THE EFFECT OF PERCEIVED INTRUDER
PROXIMITY AND RESIDENT BODY SIZE ON THE
AGGRESSIVE RESPONSES OF MALE GREEN
FROGS, RANA CLAMITANS (ANURA: RANIDAE)
Behav Ecolog Sociobiol
2005
Patrick C. Owen
and
Noah M. Gordon
Introduction
Graded response
 A variety of animals modify or grade their
aggressive responses in a continuous fashion.
(Archer 1988; Bond 1989a; Hauser and Nelson 1991; Bradbury and
Vehrencamp 1998; Hurd and Enquist 2001)
 Can prevent encounter from escalating into fight.
(Martins 1994; Adams and Mesterton-Gibbons 1995;
Hurd and Enquist 2001)
 Costs
reduced mating success, greater energetic
expenditure, or greater risk of predation.
2
 These aggressive responses may vary with the
size
(Archer 1988; Andersson 1994; Pusey and Packer 1997).
 Graded responses may function as signals of
1) aggressive motivation
2) fighting ability
(Bond 1989a, b, 1992; Hauser and Nelson 1991; Hurd and Enquist 2001; Lange and Leimar 2003)
 Studies in amphibians have suggested that
graded variation may serve in both of these roles.
(Wells 1988; Schwartz 2001; Gerhardt and Huber 2002)
3
 These responses most often take the form of
discrete call types that can vary continuously
Modified item
Reference
Duration of notes
Schwartz and Wells 1984; Wells and Schwartz 1984;
Littlejohn and Harrison 1985; Wells and Bard 1987; Martins
et al. 1998; Wogel et al. 2004
The number of pulses of
notes
Pengilley 1971; Arak 1983b; Schwartz 1989; Wagner 1989b;
Grafe 1995;Wogel et al. 2004
The dominant frequency Lopez et al. 1988; Wagner 1989a; Grafe 1995
Calling rate
Given 1987; Stewart and Bishop 1994; Grafe 1995; Jehle and
Arak 1998; Grafe et al. 2000; Narins et al. 2000; Grafe 2003
Call intensity
Lopez et al. 1988
Response threshold
Rose and Brenowitz 1991; Brenowitz et al. 2001
Diversity of call types
Christensen-Dalsgaard et al. 2002
4
 Some anurans may signal body size or
fighting ability with these call responses (Davies
and Halliday 1978; Arak 1983a; Ramer et al. 1983; Robertson 1986; Given 1987;
Wagner 1989a; Bee et al. 1999, 2000),
 Less attention has been focused on their role
in signaling aggressive motivation (Burmeister et al.
2002).
 The graded nature of non-vocalization
responses such as movements or visual
displays has not been investigated.
5
 We examined graded responses in green
frogs (Rana clamitans,Ranidae).
 Eastern North America
 Part of the Rana catesbeiana species group (Austin et al.
2003).
 Prolonged summer breeders (Wells 1977a)
 Resource defense mating system in which males
defend oviposition sites (Wells 1977b).
 The quality of the oviposition site is more
important in mate choice than is calling
behavior.
6
 Aggressive behavior is well documented in
green frogs (Jenssen and Preston 1968; Wells 1978; Ramer et al. 1983;
Bee and Perrill 1996; Owen and Perrill 1998; Bee et al. 1999, 2000)
 Aggressive encounters escalate when an intruding
male approaches and enters the territory of a
resident.
 Encounters sometimes result in wrestling matches.
 Residents typically have the advantage
 Larger males usually defeat smaller males (Wells 1978).
 Green frog displays consist of a combination
of calls, movements, and other visual stimuli
such as prominently displayed yellow throats
and elevated body posture (Wells 1978).
7
 Green frog advertisement (type I) and
aggressive (type III) calls are similar (Wells 1978).
 The main consistent differences are that
aggressive calls are lower in dominant
frequency, and longer than advertisement
calls (Bee and Perrill 1996).
longer
type I
type III
lower
8
 Advertisement and aggressive calls are not
really two discretely different signal types,
but a single signal that varies continuously
along a continuum.
http://www.sonic.net/~ric/go/vgame1.htm
9
 A third, discretely different call sometimes given
during the course of aggressive encounters is the
type II “high-intensity” advertisement call (Wells
1978).
 The function of this call has never been
satisfactorily determined (Bee and Perrill 1996).
10
Dominant frequency
 In advertisement calls are stable(Bee et al. 2001),
but lower and more variable in aggressive
calls
 May function in signaling resident size to
intruders (Bee et al. 1999), and smaller green frogs
may bluff their size(Bee et al. 2000).
