MULTIMODAL COMMUNICATION IN THE PANAMANIAN GOLDEN FROG (ATELOPUS ZETEKI)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Joni McCullar Criswell, B.S., M.S.
*****
The Ohio State University 2008
Dissertation Committee:
Dr. Thomas E. Hetherington, Advisor
Dr. William M. Masters
Dr. Doug Nelson Approved by
Dr. Jill Soha ______Adviser Evolution, Ecology, and Organismal Biology Graduate Program
Copyright by Joni McCullar Criswell 2008
ABSTRACT
Animals may combine different types of signals (e.g. acoustic, visual, etc.) for multimodal communication. Anuran amphibians (frogs and toads) are well known for their use of acoustic signals in communication. Some anurans also may use visual signals for this purpose. The genus Atelopus (family Bufonidae) produces conspicuous movements of the forelimb and forefoot (semaphores) and provides an excellent opportunity to study multimodal (acoustic and visual) communication in an anuran amphibian.
Results of experiments using model frogs that can produce semaphores suggest that vocalizations and semaphoring actions do indeed function together in biomodal communication in A. zeteki. Target males produced significantly more behavioral responses when acoustic and visual signals were combined compared to signals presented individually. Results of these experiments suggest that semaphores may be important in providing information on the position of males. Data suggest that A. zeteki (which lacks a tympanic middle ear) is relatively ineffective at localizing sound sources, and that visual semaphore signals may facilitate the location of a vocalizing territorial male.
Although semaphores occur in communication in A. zeteki, male frogs responded almost equally well to any type of model motion and not just the specific semaphore action. The
ii semaphore is a modified stepping action, and potentially represents an exaptation for functioning in territorial communication occurring between resident and intruding males.
Studies examined the pattern of co-occurrence of semaphores and pulsed vocalizations in male A. zeteki. Signals were more likely to be followed by the same type of signal, and time delays between signals were quite variable. Analysis of the pattern of co-occurrence of semaphores and vocalizations did not provide any additional insight
into the role of these signals in multimodal communication.
Field observations established that juvenile A. zeteki also actively semaphore. In fact, semaphoring rates of juveniles were significantly higher than those of adults.
However no clear function of juvenile semaphoring was apparent from the field observations.
Tests also examined the potential use of other types of visual signals (vocal sac inflation and body color pattern) in this species. However, no evidence was found that such potential visual signals modify the territorial behavior of male A. zeteki.
iii
Dedicated to my husband Christopher “Cricket” Criswell whose love and support carried me through this journey and to
my parents, Ron and Jennifer McCullar, whose undying faith
have been a constant throughout my life.
iv
ACKNOWLEDGMENTS
I wish to thank my adviser, Tom Hetherington, for his support, direction, patience, and scientific insight throughout this process. I would like to thank all my committee members Doug Nelson, Mitch Masters, and Jill Soha for their statistical guidance, encouragement, and excellent insights.
I am grateful to Erik Lindquist who introduced me to the Panamanian Golden
Frog and helped develop my love for field work and Panama. In addition he provided data that was included in chapter four.
I am thankful for all my research assistants (Ben Lewis, Oniel Valdes, Ron
McCullar, Grace Diehl, and Scott Sapoznick) that endured the jungle and helped me collect data.
I would like to thank all the individuals that are involved in the Project Golden
Frog for allowing me use of equipment and for providing direction and companionship during field seasons.
I would like to thank the Columbus Zoo and Aquarium for allowing me access to captive animals and Pete Johangten for preparing aquariums for me to conduct lab studies.
v This research was supported by funds from the Smithsonian Tropical Research
Institution, Columbus Zoo and Ohio State Cooperative Grant, and Conservation
Collection Management Committee.
vi VITA
February 25, 1979……………………………………Born – Royston, Georgia
2000………………………………………………….B.S. Biology & Biochemistry Lee University
2006…………………………………………………M.S. Evolution, Ecology, and Organismal Biology; The Ohio State University
2001 – 2008……………………………………….Graduate Teaching and Research Associate, The Ohio State University
PUBLICATIONS
1. Erik D. Lindquist, Scott A. Sapoznick, Edgardo J. Griffith Rodriguez, Peter B. Johantgen, and Joni M. Criswell. 2007. Determination of Nocturnal Vertical Position in a Rain Forest Frog (Atelopus: Bufonidae), using Fluorescent Pigment. Journal of Herpetology. 6:37-44.
FIELDS OF STUDY
Major Field: Evolution, Ecology, and Organismal Biology
vii TABLE OF CONTENTS
Page
Abstract……………………………………………………………………………………ii
Dedication……………………………………………………………………………..….iv
Acknowledgments……………………………………………………………………...... v
Vita………………………………………………………………………………………vii
List of Tables……………………………………………………………………………..xi
List of Figures……………………………………………………………………………xii
Chapters
1. Introduction……………………………………………………………………….....1
1.1 Acoustic and visual signaling in anuran amphibians……………………...2 1.2 Semaphoring in the genus Atelopus…………………………………….....3 1.3 Major questions concerning multimodal signaling in Atelopus…………...6 1.3.1 Specific roles of semaphores and vocalizations…………………….6 1.3.2 Informational content specific to semaphores……………………....7 1.3.3 Temporal relationships between semaphores and vocalizations…....7 1.3.4 Semaphoring in juvenile Atelopus zeteki…………………………....8 1.3.5 Vocal sac inflation as a potential visual cue in Atelopus zeteki……10 1.3.6 Color as a potential visual cue in Atelopus zeteki………………….11 1.4 Research aims of this dissertation………………………………………..12 1.5 Summary…………………………………………………………………13
2. Visual signaling in the Panamanian golden frog (Atelopus zeteki)…………………15
2.1 Introduction………………………………………………………………15 2.2 Methods…………………………………………………………………..19 2.2.1 Study site……………………………………………………….…..19 2.2.2 Puppet models (model experiment)………………………………..19 2.2.3 Puppet models (motion experiment)………………....…………….20 2.2.4 Treatment types (model experiment)………………………………21
viii 2.2.5 Treatment types (motion experiment)………………………...……22 2.2.6 Experimental protocol……………………………………….…..…23 2.2.7 Video analysis……………………………………………..….……24 2.2.8 Statistical methods…………………………………………..….….25 2.3 Results……………………………………………………………...…….26 2.3.1 Stimulus period (model experiment)……………………..….…….26 2.3.2 Post-stimulus period (model experiment)………………...………..27 2.3.3 Stimulus period (motion experiment)…………...…………………27 2.3.4 Post-stimulus period (motion experiment)……………...………….28 2.4 Discussion………………………………………………………………..28
3. Patterns of co-occurrence of semaphores and vocalizations in the Panamanian golden frog (Atelopus zeteki)……………………………………………………….46
3.1 Introduction………………………………………………………………46 3.2 Methods…………………………………………………………………..49 3.2.1 Timing relationship between signals……………………………....49 3.2.2 Sound localization…………………………………………...…..…50 3.2.3 Statistical methods (sequential patterns of signals)………………..51 3.2.4 Statistical methods (timing relationship of signals)………………..52 3.2.5 Statistical methods (sound localization)…………………………...52 3.3 Results……………………………………………………………………52 3.3.1 Sequential patterns of signal types…………………………………52 3.3.2 Effects of stimulus treatments on sequential signal patterns….…...53 3.3.3 Timing relationships of signal types………………...……………..53 3.3.4 Effects of stimuli on co-occurrence patterns………………………54 3.3.5 Sound localization………………………………………………....54 3.4 Discussion………………………………………………………………..55 3.4.1 Sequential patterns of semaphores and vocalizations……………...55 3.4.2 Timing relationships between semaphores and vocalizations……..56 3.4.3 Sound localization………………………………………………….57 3.5 Summary…………………………………………………………………57
4. Studies on semaphoring in juvenile Panamanian golden frog (Atelopus zeteki)…...65
4.1 Introduction………………………………………………………………65 4.2 Methods…………………………………………………………………..68 4.2.1 Study sites………………………………………………………….68 4.2.2 Observational studies………………………………………………69 4.2.3 Juvenile experiments……………………………………………….69 4.2.4 Video analysis……………………………………………………...70 4.3 Results……………………………………………………………………70 4.3.1 Observational studies………………………………………………70 4.3.2 Juvenile experiments……………………………………………….71 4.4 Discussion………………………………………………………………..71
ix 5. Vocal sac inflation as a visual signal in the Panamanian golden frog (Atelopus zeteki)…………………………...……………………………………….75
5.1 Introduction………………………………………………………………75 5.2 Methods…………………………………………………………………..76 5.2.1 Study site…………………………………………………………76 5.2.2 Video Clips………………………………………………………77 5.2.3 Treatment types…………………………………………………..78 5.2.4 Experimental Design……………………………………………..78 5.2.5 Video Analysis…………………………………………………...79 5.2.6 Statistical Methods……………………………………………….80 5.3 Results……………………………………………………………………78 5.3.1 Comparison across treatment periods (pre-stimulus vs. post- stimulus)…………………………………………………….……80 5.3.2 Comparison between treatments (stimulus period)..……………..81 5.3.3 Comparison between treatments (post-stimulus period)…..……..81 5.4 Discussion………………………………………………………………..81
6. Color pattern and male size as a potential visual signal in intraspecific interactions of the Panamanian golden frog (Atelopus zeteki)…………………………………….88
6.1 Introduction………………………………………………………………88 6.2 Methods…………………………………………………………………..90 6.2.1 Study site…………………………………………………………...90 6.2.2 Puppet models……………………………………………………...90 6.2.3 Treatment types…..………………………………………………...90 6.2.4 Experimental protocol……………………………………………...92 6.2.5 Video analysis……………………………………………………...93 6.2.6 Statistical methods…………………………………………………94 6.3 Results……………………………………………………………………94 6.3.1 Comparison across treatment periods (pre-stimulus vs. stimulus) ..94 6.3.2 Comparison between treatment (stimulus period)……...………….95 6.3.3 Comparison between treatment (post-stimulus period)…...……….95 6.4 Discussion….…………………………………………………………….95
List of References………………………………………………………………………104
x LIST OF TABLES
Table Page 2.1 Total variance, eigenvalues, and principal component loadings derived in Principal Component Analysis for both the Model Experiment and the Motion Experiment………………...……………………………………33
2.2 Means and STDVs for each type of display measure during both the stimulus and post stimulus periods of the five treatments (L-, L+, H+, H-, and Ø+) in the Model Experiment.………………...…………..34
2.3 Means and STDVs for each type of display measure during both the stimulus and post stimulus periods for each treatment (GEN, LAT, SEM, ARMONLY) in the Motion Experiment.…………………...35
3.1 Number of responses for each category during various treatment types. For detail descriptions of the treatment types please see Chapter 2……………..60
3.2 Descriptive statistics for each signal category observed in this study…………...61
3.3 Results of Kruskal-Wallis tests (H) for latencies between five stimulus treatment types for each sequence dyad category……………………...62
5.1 Total variance, eigenvalues, and principal component loadings for scored behaviors……………………………………………………………...83
5.2 Mean and standard deviations of behavioral response measures from individual test periods for each treatment type…………………………….84
6.1 Total variance, eigenvalues, and principal component loadings for scored behaviors……………………………………………………………...97
6.2 Mean and standard deviations of behavioral response measures from each test period for treatment types………………………………………..98
xi LIST OF FIGURES
Figure Page 2.1 A) Frontal view of the model puppet frog used in the model experiment. B) Frontal view of the model puppet used in the motion experiment. Both puppet models are in the neutral position……………………36
2.2 A) Model puppet with forelimb in the highest position of the semaphoring movement. Both the model and motion experiments used this type of movement. B) Model puppet with forelimb in the farthest position of the lateral motion. This movement was only used in the motion experiment. C) Model puppet demonstrating the entire body motion in the neutral position. This movement was only used in the motion experiment. D) Detached arm in the neutral position but would be raised to the highest position as seen in the semaphore treatments (see A). This was only used in the motion experiment…………………………37
2.3 Boxplots of PC1 derived from response measured during the stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments……..…………38
2.4 Boxplots of PC2 derived from response measured during the stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.……………….39
2.5 Boxplots of PC1 derived from response measured during the post-stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments……….……...40
xii LIST OF FIGURES (continued)
Figure Page 2.6 Boxplots of PC2 derived from response measured during the post-stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.……………...41
2.7 Boxplots of PC1 derived from response measured during the stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box.. Horizontal lines connect significantly different treatments.…………………………………...…………………………42
2.8 Boxplots of PC2 derived from response measured during the stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.………………43
2.9 Boxplots of PC1 derived from response measured during the post-stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.……………...44
2.10 Boxplots of PC2 derived from response measured during the post-stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.……………...45
xiii LIST OF FIGURES (continued)
Figure Page 3.1 Representative signal sequences from each treatment during the 5 min stimulus period. Each line represents a different individual. “S” represents a semaphore and “V” represents a vocalization produced by a frog………………………………………………………………………….63
3.2 Boxplots for each signal sequence category. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Solid horizontal bars represent significant differences………...64
4.1 A) Adult male Atelopus zeteki. B) Juvenile Atelopus zeteki (dorsal view). C) Juvenile Atelopus zeteki (ventral view)………….……………74
5.1 Boxplots of PC1 derived from response measured during the pre-stimulus (solid box) and post-stimulus (white box) periods for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments………………………………………………………………………...85
5.2 Boxplots of PC1 derived from response measured during the stimulus period for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.………………………………………………..86
5.3 Boxplots of PC1 derived from response measured during the post-stimulus period for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.………...……..87
xiv LIST OF FIGURES (continued)
Figure Page 6.1 A) Frontal view of the model puppet used in the YELLOW treatment. B) Frontal view of the model puppet used in the PATTERN treatment. Both puppet models are in the neutral position………………………………………………………………………...…99
6.2 Puppet models with forelimb in the highest position of the semaphoring movement. The magnets were used to attach the puppet models to a counterweight thus providing stabilization during the semaphore motion…………………………………………………...100
6.3 Boxplots of PC1 derived from response measured during the pre-stimulus (white box) and stimulus (lined box) periods for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.…..101
6.4 Boxplots of PC1 and PC2 values derived from responses measured during the stimulus period of each treatment. PATTERN treatment is represented by lined boxes and YELLOW treatment is represented by white boxes. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed.…………………………………...…………………………………...102
6.5 Boxplots of PC1 and PC2 values derived from responses measured during the post-stimulus period of each treatment. PATTERN treatment is represented by lined boxes and YELLOW treatment is represented by white boxes. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed………….…….103
xv CHAPTER 1
INTRODUCTION
Animals employ many sensory modalities to communicate (acoustic, visual, chemosensory, etc). Recently, several behavioral studies have focused on communication involving more than one modality. In multimodal signaling the different component signals can carry different information and may interact in affecting the behavior of the receiver (Hebets and Papaj 2005; Partan and Marler 2005). For example, the temporal relationship of different signal types can dramatically affect behavioral responses (Hebets and Papaj 2005). Multimodal signaling may therefore provide more information during communication than unimodal signals (Partan and Marler 2005).
