Herpetology Notes, volume 10: 659-664 (2017) (published online on 23 November 2017)

Behavioural responses of Boana lundii (Anura: ) to visual and acoustic aggressive stimuli

Raíssa Furtado1,*

Abstract. Although acoustic signals are the primary mode of communication in anurans, visual signals are also important during agonistic interactions. The visual repertoire of Boana lundii was described and five males were subjected to visual (VS – male artificial model emitting ‘face wiping’) and/or acoustic (AS – playbacks of conspecific calls) stimuli. From visual (limb lifting, face wiping, throat display and vocal sac display) and acoustic (advertisement and aggressive calls) behavioural responses, only the emission of aggressive calls differed among treatments (Control, VS, AS, VS+AS). Therefore, visual displays of B. lundii do not appear to be used for communication but rather constitute displacement activities.

Keywords: Territoriality; Visual displays; Aggressive call; Displacement activities

Introduction in anurans. Multimodal signals could be used to send redundant (increasing the accuracy of the information) Anuran communication is mediated mostly by acoustic or different information at the same time (Johnstone, signals (Wells, 2010) and several types of vocalizations 1996). However, in some anuran of the family can be emitted (Toledo et al., 2014). Two types of calls Hylidae (e.g., Boana albomarginata, B. raniceps, are the focus of this study: (1) advertisement calls nanus, limellum) the visual – the most commonly emitted vocalization by males, displays emitted in social contexts probably represent functioning to attract conspecific females and determine displacement activities (Furtado and Nomura, 2014; territorial occupation in choruses; (2) aggressive calls Furtado et al., 2016). Displacement activities are – used in agonistic interactions between conspecific unintentional behaviours, elicited by motivational males for territorial defence (Wells, 2010; Toledo et conflict (fight or flight) or stress, and they are not used al., 2014). Besides acoustic signals, visual signals can for communication (Tinbergen, 1952). also be important during social interactions in anurans Hylidae is the most diverse anuran family (Frost, (Hödl and Amézquita, 2001; Hartmann et al., 2005; 2017) and visual displays have been described for Preininger et al., 2013). For example, limb lifting 21 species of ten genera: Agalychnis (Caldwell et (the most common visual cue in anurans; Hödl and al., 2010), Phyllomedusa (Jansen and Köhler, 2008), Amézquita, 2001; Hartmann et al., 2005) and vocal sac Litoria (Meyer et al., 2012), Bokermannohyla (Lima displays have been strongly associated with aggressive et al., 2014; Souza, 2014), Aplastodiscus (Hartmann et interactions, especially between males (Preininger et al., al., 2005; Toledo et al., 2007), Boana (Hartmann et al., 2013). 2005; Toledo et al., 2007; Lipinski et al., 2012; Furtado Amézquita & Hödl (2004) suggest that visual and and Nomura, 2014; Souza, 2014), Scinax (Hartmann acoustic signalling may represent multimodal signals et al., 2005; Barros and Feio, 2011), Dendropsophus (Amézquita and Hödl, 2004; Miranda et al., 2008), Acris (Horne et al., 2014) and (Gomez et al., 2009). Limb movements (e.g., limb lifting, face wiping, and leg stretching) are the most common visual displays 1 Programa de Pós-Graduação em Ecologia, Departamento de in hylids (Hödl and Amézquita, 2001; Hartmann et al., Ecologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, CEP 91501-970, Porto Alegre, Rio 2005). Grande do Sul, Although visual signalling has not been described for * Corresponding author e������ ��������������������������� Boana lundii (Burmeister, 1856), face wiping displays 660 Raíssa Furtado

aggressive calls are redundant multimodal signals; thus, the combination of visual and acoustic stimuli results in the most aggressive response. Alternatively, given the difficulty to recognize a visual signal (Hödl and Amézquita, 2001), the visual displays of B. lundii might represent displacement activities.

