tissue-specific rates of metabolism. Additional sampling locations may clarify whether Alaskan chickadees are simply an exception to this conclusion, or if the trend observed in Fig. 4 is robust throughout the BC range. Sharbaugh (2001) reported measurements of Alaskan chickadee standard metabolic rates (SMR) that were closer to our measurements from the continental United States (SMR: 0.254 W in winter 1994, 0.338 W in winter 1995).

However, the data for Alaskan birds must be interpreted with caution, as sampling protocols were not consistent with other locations: Alaskan chickadees in both of these

61 studies (Grossman and West 1977, Sharbaugh 2001) were housed in outdoor cages with ad libitum food throughout the winter. In addition, Grossman and West (1977) only used five birds for multiple measurements, indicating that at least three of the data points used in our analysis are cases of pseudoreplication.

The Ohio chickadees represent a moving hybrid zone with forces opposing gene flow between the two species. Most hybrid zones are maintained by some form of endogenous selection (Barton and Hewitt 1981, 1985), which favors alleles within a complementary genetic background and selects against individuals of mixed ancestry

(Barton and Gale 1993). Despite evidence of gene flow across the contact zone (Sattler and Braun 2000), previous studies revealed that there is selection against hybrid reproductive success (Bronson et al. 2003a). These results suggested to us that the genetic incompatibilities of hybrids might have additional physiological manifestations beyond reproduction.

Ohio chickadees provided us with the opportunity to explore the metabolic consequences of being a hybrid. One hypothesis is that there will be no observable difference between hybrids and parental populations, either because selection has already removed individuals with severe mitochondrial inefficiency during the months preceding our sampling, or because the two species’ genomes are so similar that metabolism is not penalized by hybridization. However, we predicted that Ohio hybrids would have higher

BMR and lower PMR than BC or CA chickadees in the same environment. A mismatch between proteins encoded by nuclear and mitochondrial genes, such as could be the case in hybrids, may not produce a properly functioning metabolic system due to misassembled metabolic proteins. In our study, Ohio hybrids did have a higher BMR than

62 both BC and CA chickadees, after including mass as a covariate. Although speculative, we think that the higher BMR of hybrid chickadees may be due to genetically-induced mitochondrial deficiencies.

Several recent studies have suggested that nDNA-mtDNA interactions are disrupted by hybridization events, impairing the function of metabolic enzymes - specifically those encoded in the mtDNA (Breeuwer and Werren 1995, Edmands and

Burton 1999, Sackton et al. 2003, Perrot-Minnot et al. 2004, Zeyl et al. 2005, Ellison and

Burton 2006). For example, cytochrome c oxidase (COX, complex IV of the electron transport system) contains thirteen subunits, three of which are mitochondrial encoded

(Grossman et al. 2004). In addition to the reduced fecundity and viability observed in interpopulation hybrid marine copepods ( Tigriopus californicus , Edmands 1999),

Edmands and Burton (1999) also detected reduced COX activity in individuals with mtDNA subunits from one population and a mixed (hybrid) nuclear background. Further, the low fitness and reduced ATP synthesis of F 3 hybrids can be restored through

maternal, but not paternal backcrosses, which allow the parental nuclear-mitochondrial

genomes to be reassembled. Although BC and CA chickadees have a relatively small

divergence in nuclear DNA (<0.3%, based on restriction fragment mapping), they exhibit

a mitochondrial DNA sequence divergence of about 7% (Mack et al. 1986, Sawaya

1990).

Ohio BC chickadees were larger, and they did not differ from HY or CA

chickadees with respect to PMR or cold tolerance after including mass as a covariate.

Peak metabolic rate reflects the maximum limit of oxidative respiration in muscle cells,

and for many winter passerines heat production facilitated by shivering thermogenesis is

63 energetically demanding. We proposed that Ohio could be the northern thermal limit of the CA chickadee population, due to the relationship between temperature isotherms and the hybrid zone. Thus, we predicted that higher peak metabolic rates would convey an advantage to Ohio BC chickadees over Ohio CA chickadees during periods of thermal challenge. It does appear that BC chickadees are better adapted to the cold weather found in northern areas of Ohio, and the northward movement of CA chickadees may be facilitated by the increase in winter temperatures observed over the past half century.

64

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77

CHAPTER 3

METABOLIC PERFORMANCE AND AEROBIC ENZYME ACTIVITY IN BLACK-CAPPED AND CAROLINA CHICKADEES

3.1 ABSTRACT

In cold, temperate climates, winter-acclimatized birds often increase their basal

(BMR) and peak metabolic rates (PMR) relative to summer-acclimatized birds as a response to the increased energetic demands of thermoregulation. If these responses to seasonal temperature variation can be extrapolated to geographic temperature variation, then we predict that birds living at higher latitudes should have a higher BMR and PMR compared with conspecifics at lower latitudes. Physiological features at several levels may explain variation in whole-organism BMR and PMR, including changes in muscle mass, transport capacity of oxygen and substrates to tissues, capillarity of muscles, intracellular transport to mitochondria, mitochondrial density, mitochondrial cristae surface area, and metabolic enzyme activity.

