AN ABSTRACT OF THE DISSERTATION OF
Leslie A. Dyal for the degree of Doctor of Philosophy in Zoology presented on April
8, 2005.
Title: Variation in Female Reproductive Success in Amphibians
Redacted for Privacy
Abstract approved:
My dissertation focuses on the factors that influence variation in female
reproductive success in plethodontid salamanders and in toads. Variation in
reproductive success fuels evolutionary change. Although, females often have been
overlooked in studies of reproductive success due to perceived lower levels of
variation when compared to variation in male reproductive success, understanding
factors influencing variation in female reproductive success is critical for several
reasons. First, female reproduction is usually the limiting factor on population growth.
Second, the factors affecting female reproduction provide the impetus for current and
evolving patterns of sexual dimorphism. Lastly, male reproductive success inevitably
is determined by the reproductive success of the females with which they mate.
Recent theoretical developments of sexual conflict have contributed significantly to a
renewed emphasis on studies of female reproductive success.
Sexual conflict theory elucidates important factors, from the perspective of
females, affecting female behavior and reproductive success. Sexual conflict assumes
that a female will benefit, in terms of reproductive success and offspring viability,
when she is able to freely choose among males, unconstrained by social and
environmental factors. Female choice would be constrained if a non-preferred male
(i.e., one that would be rejected by a freely choosing female) coerced a female to mate
in order to increase his own mating success. The dynamics between discriminating
females and non-preferred males will lead to a coevolutionary "arms race", referred to
as sexual conflict. From this perspective, secondary male traits may not reflect their
intrinsic quality, but rather their ability to manipulate or coerce female mating
decisions. The current debate lies in the importance and pervasiveness of sexual
conflict.
To address the current view of sexual conflict, I investigated whether sexual
conflict plays a role in the mating systems of amphibians. In particular, my results
support the main assumption that females benefit from freely expressed female mate
choice in toads. In addition, I explored the potential influence of sexual conflict in
plethodontid salamanders. During mating trials, I documented novel female courtship
behaviors. I also examined the effects of male courtship pheromones on female
reproductive success to assess the potential role of male pheromones in sexual
conflict. My results suggest that sexual conflict also may be an important factor in
plethodontid mating systems. My investigations of sexual conflict theory have
provided new insights and novel predictions for understanding sexual selection and
sexual dimorphism.
©Copyright by Leslie A. Dyal
April 8, 2005
All Rights Reserved
Variation in Female Reproductive Success in Amphibians
Leslie A. Dyal
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented April 8, 2005
Commencement June 2005
Doctor of Philosophy dissertation of Leslie A. Dyal presented on April 8, 2005
APPROVED:
Redacted for Privacy
Major
Zoology
Redacted for Privacy
Chair
of Zoology
Redacted for Privacy
Dean of the Gdhat'e School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of
my dissertation to any reader upon request.
Redacted for Privacy
Leslie AAYval. Author
ACKOWLEDGEMENTS
Above all, I want thank my advisor, Lynne I-buck. Lynne was always helpful
and willing to share her time, knowledge, and money. Lynne was there when I
needed her and provided much more than financial and intellectual support. When we
were not discussing science, we could spend hours talking about our pets and their
"issues". I owe a debt of gratitude for all that Lynne has done for me over the years.
My appreciation goes out to Steve Arnold who support was critical. Steve was
always willing to listen to and appreciate my "tangential" theories. His
encouragement and knowledge were critical at the times I needed it most. I'll always
be grateful for Steve's involvement in my graduate career.
I also want to thank Joe Beatty for too many things to list. It suffices to say that
I do not think I would have made it through this endeavor without his encouragement
and support. Joe is the best!
I would also like to thank my committee members, Deanna Olson, Paul
Murtaugh, Jeff Arthur, and Bruce Coblentz. Paul and Jeff, I thank for critical
statistical counsel. Thanks to Deanna for providing the inspiration for my research
with toads. I especially enjoyed the rare occasion when we could actually do field
work together. I would like to thank Bruce for his support and perspective.
I especially appreciate the support and guidance of Bob Mason and Barb Taylor.
I want to thank the Zoology Department for the financial support and the office staff,
Sarah Cain, Traci Durrell-Khalife, Mary Crafts, and Tara Bevandich for their critical
assistance. I would like to thank my friends and lab mates, Heather Waye, Catherine
Palmer, Mollie Manier, Jerod Sapp, Doug Degross, Erika Adams, Mark Christie,
Chris Stallings, Amy Picard, Mike Westphal, and Karen Kiemnic. I'm thankful for
the theoretical debates with Adam Jones and Mike Phrender. The Ethology Writing
Workshop was helpful in developing a portion on this dissertation. I would also like
to thank Richard and Pamela Feidhoff for the pheromone contributions. I thank all the
undergraduates that helped with my research, Keeta Owens, Renee Fox, and Randa
Mills. I also owe thanks to Rebecca Hundt who drew the fabulous illustration of
Plethodon dorsalis in Chapter 2.
I would also like to thank Micheal Blouin and his lab for the chance to broaden
my horizons into population genetics. I appreciated all the chats with Becky Cooper,
Jacob Tennessan, Charles Criscione, and Roman Vilas. In addition, I would like to
thank Richard Watts for his sharing his implacable knowledge of molecular biology.
On a more personal note, I would like to thank my parents, Christa Wiggins and
Conrad Dyal, for everything including the "field work grants".
I would like to thank
my grandmother, Margaret Summerlin for her love and support. I am indebted to
Kevin Hood for his love and friendship. Without him, I would never have been able
to leave the house and do field work since he was the only affordable and competent
house and pet sitter. I also want to thank Kelly Gibbs and all her horses for my
weekly horse riding therapy. Last, but not least, I want to thank my pets, Cheese,
Bodhi, Moksha, Alex, G, Fly, Nima, Leto, Old Man, Veda, Sophie, Spea,
Clyde, Ged, Nansen, Slick and Turtie for keeping me going.
Squirt,
CONTRJBUTION OF AUTHORS
My advisor, Lynne D. Houck, provided design and financial assistance for all of the
projects included in this dissertation. Dr. Stevan J. Arnold also helped with project
design and provided ideas for side projects that ended up being critical components of
my dissertation.
TABLE OF CONTENTS
CHAPTER 1. INTRODUCTION
CHAPTER 2. NOVEL COURTSHIP BEHAVIOR
IN THREE SMALL EASTERN PLETHODON SPEICES
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CHAPTER 3. MALE COURTSHIP PHEROMONES AND FEMALE
REPRODUCTIVE SUCCESS
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CHAPTER 4. FEMALES BENEFIT FROM FREELY EXPRESSED MATE
CHOICE IN THE WESTERN TOAD, BUFO BOREAS
ABSTRACT
INTRODUCTION
METHODS
DISCUSSION
18
18
19
23
25
38
48
48
49
54
58
63
66
66
66
74
78
84
CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS
90
BIBLIOGRAPHY
93
LIST OF FIGURES
Figure
1.1
A model of social behavior in sexual species from Gowaty 1997
3
1 .2
Factors that primarily limit the reproductive success of females
and males
4
1.3
Interactions between the sexes, comprising the dialectics of sex
5
1.4
Effects of male efforts to control females on female fitness
7
1.5
Female resistance, variation in mechanisms of male-male
competition and mating systems
9
2.1
Courtship sequence for Plethodon angusticlavius
42
2.2
Character states of behavioral features mapped onto a partial
phylogeny of Plethodon and the related genus Aneides
46
Relationship between female SVL and clutch size for Plethodon shermani
in 2002
60
Relationship between female SVL and clutch size for Plethodon shermani
in 2003
62
The total number of eggs retained by female Plethodon shermani after
oviposition in each treatment group in 2003
62
The relative size of male and female Western toads represented as the
relation between snout-urostyle length and mass
72
4.2
Schematic of female choice enclosures
76
4.3
The average proportion of hatching tadpoles for preferred and nonpreferred matings for Western toads at Todd Lake and Little Three
Creeks Lake
81
3.1
3.2
3.3
4.1
LIST OF FIGURES (Continued)
Figure
4.4
4.5
Tadpole length at hatching averaged for preferred and non-preferred
matings for Western toads at Todd Lake and Little Three Creeks
84
Differences in Western toad male SUL at Little Three Creeks Lake and
Todd Lake
89
LIST OF TABLES
Table
2.1
A comparison of female and male courtship behavior for each species
31
2.2
A summary of the duration of the phases of courtship for Plethodon
cinereus
34
A summary of the duration of the phases of courtship for Plethodon
angusticlavius
36
A summary of the duration of the phases of courtship for Plethodon
richmondi
39
3.1
A summary of results for each year of the study for Plethodon shermani
58
3.2
Linear regression estimates for Plethodon sherman! clutch size
59
4.1
Clutch data for Western toads from Little Three Creeks Lake
79
4.2
Clutch data for Western toads from Todd Lake
80
4.3
Mean tadpole length per clutch for preferred and non-preferred matings
for Western toads at Little Three Creeks Lake
82
Mean tadpole length per clutch for preferred and non-preferred matings
for Western toads at Todd Lake
83
2.3
2.4
4.4
DEDICATION
This dissertation is dedicated
In memory of Keeta Owens, an adventurer, biologist, and friend
VARIATION IN FEMALE REPRODUCTIVE SUCCESS IN AMPHIBIANS
CHAPTER 1
INTRODUCTION
Variation in reproductive success is the basis of evolutionary change. Until
recently, the study of mating behavior and its effects on variation in reproductive
success has focused primarily on males. There are three main reasons for this focus.
First, male mating success and reproductive success usually are directly related. The
more often a male mates, the higher his reproductive success. In contrast, female
mating success is rarely a determinate of female reproductive success (unless males are
limited in numbers or a single male does not produce enough sperm to fertilize all of a
female's eggs). Even under scenarios where multiply-mating females incur some
fitness benefits, the consequent increase in reproductive success is considerably less
than that in males. Second, male mating success and reproductive success typically are
highly variable among males, depending on mating system dynamics. As there is less
variation in female mating success and thus a weaker correlation with reproductive
success, variation in reproductive success of females has not been a main focus of
studies of sexual selection. Third, Darwin (1871) defined sexual selection in terms of
intra-sexual selection (e.g., male-male competition) and inter-sexual selection (e.g.,
female choice). As both of these mechanisms of sexual selection influence male
variation, variation and selection in females received less attention.
The focus of sexual selection research, however, has recently broadened its
scope to include potential conflicting selective pressures acting on males and females
2
(Parker 1979, Rice 1996, Drickamer et al. 2000, Moore et al., 2000, Arnqvist and Rowe
2002, Chapman et al. 2003, Bluhm and Gowaty 2004a). While much research of
traditional sexual selection has been conducted, these findings do not exclude or clarify
the role of sexual conflict (Figure 1.1) (see reviews in Andersson 1994, Pizarri and
Snook 2003, Eberhard and Cordero 2003, Cordoba-Aguilar and Contreras-Garduno
2003). I discuss the general concept of sexual conflict first, and then describe particular
examples of well-studied systems. Sexual conflicts occur when the reproductive
interests of males and females are different (Figure 1.2). In general, sexual conflicts
can occur at many stages of reproduction, including the decision to mate, the sex ratio
of offspring, and allocation of parental care. The theoretical basis for my research is
focused on sexual conflicts over mating (also termed "sexual dialectics"; Gowaty 1997).
For simplicity, I will use the term sexual conflict. The main assumption of sexual
conflict is that "freely expressed" female mate choice is adaptive. In other words,
female reproductive success is higher when under female control (i.e., when a female
can select the mate that she prefers) than when the female is manipulated into
suboptimal mating by non-preferred males. Sexual conflict describes the behavioral
and/or physiological interactions when there is a conflict over mating between
discriminating females and non-preferred males (Figure 1.3). The conflict between
females and non-preferred males leads to selection favoring males that are able to
manipulate or coerce female mating decisions. If female reproductive success is higher
under her own control, selection will favor resistance by females.
3
Mechanisms of Sexual Selection
Legend
same-sex behavioral
competition
choice
4
'
counterchoice behavior
e
I
4
'U
Figure 1.1. A model of social behavior in sexual species from Gowaty 1997. The thin
lines represent traditional sexual selection view of male-male competition and female
choice (sexual selection on males). The thick lines represent what is usually left out of
the traditional discussion, female-female interactions and male mate choice (sexual
selection on females). The medium line with double headed arrows from male to
female represents sexual dialectics (sexual conflict over mating), mediating sexual
selection on either males or females.
Factors Primarily Limiting RS
Females
Males
Females
symmetry
Nutrients, nest sites,
refugia
"typical"
asymmetry
mammals, birds,
insects
Males
Nutrients,
nest sites,
refugia
"role-reversed"
asymmetry
symmetry
or
asymmetry
Figure 1.2. Factors that primarily limit the reproductive success (RS) of individuals
may include access to the opposite sex or to other resources for reproduction, such as
nutrients, nest sites, and refugia. In typical species, reproductive success of females is
thought to be primarily limited by access to nutrients and nest sites; males are most
limited by access to females.
5
Sexual Dialectics
Males attempt to
control females directly
(e.g., forced copulation;
affiliative conditioning)
Females resist
"nice"
"nasty"
Males attempt to control
females by brokering
females' access to resources
Females resist
Females freely choose so
that males compete through
honest signals of quality
Figure 1.3. Interactions between the sexes, comprising the dialectics of sex (i.e., sexual
conflict over mating). The dialectics of sex are engaged whenever females fail to freely
choose a particular male as a gametic partner. Selection acts on non-preferred males to
manipulate female mating decisions using "nice", "null", and "nasty" mechanisms.
When these attempts have deleterious effects on female fitness, females are under
selection to resist. When females are successful at resisting direct mechanisms, males
are under selection to manipulate females by brokering females' access to resources
necessary for reproduction. If male resource brokering is deleterious to female fitness,
females will be under selection to resist. If females are successful at resistance, females
will be free to choose among males based on honest signals of intrinsic male quality.
Under the scenario proposed by sexual conflict, non-preferred males initially
attempt to control females by using direct mechanisms. One such mechanism is
aggressive conditioning or forced copulation. Another mechanism is affiliative
conditioning (Gowaty 1997). Affiliative conditioning refers to situations in which
males manipulate female mating decisions without costs or sometimes with benefits to
females' survival (Figure 1.4). Affiliative conditioning may seem like cooperation
between the sexes when it is actually "helpful coercion" (Gowaty 1997). This kind of
conditioning occurs on a continuum, based on the effects on the survival components of
female fitness. An example of a situation in which a female's survival is lowered by
male behavior is mate guarding in which guarding makes the female more vulnerable to
predators (as in water striders Aquarius sp., Gerris sp., Rowe 1994). On the other end
of the continuum, male behavior may enhance female survival (e.g., nuptial feeding).
Females should still resist these behaviors, even though they may seem helpful, because
of the costs of suboptimal mating on the reproductive components of female fitness.
Females will inherently vary in their abilities to resist male coercion. Female resistance
may take the form of social patterns, such as kin coalitions facilitated by female
philopatry in primates (Smuts 1992, 1995). Females may resist through ecological
advantage such as niche separation and availability of refugia (Rowe et al. 1994). Also,
female resistance can take the form of morphological adaptation, including female size
relative to male size (RaIls 1976) and female genital spines that prevent forced
ii
copulation in water striders (Fairbairn et al. 2003). In addition, physiological
adaptations may include facultative control of ovulation, implantation, and sperm
storage.
Effects of
Males' Efforts to Control Females
on Females' Fitness
Effects on Females' Survival
+
0
Effects on
Females'
RS
-
-
0
-
+
0
0-000+
+
+o'++
Figure 1.4. Matrix of the effects of males' efforts to manipulate or coerce females.
Effects may be on either the survival component or reproductive success (RS)
component of a female's fitness. Because manipulation of females' reproductive
capabilities theoretically is almost always costly to the reproductive success component
of a female's fitness, the continuum of manipulation efforts is defined in terms of their
effects on the survival component of a female's fitness only, and ranges from "nasty" to
"null" to "nice." The full matrix is present to show the entire range of possible
interactions, even though this discussion is based on the first row of the matrix.
8
If females are able to resist direct male attempts to control mating, selection
should favor males that indirectly attempt to control females by brokering females'
access to critical resources. Such males will benefit through increased mating success
(Figure 1.2). Female success at resisting indirect control will depend on the
environmental potential for females to resist resource brokering. This environment
potential for resistance includes factors such as the spatial distribution of resources,
male-female niche separation and arrival times at critical resources. If the
environmental potential favors female resistance, females will freely choose so that
males compete through honest signals of quality (Figure 1.5).
Sexual conflict and the consequent male-female coevolution often result in male
traits that stem from females discriminating against non-preferred males. This
discrimination can lead to male-male competition as a mechanism to manipulate female
mating decisions (Gowaty 1997). When considering the potential conflict between
males and females, we cannot assume that females prefer male traits that function in
male-male competition. For example, males may compete for territories that have
nesting sites needed by females. Thus, a male can monopolize a resource needed by
females. In this scenario, a female's inherent preferences that confer a selective
advantage, the "freely expressed choice", are constrained by her need for a resource
monopolized by males. The resulting "choice" is a compromise between mate
compatibility and access to a needed resource. In fact, there is evidence of the
conflicting nature of male-male competition and free female choice in both invertebrate
Female Resistance, Variation In
Mechanisms of Male-Male Competition and Mating Systems
gh
Environmental
potential for female
resistance to
resource brokering
Success of female
resistance to direct
control by males
II
Honest signals of
male intrinsic
quality
I
1'ree Female
Choice
I
Territoriality
Female-male
contests for
resources
Malemale contests
for resources
Exploitation of sensory bias
Affiliative conditioning
Helpful coercion
Mate herdlng."guarding"
Aggressive coercion
Forced copulation
Male ResourceBrokering
Mating Systems
Direct Control of
Females Mating
Systems
Figure 1.5. The success of females at resisting direct mechanisms of manipulation by
males, coupled with the environmental potential for females to resist resource brokering
by males, determines the type of mating system. Note that the success or failure of
females' resistance determines the type of male-male competitive interactions and thus
affects variance in male reproductive success. From a female's perspective, there are
three types of mating systems: free female choice mating systems, male resource
brokering mating systems, and direct control of females mating systems.
10
and vertebrate systems (discussed below). Male traits are potentially influenced by
sexual conflict if these traits directly or indirectly manipulate or coerce females into
mating. The consequent patterns of intra- and inter-specific mating system variation are
generated through variation in females and their interaction with the environment
(Figure 1.5). The sources of mating system variation are (1) the female's ability to
physically resist attempts to mate with a manipulative, non-preferred male, and (2) the
environmental and social factors that indirectly affect females' ability to resist matings.
These factors include the availability of food, niche separation between males and
females, refugia, the operational sex ratio experienced by females, and the density of
individuals (Rowe et al. 1994, Lauer et al., 1996, Gowaty 1997). The accumulating
evidence suggests that sexual conflict is more pervasive than previously thought and
that determining the presence and role of conflict will expand our understanding of
male-female coevolution and the resulting sexual dimorphism.
Much debate centers on what role is played by sexual conflict, particularly in
terms of the evolution of mating systems and sexual dimorphism (Gowaty 1997,
Chapman et al. 2003, Cordoba-Aguilar and Contreras-Garduno 2003, Eberhard and
Cordero 2003, Kokko et al. 2003, Pizarri and Snook 2003). Most researchers agree that
sexual conflict and traditional sexual selection are not mutually exclusive. The
behavioral manifestations of intra- and inter-sexual selection are often behaviorally and
theoretically interconnected, and thus are difficult to distinguish (Gowaty 1997,
Chapman et al. 2002). The method of untangling the most important mechanisms of
selection requires predictions appropriate to the mating system of interest, while
11
addressing all potential behavioral pathways of intra- and inter-sexual selection. Recent
studies revealed that conflicts over whether or not to mate often play a pivotal role. In
certain well-studied systems, sexual conflicts are the causative pathway of sexual
selection acting on male traits (Rowe et al. 1994, Crean and Gilburn 1998, Shukar and
Day 2002, Bonduriansky and Rowe 2003). These systems are discussed below.
Sexual conflict has been most thoroughly investigated in several invertebrate
systems. The phylogenetic footprint of sexual conflict and the consequent coevolution
of male and female traits have been confirmed in water striders (Gerris sp., Rowe et al.
