Research ArticleORGANISMAL BIOLOGY

High-frequency hearing in a hummingbird

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Science Advances  17 Jul 2020:
Vol. 6, no. 29, eabb9393
DOI: 10.1126/sciadv.abb9393

Abstract

Some hummingbirds produce unique high-frequency vocalizations. It remains unknown whether these hummingbirds can hear these sounds, which are produced at frequencies beyond the range at which most birds can hear. Here, we show behavioral and neural evidence of high-frequency hearing in a hummingbird, the Ecuadorian Hillstar (Oreotrochilus chimborazo). In the field, hummingbirds responded to playback of high-frequency song with changes in body posture and approaching behavior. We assessed neural activation by inducing ZENK expression in the brain auditory areas in response to the high-frequency song. We found higher ZENK expression in the auditory regions of hummingbirds exposed to the high-frequency song compared to controls, while no difference was observed in the hippocampus between groups. The behavioral and neural responses show that this hummingbird can hear sounds at high frequencies. This is the first evidence of the use of high-frequency vocalizations and high-frequency hearing in conspecific communication in a bird.

INTRODUCTION

Vocal communication is a fundamental component of diverse social contexts such as aggression, territoriality, courtship, and parental care (1). For these vocal signals to be effective, the intended receiver should be able to detect and discriminate them (2, 3). Therefore, auditory sensitivity of the intended receiver often coevolves with the vocal production of the sender of the signal (2). The Ecuadorian Hillstar (Oreotrochilus chimborazo) is a hummingbird that lives in Andean high-altitude grasslands. It produces a high-frequency (HF) song with a fundamental frequency of 13.4 kHz, the highest in any bird vocalization known to date (4). The frequency content of this song is also far beyond the hearing range in most birds (2 to 8 kHz) (5). It has never been shown that hummingbirds or any other bird, except for some species of owls (6), can hear these sounds. While owls use specialized ears for hunting their prey, the Ecuadorian Hillstar seems to use its HF song for conspecific communication.

O. chimborazo produces the most complex HF vocalization among the species of hummingbirds producing these extraordinary signals (4). This stereotyped song, which only males produce, consists of introductory motifs followed by trills (Fig. 1A). Males change their behavior depending on the social context in which they produce the song. If a male is vocalizing in its own territory, then it usually perches at a preferred high branch while patrolling the site, suggesting that, in this context, the HF song is used as a territorial signal (4). In addition, we have observed that when a male visits a female’s territory, he sings the HF song while displaying iridescent feathers from his hood to the female. While singing, the throat inflates, generating a wave-like motion on the iridescent feathers (Fig. 1B). If the female is interested, then both hummingbirds engage in a dance-like chase, in which the female briefly chases the male and vice versa. This behavior suggests that the Ecuadorian Hillstar uses its HF song as a courtship signal, in addition to its previously suspected territorial function.

Fig. 1 HF vocalizations and behavioral responses to conspecific song.

Spectrogram and power spectrum of (A) a representative HF vocalization. (B) Picture of a male O. chimborazo producing the HF song. Feathers on the cheeks flare while singing, and throat inflates eliciting waves of iridescent feathers moving along the purple hood. Photo credit: Fernanda G. Duque, Georgia State University. (C) Hummingbirds in the field that responded to playback of HF song and of ambient noise. When we played HF song, all observed hummingbirds (n = 13) exhibited changes in body posture, head tilts with neck extensions, and approached the area surrounding the playback speaker. Only a few of these hummingbirds also elicited some of these behaviors during playback of ambient noise. Hummingbirds that elicited behaviors during both stimuli (ambient noise and HF song) are shown in blue (approach: 2 hummingbirds; body posture, 3; head/neck, 1), while hummingbirds that only responded to the playback of HF song are shown in pink (approach, 11; body posture, 10; head/neck, 12).

These behavioral patterns of vocal production suggest that O. chimborazo can hear the HF songs produced by conspecifics. Therefore, we wanted to test whether O. chimborazo has evolved HF hearing consistent with the production of its HF song. Studies with songbirds have shown that individuals respond to the playback of conspecific song by vocalizing, approaching the speaker, or producing aggressive displays (711). It has also been observed that the protein expression of the immediate-early gene zenk [also known as zif268, early growth response protein 1 (EGR-1), nerve growth factor-inducible protein A (NGFI-A), and krox24] is a consistent marker for neuronal activation in response to salient stimuli in the secondary regions of the avian auditory forebrain, the caudal medial mesopallium (CMM) and the caudal medial nidopallium (NCM) (1214).

