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*London J. 1903. The call of the wild. New York: Macmillian Publishers. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA The call of the (Neotropical) wild*: maned wolf long-range acoustic ecology LUANE MARIA STAMATTO FERREIRA Orientadora: Profª. Drª. Renata Santoro de Sousa Lima Co-orientador: Prof. Dr. Flávio Henrique Guimarães Rodrigues (UFMG) NATAL RN 2019

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Page 1: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

*London J. 1903. The call of the wild. New York: Macmillian Publishers.

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA

The call of the (Neotropical) wild*: maned wolf long-range acoustic

ecology

LUANE MARIA STAMATTO FERREIRA

Orientadora: Profª. Drª. Renata Santoro de Sousa Lima

Co-orientador: Prof. Dr. Flávio Henrique Guimarães Rodrigues (UFMG)

NATAL – RN

2019

Page 2: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

LUANE MARIA STAMATTO FERREIRA

The call of the (Neotropical) wild: maned wolf long-range acoustic

ecology

Tese de doutorado apresentada ao Programa

de Pós-graduação em Psicobiologia do

Centro de Biociências da Universidade

Federal do Rio Grande do Norte, como parte

dos requisitos para a obtenção do título de

Doutor.

Orientadora: Profª. Drª. Renata Santoro de Sousa Lima

Co-orientador: Prof. Dr. Flávio Henrique Guimarães Rodrigues (UFMG)

NATAL – RN

2019

Page 3: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,
Page 4: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

Título da tese: The call of the (Neotropical) wild: maned wolf long-range acoustic ecology

Autora: Luane Maria Stamatto Ferreira

Orientadora: Profa. Renata Sousa-Lima

Coorientador: Prof. Flávio Rodrigues

Data da defesa: 28 de fevereiro de 2019

Parecer:

Banca examinadora:

Profa. Dr. Renata Sousa-Lima (presidente)

Universidade Federal do Rio Grande do Norte (UFRN)

Prof. Dr. Jeff Podos

University of Massachusetts Amherst

Prof. Dr. Holger Klinck

Cornell University

Profa. Dra. Julie Patris

Aix-Marseille Université

Profa. Dra. Susan Parks

Syracuse University

Page 5: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Agradecimentos

A aparente solitária e monumental tarefa de escrever uma tese é, na verdade, o

trabalho de um exército e eu agradeço a todos que apoio por apoio, ideia por ideia,

construíram esse projeto. Assim como o lobo-guará parece solitário, mas está inserido

em uma rede social de comunicações, eu agradeço a vocês que foram, mesmo que

alguns à longas distâncias, minha alcateia.

Agradeço primeiramente aos meus pais, Inês e Adir, pelo excepcional cuidado

parental. Agradeço ao meu parceiro Carlos Carvalheira, que caminhou sempre ao meu

lado. Agradeço a meu irmão Alessandro e sua parceira Sarah, que já dispersaram para

Curitiba, mas sempre serão parte da alcateia. E também aos meus canídeos não

selvagens, Neblina e Luke Luke.

Agradeço à minha família estendida, o Laboratório de Bioacústica da UFRN.

Em especial aos lobos Victor Sábato (o primeiro lobo), Luciana Rocha (a primeira loba

selvagem), Danielly Duarte, Edvaldo Neto, Thiago Pinheiro, Rafael Frigo, Flávio

Rodrigues (o lobo-mor) e claro, à líder do grupo de caça, Renata Sousa Lima. Sem sua

visão e orientação esse projeto não seria possível e nenhum lobo teria chegado tão

longe.

Meus agradecimentos também vão a todos outros integrantes do LaB, mesmo os

que ficaram por tempo breve. Vocês foram mais que colegas, foram amigos e família.

Daqueles de mar e de terra, de rio e de ar, desde as intérpretes de baleiês, aos “ouvidores

de tudo” (paisagem acústica), as lontras (e ariranhas), tartarugas, golfinhos, focas,

saguis, passarinhos e gaviões. Meus agradecimentos especiais aos companheiros de

aventura: Eliziane, Divna, Lara, Marcos e Letícia. E a todos colaboradores, Milagros

Villavicencio, Júlio Baumgarten, Eduardo Venticinque e Mauro Pichorim.

Agradeço as pessoas que se disponibilizaram a ler e também contribuir com suas

vozes no trabalho: Jeff Podos, Holger Klink, Julie Patris, Paulo Cordeiro, Susan Parks e

Gustavo Zampier.

Agradeço as pessoas cujo apoio logístico e de know-how foi essencial ao projeto,

em particular Jean Piere (que é afiliado do Dietz!), Ricardo, Marcello, Júlia Simões

Page 6: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

(time 3m), Rogério Cunha de Paula e Flávio Rodrigues. Agradeço as pessoas fantásticas

que conheci em São Roque de Minas, incluindo Adriano Gambarini e Flávia Ribeiro.

Também aqueles que me ofereceram estadia nos momentos mais críticos: Renilda e

funcionários da pousada Chapadão da Canastra, Pavel (melhor flash-touristic-guide de

Ilhéus), Maria Inês Santoro (a mãe da Renata, que tem o mesmo nome da minha!), e

Rachel e Gustavo, amigos de longa data (saudades). Me senti acolhida em todos

sentidos.

Agradeço a CAPES pela bolsa, ao Programa de Pós-Graduação em

Psicobiologia da UFRN pelo auxílio financeiro, ao ICMBio pela licença concedida e

aos funcionários do centro administrativo do PNSC, em São Roque de Minas, por terem

sido tão compreensivos e prestativos.

Agradeço de coração aos amigos que me mantiveram sã no processo, os grupos

do RPG, boardgames, aikidô, séries (Lúcifer!), biologia (As Cobaias) e tantos outros.

Um obrigada muito especial a Rodrigo, Thieza, Amanda, Aja, Fred Dimitrius, Geórgia,

Girão, Ramon, Moal, Nicolau, Walles, Dinara, Suelen, Naíra, Carol e Nelson. E um

domo arigato gozaimashita ao meu sensei James!

Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

mesmo sem terem assinado termo de consentimento livre esclarecido, tiveram suas

conversas pessoais gravadas, dando, literalmente, voz a esse projeto. (Também espero

que um dia eles tragam meu celular de volta).

Enfim, obrigada a todos.

Passamos por fogo e foi preciso ser ninja,

mas esta etapa está concluída!

Luane Maria Stamatto Ferreira

2019

Page 7: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

For the strength of the Pack is the Wolf, and the strength of the Wolf is the Pack.

– Rudyard Kipling

Page 8: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Abstract

Maned wolves are difficult to observe in the wild because of their low densities

and their cryptic and crepuscular-nocturnal habits. Exploring their long-range call – the

roar-bark – is an efficient alternative for studying the species. We used a combination of

methodologies: we played back roar-barks in the wolves’ natural habitat to test how

free-ranging animals would respond and to understand the propagation properties of this

vocalization in the wild; we recorded spontaneous roar-bark sequences of wild maned

wolves using a grid of autonomous recorders for eight months to reveal long term

temporal patterns; and we used captive records to access sex and individuality in the

roar-bark and to test its application to natural recordings. We found that maned wolves

vocalize more during the beginning of the night, and this was the only period we

obtained responses during the playback experiment, despite both twilights having

efficient propagation of roar-barks. Social factors may be influencing the timing of the

wolves’ long-range vocal activity. We suggest that roar-barks may be an honest

advertisement of quality for territorial defense. Maned wolves vocalize more on better

moonlit nights, especially when the first half of the night is illuminated, likely as a

consequence of reduced foraging time and therefore having more time to invest in

acoustic communication. It was possible to identify the mating and circa-parturition

period in our natural recordings by an increase in solo and group vocal activity, which

suggests a role of roar-barks in partner attraction/guarding and intra-familiar-group

communication. In captivity, male roar-barks were distinguishable by their longer

duration, also indicating a sexual function and suggesting a higher energy investment to

advertise motivation. Roar-barks were also individually distinct. However, site

characteristics, such as presence of vegetation, drastically affected both the propagation

of broadcasted roar-barks and most identity and sexual parameters’ transmission in the

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wild. Elevating the speaker 45° upward to simulate the head/muzzle position during

vocalization lead to lower recorded sound intensities, but partially counteracted the

negative effects of vegetation on signal transmission. The few stable parameters were

able to discriminate individuals, although with lower success rate. In wild recordings

the variation of parameters due to propagation was larger than the variation due to

individual differences, therefore limiting passive acoustic monitoring as a means of

counting individuals in their natural habitats. Despite the present limitation of vocal

identification in the wild, bioacoustic tools proved efficient in revealing the secretive

behavior ecology of maned wolves.

Key-words: Chrysocyon brachyurus, canid, vocalization, sound propagation, passive

acoustic monitoring, temporal patterns, playback.

Page 10: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Resumo

Os lobos-guará são difíceis de serem observados na natureza devido as suas

baixas densidades e hábitos crípticos e noturno-crepusculares. Explorar seu chamado de

longa distância – o aulido – pode ser uma alternativa eficiente para estudar a espécie.

Usando uma combinação de metodologias: reproduzindo aulidos no ambiente natural da

espécie para testar como animais de vida livre responderiam e para entender as

propriedades de propagação dessa vocalização; gravando sequências de aulidos

espontâneas de lobos-guará selvagens através de uma rede de gravadores autônomos por

oito meses para revelar padrões temporais de longo prazo; e registramos os sons

produzidos em cativeiro para conferir a discriminação de gênero e individualidade no

aulido e testar sua aplicação em gravações de ambiente natural. Nós descobrimos que os

lobos-guará vocalizam mais no início da noite, e esse foi o único período em que

obtivemos respostas durante o experimento de playback, apesar de ambos crepúsculos

apresentarem uma propagação eficiente deste tipo de som. Fatores sociais podem estar

influenciando esse padrão temporal, como o anúncio honesto de qualidade para defesa

territorial. Lobos-guará vocalizam mais em noites de maior iluminação lunar,

especialmente quando a primeira metade da noite está iluminada, provavelmente como

consequência de uma redução no tempo de forrageio e, portanto, mais tempo para

investir na comunicação acústica. Foi possível identificar o período de acasalamento e

aquele em torno do parto nas nossas gravações de ambiente natural através do aumento

na atividade vocal solo e de grupo, o que indica um papel dos aulidos na atração/guarda

de parceiros e na comunicação intra grupo familiar. Em cativeiro, os aulidos dos

machos foram distinguíveis principalmente por sua duração mais longa, também

indicando uma função sexual e sugerindo um investimento energético mais alto para

anunciar motivação. Aulidos também foram distintos individualmente. Porém,

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características locais afetaram dramaticamente tanto a propagação dos aulidos

reproduzidos quanto quase todos parâmetros que conferem identidade e gênero aos sons

emitidos. Elevar a caixa de som 45° para cima para simular a posição da cabeça/focinho

durante a vocalização resultou em intensidades sonoras mais baixas nas gravações, mas

compensou parcialmente os efeitos negativos da vegetação na transmissão do sinal

acústico. Os poucos parâmetros estáveis durante a propagação em ambiente natural

foram capazes de discriminar indivíduos, embora com menor taxa de sucesso.

Infelizmente, nas gravações obtidas na natureza a variação dos parâmetros devido à

propagação foi maior que as diferenças individuais observadas. Apesar da presente

inaplicabilidade da identificação vocal em gravações de aulidos na natureza, as

ferramentas bioacústicas se provaram eficientes em revelar a elusiva ecologia

comportamental dos lobos-guará.

Palavras-chave: Chrysocyon brachyurus, canídeo, vocalização, propagação sonora,

monitoramento acústico passivo, padrões temporais, playback.

Page 12: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Figure list

Figure A. Maned wolf. From: Paula & Gambarini 2013 (book) ………………………….……………. 20

Figure B I. Maned wolf roar-barking. From: Paula & Gambarini 2013 (book) ……….……………..… 23

Figure B II. Maned wolf (male “Nopal”) roar-barking at the Endangered Wolf Center in St. Louis,

MO/U.S.A. Photo: Michelle Steinmeyer, 2015. ……………………………………….………….…….. 24

Figure B III. Radio collared maned wolf roar-barking. Photo: Flávio H. G. Rodrigues, 2011. .............. 25

Figure C. Broadcasting (Pioneer S-DJ50X speaker) maned wolf roar-barks and re-recording them

(SongMeter SM2+) at different distances (top: 03/07/2017) and deploying an autonomous recorder (from

13) to passively register spontaneous roar-barks sequences (bottom: 03/09/2016). Serra da Canastra

National Park, Minas Gerais, Brazil. …………………………………………………………….……… 27

Chapter 1

Figure 1. Location of passive autonomous recorders and playback sites to study maned wolves at Serra

da Canastra National Park, Minas Gerais, Brazil. Imagery ©2018 CNES / Airbus, Map data ©2018

Google…………...………………………………………………………………………………….……. 34

Figure 2. Edited maned wolf roar-bark sequences used as stimuli for playback studies of maned wolves

in the wild (Serra da Canastra National Park, Brazil). GA and SH are males, SA and JU are females. Top

spectrograms are the original files (96 kHz sample rate, 32 bit wav, 4000 windows size, 56% brightness

and 50% contrast) and the bottom a recording extracted from one autonomous recorder (Song Meter

SM2+; Wildlife Acoustics) 80 meters from the playback speaker (8 kHz sample rate, 16 bit wav, 512

windows size, 50% brightness and contrast). Spectrogram made on Raven Pro 1.5…………….…..….. 37

Figure 3. Distribution of wild maned wolf roar-bark sequences registered between March 04 and 11 2017

during a playback experiment at Serra da Canastra National Park, MG/Brazil. Each sequence is named by

its start time and the size of the bar shows the time elapsed from the last broadcasted playback sequence.

This time is also discriminated on tags above the sequences considered responses to the playback, i.e.

those within 10 minutes after the end of any broadcasted sequence. ……………………….…..…......... 42

Figure 4. Temporal distribution of maned wolf roar-bark sequences recorded at Serra da Canastra

National Park, MG/Brazil, with autonomous recorders (Song Meter SM2+; Wildlife Acoustic). March

2017 (black line, left axis): percentage relative to the total (30 sequences) of vocal activity registered on

continuous recordings of the 6 days in which the roar-bark playback experiment was conducted. March

2016 (dark gray bars, right axis): absolute number of sequences (total 224), 13 recorders, 20 nights, from

5 PM to 5 AM. April 2014 (light gray bars, right axis): absolute number of sequences (total 192), 12

recorders, 25 nights, from 6 PM to 6 AM (Rocha et al. 2016 dataset, used with permission). …...….…. 47

Supplementary Data SD1.Wild maned wolf roar-bark sequences recorded at Serra da Canastra National

Park, MG/Brazil. a and b: sequence in response to a playback stimulus registered on 18:55:37 March 09

2017. c and d: two individuals (note the change in spectral characteristics after 33 s) recorded passively

on 19:15:00 March 17 2016. Recordings made with autonomous recorders (Song Meter SM2+; Wildlife

Acoustic), at 8 kHz sample rate and 16-bit wav file format. Spectrogram made on Raven pro 1.5 (Cornell

Bioacoustics Lab, Ithaca, NY, USA), Hann window, 512 window size, 50% brightness and contrast, 50%

overlap, smoothing on. .…………………………………….. ……………………………………...…… 59

Chapter 2

Figure 1. Study site at Serra da Canastra National Park, MG, Brazil. a - site Flat; b - site Low to high; c -

site Vegetation; d - site High to low. Horizontal distance to the speaker is discriminated on the left side of

the / and altitude on the right side. Maps constructed with QGIS 3.4.0-Madeira (QGIS Development

Team, 2018. QGIS Geographic Information System. Open Source Geospatial Foundation

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Project. http://qgis.osgeo.org) and Google Satellite images (Map data ©2018 Google, Imagery ©2018

TerraMetrics). ……………………………………………………………………………..…………….. 70

Figure 2. Captive maned wolves roar-barks sequences broadcasted at Serra da Canastra National Park,

MG/Brazil. GA and SH are males, SA and JU females. Red selection boxes on the first roar-bark of each

animal exemplifies the ones used to measure roar-bark intensity (peak power, dB). Selections near the

second roar-bark of each animal exemplifies the ones used to measure noise intensity (average power,

dB). Spectrograms and measures were made on Raven Pro 1.5 (Cornell Bioacoustics Lab, Ithaca, NY,

USA), Hann window, 512 window size, 50% brightness and contrast, 50% overlap, smoothing “on”... 76

Figure 3. Propagation of broadcasted roar-barks from captive maned wolves at Serra da Canastra

National Park, MG/Brazil. Re-recordings made with autonomous recorders (Song Meter SM2+; Wildlife

Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the re-recording at 1.25m …. 80

Figure 4. Propagation of broadcasted captive records of maned wolves roar-barks at 4 sites at Serra da

Canastra National Park, MG/Brazil. Re-recordings made with autonomous recorders (Song Meter SM2+;

Wildlife Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the re-recording at

1.25m. ……………………………………………...…………………………...………...…………..…. 82

Figure 5. Propagation of broadcasted captive records of maned wolves roar-barks at 4 sites at Serra da

Canastra National Park, MG/Brazil. We conducted broadcasts with the speaker box positioned straight

forward (Straight) and with the speaker box inclined 45o upward (Inclined) to simulate the inclination of

the head/muzzle seen when animals roar-bark. Re-recordings made with autonomous recorders (Song

Meter SM2+; Wildlife Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the

straight re-recording at 1.25m. …………………………………………………………………..….…… 84

Figure 6. Propagation of broadcasted captive records of maned wolves roar-barks at 6 time intervals at

Serra da Canastra National Park, MG/Brazil. The time shown is the beginning of a 1 hour interval in

which broadcasts were made. Re-recordings made with autonomous recorders (Song Meter SM2+;

Wildlife Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the re-recording at

1.25m……………………………………………….…………………………...……………………….. 85

1 – Preliminaty data exploration (supplementary material) ……………………..…………………….… 94

2 – Normality and homogeneity of residuals (supplementary material) ……………………...…………. 95

3 – Predicted x observed values of the model (supplementary material) …………………...……..……. 96

Chapter 3

Figure 1. Study region at the Serra da Canastra National Park, MG/Brazil. Yellow squares indicate

autonomous recorder (SongMeter SM2+) sites used only in 2014, pink triangles sites used only in 2016,

and white circles sites used in both years. …………...……………………………………….….......… 107

Figure 2. Wild maned Wolf roar-bark sequences recorded passively with a grid of 12/13 autonomous

recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. a) solo roar-bark sequence

with detection boxes from XBAT (Figueroa 2007) extension for Matlab (MathWorks, Inc.). Spectrogram

parameters: 512 window size, Hann window, 100% brightness, 43% contrast, 50% overlap on an 8 kHz

sampling rate wav file. b) group vocalization consisting of two animals alternating roar-barks.

Spectrogram made on Raven pro 1.5 (Cornell Bioacoustics Lab, Ithaca, NY, USA), Hann window, 512

window size, 50% brightness and contrast, 50% overlap, smoothing on, on an 8 kHz sampling rate wav

file. ...…………………………………………………………………................................................… 111

Figure 3. Histogram of the number of roar-barks on each sequence of maned wolves’ vocalizations

recorded passively with a grid of 12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra

National Park, MG/Brazil. One sequence was defined by one or a bout of roar-barks not separated by

more than 10 seconds. ………………………………………………………………………….....……. 115

Page 14: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Figure 4. Seasonal variation in the maned wolf vocal activity recorded passively with a grid of 12/13

autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. Each point is

a sum of 5 nights. Photos: Endangered Wolf Center, St.Louis, and Adriano Gambarini ……………… 116

Figure 5. Maned wolf roar-bark sequences distribution over the lunar phases (gray = total). Records were

made from April to July on 2014 (blue) and from March to June on 2016 (red) with a grid of 12/13

autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. The radial line

represents the mean angle and the concentric bar at the end of the line the 95% confidence interval. …119

Figure 6. Maned Wolf roar-barks registered between 17-19h on passive audio recordings made with a

grid of 12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil.

In 2014 there was no recordings in March, and in 2016 no recordings in July. ………….……….…… 121

Figure 7. Maned wolf nightly vocal activity relative to sunset. Recordings were made with a grid of

12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil…… 122

Figure 8. Maned wolf roar-bark sequences recorded passively with 12 autonomous recorders (Song

Meter SM2+) at Serra da Canastra National Park, MG/Brazil (gray contour). Circles have approximately

0.5 km radius with the center point being the recorder site. Heat colors represent the intensity of vocal

activity (number of sequences). Letters indicates the site name and numbers following them on the circles

indicates the amount of group vocalizations. * Indicates at least one sequence involved 3 animals

(otherwise group vocalizations involve 2 animals).…………………………………………...…...…... 124

Figure 9. Maned wolf roar-bark sequences recorded passively with 13 autonomous recorders (Song

Meter SM2+) at Serra da Canastra National Park, MG/Brazil (gray contour). Circles have approximately

0.5 km radius with the center point being the recorder site. Heat colors represent the intensity of vocal

activity (number of sequences). Letters indicates the site name and numbers following them indicates the

amount of group vocalizations (sequences involving 2 animals). ….…………...…………………..…. 125

Figure S1. Nightly maned wolf roar-barks recorded passively with 12 autonomous recorders (Song Meter

SM2+) at Serra da Canastra National Park, MG/Brazil (gray contour). From top-left to bottom-right:

April 05/06/07/08 2014, April 15/16/17/18 2014, March 19/20/21/22/23 2016, and May 01/02/03/04

2016. **indicates at least one sequence of roar-barks involved two animals. *** indicates at least one

sequence of roar-barks involved three animals. ……………………………………………...………… 140

Chapter 4

Figure 1. Maned wolves roar-barks recorded in Minas Gerais, Brazil. a. One example of roar-bark from

each of 10 individuals (letters) recorded with unidirectional microphone and a hand recorder in two

captivity facilities. b. Roar-barks of GA and JU broadcasted and re-recorded with autonomous recorders

at 7 different distances from the speaker on site “Flat” at the Serra da Canastra National Park. c. Free-

ranging animals spontaneous roar-bark sequences recorded with autonomous recorders at the same park:

top spectrogram shows some roar-barks (numbers) from a sequence involving two animals (letters);

bottom spectrogram shows the same sequence recorded by another autonomous recorder 2.41 km away,

roar-barks from “b” reach the recorders on different times because animals are at different positions.

Spectrogram parameters: a. 96 kHz sample rate, 3080 window size, Hann window, 55% brightness, 60%

contrast, 24-bit wav; b. and c. 8 kHz sample rate, 512 window size, Hann window, 50% brightness, 60%

contrast, 16-bit wav. ……………………………………………………………….............................… 152

Figure 2. First 3 linear discriminant functions for identity discrimination of 10 captive maned wolves

(colors) roar-barks recorded from two facilities at Minas Gerais, Brazil. …………………....…..……. 159

Figure 3. Differences in the roar-barks parameters between females and males maned wolves recorded in

two facilities in Minas Gerais, Brazil. ……………………………………………………..………..…. 161

Figure 4. Variation of 8 selected parameters of broadcasted maned wolves roar-barks re-recorded at 7

distances (1.25-640m) at the Serra da Canastra National Park, MG/Brazil. ……………….….………. 163

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Figure 5. LDA percentage of correctly identity classification of broadcasted roar-barks of 4 maned

wolves (bottom) re-recorded at 7 distances (1.25-640m) in 4 sites (top) at the Serra da Canastra National

Park, MG/Brazil. ……………………………………………………………………..…...............……. 164

Figure 6. Figure 6. Signal-to-noise ratio of broadcasted roar-barks of maned wolves re-recorded at 7

distances (1.25-640m) in 4 sites at the Serra da Canastra National Park, MG/Brazil. The signal-to-noise

ratio was calculated subtracting from the in-band power of each roar-bark (150-2000 Hz) and the same

measurement taken from an equal sized spectrogram portion immediately before the vocalization

(measure of the background noise level). ………………………………………………...………….… 165

Figure 7. Two different roar-bark sequences involving the same two maned wolves each (top), and their

roar-bark parameters (bottom). Recordings made passively by two different autonomous recorders

(SongMeter SM2+) at the Serra da Canastra National Park, MG/Brazil …………..…………….…….. 168

Appendix I

Maned wolves do not emit more roar-barks than expected by chance during the illuminated versus the

non-illuminated portion of the night, except from new to waxing crescent phase. In this phase only the

first part of the night is illuminated, and thus the difference may be caused by the species preference to

vocalize on this time. *t=2.906, df=33, p=0.006. Graph extracted and translated from: ÁRAUJO, D.D.,

FERREIRA, L.S., ROCHA, L.H.S., & SOUSA-LIMA, R.S. 2016. Influência do ciclo lunar nas

vocalizações de lobo guará. Abstract and poster presentation at the III Conferência e VIII Simpósio de

Psicobiologia, UFRN, Natal, Rio Grande do Norte, Brazil. ................................................... ................. 191

Appendix II

Individual variation of the time interval between the start of one maned wolf roar-bark to the next one in

the sequence. Potential for Identity Coding (PIC; as in Robisson et al. 1993) for this parameter is 1.04.

Data from 10 captive maned wolves, 24-124 roar-barks by individual, 897 roar-barks in total, recorded in

2010 by V.S. Rocha, at 2 facilities in Minas Gerais, Brazil …………………………………………… 192

Annex I

A sedated lactating maned wolf being examined by the Lobos da Canastra team. This female (known as

“Rose”) was captured on the night between July 12-13 2016 at the Serra da Canastra National Park,

around site F (Figure 1, Chapter 3). At this year she was already without a VHS collar, but her data shows

she lived on the present study area since at least 2014, when she was first captured, also lactating. Photo:

Rogério Cunha de Paula. Used with permission. ………………………………………………...……. 193

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Table list

Chapter 1

Table 1. Wild maned wolves’ vocal activity recorded by autonomous recorders (Song Meter SM2+;

Wildlife Acoustics) at Serra da Canastra National Park, MG/Brazil, during the playback experiment days

on 2017. Each row is a different roar-bark sequence. Those considered playback responses are

underlined. Each playback session consisted of 8 broadcasted roar-bark sequences. The column “Heard?”

indicates if the researchers, when present in loco, heard maned wolves’ calls …………………………. 43

Chapter 2

Table 1. ANOVA test for the fixed factors of the main model for the intensity (dB) loss of maned wolves

roar-barks broadcasted on their natural environment. ...…………...……………………………...…….. 78

Table 2. Approximate 95% confidence intervals for the estimate factor effects of the main model for the

intensity (dB) loss of maned wolves roar-barks broadcasted on their natural environment. Base categories

are specified under parenthesis. Factors/levels with positive and negative estimates, which indicates they

are not influential on the model or not significantly different from the base category, are

underlined………………...…………………………………………………………………..…...…...… 79

Table 3. Simultaneous tests for general linear hypotheses using Tukey contrasts for multiple comparisons

of means. The reported p values are adjusted by single-step method. Significance codes: 0 '***', 0.001

'**', 0.01 '*', 0.05 '.', 0.1 ' '. Only comparisons of consecutive distances are shown. ………...………… 81

Table S2. ANOVA test for the fixed factors of the secondary model for the intensity propagation of

maned wolves roar-barks broadcasted on their natural environment. …………………………….…….. 97

Table S3. Secondary model: simultaneous tests for general linear hypotheses using Tukey contrasts for

multiple comparisons of means. The reported p values are adjusted by single-step method. Significance

codes: 0 '***', 0.001 '**', 0.01 '*', 0.05 '.', 0.1 ' '. Only com comparisons of consecutive distances are

shown. ………………………………………………………………………...……………….……..….. 97

Chapter 3

Table 1. Summary of maned wolf’s vocal activity recorded passively with a grid of 12/13 autonomous

recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. ………………...…..… 114

Table 2. Maned wolf vocal activity recorded passively with a grid of 12/13 autonomous recorders (Song

Meter SM2+) at Serra da Canastra National Park, MG/Brazil. Values reported are mean by night ± SD.

…………………………………………………………………………………………………………... 117

Table 3. Concentration of maned wolf vocal activity on each of eight moon cycles recorded passively

with a grid of 12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park,

MG/Brazil. When the concentration is significant, the mean moon phase, mean angle ± circular standard

deviation, and Rayleigh test statistics are reported. ……………………………………….....……....… 120

Chapter 4

Table 1. Maned wolves recorded on 2010 at two facilities in Minas Gerais, Brazil. *estimated age. mx are

non-participant males. m3 is GA/GI half-brother. ………………………………………………..……. 148

Table 2. Selected parameters on maned wolves roar-barks (LSF analyst). “Full” measures refer to the

roar-bark from 150 Hz to 2000 Hz, while “First” and “Second” bands refer to portions from lower to

higher frequencies. Means±SD are for all 10 individuals, 20 roar-barks each. PIC = Potential for Identity

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Coding (Robisson et al. 1993). (A) = unitless: proportion relative to entire duration. Parameters detailing

can be found on Raven’s manual (Charif et al. 2010). ……………………………………………….... 158

Table 3. Confusion matrix for the cross validation classification of 10 captive maned wolves by their

roar-barks. Average from the classification results of two analysts (LF, VS), each constructed by means

of the results from 1000 randomizations of 100 roar-barks (10 for each individual) from a total of 200.