In black-capped ( Poecile atricapillus) and Carolina ( P. carolinensis) chickadees, heat production during cold exposure is primarily achieved through shivering

78 thermogenesis, an energetically expensive process that requires high rates of ATP production. Thus, we predicted that northern populations would exhibit higher PMR compared to southern populations, facilitated by an elevation in enzyme activity of oxidative pathways. To explore this relationship, we measured in vitro activity of three

pectoral muscle metabolic enzymes that catalyze non-equilibrium, highly exergonic

reactions: citrate synthase (CS), phosphofructokinase (PK), and L-3-hydroxyacyl-CoA-

dehydrogenase (HOAD).

While we did not find any differences in mass- or protein-corrected enzyme activities

between the two species, we did observe a higher level of total muscle CS activity in BC

chickadees. In addition, PMR in BC chickadees was significantly correlated with

pectoralis mass and total muscle enzyme activity of CS, PFK, and HOAD. We concluded

that among BC chickadees, higher metabolic rates were primarily a result of greater

pectoralis mass, rather than an increase in tissue-specific metabolic enzyme activity.

3.2 INTRODUCTION

Variation in avian metabolism at the individual level has received a great deal of attention from physiological ecologists in recent decades. A majority of these studies have focused on changes that occur in birds’ metabolisms corresponding to seasonal change and cold acclimatization (e.g. West, 1972; Marsh and Dawson, 1989; Swanson

1991; Cooper and Swanson, 1994; Dawson and O’Connor, 1996; Liknes and Swanson,

1996) or preparation for migratory flight (e.g. Piersma et al., 1995; Weber and Piersma,

79 1996; Swanson and Dean, 1999). However, intraspecific variation in metabolism along a thermal gradient has been virtually ignored (Swanson, in press ). Many bird species have

populations that are distributed across a large range of thermal environments, which

prompts the question: have populations in colder regions developed energetic adaptations

not present in more southerly conspecifics in order to meet this physiological challenge?

In temperate passerines, winter acclimatization is accomplished through several

methods, including elevated fattening (Dawson et al. 1983, Blem 1990, Rogers and Smith

1993), increased shivering endurance (Dawson and Marsh 1989), increased peak

metabolic rate (Dawson and Marsh 1986, Swanson 1990, Cooper and Swanson 1994,

O’Connor 1995, Liknes and Swanson 1996, Liknes et al 2002, Cooper 2002), and

changes in catabolic enzyme activity and substrate metabolism (Marsh and Dawson 1982,

Carey et al. 1989, Marsh et al. 1990, Swanson 1991, O’Connor 1995). However, relative

contributions of the above mechanisms to an increased cold tolerance are not well

understood (Liknes 2005).

Basal metabolic rate (BMR) reflects the minimum amount of energy required to sustain basic cellular functions in a post-absorptive, thermally neutral state, and it is largely a function of central organs (e.g. brain, heart, kidneys and gut) (Swanson, in press ; Hoppeler and Weibel, 1998; Bennett, 1991; Taigen, 1983), while peak metabolic rate (PMR) reflects the maximum limit of oxidative respiration. BMR and PMR can vary seasonally in small birds (Dawson and Smith 1986, Cooper and Swanson 1994, Liknes and Swanson 1996, Swanson and Olmstead 1999), though not necessarily in tandem

(Dawson and Smith 1986). Physiological features at several levels may explain variation in whole-organism basal and peak metabolic rates, including changes in muscle mass,

80 transport capacity of oxygen and substrates to tissues, capillarity of muscles, intracellular transport to mitochondria, mitochondrial density, mitochondrial cristae surface area, and metabolic enzyme activity (Swanson, in press ).

North American chickadee species offer researchers the opportunity to investigate

this metabolic variation across a range of environments. Black-capped chickadees

(Poecile atricapillus , BC) are year-round residents in most of Canada and the northern

half of the United States, whereas Carolina chickadees ( Poecile carolinensis , CA ) are

found exclusively in the southeastern United States. The ranges of these two species meet

along a narrow band extending from Kansas to New Jersey, and hybridization occurs

throughout this contact zone, including a region located in Ashland County, Ohio (e.g.,

Missouri – Braun and Robbins 1986, Sawaya 1990, Ohio – Grubb et al. 1994, Virginia

and West Virginia – Sattler 1996, Sattler and Braun 2000, Pennsylvania – Cornell 2001).

In cold, temperate climates, winter-acclimatized birds often increase their BMR

and PMR relative to summer-acclimatized birds as a response to the increased energetic

demands of thermoregulation (Dawson and Marsh 1989, Swanson 1990, Cooper and

Swanson 1994). If these responses to seasonal temperature variation can be extrapolated

to geographic temperature variation, then we predict that birds living at higher latitudes

should have a higher BMR and PMR compared with conspecifics at lower latitudes. In

chickadees, heat production during cold exposure is primarily achieved through shivering

thermogenesis, an energetically expensive process that requires high rates of ATP

production. This increased metabolic rate may be facilitated by an elevation in enzyme

activity of oxidative pathways. Candidates include enzymes that catalyze non-

equilibrium, highly exergonic reactions. Previous studies that have investigated variation

81 in avian metabolic enzymes have used phosphofructokinase, L-3-hydroxyacyl-CoA- dehydrogenase, and citrate synthase as indices of the muscle’s capacity for carbohydrate oxidation, lipid oxidation, and overall oxidative capacity, respectively. Citrate synthase

(CS) catalyzes the first reaction of the tricarboxylic acid cycle (TCA): the condensation of acetyl-CoA and oxaloacetate to form citrate. This enzyme is often used as an index for tissue aerobic capacity (see Marsh 1981). Phosphofructokinase (PFK) catalyzes the third step of the glycolytic pathway, which is generally considered the rate-limiting reaction of glycolysis. The activity PFK is an indicator of a tissue’s ability to mobilize blood glucose

(Crabtree and Newsholme 1975). L-3-hydroxyacyl-CoA-dehydrogenase (HOAD) catalyzes the third step of the β-oxidation pathway, and although it is not a rate-limiting step, HOAD is often employed as an indicator of the tissue’s capacity for β-oxidation of

fatty acids (Marsh 1981).