1994), dragonflies and damselflies (Odonata: Anisoptera and Zygoptera, Finke 1997),
diving beetles (Coleoptera: Dystiscidae, Soulier-Perkins 2001) and planthoppers
(Hemiptera: Lophopidae, Miller 2003). Additionally, the interplay between traditional
forms of sexual selection (e.g., female choice, male-male competition) and sexual
conflict has been clarified in several insect systems (Bonduriansky and Rowe 2003,
Rice 1996), and also in meadow voles (Spritzer et al. 2005)). For example, in the
cockroach (Nauphoeta cinerea), males produce a social pheromone that has multiple
components. One component of this pheromone indicates social dominance amongst
males, but another component (usually absent in dominant males) is most attractive to
females (Moore and Moore 1999). In cockroaches, therefore, the outcome of malemale competition is in conflict with female mating preferences (Moore et al. 2000).
Also, water striders exhibit a premating struggle, a behavior that has led to this system
being thoroughly studied in terms of a conflict over mating (Arnqvist et al. 1997,
Arnqvist and Rowe 1995, Rowe et al. 1994, Fairburn et al. 2003). Variables thought to
12
influence the outcome of the pre-mating struggles included: morphology, refugia, sex
ratio, density, diet and mating status. Results from experimental manipulation of each
of these variables supported the predictions of sexual conflict as determined by female
resistance behavior (Rowe et al. 1994, Watson et al. 1998, Ortigosa and Rowe 2003,
Arnqvist and Rowe 2002). The possibility that sexual conflict drives traditional sexual
selection also has been supported by research in the seaweed fly (Coleopafrigida).
Selection favoring large male size occurs as a result of sexual conflict over mating
(Crean and Gilburn 1998, Shukar and Day 2002). The seaweed fly mating system is
characterized by scramble competition without obvious active mate choice. Although
females do not actively choose males, female rejection behaviors select for large males
that are best able to overcome female resistance and force copulations (Shukar and Day
2002).
In addition to identifying examples of sexual conflicts, the costs and benefits of
many aspects of sexual conflict have been investigated in the previously mentioned
invertebrate systems (also Drosophila, see Rice 1996). There also is accumulating
evidence of sexual conflict in a few vertebrate systems, including mice (Mus musculus,
see Drickamer et al. 2000), mallards (Anas platyrhinos, see Bluhm and Gowaty
2004a,b), and fish (Gadus morhua, Rudolfsen et al. 2005; Poecilia reticulata,
Rosenqvist et at., 2001; Symphodus ocellatus, Alonzo and Warner 2000). In mice and
guppies, females benefited from "freely expressed choice" in many reproductive
components of fitness, including offspring survival, viability, and performance
(Drickamer et al. 2000, Rosenqvist et al. 2001). In another example, female mallards
13
incurred reproductive costs associated with non-preferred matings occurring as a result
of social constraints on female preferences (Bluhm and Gowaty 2004a). Also, with
increasing female age and experience, female mallards were able to compensate for
costs associated with non-preferred matings by increasing investment in offspring
(Bluhm and Gowaty 2004b; also found in guppies, Reynolds and Gross 1992, Nicoletto
1995, Nicoletto et al. 2001). In another vertebrate study, compatible male-female
crosses in Atlantic cod resulted in higher offspring survival, and this higher suvivorship
was not correlated with male traits such as sexual ornaments or sperm quality
(Rudolfsen et al. 2005). Lastly, sexual conflict has been shown to influence costs of
courtship in female garter snakes (Shine et al. 2003, 2004). In garter snakes, female
resistance behavior may explain size-assortative mating, temporal patterns in dispersal
from overwintering dens, and female mimicry (Shine et al. 2000). Since most studies of
sexual conflict focused on invertebrates as study organisms, it will be necessary to
include more vertebrate systems in studies of sexual selection and sexual dimorphism to
understand the pervasiveness of sexual conflict. The work reported below addresses
this need.
My research has primarily focused on variation in female mating behavior and
corresponding effects on female reproductive success. Amphibians provide an
opportunity to examine the nature of variation in reproduction in a range of mating
systems, from systems having prolonged courtships and breeding seasons (plethodontid
salamanders) to systems characterized by explosive breeding with very brief breeding
seasons (Western toads). I focused on amphibians as a model for addressing three
14
questions not usually considered within the context of reproductive success. The
questions are described in Chapters 2, 3, and 4.
In Chapter 2, I ask whether females can play an active role in courtship interactions
in plethodontid salamanders. Courtship in this family is highly stereotyped and
courtship behaviors have been well documented for many species. The general
observation is that the male plays the more active role in courtship (Noble and Brady
1930; Organ 1960; Stebbins 1949; Arnold 1972, 1976, 1977). In addition, Plethodon
males deliver courtship pheromones by two methods, vaccination and olfaction (Arnold
1977, Houck and Sever 1994). These courtship pheromones alter female reluctance to
mate (Houck 1986, Houck and Reagan 1990, Houck et al. 1998, Rollmann et al. 1999),
and thus may reflect sexual conflict. If courtship pheromones were involved with
sexual conflict, I would expect to observe variation in female courtship behavior within
the genus Plethodon, since variation in female behavior is a critical tenet of sexual
conflict theory. I investigate courtship behavior in three salamander species to address
the potential for variation in the female's role in courtship. Previous studies of social
interactions in one of the study species (Plethodon cinereus) found that females
defended territories, were socially monogamous, and exhibited differential aggressive
behavior between familiar and unfamiliar individuals (Gabor and Jaeger 1995, Jaeger
and Peterson 2002, Lang and Jaeger 2000, Gillette et al. 2000). Thus, the potential for
active female participation in courtship seemed possible for P. cinereus since these
females were, in general, active participants in social situations. I chose P. richmondi
as a study species due to a lack of behavioral information and their close relationship to
15
P. cinereus. Plethodon angusticlavius was chosen because they are phylogenetically
intermediate between species using olfactory and vaccination courtship pheromone
delivery. I made observations of courtship behavior for each of these three salamander
species. I documented novel female courtship behaviors in all three species. The active
role of these females may reflect a more complex social structure within these species.
The use of courtship pheromones that alter female resistance in plethodontid
salamanders may reflect sexual conflict if it is used to manipulate female mating
decisions. If sexual conflict were occurring, I would expect to see variation in female
behaviors reflecting attempts by females to resist manipulation by males. I found that
females actively attempted to persuade males to mate, and both males and females
played a leading role in courtship interactions. My observations of courtship broaden
the range of documented female courtship behavior and provide insight into the
potential for sexual conflict in plethodontid salamanders.
In Chapter 3, I address more directly the potential role of courtship pheromones in
sexual conflict. In particular, I test the hypothesis that female reproductive success is
affected by male pheromone delivery in plethodontid salamanders. I focused on a
single well-studied species, Plethodon shermani. For P. shermani, the male courtship
pheromone has already been shown to alter female reluctance to mate, as indicated by a
decreased duration of courtship (Rollmann et al. 1999, Rollmann et al. 2003).
Precedence for courtship chemicals to have multiple effects on females has been
documented in several insects (Drosophila, Rice 1996, cockroaches, Moore et al. 2003).
Consequently, I investigated other potential consequences of male courtship pheromone
16
delivery on female reproductive success in P. shermani.
I
examined male courtship
pheromone delivery from the perspectives of sexual conflict. If female choice is
adaptive and is operating in this system, then the pheromone may function to overcome
female resistance to mating with a non-preferred male. In this case, the pheromone
should negatively affect female reproduction, even if the pheromone is beneficial in
terms of survival (e.g., decreased exposure to predators due to shorter courtships). If,
on the other hand, the male pheromone is an honest signal of quality or only decreases
courtship time in response to predator exposure, then the effects on female reproduction
will be neutral or positive. I discovered that distinguishing between these two
alternatives is difficult, but my results provide incentive for more research.
In Chapter 4, I test the main assumption of sexual conflict theory that freely
expressed female choice is selectively advantageous. In particular, is female choice
important in a species of toad (Bufo
boreas)
in which a female's mating decisions
typically are constrained by male behavior? I conducted an experiment to determine if
females benefit from freely expressed mate choice, even when the mating systems
seems to prevent the expression of the preferences of females. In addition, I conducted
a post hoc analysis of male traits that females may be using to differentiate among
males. My results support the assumption that females benefit from mate choice. The
benefits were independent of particular male traits and occurred in a mating system that
constrains free female mate choice. My post-hoc analysis of male traits indicates that
preferred males are often smaller than are non-preferred males or the original male with
17
which a female was amplexed. My results suggest that the observed mating system
patterns of B.
boreas
are influenced by female choice and sexual conflict.
In conclusion, the theoretical developments based on sexual conflict theory now
allow us to evaluate reproductive success from the perspective of females. These
developments help to identify potential sources of variation in female reproductive
success. I have addressed three different amphibian examples in which female variation
in reproductive success may be affected by sexual conflict. In addition, a new
perspective on the role of females clarifies what factors may influence male and female
traits involved with male-female coevolution, sexual dimorphism, and speciation.
18
CHAPTER 2
NOVEL COURTSHIP BEHAVIOR IN THREE SMALL EASTERN PLETHODON
SPECIES
Leslie A. Dyal
ABSTRACT
Plethodontid salamanders provide an opportunity to investigate mating
behaviors and pheromonal communication within the context of well-established
phylogenetic relationships. Courtship in many members of the genus Plethodon has
been well studied, but critical gaps exist in observations of pheromone delivery methods
(vaccination or olfactory) in transitional species, those species phylogenetically
intermediate between those having the two pheromone delivery methods. I observed
courtship interactions in three small eastern plethdontids, P. cinereus, P.
angusticlavius, and P. richmondi. I documented several novel female behaviors, as
well as interspecific variation in courtship patterns that otherwise have been highly
conserved for over 70 million years. I confirmed that olfactory pheromone delivery
occurs in P. angusticlavius, a species that is phylogenetically in the transition from
vaccination to olfactory pheromone delivery within the genus Plethodon. The deviation
from typical courtship patterns observed for the three study species revealed that
females play a more active role in courtship than has previously been documented for
Plethodon species.
INTRODUCTION
Studies of the evolutionary dynamics of courtship behavior have been facilitated
by molecular data and other independent information providing the basis for wellestablished phylogenies (Hoikkala and Kaneshiro 1984, Rowe 1994, Greenspan and
Ferveur 2000). Mapping behaviors and associated morphologies onto phylogenies
often can provide insight into the evolution of mating patterns by elucidating how
differences among species could have resulted from sexual selection and/or sexual
conflict (Rowe 1994, Fincke 1997, Weller et al. 1999, Soulier-Perkins 2001, Miller
2003). Invertebrates, in particular, have contributed significantly to our understanding
of these processes of selection. In water striders (Aquarius sp., Gerris sp.) and diving
beetles (Dytiscidae), for example, independently established phylogenetic patterns
reveal the order and alternation of traits that are related to a sexual conflict over mating
(Rowe 1994, Miller 2003). In tiger moths (Arctiidae), a phylogenetic study of mating
signals and displays showed that these signals originated as visual and chemical
defenses against predators (Weller et al. 1999). Studies based on robust phylogenies
also reveal behavioral trends in vertebrates, particularly in terms of female mate choice.
The determination that pre-existing sensory biases affect female mating behaviors has
been supported by phylogenetic information in swordtail fish (Xiphophorus) (Basolo
1990).
Less attention has been devoted to the evolution of courtship behavior and
chemical reproductive signals in other vertebrate taxa. For salamanders, however, a
20
recent summary (Houck and Arnold 2003) reviewed both stability and dynamic changes
in basic courtship patterns across all nine families. More basal families of salamanders
(Sirenidae, Cryptobranchidae and Hynobiidae) have external fertilization, while the
remaining, more recently derived families have internal fertilization that presumably
enhances fertilization success (Houck and Arnold 2003). Another trend is the long-term
stability of particular aspects of courtship behavior. For example, male egg-guarding in
Cryptobranchidae and Hynobiidae (Kerbert 1904, Smith 1907, Sato 1992, Usuda 1993,
Hasumi 1994, Kawamichi and Ueda 1998, Park and Park 2000) and ventral amplexus
with "pinwheel" sperm transfer in Salamandridae (Arnold 1977, Rehberg 1986, Fleck
1992) are behaviors conserved for tens of millions of years. Similarly, a strikingly
conserved behavior is seen in the plethodontid salamanders (Houck and Arnold 2003).
All plethodontid species for which courtship has been observed are characterized by a
linear "tail-straddling walk" (Noble and Brady 1930, Stebbins 1949, Arnold 1977,
described below). Phylogenetic evidence supports the estimate that the linear tail-
straddling walk has been retained for over 70 million years (Highton et al. 2000). The
stability of the linear tail-straddling walk behavior is most likely attributable to the
increased success of sperm transfer since courtship in the Plethodontidae involves the
deposition of only one or very few spermatophores. In other families, a male may
deposit several or scores of spermatophores per courtship (Houck and Arnold 2003).
Courtship in plethodontid salamanders, and particularly in the genus
Plethodon,
is typically initiated when the male orients to and approaches the female (Organ 1961,
Arnold 1977). Initially, the male will make head contact with the female, and the pair
21
later engages in tail-straddling walk. During tail-straddling walk, the female follows the
male with her chin on the base of his tail while having her forelimbs on either side of
the male's tail (Noble and Brady 1930, Stebbins 1949, Arnold 1977; see Figure 2.2).
During the courtship season, the male develops a mental gland that secretes pheromone
proteins that can alter female behavior (Houck and Reagan 1990, Rollmann et al. 1999,
Rollmann et al. 2003). Courtship pheromones are delivered to the female before or
during the tail-straddling walk. Most plethodontids maintain the ancestral mode of
pheromone delivery by "vaccination." Vaccination occurs when the male uses his premaxillary teeth to scratch and abrade the skin on the female's dorsum, and rubs his
mental gland over the same area (Organ 1961, Arnold 1972, Houck and Arnold 2003).
Also, males of several species use a rapidly executed snapping or puUing behavior
(described below) to deliver pheromone to the female (Arnold 1972, Marvin and
1-lutchison 1996). These vaccination behaviors presumably allow the pheromone
produced by the mental gland to enter the female's superficial circulatory system
(Organ 1961, Arnold 1972, Houck and Arnold 2003). In contrast, large eastern
plethodontids have a derived mode of pheromone delivery in which the male "slaps" his
mental gland directly on the female's nares (Arnold 1976, 1977; and Figure 1 in
Rollmann et al. 2000). Pheromone stimulation by direct olfactory delivery is processed
by the female's accessory olfactory system (Wirsig-Wiechmann et al. 2002).
Regardless of the delivery mode, the majority of pheromone delivery occurs during tailstraddling walk (1-buck and Arnold 2003).
22
During courtship, the male is traditionally considered the more active participant
with female behavior being more subtle. For example, the female initially demonstrates
receptivity by no longer fleeing when the male approaches her and contacts her with his
head. When the male initiates tail-straddling walk, the female steps over his tail and
places her chin on the base of his tail. Spermatophore deposition timing presumably is
coordinated by the ability of the male to assess the female's forward movement during
linear tail-straddling walk (Arnold 1976). If a female moves in a coordinated fashion
with the male (i.e., does not lag behind), the male is more likely to deposit a
spermatophore without further courtship interactions. After spermatophore deposition,
the female obtains the sperm mass by moving forward and lowering her cloaca onto the
spermatophore, thus lodging the sperm mass in her cloaca.
Courtship in the Plethodontidae is highly stereotyped in that the same behaviors
are observed in many species (Arnold 1977, Marvin and 1-lutchison 1996, Houck and
Arnold 2003). However, more recent observations of previously unstudied species have
revealed novel variations in courtship behavior between species (Sapp 2002). In
particular, these recent studies have revealed unsuspected variation in the tail-straddling
walk. Species of Aneides, for example, engage in a tail-straddling walk that proceeds in
a circle, instead of in the traditional straight line (Sapp 2002).
Aneides
females also
exhibit behaviors previously considered to be stereotypical male behaviors. For
example, a female will rub the male's back with her chin, a behavior that in other
species is typically performed only by males. Adding to the examples of variation in
23
tail-straddling walk and more active female behavior in Aneides, I describe below the
courtship behavior of P. cinereus, P. angusticlavius, and P. richmondi.
MATERIALS AND METHODS
Animals were collected from two locations in Virginia, USA and one location in
Arkansas, USA in August 2000 and February 2001. Twenty-eight P. richmondi and
eight P. cinereus were collected from Grayson County, VA (36 37.73 N, 81 35.23).
Another twelve P. cinereus were collected from a second site in Grayson County, VA,
on Mount Rogers (36 47' N, 81 32.96' W). Five P. angusticlavius were collected from
Madison County Wildlife Management Area in Arkansas (36 14' 42"N, 93 40'45W).
Animals were sexed using the "candling" method (Gillette and Peterson 2001). There
were 14 P. richmondi of each sex and 14 females and 6 males of P. cinereus. For P.
angusticlavius, there were 4 males and 1 female. Although the sample size for P.
angusticlavius observations are small, the highly stereotyped nature of behavior in these
salamanders increases the likelihood that behaviors observed are robust. In courtships
of P. angusticlavius and P. richmondi, males and females used in trials were from the
same population. Since there were two populations of P. cinereus, males and females
may or may not have been from the same population due to the shortage of males.
Salamanders were transported to Oregon State University, Corvallis, Oregon,
USA and housed in a climate-controlled room (13 - 16 °C) with a natural photoperiod.
Animals were housed individually in plastic shoeboxes (12 x 17 x 6 cm). Boxes were
lined with moist paper towels and several crumpled paper towels were added to provide
24
cover. The animals were fed white worms (Galleria ne/Ia) and/or small crickets
(Acheta domestica) weekly.
Separate from observed courtship stagings, males and females were paired
randomly within each species and allowed to interact overnight to maintain levels of
reproductive and social behavior in the laboratory. These unobserved courtships were
conducted on nights when staged courtships were not recorded on video. Infrequently,
and in all species, spermatophores were found the morning following unrecorded social
interactions, indicating the maintenance of reproductive condition. Staged video taped
courtship trials began in October 2000 and continued until behavioral interactions
diminished (mid-December 2000 for P. cinereus, P. richmondi, through March 2001
for P. angusticlavius). The trials were staged at night, beginning at 1500-1900 hr
,
in
the same room in which the animals were housed. Each courtship arena consisted of
two glass plates separated by a plexiglass frame (17 x 17 x 5 cm). The lower glass
sui-face was covered with a moist paper towel. Trials lasted until the following morning
(0700-0900 hr). Animals were given at least 3 days of rest between staged interactions
or after successful unrecorded courtships occurring between video taped encounters.
Courtships were recorded using a low light camera (Panasonic VW BD400) and
a closed circuit time-lapse video tape recorder (Panasonic AG 6540). Up to four
different pairs of animals were recorded each night, each at four images per second.
Thirty-seven courtship interactions were recorded, including 13 spermatophore
depositions. Behavioral observations are described using summary statistics (mean,
standard deviation, and range).
25
RESULTS
Courtship behaviors are cataloged below in ethogram format for P. cinereus, P.
richmondi, and P. angusticlavius. The catalog describes in detail all behaviors
observed in any of the three species and generally follows nomenclature in previous
ethograms from Arnold (1972, 1976). Following the catalog, I clarified which
behaviors were performed by a particular species and by sex (Table 2. 1). I also
described the temporal relations of behaviors observed during courtship interactions for
each species, and compare the duration of each stage of courtship among species
(Tables 2.2-2.4).
Behaviors performed by males or females
Approach
Retreat
Nudging
One individual moves toward the other (within 2 cm).
One individual moves away after an approach by the other.
One individual moves its head along the sides of the other's body.
Rubbing and sliding
One individual moves its head in a side-to-side (rubbing) or
forward-to-backward (sliding) motion while in contact with body/dorsum. When
rubbing is performed by males, the pre-maxillary teeth may abrade the female's skin,
accomplishing a type of vaccination pheromone delivery.
Foot shuffling
While stationary, the animal lifts one foot as the other descends. This
behavior is confined to rear legs. It is similar to foot dancing (Arnold 1976), which
includes all four legs.
