Behav Ecolog Sociobiol 2005 Patrick C. Owen and Noah M. Gordon

1. Behav Ecolog Sociobiol 2005 Patrick C. Owen and Noah M. Gordon The effect of perceived intruder proximity and resident body size on the aggressive responses of male green frogs, Rana clamitans(Anura: Ranidae)

2. Introduction Graded response • A variety of animals modify or grade their aggressive responses in a continuous fashion. • Can prevent encounter from escalating intofight. • Costsreduced matingsuccess, greater energetic expenditure, or greater riskof predation. (Archer 1988; Bond 1989a; Hauser and Nelson 1991; Bradbury and Vehrencamp 1998; Hurd and Enquist 2001) (Martins 1994; Adams and Mesterton-Gibbons 1995; Hurd and Enquist 2001)

3. These aggressive responses may vary with the size (Archer 1988; Andersson 1994; Puseyand Packer 1997). • Graded responses may function as signals of1) aggressive motivation2) fighting ability • Studies in amphibians havesuggested that graded variation may serve in both of these roles. (Bond 1989a, b,1992; Hauser and Nelson 1991; Hurd and Enquist 2001;Lange and Leimar 2003) (Wells 1988; Schwartz2001; Gerhardt and Huber 2002)

4. These responses mostoften take the form of discrete call types that can varycontinuously

5. Some anurans may signalbody 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 ontheir role in signaling aggressive motivation (Burmeisteret al. 2002). • The graded nature of non-vocalization responsessuch as movements or visual displays has not beeninvestigated.

6. We examined graded responses in green frogs (Ranaclamitans,Ranidae). • EasternNorth 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.

7. 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).

8. 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

9. 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

10. 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).

11. 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 • informationabout fighting ability. • information about signaler motivation,but this has not been previously investigated.

12. Objectives • To determine whetherthese aggressive responses are graded accordingto increases in stimulus level and to examine howthese responses vary with resident body size. • We investigatedthe components of aggressive responses • rates of response calls • temporal (duration)and spectral (dominant frequency) characteristics • We also examined rates of response movements.

13. The null expectation • Resident would not respond differently to stimuli representing different level of threat Progressive changes inresponses wouldprovide evidence that aggressive responses are graded asa function of the perceived level of threat • Frogs would give the same level ofresponse regardless of body size If larger malesgive proportionally greater responses to stimuli, then thissuggests that larger males employ potentially more costlyresponses than do smaller males

14. Methods • We conducted experiments in a large pond in Brooklyn,Connecticut, USA, during the summer of 1999. • Time: 2200 to0300 h • Measurement: SVL and body mass • Individual recognition • spot patternsand scars were recorded • avoid pseudo-replication. • Water temperature did not vary a lot.

15. 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). • territorysizes were typically 1–2m in radius. • Simulate an encroaching intruder

16. Playback • Speaker mounted on aStyrofoam platform. • 1mfrom 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 stimulusproperties.

17. Playback • We used a graded playback design similar to Schwartz(1989) and Wagner (1989b) with the following modifications. • Pre-experimental period ofat least 5 min. • If the maleinterruptedduring the pre-experimental or experimental periods, thenwe stopped • We ultimately excludedfive males • However,relocate disturbed males was not an ideal solution (Gordon 2004).

18. Playback Record 3-5 calls→S1→S2→S3 • Each experiment lasted for at least 35 min. • A balanced designthat controlled for possible carry-over effects (Quinn andKeough 2002). • Movements were assessed visually. Pre-experimental period Experimental period

19. 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 noteduration • Response latency (reaction time).

20. Repeated measures multivariateanalysis of variance (MANOVA) and covariance(MANCOVA) • dependent variables • mean dominant frequency • mean noteduration • calling rate (calls/5-min period) • movement rate(moves/5-min period) • calling latency, and movement latency • SVL wasused as the covariate.

21. We used the rank transform approach inwhich response data were converted to ranks within eachvariable, and the resulting ranked data were analyzed usingstandard parametric methods (Quinn and Keough 2002). • Linearcontrasts were used to test the hypothesis of responsechange trends for each significant ANOVA. • Critical significancelevels of ANOVAs and linear contrasts were adjustedusing the sequential Bonferronimethod (Quinn and Keough 2002).

22. Results Aggressive response • 17 individualsresponded to the stimulus with a combination of callsand movements. • Gave a response call first (69% of broadcasts), the firstresponse movement was given an average of 21.1 s later. • When a movement was given first (31% of broadcasts), thefirst response call was given an average of 15.3 s later. • Inone instance the male mounted the speaker platform andattempted to wrestle with the speaker. • Normally, the frogsapproached no closer than the water in front of the speakerplatform. • Returned totheir original positions by the end of each intervening5-min silent period

23. Type II high-intensity advertisementcalls • occasionally given by 10 males. • Three of theseindividuals gave type II calls in response to our broadcasts • The other seven individuals gave type II callsin response to other frogs what were producingtype II calls. • Most type II calls were produced duringbroadcast periods (83 dB = 6 calls, 90 dB = 5 calls, 97 dB = 5 calls, silent periods = 2 calls). • Only 17of 940 total response calls for all individuals were type IIcalls.

24. Male body size (SVL) of the subjects ranged from 69 to91 mm encompassing the range of sizes in previous studiesthat addressed fighting ability (Wells 1977b, 1978; Rameret al. 1983; Bee et al. 1999, 2000).

25. A preliminary analysisusing Spearman rank-order correlations indicated thatSVL was significantly correlated with some of the responsevariable difference scores at some broadcast intensities.