In order to explore the mechanistic correlates to whole-organism metabolism, we measured in vitro activity of these three rate-limiting enzymes in BC and CA pectoral muscle. Given winter chickadees’ reliance on shivering as the primary means for thermoregulation, we predicted that northern populations would exhibit higher PMR, facilitated by higher metabolic enzyme activity, compared to southern populations.

82 3.3 MATERIALS AND METHODS

Capture of birds

We trapped both black-capped and Carolina chickadees in Ashland County, Ohio

(40 o51’N, 82 o10’W) between December 2005 and February 2006 using remote

controlled feeder traps located on privately owned woodlots. In addition, we used mist

nets to capture black-capped chickadees in Oshkosh, WI (44 o01’N, 88 o33’W) and

Carolina chickadees in Paris, TN (36 o18’N, 88 o19’W) during February 2006.

We housed birds in 30 cm x 25.5 cm x 35.5 cm wire mesh cages and provided them with sunflower seeds, mealworms, and water ad libitum . Cages were kept in a university-approved animal housing room at ambient indoor temperatures with lights turned on during daylight hours (9L:15D). Experiments were approved by the ILACUC committee of Ohio State University (Protocol 2004A0065) .

Measurements of metabolic rate

We completed all metabolic measurements within one week of capture, and I

removed food from birds 2 hrs prior to any measurements in order to assure that they

were postabsorptive. Mass (±0.1g) and cloacal temperature (±0.1 oC) of each bird were

recorded before and after each trial.

Measurements of BMR began at 20:00 hrs and continued throughout the night.

We placed birds in one liter stainless-steel chambers lined with dark brown Teflon to

reduce reflective radiation and minimize adherence of water molecules to the chamber

(Porter 1969). Each chamber contained a wire mesh platform and a plastic perch above a

83 layer of mineral oil to capture feces, thus eliminating them as a source of water vapor.

Rubber gaskets rendered the lids airtight and excluded light. To monitor Ta within the chambers, we inserted 30-gauge copper-constantan thermocouples into each chamber.

We placed four chambers into a temperature-controlled cabinet that was maintained at

32 oC using a Peltier device (Sable Systems Pelt 4). Before entering the chamber, compressed air was directed through a column of Drierite to remove water, and then through mass flow controllers (Tylan/Mykrolis, Chaska, MN) that we had previously calibrated using a glass bubble meter (500 mL-Levy 1964). An automated system of solenoids sequentially sampled the outflow air from each chamber. Excurrent air passed through a Dew Prime II hygrometer to measure dewpoint (EdgeTech, Marlborough,

Massachusetts), and then through tubes containing silica gel, ascarite, and silica gel to remove water and CO 2. Finally, the excurrent air advanced to an Ametek S3AII oxygen analyzer to measure fractional concentration of oxygen. We directed outputs from the oxygen analyzer, dew point hygrometer, and thermocouples to a CR23X Micrologger®

(Campbell Scientific, Logan, Utah) with a one minute sampling rate. Data from each chamber were recorded for 12 minutes before the solenoid system switched to the next chamber. We averaged oxygen consumption over 10 minute intervals, chosen by visual examination of low and constant values during the sampling period. We calculated rate of oxygen consumption, V& , using equation 4 from Hill (1972). O2

One to two days after measurement of BMR we measured PMR between 08:00 and 18:00 hrs. PMR was measured using a system similar to that for BMR, except that the temperature of the chamber was controlled by a mixture of antifreeze circulating in copper coils that surrounded the chamber. The entire assembly was insulated by 84 Styrofoam, and the temperature of the antifreeze was controlled by a refrigerated water bath (NESLAB RTE-140, Pittsburgh, PA). Incurrent air was a mixture of 79% helium-

21% oxygen (heliox) (Rosenmann and Morrison 1974). We set the initial water bath temperature at 32 oC, and birds were allowed a minimum of 30 min to adjust to the

o chamber before we began decreasing Ta by about 1.0 C per min. We terminated

measurements when oxygen consumption no longer increased with a decrease in chamber

temperature (Swanson et al. 1996). For PMR, we calculated instantaneous rates of

oxygen consumption (Bartholomew et al. 1981) by averaging values of oxygen

consumption over 10 minute intervals, and took the highest 10 minute mean over the

entire test period as PMR.

Assays of metabolic enzyme activity

Following BMR and PMR measurements, we sacrificed birds by cervical

dislocation and immediately excised the pectoral muscles. We weighed both organs, snap

froze them in liquid nitrogen, and placed them in a -80 oC freezer, where they were stored

until the time of assay.