Extracted by the pDFA R function written by Roger Mundry (2015 version). ……………..…………. 160

Table 4. ANOVA effect sizes (F) for the 8 selected parameters of 20 roar-barks from 4 maned wolves

broadcasted and re-recorded at 5 distances (1.25-160m) in 4 sites. (A) = unitless: proportion relative to

entire duration. α=0.0012. ……………………………………………………………………..…….…. 162

Table 5. Effect size (t) for the absolute difference between parameters of roar-barks from two different

maned wolves vocalizing together (2 wolves) and roar-barks simultaneously recorded by two different

autonomous recorders (2 recorders; SongMeter SM2+). 2 wolves: one sample t-test, df=91. 2 recorders:

paired t-test, df=82. α=0.0012. Cor. SNR: Pearson’s correlation coefficient with the signal-to-noise ratio

difference. (A) = unitless: proportion relative to entire duration. The vocalizations were recorded

passively with a grid of 12/13 autonomous recorders at Serra da Canastra National Park, MG/Brazil... 167

Table 6. Mean±SD difference of the absolute difference between parameters of roar-barks from two

different maned wolves vocalizing together (2 wolves) and roar-barks simultaneously recorded by two

different autonomous recorders (2 recorders; SongMeter SM2+). Those differences were compared with a

Welch two sample t-test. α=0.0012. (A) = unitless: proportion relative to entire duration. The

vocalizations were recorded passively with a grid of 12/13 autonomous recorders at Serra da Canastra

National Park, MG/Brazil. ………………………………………...…………...…………………....…. 167

Table S1. All measured parameters on maned wolves roar-barks (LSF analyst). “Full” measures refer to

the roar-bark from 150 Hz to 2000 Hz, while “First” and “Second” bands refer to portions from lower to

higher frequencies. Means±SD are for all 10 individuals, 20 roar-barks each. PIC = Potential for Identity

Coding (Robisson et al. 1993). # selected for the identity classification. (A) = unitless: proportion relative

to entire duration. *the selection box of the 2nd band is limited below by the selection box of the 1st band,

therefore we manually measured the 2nd band true lower frequency. Parameters detailing can be found on

Raven’s manual (Charif et al. 2010). …………………………………..………..……………..….…… 183

Table S2. Explained variance and coefficients of each linear discriminant function for identity

discrimination of 10 captive maned wolves roar-barks. Non-normal parameters were transformed (Yeo

Johnson). All parameters were centralized and scaled. ………………………....………….………….. 184

Table S3. ANOVA effect sizes (F) for all measured parameters on maned wolves roar-barks broadcasted

and re-recorded at 5 distances (1.25-160m). “Full” measures refer to the entire roar-bark (from 150 Hz to

2000 Hz), while “First” and “Second” bands refer to portions from lower to higher frequencies. (A) =

unitless: proportion relative to entire duration. α=0.0012. ……………………………………...……... 185

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Summary

General introduction ....................................................................................................... 20

Chapter 1......................................................................................................................... 28

Using playbacks to monitor and investigate the behavior of wild maned wolves ......... 28

Abstract ....................................................................................................................... 29

Resumo ....................................................................................................................... 30

Introduction ................................................................................................................. 31

Materials and methods ................................................................................................ 34

Results ......................................................................................................................... 42

Discussion ................................................................................................................... 49

References ................................................................................................................... 55

Supplementary data ..................................................................................................... 59

Chapter 2......................................................................................................................... 60

Maned wolf long range call propagation and its implication for the species’

communication ............................................................................................................... 60

Abstract ....................................................................................................................... 61

Resumo ....................................................................................................................... 62

Introduction ................................................................................................................. 64

Materials and methods ................................................................................................ 69

Results ......................................................................................................................... 78

Discussion ................................................................................................................... 86

References ................................................................................................................... 90

Supplementary material .............................................................................................. 94

Chapter 3......................................................................................................................... 99

Temporal and spatial patterns of the long-range calls of maned wolves ........................ 99

Abstract ..................................................................................................................... 100

Resumo ..................................................................................................................... 101

Introduction ............................................................................................................... 103

Material and Methods ............................................................................................... 107

Results ....................................................................................................................... 114

Discussion ................................................................................................................. 127

References ................................................................................................................. 136

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Supplementary material ............................................................................................ 140

Chapter 4....................................................................................................................... 141

Identity and sex discrimination of roar-barks for captive and free-ranging maned wolves

...................................................................................................................................... 141

Abstract ..................................................................................................................... 142

Resumo ..................................................................................................................... 143

Introduction ............................................................................................................... 144

Material and Methods ............................................................................................... 148

Results ....................................................................................................................... 158

Discussion ................................................................................................................. 169

References ................................................................................................................. 177

Supplementary material ............................................................................................ 183

Final remarks ................................................................................................................ 186

Extra-textual references ................................................................................................ 188

Appendix I .................................................................................................................... 191

Appendix II ................................................................................................................... 192

Annex I ......................................................................................................................... 193

Annex II ........................................................................................................................ 194

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General introduction

The maned wolf (Chrysocyon brachyurus, Illiger, 1815; Figure A) is the only

large canid of South America (Queirolo et al. 2007). The species is listed as Near

Threatened by IUCN (IUCN 2015), Vulnerable in the Brazilian list of endangered

species (Paula et al. 2013), and models predict a 30% reduction in populations in only 3

generations (21 years; Paula et al. 2008). The main threat to the species is the

destruction of its habitat, the Cerrado neotropical savanna, which is one of the world’s

most important biodiversity hotspots (Silva & Bates 2002).

Figure A. Maned wolf. From: Paula & Gambarini 2013 (book).

The maned wolfs’ iconic characteristics make it appealing to the public and a

potential flagship species for the conservation of this biome (Myers et al. 2000). Its

slender constitution and long legs are adapted to walk over the bushes and tall grass of

its environment, while its large ears are used to detect hidden rodents and birds (Rodden

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et al. 2004). This last characteristic also suggests that acoustic communication among

individuals of this species over long distances might play a significant role.

Maned wolves present many typical canid characteristics, including

nocturnal/crepuscular habits, some degree of territoriality, monogamous breeding with

the formation of stable pairs, and biparental care of young (Rodden et al. 2004).

However, “canid rules” state that larger canids (>13 kg) tend to be: more social,

forming larger groups composed of the breeding pair and grown offspring; to have

young cared by parents and helpers; to have large litter sizes; and to hunt cooperatively

fot larger preys (Kleiman & Eisemberg 1973; Moehlamn 1987, 1989). Despite their size

(20-30 kg, 70-90 cm; Silveira 1999), maned wolves tend to behave opposite than

expected (Rodden et al. 2004): they forage alone for small vertebrates and fruits, the

pair is rarely seen together, the presence of helpers have never been confirmed, and the

litter size is small (captivity mean is 3; Maia & Gouveia 2002). They are normally

described as less social than many small canids for which at least the breeding pair

associates extensively, as with artic foxes (Frommolt et al. 2003), and crab eating foxes

(Courtenay & Maffei 2004).

In comparison, the maned wolf closest living relative, the bush dog (Speothos

venaticus; Slater et al. 2009), is highly social and also an exception to canid rules, in the

other direction (as it is small: 4-7 kg; Beisiegel & Ades 2002). They are hypercarnivores

and hunt cooperatively for large prey (Kleiman 1972; Beisiegel & Ades 2002), which

highlights the relationship of feeding habits and canid social systems and indicates

canid rules may not be widespread. The small items’ diet of the maned wolf makes

sharing unprofitable and the presence of conspecifics may interfere in foraging (McNab

1986). Accordingly, Melo et al. (2007) reports that a breeding pair and a juvenile slept

relatively close during the day but stayed far apart during the active hours (i.e., night).

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This last study also suggests a higher intra-group (pair and occasional offspring)

association than previously though. Despite telemetry studies usually not showing so

(Jácomo et al. 2009), there are several instances of group bonding/socialization: reports

of a breeding pair sleeping together (Melo et al. 2009; Emmons 2012); cooperative

hunting (Jácomo et al. 2009); males accompanied by young (Rodrigues 2002); males

providing or regurgitating to the female and pups (Dietz 1984; Jácomo et al. 2009),

including regurgitation to 5-9 month young in captivity (Rasmussen & Tilson 1984);

and a group composed of the breeding pair and 3 juveniles that interacted often

(Emmons 2012). On Emmons’ (2012) work, the long-range acoustic communication

was very important for these interactions.

Maned wolves use multiple communication modalities (Rodden et al. 2004), as

visual (e.g. piloerection, gape, ear and tail positioning) and chemical (e.g. urine, feces,

scent marks). The acoustic communication channel is also expected to be exploited by

the species, especially considering the limitations of visual signaling in a

crepuscular/nocturnal and solitary animal (Fox 1975). The species has a complex

acoustic repertoire, including at least 10 types of vocalizations and combinations of

vocalizations (Sábato 2011). Maned wolves’ vocal repertoire comprises almost all canid

broad categories of vocalizations (Tembrock 1976), which points to complex social

interactions, as social and vocal complexity are generally linked (Freeberg 2006). The

most frequent type of vocalization recorded in captivity is the long-range roar-bark

(Sábato 2011; Figure B I-III), a call heard throughout the year in the wolves’ natural

habitat (Rodden et al. 2004). Thus, maned wolves may be solitary, but seem to maintain

social acoustic contact over distance.

Roar-barks are emitted in sequences of 5-15 units, spaced by 3-5 seconds, and

are proposed to have multiple functions. One of the most cited is territorial

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announcement (Kleiman 1972; Rocha 2011), especially for same-sex spacing (Brady

1981). Maned wolves from adjacent ranges have been heard exchanging roar-barks and

emitting them when facing threats (conspecifics and humans; Dietz 1984), and in

captivity, same sex individuals often exchange roar-barks (Brady 1981; Sábato 2011).

Figure B I. Maned wolf roar-barking. From: Paula & Gambarini 2013 (book).

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Figure B II. Maned wolf (male “Nopal”) roar-barking at the Endangered Wolf Center in St. Louis,

MO/U.S.A. Photo: Michelle Steinmeyer, 2015.

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Figure B III. Radio collared maned wolf roar-barking. Photo: Flávio H. G. Rodrigues, 2011.

The other function often cited for the roar-bark is in intra-pair communication

(Rocha et al. 2016; Balieiro & Monticelli 2019), being more important during the

breeding season (Dietz 1984), even in captivity (Sábato 2011). Researchers report that

between roar-barks maned wolves aurally attend to answers (Emmons 2012) and search

visually for the partner (Bestelmeyer 2000), that during estrous they emit roar-barks

whenever the partner is outside of visual range (Rodden et al. 2004), that often the

partner appears after the vocalization or move towards it (Bestelmeyer 2000; Emmons

2012), and pairs have been heard exchanging roar-barks many times (Dietz 1984;

Sábato 2011; Emmons 2012; Balieiro & Monticelli 2019). Some authors thus propose

the intra-group (pair and occasional offspring) communication would be the main roar-

bark function (Emmons 2012).

Roar-barks are considered far ranging, with Brady (1981) stating a human could

discriminate individuals over 1 km. Indeed, it was the only maned wolf vocalization we

could detect with passive autonomous recorders in the wild (Rocha et al. 2015, 2016).

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Maned wolves are more easily heard than visualized (Emmons 2012; personal

observation), and usually very hard to follow due to their shyness, habitat composition,

and crepuscular/nocturnal habits (Rodden et al. 2004). Thus, exploring their long-range

acoustic communication might be an efficient alternative to monitor populations and

elucidate the species behavior ecology. The potential for vocal individualization makes

the bioacoustics approach even more interesting.

In this work I explored the maned wolf long range acoustic communication to

better understand the species’ behavioral ecology. I did it through three complimentary

methodologies (Figure C). First, I conducted a roar-bark playback experiment in the

wolves’ natural habitat. The experiment had a dual objective: to test if, and how, wild

maned wolves would respond to roar-bark playbacks, assessing both evidence of its

function and monitoring applicability (chapter 1); and to understand how the roar-bark

propagates over distance, also testing if some behaviors, like period of the day and night

and head elevation, were related to acoustic propagation (chapter 2). Second, I passively

recorded spontaneous (non-playback elicited) roar-barks sequences of wild maned

wolves using 12/13 autonomous recorders during eight months over two years (2014

and 2016). Those recordings generated an enormous dataset (over 3.5 TB), that was

processed by automatically detecting roar-barks through a previously established

method (Rocha et al. 2015: 100% of detections in half the time x 92% for visual

inspection of spectrograms). The primary goal of those wild recordings was to

characterize the maned wolf seasonal, lunar and nightly roar-bark emission patterns

(chapter 3). Finally, I used the experimentally broadcasted roar-barks, the naturally

spontaneous vocalization recordings, and roar-barks recorded in captivity by V. Sábato

(2011) to confirm roar-bark individual and sexual discriminability through permuted

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discriminant analyses and, more important, test the applicability of vocal

individualization in recordings from the wild (chapter 4).

Figure C. Broadcasting (Pioneer S-DJ50X speaker) maned wolf roar-barks and re-recording them

(SongMeter SM2+) at different distances (top: 03/07/2017) and deploying an autonomous recorder (from

13) to passively register spontaneous roar-barks sequences (bottom: 03/09/2016). Serra da Canastra

National Park, Minas Gerais, Brazil.

Examples of maned wolf roar-bark sequences recorded in this work can be heard

in https://soundcloud.com/luane-ferreira-327256713. Visual and acoustic data collection

at this park was authorized by Instituto Chico Mendes de Conservação da

Biodiversidade (ICMBio; SISBIO license number 41329-2, annexed in the end of this

text).

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Chapter 1

Using playbacks to monitor and investigate the

behavior of wild maned wolves

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Using playbacks to monitor and investigate the behavior of wild maned

wolves

Luane Stamatto Ferreira, Júlia Simões Damo, Victor Sábato, Júlio Baumgarten, Flávio

H. G. Rodrigues, Renata S. Sousa-Lima

Intended for submission to: Mastozoologia neotropical

Abstract

Maned wolves are difficult to observe in the wild because of their low densities

and their cryptic and crepuscular-nocturnal habits. Exploring their long-range acoustic

communication may offer an efficient alternative to study the species. Here we

evaluated the applicability of playbacks to study maned wolves in the wild and compare

the results with 20 nights of passive recordings on the same area and month during the

previous year. We obtained vocal responses on 3 of 6 nights tested, including responses

involving two animals and an approach after an interactive playback. Although we

conducted 3-6 playback sessions each day at different times, we only obtained vocal

responses during sessions between 17:00 and 19:40. During our passive recordings we

detected on average 0.86 roar-bark sequences per recorder per night, mostly during the

first half of the night. Vocal activity – responses and spontaneous roar-bark sequences –

during playback nights was nearly 4 times greater than during the passive recordings.

We conclude that playbacks stimulate maned wolves to emit roar-barks and that this

method is applicable to test hypotheses about maned wolf behavior and aid in their

monitoring.

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Resumo

Lobos guará são difíceis de serem observados na natureza devido às suas baixas

densidades e hábitos crípticos e crepusculares-noturnos. Explorar sua comunicação

acústica de longo alcance pode oferecer uma alternativa eficiente para estudar a espécie.

Neste trabalho nós avaliamos a aplicabilidade de usar playbacks para estudar lobos

guará na natureza e comparamos estes resultados com 20 noites de gravações passivas

na mesma área e mês durante o ano anterior. Obtivemos respostas vocais em 3 das 6

noites testadas, incluindo respostas envolvendo dois animais e uma aproximação depois

de um playback interativo. Apesar de termos conduzido 3-6 sessões por dia em

diferentes horários, nós só obtivemos respostas vocais em sessões entre 17:00 e 19:40.

Durante as gravações passivas nós detectamos em média 0.86 sequências de aulidos por

gravador por noite, a maioria na primeira metade da noite. A atividade vocal – respostas

e sequências de aulidos espontâneas – durante as noites de playback foi quase 4 vezes

maior que durante as gravações passivas. Nós concluímos que playbacks estimulam os

lobos guará a emitir aulidos e que o emprego deste método e viável para testar hipóteses

sobre o comportamento do lobo guará e auxiliar seu monitoramento.

Key words: Chrysocyon brachyurus, maned wolf, passive acoustic monitoring,

playback, roar-bark, vocal activity, vocalization time

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Introduction

The maned wolf (Chrysocyon brachyurus; Illiger, 1815) is South America's

largest canid (80-90 cm shoulder height and 20-30 kg in weight; Rodden et al. 2004;

Jácomo et al. 2009). These animals forage alone for fruits and small vertebrates

(Queirolo and Motta-Junior 2007). Maned wolves form stable breeding pairs that share

an extensive home range (15-115 km2; Rodrigues 2002; Azevedo 2008). Unlike other

large canids, the pair is thought to rarely interact outside the breeding season (Dietz

1984; Rodden et al. 2004; Jácomo et al. 2009). From estrus to the weaning of young, the

pair may sleep together (Melo et al. 2007), encounter frequently, and travel or forage

together for several hours (Rodden et al. 2004; Emmons 2012).

Maned wolves communicate acoustically throughout the year using a long-

distance vocalization called the roar-bark, normally uttered in sequences (bouts) of 5-15

repetitions (see spectrograms in Materials and Methods; Rodden et al. 2004; Rocha et

al. 2016). The proposed functions of roar-barks are intra-pair and parent-offspring

communication, opposite-sex attraction and same-sex repelling, and/or territorial

announcement (Brady 1981; Dietz 1984; Sábato 2011; Emmons 2012; Rocha et al.

2016).

Few studies have been conducted observing maned wolves in the wild because

of their low densities and of their cryptic and crepuscular-nocturnal habits (Jácomo et al.

2004; Melo et al. 2007; Trolle et al. 2007). As the species is difficult to visualize and

follow, exploring their long-range acoustic communication may be an efficient

alternative to fill the many gaps in the knowledge of their behavior, and to monitor their

distribution and population trends (Marion et al. 1981; Rodden et al. 2004; Blumstein et

al. 2011).

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Grids of autonomous audio recorders mounted for extended periods have been

used to investigate many aquatic species that cannot be easily observed, as most

cetaceans (e.g. humpback whales - Megaptera novaeangliae: Sousa-Lima and Clark

2008; beaked whales - Mesoplodon densirostris: Marques et al. 2009; minke whales -

Balaenoptera acutorostrata: Risch et al. 2013). For terrestrial environments, birds

dwelling in dense forests (Mennill and Vehrencamp 2008), nocturnal remote-nesting

birds (Oppel et al. 2014), and widely dispersed forest mammals (e.g. elephants -

Loxodonta africanacyclotis: Thompson et al. 2010), have also been studied with passive

acoustic monitoring. This methodology has already proven useful in the investigation of

wild maned wolves. The recording of 32 nights (8/month) suggests they vocalize more

often in the mating season, in the beginning of their activity period (first hour of the

night), and less often during new moon nights (Rocha et al. 2016).

Playbacks, different from passive acoustic monitoring, offers a more direct way

of testing hypothesis about the species behavior, and can possibly reduce monitoring

effort by stimulating immediate and higher vocal activity and inducing approaches to

facilitate detection or captures. Playbacks have long been used to study the behavior and

monitor populations of primates (Radick 2005) and birds (e.g. Lanyon 1963; Marion et

al. 1981). Today its use is widespread across taxa including insects, anurans (Greenfield

1994), and carnivores, such as lions and hyenas (Panthera leo, Crocuta crocuta; Cozzi

et al. 2013). This technique has been successfully employed to study other canids in the

wild, including wolves (Canis lupus; Harrington 1987; Brennan et al. 2013), coyotes

(Canis latrans; Petroelje et al. 2013), swift foxes (Vulpes velox; Darden and Dabelsteen

2008), and bush dogs (Speothos venaticus; DeMatteo et al. 2004), but remained untested

for maned wolves.

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Here we evaluated the applicability of playbacks to study maned wolves in the

wild through the broadcast of roar-bark sequences to elicit responses from free-ranging

animals. We tested different broadcasting hours aiming to guide the planning of future

playback efforts, and to identify the best period of the day for conducting an interactive

playback aiming to escalate the animals’ response and induce approaches to the speaker.

If maned wolves are responsive to playbacks, future investigations could test how the

species use roar-barks, e.g. if sexes respond equally to male and female broadcasted

roar-barks, or survey maned wolves’ presence and distribution in a practical way. If

maned wolves are also shown to be attracted to the playback, this can be used to permit

their detection, even if they do not respond vocally, count individuals, and improve

capture chances. Additionally, we tested the use of autonomous recorders to increase the

probabilities of registering responses and identifying local roar-bark range (and other

propagation effects whose results are not shown here). Determining the roar-bark range

is important to make playback sites independent and to estimate the surveyed area,

while estimating our roar-bark detection capabilities will serve to plan how effectively

we can survey an area.

Playback results were compared with nightly detections of roar-barks from

passive acoustic recordings in the same area on the same month of the previous year.

Although there are no residency studies for the species, it is likely that the same

individuals were residing the area. Long term research suggests that individuals occupy

the same territories from 3 to 5 years (ranging from 1 to over 9 years; Emmons 2012).

The passive acoustic pattern of detection were used to establish a baseline level of roar-

bark spontaneous emissions along the night to test if playbacks would increase vocal

activity or change the temporal emission pattern.

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34

Materials and methods

1. Study area

We conducted this study at Serra da Canastra National Park, Minas Gerais state,

Brazil (Figure 1). The park is mainly composed of Cerrado open savannas with a cold,

dry season (April-September) and a hot, rainy season (October-March; Queirolo and

Motta-Junior 2007).

Wild maned wolf acoustic data collection at this park was authorized by Instituto

Chico Mendes de Conservação da Biodiversidade (ICMBio; SISBIO license number

41329-2) and playback experiments were done in accordance with the ASM guidelines

(Sikes et al. 2016).

Figure 1. Location of passive autonomous recorders and playback sites to study maned wolves at Serra

da Canastra National Park, Minas Gerais, Brazil. Imagery ©2018 CNES / Airbus, Map data ©2018

Google.

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35

2. Playbacks

Roar-barks used as stimuli were obtained from two facilities in Minas Gerais

state that keeps captive maned wolves: Criadouro Científico de Fauna Silvestre para

Fins de Conservação da Companhia Brasileira de Metalurgia e Mineração, and

Zoológico da Associação Esportiva e Recreativa dos Funcionários das Usinas

Siderúrgicas de Minas Gerais. Recordings were conducted between April and June

2010 and November 2010, respectively. The sounds were recorded 5-8 m from the

animals with a unidirectional microphone Sennheiser K6 coupled to a Sennheiser ME-

66 module and connected with a solid-state recorder Marantz PMD-661, using a 96 kHz

sample rate, and a 24-bit wav coding form.

To set the intensity level we used the playback equipment (described below) to

broadcast the captivity recordings and then re-record the played back sounds with the

same equipment and settings used for the original captivity recordings at the same

distance the focal animals were. We then changed the speaker volume until the roar-

bark intensity measured in the re-recordings matched the intensity measured in the

original captivity recordings (measures made in Raven Pro 1.5 software: Bioacoustics

Research Program, 2014. Raven Pro: Interactive Sound Analysis Software. Ithaca, NY:

The Cornell Lab of Ornithology). This was our solution to achieve a playback intensity

level as similar as maned wolves' roar-bark emission. During the recordings in captivity

we did not have a direct way to measure sound pressure levels, and thus no means of

calculating absolute source intensity levels. Two experienced researchers, including the

author of the original captivity recordings (VS), reported the playback sounded as

strong as heard from the animals in captivity and in the wild (FHR).

Playbacks sessions were conducted in three different sites in the park (Figure 1)

between March 4 and 9, 2017. We used an Acer AspireOne notebook to broadcast the

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36

sounds using Raven Pro 1.5 software and a Pioneer S-DJ50X speaker (class A/B Bi-

amp, 80 W output, 50-20000 Hz frequency range) 86 cm above the ground to simulate

the height of a maned wolf.

We used 4 edited roar-bark sequences including sounds from two males and two

females (Figure 2). Recordings of both sexes were used to maximize the chance of

response. Each sequence was composed of 5 roar-barks separated by 2.9-6.2 seconds

(similar to the natural emission for an individual) and intervals between sequences

varied from 10 seconds to 10 minutes depending on weather conditions. In each

playback session, all four individual sequences were played in random order and then

repeated once, resulting in complete sessions being composed of 8 sequences of 5 roar-

barks each and lasting 5-25 minutes in total. If we heard an answer from a wild maned

wolf before finishing the broadcast of all sequences we broadcasted the next sequence

right after the response aiming to create an interactive playback. This was done trying to

stimulate the responsive animal’s approach.

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Figure 2. Edited maned wolf roar-bark sequences used as stimuli for playback studies of maned wolves

in the wild (Serra da Canastra National Park, Brazil). GA and SH are males, SA and JU are females. Top

spectrograms are the original files (96 kHz sample rate, 32 bit wav, 4000 windows size, 56% brightness

and 50% contrast) and the bottom a recording extracted from one autonomous recorder (Song Meter

SM2+; Wildlife Acoustics) 80 meters from the playback speaker (8 kHz sample rate, 16 bit wav, 512

windows size, 50% brightness and contrast). Spectrogram made on Raven Pro 1.5.

We conducted playbacks on two nights at each site, except on site A where we

could only do it once (March 04 to 05) due to logistical issues. Sessions were conducted

three times each night at the following moments: After Sunset (between 15 and 75

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38

minutes after sunset), Midnight (between 23:00 and 00:00), and Before Sunrise

(between 15 and 75 minutes before sunrise). Due to weather conditions, on the first

night (March 07 to 08) at site C we could not do the After Sunset session, and

compensated for it by conducting a session on a third consecutive night (March 09). At

site C we conducted additional diurnal sessions on two days (March 08 and 09) three

times each day to tests the animals’ responsiveness during the light period: After

Sunrise (between 15 and 75 minutes after sunrise), Midday (between 11:00 and 12:00),

and Before Sunset (between 15 and 75 minutes before sunset). Mean local sunset was

18:28 and sunrise 06:05 during the playback days (calculated on

https://www.sunearthtools.com/pt/solar/sunrise-sunset-calendar.php; access Set/16

2018). This experimental design resulted in a total of 21 playback sessions over 6 days.

During the conducted playback sessions wind speed was 0.37 ± 0.61 m/s (mean ± SD,

maximum 2.4 m/s), which based on our previous work should not significantly impact

roar-bark detection and propagation (Rocha et al. 2016).

Roar-bark sequences of free-ranging maned wolves detected up to 10 minutes

after the end of any broadcasted sequence were considered responses to the playback.

Although it is impossible to differentiate a vocal response from a spontaneous

vocalization, we consider 10 minutes a plausible interval to assume the vocalization is

an playback-elicited answer as maned wolves vocalize 0.41 roar-bark sequences per

night per recorder in the wild (Rocha et al. 2016) and 0.68 sequences per individual per

night in captivity (Sábato 2011). The chance of a maned wolf spontaneously emitting a

roar-bark sequence within any given 10 minutes each night is less than 1% (0.57%

based on Rocha et al. 2016; 0.94% based on Sábato 2011).

For each response we recorded the time of emission and, based on the

vocalization source direction determined by ear, if there were more than one animal

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39

vocalizing. Additionally, we waited at least 30 minutes after the end of each session

near the playback site to verify possible approaches by responsive animals.

3. Autonomous recordings during the playback experiment

We successively mounted at each playback site a line of 8 autonomous recorders

(Song Meter SM2+; Wildlife Acoustics, Inc., Concord, Massachusetts) with one

omnidirectional weatherproof microphone each (SMX-II; Wildlife Acoustics, Inc.;

sensitivity -36±4dB [0dB=1V/pa@1kHz]; 20Hz-20kHz flat response frequency).

Recorders were set on the road side in a single direction from the playback speaker

positioned at distances of 1.25 m, 20 m, 40 m, 80 m, 160 m, 320 m, 640 m, and 1280 m.

Distances were measured using a tape measure (1.25 to 80 m) and a GPS (Garmin

GPSMAP® 76S; accuracy < 15 m). The autonomous recorders were attached on stakes

of the same height of the speaker (86 cm) with the omnidirectional microphone in a

perpendicular position in relation to the speaker. Recordings were made continuously,

with an 8 kHz sample rate, 16-bit wav files, and partitioned in 30 minutes files.

At site A the autonomous recorders were active from 16:34 March 04 to 06:04

March 05 (total 13 h 30 min), at site B from 16:03 March 05 to 08:33 March 07 (total

40 h 30 min), and at site C from 19:03 March 07 to 06:33 March 11 (total 83 h 30 min).