Many examples of multimodal communication employing a variety of signal combinations exist in a range of taxa. Wolf spiders (family Lycosidae) use simultaneous visual and vibratory signals in sexual interactions (Uetz and Roberts 2002). Male wolf spiders use a combination of leg raises and waves to display pigmentation and/or tufts of bristles and in addition provide while simultaneously providing percussive drumming through the substrate (Uetz and Roberts 2002). Nestling birds display visually (posturing and/or gaping) as well as vocally during begging behavior to influence parental feeding efforts
1 (Leonard et al. 2003). During courtship of the magnificent frigatebird (Fregata magnificens) the male produces a drumming sound and also inflates a brightly colored gular pouch (Madsen et al. 2004).
Several hypotheses have been proposed to explain the function of multimodal signals (Grafe and Wagner 2007). One group of hypotheses states that signals are redundant and act only to convey the same information with greater certainty. In other words, the redundant hypothesis suggests that different signals will convey the same information which can increase the accuracy of information transferred to the receiver
(Johnstone 1996; Møller and Pomiankowski 1993). Another group of hypotheses states that signals are nonredundant and convey different information during signaling (Grafe and Wagner 2007). For example the multiple message hypothesis suggests that each modality provides different information to the receiver (Grafe and Wagner 2007;
Johnstone 1996; Møller and Pomiankowski 1993).
1.1 ACOUSTIC AND VISUAL SIGNALING IN ANURAN AMPHIBIANS
Acoustic signals (vocalizations) play a prominent and extensively-studied role in anuran amphibians (frogs and toads). However, evidence suggests that visual signals also may be used in communication in this group (reviewed by Hodl and Amezquita 2001) and that bimodal communication involving both acoustic and visual signals may be
required for certain behavioral responses (Narins et al. 2003). One of the first reports of
conspicuous visual displays in anurans concerned forelimb movements in the genus
Atelopus (Crump 1988). This genus is known for its forefoot and forelimb waving
actions, which are thought to be involved in territorial defense and aggression (Crump
2 1988, Lindquist and Hetherington 1996, 1998). The Brazilian torrent frog (Hylodes
asper) vocalizes and also extends its hindlimbs while spreading the silvery-colored foot
to produce a conspicuous visual signal (Haddad and Giaretta 1999; Hodl and Amezquita
2001). Such foot-flagging actions are hypothesized to play an important role in male-
male interactions (Haddad and Giaretta 1999). The Bornean frog Staurois guttatus also
displays foot flagging and may respond to playback vocalizations with foot flagging. On
the whole, foot flagging is more commonly observed during daylight hours and
vocalizations are produced more frequently at night (Grafe and Wanger 2007). There
also is evidence that movements associated with inflation of the vocal sac can function as signals in male-female interactions in Physalaemus pustulosus. In studies using modified videotapes of males and playback vocalizations, females were more attracted to male images with vocal sac inflation during vocalization than to male images without vocal sac inflation but with playback vocalizations, suggesting that the vocal sac can serve as a visual cue (Rosenthal et al. 2004).
1.2 SEMAPHORING IN THE GENUS ATELOPUS
As mentioned above, members of the genus Atelopus (family Bufonidae) produce conspicuous movements of the forelimb and forefoot and provide an excellent
opportunity to study multimodal communication in an anuran amphibian. In addition to
this visual display, members of this genus produce a variety of vocalizations. The most
common type of vocalization consists of a series of pulsed calls and is called a pulsed
vocalization. Pulsed vocalizations are frequently made by resident males sitting in their
territories and appear to function in territorial behavior (Lindquist and Hetherington
3 1996, 1998). The visual display of Atelopus (termed here a semaphore) typically consists
of one to three forelimb rotations made while the body is braced by the opposing
forelimb (Lindquist and Hetherington 1996). Males often produce semaphores when
vocalizing or when involved in intrasexual aggression (Crump 1988; Lindquist and
Hetherington 1996, 1998).
Several functions have been hypothesized for semaphores. Much attention has
been paid to their potential role as visual signals in intraspecific communication. A role
in communication has been stressed because the Panamanian golden frog, A. zeteki, lacks
a tympanic middle ear and lives in noisy, cascading stream environments. Lack of a
tympanic middle ear has been assumed to result in reduced hearing sensitivity, although
experimental studies have found only a slight decrement in hearing thresholds at most
frequencies (Lindquist et al. 1998). It also has been suggested that the loss of a
tympanic middle ear evolved in response to the noisy stream environment that made
hearing and acoustic communication problematic. A reduction in the efficacy of acoustic
signaling potentially could lead to the evolution of the increased use of visual signals in
communication.
A dominant hypothesis concerning the role of semaphores in communication
concerns intraspecific communication between territorial males. Evidence from field
studies on adult males holding territories along streams suggests that semaphoring does
represent a form of communication in A. zeteki (Lindquist and Hetherington 1996, 1998).
Semaphoring behavior can be elicited by playback vocalizations of other males and also
by mirrored self-images, suggesting that this behavior can be triggered by the perception of other males and is directed toward specifically targeted individuals (Lindquist and
4 Hetherington 1998). Preliminary experiments using a small yellow flag attached to a
pole found that semaphoring could be elicited by rotational movements of this simple
model (Hetherington and Lindquist, pers. obs.). This evidence suggests that semaphoring does function in intraspecific communication, particularly male-male territorial defense.
Semaphoring may also function in intersexual communication prior to mate
selection. In field observations, five of seven gravid females were observed semaphoring
toward calling males, possibly as a positive response to courtship advances, as each
semaphoring female (as well as non-semaphoring females) eventually was amplexed by
the calling male (Lindquist, pers. obs.). However, obtaining information on intersexual
interactions in A. zeteki is difficult. Whereas males hold territories along streams year
round, females live in the surrounding forest and only infrequently and unpredictably come to streams when they are ready to mate. This pattern of behavior minimizes the chance of studying large numbers of intersexual interactions.
Functions other than those related to intraspecific communication also have been
proposed for semaphores. For example, semaphores have been suggested to function as
aposematic displays. Skin glands in A. zeteki produce highly toxic secretions (Fuhrman
et al. 1969), and the bright yellow body coloration of this species likely evolved as
warning coloration. Semaphores might reinforce this aposematic warning by drawing the
attention of a potential predator to the brightly colored frog. In certain species of
Atelopus (e.g., Atelopus spumarius), the body is cryptically colored (green and black)
whereas the feet, which are exposed during semaphoring, are bright red. Conspicuous
warning coloration in other animals is often accompanied by other types of signals which
include odors, rattles, shaking of wings, body movement, or even a combination of
5 several movements (Rowe and Guilford 1999). Many highly toxic amphibians (e.g.,
poison arrow frogs of the genus Dendrobates, newts of the genus Taricha, etc.) display
conspicuous body postures and movements (i.e. the unken reflex, head butting, tail
lashing, etc.) that presumably serve as warning behaviors (Duellman and Trueb 1996).
Semaphores potentially could have additional functions. For example,
semaphores could flush potential prey by startling them into moving so that frogs could
visually detect and capture them. These proposed functions are not mutually exclusive
and selection for more than one function may explain the evolution of semaphoring
behavior in the genus Atelopus. However, the only function thus far supported by
experimental data is that of a signal used in male-male territorial interactions. More
comprehensive studies are needed to determine the full function of semaphoring in this
species, and its significance compared to acoustic signaling.
1.3 MAJOR QUESTIONS CONCERNING MULTIMODAL SIGNALING IN
ATELOPUS
1.3.1 Specific Roles of Semaphores and Vocalizations
As discussed in the preceding section, there are many hypotheses about the
function of semaphores. The general consensus is that semaphores function as a male
aggressive signal (Hetherington and Lindquist 1996, 1998; Crump 1988). However, no
studies to date have focused on the specific role of semaphores vis-a-vis vocalizations during a male-male interaction. To tease apart any specific functions of these two types
6 of signals, males need to be presented with various combinations of semaphoring actions
and playback vocalizations and their responses ascertained.
1.3.2 Informational content specific to semaphores
It is not known if the semaphore motion of A. zeteki contains unique features that
convey specific information to other conspecifics. Potentially, any type of body motion
could fulfill the function of the semaphore action. The semaphore action appears to be a
modified stepping action (Lindquist and Hetherington 1998), and therefore is not a highly
derived motion. Selection for any type of generalized movement as a visual signal may
have led to the eventual use of a basic motion of this type rather than the evolution of a
highly derived movement. Therefore, any type of body motion might be interpreted as an
effective visual signal, but the use of the semaphore action evolved because it already
existed as a locomotory movement. Analysis of whether other kinds of body movements
can relay the same type of information as a semaphore would provide insight into the
evolution and precise function of the semaphore signal. For example, if males respond
equally to a more general type of movement (e.g. general shifting of the body), this would suggest that the semaphore motion rather than relaying specific types of information primarily serves to attract the attention of other males.
1.3.3 Temporal relationships between semaphores and vocalizations
The interpretation of multimodal signaling depends on the duration and frequency
of the component signals and their co-occurrence in nature (Ryan and Rand 2003).
Analysis of the pattern of co-occurrence of multimodal signals can provide insight into
7 their functional relationship. For example, in the alerting signal hypothesis, an initial
signal is used to draw the receiver’s attention to a more informative second signal (Grafe and Wagner 2007). Alternatively, A. zeteki may use visual and acoustic signals simultaneously to increase the probability of any signal being detected (redundant signals). Analysis of the sequential relationship of semaphores and vocalizations potentially could test which hypothesis applies to the visual and acoustic signaling of A. zeteki.
Although multimodal communication may be used during courtship, aggression, and predator avoidance, studies dealing with the frequency and temporal spacing of signals have mainly focused on female-male interactions (reviewed by Ryan and Rand
2003). For example, as discussed above, both vocal sac inflation and vocalizations affect female choice in P. pustulosus (Rosenthal et al. 2004). Because studies of intersexual communication are difficult to perform with A. zeteki, study of multimodal
communication in a natural setting for this species will be restricted to male-male
interactions.
1.3.4 Semaphoring in juvenile Atelopus zeteki
Until now all detailed observations and experiments on semaphoring behavior
have focused on adult frogs. However, semaphores sometimes are produced by juvenile
A. zeteki (pers. obs.). The occurrence of semaphoring behavior in juveniles may help us
understand the function of semaphoring behavior in general. Juvenile A. zeteki frequently
are observed living in the vicinity of streams, but they do not hold territories as adult
males do, and they sometimes are found clustered in small groups (pers. obs.).
8 Therefore, besides being non-reproductive, juveniles also do not display territorial aggressive behavior. Juvenile A. zeteki also differ from adults in their body coloration.
Metamorphic juveniles are cryptically colored with bands of black and green and effectively blend into a mossy background. As individuals grow and age, areas of green coloration become yellow and areas of black become proportionately smaller (Lindquist and Hetherington 1998), resulting in adults that in some cases are entirely yellow in coloration. Although juveniles, especially very young ones, are more cryptically colored than adults, they do possess bright yellow palmar and plantar surfaces. When juveniles semaphore, the bright yellow coloration is effectively displayed. Because juveniles do not maintain territories or engage in mating behavior (and adults seem to use semaphoring as an aggressive signal) such conspicuous semaphoring actions that display brightly colored feet likely do not function in intraspecific communication. However, a function as an aposematic warning signal or as a mechanism to flush prey remains feasible in such young individuals.
Observational studies need to be performed to confirm that semaphores are consistently produced by juveniles. Observations of juvenile behavior when semaphoring can provide insight on the function of semaphoring in juveniles. For example, if juveniles tend to catch prey after semaphoring this might suggest that juveniles use the semaphore to flush prey. If juveniles respond to a threat with a semaphore this might suggest that semaphores represent aposematic signals to potential predators.
9 1.3.5 Vocal sac inflation as a potential visual cue in Atelopus zeteki
In addition to studying limb movements, much attention has been paid to how vocal sac inflations are a visual signal accompanying vocalizations. In playback experiments on Epipedobates femoralis, physical attacks can be elicited from territorial males only when a bimodal stimulus involving both vocalizations and vocal sac pulsations is provided (Narins et al. 2003). Males of the species Phrynobatrachus krefftii possess a yellow vocal sac that is highly visible during vocal sac inflation, but males also inflate and deflate their vocal sac without sound production (Hirschmann and Hodl
2006), potentially providing a visual signal in the absence of acoustic cues. In studies on tungara frogs (Physalaemus pustulosus) video stimuli were produced that allowed vocal sac inflation to be disconnected from vocalizations. In these studies female tungara frogs preferred males producing calls synchronized with vocal sac inflation (Rosenthal et al.
2004).
Species of Atelopus also produce conspicuous inflations and deflations of their vocal sacs during sound production, and the vocal sacs often are brightly colored. For example, the body of Atelopus flavescens is yellow but the vocal sac is bright pink, and in some populations of A. varius, the body is yellow and black but the vocal sac is bright red
(pers. obs.). Given the evidence that the genus Atelopus employs visual signals (i.e., semaphores) during communication, this genus is a likely prospect for the use of vocal sac motion as a visual signal as well. For example, if vocal sac inflation can elicit a behavioral response during a male-male interaction this would suggest males use this motion as a signal.
10 1.3.6 Color as a potential visual cue in Atelopus zeteki
Intraspecific visual communication may not be limited to movement displays.
Many species utilize color to influence social interactions (Fodgen and Fodgen 1974).
Colored ornaments typically function in signaling individual quality in intra- and intersexual interactions (Solis et al. 2008). However color is not solely limited to the expression of quality. It can be used to signal warnings or aggressive intent, and even to attract prey (Drickamer et al. 2002). For example, the red belly of male three-spined stickleback fish (Gasterosteus aculeatus) appears to advertise a territorial claim and can elicit aggressive behavior in other males (Bakker 1986). The role of coloration has been well studied in many species including anurans. Non-cryptic coloration in anurans is typically considered to function as aposematic (warning) coloration (e.g., poison arrow frogs of the family Denbrobatidae). But, as discussed earlier, there is also evidence that coloration functions as a visual signal among conspecifics of certain species, such as
Phyrnobatrachus krefftii. In these species males are reported to perform nonaudible vocal-sac inflations during intraspecific interactions suggesting that vocal sac color may play a role in male-male interactions (Hirschmann and Hodl 2006).
Most species of Atelopus, including A. zeteki, are brightly colored, typically considered aposematic coloration related to their high skin toxicity. Coloration and patterning in A. zeteki is variable and changes dramatically during growth (Lindquist and
Hetherington 1998). There is an ontogenetic shift toward more yellow coloration with increasing body size, and this may be related to increased toxicity associated with incorporation of food-based toxins (Lindquist and Hetherington 1998). However, it is possible that coloration also may function in intraspecific communication. Males may
11 recognize and be intimidated by larger males with more yellow coloration, and female
mate choice may involve selection for more yellow coloration as an indicator of male
fitness. In an experimental setting, if males responded differently to models of varying
size and coloration it would suggest that males use coloration during intrasexual
interactions.