Materials and Methods Study site.—The study was conducted in January and February 2013, at a lotic water body in Cerrado biome in the National Forest of Silvânia, Goiás, Brazil (16º39’32” S, 48º36’29” W). The climate of the region is tropical with two well-defined seasons: a rainy season (October-March) and a dry season (April-September) (Morais et al., 2012). Experimental design.—Males of Boana lundii were observed (100 min of recordings; 5 males) in natural conditions between 20:30h and 23:30h. Calling individuals were arbitrarily selected using a flashlight, after which the flashlight was turned off to avoid manipulation stress to the focal . Behaviours were recorded with a video camera (Sony DCRDVD910) equipped with an infrared light, positioned at least 1 m from the focal animal. To simulate the presence of a conspecific intruder male, an artificial model with a colour pattern similar to that of the species (Figure 1), was positioned approximately 30 cm from the focal subject. To construct artificial models resembling real , a male specimen of Figure 1. Male (top) and artificial model (bottom) of Boana lundii in the National Forest of Silvânia, Goiás, Brazil. B. lundii deposited in the Zoological Collection from Universidade Federal de Goiás (ZUFG) was used to make a plaster mould. The plaster mould was filled with a white plastic polymer, which was then painted with oil paint to resemble the colour pattern of the have been observed during male-male interactions (pers. species (Figure 1; for more details about artificial model obs.). Boana lundii (Figure 1) is territorial and nocturnal, construction see Souza, 2014). After complete drying, and reproduces in lotic water bodies or temporary ponds the oil paint was almost odourless to human sense of in the Cerrado (Brazilian savannah; Mazzarelli, 2015; smell. The artificial model was positioned with the help Frost, 2017). During the rainy season, males call from of a malleable green metallic structure that allowed the ground or high perches on the margins of water aligning the artificial model with the position of the bodies (Mazzarelli, 2015). Five types of calls have focal animal. been described for B. lundii: advertisement, courtship, To test the hypothesis that visual and/or acoustic aggressive (territorial 1 and territorial 2) and distress stimuli modulate the behavioural repertoire of B. (Toledo and Haddad, 2009; Mazzarelli, 2015). lundii in an agonistic context, the focal animals were In this context, the objective of this study was to: (1) submitted to four treatments: (1) control (artificial describe the visual repertoire of males of B. lundii during model in passive posture; Figure 1); (2) visual stimulus agonistic interactions; and (2) test the hypothesis that (VS – artificial model emitting face wiping behaviour, males modify their behavioural response according to with help of a transparent nylon line); (3) acoustic visual and/or acoustic stimuli displayed by a conspecific stimulus (AS – playbacks of conspecific calls, with a male intruder. I predicted that visual displays and speaker positioned behind the artificial model in passive Behavioural responses of Boana lundii to visual and acoustic aggressive stimuli 1 661

1 Table 1. Two-way ANOVAs comparing the frequencies of behavioural responses by males of Hypsiboas lundii Table 1. Two-way ANOVAs comparing the frequencies of behavioural responses by males of Boana lundii subjected to four treatments:2 Control,subjected Visual to four Stimulus treatments: (VS), Control, Acoustic Visual Stimulus (VS), (AS), Acoustic and VS Stimulus and AS (AS), simultaneously and VS and AS (VS+AS). simultaneously DF and MS mean degrees of freedom and mean square, respectively. 3 (VS+AS). DF and MS mean degrees of freedom and mean square, respectively.

Behaviour Predictor Variable DF MS F P VS 1 0.12 0.61 0.45 Limb lifting AS 1 0.54 2.58 0.13 VS:AS 1 0.13 0.61 0.45 VS 1 0.02 0.32 0.57 Face wiping AS 1 0.18 3.56 0.08 VS:AS 1 0.02 0.32 0.57 VS 1 0.30 0.90 0.36 Throat display AS 1 0.42 1.27 0.28 VS:AS 1 0.30 0.90 0.36 VS 1 0.003 0.29 0.60 Vocal sac display AS 1 0.01 1.19 0.29 VS:AS 1 0.002 0.19 0.67 VS 1 103.6 3.41 0.09 Advertisement call AS 1 22.3 0.73 0.41 VS:AS 1 3.2 0.10 0.75 VS 1 0.36 0.63 0.44 Aggressive call AS 1 3.24 5.70 0.03* VS:AS 1 0.49 0.86 0.37 4 * Significant values.

posture), and (4) VS and AS simultaneously. The calls repetition rate of each behavioural response (response used in the AS treatment were previously recorded at variable) differed among treatments (two predictor the study site, and the repetition rate for playback was variables – VS and AS – with two levels each – stimulus four calls per minute (based on previous observations). offered or not offered to focal animals). I used the I used Cool Edit Pro 2.0 software to make the call edits function ‘aov’ of {stats} package to perform a two-way and a speaker (model GT Power Music, Goldentec) with analyses of variance in block (Sokal and Rohlf, 1995). frequency response of 90 Hz to 20 kHz to broadcast the Each individual represented a block and the statistical playbacks. The intensity of the playbacks was adjusted tests were performed in the software R, version 3.4.0 (R at 1 m of distance to 70 dB with a decibelimeter (model Development Core Team, 2017). DEC – 460). Each male was submitted to all treatments; however, the order of treatments was randomized for Results each focal animal. Each treatment lasted 5 min, with 2 Males of B. lundii were found calling on the ground min intervals between them. (N = 2 males) or on perches (up to 1.5 m above ground, Data analysis.—Behaviours were classified as (1) N = 3 males) on the margin of a lotic water body with visual displays (sensu Hödl and Amézquita, 2001; slowly-flowing water. All focal males were observed Hartmann et al., 2005; de Sá et al., 2016); and (2) calling individually, and not as part of a chorus. We acoustic signals (sensu Mazzarelli, 2015). The mean recorded two types of vocalizations – advertisement and emission rate per minute was calculated for each aggressive calls – emitted by all focal males. Aggressive behaviour in each treatment, except for vocal sac calls were not distinguished between ‘territorial call 1’ display, which was quantified as the proportion of time and ‘territorial call 2’ due to the great similarity between that the animal kept the vocal sac inflated during the them (Mazzarelli, 2015). treatment. For statistical analysis, the data for one focal Although the total number of visual displays (47 animal that did not respond visually or acoustically events) was much lower than the number of acoustic to the experiment was removed. Finally, to test if the signals (557 events), we recorded four types of visual 662 Raíssa Furtado

disposition, measured by the increase in the rate of aggressive calls, of B. lundii males.