After the end of the experiment we left the recorders active for an extra day and night

(March 10 to 11), resulting in the larger amount of recording hours at site C. We used

this extra day and night to evaluate if maned wolves would continue to vocalize

spontaneously without the playback stimuli.

We automatically detected roar-barks on the files using the methodology

detailed in Rocha et al. (2015). This methodology uses XBAT_R7 (Extensible

Bioacoustics Tool; Figueroa 2007) extension for MATLAB (R2010a version;

MathWorks, Inc., Natick, MA, USA) to generates spectrograms of the files that are

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40

subjected to a cross correlation tool employing 4 roar-bark templates. Matches above a

pre-defined threshold (0.21) are then manually verified for false positives and

undetected roar-barks within 24 seconds of the detected ones.

We detected both free-ranging maned wolves roar-barks and the roar barks we

broadcasted during the playback. For the broadcasted roar-barks we noted all recorders

that registered the calls (e.g. 1.25 m, 20 m, 40 m, etc.). We used this information to

estimate roar-bark range. To do so we used only the playbacks conducted at night, as

maned wolves rarely vocalize during the daylight period (Brady 1981; Emmons 2012).

For free-ranging maned wolves roar-bark sequences we noted the time of

emission, time from the end of the last broadcasted roar-bark sequence (equivalent to

the latency in the cases considered responses), the number of roar-barks in the sequence,

the distance of the autonomous recorder that the roar-bark recording was most intense

(measured by the peak power function of Raven pro 1.5), and if the vocalizations were

heard by us in the cases we were present at the site near the speaker. The maximum

interval between roar-barks that we considered a single sequence was 10 seconds,

longer intervals were considered the beginning of a different sequence (based on Rocha

et al. 2016 dataset and Bender et al. 1996).

4. Comparative passive acoustic monitoring

In 2016 we deployed 13 autonomous recorders (Song Meter SM2+, Wildlife

Acoustics; 8 of which were the same ones used during the 2017 playback experiment) at

Serra da Canastra National Park on the same region that we conducted the playback

experiments (Figure 1). Mean distance between recorders was 3.03 km (minimum 2.16

km, maximum 3.90 km). Recorders were attached onto 1.4 m wooden stakes to

maximize detections of roar-barks and were set with the same recording configurations

as the playback experiment but programed to record from 5 PM to 5 AM each day.

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Considering our previous study in the area (Rocha et al. 2016) we expected this period

to comprise most of the maned wolf vocal activity. Recordings were made during 20

consecutive nights between March 09 and 28, 2016.

Roar-bark sequences were automatically detected on the audio files the same

way we did with the playback recordings. For each roar-bark sequence found we noted

time of emission, the number of roar-barks in the sequence, recorder location, and if the

sequence was recorded by more than one recorder. That last information was verified

comparing the sequences´ time of emission and the inter roar-barks intervals, which is

unique for each sequence and ensures it is the same sequence recorded in two (or more)

recorders and not two independent sequences of different animals vocalizing at the same

moment. We only counted the most intense record of a sequence that has been detected

in multiple sensors. Finally, we measured the time interval between sequences emitted

in the same recorder during the same night.

In some cases we could identify a second animal started a roar-bark sequence

before the end of another animal sequence, resulting in intercalated roar-barks. We

could recognize the presence of a second animal based on differences in roar-bark

cadence, intensity, spectral shape, and occasional overlap of roar-barks. If roar-bark

sequences were separated (no intercalated roar-barks) and there was no striking spectral

shape difference between them we could not tell if the sequences were emitted by the

same or different animals. In cases of two animals vocalizing at the same moment we

considered the interval between the sequences as 0 seconds.

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Results

1. Playback experiment and recordings

The results from the playback experiment are summarized in Table 1 and

Figure 3. All roar-bark sequences heard in loco by the researchers were recorded by at

least one autonomous recorder. We were not able to visualize the animals.

We obtained vocal responses in only 4 of 21 playback sessions (see

Supplementary data SD1 and SD 2 for examples of such responses). Responses

occurred in 3 of 6 nights in which there was at least one playback session. On 2 nights

the “After Sunset” playback session was answered and on one night both the “Before

Sunset” and “After Sunset” sessions elicited vocal responses. Responses consisted of 1-

3 roar-bark sequences on average 02:12 ± 01:56 minutes (X ± SD; N = 8) after the end

of the broadcasted sequence.

Figure 3. Distribution of wild maned wolf roar-bark sequences registered between March 04 and 11 2017

during a playback experiment at Serra da Canastra National Park, MG/Brazil. Each sequence is named by

its start time and the size of the bar shows the time elapsed from the last broadcasted playback sequence.

This time is also discriminated on tags above the sequences considered responses to the playback, i.e.

those within 10 minutes after the end of any broadcasted sequence.

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44

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In 4 of 6 nights there was additional vocal activity initiating around 30 minutes

to 1 hour after the playback and consisted of 2-7 shortly spaced sequences (Figure 3).

We did not always register a response to the preceding playback session, for instance,

the March 06-07 Midnight session was not answered (to our best detection capabilities)

despite the vocal activity one hour later. We did not register any roar-bark sequence on

the extra day and night recorded at site C following the end of the playback experiment.

Based on differences in the direction of the vocalization source detected aurally

we could identify two occasions in which the vocal response involved two different

wild maned wolves. On March 08, 10 seconds after one animal responded to the “After

Sunset” playback session (sequence no. 14, Table 1), a second animal emitted a

sequence (no. 15, Table 1) and then another sequence 04:39 minutes thereafter (no. 16,

Table 1). Although very close to the pre-established 10 minutes window (10:09 minutes

after the playback), this last roar-bark sequence was not considered a playback response

to our criterion. It was, however, emitted 05:22 minutes after the first animal response

and could have been an answer to that animal.

The second occasion occurred following the interactive playback we were able

to produce during our last playback session (March 09 “After Sunset”). There were

three callback response sequences, after the first, second and fourth sequence we

broadcasted (no. 17-19, Table 1). This playback timing scheme elicited the most intense

behavioral response: 40 minutes later we heard two animals exchanging roar-bark

sequences and moving fast toward the playback site. They reached the location and then

continued moving past it, still exchanging roar-bark sequences (no. 20-26, Table 1).

After a one-hour interval there was another roar-bark sequence nearby (no. 27, Table 1;

not heard, we left the site soon thereafter) and 20 minutes another three (no. 28-30,

Table 1; not present). The last sequence was most intense at the autonomous recorder

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1.25 m from the position where the speaker was during the playback. The time pattern

sequence of this night can be seen in black on Figure 3.

For comparison purposes, we considered the area covered by the 8 autonomous

recorders (1280 m in line) during the playback experiment as equivalent of the area

covered by 1.5 autonomous recorders during the 2016 passive recordings, as the average

distance between then was 3 km. That results in 3.33 roar-bark sequences per

‘autonomous recorder’ (1.5) per night (6).

All broadcasted roar-barks were detectable in the recordings up to 80 m, 96.2%

were detectable in the 160 m autonomous recorder, 47.7% in the 320 m recorder, 26.7%

in the 640 m recorder, and 0.8% in the 1280 m recorder. From the 21 recorded

sequences emitted by free-ranging animals while we were present at the site, 7 we had

not heard. Six of those sequences had the most intense register in the recorder at 1280

m.

2. Comparison with passive acoustic monitoring detections

We registered 2610 roar-barks distributed in 224 sequences (11.65 ± 7.09 roar-

barks per sequence, X ± SD; minimum 1, maximum 50) during the 20-night recording

period in 2016. Each night had an average of 11.20 ± 8.45 sequences (X ± SD;

minimum 0, maximum 25). The mean number of autonomous recorders that registered

roar-bark sequences each night was 3.65 ± 2.01 (X ± SD; minimum 0, maximum 9),

each of those with an average of 3.07 ± 2.64 sequences (X ± SD; minimum 1, maximum

13). Overall, we obtained an average of 0.86 roar-bark sequences (total 224) per

autonomous recorder (13) per night (20). Supplementary data SD1 and SD3 contains

an example of sequence recorded passively.

We measured a total of 173 intervals between sequences, including 22 times

when there was overlap of sequences, i.e., when the second animal vocalized before the

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first animal ended its sequence (considered as 0 second interval). Sequences on a single

recorder and night were in general separated by short intervals (6 min 40 s, median; 51 s

– 21 min 47 s, 1st – 3rd quartiles). A recurrent emission pattern consisted of 1-3

sequences separated by short intervals followed by a longer interval (20-80 min) and

another set of 1-3 sequences separated by short intervals.

The diel roar-bark emission temporal pattern found is shown in Figure 4. A

small percentage (8.04%) of the sequences occurred between 17:00 (recordings start)

and sunset (sunset time varied from 18:09 to 18:26), 34.82% in the first 3 hours of the

night, 68.75% on the first half of the night, and 23.21% on the second half of the night.

Except for a peak between 22:00-23:00 hours, the pattern was very similar to the April

2014 temporal pattern described by Rocha et al. (2016; recordings started at 18:00 and

sunset varied from 17:44 to 18:03 in April 2014).

Figure 4. Temporal distribution of maned wolf roar-bark sequences recorded at Serra da Canastra

National Park, MG/Brazil, with autonomous recorders (Song Meter SM2+; Wildlife Acoustic). March

2017 (black line, left axis): percentage relative to the total (30 sequences) of vocal activity registered on

continuous recordings of the 6 days in which the roar-bark playback experiment was conducted. March

2016 (dark gray bars, right axis): absolute number of sequences (total 224), 13 recorders, 20 nights, from

5 PM to 5 AM. April 2014 (light gray bars, right axis): absolute number of sequences (total 192), 12

recorders, 25 nights, from 6 PM to 6 AM (Rocha et al. 2016 dataset, used with permission).

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Some of the sequences (54 of 224) were detected in more than one autonomous

recorder. All of those cases (except 1) happened on 5 combinations of two autonomous

recorders that were on average 2.40 ± 0.15 km (X ± SD) apart. On average 45.0 ± 18.6

% (X ± SD) of the times one of those recorders registered a sequence it was also

detected on another recorder. The maximum distance between autonomous recorders

that registered a given sequence was 4.85 km (distance from the first and third recorder

that registered that particular sequence). Considering the case of an animal exactly in

the midpoint between those recorders, that means the calls from this sequence

propagated at least 2.4 km before reaching the recorders.

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Discussion

Here we tested the playback of roar-barks in a protected area where wild maned

wolves inhabit. They responded vocally, proving this technique is applicable to monitor

and study the species. We were able to describe their response and compare it to a 20-

night passive acoustic monitoring effort on the same area and period of the year. We

also identified the best broadcasting time and feasibility of conducting interactive

playbacks. Although we could not define with precision the roar-bark range, we were

able to acquire some information on the response distance and our hearing capabilities.

The vocal response rate obtained (4/21 sessions) may seem low, but maned

wolves are not highly vocally active (Rocha et al. 2016; see also Results), occur in very

low densities (Trolle et al. 2007), and we did not know a priori if there were any

animals on the region at that time. Considering the aforementioned factors, we evaluate

that responses in 3 out of the 6 nights tested was a high response rate.

The average of 0.86 sequences per recorder per night obtained in our

comparative passive recordings of March 2016 was greater than the previous values

obtained in the 2014 passive recording study (April to July 0.41 and only April 0.52;

Rocha et al. 2016). Even so, the average vocal response recorded during our playback

experiment in 2017 (estimated 3.33 sequences per recorder per night, including

responses and non-responses) was near 4 times higher than the spontaneous vocalization

rate one obtained in March 2016.

This higher vocal activity and the complete lack of roar-bark sequences on the

night after the playback suggests most of the vocal activity recorded was at least

indirectly influenced by the playback. That may be true even for sequences emitted

several minutes later (30-60 min). The animals´ approach to the speaker’s site following

the interactive playback vocal responses, although evidently influenced by it, happened

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40 minutes later with a second set of uttered sequences. A third set of roar-bark

sequences was registered 1 hour after the second set and the last sequence was most

intense on the 1.25 m recorder, suggesting the emitter(s) may still be investigating the

playback area after the stimuli had ceased. Sets of sequences separated by similar

intervals (20-80 min) on the same night were also observed during the passive acoustic

monitoring. Although we do not know what motivated the vocalizations recorded

passively, this suggests that when there is a second set of sequences within such time

intervals, they may have been elicited by the same stimulus (similar motivation) than

the first set, i.e., a redundant message, a reiteration.

We cannot rule out the possibility the roar-bark sequences we are calling

responses were, in fact, spontaneous vocalizations. However, we reinforce that maned

wolves emit on average less than one roar-bark sequence each night (Sábato 2011;

Rocha et al. 2016), making a spontaneous vocalization within 10 minutes of the

playback unlikely. Even considering the 0.86 sequences per recorder per night obtained

here and the increased detection probability between 18:00 and 19:00 (12.05% on the

passive monitoring), the probability of a spontaneous roar-bark sequence being emitted

during our 10-minute response time window would be 1.73%. Additionally, during the

playback experiment, there were instances when more than one sequence within the

same 10 minutes were detected and the maned wolves that emitted those sequences

intercalated them with our playback sequences, which would make the time coincidence

even more unlikely.

We registered two occasions where vocal responses involved two animals, but it

was not possible to confirm their sexes or territory ownership. As maned wolves form

stable breeding pairs that share the same area (Azevedo 2008), and it was the beginning

of the mating season (March to May on this park; Rodden et al. 2004), which mean the

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pair could be maintaining proximity (Emmons 2012), we speculate it was a mated male

and female. However, it could have been two non-mated individuals from the same or

different territories, or even non-residents, independently responding to the playback.

During a pilot playback experiment at the same park we also obtained delayed

responses involving two animals (conducted in May 2016), indicating this behavior is

not uncommon. Further studies are needed to clarify which animals participate in such

responses.

Although the species increase its physical activity around both twilights (Jácomo

et al. 2004; Melo et al. 2007), responses and vocal activity were concentrated only

closer to dusk (1h before, 3h later). Our passive acoustic monitoring confirms that

animals naturally vocalize more on the first half of the night and thus could be more

prompt to respond also during that period. Despite our playback sessions near dawn

(05:00-06:00 and 06:00-07:00), there was no response or vocal activity on this period.

We cannot know if no animal heard the playback or if they just chose not to respond.

More studies are needed to verify if there is a biological reason for this lack of

responsiveness. Nevertheless, for studies not aiming at this question, it seems

reasonable to concentrate playback efforts on the 17:00 to 22:00 period.

While less than half of the broadcasted roar-barks were detectable at 320 m, a

large portion of naturally emitted roar-bark sequences were detectable in two or more

recorders 2.16 to 4.85 km apart during the passive monitoring (meaning the maned wolf

was at least halfway between those distances). We believe this discrepancy is a product

of the recording height. The 86 cm used during the playback was much lower than the

1.4 m used during the passive monitoring. We choose this lower height to simulate a

maned wolf position, but the range obtained this way does not seem to reflect neither

the maned wolf's nor our own roar-bark hearing capability (see below). Another non-

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exclusive explanation for this discrepancy in detectability would be that the captive

individuals, whose recordings were used to set the playback intensity, roar-barked at a

lower intensity level than wild maned wolves normally do. This could have happened

because in captivity individuals are held in much closer proximity to conspecifics than

in natural conditions and thus, they would not need to vocalize as loud to reach others,

optimizing their vocal output.

We obtained responses that were most intense in the 1280 m recorder,

suggesting the animal was at least 960 m away (between the 640 m and 1280 m

recorders; sequences no. 4 and 9, Table 1). This was slightly greater than our own

hearing limit as no sequence with the most intense signal in the 1280 m recorder was

heard by us, while all most intense signals on the 640 m were. Some caution with those

distances is needed because terrain composition and wind currents and turbulence can

drastically affect the way the sound reach each recorder, especially in open fields

(Brown and Handford 2000). Besides, the autonomous recorders were not calibrated

together so some intensity discrepancies between the recordings is expected.

Nevertheless, those distances are in agreement with Brady (1981) study that

states a roar-bark could be heard 1 km away, although the author mentioned they could

be even discriminated individually at this distance. Considering the above, future

playbacks should be conducted at sites 2 km apart at minimum (1 km for each side).

Greater distances would be ideal to ensure site independency as we obtained evidence

for the minimum maned wolf roar-bark hearing range, but not the maximum. If it is as

good as a well-positioned autonomous recorder, then minimum site distances should be

4.85 km.

Playbacks could be used to survey maned wolf presence and distribution in a

relatively fast way, considering the increase in vocal activity and smaller data volume to

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analyze compared to passive acoustic monitoring (Rocha et al. 2015). Playback

methodology has been used, for instance, to monitor the expansion of golden jackals’

range in Europe and count territorial groups (Acosta et al. 2018). Acoustic stimulation

can also be used to attract maned wolves to facilitate their capture, as suggested for

African wild dogs and bush dogs (Robbins and McCreery 2003; DeMatteo et al. 2004),

to detect their presence even without vocal responses (directly or indirectly, e.g. by

footprints: Brennan et al. 2013), or to visually identify individuals for counting, as done

with hyaenas and lions (Cozzi et al. 2013). To estimate absolute population numbers,

we need to conduct future research on the maximum distance, probabilities, and other

factors influencing the emission of maned wolves’ vocal responses and approaches, as

Cozzi et al. (2013) did for their target species. Another possibility is using playback-

elicited vocal responses to create a vocal identity catalog to then count and recognize/

“recapture” individuals on recordings, as for African wild dogs (Hartwing 2005). Roar-

barks have been demonstrated to be individually distinct (Sábato 2011), however, to

date, our efforts have been insufficient to identify individuals on recordings in their

natural habitat (see Chapter 4). Finally, different from the passive acoustic monitoring,

playbacks allow direct test of hypothesis (e.g. Marshall-Ball et al. 2006). This technique

could be used, for instance, to test if maned wolves indeed use roar-barks to defend

territories and/or mates, and what is the male and female role in territorial defense.

On the other hand, passive acoustic monitoring is a non-invasive approach that

result in a less biased index of population size. By counting acoustic cues (e.g. number

of roar-bark sequences) and comparing different years it is possible to monitor relative

population trends (e.g. seabirds: Oppel et al. 2014). Cue counting also depends on

auxiliary information about vocal behavior, sender identity and signal ranging that is not

yet available for maned wolves (Marques et al. 2013). To monitor absolute population

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size, we would also need more information, such as individual emission rate,

estimatives of caller distance, and/or vocal individual identification. Nonetheless,

passive acoustic monitoring has the advantage of revealing natural multi-individual

activity and interaction patterns (Blumstein et al. 2011). Field time and costs are smaller

than other methodologies (Jorge et al. 2018), allowing for long term studies covering

larger areas.

The total investment cost, however, can be high for both approaches.

Autonomous recorders are expensive (for most underdeveloped countries) and can

represent over 90% of a passive monitoring study cost (Jorge et al. 2018). Maned

wolves occur in low densities on very extensive areas and thus playbacks would need to

be done over many distant sites to have reliable sample size, which can increase field

time and cost manifold. In the end the method of choice will depend on the question to

be answered or problem to be solved. Combining methodologies will be advantageous

as no single approach can reveal every aspect of a given system.

Is conclusion, for future playbacks to maned wolves we recommend: waiting or

recording at least one hour after the playback as vocal activity and approaches may be

delayed; using interactive playbacks for maximum response and chance of approach;

expect multi individual responses; concentrating efforts on the first portion of the night;

choosing distant sites (2-5 km minimum); and consider using autonomous recorders to

increase detection of responses. Our work shows that playbacks, and also passive

acoustic monitoring, have good potential to be used in conjunction during investigations

of behavior ecology even if the target species is not seen in the field and is not highly

vocal, as is the case for many other mammals.

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Conservation, 16, 1197-1204.

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Supplementary data

Supplementary Data SD1.Wild maned wolf roar-bark sequences recorded at Serra da Canastra National

Park, MG/Brazil. a and b: sequence in response to a playback stimulus registered on 18:55:37 March 09

2017. c and d: two individuals (note the change in spectral characteristics after 33 s) recorded passively

on 19:15:00 March 17 2016. Recordings made with autonomous recorders (Song Meter SM2+; Wildlife

Acoustic), at 8 kHz sample rate and 16-bit wav file format. Spectrogram made on Raven pro 1.5 (Cornell

Bioacoustics Lab, Ithaca, NY, USA), Hann window, 512 window size, 50% brightness and contrast, 50%

overlap, smoothing on.

Supplementary data SD2.Playback response audio file.

Supplementary data SD3.Passive recorded audio file.

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Chapter 2

Maned wolf long range call propagation and its

implication for the species’ communication

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Maned wolf long range call propagation and its implication for the

species’ communication

Luane Stamatto Ferreira, Lourdes M.M. Villavicencio, Júlio Baumgarten, Flávio H.

Rodrigues, Renata S. Sousa-Lima

Intended for submission on: Journal of the Acoustic Society of America

Abstract

There are several major open questions about the behavior of maned wolves due

to the difficulties in finding and following individuals in the wild. The investigation of

the species’ acoustic communication has a great potential to fill those gaps.

Understanding the propagation properties of the species’ long-range roar-bark call is the

first step to accomplish this fascinating challenge of learning about their acoustic

communication. We broadcasted roar-barks and re-recorded them simultaneously at

different distances at the Serra da Canastra National Park, MG/Brazil. Roar-barks

broadcasted and re-recorded from a higher to a lower altitude (down-slope) lost more

intensity than when propagated from a lower to a higher altitude (up-slope). We argue

this could have been wave interaction effects and/or an effect of acoustic shadows. On

the site with more vegetation sound attenuated more than at all other sites, which was in

accordance with our excess attenuation prediction as there are more objects that can

absorb sound energy. We found that inclining the speaker 45o upward to simulate the

head/muzzle position of maned wolves during vocalization had a negative effect on

sound intensity instead of enhancing propagation. However, this angle did counteract

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partially the negative effects of vegetation, and, coupled with other possible positive

effects of this position, may compensate the observed intensity loss. Finally, we found

that around dusk and before dawn transmission loss was lower. Maned wolves vocalize

more on the first hours of the night, but only rarely near dawn. Thus, the species’ time

choice of vocalization is not only influenced by sound propagation, as our results

suggest factors such as social interactions and weather may explain why they do not

take advantage of both twilights to communicate acoustically.

Resumo

Existem muitas perguntas em aberto em relação ao comportamento do lobo-

guará devido às dificuldades em encontrar e acompanhar os indivíduos na natureza. A

investigação da comunicação acústica da espécie tem grande potencial de preencher

essas lacunas. Entender como o aulido, o chamado de longa distância da espécie, se

propaga em ambientes naturais é o primeiro passo para compreender esse sistema de

comunicação. Nós reproduzimos aulidos e os re-gravamos simultaneamente à diferentes

distâncias no Parque Nacional da Serra da Canastra, MG/Brasil. Diferente do que

previmos, aulidos propagados de uma maior altitude para menor (morro abaixo)

perderam mais intensidade que os propagados de uma menor altitude para uma maior

(morro acima). Nós argumentamos que isso pode ter sido resultado de sombras

acústicas e/ou efeitos da interação entre ondas. No local com mais vegetação o som

atenuou mais do que em todos os outros locais, o que estava de acordo com nossa

predição já que há mais atenuação excessiva devido a presença de mais objetos que

podem absorver energia acústica. Nós descobrimos que inclinar a caixa de som 45o para

cima, para simular a posição do focinho dos lobos-guará durante a vocalização, teve um

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efeito negativo na intensidade do som, ao invés de melhorar a sua propagação.

Entretanto, esse ângulo neutralizou parcialmente os efeitos negativos da vegetação e,

em conjunto com outros possíveis efeitos positivos dessa posição, pode compensar a

perda de intensidade observada. Por fim, encontramos que em torno do entardecer e

logo antes do amanhecer a atenuação foi menor. Lobos guará vocalizam mais nas

primeiras horas da noite, mas apenas raramente perto do amanhecer. Logo, a escolha de

horário de vocalização da espécie não é influenciada apenas pela propagação do som e

os resultados sugerem que fatores como interações sociais e clima, podem explicar por

que lobos-guará não aproveitam ambos crepúsculos para comunicarem-se

acusticamente.

Key-words: Chrysocyon brachyurus, bioacoustic, acoustic adaptation, sound

propagation

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Introduction

Maned wolves (Chrysocyon brachyurus; Illiger 1815) are nocturnal/crepuscular

and solitary foragers (Rodden et al. 2004). As other canids, the species is monogamous

and the breeding pair shares the same home range, which is usually very extensive

(mean ± SD: 80 ± 53 km2; Jácomo et al. 2009). However, it is believed that the pair

rarely interacts physically, getting close only during the reproductive season (Dietz

1984). Those habits make visual communication impractical, and a compensation by

other sensory channels is expected, as acoustic communication (Fox 1975). Studies in

wild population indicates that maned wolves indeed interact vocally at long distances in

a regular basis (Rocha et al. 2016). There are many gaps in our knowledge of the

species’ behavior (Rodden et al. 2004), and few studies on natural environments due to

the difficulties in finding and following individuals (Bestelmeyer 2000). Therefore, the

investigation of the species acoustic communication has a great potential to fill those

knowledge gaps.

Understanding the propagation of the acoustic signals in the natural environment

is one way to investigate this communication modality. According to the Acoustic

Adaptation Hypothesis (AAH) long range acoustic signals are shaped by selective

pressures to effectively propagate through the natural habitat of the species (Morton

1975; reviewed in Ey & Fischer 2009). The maned wolf inhabits mainly open

neotropical savannas (Cerrado biome; Coelho et al. 2008). The predicted sound

characteristics for effective propagation in this type of environment are high intensity,

low frequency, and tonal structure (Wiley & Richards 1978, 1982). In agreement, the

long-range vocalization of the maned wolf, the roar-bark (Brady 1981; Sábato 2011),

has high intensity and low frequency. However, the signal energy occupies a broad

frequency band which gives the call a harsh, noisy quality. This characteristic can favor

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the localization of the emitter (Naguib & Wiley 2001), while redundancy, in the form of

bouts of roar-barks (sequences), can favor signal reception (Morton 1975; Brown &

Handford 2000).

A recurrent point cited in terrestrial sound propagation studies is that the vertical

positioning of the emitter influences signal transmission, with higher positions generally

improving emission and reception (Morton 1975; Brown & Handford 2000; Kime at al.

2000; Schwartz et al. 2015). Singing at positions higher than 1 meter from the ground is

enough to minimize ground effects (Marten & Marler 1977) such as destructive

interference (Wiener & Keast 1959). The maned wolf height ranges from 70 to 90

centimeters at the shoulder (Silveira 1999), with the adult average closer to 84

centimeters (Jácomo et al. 2009). That places the species near the limit to experience

those ground effects (or not). To counter those effects a maned wolf could vocalize on

higher grounds or raise its head the maximum possible while vocalizing. In fact,

captivity studies describe that the species elevate the head (pointing the nose upward)

while emitting roar-barks (Sábato 2011; Balieiro 2015; see Figure B in the General

Introduction).

Additionally, vocalizing with the head/muzzle elevated can broadcast body size

information. There are some cues on the vocalization of animals that are correlated with

the vocal tract length, which is normally correlated to body size (like formants; Fitch

1997). Pointing the nose upward maximize the vocal tract length at the moment of

vocalization, and this can pass a larger body acoustic impression (Charlton & McComb

2007; Pisanski & Rendall 2011).

Another form of understanding a communication system is identifying its

temporal pattern. Classical examples are the dawn and dusk choruses, consisting of a

concentration of vocal activity around twilight (Burt & Vehrencamp 2005; Berg et al.

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2006). This phenomenon is typical of birds (Sedgwick 1941) but has been described in

several other taxa as primates (Colobus guereza—Schel & Zuberbühler 2012) and

invertebrates (Cato 1978). There are at least 12 non-exclusive hypotheses to explain this

time choice (Staicer et al. 1996), especially a possibly optimized sound propagation

(Brown & Handford 2003), and a period not yet ideal to forage due to illumination but

good to communicate while avoiding predators (Berg et al. 2006).

Temperature gradients are important to define sound propagation properties

during transmission. When the sun is up it heats the surface and the soil heats the above

air layers by irradiation, creating a temperature gradient where the ground and lower air

layers are hotter than the above air layers. As sound travel faster in higher temperatures,

this gradient refracts the sound upward making it harder to be detected by a receptor at

ground level (Wiley & Richards 1978; Embleton 1996). When the sun sets the ground

loses heat irradiating to the air above until eventually the ground becomes cooler than

the air. Thus, at night the sound will bend downward and more of its energy will reach a

ground level receptor (Wiley & Richards 1978; Embleton 1996). Finally, when the

night is ending the ground is at its coldest moment and the rising sun heats the above air

layers, amplifying this gradient and in this case its positive effects on sound

transmission (Wiley & Richards 1978; Embleton 1996).