Hence, to determine the behavioral and neural responses of O. chimborazo hummingbirds to the playback of conspecific HF song, we conducted playback experiments in the field and assessed neural responses in auditory regions in the forebrain of these hummingbirds. We hypothesized that O. chimborazo hummingbirds can hear the HF song of conspecifics; therefore, we predicted that they will exhibit behavioral responses to the playback of HF song in the field. We also predicted that the auditory regions in the brains of hummingbirds exposed to HF song will express higher levels of ZENK protein compared to control hummingbirds. Together, the behavioral and neural responses will determine whether this species of hummingbirds can hear frequencies above 10 kHz, allowing them to use their HF song for communication and social interactions.

RESULTS

First, we evaluated the behavioral responses of O. chimborazo to the playback of the conspecific HF song in the field. We identified O. chimborazo individuals with defined territories and placed a speaker in one of the hummingbird’s preferred perches to simulate an intrusion by another male. We played two different sound stimuli: (i) ambient noise, which mostly consisted of wind noise, and (ii) conspecific HF song. We then recorded behavioral responses to each stimulus. Table S1 shows an ethogram describing all the behaviors that we assessed in response to playback. We reported only those individuals that were exposed to both experimental conditions (ambient noise and HF song) (n = 13).

The hummingbirds exhibited mainly three behavioral responses to the playback of conspecific HF song: approach to the area surrounding the speaker, head tilts and neck extensions, and changes in body posture (Fig. 1C). These behaviors were produced only by a few birds during the playback of ambient noise. For each behavior, we used a McNemar chi-square test to compare responses to HF song versus ambient noise. Regardless of the type of behavior that was scored, more hummingbirds responded to playback of HF song than to that of ambient noise (approach: χ2 = 9.09, P = 0.003; head tilts and neck extensions: χ2 = 8.1, P = 0.004; body posture: χ2 = 10.083, P = 0.002). At the onset of playback of a block of HF song, hummingbirds moved their heads in the direction of the speaker while extending their necks. After a few seconds, they approached the area near the speaker, perching at a nearby perch from which they inspected the speaker for the rest of the experiment (total of 5 min). After approaching, the hummingbirds continued responding with head tilts and neck extensions to the onset of playback and often shifted their body posture toward the speaker if necessary. Only one hummingbird flew over the speaker in response to the playback. In contrast, during the playback of ambient noise, hummingbirds were foraging undisturbed, maintaining a considerable distance from the perch where the speaker was placed. These behavioral responses show that O. chimborazo attends to and responds to the playback of HF song, demonstrating that these hummingbirds can detect this signal in their habitat.

We then assessed auditory responses in the hummingbird brain to frequencies above 10 kHz. We collected individuals at their roosting site before sunrise (4:30 a.m. to 5:00 a.m.) and isolated them to prevent exposure to external sounds before the experiment. At the beginning of the experiment, each hummingbird was placed in a sound-attenuating chamber and allowed to acclimate for 30 min. Then, it was exposed to either the HF song (frequencies of >10 kHz) (n = 4) or silence (n = 4), respectively, for 25 min (14). The hummingbirds were euthanized 90 min after the onset of playback (Fig. 2) (1517). Using immunohistochemistry (IHC), we measured the protein expression of the immediate-early gene zenk, a marker for neuronal activity (1719). We evaluated the secondary auditory regions CMM and NCM, which exhibit robust ZENK expression in response to salient auditory stimuli, particularly to conspecific song (16, 20, 21). In addition, in individuals from both groups, we measured ZENK expression in the hippocampus (HP), which we used as a control region (Fig. 3A) (21, 22).

Fig. 2 Induction of ZENK expression in the brains of hummingbirds.

Timeline showing the experimental design for inducing ZENK protein expression in the hummingbird brain in response to a sound stimulus. The control group (n = 4) was exposed to a playback of silence, while the experimental group (n = 4) was exposed to playback of HF song.

Fig. 3 Neural responses to HF song.

Top left panel shows (A) the representative section of the brain showing the secondary auditory areas CMM (a) and NCM (b) and the HP (c) in hummingbirds exposed to HF song. The blue square delineates the area magnified 10 times in the picture below to show the two secondary auditory areas and, as landmark, L2, the primary auditory region, which expresses little to no ZENK (49). Additional panels show representative magnified images of each region from brains exposed to silence and to HF song. (B) ZENK protein expression in the brains of hummingbirds exposed to HF song (n = 4) (pink) compared to hummingbirds exposed to silence (n = 4) (blue). The boxplot was produced using median and interquartile range (IQR) as measures of centrality. Upper and lower whiskers in the graph extended to the highest and lowest value, respectively, with a threshold set at 1.5 × IQR; any values beyond this threshold were plotted as outliers (48). Statistical analysis was done using a two-way ANOVA, Tukey’s post hoc test. ***P < 0.001.