26
Cloacal nudging
The snout of one animal contacts the cloacal region of the other
salamander.
Head contact
Contact to the head is initiated by one individual (cheek to cheek, snout
to cheek, snout to snout).
Head rubbing
I-lead to head contact is performed by one individual by making a side-
to-side or forward-to-backward motion while its head is on top of the other's head.
Turning back
During female-first tail-straddling walk (ffTSW) or during linear tail-
straddling walk, the female or male turns back towards the other's dorsal and/or sacral
region. When performed by a female, her body remains in contact with (or close to) the
substrate. In contrast, a male turning back raises his body off the ground to better reach
the female's nares for olfactory pheromone delivery.
Tail wagging
One individual will undulate the distal portion of its tail (one-half to
one-third of the tail). Tail wagging usually occurs throughout the courtship, often (but
not necessarily) in contact with the other salamander Isimilar to tail undulation (see
below) but faster and with only the tail tip moving
I.
Behaviors performed by females only
Cloacal rubbing
The female crawls over the male and rubs her cloaca with quick
forward-backward movements while in contact with the male dorsuni (usually toward
the rear of the male).
High amplitude tail undulations
When the male is in contact with female's tail,
usually with his chin touching her tail, she will undulate her tail in a slow, high
27
amplitude wave-like motion while her tail is either in full contact with the substrate or
with the tail base slightly arched.
High amplitude head swinging
The female swings her head side to side at
approximately 45-degree arcs. She may be a short distance from the male (<5cm) and
in visual contact, or the male may be rubbing or nudging the female's back or tail.
Pulling
The female turns back towards the male, places her head on the posterior
portion of the male's body, and pulls her head quickly over the male's back. This
behavior is similar to snapping (see male behaviors), except the female is not displaced
from the male. Pulling usually occurs during female-first tail-straddling walk.
Stop and lower vent
The female halts her forward movement and lowers her vent
close to (or over) the spermatophore. She may or may not retrieve the sperm mass.
Pass over spermatophore
After the male has deposited a spermatophore, the female
walks forward while elevating her body over the spermatophore. At this point, the male
has moved his tail to one side, but the female continues forward with her chin on his tail
base.
Transfer sperm mass
After the female lowers her vent on to the spermatophore, she
will undulate her tail and sway forward and backward.
Lift off The female lifts her cloaca upward after retrieving sperm and moves away.
Resume tail straddling walk The female does not move away after spermatophore
deposition, but re-engages in tail-straddling walk. This re-engagement occurs when the
female is not inseminated and usually leads to additional spermatophore depositions (up
to 4).
28
Behaviors performed by males only
Snapping
The male's head moves backward over the female's dorsum in a very rapid
pulling motion, either at the base of the female's tail (from ffl'SW position) or
perpendicular to the female's body. This usually places male 1-4cm from the female
(Arnold and Houck 1982). Snapping is one type of "vaccination" pheromone delivery
in which the male's pre-maxillary teeth abrade the skin of the female.
Slapping
While in tail-straddling walk, the male curls back toward the female and
'slaps' his mental gland on the female's nares (olfactory pheromone delivery).
Crossing under
The male walks under the female's chin until her chin is at the base of
his tail. When the male positions himself so that his tail base is under her chin, he may
straighten his hind limbs to lift up the female's chin. If the female is receptive, she will
enter tail-straddling walk from this position.
Spermatophore deposition and sperm transfer The male stops, lowers his vent to the
substrate, and undulates his tail. When deposition is complete several
mm
later, the
male flexes his tail to one side, raises his body off the spermatophore and resumes
forward motion with the female's chin on his tail base. When the female stops to obtain
the sperm mass, the male pushes backward under the female's chin by entending his
hind limbs.
Tail undulation
The male arches and undulates his tail, which involves the full length
of the tail. This is usually performed when the base of the male's tail is in contact with
any part of the female's body.
Head swinging
The male moves head laterally in a side-to-side motion slightly above
the substrate, alternating sides at a 15-degree arc.
Behaviors performed jointly by males and females
Linear Tail straddling walk (TSW)
The female follows the male with her head on his
tail base, straddling his tail.
Female-first tail straddling walk (ffTSW)
The male follows the female, initially with
his head on the distal portion of her tail but moving towards the base of her tail, the
male straddles her tail as he moves forward. An unreceptive female will pull her tail
away from the male or walk fast to prevent ffTSW.
Joint head swinging The male and female sway heads laterally, usually alternating
sides relative to each other. The female's head moves in +I 45 degree arcs while the
male's head swings at +1- 15 degree arcs. The pair either faces one another or the male
is on top of the female and facing the same direction.
Circling
From female-first tail-straddling walk, the female turns back towards the
male and the pair forms a circle. The female may or may not straddle the male's tail. If
the female does straddle his tail, a circular form of tail-straddling walk ensues later
leading to the traditional linear tail-straddling walk.
Temporal Relations
Plethodon cinereus
I observed 11 complete courtship sequences (i.e., each sequence included
spermatophore deposition and sperm transfer). Also, 13 observations were made of
30
initial courtship interactions not leading to spermatophore deposition (Table 2.2). In
total, these observations involved 6 males and 13 females and included over 90 hours of
courtship. Below, I categorize the courtship interactions using the three stages of
courtship previously described for members of the plethodontid family (Organ 1960,
Arnold 1972): (1) orientation (approach); (2) persuasion (head contact and pheromone
delivery); and (3) sperm transfer (tail-straddling walk, pheromone delivery and
spermatophore deposition).
When first introduced into the courtship enclosure, both the male and the female
explored the area. During the orientation stage, contact was initiated by males in 43%
and by females in 56% of the staged encounters (N=24). During orientation, the male
and female usually made contact by nudging, head contact, and, in one case, cloacal
rubbing. This stage of the courtship interactions lasted, on average, 22 mm (SD=62.1;
N24).
The persuasion stage of courtship began after initial contact and both the male
and female maintained interest in the courtship by slowing their movements around the
arena, allowing for more direct contact. The main behavioral components of this stage
were rubbing, tail wagging, and female-first tail-straddling walk. The male generally
contacted the female's body, moving his head from posterior to anterior in a variety of
ways, including rubbing, nudging, cloacal nudging, and snapping. Tail wagging by
males usually occurred simultaneously with these behaviors, either while in contact
with, or while following, the female. These male behaviors were often performed while
in female-first tail-straddling walk. Snapping occurred sporadically, either while in the
31
Table 2. 1. A comparison of female (F) and male (M) courtship behaviors for each
species. See text for description of the behaviors. + = behavior present, = behavior
not present 0 = behavior not observed, and ? = uncertainty due to limited observations.
Behaviors
Female or Male
Approach
Retreat
Nudging
Rubbing/Sliding
Cloacal nudging
Head contact
Head rubbing
Foot shuffling
Tail wagging
Turning back
Female Only
Cloacal rubbing
High amplitude tail
undulations
Tail wagging
High amplitude head
swinging
Pulling
Stopandlover
cloaca
Pass over
spermatophore
Retreive sperm
Resume tailstraddling walk
P. angusliclavius
F
M
P.
richmondi
F
M
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
+
+
+
0
?
+
?
+
+
+
+
+
-
+
+
+
0
+
+
0
+
+
+
0
0
Male Only
Snapping
Pulling
Slapping
Head swinging
Spermatophore
deposition
Eat spermatophore
Tail undulation
Tail wagging
Crossing under
Male and Female
Linear tailstraddling walk
Female first tailstraddling walk
Circling
Joint head swinging
P. cinereus
F
M
+
0
+
+
+
0
+
-
+
+
+
0
0
0
+
0
+
+
+
+
+
+
+
+
+
+
+
+
0
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
32
female-first tail-straddling walk position or from a position perpendicular to the female.
While in female-first tail-straddling walk, a female may arch and undulate her tail under
the male's chin. Females also may perform high amplitude tail undulations, usually
when in contact with the male. A female occasionally turned back towards the male
while in female-first tail-straddling walk. In one courtship interaction, the female
repeatedly turned back towards the male and the pair walked briefly in a circle. During
the persuasion stage, females initiated rubbing, nudging, head contact and head rubbing.
In courtship interactions that did not result in spermatophore deposition, the persuasion
stage accounted for most of the time spent in courtship. In the 11 complete courtship
sequences, the persuasion stage (as well as the entire courtship) was relatively short and
many of the behaviors described above were not performed.
The sperm transfer stage began when the male crossed under the female's chin,
moved forward, and the female entered tail-straddling walk (TSW). While in TSW, the
male undulated his tail under the female's chin and body as the pair walked forward in
tandem. The amount of time in TSW ranged from 2 to 240 mm. The mean amount of
time in TSW was 35 mm (SD=62.6; N=14) when courtships with more than two
spermatophore depositions were included. Since the length of TSW increased with
each successive spermatophore deposition, a few observations of multiple
spermatophore deposition increased the mean substantially. If courtships with more
than two spermatophore depositions are excluded, the mean amount of time in TSW
was 5.7 mm (SD=4.82; N=7). After the TSW, the pair ceased moving forward as the
male lowered his vent. While the male's vent was lowered, tail undulations increased in
33
speed, particularly as spermatophore deposition was nearing completion, which was
within 6 mm. The male then raised his vent and shifted his tail to one side at a 90degree angle to his body (cf Arnold 1976). The pair moved forward until the female
was positioned over the spermatophore. The female lowered her cloaca and
swayed/rocked on the spermatophore to lodge the sperm mass in her cloaca. While the
female retrieved sperm, she foot shuffled. In one instance, the female then crawled over
the male and rubbed her cloaca on the base of the male's tail, dislodging the sperm mass
from her cloaca. A male typically deposited one spermatophore, but occasionally two
to four spermatophores were deposited in the course of a single courtship interaction.
Plethodon angusticlavius
I observed four courtship sequences with spermatophore deposition occurring in
two of these courtships. Two of the four courtship sequences involved the same male
and I had only one female in the study. Observations include over 4.5 hours of
courtship (Table 2.3).
When introduced into the courtship arena, a pair initially remained stationary
and in close proximity, usually with one animal on top of the other. Once the pair
began exploring the area, both the male and female would approach one another
repeatedly. The male would attempt to make head contact with the female while tail
wagging, in a manner similar to that described above for P. cinereus. The approach
34
Table 2.2. A summary of the duration (mm) of the phases of courtship for Plethodon
cinereus. The phases are separated into seven categories: approach, HC/TW= head
contact and tail wagging, fITS W=female-first tail-straddling walk, ITSW = linear tailstraddling walk, SD=spermatophore deposition, ST=sperm transfer. See text for a
more detailed description of these phases. The total time of each courtship sequence is
presented (far right column), along with means, standard deviations, and percent of total
for each courtship component (zeros were not included in calculations). The number in
parentheses next to the sequence number denotes the number of multiple courtship
sequences by the same pair that occurred sequentially on the same night. A blank in
the ST column reflects a courtship without SD.
Plethodon
cinereus
Phases of Courtship
Approach
HCTTW
ffTSW
ITSW
SD
ST
1(1)
14
103
0
2
5
2(1)
3(1)
4(1)
5(1)
6(2)
7(2)
8(3)
9(4)
0
0
0
5
0
5
5
0
0
0
9
0
0
0
0
25
22
35
240
6
6
0
6
0
0
0
0
0
0
No
No
No
No
Sequence
10(5)
11(6)
12(7)
13(8)
14(8)
15(8)
16(9)
17(9)
18(9)
19(9)
20(9)
21(10)
22(11)
23(12)
24(13)
240
2
2
1
1
I
1
1
60
0
0
450
150
287
520
62
243
30
0
420
10
8
0
0
0
0
1
6
0
0
690
291
245
195
238
Mean
337
22.5
3952
219.6
SD
62.1
%
6.2
199.5
73.1
Total
of total
1
1
1
54
39
I
1
1
0
0
0
0
0
0
0
0
6
83
249
1
460
46
76.6
8.5
3
0
0
0
0
0
0
2
4
II
5
0
3
0
6
14
5
26
58
64
0
0
0
0
5
485
34.6
62.6
9.0
6
0
0
0
0
0
59
5.4
0.7
1.1
Yes
Total (mm)
124
15
28
41
480
22
477
208
332
750
No
Yes
No
No
Yes
No
75
333
96
18
420
27
25
33
64
754
346
298
195
246
5407
225.3
224.5
35
phase of courtship accounted for 29% of courtship duration (mean=27 mm; SD 36
mm, N=4).
When the female became more receptive, the male began head contact by
rubbing and sliding his head on the female, beginning at the tip of her tail and working
his way to her head. While the male rubbed the female's head, he seemed to focus on
and around the nares of the female, perhaps accomplishing olfactory pheromone
delivery. The female would slow her walk, allowing for female-first tail-straddling
walk. The mean time spent in female-first tail-straddling walk was 10.25 mm (SD=7.6
mm; N=4). While the male straddled the female's tail, she undulated her tail in
response to the male's contact.
From female-first tail-straddling walk, either the male
would break contact and move axially along the female and attempt to cross under her
chin, or else the female curled back towards the male's tail base (forming a circle) and
then the pair entered linear tail-straddling walk. If the male broke contact, he made
several attempts to initiate tail-straddling walk by crossing under the female's chin
while walking forward to give the female an opportunity to step over his tail and enter
tail-straddling walk.
Tail-straddling walk lasted an average of 6.3 mm (SD=2.9 mm) for the two
complete sequences with spermatophore deposition. During tail-straddling walk, the
male would undulate his tail under the female's body. In one sequence, the male turned
back towards the female's head and seemed to tap her nares in a manner similar to that
observed for pheromone delivery in Plethodon shermani (see Figure 1, Rollmann et al.
2000). However, due to the angle of the view, I was unable to confirm if the male made
36
contact with the female's nares. I also did not observe snapping or pulling in P.
angusticlavius. After a short period of linear tail-straddling walk, the male stopped
forward movement and lowered his cloaca onto the substrate and deposited a
spermatophore. Spermatophore deposition consistently took 6 mm. Once the
spermatophore was deposited, the pair moved forward. The female in these two
courtships did not attempt to become inseminated (i.e., in both cases she did not move
her cloaca down onto the spermatophore).
Table 2.3. A summary of the duration (mm) of each phase of courtship for Plethodon
angusticlavius. The phases are separated into seven categories: approach, HC!TW=
head contact and tail wagging, fffSW=female-first tail-straddling walk, ITSW = linear
tail-straddling walk, SD=spermatophore deposition, ST=sperm transfer. See text for a
more detailed description of the courtship phases. The total time of each courtship
sequence is presented (far right column) along with means, standard deviations, and
percent of total of each courtship component. The number in parentheses next to the
sequence number denotes the number of multiple courtship sequences by the same pair
that occurred sequentially on the same night. A blank in the ST column reflects a
courtship without SD.
Plethodon
angusticlavius
Phases of Courtship
Approach
HC/TW
1(1)
2(1)
3(2)
68
64
4
9
3
3
59
4(2)
0
8
21
8
Total
81
Mean
SD
27
35.6
29.0
134
33.5
32.5
41
10.3
48.0
14.7
Sequence
% of total
ffTSW
9
7.6
ITSW SD
6
8
0
3
8
0
6
0
19
12
6.3
2.9
6.8
6
0
4.3
ST
No
No
Total
(mm)
155
10
90
24
279
69
66.7
37
Plethodon richinondi
I observed five initial courtship interactions. Spermatophore deposition only
occurred during courtship encounters that were not recorded on video-tape. These
observations involved two females and two males and included over 27 hours of
courtship (Table 2.4). I describe below the male-female interactions during the
orientation and persuasion courtship stages since sperm transfer behaviors were not
observed.
A male and female were equally likely to initiate contact by approaching the
other. During this stage, the male and female made contact by nudging or head contact.
This phase of courtship was relatively brief lasting on average 5.6 mm (SD=7.4 mm;
N=5).
Most of the courtship interactions occurred during the persuasion stage and
included head contact and tail wagging. On average, this stage lasted 257 mm (SD= 172
mm; N=5) and included behaviors such as male rubbing and sliding, head rubbing, male
and female tail undulations, high amplitude head swinging, joint head swinging, cloacal
nudging, female-first tail-straddling walk, and snapping. A male initiated the head
contact stage by following the female axially along her dorsum. A male began rubbing
and sliding on all parts of the female's body, approaching the female's head last. Once
the male reached the female's head, the female often performed high amplitude head
swinging and, in one courtship, the pair performed joint head swinging. The male
focused his rubbing around the cloacal region of the female. During this stage the male
and female undulated their tails when contact occurred.
Female-first tail-straddling walk also occurred during the persuasion phase of
courtship. A receptive female allowed the male to rub and slide while straddling her
tail. An unreceptive female kept her tail curled away from the male in response to his
rubbing her tail, preventing ffTSW. The male moved forward over her tail until he
reached the tail base. Snapping typically occurred while in this position, although
males infrequently will snap while perpendicular to the female. During female-first
tail-straddling walk, the female sometimes turned back towards the male's tail base and
nudged the male. The male arched and undulated his tail, presumably in an attempt to
persuade the female to enter linear tail-straddling walk. When not in female-first tailstraddling walk, the male occasionally lifted his tail base under the female's chin and
arched and undulated his tail. The female usually broke contact with the male and he
then ceased tail undulations. On average, female-first tail-straddling walk lasted 21 mm
(SD=36 mm; N=5).
DISCUSSION
Courtship behaviors observed in three Plethodon species represent a significant
deviation from the typical courtship pattern previously described for this salamander
genus. The typical pattern involves a linear, tail-straddling walk led by the male, a
pattern found in all major clades of the family (Houck and Arnold 2003). This type of
tail-straddling walk is consistently linear except when the male turns back for
pheromone delivery (e.g., Fig. 1 Rollmann et al. 2000) or turns for obstacle avoidance.
In contrast, the courtship behaviors of Plethodon cinereus, P. angusticlavius, and P.
39
Table 2.4. A summary of the duration (mm) of the phases of courtship for Plethodon
richrnondi. The phases are separated into seven categories: approach, HC/TW= head
contact and tail wagging, fffSW=female-first tail-straddling walk. See text for a more
detailed description of the courtship phases. The total time of each courtship sequence
are presented (far right column) along with means, standard deviations, and percent of
total for each courtship component. Tail-straddling walk, spermatophore deposition,
and sperm transfer were not observed in this species.
Plethodon
richmondi
Sequence
1
2
3
Phases of Courtship
Approach
1
I
1
4
18
5
7
Total
Mean
HC/TW
447
299
377
33
132
ffVSW
84
1
17
2
1
Total (mm)
519
330
577
61
139
28
1288
105
1626
257.6
21
SD
5.6
7.4
171.8
% of total
1.7
79.2
35.9
6.5
325.2
226.6
richmondi all included a novel, female-led tail-straddling walk. Moreover, females in
these species more actively participated in courtship than did females of other
Plethodon species. The females I observed initiated courtship as often as did males,
performed tail undulations or tail wagging, and also approached, nudged, and rubbed
males. Additionally, these females occasionally turned back towards the male while in
female-first tail-straddling walk. Other notable female behaviors seen in at least one of
these species included head swinging (P. cinereus and P. richmondi), cloacal rubbing
(P. cinereus) and pulling (P. richmondi). While small sample sizes in two species, P.
angusticlavius and P. richmondi, provided limited observations, plethodontid
salamanders exhibit highly stereotyped behavior within a species. Consequently, my
results provide strong predictions of female behavior under average conditions.
The female-first tail-straddling walk seen in these three species may represent a
less frequent segue into the male-led linear tail-straddling walk (Figure 2.1), which
typically leads to spermatophore deposition. Such a sequence of behavior happened
when, during female-first tail-straddling walk, the female turned back towards the male
(forming a circle) and placed her chin on the base of his tail. The male then began the
linear male-led tail-straddling walk. I did not observe this complete sequence in each of
the three species, but P. angusticlavius performed this sequence of behaviors and the
courtship led to insemination. Although P. richmondi performed female-first tailstraddling walk, I did not observe the male-led tail-straddling walk or insemination. In
P. cinereus, pairs that performed female-first tail-straddling walk either did not engage
in male-led tail-straddling walk or terminated the female-first tail-straddling walk and
only later entered the male-led tail-straddling walk. However, females in all three
species turned back towards the male during female-first tail-straddling walk. Recent
observations of plethodontids in the genus Aneides also revealed a circular tail-
straddling walk that led to the traditional male-led linear form (Sapp 2002). Based on
the phylogeny of these salanianders (Larson et al. 2003), it seems likely that the
circular form of tail-straddling walk evolved twice within this group (Figure 2.2).