Many canids vocalize most often during twilight, including grey wolves (Canis

lupus; Harrington & Mech 1979), coyotes (Canis latrans; Walsh & Inglis 1989), and

dingos (Canis dingo; Corbett 2001). Maned wolves are most physically active during

twilight (Melo et al. 2007; Jácomo et al. 2004) but seem to concentrate their vocal

activity during the beginning of the night (Rocha et al. 2016). Nocturnal recordings of

four months indicated that the first hour of the night is when they vocalize most (Rocha

et al. 2016).

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Here we investigate how the long-range maned wolf roar-bark propagates

through its environment by broadcasting calls and re-recording them at different

distances. We tested the propagation in four different sites to investigate a possible

terrain effect. Our prediction was that on the site with more vegetation the propagation

would be less effective due to the increased number of obstacles (leaves) absorbing and

deflecting sound in multiple directions (Bradbury & Vehrencamp 1998). We also

broadcasted sound from a higher to a lower altitude (down-slope) and vice-versa (up-

slope). In these situations, there are two possible predicted outcomes (Bradbury &

Vehrencamp 1998): 1) that sound will propagate more effectively up-slope, due to

positive interference of the sound wave reflected in the same phase by the smooth

ground surface, contrasting with a sound over-spreading in the open down-slope

situation; or 2) that sound will propagate less effectively up-slope, due to ground effects

as negative interference of the sound wave reflected in the opposite phase and

absorption by the ground and obstacles, contrasted with a sound path free of ground

effects and obstacles in the open down-slope situation.

Although we have no information on the directionality of maned wolves roar-

barks, here we assumed both the natural vocalization and the speaker have a broad

conical-like shape sound volume (similar to Lehner 1982). Therefore, by inclining the

speaker 45o upward we also tested whether the head positioning seen in captivity

enhances propagation, and if the position interacted with site differences such as the

presence of vegetation.

Finally, we tested if the maned wolves’ time choice of vocalization was related

to sound propagation. Our hypothesis was that maned wolves vocalize more in the

beginning of the night (Rocha et al. 2016) because this is the period in which roar-barks

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propagates more efficiently in their habitat. Therefore, we predict playbacks conducted

at this time would propagate more efficiently than at other times of the day.

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Materials and methods

We conducted this study at Serra da Canastra National Park, located southwest

of Minas Gerais state, in Brazil (Figure 1). The habitat consists mainly of Cerrado

biome, with morphologies varying from open grasslands, mix compositions of tall grass

and shrubs, and areas with dense shrub and low trees (MMA/IBAMA2005). The park

has many rocky outcrops and water bodies, including souterrain rivers, that are also

typical of Cerrado biomes (MMA/IBAMA 2005). Audio data collection in this park

was authorized by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio;

SISBIO license number 41329-2).

1. Playback and re-recording procedure

The roar-barks used were recordedby Sábato (2011) at two captive facilities in

Minas Gerais: the Criadouro Científico de Fauna Silvestre para Fins de Conservação

da Companhia Brasileira de Metalurgia e Mineração at Araxá; and the Zoológico da

Associação Esportiva e Recreativa dos Funcionários das Usinas Siderúrgicas de Minas

Gerais S/A at Ipatinga. Recordings were made using a Marantz PMD-661 solid state

recorder, a Sennheiser ME-66 module, and a directional Sennheiser K6 microphone.

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We used sounds recorded from 4 captive individuals, 2 males and 2 females

(Figure 2), which were 6-8 meters away from the microphone. We chose 5 good quality

roar-barks coming from the same sequence (bout) for each of the 4 individuals (total: 20

roar-barks) aiming to include roar-barks that were as spectrally diverse as possible. All

roar-barks were broadcasted on each site and condition (time and speaker position; see

below) with a 2.9-6.2 second interval between roar-barks of the same animal and 10

seconds to 10 minutes between animals. The 5 roar-barks of each animal were always

broadcasted together and in the same order (they were on the same file), but the 4

animals order was randomized for each site.

To set the intensity level we used the playback equipment (described below) to

broadcast the captivity recordings and then re-record the played back sounds with the

same equipment and settings used for the original captivity recordings at the same

distance the focal animals were. We then changed the speaker volume until the roar-

bark intensity measured in the re-recordings matched the intensity measured in the

original captivity recordings (measures made in Raven Pro 1.5 software: Bioacoustics

Research Program, 2014. Raven Pro: Interactive Sound Analysis Software. Ithaca, NY:

The Cornell Lab of Ornithology). This was our solution to achieve a playback intensity

level as similar as maned wolves' roar-bark emission. During the recordings in captivity

we did not have a direct way to measure sound pressure levels, and thus no means of

calculating absolute source intensity levels. Two experienced researchers, including the

Figure 1. Study site at Serra da Canastra National Park, MG, Brazil. a - site Flat; b - site Low to high; c -

site Vegetation; d - site High to low. Horizontal distance to the speaker is discriminated on the left side of

the / and altitude on the right side. Maps constructed with QGIS 3.4.0-Madeira (QGIS Development

Team, 2018. QGIS Geographic Information System. Open Source Geospatial Foundation

Project. http://qgis.osgeo.org) and Google Satellite images (Map data ©2018 Google, Imagery ©2018

TerraMetrics).

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author of the original captivity recordings (VS), reported the playback sounded as

strong as heard from the animals in captivity and in the wild (FHR).

It should be noted that Raven do not give absolute sound levels, instead

amplitude is displayed in a decibel measurement relative to an arbitrary reference value

of 1 (Charif et al. 2010). However, spectrograms generated by recordings made with the

same equipment, configurations, and broadcasted files, should render comparable

amplitudes (although small variations could happen). That is, the amplitude of the

captivity records and calibration can be compared among them, but they cannot be

compared to the files of the re-recorded broadcasts using autonomous recorders

(detailed below). In the same way, the amplitude of the re-recordings can be compared

among them, but not to the captivity and calibration records.

We used an Acer AspireOne notebook to broadcast the sounds using Raven pro

1.5 software and a Pioneer S-DJ50X speaker (class A/B Bi-amp, 80 W output, 50-

20000 Hz frequency range) 86 centimeters above the ground to simulate the height of a

maned wolf.

To re-record the broadcasts we successively mounted at each site a line of 7

autonomous recorders (Song Meter SM2+; Wildlife Acoustics, Inc., Concord,

Massachusetts) with one omnidirectional weatherproof microphone each (SMX-II;

Wildlife Acoustics, Inc.; sensitivity -36±4dB [0dB=1V/pa@1kHz]; 20Hz-20kHz flat

response frequency). Recorders were set on the road side on the most straight and open

portion possible (except site “Vegetation”, see below) in a single direction from the

speaker position at distances 1.25 m, 20 m, 40 m, 80 m, 160 m, 320 m, and 640 m

(Figure 1a-d). Distances were measured by measuring tape (1.25 to 80 m) and GPS

(Garmin GPSMAP® 76S; accuracy < 15 m). The autonomous recorders were attached

on stakes of the same height of the speaker (86 cm) with the omnidirectional

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microphone in a perpendicular position in relation to the speaker. Recordings were

made continuously, with an 8 kHz sample rate, 16 bits wav files, 36dB gain, and

partitioned in 30 minutes files (same configuration used in Rocha et al. 2015, 2016).

We had an eighth recorder placed at and 1280 m, but as only once it registered

the roar-barks broadcasted we decided to exclude it from all analysis.

Each recorder unit was placed at the same distance at all sites, e.g. the recorder

placed at 160 m at the first site was always placed at that distance at the following sites.

This was done to render the measures among the same distance more comparable, as the

units were not calibrated together and present some variation between their intensity

reading. A field test indicated our SongMeters units varied 1.91 ± 1.62 dB (mean ± SD)

in their intensity measures.

2. Broadcast sites

The sites were chosen based on the points with the highest spontaneous roar-barks

records of Rocha et al. (2016) study. Those sites also had the different characteristics we

were looking to test the terrain influence.

From March 04 to 05 2017 we conducted the broadcasts on the first site (Figure

1a: “Flat”). From March 05 to 06 2017 we conducted the broadcasts on the second site

(Figure 1b: “Low to High”). After we transferred the autonomous recorders 20 meters

laterally, so they would be outside the road and inside the tall grass, shrubs, and rocky

outcrop area, and conducted broadcasts from March 06 to 07 2017 (Figure 1c:

“Vegetation” site; see top photo in General Introduction Figure C). The vegetation

height was, in general, slightly below the speaker (86 cm). Altitudes differed

approximately 0-2 m from the “Low to High” site. From March 07 to 09 2018 we

conducted the broadcasts on the last site, “High to Low” (Figure 1d).

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3. Playback times

At each site we conducted playbacks three times a night. We chose the following

moments: between 18:40 and 19:40 (15-75 minutes after sunset), when wild maned

wolves are more vocally active (Rocha et al. 2016); between 23:00 and 00:00, an

intermediary time point; and between 05:00 and 06:00 (15-75 minutes before sunrise),

when wild maned wolves are less vocally active (Rocha et al. 2016).

At site “High to Low” we conducted extra broadcasts to test roar-bark

propagation during the light period: between 06:00 and 07:00 (15-75 minutes after

sunrise); between 11:00 and 12:00; and between 17:00 and 18:00 (15-75 minutes before

sunset). At this site we also conducted one replicate of all broadcasts (day and night) on

the next day.

Mean local sunset was 18:28 and sunrise 06:05 during the experiment days

(calculated on https://www.sunearthtools.com/pt/solar/sunrise-sunset-calendar.php;

access Set/16 2018).

4. Speaker position

To simulate the maned wolf inclination of the head/muzzle seen on captivity we

conducted broadcasts with the speaker box inclined 45o upward. This position was

named “Inclined” while the normal straight forward position was referred as “Straight”.

We conducted a “Straight” and “Inclined” broadcast, in random order, for all sites and

times mentioned above.

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5. Measurements

All recordings were analyzed on spectrograms generated by Raven Pro 1.5

software with the following characteristics: Hann window type, 512 window size,

grayscale, 50% of brightness and contrast, 50% overlap, and smoothing on.

We created selection boxes including the first two frequency bands

(Figure 2) as this is the only portion normally visible on wild maned wolf recordings

(Rocha et al. 2015). We extracted the roar-bark intensity through the peak power (dB)

measure on Raven Pro 1.5. We then calculated the relative loss in dB compared to the

measure of the same roar-bark re-recorded at 1.25 m. The dB loss was used as the

response variable of the model (see next section). A box of equal dimension was made

immediately before each roar-bark and the average power (dB) measure was taken on

Raven. This was used as a measure of the background noise level to be inserted in the

model as a control.

The temperature (oC), relative air humidity (%RH), and wind speed (m/s) was

measured for each playback session with a digital termo-higro-anemo-luximether

(SKTHAL 01, Skill-tec) at the speaker position and height. We used a single measure

for the “Straight” and “Inclined” broadcasts as they were done in succession (both

broadcasts together lasted between 5 and 25 minutes).

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Figure 2. Captive maned wolves roar-barks sequences broadcasted at Serra da Canastra National Park,

MG/Brazil. GA and SH are males, SA and JU females. Red selection boxes on the first roar-bark of each

animal exemplifies the ones used to measure roar-bark intensity (peak power, dB). Selections near the

second roar-bark of each animal exemplifies the ones used to measure noise intensity (average power,

dB). Spectrograms and measures were made on Raven Pro 1.5 (Cornell Bioacoustics Lab, Ithaca, NY,

USA), Hann window, 512 window size, 50% brightness and contrast, 50% overlap, smoothing “on”.

6. Statistical analysis

All statistical analyses were conducted with R software (R project version 3.4.4).

The script used and the resulting output is in the supplementary material.

To evaluate the effect of the different sites, times, and speaker position on sound

propagation we constructed a linear mixed model. We opted for a linear model as the

sampled distances increased in doubles resulting in a predicted loss of 6 dB by spherical

spreading only at each distance of re-recording (although some variation in the

measures are expected between different recording units). We used a mixed model to

control for the individual and particular roar-bark differences as they repeated

themselves across distance, sites, times, and speaker position. The method of restricted

maximum likelihood was used in order to produce unbiased variance estimates.

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The main model was a function of the relative dB loss and we included as fixed

factors distance (7), site (4), speaker position (2), an site*speaker.position interaction,

time (6), wind speed (m/s), and background noise (dB). Neither temperature nor

humidity were included on the main model as they had a conspicuous daily cycle, and

thus would be highly related to the broadcasting time. As our goal was to evaluate

propagation on the different broadcasting times in general, we chose to maintain all

daily cycles variation inside the factor “time” instead of separating the effect of time

and temperature/ humidity. For comparative purposes, we build a secondary model

including temperature (°C) on the fixed factors to control for days with overall different

temperatures, which would bias the factor “site” as each site was tested on a different

day. Only temperature was included on the secondary model as it was significantly

correlated with humidity (-0.738 spearman correlation coefficient). As random factors

for the intercept we included in the models the individual (4) and as sublevels the

different roar-barks (5) of each individual.

To validate the main model, we tested if the residuals had a normal distribution

and if they were symmetrically distributed in relation to the fitted values. We further

evaluated the model by comparing predicted and observed values. We used an ANOVA

to test if the models fixed factors had a significant effect. To test for differences

between levels of the fixed factors we compared multiple means on the models with

Tukey contrasts.

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Results

An exploratory analysis of the continuous factors (Supplementary material 1)

revealed that temperature and humidity fluctuated according to time: the 11-12h period

had the highest temperature and lower humidity, and 23-00h period the contrary. The

night at the site “Low to High” was on average colder and more humid than the other

nights. Wind speed was very low (0.37 ± 0.61 m/s, mean ± SD), being higher during the

day (maximum 2.4 m/s). The site High-to-low had more intense background noise than

other sites and there was a tendency for recorders positioned farther away to have less

intense background noise.

The main model residuals had a normal distribution and were symmetrically

distributed in relation to the predicted values (Supplementary material 2). The

predicted and the observed values correlated at a coefficient of 0.946 (Supplementary

material 3). An ANOVA revealed the intercept and all fixed factors were significant on

the model, including the interaction (Table 1). Confidence intervals for fixed and

random factors are reported on Table 2.

Table 1. ANOVA test for the fixed factors of the main model for the intensity (dB) loss of maned wolves

roar-barks broadcasted on their natural environment.

numDF denDF F-value p-value

Intercept 1 3736 41584.23 <.0001

Distance 5 3736 14.82 <.0001

Site 3 3736 11114.84 <.0001

Speaker position 1 3736 132.76 <.0001

Site:Speaker.position 3 3736 86.25 0.0008

Time 5 3736 1144.79 <.0001

Wind speed 1 3736 3335.92 0.0001

Background Noise 1 3736 5.57 <.0001

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Table 2. Approximate 95% confidence intervals for the estimate factor effects of the main model for the

intensity (dB) loss of maned wolves roar-barks broadcasted on their natural environment. Base categories

are specified under parenthesis. Factors/levels with positive and negative estimates, which indicates they

are not influential on the model or not significantly different from the base category, are underlined.

FIXED EFFECTS:

lower est. upper

Intercept -36.709 -34.9073 -33.1052

-36.709 -34.9073 -33.1052

-36.709 -34.9073 -33.1052

Distance (x 20m)

40m -6.955 -6.474 -5.992

80m -12.877 -12.336 -11.794

160m -27.953 -27.375 -26.798

320m -34.880 -34.183 -33.485

640m -39.183 -38.283 -37.384

Site (x Flat)

Low to High -4.691 -3.906 -3.121

Vegetation -10.985 -10.220 -9.454

High to Low -6.229 -5.476 -4.723

Speaker position (x Inclined)

Straight -0.878 -0.176 0.527

Site:Speaker.position (x Inclined:Flat)

Straight:Low to High -1.365 -0.265 0.835

Straight:Vegetation -2.953 -1.891 -0.830

Straight:High to Low -2.061 -1.241 -0.421

Time (x 05-06h)

06-07h -2.138 -1.451 -0.764

11-12h -3.560 -2.667 -1.774

17-18h -0.793 0.335 1.463

18:40-19:40h -0.680 -0.241 0.199

23-00h -2.933 -2.489 -2.045

Wind speed -1.396 -0.860 -0.324

Background Noise 0.532 0.592 0.652

RANDOM EFFECTS:

Between-group: Individual 0.018 0.227 2.884

Between-group: Roar-bark 0.407 0.634 0.988

Within-group standard error 4.695 4.802 4.913

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When compared to the main model, the secondary model had minimal

differences of estimated values for all factors except Time (Supplementary material

4). This mean days with overall different temperatures did not biased the site differences

result.

As expected, Distance had the larger effect on the model sound intensity (Table

2). Mean estimate difference between consecutive distances was -7.66. The values were

very close to the predicted for spherical spreading loss alone (-6dB), except for the

difference between 80m and 160m (Figure 3 and Table 3). That mean the factor

Distance on the model was able to reflect almost only the spherical loss, leaving the

excess of attenuation (i.e. other negative influences besides the spherical spreading) to

be explained by the remaining factors, for instance Site and Time.

Figure 3. Propagation of broadcasted roar-barks from captive maned wolves at Serra da Canastra

National Park, MG/Brazil. Re-recordings made with autonomous recorders (Song Meter SM2+; Wildlife

Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the re-recording at 1.25m.

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Table 3. Simultaneous tests for general linear hypotheses using Tukey contrasts for multiple comparisons

of means. The reported p values are adjusted by single-step method. Significance codes: 0 '***', 0.001

'**', 0.01 '*', 0.05 '.', 0.1 ' '. Only comparisons of consecutive distances are shown.

Hypothesis Estimate Std. Error z value Pr(>|z|)

D2(40m) - D1(20m) = 0 -6.474 0.246 -26.350 <2e-16 ***

D3(80m) - D2(40m) = 0 -5.862 0.253 -23.202 <2e-16 ***

D4(160m) - D3(80m) = 0 -15.040 0.240 -62.748 <2e-16 ***

D5(320m) - D4(160m) = 0 -6.808 0.318 -21.394 <2e-16 ***

D6(640m) - D5(320m) = 0 -4.100 0.471 -8.707 <2e-16 ***

Low to High - Flat = 0 -3.906 0.400 -9.760 <0.001 ***

Vegetation - Flat = 0 -10.220 0.390 -26.182 <0.001 ***

High to Low - Flat = 0 -5.476 0.384 -14.258 <0.001 ***

Vegetation - Low to High = 0 -6.314 0.419 -15.064 <0.001 ***

High to Low - Low to High = 0 -1.570 0.411 -3.824 <0.001 ***

High to Low - Vegetation = 0 4.743 0.414 11.450 <0.001 ***

Straight - Inclined = 0 -0.176 0.359 -0.490 0.624

06h - 05h = 0 -1.451 0.351 -4.139 <0.001 ***

11h - 05h = 0 -2.667 0.456 -5.854 <0.001 ***

17h - 05h = 0 0.335 0.576 0.582 0.991

18h - 05h = 0 -0.241 0.224 -1.074 0.878

23h - 05h = 0 -2.489 0.227 -10.991 <0.001 ***

11h - 06h = 0 -1.216 0.418 -2.910 0.036 *

17h - 06h = 0 1.786 0.495 3.612 0.004 **

18h - 06h = 0 1.211 0.351 3.451 0.006 **

23h - 06h = 0 -1.038 0.348 -2.986 0.029 *

17h - 11h = 0 3.002 0.430 6.986 <0.001 ***

18h - 11h = 0 2.426 0.444 5.459 <0.001 ***

23h - 11h = 0 0.177 0.446 0.398 0.998

18h - 17h = 0 -0.576 0.563 -1.022 0.898

23h - 17h = 0 -2.824 0.562 -5.029 <0.001 ***

23h - 18h = 0 -2.249 0.231 -9.727 <0.001 ***

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Regarding sites (Table 3 and Figure 4), Tukey contrasts revealed that sound

propagated more efficiently, i.e. lost less intensity, at the Flat site than all other sites.

Sound propagated more efficiently at the Low-to-high site than at the High-to-low site,

in accordance with our first predicted outcome that positive interference would be more

influential than negative ground effects and obstacles. In accordance with our

prediction, sound propagated less efficiently at the Vegetation site than all other sites.

The estimate difference when compared with the same place at the road (site Low to

High) was -6.31 dB. The negative effect of vegetation was less noticeable beyond 160

m (Figure 4).

Figure 4. Propagation of broadcasted captive records of maned wolves roar-barks at 4 sites at Serra da

Canastra National Park, MG/Brazil. Re-recordings made with autonomous recorders (Song Meter SM2+;

Wildlife Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the re-recording at

1.25m.

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Regarding the Speaker Position effect (Table 3), Tukey contrasts revealed that

the there was no difference in the sound broadcasted in the Inclined position compared

to the Straight position. However, different from the other factors tested, the Speaker

position caused an intensity difference already in the 1.25m reading (Figure 5 shows

the dB loss relative to the 1.25m Straight), with the Inclined position being on average

2.50 dB (± 2.22 dB) lower in intensity than the Straight position at this initial distance.

This difference is maintained at greater distances (-1.44 ± 3.36 dB x the Straight). As

our measure of intensity loss is relative to the 1.25m re-recording of each roar-bark, no

difference in the Speaker Position was detected. This indicates that once the initial

lower intensity of the Inclined position is taken into account, both positions propagate

similarly.

The general lower intensity of the Inclined broadcast contradicted out theory that

the head/muzzle elevation enhanced propagation. At the site Low to High, and specially

at the site Vegetation (Figure 5), the mean intensity difference between Inclined and

Straight positions was smaller, and at 160 m even positive (i.e. the Inclined position was

better than the Straight). This can also be seen on the model on the interaction between

site and position (Table 2). The interaction effect is negative for the position Straight on

the site Vegetation and larger than the other combinations. This suggests that on this site

the Straight speaker position had a much smaller effect than on other combination.

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Figure 5. Propagation of broadcasted captive records of maned wolves roar-barks at 4 sites at Serra da

Canastra National Park, MG/Brazil. We conducted broadcasts with the speaker box positioned straight

forward (Straight) and with the speaker box inclined 45o upward (Inclined) to simulate the inclination of

the head/muzzle seen when animals roar-bark. Re-recordings made with autonomous recorders (Song

Meter SM2+; Wildlife Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the

straight re-recording at 1.25m.

Finally, regarding the effect of the broadcasting time (Table 3 and Figure 6),

Tukey contrasts revealed that the propagation at 17h, 18h and 05h were more efficient

than at 11h, 23h and 06h, and indistinguishable among them. Those results were

partially in accordance with our prediction of the beginning of the night being the

period with the most efficient propagation of roar-barks, even though our results show

the end of the night as an equally efficient period to call. Propagation at 23h and 11h

were less efficient than all other times and not significantly different. An impaired

propagation at midday was expected, but not at midnight, which was not expected.

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The secondary model revealed fewer differences between broadcasting times

(Supplementary material 4), suggesting temperature and its inverse correlate humidity

are the main influence on propagation differences. Some differences remained, as

between the 05h and 06h broadcasts and 18h and 23h broadcasts, indicating other

characteristics of those periods are driving the differences, potentially the direction of

the air temperature gradient and wind masses.

Figure 6. Propagation of broadcasted captive records of maned wolves roar-barks at 6 time intervals at

Serra da Canastra National Park, MG/Brazil. The time shown is the beginning of a 1 hour interval in

which broadcasts were made. Re-recordings made with autonomous recorders (Song Meter SM2+;

Wildlife Acoustics, Inc., Concord, Massachusetts). The intensity loss is relative to the re-recording at

1.25m.

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Discussion

In this work we investigated how roar-barks, the long-range maned wolf call,

propagate through the species’ natural environment. Roar-barks broadcasted and re-

recorded from a higher to a lower altitude (down-slope) lost more intensity than when

propagated from a lower to a higher altitude (up-slope). At the site with more vegetation

sound attenuated more than at all other sites, which was in accordance with our

prediction. We found that inclining the speaker 45o upward to simulate the head/muzzle

position during vocalization had a negative effect on sound intensity instead of

enhancing transmission. However, this position did counteract partially the negative

effects of vegetation. Finally, we found that the maned wolf roar-bark calling time

choice is partially correlated with sound propagation properties of its habitat, since

transmission loss is lower in the beginning of the night. However, attenuation was also

minimal between 05 and 06 AM and these are times when the species rarely vocalizes.

Better sound propagation (measured as a smaller intensity loss) when

broadcasting from a lower to a higher position was consistent with our first predicted

scenario (and opposite to our second) which is likely due to positive interference from

sound waves reflected from the surface of the bare soil road (Bradbury & Vehrencamp

1998).

On the site with vegetation, although it was also a site where sound was

propagated from a lower to a higher position, any interference is less likely as the

uneven leaf surface would reflect sound in many different directions. This fact,

combined with the presence of more obstacles to attenuate the sound, resulted in a

worse sounds transmission at this site compared to all others. It is worth to note that the

effect of this site was the larger on the model (larger estimate except for distance) even

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considering that the grass and bushes were just below the speaker. Beyond 160 m the

vegetation tended to get lower, and the difference between this and other sites became

less evident.

Captivity studies describe that maned wolves usually emit roar-barks with the

nose pointed upward (Sábato 2011; Balieiro & Monticelli 2019; see Figure B in the

General Introduction). However, here we found that broadcasting those calls mimicking

this position with the speaker lead to lower registered intensities. At least for the Flat

site and where the broadcast was done from a higher to a lower place, this difference

was very consistent, appearing in the first recorder (1.25m) and maintaining itself

through distance. We can only assume in general situations there is a tradeoff between

signal intensity and other positive effects of this posture.

One possibility is the potential of this posture to counteract the negative effects

of the vegetation, as seen in this work. On the site with vegetation the upward inclined

speaker position could have reduced the amount of obstacles between the sound source

and the recorders, in some distances resulting in a smaller loss of intensity. It is worth

noting this site probably represents the normal vocalization scenario for maned wolves

on their natural environment. That would indicate a behavioral adaptation rather than an

acoustic signal adaptation for better propagation on the species’ natural habitat

(Acoustic Adaptation Hypothesis; Morton 1975).

Some other possible advantages of the nose/muzzle elevation could be that

emitting the sound upward makes it more omnidirectional, provides a larger body

impression through maximization of the vocal tract length, or that it enhances the

maintenance of signal integrity despite the intensity loss. Another related hypothesis is

that this posture can make it easier for the animal to produce the call, being a

physiological requirement.

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Regarding the time of the day, the times when propagation was substantially

impaired was between 11h and 12h, which was expected and reported elsewhere

(dingos’ long-range vocalizations; Déaux et al. 2016c), and between 23h and 00h,

which was unexpected. Different from the day, during the night the colder surface and

hotter air would refract the sound downward enhancing propagation (Embleton 1996). It

is possible, however, that the air at the Serra da Canastra National Park at that period

had already lost its heat and there was no temperature gradient.

Propagation of the broadcasted roar-barks was more efficient near dusk (17h to

19:40h) and before dawn (between 05h and 06h). The efficient sound propagation

before dawn is one of the most cited theories explaining the existence of the dawn

chorus (Brenowitz 1982; Brown & Handford 2003). Those studies are in general

conducted in temperate zones and sometimes this effect is not found in the tropics

where other factors may be prevalent (Berg et al. 2006). In this study we did find a

better sound propagation before dawn and this could mean the Serra da Canastra

National Park may behave more like a temperate than a tropical area in relation to sound

transmission.

Maned wolves are crepuscular, being more active during twilight (Melo et al.

2007; Jácomo et al. 2004). However, they rarely vocalize around dawn (Rocha et al.,

2016; Ferreira et al., unpublished; Chapter 3). That indicates that propagation properties

alone are not the main reason for the species calling period, or they would vocalize

equally on both twilights instead of concentrating the vocal activity in the beginning of

the night.

Again, there may be other acoustic factors not tested that could explain why

maned wolves prefer to vocalize in the beginning of the night, as signal integrity and

transmission consistency (Brown & Handford 2003). There may be also social factors

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involved. Rocha et al. (2016) proposes that the beginning of the activity would be the

most important period to acoustically announce/ defend territories, as vagrant

individuals would also be starting their activities and the probability of invading a

territory while searching for food would be highest.

Finally, there may be a weather influence. Wind speed has been previously

found to affect roar-bark detection and to increase throughout the night for the same

area between April and July (Rocha et al. 2016). Although the wind speed registered

during this experiment were very low and apparently had little influence on the model,

general year-round patterns may be influencing the evolution of the maned wolf time

choice for long distance calling.

We expected more intense backgrounds to impair the signal, but the background

noise level fluctuated with the signal intensity, having a small positive effect on the

model. This could mean our measure was mainly related to the equipment sensitivity. In

accordance, we did not include on the selection boxes wind noise or any noticeable

zoophony. As the recorders were not calibrated together, our measure probably acted on

the model as a control for equipment variance, which was a positive feature. It could

also act as a control for differences in the background noise levels between sites.

In conclusion, maned wolves have to deal with dynamic propagation scenarios

that will dramatically influence their vocalization active space, which explains why the

signal must be redundant. Their simultaneous listening might enable behavioral

adjustments to local propagation scenarios, i.e., moving their head. Finally, we can also

expect receiver maned wolves to have evolved characteristics and behaviors that

enhance signal reception, and further studies will clarify other aspects of this

communication system.