1.4 RESEARCH AIMS OF THIS DISSERTATION
This dissertation is organized into the following chapters that together aim to
provide a general understanding of multimodal (acoustic and visual) communication in A.
zeteki.
Chapter 2: This study aimed to test whether semaphores can elicit a change of
behavior in a receiver and how semaphores are functionally related to vocalizations. To
test these questions resident males were presented with a puppet model that displayed at
various semaphore rates with or without playback vocalizations. Tests were also
conducted to determine whether A. zeteki responds specifically to the semaphore
movement or whether general body movement can elicit the same behavioral response
from males.
Chapter 3: This study examined the pattern of co-occurrence of semaphores and
pulsed vocalizations in male A. zeteki. Information on the pattern of co-occurrence of
these two different signals may provide insight into how semaphores and vocalizations
function in multimodal communication. This study also included an investigation of the
ability of male A. zeteki to localize conspecific vocalizations, as this information may provide insight into the functional significance of the visual semaphore signal.
12 Chapter 4: This study examined the role of semaphoring in juvenile A. zeteki to further understand the function of semaphoring in this species. Observational studies were performed in the field to determine the relative frequency of semaphoring behavior in different sized (aged) individuals. Videotapes of naturally behaving frogs also were analyzed to examine whether semaphores are used to flush prey. Lastly, to test for a possible role of semaphoring as an aposematic behavior, field observations of the responses of juvenile frogs to a potential threat were performed.
Chapter 5: This study examined the potential role of vocal inflation and deflation as a visual signal during territorial interactions between male A. zeteki. Males were
presented with edited video clips of various combinations of vocal sac inflations and/or
playback vocalizations.
Chapter 6: This study examined whether color pattern and body size are involved
in visual communication in A. zeteki. Males were presented with varying sized puppet
models with different color patterns to see if color and body size could modify behavioral
responses.
1.5 SUMMARY
Behavioral experiments described in this dissertation demonstrate that
vocalizations and semaphoring actions do indeed function together in bimodal
communication in A. zeteki. However, although a combination of acoustic and visual
signals appear to be the most effective in increasing territorial male behaviors, acoustic
signals (vocalizations) remain the primary modality for communication in this species. A
variety of studies found no additional role of semaphores in multimodal communication
13 in adult male A. zeteki. Field studies confirmed that juvenile A. zeteki do semaphore as well, although the function of these visual signals in juveniles remains unclear. This dissertation also describes tests examining the potential use of other types of visual signals (vocal sac inflation and body color pattern) in this species. However, no evidence was found that such potential visual signals modify behavior of male A. zeteki.
14 CHAPTER 2
VISUAL SIGNALING IN THE PANAMANIAN GOLDEN FROG (ATELOPUS
ZETEKI)
2.1 INTRODUCTION
Animals employ several sensory modalities to communicate (acoustic, visual, chemosensory, etc). Recent studies have begun to focus on communication involving more than one modality. In multimodal signaling the different component signals can carry different information and can interact in affecting the behavior of the receiver
(Hebets and Papaj 2005; Partan and Marler 2005). For example, the temporal
relationship of different signal types can dramatically affect behavioral responses (Hebets
and Papaj 2005). Multimodal signaling may therefore provide more information during
communication than unimodal signals (Partan and Marler 2005).
Many examples of multimodal communication employing a variety of signal
combinations exist in a variety of taxa. Wolf spiders (family Lycosidae) use
simultaneous visual and vibratory signals in sexual interactions (Uetz and Roberts 2002).
Male wolf spiders use a combination of leg raises and waves to display pigmentation
and/or tufts of bristles while simultaneously providing percussive drumming through the substrate (Uetz and Roberts 2002). Nestling birds display visually (posturing and/or
15
gaping) as well as vocally during begging behavior to influence parental feeding efforts
(Leonard et al. 2003). During courtship of the magnificent frigatebird (Fregata magnificens) the male produces a drumming sound and also inflates a brightly colored gular pouch (Madsen et al. 2004).
Acoustic signals (vocalizations) play a prominent and extensively studied role in anuran amphibians (frogs and toads). However, evidence suggests that visual signals also may be used in communication (reviewed by Hodl and Amezquita 2001) and that bimodal communication involving both acoustic and visual signals may be required for certain behavioral responses (Narins et al. 2003). One of the first reports of conspicuous visual displays in anurans concerned forelimb movements in the genus Atelopus (Crump
1988). This genus is known for its forefoot and forelimb waving actions which are thought to be involved in territorial defense and aggression (Crump 1988, Lindquist and
Hetherington 1996, 1998). The brazilian torrent frog (Hylodes asper) vocalizes and also
extends its hindlimbs while spreading the silvery-colored foot to produce a conspicuous
visual signal (Haddad and Giaretta 1999; Hodl and Amezquita 2001). Such foot-flagging
actions are hypothesized to play an important role in male-male interactions (Haddad and
Giaretta 1999). The Bornean frog Staurois guttatus also displays foot flagging and
responds to playback vocalization with foot flagging. Foot flagging often occurs soon
after vocalization and vocalization shows a greater latency after a foot flag. Foot
flagging is more commonly observed during daylight hours and vocalizations more
commonly at night (Grafe and Wanger 2007).
Much attention also has been paid to how inflation and deflation of a vocal sac
may provide a visual signal to accompany vocalizations. In playback experiments with
16
Epipedobates femoralis, physical attacks can be elicited from territorial males only when
a bimodal stimulus involving both vocalizations and vocal sac pulsations is provided
(Narins et al. 2003). Males of the species Phrynobatrachus krefftii possess a yellow
vocal sac that is highly visible during call production, but males also inflate and deflate
their vocal sac without sound production (Hirschmann and Hodl 2006), potentially
providing a visual signal in the absence of acoustic cues. In studies on tungara frogs
(Physalaemus pustulosus) video stimuli were produced that allowed vocal sac inflation to
be disconnected from vocalizations. In these studies female tungara frogs preferred
males producing calls synchronized with vocal sac inflation (Rosenthal et al. 2004). All
of these observations and studies suggest that multimodal communication involving both
acoustic and visual signals may play an important role in anuran communication.
As mentioned above, the genus Atelopus (family Bufonidae) produces
conspicuous movements of its forelimb and forefoot and provides an excellent opportunity to study multimodal communication in an anuran amphibian. In addition to visual displays, members of this genus produce a variety of vocalizations. The most common type of vocalization consists of a series of sound bursts and is called a pulsed vocalization. Pulsed vocalizations are frequently made by resident males sitting in their territories and appear to function in territorial behavior (Lindquist and Hetherington
1996, 1998). The visual display of Atelopus, termed here semaphores, typically consists of one to three forelimb rotations made while the body is braced by the opposing forelimb (Lindquist and Hetherington 1996). Males often produce semaphores when vocalizing or when involved in intrasexual aggression (Crump 1988; Lindquist and
Hetherington 1996, 1998).
17
Evidence from field studies on male behavior suggests that semaphoring does
represent a form of communication in the panamanian golden frog A. zeteki (Lindquist
and Hetherington 1996, 1998). These studies were performed on males holding
territories along streams. Semaphoring behavior can be elicited by playback of
vocalizations of other males and also by mirrored self-images, suggesting that this
behavior can be triggered by the perception of other males in the area and is directed
toward specifically targeted individuals (Lindquist and Hetherington 1998). Preliminary
experiments using a small yellow flag attached to a pole found that semaphoring could be
elicited by rotational movements of this simple model (Hetherington and Lindquist, pers. obs.) In addition, forelimb movement of a “puppet” model of A. zeteki resulted in a frog
attacking this model (Lindquist, pers. obs.). This evidence suggests that semaphoring does function in intraspecific communication, particularly in male-male territorial defense. However, the full function of semaphoring in this species and its significance compared to acoustic communication have not previously been investigated.
The present study aimed to test whether semaphores in A. zeteki can elicit a
change of behavior in a receiver, and how semaphores are functionally related to
vocalizations. To test these questions we presented resident males with a puppet model
(Figure 2.1) that displayed at various semaphore rates with or without playback
vocalizations. We also set out to test if A. zeteki responds specifically to the semaphore
movement or if any general type of body movement can elicit the same behavioral
response from males. Recent evidence shows that a specific movement (vocal sac
inflation) can be important in male-female interaction in Physalaemus pustulosus.
Females significantly prefer vocal sac inflation over vocalization alone without vocal sac
18
inflation, suggesting that the vocal sac can serve as a specific visual cue (Rosenthal et al.
2004). Since the semaphore is unique to A. zeteki, a response to a specific semaphore
motion would suggest that semaphores may be used for species identification. If males
respond equally to a more general type of movement, this would suggest that the
semaphore motion primarily serves to attract the attention of other males. Tests on the
specificity of responses to semaphore involved a variety of model movements combined
with playback vocalizations.
2.2 METHODS
2.2.1 Study Site
Field observations were carried out on a population of Atelopus zeteki in Parque
Nacional General de División Omar Torrijos Herrera in the Coclé province of Panama at
the Rio Marta (elevation approximately 300m) between 08:00 and 17:00 hours from July
to September 2002 between (model experiment) and from December to February 2004
(motion experiment).
2.2.2 Puppet Models (Model Experiment)
Puppet frog models were made from plastic frogs of about the same size as adult
male A. zeteki (approximately 35 mm snout-to-vent length). The models were painted to
resemble a typical male adult frog – goldenrod yellow with narrow black bands. A gray
metal rod (2 mm diameter by 288 cm) penetrated the model’s body transversely, running
from one side and connecting into the opposite arm (Figure 2.1). To approximate a
semaphore, the rod was rotated slightly, lifting the forelimb with the hand facing forward
19
(Figure 2.2). The frog puppet was glued by its belly to a small, thin stone (collected from
a local stream) to provide sufficient stability to allow rotation of the rod. Five models
were used to minimize pseudoreplication; in four models the right arm moved and in one
model the left arm moved.
2.2.3 Puppet Models (Motion Experiment)
The same types of puppet frog models were used as in the model experiment except that minor changes were made to allow for different types of movements. Four
types of movements were tested: (1) the standard semaphore motion, (2) a motion in
which the entire arm was pushed laterally away from the body, (3) side-to-side motion of
the entire model body, and (4) semaphore motion of a detached arm. To approximate a
semaphore the same type of movement was made as described above. A lateral
movement of the forelimb was produced by pushing the rod in and out of the model. A
counterweight was required to stabilize the puppet model during the production of semaphore and lateral arm motions. Puppet models had a small magnet glued on their undersides that attached to a rock with its own attached magnet (Figure 2.1). No counterweight was required during side-to-side movements of the entire frog model
(Figure 2.2). For the test involving isolated arm movement, arms were removed from the puppet body (Figure 2.2). Five models were used in order to minimize pseudoreplication; in three models the right hand moved and in two models the left hand moved.
20
2.2.4 Treatment Types (Model Experiment)
Five treatments were conducted to test male responses to different combinations
of model semaphores and playback vocalizations. These treatments included two rates of
semaphoring (a low rate of 1 min-1 and a high rate of 5 min-1) that cover a typical range
of semaphoring rates seen in males interacting in the field. Treatments are coded based on their semaphore rate (L = low, H = high, Ø = none) and presence of playback vocalization (+ = present, - = absent). All playback periods consisted of a continuous series of pulsed vocalizations on an endless cassette loop played through a Sony TCD-D5 cassette recorder. Pulsed call recordings were obtained from males in the Rio Marta population. Because of the high background noise of cascading-stream habitats, good quality recordings (with minimal background noise) were obtained from only two males.
The bouts of calling for each male lasted about 30 sec with a 10 sec pause between each call. Two bouts of calling were recorded from each male (2 exemplars), thereby providing four calls for use in playback tests. The four calls were distributed randomly but equally throughout those tests that included playback vocalizations. To address concerns about pseudoreplication, an ANOVA was performed to determine if there was any significant difference between response measures to the two stimulus males’ vocalizations; none was found (p = 0.909). Sound amplitude was set at 86 dB at 1 m from the speaker (linearly calibrated with a Quest 215 sound level meter). This sound pressure is comparable to that produced by a male calling at 1 m.
In treatment L- models semaphored at a low rate (1 min-1) without playback
vocalizations, in treatment H- models semaphored at a high rate (5 min-1) without
playback vocalizations, in treatment H+ models semaphored at a high rate (5 min-1) with
21
playback vocalizations, in treatment L+ models semaphored at a low rate (1 min-1) with
playback vocalizations, and in treatment Ø+ models produced no semaphores with
playback vocalizations. Treatments were randomly assigned to individual males.
2.2.5 Treatment Types (Motion Experiment)
Four types of treatments were conducted to test male responses to different combinations of model movements and playback vocalizations. All treatments used a relatively high rate of movement (5 min-1) that is nonetheless within the typical range of
semaphoring behavior seen in males interacting in the field. This rate of movement,
along with playback vocalizations, elicited the greatest behavioral response in the initial
model experiment.
All stimulus periods contained a continuous series of pulse vocalizations on an
endless cassette loop played through a Sony T-CD-D5 cassette recorder. Pulsed call
recordings were obtained from the Borror Laboratory of Bioacoustics at The Ohio State
University. Five male recordings with minimal background noise from a nearby
population of A. zeteki (~ 10 km away) were used to make playback stimuli. The bouts
of calling for each male lasted about 30 sec with a 10 sec pause between each calling
bout. Five exemplars from five different males were created to use in playback tests. The
five exemaplars were distributed randomly but equally throughout all tests. Sound
amplitude was set at 86 dB at 1 m from the speaker (linearly calibrated with a Quest 215
sound level meter). This sound pressure is comparable to that produced by a male calling at 1 m.
22
In treatment GEN, the entire model were moved side to side over a range of
approximately 2 cm at a rate of 5 min-1 (Figure 2.2). In treatment LAT, one arm was
moved laterally away from the body approximately 1 cm (total range of movement 2 cm) at the rate of 5 min-1. This lateral motion contrasts with the semaphore action, which
mostly involves limb rotation around a horizontal axis (Figure 2.2). In treatment SEM,
models produced the typical semaphore motion (5 min-1) (Figure 2.2). In treatment
ARMONLY, the typical semaphore motion was produced by isolated arms detached from model bodies (5 min-1) (Figure 2.2).
2.2.6 Experimental Protocol
All tests were performed on resident males sitting on territories along their rocky
stream habitat. After a male frog was located, a full range Audix PH-3 speaker was
placed approximately 1 m from, and directed toward, the individual. Speaker position
and distance varied due to cascade topography and location of each animal. A puppet
model of a male golden frog was placed directly (between 0.3 to 0.5m) in front of the sound speaker. The observer who operated the puppet sat about 0.5 m behind the speaker, and another observer, who videotaped the trials, sat at least 3 m away from the frog and speaker. Both observers remained silent and motionless (expect for the motion needed to operate the model) throughout the experimentation period. Tests were conducted in the following manner. A 5-min pre-stimulus period allowed the test subject to acclimate to the presence of the speaker and observers. This was followed by a 5-min stimulus period during which signals (semaphores, movements and/or vocalizations) were presented to the test subject, followed by a 5-min post-stimulus period. All
23
behavioral responses during each period were videotaped. Twenty different male frogs
were tested in each treatment for both experiments, expect for treatment ARMONLY of
the motion experiment, which included 15 test males. Treatments were randomly
distributed throughout the field season. Male frogs typically occupy discrete and stable
territories along the stream. Body size and color pattern characteristics were recorded for
each test animal, and each male territory also was flagged, to ensure that no individual
was tested more than once. In the motion experiment animals were photographed to help
with individual identification.