The delivery of pheromones by male Plethodon salamanders typically occurs
during the linear tail-straddling walk (Arnold and Houck 1982, Houck 1986). In these
three species, however, most pheromone delivery occurred during female-first tail-
straddling walk. The male scratched the female's tail from the tip until he arrived at the
base of her tail. In this position, the male snapped (a quick pulling back of his teeth
across her dorsum), which is another type of vaccination pheromone delivery. As a
courtship progressed, the female walked more slowly, allowing for the maintenance of
contact during the female-first tail-straddling walk. In P. angusticlavius, one courtship
interaction included a "slap" from the male as he led tail-straddling walk. Also, P.
richmondi and P. angusticlavius exhibited persistent head-head rubbing, which may
result in olfactory pheromone delivery to the female's nares. The earlier-timed
pheromone delivery in these species may indicate the pheromone's function to persuade
reluctant females to mate suboptimally, perhaps indicative of a sexual conflict over
mating.
In an earlier description of courtship in P. cinereus, Gergits and Jaeger (1990)
observed 10 courtships and four inseminations in the field. They concluded that the
courtship sequence of this species generally followed the pattern of other plethodontid
salamanders. In contrast, my observations show that female behavior in this species is
much more variable than previously thought. The difference between field observations
and my laboratory observations of courtship behaviors may be due to laboratory
artifact. However, studies of agonistic behavior in P. cinereus conducted in the field
and laboratory indicate that laboratory-based inferences are a reasonable representation
of field behavior, at least in P. cinereus (Gergits and Jaeger 1990). Another
explanation for the differences between my observations and those of Gergits and
Jaeger (1990) may be the unusual weather conditions that led to their observation of 10
pI
Figure 2.1. Courtship sequence for P. angusticlavius. This sequence began with
female-first tail-straddling walk (top). The female (lighter animal) turned around
towards the male, formed a circle (bottom right). From this position, the female
entered tail-straddling walk (bottom left). This drawing has been created from
videotaped footage of P. angusticlavius. The depicted courtship led to spermatophore
deposition.
43
coiirtships in one night. Prior to their field observations, multiple weeks of dry
conditions led to limited courtship opportunities. After one rainy day, the density and
activity of salamanders on the surface dramatically increased and the females
apparently were highly receptive. Courtships in which the female is highly receptive
often do not include pheromone delivery (Arnold 1977, Halliday 1990, Houck and
Reagan 1990). Thus, it is not surprising that the courtship observations of Gergits and
Jaeger (1990) did not include pheromone delivery and the associated behaviors of
snapping and female-first tail-straddling walk.
Previous attempts to observe the complete courtship of these species have had
limited success (Arnold 1972). In general, my observations of male behavior in P.
cinereus are in agreement with earlier observations of Arnold (1972). However, the
female behaviors I observed in this species have not been reported previously.
Similarly in P. richmondi, many of my observations are novel, but in general my
observations of male behavior agree with those of Arnold (1972). Interestingly, the
courtship observations of P. vandykei, a western species, reveal some similarities to
those of P. richmondi (Lynch and Wallace 1987). In particular, these are the only two
Plethodon species that exhibit joint head swinging. Similar to my observations and
those of Aneides, female P. vandykei, also have a more active role in courtship than
most plethodontids (Sapp 2002, Lynch and Wallace 1987).
Prior observations of P. angusticlavius were not available, but following my
observations, a colleague observed courtship in a northern population of a closely
related species P. dorsalis, both species are within the P. dorsalis complex (Picard
2005). These observations are in general agreement with those presented here. In
particular, slapping was observed as the mode of pheromone delivery, but occurred
prior to, instead of during, the male-led tail-straddling walk. In addition, female-first
tail-straddling walk was observed, but females did not turn back towards the males.
These populations are now considered to be different species within the P. dorsalis
complex (Highton 1997), perhaps accounting for differences between courtship
behaviors. The courtship of P. welleri, closely related to P. angusticlavius, has been
observed, but the mode of pheromone delivery is still unclear (Organ 1960b, Arnold
1972). Based on observations of pheromone delivery in P. angusticlavius, I would
expect to see olfactory delivery in P. welleri as well.
Documentation of courtship pheromone delivery suggests that most small
eastern Plethodon species have the ancestral delivery mode of "vaccination" while large
eastern Plethodon have a derived mode of olfactory pheromone delivery. My additional
observations of pheromone delivery correspond to the phylogeny established from
molecular data for the eastern Plethodon species (Figure 2.2) (Highton and Larson
1979, Larson et al. 1981, Highton 1991, Mahoney 2001). In particular, P. cinereus and
P. richrnondi are among the vaccinating Plethodon species, and my observations of
scratching, pulling and snapping in these species confirm their more basal placement in
the group. Plethodon angusticlavius lies in an intermediate position on the phylogeny
in that this species is more closely related to large eastern Plethodon species than to
other small eastern Plethodon species. Until now, the method of pheromone delivery in
this intermediate group had been undetermined. My observations suggest that the P.
45
angusticlavius method of pheromone delivery is intermediate between those having
vaccination and olfactory delivery since males perform behaviors associated with both
delivery modes (i.e., rubbing and slapping, respectively). In addition, mental gland
morphology, pre-maxillary teeth, and behavioral observations of slapping prior to linear
tail-straddling walk supports the placement of this species as intermediate between
vaccination and olfactory pheromone delivery (Highton 1962, Coss 1974, Picard 2005).
As compared to other well-studied Plethodon, females of these three species participate
more actively in courtship during the course of this study. The more assertive courtship
behaviors of these females could have social and phylogenetic implications. In
particular, courtship behaviors performed by females of these species may represent a
derived condition, one reflected in salamanders that have a more complex social
structure relative to other conspecifics. Many laboratory and field studies have revealed
such a complex social structure for P. cinereus. Females and males each maintain
territories that often overlap, adults are socially monogamous, both males and females
co-defend territories during the breeding season and females are less aggressive towards
familiar females than towards unfamiliar females (Gabor and Jaeger 1995, Gillette et al.
2000, Lang and Jaeger 2000, Jaegar et al. 2001, Jaeger and Peterson 2002). Male P.
(inereus are more aggressive towards polyandrous females than towards monogamous
females during the courtship and non-courtship season (Jaegar et al. 2002). In addition,
individuals in pairs co-defending territories are not aggressive towards opposite-sex
intruders, suggesting a potential conflict between male and female remating strategies
(Jaeger et al. 2002). If more active female participation in courtship is a derived
jHS
SN
P
IH
f1-TSW
SL
Aneide aeneu
D D
Aneidex/erreus.
E
flaipunciatus.
lugubris
0
0 0
Plethodon
OlE 1E1 lEE 1
(largeeastern)
Plethodon angusticlavius
Plethodon dorsalic
I 0
I
Plethodon welleri
Plethodoncinereus
Plethodon richmondi
I
0
0 IU I0
I0 IU IU
0 0 0
0 U U
0 0 U 0 0
0
IU
i
I
i
I
U U U U U U
Plethodon vandykei
fl
Figure 2.2. Character states of behavioral features mapped onto a partial phylogeny of
Plethodon and the related genus Aneides: P (pulling), I-TSW (linear tail-straddling
walk), jHS (joint head swinging), SN (snapping), SL (slapping), ff-TSW (female-first
tail-straddling walk), fH (female head rubbing), and fHT (female head rubbing and/or
tail wagging). Behaviors are described in the text. Minus sign denotes evolutionary
loss. This tree is based on information in Mahoney (2001) and Larson et al. (2003).
Dark squares denote observed behaviors, gray squares denote hypothesized behavior
presence, and open squares indicate a lack of observations or absence of behavior. Gray
rectangles represent proposed phylogenetic transitions.
condition corresponding to a more complex social structure, I predict that western
species of the genus Aneides would have independently evolved more active female
behaviors (e.g., Sapp 2002) and a complex social structure similar to that found in P.
47
cinereus. Future studies of social and courtship behavior in Aneides could test this
prediction.
My observations of courtship in P. cinereus, P. angusticlavius, and P.
richmondi revealed novel behaviors that have implications for the phylogeny and
sociality of plethodontid salamanders. In particular, the method of pheromone delivery
in P. angusticlavius is behaviorally intermediate between courtship behaviors for
Plethdon species having vaccination or olfactory delivery methods. In addition, more
active female participation in courtship is present in at least one species (P. cinereus)
that has well-documented, complex social structure. Documenting previously unknown
courtship behaviors contributes to a broader foundation of courtship observations in
these salamanders and facilitates the generation of novel, testable predictions. For
example, Plethodon species that are closely related to P. angusticlavius (P. wehrlei, P.
welleri, and P. punctatus) should also perform slapping as a method of pheromone
delivery. Consequently, courtship descriptions enhance our understanding of the
relationships between closely related species and provide insight into the direction and
impetus for behavioral evolution.
CHAPTER 3
MALE COURTSHIP PHEROMONES AND FEMALE RERPRODUCTIVE
SUCCESS
Leslie A. Dyal, Lynne D. Houck, and Amy Picard
ABSTRACT
Male plethodontid salamanders produce a pheromone that is delivered to
females during courtship. Courtship pheromone communication between males and
females provides an opportunity to investigate the costs and benefits of a signaling
system from the perspective of sexual conflict. The courtship pheromone alters a
female's reluctance to mate, leading to courtships that are shorter in duration. If the
pheromone functions to manipulate female mating decisions, it may negatively affect
some aspect of female reproductive success. We examined the consequences of
pheromone delivery on clutch size, egg size, and early mortality in Plethodon shermani.
Our results do not provide conclusive evidence that male courtship pheromones
negatively affect female reproductive success. However, the pheromone appears to
alter clutch size in some females under particular circumstances. In addition, the
pheromone treatment is correlated with higher egg retention in females. Our results
provide incentive for more research on whether the male courtship pheromones mediate
sexual conflicts over mating.
INTRODUCTION
The evolution of signaling systems has long been a popular area of research and
a subject of considerable controversy (Darwin 187!, Huxley 1914, Tinbergen 1959,
Johnstone 1996). The debate centers on the degree of honesty and cheating between
signal senders and receivers (Grafen 1990, Zuk et al. 1990, Guilford and Dawkins 1991,
Dawkins and Guilford 1991, Michod and Hasson 1990, Krebs and Dawkins 1984, Burk
1988). If signals are predominately honest, both the sender and receiver will benefit
from communication. However, if signals are dishonest and manipulative, then the
benefits of communication may predominantly lie with the sender. The concept of
honest or manipulative signals can be examined in communication systems involved
with female choice of mates with exaggerated secondary sexual traits. Understanding
the evolution of signaling between males and females for the purposes of reproduction
requires addressing the theoretical perspectives of both sexual selection and signal
systems (honesty vs. manipulation).
Proponents of the view that signaling systems are predominately honest suggest
that, if the signal is costly, then cheating will be rare. In this scenario, the signal is an
honest indicator of quality due to the costs of producing the signal (Williams 1966,
Trivers 1972, Emlen 1973, Zahavi 1975, Grafen 1990, Kodric-Brown and Brown 1984,
Rich and Hurst 1998). Costs associated with signal production and maintenance ensure
that only individuals of high quality can handle the costs (Zahavi 1975, Clutton-Brock
and Albon 1979, Hamilton and Zuk 1982, Zuk et al. 1990). The honest signaling
perspective focuses mainly on the fitness consequences of the signal on its sender (e.g.,
50
males) (Fisher 1958, Michod and Hasson 1990, Arnold 1983, Kirkpatrick 1982, Lande
1981, Grafen 1990, Kodric-Brown and Brown 1984). The frequency of cheating in
such systems is rare due to the phenotypic constraints associated with signal production
(e.g., Hill 1992, Hill and Montgomerie 1994). While the idea of condition-dependent
costs of signal production seem compelling, the experimental evidence linking
condition-dependent male traits to female mate choice based on mate quality is lacking
in most animal systems (Johnstone 1996, Navara et al. 2001, Mays and Hill 2004, Hill
et al. 2004).
Alternatively, the manipulative or dishonest signaling perspective addresses the
fitness consequences on signal receivers (typically, females) and argues that costs to
receivers are essential to understanding the evolution of communication systems
(Guilford and Dawkins 991). At the very least, receivers incur costs that include the
time spent determining the honesty of a signal, as well as predation and disease risks
arising from proximity of the sender (Krebs and Dawkins 1991). In other words, if the
signal is costly to produce and send, it may also be costly to receive. When receiver
costs are included, the honesty of communication systems becomes questionable and is
likely to be less frequent than expected (Dawkins and Krebs 1978, Krebs and Dawkins
1984). Thus, females may settle for less informative signals that are less costly to
receive, even when reception costs are less than signaling costs. These receiver
dynamics are predicted to lead to a less honest, but more conventional, signaling system
(Guilford and Dawkins 1991).
Conventional signals occur when signals do not reflect an underlying quality in
the signaler (even if the signal is somewhat costly to produce) because of selection
pressures to reduce costs on both signaler and receiver (Dawkins and Guilford 1991).
One common example is prey with warning coloration associated with distastefulness.
The predator learns that prey with warning colors are distasteful (i.e., category
recognition). Asssessing the honesty of this signal is costly to the receiver such that
cheats in the form of Batesian mimics exist in low frequencies (Guilford and Dawkins
1991). Conventional signals may or may not be limited by phenotype, depending on the
costs of cheating, in addition to signal production costs. If cheaters can avoid
punishment, their frequency may increase (Barnard and Burk 1979). In fact, the high
diversity of signals can be attributed to variation in the receivers of conventional signals
(Dawkins and Guilford 1991). Additionally, if receivers benefit from of an accurate
assessment of male quality, selection on receivers would need to be incorporated to
determine the cooperative or conflicting nature of the communication system (Trivers
1972, Clutton-Brock and Vincent 1991, Clutton-Brock and Parker 1992, Johnstone et
al. 1996, Johnstone 1996).
Proponents of the conventional signaling perspective suggest that an honest
indicator of quality in the form of one clear signal would be beneficial to both senders
and receivers. However, most signals have multiple components that provide visual,
tactile, acoustic, and/or olfactory information. Conventional signaling theory predicts
that multi-component signals contain no strategic message about sender quality
(Guilford and Dawkins 1991). Instead, multi-component signals are constrained by
efficiency and are designed to enhance the effectiveness in eliciting the appropriate
receiver response (Johnstone 1996). Communication systems with conventional, multicomponent signals are more likely to be invaded by cheaters.
52
Predictions about dishonest or manipulative signals are similar to those proposed
by sexual dialectics theory, which addresses conflicts over mating decisions (Gowaty
1997). Sexual dialectics suggests that a conflict between discriminating females and
non-preferred males will result in sexually dimorphic traits in males that function to
manipulate or coerce female mating decisions. The main assumption of the theory is
that freely expressed female mating preferences are adaptive, such that female
reproductive success is higher when mating is under female control than when coerced
or manipulated by males. Sexual dialectics theory predicts that a signal that alters a
female's mating decisions could negatively affect female reproductive success and/or
offspring fitness. This is a key point. Thus, the most direct method of distinguishing
between the alternative theories of the evolution of animal communication systems lies
in the fitness consequences to signal receivers (Guilford and Dawkins 1991, Dawkins
and Guilford 1991, Krebs and Dawkins 1984, Gowaty 1997).
Plethodontid salamanders have a sexual communication system that permits
examination of the fitness consequences of signal reception. During the breeding
season, a male produces a multi-component pheromone cocktail from a seasonally
enlarged gland under its chin (I-buck and Sever 1994, Feldhoff et al. 1999, Rollmann et
al. 2000). The courtship chemical signal is delivered only after courtship has begun
(Arnold 1977, Houck 1986). This direct male-to-female delivery within the context of
courtship means that there are no unintended receivers of the pheromone signal. Males
within a population vary significantly in the relative composition of pheromone
components (Rollmann et al. 2000). This variation is a means by which a female might
prefer one male over others. Experimental delivery of pheromone to females decreases
53
the length of courtship, interpreted as increasing female receptivity (Houck and Reagan
1990, Rollmann et al. 1999). Alternatively, sexual dialectics and conventional signaling
theories would suggest that the pheromone could manipulate female mating decisions.
Based on the theories discussed above, the chemical communication system of
plethodontids may be one that is vulnerable to dishonest or manipulative signaling for
several reasons. First, signal production is limited to the breeding season so that any
costs of signal production are limited in time, which would make cheating easier.
Second, females selecting mates would only discriminate against a subset of males, so
that the number of males under selection to cheat would be smaller than the total
number of males in the population. In addition, if females vary in their preferences,
then the pool of non-preferred males would not be fixed. As a consequence, honest
signaling males would predominate over cheaters, preventing the collapse of the
communication system. The frequency of courtships that lack pheromone delivery, but
contain visual and tactile signals (Arnold 1976), supports the idea that some males are
successful without attempts to alter female receptivity to mating. The lack of
pheromone delivery may occur because these particular females are initially receptive
to mating with preferred males. In addition, the pheromone signal itself has multiple
components that are highly variable and often combined with tactile cues. Thus, this
pheromone signal is likely designed for effectiveness in eliciting the preferred responses
from receivers (e.g., receptivity to mating) (Krebs and Dawkins 1984, Guilford and
Dawkins 1991). Finally, the cost of producing the pheromone is likely to be small due
to the seasonal constraints on production relative to benefits in terms of increased
mating success experienced by males that use the pheromone.
54
The male courtship pheromone of plethodontid salamanders may manipulate
female mating decisions. Accordingly, we experimentally addressed the fitness
consequences of signal reception in terms of female reproductive success in Plethodon
sherinani. The main goal was to distinguish between the alternative functions of the
pheromone signaling system. If the courtship pheromone functions as a dishonest
signal that manipulates female mating decisions due to a sexual conflict over mating,
then female reproduction should be negatively affected. On the other hand, if the
pheromone is an honest signal of male quality, then it should be beneficial to both males
and females or at least neutral in its effects on female reproductive success. We
measured clutch size, egg size, egg retention, and early mortality to assess whether
pheromone delivery affected female reproductive success.
METHODS
Adult male and female P. shermani were collected from a single site in Macon
County, North Carolina, USA, during August and early September in 2001,
2003.
2002,
and
The year of collection was used to refer to results for each year, although
treatment results were measured in the following January or February. Females were
determined to be gravid by the presence of enlarged ova, visible through the body wall.
Each male had an enlarged mental gland, known to produce courtship pheromones.
Following collection, all salamanders were transported to Oregon State University
(Corvallis, OR) and housed in a climate-controlled room at
13
16° C
photoperiod. Animals were housed individually in plastic shoeboxes
with a natural
(9x13x17 cm).
Boxes were lined with moist paper towels and several moist, crumpled paper towels
55
were added to provide cover. Each salamander was fed two waxworms (Galleria
mellonella) once per week.
Total sample sizes for control and experimental treatments were: 29, 72, and 76
females in 2001, 2002, and 2003, respectively. Beginning in August and ending in mid
September, each female was repeatedly paired with a randomly selected male until
insemination was confirmed by observation of spermatophore deposition and sperm
transfer (from one to five pairings) beginning in August and ending in mid-September.
In 2002, each female that mated more than once was inseminated by the same male
during subsequent matings, thus decreasing the potential for multiple paternity within a
clutch. The size measurements of all males in 2002 were collected as a covariate in the
analysis of factors influencing fecundity. Additionally, the females from 2003 were not
randomly chosen. Females were selected in 2003 such that there were relatively similar
numbers of females distributed across the size range. This selection procedure was
inspired by results from 2002, in which a relatively truncated distribution of females
may have affected the assessment of treatment effects.
We prepared a pooled whole pheromone extract by anesthetizing males in 4%
ether and excising their mental glands. Glands were placed in a vial containing a
solution of 0.8 mM acetylcholine chloride in amphibian Ringer's solution for 30 mm.