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WILEY, R.H., & RICHARDS, D.G. 1978. Physical constraints on acoustic

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Supplementary material 1 – Preliminary data exploration

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2 – Normality and homogeneity of residuals

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3 – Predicted x observed values of the model

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4 – Secondary model (including temperature):

Table S2. ANOVA test for the fixed factors of the secondary model for the intensity propagation of

maned wolves roar-barks broadcasted on their natural environment.

numDF denDF F-value p-value

Intercept 1 3735 41460.96 <.0001

Distance 5 3735 3347.82 <.0001

Site 3 3735 1148.00 <.0001

Speaker position 1 3735 85.66 <.0001

Site:Speaker.position 3 3735 5.63 0.0008

Time 5 3735 125.37 <.0001

Wind speed 1 3735 14.87 0.0001

Background Noise 1 3735 11095.45 <.0001

Temperature 1 3735 103.66 <.0001

Table S3. Secondary model: simultaneous tests for general linear hypotheses using Tukey contrasts for

multiple comparisons of means. The reported p values are adjusted by single-step method. Significance

codes: 0 '***', 0.001 '**', 0.01 '*', 0.05 '.', 0.1 ' '. Only com comparisons of consecutive distances are

shown.

Hypothesis Estimate Std. Error z value Pr(>|z|)

D2(40m) - D1(20m)= 0 -6.497 0.245 -26.480 <2e-16 ***

D3(80m) - D2(40m) = 0 -5.851 0.252 -23.193 <2e-16 ***

D4(160m) - D3(80m) = 0 -15.047 0.239 -62.879 <2e-16 ***

D5(320m) - D4(160m) = 0 -6.873 0.318 -21.600 <2e-16 ***

D6(640m) - D5(320m) = 0 -4.049 0.470 -8.608 <2e-16 ***

Low to High - Flat = 0 -5.888 0.679 -8.675 <0.001 ***

Vegetation - Flat = 0 -10.860 0.428 -25.368 <0.001 ***

High to Low - Flat = 0 -6.558 0.487 -13.479 <0.001 ***

Vegetation - Low to High = 0 -4.972 0.560 -8.884 <0.001 ***

High to Low - Low to High = 0 -0.670 0.480 -1.396 0.485

High to Low - Vegetation = 0 4.302 0.431 9.974 <0.001 ***

Straight - Inclined = 0 -0.176 0.358 -0.491 0.623

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06h - 05h = 0 -1.110 0.363 -3.062 0.019 *

11h - 05h = 0 1.134 1.146 0.989 0.8729

17h - 05h = 0 2.303 0.792 2.908 0.0301 *

18h - 05h = 0 1.081 0.429 2.521 0.0853

23h - 05h = 0 -1.517 0.352 -4.316 <0.001 ***

11h - 06h = 0 2.244 1.045 2.148 0.1974

17h - 06h = 0 3.413 0.668 5.107 <0.001 ***

18h - 06h = 0 2.191 0.443 4.945 <0.001 ***

23h - 06h = 0 -0.407 0.389 -1.047 0.8453

17h - 11h = 0 1.169 0.664 1.759 0.3962

18h - 11h = 0 -0.054 0.817 -0.066 1

23h - 11h = 0 -2.651 0.901 -2.944 0.027 *

18h - 17h = 0 -1.222 0.590 -2.072 0.2298

23h - 17h = 0 -3.820 0.625 -6.114 <0.001 ***

23h - 18h = 0 -2.598 0.250 -10.382 <0.001 ***

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Chapter 3

Temporal and spatial patterns of the long-range calls

of maned wolves

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Temporal and spatial patterns of the long-range calls of maned wolves

Luane Ferreira, Luciana Rocha, Danielly Duarte, Edvaldo Neto, Júlio Baumgarten, Flávio

Rodrigues, Renata Sousa-Lima

Intended for submission on: Biota neotropica

Abstract

Passive acoustic monitoring has a great potential for aiding conservation efforts

and elucidating the behavior and ecology of nocturnal/crepuscular secretive species, like

the maned wolf. Here we characterize the seasonal, lunar, and diel patterns in the long-

range vocalizations (roar-barks) of free ranging maned wolves at Serra da Canastra

National Park (Brazil) throughout eight months of recordings over two years with a grid

of 12/13 autonomous recorders. We found it is possible to identify the mating season

and probably the circa-parturition period through an increase in vocal activity of the

species. Those peaks in vocal activity indicate a role of roar-barks in partner attraction

and mate guarding, and also in intra-familiar-group communication. Additionally, vocal

activity happened throughout all recorded period and was much higher at some sites

than at others, corroborating that the species uses roar-barks to announce territorial

ownership and defense. Maned wolves vocalize more around the waxing gibbous lunar

phase, and after dusk until mid-night, following the seasonal variation in sunset time.

Moonlight likely reduces foraging time, resulting in more time available to invest in

acoustic signaling for communication, while vocalizations early on the onset of activity

suggest a territorial announcement function similar to bird dawn chorus. Group

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vocalizations follow the seasonal variation of the general vocal activity, but not always

the lunar or nightly vocal activity pattern. This suggests that social events may require:

response independent of illumination and hour, as territorial contests; and/or

simultaneous location of animals, as mate guarding and joint territorial defense. Based

on spatial patterns, we estimate between 6 and 11 individuals contributed to the

recordings, and found the vocal activity varies among sites and nights as results of a

spatial-temporal dynamic that still needs to be further explored.

Resumo

O monitoramento acústico passivo tem grande potencial para ajudar esforços

para conservação e elucidar o comportamento e ecologia de espécies evasivas noturno-

crepusculares, como o lobo-guará. Aqui nós caracterizamos os padrões sazonais, lunares

e nictemerais nas vocalizações de longo alcance (aulidos) de lobos-guará de vida livre

no Parque Nacional da Serra da Canastra (MG, Brasil) através de oito meses de

gravações ao logo de dois anos com uma rede de 12/13 gravadores autônomos. Nós

descobrimos que é possível identificar a estação de acasalamento, e possivelmente o

período em torno do parto, através de um aumento na atividade vocal. Esses picos de

atividade vocal sugerem que os aulidos têm um papel na atração e guarda de parceiros e

também na comunicação intra grupo familiar. Contudo, houve atividade vocal durante

todo o período amostral e muito mais em alguns locais do que em outros, corroborando

a hipótese de que a espécie usa aulidos para anúncio e defesa territorial. Lobos-guará

vocalizam mais na Lua crescente gibosa e depois do anoitecer até meia-noite, seguindo

a variação sazonal no pôr do Sol. A luz da Lua provavelmente reduz o tempo de

forrageio, resultando em mais tempo disponível para investir na sinalização acústica

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para comunicação, enquanto vocalizações logo no início da atividade sugerem uma

função de anúncio territorial. Vocalizações de grupo seguiram a variação sazonal da

atividade vocal geral, mas nem sempre o padrão lunar ou noturno de atividade vocal.

Essas vocalizações em grupo podem ocorrer devido a eventos sociais que requeiram:

resposta independente de iluminação ou hora, como disputas territoriais; e/ou

localização simultânea dos animais, como para guarda de parceiro e defesa territorial

conjunta. Baseado nos padrões espaciais, nós estimamos entre 6 e 11 animais

contribuíram para as gravações, e descobrimos que a atividade vocal varia entre os

locais e noites como resultado de uma dinâmica espaço-temporal que ainda precisa ser

explorada.

Key-words: maned wolf, Chrysocyon brachyurus, passive acoustic, vocalization,

seasonal, moon, diel.

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Introduction

The majority of mammals are nocturnal, crepuscular or cathemeral (Jones et al.

2009), with nocturnality assumed to be the ancestral condition (Gerkema at al. 2013).

Nocturnality favors communication modalities other than vision, such as chemical and

acoustic (Fox 1975). Most mammals are also solitary (Lukas & Clutton-Brock 2013),

which implies that an important part of their social interaction is mediated by long range

signals to maintain spacing among individuals (Kleiman 1972; Morton 1977).

For those reasons, passive acoustic monitoring of terrestrial mammals has great

potential in aiding conservation efforts and in elucidating their behavior and ecology

(Blumstein et al. 2011). In fact, many mammals are more easily heard than seen at

distance (Marques et al. 2013). Besides allowing monitoring of species hard to visually

follow, passive acoustic monitoring has the advantage of enabling behavioral sampling

over large temporal and spatial scales (Van Parijs et al. 2009; Blumstein et al. 2011).

This feature is crucial for investigating seasonal patterns in animal behavior. For

instance, some species can have the breeding season tracked by increased number of

vocalizations, as most cervids, e.g. the red deer (Clutton-Brock & Albon 1979; Bocci &

Laiolo 2013), and in birds, as the critically endangered Araripe Manakin (Girão &

Souto 2005).

The lunar cycle is also an important driver of the activity of nocturnal animals

(Kronfeld-Schor et al. 2013), and variations over this cycle can also be acoustically

investigated. For instance, coyotes emit more group vocalizations on new moon nights,

when territorial pressure is higher and/or pack coordination for hunting large prey is

required (Bender et al. 1996). As another example, eagle owls are more active and

vocalize more on bright moonlit nights and during twilight (Penteriani et al. 2009),

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although they also have less success in capturing prey during those periods (Penteriani

et al. 2011). A meta-analysis found that the majority of terrestrial mammals reduce their

activity around full moon nights, probably as a predator avoidance strategy and as an

energy conserving response to lower prey availability (Prugh & Golden 2014).

Information on the relationship between terrestrial mammals’ locomotion/physical

activity and vocal activity over the moon cycles is lacking (except for the echolocation

of bats, which sometimes is used as a proxy for activity per se; Hecker & Brigham

1999; Kronfeld-Schor et al. 2013).

Finally, the diel vocalization pattern is also frequently investigated and can

reveal times better suited for acoustic communication. One of the most prominent

examples of such times are the dawn and dusk choruses, the latter being especially

relevant for crepuscular avian species. There are over 12 non-exclusive hypotheses for

this phenomenon (Staicer et al. 1996), including that this is a period of better acoustic

propagation (Brown & Handford 2003). Another explanation states that those would be

moments when individual quality would be honestly advertised due to the energetic

constraint of having passed through a period without food (Zahavi 1975; Montgomerie

1985; Cuthill & Macdonald 1990). Accordingly, nocturnal birds vocalize more during

the dusk chorus than in the dawn chorus (Hardouin et al. 2008). Yet another explanation

points that dawn and dusk are not ideal for foraging but are good to communicate while

avoiding predators (Berg et al. 2006). For nocturnal animals those are the more

illuminated periods of their activity, improving visual acuity (Prugh & Golden 2014).

This is the case of the afore mentioned eagle owl, which couples visual and vocal

displays on dim light (Penteriani et al. 2009).

The South American maned wolf (Chrysocyon brachyurus, Illiger 1815) is a

promising species for acoustic monitoring. They are large (70-90 cm, 20-30 kg; Silveira

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1999), but very shy and cryptic, crepuscular/nocturnal canids (Mello et al. 2007).

Monogamous breeding pairs share and defend the same extensive home range (15-115

km2; Rodrigues 2002; Azevedo 2008), yet rarely meet, living largely solitary lives

(Dietz 1984). They forage for small vertebrates and fruits (Rodden et al. 2004), and thus

conspecific presence may interfere (Jácomo et al. 2009). All of which makes them hard

to observe and visually follow in the wild (personal [non] observation), even when

tagged with GPS collars (Emmons, 2012). Hence, many aspects of their behavior are

poorly understood (Rodden et al. 2004). To our benefit, however, they communicate

throughout the year with a long-range explosive call (the roar-bark, Kleiman 1972;

same as “extended bark”, Balieiro & Monticelli 2019). Roar-barks are emitted in

sequences (bouts) of 5-15 roar-barks separated by 2-4 seconds (see spectrograms in next

session) and are proposed to function as: territorial announcement, especially for intra-

sexual spacing; partner attraction and guarding; and intra-group (partner and offspring)

communication (Kleiman 1972; Brady 1981; Dietz 1984; Bestelmeyer 2000; Sábato

2011; Emmons 2012; Rocha et al. 2016; Balieiro & Monticelli 2019). Animals from the

same group or from adjacent territories can exchange vocalizations, creating a group

sequence of alternating roar-barks (Dietz 1984; Emmons 2012).

Our goal was to characterize the maned wolf seasonal, lunar, and diel long-range

acoustic communication pattern at a protected Brazilian area with passive audio

recordings of eight months over two years. Our first specific objective was to test if the

vocalization increase registered in captivity during the mating season (Sábato 2011)

could be identified on natural recordings. Our second specific objective was to confirm

the lunar and diel patterns suggested by our previous small sample record (32 nights)

that suggested that maned wolves vocalize more on the waxing gibbous phase and

between 18h and 19h (Rocha et al. 2016). Our third specific objective was to test if

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group vocalizations followed the seasonal, lunar, and diel patterns of solo vocalizations

or if they varied independently as in coyotes (Bender et al. 1996). Finally, we wanted to

detect spatio-temporal patterns that could contribute evidence of roar-bark functions,

and to help in estimating the number of animals recorded.

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Material and Methods

1. Study área

The study was conducted at Serra da Canastra National Park, Minas Gerais state,

Brazil (Figure 1). The park is mainly composed of Cerrado open savannas with a cold,

dry season (April-September) and a hot, rainy season (October-March; Queirolo &

Motta-Junior 2007). Maned wolf density at the park is considered high (0.08

individuals/km2; Paula et al. 2013).

Figure 1. Study region at the Serra da Canastra National Park, MG/Brazil. Yellow squares indicate

autonomous recorder (SongMeter SM2+) sites used only in 2014, pink triangles sites used only in 2016,

and white circles sites used in both years.

2. Recordings

Recordings were made with autonomous recorders (SongMeter SM2+; Wildlife

Acoustics, Inc., Concord, Massachusetts) coupled with a single SMX-II weatherproof

microphone each (Wildlife Acoustics, Inc.). Autonomous recorders were programed to

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record continuously for 12 hours each night, partitioning samples in 30 minutes files,

with an +36dB gain, 8 kHz sample rate, and 16-bit wav coding. Recorders were

attached to 1.4m high wooden stakes and distributed in areas where tracks, scats, and

reported observations of maned wolves had been made. We aimed to sample high

elevation sites (1.373±56.65 m; all measures are reported in mean±SD, unless noted

otherwise) and the broadest distribution possible, but accessibility was a major

constraint.

In 2014 we deployed 12 autonomous recorders, with the linear distance between

them of 2.27 km (±0.72 km). Recorders remained active between April 05 to August 08,

recording from 18h to 06h. In 2016, we deployed 13 autonomous recorders (8 were the

same from 2014), with the linear distance between them of 3.03 km (±0.78 km).

Recorders remained active between March 09 to July 01, recording from 17h to 05h.

Due to a technical problem the equipment failed to record between March 29 and April

03 2016.

3. Audio processing and measures

The most recent vocal repertoire of the maned wolf (Sábato 2011) describes 10

different vocalization types (only for adult wolves, see Brady 1981 for vocalizations

during development). From those, the long-range types described are the roar-bark and

the similar, but slightly shorter and higher in frequency, single bark. During our pilot

study we manually searched for any maned wolf vocalization and the only type found

was the roar-bark. Therefore, the present study was focused in this vocalization type.

Roar-barks were detected automatically using XBAT (Extensible Bioacoustic

Tool; Figueroa 2007) extension for Matlab (R2010a version; MathWorks, Inc., Natick,

MA, USA) following the methodology detailed by Rocha et al. (2015). In summary,

spectrograms are scanned with a mobile cross correlation using 4 roar-barks templates.

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Matches above a threshold (0.21) are then manually verified for false positives and

undetected roar-barks within 24 seconds of the detected ones (Figure 2a). We used 4

different templates, including different frequency portions, and a very low threshold to

guarantee that even very faint, partially masked, or uncommon frequency shaped roar-

barks were detected, at the cost of increasing the number of false positives. In our test

data, this methodology resulted in 100% of roar-bark sequences being detected in half

the processing time. This method yielded even more detections than found by manually

scanning spectrograms (93%; Rocha et al. 2015).

We considered a single sequence when roar-barks were not separated by more

than 10 seconds (based in Bender et al. 1996, and preliminary observation of the data).

Sequences with a single roar-bark are possible. For each roar-bark sequence we noted

the recording site, date, absolute start time (17h to 06h), start time in relation to sunset

(± 0-12h), number of roar-barks, and the number of animals vocalizing at the same

moment (solo x group sequences, detailed below).

Sunset times used to calculate the start time in relation to sunset were extracted

from https://www.sunearthtools.com/pt/solar/sunrise-sunset-calendar.php, which allows

for GPS location specification.

The number of vocalizing animals was detected inspecting Raven’s pro 1.5

spectrograms (Bioacoustics Research Program, 2014. Ithaca, NY: The Cornell Lab of

Ornithology. http://www.birds.cornell.edu/raven. Configurations: grayscale, 50%

brightness and contrast, 50% overlap, 512 points Hann window, smoothing active;

Figure 2). The presence of a second, and very rarely a third, animal could be verified by

differences in the spectral shape of roar-barks, cadence, intensity, and eventual overlaps.

When two or more animals intercalated roar-barks on the same sequence we termed it a

group vocalization (Figure 2b). When only one animal could be detected on the

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sequence, we termed it a solo vocalization (Figure 2a). General vocal activity refers to

both number of sequences and roar-barks, with no separation between solo and group

vocalizations.

In some cases, the same sequence was recorded on more than one Song Meter.

This was verified by temporal proximity and comparison of inter roar-bark intervals,

guarantying that it was in fact the same sequence in two different sensors and not two

animals vocalizing at the same moment. Only the most intense sequence (measured with

Raven’s peak power function) was counted for the analysis. The autonomous recorders’

clocks were not exactly synchronized, therefore triangulation of the emitter position

based on the time difference in the signal arrival at different sensors was not possible.

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The total area recorded by Song Meters – in the sense of roar-bark detectability

– was estimated in two ways: first a ‘lower estimate’ by using half the mean distance

between recorders as the radius for each recorder (we used 2014 mean recorders’

distance for both year so values would be comparable); and second a ‘maximum

estimate’ by using half the distance between the most distant recorders that ever

registered the same sequence as the radius for each recorder (overlapping areas were

discounted).

We mapped the vocal activity distribution over the months according to recorder

sites, identifying regions with concentrated activity. This was done to verify if space

and roar-bark use were associated, which would indicate a territorial or resource defense

function for this vocalization. We also wanted to see if the vocal activity during the

mating season occurred at the same sites as during other periods.

We also used the spatial information to estimate the number of animals recorded.

We calculated the radius (5.36 km) of the mean home range (90.29 km2) from a recent

study in the area (Paula 2016) and looked for roar-bark sequences on sites farther away

than this value within 1-3 consecutive nights. Those sequences were considered having

been emitted by different animals, or group of animals (in the cases of group

vocalizations). We estimated the number of animals in two other ways: by dividing the

mean number of roar-bark sequences per night by the mean number of sequences

emitted per individual per night in captivity (Sábato 2011; 0.68 proestrous, 0.28

anestrous); and by multiplying our estimate of total area recorded by the maned wolf

density in the studied park (0.08/km2; Paula et al. 2013).

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4. Statistical analyses:

The monthly vocal activity was compared with ANOVAs followed by Tukey

contrasts on R software (R version 3.5.1 [2018-07-02], The R Foundation for Statistical

Computing). For the circular data, moon phases and hours of the day, we used

Rayleigh’s test on Oriana software (version 4; Kovach Computing Services, Anglesey,

Wales; https://www.kovcomp.co.uk/oriana/). In an effort to eliminate the seasonal

variation as a confounding effect, we standardized the vocal activity by each lunar

month and then compared moon phases with ANOVAs.

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Results

1. Roar-bark calling general characterization

We detected a total of 13180 roar-barks distributed in 1210 sequences over the

233 nights of recording (2014 and 2016). There were fewer sequences and roar-barks in

2014 than in 2016 (Table 1 shows only the vocal activity of entire months). Few

sequences were emitted by more than one animal (12%), and there were more group

vocalizations in 2014 than 2016 (Table 1). On five occasions the group vocalization

involved three animals (3.3% of group vocalizations). All other group vocalizations

involved only two animals (96.7%, 145 sequences).

Overall the mean number of roar-barks per sequence was 10.89 (± 8.07 SD). The

sequences were shorter in 2014 than in 2016 (Table 1 and Figure 3). Sequences with a

single roar-bark in general preceded or followed other sequences (60% within 1

minute). The longest sequence (highest number of roar-barks) of 2014 involved two

animals and had 91 roar-barks, followed 10 seconds later by a 56 roar-bark sequence

and right after that 6 more sequences summing 31 roar-barks. In 2016 the longest

sequence had 50 roar-barks uttered by a single animal.

Table 1. Summary of maned wolf’s vocal activity recorded passively with a grid of 12/13 autonomous

recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. Year Total March April May June July

Sequences of roar-barks 2014 478 - 193 86 118 81

2016 708 229 184 117 178 -

Number of roar-barks 2014 4748 - 1950 819 1253 726

2016 8177 2655 2246 1306 1970 -

Group vocalizations 2014 79 - 26 16 21 16

2016 65 25 14 11 15 -

% of group vocalizations 2014 16.5 - 13.5 18.6 17.8 19.8

2016 9.2 10.9 7.6 9.4 8.4 -

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Figure 3. Histogram of the number of roar-barks on each sequence of maned wolves’ vocalizations

recorded passively with a grid of 12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra

National Park, MG/Brazil. One sequence was defined by one or a bout of roar-barks not separated by

more than 10 seconds.

In 2014, 23.1% of roar-bark sequences were detected by more than one

autonomous recorder, with 8% of those being detected by three or more (up to five)

recorders. In 2016 we obtained the same percentage of sequences detected by more than

one recorder (23.9%), but only 3.2% of those were detected by three recorders and none

in four or more.

In 32 cases, group sequences were registered in more than two recorders. In all

but two of those events, the time difference between the same vocalization of each

individual differed by 0.5-8.8 seconds between sensors. Therefore, the relative position

of each emitter suggest animals were between 86 m and 1517 m away from each other

(considering a sound speed of 343 m/s). Those were the only situations in which we

could estimate distances (and not from the recorder).

2. Seasonal variation

The seasonal fluctuation in maned wolf vocal activity was very similar in both

years (Figure 4). During March and April, the vocal activity was at its maximum,

dropping in mid-April and remaining low until the beginning of June. There was a

second smaller increase in vocal activity in June, dropping to the previous levels again

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at the end of the month. There was no visible seasonal pattern on the number of roar-

barks by sequence (the sequence size), neither on the percentage of group vocalizations.

The number of group vocalization in general followed the vocal activity, except for a

marked peak in the middle of June on 2014 (Figure 4, bottom right).

Figure 4. Seasonal variation in the maned wolf vocal activity recorded passively with a grid of 12/13

autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. Each point is

a sum of 5 nights. Photos: Endangered Wolf Center, St.Louis, and Adriano Gambarini.

In 2014 there was a significant monthly variation on the mean number of

sequences by night (F=4.4978, df=3, p=0.005; Table 2). There were more sequences by

night in April than in other months (p<0.05), except for June (p=0.0954). The result was

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the same for the monthly mean number of roar-barks by night (F=7.5311, df=3,

p=0.0001), except that there were also more roar-barks by night in April than June

(p=0.0317). No significant differences were found for the size of the sequences

(F=0.9681, df=3, p=0.4115), number of group vocalizations (F=1.0522, p=0.3724), and

percentage of group vocalizations (F=0.2698, df=3, p=0.847) by night.

In 2016 there were also a significant monthly variation on the mean number of

sequences by night (F=5.4306, df=3, p=0.0016; Table 2). There were more sequences

by night in March than in other months (p<0.05), except for April (p=0.0953). The

results were the same for the monthly number of roar-barks (F=4.9486, df=3, p=0.003).

There was a marginal trend in the monthly number of group vocalizations by night

(F=2.6757, df=3, p=0.051), with March mean higher than May’s (p=0.0461). No

significant difference was found in the size of the sequences (F=0.8459, df=3,

p=0.4725), and percentage of group vocalizations (F=0.2068, df=3, p=0.8915) by night.

Table 2. Maned wolf vocal activity recorded passively with a grid of 12/13 autonomous recorders (Song

Meter SM2+) at Serra da Canastra National Park, MG/Brazil. Values reported are mean by night ± SD. Year Total March April May June July

Sequences of

roar-barks

2014 4.0±5.7 - 7.4±8.7 2.8±3.9 4.0±4.0 2.6±4.6

2016 6.6±7.1 11.5±8.6 6.8±8.9 3.8±4.0 5.9±5.0 -

Number of

roar-barks

2014 39.9±49.0 - 75.0±71.3 26.1±29.5 41.3±39.6 23.6±34.0

2016 73.8±83.4 127.6±100.5 80.0±111.2 42.0±39.1 65.0±56.5 -

Roar-barks /

sequences

2014 10.8±5.7 - 12.7±5.6 10.1±5.4 10.9±4.4 10.1±7.1

2016 11.9±4.7 11.5±3.6 12.0±4.8 13.0±5.9 10.9±3.9 -

Group

vocalizations

2014 0.7±1.3 - 1.0±1.3 0.5±0.8 0.7±1.6 0.5±1.0

2016 0.6±1.2 1.3±1.9 0.5±1.1 0.4±0.8 0.5±0.9 -

% of group

vocalizations

2014 18.5±26.8 - 15.0±18.4 20.9±27.3 17.5±27.0 21.1±3.7

2016 9.4±20.5 8.9±13.1 7.2±15.1 11.9±28.1 9.5±21.6 -

3. Lunar pattern

Overall (2014 and 2016) vocal activity was concentrated on the waxing gibbous

phase (Figure 5). This was true for the number of sequences (122.9±98.4° [mean angle

± Circular Standard Deviation]; Z=63.125, p<0.0001, N=1206), for total number of

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roar-barks (124.7±99.3°; Z=623.101, p<0.0001, N=12556), and also for the number of

group vocalizations (114.2°±111.8°; Z=3.325, p=0.036, N=150).

In 2014 the roar-bark sequences were significantly concentrated in the waxing

gibbous phase (Figure 5; Z=31.225, p<0.0001, N=501), with a mean angle of 158.9° (±

95.5°). The total number of roar-barks followed the same pattern (152.6±101.1°

[waxing gibbous]; Z=221.87, p<0.0001, N=4989). The number of group vocalization

was not significantly concentrated in any moon phase (Z=2.336, p=0.097, N=87).

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Figure 5. Maned wolf roar-bark sequences distribution over the lunar phases (gray = total). Records were

made from April to July on 2014 (blue) and from March to June on 2016 (red) with a grid of 12/13

autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil. The radial line

represents the mean angle and the concentric bar at the end of the line the 95% confidence interval.

In 2016 there were significantly more roar-bark sequences during the waxing

crescent and waxing gibbous phases (Figure 5), with a mean angle of 95.3° (±90.9° [1st

quarter]; Z=57.097, p<0.0001, N=707). The total number of roar-barks followed a

similar pattern, with a slightly greater angle (mean 104.6±93.4° [waxing gibbous];

Z=530.156, p<0.0001, N=7578). The number of group vocalizations was concentrated

during the waxing crescent phase (mean 74.4±92.6°; Z=530.156, p=0.01, N=63).

Higher vocal activity during March and April could have biased the moon

concentration results, therefore we tested each moon cycle separately (Table 3). Of

eight cycles recorded on both years, for three the number of roar-bark sequences was

concentrated during the waxing gibbous phase, two during the waxing crescent, one

during the waning gibbous, and two were not concentrated on any moon phase. Results

were similar for the number of roar-barks, and only in March and April 2016 the

number of group vocalizations was concentrated on the waxing gibbous and waxing

crescent phases, respectively (Table 3).

After standardizing vocal activity by each lunar month, the mean values seemed

to decrease from the waxing crescent (e.g. number of roar-barks: 0.732) to the new

moon phase (e.g. number of roar-barks: -0.512). However, the difference was not

significant for any variable (ANOVAs; number of sequences: p=0.3714; number of

roar-barks: p=0.0809; group vocalizations: p=0.4055).

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Table 3. Concentration of maned wolf vocal activity on each of eight moon cycles recorded passively

with a grid of 12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park,

MG/Brazil. When the concentration is significant, the mean moon phase, mean angle ± circular standard

deviation, and Rayleigh test statistics are reported.