Frequency alteration
 information about fighting ability.
 information about signaler motivation, but
this has not been previously investigated.
11
Objectives
 To determine whether these aggressive
responses are graded according to increases in
stimulus level and to examine how these
responses vary with resident body size.
 We investigated the components of aggressive
responses
 rates of response calls
 temporal (duration) and spectral (dominant frequency)
characteristics
 We also examined rates of response movements.
12
The null expectation
1.
Resident would not respond differently to stimuli
representing different level of threat
Progressive changes in responses would provide
evidence that aggressive responses are graded as a
function of the perceived level of threat
2.
Frogs would give the same level of response
regardless of body size
If larger males give proportionally greater responses to
stimuli, then this suggests that larger males employ
potentially more costly responses than do smaller
males
13
Methods
 We conducted experiments in a large pond in
Brooklyn, Connecticut, USA, during the
summer of 1999.
 Time: 2200 to 0300 h
 Measurement: SVL and body mass
 Individual recognition
 spot patterns and scars were recorded
 avoid pseudo-replication.
 Water temperature did not vary a lot.
14
Stimulus call intensity
 We recorded the intensity of four calls for
each subject, measured the distance
 mean sound intensity (y) as a function of
distance (x) in cm
(n=20, r2=0.884, P<0.0005, y=−0.07x+96.815).
 territory sizes were typically 1–2m in radius.
 Simulate an encroaching intruder
Distance (m)
2
1
0.5
Intensity (dB)
83
90
97
15
Playback
 Speaker mounted on a Styrofoam platform.
 1m from a calling male.
 Calls were recorded using a tape cassette
recorder.
 Average characteristics
 327 Hz in dominant frequency
 250 ms in duration
 presented at a rate of 2 calls per min).
 Our focus for this experiment was on responses
to an “average” intruder.
 Battery power can affect stimulus properties.
16
Playback
 We used a graded playback design similar to
Schwartz (1989) and Wagner (1989b) with the
following modifications.
 Pre-experimental period of at least 5 min.
 If the male interrupted during the pre-
experimental or experimental periods, then we
stopped
 We ultimately excluded five males
 However, relocate disturbed males was not
an ideal solution (Gordon 2004).
17
Playback
Record 3-5 calls→S1→S2→S3
Pre-experimental period
Experimental period
 Each experiment lasted for at least 35 min.
 A balanced design that controlled for possible
carry-over effects (Quinn and Keough 2002).
 Movements were assessed visually.
18
Sound analyses
 Digitized recordings at 48 kHz (16 bit).
 Only the first note of multi-note calls was
analyzed (Bee and Perrill 1996).
 Call variables included
 dominant frequency
 call note duration
 Response latency (reaction time).
19
Repeated measures multivariate analysis of
variance (MANOVA) and covariance
(MANCOVA)
 dependent variables
 mean dominant frequency
 mean note duration
 calling rate (calls/5-min period)
 movement rate (moves/5-min period)
 calling latency, and movement latency
 SVL was used as the covariate.
20
 We used the rank transform approach in which
response data were converted to ranks within
each variable, and the resulting ranked data
were analyzed using standard parametric
methods (Quinn and Keough 2002).
 Linear contrasts were used to test the hypothesis
of response change trends for each significant
ANOVA.
 Critical significance levels of ANOVAs and linear
contrasts were adjusted using the sequential
Bonferroni method (Quinn and Keough 2002).
21
Results
Aggressive response
 17 individuals responded to the stimulus with a combination
of calls and movements.
 Gave a response call first (69% of broadcasts), the first
response movement was given an average of 21.1 s later.
 When a movement was given first (31% of broadcasts), the
first response call was given an average of 15.3 s later.
 In one instance the male mounted the speaker platform
and attempted to wrestle with the speaker.
 Normally, the frogs approached no closer than the water in
front of the speaker platform.
 Returned to their original positions by the end of each
intervening 5-min silent period
22
Type II high-intensity advertisement calls
 occasionally given by 10 males.
 Three of these individuals gave type II calls in response
to our broadcasts
 The other seven individuals gave type II calls in
response to other frogs what were producing type II
calls.
 Most type II calls were produced during broadcast
periods (83 dB = 6 calls, 90 dB = 5 calls, 97 dB = 5 calls,
silent periods = 2 calls).
 Only 17 of 940 total response calls for all individuals
were type II calls.
23
 Male body size (SVL) of the subjects ranged
from 69 to 91 mm encompassing the range of
sizes in previous studies that addressed
fighting ability (Wells 1977b, 1978; Ramer et al. 1983; Bee et al. 1999,
2000).