A two-way analysis of variance (ANOVA) showed statistically significant effects of condition (HF song versus silence), brain area, and the interaction between condition and brain region (condition: F1,162 = 100.652, P < 0.001; brain area: F2,162 = 42.420, P < 0.001; interaction: F2,162 = 5.631, P = 0.004). Birds exposed to HF song showed higher ZENK expression in CMM and NCM compared to the controls (CMM: exp, x¯ = 187.2344 ± 10.2705; cont, x¯ = 102.7333 ± 7.9956, P < 0.001; NCM: exp, x¯ =168.4107 ± 7.3816; cont, x¯ = 104.0689 ± 7.3816, P < 0.001) (Fig. 3B). There were no statistically significant differences in ZENK expression in the HP between the experimental and control groups (exp, x¯ = 82.1053 ± 13.4020; cont, x¯ = 54.7167 ± 3.7703, P = 0.2817). These results show that the auditory regions in the brain of O. chimborazo responded robustly to the HF song of conspecifics, demonstrating that this hummingbird species is adapted for HF hearing.

DISCUSSION

To our knowledge, this is the first evidence of HF hearing in a bird except for some owls (6), which are specialized predators. Unlike owls, which have evolved HF hearing for predation, O. chimborazo evolved this feature for conspecific communication. Here, we showed that the Ecuadorian Hillstar (O. chimborazo) is adapted to hear its HF song (23). Individuals showed behavioral and neural responses to the HF content of conspecific song (frequencies of >10 kHz). We also report on the use of these vocalizations as part of the courtship displays that males present to females.

During the behavioral assessment in the field, hummingbirds changed their attention and approached the speaker in response to playback, showing that they can hear conspecific HF song. However, O. chimborazo hummingbirds did not show strong aggressive responses to the playback of the HF song, which suggests that a sound stimulus alone is not enough to evoke aggressive behavior. This pattern is consistent with our field observations in which O. chimborazo males and females do not engage in aggressive encounters with nearby individuals, unless the latter trespass the territory of the former. The use of multiple sensory signals in hummingbird communication has been documented in agonistic and courting contexts (24, 25). Therefore, in this species, the HF song alone may not be enough to elicit a strong aggressive behavioral response in other males. This behavior may help conserve energy so that the Hillstars engage in aggression with other birds only after they have visually confirmed the intrusion or aggression from another individual (24, 26).

Our evidence on the neural responses in the secondary auditory regions, which are sensitive to species-specific sounds (16, 18), also shows that O. chimborazo can hear its HF song. These findings are aligned with electrophysiological evidence in other species showing that specialized neurons in the brain auditory nuclei are tuned to spectral and temporal parameters of conspecific songs (27, 28). Studies in other animals have shown that the best frequency sensitivity for hearing in a species usually matches the dominant frequency in its vocal repertoire (2, 27, 29, 30). Similar findings have been reported for some anurans that produce ultrasonic sounds and whose hearing matches vocal production (31, 32). A study evaluated the hearing curve of the Blue-throated Hummingbird (Lampornis clemenciae) (33), which produces HF sounds as part of its song. The best frequency sensitivity was between 2 and 3 kHz in this hummingbird, rapidly declining after that and not reaching the HFs in some components of the song. Nonetheless, the auditory sensitivity in L. clemenciae matched the dominant frequency of the low-frequency notes in its song, still consistent with previous findings. In contrast to the song of L. clemenciae, the song of the Ecuadorian Hillstar is produced almost entirely in the HF range, with only a single note reaching as low as 7 kHz (Fig. 1A), while all other elements are produced above 10 kHz. We showed neural responses in the forebrain auditory regions of these hummingbirds, which demonstrates that they can hear the HF song; however, the best frequency for auditory sensitivity in the Ecuadorian Hillstar (O. chimborazo) remains to be determined. Characterizing the hearing curve in this species will allow us to test for trade-offs in hearing capabilities between low and high frequencies in species producing HF vocalizations as part of their vocal repertoire (34).