26. Arepeated measuresMANCOVAwas performed with SVL as thecovariate. • In contrast to the analysis of Beeet al. (1999), SVL did not have a significant effect on responselevel (F1,15=3.74, P=0.072), • and including SVL asa covariate only resulted in a decrease of 0.9% in total unexplainedvariation compared to a MANOVA (see below)on the same data. • Thus, SVL was not included in furtheranalysis of stimulus level effect.

27. Repeated measures ANOVAon response level • movement rate • (F2,32=18.42, Pcrit=0.050,P<0.0001) • calling rate • (F2,32=10.071, Pcrit=0.013, P=0.001)

28. Stimulus levels did not have a significanteffect on response latencies movements F2,32=0.14, Pcrit=0.001, P=0.849 calls F2,32=0.14,Pcrit=0.001, P=0.849

29. stimuluslevel had a significant effect on call dominant frequency(F2,32=6.66, Pcrit=0.006, P=0.004; Fig. 4A) and on callduration (F2,32=13.45, Pcrit=0.025, P<0.0001; Fig. 4B).

30. Linear contrasts indicated significant changes for • movementrate (F1,16=37.09, Pcrit=0.050, P<0.0001) • callduration (F1,16=13.452, Pcrit=0.025, P<0.0001) • callingrate (F1,16=10.07, Pcrit=0.013, P=0.001) • call dominantfrequency (F1,16=13.649, Pcrit=0.006, P=0.002) • Repeated measures MANOVA showed that the orderingof stimuli did not significantly influence response levels(F17,272=0.42, P=0.832).

31. Discussion Graded Response • Male green frogs graded their aggressive responses as afunction of stimulus intensity. With increasing intensity, males increased both their calling and movement rates • Latencies for these responses did not significantlychange with increasing stimulus intensity. • In somefrogs 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 differaccording to response type. Although most individuals gavea call before the first movement.

32. Graded Response • As callingrate increased, call duration and dominant frequency decreased • Longercalls with a dominant frequency at least 20–30 Hz belowthat of advertisement calls to be aggressive calls (Beeand Perrill 1996; Owen and Perrill 1998).

33. However, therewas no obvious clustering that would indicate two distinctcall types according to those measures (Fig. 5).

34. Thus, advertisement(type I) and aggressive (type III) calls aregraded continuum. • Similar in Acriscrepitans(Wagner 1989a,b) and Hyperoliusmarmoratus(Grafe 1995).

35. type II high-intensityadvertisement calls • not commonly employed in closerange aggressive interactions. • in most instances they appeared to be given in response totype II calls from other males in the chorus. • In contrast,the other types of response calls and movements were exclusivelydirected at stimulus broadcasts or real intruders. • Wells (1978) and Ramer et al. (1983) suggested that typeII calls played an intermediate role in the escalation of aggressiveencounters. • However, in the context of this study,type II calls do not appear to be used as a close-rangethreat display, and their function may have more to do withlong-range communication.

36. Movement respond • Most previous studies of graded responses in frogs havedemonstrated that males increase their calling rate with increasingstimulus intensity (Brzoska et al. 1982; Schwartzand Wells 1984; Wells and Schwartz 1984; Schwartz andWells 1985; Given 1987; Schwartz 1989; Stewart andBishop 1994; Grafe 1995; Jehle and Arak 1998; Grafe et al.2000). • Occasional phonotaxis toward an intruderstimulus has been previously observed in a number ofspecies, but this occurs infrequently during encounters(Brzoska et al. 1982; Harrison and Littlejohn 1985;Littlejohn and Harrison 1985; Brenowitz 1989; Wagner1989b; Stewart and Bishop 1994; Jehle and Arak 1998;Grafe et al. 2000). • Most of our subject green frogsapproached our intruder stimulus, and they appear morelikely to use movements as a threat display than otherspecies of frogs.

37. Graded changes in temporalproperties • males generally increased theduration of the calls or call notes in response to increasingstimulus intensity (Schwartz and Wells 1984; Wells andSchwartz 1984;Littlejohn and Harrison 1985; Schwartzand Wells 1985; Schwartz 1989; Wagner 1989b; Grafe1995). • In contrast, we found that green frogs decreasedthe duration of call, also differs from the results ofBee and Perrill (1996). • A possible explanation • increase call durationin response to calls of a more distant intruder • shorten call duration if that intruder approaches.

38. Graded changes in spectraproperties • Several speciesof frogs are known to alter dominant frequency duringaggressive 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 afew species are known to do this in a graded context. • Leptodactylusalbilabris(Lopez et al. 1988) tended to increase ordecrease frequency to match that of a stimulus. • In contrast,both Acriscrepitans(Wagner 1989a) and Hyperoliusmarmoratus(Grafe 1995) decreased their dominant frequencyregardless of the frequency of the stimulus.

39. Graded changes in spectraproperties • Green frogsresponded in a similar manner by progressively decreasingthe dominant frequency • Dominant frequency changes on theorder of 50 Hz play a potentially importantbehavioral role in green frog communication (Beeet al. 1999, 2000). • Forall calls, the mean differencebetween highest and lowest frequencies was 43 Hz (range8–68 Hz) • For response calls alone, themean difference was 26 Hz (range 2–50 Hz).

40. the mechanism for lowering dominant frequency does notrelated to the shortening of call note duration.

41. 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.

42. 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

43. 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.

44. 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.

45. 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 (Nerodiasipedon), • 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.

46. 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.

47. Trade off • Stronger responses • less appealing to females • more energetically costly • attract predators • Weaker responses • less likely to repel an intruder.

48. 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.

49. 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).

50. 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