SOCIAL FACILITATION OF REPRODUCTION:
POTENTIAL FOR ALLEE EFFECTS IN A DECLINING
AMPHIBIAN
Flavia Papini
Department of Biology
McGill University, Montreal
August 2016
A thesis submitted to McGill University in partial fulfilment of the
requirements of the degree of Master of Science.
©Flavia Papini, 2016
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………………...1
RÉSUMÉ…………………………………………………………………………………………2
ACKNOWLEDGEMENTS……………………………………………………………………..4
CONTRIBUTIONS OF AUTHORS…………………………………………………………....6
CHAPTER I: GENERAL INTRODUCTION & LITERATURE REVIEW……………...…7
ABUNDANCE & FITNESS: THE ALLEE EFFECT…………………………………….8
TEMPERATE ANURAN MATING SYSTEM & REPRODUCTIVE PHYSIOLOGY
……………………………………………………………………………………………12
STUDY SPECIES & RESEARCH GOALS…………………………………………….15
LITERATURE CITED…………………………………………………………………..17
FIGURE………………………………………………………………………………….23
CHAPTER II: MALE ABUNDANCE AND BREEDING EFFORT……………….………..24
LINKING STATEMENT………………………………………………………………..25
ABSTRACT……………………………………………………………………………...26
INTRODUCTION……………………………………………………………………….27
MATERIALS AND METHODS………………………………………………………...29
RESULTS……………………………………………………………………………......32
DISCUSSION……………………………………………………………………………34
LITERATURE CITED…………………………………………………………………..37
TABLES………………………………………………………………………………....41
FIGURES………………………………………………………………………………...42
CHAPTER III: BREEDING EFFORT AND TESTOSTERONE LEVEL....……………….46
LINKING STATEMENT………………………………………………………………..47
ABSTRACT……………………………………………………………………….……..48
INTRODUCTION……………………………………………………………………….49
MATERIALS AND METHODS………………………………………………………...50
RESULTS………………………………………………………………………………..53
DISCUSSION……………………………………………………………………………55
LITERATURE CITED………………………………………………………………..…58
TABLES………………………………………………………………………………....62
FIGURES…………….…………………………………………………………………..63
CHAPTER IV: CONCLUSIONS AND CONSERVATION IMPLICATIONS......................65
LITERATURE CITED…………………………………………………………………..68
LIST OF TABLES
Chapter 2
Table 1. Summary of male abundance, density, and sample sizes used in comparison of male
Fowler’s Toad breeding effort across three Long Point breeding sites in 2015............................41
Chapter 3
Table 1. Summary of urinary testosterone statistics of male Fowler’s Toads from Long Point and
Nickel Beach during the 2015 peak and post-breeding seasons....................................................62
LIST OF FIGURES
Chapter 1
Figure 1. Strong and weak demographic Allee effects, compared to logistic population growth,
adapted from Berec et al., 2007.....................................................................................................23
Chapter 2
Figure 1. Location of study sites in southern Ontario....................................................................42
Figure 2. Fowler’s Toad calling phenology at two sites differing significantly in abundance......43
Figure 3. Comparison of population and individual-level breeding effort indices across three
low-abundance sites at Long Point (2015).....................................................................................44
Figure 4. Relationship between male abundance and breeding effort in Fowler’s Toads at Long
Point, Ontario (data from 1998-2015)...........................................................................................45
Chapter 3
Figure 1. Urinary testosterone concentrations of male and female Fowler’s Toads at Long Point
during the 2015 breeding season....................................................................................................63
Figure 2. Urinary testosterone concentrations of male Fowler’s Toads across three breeding sites
at Long Point during the 2015 breeding season.............................................................................63
Figure 3. Urinary testosterone concentrations of male Fowler’s Toads at Long Point and Nickel
Beach during the 2015 peak and post-breeding seasons................................................................64
Figure 4. Urinary testosterone concentrations of male Fowler’s Toads found either attending or
away from a chorus in Long Point during the 2015 breeding season............................................64
ABSTRACT
The Allee effect refers to reduced population fitness (negative per capita growth rate) at
very small abundances due the positive relationship between some component of individual
fitness and number or density of conspecifics. One causal mechanism of Allee effects is ‘social
facilitation of reproduction’, where individuals are less likely to reproduce if not perceived or
stimulated by others. Considering global amphibian declines, chorusing anurans are likely
candidates for this kind of Allee effect because of their reliance on social cues (calls) to enhance
and synchronize reproductive effort among conspecifics, and maintain elevated gonadal activity
throughout the breeding season. Interference of these essential processes from insufficient
individual participation should negatively affect recruitment, and ultimately population
persistence. I test this prediction in an endangered population of Fowler’s Toads (Anaxyrus
fowleri) in Long Point, Ontario, whose decline, initially triggered by invasion of a non-native
plant, has led to reduced chorus formation. To test for a positive abundance-benefit relationship,
I first compared male breeding effort and phenology across small (Long Point) and large (Nickel
Beach) toad populations. As expected, low abundance conditions resulted in a shortened
breeding season with fewer and significantly smaller choruses. Long-term data confirmed that
male breeding effort is disproportionately lower at smaller abundances, and that groups may
attain a critical behavioural threshold (circa four males) below which individuals may not breed.
This constitutes the second case of empirical evidence for a behaviourally-mediated component
Allee effect in an endangered amphibian. I then ask whether endocrine activity can explain this
variation in breeding effort. Contrary to expectations that testosterone amplifies expression of
reproductive behaviours, male toads not attending breeding aggregations had slightly higher
urinary testosterone levels compared to participatory males. However, in agreement with a social
enhancement of reproduction, I found that the prolonged breeding season exhibited by males at
Nickel Beach was mirrored by a sustained hormonal state after calling ceased. This was not the
case at Long Point, where testosterone levels dropped abruptly post-breeding. Based on these
results, I conclude that declines of Fowler’s Toads at Long Point may be fuelled by progressive
loss of facilitative breeding behaviours, but that urinary testosterone levels do not fully explain
this phenomenon. Through use of detailed long-term and local-scale data we can better detect
component Allee effects and infer their potential to catalyse demographic change, both
improving our understanding of population-specific risks and informing conservation strategies.
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RÉSUMÉ
L’effet Allee est un principe fondé sur la relation positive entre un élément de fitness
(valeur sélective) individuelle et le nombre ou densité de congénères, tel que la fitness d’une
population (taux de croissance négatif) est réduite quand elle devient très petite. Un mécanisme à
la base de l’effet Allee est la ‘facilitation sociale de reproduction’, où les évènements
d’accouplement ont lieu seulement si les individus se perçoivent ou s’incitent à reproduire. Étant
donné que les amphibiens déclinent au niveau mondial, les anoures devraient être encore plus
sensibles à ce type d’effet Allee à cause de leur dépendance sur les signaux sociaux (vocalises)
pour amplifier et synchroniser l’effort reproductif entre congénères, et maintenir l’état actif des
organes sexuelles pendant la saison reproductive. L’entrave de ces comportements de
rassemblement devrait négativement affecter la probabilité d’accouplement et en conséquence, la
quantité de jeunes produits. Je mets à l’essai cette prédiction en étudiant une population menacée
de crapauds Fowler (Anaxyrus fowleri) à Long Point, en Ontario, dont son déclin a été incité par
l’établissement d’une plante invasive, et qui par conséquence devient de plus en plus diminuée.
Je commence par démontrer la dépendance positive entre le nombre de congénères et la
manifestation des comportements bénéfiques en comparant le degré et la phénologie d’effort
reproductive entre une petite (Long Point) et une grande (Nickel Beach) population. Comme
prévu, les milieux de faible abondance ont démontré une saison reproductive plus courte, avec
des chœurs plus petits et moins fréquents. Les données historiques confirment que l’effort
reproductif est disproportionnellement réduit quand les congénères sont moins nombreux et que
les groupes peuvent atteindre un seuil critique (environs quatre mâles) au-dessous duquel il n’y
aura aucun effort reproductif. Ceci représente la deuxième preuve d’un effet Allee élémentaire
réglé par la reproduction coopérative dans une population d’amphibiens. Je continue en
demandant si le manque d’effort reproductif à Long Point est lié au fonctionnement du système
endocrinien. Contrairement aux avis que le testostérone amplifie l’expression des comportements
sexuels, les niveaux de testostérone urinaire des mâles non-participants étaient plus élevés que
ceux des mâles présents au chœur. Cependant, j’ai trouvé un maintien des niveaux de
testostérone élevés après la fin de la période d’accouplement à Nickel Beach, ce qui correspond à
la saison reproductive prolongée observée au même site. Les niveaux de testostérone des mâles à
Long Point se sont baissés rapidement après les derniers chœurs. Ces observations sont en accord
avec l’hypothèse d’un renforcement social de reproduction. Selon ces résultats, je conclu que les
2
déclins de crapauds Fowler à Long Point peuvent être encouragés par la perte successive
d’interactions sociales, mais que le niveau de testostérone urinaire n’explique pas entièrement ce
phénomène. En utilisant les données détaillées provenant d’enquête locale à long-terme, on peut
mieux détecter les effets Allee élémentaires et déduire leur potentiel pour catalyser des
changements démographiques, tout en améliorant notre compréhension des risques uniques aux
populations d’intérêt et informant des stratégies pour les conserver.
3
ACKNOWLEDGEMENTS
Over four years ago, I joined the Toad Forces as an eager undergraduate student
volunteer. Little did I know, it would only be the beginning of my residence with the Fowler’s
Toad Team. First, I must thank my supervisor, Dr. David M. Green, for offering me the
opportunity to do meaningful field research, and the hours spent reviewing this thesis. Thanks to
my committee members, Dr. Simon Reader and Dr. Lauren Chapman, whose novel perspectives
and thoughtful recommendations were greatly appreciated. Kind regards also to Dr. Jon Sakata
for his mentorship and guidance regarding data analysis. Honoured thanks to Le Fonds de
recherche du Québec – Nature et technologies (FRQNT) for financial support through their
Master of Science Research bursary.
I will forever be indebted to my fellow “toad girl”, Katharine Yagi, for her lasting
friendship and unparalleled involvement in every aspect of this project. With four field seasons
under our belts, we’ve seen it all! I look back fondly on our (mis)adventures. A heartfelt thank
you to Anne Yagi (OMNR) for her immense generosity and hospitality over the years. From
finding me a supply of liquid nitrogen to collecting urine samples when I could not be on-site,
this project would not have been possible without her efforts. In fact, thank you to the entire
Yagi family for feeding and sheltering me as one of their own!