Our results suggest that hummingbirds producing HF vocalizations (4, 35) have evolved adaptations for the production and perception of HF sounds, which may not be present in other birds. Recently, two independent research groups (36, 37) reported on the unusual position of the avian vocal organ, the syrinx, in hummingbirds. While in most birds the syrinx is located inside the thoracic cavity, hummingbirds have a syrinx that is located outside the thorax, a feature that we have also observed in O. chimborazo. The position of the syrinx (36) paired with other musculature differences (37) may change the mechanical properties of the vocal organ and air pressure while vocalizing, facilitating the production of HF sounds. In some species of hummingbirds, which have also encountered environmental pressures to avoid signal masking in acoustically challenging environments (4), these conditions may have come together to promote the evolution of HF vocalizations as part of the vocal repertoire of these species.

Here, we also reported that O. chimborazo males produce their HF song while courting females in the female’s territory. Our observations of the natural history of O. chimborazo show that most females tend to roost, forage, breed, and nest near creeks at lower elevations, in contrast to most males, which are usually found at higher elevations (26). The running water in these sites creates additional ambient noise, which can reach higher frequencies than those in the wind-dominated noise characteristic of the high-altitude grasslands (4). In other species of Andean hummingbirds, which also produce HF vocalizations, the profile of the ambient noise suggests that these species evolved vocalizations at HFs to avoid signal masking in their noisy habitat (4). This evidence paired with behaviors produced while vocalizing suggests that O. chimborazo males evolved a HF song to avoid ambient noise in the female’s territory during the courtship process. Just like it occurs in other species, songs can also be used to signal territoriality (1), which seems to be the case in this species when males vocalize in their own territories at higher elevations (4).

Together, the behavioral responses and the neural activation of critical auditory regions in response to HF song as well as the context in which these vocalizations are produced point to the use of HF vocalizations in conspecific communication in O. chimborazo. Our results constitute the first evidence of the use of HF vocal signals and HF hearing in conspecific communication in hummingbirds and any birds. The presence of HF vocalizations in hummingbirds offers a new avenue to study the morphological, peripheral, and central sensory adaptations that facilitate communication and social interactions. These findings also contribute to broadening our understanding of the coevolution of vocal signals and auditory perception to facilitate social interactions in acoustically challenging conditions.

MATERIALS AND METHODS

Playback recordings

Stimuli for field playback experiments were assembled using high-quality recordings of the HF song of O. chimborazo, which were recorded at 1 m from naturally vocalizing birds using a TASCAM DR-40 recorder (TEAC American Inc., CA, USA) (4). These recordings were collected at Cunugyacu and Chimborazo Lodge for the subspecies O. chimborazo chimborazo and at Rucu Pichincha for the subspecies O. chimborazo jamesonii. We used a single vocalization from each location to generate each of the HF song playbacks: two for O. chimborazo chimborazo and one for O. chimborazo jamesonii. Although the HF song of the two subspecies differs in structure, the frequency range remains the same (4).

Recordings of the selected vocalizations were normalized to 70 dB using Audacity (38). This amplitude level was confirmed using a sound pressure level meter (Brüel & Kjaer, Denmark) at 1 m in a sound-attenuating chamber. Using the seewave package (39) in R (40), we created a block of 10 vocalizations, leaving 1-s intervals between vocalizations. We applied a band-pass filter (10 to 20 kHz) to delete from the playback any sound outside of this range. Blocks were repeated at random intervals until the playback stimulus reached 5 min. To create playbacks of ambient noise, we collected multiple recordings of environmental noise at the same locations where we recorded HF song (4). Then, we selected a section of the recordings that contained only abiotic noise and was not contaminated with sounds from other animals. A similar procedure for generating the HF song playback was conducted to assemble the playback of ambient noise composed of frequencies below 10 kHz, which is the natural range for noise in this habitat (4).

For the controlled ZENK experiment, we followed the procedure described above; however, vocalizations were assembled in pairs. Blocks of these pairs were repeated at random intervals until the stimulus had a total duration of 25 min. Using Audacity (38), we built a 120-min stimulus, including silence before and after the experimental condition (Fig. 2). For the control group, which was not exposed to any sound stimuli, we used Audacity (38) to build a 120-min audio file containing silence. For the ZENK experiment, we only generated two playbacks of HF song for the subspecies O. chimborazo chimborazo, since the experiment was only conducted with this subspecies at two locations on the slopes of Mount Chimborazo. Hummingbirds were exposed to a playback of HF song recorded at a different locale from where the experiment was conducted.