2.2.7 Video Analysis
In scoring the videotaped experiments, we recorded all behavioral displays
exhibited by the test subject, but only behaviors that were typically observed and believed
to be biologically significant in territorial interactions were analyzed. These included the
following:
(1) Semaphores - the conspicuous forelimb rotation described by Lindquist and
Hetherington (1998). Although some males seemed to produce semaphore-like actions with their hindlegs, these were rare and the numbers were insufficient for statistical analysis.
(2) Vocalizations (pulsed calls) - see Introduction for description.
(3) Attacks - approach and contact with the puppet model. A male frog typically first
contacted the model with its forelimbs and then climbed on top of the model’s back. The frog would subsequently dismount and often begin to walk around the model. In natural
conditions, male A. zeteki sitting on territories will engage interloping males in combat,
24
with “winners” often climbing on the backs of “losers” and pushing the latter toward the
ground. In our study test frogs did not show such “fighting” behavior, likely because the
puppet models, once mounted, did not provide sufficient cues to trigger further
aggressive actions. Approaches did not always result in an attack. However, when target
males came within ≤5 cm of the puppet model they typically attacked the model.
(4) Orienting movements - males turned their body. This movement could be directed
toward or away from the puppet model or speaker.
2.2.8 Statistical Methods
We used Principal Component Analysis (PCA) to reduce the four behavioral
variables to a smaller set of PC scores for statistical analysis (Cooley and Lohnes 1971).
We conducted PCAs separately on the stimulus and post stimulus periods of treatments
(total of 4 PCAs across both experiments). Mann-Whitney U tests were then used to test
for the influence of treatment type (α = 0.025 and α = 0.0125). Comparisons for the
MODEL experiment included semaphore rate only, vocalization only, and combined
signals. Comparisons for the MOTION experiment included SEM vs. other movements,
ARMONLY vs. other movements, GEN vs. LAT. All statistical calculations were
conducted using SPSS 16.0 (2008). Bonferroni procedures were used to adjust for the
alpha in multiple comparisons (Chandler 1995).
25
2.3 RESULTS
2.3.1 Stimulus Period (Model Experiment)
Principal Component Analysis reduced four behavioral display measures to two
composite measures explaining 71% of the variance (Table 2.1) for the stimulus period.
The first axis (PC1) was strongly correlated with the number of semaphores, orienting
movements, and attacks (Table 2.1). Mann-Whitney tests showed a significant difference
between treatment types L- and Ø+ (p=0.001, U=83.5, Figure 2.3) and H- and Ø+
(p=0.000, U=68.0, Figure 2.3). In both cases subjects gave more semaphores, orienting
movements, and attacks in treatment Ø+ than treatment L- or H- (Table 2.2). There was
also a significant difference between treatment types H+ and L- (p=0.000, U=48.0,
Figure 2.3) and a significant difference between treatment types H+ and H- (p=0.000,
U=36.5, Figure 2.3). Treatment H+ elicited more semaphores, orienting movements, and
attacks than either treatment L- or H- (Table 2.2).
The second axis (PC2) was strongly correlated with the number of vocalizations
produced during the stimulus period (Table 2.1). There was a significant difference
between treatment types L- and Ø+ (p=0.021, U=115.5, Figure 2.4) and treatment types
H- and Ø+ (p=0.000, U=71.0, Figure 2.4). In both cases subjects produced more
vocalizations in L- and H- than Ø+ (Table 2.2). There was also a significant difference between treatment types H+ and L- (p=0.010, U=115.5, Figure 2.4) and treatment types
H+ and H- (p=0.000, U=61.5, Figure 2.4). Treatments L- and H- elicited a greater number of vocalizations than treatment H+ (Table 2.2).
26
2.3.2 Post-stimulus Period (Model Experiment)
The PCA for the post stimulus period reduced the four behavioral display
measures to two composite measures (Table 2.1) explaining 71% of the variance (Table
2.1). The first axis (PC1) was strongly correlated with the number of vocalizations,
semaphores, and orienting movements and the second axis (PC2) was strongly correlated
with the number of attacks (Table 2.1). No significant differences were found between
treatments for either of these component measures (Figure 2.5 and 2.6).
2.3.3 Stimulus Period (Motion Experiment)
Principal Component Analysis reduced the four behavioral display measures to
two composite measures explaining 68% of the variance (Table 2.1) for the stimulus
period. The first axis (PC1) was strongly correlated with the number of orienting
movements and semaphores (Table 2.1). Mann-Whitney tests showed a significant
difference between treatment types SEM verses LAT (p=0.012, U=33.5, Figure 2.7).
Treatment SEM produced more orienting movements and semaphores than treatment
LAT (Table 2.3).
The second axis (PC2) was strongly correlated with the number of attacks (Table
2.1). There was a significant difference between treatment types LAT and ARMONLY
(p=0.025, U=83.5, Figure 2.8). Treatment LAT elicited more attacks than treatment
ARMONLY (Table 2.3).
27
2.3.4 Post-stimulus Period (Motion Experiment)
The PCA for the post stimulus period reduced the four behavioral display
measures to two composite measures explaining 78% of the variance (Table 2.1). The
first axis (PC1) was strongly correlated with the number of orienting movements,
vocalizations, and attacks and the second axis (PC2) was strongly correlated with the
number of semaphores (Table 2.1). There were no significant differences between any
treatment types in either of these components (Figure 2.9 and 2.10).
2.4 DISCUSSION
The results of the model experiment suggest that vocalizations and semaphoring
actions do function together in bimodal communication in A. zeteki. Target males
produced significantly more semaphores, vocalizations, attacks, and orienting movements
when playback vocalizations were combined with semaphores than when semaphores
were presented alone. In some cases the combined stimuli had effects comparable to
playback vocalizations alone (e.g., orienting movements). Semaphores alone elicited the
most vocalizations during the stimulus period; however, the latter effect was due to the
behavior of only a few males. In treatment L- three males gave vocalizations (N=24, 7, 4
respectively) and in treatment H- one male produced all of the vocalizations (N=16). So
although the behavior of target males could be changed by either semaphores or playback
vocalizations alone, effects on behavior typically were greater when both signals were
combined. For most of the behavioral responses (e.g., semaphoring, attacks, and
orienting movements) the responses of male frogs receiving a combination of both types
of signals was greater to either signal alone and also greater than one would expect from
28
an arithmetic sum of responses to the signals presented separately. For example, the
number of semaphores produced by target males during the H- and Ø+ treatments was 23
and 66, respectively, whereas the H+ treatment had a total of 169. Furthermore, the most
aggressive behavior, actual attacks on the puppet models, occurred only in the presence
of both semaphores and vocalizations, and occurred most when vocalizations were
combined with high rates of semaphoring by the model.
Multimodal communication typically requires that the different signal
components occur within a relatively short time frame (Hebets and Papaj 2005). This
study did not explore the effects of the temporal relationship between semaphoring and
vocalizations on the behavioral responses of a receiver however this will be addressed in
Chapter three. In addition there are no data on the temporal relationship of these signals
in naturally behaving male frogs. Nonetheless, the finding that a combination of
semaphores and vocalizations produces a greater effect on the behavior of target frogs
than either stimulus alone certainly suggests that bimodal communication is occurring in
A.zeteki.
Although a combination of acoustic and visual signals appears to be most
effective in eliciting territorial behavior in A. zeteki, this study suggests that acoustic
signals (vocalizations) are an especially important modality for communication in this
species. Compared to semaphores only, vocalizations alone produced significantly more
semaphore responses during the stimulus period and more orienting movements during
both the stimulus and post-stimulus periods. Given the fundamental role that
vocalizations play in many aspects of anuran reproductive and territorial behavior, this is
29
not surprising. The visual semaphoring signal of this genus is almost certainly a more
recent, derived behavior compared to vocalizations.
One interesting pattern observed in these studies was the low number of vocalizations made by target males during the stimulus period of experiments with playback calls. In trials with playback calls males possibly waited and/or produced other non-acoustic behaviors until the end of calling bouts before responding with vocalizations of their own. This is supported by the observed increase in number of vocalizations during the post stimulus period of these tests.
The results of this study suggest that semaphores may be especially important in providing information on the position of aggressively interacting males. Target males showed an increase in orienting movements during the stimulus period of trials L+ and
Ø+, suggesting they had more difficulty localizing a potentially intruding male when semaphores of the puppet model were minimal or absent. Also, orienting movements were more frequently observed in the post stimulus period in treatment Ø+. In this case target males aroused by playback vocalizations may have continued to look for the calling male in the absence of any identifying semaphoring movements by the model during the stimulus period. In all other treatments semaphores were displayed by the puppet model, possibly helping to direct target males toward the “intruder.”
A possible interpretation of these experiments is that male A. zeteki identify the presence of a potentially intruding male by hearing vocalizations of the latter, but the males may be poor at localizing the source of the vocalizations. These frogs live in noisy cascading stream habitats with significant background noise (Crump 1988; Lindquist and
Hetherington 1996), and they also lack tympanic middle ears. Although this species can
30
hear fairly well without a tympanic middle ear (Lindquist and Hetherington, 1998),
absence of such auditory structures may impair their abilities to localize the sources of
sounds. Without a conspicuous visual signal (i.e., semaphore) from a calling intruder, the resident males may have difficulty determining the position of the caller.
Results of the body motion experiment demonstrated that male frogs responded
almost equally to any type of model motion and not just to the specific semaphore action.
A significant difference was seen in the first principal component during the stimulus
period between treatment types LAT and SEM with treatment SEM eliciting more
semaphores and orienting movements (Table 2.3). In contrast to our results with
Atelopus semaphoring, Phsalaemus pustulosus males responded more specifically to
normal vocal sac inflation than to manipulated images of vocal sac motion (Rosenthal et
al. 2004).
The lack of a tympanic middle ear (with possibly decreased directional resolution)
and the presence of environmental noise might provide a scenario that could explain the
adaptive advantage of semaphoring in this species. Although vocalizations are key in triggering territorial behaviors, the limited ability to localize sound sources may have
provided selective pressure for the production of a conspicuous visual signal (i.e.,
semaphore). Semaphoring by a territorial male could provide information on his precise
position to intruders and potentially stop an intruder from entering the territory and
initiating an aggressive encounter. The semaphore motion most likely arose from the walking motion of this species (Lindquist and Hetherington). Any type of easily visible motion potentially can serve this signaling function, but the semaphore as an exaggerated stepping motion could readily be modified to produce a conspicuous visual signal.
31
This study provides clear evidence that the visual signal represented by semaphoring functions in communication in A. zeteki. Other visual signals may also be
used by this species. Specifically, calling males also inflate and deflate their brightly
colored (yellow) vocal sacs, and this action could also provide a visual signal as demonstrated in Epipedobates (Narins et al. 2003) and Physalaemus pustolosus
(Rosenthal et al. 2004). It would interesting to incorporate both movements (semaphores
and vocal sac inflations) into an experiment to determine whether if both visual actions
combine to have a more pronounced effect on male behavior.
32
PC 1 PC 2
Stimulus Period
Eigenvalues 1.85 0.99
% of Variance 46.2 % 24.7 %
Semaphores 0.801 0.016
Vocalizations -0.166 0.985 Model Attacks 0.754 0.105 Experiment Orienting Movements 0.783 0.092
Post-Stimulus Period Eigenvalues 1.82 1.02 % of Variance 45.5 % 25.4 %
Semaphores 0.791 -0.051
Vocalizations 0.815 -0.183 Attacks 0.091 0.981 Orienting Movements 0.723 0.139
Stimulus Period Eigenvalues 1.71 1.01 % of Variance 42.7 % 25.1 %
Semaphores 0.785 -0.269 Motion Vocalizations 0.584 -0.231 Experiment Attacks 0.286 0.932
Orienting Movements 0.817 0.097 Post-Stimulus Period Eigenvalues 2.18 0.93
% of Variance 54.4 % 23.2 % Semaphores 0.480 0.848 Vocalizations 0.788 0.027
Attacks 0.779 -0.445
Orienting Movements 0.847 -0.096
Table 2.1: Total variance, eigenvalues, and principal component loadings derived in Principal Component Analysis for both the Model Experiment and the Motion Experiment.
33
Behavior (Stimulus) L- L+ H+ H- Ø+ Semaphores 0.95 ± 3.35 0.05 ± 0.22 7.5 ± 7.17 4.7 ± 5.08 2.2 ± 4.42 Vocalizations 1.75 ± 5.52 0.8 ± 3.58 0.1 ± 0.31 0.65 ± 1.76 0.05 ± 0.22 Attacks 0 0 0.75 ± 1.16 0.35 ± 0.99 0 Orienting Movements 0.4 ± 0.82 0.35 ± 0.99 3.8 ± 3.05 6 ± 4.67 4.85 ± 4.82 Behavior (Post Stimulus) L- L+ H+ H- Ø+ Semaphores 0.4 ± 1.19 1.2 ± 3.44 0.95 ± 1.54 1.8 ± 3.14 0.35 ± 0.75 Vocalizations 0.25 ± 1.12 1.7 ± 4.37 4.25 ± 7.83 3.7 ± 6.65 1.5 ± 2.96 Attacks 0 0 0.15 ± 0.49 0 0 Orienting Movements 0.1 ± 0.31 0.2 ± 5.2 0.9 ± 1.41 0.95 ± 1.23 1.55 ± 2.06
34 Table 2.2: Means and STDVs for each type of display measure during both the stimulus and post stimulus periods of the five treatments (L-, L+, H+, H-, and Ø+) in the Model Experiment.
Behavior (Stimulus) GEN LAT SEM ARMONLY Semaphores 6.1 ± 8.39 2.25 ± 4.30 6.3 ± 7.60 7.20 ± 8.01
Vocalizations 0.75 ± 2.31 0 0.3 ± 0.73 0
Attacks 0.4 ± 1.14 0.5 ± 1.15 0.4 ± 1.10 0 Orienting Movements 3.2 ± 3.85 3.5 ± 3.89 4.65 ± 4.03 7.67 ± 6.68 Behavior (Post Stimulus) GEN LAT SEM ARMONLY Semaphores 2.05 ± 4.59 1.5 ± 3.35 4.5 ± 10.09 3.20 ± 7.18 Vocalizations 4.9 ± 7.67 3.25 ± 9.70 2.75 ± 5.27 4.20 ± 8.54 Attacks 0 0.05 ± 0.22 0.1 ± 0.45 0 35 Orienting Movements 1.3 ± 2.13 1.8 ± 3.38 2 ± 4.10 2.73 ± 3.88
Table 2.3: Means and STDVs for each type of display measure during both the stimulus and post stimulus periods for each treatment (GEN, LAT, SEM, ARMONLY) in the Motion Experiment.