Lunar cycle Sequences Roar-barks Group vocalizations

2014 April

4/5 – 5/4

Waxing gibbous

147.3±74.6°

Z=36.72, df=199, p<0.0001

Waxing gibbous

145.9±77.7°

Z=321.103, df=2019, p<0.0001

-

2014 May

(5/5 – 6/2) - - -

2014 June

(6/3 – 7/2)

Waning gibbous

193.0±108.3°

Z=5.564, df=122, p=0.004

Full

178.9±110.8°

Z=30.225, df=1269, p<0.0001

-

2014 July

(7/3 – 7/31) -

Waning crescent

313.7±94.0°

Z=45.021, df=665, p<0.0001

-

2016 March

(3/9 – 4/7)

Waxing gibbous

116.3±77.7°

Z=41.109, df=258, p<0.0001

Waxing gibbous

125.9±76.1°

Z=499.409, df=2915, p<0.0001

Waxing gibbous

106.9±47.8°

Z=12.958, df=25, p<0.0001

2016 April

(4/8 – 5/6)

Waxing crescent

44.3±77.5°

Z=30.472, df=189, p<0.0001

Waxing crescent

43.8±76.9°

Z=298.245, df=1809, p<0.0001

Waxing crescent

26.6±62.8°

Z=4.811, df=15, p=0.006

2016 May

(5/7 – 6/5)

Waxing crescent

43.1±102.4°

Z=3.978, df=96, p=0.019

Waxing crescent

33.6±108.6°

Z=30.738, df=1116, p<0.0001

-

2016 June

(6/6 – 6/30)

Waxing gibbous

135.3±81.6°

Z=21.146, df=160, p<0.0001

Waxing gibbous

144.1±83.7°

Z=205.843, df=1734, p<0.0001

-

4. Nightly pattern

Vocal activity during the first two recording hours (17-19h) revealed a seasonal

variation correlated with sunset (Figure 6). During March and April, when sunset was

after or close to 18h, the vocal activity was lower between 17-18h and higher between

18-19h. The inverse pattern is seen when sunset was before 18h, in May and June. In

July the sunset starts to get later again and the vocal activity between 18-19h starts to

increase again. Because of this variation we decided to report the nightly vocalization

pattern as time relative to sunset (Figure 7).

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Figure 6. Maned Wolf roar-barks registered between 17-19h on passive audio recordings made with a

grid of 12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil.

In 2014 there was no recordings in March, and in 2016 no recordings in July.

In both years the vocal activity was concentrated in the first half of the night

(Figure 7). In 2014 the first hour after sunset alone comprised 18% of roar-barks, and

the first 3 hours 44%. In 2016 there was a moderate vocal activity already during the

hour preceding sunset, and this level doubled during the hour after. Although there was

a tendency for vocal activity to decrease throughout the night, on 2016 it continued

relatively high until 5-6h after sunset.

Group vocalizations did not always follow vocal activity (number of sequences

and roar-barks). In 2014 there was an increase in group vocalizations after 7 hours after

sunset despite the overall low vocal activity on this period. That resulted in a higher

percentage of group vocalization between 7 and 12 hours after sunset in 2014 (Figure

7). In 2016, while the vocal activity was high between 2 and 4 hours after sunset, the

number of group vocalizations decreased markedly during this period (Figure 8). On 3

occasions a peak in the vocal activity was followed by a peak in group vocalizations in

the next hour (Figure 7): in 2014 6-7 hours (sequences) and 7-8 hours (group

vocalizations), in 2016 0-1 hours and 1-2 hours, and on 2016 3-4 hours and 4-5 hours.

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17-18h 2016

18-19h 2016

18-19h 2014

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Figure 7. Maned wolf nightly vocal activity relative to sunset. Recordings were made with a grid of

12/13 autonomous recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil.

The mean hour of roar bark sequence start for the two combined years was 22:02

(±03:16 [mean hour ± Circular Standard Deviation]). In 2014 the mean hour of

sequence emission was 22:14 (±03:27; Z=220.97, p<0.0001, N=503), and in 2016 21:51

(±03:05; Z=317.911, p<0.0001, N=614). Some caution with the Rayleigh test is needed

in this case, as the recording period of the two years differed in one hour and on both

years half of the 24h cycle was not sampled (only night records). Despite this bias, the

result showing a concentration of vocal activity in the first half of the night is robust.

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5. Spatial patterns and number of recorded animals

The mean distance between Song Meters in 2014 was 2.27 km, giving a 1.14 km

radius for the total area recorded ‘low estimate’. The most distant recorders to register

the same sequence were 4.86 km away (K and N), giving a radius for the total area

recorded ‘maximun estimate’ of 2.43 km. That resulted in a estimated area covered of

48.97 to 139.90 km2 (low to maximun) for 2014 and of 53.05 to 195.25 km2 for 2016.

The spatial distribution of vocal activity revealed 4 main regions with

concetrated activity in 2014: C/D sites, H/J sites, K/L/M sites, and O/P sites (Figure 8

and supplementary Figure S1). There was high vocal activity at the sites between C/D

and H/I, making it unclear if this larger area was the home range of two animals that

moved around, three animals with one moving between the two areas, or four (or

more?) different animals. There were many group vocalizations in all those regions

(Figure 8).

In April 2014 there was high and widespread vocal activity. In May the vocal

activity reduced and becomes more concetrated at the eastern portion of the park. In

June 2014 the vocal activity increased again and was concentrated at 3 points (C, H, and

O). Finally, on July 2014 the vocal activity was low and concentrated on the western

and eastern sides.

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Figure 8. Maned wolf roar-bark sequences recorded passively with 12 autonomous recorders (Song

Meter SM2+) at Serra da Canastra National Park, MG/Brazil (gray contour). Circles have approximately

0.5 km radius with the center point being the recorder site. Heat colors represent the intensity of vocal

activity (number of sequences). Letters indicates the site name and numbers following them on the circles

indicates the amount of group vocalizations. * Indicates at least one sequence involved 3 animals

(otherwise group vocalizations involve 2 animals).

Based on the vocal activity of distant (> 5.36 km) sites in consecutive 1-3 nights,

we estimate tha between 6 and 9 different animals were recorded during 2014 (example

in Figue S1 top). Based on Sábato (2011) captivity report of mean sequences by animal

by night (0.68 proestrous, 0.28 anestrous) and our mean sequences by night (Table 1

and 2), our estimative would be 10.88 (April) and 11.17 (May-July) recorded animals in

2014. Based on our estimmates of recorded area and the reported maned wolf density in

this park (0.08/km2; Paula et al. 2013) the number of animals would range from 3.92 to

11.19 individuals (low and maximum area estimate).

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The spatial distribution of vocal activity revealed 5 not well separated regions of

concentrated activity in 2016: A/B sites, C/D/F sites, H/I sites, J site, and M/N sites

(Figure 9 and supplementary Figure S1b). Compared to 2014, the K/L/M focus seem

to have shifted south and the O/P focus seem to have become much less active (only

recorder P is present on 2016). It was not clear if the activity on sites J and G were from

animals of near by sites (I,K and E,H), or from different animals. Although this year had

fewer group vocalizations than 2014, there were still group vocalizations on most

regions (Figure 9).

Figure 9. Maned wolf roar-bark sequences recorded passively with 13 autonomous recorders (Song

Meter SM2+) at Serra da Canastra National Park, MG/Brazil (gray contour). Circles have approximately

0.5 km radius with the center point being the recorder site. Heat colors represent the intensity of vocal

activity (number of sequences). Letters indicates the site name and numbers following them indicates the

amount of group vocalizations (sequences involving 2 animals).

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In 2016 the vocal activity on sites C, F, H, and J was high during all recorded

months. This included a single night (from 17:06 to 00:24) in April with 41 sequences,

totaling 510 roar-barks, involving at least two animals. From the vocal activity on this

night, it seems there were an individual near site C and one or two near site J. After

some hours of the J animal/s vocalizing the animal from C comes between H and J and

they utter group vocalizations (only after 23:45).

Based on the vocal activity of distant sites in consecutive 1-3 nights, we estimate

that between 7 and 11 different animals were recorded during 2016 (example in Figue

S1 bottom). Based on the captivity report of mean sequences by animal by night and our

mean sequences by night (Table 1 and 2), our estimative would be 13.20 (March-April)

and 17.28 (May-June) recorded animals in 2016. Based on our estimmates of recorded

area and the reported maned wolf density in this park the number of animals would

range from 4.24 to 15.62 individuals (low and maximum area estimate).

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Discussion

We passively recorded spontaneous long-distance calls (roar-barks) of maned

wolves during eight months over two years in a protected park with the aim of

elucidating the maned wolf’s long-range acoustic behavior. We found that there is an

increase in vocal activity during March and April, indicating that it is possible to

identify the species mating period in the wild by the higher number of roar-barks. We

also found a second smaller increase in June, coinciding with the peak period of

parturition, which suggest that this reproduction period can also be monitored

acoustically. We found that maned wolves vocalize more on more moon illuminated

nights, especially during the waxing gibbous lunar phase. They concentrate their

vocalizations from dusk to mid-night, adjusting to the seasonal variation in sunset time.

Group vocalizations (detected vocal activity coming from 2 or more animals) did follow

the seasonal variation of the general vocal activity, but not always the lunar or nightly

vocal activity pattern, suggesting that environmental cues are less important than social

interactions. Vocal activity was much higher at some recording sites than at others over

all months, corroborating that the species uses roar-barks as territory announcement and

defense besides reproductive related purposes. Based on the vocal activity and group

vocalizations on consecutive nights, we that estimate between 6 and 11 different

animals contributed to recordings.

The peak in vocal activity in March and April coincides with the reported mating

season for the species (Carvalho & Vasconcellos 1995; Rodden et al. 2004). Female

maned wolves are monoestral and stay fertile for only 5 days (Rodden et al. 2004). In

this scenario, vocalizations could play an important role for normally solitary wolves to

quickly locate their potential mate or partner on an extensive home range. Reports from

captivity describe the frequency of vocalizations increase weeks prior to estrous up to

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the end of the mating season (Brady 1981; Dietz 1984; Sabato 2011). A report from the

wild describes that pairs stay together during one or two nights, copulating often, and

foraging close to each other (Rodden et al. 2004). This report showed that animals

emitted roar-barks whenever the partner was out of sight (Rodden et al. 2004). Our

findings are in accordance with those reports and reinforce the role of roar-barks in

partner attraction and possibly mate guarding.

We also found an increase in vocal activity in June, approximately two months

after the mating period, which coincides with the species’ gestation period (65 days;

Carvalho & Vasconcellos 1995), and the reported peak in births both in captivity (Maia

& Gouveia 2002; Rodden et al. 2004) and for the Serra da Canastra park (Dietz 1984;

de Mello et al. 2007, 2009). We have confirmation that at least one female on the area

was lactating in July 2014 and 2016 (R. C. de Paula, personal communication; Annex

I). The increase in vocalizations was smaller and, although clear in the 5-night sum of

both years (Figure 4), was not detected on the statistics of monthly rate of vocalization

by night (Table 2). Probably the time scale used, an entire month, was too long for the

event to capture these sudden and more isolated increases in calling behavior.

Mating has been reported up to June (Dietz 1984), which raises the possibility

that the second peak observed in June was related to other pairs (different than the ones

of the first peak) vocalizing near estrous, instead of related to vocal activity around

parturition as proposed above. However, this second vocal activity peak happened at

similar sites from those during the first vocal peak in March and April, which suggests

it involves the same individuals, unless the first breeding pair has lost its territory (e.g.,

due to reproductive failure). Maned wolves enter estrous only once per year, pairs do

not mate twice in the same year. Thus, we argue the second increase in vocal activity is

likely related to the birth of pups.

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GPS tracking results indicate that males reduce their activity and stay closer to

females on the days around parturition (de Mello et al 2007; Emmons 2012). The male

may rest all daylight hours with the female and pup, and at night visit the den regularly

(de Mello et al. 2007, 2009). Extensively in captivity and occasionally in the wild,

males maned wolves have been observed to guard, defend, regurgitate, and carry food to

the mother and pups, and also groom, play with, and accompany their young (Dietz

1984; Carvalho & Vasconcellos 1995; Bestelmeyer 2000; Rodrigues 2002; Jácomo et

al. 2009).

Emmons (2012) reports maned wolves vocalize more when pups are present and

suggest an intra group (pair and offspring) communication function. However, for the

reported cases pups/juveniles were older (3-15 months) and had already left the den

(Emmons 2012). Only Dietz (1984) reports roar-barks around parturition, which

suggests a more prominent role of this vocalization in intra-pair communication. The

use of roar-barks for intra-pair vocal communication is poorly discussed, but we

speculate that around parturition while pups are still dependent of parental care acoustic

communication may mediate family coordination, e.g. signal the location of females

and their den to males, since females are known to shift pups’ location often (Dietz

1984; Bestelmeyer 2000), or mediate negotiation/manipulation of parental care

(Wachtmeister 2001). Another possible cause would be an increased urge for

announcing and defending the territory, as other wolves, pumas, and feral dogs could be

a threat to the pups (Dietz 1984; de Mello et al 2007).

While maned wolves have been shown to decrease movement around full moon

nights (Sábato et al. 2009), here we found they vocalize more on bright nights. This

highlights the importance of coupling the investigation of activity levels and

vocalization levels, as they may not fluctuate together. The most accepted explanation

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of decreased activity for mammal predators is a strategy to minimize energy loss due to

reduced prey availability (Prugh & Golden 2014). Alternatively, there may be an

increase in foraging efficiency if: the increase in the ability of maned wolves to visually

detect preys surpass both the low prey availability and the increase in the ability of

preys to detect the maned wolves (Prugh & Golden 2014); and/or they have a gain in

fruit detectability. It should be noted that periodicity of spatial movement was not

correlated with the lunar cycle on maned wolves of the same region (Péron et al. 2017),

indicating they do not change habits. In either case, i.e. energy saving or increased

foraging efficiency, would result in less time foraging and therefore more time to

announce territory and interact with conspecifics, increasing vocalization levels.

Other explanation for the lunar pattern observed is that roar-barks reveal the

emitter position to prey and other predators. This could be particularly detrimental in

darker nights compared to brighter nights, when the emitter is already more easily

detected independent of sound and can itself detect threats more easily (as poorwills:

Woods & Brigham 2008). The problem with this line of thought is that maned wolves

vocalize too rarely to seriously impair hunting or expose themselves to risks (mean 0.68

sequences/individual/night: Sábato 2011; 4-7 sequences/night/12-13 recorders: Table

2). Yet another hypothesis would be that coupled visual and vocal communication is

important for the species, and they would take advantage of better iluminated periods to

increase those displays (as eagle owls: Penteriani et al. 2011). However, maned wolves

show only a small concentration of vocalizations on the minutes of dusk twilight

(30.6% x 18.3% chance on the ±1h sunset; data not shown), and not at all on the dawn

twilight minutes (8.9% x 18.3% chance -1h sunrise; data not shown). Besides, in 2014,

vocalizations during the moonlit versus dark portion of the night were not different than

expected by chance (Duarte et al. unpublished; Appendix I). Finally, the wolves’ typical

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habitat has many tall grass/bushes and most of the time wolves are far enough that they

would not see each other even in bright daylight (Jácomo et al. 2009).

On the other hand, we found more vocalizations during the waxing gibbous

phase, but also during the waxing crescent and the waning gibbous (with an angle near

full). For those moon phases the first half of the night is illuminated, and this period is

when they vocalize more. That fact corroborates the idea that light has a direct influence

on the species vocalization, and that deserves further investigation. It also indicates that

it is best to describe maned wolves vocalize less on darker nights than the other way

around.

To announce the territory ownership right at the start of the activity may be

especially important as other wolves will also start moving around at this time and may

decide to trespass or not (also suggested by Rocha et al. 2016). This is also the most

constraining hour for communication, as they haven’t feed over the day. In this situation

the advertisement of the individual quality should be honest (Zahavi 1975; Cuthill &

Macdonald 1990). This will influence even monogamous territorial species, as, beside

mate guarding, territorial intrusions can be presumably avoided by advertising body

condition (Cuthill & Macdonald 1990; Hardouin et al. 2008). Territory pressures may

be high, and there are several reports of resident maned wolves extending their home

range shortly after a neighbor death/disappearance (Dietz 1984; Rodrigues 2002;

Jácomo et al. 2009).

Conversely, the peak in vocal activity on the first hour of the night in 2016 was

not accompanied by an increase in group vocalizations. Those interactions only appear

latter, sometimes one hour after a previous increase in vocal activity (Figure 7). This

can indicate the first roar-bark display is used to announce territory, before others can

trespass. Later the partner can respond, or another wolf may dispute the area, creating

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group vocalizations. Also corroborating this idea, maned wolves only responded to

playbacks between 17-19:40h (Ferreira et al. unpublished; Chapter 1).

Although peaks in activity (Jácomo et al. 2004; Melo et al. 2007) and

vocalizations (Brady 1981; Balieiro & Monticelli 2019) have been reported on both

dusk and dawn, here we registered very few roar-bark sequences preceding dawn (05-

06h of 2014). Maybe there is a second peak in vocalizations after dawn that we did not

record. However, 24h recordings in the same park, including periods when roar-bark

playbacks were conducted before and after dusk, did not reveal any vocalization around

dawn or morning (8 days of continuous recordings and playbacks: Chapter 1; 5

recorders, 13 days of continuous recordings in 2015: Ferreira et al. unpublished).

Group vocalizations occur in a variety of contexts, including interactions

between neighbors, breeding pairs, and parent offspring (Kleiman 1972; Brady 1981;

Dietz 1984; Emmons 2012; Balieiro & Monticelli 2019). In captivity group

vocalizations are common and roar-barks of one wolf often induce vocalizations of

other wolves (Sábato 2011). Emmons (2012) reports a particularly familiar group of

maned wolves (5) that responded sequences to each other 31% of times on average.

In our study, there were more group vocalizations on 2014 than in 2016, when

recorders were spaced over a larger area (49-140 km2 x 53-195 km2 estimated recorded

area). This suggests we could have failed to detect the second animal on some

sequences classified as solo in 2016. However, considering Emmons and our own

personal observations of the data, we still think those are low percentages to believe all

sequences heard by another maned wolf are actually answered. For instance: on several

occasions, sequences appearing to come from different individuals were separated by 5-

20 minutes, a time interval short enough to assume the second wolf heard the first wolf

but choose not to intercalate roar-barks with it. Secondly: on a playback experiment

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(Chapter 1), wolves never intercalated the response with the broadcasted roar-barks,

even during an interactive playback (but intercalated entire sequences; Ferreira et al.

unpublished). This suggest wolves intentionally choose to intercalate roar-barks on

some occasions but not on others. Our results suggest solo and group vocalizations may

have slightly different functions as they do not always fluctuate together.

We hypothesize group vocalizations happen more often when the response to a

conspecific vocalization must be immediate, as territorial defense (versus passive

territory ownership announcement), and/or when pair members must be located

simultaneously, as for mate guard and joint territorial/offspring defense. Accordingly,

group vocalizations increased in the mating period, and in 2014 near parturition, when

those situations would happen more often. On the other hand, group vocalizations did

not follow the lunar cycle (except for, and probably biased by, the mating season of

2016), neither the diel solo vocalization pattern (especially on 2016). Those facts

indicate events of social interaction not related to breeding occur at any time and must

be addressed independent of illumination and hour. As a final remark on this topic,

animals emitting group vocalizations were still several meters apart, that is, vocal

interactions do not mean close physical interaction.

Further studies on solo and group vocalizations are needed to clarify if they have

indeed distinct functions, which was not the focus of this work. As a recommendation,

clearly separating sequences with and without intercalating roar-barks would help in

this matter, something that has not been done previously (Kleiman 1972; Brady 1981;

Dietz 1984; Balieiro & Monticelli 2019).

Our estimates of number of animals that contributed to the recordings ranged

from 4 to 17. While we are certain that at least 4 different animals were recorded (in

March 20, 2016 there were group vocalizations on both extremes of the park), we think

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17 animals is an overestimate. Data from telemetry tracking show that long straight

trajectories that are bigger than their territories are uncommon (Paula 2016), thus it is

likely that we have recorded 6 individuals because on March 22, 2016 (two days after

detection of group vocalizations on the limits of the park) there was another group

vocalization in the middle of the park.

The higher estimate of 17 individuals was obtained based on captivity data of

mean roar-bark sequences by individual by night (Sábato 2011). Interestingly, we

imagined animals in captivity would vocalize more than those in the wild, as they are

closer and roar-barks from one individual stimulate responses of other individuals,

which would result in lower estimative for the wild. Therefore, maned wolves appear to

vocalize less in captivity, maybe as a combined result of being habituated to each

other’s roar-bark sequences, favoring close-range communication types as they are in

proximity, and/or a reduced pressure to defend a territory/mate.

Nevertheless, based on telemetry data obtained in our study area (Paula 2016)

we estimate that 5 ranges could have been recorded, considering generally ranges do not

change drastically (Dietz 1984; Emmons 2012). Each range would have a breeding pair,

and occasionally up to 3 juveniles or adult offspring (maximum group size reported:

Azevedo 2008, Emmons 2012). Additionally, transient animals may also vocalize and

contribute to the maximum estimate, so it would be possible that 17 different animals

were recorded.

As conclusion, maned wolves use roar-barks in many contexts, and more in the

mating season, on better moonlit nights (or less on darker ones), at the onset of their

activity in the first part of the night. All of these findings support the role of this

vocalization in territorial announcement and defense, partner attraction and guarding,

and intra-group communication. This multi functionality attests that maned wolves

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interact in complex ways and more frequently than previously thought. Additionally, we

were able to estimate the number of vocalizing individuals in a way useful to monitor

populations and detect large scale fluctuations in the number of individuals at a low cost

(passive acoustic monitoring). Those fluctuations would indicate serious environmental

problems requiring urgent decision making and action.

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Supplementary material

Figure S1. Nightly maned wolf number of roar-barks recorded passively with 12/13 autonomous

recorders (Song Meter SM2+) at Serra da Canastra National Park, MG/Brazil (gray contour), in

2014/2016. From top-left to bottom-right: April 05/06/07/08 2014, April 15/16/17/18 2014, March

19/20/21/22/23 2016, and May 01/02/03/04 2016. **indicates at least one sequence of roar-barks

involved two animals. *** indicates at least one sequence of roar-barks involved three animals.

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Chapter 4

Identity and sex discrimination of roar-barks for

captive and free-ranging maned wolves

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Identity and sex discrimination of roar-barks for captive and free-

ranging maned wolves

Ferreira, L.S., Sábato V.R., Baumgarten, J., Rodrigues, F.H., Sousa-Lima, R.S.

Intended for submission on: Biological Conservation

Abstract

Although vocal identity is a widespread trait, it is rarely tested for application in

natural conditions. Such methodology could aid in the understanding and conservation

of elusive nocturnal/crepuscular species, especially those whose populations are

predicted to decline, as are maned wolf populations. Here we recorded captive maned

wolves and found that their long-range call, the roar-bark, is individually and sexually

distinct. Roar-barks could be correctly assigned to individuals 72.6% of times, and to

sex 78.6%. The roar-bark duration and the concentration of energy in lower frequencies

were the most important parameters. However, when roar-barks were experimentally

broadcasted on natural habitat most parameters were not stable, even at distances as

short as 160 m. The few stable parameters were mediocre in discriminating among

individuals (42.5% success x 13.0% chance). Site characteristics, as vegetation and

relief, and individual differences, were more influential than distance, suggesting some

identity information may be ranging far given the appropriate conditions. Unfortunately,

we also found that in passive records of spontaneous roar-barks sequences of free-

ranging maned wolves the variation in parameters due to propagation is larger than the

individual differences, which seriously compromises the applicability of vocal

identification of individuals of the species on natural conditions.

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Resumo

Apesar da identidade vocal ser um traço bastante difundido em animais, ela é

raramente testada para aplicação em condições naturais. Tal metodologia poderia ajudar

na compreensão e conservação de espécies elusivas noturno-crepusculares,

especialmente aquelas em que há previsão de declínio populacional, como o lobo-guará.

Neste trabalho nós gravamos lobos-guará em cativeiro e descobrimos que seus

chamados de longa distância, os aulidos, são individual e sexualmente distintos. Aulidos

puderam ser corretamente designados aos indivíduos em 72,6% dos casos, e aos sexos

78,6%. A duração do aulido e a concentração de energia em frequências baixas foram os

parâmetros mais importantes. Entretanto, quando os aulidos foram experimentalmente

propagados em ambiente natural, a maioria dos parâmetros variaram, mesmo em

distâncias curtas (160 m). Os poucos parâmetros que se mantiveram estáveis foram

capazes de discriminar entre indivíduos, mas com sucesso medíocre (42,5% x 13,0% ao

acaso). Características locais, como vegetação, e diferenças individuais, foram mais

influentes do que a distância, sugerindo que alguma informação de identidade pode ter

longo alcance em condições propícias. Infelizmente, nós também descobrimos que em

gravações passivas de sequências de aulidos espontâneas de lobos-guará em vida livre a

variação nos parâmetros causada pela propagação é maior do que as diferenças

individuais identificadas, o que compromete seriamente a aplicação da identificação

vocal individual da espécie em condições naturais.

Key-words: vocal identity, Chrysocyon brachyrurus, propagation, passive monitoring

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Introduction

Maned wolves (Chrysocyon brachiurus, Illiguer 1815) are an exception among

large canids (70-90cm, 20-30kg; Silveira 1999). They forage alone for fruits and small

vertebrates and rarely interact with conspecifics (Dietz 1984), despite the similar sized

family members being highly social (Kleiman & Eisenberg 1973; Mohelman 1987,

1989). The monogamous mated pair share the same extensive home range (15-115 km2;

Rodrigues 2002; Azevedo 2008), being seen together almost exclusively in the breeding

season (Dietz 1984). Females are monoestral and the litter size is small (captivity

average is 3; Maia & Gouveia 2002). Both sexes provide parental care (Bestelmeyer

2000; Mello et al. 2009) and the young stay in their natal range for 1 or more years

before dispersing (Rodden et al. 2004; Emmons 2012). The species has mainly

nocturnal/crepuscular habits (4pm-8am; Paula 2016) and delimit/announce their

territory through urine, scats (Bestelmeyer 2000; Rodden et al. 2004), and long-range

calls (the roar-bark; Kleiman 1972; Rocha et al. 2016).

More extensive studies on the behavior ecology of this peculiar species could

help the understanding of evolutionary patterns on the Canidae family (Mohelman

1987, 1989). Additionally, the species’ numbers are predicted to decline 30% over 3

generations (21 years) due to habitat loss alone (Paula et al. 2008) and additional

populational estimates are needed (Paula et al. 2013). Unfortunately, many of the

characteristics cited above, coupled with the fact that maned wolves are shy, occur in

low densities, in difficult terrains (Melo et al. 2007; Trolle et al.2007), makes finding

and monitoring the species a challenge. This is a common scenario for many terrestrial

mammals, and therefore, methods for identifying individuals in the wild without need of

visual confirmation would be an extremely useful tool and would allow for less invasive

studies.

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For many species long range vocalizations are ideal for use in identification

(Terry et al. 2005). The presence of identity information in vocalizations, referred to as

vocal signature, is a well-documented phenomenon in birds (e.g. Falls 1982), primates

(e.g. Marler & Hobbett 1975), ungulates (e.g. Reby et al. 2001), cetaceans (e.g. Sayighet

al.1998), proboscids (e.g. McComb et al. 2003), rodents (e.g. Kober et al. 2008), canids

(e.g. Durbin 1998; Darden et al. 2003; Mitchell et al. 2006), and many others, indicating

this is a widespread trait (Falls 1982). Some studies have additionally investigated sex

and age cues on vocalizations, for instance in manatees (Sousa-Lima et al. 2002, 2008)

and dingos (Déaux et al. 2016a).

While vocal signatures are a widespread trait, it does not mean all species show

them, or are capable of detecting or use those clues to recognize conspecifics (Terry et

al. 2005). Vocal discrimination in general evolves in situations in which failing to

identify the emitter – offspring, partner, competitor – generates a loss in the receptor

fitness, and, simultaneously, not being correctly identified generates a loss in the emitter

fitness (Bradbury & Vehrencamp 1998). Those situations normally involve: the risk of

misdirecting parental care, common in colonial species, as penguins (Jouventinet al.

1999), bats (Fanis & Jones 1996), and pinnipeds (Charrier & Harcourt 2006) and in

species that invest highly in parental care, such as manatees (Sousa-Lima et al. 2001,

2009). The importance of discriminating established neighbors from trespassing

vagrants to optimize the energetical investment in territorial defense is also dependent

on individual recognition, as the observed in artic foxes (Frommolt et al. 2003). Besides

artic foxes, vocal discrimination by canids has been demonstrated in gray wolves

(Palacios et al. 2015), domestic dogs (Molnáret al. 2009), and dingos (Déaux et al.

2016b).

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Therefore, vocal signatures and individual discrimination and recognition in

maned wolves may have evolved to maintain pups close to parents and hidden from

strangers, juveniles inside their parents’ range, to optimize the energetic investment in

territorial defense, or to find a mate or reunite a previously established pair during the

breading season. Long ago, Brady (1981) reported that a human would be able to

discriminate individual maned wolves from 1 kilometer, and, recently, it has been

demonstrated that captive maned wolves discriminate between roar-barks of familiar

versus unfamiliar individuals (Balieiro & Monticelli 2019). Although neither work

quantified individual characteristics of the roar-bark (but see Sabato 2011), they suggest

maned wolves have vocal signatures that could be used by researchers to identify

individuals.