Glands were kept on ice for 30 mm, after which the gland was removed and the solution
containing pheromone components was centrifuged (14,000 g) for 10 mm at 4 °C. The
pooled extract was frozen at 80°C. The pheromone solution was later processed in the
laboratory of Richard Feldhoff (Univ. of Louisville School of Medicine, Louisville,
KY), as described in Rollmann etal. (1999). This processing involved removal of the
56
acetylcholine chloride, and standardizing the protein content of multiple aliquots that
were subsequently thawed and used for treating females.
Pheromone treatment began towards the end of the breeding season (November
5 in 2001 and in 2002, November 13 in 2003). Females were randomly assigned to a
treatment type (pheromone or saline control), with the condition that both treatment
types were represented in approximately equal numbers. A treatment was applied to
each female in the box in which it was housed. Males were not present during the
experiment to ensure that male behavior would not influence the outcome. Pheromone
or saline was applied to a female's nares using a pipette. A treatment solution was
delivered every other day for one hour. During this hour, a female received the
appropriate solution at 10-minute intervals. In 2001 and 2002, females received
treatments during the first two weeks of November (5 drops of 3tL for each of 5 days
for a total of 751iL per female). Due to limited availability of pheromone in 2003,
pheromone delivery was altered to the following scheme (each delivery occurring
during one hour): Day 1 = 3 drops of 3tL, Day 2 = 3 drops of 3tL, Day 3 = 4 drops of
3tL, Day 4 = drops of 3tL, Day 5 = 5 drops of 3tL (total volume of pheromone or
saline was 57jiL per female).
Female P.
shermani
typically do not oviposit spontaneously in the laboratory, so
each female was injected with gonadotropin (human pregnancy urine) every third day,
beginning in mid-December, until oviposition occurred. If a female failed to oviposit
by the
6Ih
injection, she was excluded from subsequent analyses. Females that failed to
oviposit included 9, 12, and 10 females in 2001, 2002, and 2003, respectively. Sample
sizes reported in Table 3. 1 exclude females that did not oviposit. Injections occurred
57
between December 11 and 20 of each year. Gonadotropin (1mg) was diluted with 2mL
of Ringers solution (500m1 DI water, 2.98g NaCL, 0.19g KCI, 0.12g CaCI). Eggs were
collected from each female immediately following oviposition. Eggs were counted and
yolk diameters were measured at the widest point using digital calipers. The
relationship between female size and clutch size for treatment groups in 2002 suggested
that treatment differences might affect the number of ova that were oviposited, with
some females retaining more ova than did others. Accordingly, in the protocol for
2003, retained ova (visible through the body wall) were counted in addition to the
number of eggs actually oviposited. In 2001 and 2002, each clutch was maintained in a
separate jar under a water drip system (constant water movement minimized fungal
growth). Clutches were recounted once per week for the first three weeks to determine
if the treatment affected mortality rates during this time.
Clutch size and mean egg size for each clutch were analyzed using linear
regression (JMP by SAS), beginning with a model that included all factors believed to
potentially influence these measures. Factors were dropped from the regression model
if the p-value was greater than 0.05. The explanatory variables initially included female
snout-vent length (SVL), male SVL, treatment (pheromone or saline), and the pairwise
interactions between these variables. Early estimates for mortality (collected in 2002)
and the proportion of eggs retained after oviposition (collected in 2003) were compared
between treatments with logistic regression (alpha = 0.05).
58
RESULTS
Measures of female body size (SVL), clutch size, and egg size are summarized
in Table 3.1 for females in each treatment group. For all years combined, the mean
female size was 62.49mm in SVL (range = 52 74mm), the mean clutch size was 18
eggs (range = 10- 38 eggs), and the mean egg size was 4.3mm in diameter (range = 3.5
5.1mm).
Table 3.1. A summary of results for each year of the study for Plethodon shermani.
Means were calculated over both treatment groups (0) and separately for each treatment
group (P=pheromone; S=saline). All SVL data is in mm. Egg size was measured at the
widest diameter in mm.
Year
2001
n=20
n=59
2002
2003
n=75
0
P
S
0
P
S
o
P
s
63.2
63.1
63.3
61.8
62.6
61.0
62.5
62.0
63
Largest
Female SVL
70.3
69.0
70.3
71.8
67.8
71.8
74.0
71.0
74.0
Smallest
Female SVL
56.9
59.6
56.9
52.6
58.9
52.6
52.0
52.0
54.0
Mean Clutch
Size
15
15
15
19
19
19
21
21
20.
4.00
4.01
3.99
4.62
4.64
4.59
na
na
na
Mean Female
SVL
Mean Egg
59
Clutch Size
In 2001, there was no correlation between female SVL and clutch size, and no
treatment effects on clutch size. In 2002, the sample size was increased (from 20
females in 2001) to 59 females. There was a significant positive relationship between
Table 3.2. Linear regression anlysis of Plethodon shermani clutch size. The
explanatory variables are female snout-vent length (SVL), treatment (saline or
pheromone), and the interaction between female snout-vent length and treatment.
YEAR
2001 (n=20)
2002 (n=59)
2003 (n=75)
Variable
Coefficient
SE
t-stat
p-value
SVL
0.39
0.41
-0.94
0.362
Treatment
-0.33
1.41
-0.24
0.81
SVLxTreatment
-0.19
0.41
-0.46
0.65
SVL
1.073
0.223
4.81
<0.0001 *
Treatment
55.584
24.623
2.26
0.028 *
SVLxTreatment
-0.909
0.396
-2.29
0.0257 *
SVL
0.4997
0.158
3.17
0.0022*
Treatment
0.2703
13.59
0.02
0.984
SVLxTreatment
0.0167
0.2168
0.08
0.939
I
In particular, the female SVL coefficient was 1.07 and 0.97 with and without two
females, respectively, and the p-value for this variable went from 0.003 to 0.019.
Similarly, the p-values for treatment and the interaction between treatment and female
SVL were 0.028 and 0.026 with all females, and 0.095 and 0.093 when these females
were omitted. In addition, the overall variance explained by the regression model
decreased from 26% to 10% when the smallest and largest females were excluded (p-
value increased from 0.0002 to 0.04). In 2002, sire SVL was not correlated with clutch
size.
The 2003 data were analyzed separately due to differences in female selection
criteria and quantity of pheromone available for treatment. In 2003, there was a
significant positive relationship between female SVL and clutch size (Figure 3.2).
However, there was no significant treatment effect on clutch size. Overall, females
retained from 1-9 eggs (mean 2.7 eggs). Pheromone-treated females retained
proportionally more ova than did saline-treated females (t-ratio=-2.39; p-value=0.02;
Figure 3.3). The number and size of pheromone-treated and saline-treated females that
retained eggs was not significantly different. Of females that retained ova, pheromonetreated females averaged 60.9mm in SVL (n = 20) and oviposited 19.5 eggs and saline-
treated females averaged 62.1mm (n = 13) and laid 18.6 eggs. Females (n = 15
pheromone-treated, n = 13 saline-treated) that oviposited an entire clutch averaged
64mm in SVL and oviposited an average of 22 eggs.
62
40
35
A
S
30
w 25
-
N
.5 20
15
10
5
0
50
55
60
65
70
75
Female SVL
Figure 3.2. Relationship between female SVL (mm) and clutch size (number of eggs)
for Plethodon shermani in 2003. The dark circles represent the pheromone-treated
females and light triangles represent saline-treated females. The solid line represents the
group of females receiving pheromone treatment. The dotted line represents the
females receiving saline.
800
F,
700
600
500
400
300
200
100
0
Treat
Figure 3.3. The total number of eggs retained by female Plethodon shermani after
oviposition in each treatment group in 2003. Pheromone-treated females retained a
significantly larger proportion of eggs than did saline-treated females (t-ratio=-2.39, p =
63
0.02, df=42, N=46). Lighter bars represent the total number of eggs oviposited and the
dark bars represent retained ova.
Egg Size
In 2001, there was no significant effect of any explanatory variable on egg size,
presumably due to high variation and low sample sizes. In 2002, there was no
significant treatment effect. However, female size was positively correlated with mean
egg size (effect estimate = 0.029, p
0.0009). In addition, clutch size was negatively
correlated with mean egg size (effect estimate
0.014, p = 0.0083). In 2002, male
SVL was not correlated with egg size. In 2003, we did not measure egg sizes due to
lack of treatment effects in a previous experiment.
Early Mortality
We monitored early mortality in 2001 and 2002. Early mortality was not related to any
explanatory variable.
DISCUSSION
Tests for the influence of male courtship pheromones on measures of female
reproductive success revealed different effects in different years. In 2002, smaller
females treated with pheromone had an increased clutch size, while larger females had a
smaller clutch size. This effect is puzzling in that there is no obvious physiological
mechanism that would account for differential effects due to female body size. The
pheromone is detected by the vomeronasal system (Wirsig-Wiechmann et al., 2002)
and there is no reason to think that smaller females have a different response to
vomeronasal stimulation than would larger females It is possible that the observed
pheromone effect is spurious because, a few "outlier" females representing the tails of
the female size distribution may have substantially affected the results.
In contrast, the 2003 data documented a lack of pheromone effect on clutch size.
The amount of pheromone delivered to each female was lower than that in the 2002
experiment. However, the 57 tL delivered to each female was likely a substantial
increase over amounts normally delivered by males during the mating season. While it
is possible that multiple paternity in 2003 altered within-clutch variation, there is no
evidence in plethdontid salamanders that males influence clutch size (no significant
effect of male body size on any results in 2002). While there was no treatment effect on
clutch size in 2003, pheromone-treated females retained more eggs than did saline-
treated females. It is possible that the decrease in clutch size observed for larger,
pheromone-treated females in 2002 resulted from egg retention, similar to that found in
2003.
The lack of correlation in 2001 between female SVL and clutch size was
unusual because a positive correlation typically is seen in plethodontid salamanders
(Houck 1977a, b). Clutch size is highly variable in plethodontid salamanders, however,
such that there can be a wide range of clutch size for females of a given SVL (Houck
1977a, b). Thus, small sample size (N=20 females) and relatively narrow range of SVL
probably precluded seeing any positive correlation between female SVL and clutch size,
as well as any effect of pheromone treatment. However, after combining 2001 and
2002 data for analysis, we found the same significant interaction between female size
and pheromone treatment, similar to 2002 alone, but slightly higher p-values (combined
analysis not included). A significant interaction between year of the study and female
size in addition to a year effect on clutch size suggests that these years are different and
should be analyzed separately. Larger sample sizes in 2002 and 2003 facilitated the
demonstration of the expected female SVL and clutch size relationship. The positive
correlation between female size and clutch size was consistent with that found in other
plethodontid studies that had a sufficient sample size and range of female SVL (Houck
1977a, b).
Drawing conclusions based on sexual conflict theory is difficult due to the
inconsistency of results. If the male courtship pheromone functions to manipulate or
coerce female mating decisions, then we would expect it to negatively affect female
reproductive success. In fact, the pheromone did alter clutch size and egg retention for
particular females in particular years. It may be that a female can alter the number of
eggs oviposited in response to receiving pheromone due to perceived suboptimal mating
with non-preferred males. Evidence that females can alter clutch characteristics relative
to her or her mate's condition and quality has been found in many taxa (Blaxter 1986,
Rothchild 1986, Bailey and Houde 1989, Chambers et al. 1989, Kaplan 1992, Leggett
and Deblois 1994, Clark and Galef 1995, Parichy and Kaplan 1995, Gil et al. 1999,
Olsson et al. 2002, Rutkowska and Cichon 2002, Pilz et al. 2003). Based on the results
of this study, the possibility that male courtship pheromones function in the context of
sexual conflict warrants further investigation.
66
CHAPTER 4
FEMALES BENEFIT FROM FREELY EXPRESSED MATE CHOICE IN THE
WESTERN TOAD (BUFO BOREAS)
Leslie A. Dyal
ABSTRACT
The fitness benefits of freely expressed female mate choice are assumed to drive
sexual conflict over mating, also termed sexual dialectics (Gowaty 1997). I tested this
assumption in two populations of the Western Toad,
Bufo boreas
because male-male
scramble competition and forceful, indiscriminate amplexus by males seem to preclude
the opportunity for a female to exercise her mating preferences. I asked whether female
toads that mate with preferred males benefit in terms of offspring survivorship and size
at hatching. Females mated to preferred males had a higher proportion of offspring
surviving to hatching at one population. In addition, females mated to preferred males
had longer tadpoles at hatching than did females that mated to non-preferred males at
both populations. My results provide the first evidence in amphibians, anurans in
particular, that freely expressed female mate choice is selectively advantageous. I
discuss these results in terms of female resistance behavior and consequent mating
system patterns.
INTRODUCTION
The theories on the evolution of female mate choice have undergone
considerable transformation since they were recognized as one mechanism of sexual
selection explaining the evolution of elaborate male traits (Darwin 1871, review in
Andersson 1994). Darwin also proposed male-male competition as another force of
sexual selection on males. The initial focus of both mechanisms was on how sexual
selection affected male traits. The evolution of female preferences, however, was not
considered in depth until Fisher's 1958 publication. Fisher argued that female
preferences could be genetically correlated to the male traits that were preferred. Thus,
the evolution of female preference could be driven by selection on males (Lande 1980,
Kirkpatrick 1982). Over time, other work suggested that selection on female
preferences might indirectly benefit females through genetic benefits to their offspring
from mating with particular males (Fisher 1915; Williams 1966; Trivers 1972; Emlen
1973; Zahavi 1975; Partridge 1980, 1983; Hamilton and Zuk 1982; Kodric-Brown and
Brown 1984; Andersson 1986; Pomiankowski 1988; Grafen 1990a,b; Iwasa etal. 1991;
review in Andersson 1994). Studies also have suggested that female mate choice may
be beneficial through non-genetic effects on female survival and fecundity (review in
Andersson 1994). Most recently, the development of sexual dialectics theory suggests
that female choice is adaptive through direct effects on female reproductive success and
indirectly by accruing fitness benefits for offspring (Gowaty 1997).
The main assumption of sexual dialectics theory is that a female mated with a
male of her choice will benefit more than if she mated with a non-preferred male
(Gowaty 1997). If females benefit from freely expressed mate choice, then male-female
interactions that result in females mating with a non-preferred male will reflect sexual
conflict. The behavioral manifestations of this conflict between potential mates, also
termed sexual dialectics, results from selection acting on non-preferred males to
68
manipulate or coerce females' mating decisions. In turn, selection will favor females
that are able to resist manipulation, if these females are better off, in terms of
reproductive success and/or offspring viability, when mating decisions are under their
own control. As a consequence, mating system variation, both within and among
species, should be a function of females' abilities to resist direct and indirect attempts of
manipulation or coercion (see Gowaty 1997).
Recent research has revealed the importance of sexual conflicts over mating
decisions. In certain species, conflicts over mating are critical in determining many
significant factors: individual fitness, selection on males and females, physiological,
behavioral, and morphological evolution, mating system variation and speciation (e.g.,
Rowe et al. 1994, Parker and Partridge 1998, Arnqvist et al. 2000, Drickamer et al.
2000, Moore et al. 2001, Arnqvist and Rowe 2002,). Studies of invertebrates, in
particular, have contributed significantly to our understanding of sexual conflicts (Rice
1996, Finke 1997, Holland and Rice 1999, Arnqvist et al. 2000, Shukar and Day 2001,
Soulier-Perkins 2001, Blankenhorn et al. 2002, Miller 2003). In water striders
(Aquarius sp., Gerris sp.),
planthoppers (Lophopidae), and diving beetles (Dystiscidae),
for example, independently established phylogenetic patterns reveal the order and
alternation of traits that are related to sexual conflict over mating (Rowe et al. 1994,
Soulier-Perkins, 2001, Miller 2003).
Water striders, in particular, are one of the most studied systems in terms of
sexual conflict over mating. The focus on these insects can be attributed to the visually
obvious conflict over mating as seen in pre- and post- mating struggles between males
and females (Rowe et al. 1994, Watson et al. 1998, Ortigosa and Rowe 2003). Research
on mating conflicts in water striders has focused on the costs and benefits to each sex,
environmental and social factors influencing conflict outcomes, behavioral and
morphological evolution, and phylogenetics (Fairbairn 1990, Rowe 1992, Arnqvist and
Rowe 1995, Watson et al. 1998, Arnqvist and Danielsson 1999, Fairbairn et al. 2003).
In the water strider Gerris buenoi, mating is costly to a female in terms of slower
skating speed due to carrying a male (Rowe 1992). The slower speed leads to decreased
foraging efficiency and increased risk of predation. Consequently, females frequently
are reluctant to mate and this reluctance leads to pre-mating struggles (Rowe et al.
1994). However, when the sex ratio is highly skewed towards males, a female is more
likely to accept a mate to offset the costs of repeated harassment by males while being
single. The results of manipulating social and environmental variables (density, sex
ratio) follow the prediction of increased female reluctance as a function of declining
male harassment rate (Rowe 1992). In another water strider, G. odontogaster, males
have enlarged clasping appendages that function to overcome female resistance during
pre-mating struggles (Arnqvist 1989, Andersen 1991). Female resistance biases mating
frequency according to male clasper size (Arnqvist 1992). Traditionally, clasper size
would be viewed as a trait under sexual selection by female mate choice, even though
clasper size is driven by sexual conflict and female resistance behavior. In addition,
other components of male size appear to be related to the ability to subdue females,
resulting in nonrandom mating patterns by body size and leg size in several other water
strider species (Rubenstein 1984, J-Jayashi 1985, Fairbairn 1988, 1990, Kaitala and
Dingle 1993, Preziosi and Fairbairn 2000).
70
Studies of sexual conflict are increasing, but the importance and taxonomic
distribution of sexual conflicts is unclear (Gowaty 1997, Chapman et al. 2003, CordobaAguiler and Contreras-Garduno 2003, Eberbard and Cordero 2003). We need concise
and well-controlled experiments that allow us to differentiate between traditional forms
of sexual selection (e.g., unhindered mate choice) and sexual conflict in a broader
variety of taxa (Chapman et al. 2003, Cordoba-Aguiler and Contreras-Garduno 2003,
Eberbard and Cordero 2003). The distinction between these selective forces hinges on
determining the fitness consequences of mating for females and their offspring (Watson
et al. 1998, Chapman et al. 2003, Cordoba-Aguilar and Contreras-Garduno 2003,
Eberhard and Cordero 2003, Pizarri and Snook 2003). In particular, the confounding
effects of potential benefits associated with male traits (versus the benefits from free
choice) should be controlled without a priori assumptions about traits in males that
females may use to differentiate mates (Gowaty 1997, Drickamer et al. 2000). Other
important factors such as male-male competition, male mate choice, male-female
competition, and male coercion of females need to be experimentally controlled or
eliminated if these elements constrain freely expressed female choice (Zuk et al. 1990,
Gowaty 1997, Drickamer et al. 2000).
Although research on sexual conflict has increased in the past several years,
amphibians are a group that has been overlooked (but see Roesli and Reyer 2000).
Anurans, in particular, provide an opportunity to assess fitness consequences of freely
expressed female choice while limiting other selective events that may potentially
influence reproductive success and offspring viability. Anurans typically have external
fertilization, so factors that may alter reproductive success such as sperm competition
71
and cryptic female choice are absent or limited. Additionally, in most North American
anurans, parental care is not provided by either sex.
Aspects of the mating system of the Western toad
(Bufo boreas)
make this
species a promising candidate for studies of sexual conflict. While many anuran males
display and chorus, the Western toad does not. In non-chorusing, non-displaying
anurans, there are no obvious traits that females may use to differentiate males, except
for male body size and clasping vigor. Western toads are explosive breeders with a
very limited breeding season (Olson 1988). Consequently, the likelihood of sexual
conflicts over mating seem high due to male-male scramble competition and male
clasping behaviors (discussed below) that preclude females' abilities to exercise their
preferences. In this sense, the Western toad mating system is similar to other systems in
which sexual conflicts over mating have been documented (e.g., water striders-Rowe
1994, seaweed flies-Shukar and Day 2001, Crean and Gilburn 1998, and mosquito fishWatson et al. 1998).
One aspect of the mating system that likely contributes to sexual conflict is that
male
B. boreas
amplex other individuals vigorously and indiscriminately, without prior
courtship behavior. Males appear to exhibit preference for larger individuals, whether
male, female, dead, alive, or inanimate floating objects (Marco et al. 1998, pers. ohs.).