I extend my admiration and gratitude to Dr. Gabriela Mastromonaco and Christine
Gilman from the Reproductive Biology lab at the Toronto Zoo for all urine-related advice, and
conducting the assays. Their patience and professionalism was remarkable. Thanks to Dr. Glenn
Tattersall at Brock University, for coming to the rescue and graciously lending his liquid
nitrogen dewar (fondly nicknamed R2N2) for sample transport. Since one cannot be two places at
once, thanks also to Jeff Houlahan and Paul Crump from the University of New Brunswick, for
lending their outdoor sound recorders and allowing me to have a pair of ears at Nickel Beach.
Credit is also due to the many undergraduate volunteers whom assisted with the annual
population survey while I chased toads for pee: Adam Lisi Verillo, Elisabeth Belanzaran,
Stephen Lee, Edith Shum, Nadia Dalili, Owen Tao, Kristen Crandall and Andrea Cherry.
Deepest thanks to my closest friends and peers: Stephanie Shooner, Lauren Mechak, and
Natasha Salter, for unknowingly helping me find my niche over the years, and reminding me to
have fun along the way. Their individual accomplishments in the field continue to inspire me. I
4
am also grateful for my brilliant lab-mates, Dan Greenberg, Brandon Varela, and David
O’Connor for sharing ideas, laughs, and innumerable venting sessions about the perils of grad
school.
Very special thanks to my high-school science teachers Pardo Pannunzio and Steve
Spetsieris, for recognizing my curiosity about the natural world and encouraging me to pursue
research (beyond our annual Science Fair).
Of course, infinite gratitude to my mother and father, Susan and Marco, for their
unwavering support and confidence over the years. Through nurturing my love of learning from
a young age, they instilled in me a work ethic that has carried me to this point. Thank you for
embracing my insatiable passion for all living things, big and small. I am equally obliged to my
sister, Monica, for her vital positivity and necessary bouts of distraction. Grazie to my
grandparents, my self-proclaimed number one fans, for cheering me on from the side-lines. And
finally, to my partner Quinn: thank you for always believing in me.
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CONTRIBUTIONS OF AUTHORS
Chapter 1
I reviewed relevant literature with editorial support from Dr. David M. Green.
Chapter 2
I analysed field data that I collected in addition to historical data originating from Dr. David M.
Green’s long-term population study. I wrote this chapter with editorial support from Dr. David
M. Green.
Chapter 3
I collected the data and analysed the results. Enzyme immunoassays were conducted by the
Toronto Zoo’ Reproductive Biology Laboratory. I wrote this chapter with editorial support from
Dr. David M. Green.
Chapter 4
I wrote this chapter with editorial support from Dr. David M. Green.
6
CHAPTER I: General Introduction & Literature Review
7
Population size and conspecific density can have profound effects on an organism’s
biology. High density conditions can lead to intraspecific competition for space and/or resources
(Fowler, 1981), which may limit growth and development, ultimately having consequences on
condition, life-history processes, and individual fitness (Semlitsch and Caldwell, 1982; Stewart
et al., 2005). Density-dependent processes exist among plant and animal taxa. For example, corn
planted above optimal density will experience delayed structure differentiation resulting in
reduced growth rate, fertilization, and yield (Sangoi, 2001). Similarly, increased foraging
competition due to high density environments may lead to reduced body condition and higher
rates of juvenile mortality in feral donkeys (Choquenot, 1991). In contrast, low abundances or
densities can result in inbreeding and loss of genetic diversity in plants (Jimenez et al., 2013),
commercially important fish (O’Leary et al., 2013; Pinski and Palumbi, 2014) and mammals
(Gaines et al., 1997).
Abundance is commonly discussed in terms of carrying capacity (K), a quantity above
which the population cannot sustain its size because of finite local resources (Fowler, 1981).
When carrying capacity is reached, a negative density dependence mechanism should cause
negative population growth rate, reducing numbers back below K, whereas smaller but healthy
populations benefit from low enough levels of intraspecific competition, allowing positive
growth rate. Alternatively, in light of recent global trends of population decline and biodiversity
loss, effort has shifted to studying consequences of extremely low abundances or densities
(Stephens and Sutherland, 1999; Courchamp et al., 1999). It is well known that small populations
are particularly susceptible to demographic and environmental stochasticity, however at critically
low abundances, populations may also become vulnerable to inverse density dependence, or
Allee effects (Courchamp et al., 1999; Stephens et al., 1999).
Abundance and Fitness: The Allee Effect
The Allee effect is a trend that manifests itself through mechanisms that rely on the
positive relationship between population size and individual fitness, resulting in higher survival
and/or reproduction at larger abundances and a concomitant reduction of per capita growth rate
at very low abundances or densities (Stephens et al., 1999). This is based on the idea that the
presence of conspecifics confers some fitness benefit, which is significantly reduced or lost when
8
individuals become too few or too sparse (Allee, 1931). The Allee effect can be modeled by
introducing a lower unstable equilibrium (K_ ) to the classic logistic equation of population
growth (Courchamp et al., 1999):
𝑑𝑁
𝑁 𝑁
= rN (1 − ) ( − 1)
𝑑𝑡
𝐾 𝐾−
where per capita growth rate (dN/dt) follows a hump-shaped curve, being negative both above
carrying capacity (K) and below a critical population threshold (K_ ). Below this point, K_,
inverse density-dependence will drive a declining population to extinction (Figure 1).
Allee effects can also exist at smaller scales without affecting population persistence.
Accordingly, Stephens et al. (1999) distinguished between two kinds of Allee effects: component
and demographic. A component Allee effect is based on a positive relationship between
population size and any component of individual fitness, while demographic Allee effects result
from positive relationships between overall population fitness and size (Stephens et al., 1999).
Demographic Allee effects will manifest themselves at the population level only if additive
component Allee effects are strong and abundant enough to result in a net negative population
growth rate at some critical threshold (Stephens et al. 1999). Based on magnitude of opposing
intraspecific competition (negative density dependence) and intraspecific facilitation (positive
density dependence), the resulting demographic Allee effect will vary in strength, and therefore,
detectability (Berec et al., 2007; Kramer et al., 2009). The severity of the effect will also
influence the exact shape of the curve (Figure 1). Weak demographic Allee effects result in
reductions in growth rate over a wide range of population sizes, while strong effects result in
sharper reductions over a smaller range of population sizes, and are characterised by the presence
of a lower critical threshold (Berec et al., 2007, Stephens and Sutherland, 1999).
Any mechanism related to reproduction and/or survival can generate Allee effects
(reviewed by Berec et al., 2007; Kramer et al., 2009). These include anthropogenic exploitation,
cooperative defense, foraging efficiency, mate limitation, and reproductive facilitation, among
others. When any of these strategies become deleterious or inefficient at small population sizes,
an Allee effect may increase risk of population collapse. For example, meerkats exhibit
cooperative anti-predator behaviours when threatened, and therefore suffer higher juvenile
9
mortality rates and extinction risk when living in very small groups (Clutton-Brock et al., 1999).
Similarly, desert bighorn sheep engage in lesser collective vigilance when in smaller groups (<5)
and are more subject to predation when separated from conspecifics (Mooring et al., 2004).
Component Allee effects have also been identified in the black-browed albatross whose foraging
success (fitness correlate) declined significantly at low densities due to their dependence on
observing conspecifics utilizing resource patches (Grunbaum and Veit, 2003). Plants may also be
subject to Allee effects if pollination services are insufficient at low population size or density,
such that individuals experience reduced reproductive success in low flowering years (Forsyth,
2003) or at leading edges of expansion (Davis, 2004).
From the definition proposed by Stephens et al. (1999), it is important to note that not all
aspects of demographic stochasticity can act as mechanisms of the Allee effect. Though rates of
birth, death, or recruitment can increase extinction risk with decreasing population size, they do
not reduce individual fitness, and therefore cannot be considered mechanisms of the Allee effect
(Stephens et al., 1999). Fluctuations in sex ratio, however, do qualify, because sex ratios tend to
skew at low population sizes/densities, decreasing probability of mate encounters and reducing
fitness (Stephens et al., 1999). As such, direct correlations with individual reproductive success
make mating systems particularly sensitive to Allee effects (Kokko and Rankin, 2006; Gascoigne
et al., 2009). Furthermore, most mating systems are regulated in part by abiotic cues, such that
unfavourable environmental events may also severely limit reproduction. Accordingly, among
the Allee effects identified in animal populations, the most commonly cited mechanisms of
action are mate finding/limitation, and social facilitation of reproduction (Kramer et al., 2009).
The first refers to a purely spatial effect, where fewer individuals simply reduce chances
encountering a mate. For example, Kussaari et al., (1998) found that proportion of mated female
Glanville fritillary butterflies decreased with decreasing density, suggesting that declines
hindered mate-finding. On the other hand, social facilitation of reproduction results from some
species-specific behaviour(s). A classic example of this was demonstrated in sexual and
unisexual whiptail lizards, where presence of a sexually active male (or individual displaying
male-like pseudosexual behaviours) can increase the number of ovulating females, thereby
enhancing receptivity and mating probability (Crews et al., 1986).
10
Related to this is the problem of population size versus population density. Though the
terms are often used interchangeably, the distinction between number and density of conspecifics
is an important one (Stephens et al., 1999). Whether an Allee effect is caused by low numbers or
low densities depends on the scale and taxon-specific mechanism in question (Stephens et al.,
1999). For example, considering a closed system, increasing number of individuals without
changing density would still lead to increased mate encounters, potentially easing Allee effects
caused by skewed sex ratios. However, this would not help if the causal mechanism was
environmental modification, for example, in the case of plants that improve local growth
conditions, or sessile animals that better thermoregulate when aggregated (Bertness and
Grosholz, 1985). In the latter context, individuals would only experience a fitness benefit with
increases in density specifically.
Historically abundant but currently declining populations are most vulnerable to Allee
effects, as opposed to those who have always remained stable at low sizes (Courchamp et al.,
2008). With current levels of species loss, understanding population risk to Allee effects is more
important now than ever. Behaviours with fitness outcomes should be represented in population
growth rate, and resulting population densities will directly re-inform the success of those
behaviours (Kokko and Rankin, 2006). Allee effects amplify this principle such that a declining
and critically small or sparse population may fuel its own decline through progressive loss of
beneficial behaviours, catalysing a sort of “extinction vortex” (Courchamp et al., 1999).