24
 A preliminary analysis using Spearman rank-
order correlations indicated that SVL was
significantly correlated with some of the
response variable difference scores at some
broadcast intensities.
Broadcast Move rate Call rate
level (dB)
Call
duration
Dominant Move
frequency latency
Call
latency
83
0.371
0.162
-0.203
0.147
-0.402
0.086
90
0.477
0.148
0.067
0.391
-0.055
-0.083
97
0.522
0.419
0.125
0.268
0.124
-0.187
25
 A repeated measures MANCOVA was performed
with SVL as the covariate.
 In contrast to the analysis of Bee et al. (1999),
SVL did not have a significant effect on response
level (F1,15=3.74, P=0.072),
 and including SVL as a covariate only resulted in a
decrease of 0.9% in total unexplained variation
compared to a MANOVA (see below) on the
same data.
 Thus, SVL was not included in further analysis of
stimulus level effect.
26
 Repeated measures ANOVA on response level
calling rate
movement rate
(F2,32=10.071, Pcrit=0.013, P=0.001)
(F2,32=18.42, Pcrit=0.050, P<0.0001)
27
 Stimulus levels did not have a significant
effect on response latencies
movements
calls
F2,32=0.14, Pcrit=0.001, P=0.849
F2,32=0.14, Pcrit=0.001, P=0.849
28
 stimulus level had a significant effect on call
dominant frequency (F2,32=6.66, Pcrit=0.006, P=0.004; Fig. 4A)
and on call duration (F2,32=13.45, Pcrit=0.025, P<0.0001; Fig. 4B).
29
 Linear contrasts indicated significant changes
for
 movement rate (F1,16=37.09, Pcrit=0.050, P<0.0001)
 call duration (F1,16=13.452, Pcrit=0.025, P<0.0001)
 calling rate (F1,16=10.07, Pcrit=0.013, P=0.001)
 call dominant frequency (F1,16=13.649, Pcrit=0.006, P=0.002)
 Repeated measures MANOVA showed that
the ordering of stimuli did not significantly
influence response levels (F17,272=0.42, P=0.832).
30
Discussion
Graded Response
 Male green frogs graded their aggressive responses
as a function of stimulus intensity. With increasing
intensity, males increased both their calling and
movement rates
 Latencies for these responses did not significantly
change with increasing stimulus intensity.
 In some frogs different types of responses have
different latencies (Martins et al. 1998; Christensen-Dalsgaard et al.
2002).
 However, in green frogs the latencies did not greatly
differ according to response type. Although most
individuals gave a call before the first movement.
31
Graded Response
 As calling rate increased, call duration and
dominant frequency decreased
 Longer calls with a dominant frequency at
least 20–30 Hz below that of advertisement
calls to be aggressive calls (Bee and Perrill 1996;
Owen and Perrill 1998).
32
 However, there was no obvious clustering
that would indicate two distinct call types
according to those measures (Fig. 5).
33
 Thus, advertisement (type I) and aggressive
(type III) calls are graded continuum.
 Similar in Acris crepitans (Wagner 1989a, b) and
Hyperolius marmoratus (Grafe 1995).
34
type II high-intensity advertisement calls
 not commonly employed in close range aggressive
interactions.
 in most instances they appeared to be given in
response to type II calls from other males in the
chorus.
 In contrast, the other types of response calls and
movements were exclusively directed at stimulus
broadcasts or real intruders.
 Wells (1978) and Ramer et al. (1983) suggested that
type II calls played an intermediate role in the
escalation of aggressive encounters.
 However, in the context of this study, type II calls do
not appear to be used as a close-range threat display,
and their function may have more to do with longrange communication.
35
Movement respond
 Most previous studies of graded responses in frogs
have demonstrated that males increase their calling
rate with increasing stimulus intensity (Brzoska et al. 1982;
Schwartz and Wells 1984; Wells and Schwartz 1984; Schwartz and Wells 1985;
Given 1987; Schwartz 1989; Stewart and Bishop 1994; Grafe 1995; Jehle and
Arak 1998; Grafe et al. 2000).
 Occasional phonotaxis toward an intruder stimulus
has been previously observed in a number of species,
but this occurs infrequently during encounters (Brzoska
et al. 1982; Harrison and Littlejohn 1985; Littlejohn and Harrison 1985;
Brenowitz 1989; Wagner 1989b; Stewart and Bishop 1994; Jehle and Arak
1998; Grafe et al. 2000).