Despite the potential for the use of vocal signature on animal research and

conservation, very few studies in fact apply the vocal identity after its description

(Gilbert et al. 2002; Delport et al. 2002), a critic highlighted by Terry et al. (2005).

From all studies of vocal signature in canids, only 3 uses data from natural habitat

besides or instead of captivity (Frommolt et al. 2003; Hartwig 2005; Root-Guteridge et

al. 2014b), and none apply identification protocols in vocalizations of unknown

emitters.

The main problem in the classification of vocalizations recorded from natural

habitat is the degradation of acoustic parameters used in the discrimination when the

sound propagates through long distances (Mitchell et al. 2006). Frommolt et al. (2003) e

Hartwig (2005) results stem from data acquired at small distances, between 20 and 100

meters. However, in the work of Root-Guteridge et al. (2014b) recordings were of

unknown distance, with low signal-to-noise ratios, and, still, discriminant functions

attained 100% of correct classification of individual grey wolves.

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Here we used a combination of maned wolf captivity recordings, field sound

propagation experiment re-recordings, and recordings from wild animals, to (i) test the

individual distinctiveness of roar-barks, (ii) test the sexual distinctiveness of roar-barks,

(iii) test the acoustic distinctive parameters’ stability over distance, and (iv) test the

applicability of vocal identification on natural habitat recordings.

We previously used a grid of autonomous recorders to passively monitor maned

wolves, detecting their presence by roar-barks (Rocha et al. 2016; see Chapter 3).

Therefore, we were interested in knowing if a vocal identification of maned wolves

could be applied in natural habitat recordings of this kind. Roar-barks recorded this way

rarely have a high signal-to-noise ratio and the emitter identity, distance, and location in

the habitat are unknown. Thus, for an acoustic parameter to be reliably used to

distinguish between individuals, its value must remain unchanged (or change less than

within individuals) at distance and in different habitats. If parameter values change the

identity classification will change for the same individual depending on where it is

calling from, making long-term monitoring impossible. Also, vocal identification must

apply to typical low signal-to-noise ratio recordings, otherwise it will only be useful for

a small amount of fortuitous high-quality recordings. For those reasons we conducted

the roar-bark propagation experiment so we could test how much the acoustic parameter

values of known individuals would change over controlled distances and habitats. The

final goal was to test the resulting vocal identification method on a 8-month passive

acoustic monitoring dataset at the Serra da Canastra National Park (MG/BR).

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Material and Methods

1. Data acquisition in Captivity

We recorded 10 sexually mature maned wolves (Table 1, Figure 1a), from two

facilities in Minas Gerais state: Criadouro Científico de Fauna Silvestre para Fins de

Conservação da Companhia Brasileira de Metalurgia e Mineração (CC-CBMM), and

Zoológico da Associação Esportiva e Recreativa dos Funcionários das Usinas

Siderúrgicas de Minas Gerais (ZOO-USIPA). Recordings on CC-CBMM were made

from April 17 to June 03 2010, during the species breeding period (proestrous was

confirmed by swabs), and on ZOO-USIPA from November 12 to 27 2010, which

correspond to the non-breeding period (anestrous). Enclosures had between 1500 and

6000 m2 on CC-CBMM, and between 175 and 185m2 on ZOO-USIPA. All recordings

were made between 17:30h and 7:00h, from 5 to 200 meters away from the animals.

Table 1. Maned wolves recorded on 2010 at two facilities in Minas Gerais, Brazil. *estimated age. m# are

non-participant males. m3 is GA/GI half-brother.

ID Sex Age Facility Origin Relatedness Roar-barks

Recorded Selected

SH male 3y9m* CC-CBMM Nature ? 827 20

FI female 3y6m* CC-CBMM Nature ? 354 20

RO female 13y8m* CC-CBMM Nature JU/NE mother 148 20

JU female 9y6m CC-CBMM Captivity m1+RO offspring 353 20

NE male 5y6m CC-CBMM Captivity m2+RO offspring 297 20

SA female 6y4m CC-CBMM Captivity m3+JU offspring 33 20

GA male 5y6m ZOO-USIPA Captivity GI litter mate 137 20

GI male 5y6m ZOO-USIPA Captivity GA litter mate 29 20

LU female 12y ZOO-USIPA Captivity ? 73 20

BA female 4y* ZOO-USIPA Nature ? 70 20

Total 2321 200

Roar-barks were recorded with a unidirectional microphone Sennheiser K6

coupled with a Sennheiser ME-66 module (40-20000 Hz ± 2.5 dB flat response

frequency), protected by a windscreen, and connected to a Marantz PMD-661 solid state

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recorder using 96 kHz sampling rate and 24-bit wav coding format. Recordings were

monitored with a headset Sony MDR-7506 and the gain was manually set to maximize

the signal-to-noise ratio while avoiding clipping.

Using R program (R version 3.5.1 [2018-07-02], The R Foundation for

Statistical Computing) we randomly selected 20 good quality non-overlapping roar-

barks for each of the 10 animals, totaling 200 roar-barks from captivity.

2. Propagation experiment

We selected 5 good quality roar-barks of each of 4 individuals (GA, SH, JU, SA)

that were recorded close (5-8 m) and that we judged encompassed the variation

observed in captivity (Figure 1b). Broadcasts were done at the Serra da Canastra

National park, representing a typical maned wolf habitat (Dietz 1984), between 18:44

and 19:41h, the period of highest vocal activity for the species (Rocha et al. 2016), from

March 4 to 9, 2017. All 20 roar-barks were broadcasted in 4 different sites at the park,

resulting in a total of 80 playbacks. The most prominent features of sites were: flat

terrain on open bare soil road (“Flat” site; -20.22401 -46.55861 [decimal degrees

WGS84]); broadcast from a lower to a higher position on open bare soil road (“Low-to-

high” site; -20.26104 -46.42372); 20 m from Low-to-high but on a tall grass and shrubs

area (“Vegetation” site; see General Introduction Figure C); and broadcast from a higher

to a lower position on a sinuous bare soil road (“High-to-low” site; -20.25403 -

46.4188).

We used an Acer AspireOne notebook to broadcast the sounds using Raven Pro

1.5 software (Bioacoustics Research Program, 2014. Ithaca, NY: The Cornell Lab of

Ornithology; http://www.birds.cornell.edu/raven) and a Pioneer S-DJ50X speaker (class

A/B Bi-amp, 80 W output, 50-20000 Hz frequency range) 86 cm above the ground to

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simulate the height of a maned wolf. The sound level for the playback was calibrated to

be similar to a natural emission based on captivity recordings of known distance.

Broadcasted roar-barks were re-recorded simultaneously by 7 autonomous

recorders (Song Meter SM2+; Wildlife Acoustics, Inc., Concord, Massachusetts) with

one omnidirectional weatherproof microphone each (SMX-II; Wildlife Acoustics, Inc.;

sensitivity -36±4dB [0dB=1V/pa@1kHz]; 20-20000 Hz flat response frequency).

Recorders were set on the road side in a single direction from the speaker positioned at

distances of 1.25 m, 20 m, 40 m, 80 m, 160 m, 320 m, and 640 m. Distances were

measured using a measuring tape (1.25 to 80 m) and a GPS (Garmin GPSMAP® 76S;

accuracy < 15 m). The autonomous recorders were attached to stakes of the same height

of the speaker (86 cm) with the omnidirectional microphone in a perpendicular position

in relation to the speaker. Recordings were made continuously, with an +36dB gain, 8

kHz sample rate, and 16-bit wav files (same configurations used for recordings of

spontaneous vocalizations in the wild).

At some sites the re-recorded roar-bark quality sharply decreased after 160 m or

there were no detectable roar-bark. We used all re-recordings of the 80 broadcasted

roar-barks in which we could distinguish the two first bands (around 250-1000 Hz,

detailed below).

3. Data acquisition in maned wolves’ natural habitat

We recorded for a total of 233 nights, from April 05 to August 08 2014 and from

March 09 to July 01 2016, with a grid of autonomous recorders (SongMeters SM2+) at

the Serra da Canastra National Park, MG/Brazil. Autonomous recorders were

programed to record continuously for 12 hours each night, partitioning samples in 30

minutes files, with an +36dB gain, 8 kHz sample rate, and 16-bit wav coding (same

configurations as Rocha et al. 2015).

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In 2014, 12 recorders were active from 18:00h to 06:00h with a linear distance

between them of 2.27 ± 0.72 km, and in 2016, 13 recorders were active from 17:00h to

05:00h with a linear distance between then of 3.03 ±0.78 km.

Roar-barks were detected automatically using XBAT (Extensible Bioacoustic

Tool; Figueroa 2007) extension for Matlab (R2010a version; MathWorks, Inc., Natick,

MA, USA) following the methodology detailed by Rocha et al. (2015). In summary,

spectrograms are scanned with a mobile cross correlation of 4 roar-barks templates.

Matches above a threshold (0.21) are then manually verified for false positives and

undetected roar-barks within 24 seconds of the detected ones.

We inspected the detected roar-bark sequences on Raven’s spectrograms and

noted if there was more than one animal emitting roar-barks on the same sequence

(Figure 1c top), and if the same sequence was recorded by more than one autonomous

recorder (Figure 1c bottom). The presence of a second animal could be verified by

differences in the spectral shape of roar-barks, cadence, intensity, and eventual overlaps.

Sequences recorded by more than one recorder were verified by comparing inter roar-

bark intervals, besides emission time and spectral similarity, to ensure that it was in fact

the same sequence and not two different animals vocalizing at the same moment in two

different recorders.

For the prospect of applying vocal identity we used only sequences with two

animals vocalizing together and those recorded simultaneously by two recorders. We

randomly selected sequences among good quality ones (with the two first frequency

bands visible, see below) and selected up to 5, minimum 2, roar-barks until we had at

least 80 roar-barks of each of the two animals and two recorders.

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Figure 1. Maned wolves roar-barks recorded in Minas Gerais, Brazil. a. One example of roar-bark from

each of 10 individuals (letters) recorded with unidirectional microphone and a hand recorder in two

captivity facilities. b. Roar-barks of GA and JU broadcasted and re-recorded with autonomous recorders

at 7 different distances from the speaker on site “Flat” at the Serra da Canastra National Park. c. Free-

ranging animals spontaneous roar-bark sequences recorded with autonomous recorders at the same park:

top spectrogram shows some roar-barks (numbers) from a sequence involving two animals (letters);

bottom spectrogram shows the same sequence recorded by another autonomous recorder 2.41 km away,

roar-barks from “b” reach the recorders on different times because animals are at different positions.

Spectrogram parameters: a. 96 kHz sample rate, 3080 window size, Hann window, 55% brightness, 60%

contrast, 24-bit wav; b. and c. 8 kHz sample rate, 512 window size, Hann window, 50% brightness, 60%

contrast, 16-bit wav.

4. Acoustic measurements

Although some individuals’ roar-barks have harmonic-like structure (Figure 1a

SH), often roar-barks are very harsh, noisy, broad-banded (Figure 1a GI), sometimes

varying in structure for the same individual (Figure 1a JU x 1b JU). Sub-harmonics

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may also appear (Figure 1a LU), generally less intense and incomplete, i.e. not lasting

the entire roar-bark duration. The presence of non-linear phenomenon (Fitch et al. 2002)

also occurred, as SH (Figure 1a) abrupt loss of sub-harmonics and JU (Figure 1b)

abrupt transition from noisy to a more tonal signal. Finally, formants (Fitch 1997)

appear to be present in at least some roar-barks (Figure 1a SA), making the distinction

between harmonics and sub-harmonics unclear.

Those characteristics made it difficult to identify harmonic correspondences and

we opted to use “frequency bands” instead. Using the natural habitat roar-barks as base

(Figure 1c), we chose not to use signal energy below 150 Hz and above 2000 Hz since

these portions of the signal are rarely conserved at distance. Still based on the natural

habitat roar-barks, we identified two frequency bands that frequently were the only

visible portion of the signal at distance (Figure 1c a7), the lower around 250-620 Hz

and the next one above it around 620-1000 Hz. We termed them 1st and 2nd Bands and

made separated selection boxes on Raven Pro 1.5 spectrograms for each, in addition to a

selection box of the entire roar-bark (150-2000 Hz), referred as “Full”.

In those 3 spectrogram selection boxes we measured 41 parameters that we

considered biologically relevant and with potential for coding identity and sex on the

roar-barks (Table S1). The choice was made based on literature on canid that looked for

vocal identity (Darden et al. 2003; Hartwig 2005) and on previous work on maned

wolves (Sabato 2011). We also included some parameters considered “robust

measurements” on Raven’s manual (Charif et al. 2010). For the propagation experiment

we measured the signal-to-noise ratio of re-recorded roar-barks and roar-barks recorded

in the maned wolves’ natural habitat. We used the Full roar-bark in-band power (given

in dB in Raven pro 1.5) as the signal level and the in-band power of an equal dimension

box immediately before the roar-bark as the background noise level. As the autonomous

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recorders were not calibrated, this signal-to-noise ratio should be regarded as a relative

measure of the signal quality instead of an absolute intensity measure. Detailed

description of the variables can be found on the Raven’s manual (Charif et al. 2010).

For the captivity roar-barks, we made selection boxes on spectrograms with the

following parameters: Hann window, 4096 window size, 50% overlap, 50% brightness,

75% contrast, smoothing “off”, which resulted in pixels of 23.4 Hz x 0.022 s. For the

propagation experiment and natural habitat roar-barks we used the following

parameters: Hann window, 512 window size, 50% overlap, 45-55% brightness, 60%

contrast, smoothing “off”, which resulted in pixels of 15.6 Hz x 0.032 s. For the

propagation experiment and natural habitat, if the Full 1st frequency quartile or Full

peak frequency were on a noise (mostly wind) on lower frequencies, we elevated the

Full selection box inferior limit (150 Hz) until the measured frequency parameters were

placed on the roar-bark.

Before starting to create the selection boxes, three analysts preliminary observed

roar-barks and agreed on the general position of frequency bands and standardized the

scales and size of the spectrogram while making measures.

5. Procedure and statistical analyses

The two analysts (LSF, VS) each created measuring boxes for all 200 captivity-

selected roar-barks. We calculated for all variables the difference between analysts’

measurements, using the Spearman’s correlation (in R program), and the Potential for

Identity Coding (PIC; as in Robisson et al. 1993). Beside the absolute difference

between analysts’ measurements, we also compared the difference with the parameter

overall standard deviation as a way to predict the potential impact on the discrimination

analysis. We looked for parameters with low difference between analysts, low

correlation among them, and high PIC values, as well as biological relevant

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combinations in our opinion, to select among the original 41 parameters those that

would be included in the identity and sexual discriminant analyses (Table S1). The

number of variables included to create discriminant functions should be smaller than the

number of samples in the smaller group (Tabachnick & Fidell 2001), in our case a

maximum of 9 parameters for identity and 3 for sex.

We choose 8 parameters among the 41 (Table 2) and tested their distribution

with the Shapiro-Wilk test, transforming the non-parametric to normal-like distribution

with the Yeo-Johnson transformation, and them centralized and scaled them with the R

function preprocess{caret}.

We used the permuted Discriminant Function Analysis (pDFA; Mundry &

Sommer 2007) which balances the tendency of traditional DFA to overestimate

discriminability by permuting elements between groups and comparing the

discriminability of random versus real grouping (Mundry & Sommer 2007). We set the

R function written by Roger Mundry (2015 version) to randomly select 100 of the 200

roar-barks, balancing individuals (10 of each), and ran the discriminant analysis for

1000 permutations. For the identity pDFA we used sex as a control factor for

permutation.

We conducted a separate pDFA for the data collected by each analyst, and, as a

further measure of difference between analysts, conducted a pDFA on 100 random

combinations of captivity roar-barks measured by both analysts and took the average

percentage of correctly cross classified individuals. We further used a homogeneous

combination of data from both analyst (i.e. equal number of roar-barks measured by

each analyst for each animal), which is referred here as the “combined dataset”.

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To evaluate the importance of functions and each parameter on the model we

used the combined dataset to conduct a normal DFA, and observed the cumulative

variance explained by functions and standardized coefficients.

For the sexual discrimination, we used the combined dataset and tested all

combinations of 3 parameters among the 8 we had chosen and analyzed combinations

that were statistically significant on the pDFA (p<0.05). We used Welch’s t-test (for

unequal sample sizes and/or variances) to test if sexes differed on the most recurrent

parameters.

For the propagation experiment each analyst measured half of the data, with

each re-recording file (containing all 20 roar-barks on a specific site and distance) being

randomly assigned to each of the 2 analysts. We conducted a MANOVA, followed by

Bonferroni p-corrected ANOVAs, for all 41 parameters (α=0.0012). We then observed

if the effect size (F) of the distance on the parameters was at least 2 times smaller than

the effect size of the individual (as in Mitchell et al. 2006). The parameters attending

this requisite were termed “Stable parameters”. As we used only 4 animals on the

propagation experiment, we choose 3 parameters among stable ones from the 8 selected

for captivity discrimination. As we had only 2 males and 2 females and based on the

captivity sexual discrimination results (see below), we decided not to conduct further

sexual analysis.

With those 3 parameters we trained a DFA for identity calls using the measures

from 1.25 m of each site and them predicted the identity of the roar-barks for the other

distances. Additionally, we re-conducted the pDFA on the captivity dataset including

only the 3 selected stable parameters to compare the success of discrimination.

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For the natural habitat data, on sequences involving two animals, we measured

the absolute difference between roar-barks of different animals of the same sequence.

We then tested if the animals’ parameters differed with a one-way t-test of the absolute

difference. We also tested, with Pearson’s correlation, if the size of the difference was

correlated with the absolute difference in signal-to-noise ratio between animals.

For sequences simultaneously registered by more than one recorder, we

measured the difference between parameters from the sound with the higher signal-to-

noise ratio to the one with the lower signal-to-noise ratio. We then tested if the

difference was significant with a paired t-test. We also tested, using Pearson’s

correlation, if the absolute difference was correlated with the absolute difference in the

signal-to-noise ratio among roar-barks.

Finally, to test if the absolute difference between animals was greater than the

absolute difference caused by sound degradation, we compared the two differences with

Welch’s t-test.

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Results

1. Captivity

All parameters had PIC values above 1, indicating they could be used for

identity discrimination, but none had high values, i.e. above 2 (Robisson et al. 1993;

Table 2 and S1). The parameters that varied most between analysts were related to the

box limits, such as the 2nd Band higher frequency (70.14% relative to the SD), or more

subjective, such as the 2nd Band “true” lower frequency (120.59% relative to the SD)

which we measured manually. Raven’s robust parameters were in fact very similar

between analysts. Among the 8 parameters chosen, none had correlation coefficients

higher than 0.660 (1st and 2nd Band peak frequency).

Table 2. Selected parameters on maned wolves roar-barks (LSF analyst). “Full” measures refer to the

roar-bark from 150 Hz to 2000 Hz, while “First” and “Second” bands refer to portions from lower to

higher frequencies. Means±SD are for all 10 individuals, 20 roar-barks each. PIC = Potential for Identity

Coding (Robisson et al. 1993). (A) = unitless: proportion relative to entire duration. Parameters detailing

can be found on Raven’s manual (Charif et al. 2010).

Parameter Mean±SD Dif. to VSR analyst PIC

Full duration (s) 0.591±0.112 -0.032±0.051 1.817

Full position of 1st time quartile (A) 0.375±0.056 0.013±0.039 1.039

Full position of 3rd time quartile (A) 0.635±0.070 0.025±0.044 1.154

Full 1st frequency quartile (Hz) 609.8±169.7 1.1±7.2 1.997

Full 3rd frequency quartile (Hz) 905.2±220.2 1.4±7.7 1.657

Full average entropy (bits) 3.765±0.584 -0.015±0.056 1.691

1st band peak frequency (Hz) 528.2±60.9 -8.4±59.5 1.627

2nd band peak frequency (Hz) 848.4±139.0 -35.5±113.6 1.641

The pDFA for identity discrimination resulted, for each analyst (LSF/VS), in

74.5%/71.80% of correctly cross classified roar-barks, which is significantly (p=0.001)

higher than expected by chance on the cross classification (17.24%/15.98%). The 100

random combination of both datasets resulted in a mean 72.58% (±0.98%) of correctly

cross classified roar-barks, being always significant above chance (p=0.001).

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The LDA resulted in 8 discriminant functions, with the first 3 explaining 89% of

the variance (LD1 44.22%, LD2 29.08%, LD3 15.59%; Table S2). The most important

parameters were: for the first function Full duration and Full 1st frequency quartile; for

the second, Full average entropy; and for the third, Full 1st frequency quartile and Full

duration. PIC measure predicted accurately the best discriminant parameters (Table 2).

Figure 2 shows the prediction of identity for the first 3 discriminant functions.

Figure 2. First 3 linear discriminant functions for identity discrimination of 10 captive maned wolves

(colors) roar-barks recorded from two facilities at Minas Gerais, Brazil.

Table 3 shows the mean classification results for all individuals (confusion

matrix). We could not identify any relationship of the errors with the individuals’

relatedness or origin (nature/captivity; see Table 1). All mistakes above 1 roar-bark

(From 10. Values are means) involve non-kin animals (e.g. FI and GI).

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Table 3. Confusion matrix for the cross-validation classification of 10 captive maned wolves by their

roar-barks. Average from the classification results of two analysts (LSF, VS), each constructed by means

of the results from 1000 randomizations of 100 roar-barks (10 for each individual) from a total of 200.

Extracted by the pDFA R function written by Roger Mundry (2015 version).

Individual BA FI JU LU RO SA GA GI NE SH

BA 8.09 0.33 0.83 0.27 0.42 0.05 0.00 0.00 0.03 0.00

FI 0.05 5.18 0.01 0.00 2.74 0.00 0.12 1.86 0.06 0.00

JU 0.01 0.00 9.30 0.43 0.03 0.02 0.00 0.00 0.23 0.00

LU 0.23 0.00 0.55 9.22 0.00 0.00 0.00 0.00 0.00 0.00

RO 0.41 1.29 0.93 0.00 6.32 0.05 0.11 0.36 0.37 0.18

SA 0.27 0.00 0.08 0.01 0.75 8.48 0.16 0.00 0.18 0.09

GA 0.18 0.29 0.00 0.00 0.23 0.03 6.16 0.11 0.12 2.90

GI 0.17 2.69 0.00 0.00 0.11 0.00 0.00 6.70 0.03 0.31

NE 0.46 0.42 0.14 0.24 0.65 0.21 0.11 0.00 7.71 0.09

SH 0.22 0.65 0.00 0.00 0.18 0.12 2.30 0.07 0.45 6.03

For the sex classification, the only combination of parameters that significantly

discriminated sex better than chance (p=0.048) was Full duration, Full 1st frequency

quartile, and 1st Band peak frequency. This combination yielded 81.93%/75.27%

(LSF/VS) of correctly cross classification of sexes, compared to the expected

63.37%/62.48%. Other two combinations yielded marginally significant results: Full

duration, Full 1st frequency quartile, and 2nd Band peak frequency (p=0.063); and Full

duration, Full 1st frequency quartile, and Full position of 1st time quartile (p=0.066).

Increasing the permutations to 10,000 (as suggested by Mundry & Sommer 2007) did

not changed results (p=0.049, p=0.061, p=0.058, respectively).

Of those 5 different parameters, only Full duration was significantly different

between sexes (t=-9.108, df=172.38, p<0.0001), with the male roar-bark being longer

(difference: 0.122±0.022 s; Figure 3). Full 1st frequency quartile (t= -2.612, df=117.44,

p=0.0102) and the 2nd Band peak frequency (t=2.484, df=184.94, p=0.0139) were

marginally significant (α=0.01; Bonferroni correction).

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Figure 3. Differences in the roar-barks parameters between females and males maned wolves recorded in

two facilities in Minas Gerais, Brazil.

2. Propagation experiment

Only roar-barks broadcasted on the Flat site could be detected and measured up

to 640 m, while on the other sites roar-barks were too degraded or completely

undetectable at 320 m and beyond. Therefore, to balance the MANOVA and subsequent

ANOVAs we used only the data up to 160 m.

The repeated measures MANOVA for the 41 original parameters showed

significant individual (F=40.35, Pillai=2.9047, df=3, p<0.0001), distance (F=5.1061,

Pillai=1.5514, df=4, p<0.0001), and interaction (F=2.9636, Pillai=3.1590, df=14,

p<0.0001) effects. Individual effects for each parameter are reported on Table 4 and S3.

From the 8 selected parameters, only Full position of 3rd time quartile, Full 1st frequency

quartile, and 2nd Band peak frequency were significantly affected by the individual but

not by distance (Table 4). The other 5 parameters did not have individual effects two

times greater than the distance effect. An equal number of Raven’s robust measures

were stable (Full position of 3rd time quartile, Full 1st frequency quartile) but others so

called were not (Full position of 1st time quartile, Full 3rd frequency quartile). The

parameters variation with distance are shown in Figure 4.

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Table 4. ANOVA effect sizes (F) for the 8 selected parameters of20 roar-barks from 4 maned wolves

broadcasted and re-recorded at 5 distances (1.25-160m) in 4 sites. (A) = unitless: proportion relative to

entire duration. α=0.0012.

Parameter Fwolf /

Fdist

Wolf Distance Interaction

F P F p F p

Full duration (s) 0.32 39.57 <0.0001 124.60 <0.0001 13.03 <0.0001

Full position of 1st time quartile (A) 0.56 20.00 <0.0001 35.69 <0.0001 2.96 0.0325

Full position of 3rd time quartile (A) 11.60 39.48 <0.0001 3.40 0.0660 6.70 0.0002

Full 1st frequency quartile (Hz) 66.94 93.07 <0.0001 1.39 0.2392 0.70 0.5501

Full 3rd frequency quartile (Hz) 1.07 21.60 <0.0001 20.23 <0.0001 2.66 0.0484

Full average entropy (bits) 0.02 6.42 0.0006 272.20 <0.0001 0.52 0.6672

1st band peak frequency (Hz) 1.00 18.73 <0.0001 18.81 <0.0001 3.81 0.0105

2nd band peak frequency (Hz) 1459 149.44 <0.0001 0.10 0.7492 1.49 0.2168

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Figure 4. Variation of 8 selected parameters of broadcasted maned wolves roar-barks re-recorded at 7

distances (1.25-640m) at the Serra da Canastra National Park, MG/Brazil.

LDA for re-recorded roar-barks with the 3 stable parameters had an overall

mean percentage of correct identity classification of 73.96% (±8.96%, not including the

1.25 m training set, Figure 5). On site Vegetation the classification success dropped

rapidly, while on site Flat it remained relatively high across all distances. There was a

great difference among individuals for the classification success, with the animal JU

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achieving the lowest rates, while the other 3 achieved 100% of correct classification at

320 m, and GA and SA remaining at this level at 640 m (Figure 5).

Figure 5. LDA percentage of correctly identity classification of broadcasted roar-barks of 4 maned

wolves (bottom) re-recorded at 7 distances (1.25-640m) in 4 sites (top) at the Serra da Canastra National

Park, MG/Brazil.

The pDFA for the captivity dataset with all 10 animals and only the 3 stable

parameters achieved 43.68%/41.39% (LSF/VS) of correctly cross validated

classification. Although much lower than the results using all 8 selected parameters,

discrimination was significantly (p=0.001) above the expected by chance on the cross

classification (13.75%/12.22%).

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Signal-to-noise ratio decayed over distance as predicted (Figure 6). Although

not so clear on the LDA results, 160 m seem to be a limit that affects most parameters

(Figure 4). Mean signal-to-noise ratio on 160 m was 11.73 (±6.06 dB; Figure 6), with

all sites having significantly different means (F=139.74, df=3, p<0.0001), except

Vegetation and High-to-low sites (Tukey contrasts: p=0.4140). Despite the great

individual difference in classification success at 160 m, there was no difference in

signal-to-noise ratio among them (F=2.22, df=3, p=0.0927).

Figure 6. Signal-to-noise ratio of broadcasted roar-barks of maned wolves re-recorded at 7 distances

(1.25-640m) in 4 sites at the Serra da Canastra National Park, MG/Brazil. The signal-to-noise ratio was

calculated subtracting from the in-band power of each roar-bark (150-2000 Hz) and the same

measurement taken from an equal sized spectrogram portion immediately before the vocalization

(measure of the background noise level).

3. Natural habitat

We detected a total of 1210 roar-bark sequences over the 233 nights of

recordings. Of those, 150 involved two (very rarely three) different animals exchanging

roar-barks and 284 were registered by two (or more) recorders.