At the time of assay, tissue samples were thawed, weighed to the nearest 0.1g,

minced on a chilled glass Petri dish, and processed in 10 volumes (vol/wt) of

homogenization buffer (20 mM HEPES, 1.0 mM EDTA, 1.0 mM DTT, 0.2% BSA, 0.1%

TritonX-100, pH 7.3). The homogenization process included a three second immersion in

a Polytron tissue processor (Brinkmann Instruments, USA) followed by ten passes in a

85 glass-glass homogenizer. We then sonicated the homogenate in three 15-second intervals separated by 45-second pauses. Samples were kept on ice throughout the homogenization procedure.

We performed all enzyme assays at 25 oC on a Shimadzu UV-2450 spectrophotometer (Columbia, MD, USA) with a temperature controlled chamber. CS

(E.C. 2.7.1.11) activity was measured at 412nm following Dawson and Olson’s (2003) modified method of Srere (1969). This procedure observes the formation of mercaptide ions from 5,5-dinitrobenzoic acid (DTNB) as a result of the condensation of oxaloacetate with acetyl CoA. The CS assay medium included 200 mM Tris-HCl, 5 mM EDTA, 0.1 mM DTNB, 0.2 mM s-acetyl-CoA, 0.5 mM cis-oxaloacetate, and 0.1 ml diluted homogenate at a pH of 7.5 in a final volume of 1.0 ml.

HOAD (E.C. 1.1.1.35) activity was assayed using Dawson and Olson’s (2003) modified method of Bass et al. (1969), which follows the absorbance reduction at 340 nm due to the oxidation of NADH to NAD +. The final reaction volume of 1.0 ml contained

200 mM TEA-HCl, 10 mM EDTA, 0.3 mM NADH, 0.1 mM s-acetyl-CoA, and 0.1 ml diluted homogenate at a pH of 7.0.

We assayed PFK (E.C. 4.1.3.7) activity using Dawson and Olson’s (2003) modified method of Crabtree and Newsholme (1975). This assay observes a change in absorbance at 340 nm due to the oxidation of NADH after the addition of fructose-6- phosphate. The PFK assay medium (pH=8.2) included 0.4 M Tris-Cl, 50 mM MgCl 2, 2.0

M KCl, 30 mM KCN, 2 mM ATP, 4 mM AMP, 0.3 mM NADH, 2 mM F6P, 0.1 ml

86 diluted homogenate, and an excess of aldolase, glycerol-3-phosphate dehydrogenase, and triosephosphate isomerase to ensure a complete reaction. Each sample was run in triplicate and averages were used in calculations.

Total protein concentrations were determined using the Folin phenol reagent method of Lowry et al. (1951), with bovine serum albumin (BSA) as the standard.

Maximum enzyme activities are reported as both µmoles min -1 g-1 wet tissue and µmoles

1 -1 min- g protein, and total muscle enzyme activity was calculated based on organ wet mass.

Species identification

In regions such as Ohio where black-capped and Carolina chickadees coexist,

morphological similarities make species identification difficult (Sattler and Braun 2000).

We designated Ohio birds as BC, CA, or hybrid based on amplified fragment length

polymorphism (AFLP) analysis (Vos et al. 1995), with modifications proposed by

Kingston and Rosel (2004). At the time of capture, we collected blood from the bird’s

brachial vein using a 25-gauge needle and a heparinized capillary tube, placed the sample

in lysis buffer (100 mM Tris at pH 8.0, 100 mM EDTA, 10 mM NaCl, 0.5% SDS;

Longmire et al. 1988), and stored it at 4 oC.

We extracted DNA from blood samples using the methods of Bronson et al.

(2003). Extracted DNA was digested with restriction enzymes Eco RI and Taq I, followed

by two rounds of PCR (preselective and selective) to both amplify fragments and

fluorescently label them for detection. In addition to the birds collected above, we also

incorporated extracted DNA from additional chickadees captured between December

87 2003 and February 2006 in our analysis (see Chapter 2). We sized restriction fragments using GeneMapper v3.7 software (Applied Biosystems, Foster City, CA) and assigned individuals to populations using STRUCTURE v2.1 (Pritchard et al. 2000). Including all samples, we analyzed 245 loci from 218 individuals using 100,000 estimation steps after

50,000 burn-in steps, and each value of K (the number of clusters) 1 to 5. We achieved the most logical population structure using a K of 2, and we classified birds as hybrids if they were assigned a 25-75% probability of belonging to either parental species. We identified only one hybrid among the Ohio samples and eliminated this individual from all subsequent analyses.

Statistical analysis

Prior to analysis, all data were tested for normality and homogeneity of variance using Shapiro-Wilk’s test and Levene’s test, respectively. In the instances where assumptions of normality were not met, data were either log- or reciprocal- transformed to meet assumptions. We assessed whole-organism metabolic rates both across and within species using an analysis of variance (ANOVA) with species or population as a fixed factor. Maximum enzyme activity per gram of wet tissue and protein, as well as total muscle enzyme activity, was analyzed in a similar fashion.

Because mass and metabolic rate were not statistically correlated within each species, we could not use an analysis of covariance (ANCOVA) to control for the effect of body size on metabolic rate as we have in the past (see Packard and Boardman 1988, 1999, Hayes

2001). Thus, we also assessed mass-corrected metabolic rates both across and within species using an analysis of variance (ANOVA) with species or population as a fixed

88 factor. We compared total muscle enzyme activity across and within species using an

ANCOVA with species or population as the fixed factor and body mass-organ mass as the covariate, in order to correct for body mass order while eliminating part-whole correlations (Christians 1999).