According to theory, Allee effects should be common among highly social and/or
endangered species (Courchamp et al., 2008), however some have debated the prevalence Allee
effects in nature (Kramer et al., 2009). While there have been several empirical studies aiming to
describe Allee effects in natural populations, good data are generally lacking (reviewed by
Kramer et al., 2009). Due to limitations in methodology, and therefore, the type of data able to be
collected, many authors often cannot distinguish between component and demographic effects,
or present ambiguous results, or may find patterns of inverse density dependence without
identifying the causal mechanism (Kramer et al., 2009). Most studies reporting Allee effects
have been observational, perhaps because they allow accumulation of large long-term data sets,
difficult to obtain from experimental approaches (Kramer et al., 2009). Allee effects might not be
detected at all when more than one component is involved or if there is an insufficient range of
11
population sizes/densities (Kramer et al., 2009; Gascoigne et al., 2009). Additionally, review of
taxonomic focus within the literature shows uneven investigation: most in terrestrial arthropods,
mammals and birds, less in fish and plants, and notably scarce amongst herpetofauna (Kramer et
al., 2009). Considering all taxa and study designs, component effects are identified twice as often
as demographic Allee effects in nature (Kramer et al., 2009). Aside from sampling limitations
and discrepancies, it may simply be difficult to “catch” populations near their critical thresholds
(Kramer et al., 2009), or demographic effects may be hidden by the spatial context (scale and
heterogeneity) considered (Terui et al., 2015).
Susceptibility to Allee effects is certainly species-specific, depending on the fitnessrelated mechanism involved. Conservationists have begun to consider Allee effects, especially
potentially synergistic component effects, in their assessments of at-risk taxa in order to
prioritize populations and inform management strategies. One example of this application was in
the conservation of shearwaters (birds) in New Zealand. Historic shearwater breeding range
initially contracted due to nesting habitat destruction by feral pigs. Subsequent inverse densitydependent predation by stoats led to the extinction of several bird colonies (Cuthbert, 2002).
These findings recommended focusing on protecting the few remaining colonies, including
hunting pigs whose home ranges overlap with shearwater breeding sites, and restoring old
breeding habitats along with stoat trapping until colony numbers are sufficiently large enough to
benefit from a predator satiation mechanism (Cuthbert, 2002).
Recently, investigators have also sought to exploit Allee effects to mitigate biological
invasions (reviewed in Tobin et al., 2011). By taking advantage of survival and reproductive
mechanisms specific to the non-native invader, management tactics such as culling, predator
augmentation, disruption of mating, or increasing host vigour, could significantly hinder its
spread (Tobin et al., 2011).
Temperate anuran mating system and reproductive physiology
Anuran amphibians are excellent but underused candidates for studying demographic
Allee effects for several reasons. Anuran populations are declining globally (Collins and Storfer,
2003), and the increasing number of reported frog and toad species extirpations suggests critical
abundances can be attained rapidly in this taxon. Additionally, for most temperate species,
12
successful breeding relies on social cues and behavioural synchronicity. Formation of such
discreet breeding aggregations not only makes them at risk for reproductive facilitation-mediated
Allee effects (Gaston et al., 2010), but is also practical for quantifying local effects of abundance
in the field.
In general, anuran breeding is characterised by male production of advertisement calls to
attract conspecific females (Wells, 1977). In temperate species specifically, anuran mating
systems take the form of annual choruses, where adult males aggregate and call from aquatic
habitats for nights or weeks of the active season. It has been suggested that aggregative
behaviour confers benefits over solitary calling because cumulative calls favour audibility,
thereby increasing female choice and/or male mating probability (Alexander, 1975; Wagner and
Sullivan, 1992; Tejedo, 1993; Gaston et al., 2010). Larger choruses do tend to attract more
females (Wagner and Sullivan, 1992), likely explained by an innate preference for calls of higher
frequency and rate (Sullivan, 1992). Empirical evidence also supports that male anurans
attending larger choruses experience greater mating success compared to those attending smaller
aggregations (Gerhardt et al., 1987; Tejedo, 1993; Gaston et al., 2010).
Chorus formation is regulated by a combination of biotic and abiotic factors, generalized
by environmental conditions favouring female arrival and the energetic costs of chorus
attendance (Lucas and Howard, 1995; McCauley et al., 2005; reviewed by Rastogi et al., 2011).
Temperature, precipitation and even lunar cycle can act as cues to initiate and/or maximize male
calling (Wagner and Sullivan, 1992; Parua et al., 1998; Kopp and Eterovick, 2006; Walpole et
al., 2012). Metabolic costs of calling are mostly related to aggregation density, which can
influence individual male behaviour and chorus structure (Tejedo, 1993; Hobel, 2015). On the
most basic level, calls function as intraspecific signals, transmitting information to both sexes
across large distances (Wells, 1977), though recent work has shown that vocal cues are essential
in the onset and maintenance of most anuran reproductive processes (reviewed by Arch and
Narins, 2009). Exposure to male calls has physiological and behavioural consequences,
independently influencing neuroendocrine pathways and reproductive actions, such that call
reception, androgen production, and the manifestation of sexual behaviours are each linked in
complex multidirectional feedback loops (Burmeister and Wilczynski, 2000; Chu and
Wilczynski, 2001). This interplay between abiotic, social, and hormonal cues ensures
13
synchronization of the central nervous system with optimal environmental conditions (Crews and
Moore, 1986).
In anurans, received calls are first translated from tympanic membrane vibrations to
neurological signals in the midbrain torus semicircularis (TS). Among the TS’ sub-nuclei, the
laminar nucleus (LN) is thought to be involved in integrating sex hormones and auditory cue
reception (Wilczynski and Endepols, 2007), because it is comprised of cells containing estrogens
and androgens (di Meglio et al., 1987) and connects extensively to motor areas of the brain
(Endepols and Walkowiak, 2000). Males exposed to chorus sounds experience elevated plasma
testosterone and dihydrotestosterone (DHT) levels (Burmeister and Wilczynski, 2000; Chu and
Wilzcynski, 2001), as well as prolonged gonadal maturity and greater testis mass (Brzoska and
Obert, 1980) compared to no sound or control tones. Exposure to conspecific calls also evokes
increased calling behaviour in male receivers (Solis and Penna, 1997; Burmeister and
Wilczynski, 2000; Emerson, 2001), while callers themselves experience higher testosterone and
DHT levels compared to non-attendees and post-breeders (Harvey et al., 1997). Each of these
effects enhances male mating probability by inciting conspecific males to call, amplifying local
effort, and maintaining elevated gonadal activity for a longer period, thereby facilitating overall
reproduction (Wilczynksi et al., 2005). As such, we would predict that some minimal chorus size
is necessary for instigating sufficient effort, especially considering the metabolic costs associated
with calling. The Energetics-Hormone Vocalization (EHV) model (Emerson, 2001) proposes that
chorusing stimulates androgen production and greater calling effort in participating males, which
should consume lipids and glycogen (Grafe, 1997) and increase corticosteroid (CORT)
production. CORT accumulation should then feed back to inhibit androgen production and
associated calling, resulting in occasional pauses in vocal effort (Emerson, 2001). In small
choruses, resting callers would have a disproportionate effect on the overall chorus volume,
resulting in more intermittent calling and presumably, a less attractive chorus.
Male advertisement calls also influence female behaviour and reproductive physiology.
Exposure to male vocalizations are often required to initiate breeding (Gramapurohit and Radder,
2013), triggering phonotaxis and increasing mate receptivity (Wilczynski and Lynch, 2011) by
stimulating production and maintenance of elevated estradiol levels (Lynch and Wilczynski,
2006; Gramapurohit and Radder, 2013; Gordon and Hellman, 2015), as well as progesterone and
14
testosterone (Gramapurohit and Radder, 2013; Gordon and Hellman, 2015). Presence of male
cues also stimulates egg maturation and maintenance of ovarian development (Lea et al., 2001;
Gramapurohit and Radder, 2013) resulting in continued investment in eggs instead of
reabsorption (Lea et al., 2001). Thus, not only do male vocalizations ensure sufficiently balanced
sex ratios at breeding sites, but are essential in facilitating sexual readiness in female
conspecifics and improving probability of successful mating.
When considering the independent and potential additive roles of vocalizations and
androgens in enhancing anuran breeding effort, any significant reduction or complete loss of
social signalling should have detrimental effects on reproduction. This negative fitness
consequence at small breeding population sizes suggests that behavioural and physiological
processes involved in chorus formation constitute ‘social facilitation of reproduction’
mechanisms (Gaston et al., 2010). Dependence on particular environmental conditions only
makes chorus formation more vulnerable to Allee effects at extremely low abundances because
weakened signalling must coincide with occurrence of appropriate aquatic habitat and ambient
temperatures.
Study species and research goals
The Fowler’s Toad (Anaxyrus fowleri) is a nocturnal species of toad inhabiting most of
eastern U.S.A, and regions of Southern Ontario along the shores of Lake Erie, at the species’
most northern range (Green, 2005). As the Great Lakes form a barrier between the American and
Canadian populations, the latter are particularly sensitive to range-edge effects. Long-term
monitoring of Fowler’s Toads at Long Point, one of the occupied regions in Ontario, revealed
significant declines in abundance (Greenberg and Green, 2013). The species’ Canadian status
was elevated from Threatened to Endangered in 2010. Changes in landscape ecology, namely the
introduction and invasion of the non-native Common reed Phragmites australis, was identified
as the main contributor to loss of breeding habitat and subsequent negative population growth
(Greenberg and Green, 2013). Only a fraction of known historical calling sites at Long Point are
still utilized by the toads, and the frequency and size of choruses have decreased notably (Green,
2013).
15
Since declining populations of species reliant on conspecific cues to ensure reproduction
should be doubly vulnerable to inverse density-dependence (Gascoigne et al., 2009), there is
sufficient reason to hypothesize that a demographic Allee effect is fueling continued declines of
Fowler’s Toads at Long Point. In this thesis, I take a multidisciplinary approach, merging
traditional capture-recapture survey methods with behavioural endocrinology, to determine
whether there are significant fitness-abundance relationships consistent with a proposed
demographic Allee effect. In Chapter 2, I test the basic predictions of a component Allee effect
by comparing male breeding effort (fitness correlate) across groups of differing sizes. If an
extremely low number of conspecifics is severely interfering with the toads’ reproductive
processes, we would expect to find (1) a shortened breeding season composed of smaller and
fewer choruses, (2) disproportionately lower breeding effort at low abundances, and (3) the
presence of a lower critical threshold, in the focal low-abundance population. In Chapter 3, I
investigate whether endocrine activity explains any variation in individual and seasonal breeding
effort by comparing urinary testosterone levels of males from differing abundance conditions,
before and after the breeding period. Knowing that testosterone plays important roles in
expression of reproductive behaviours and sexual readiness, we would expect that (1) the
duration of the population’s mating season should be reflected at the hormonal level as a
respective drop or maintenance of testosterone post-breeding, and (2) testosterone levels among
males not present at breeding sites are lower than those of participatory males. In Chapter 4, I
synthesize my findings in the context of Long Point’s Fowler’s Toad population and anuran
conservation in general.