 Most of our subject green frogs approached our
intruder stimulus, and they appear more likely to use
movements as a threat display than other species of
frogs.
36
Graded changes in temporal properties
 males generally increased the duration of the
calls or call notes in response to increasing
stimulus intensity (Schwartz and Wells 1984; Wells and Schwartz 1984;
Littlejohn and Harrison 1985; Schwartz and Wells 1985; Schwartz 1989; Wagner 1989b;
.
 In contrast, we found that green frogs decreased
the duration of call, also differs from the results
of Bee and Perrill (1996).
 A possible explanation
Grafe 1995)
 increase call duration in response to calls of a more
distant intruder
 shorten call duration if that intruder approaches.
37
Graded changes in spectra properties
 Several species of frogs are known to alter
dominant frequency during aggressive
interations (Lopez et al. 1988; Wagner 1992; Bee and Perrill 1996; Howard and
Young 1998; Bee et al. 1999, 2000; Given 1999; Bee and Bowling 2002).
 Only a few species are known to do this in a
graded context.
 Leptodactylus albilabris (Lopez et al. 1988) tended to increase
or decrease frequency to match that of a stimulus.
 In contrast, both Acris crepitans (Wagner 1989a) and
Hyperolius marmoratus (Grafe 1995) decreased their
dominant frequency regardless of the frequency of the
stimulus.
38
Graded changes in spectra properties
 Green frogs responded in a similar manner by
progressively decreasing the dominant
frequency
 Dominant frequency changes on the order of 50
Hz play a potentially important behavioral role in
green frog communication (Bee et al. 1999, 2000).
 For all calls, the mean difference between
highest and lowest frequencies was 43 Hz (range
8–68 Hz)
 For response calls alone, the mean difference
was 26 Hz (range 2–50 Hz).
39
 the mechanism for lowering dominant
frequency does not related to the shortening
of call note duration.
40
Cost and benefit in graded response
 Potential cost- Reduced mating success
 Females of other frog species prefer
advertisement to aggressive calls (Wells 1988; Gerhardt and
Huber 2002).
 In green frogs, aggressive (response) calls are
produced at lower sound intensities (Bee and Perrill 1996)
 This may decrease the chance of long-range
detection by females.
41
 Since advertisement and aggressive calls form a
graded continuum in green frogs, graded
response calls could be “bi-functional” (Wells 1988; Schwartz
2001).
 Female choice in green frogs is based on territorial
quality.
 Thus, males may not suffer reduced mating
success
42
 Potential cost- energetic costs
 Energetic costs are associated with aggressive
responses in green frogs, and these costs increase
as responses increase in intensity.
 The energetic cost of advertisement calling is
probably very low, especially when compared to
species of hylid treefrogs(Wells 2001).
 However, aggressive response behaviors in green
frogs are more costly than advertisement
behaviors.
 Males giving the most intense aggressive
responses produced their calls at rates more than
three times.
43
 The increase in movement rates was even more
dramatic.
 Movements are most likely primarily fueled by
anaerobic metabolism (Gatten et al. 1992), and green frogs
lack the large lipid stores in their trunk muscles (Wells
2001).
 Thus, high rates of movement or calling cannot be
sustained for long periods.
44
 Potential cost- predation risk
 Elevated levels of responses may make male green frogs
more visible to predators.
 Predation was the most obvious cost in our study.
 A wide variety of vertebrates are known to prey upon adult
green frogs (Martof 1956), and predators are attracted to calls in
other species of frogs (Gerhardt and Huber 2002).
 Two male green frogs did not appear in our final analysis of
graded responses.
 While responding to our playbacks, one male was attacked and
injured by a water snake (Nerodia sipedon),
 The other male was devoured by a female bullfrog (Rana
catesbeiana).
 In contrast, we did not witness any attacks on males that
were merely advertising during our experimental trials.
45
 Graded responses are favored if they are on
average less costly than producing discrete
“all-or-nothing” responses.
 If graded responses reduce the chances of an
encounter escalating into a fight (i.e. they act
as signals), then the responses may also
function as threat displays.
46
 Trade off
 Stronger responses
 less appealing to females
 more energetically costly
 attract predators
 Weaker responses
 less likely to repel an intruder.
47
 Since male green frogs defend calling
positions that are used as oviposition sites by
females, and females enter breeding
aggregations sporadically throughout the
breeding season(Wells 1977b; Gordon 2004).
 Thus, the cost of losing a territory in an
encounter is potentially very high for a
resident.
 Graded responses may represent a
compromise in which the costs of producing
the response are balanced with the potential
costs of territory loss.