Behavioral experiments

Field experiments were conducted at several locations along the Ecuadorian Andes, namely, at the grasslands in Cotacachi, Pichincha, Antisanilla, Cotopaxi, and Cajas for the subspecies O. chimborazo jamesonii and in two locations in the slopes of Mount Chimborazo for the subspecies O. chimborazo chimborazo. After identifying a hummingbird with a defined territory, we placed a rechargeable JBL Clip 2 speaker (Harman, CT, USA) at one of the individual’s preferred perches. We followed a within-subjects design, in which all hummingbirds were exposed to the playback of ambient noise and HF song. We allowed the hummingbird to approach, inspect, and acclimate to the presence of the speaker for at least 30 min. After acclimation, we played back the ambient noise to establish a behavioral baseline for 5 min. Then, we broadcast the playback of HF song for another 5 min. Playback of HF song matched the subspecies living in each field site where we conducted the behavioral experiment; however, the field playback experiments were conducted at different locations from where the original recordings were made.

Two scorers were positioned at least 5 m away from the speaker and the hummingbird, with clear views of both. Scorers independently scored the behaviors of the hummingbird according to an ethogram (table S1). If a behavior could be attributed to the presence of another animal, then it was not recorded by the scorers. Scorers only recorded those behaviors produced by the experimental animal at the onset of each block of sound stimulus for the duration of the playback (5 min). Any behavior that was produced during the silent intervals between blocks was not included. After the experiment, annotations of the two scorers were compared, and only those behaviors that were consistently reported by the two scorers were selected for analysis.

Induction of ZENK expression in the brain

For the controlled ZENK experiment, we collected O. chimborazo hummingbirds (n = 8) at two locations around Mount Chimborazo in Ecuador, at the beginning of the breeding season in 2018 and 2019. They were isolated from each other and hand-fed 20% sucrose solution every 15 min until the beginning of the experiment. We built a hardwood sound-attenuating chamber (45.72 cm by 45.72 cm by 45.72 cm), which had the inner walls covered with acoustic foam. It contained a perch and two small hand feeders for the hummingbirds to feed during the experiment. Two small lights were provided to facilitate feeding during the experiment. The speaker in a TASCAM DR-40 recorder (TEAC American Inc., CA, USA) was used for playback; a second TASCAM DR-40 recorder was placed inside the chamber to record any vocalizations that the hummingbird produced. Hummingbirds were exposed to 25 min of playback stimulus, either silence or HF song, and rested for additional 65 min inside the chamber to reach peak ZENK protein levels in the brain (Fig. 2) (41).

Upon completion of the experiment, hummingbirds were anesthetized using 4% isoflurane and rapidly decapitated. Brains were collected immediately after and preserved in 4% paraformaldehyde solution for 24 hours. Then, the brains were submerged in 10, 20, 30, and 40% sucrose solution with 0.01% sodium azide as a preservative to prevent fungal growth. The brains were then transported to our laboratory at Georgia State University, where they were embedded in optimal cutting temperature medium and stored at −80°C until sectioning. Brains were sectioned in a cryostat in 20-μg coronal sections and placed on slides in a sequential order so that the first section of slide A was adjacent to the first section in slide B. All procedures were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) (protocol no. A18049).

Immunohistochemistry

For IHC, we followed standard protocols as described in Shahbazi et al. (42). We used two sets of slides for each brain (A and B). Set A received the primary antibody and was used to assess our experimental conditions, while set B was used as a control for background and nonspecific staining during the IHC procedure. We bathed the sections in multiple washes of tris buffer solution (TBS) followed by a bath in Triton X-100 (detergent) and trypsin (porcine enzyme). Then, sections were submerged in hydrogen peroxide and methanol for 15 min. After additional washes in TBS, normal rabbit serum (5560-0008, SeraCare, MA, USA) was applied on the tissue, which was incubated at room temperature (RT) for 30 min. Then, we applied the primary antibody to the tissue in set A while leaving the tissue in set B with additional normal serum. We used an EGR-1 polyclonal antibody (dilution, 1:500; AF2818, Novus Biologicals, CO, USA) and let it incubate for 48 hours. Then, we applied a biotinylated rabbit anti-goat secondary antibody (5570-0009, SeraCare, MA, USA) and let it incubate for 90 min at RT. Peroxidase-labeled streptavidin (5550-0001, SeraCare, MA, USA) was then applied, and the tissue was incubated for 60 min in a dark chamber at RT. Last, we applied 3,3′-diaminobenzidine (SK-4100, Vector Laboratories, CA, USA) and let the sections rest at RT for 15 min for staining. We then rinsed the slides and dehydrated the tissue by submerging it in ethanol at increasing concentrations (40, 70, 90, and 100%). Last, we submerged the slides in a clearing agent before covering them.