A
B
Figure 2.1: A) Frontal view of a model puppet frog used in the model experiment. B) Frontal view of a model puppet used in the motion experiment. Both puppet models are in the neutral position. .
36
A B
C D
Figure 2.2: A) Model puppet with forelimb in the highest position of the semaphoring movement. Both the model and motion experiments used this type of movement. B) Model puppet with forelimb in the farthest position of the lateral motion. This movement was only used in the motion experiment. C) Model puppet demonstrating the entire body motion in the neutral position. This movement was only used in the motion experiment. D) Detached arm in the neutral position but would be raised to the highest position as seen in the semaphore treatments (see A). This was only used in the motion experiment.
37
Figure 2.3: Boxplots of PC1 derived from response measured during the stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.
38
Continue to 7.49
Figure 2.4: Boxplots of PC2 derived from response measured during the stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments. .
39
Figure 2.5: Boxplots of PC1 derived from response measured during the post-stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.
40
Continues to 8.6
Figure 2.6: Boxplots of PC2 derived from response measured during the post-stimulus period of each treatment in the Model Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.
41
Figure 2.7: Boxplots of PC1 derived from response measured during the stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box.. Horizontal lines connect significantly different treatments.
42
Figure 2.8: Boxplots of PC2 derived from response measured during the stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.
43
Figure 2.9: Boxplots of PC1 derived from response measured during the post-stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.
44
Figure 2.10: Boxplots of PC2 derived from response measured during the post-stimulus period of each treatment in the Motion Experiment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed between treatments.
45 CHAPTER 3
PATTERNS OF CO-OCCURRENCE OF SEMAPHORES AND
VOCALIZATIONS IN THE PANAMANIAN GOLDEN FROG (ATELOPUS
ZETEKI)
3.1 INTRODUCTION
Animals employ many sensory modalities to communicate (acoustic, visual, chemosensory, etc.). Many recent studies have focused on communication involving
more than one modality. In multimodal signaling, the different component signals can
carry different information and can interact in affecting the behavior of the receiver
(Hebets and Papaj 2005; Partan and Marler 2005). For example, the temporal
relationship of different signal types can dramatically affect behavioral responses (Hebets
and Papaj 2005). Many species in the anuran amphibian genus Atelopus (family
Bufonidae) produce both vocalizations and conspicuous movements of the forelimb and
forefoot, thus providing an excellent opportunity to study multimodal communication and
interactions between acoustic and visual signals. The most common type of vocalization
consists of a series of sound bursts and is called a pulsed vocalization. Pulsed
vocalizations are frequently made by resident males sitting in their territories and appear
to function in territorial behavior (Lindquist and Hetherington 1996, 1998). The visual
display of Atelopus (termed here semaphores) typically consists of one to three forelimb
46 rotations made while the body is braced by the opposing forelimb (Lindquist and
Hetherington 1996). Males often produce semaphores when vocalizing or when involved
in intrasexual aggression (Crump 1988; Lindquist and Hetherington 1996, 1998).
Many species of Atelopus, including A. zeteki, live in cascading stream habitats
with significant background noise (Crump 1988; Lindquist and Hetherington 1996), and lack tympanic middle ears. Although this species can hear fairly well without a tympanic middle ear (Lindquist and Hetherington 1998), absence of such auditory structures may
impair their abilities to localize the sources of sounds. The visually conspicuous
semaphore actions might aid localization of calling individuals during territorial
interactions.
Several hypotheses have been proposed to explain the function of multimodal
signals (Grafe and Wagner 2007). One group of hypotheses states that signals are
redundant and act only to convey the same information with greater certainty. In other
words, the redundant hypothesis suggests that different signals will convey the same
information which can increase the accuracy of information transferred to the receiver
(Johnstone 1996; Møller and Pomiankowski 1993). Another group of hypotheses states
that signals are nonredundant and convey different information during signaling (Grafe
and Wagner 2007). For example the multiple message hypothesis suggests that each
modality provides different information to the receiver (Grafe and Wagner 2007;
Johnstone 1996; Møller and Pomiankowski 1993). Both types of hypotheses could
explain the use of multimodal signaling in A. zeteki. For example, in the alterting signal
hypothesis (nonredundant), an initial signal is used to draw the receiver’s attention to a
more informative second signal (Grafe and Wagner 2007). A. zeteki might use one signal
47 modality to draw the attention of a male to another signal that could potentially convey aggressive intent. If male A. zeteki employ semaphores to increase the ability to localize each other, and vocalizations to identify conspecifics, the two types of signals would best fit the multiple message hypothesis. Alternatively, A. zeteki may use both visual and acoustic signals to increase the probability of detection during territorial disputes (i.e., as redundant signals).
Interpretation of multimodal signaling depends on the duration and frequency of the component signals and their co-occurrence in nature (Ryan and Rand 2003).
Although multimodal communication are used in many taxa during courtship, aggression, and predator avoidance, studies dealing with the frequency of and timing relationships between signals in anuran amphibians have focused on female-male interactions
(reviewed by Ryan and Rand 2003). There have been no studies that have explored the pattern of co-occurrence of components in aggressive multimodal signals in anurans.
The present study examined the pattern of co-occurrence of semaphores and pulsed vocalizations in male A. zeteki. Information on the pattern of co-occurrence of these two different signals may provide insight into how they might function in multimodal communication. Analysis of the sequential relationship of semaphores and vocalizations potentially could test which multimodal hypothesis (multiple message versus redundant) applies to the visual and acoustic signaling of A. zeteki. This study also included an investigation of the ability of male A. zeteki to localize conspecific vocalizations, as this information may provide insight into the functional significance of the visual semaphore signal.
48 3.2 METHODS
3.2.1 Timing relationship between signals
Videotaped data from behavioral studies conducted in the summer of 2002 were
analyzed to determine the pattern of co-occurrence of semaphores and pulsed
vocalizations in male A. zeteki (see chapter 2 for detailed description of 2002 study).
Field observations were carried out on a population of A. zeteki in Parque Nacional
General de División Omar Torrijos Herrera in the Coclé province of Panama at the Rio
Marta (elevation approximately 300m) from July to September 2002 between 08:00 and
17:00 hours. During this study, videotaped records were made of males exposed to one of five treatments consisting of different combinations of model semaphores and playback vocalizations (see chapter 2 for experimental details). The five different treatments included: a low rate of semaphores (1 min-1) without playback vocalizations
(L-), a high rate of semaphores (5 min-1) without playback vocalizations (H-), a high rate
of semaphores (5 min-1) with playback vocalizations (H+), a low rate of semaphores (1
min-1) with playback vocalizations (L+), and no semaphores with playback vocalizations
(0+). The low and high rates of semaphoring used in the treatments bracket the typical
range of semaphoring behavior seen in males interacting in the field.
Analysis of the temporal relationship between semaphores and vocalizations was
restricted to events in which one signal was produced within 60 sec of another signal.
Responses, either semaphore or vocalization, usually occurred within 60 sec of a
preceding response. There were cases in which a signal was not followed by a response
within 60 sec; however, these were not considered frequent enough to increase the 60 sec
time frame. There were 57 cases in which a semaphore was not followed by another
49 signal and 14 cases in which a vocalization was not followed by another signal within the
60 sec time frame. Time was measured beginning with the first semaphore or
vocalization produced by a videotaped male. The time of the next signal was recorded
and a new 60 s time period was started when another signal was observed. There were a
few cases where signals appeared to be given simultaneously; however, these cases were
excluded because it was unclear which signal preceded the other. The patterns of co-
occurrence and timing relationships between visual and acoustic signals were analyzed in a total of 100 males over a 5-min stimulus time frame.
3.2.2 Sound localization
Tests were carried out on a population of Atelopus zeteki in Parque General de
Division Omar Torrijos-El Cope (elevation approximately 300m) in February 2004 between 08:00 and 17:00 hours. All tests were performed on resident males sitting on territories along their rocky stream habitat. After male frogs were located, a full range
Audix PH-3 speaker was placed approximately 1 m to the right or to the left of, and
directed toward, an individual. Left or right speaker position was randomly assigned for
each animal. After positioning of the playback speaker another silent speaker was placed
on the opposite side of the individual. Speakers were initially positioned in the same
horizontal plane as the frog, thus, by turning, a target male could directly face either
speaker. No tests were conducted in which a frog was oriented directly toward or away from either speaker, and experiments were terminated if a frog moved into such a
position during the test. Pulsed call recordings were obtained from the Borror Laboratory
of Bioacoustics at The Ohio State University. Recordings of 5 males with minimal
50 background noise from a nearby population of A. zeteki (~ 10 km away) were used to
make playback stimuli. The playback bouts of calling lasted about 30 sec with a 10 sec
pause between each bout. The calls from the five examplars were distributed randomly
but equally throughout the tests. Two observers were positioned approximately 3 m
away from the frog and speaker. Both observers remained silent and motionless throughout the experimentation period. Tests were conducted in the following manner. A
3 min pre-stimulus period allowed the test subjects to acclimate to the presence of the speaker and observers. This was followed by a 3 min stimulus period during which playback vocalizations were presented to the test subject. The direction of the first orientation movement (either toward or away from the playback speaker) was documented after the playback vocalization started. An orientation movement included any full body turn that would, if continued, make the target frog directly face an individual speaker. Although the entire 3 min stimulus period was observed and all behavioral responses were recorded, only the first orientation movement was analyzed in this study. No consistent movement toward or away from playback speakers was observed during the stimulus period. Twenty-one male frogs were tested.
3.2.3 Statistical Methods (Sequential patterns of signals)
A Fisher’s exact test was performed to determine if there was any overall significant difference between patterns of signal types. Chi-squared (X2) tests were
conducted to determine significance between specific signal patterns. In the X2 test concerning semaphore patterns the number of semaphores not followed by another signal within the 60 s time frame was used as the expected value (n=57). Similarly, the X2 test
51 conducted on vocalization patterns use the number of vocalizations not followed by
another signal within the 60 s time frame as the expected value (n=14). In all other X2
tests the expected values were calculated. Finally, X2 tests were performed to determine
the significance of differences in patterns of signal co-occurrence between different stimulus treatments.
3.2.4 Statistical Methods (Timing relationship of signals)
Mann-Whitney tests were performed to determine the significance of differences in timing between different and similar signal types. To examine the effect of context on signaling, Kruskal-Wallis tests were performed to determine if there were significant differences between signal latencies measured for different stimulus treatments.
3.2.5 Statistical Methods (Sound localization)
A X2 test was performed to determine whether there was any significant
difference in the direction of initial orienting movements of the males.
3.3 RESULTS
3.3.1 Sequential patterns of signal types
There were 160 cases (involving 45 males) of a semaphore followed by another
semaphore within the 60 s time window, whereas there were 53 cases (involving 23
males) of a semaphore followed by a vocalization. There were 125 cases (involving 24
males) of a vocalization followed by another vocalization, whereas there were 20 cases
52 (involving 15 males) of a vocalization followed by a semaphore. There was a significant
difference in the sequential pattern of signal types (p = 0.01 Fisher’s exact test, Table
3.2).
Semaphores were more often followed by another semaphore than a vocalization
(X2 = 254.1, p < 0.0001, 1 df). Similarly, vocalizations were more often followed by
another vocalization than by a semaphore (X2 = 974.1, p < 0.0001, 1 df). Concerning sequences of different signal types, significantly more semaphore-vocalization sequences
(N=53) were observed than vocalization-semaphore sequences (N=20) (X2 = 14.9, p <
0.0001, 1 df). All descriptive statistical data are summarized in Table 3.1.
Figure 3.1 displays some representative signal sequences of male frogs. Visual
inspection supports the finding reported above that similar signals are more likely to
follow each other, and there is no evidence of any pattern outside the 60 sec time frame
used to analyze sequential relationships.
3.3.2 Effects of stimulus treatments on sequential signal patterns
Stimulus treatments had no affect on the frequency with which a semaphore was
followed by another semaphore (X2 = 3.08, p = 0.5, 4 df). Likewise stimulus treatment did not affect the frequency with which a vocalization was followed more often by another vocalization (X2 = 4.73, p = 0.3, 4 df). (See Table 3.2).
3.3.3 Timing relationships of signal types
Figure 3.2 displays the time delay observed between consecutive pairs of signal
combinations that occurred within 60 sec of each other. The shortest interval was
53 observed between vocalizations (6.4 ± 8.4 s; range 2 – 60 sec) and the longest latency
was observed between vocalizations followed by semaphores (16.7 ± 18.3 sec; range 1 –
59 sec). Semaphores followed semaphores by an average of 11.2 ± 11.0 s (range 1 – 60 sec) and semaphores followed vocalizations by an average of 11.3 ± 14.9 s (range 1 – 60 sec). The interval between semaphores was significantly different from the interval between vocalizations (p< 0.0001, U= 5891, Mann-Whitney), and semaphore interval was significantly different from the interval between semaphores and vocalizations (p <
0.04, U=3451, Mann-Whitney). There was no significant difference in the interval between vocalizations and semaphores and the other intervals (SEM vs VOC/SEM: p =
0.5, U= 1464, Mann-Whitney, VOC vs VOC/SEM: p = 0.3, U= 3050, Mann-Whitney, and SEM/VOC vs VOC/SEM: p = 0.2, U= 440.5, Mann-Whitney).
3.3.4 Effects of stimuli on co-occurrence patterns
The different stimulus treatments had no statistically significant effect on the
interval between any signal types (Table 3.3).
3.3.5 Sound localization
Of the 21 males tested, nine males first oriented toward the playback speaker,
nine males first oriented away from the playback speaker, and 3 males did not move.
There was no significant difference in turning direction (X2 = 0, p = 1, 1 degree of
freedom).
54 3.4 DISCUSSION
3.4.1 Sequential patterns of semaphores and vocalizations
Both semaphores and vocalizations were more likely to be followed by the same
type of signal than be followed by the other signal type in A. zeteki. There were
significantly more cases of same signal type sequences among the 100 males observed in
this study, and also more males responded with the same signal type than different signal
types. With regard to different signal type sequences, semaphore-vocalization sequences
were significantly more common than vocalization-semaphore sequences. The different
stimulus treatments used in the behavioral experiments analyzed in this study (including
the presence or absence of playback vocalizations and frog model semaphores, and
different rates of model semaphoring) had no significant effect on sequential patterns of
signals. This study therefore found no evidence of context-specific changes in signal type sequences. Because each type of signal tended to be associated with signals of the same type, this study also found little evidence of any consistent pattern of intermixing of semaphores and vocalizations.