3.4 RESULTS

Body mass and metabolic rate

BC chickadees were significantly larger than CA chickadees ( F1,30 = 25.1,

P<0.001; Figure 3.1, Table 3.1). There was no significant difference in mass between WI and OH BC (P=0.78), or between TN and OH CA (P=0.95). BC birds (mean=1.53 g) also

had a significantly larger pectoral muscle mass than CA (mean = 1.21 g) ( F1,30 =19.6,

P<0.001), however, this difference was no longer significant after accounting for total

body mass ( P=0.07). There was also no significant difference in pectoralis mass between

WI and OH BC (P=0.12), or between TN and OH BCA (P=0.90). After adjusting for body

mass WI birds did have larger pectoral muscles than OH BC birds ( P=0.03), but this

difference was no longer significant after correcting for multiple comparisons in Sidak

post-hoc tests ( P=0.16).

89

Figure 3.1. Mean total body mass (A - species, B - populations) and pectoralis mass (C -

species, D - populations) for chickadee species and populations. Sample sizes: BC n=22,

CA n=16, WI n=11, OH BC n=11, OH CA n=5, TN n=5. Pairs designated with a ( ∗) are

significantly different (P<0.05). Pairs designated with a (**) are significantly different

(P<0.05) after correcting for body mass. 90 Table 3.1. Results of ANOVA and ANCOVA comparing total body mass and pectoral mass across species and populations. a

All BC vs All CA BC: WI vs. OH BC

BC CA P-value WI OH BC P-value Body mass 10.9 ± 0.2 9.6 ± 0.1 < 0.001 11.1 ± 0.3 10.7 ± 0.2 0.78 Pectoral muscle mass 1.53 ± 0.04 1.21 ± 0.05 < 0.001 1.62 ± 0.07 1.44 ± 0.04 0.12 Pectoral muscle mass b (adj.) 0.07 0.16

CA: TN vs. OH CA OH BC vs. OH CA

TN OH CA P-value P-value Body mass 9.8 ± 0.2 9.4 ± 0.2 0.95 0.008* Pectoral muscle mass 1.27 ± 0.05 1.16 ± 0.08 0.90 0.03* Pectoral muscle mass b (adj.) 0.98 0.69

a All data are reported as mean ± 1 S.E. b P-values for pectoral muscle comparisons are from ANCOCA adjusting for total body mass * Statistically significant difference between species or populations being compared

91 With data pooled from all individuals, both basal and peak metabolic rates increased significantly with mass (Figure 3.2). The relationship between mass and BMR was described as log BMR (W) = -1.70 + 1.06 log Mass (g) ( r=0.67, P<0.001), and the

relationship between mass and PMR was described as log PMR (W) = -0.84 + 0.97 log

Mass (g) ( r=0.63, P<0.001).

We did not obtain the same significant relationship between mass and metabolic

rate when analyzing the two species independently. Mass was only a significant predictor

of BMR in BC ( r=0.45, P=0.04). Mass was not significantly related to BMR in CA ( r=

-0.18, P=0.65) or to PMR in either BC or CA ( r=0.17, P=0.47 and r=0.52, P=0.15, respectively). Thus, further comparisons of metabolic rate between species or populations were done using ANOVA with mass-corrected metabolic rate as the dependent variable, rather than using ANCOVA with whole-organism metabolic rate as the dependent variable and including mass as a covariate.

Metabolic rate across and within species

Mean whole-organism BMR of BC was significantly greater than that of CA ( F1,29 =27.6,

P<0.001; Figure 3.3a, Table 3.2).This result remained significant after controlling for mass ( F1,29 =5.0, P=0.033). Mean whole-organism PMR was also significantly greater in

BC ( F1,27 =25.4, P<0.001; Figure 3.3b). However, there was no significant difference

between the mass-corrected PMR of BC and CA chickadees ( F1,27 = 3.4, P = 0.08).

92

Figure 3.2. The relationship of BMR (A) and PMR (B) to mass in BC (filled circles) and

CA (open circles). Trend lines are plotted solid for BC and dashed for CA. Sample sizes:

BC BMR n=22, CA BMR n=9, BC PMR n=20, CA PMR n=9.

93

Figure 3.3. Mean whole-organism BMR (A) and PMR (B) for CA and BC chickadees.

Sample sizes: BC BMR n=22, CA BMR n=9, BC PMR n=20, CA PMR n=9. Pairs designated with a ( ∗) are significantly different ( P<0.05).