To evaluate the specificity of the primary antibody, we conducted protein sequence alignments of the immunogen sequence of the EGR-1 antibody (UniProt, accession no. P18146) (43) and the coding sequence (CDS) of the zenk gene in the zebra finch (Taeniopygia guttata) (GenBank, accession no. EF052676.1) (44) using protein-protein BLAST (45). We found 93% alignment of amino acid sequences between the antibody immunogen and the zenk CDS. Then, we used the SVMTriP online software (46) to predict putative epitopes in each sequence to which the EGR-1 polyclonal antibody could bind. SVMTriP provides a total of 10 putative epitopes for each sequence. Our results showed that at least five putative antigenic epitopes in the EGR-1 immunogen correspond to putative antigenic epitopes in the CDS of the zenk gene in the zebra finch.

Cell counting

To quantify ZENK expression, two scorers blinded to experimental condition and brain region counted immune-positive cells in coded pictures. Pictures were modified using GIMP (47) to superpose a 5 × 5 grid so that scorers counted cells only within the 3 × 3 inner grid on each picture, as shown in fig. S1A. This method guaranteed that only immune-positive cells in the region of interest were counted while not including cells that could potentially belong to an adjacent region. Scorers counted multiple alternate sections for each brain region in each animal.

A coefficient of variation (CV) (SD/mean) < 0.25 between the two scorers indicated valid counts; otherwise, the pictures were recounted by both scorers. In the final dataset, the CV between the scorers was low (mean CV = 0.0906, SD = 0.0611), and the correlation was strong (R2 = 0.9604, P < 0.001) (fig. S2B), demonstrating that the cell counts were reliable. The average of the cell counts from the two scorers was then used for our statistical analysis.

Statistical analysis

For the analysis of behavioral responses, we used a McNemar chi-square test to evaluate the effect of HF song in the behavioral responses of hummingbirds in the field compared to the behaviors exhibited during a playback of ambient noise. To evaluate the effect of the presentation of HF song in forebrain auditory regions, a two-way ANOVA was used. Tukey’s post hoc analysis was used to assess pairwise comparisons. All statistical analyses were conducted in R (40). All plots were generated using the ggplot2 v.3.3.0 package (48) in R (40).

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/29/eabb9393/DC1

https://creativecommons.org/licenses/by-nc/4.0/

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

REFERENCES AND NOTES

Acknowledgments: We thank E. Bonaccorso for assistance with logistics and permits; S. L. Punina, M. Cruz and Chimborazo Lodge, and Fundacion de Conservacion Jocotoco for access to field sites; M. “Pancho” Cuichan and P. Chanaba for assistance in the field; and Brigit McGuinness for assistance with IHC procedures. This research was covered by collection permits from the Ecuadorian Ministry of Environment (MAEDNB-CM-2015-0017 and MAEDNB-CM-2018-0105). Funding: This research was supported by Research Exchange Awards to F.G.D. from NSF Sociogenomics Research Coordination Network grant IOS 1256839, the Center for Behavioral Neuroscience (CBN) at Georgia State University (GSU), and the Konishi Neuroethology Research Award to F.G.D. by the International Society for Neuroethology (ISN). Author contributions: Conceptualization: F.G.D., W.W., and L.L.C. Methodology: F.G.D., C.A.R.-S., W.W., and L.L.C. Investigation: F.G.D., C.A.R.-S., and M.F.M. Data analysis: F.G.D., C.A.R.-S., S.U., and I.N. Writing (original draft): F.G.D. Writing (reviewing and editing): F.G.D., C.A.R.-S., S.U., I.N., M.F.M., W.W., and L.L.C. Funding acquisition: F.G.D. and W.W. Supervision: W.W. and L.L.C. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Protocols to conduct this research were approved at GSU under IACUC protocol no. A18049. Tissue samples were transferred to the United States under material transfer agreement (MTA) MAE-DNB-CM-2015-ATM-2017-003 between Universidad Tecnologica Indoamerica (UTI) and the Neuroscience Institute at GSU and MTA ATM-CM-2018-0105-2019-002 between Universidad San Francisco de Quito (USFQ) and CBN at GSU, appropriate CITES permits provided by the Ecuadorian Ministry of Environment, and in compliance with the U.S. Fish and Wildlife Service (USFWS) and the U.S. Department of Agriculture (USDA). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
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