In previous experiments (see Chapter 2), territorial male A. zeteki responded more strongly to a combination of acoustic and visual signals than to either type of signal alone. However, the results presented here suggest that this effect is not dependent upon any exact temporal relationship between the two types of signals. It is possible that the
frogs are integrating signals over a longer time frame than 60 s. However, visual
inspection of the pattern of vocalizations and semaphores produced over the full 5 min
stimulus period suggested no pattern of signal production (Figure 3.1).
55 3.4.2 Timing relationships between semaphores and vocalizations
The time intervals between signals were quite variable. Although significant differences in the intervals associated with different signal pairs were found, the functional significance of these differences is not clear. There was a shorter mean interval between vocalizations than between semaphores (6.4 s and 11.4 s, respectively).
This difference is likely not related to constraints associated with the mechanical actions themselves. In fact, semaphores were sometimes observed to immediately occur after another semaphore (1 s latency), whereas the shortest delay between vocalizations was 2 s. Therefore, if mechanical factors acted to constrain the frequency of signaling for any of the signal types they would have affected vocalizations, but in fact the latter signals showed on average lower latencies than semaphores. Overall, the variation in latencies observed in this study does not suggest any organized pattern of temporal relationship between semaphores and vocalizations.
3.4.3 Sound localization
In nature territorial males will typically confront interlopers (per obs.). Therefore
we assumed that territorial males would actively orient toward the playback speaker. In
this study, the first movements of males exposed to playback vocalizations was random with respect to the position of the playback speaker, suggesting that A. zeteki is poor at
sound localization. Given that sound localization in anuran amphibians has been linked
at least in part to function of the tympanic middle ear as a pressure gradient receiver
(Eggermon 1988), the lack of such a middle ear in A. zeteki may explain the poor localizing ability of this species. The loss of a tympanic middle ear in the genus Atelopus
56 has been attributed to the high noise background associated with the montane streams that many species inhabit. Regardless of the cause of the evolutionary loss of a tympanic middle ear, any associated loss in the ability to localize sound sources, such as vocalizing males, might produce strong selective pressures for the evolutionary development of a visual signal such as a semaphore. The conspicuous semaphore action certainly would provide unambiguous information about the location of vocalizing males during interactions between territorial males and potentially between male and females individuals at times of mating.
3.5 SUMMARY
Overall, the analysis of the patterns of co-occurrence and timing of semaphores
and vocalizations does not fit into one specific multimodal communication hypothesis.
If the major function of a semaphore is to alert an intruder to the position of a territorial
male (i.e., the attention-alerting hypothesis), it would seem likely that semaphores should
follow vocalizations. Vocalizations could inform an intruding male that it is entering
another male’s territory and the intruder could then scan the environment to detect
semaphores signaling the actual position of the territorial male. As discussed in Chapter
1, vocalizations affect male behavior more than semaphores, and the importance of
vocalizations in stimulating male responses fits into the scenario that males should
vocalize first and semaphore later. However this study found that semaphores were more
likely to be followed by another semaphore and that vocalizations were more likely to be
followed by another vocalization. Potentially, this redundant signaling could ensure that
the receiver interprets the information accurately and can locate the signaler in the
57 environment. However, in different signal type sequences, semaphores were followed
more often by vocalizations than vice-versa. So vocalization-semaphore sequences are
the least common signaling seen in this species.
Based on the data observed in this study the communication system of A. zeteki
seems to fit both the attention-alerting and redundant hypotheses. Although there was no
consistent pattern of co-occurrence (i.e., vocalizations were not typically followed by semaphores or vice-versa), other studies (see Chapter 2) suggest that vocalizations alert intruders to the presence of a male and semaphores aid in localization of the exact position of the signaler. This scheme potentially explains why the semaphore evolved.
As A. zeteki lacks a tympanic middle ear, their ability to localize sound is reduced and selection might favor the use of a visual signal for precise localization of a calling individual.
On the other hand, the redundant signaling hypothesis is also supported by the observed data. Semaphores were more likely followed by semaphores and vocalizations were more likely followed by vocalizations. This pattern of co-occurrence suggests that signals are potentially used to ensure a receiver receives the information. Since both the visual and acoustic signals are used in male-male interactions, repeating these signals ensures the receiver of the signaler’s aggressive intent. This can be seen in other studies in which combining the two modalities at a high rate elicited the greatest behavioral response from males (see Chapter 2). In addition, repeating the same signal can increase the intensity of the interaction.
This study only analyzed the temporal relationships of semaphores and vocalizations given by signaling males. It did not analyze the pattern of semaphores and
58 vocalizations given by a male while interacting with another individual. The latter
analysis would best involve studies of males interacting in natural conditions. Also, this
study was done on responses of males to artificial male models and playback
vocalizations. Although the behavioral experiments that formed the foundation of this
study used a normal range of vocalizing and semaphoring behavior observed in the field,
males interacting with each other in nature may employ different signaling patterns. The frog models in this study may not have interacted with the target frogs in a naturalistic manner.
59
Treatment Type Category Low semaphore High High Low Playback Only rate semaphore rate semaphore rate + semaphore rate + playback playback Semaphore followed by semaphore 16 7 71 40 26
Vocalization followed by vocalization 20 21 37 34 13
Semaphore followed by vocalization 3 1 30 12 7
Vocalization followed by semaphore 3 1 5 6 5
60
Table 3.1: Number of responses for each category during various treatment types. For detailed descriptions of the treatment types please see Chapter 2.
Category # # Interval (s) Interval Range Males Events (s) Semaphore followed by 45 160 11.2 ± 11.0 1 – 60 semaphore Vocalization followed 21 125 6.4 ± 8.4 1 – 59 by vocalization Semaphore followed by 24 53 11.3 ± 14.9 2 – 60 vocalization Vocalization followed 15 20 16.7 ± 18.3 1 – 60 by semaphore
Table 3.2: Descriptive statistics for each signal category observed in this study.
61
Category H p-value N df Semaphore followed by 3.563 0.468 160 4 semaphore Vocalization followed by 6.184 0.186 125 4 vocalization Semaphore followed by 2.742 0.602 53 4 vocalization Vocalization followed by 2.541 0.637 20 4 semaphore
Table 3.3: Results of Kruskal-Wallis tests (H) for latencies between five stimulus treatment types for each sequence dyad category.
62
Treatment
Ø+ V VVV2VS S
L+ S S S SSS VVVV VVVVV VVVV V V V
H+ S S SS S S S V V S V V VV V
VVV V VV VVVV V VV H-
63 L- V VV VV V V V
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 Time (sec)
Figure 3.1: Representative signal sequences from each treatment during the 5 min stimulus period. Each line represents a different individual. “S” represents a semaphore and “V” represents a vocalization produced by a frog.
N=160 N=125 N=53 N=20
Figure 3.2: Boxplots for each signal sequence category. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Solid horizontal bars represent significant differences.
64 CHAPTER 4
STUDIES ON SEMAPHORING IN JUVENILE PANAMANIAN GOLDEN FROG
(ATELOPUS ZETEKI)
4.1 INTRODUCTION
Social communication in anuran amphibians (frogs and toads) relies largely on acoustic signals (vocalizations). However, evidence suggests that visual signals also may
be used in communication (reviewed by Hödl and Amezquita 2001). One of the first
reports of conspicuous visual displays in anurans concerned forelimb movements in the
genus Atelopus (Crump 1988). Many species of Atelopus (family Bufonidae) produce
forefoot and forelimb waving actions (termed here semaphores) that typically consist of
lifting and rotating a forelimb one to three times while the body is braced by the opposing
forelimb (Crump 1988; Lindquist and Hetherington 1996, 1998). Adult males often
produce semaphores when vocalizing or when involved in intrasexual aggression (Crump
1988; Lindquist and Hetherington 1996, 1998).
Several functions have been hypothesized for semaphores. A dominant
hypothesis is that the semaphore functions in intraspecific communication between
territorial males. Evidence from field studies on adult males holding territories along streams suggests that semaphoring does represent a form of communication in the
65 Panamanian Golden Frog, A. zeteki (Lindquist and Hetherington 1996, 1998, chapter 2).
Semaphoring behavior can be elicited by playback of vocalizations of other males and
also by mirrored self-images, suggesting that this behavior can be triggered by the
perception of other males and is directed toward specifically targeted individuals
(Lindquist and Hetherington 1998). In addition, forelimb movement of a “puppet” model of A. zeteki increases the rate of vocalizing and semaphoring of target males (see Chapter
2). This evidence suggests that semaphoring does function in intraspecific
communication, particularly involving male-male territorial defense.
Semaphoring also may function in intersexual communication prior to mate
selection. Under field conditions, five of seven gravid females were observed
semaphoring toward calling males, apparently inviting courtship advances (Lindquist,
pers. obs.). All seven females were eventually amplexed by the males. However,
obtaining information on intersexual interactions in A. zeteki is difficult. Whereas males
hold territories along streams year round, females live in the surrounding forest and only
infrequently come to streams when they are ready to mate.
Semaphores also have been proposed to function as aposematic displays. Skin
glands in A. zeteki produce highly toxic secretions (Fuhrman et al., 1969), and the bright
yellow body coloration of this species likely evolved as warning coloration. Semaphores
potentially could reinforce this aposematic warning by drawing the attention of a
potential predator to the brightly colored frog. In certain species of Atelopus (e.g.,
Atelopus spumarius), the body is cryptically colored (green and black) whereas the feet
exposed during semaphoring are bright red. Conspicuous warning coloration in other
animals is often accompanied by other types of signals which include odors, rattles,
66 shaking of wings, body movement, or even a combination of several movements (Rowe and Guilford 1999). Many highly toxic amphibians (e.g., poison arrow frogs of the genus
Dendrobates, newts of the genus Taricha, etc.) display conspicuous body postures and movements (i.e. the unken reflex, head butting, tail lashing, etc.) that presumably serve as warning behaviors (Duellman and Trueb 1996).
Semaphores potentially could have additional functions. For example, semaphores could flush potential prey by startling them into moving so that frogs could visually detect and capture them. These proposed functions are not mutually exclusive and selection for more than one function may explain the evolution of semaphoring behavior in the genus Atelopus. However, the only function thus far supported by experimental data is that of a signal used in male-male territorial interactions.
Until now all detailed observations and experiments on semaphoring behavior have focused on adult frogs. However, semaphores sometimes are produced by juvenile
A. zeteki (pers. obs.). The occurrence of semaphoring behavior in juveniles may help us understand the function of semaphoring behavior in general. Juvenile A. zeteki frequently are observed living in the vicinity of streams, but they do not hold territories as adult males and they sometimes are found clustered in small groups (pers. obs.). Therefore, besides being non-reproductive, juveniles also do not display territorial aggressive behavior. Juvenile A. zeteki also differ from adults in their body coloration.
Metamorphic juveniles are cryptically colored with bands of black and green that effectively blend into a mossy background. As individuals grow and age, the areas of green coloration become yellow and areas of black become proportionately smaller
(Lindquist and Hetherington 1998), resulting in adults that in some cases are entirely
67 yellow in coloration. Although juveniles, especially very young ones, are more
cryptically colored than adults, they do possess bright yellow palmar and plantar surfaces
(Figure 4.1). When juveniles semaphore, the bright yellow coloration is effectively
displayed. Because juveniles do not maintain territories, display aggressive behaviors
(e.g., male-male combat), or engage in mating behavior, such conspicuous semaphoring actions that display brightly colored feet likely do not function in intraspecific communication. Rather, it could serve as a type of aposematic signal to potential predators or as a mechanism to flush prey.
This study aims to examine the role of semaphoring in juvenile A. zeteki to further understand the function of semaphoring in this species. Observational studies were performed in the field to determine the relative frequency of semaphoring behavior in different sized (aged) individuals. Videotapes of naturally behaving frogs also were analyzed to examine whether semaphores are used to flush prey. Lastly, to test for a possible role of semaphoring as an aposematic behavior, field observations of the responses of juvenile frogs to a potential threat were performed.
4.2 METHODS
4.2.1 Study Sites
All observations were made along tributary streams in the Parque Nacional
General de División Omar Torrijos Herrera in the Coclé province of Panama from July to
August 2004 between 08:00 and 17:00 hours. All study sites were within 3 km of each
other.
68 4.2.2 Observational studies
In observational studies conducted by E. Lindquist and students, observers
approached to within about 3m of individual frogs (both juvenile and adult) and, after a 5
min acclimation period, videotaped their behavior for 15 min. Thirty-seven individuals,
including 18 juveniles (under 25 mm SVL) and 19 male adults (> 25 mm SVL), were
videotaped. Most territorial (i.e., reproductive) male adults have SVLs > 32 mm (pers. obs.), so individuals smaller than 25 mm likely are juveniles that do not hold territories along the streams. In most cases, observed individuals were alone. In two cases the observed juvenile individual was near an adult, and in four cases the observed juvenile was near other juveniles. In the case of one observed adult, vocalizations from a different male were heard in the distance. Videotaping was done using a Sony DCR-TRV350
Digital Handycam camcorder (or Sony DCR-TRV250 as a backup) and Fujifilm P6-120
Hi8 videocassettes. Following each recording individuals were captured, weighed, measured, and photographed, then released.
4.2.3 Juvenile experiments
In mock threat experiments, juvenile frogs were located as they walked along streams. Observers produced a mock threat by extending a fist to approximately 25 cm in front of the target juvenile and moving it back and forth for one minute. The number of semaphores produced by the target juvenile during that minute was counted. If the target juvenile moved, the observer moved with the animal to maintain hand position at the approximate 25 cm distance. After the one minute period target juveniles were captured and their SVLs measured. Forty-three individuals (< 22 mm SVL) were tested in these
69 experiments. No comparable tests were done on adult frogs at the site because other
experiments were being conducted on those individuals and we did not wish to introduce
the confounding factor of multiple tests on individual frogs.
4.2.4 Video Analysis
In scoring the videotaped experiments and observational studies, we recorded all semaphores (conspicuous forelimb rotation as described by Lindquist and Hetherington
1998). In addition, we recorded all tongue protrusions, defined as any movement of the tongue that usually results in the capture of prey.
The percentage of individuals exhibiting semaphores and mean number of semaphores for both groups (adults and juveniles) were calculated. To test if juveniles were semaphoring at the same rate as adults a binomial test using the probability value of
the adult semaphore rate (p=0.47) was performed. A two-tailed two sample t-test was
performed to compare mean semaphore numbers for each group.
4.3 RESULTS
4.3.1 Observational studies
Seventy-two percent of the 18 observed juveniles (< 25 mm SVL) produced
semaphores during the 15 minute observation period. The mean number of semaphores
produced was 6, and the most semaphores produced by a juvenile was 26. Forty-seven
percent of the 19 observed adults (> 25 mm SVL) produced semaphores. The mean
number of semaphores produced was 5, and the most semaphores produced by an
individual was 44. While juveniles were more likely to semaphore than adults during the
70 observation period (p=0.028 binomial test) the mean number of semaphores produced by
adults and juveniles was not significantly different (p=0.84, two-tailed two sample t-test).
In the two cases where the target juvenile was near an adult frog, only one and
two semaphores were produced. In the four cases where the target juvenile was in the
company of other juveniles, 0,0,10, and 16 semaphores were produced. In the one case
where the target adult was near a juvenile, no semaphores were produced. Only once was
a target adult within hearing range of another male that called in the background, and in
that case 10 semaphores were produced.