Discussion This study contributes to the understanding of visual behaviour in hylid and demonstrates that it is possible to make discoveries about natural history even with limited sample sizes. Here I reported, for the first time, four types of visual displays for B. lundii that were already known for the genus Boana (Hödl and Amézquita, 2001; Hartmann et al., 2005; Toledo et al., 2007; Lipinski et al., 2012; Furtado and Nomura, 2014; Souza, 2014; Furtado et al., 2016). However, it is prudent not to exclude the possibility that males of B. lundii may be using other visual displays (29 different Figure 2. Rate of aggressive calls emitted by males of Boana visual cues have been reported for anurans (Hödl and lundii subjected to four treatments: Control, Visual Stimulus Amézquita, 2001; Hartmann et al., 2005; Souza, 2014; (VS), Acoustic Stimulus (AS), and VS and AS simultaneously de Sá et al., 2016)) during agonistic interactions. (VS+AS). The lowest rate of aggressive calls occurred in Although visual signals have been associated with the Control follow by VS treatment. The rate of aggressive aggressive contexts in some hylid species (e.g., calls in the AS treatment was significantly higher than in the Dendropsophus parviceps (Amézquita and Hödl, Control, VS and VS+AS treatments (F =5.7, p=0.03). Boxes 1,3 2004); Agalychnis callidryas (Caldwell et al., 2010); represent real values; horizontal lines represent mean values; Bokermannohyla sapiranga (Souza, 2014)), the findings and vertical bars represent standard deviations. here indicate that behaviours typically associated with visual communication in B. lundii were not correlated with visual and/or acoustic stimulation. Therefore, the visual displays – limb lifting, face wiping, throat display displays for the first time for B. lundii: (1) limb lifting and vocal sac display – performed by B. lundii males – rapid up-and-down movements of one or more limbs may represent unintentional behavioural responses, (14 events, 2 males); (2) face wiping – lifting an arm which are not used for the purpose of communication and touching the head (eye, snout and/or mouth) with (Tinbergen, 1952). Displacement activities during the hand (7 events, 2 males); (3) throat display – rapid agonistic contexts have been reported for other species pulsation (inflation and exhalation) of the throat with of genus Boana (B. goiana, B. albopunctata (Souza, or without sound production (13 events, 2 males); we 2014); B. albomarginata (Furtado and Nomura, 2014); did not consider males emitting one of the previously B. raniceps (Furtado et al., 2016)), which suggests that described calls with a communication function (e.g., visual displays may not have evolved as aggressive advertisement and aggressive calls) to be performing signals for territorial defence in this particular anuran a “throat display” behaviour; and (4) vocal sac display group. – inflation of the vocal sac, with or without sound Males of B. lundii exclusively responded to acoustic production, and maintaining it inflated for some time stimuli with aggressive calls toward the simulated (13 events, 4 males). intruding conspecific male during the experiments, The emission of visual behaviours and advertisement which corroborates the hypothesis that anuran calls by males of B. lundii did not differ among communication is mediated mostly by acoustic signals treatments (Table 1). Only the frequency of emission of (Wells, 2010). However, although Furtado and Nomura aggressive calls in the acoustic stimulus treatment was (2014) reported acoustic behavioural responses of B. significantly higher than in the other treatments (Table 1; albomarginata males to visual stimuli, the prediction Figure 2). The lowest rates of aggressive calls occurred that the combination between visual and acoustic stimuli in the control and visual stimulus treatment (Table 1; results in the most aggressive response was not found Figure 2). Thus, acoustic stimulus not associated with in B. lundii. On the other hand, the aggressive acoustic visual stimulus was required to increase the aggressive response of B. lundii males to playback experiments has Behavioural responses of Boana lundii to visual and acoustic aggressive stimuli 663 been reported for many other anuran species in the past Hartmann, M.T., Giasson, L.O., Hartmann, P.A., Haddad, C.F.B. few decades (e.g., Dendropsophus ebraccatus (Wells, (2005): Visual communication in Brazilian species of anurans 1989; Dryophytes versicolor, Reichert, 2014)). from the Atlantic forest. Journal of Natural History 39(19): 1675–1685. I conclude that communication between males of B. Hödl, W., Amézquita, A. (2001): Visual signaling in anuran lundii during territorial disputes occurs exclusively . In Anuran Communication (ed. M.J. Ryan), pp. by acoustic signals. In the present study, the acoustic 121–141, Washington DC, USA, Smithsonian Inst. 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Accepted by Simon Maddok