16
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Figures
Figure 1. Comparison of classic logistic growth (dotted line), with an Allee effect characterised
by reduced fitness at low abundances (adapted from Berec et al., 2007). Strong demographic
effects (solid) feature a lower unstable equilibrium point (K_ ), below which the population will
be driven to extinction. This critical “Allee threshold” is absent when the effects are weak at the
population level (dashed). The severity, and therefore curve shape, of demographic Allee effects
should vary according to the relative strength of underlying component effects and intraspecific
competition.
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CHAPTER II: Male Abundance and Breeding Effort
24
Linking Statement
Allee effects may manifest themselves as dramatic reductions in mate encounter rate,
negatively affecting breeding, and ultimately population growth. A potential causal mechanism
in (temperate) anuran mating systems is the loss of male calling behaviour, as it is essential in
bringing together mature males and females for a period of their brief active season. Here I
investigate the role of male abundance on breeding effort, and test for evidence consistent with
an Allee effect in a declining population of Fowler’s Toads in Southern Ontario.
25
Abstract
Declining populations are particularly vulnerable to demographic stochasticity, including
inverse density-dependence, or Allee effects. Identifying possible mechanisms behind such
phenomena is necessary in recognizing and managing high-risk populations. I investigated
whether Allee effects can influence population persistence specifically through changes in
breeding behaviour of endangered Fowler’s Toads. By combining intensive capture-recapture
methods and automated recorder data, I tracked calling trends and estimated male breeding effort
by quantifying chorus attendance among populations of Fowler’s Toads varying in abundance. If
an Allee effect were regulating male aggregation, I expected to find lower breeding effort within
smaller groups compared to larger groups. The proportion of total or emerged males that
attended choruses at Long Point were not highly predictable by male abundance or density, nor
was the proportion of total nights a given male would attend a chorus. This high variability in
local breeding effort was likely due to the small range of abundances among the sites monitored
(7, 10, and 22 males). At the population scale, I found significantly larger, more frequent
choruses, and a longer breeding season at the high abundance site Nickel Beach, compared to the
low abundance site Hastings 1 (in Long Point). Similarly, long-term survey data from breeding
groups at Long Point revealed disproportionately reduced breeding effort at lower abundances.
These results are consistent with a ‘social enhancement of reproduction’ component Allee effect.
Breeding effort in Fowler’s Toads appears to become erratic, and therefore more vulnerable to
decline, when groups were smaller. Further, abundances of 4 or fewer males are likely to make
no breeding effort at all. This could represent preliminary evidence for a lower critical
abundance-behaviour threshold, suggesting that a demographic Allee effect may be aggravating
recent declines of Fowler’s Toad at Long Point.
26
Introduction
The mating system of most temperate anuran species involves seasonal aggregative
behaviour, where males call in groups from aquatic oviposition sites (Wells, 1977). The male
advertisement call acts as an acoustic signal, transmitting information to conspecifics of both
sexes over large distances. The calls function to attract females to local breeding sites, while
simultaneously informing other males when and where it is appropriate to sing (Arch and Narins,
2009). Chorusing behaviour confers benefits compared to calling in isolation because cumulative
calls are louder and better heard, attracting more females and increasing mating probability,
while male aggregation also allows greater female choice (Alexander, 1975; Wagner and
Sullivan, 1992; Tejedo, 1993; Gaston et al., 2010).
Anuran mating system structure is influenced seasonally by both abiotic and biotic
conditions. Drought, for example, will limit local abundance and breeding effort (Wagner and
Sullivan, 1992). Chorus density can also influence the arrangement and effort of attending males
in many anuran species (Woolbright et al., 1990; Tejedo, 1993; Byrne and Roberts, 2004; Kokko
and Rankin, 2006; Buzatto et al., 2015). Male tactics for monopolizing females at high densities
has been well studied, but behavioural consequences of extremely low male abundance has not
received the same attention, despite global amphibian declines. Since chorusing is fundamental
in bringing together males and females for a portion of the brief active season, some minimal
chorus size should be necessary to support sufficient mate encounters and successful breeding.
Central to this reasoning is that females prefer high-effort calls (rate x duration) and are therefore
more disposed to attend louder choruses (Sullivan, 1992). Additionally, male receivers exhibit
increased calling behaviour when exposed to conspecific calls (Solis and Penna, 1997;
Burmeister and Wilczynski, 2000). In these ways, calls act as cues for local male participation,
thereby regulating temporal synchronicity of effort, amplifying and maintaining the signal, and
enhancing reproductive success (Crews and Moore, 1986; Wilczynski et al., 2005; Lynch and
Wilczynski, 2006).
Since chorus attendance is strongly predictive of a male’s fitness, chorus formation may
be subject to Allee effects (Gaston et al., 2010). The Allee effect is a positive relationship
between some component of individual fitness and population size, and the concomitant
reduction of population growth at very low abundances (Stephens et al., 1999). Allee effects
27
typically function through loss of mechanisms that depend on sufficient number of conspecifics
and that maintain balanced sex ratios. Aspects of animal mating systems, such as the degree of
aggregation, are also determined by the strength of these processes (Stephens and Sutherland,
1999). As such, chorusing amphibians should make good candidates for studying Allee effects
(Kramer et al., 2009, Gaston et al., 2010). Thus far, Gaston et al. (2010) provide the only
empirical evidence for component Allee effects in an (endangered) anuran species, finding
greater reproductive success among larger choruses of Houston Toads (Anaxyrus houstonensis).
At extremely high chorus sizes, many species of male anurans will adjust their behaviour
to optimize effort-benefit gains (Tejedo, 1993; Byrne and Roberts, 2004; Kokko and Rankin,
2006). Therefore, within a small population with fewer competitors, all males should exhibit
maximal breeding effort (Lucas and Howard, 1995). However, in a critically small population,
the low number of breeding-age males may not be sufficient to initiate coordinated aggregation
and calling. This interference of essential synchronous behaviour could fuel trends of low
recruitment and have population-level consequences (Gaston et al., 2010).
I used the endangered Fowler’s Toads from Southern Ontario as a study system to test
these basic hypotheses about Allee effects. Long-term population monitoring of Fowler’s Toads
in Long Point, Ontario, has shown marked declines in both chorus size (Green, 2013) and
population size (Greenberg and Green, 2013). Since recently declining populations should be
most vulnerable to Allee effects (Courchamp et al., 2008), and chorus attendance is the main
factor determining mating success of anurans (Gerhardt et al., 1987; Tejedo, 1993; Given, 2002;
Gaston et al., 2010), a demographic Allee effect may be acting on Fowler’s Toads populations at
Long Point. Using chorus attendance as a fitness correlate, I was able to quantify and compare
indicators of male breeding effort between this site and Nickel Beach, a significantly larger and
denser population. If an Allee effect is present in these toads, there should be disproportionately
lower breeding effort in small groups compared to large groups. More specifically, where there
are larger choruses, there should be a longer breeding season, and greater individual and
population-level male effort compared to low-calling environments.
28
Materials and Methods
Study Sites
I studied Fowler’s Toad populations at two locations in Southern Ontario. Nickel Beach,
a high abundance site, is a 2 km beach on the northern shoreline of Lake Erie (42.875878,
-79.238608). Fowler’s Toads breed in an expanse of rocky pools located at the eastern end of
the beach. This population has been consistently characterised as relatively large, with
population estimates in the hundreds of individuals (Anne Yagi, personal communication). Long
Point (42.580506, -80.419045), the low abundance area, featured three acoustically isolated
sites (in 2015) that I considered separate breeding groups. Hastings 1 was a 450 m stretch of
sandy beach between two residential properties where there formed a natural beach pool.
Hastings 3.75-4 was a 290 m section of pebbly beach lined with sand dunes and marsh.
Thoroughfare was a 1.5 km stretch of beach with adjacent back-dune marshland and artificial
amphibian ponds (Figure 1).
Monitoring calling activity
I studied calling behaviour throughout the toads’ entire breeding season in 2015 (May to
mid-June/July). Fowler’s Toads are nocturnal, so all call surveys and chorus monitoring took
place between 9:30 PM and 2:00 AM, depending on time of sunset.
I deployed an outdoor sound recorder, Song Meter SM2+ (Wildlife Acoustics, Inc.,
Maynard, MA, USA), at each of the four breeding sites. All recorders were secured with bungee
cords to trees or dune vegetation four meters above ground, within 50 meters from the local
breeding site: Nickel Beach (42.868912, -79.221158), Thoroughfare (42.57614, -80.3705),
Hastings 1 (42.57722, -80.44805), and Hastings 3.75-4 (42.57558, -80.47235). The devices
were set to record five minutes of audio every 30 minutes from 7 P.M. to 7 A.M. I deployed the
recorder at Nickel Beach May 6th 2015 and those at Long Point, May 8th 2015. They were left to
record for three months and retrieved approximately two weeks after the last night of local
calling, on July 25th and July 7th 2015, respectively.
Exceptionally at Long Point, I was able to ground truth the audio recordings. The three
Long Point sites were monitored nightly from May 1st to June 13th. If at least one male was heard
29
calling, I approached and estimated chorus size. Since an individual’s overall attendance (also
known as chorus tenure) is strongly correlated to mating success in Fowler’s Toads (Given,
2002), I estimated chorus size as the total number of males observed sitting in or around the pool
on nights with at least one caller, whether the individual was actively calling or not. As calling
behaviour is influenced by weather conditions (Oseen and Wassersug, 2002; Saenz et al., 2006),
I recorded air and breeding water temperatures, as well as any heavy wind or rain events. For the
nights when surveying was not possible, chorus sizes were estimated strictly from the audio
recordings.