There was no evidence that juvenile or adult frogs employed semaphores to
“flush” potential prey. Both juvenile and adults were observed to feed during the
observation period, but feeding was never preceded by semaphoring actions.
4.3.2 Juvenile experiments
Twenty-three percent of the 43 juvenile frogs tested produced semaphores in
response to the mock threat. Most of these juveniles produced only one semaphore
during the one-minute test period, and the most produced by any single individual was
three.
4.4 DISCUSSION
Our data confirm that juvenile A. zeteki do semaphore. In fact, observations of
free ranging individuals found that more juveniles (72%) semaphored during the 15
minute observation period than adults (47%). This finding suggests that semaphore function is not restricted to intraspecific communication between adults.
71 Twenty-three percent of the juvenile frogs tested produced semaphores in response to an extended fist stimulus. Assuming that the hand stimulus was interpreted as a potential predator, this finding provides support for the hypothesis that semaphores can function as aposematic signals. Juveniles are largely cryptic in coloration, and the semaphoring action reveals the brightly colored palmar surface of the hands of the individuals. This is comparable to the unken reflex behaviors of various amphibian species (e.g., Bombina, Taricha, etc.) that serve to display bright colors on the underside of the belly or tail in response to a potential threat (Duellmand and Trueb, 1996).
Although comparable experiments were not performed on adult frogs at the study site (see Materials and Methods), adult males have been observed to semaphore in response to the presence of a human observer when no other likely stimulus (e.g., another conspecific male, etc) was in the immediate vicinity (pers. obs.). Therefore, it is likely that adult A. zeteki also produce semaphores in response to a potential threat. During most of the observational studies no conspecifics were present or vocalizing nearby, and in the few cases that were exceptions to this rule there was no clear relationship between the presence of conspecifics and semaphoring behavior. In the one case where an adult was observed while another male called in the background, 10 semaphores were produced, an above average number that may suggest an influence of the background vocalizations. Indeed, playback vocalizations have been found to increase the rate of semaphoring in target males (see chapter 2).
Overall, it seems likely that the semaphores of both juveniles and adults recorded during the observational studies served as aposematic signals toward the human observer.
Panamanian Golden Frogs often respond to approaching human observers with orienting
72 movements and semaphores (pers.obs.), and likely remain aware of their presence even when the observers sit quietly nearby. The functional significance of the greater likelihood of semaphoring observed in juveniles is uncertain. Semaphores may be more important as aposematic signals in juveniles because of their smaller size and reduced amount of bright yellow coloring.
If semaphores can function as both aposematic and communicative signals, the question arises as to whether one function is ancestral and the other more recently derived. Given that many amphibians employ bright colors as warning signals, the most likely scenario may be that the behavior first evolved because of selection for an aposematic display and then later became associated with intraspecific communication.
However, the determination of the ancestral or derived status of the two semaphore functions will require further comparative study of the presence and role of semaphoring behavior in both juvenile and adults of many different species of the genus Atelopus.
Perhaps the most intriguing aspect of semaphoring in A. zeteki is that juveniles actively signal at all. No reports of visual or acoustic (interspecific or intraspecific) communication in juveniles exist in the anuran literature. Hence, this study provides the first documentation of visual signaling in juvenile frogs.
73
A
B
C
Figure 4.1: A) Adult male Atelopus zeteki. B) Juvenile Atelopus zeteki (dorsal view). C) Juvenile Atelopus zeteki (ventral view).
74 CHAPTER 5
VOCAL SAC INFLATION AS A VISUAL SIGNAL IN THE PANAMANIAN
GOLDEN FROG (ATELOPUS ZETEKI)
5.1 INTRODUCTION
The role of the vocal sac of anuran amphibians in sound production is well known. Recent work has studied how inflation and deflation of a vocal sac may also provide a visual signal accompanying acoustic signals. In the dendrobatid frog
Epipedobates femoralis physical attacks can be elicited from territorial males only when a bimodal stimulus involving both vocalizations and vocal sac pulsations is provided
(Narins et al. 2003). In the bufonid Phrynobatrachus krefftii males possess a yellow vocal sac that is highly visible when inflated. Males also inflate and deflate their vocal sac without sound production (Hirschmann and Hodl 2006), potentially providing a visual signal in the absence of acoustic cues. In studies on tungara frogs (Physalaemus pustulosus) video stimuli were produced that allowed vocal sac inflation to be disconnected from vocalizations. In these studies female tungara frogs preferred males producing calls synchronized with vocal sac inflation (Rosenthal et al. 2004). All of these observations and studies suggest that vocal sac motion may function as a visual
75 signal in many frogs and toads. In addition, these observations and studies suggest that multimodal (visual and acoustic) signaling may be important in anuran communication.
Many species of the genus Atelopus (family Bufonidae) produce conspicuous forelimb movements (termed semaphores) that appear to function as visual signals. In the Panamanian golden frog A. zeteki, semaphores can modify the behavior of target males during territorial interactions (Crump 1988, Lindquist and Hetherington 1996,
1998, Chapter 2). Species of Atelopus also produce conspicuous inflations and deflations of their vocal sacs during sound production, and the vocal sacs often are brightly colored.
For example, the body of Atelopus flavescens is yellow but the vocal sac is bright pink,
and in some populations of A. varius, the body is yellow and black but the vocal sac is bright red (pers. obs.). Given the evidence that the genus Atelopus employs visual signals
(i.e., semaphores) during communication, this genus is a likely prospect for the use of vocal sac motion as a visual signal as well. In this study we analyze the potential role of vocal sac inflation and deflation as a visual signal during territorial interactions between male A. zeteki.
5.2 METHODS
5.2.1 Study Site
Field observations were carried out on a population of Atelopus zeteki in Parque
Nacional General de División Omar Torrijos Herrera in the Coclé province of Panama at
the Rio Marta (elevation approximately 300m) from July to August 2004 between 08:00
and 17:00 hours.
76 5.2.2 Video Clips
Video clips were obtained from video recordings of males in a previous 2002
experiment (See Chapter 2). Video clips were edited to make male individuals appear about the same size as adult male A. zeteki (approximately 35 mm snout-to-vent length)
on the Sony Handycam camcorder video monitor used during experiments. Each video
clip consisted of 15 min of manipulated video. Videos were divided into three 5-min
segments. The first and last five minutes consisted of video footage of the selected male
in a motionless position. Video clips approximately 45 sec in length were spliced
together to equal 5 min. Rather than displaying a frozen frame of the males, these clips
were aligned to make the males appear as silent, resting individuals with normal
respiratory movements, etc. The middle five minute period represented the stimulus
period and consisted of video footage of the selected male producing the desired behavior
(i.e., vocal sac inflation) with or without playback vocalizations. Again video clips of
approximately 45 sec in length were spliced together to form 5 min. When playback
vocalizations were added, these were synchronized to match the audio signals with vocal
sac inflation. Pulsed call recordings were obtained from the Borror Laboratory of
Bioacoustics at The Ohio State University. Recordings of five males with minimal
background noise from a nearby population of A. zeteki (~ 10 km away) were used to
make playback stimuli. The five recordings were randomly and evenly assigned to
manipulated video clips. All video and audio clips were edited with Showbiz software
and a Sony VIAO computer. Five exemplars were produced for each treatment type.
77 5.2.3 Treatment Types
Three stimulus treatments were conducted to test male responses to different
combinations of vocal sac inflation and playback vocalizations. In treatment V video
clips displayed a motionless male with playback vocalizations, in treatment I video clips
displayed a male producing vocal sac inflations without playback vocalizations, and in
treatment VI video clips displayed a male producing vocal sac inflations with playback
vocalizations. Treatments were randomly assigned to individual males. The pre-stimulus
and post stimulus periods (no motion, no playback vocalizations) provided the control treatments for all of the experiments.
5.2.4 Experimental Design
All tests were performed on resident males sitting on territories along their rocky
stream habitat. After male frogs were located, a Sony Handycam camcorder and a full range Audix PH-3 speaker were placed approximately 1 m from, and directed toward, an
individual. Video and speaker position and distance varied due to cascade topography
and location of each animal. The observer operating the camcorder sat about 0.5 m
behind the speaker, and another observer (who videotaped the trials) sat at least 3 m away
from the frog and speaker. Both observers remained silent and motionless (with the
exception of the video operator) throughout the experimentation period. Tests were
conducted in the following manner. A 5-min pre-stimulus period allowed the test subjects
to acclimate to the presence of the equipment and observers. This was followed by a 5-
min stimulus period during which signals (vocal sac inflation and/or vocalizations) were presented to the test subject, followed by a 5-min post-stimulus period of silence. All
78 behavioral responses during each period were videotaped. Twenty-four male frogs were tested with all three treatment types. Male frogs typically occupy discrete territories along the stream. Body size, color pattern characteristics, and digital pictures were recorded for each test animal (after all treatments were conducted) and each male territory also was flagged, thereby ensuring that the same individual was tested with each treatment. Males were tested with only one treatment daily and on average two days separated treatments for each male.
5.2.5 Video Analysis
In scoring the videotaped experiments, we recorded all behavioral displays exhibited by the test subject that we deemed biologically significant for territorial
behavior. These behaviors included:
(1) Semaphores - the conspicuous forelimb rotation as described by Lindquist and
Hetherington, 1998. Although some males seemed to produce semaphore-like actions
with their hindlegs, these were rare and the numbers were insufficient to warrant
statistical analysis.
(2) Vocalizations (pulsed calls) – The most common type of vocalization consisted of a
series of sound bursts. Pulsed vocalizations are frequently made by resident males sitting in their territories (Lindquist and Hetherington 1996, 1998).
(3) Approaches - males moved directly toward the video monitor, usually coming within about 10 cm of it. Males either approached the monitor head-on or from the side.
(4) Orienting movements - males turned their body. This movement could be directed toward or away from the video monitor or speaker.
79 5.2.6 Statistical Methods
We used Principal Component Analysis (PCA) to reduce the four behavioral
variables to a smaller set of PC scores for statistical analysis (Cooley and Lohnes 1971).
We conducted a single PCA on data from all test periods (pre stimulus, stimulus, and post stimulus) for the three treatment types. Wilcoxon Signed Rank tests were then used to test for the influence of treatment type during the stimulus and post stimulus periods. In addition, Signed Rank tests were performed to determine if video images had an overall affect on behavior during treatment types (pre stimulus vs. post stimulus). All statistical calculations were conducted using SPSS 16.0 (2008) and Microsoft Excel (2007).
5.3 RESULTS
Principal Component Analysis reduced the four behavioral response measures to
one composite measure explaining 41% of the variance (Table 5.1) for the treatment
periods. This composite measure (PC1) was positively correlated with semaphores,
orienting movements, and approaches (Table 5.1).
5.3.1 Comparison across treatment periods (pre-stimulus vs. post-stimulus)
Signed-rank tests showed a significant difference between the pre-stimulus and
stimulus period for the V treatment (p=0.0008, N=16, W=130) and VI treatment
(p=0.0002, N=19, W=168). In both cases more semaphores, orienting movements, and
approaches occurred during the stimulus period (Table 5.2). Signed-rank tests showed a
significant difference for all treatment types between the pre-stimulus and post-stimulus
periods (V: p=0.016, N=16, W=91; I: p=0.02, N=9, W=39; VI: p=0.007, N=15, W=92
80 Figure 5.1). In all treatments the post-stimulus period showed more semaphores, orienting movements, and approaches than the pre-stimulus period (Table 5.2).
5.3.2 Comparison between treatments (stimulus period)
Signed-rank tests showed a significant difference between treatment types V and I
(p=0.0003, N=16, W=126) and I and VI (p<0.001, N=18, W=171) (Figure 5.2). Fewer semaphores, orienting movements, and approaches occurred in treatment I (vocal sac inflation alone) than treatment V (vocalization alone) or VI (vocal sac inflation and vocalizations) (Table 5.2).
5.3.3 Comparison between treatment (post-stimulus period)
Signed-rank tests showed a significant difference between treatment types V and I
(p=0.006, N=16, W=101) and I and VI (p=0.001, N=15, W=106) (Figure 5.3). Again, fewer orienting movements, semaphores, and approaches occurred in treatment I (vocal sac inflation alone) than treatment V (vocalization alone) or VI (vocal sac inflation and vocalizations) (Table 5.2).
5.4 DISCUSSION
Results of this study suggest that vocal sac inflation and deflation does not represent a potent visual signal in A. zeteki during territorial interactions. Males
displayed fewer semaphores, orienting movements, and approaches during the vocal sac
inflation alone (I) treatment than during the vocalization alone (V) or vocal sac inflation
and vocalization (VI) treatment. There also was no significant difference in male
81 behavior during the vocal sac inflation alone (I) treatment and the pre-stimulus period
that lacked any vocal sac movement or vocalization. The only potential effect of vocal
sac motion alone was observed when comparing male behavior during the pre-stimulus
period in the vocal sac motion alone treatment to behavior during the post-stimulus
period. There were significantly more behavioral responses (semaphores, orienting
movements, and approaches)observed in the post-stimulus period, possibly suggesting a delayed reaction to vocal sac motion presented to the target males during the stimulus period.
Given that male A. zeteki respond to the semaphore visual signal, the general lack of response to vocal sac motion is surprising, especially as other anuran species have been shown to respond to vocal inflation and deflation. Previous work using a similar experimental design had established a clear effect of semaphore signals on male behavior
(chapter 2). However, the method of stimulus presentation differed in the two studies. In the semaphoring study, forelimb motion was generated using a model frog, whereas in this study vocal sac motion was generated using video images. Male A. zeteki potentially may perceive model arm motion and video image motion differently. However, at least one other study has successfully used video images in anuran behavioral experiments
(Rosenthal et al. 2004), so it seems likely that A. zeteki can perceive the video images.
Nonetheless, a video study using both vocal sac and semaphore motions might resolve any question that the vocal sac provides a significant visual signal in this species. Also, vocal sac inflation might be more important as a visual signal in another context, such as mate attraction.
82
PC 1 Eigenvalue 1.643 % of Variance 41.1 Semaphores 0.782 Vocalizations 0.339 Approaches 0.572 Orienting Movements 0.768
Table 5.1: Total variance, eigenvalue, and principal component loadings for scored behaviors.
83
Treatment V (playback only) Pre-stimulus Stimulus Post-stimulus Semaphores 0.667 ± 1.523 3.25 ± 5.093 2.25 ± 3.578 Vocalizations 0 1.292 ± 3.569 1.583 ± 3.988 Approaches 0 0.375 ± 0.924 0.083 ± 0.282 Orienting 0.708 ± 1.083 3.583 ± 4.671 2.167 ± 3.749 Movements Treatment I (vocal sac inflation only) Pre-stimulus Stimulus Post-stimulus Semaphores 0.083 ± 0.408 0 0.375 ± 1.245 Vocalizations 0 0.792 ± 3.878 0 Approaches 0 0 0 Orienting 0 0.167 ± 0.637 0.708 ± 1.42 Movements Treatment VI (playback and vocal sac inflation) Pre-stimulus Stimulus Post-stimulus Semaphores 0 3.708 ± 7.0 1.208 ± 1.888 Vocalizations 0 0.125 ± 0.612 0.5 ± 1.865 Approaches 0 0.375 ± 0.824 0.042 ± 0.204 Orienting 0.375 ± 1.056 3 ± 4.754 1.917 ± 2.903 Movements
Table 5.2: Mean and standard deviations of behavioral response measures from individual test periods for each treatment type.