94 Table 3.2. Results of ANOVA comparing whole-organism and mass-corrected metabolic rates across species and populations. a

All BC vs All CA BC: WI vs. OH BC

BC CA P-value WI OH BC P-value BMR (W) 0.257 ± 0.01 0.205 ± 0.004 <0.001* 0.266 ± 0.01 0.247 ± 0.001 0.32 BMR (W/g) 0.024 ± 0.001 0.022 ± 0.001 0.033* 0.024 ± 0.001 0.023 ± 0.001 0.83 PMR (W) 1.50 ± 0.03 1.22 ± 0.04 <0.001* 1.54 ± 0.04 1.46 ± 0.05 0.72 PMR (W/g) 0.137 ± 0.003 0.127 ± 0.003 0.08 0.139 0.005 0.136 ± 0.005 0.99

CA: TN vs. OH CA OH BC vs. OH CA

TN OH CA P-value P-value BMR (W) 0.205 ± 0.01 0.205 ± 0.01 0.99 0.021* BMR (W/g) 0.021 ± 0.001 0.021 ± 0.001 0.99 0.94 PMR (W) 1.24 ± 0.03 1.20 ± 0.08 0.99 0.02* PMR (W/g) 0.126 ± 0.004 0.128 ± 0.01 0.99 0.90 a All data are reported as mean ± 1 S.E. * Statistically significant difference between species or populations being compared

95

Figure 3.4. The relationship of BMR (A) and logPMR (B) to pectoralis mass in BC (filled circles) and CA (open circles). Trend lines are plotted solid for BC and dashed for CA.

Sample sizes: BC BMR n=22, CA BMR n=9, BC PMR n=20, CA PMR n=9.

96 There was a significant effect of chickadee species and location on whole- organism BMR ( F3,27 =11.04, P<0.001; Figure 3.4a). However, post hoc tests revealed that there was no significant difference between WI and OH BC (P=0.32) or between TN and OH CA (P>0.99), specifically. Although there was also a significant effect of species

and population on whole-organism PMR ( F3,25 =9.10, P<0.001; Figure 3.4b), post hoc

tests returned no significant difference between WI and OH BC (P=0.72) or between TN

and OH CA (P>0.99). After correcting for mass, there was no significant effect of species

and location on PMR ( P=0.36).

Metabolic enzyme activity

There was no significant difference between BC and CA chickadees in mass-

corrected or protein-corrected activity of any enzyme ( P > 0.05 for all enzymes, Table

3.3). We also used organ mass and mass-specific enzyme activity to calculate the total

muscle activity of each enzyme, and we found a significant difference in total pectoralis

enzyme activity after adjusting for body size. BC had significantly higher pectoralis CS

activity after adjusting for pectoral muscle size relative to total body mass ( F1,29 =5.2,

P=0.03) The difference in total HOAD and PFK enzyme activity was also not significant

(P > 0.17 for both).

Whole-organism metabolic rate was not significantly correlated with total activity of any enzyme in BC or CA ( P>0.05 for all correlations). There was a significant relationship between whole-organism PMR and total pectoralis activity of all three enzymes in BC (Figure 3.5), but the same correlations were not significant in CA. We

97 Table 3.3. Results of ANOVA and ANCOVA comparing metabolic enzyme activity across species and populations. a

All BC vs All CA BC: WI vs. OH BC

Enzyme BC CA P-value WI OH BC P-value

CS (activity per g muscle) 112.6 + 3.3 100.8 ± 4.9 0.59 110.0 ± 2.8 114.3 ± 4.7 0.99 CS (activity per g protein) 309.3 ± 9.2 296.7 ± 17.4 0.36 305.4 ± 8.2 302.6 ± 13.7 0.98 b CS (total muscle activity) 171.2 ± 6.3 124.2 ± 10.1 0.03* 177.4 ± 9.4 164.9 ± 8.3 0.96

HOAD (activity per g muscle) 13.5 ± 1.2 18.6 ± 2.8 0.76 14.9 ± 1.9 12.0 ± 1.5 0.55 HOAD (activity per g protein) 37.1 ± 3.1 55.7 ± 9.2 0.54 42.2 ± 4.4 32.0 ± 4.0 0.16 b HOAD (total muscle activity) 20.9 ± 2.3 23.4 ± 4.1 0.52 24.5 ± 3.9 17.4 ± 2.2 0.40

PFK (activity per g muscle) 24.0 ± 1.9 27.7 ± 2.8 0.30 24.7 ± 3.0 23.4 ± 2.4 >0.99 PFK (activity per g protein) 66.2 ± 4.9 82.0 ± 8.9 0.19 71.2 ± 4.5 62.1 ± 6.6 0.90 b PFK (total muscle activity) 37.5 ± 3.8 34.3 ± 4.1 0.17 41.1 ± 6.5 33.9 ± 4.0 0.91

CA: TN vs. OH CA OH BC vs. OH CA

Enzyme TN OH CA P-value P-value

CS (activity per g muscle) 107.3 ± 5.7 94.3 ± 7.2 0.52 0.82 CS (activity per g protein) 317.8 ± 31.0 275.7 ± 13.4 0.78 0.86 b CS (total muscle activity) 137.1 ± 11.5 111.3 ± 15.6 0.40 0.004*

HOAD (activity per g muscle) 23.5 ± 3.9 13.8 ± 2.7 0.20 0.98 HOAD (activity per g protein) 70.3 ± 14.4 41.1 ± 8.2 0.33 0.71 b HOAD (total muscle activity) 30.4 ± 5.9 16.4 ± 4.1 0.13 0.99

PFK (activity per g muscle) 33.7 ± 3.0 21.8 ± 3.0 0.17 0.99 PFK (activity per g protein) 98.9 ± 10.5 65.2 ± 10.0 0.31 0.99 b PFK (total muscle activity) 42.9 ± 4.6 25.6 ± 4.1 0.15 0.67

a All data are reported as mean ± 1 S.E. b ANCOVAs comparing total muscle activity included body mass-organ mass as a covariate * Statistically significant difference between species or populations being compared

98

Figure 3.5. Scatterplots of total pectoralis enzyme activity for CS (A), HOAD (B), and

PFK (C) versus logPMR for BC (filled circles) and CA (open circles). Trend lines are plotted solid for BC and dashed for CA. Correlation coefficients and significance levels are presented next to each plot. Sample sizes: BC n=20, CA n=9.