To determine how abundance and chorus size affect seasonal breeding effort, I compared
calling phenology between two populations of differing size. I chose Hastings 1 as the lowabundance group of interest because it was the sole site of chorus formation at Long Point (in
2015), and Nickel Beach as the high-abundance group. I reviewed survey data and audio
recordings and determined which nights Fowler’s Toads were heard or observed calling. To
quantify seasonal breeding effort, I measured the length of breeding season as the number of
days between the dates of first and last calling, and calculated proportion of total nights that
calling (at least one male) took place. To estimate aggregation size, I designed a species-specific
index to rank chorus size categorically from the audio recordings (Crouch and Paton, 2002;
Stevens and Paszkowski, 2004; Nelson and Graves, 2004). My ranking system was informed by
ground-truthed counts from Long Point survey data, along with the degree of call overlap and
intensity: 0 (0 males), 1 (1-3 males), 2 (4-9 males), 3 (10-15 males), 4 (16-20 males), 5 (21+).
Each rank number designated a range of chorus sizes, and these intervals were not scaled
metrically. I chose not to follow the commonly used NAAMP Calling Index (CI), where a rank
of 0 = no calling, 1= individual calls without overlap, 2= distinguishable individual calls with
overlap, 3= full chorus: continuous and overlapping calling (Weir and Mossman, 2005), because
that gradient did not provide enough detail. I reviewed all recordings myself to eliminate intraobserver variation (Corn et al., 2011). Median chorus size ranks from low abundance Hastings 1
and high abundance Nickel Beach were compared with a non-parametric Wilcoxon rank-sum test
(also known as Mann-Whitney U test). Ranks of zero were not included. All statistical analyses
were performed using JMP 11.0 statistical software (SAS Institute Inc., Cary, NC, USA).
30
Quantifying local breeding effort at Long Point (2015)
To investigate the relationship between local breeding effort and male abundance, I
surveyed the three breeding sites at Long Point using nightly capture-recapture methods from
May 1st to June 13th. Regardless of calling activity, I surveyed each breeding site and captured all
adult Fowler’s Toads present. In order to track individual male participation over the breeding
season, I took a photo of each toad’s unique dorsal spot pattern and used custom photorecognition software, FotoSpottr (Schoen et al., 2015), to identify individuals upon recapture.
The rest of the site was surveyed so that any males not present at the breeding pool, but
positioned elsewhere along beach, were also identified. Instead of quantifying breeding effort by
some derivative of Calling Index (Tupper and Cook, 2008), I calculated proportions of male
participation to investigate population and individual-levels of breeding effort using three simple
“breeding effort indices”:
(1) Nightly population effort: number of males present at the breeding site on a given
night, divided by the total number of males known to frequent the area (i.e.“population”).
(2) Nightly emerged effort: number of males present at the breeding sites, divided by the
number of males emerged (at either foraging or breeding sites) on a given night.
(3) Individual effort: number of nights a given male attended a chorus, divided by the
total number of nights calling took place at that site.
Only adult males present during the spring breeding season were considered (i.e. males that
emerged post-breeding were not part of total population abundance). To investigate the
sensitivity of breeding effort to abundance, I calculated indices (1) and (2) for each night of
calling (that I was present to confirm caller identity), and index (3) for each individual at a given
site. I then compared the resulting index values across Hasting 1, Hastings 3.75-4, and
Thoroughfare groups using a nonparametric Kruskal-Wallis test with Bonferroni correction
(alpha value adjusted to 0.0167 due to multiple comparisons). As the sites differed in area and
therefore male density, I repeated the tests using index values standardized by length of site
shoreline (in meters).
31
Yearly male abundance and breeding effort at Long Point (1998-2015)
I conducted an additional analysis with a larger range of population sizes as a more
robust approach to investigating the relationship between local male abundance, density, and
breeding effort. I reviewed 23 years of raw historical data from the Long Point population survey
(see Greenberg and Green, 2013) and compiled local male abundance and the number of males
found at least once at a breeding site, for as many sites possible from 1998 to 2015. Data from
years 1999, 2000, 2001, 2008 and 2010 were omitted because the sites were not monitored
nightly and/or the locations of captures were not noted and/or the individual was not identified.
Each acoustically isolated site was treated as a separate breeding group.
To determine if breeding effort was disproportionately lower at smaller population sizes,
and test the main prediction of the Allee effect, I plotted the number of males found at a breeding
site at least once during the breeding season against the corresponding local male abundance. I
tested the role of male abundance specifically using an ANCOVA model, accounting for
different male density (covariate) across sites.
Results
Male abundance
In 2015, male Fowler’s Toad abundance and density varied among the three groups at
Long Point (Table 1). A total of 22 males occupied Hastings 1 (breeding pool and/or beach) at a
density of 0.049 ♂/m. Ten adult males occupied Hastings 3.75-4 (marsh and/or adjacent beach)
at a density of 0.034♂/m. Seven adult males occupied Thoroughfare (ponds and/or adjacent
beach) at a density of 0.0045 ♂/m. At Nickel Beach, I estimated male abundance and density to
be 147 males and 0.074 ♂/m, respectively.
32
Length of breeding season
The Fowler’s Toad (2015) breeding season was 62 days at Nickel Beach, and 47 days at
Hastings 1 (Figure 2). At least one toad called on a total of 42 nights out of the 62 at Nickel
Beach (67%), and 23 out of the 47 at Hastings 1 (49%). There were periods of time when calling
was absent at both sites. These were associated with occurrence of cold temperatures (below 10
degrees) and stormy weather (Figure 2).
Average chorus size
Maximum chorus size at Nickel Beach was rank 5 (21+ males), some of which I
estimated to involve well over 50 males. The mean chorus size rank was 3.6, (rounded to rank 4
~ 16-20 males). Maximum chorus size rank at Hastings 1 reached 12-13 males (rank 3), but the
mean was rank 1.92 (rounded to rank 2 ~ 4-9 males). Median chorus size rank was significantly
larger at Nickel Beach than at Hastings 1 (Z= -3.49, p = 0.0005) (Figure 2).
Male breeding effort at Long Point (2015)
The three breeding effort indices I tested varied differently across Hastings 1, Hastings
3.75-4 and Thoroughfare groups. The proportion of emerged males that attended the local chorus
(nightly emerged effort) did not differ across breeding sites (Figure 3.a). The proportion of total
males that attended the local chorus (nightly population effort) was significantly lower at
Hastings 3.75-4 than at Hastings 1 (Z = -3.52392, p = 0.0004) and Thoroughfare (Z = 3.46045,
p = 0.0005) (Figure 3.b). Regarding individual effort, the proportion of calling nights a given
male attended the chorus was significantly lower at Hastings 3.75-4 compared to Hastings 1 (Z=
-2.61002, p = 0.0091), but the same as those from Thoroughfare (Figure 3.c).
Standardizing male breeding effort indices by site length yielded slightly different results.
The standardized nightly emerged effort at Thoroughfare was lowest compared to both Hastings
1 (Z= -3.75389, p = 0.0002) and Hastings 3.75-4 (Z = -3.56788, p = 0.0004). Hastings 1 and
Hastings 3.75-4 also differed significantly from each other (Z= 3.02004, p = 0.0025) (Figure
3.d). Hastings 1 showed significantly higher standardized nightly population effort than
Thoroughfare (Z = -3.74388, p = 0.0002) and Hastings 3.75-4 (Z= -3.35409, p = 0.0008) (Figure
33
3.e). The latter two groups did not differ. Finally, standardized individual effort did not vary
significantly across groups after applying the Bonferroni correction (Figure 3.f).
Yearly breeding effort and male abundance at Long Point (1998-2015)
Overall group breeding effort differed across Long Point sites in 2015 (Table 1): 91%,
40%, and 71% of resident males at Hastings 1, Hastings 3.75-4, and Thoroughfare attended a
chorus at least once during the breeding season, respectively. Including a larger range of
abundance values from long-term monitoring showed that the number of males found at least
once at a breeding site declined disproportionately at low abundances (Figure 4). This
relationship was statistically significant, even when controlling for differences in male density
and abundance-density interactions (F2, 16 = 42.24, R2 = 0.89, p < 0.0001). The x-intercept was 4,
an abundance below which breeding effort became zero (y = 0.8524x - 3.5182).
Discussion
My findings demonstrate that at lower abundances, a smaller subset of total adult male
Fowler’s Toads actively joined local choruses, which is consistent with the hypothesis that
vocalizations facilitate chorus participation among conspecifics, whether it be active calling or
minimally, as satellites. This relationship is consistent with trends predicted by a component
Allee effect which appears to be acting on Fowler’s Toads at Long Point, Ontario. This is only
the second example of empirical evidence for reduced breeding effort at extremely low
abundances in an amphibian population. Gaston et al. (2010) reached comparable conclusions
looking at the endangered Houston Toad (Anaxyrus houstonensis), finding a significantly higher
probability of reproduction at sites of higher calling activity.
The significantly larger choruses and prolonged breeding season exhibited at Nickel
Beach also suggests that abundant male vocalizations may be essential in instigating
participation and maximizing population breeding capacity. Males from Hastings 1 formed much
smaller choruses and called less frequently, resulting in a breeding season two weeks shorter
than that at Nickel Beach. This exemplifies social facilitation of reproduction, whereby high34
abundance populations benefit from extended breeding periods, potentially increasing mating
probability (Wilczynski et al., 2005).
Comprehensive historical Fowler’s Toad survey data not only reinforce the central
relationship of effort increasing with local group size, but also show that it decreases
disproportionately at very low abundances. Figure 4 illustrates smaller and more scattered (risky)
values of breeding effort when male abundance is lower, while the regression line’s x-intercept
suggests that groups of four or fewer males may make no breeding effort at all. Due to lack of
very high male abundance values (i.e. > 60 males) in the post-1998 records, the upper limit of the
curve was undetermined. This is consistent with reports that individual benefit from chorus
participation only plateaus at very high densities (Tejedo, 1993). Based on historical accounts of
Fowler’s Toads at Long Point, it is possible that choruses likely become saturated in the
hundreds of individuals, though this threshold is certainly species-specific.
In general, the individual-scale effort indices did not differ across the three breeding sites
at Long Point. In the few cases that significant differences were detected, the more abundant
group did not consistently exhibit higher degrees of effort, or vice versa. Only the average
proportion of total males that attended a chorus on a given night (nightly pop. effort) increased
with abundance when standardized for site size. This high variability in response is likely
attributable to the similarly low male abundances at the compared sites. Despite Hastings 1
having double the number of males than Hastings 3.75-4 and Thoroughfare, there was no reliable
increase in breeding effort made by 22 males compared to 7 - 10 males. These results are
nonetheless consistent with the erratic degree of breeding effort exhibited under low abundance
conditions shown by long-term monitoring. The relative success of this approach highlights the
importance of full-range data: much larger populations (such as Nickel Beach) would have been
ideal as a comparative group, but fine-scale data from especially low-abundance populations is
essential for detecting minimal thresholds (Ray and Hastings, 1996).