48
Signaling system
 Graded signaling system potentially provides
males with the ability to convey information
on motivation to fight or on fighting ability
itself (Hurd and Enquist 2001).
 Spectral components of calls may be used to
assess fighting ability in several anurans
including green frogs (Davies and Halliday 1978; Arak 1983a;
Ramer et al. 1983; Dyson and Passmore 1992; Wagner 1992; Bee et al. 1999,
2000; Burmeister et al. 2002)
.
49
Signaling system - body size or fighting ability
 In this experiment, some of the difference scores
for responses were significantly and positively
correlated with body size (Table 1)
 The intensity of these responses between
individuals potentially could be used by
opponents to assess body size or fighting ability.
 Movement rates were most strongly correlated
with body size in this experiment
 In the current experiment, body size was not a
significant predictor of male response level
50
Signaling system - motivation to fight
 Alternatively, the differential changes in response
levels within individuals may signal motivation to
fight (Hauser and Nelson 1991; Hurd and Enquist 2001; Burmeister et al.
2002; Lange and Leimar 2003).
 Motivation is expected to vary over a much shorter
time scale than fighting ability.
 Since the responses of the frogs in our study were
highly variable during the 35-min timeframe, this
variation more likely reflects motivation to fight
rather than just simply fighting ability.
 An intruder potentially could use pre-response or
initial response levels of calling and movements as
well as the dominant frequency of advertisement
calls to assess fighting ability and size of a resident.
51
 The intruder then could use these changes in the
number of calls, movements, and other
information as the encounter escalated to assess
the aggressive motivation of the resident.
 Calls are well documented as threat displays, and
presumably movements (and possibly other
visual displays) serve in this capacity as well.
 However, a definitive test of the role of
motivation in these responses requires assessing
threat displays as well as the responses to them
in both signalers and receivers (Hauser and Nelson 1991).
52
 Our data show that male green frogs increase
their aggressive responses in relation to
perceived intruder threat.
 However, the exact costs involved and the
functional significance of these responses
remains uncertain.
 No studies have quantified the physiological
costs of graded responses in frogs.
 Only a few studies of frogs have examined
whether differences in graded aggressive calls
are detected by receivers (Schwartz 1989, 1994; Wells 1989;
, and other kinds of graded
responses such as movements have not been
investigated.
Burmeister et al. 1999, 2002)
53
 It is likely that graded responses in green
frogs reduce energetic and other potential
costs of aggression.
 However, it is not known whether they serve
as graded threat displays to conspecific males.
 Future work will examine whether these
observed changes in aggressive responses are
perceived by receivers and represent
information that can be used to assess
fighting ability or aggressive intent.
54
The end
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55
 Dominant frequency changes were quite
variable among individuals (see Appendix).
56
57
Response thresholds
 Green frogs in our study may have had thresholds
for aggressive responses, and it is possible that our
stimulus broadcasts did not exceed the aggressive
thresholds of the frogs in all instances.
 Thus, the overall picture of graded responses could
be an artifact of proportionally more frogs giving
responses at each successive stimulus level due to
individual differences in aggressive thresholds.
 Studies of both Pacific treefrogs (Hyla regilla) and
spring peepers (Pseudacris crucifer) used the
production of aggressive calls that were discrete in
structure from advertisement calls to determine the
exact onset of an aggressive response (Brenowitz et al.
2001; Marshall et al. 2003).
58
 In almost all cases in our study, individual green
frogs reacted to the stimulus at all broadcast levels
with movements and call responses (see Appendix).
 Since advertisement and aggressive calls in this
species are continuous in structure, they are not
ideal for determining the threshold onset of an
aggressive response.
 Movements could be a better indicator since the
frogs rarely moved during non-broadcast periods.
 Four individuals did not move in response to the 83
dB stimulus, and of those four, three did move in
response to the 90 dB stimulus.
59
 All of our subject frogs moved in response to the 97
dB stimulus.
 However, in five of the six total cases in which the
frogs were not moving, the calling rate was elevated
or the dominant frequency and duration of calls had
decreased compared to the baseline.
 Thus, there were really only two instances where our
stimulus had not exceeded the aggressive thresholds
of the subjects for at least one measure of response,
and the instances where we did not exceed their
thresholds probably exert little influence on the
overall interpretation of individuals showing graded
responses.
60
 This issue is not easily resolved within the
context of a balanced experimental design,
and additional work is needed to determine
whether green frogs have aggressive
thresholds and whether these thresholds act
in concert with graded response levels during
the escalation of encounters.
61