99 Figure 3.5

100

Figure 3.6. Scatterplots of total pectoralis enzyme activity for CS (A), HOAD (B), and

PFK (C) versus logPMR for WI (filled triangles) and OH BC (open triangles). Trend lines are plotted solid for WI and dashed for OH BC . Correlation coefficients and significance levels are presented next to each plot. Sample sizes: WI n=9, OH BC n=11.

101 Figure 3.6

102 also examined the correlation between whole-organism PMR and total pectoralis enzyme activity within each species. In chickadees from WI, PMR was positively correlated with both HOAD ( r=0.87, P=0.001) and PFK ( r=0.87, P=0.001), but not with CS ( P=0.21,

Figure 3.6). There were no significant correlations between PMR and any enzyme in chickadees from TN, OH CA , or OH BC .

3.5 DISCUSSION

This study compared the activity of three metabolic enzymes and their

relationships to whole-organism metabolic rate in multiple populations of black-capped

and Carolina chickadees. Combining conspecifics populations, BC chickadees did have

higher whole-organism BMR and mass-corrected BMR when compared to CA

chickadees. These results conflict with our previous work (see Chapter 2), in which mass-

corrected BMR did not differ between species, a potentially confounding result because

the birds included in this study are merely a subset of the larger sample presented in

Chapter 2 (Sample sizes in Chapter 2: BC BMR: 66, CA BMR: 28, BC PMR: 58, CA

PMR: 17). In addition, when we pooled all birds both whole-organism BMR and whole-

organism PMR were positively correlated with mass. However, these correlations were

no longer significant when the two species were examined separately. All of these

disparities may be due to the much smaller sample size presented here, and thus,

subsequent results should be interpreted with this discrepancy in mind.

103 The pectoral muscle is the primary thermogenic organ in birds (Carey et al. 1978,

Marsh and Dawson 1982), and multiple studies have found a correlation between pectoralis size and an increase in thermogenic capacity in winter passerines (Swanson

1991, Cooper 2002). For example, mountain chickadees in Utah had a 33% increase in pectoralis mass in winter over summer conspecifics (Cooper 2002), and BC chickadees from South Dakota had pectoral muscles that were 13% larger in winter (Liknes 2005).

Both of these increases in muscle mass were accompanied by a higher PMR. In this study, BC chickadees did not differ from CA with respect to mass-corrected PMR or pectoralis mass. While this is consistent with the notion that the pectoral muscle is the primary site for shivering thermogenesis and a primary contributor to PMR, it does not provide positive evidence for this association.

Compared to CA chickadees, BC chickadees have a higher mass-corrected BMR, suggesting a difference in tissue metabolic rate between the two species. While we did not find any differences in mass- or protein-corrected enzyme activities between the two species, we did observe a higher level of total muscle CS activity in BC. However, BMR is largely a function of central organs, and because we did not collect data on these additional organs, we can not eliminate variation in their mass or enzymatic activity as a contributing factor to the higher BMR of BC chickadees.

Avian studies have produced mixed results with regards to the relationship between high CS activity and high levels of aerobic metabolism, such as those required for migration and shivering thermogenesis. Some studies have found significant increases in pectoralis CS associated with migration (Marsh 1981, Lundgren and Kiessling 1985,

1986; Lundgren 1988 ) or winter acclimatization (Dawson and Olson 2003), while other

104 studies failed to observe significant trends ( Marsh and Dawson 1982, Vézina and

Williams 2005). Thus, variation in muscle CS activity is not a universal adjustment promoting elevated aerobic capacity under energetically challenging conditions.

The data for mass-specific CS activity is this study (BC: 112 µmol min -1 g-1, CA:

101 µmol min -1 g-1) fell at the lower end of values reported in the literature for avian

pectoral muscle (approximate range: 90-275 µmol min -1 g-1; Marsh 1981, Marsh and

Dawson 1982, Lundgren and Kiessling 1985, Lundgren and Kiessling 1986, O’Connor and Root 1993, Dawson and Olson 2003, Vézina and Williams 2005). The values of mass-specific PFK activity in this study (BC: 24 µmol min -1 g-1, CA: 28 µmol min -1 g-1)

are also at the lower end of those reported in the avian literature (22-75 µmol min -1 g-1;

Marsh 1981, Marsh and Dawson 1982, Yacoe and Dawson 1983, Lundgren and Kiessling

1985, Lundgren and Kiessling 1986, Carey et al. 1989, Dawson and Olson 2003). Due to its regulatory role, PFK is used to characterize the glycolytic potential of avian muscle,

(see Dawson and Olson 2003, Krijgsveld et al. 2001, Marsh 1981, Marsh and Dawson

1982) which represents the provisioning of carbohydrates to aerobic respiration. Studies specifically on winter-acclimatized birds have demonstrated high levels of PFK in the pectoral muscles relative to other muscles, or relative to the same muscles in summer birds, suggesting further evidence that the pectoral muscles are the primary thermogenic tissue (Marsh and Dawson 1982, Dawson and Olson 2003). Our results suggest that this reliance on carbohydrates for aerobic respiration does not change among the winter populations of chickadees that we studied.