Though I did not measure breeding effort against per capita growth rate over time, the
highest abundances at Long Point (30 - 60 males) were recorded between 1998 and 2004, while
the lowest counts were found in more recent years. This suggests that the component Allee effect
identified may be manifesting itself at the population level and intensifying declines of Fowler’s
Toads at Long Point. In this context, my preliminary behavioural threshold could be analogous to
35
an unstable lower equilibrium point. Small populations of Fowler’s Toads will not only form
weaker choruses but also experience reduced mating probability because of progressive loss of
beneficial chorusing behaviour. Numbers are at risk of declining until reaching an abundance of
circa four, at which point males may not call at all, likely suppressing the remnant population’s
recruitment beyond rescue.
Behaviours interrupted by skews in operational sex ratio are most susceptible to Allee
effects at critically low abundances (Stephens and Sutherland, 1999). There is already some
evidence consistent with this. Annual population surveys indicate fewer female toads at Long
Point are moving to active breeding sites, suggesting that they are not receiving sufficient vocal
cues to induce phonotaxis. Even with minimal search effort, I found 15 amplectant pairs after a
single night of average chorusing at Nickel Beach, compared to 5 total amplectant pairs during
the entire breeding season at Long Point (Hastings 1). Gaston et al. (2010) reasoned that
increased spatial separation or particular landscape features could hinder reception of vocal cues
in Houston Toads, thereby interfering with mechanisms underlying aggregative behaviour. My
results also suggest that interactions between call and environment acoustics are important
factors in breeding habitat quality for Fowler’s Toads. The only study sites supporting mating,
Nickel Beach and Hastings 1, both hosted calling and breeding in rocky or sandy beach pools.
This could indicate that open beach habitat, in particular, favours audibility of calls and
aggregation of both sexes. In fact, recent observations that females do not respond well to backdune callers suggests that marsh choruses must be very large to overcome topographic
interference.
Many studies of amphibian populations over the past two decades have been quick to
identify direct environmental, as well as indirect climate-mediated, and pathogenic factors
responsible for global declines (Kiesecker et al., 2001; reviewed by Collins and Storfer, 2003).
While habitat change or loss can trigger population declines, my results suggest that behavioural
(component) mechanisms affected by these modifications can manifest themselves as
demographic Allee effects in the aftermath of such events, pushing already vulnerable
populations to their brink. It is crucial for those studying anuran declines to consider such
processes when interpreting survey data. For example, Whitfield et al. (2007) reported steady
declines without identifying a specific threat. There was no evidence of dramatic mortality
36
events typical of viral infection, nor of habitat fragmentation. They instead speculated that a
reduction in litter mass, indirectly due to climate-change-related increases in temperatures and
precipitation led to increased biomass decomposition rates. While this is a viable hypothesis, the
authors did not consider breeding site quality nor seasonal aggregation size, potentially missing
significant inverse density-dependent processes influencing continued declines.
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40
Tables
Table 1: Male demographics of groups of Fowler’s Toads in Southern Ontario, spring 2015.
Only nights* where caller identity was confirmed were considered. Only males who were
breeding-age at spring and who frequented the site at least once (at any point during the breeding
season) were included.
Group/Site
Male
abundance
Male density
(♂/m)
Number
of nights*
Hastings 1
22
0.049
11
0.91
Hastings 3.75-4
10
0.034
8
0.40
Thoroughfare
7
0.0045
9
0.71
Nickel Beach
147
0.0735
---
---
Pop. breeding
effort
41
Figures
Figure 1: Location of study sites in Southern Ontario, Canada.
42
Figure 2. Breeding phenology of two populations differing in male abundance. Calling activity of Hastings 1 (low abundance, ○) and
Nickel Beach (high abundance, ●) populations were ranked nightly from date of first to last calling in spring 2015. Light blue shading
represents periods of more than three consecutive days of temperatures below 10C and/or stormy weather in both study regions. The
solid and dashed lines illustrate the general calling trends at Nickel Beach and Hastings 1, respectively.
43
Figure 3. Three indices of local breeding effort were mostly unpredictable across breeding
groups at Long Point, even when standardizing for site size (density). Local male abundance was
7, 10, and 22, respectively.
44
Number males found at breeding sites
60
50
40
30
20
10
0
0
10
20
30
40
50
60
-10
Male abundance
Figure 4. The number of males that made minimal breeding effort was disproportionately lower
at smaller abundances and reached zero when abundance was ≤ 4 males. Each point represents a
breeding group/site in a given year at Long Point (data are from 1998 to 2015, omitting years
1999, 2000, 2001, 2008, 2010). The relationship was highly predictive, even when accounting
for discrepancies in male density across 5-6 different sites (F2, 16 = 42.24, R2 = 0.89, p < 0.0001,
y = 0.8524x - 3.5182).
45
CHAPTER III: Breeding Effort and Testosterone Level
46
Linking Statement
Identifying the causal mechanism of an Allee effect is key in understanding how specific
populations become vulnerable at small sizes. Results from Chapter 2 suggest that abundancelimited chorusing behaviour is responsible for loss of beneficial aggregation behaviour and
reduction of breeding probability. The next step is to test hypotheses regarding underlying
physiological pathways. Here, I investigate whether endocrine activity, specifically urinary
testosterone level, corresponds to the manifestation or absence of breeding behaviours in male
Fowler’s Toads.
47
Abstract
Advertisement calls are important stimuli for initiating and regulating essential
reproductive behaviours of both male and female anurans. As calls are known to have
physiological consequences on the receiver’s endocrine system, I reasoned that the loss of
beneficial social behaviours seen at critically low densities (Allee effect) may be related to
reduced circulating testosterone, such that non-attendee males may have significantly lower
levels than their participatory counterparts. Accordingly, if social cues work in a positive
feedback loop with testosterone to enhance breeding effort (‘conspecific enhancement of
reproduction’), males from high-chorusing sites should maintain raised testosterone levels longer
into the post-breeding season, while levels at low-calling sites should decline more abruptly. I
collected urine samples from male Fowler’s Toads at a low abundance (Long Point) and high
abundance (Nickel Beach) site, sampling both during the spring breeding period and the late
summer post-breeding period. I then compared urinary testosterone concentrations across
seasons and groups. As expected, I found a significant post-breeding season drop in testosterone
levels at the low-abundance site, while males from the high-chorusing environment maintained
similar levels even after calling ceased. The prolonged hormonal state of males from Nickel
Beach was likely correlated to a longer breeding season observed at that locality. This was
consistent with a hypothesis of ‘conspecific enhancement of reproduction’. Surprisingly, I found
slightly lower testosterone levels in chorus attendees versus non-attendees, suggesting that
insufficient testosterone levels may not be responsible for low effort among non-participatory
males at Long Point. These results contradict reports of increased testosterone production in
males exposed to chorus sounds, but agree with others showing that callers experience lower
androgen levels than non-callers. These inconsistencies highlight the complexity and partial
independence of androgen activity and expression of reproductive behaviours, in addition to
potential limitations of using urinary testosterone concentrations to estimate levels acting in the
brain.
48
Introduction
Classically, two breeding patterns have been defined among vertebrates: associate, where
elevated circulating androgens and gametogenesis are synchronized with reproductive
behaviours, and dissociate, where these events occur at different times (Crews and Moore,
1986). Amphibians adapted to temperate environments are usually described as dissociate
breeders because overwintering represents a pause in the hormone cycle. As hormone phenology
is sensitive to abiotic conditions, the endocrine system is also responsive to immediate social
cues (Wilczynski et al., 2005). Loss of essential stimuli can interfere with gametogenesis and
result in absence of appropriate reproductive behaviours (Lea et al., 2001). Anuran mating calls
are one such signal: exposure to calls is known to stimulate the nervous and endocrine systems,
modulating the behaviours of both male and female receivers (reviewed by Arch and Narins,
2009). Males exposed to chorus sounds experience elevated testosterone (and
dihydrotestosterone) levels (Burmeister and Wilczynski, 2000; Chu and Wilzcynski, 2001), and
prolonged gonadal maturity (Brzoska and Obert, 1980). Exposure to conspecific calls also
evokes increased calling behaviour in the receiver (Solis and Penna, 1997; Burmeister and
Wilczynski, 2000; Emerson 2001). In females, exposure to male vocal signals can stimulate
production and maintenance of elevated estrogen levels (Lynch and Wilczynski, 2006), as well
as increased mate receptivity (Wilczynski and Lynch, 2011).
It this way, vocal cues in anurans function in a positive feedback loop with the endocrine
system to synchronize and amplify breeding effort, thereby facilitating reproduction (Brzoska
and Obert 1980; Chu and Wilzcynski 2001). This mutual reinforcement between sex hormones
and calling behaviour, specifically, is directly related to mating probability, emphasising the
overall centrality of chorus formation in anuran fitness (Burmeister and Wilczynski, 2000;
Wilczynski et al., 2005; Lynch and Wilczynski, 2006). Accordingly, and in light of current
anuran declines, it could be possible that low levels of social signalling (calling effort and
exposure) would interfere with essential hormone-behaviour interactions, fueling loss of cues
that favour high operational sex ratios, thereby leaving populations vulnerable to Allee effects
(Stephens et al. 1999).
As a temperate species that undergoes a distinct annual mating season, Fowler’s Toads
likely have features of both associated and disassociated patterns, such that levels are highest
49
leading up to and during mating, decrease after breeding, and rise again prior to hibernation
(Jorgensen 1981, 1982). Because the timing and duration of hormonal peaks are susceptible to
presence of vocal cues, comparatively longer or shorter breeding seasons should be reflected in
underlying androgen cycling. Similarly, local-scale differences in male participation due to
abundance should also be detectable at the hormonal level. I collected urine samples from male
Fowler’s Toads from two populations differing significantly in abundance, and tested predictions
about their urinary testosterone levels in relation to their respective breeding behaviours and
phenology (Chapter 2). Where the higher degree of chorusing activity leads to a longer breeding
season (Chapter 2) there should be a delayed drop of male urinary testosterone level after
breeding, compared to a more abrupt decline in males from a site where the calling season was
shorter. If the disproportionately lower participatory behaviour among males within a critically
low-abundance population (Chapter 2) is related to inadequate testosterone levels (from the lack
of social cues), we should find lower urinary testosterone concentrations in non-attendee males
compared to chorus attendees.