This preference presumably increases the likelihood of amplexing a female, as most
adult females are larger than males (Marco et al. 1998, see Figure 4.1). Amplexus is
prolonged (longer than required for oviposition) and resembles pre-copulatory mate
guarding in arthropods (e.g., water striders Rowe et al. 1994, seaweed flies
and Day 2001, amphipods
Shukar
Cothran 2004). No direct investigations of female mating
72
preferences have been made because male-male competition and a highly male biased
sex ratio seem to preclude the opportunity for female mate choice.
160
140
.
.
120
100
40
(0
80
60
.
55
S
40
S $S
S
S
20
0
5
5 5
6
6.5
7
7.5
8
8.5
9
9.5
SUL
Figure 4.1. The relative size of male and female Western toads represented as the
relation between snout-urostyle length and mass. Females (large diamonds) generally
are larger than males (small circles), although there is overlap between large males and
small females in this population at Little Three Creeks Lake.
Previous research with
B. boreas
confirmed that females can influence male
clasping success, and that females are not passive during the clasping process,
appearing to avoid encounters (Olson 1988). In order to evade an approaching male, a
female B.
boreas
can exhibit a suite of behaviors, including kicking, diving, swimming
away, and retracting (slight backward movement on the water surface) when
approached by a male (Olson 1988). In experimental closures at the natural breeding
site, male clasping success varied depending on which behavior the female performed
73
(Olson 1988). For example, when a female dived away from a male, amplexus was less
frequent (28.9%). However, retracting from the male often led to amplexus (64.3%).
In experimental enclosures, the consequences of female behavior during an encounter
were dependent on male size (Olson 1988). Successful clasps by small males were
frequently associated with female retractions, whereas large males were more
successful when females responded to the encounter by diving, swimming, or kicking.
Also, most females initially attempted to dislodge males when amplexed (pers. obs.).
These behaviors may be attempts by females to express mate choice.
In this paper, I test the main assumption of sexual dialectics theory that freely
expressed female choice is selectively advantageous to female Western toads.
Specifically, females that mate with preferred males should benefit in terms of higher
reproductive success as compared to females that mate with non-preferred males. In
measuring this selective advantage, care must be taken so the investigator does not bias
a female's options (Gowaty 1997, Drickamer 2000). A bias could be introduced, for
example, by only offering females a choice between toads that differed greatly in body
mass. The assumption here is that a female selects a male based on phenotypic
attributes, which may include traits (e.g., odor) not easily measured by the investigator.
To test the sexual dialectics assumption, therefore, I first evaluated female preferences
using randomly selected males. I then staged matings between females and preferred or
non-preferred males. For all mating pairs, I recorded offspring survivorship to hatching
(a measure of reproductive success) and average size at hatching (a measure of
offspring viability).
74
METHODS
Study Species
I studied two populations of B. boreas: Little Three Creeks Lake (Deschutes
County, elevation 2000m) and Todd Lake (Deschutes County, 1 860m) in the Cascade
Range of Oregon during 2003. The length of the breeding season ranged from 5 - 15
consecutive days and occurred between May and July. Breeding population size at
Little Three Creeks Lake ranged from 260-438 individuals and at Todd Lake from 131
to 497 individuals. Over the breeding season, the sex ratio of males to females ranged
from 2.5 to 3.4 at Little Three Creeks Lake and 0.72 to 2.25 at Todd Lake (Olson 1988).
However, on any day during breeding, there can be up to 9.5 males per female at Little
Three Creeks Lake and 3.3 males per female at Todd Lake (personal obs., Olson 1988).
Both populations had significant large male mating advantages (larger males experience
increased mating success, Olson 1988). However, the pattern was not consistently
significant across days or years. Futhermore, the apparent size assortative mating
pattern was not related to fertilization success, as is the case in other anurans (Olson et
al. 1986, Olson 1988). Oviposition is communal, usually occurring in an area
approximately Sm x Sm.
Female Choice Trials
At each lake, animals were collected from the periphery of the oviposition site to
minimize disturbance of the breeding aggregation. I captured gravid females in
75
amplexus, removed the male, and immediately placed the female in a choice enclosure
(described below) to acclimate. The original male was measured and released. Two
single males were randomly collected from the aggregation and assigned as mate choice
candidates. Trials were conducted one at a time to decrease handling stress. By
selecting males at random, I did not bias the male phenotypic traits that characterized
the mate choice candidates. As the focus was on the benefits of choice, not on a
particular male trait (such as body size), it was necessary to eliminate potentially
confounding variables by randomization.
The choice enclosure consisted of a PVC frame (150 x 50 x 100cm) covered
with nylon mesh (see Figure 1). The enclosure had three compartments created by mesh
dividers. One male was placed in each of the two end compartments and the female
was allowed to move freely throughout the center compartment. The female could
smell, hear, and make some tactile contact with each male. The divisions within each
enclosure excluded other forms of reproductive competition, such as male-male
competition, male-female competition, male mate choice, and male coercion of females
(cfZuk et al. 1990). Each trial consisted of a 10-minute focal period. Females used in
the experiment met the following criteria for choice: (a) the female spent at least 300
seconds inspecting males (i.e., the female was < 15 cm from a male) and (b)
60% of
the inspection time was spent in close proximity (<15 cm) to one male, defined as the
preferred male. Twenty-two trials were conducted at Little Three Creeks Lake and 18
trials at Todd Lake. All 40 females met both criteria. In all trials, I recorded standard
measurements for each individual, snout-urostyle length and body mass. Trials were
conducted at peak breeding activity time, between 1000 and 1800 hr.
76
Figure 4.2. Schematic of female choice enclosures (150 x 50 x 100 cm). The dash-dot
lines represent the mesh divisions within the enclosure. The dotted lines represent the
proximity threshold (15cm) for demarking female preference. The female's head must
cross this line to be considered in close proximity to a male.
Mating Pair Manipulations
Once a female met the choice criteria described above, she was paired with a
male and allowed to oviposit. The type of male was assigned such that each female was
paired with either the preferred or non-preferred male from her trial. On a given day, a
coin flip determined whether the first female to be paired was assigned the preferred or
non-preferred treatment. The remaining treatment assignments on that day alternated to
assure equal sample sizes in each treatment group. Each pair was placed in an
oviposition enclosure 5-lOm from the oviposition site. These oviposition enclosures
were mesh baskets suspended from a Styrofoam flotation frame (individual baskets: 20
x 30 x 30cm). All paired females oviposited within 48 hours, except for one female in a
non-preferred mating. This pair was released into the lake and no data from this pair
was included in the analysis of treatment effects. Toads were released from the
77
enclosures immediately following the completion of oviposition, as indicated by the
pair no longer being in amplexus.
Survivorship and Hatching Size
Following oviposition, I randomly collected a subset of eggs from each clutch
due to logistical problems counting entire clutches that usually contain 12,000 eggs.
Depending on location, the subset consisted of 200-300 eggs per mating pair. Eggs
from each pair were placed in Styrofoam-framed, floating mesh baskets (similar to
oviposition enclosures) at the main oviposition site. Embryos were reared in these
enclosures. During the embryonic period, I closely monitored the enclosures until
hatching. Eggs from all clutches were reared at the same depth of water, equidistant
from the shore, and the mesh enclosure allowed water flow between clutches. I recorded
survivorship to hatching and hatching size, measured as total length. I released all
tadpoles at the oviposition sites after hatching to minimize disturbance to the breeding
population. As toad clutches contain about 12,000 eggs and mortality is usually high,
survivorship to hatching may be a better estimate of female reproductive success than is
the number of eggs laid for this species.
Statistical Analyses
From each study population, I obtained two sets of dependent variables for
preferred and non-preferred matings, survivorship to hatching and size at hatching.
I
averaged tadpole size for each mated pair (N21 pairs at Little Three Creeks Lake and
N=l 8 pairs at Todd Lake). I analyzed the body size data (tadpole body length) with
linear regression, including treatment (preferred or non-preferred) and
parental size as
covariates. I used logistic regression to analyze treatment effects on the proportion of
tadpoles that survived until hatching.
RESULTS
Reproductive Success: survivorship to hatching
There was no significant difference in the percentage of eggs surviving to
hatching between preferred (3 9%) and non-preferred (42%) matings at Little Three
Creeks Lake (t-ratio-O.21, pO.84, df
19, N21; Table 4.1; Figure 4.3). However,
the percentage of eggs surviving to hatching at Todd Lake was significantly higher in
preferred matings (61%) than in non-preferred (33%) matings (t-ratio-3.Ol, p = 0.009,
df 16, N18; Table 4.2; Figure 4.3). Parental size did not explain
a significant
amount of variation in survivorship at either population. Overall, mortality was higher
at Little Three Creeks Lake. The source of mortality is unknown, although spotted
sandpipers (Actitus macularia) were regularly seen perched on my floating enclosures.
78
79
Table 4.1. Clutch data for Western toads from Little Three Creeks Lake. The total
number of eggs sampled and number of tadpoles present at hatching are given for
preferred and non-preferred matings.
Initial Egg
No.
Clutch
No.
Hatched
1
315
315
328
308
309
342
358
339
350
309
348
330
338
358
334
434
313
356
339
351
315
81
2
3
4
5
6
7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
PAvg
NP Avg
3787
3302
96
328
60
179
216
210
137
136
193
81
86
215
224
115
99
299
6
2
7
84
1548
1304
Proportion
Hatched Treatment
0.26
p
0.30
np
1.00
p
0.19
np
0.58
p
0.63
np
0.59
p
0.40
p
0.39
np
0.62
p
0.23
np
0.26
p
0.64
np
0.63
p
0.34
np
0.22
p
0.96
np
0.02
p
0.01
np
0.02
p
0.27
np
0.42
0.39
p
np
80
Table 4.2. Clutch data for Western toads from Todd Lake. The total number ofeggs
sampled and number of tadpoles present at hatching are given for preferred and nonpreferred matings.
Initial Egg
No.
Clutch
No.
1
227
232
249
258
245
238
255
257
239
234
219
231
244
233
211
226
237
214
Hatched
114
112
201
77
108
97
172
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
PAvg
NP Avg
2126
2123
14
131
102
206
45
28
141
199
82
116
23
1023
827
Proportion
Hatched Treatment
0.50
p
0.48
np
0.81
p
0.30
np
0.44
p
0.41
np
0.67
p
0.05
np
0.55
p
0.44
np
0.94
p
0.19
np
0.11
p
0.61
rip
0.94
p
0.36
np
0.49
p
0.11
np
0.61
0.33
p
np
81
0
0.6
0.5
VI
C
0.3
ox
0
0.2
o
I-
0.1
0.
Little Three Creeks Lake
Todd Lake
Location
Figure 4.3. The average proportion of hatching tadpoles for preferred (dark bars on the
right) and non-preferred matings (light bars on the left) for Western toads at Todd Lake
and Little Three Creeks Lake. ** denotes p-value below 0.05.
Offspring Viability: Hatching Size
At Little Three Creeks Lake, the average length of newly hatched tadpoles was
significantly greater from preferred matings than from non-preferred matings (t-ratio =
-
2.35, p-value = 0.03 18, df19, N21; Table 4.3; Figure 4.4). Tadpoles from preferred
matings averaged 8.58mm (SD = 0.23mm) in length and tadpoles from non-preferred
matings averaged 7.66mm (SD
0.23mm) in length. Similarly, at Todd Lake, tadpole
length at hatching was significantly greater in preferred matings than in non-preferred
matings (t-ratio = -4.82, p-value = 0.0002, df16, N18; Table 4.4; Figure 4.4). On
average at Todd Lake, tadpoles from preferred matings were 7.72mm (SD = 0.17mm) in
length and tadpoles from non-preferred matings were 6.8 8mm (SD
0.17mm). Female
and male size (snout-urostyle length) did not explain a significant amount of variation
in tadpole size at hatching.
82
Table 4.3. Mean tadpole length per clutch for preferred and non-preferred matings for
Western toads at Little Three Creeks Lake.
Clutch
1
2
3
4
5
6
7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
PAvg
NP Avg
Mean Tadpole
Length
9.15
7.71
8.58
7.54
8.38
7.72
8.32
7.75
8.59
8.33
8.01
7.44
7.72
7.92
6.91
7.42
7.6
7.7
7.1
8.61
7.65
8.58
7.66
Treatment
p
np
p
np
p
np
p
p
np
p
np
p
np
p
np
p
np
p
np
p
np
p
np
Table 4.4. Mean tadpole length per each clutch from preferred and non-preferred
matings for Western toads at Todd Lake.
Clutch
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
PAvg
NP Avg
Mean Tadpole
Length
7.8
6.29
6.92
7.2
8.11
6.93
7.76
6.24
7.95
7.29
7.3
7.12
7.58
7.02
8.2
6.96
7.88
6.89
7.72
6.88
Treatment
p
np
p
np
p
np
p
np
p
np
p
np
p
np
p
np
p
np
p
np
83
84
Figure 4.4. Tadpole length at hatching averaged for preferred (dark bar on right) and
non-preferred (light bar on left) matings for Western toads at Todd Lake and Little
Three Creeks Lake. ** denotes p-values below 0.05
DISCUSSION
Two main differences were found when female Western toads were allowed to
select their mates. Female toads that mated with preferred males (a) oviposited eggs
that were more likely to survive until hatching and (b) produced tadpoles that had larger
average size at hatching. It is possible that fertilization success may have contributed
to differences in hatching success. However, a previous study of B.
boreas
found that
fertilization success was nearly 100% for all pairs. Thus, the successful development of
zygotes to hatching is inferred as the mechanism by which females mating with
preferred males produced proportionally more hatchlings. These differences in
hatchling survivorship and body size presumably translate into fitness benefits for
females that freely express mate choice. Thus, the benefits of free female choice can be
85
found even in a mating system that normally constrains a female's ability to
discriminate between potential mates. Additionally, these results provide the first
evidence in amphibians, and anurans in particular, that females benefit from mate
choice that is independent of any specific male trait (e.g., body size) that females may
use to differentiate among males. Together, these results are consistent with the main
assumption of sexual dialectics theory that females benefit from freely expressed mate
choice.
The assumption that survivorship and hatching size are beneficial is supported
by evidence from other anuran studies. These studies have shown that larger tadpoles
can have higher competitive ability, better predator avoidance, increased survival, and
shorter larval periods (Wilbur and Collins, 1973, Smith-Gill and Gill 1978,
Steinwascher 1979, Travis et al. 1987, Woodward 1987). In addition, reduced larval
periods in anurans are linked to other fitness measures, such as age at first reproduction,
overwintering survival, and survival to adulthood (CaIdwell et al. 1980, Morin 1983,
Berven and Gill 1983, Smith 1987, Travis et al. 1987, Woodward et al. 1988, Mitchell
1990, Berven 1990, Blouin 1992). These offspring fitness measures may be especially
important in toad populations since tadpoles experience selection to metamorphose
quickly due to drought and lake drying at breeding sites (Blaustein and Olson 1991).
My experimental design was conservative with respect to observing potential
differences between preferred and non-preferred males (cf Drickamer 2000). First, only
two males were used in each choice trial, which limits the potential differences that
were the basis for choice by a given female. Another conservative feature of the
experimental design was that eggs were kept in mesh enclosures, which limited egg
86
predation from aquatic predators and potentially decreased pathogen exposure. These
design features increased the likelihood that observed differences in survivorship and
hatching size were related primarily to preferred versus non-preferred matings.
The focus of this study was to determine whether preferred matings provided
benefits. No attempt was made, a priori, to determine the mechanism by which females
might benefit from mate choice. However, several possible mechanisms that might be
responsible for the observed results. Other studies have identified two agents as
responsible for high embryo mortality in the Western toad: exposure to ultraviolet-B
light and S.
ferax
(Kiesecker and Blaustein 1995). A female might choose a male such
that offspring are more resistant to evolving pathogens or better suited to changing
environmental conditions. Hamilton and Zuk (1982) and Gowaty (1997) propose that
mate choice is based on evolving resistance to disease or adaptation to changing
environmental conditions, as opposed to a static trait (e.g., ozone depletion causing
higher levels of ultraviolet B or drought leading to premature lake drying). In addition,
if mate choice is based on genetic compatability between males and females, then nonpreferred matings could result in incompatable genetic recombinations in some
proportion of offspring. In contrast, food availability and competition for food are not
potential explanatory factors as the experiment was terminated at hatching (before
tadpoles begin to feed) and adult toads do not forage while at the breeding aggregation.
Another possible mechanism that may explain the advantages of preferred
mating is that mating with non-preferred males could be stressful and result in increased
cortisol levels. Numerous studies of other vertebrate taxa have revealed negative
effects of stress on offspring survivorship or size (Weiner et al. 1986, Tam et al. 1990,
87
Foo and Lam 1993, Campbell et al. 1994, Pankhurst et al. 1995, Kerrigan 1996).
McCormick (1998), for example, determined that cortisol from stress influenced yolk
size of larval progeny and larval morphology in a tropical damselfish. Small effects on
larval stages are of critical importance because: (a) these effects can alter
developmental trajectories, and (b) small changes in mortality can have large effects on
recruitment into the breeding population (Houde 1987, Underwood and Fairweather
1989, De Jesus et al. 1990, Galton 1990, Leggett and Deblois 1994, Bernardo 1996,
McNamara and Houston 1996, McCormick 1998). In addition, females of many
species can alter nutrient and hormone allocations to their clutches, and even to
different sexes within the clutch, based on her or her mate's quality (Pilz et al. 2003,
Rutkowska and Cichon 2002, Olsson et al. 2002, Gil et al. 1999, Clark and Galef 1995,
Leggett and Deblois 1994, Blaxter 1986, Bailey and Houde 1989, Rothschild 1986,
Chambers et al. 1989). Studies of maternal investment and offspring development in
anurans suggest that differential investment may have consequences for offspring
fitness (Parichy and Kaplan 1995, Kaplan 1992). These non-genetic maternal effects
also may be responsible for the observed differences in toad progeny from preferred
versus non-preferred matings.
The benefits of free female mate choice found in Western toads have
implications in terms of how a female may attempt to control mating with a non-
preferred male. A female already in amplexus is not easily able to resist mating with a
non-preferred male due to the strength of male clasping. However, females control the
movements of the pair prior to and during oviposition. Consequently, some females
oviposit in close proximity to other ovipositing pairs, while others oviposit at the
88
periphery of the breeding sites where other pairs are not abundant. Since most anurans
have external fertilization, it may be possible that a female amplexed by a non-preferred
male can position herself at the oviposition site to increase the chances of multiple
paternity. In addition, females are rarely found single at the breeding site where males
amplex forcefully and indiscriminately and the operational sex ratio is highly male
biased. It is possible that females could choose males at a distance from the actual
breeding site to increase their ability to exercise their preferences. Similary, females
that arrive at the lake early experience a less skewed sex ratio and lower density of
individuals than do late arriving females because late in the season most males remain
at the lake and most females have oviposited and left the lake.
In addition to predictions of female resistance behavior, my results have
implications for observed mating system patterns. In particular, Western toads have
prolonged amplexus (similar to pre-copulatory mate guarding in invertebrates) where
the female carries the male on her back. Prolonged amplexus is likely to incur energetic
costs, decrease mobility, and increase her risk of predation (as evidence by higher
mortality of females from raven predation, Olson 1988). As mentioned earlier, Western
toad populations exhibit a significant, but inconsistent size-based mating pattern, this
pattern does not necessarily reflect female choice (see Figure 4.5). Larger males may
be better able to overcome female resistance behaviors, as shown by Olson (1988).
Similar size-based mating patterns reflecting sexual conflicts over mating have been
found in water striders and seaweed flies (Ortigosa and Rowe 2003, Fairburn et al.
2003, Shukar and Day 2001, Rowe et al. 1994, Crean and Gilburn 1998, Watson et al.
1998).
89
Todd Lake
Little Three Creeks Lake
D--
7.6
ab
7.4
7.2
a
7
68
'1±.
62
6
5.8
Preferred
Non-preferred
Original
Preferred
Non-preferred
Original
Male Category
Figure 4.5. Differences in Western toad male SUL at Little Three Creeks Lake and
Todd Lake. Preferred males were smaller on average than non-preferred males and the
original males with which females were amplexed. Significant differences are denoted
with different letters.