105 The data for mass-specific HOAD activity is this study (BC: 14 µmol min -1 g-1,

CA: 19 µmol min -1 g-1) also fell at the lower end of values reported in the literature

(approximate range: 10-75 µmol min -1 g-1; Marsh 1981, Marsh and Dawson 1982, Yacoe and Dawson 1983, Lundgren and Kiessling 1985, Lundgren and Kiessling 1986, Carey et al. 1989, O’Connor and Root 1993, O’Connor 1995, Dawson and Olson 2003), and as with CS and PFK, we saw no difference in enzyme activity between any populations.

Triglycerides (fatty acids) are the primary source of metabolic energy storage, the oxidation of which yields up to eight times more energy than an equal dry weight of carbohydrates (Hochachka et al. 1977). Several studies have measured increased HOAD activity in the muscle tissue of animals that are under increased energetic demands. The pectoral muscles of small winter-acclimatized birds rely predominantly on the β- oxidation of fatty acids for production of ATP during prolonged cold exposure (Dawson and Olson 2003, Marsh and Dawson 1982, Yacoe and Dawson 1983). In American goldfinches ( Carduelis tristis ), HOAD activity in the pectoral muscle increased 52% from summer to winter, without any corresponding increase in muscle size (Marsh and

Dawson 1982). This increase coincided with a reduced reliance on carbohydrates during times of cold stress, suggesting that cold resistance of winter birds coincides with a increased reliance on fats as energetic fuel. Similar results were observed in the pectoral muscle of the Grey Catbird ( Dummetella carolinensis ), where HOAD activity doubled as a response to premigratory fattening in preparation for sustained flight (Marsh 1981).

Although our results show no difference in mass-specific enzyme activity between any chickadee populations, some researchers have observed such geographical variation (O’Connor 1995, O’Connor 1996); winter house finches (Carpodacus

106 mexicanus ) from Ann Arbor, MI (42.27 oN) had significantly higher CS and HOAD activity that those from Irvine, CA (33.67 oN). This study noted that the trend in HOAD

activity is consistent with seasonal changes in body fat content. While California house

finches exhibit no seasonal change in body fat, Michigan birds carry twice the fat stores

in winter than they do in summer (Dawson et al. 1983), possibly due to differences in

food predictability, though the same trend may not be predicted for chickadee

populations. Comparisons of enzymes specifically associated with fat metabolism may

not be appropriate when comparing primarily ground-foraging (e.g. house finches, Hill

1993) with primarily tree-foraging, food-caching birds such as chickadees (Bent 1946,

Haftorn 1956). Body mass and fat levels of tree-foraging birds typically show less

seasonal variation compared with ground-foraging birds, due to more predictable food

supplies for tree-foraging birds than for ground-foraging birds (Rogers 1987, Rogers and

Smith 1993). With the exception of BC chickadees from Fairbanks, Alaska (Sharbaugh

2002), North American Parids do not appear to undergo true winter fattening (Cooper

2007), so it may not be surprising that mass-specific, fat-catabolizing enzyme activity

does not vary seasonally either. Thus, although chickadees may show an increased

reliance on lipid metabolism in winter (Liknes 2005), there is no reason to expect that this

response varies among chickadees at different latitudes as it does with house finches.

We also examined the relationship between metabolic enzyme activity and

metabolic rate among individuals of both chickadees species. Although whole-organism

PMR was not correlated with total body mass in either species, PMR in BC was

significantly correlated with pectoralis mass and total muscle enzyme activity of CS,

PFK, and HOAD. However, this trend was not consistent among all BC. We did not find

107 any significant correlations between PMR and total activity of any enzyme among OH BC ,

yet in WI birds, PMR was strongly correlated not only with pectoralis size, but also with

total HOAD and PFK activity. We conclude that among BC, higher metabolic rates are

primarily a result of greater pectoralis mass, rather than an increase in tissue-specific

metabolic enzyme activity. However, as these are strictly correlational analyses, further

study will be necessary to determine if these relationships are associated with

thermogenic capacity.

While many studies have used CS, HOAD, and PFK activities to make functional

connections between cellular processes and whole-organism metabolic rates, it should be

noted that in vitro enzyme activities do not necessarily equate with in vivo metabolic fluxes. Unfortunately, we know little about how metabolic enzymes and mitochondria actually function in vivo (Suarez 1996), especially with regards to how they govern

whole-organism metabolic output. Further studies exploring enzymatic correlates to

whole-organism metabolic rate would benefit not only from a refinement of these enzyme

assays, but also from the inclusion of additional processes that may bridge the gap

between enzyme activity and tissue metabolism, such as measuring the oxygen

consumption of whole mitochondria or exploring the roles that regulators, such as

transcriptional coactivators (Wende et al. 2007), have on mitochondrial function. Finally,

determining the direction of causality between cellular processes and chickadee

distribution will require not only broad geographical sampling, but also the inclusion of

studies that incorporate experimental acclimatization to multiple thermal environments.

108

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