84
Figure 5.1: Boxplots of PC1 derived from response measured during the pre-stimulus (solid box) and post-stimulus (white box) periods for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.
85
Figure 5.2: Boxplots of PC1 derived from response measured during the stimulus period for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.
86
Figure 5.3: Boxplots of PC1 derived from response measured during the post-stimulus period for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.
87 CHAPTER 6
COLOR PATTERN AND MALE SIZE AS A POTENTIAL VISUAL SIGNAL IN
INTRASPECIFIC INTERACTIONS OF THE PANAMANIAN GOLDEN FROG
(ATELOPUS ZETEKI)
6.1 INTRODUCTION
Many diurnal as well as many nocturnal species utilize color both to conceal themselves and blend in with their environment as well as influence social interactions
(Fodgen and Fodgen 1974). Colorful ornaments typically function in signaling individual quality in intra- and intersexual interactions (Solis et al. 2008). However color is not solely limited to the expression of quality. It can be used to signal warnings or aggressive intent, and even to attract prey (Drickamer et al. 2002). For example, male three-spined stickleback fish (Gasterosteus aculeatus) possess a red belly that can intimidate rivals (Bakker 1986).
The functional role of coloration has been studied in many species of anuran amphibians. Non-cryptic coloration in anurans is typically considered to function as aposematic (warning) coloration (e.g., poison arrow frogs of the family Dendrobatidae)
(Myers and Daly 1983; Pough et al 2001; Summers and Clough 2001). However, there is evidence that coloration functions as a visual signal among conspecifics of certain species, such as Phyrnobatrachus krefftii. P. krefftii is a brown frog with a bright yellow
88 vocal sac. In experiments males were reported to perform nonaudible vocal-sac inflations during intraspecific interactions, suggesting that vocal sac color may play a role in male-male interactions for this species (Hirschmann and Hodl 2006).
Most species of Atelopus, including A. zeteki, are brightly colored, and this is typically considered aposematic coloration related to their high skin toxicity. Coloration and patterning in A. zeteki is variable and changes dramatically during growth (Lindquist and Hetherington 1998). There is an ontogenetic shift toward more yellow coloration with increasing body size, and this may be related to increased toxicity associated with incorporation of food-based toxins (Lindquist and Hetherington 1998). However, it is possible that coloration also functions in intraspecific communication. Males may recognize and be intimidated by larger males with more yellow coloration, and female mate choice may involve selection for more yellow coloration as an indicator of male fitness. Even though there is no physiological or behavioral evidence that these frogs can detect differences in color, most anurans possess two types of cone photoreceptors which suggests that they likely can perceive some color (Duellman and Trueb 1994). However, even lacking color vision they could perceive the clear difference in proportion of dark versus light (yellow) areas in the pattern of small and large males. The aim of this study was to test whether color pattern is involved in visual communication in A. zeteki.
89 6.2 METHODS
6.2.1 Study Site
Field observations were carried out on a population of Atelopus zeteki in Parque
Nacional General de División Omar Torrijos Herrera in the Coclé province of Panama at
the Rio Marta (elevation approximately 300m) from December 2003 to February 2004
between 08:00 and 17:00 hours.
6.2.2 Puppet Models
Puppet frog models consisted of two different types: a small patterned (yellow
and black) model and a large completely yellow model (Figure 6.1). Males holding territories along Rio Marta range in size from about 32 mm to 49 mm snout-vent length
(mean 43.9). Accordingly, body lengths for the small and large models were 32 mm and
49 mm respectively. The models were painted to resemble a typical male adult frog – goldenrod yellow with narrow black bands, or solid goldenrod yellow. A 2 mm diameter gray metal rod penetrated the model’s body transversely, running from one side and connecting to the opposite arm. To approximate a semaphore (visual display of Atelopus that consists of one to three forelimb rotations made while the body is braced by opposing forelimb as defined by Hetherington and Lindquist 1996), the rod was rotated slightly, lifting the forelimb with the hand facing forward. A weight was required to stabilize the puppet model during the production of semaphore motions (Figure 6.2).
Puppet models had a small magnet glued to their bottom side that attached to a rock via its own attached magnet. Five different models of each type (small black and yellow
90 patterned or large solid yellow) were used to minimize pseudoreplication; in four models
of each type the right arm moved and in one model of each type the left arm moved.
6.2.3 Treatment Types
Experiments consisted of presenting target male frogs with a model (either small
and patterned or large and yellow) producing semaphores and accompanied by playbacks of territorial vocalizations. A rate of semaphoring (5 min-1) toward the high end of rates
typically observed in males interacting in the field was used. This high rate of semaphoring together with playback vocalizations elicited the greatest behavioral
responses in target males in earlier experiments (see chapter 2). In the PATTERN
treatment target males were presented with small, semaphoring patterned (black and
yellow) models accompanied by playback vocalizations. In the YELLOW treatment males were presented with large, semaphoring all-yellow models accompanied by playback vocalizations. Treatments were randomly assigned to individual males. Each target male (N=32) received only one treatment (PATTERN or YELLOW). Digital pictures were taken of each test animal and each male’s territory also was flagged, thereby ensuring that no individual was tested more than once.
All stimulus periods contained a continuous series of pulse vocalizations on an endless cassette loop played through a Sony TCD-D5 cassette recorder. Pulsed call recordings were obtained from the Borror Laboratory of Bioacoustics. Five male recordings with minimal background noise from a nearby population of A. zeteki (~ 10
km away) were used to make playback stimuli. The bouts of calling for each male lasted
about 30 seconds with a 10 sec pause between each calling bout. Five exemplars from
91 five different males were created to use in playback tests. The five calls were distributed randomly but equally throughout all tests and across frog model types. Sound amplitude was set at 86 dB at 1 m from the speaker (linearly calibrated with a Quest 215 sound level meter). This sound pressure is comparable to that produced by a male calling at 1 m.
6.2.4 Experimental protocol
All tests were performed on resident males sitting on territories along their rocky stream habitat. After male frogs were located, a full range Audix PH-3 speaker was placed approximately 1 m from, and directed toward, an individual. Speaker position and distance varied due to cascade topography and location of each animal. A puppet model of a male golden frog was placed directly (between 0.3 to 0.5m) in front of the sound speaker. The observer who operated the puppet sat about 0.5 m behind the speaker, and another observer (who videotaped the trials) sat at least 3 m away from the frog and speaker. Both observers remained silent and motionless (with the exception of the puppet operator) throughout the experimentation period. Tests were conducted in the following manner. A 5-min pre-stimulus period allowed the test subjects to acclimate to the presence of the speaker and observers. This was followed by a 5-min stimulus period during which the test subject was presented with model semaphores and playback vocalizations, followed by a 5-min post-stimulus period of silence and no model semaphoring. All behavioral responses during each period were videotaped. Body sizes of the target males in the two treatments were similar (patterned SVL 44.1 ± 1.50 mm; yellow SVL 43.7 ± 2.74 mm).
92 6.2.5 Video Analysis
In scoring the videotaped experiments, we recorded all behavioral displays exhibited by the test subject. Only responses that were typically observed and involved in territorial behavior were analyzed. These included the following:
(1) Semaphores - the conspicuous forelimb rotation as described by Lindquist and
Hetherington, 1998. Although some males seemed to produce semaphore-like actions with their hindlegs, these were rare and the numbers were insufficient to warrant statistical analysis.
(2) Vocalizations - The most common type of vocalization consisted of a series of pulsed calls and is called a pulsed vocalization. Pulsed vocalizations are frequently made by resident males sitting in their territories and appear to function in territorial behavior
(Lindquist and Hetherington 1996, 1998).
(3) Attacks – males approached and contacted the puppet model. A male frog would typically first contact the model with its forelimbs and then climb on top of the model’s back. The frog would subsequently dismount and often begin to walk around the model.
In natural conditions, male A. zeteki sitting on territories will engage interloping males in combat, with “winners” often climbing on the backs of “losers” and pushing the latter toward the ground. In our study, test frogs did not show such “fighting” behavior, likely because the puppet models, once mounted, did not provide sufficient cues to trigger further aggressive actions. Approaches did not always result in an attack. However, when target males came within ≤5 cm of the puppet model they typically attacked the model.
93 (4) Orienting movements - males turned their body either to the right or left. This movement could be directed toward or away from the puppet model or speaker.
6.2.6 Statistical Methods
We used Principal Component Analysis (PCA) to reduce the four behavioral variables to a smaller set of PC scores for statistical analysis (Cooley and Lohnes 1971).
We conducted a single PCA on data from all test periods (pre-stimulus, stimulus, post- stimulus) for the two treatments. Mann-Whitney U tests were then used to test for the influence of treatment type (PATTERN vs YELLOW). All statistical calculations were conducted using SPSS 16.0 (2008).
6.3 RESULTS
Principal Component Analysis reduced the four behavioral response measures to two composite measures explaining 74% of the variance (Table 6.1) for the treatment periods. The first composite measure (PC1) was positively correlated with semaphores, orienting movements, and attacks (Table 6.1). The second composite measure (PC2) was positively correlated with vocalizations (Table 6.1).
6.3.1 Comparison across treatment periods (pre-stimulus vs. stimulus)
Mann-Whitney tests showed a significant difference between the pre-stimulus and stimulus period for PC1 values in both treatments (YELLOW: U = 11.5, p < 0.0001
PATTERN: U = 46. 5, p < 0.0001, Figure 6.3). There were more semaphores, orienting movements, and attacks in the stimulus period for both treatments (Table 6.2).
94 Mann-Whitney tests did not show a significant difference between the pre-stimulus and
stimulus period for the second axis (PC2) for either treatment (YELLOW: U = 93.5, p =
0.181 PATTERN: U = 78.0, p = 0.06) (Table 6.2).
6.3.2 Comparison between treatments (stimulus period)
Mann-Whitney tests did not show a significant difference for PC1 values between
treatment types (U = 112.0, p = 0.558, Figure 6.4). In addition, Mann-Whitney tests did not show a significant difference for PC2 values between treatment types (U = 116.5, p =
0.664, Figure 6.4). Means for each response behavior for the stimulus period can be viewed in Table 6.2.
6.3.3 Comparison between treatments (post-stimulus period)
Mann-Whitney tests did not show a significant difference for PC1 values between treatment types (U = 113.0, p = 0.564, Table 6.2). In addition, Mann-Whitney tests did not show a significant difference for PC2 values between treatment types (U = 102.0, p =
0.317, Figure 6.5). Means for each response behavior for the post-stimulus period can be viewed in Table 6.2.
6.4 DISCUSSION
The results of this study provide no evidence that color pattern or male body size play a role in communication in male-male interactions in A. zeteki. Males responded in the same manner to both small patterned models and large completely yellow models.
This suggests that aggressive interactions are not affected by color pattern or male body
95 size and males are not using these cues in assessing the quality of intruding males.
Although males do not seem to use coloration or size during aggressive interactions, color pattern and male body size may still be important in intersexual communication.
Females might use color pattern and/or size as an indicator of male quality. Since males shift toward a more yellow coloration with increasing body size, females may choose males that are less patterned and larger. However, this type of experimentation is difficult to perform in A. zeteki as females spend the majority of their lives in the surrounding forests and only infrequently come to streams when they are ready to mate.
This study differed from other studies examining the role of color in communication in anuran amphibians in that it focused on overall body color pattern.
Other studies have examined moving structures that are brightly colored (vocal sac inflation, forelimb semaphores, hindfoot flagging, etc.) (Rosenthal et al 2004,
Hetherington and Lindquist 1998, Hirschmann and Hodl 2006). In those studies the effects of the motion alone or color alone were not teased apart, so it is unclear if color actually plays any role. Presumably the bright coloration of a moving body part represents an adaptation for increasing visibility of the structure, especially if the species can perceive color. Nonetheless, to date there is no unamibguous evidence that color functions as an important visual signal to conspecifics in anuran amphibians.
Experiments employing video images or models with different colored structures (vocal sacs, limbs, etc.) could unambiguously determine the functional role of color in amphibian communication.
96
PC 1 PC 2
Eigenvalues 1.933 1.018
% of Variance 48.3 25.4
Semaphores 0.865 0.031
Vocalizations 0.132 0.968 Attacks 0.704 -0.278
Orienting Movements 0.820 0.050
Table 6.1: Total variance, eigenvalues, and principal component loadings for scored behaviors.
97 Treatment YELLOW Pre-stimulus Stimulus Post-stimulus Semaphores 0.19 ± 0.40 9.56 ± 8.52 1.69 ± 2.65 Vocalizations 0 1.62 ± 4.50 6.69 ± 10.21 Attacks 0 0.56 ± 0.89 0 Orienting 0.44 ± 0.89 2.94 ± 2.96 0.81 ± 1.11 Movements Treatment PATTERN Pre-stimulus Stimulus Post-stimulus Semaphores 0.31 ± 0.602 8.50 ± 8.66 2.25 ± 3.96 Vocalizations 0 0.19 ± 0.75 3.12 ± 5.63 Attacks 0 0.31 ± 0.70 0.12 ± 0.34 Orienting 0.25 ± 0.58 3.94 ± 3.61 1.56 ± 2.19 Movements
Table 6.2: Mean and standard deviations of behavioral response measures from each test period for treatment types.
98
A B
Figure 6.1: A) Frontal view of the model puppet used in the YELLOW treatment. B) Frontal view of the model puppet used in the PATTERN treatment. Both puppet models are in the neutral position.
99
magnet
Figure 6.2: Puppet models with forelimb in the highest position of the semaphoring movement. The magnets were used to attach the puppet models to a counterweight thus providing stabilization during the semaphore motion.
100
Figure 6.3: Boxplots of PC1 derived from response measured during the pre-stimulus (white box) and stimulus (lined box) periods for each treatment. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. Horizontal lines connect significantly different treatments.
101
Figure 6.4: Boxplots of PC1 and PC2 values derived from responses measured during the stimulus period of each treatment. PATTERN treatment is represented by lined boxes and YELLOW treatment is represented by white boxes. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed.
102
Figure 6.5: Boxplots of PC1 and PC2 values derived from responses measured during the post-stimulus period of each treatment. PATTERN treatment is represented by lined boxes and YELLOW treatment is represented by white boxes. The “o” symbols represent outliers that are between 1.5 to 3 box lengths from the upper or lower edges of the associated box. The “*” symbols represent extremes that are over 3 box lengths from the upper or lower edges of the associated box. No significant difference was observed.
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