The previously discussed predictions of female resistance behavior and
consequent mating patterns in B.
boreas
are novel and testable. Female mate choice in
explosive breeding anurans has often been overlooked due to social constraints (e.g.,
male clasping behavior and male-biased sex ratios) and seasonal constraints (short
breeding season) within the system. My findings suggest that free mate choice by
females can play a role in the dynamics of social behavior and mating patterns and,
consequently, can be an important source of evolutionary change. To my knowledge,
this is the first study in anurans to address the critical assumption of sexual conflict that
freely expressed female mate choice is selectively advantageous. Consequently, this
work contributes to our understanding of the pervasiveness of sexual conflicts over
mating across taxa.
CHAPTER 5
GENERAL CONCLUSIONS AND FUTURE DIRECTIONS
The research presented here addresses the causes and consequences of variation
in female mating behavior and female reproductive success in plethodontid salamanders
and toads. Sexual conflict theory provided the theoretical background from which to
address variation in female reproductive success from the perspective of females.
In Chapter 2, I assessed the potential for sexual conflict to play a role in
plethodontid courtship behavior and male courtship pheromone delivery. The results of
this study provided evidence that, even in plethodontid salamanders with highly
stereotyped courtship behavior, there is significant variation among species in the roles
females play in courtship interactions. Female Plethdon cinereus, P. angusticlavius,
and P. richmondi exhibited behaviors previously considered as male behaviors. In
addition, females of these species performed behaviors that had never been reported
prior to this study. The timing of male courtship pheromone delivery occurred earlier in
the courtship interactions (prior to tail-straddling walk) than had been observed for
other well-studied plethodontids (pheromone delivery during tail-straddling walk).
I
confirmed that P. angusticlavius males deliver courtship pheromones by "slapping,"
and that "vaccination" pheromone delivery occurs in P. cinereus and P. richmondi. A
more active female role in courtship interactions and pheromone delivery occurring
during early stages of courtship are consistent with, but do not confirm, the predictions
that sexual conflict may be operating in these salamanders. My observations provide
incentive for more research from the perspective of females and sexual conflict.
91
In Chapter 3, I address more directly the effects of male courtship pheromone
delivery on selected measures of female reproductive success. My results suggest that
male courtship pheromones may have other effects on females, in addition to decreasing
female reluctance to mate. The overall results vary between years, and thus are not
conclusive. In one year of the study, the pheromone altered the positive relationship
between female size and clutch size. In particular, the normal slope of the relationship
between female size and clutch size is depressed in the group of females that received
pheromone. However, the second year of the study failed to support this result, and
there was no treatment effect on clutch size/female size relationships. At least in certain
circumstances and in particular females, however, the male courtship pheromone
apparently can alter female clutch size. My results suggest that the male courtship
pheromone may function to overcome female resistance in interactions between a
female and a non-preferred male. The potential for male courtship pheromones to
function in sexual conflict provides incentive for more research to clarify the role
courtship pheromones play in plethodontid salamanders.
Chapter 4 addressed the main assumption of sexual conflict theory that a female
accrues benefits for herself and her offspring through freely expressed mate choice. My
results provided the first evidence in amphibians, and anurans in particular, that female
reproductive success (offspring survivorship to hatching) and offspring viability (size at
hatching) is higher in females that mated with preferred males. In a post-hoc analysis of
male traits, I found that, contrary to observed mating patterns favoring large males
(large male mating advantage), preferred males were often the smaller male used in the
choice trial. Thus, the large male mating advantage did not occur as a result of female
92
mate choice. Instead, larger, non-preferred males were better able to maintain amplexus
with reluctant females. In addition, my results provide the basis for novel predictions
of female resistance behaviors that enable a female to more easily exercise her mating
preferences.
In general, my research broadens the scope of sexual conflict by investigating its
role in the mating systems of amphibians. Behavioral information critical to
reproductive success may contribute to future management strategies for threatened or
endangered species. Also, my research provides incentive for a new perspective on
quantitative genetic models of sexual selection. Models of sexual selection previously
assumed that female mate choice evolved as a by-product of selection on male traits or
at a cost to females that spent time discriminating among males (Lande 1981; Arnold
1982; Iwasa and Pomiankowski 1995; Pomiankowski and Iwasa 1998; Mead and
Arnold 2004). My research supports the incorporation of substantial fitness benefits for
females into models of sexual selection. The addition of variation in sexual selection
attributable to female mate choice yields models that more accurately represent the
natural variation in animal mating systems. Lastly, by broadening the taxa studied from
the perspective of sexual conflict, my research further supports ideas that sexual conflict
plays a critical role in sexual dimorphism, reproductive isolation, and speciation
(Chapman and Partridge 1996; Parker and Partridge 1998; Arnqvist et al., 2000;
Gavrilets 2000a, b; Blankenhorn et al. 2002). Freely expressed mate choice and
subsequent sexual conflict are important in many vertebrate and invertebrate mating
systems.
BIBLIOGRAPHY
Alonzo, S. H. and R. R. Warner (2000). "Female choice, conflict between the sexes,
and the evolution of male alternative reproductive behaviors." Evolutionary Ecological
Research 2: 149-170.
Andersson, M. (1986). "Evolution of condition-dependent sex ornaments and mating
preferences: sexual selection based on viability differences." Evolution 40: 804-8 16.
Andersson, M. (1994). Sexual Selection. Princeton, NJ, Princeton University Press.
Andersson, 5. (1991). "Bowers on the savanna: Display courts and mate choice in a
lekking widowbird." Behavioral Ecology 2: 210-218.
Arnold, S. J. (1972). The evolution of courtship behavior in salamander. Ann Arbor,
University of Michigan.
Arnold, S. J. (1976). "Sexual behavior, sexual interference, and sexual defense in the
salamanders Ambystoma maculatum, Ambystoma tigrinum, and Plethodonjordoni."
Zeitschrift fur Tierpsychologie 42: 247-3 00.
Arnold, S. J. (1977). The evolution of courtship behavior in new world salamanders
with some comments on old world salamanders. The Reproductive Biology of
Amphibians. D. H. Taylor and S. I. Guttman. New York, Plenum Press: 141 - 183.
Arnold, S. J. and L. D. Houck. (1982). Courtship pheromones: evolution by natural and
sexual selection. Biochemical Aspects of Evolutionary Biology. M. Nitecke. Chicago,
University of Chicago Press: 173-211.
Arnold, S. J. (1983). Sexual selection: the interface of theory and empiricism. Mate
Choice. Bateson. Cambridge, Cambridge University Press: 67-107.
Arnqvist, G. (1989). "Sexual selection in a water strider: the function, mechanism of
selection and heritability of a male grasping apparatus." Oikos 56: 344-350.
Arnqvist, G. (1992). "The effects of operational sex ratio on the relative mating success
of extreme male phenotypes in the water strider Gerris odontogaster." Animal Behavior
43: 68 1-683.
Arnqvist, G. and L. Rowe (1995). "Sexual conflict and arms races between the sexes: a
morphological adaptation for control of mating in a female insect."Proceedings of the
Royal Society of London Series B 261: 123-127.
Arnqvist, G., R. Thornhill and L. Rowe (1997). "Evolution of animal genitalia:
Morphological correlates of fitness components in a water strider. "Journal of
Evolutionary Biology 10(4): 613-640.
Arnqvist, G. and I. Danielsson (1999). "Copulatory behavior, gential morphology, and
male fertilization success in water striders." Evolution 53: 147-156.
Arnqvist, G., M. Edvardsson, U. Friberg, and T. Nilsson (2000). "Sexual conflict
promotes speciation in insects." Proceedings of the National Academy of Science 97:
10460-10464.
Arnqvist, G. and L. Rowe. (2002). "Antagonistic coevolution between the sexes in a
group of insects." Nature 415: 787-789.
Bailey, K. M., and E. D. Houde (1989). "Predation on eggs and larvae of marine fishes
and the recruitment problem." Advances in Marine Biology 26: 1-83.
Barnard, C. J. and T. Burk (1979). "Dominance hierarchies and the evolution of
"individual recognition"." Journal of Theoretical Biology 81: 65-73.
Basolo, A. L. (1990). "Female preference predates the evolution of the sword in
swordtailfish." Science 250: 808-8 10.
Bernardo, J. (1996). "Maternal effects in animal ecology." American Zoologist 36: 83105.
Bervin, K. A. (1990). "Factors affecting population fluctuations in larval and adult
stages of the wood frog (Rana sylvatica)." Ecology 71: 1599-1608.
Berven, K. A. and D. E. Gill (1983). "Interpreting geographic variation in life history
traits." American Zoologist 23: 85-97.
Blanckenhorn, W. U., D. J. Hosken, 0. Y. Martin, C. Reim,Y. Teuschl, and P. I. Ward
(2002). "The costs of copulating in the dung fly Sepsis cynipsea." Behavioral Ecology
13: 353-358.
Blaustein, A.R. and D.H. Olson. 1991. "Amphibian population declines."
Science 253: 1467.
Blaxter, J. H. 5. (1986). "Development of sense organs and behaviour in teleost larvae
with special reference to feeding and predator avoidance." Transactions of the American
Fisheries Society 115: 98-114.
Blouin, M. S. (1992). "Genetic correlations among morphometric traits and rates of
growth and differentiation in the green tree frog, Hyla cinerea." Evolution 46: 735-744.
Bluhm, C. K. and P. A. Gowaty (2004). "Reproductive compensation for offspring
95
viability deficits by female mallards, Anasplatyrhynchos." Animal Behavior 68: 985992.
Bluhm, C. K. and P. A. Gowaty (2004). "Social constraints on female mate preferences
in mallards, Anas platyrhynchos, decrease offspring viability and mother productivity."
Animal Behavior 68: 977-983.
Bonduriansky, R. and L. Rowe (2003). "Interactions among mechanisms of sexual
selection on male body size and head shape in a sexually dimorphic fly." Evolution 57:
2046-2053.
Burk, T. (1988). "Acoustic signals, arms races and the costs of honest signalling."
Florida Entomologist 71: 400-409.
Caidwell, J. P., J. H. Thorp, and T. 0. Jervey (1980). "Predator-prey relationships
among larval dragonflies, salamanders, and frogs." Oecologia 46: 285-289.
Campbell, P. M., R. G. Pottinger, and J. P. Sumpter (1994). "Preliminary evidence that
chronic confinement stress reduces quality of gametes produced by brown and rainbow
trout." Aguaculture 120: 151-169.
Chambers, R. C., W. C. Leggett, and J. A. Brown (1989). "Egg size, female effects, and
the correlation between early life history traits of capelin, Mallotus villosus: an
appraisal at the individual level." Fisheries Bulletin 87: 5 15-523.
Chapman, T. G. and L. Partridge (1996). "Sexual conflict as fuel for evolution." Nature
381: 189-190.
Chapman, T., G. Arnqvist, J. Bangham, and L. Rowe (2003). "Sexual conflict." Trends
in Ecology and Evolution 18(1): 41-47.
Clark, M. M. and B. G. Galef (1995). "Prenatal influences on reproductive life history
strategies." Trends in Ecology and Evolution 10: 15 1-153.
Clutten-Brock, T. H. and S. D. Albon (1979). "The roaring red deer and the evolution of
honest advertisement." Behaviour 69: 145-170.
Clutten-Brock, T. H. and G. A. Parker (1992). "Potential reproductive rates and the
operation of sexual selection." Quarterly Review in Biology 67: 437-455.
Clutton-Brock, T. H. and A. C. J. Vincent (1991). "Sexual selection and the potential
reproductive rates in males and females." Nature 352: 5 8-60.
Cordero, A. and J. A. Andres (2002). "Male coercion and convenience polyandry in a
calopterygid damselfly." Journal of Insect Science 2: 1-7.
Cordoba-Aguilar, A. and J. Contreras-Garduno (2003). "Sexual conflict." Trends in
Ecology and Evolution 18: 48.
Coss, H. T. (1974). Maxillary and premaxillary dentition of salamanders of the tribe
Plethodontini. Clemson, Clemson University.
Cothran, R. D. (2004). "Precopulatory mate guarding affects predation risk in two
freshwater amphipod species." Animal Behavior 68: 1133-1138.
Crean, C. S. and A. S. Gilburn (1998). "Sexual selection as a side-effect of sexual
conflict in the seaweed fly." Animal Behavior 56(6): 1405-1410.
Darwin, C. (1871). The Descent of Man and Selection in Relation to Sex. London, John
Murray.
Dawkins, R. and J. R. Krebs (1978). Animal signals: information or manipulation?
Behavioural Ecology: an Evolutionary Approach. J. R. Krebs and N. B. Davies. Oxford,
Blackwell Scientific Publications: 282-309.
Dawkins, M. S. and T. Guilford (1991). "The corruption of honest signalling." Animal
Behavior 41: 865-873.
De Jesus, E. G., Y. Inui, and T. Hirano (1990). "Cortisol enhances the stimulating
action of thyroid hormones on dorsal fin-ray resorption of flounder larvae in vitro."
General and Comparative Endocrinology 79: 167-173.
Drickamer, L. C., P. A. Gowaty, and C. M. Holmes (2000). "Free female mate choice in
house mice affects reproductive success and offspring viability and performance."
Animal Behavior 59: 37 1-378.
Eberhard, W. G. and C. Cordero (2003). "Sexual conflict and female choice." Trends in
Ecology and Evolution: 18: 122.
Emlen, J. M. (1973). Ecology: An Evolutionary Approach. Reading, Mass, AddisonWesley.
Fairbairn, D. J. (1988). "Sexual selection for homogamy in the gerridae: an extensionof
Ridley's comparative approach." Evolution 42; 1212-1222.
Fairbairn, D. J. (1990). "Factors influencing sexual dimorphism in temperate water
striders." American Naturalist 136: 6 1-86.
Fairbairn, D. J., R. Vermette, N. Kapoor and N. Zahiri (2003). "Functional morphology
of sexually selected genetalia in the water strider Aquarius remigis.' Canadian Journal
97
of Zoology 81(3): 400-4 13.
Feldhoff, R. C., S. M. Rollmann, and L. D. Houck (1999). Chemical analysis of
courtship pheromones in a plethodontid salamander. Advances in Chemical signals in
vertebrates. R. E. Johnson, D. Muller-Schwarze, and P. W. Sorenson. New York,
Kluwer Academic / Plenum Press.
Fincke, 0. M. (1997). "Conflict resolution in the Odonata: Implications for
understanding female mating patterns and female choice." Biological Journal of the
Linnean Society 60: 20 1-220.
Fisher, R. A. (1915). "The evolution of sexual preference." Eugenics Review 7: 184192.
Fisher, R. A. (1958). The genetical theory of natural selection. New York, Dover
Publications, Inc.
Fleck, J. (1992). "Haithung und Nachzucht von Tylototriton kweichowensis."
Salamandra 28: 97-105.
Foo, J. T. W. and T. J. Lam (1993). "Retardation of ovarian growth and depression of
serum steroid levels in the tilapia, Oreochromis mossambicus, by cortisol implantation."
Aquaculture 115: 133-143.
Gabor, C. R. and R. G. Jaeger (1995). "Resource quality affects the agonistic behavior
of territorial salamanders." Animal Behavior 49: 71-79.
Galton, V. A. (1990). "Mechanism underlying the acceleration of thyroid hormoneinduced tadpole metamorphosis by corticosterone." Endocrinology 127: 2997-3 002.
Gavrilets, S. (2000). "Rapid evolution of reproductive barriers driven by sexual
conflict." Nature 403: 886-889.
Gavrilets, S. (2000). "Sexual conflict and speciation." Nature 407: 150.
Gergits, W. F. and R. G. Jaeger (1990). "Field observations of the behavior of the Redbacked Salamander (Plethodon cinerus): courtship and agonistic interactions." Journal
of Herpetology 24: 93-95.
Gil, D., J. Graves, N. Hazon, and A. Wells (1999). "Male attractiveness and differential
testosterone investment in zebra finches." Science 286: 126-128.
Gillette, J. R., R. G. Jaeger, and M. C. Peterson (2000). "Social monogamy in a
territorial salamander." Animal Behavior 59: 124 1-1250.
Gillette, J. R. and M. C. Peterson. (2001). "The benefits of transparancy: candling as a
simple method for determining sex in red-backed salamanders (Plethodon cinereus)."
Herpetological Review 32: 233-235.
Gowaty, P. A. (1997). Sexual dialectics, sexual selection, and variation in reproductive
behavior. Feminism and Evolutionary Biology: Boundaries, Intersections, and Frontiers.
P. A. Gowaty. New York, Chapman and Hall: 35 1-384.
Grafen, A. (1990a). "Biological signals as handicaps." Journal of Theoretical Biology
144: 5 17-546.
Grafen, A. (1990b). "Sexual selection unhandicapped by the Fisher process." Journal of
Theoretical Biology 144: 473-5 16.
Greenspan, R. J. and J. F. Fervour. (2000). "Courtship in Drosophila." Annual Review
in Genetics 34: 205-32.
Guilford, T. and M. S. Dawkins (1991). "Receiver psychology and the evolution of
animal signals." Animal Behavior 42: 1-14.
Halliday, T. R. (1990). "The evolution of courtship behavior in newts and salamanders."
Advanced Studies of Animal Behavior 19: 137-169.
Hamilton, W. D. and M. Zuk. (1982). "Heritable true fitness and bright birds: A role for
parasites?" Science 218: 384-387.
Hasumi, M. (1994). "Reproductive behavior of the salamander Hynobius nigrescens:
monopoly of egg sacs during scramble competition." Journal of Herpetology 28: 264267.
Highton, R. (1962). "Revision of North American salamanders of the genus Plethodon."
Bulletin of the Florida State Museum 6: 235-367.
Highton, R. and A. Larson (1979). "The genetic relationship of the salamanders of the
genus Plethodon." Annual Review of Ecological Systematics 26: 5 79-599.
Highton, R. (1991). "Molecular phylogeny of plethodontine salamanders and hylid
frogs: statistical analysis of protein comparisons." Molecular Biology & Evolution 8:
796-8 18.
Highton, R. (1997). "Geographic protein variation and speciation in the Plethodon
dorsalis complex." Herpetologica 53: 345-356.
Highton, R. and R. B. Peabody (2000). Geographic protein variation and speciation in
salamanders of the Plethodonjordoni and Plethodon glutinosus complexes in the
99
southern Appalachian Mountains with descriptions of four new species. The Biology of
Plethodontid Salamanders. R. G. Jaeger, R. C. Bruce, and L. D. Houck. New York,
Plenum Press: 3 1-94.
Hill, G. E. (1992). "Proximate basis of variation in carotenoid pigmentation in male
house finches." Auk 109: 1-12.
Hill, 0. E. and R. Montgomerie (1994). "Plumage colour signals nutritional condition in
the house finch." Proceedings of the Royal Society of London Series B 258: 47-52.
Hill, G. E. and R. Montgomerie. (2002). "Dietary carotenoids predict plumage
coloration in wild house finches." Proceedings of the Royal Society of London Series B
269: 1119-1124.
Hill, G. E., K. L. Farmer, and M. L. Beck (2004). "The effect of mycoplasmosis on
carotenoid plumage coloration in male house finches." Journal of Experimental Biology
207: 2095-2099.
Hiyashi, K. (1985). "Alternative mating strategies in the water strider Gerris thoracicus
as an adaptation to food and habitat variation." Behavioral Ecology and Sociobiology
16: 301-306.
Hoikkala, A. and K. Y. Kaneshiro (1994). "Courtship songs of the picture-winged
Drosophila plantibia subgroup species." Animal Behavior 47: 1363-1374.
Holland, B. and W. R. Rice (1999). "Experimental removal of sexual selection reverses
intersexual antagonistic coevolution and removes a reproductive load." Proceedings of
the National Academy of Science 96: 5083-5088.
Houck, L. D. (1 977a). Reproductive patterns in neotropical salamanders. Department of
Zoology. Berkley, University of California.
Houck, L. D. (1977b). Life history patterns and reproductive biology of neotropical
salamanders. Reproductive Biology of Amphibians. D. H. Taylor and S. I. Guttman.
New York, Plenum Press: 43-72.