Materials and Methods
Collection of urine samples
To estimate testosterone levels in breeding toads, I collected urine samples as a noninvasive alternative to blood plasma extraction. Urinary hormone analysis is an increasingly used
method for tracking seasonal fluctuations in reproductive hormone levels and for sexing
(monomorphic) species where males and females are indistinguishable (Narayan et al., 2010). I
collected urine samples from adult males at the four study sites: Nickel Beach, Hastings 1,
Hastings 3.75-4, Thoroughfare (Chapter 2, Figure 1). To investigate seasonal shifts in
reproductive hormone concentration, I sampled during the spring breeding season and
throughout the later summer post-breeding season. I waited one week after the last calling event
(at each site, independent of date) to differentiate between peak and post sampling periods. Since
it could not be guaranteed that a given toad would release urine, I attempted sampling from every
adult male encountered. I also collected 30 urine samples from female toads during the breeding
season at Long Point (some individuals were sampled more than once). This was done to ensure
50
appropriate assay sensitivity, as females should necessarily have lower peak-breeding
testosterone levels than males (Germano et al., 2009). Adult individuals were distinguished by
secondary sex characteristics. Juveniles (i.e. non-breeding age) were not sampled.
To gather urine, I wore vinyl gloves and held the toad over a disposable weigh-dish.
Gently pressing their digits usually elicited a fright response, and the release of urine. I directly
transferred the urine to cryogenic vials (Thermo Scientific Nunc CryoTubes, 1.8 mL, free
standing round bottom) using disposable transfer pipettes. New gloves, weigh-dishes and
pipettes were used for each individual sampled, to avoid cross contamination. For each toad
encountered, I also recorded snout-vent length (SVL) using calipers and took a photograph of
their unique dorsal spot pattern for individual identification. These were performed strictly after
urine collection, as excessive handling could lead to urination response and loss of the sample. If
toads were calling, I collected urine samples from one male at a time, so as to minimize
disturbance to the chorus. Handling time was less than four minutes and most toads re-started
calling soon after release. Tubes were labeled and stored on ice in a cooler while in the field. At
the end of a sampling night, vials were moved to longer-term storage at -20C until analysis.
Estimating testosterone levels
Urine samples were transported in a liquid nitrogen vapour dewar to the Toronto Zoo’s
Reproductive Biology Laboratory for analysis. Urinary testosterone concentrations were
measured with testosterone-specific enzyme immunoassays described in detail by Narayan et al.
(2010). Briefly, this technique involved transferring urine samples (minimum volume: 0.3 mL)
to wells lined with polyclonal anti-testosterone antiserum, adding a horseradish peroxidaseconjugated testosterone label, then washing with hydrogen peroxide (substrate solution) such that
specific binding led to high absorbance readings. The testosterone standards used were 0.78 –
200 pg/well. Standard curves were fit with a regression line, whose equation was used to
determine testosterone concentrations. These were also measured relative to creatinine (Cr)
concentration as an estimate of glomerular filtration rate, accounting for any confounding
re-absorbance of water through the bladder wall (Germano et al., 2012). Resulting values were
flagged as risky either due to dilution effects or low creatinine content. If these were withinrange, they were used in subsequent statistical analyses. Obvious outliers were not included.
51
Statistical analyses
Testosterone concentrations were not normally distributed, instead being highly skewed
toward lower values. For this reason, I chose to use non-parametric tests on untransformed data
for all statistical analyses except for the inter-population comparison.
I first compared breeding season levels of urinary testosterone from males across sites at
Long Point (Hastings 1, Hastings 3.75, and Thoroughfare) using a Kruskal-Wallis test with
Bonferroni correction. If they did not differ significantly, I pooled data from these sites for
subsequent statistical analyses.
As a technical control for assay sensitivity, I compared breeding season testosterone
levels of males and females from Long Point (pooled sites), using a Wilcoxon rank-sum test
(also known as Mann-Whitney U test).
To test whether testosterone levels are maintained or drop after the breeding season, I
compared breeding levels to post-breeding levels within each population with separate Wilcoxon
rank-sum tests.
To confirm that some minimal level of circulating testosterone is necessary for calling, I
compared peak breeding season testosterone levels of males from Nickel Beach and Long Point
with an ANCOVA model. Testosterone values were log-transformed to meet the test’s
assumptions of normality. As body size negatively correlates with population density in these
toads (Green and Middleton, 2013), I included SVL as a continuous covariate. Each urine sample
was considered independent, regardless of whether it was taken from the same individual at a
past sampling event.
To test the hypothesis that testosterone is both a product of and an inducer of breeding
behaviour in males, I categorized urine samples as chorus “attendees” and “non-attendees”.
Attendee samples were collected from males present at a chorus, regardless of individual calling
activity. Non-attendee samples were collected from males situated away from breeding sites on
nights of calling or nights where calling conditions were ideal. Attendee and non-attendee
samples were each pooled across the Long Point breeding groups and then compared with a
Wilcoxon rank-sum test.
52
Results
Controls
During the Long Point breeding season, male urinary testosterone ranged from 10 to
500 ng testosterone (T)/mg creatinine (Cr), with a mean concentration ( SE) of 118.21  11.67
ng T/mg Cr (Table 1). Female toads had urinary testosterone levels ranging from 4.83 to 93.99
ng T/ mg Cr, with a mean concentration of 27.93  5.52 ng T/mg Cr. Assays were sensitive
enough to detect significantly higher median reproductive levels in males (n = 77 samples, from
27 individuals) compared to females (n = 20 samples, from 13 individuals) from Long Point
(Z = -4.02, p < 0.0001) (Figure 1).
As most urine samples were collected at Hastings 1 (n = 63), this group showed the
highest variation of testosterone levels, compared to Hastings 3.75-4 (n = 6) and Thoroughfare
(n = 8) (Table 1). Mean ( SE) testosterone concentrations were 120.23  13.54, 122.16  27.00,
and 99.35  31.39 ng T/mg Cr, respectively. Pairwise comparisons showed no significant
differences in median breeding-season testosterone concentrations in males across the three sites
(Figure 2): Hastings 1 versus Hastings 3.75-4 (Z = 0.48, p = 0.6318), versus Thoroughfare
(Z = -0.45, p = 0.6559), and the latter two (Z = -0.45, p = 0.6514). This justified using the three
relatively low-abundance breeding groups as a single sampling population in subsequent
statistical analyses.
Hormone phenology
Breeding-season testosterone levels in males from Long Point ranged from 10 to 500 ng
T/mg Cr, with a mean ( SE) concentration of 118.21  11.67 ng T/mg Cr and median value of
98.94 ng T/ mg Cr (sampled May 8th – June 19th) (Table 1). Testosterone levels of breeding
males at Nickel Beach ranged from 5.17 to 477.16 ng T/mg Cr, with a mean of 87.54  15.98 ng
T/mg Cr and median value of 45.19 ng T/mg Cr (sampled May 29th - July 2nd). After calling
ceased, testosterone levels ranged from 2.73 to 263.93 ng T/mg Cr in males from Long Point,
with a mean of 41.02  5.87 ng T/mg Cr and median of 28.46 ng T/mg Cr (sampled June 25th to
July 30th). Post-breeding testosterone levels ranged from 3.82 to 145.08 ng T/mg Cr in males
53
from Nickel Beach with a mean of 47.62  6.026 ng T/mg Cr and a median value 36.49 ng T/mg
Cr (sampled July 8th to July 25th).
Males from Long Point exhibited a marked decline in median testosterone levels from the
breeding (n = 77 urine samples) to post-breeding (n = 48 urine samples) season, as early as one
week after the last calling event (Z = -4.58, p < 0.0001) (Figure 3). The peak (n = 42 urine
samples) to post-breeding (n= 39 urine samples) comparison of median urinary testosterone
concentration in males from the Nickel Beach did not show significant differences (p = 0.2245)
(Figure 3).
Male abundance and peak testosterone levels: ANCOVA
Median breeding-season levels of urinary testosterone did not differ significantly across
Long Point and Nickel Beach populations when considering SVL (p = 0.0619). Model effect
tests indicated no difference between levels of local male abundance (p = 0.3084), nor body size
(p = 0.2461).
Chorus attendance and testosterone levels
Long Point males who exhibited aggregation or breeding behaviours at time of sampling
(n = 39 urine samples) had testosterone levels ranging from 5.36 to 463.02 ng T/mg Cr, with
mean ( SE) of 98.47  15.38 ng T/mg Cr. Non-attendees (n = 24 urine samples) had urinary
testosterone concentrations of 10.69 to 407.39 ng T/mg Cr with a mean of 155.31  23.53 ng
T/mg Cr. Median testosterone levels of non-attendees (131.77 ng T/mg Cr) were marginally
higher than those of attendees (82.83 ng T/mg Cr) (Z = 2.04, p= 0.0408) (Figure 4).
54
Discussion
The lack of discernable difference in breeding-season testosterone levels across two
populations differing significantly in male abundance suggests that males need some baseline
level of circulating androgens for expression of calling behaviour without social cues
(Burmeister and Wilczynski, 2000). This may be because call reception affects calling behaviour
and endocrine pathways independently, such that hormonal activity is necessary but not always
sufficient to evoke calling (Burmeister and Wilczynski, 2000). After the minimum androgen
threshold is attained, then social cues can act as facilitators of reproductive processes through
positive feedback mechanisms (Burmeister and Wilczynski, 2001).
The absence of an expected drop in testosterone levels in males from Nickel Beach is
likely a direct correlate of the longer calling season observed at this site (Chapter 2). This may,
in part, be an artifact of the sampling gap earlier on in the breeding season (May 6th to 29th), such
that I could have missed additional high testosterone values expressed closer to the onset of
calling. Nonetheless, the samples used in the peak/post comparisons were collected over a
month’s period (May 29th - July 2nd), and the apparent maintenance of elevated hormonal state
beyond the breeding period is notable. Because males from Nickel Beach called for two weeks
longer than those at Long Point, their delayed decline of gonadal activity is consistent with the
predicted benefits of more numerous conspecifics in enhancing reproductive capacity
(Wilczynski et al., 2005). Correspondingly, males from Long Point exhibited a shorter breeding
season and a concomitant drop in testosterone levels, in keeping with loss of the potential benefit
conferred by abundant social signalling.
Contrary to predictions, though, I found no evidence that lack of chorus participation was
correlated to unusually low testosterone levels. Grouping male urine samples by their associated
behaviours showed that individuals exhibiting any degree of breeding effort actually had slightly
lower testosterone levels compared to their non-attendee counterparts. This result can be
interpreted in several ways. First, in contrast to Burmeister and Wilczynski’s (2000) findings,
upon which I based my predictions, Leary (2014) found that exposure to advertisement calls
caused decreased testosterone and DHT in male receivers. Accordingly, calling Lithobates
catesbeianus, Bufo melanostictus and Hyla cinerea males exhibited lower plasma testosterone
levels (Mendoca et al., 1985; Gramapurohit and Radder, 2013; Leary and Harris, 2013) and
55
higher CORT levels (Mendoca et al., 1985; Leary and Harris, 2013) than non-callers, suggesting,
similarly to my results, that lack of testosterone is not consistently responsible for low breeding
effort and therefore, that elevated testosterone levels may be necessary but not sufficient to
induce reproductive behaviours in male anurans (Mendoca et al., 1985). The disagreement may
be explained by differing methods used across the studies, as Leary (2014) used isolated call
recordings while Burmeister and Wilzynski (2000) used full chorus sounds. Such contextual
differences may have consequences on auditory processing (Velez and Bee, 2010). These results
may also be explained by negative feedback mechanisms involving the glucocorticoid stress
hormone, corticosterone (CORT). In agreement with the Energetics-Hormone Vocalization
(EHV) model, CORT decreases male propensity to call (Leary et al., 2006), however its assumed
antagonistic effect on testosterone has not been consistently confirmed (Burmeister et al., 2001).
In this context, the lower levels of testosterone I observed in chorus attendees may be a result of
regulating stored energy necessary for calling and extended amplexus (Emerson, 2001; Moore
and Jessop, 2003). Furthermore, close-range aggression calls have been found to suppress
testosterone production in rival Hyla cinerea males via increased CORT levels, as means of
countering an androgen conflict (Wingfield et al., 1990), though advertisement calls did not have
the same effect (Leary, 2014). A second and more probable interpretation is that my hypothesis
centered on testosterone may have been too simplistic. Numerous interacting factors besides
testosterone, (including other androgens), also play regulatory roles in the assimilation of signals
and expression of breeding behaviours (Burmeister and Wilczynski, 2001), some of which work
independently (Propper and Dixon, 1997) and whose effects are species and sex specific (Mann
et al., 2010; Roth et al., 2010). For example, the neuropeptide arginine vasotocin (AVT) induces
calling in male Bufo cognatus and Hyla cinerea (Propper and Dixon, 1997; Burmeister et al.,
2001), territorial behaviour in Eleutherodactylus coqui (Ten Eyck, 2005) and increased
aggressiveness during antagonistic interactions between Acris crepitans males (Chu et al., 1998).
Notably, AVT may specifically mediate calling behaviour within different social contexts. For
example, AVT only increased calling of Hyla versicolor the presence of conspecific males
(Trainor et al., 2003), while it induced calling in Engystomops pustulosus, regardless of social
context (Kime et al., 2007). Additionally, Gonadotropin-releasing protein (GnRH, also known as
LHRH, luteinizing hormone-releasing hormone) induces calling (Mann et al., 2010), amplexus
behaviour (Propper and Dixon, 1997; Mann et al., 2010) and increases spermiation (Mann et al.,
56
2010) through direct action on the brain (Yang et al., 2007). These same factors also influence
the reproductive physiology and behaviour of mature female anurans (see Moore et al., 1992;
Polzonetti-Magni et al, 1998; Browne et al., 2006).
In sum, there is little evidence for testosterone action being the primary underlying
mechanism for the apparent behaviourally-mediated Allee effect in Fowler’s Toads. It is clear
that social cues are necessary to instigate calling in receiver male anurans, but their influence is
not necessarily mirrored at the hormonal level. This observation may, in part, be due to the
methods used. Urinary hormone analysis is a relatively easy, non-invasive technique well-suited
to studying hormone phenology and to sexing individuals, especially in endangered species
where customary blood plasma extractions would be inappropriate (Germano et al., 2009).
Despite these advantages, my conflicting results suggest that urinary testosterone metabolite
concentrations may be poor indicators of testosterone levels in the brain. The temporal delay in
metabolite production and accumulation may also be involved in blurring any nightly
fluctuations due to chorus exposure, especially since it is not known how quickly these are
manifested in toad urine. Based on my preliminary investigation, the strength of this approach is
in detecting broad trends in seasonal hormone levels across populations or sexes, rather than
immediate, transitory responses.
57
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61
Tables
Table 1. Summary of male urine collection effort and urinary testosterone (T) concentrations
from high and low abundance populations of Fowler’s Toads in Southern Ontario.
Long Point
Nickel Beach
Breeding
Post-breeding
Breeding
Post-breeding
Sampling period
May 8 – June 19
June 25 – July 30
May 29 – July 2
July 8 – July 25
No. urine samples (n)
77
48
42
39
98.94
28.46
45.19
36.49
118.21  11.67
41.02  5.87
87.54  15.98
47.62  6.03
Median T concentration
(ng T/mg Cr)
Mean T concentration
(ng T/mg Cr)  SE
62
Figures
*
Testosterone (ng/mg Cr)
500
400
300
200
100
0
Females
Males
Figure 1. Urinary testosterone concentration of male and female Fowler’s Toads at Long Point
during the 2015 breeding season. Detection of significantly lower testosterone levels in females
served to ensure appropriate assay sensitivity (p < 0.0001).
600
Testosterone (ng/mg Cr)
500
400
300
200
100
0
Hastings 1
Hastings 3.75-4
Thoroughfare
Figure 2. Urinary testosterone levels of males across three Fowler’s Toads breeding sites at
Long Point, during the 2015 breeding season. Similar levels among groups justified subsequent
pooling of data.
63
*
Testoserone (ng/mg Cr)
500
400
300
200
100
0
NB Breeding
LP Breeding
NB Postbreeding
LP Postbreeding
Figure 3. Urinary testosterone concentrations in males from Long Point and Nickel Beach
during peak and post-breeding seasons. Males from Long Point exhibited significant declines in
testosterone after calling ceased (p < 0.0001), but no such drop was detected in toads at Nickel
Testosterone (ng/mg Cr)
Beach, where the breeding season lasted two weeks longer (Chapter 2).
500
*
400
300
200
100
0
Non-Attendee
Attendee
Figure 4. Urinary testosterone concentrations of (Long Point) males that actively attended a
chorus (or called alone), versus those who did not participate in or initiate aggregations when
conditions allowed. Non-attendees had marginally higher testosterone levels than attendees
(p= 0.0408).
64
CHAPTER IV: Conclusions and Conservation Implications
65
Historically abundant but currently declining species should be particularly susceptible to
Allee effects (Courchamp et al., 2008). Long-term monitoring of Fowler’s Toads in Southern
Ontario revealed concerning population declines and concomitant changes in breeding ecology
(Greenberg and Green, 2013). Empirical evidence from increasingly threatened taxa such as
anurans are lacking, in part due to the many difficulties inherent to studying Allee effects in
nature (Kramer et al., 2009). By using local-scale long-term data (allowing the key sufficient
range of abundances), I was able to meet the challenges of quantifying a measure of fitness,
identifying the causal mechanism, and suggesting an approximate population-specific critical
threshold.
Evidence consistent with a ‘social facilitation of reproduction’ component Allee effect in
Fowler’s Toads at Long Point exemplifies how inverse density-dependence can manifest itself
after some detrimental environmental event (in this case, the invasion of Phragmites australis,
Greenberg and Green, 2013), and that behavioural mechanisms behind chorus formation can be
destabilized by resulting low abundances, fueling further decline through continually lowered
recruitment. This kind of self-catalysed extinction vortex may affect chorusing amphibians
disproportionately because of their reliance on social cues to facilitate reproduction and maintain
favourable sex ratios at breeding sites (Gaston et al., 2010). This study confirms that Allee
effects should be taken into consideration by all investigators working with at-risk amphibian
populations. Additionally, by bettering our understanding of taxon-wide reproductive biology,
these findings can inform the implementation of conservation strategies for anurans in general
(Loyola et al., 2008). In cases such as this, where fragmented remnant populations are so small
and amplexus events so few, focusing on breeding habitat is key in boosting mating probability
and recruitment. However, simply adding aquatic habitat may not be sufficient in luring the
animals, as local male abundance (density) resulting from the proposed aquatic spaces must be
considered. Because larger choruses tend to attract more females and result in higher mating
success, pond design must first favour male aggregation, not spread (Gaston et al., 2010). For
Fowler’s Toads and other chorusing anurans preferring open breeding sites, digging or clearing a
large, shallow pond or beach pool would be more beneficial than creating numerous, small and
widely spaced ponds/pools. This approach would support a few nuclei of high male
concentration as opposed to multiple sites each hosting only one or two callers. After several
seasons, or when abundance has increased, optimizing pond density may not be as important.
66
In theory, identifying physiological processes responsible for the expression of inverse
density-dependent behaviours could aid in recognizing potential mechanisms of Allee effects in
other taxa. In the case of chorusing, the social enhancement of reproductive behaviour I
identified under high abundance conditions was only mirrored at the hormonal level on a broad
temporal scale and not within a given night. The lack of support for low testosterone levels being
responsible for reduced breeding effort among males at Long Point points to the shortsightedness of focusing on testosterone alone (Burmeister and Wilczynski, 2000; Woodley,
2011) and the potential limitations of using urinary metabolites for detecting short-term hormone
fluctuations produced in the brain.
Anurans currently face a plethora of threats: climate-change caused shifts in temperature
and season onset, habitat loss and fragmentation due to invasive species and human
development, as well as epidemic disease (Collins and Storfer, 2003). Along with increased risks
associated with natural environmental and demographic stochasticity, weak calling activity can
evidently result in disproportionately low or inconsistent male breeding effort in extremely small
populations. Interruption of essential aggregative behaviour should catalyse population decline,
potentially leading to local extinction (Gaston et al., 2010). This thesis highlights an additional
and perhaps overlooked vulnerability of anuran populations in response to increasingly unstable
environments. For example, significant declines in breeding aggregation size and reproductive
success in populations of endangered Yosemite Toads (Anaxyrus canorus) was attributed to
drought, disease, and predation (Kagarise Sherman and Morton, 1993), while an associated
reduction in social signalling should have also been considered as a potential catalyst of decline.
67
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68