Houck, L. D. (1986). The evolution of salamander courtship pheromones. Chemical
Signals in Vertebrates: IV : Ecology, Evolution, and Comparitive Biology. D. Duvall,
D. Muller-Schwarze, and R. M. Siverstein. New York, Plenum Press: 173-190.
Houck, L. D. and N. L. Reagan (1990). "Male courtship pheromones increase female
receptivity in a plethodontid salamander." Animal Behavior 39: 729-734
Houck, L. D. and D. M. Sever. (1994). The role of the skin in reproduction and
behavior. Amphibian Biology, Vol. 1, The Integument. H. Heatwole and G.
100
Barthalamus. Chipping Norton, Australia, Surrey Beatty and Sons: 351-381.
Houck, L. D., A. M. Bell, N. L. Reagan-Wallin, and R. C. Feidhoff (1998). "Effects of
experimental delivery a male courtship pheromones on the timing of courtship in a
terrestrial salamander, Plethodonjordani." Copeia 1998(1): 214-219.
Houck, L. D. and S. J. Arnold. (2003). Courtship and mating. Phylogeny and
Reproductive Biology of Urodela. D. M. Sever. Enfield, New Hampshire, Science
Publishers: 3 83-424.
Houde, F. D. (1987). "Fish early life dynamics and recruitment variability." American
Fisheries Society Symposium 2: 17-29.
Huxley, J. S. (1914). "The courtship-habits of the great crested grebe (Podiceps
cristatus); with an addition to the theory of sexual selection." Proceedings of the
Zoological Society of London 35: 491-562.
Iwasa, Y., A. Pomiankowski, and S. Nee (1991). "The evolution of costly mate
preferences. II. The "handicap" principle." Evolution 45: 143 1-1442.
Iwasa, Y. and A. Pomiankowski. (1995). "Continual change in mate preferences."
Nature 377: 420-423.
Jaeger, R. G., M. G. Peterson, G. Gollmann, B. Gollmann, and V. R. Townsend Jr.
(2001). "Salamander social strategies: living together in male-female pairs." Journal of
Herpetology 35: 335-338.
Jaeger, R. G. and M. G. Peterson. (2002). "Familiarity affects agonistic interactions
between female red-backed salamanders." Copeia 2002: 865-869.
Jaeger, R. G., J. R. Gillette, and R. C. Cooper (2002). "Sexual coercion in a territorial
salamander: males punish socially polyandrous female partners." Animal Behavior 63:
87 1-877.
Johnstone, R. A. (1996). "Multiple displays in animal communication: 'backup signals'
and 'multiple messages'." Philosophical Transactions of the Royal Society of London
Series B 351: 328-338.
Johnstone, R. A., J. D. Reynolds, and J. C. Deutsch (1996). "Mutual mate choice and
sex differences in choosiness." Evolution 50: 1382-1391.
Kaitala, A. and H. Dingle (1993). "Wing dimorphism, territoriality, and mating
frequency of the waterstrider Aquarius remigis." Ann. Zool. Fenn. 30: 163-168.
Kaplan, R. H. (1992). "Greater maternal investment can decrease offspring survival in
101
the frog Bomb/na oriental/s." Ecology 73: 280-288.
Kawamichi, T. and H. Ueda (1998). "Spawning at nests of extra-large males in the
Giant Salamander A ndrias japonicus." Journal of Herpetology 32: 133-136.
Kerbert, G. (1904). "Zur Fortpflanzung von Megalobatrachus maximus Schiegel."
Zoologischer Anzeiger 27: 305-320.
Kerrigan, B. A. (1996). "Variability in larval development of a tropical reef fish
(Pomacentridae: Pomacentrus amboinensis): the parental legacy." Marine Biology 127:
395-402.
Kiesecker, J. M. and A. R. Blaustein (1995). "Synergism between UV-B radiation and a
pathogen magnifies amphibian embryo mortality in nature." Proceedings of the National
Academy of Science 92: 11049-11052.
Kirkpatrick, M. (1982). "Sexual selection and the evolution of female mate choice."
Evolution 36: 1-12.
Kodric-Brown, A. and J. H. Brown (1984). "Truth in advertising: the kinds of traits
favored by sexual selection." The American Naturalist 124: 309-323.
Kokko, H., R. Brooks, M. D. Jennions, and J. Morley (2003). "The evolution of mate
choice and mating biases." Proceedings of the Royal Society of London Series B 270:
653-664.
Krebs, J. R. and R. Dawkins (1984). Animal signals: Mind-reading and manipulation.
Behavioral Ecology - An evolutionary approach. J. R. Krebs and N. B. Davies,
Blackwell Scientific.
Krebs, J. R. and A. Kacelnik (1991). Decision-making. Behavioural Ecology: An
Evolutionary Approach. J. R. Krebs and N. B. Davies. Oxford, Blackwell Scientific
Publications: 105-136.
Lande, R. (1980). "Sexual dimorphism, sexual selection, and adaptation in polygenic
characters." Evolution 34: 292-305.
Lande, R. (1981). "Models of speciation by sexual selection on polygenic traits."
Proceedings of the National Academy of Science 78: 3721-3725.
Lang, C. and R. G. Jaeger. (2000). "Defense of territories by male-female pairs in the
Red-backed Salamander." Copeia 2000: 169-177.
Larson, A., D. B. Wake, L. R. Maxson, and R. Highton (1981). "A molecular
phylogenetic perspective on the origins of morphological novelties in the salamanders
102
of the tribe Plethodontini." Evolution 35: 405-422.
Larson, A., D. W. Weisrock, and K. H. Kozak (2003). Phylogenetic systematics of
salamanders, a review. Phylogeny and Reproductive Biology of Urodela. D. M. Sever.
Enfield, New Hampshire, Science Publishers: 3 83-424.
Lauer, M. J., A. Sih, and J. J. Krupa (1996). 'Male density, female density, and intersexual conflict ma stream-dwelling insect." Animal Behavior 52: 929-939.
Leggett, W. C. and E. Deblois (1994). "Recruitment in marine fishes - is it regulated by
starvation and predation in the egg and larva! stages." Netherlands Journal of Sea
Research 32: 119-134.
Mahoney, M. (2001). "Molecular systematics of Plethodon and Aneides: phylogenetic
analysis of an old and rapid radiation." Molecular Phylogenetics and Evolution 18: 174188.
Marco, A., J. M. Kiesecker, D. P. Chivers, and A. R. Blaustein (1998). "Sex recognition
and mate choice by male western toads, Bufo boreas." Animal Behavior 55: 163 1-1635.
Marvin, G. A. and V. H. Hutchison. (1996), "Courtship behavior of the Cumberland
Plateau Woodland Salamander, Plethodon kentucky, with a review of courtship in the
genus Plethodon." Ethology 102: 285-303.
Mays, H. L. J. and G. E. Hill (2004). "Choosing mates: good genes versus genes that are
a good fit." Trends in Ecology and Evolution 19: 554-559.
McCormick, M. 1. (1998). "Behaviorally induced maternal stress in a fish influences
progeny quality by a hormonal mechanism." Ecology 79: 1873-1883.
McNamara, J. M. and A. I. Houston (1996). "State-dependent life histories." Nature
380: 215-221.
Michod, R. E. and 0. Hasson. (1990). "On the evolution of reliable indicators of
fitness." The American Naturalist 135: 788-808.
Miller, K. A. (2003). "The phylogeny of diving beetles and the evolution of sexual
conflict." Biological Journal of the Linnean Society 79(3): 359-388.
Mitchell, S. L. (1990). "The mating system genetically affects offspring performance in
Woodhouse's toad (Bufo woodhousei)." Evolution 44: 502-5 19.
Moore, A. J. and P. J. Moore. (1999). "Balancing sexual selection through opposing
mate choice and male competition." Proceedings of the Royal Society of London B 266:
711-716.
103
Moore, A. J., P. A. Gowaty, W. G. Wallin, and P. J. Moore (2000). "Sexual conflict and
the evolution of female mate choice and male social dominance." Proceedings of the
Royal Society of London Series B 268: 517-523.
Moore, A. J., P. A. Gowaty and P. J. Moore (2003). "Females avoid manipulative males
and live longer." Journal of Evolutionary Biology 16: 523-530.
Morin, P. J. (1983). "Predation, competition, and the composition of larval anuran
guilds." Ecological Monographs 53: 119-138.
Navara, K. J., G. E. Hill, and M. T. Mendonca (2001). "Are there immunological costs
to carotenoid display in songbirds?" American Zoologist 41: 1535-1536.
Nicoletto, P. F. (1995). "Offspring quality and female choice in the guppy, Poecilia
reticulata." Animal Behavior 49: 377-387.
Nicoletto, P. F. and A. Kodric-Brown (2001). "Age and experience affect female choice
in the guppy (Poecilia reticulata)." The American Naturalist 157: 3 16-323.
Noble, G. K. and M. K. Brady. (1930). "The courtship of plethodontid salamanders."
Copeia 1930: 100-117.
Olson, D. H. (1988). The ecological and behavioral dynamics of breeding in three
sympatric anuran amphibians. Zoology. Corvallis, Oregon State University.
Olsson, M., E. Wapstra, and C. Olofsson (2002). "Offspring size-number strategies:
experimental manipulation of offspring size in a viviporous lizard." Functional Ecology
15: 135-140.
Organ, J. A. (1960a). The courtship and spermatophore of the salamander Plethodon
glutinosus." Copeia 1960: 34-40.
Organ, J. A. (1960b). "Studies on the life history of the salamander, Plethodon welleri."
Copeia 1960: 287-297.
Ortigosa, A. and L. Rowe. (2003). "The role of mating history and male size in
determining mating behaviors and sexual conflict in a water strider." Animal Behavior
65: 851-858.
Pankhurst, N. W., G. Van Der Kraaak, and R. E. Peter (1995). "Evidence that the
inhibitory effects of stress on reproduction in teleost fish are not mediated by the action
or cortisol on ovarian steroidogenesis." General and Comparative Endocrinology 99:
249-257.
104
Parichy, D. M. and R. H. Kaplan. (1995). "Maternal investment and developmental
plasticity: functional consequences for locomotor performance of hatchling frog larvae."
Functional Ecolov 9: 606-6 17.
Park, D. and S. R. Park. (2000). "Multiple insemination and reproductive biology of
Hynobius leechii." Journal of Herpetology 34: 594-598.
Parker, G. A. (1979). "Seuxal selection and sexual conflict." Sexual Selection and
Reproductive Competition in Insects. M. S. Blum and N. A. Blum. New York,
Academic Press.
Parker, G. A. and L. Partridge. (1998). "Sexual conflict and speciation." Philosophical
Transactions of the Royal Society of London Series B 353: 26 1-274.
Partridge, L. (1980). "Mate choice increases a component of offspring fitness in
fruitflies." Nature 283: 290-291.
Partridge, L. (1983). "Non-random mating and offspring fitness." Mate Choice. P.
Bateson. Cambridge University Press, Cambridge, UK
Picard, A. (2005). "Courtship in the zig-zag salamander (Plethodon dorsalis): insights
into a transition in pheromone delivery behavior." Ethology. In press.
Pilastro, A., B. Stefano and A. Bisazza (2003). "Female aggregation and male
competition reduce the costs of sexual harassment in the mosquitofish Gambusia
hoibrooki." Animal Behavior 65: 1161-1167.
Pilz, K. M., H. G. Smith, M. I. Sandell, and H. Schwabl (2003). "Interfemale variation
in egg yolk androgen allocation in the European starling: do high-quality females invest
more." Animal Behavior 65: 84 1-850.
Pizzari, T. and R. R. Snook. (2003). "Perspective: sexual conflict and sexual selection:
chasing away paradigm shifts." Evolution 57: 1223-1236.
Pomiankowski, A. (1988). "The evolution of female mate preferences for male genetic
quality." Oxford Surv. Evol. Biol. 5: 136-184.
Pomiankowski, A. and Y. Iwasa (1998). "Runaway ornament diversity caused by
Fisherian selection." Proceedings of the National Academy of Science 95: 5206-5 111.
Preziosi, R. F. and D. J. Fairbairn (2000). "Lifetime selection on adult body size and
components of body size in a waterstrider: Opposing selection and maintenance of
sexual size dimorphism." Evolution 54: 558-566.
Rails, K. (1976). "Mammals in which females are larger than males." Quarterly Review
105
in Biology 51: 245-276.
Rehberg, F. (1986). "Haltung und Zucht der Krokodilmolches Tylototriton verrucosus
Anderson 1871." Herpetofauna8: 11-17.
Reynolds, J. D. and M. R. Gross (1992). "Female mate preference enhances offspring
growth and reproduction in a fish, Poecilia reticulata." Proceedings of the Royal
Society of London Series B 250: 57-62.
Rice, W. R. (1996). "Sexually antagonistic male adaptation triggered by experimental
arrest of female evolution." Nature 361: 232-2344.
Rich, T. J. and J. L. Hurst (1998). "Scent marks as reliable signals of the competitive
ability of mates." Animal Behavior 56: 727-735.
Roesli, M. and H. Reyer. (2000). "Male vocalizations and female choice in the
hybridogenetic Rana lessonae/Rana esculenta complex." Animal Behavior 60: 745-755.
Rollmann, S. M., L. D. Houck, and R. C. Feldhoff (1999). "Proteinaceous pheromon
affecting female receptivity in a terrestrial salamander." Science 285: 1907-1909.
Rollmann, S. M., L. D. Houck, and R. C. Feldhoff (2000). "Population Variation in
Salamander Courtship Pheromones." Journal of Chemical Ecology 26: 2713-2724.
Rosenqvist, G., A. N. Bordal, M. E. Aune, and P. A. Gowaty (2001). "Implications of
freely expressed mate choice in the guppy." Advances in Ethology 36: 50.
Rothchild, B. J. (1986). Dynamics of Marine Fish Populations. Harvard University
Press, Cambridge, Massachusetts, USA.
Rowe, L. (1992). "Covenience polyandry in a water strider: foraging conflicts and
female control of copulation frequency and guarding duration." Animal Behavior 44:
189-202.
Rowe, L. (1994). "The costs of mating and mate choice in water striders." Animal
Behavior 48: 1049-1056.
Rowe, L., G. Arnqvist, A. Sib, and J. J. Krupa (1994). "Sexual conflict and the
evolutionary ecology of mating patterns: water striders as a model system." Trends in
Ecology and Evolution 9: 289-293.
Rubenstein, D. J. (1984). "Resource acquisition and alternative mating strategies in
water striders." American Zoologist 24: 345-3 53.
Rudolfsen, G., L. Figenschou, I. Folstad, J. T. Nordeide, and E. Soreng (2005).
106
"Potential fitness benefits from mate selection in the Atlantic cod (Gadus morhua)."
Journal of Evolutionary Biology 18: 172-179.
Rutkowska, J. and M. Cichon (2002). "Maternal investment during egg laying and
offspring sex: an experimental study of zebra finches." Animal Behavior 64: 817-822.
Sapp, J. (2002). Courtship behavior in the salamander genus Aneides. Zoology.
Corvallis, Oregon, Oregon State University.
"Reproductive behavior in the Japanese salamander Hynobius
retardatus." Japanese Journal of Herpetology 14: 184-190.
Sato, T.
(1992).
Shine, R., O'Connor, D. and R.T. Mason (2000). "Sexual conflict in the
snake den." Behavioral Ecology & Sociobiology 48: 392-401.
Shine, R., Langkilde, T. and R.T. Mason (2003). "Cryptic forcible
insemination: male snakes exploit female physiology, anatomy, and behavior
to obtain coercive matings." American Naturalist 162: 653-667.
Shine, R., Phillips, B., Langkilde, T., Lutterschmidt, D.I., Waye, H. and
R.T. Mason (2004). "Mechanisms and consequences of sexual conflict in
garter snakes (Thamnophis sirtalis, Colubridae)." Behavioral Ecology 15:
654-660.
Shuker, D. M. and T. H. Day (2001). "The repeatability of a sexual conflict over
mating." Animal Behavior 61: 755-762.
Shuker, D. M. and T. H. Day (2002). "Mate sampling and the sexual conflict over
mating in seaweed flies." Behavioral Ecology 13: 83-86.
Smith, B. G. (1907). "The life history and habits of the mole salamander, Ambystoma
talpoideum, in southeastern Louisiana." Tulane Studies of Herpeto1oy 8: 65-82.
Smith, D. C. (1987). "Adult recruitment in chorus frogs: effects of size and date at
metamorphosis." Ecology 68: 344-350.
Smuts, B. B. (1992). "Male aggression against females in primates: Data and
implications for sexual selection theory." Human Nature 3: 1-44.
Smuts, B. B. (1995). "The origins of patriarchy: an evolutionary perspective." Human
Nature 6: 1-32.
Smith-Gill, S. J. and D. E. Gill (1978). "Curvilinearities in the competition equations:
an experiment with ranid tadpoles." American Naturalist 112: 557-570.
107
Smuts, B. B. (1992). 'Male aggression against females in primates: data and
implications for sexual selection theory." Human Nature 3: 1-44.
Soulier-Perkins, A. (2001). "The phylogeny of the Lophopidae and the impact of sexual
selection and coevolutionary sexual conflict." Cladistics 17: 56-78.
Spritzer, M. D., D. B. Meikle, and N. G. Solomon (2005). "Female choice based on
male spatial ability and aggressiveness among meadow voles." Animal Behavior. In
press.
Stebbins, R. C. (1949). "Courtship of the plethodontid salamander, Ensatini
eschsholtzii." Copeia 1949: 274-28 1.
Steinwascher, K. and J. Travis (1983). "Influence of food quality and quantity on early
larval growth of two anurans." Copeia 1983: 238-242.
Tam, W. H., J. N. Fryer, B. Valentine, and R. J. J. Roy (1990). "Reduction in oocyte
production and gonadotrope activity, and plasma levels of estrogens and vitellogenin, in
brook trout exposed to low environmental pH." Canadian Journal of Zoology 68: 24682479.
Tinbergen, N. (1939). "The behaviour of the snow bunting during spring." Transactions
of the Linnean Society 5: 1-94.
Travis, J., S. B. Emerson, and M. Blouin (1987). "A quatitative genetic analysis of
fitness-related traits in Hyla cruc/er. I. Larval development patterns." Evolution 41:
145-156.
Trivers, R. L. (1972). Parental investment and sexual selection. Sexual Selection and
the Descent of Man. B. Campbell. Chicago, Aldine: 136-179.
Underwood, A. J. and P. G. Fairweather (1989). "Supply-side ecology and benthic
marine assemblages." Trends in Ecology and Evolution 4: 16-20.
Usuda, H. (1993). "Reproductive behavior of Hynobius nigrescens, with special
reference to male midwife behavior." Japanese Journal of Herpetology 15: 64-70.
Watson, P. J., G. Arnqvist, and R. R. Stallmann (1998). "Sexual conflict and the
energetic costs of mating and mate choice in water striders." The American Naturalist
151: 46-58.
Weiner, C. S., C. B. Schreck, and H. W. Li (1986). "Effect of low pH on reproduction
in rainbow trout." Transactions of the American Fisheries Society 115: 75-82.
Wilbur, H. M. and J. P. Collins (1973). "Ecological aspects of amphibian
108
metamorphosis." Science 182: 1305-13 14.
Williams, G. C. (1966). Adaptation and Natural Selection: A Critique of Some Current
Evolutionary Thought. Princeton, NJ, Princeton University Press.
Wirsig-Wiechmann, C. R., L. D. Houck, P. W. Feldhoff, and F. C. Feldhoff (2002).
"Pheromonal activation of vomeronasal neurons in plethodontid salamanders." Brain
Research Interactive 952: 335-344.
Woodward, B. D. (1987). "Paternal effects on offspring traits in Scaphi opus couchi
(Anura: Pelobatidae." Oecologia 73: 626-629.
Woodward, B. D., J. Travis, and S. Mitchell (1988). "The effects of mating system on
progeny performance in Hyla crucfer." Evolution 42: 784-794.
Woodward, B. D., J. Travis, and S. L. Mitchell (1988). "Nonrandom genetic
consequences of the spring peeper (Hyla crucfer) mating system." Evolution 42: 784794.
Zahavi, A. (1975). "Mate selection - A selection for a handicap." Journal of Theoretical
Biology 53: 205-2 14.
Zuk, M., R. Thornhill, J. D. Ligon, K. Johnson, S. Austad, S. H. Ligon, N. W.
Thornhill, and C. Costin (1990). "The role of male ornaments and courtship behavior in
female mate choice of red jungle fowl." The American Naturalist 136: 459-473.