We measured the parameters of 92 pairs of roar-barks from different animals

vocalizing together (184 roar-barks total, from 21 sequences). Mean signal-to-noise

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ratio of those roar-bark records was 11.58 (±6.93 dB). Animals were significantly

different from each other for all parameters (Table 5). The absolute difference between

animals was not generally correlated with the absolute signal-to-noise ratio difference

(Table 5), with maximum coefficients being 0.329, for the 2nd Band peak frequency,

and 0.485, for the Full average entropy.

We measured the parameters of 83 unique roar-barks registered by two recorders

(166 records total, from 17 unique sequences). Mean signal-to-noise ratio of roar-barks

from the most intense record was 11.37 (±7.77 dB), and for the last intense record 4.62

(±2.84 dB). Full duration, 1st and 2nd Band peak frequencies, and Full 3rd quartile of

frequency were all not significantly different between recorders (Table 5), although the

last presented a tendency for difference (α=0.0012, p=0.0128). The absolute difference

in parameters was also not generally correlated with the absolute difference of signal-to-

noise ratio (Table 5), except for Full 1st frequency quartile (0.594) and Full average

entropy (0.528).

The absolute parameter value difference between animals was not greater than

the difference between roar-barks registered in different recorders (Table 6), except for

Full duration. Figure 7 shows an example of two different sequences involving

apparently the same animals recorded with 4 nights of difference in recorders

approximately 5 km apart. The more stable parameters, on the propagation experiment

and the natural records, of both animals and sequences are compared in the graphs

below the spectrogram (Figure 7).

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Table 5. Effect size (t) for the absolute difference between parameters of roar-barks from two different

maned wolves vocalizing together (2 wolves) and roar-barks simultaneously recorded by two different

autonomous recorders (2 recorders; SongMeter SM2+). 2 wolves: one sample t-test, df=91. 2 recorders:

paired t-test, df=82. α=0.0012. Cor. SNR: Pearson’s correlation coefficient with the signal-to-noise ratio

difference. (A) = unitless: proportion relative to entire duration. The vocalizations were recorded

passively with a grid of 12/13 autonomous recorders at Serra da Canastra National Park, MG/Brazil.

Parameter t2wolves

/ t2rec.

Dif. 2 wolves Dif. 2 recorders

t p Cor.

SNR t p

Cor.

SNR

Full duration (s) 45.14 11.826 <0.0001 0.095 -0.262 0.7941 0.095

Full position of 1st time quartile (A) 1.94 10.618 <0.0001 0.007 5.483 <0.0001 0.037

Full position of 3rd time quartile (A) 2.75 13.411 <0.0001 0.039 -4.883 <0.0001 0.195

Full 1st frequency quartile (Hz) 2.27 11.869 <0.0001 0.066 5.228 <0.0001 0.594

Full 3rd frequency quartile (Hz) 4.17 10.610 <0.0001 0.118 -2.547 0.0128 -0.115

Full average entropy (bits) 1.64 14.425 <0.0001 0.485 -8.815 <0.0001 0.528

1st band peak frequency (Hz) 15.20 13.616 <0.0001 0.216 0.896 0.3730 0.145

2nd band peak frequency (Hz) 17.40 10.090 <0.0001 0.329 -0.580 0.5636 0.160

Table 6. Mean±SD differenceof the absolute difference between parameters of roar-barks from two

different maned wolves vocalizing together (2 wolves) and roar-barks simultaneously recorded by two

different autonomous recorders (2 recorders; SongMeter SM2+). Those differences were compared with a

Welch two sample t-test.α=0.0012.(A) = unitless: proportion relative to entire duration. The vocalizations

were recorded passively with a grid of 12/13 autonomous recorders at Serra da Canastra National Park,

MG/Brazil.

Parameter Mean±SD difference Comparison of differences

2 wolves 2 recorders t df p

Full duration (s) 0.108±0.088 0.002±0.063 5.729 126.5 <0.0001

Full position of 1st time quartile (A) 0.067±0.060 -0.037±0.061 1.255 165.5 0.2113

Full position of 3rd time quartile (A) 0.082±0.059 0.044±0.082 0.702 173 0.4835

Full 1st frequency quartile (Hz) 123.0±99.4 -98.1±170.9 -0.171 139.4 0.8641

Full 3rd frequency quartile (Hz) 161.2±145.7 87.7±313.8 -0.861 124.6 0.3910

Full average entropy (bits) 0.819±0.545 0.610±0.631 1.399 172.5 0.1637

1st band peak frequency (Hz) 40.9±28.8 -4.7±47.8 1.968 155.8 0.0508

2nd band peak frequency (Hz) 79.1±75.2 5.8±91.7 2.309 171.3 0.0222

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Figure 7. Two different roar-bark sequences involving the same two maned wolves each (top), and their

roar-bark parameters (bottom). Recordings made passively by two different autonomous recorders

(SongMeter SM2+) at the Serra da Canastra National Park, MG/Brazil.

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Discussion

Here we found that the maned wolf long range call, the roar-bark, is individually

distinct and that captive animals can be discriminated from each other by their roar-

barks with a mean 72.6% correct classification. We also found that roar-barks carry sex

information, with sexes being correctly discriminated 78.6% of times. The roar-bark

duration and the concentration of energy in lower frequencies were the most important

parameters both for identity and sexual discrimination. The majority of acoustic

parameters were not stable when experimentally broadcasted over distance, even

through distances as short as 160 m. The few stable parameters were capable of

discriminating among individuals, although classification success rate dropped

substantially (42.54%). Interestingly, site characteristics (i.e., presence of vegetation),

and individual differences, were more important for discrimination success than

distance. Unfortunately, we found that in natural habitat the variation on parameters due

to propagation is larger than the individual difference, which seriously compromises the

applicability of vocal identification of maned wolves in the wild.

One of the most important parameters for individual discrimination was the first

frequency quartile, which sometimes can be viewed as a correlate of the fundamental

frequency. Several other works on canids (Tooze et al. 1990; Frommolt et al. 2003;

Hartwig 2005; Mitchell at al. 2006), and other mammals (Rendall et al. 1998; Sousa-

Lima et al. 2002, 2008), have similarly found frequency related parameters, especially

the fundamental frequency, to be important for individual discrimination. This was

expected, as the fundamental frequency and the energy distribution over frequencies are

highly related to the individual morphology of the vocal tract, affecting both source and

filter (Fitch 1997; Sousa-Lima et al. 2002).

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On the other hand, the importance of the roar-bark duration for identity

discrimination was unexpected. Other studies in canids have found temporal patterns to

be important for identity discrimination (Durbin 1998; Darden et al. 2003), but in those

cases the parameter was the time between the start of two consecutive elements. While

in dholes and swift fox this interval is short (0.6 s: Durbin 1998; 0.2 s: Darden et al.

2003), for maned wolves it is much longer and unrelated to vocal identity (4.0-5.4 s;

Ferreira et al. unpublished; Appendix II).

The vocalization duration can be related to lung capacity (Fitch 2002), and

therefore indirectly related to body size. Body size may be important to distinguish

young from adults or females from males (in sexually dimorphic species) but seems to

provide very limited information for discriminant functions to separate among many

individuals. Thus, roar-bark duration may be at least partially under the emitter control

and reflect the animals’ internal state and/or preference.

Roar-bark duration was important to discriminate sexes, as also the first

frequency quartile. Both characteristics may provide body size cues: duration because of

the lung capacity (as cited above), and the first frequency quartile because larger vocal

folds are capable of producing lower frequencies (Morton 1977), although in several

species vocal tract structures vary independent of body size (Fitch 1997). Maned wolves

do not have a pronounced sexual dimorphism. Males are on average 2 kg and 2.5 cm

larger than females (N=79; Jácomo et al. 2009). Vocal differences may be less related to

morphology and more related to the animals’ motivation to appear bigger.

In captivity males vocalize more than females during the breeding season

(Sábato 2011) and produce longer roar-barks, which require higher energy investment.

This may be used as an indicator of the emitter motivation to fight (Vehrencamp 2000;

Linhart et al. 2012), for instance to defend their partner during the breeding season.

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Independent of being more related to morphology or motivation, the sexual

difference in the roar-barks seems to be small, as the body dimorphism. Only one

combination of parameters (from 56) discriminated sexes better than expected by

chance (78.6% obtained x 62.9% chance). Also, the difference in frequency parameters

between sexes was only marginally significant (p=0.0102 and p=0.0139, α=0.01),

despite being on the expected direction (males with lower frequencies).

Randomly testing combinations of discriminating variables can generate

spurious results (Whittingham et al. 2006). We emphasize then that the 3 parameters

were drawn from a small pool of variables that were considered biologically relevant

and that the two first parameters in the 3 significant or marginally significant

combinations were the same as the most important for identity discrimination (Full

duration and Full 1st frequency quartile). All of which suggests that our result reflects a

real difference and not spurious characteristics of our data.

The LDA on the propagation experiment had a 73.96% identity classification

success. However, this value is overestimated as we did not used the permuted approach

(pDFA; Mundry & Sommer 2007), because we needed different training and test sets,

and that was not feasible due to the small sample size (included only 4 individuals).

Probably the value attained using the 3 stable parameters on the captivity data is closer

to reality (42.54%). Nevertheless, one interesting observation was that, in most cases,

the classification success did not drop progressively with distance. The two cases in

which success clearly dropped with distance was on the site with vegetation and for

individual JU. This result suggests that obstacles for sound propagation in their habitat

is more relevant in discriminating individuals than degradation. It also suggests the

identity information in some individuals’ roar-bark is more likely to travel far than

others, revealing the importance of the particular roar-bark acoustic structure for

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discrimination at long distances. In the end this suggests some identity information may

travel far, given that environment and the vocalization spectrographic structure is

appropriate.

One thing worth noting is that while in the propagation experiment few roar-

barks even reached 640 m (less than 5%), with a mean signal-to-noise ratio of 5.92

(±2.14 dB) at this distance, on natural recordings 23.5% of sequences were registered by

more than one recorder. Based on the distance between recorders of the measured pairs

of roar-barks, sound would have to travel a mean of 1.19 km (±0.39 km) before

reaching recorders (in the minimum distance situation, in which the emitter is exactly in

the middle), and the signal-to-noise ratio of the less intense signal recorded was 4.62

(±2.84 dB). Thus, our propagation experiment, while perfectly valid, underestimates

roar-barks reach. This was probably a matter of the recorder height, which was lower on

the experiment than in the natural recordings (0.86 m x 1.4 m). It could also be that

captive maned wolves vocalize with a different intensity level than wild ones, which

would compromise the level we calibrated for. Captive maned wolves, for instance, are

maintained in much closer proximity with each other and would need a lower level to

communicate efficiently among themselves.

Unfortunately, despite some parameters being stable in the propagation

experiment (3 of 8), in natural conditions they were significantly altered in the

comparison of same roar-barks on different recorders (Full position of 3rd time quartile,

Full 1st frequency quartile) or the difference between individuals was correlated with

signal-to-noise ratio (2nd Band peak frequency). Furthermore, for all parameters (except

Full duration, see below) the variation magnitude among the same roar-bark on different

recorders was equal to the magnitude of the individual difference. The consequence is

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that none of those parameters are reliable to apply vocal identity classification in free-

ranging maned wolves.

The duration of the roar-bark, one of the most important discriminatory

parameters in captivity, presented a paradoxical propagation behavior. While it

decreased steadily on the propagation experiment measures, it was the only parameter

not altered between different records of the same roar-bark in the natural recordings.

Maybe after some distance (>1km) the degradation of this parameter stabilizes. At this

moment we cannot state this is a reliable discriminatory parameter for application in the

wild, and further research will be needed to clarify this topic.

One last curious observation on the matter is that the mean duration difference

between roar-barks of animals vocalizing at the same moment (108±88 ms) was similar

to the mean difference for males and females in captivity (122±22 ms). This could

indicate that, for this sample of roar-barks (21 of 150 group sequences), this kind of

sequence is normally emitted by breeding pairs. Therefore, group vocalizations could be

a duet-like behavior, as common in some birds (Marshall-Ball et al. 2006) and primates

(Méndez-Cárdenas et al. 2009), and may have the same proposed functions: pair

bonding/coordination, joint territorial defense, mate guarding, and parental care

manipulation (Wachtmeister 2001). However, we do not know if duration is a reliable

parameter, as stated above, and a previous work from our laboratory found exactly the

opposite result (i.e. roar-barks in group vocalizations generally have similar duration,

N=15; Frigo 2016). Reports from captivity and the wild indicates that group

vocalizations occur between same and opposite sex maned wolves (Dietz 1984;

Emmons 2012).

For the captivity data, the presence of identity information in the roar-barks was

very consistent, as revealed by the pDFA, which considers the chance of random false

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groups attaining discriminability because of information noise unrelated to the tested

factor (in our case identity). Yet, our success of vocal identity classification rate

(72.6%) was low compared to other canids, as artic foxes (91.7%, Frommolt et al.

2003), swift foxes (99%, Darden et al. 2003), and gray wolves (100%, Root-Guteridge

et al. 2014b). Also, all acoustic parameters degraded over distance, in the propagation

experiment or in the natural records, or both.

The high degradation of identity and sexual cues is probably related to the noisy

(harsh, broadband) nature of roar-barks. By contrast, long (several seconds), tonal, and

frequency modulated vocalizations are the ideal for transmitting identity information

over long distances (Wiley & Richards 1978, 1982; Fitch 1997).

In fact, studies on canids recurrently find that vocalizations like howls and

whistles (Durbin 1998; Root-Guteridge et al. 2014a), that are longer and more tonal,

attain better identity discrimination rates than barks and similarly noisy vocalizations

(Yin & McCowan 2004; Hartwig 2005). Domestic dogs’ barks are very noisy and have

the lowest identity discrimination rates among canid studies (40%), but this rate

improves in contexts in which dogs make more tonal and modulated barks (Yin &

McCowan 2004). Dingoes produce “bark-howls” concatenating a noisy and a tonal part,

with that last part attaining higher identity classification success than the first (Déaux et

al. 2016c). For coyotes, howls not only present best discrimination rates than barks

(83% x 69%), but also maintain high discrimination levels at 1000 m (81%, Mitchell et

al. 2006).

Those results raise the question of why free-ranging maned wolves do not

produce more tonal roar-barks in addition to the noisy ones, since captivity animals

demonstrate the species is capable of such. One hypothesis, that would also explain the

only moderate levels of identity and sexual discrimination obtained here, would be that

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175

the selective pressure for identity discrimination by the species is low. Maned wolves

live solitary, in low densities (Trolle et al. 2007), and share their extensive home range

with very few conspecifics (the breeding partner and eventual offspring; Rodden et al.

2004). In this scenario, one animal would only need to distinguish between group-

members and non-group members, which does not demand a very fine identity coding.

Besides, maned wolves use other communication modalities, as visual (postures,

piloerection) and chemical (scent marks, urine, feces), to signalize its presence and

status over short and long distances and duration (Rodden et al. 2004). The combination

of modalities as well as redundancy (e.g. sequences of roar-barks) maximizes the

information transmission efficiency and prevent interpretation errors (Bradbury

&Vehrencamp1998).

The moderate discrimination rates, the high degradation of identity and sexual

cues, and the rarity of more tonal elements on free-ranging maned wolves’ roar-barks

could also mean that for the species the estimation of the emitter location is more

important than precise identification of sex and identity. Broadband vocalizations are

ideal to estimate the emitter distance, as they degrade predictably over distance (Wiley

& Richards 1978; Naguib & Wiley 2001). Accordingly, the roar-bark entropy steadily

increased on the propagation experiment. That would reinforce the idea that roar-barks

are vocalizations better suited for intra-group communication, for which identifying the

emitter position is important to coordinate activities, rather than for territorial

announcement, in which propagation over long distances is desired but precise location

is not.

Despite very rarely participating in cooperative activities close to conspecifics,

e.g. hunting ground birds (Jácomo et al. 2009), knowing the location of group members

may be most important for maned wolves. For instance, the breeding pair must reunite

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176

on the short 5 days of estrous (Rodden et al. 2004), and the male must know the location

of the female and pups to defend and provide for them (Bestelmeyer 2000). Breeding

pairs are reported to use the same paths, fruit plants, and hunting areas, but not

simultaneously (Dietz 1984; Carvalho & Vasconcellos 1995), and the species present

periodicity on the use of space (Péron et al. 2017). Thus, maybe a more adaptive

function of roar-barking would be for each individual to plan their movement, according

to the position of others, as other maned wolves may interfere in their foraging and

deplete foraging areas.

Finally, one plausible alternative explanation for moderate discrimination and

low propagation of identity cues is that maned wolves are using parameters others than

the ones tested here to discriminate the identity and sex of roar-bark emitters (we

measured 41 but only tested 8 considered more relevant). Besides, maned wolves

potentially hear roar-barks at much further distances that the ones recorded by our

autonomous recorders (which was also true for the field researchers; personal

observation). When used to the captivity data, analysts could generally discriminate

with ease the emitter identity on the propagation experiment by the shape of the roar-

bark on spectrograms, no matter the distance (personal observation). In Figure 7, it is

also possible to discriminate individuals by the shape of their roar-barks. Although the

animal’s points in the graph do not overlay, they are visually (to us) more distinct than

the graphs suggest. That indicates the chosen parameters may not reliably reflect roar-

bark characteristics, and maybe other approaches, such as frequency contour tracking or

spectrogram cross correlation, will result in better discriminability increasing the

applicability of vocal identification of free-ranging maned wolves.

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Supplementary material

Table S1. All measured parameters on maned wolves roar-barks (LSF analyst). “Full” measures refer to

the roar-bark from 150 Hz to 2000 Hz, while “First” and “Second” bands refer to portions from lower to

higher frequencies. Means±SD are for all 10 individuals, 20 roar-barks each. PIC = Potential for Identity

Coding (Robisson et al. 1993). # selected for the identity classification. (A) = unitless: proportion relative

to entire duration. *the selection box of the 2nd band is limited below by the selection box of the 1st band,

therefore we manually measured the 2nd band true lower frequency. Parameters detailing can be found on

Raven’s manual (Charif et al. 2010).

Parameter Mean±SD Dif. to VSR analyst PIC

# Full duration (s) 0.591±0.112 -0.032±0.051 1.817

Full duration of 1st quartile (ms) 74±25 -1±6 1.212

Full duration of 3rd quartile (ms) 78±26 -2±8 1.138

Full inter quartile duration (ms) 152±41 -3±9 1.259

# Full position of 1st time quartile (A) 0.375±0.056 0.013±0.039 1.039

Full position of 2nd time quartile (A) 0.502±0.064 0.019±0.042 1.077

# Full position of 3rd time quartile (A) 0.635±0.070 0.025±0.044 1.154

# Full 1st frequency quartile (Hz) 609.8±169.7 1.1±7.2 1.997

Full 2nd frequency quartile (Hz) 731.3±196.4 0.4±4.4 1.649

# Full 3rd frequency quartile (Hz) 905.2±220.2 1.4±7.7 1.657

Full peak frequency (Hz) 716.4±242.6 0. 1.535

# Full average entropy (bits) 3.765±0.584 -0.015±0.056 1.691

1st band duration (s) 0.551±0.112 -0.029±0.092 1.787

1st band duration of 1st quartile (ms) 65±25 -1±19 1.043

1st band duration of 3rd quartile (ms) 77±41 -2±23 1.252

1st band inter quartile duration (ms) 142±55 -3±30 1.204

1st band position of 1st time quartile (A) 0.327±0.096 0.022±0.070 1.265

1st band position of 2nd time quartile (A) 0.448±0.105 0.030±0.089 1.230

1st band position of 3rd time quartile (A) 0.588±0.112 0.037±0.091 1.245

1st band lower frequency (Hz) 255.6±86.6 18.9±70.9 1.514

1st band higher frequency (Hz) 618.1±53.9 -9.7±63.5 1.390

1st band 1st frequency quartile (Hz) 493.1±55.4 -3.2±52.9 1.647

1st band 2nd frequency quartile (Hz) 520.9±54.7 -6.1±52.3 1.822

1st band 3rd frequency quartile (Hz) 546.2±54.7 -7.4±57.9 1.726

# 1st band peak frequency (Hz) 528.2±60.9 -8.4±59.5 1.627

1st band average entropy 2.447±0.355 -0.104±0.310 1.386

2nd band duration (s) 0.520±0.092 0.004±0.089 1.665

2nd band duration of 1st quartile (ms) 76±33 -5±27 1.072

2nd band duration of 3rd quartile (ms) 78±32 -8±29 1.161

2nd band inter quartile duration (ms) 154±50 -13±46 1.192

2nd band position of 1st time quartile (A) 0.375±0.079 0.026±0.059 1.232

2nd band position of 2nd time quartile (A) 0.522±0.092 0.014±0.067 1.193

2nd band position of 3rd time quartile (A) 0.671±0.091 -0.002±0.073 1.177

2nd band lower frequency (Hz)* 623.9±53.3 -3.0±73.1 1.350

2nd band true lower frequency (Hz)* 467.5±100.7 117.0±135.5 1.193

2nd band higher frequency (Hz) 1036.3±137.8 -75.6±136.6 1.706

2nd band 1st frequency quartile (Hz) 794.2±107.1 -23.8±82.5 1.798

2nd band 2nd frequency quartile (Hz) 844.7±127.0 -37.6±106.0 1.942

2nd band 3rd frequency quartile (Hz) 891.4±139.0 -48.4±118.7 1.920

# 2nd band peak frequency (Hz) 848.4±139.0 -35.5±113.6 1.641

2nd band average entropy (bits) 2.753±0.354 -0.113±0.328 1.311

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Table S2. Explained variance and coefficients of each linear discriminant function for identity

discrimination of 10 captive maned wolves roar-barks. Non-normal parameters were transformed (Yeo

Johnson). All parameters were centralized and scaled.

Functions LD1 LD2 LD3 LD4 LD5 LD6 LD7 LD8

Proportion of trace: 44.22% 29.08% 15.59% 5.56% 3.65% 1.28% 0.61% 0.01%

Coefficients of linear

discriminants:

Full average entropy -0.276 1.283 0.544 -0.977 0.755 0.019 0.722 0.819

Full duration -1.310 -0.190 1.221 0.210 0.527 -0.226 -0.108 -0.090

Full position of the 1st

time quartile -0.327 0.177 -0.363 -0.522 0.205 0.053 0.348 -1.005

Full position of the 3rd

time quartile 0.048 -0.158 0.446 1.200 -0.126 0.027 0.503 0.357

Full 1st frequency quartile 0.909 -0.110 1.464 -0.915 -0.335 0.020 0.716 0.357

Full 3rd frequency quartile 0.421 0.418 -0.154 1.267 -0.053 -0.415 -1.136 -0.947

1st Band peak freq 0.337 -0.514 0.042 0.094 0.777 1.094 -0.372 -0.057

2nd Band peak freq 0.040 -0.261 -0.515 -0.102 0.546 -1.159 0.587 0.026

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Table S3. ANOVA effect sizes (F) for all measured parameters on maned wolves roar-barks broadcasted

and re-recorded at 5 distances (1.25-160m). “Full” measures refer to the entire roar-bark (from 150 Hz to

2000 Hz), while “First” and “Second” bands refer to portions from lower to higher frequencies. (A) =

unitless: proportion relative to entire duration. α=0.0012.

Parameter FID /

FDist

Individual Distance Interaction

F p F p F p

Full duration (s) 0.32 39.57 <0.0001 124.60 <0.0001 13.03 <0.0001

Full inter quartile duration (ms) 149.99 10.38 <0.0001 0.07 0.7926 4.06 0.0075

Full position of 1st time quartile (A) 0.56 20.00 <0.0001 35.69 <0.0001 2.96 0.0325

Full position of 2nd time quartile (A) 2.88 25.43 <0.0001 8.82 0.0032 6.39 0.0003

Full position of 3rd time quartile (A) 11.60 39.48 <0.0001 3.40 0.0660 6.70 0.0002

Full 1st frequency quartile (Hz) 66.94 93.07 <0.0001 1.39 0.2392 0.70 0.5501

Full 2nd frequency quartile (Hz) 2.46 48.07 <0.0001 19.51 <0.0001 2.45 0.0636

Full 3rd frequency quartile (Hz) 1.07 21.60 <0.0001 20.23 <0.0001 2.66 0.0484

Full peak frequency (Hz) 29.88 64.30 <0.0001 2.15 0.1434 0.26 0.8536

Full average entropy (bits) 0.02 6.42 0.0006 272.20 <0.0001 0.52 0.6672

1st band duration (s) 0.02 2.05 0.1133 103.70 <0.0001 10.89 <0.0001

1st band inter quartile duration (ms) 0.12 7.00 0.0003 57.70 <0.0001 11.14 <0.0001

1st band position of 1st time quartile (A) 1.26 15.96 <0.0001 12.67 0.0004 17.03 <0.0001

1st band position of 2nd time quartile (A) 2.25 22.51 <0.0001 10.00 0.0017 20.49 <0.0001

1st band position of 3rd time quartile (A) 0.45 25.62 <0.0001 56.82 <0.0001 19.21 <0.0001

1st band lower frequency (Hz) 5.26 146.64 <0.0001 27.89 <0.0001 8.42 <0.0001

1st band higher frequency (Hz) 0.70 27.00 <0.0001 38.61 <0.0001 6.65 0.0002

1st band 1st frequency quartile (Hz) 0.19 15.34 <0.0001 80.54 <0.0001 8.79 <0.0001

1st band 2nd frequency quartile (Hz) 0.78 21.36 <0.0001 27.26 <0.0001 3.54 0.0150

1st band 3rd frequency quartile (Hz) 5.86 27.87 <0.0001 4.75 0.0300 3.94 0.0088

1st band peak frequency (Hz) 1.00 18.73 <0.0001 18.81 <0.0001 3.81 0.0105

1st band average entropy 0.24 27.20 <0.0001 115.43 <0.0001 1.92 0.1255

2nd band duration (s) 0.15 23.49 <0.0001 157.65 <0.0001 11.67 <0.0001

2nd band inter quartile duration (ms) 89.67 22.39 <0.0001 0.25 0.6177 9.94 <0.0001

2nd band position of 1st time quartile (A) 0.16 1.74 0.1655 11.19 0.0009 5.10 0.0018

2nd band position of 2nd time quartile (A) 103.82 28.49 <0.0001 0.27 0.6008 0.83 0.4786

2nd band position of 3rd time quartile (A) 8.61 56.84 <0.0001 6.60 0.0107 6.51 0.0003

2nd band lower frequency (Hz) 0.62 21.11 <0.0001 33.90 <0.0001 6.73 0.0002

2nd band higher frequency (Hz) 5.63 106.63 <0.0001 18.92 <0.0001 3.02 0.0301

2nd band 1st frequency quartile (Hz) 12995 291.08 <0.0001 0.02 0.8812 5.77 0.0008

2nd band 2nd frequency quartile (Hz) 130.49 302.48 <0.0001 2.32 0.1289 5.40 0.0012

2nd band 3rd frequency quartile (Hz) 15802 207.00 <0.0001 0.01 0.9090 1.86 0.1363

2nd band peak frequency (Hz) 1459 149.44 <0.0001 0.10 0.7492 1.49 0.2168

2nd band average entropy (bits) 0.11 15.75 <0.0001 137.04 <0.0001 7.89 <0.0001

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186

Final remarks

Through a combination of approaches, including recordings in captivity, in the

wild, and a propagation experiment, the present work supports the hypothesis that the

maned wolf roar-bark is a multifunctional vocalization. Roak-barks are used for

territorial announcement and defense, as indicated by the observation that: wild maned

wolves vocally responded and approached roar-bark playbacks of unknown individuals

(of both sexes); and roar-barks are emitted all year, not only in the breeding season.

Maned wolves emit more roar-barks at the beginning of the night, but not near dawn,

despite both these times presenting efficient propagation. Vocalizing at the on-set of the

activity period can provide an honest advertisement of quality as it is more restrictive to

vocalize after a period without food. They also do not respond to playbacks after 20h,

which can indicate, as in some birds, this is the most important period to announce

territory occupancy and maned wolves will pay less attention to it outside this period.

On two occasions two different maned wolves responded to the playback, one

time with an apparent coordinated approach. Maybe it was a mated pair (or same group

members), which if true would show maned wolves also use roar-barks to jointly defend

their territory. The roar-bark structure favors location of the emitter instead of long-

range identity information transmission, in accordance with the idea that group-

members must coordinate their activities, even if it is only to avoid interference in

foraging. During the mating season maned wolves use roar-barks to attract new partners

or reunite previously established pairs and mate guard, as indicated by the increase in

both solo and group vocalizations. Captive males emitted longer, and thus costlier, roar-

barks, which reinforce the role of roar-barks in announcing motivation to defend sexual

partners. Around parturition solo and group vocal activity also increased, as the pair

must coordinate the parental care and/or territorial defense becomes more important.

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Besides finding support for the previously proposed functions of roar-barks, the

present work highlights some new aspects of the species vocal behavior, as the: low

vocal activity and responsiveness near dawn, despite the species being more active

around twilight; the increase in solo vocal activity during better moonlit nights; and the

possibility that group vocalizations, i.e., when animals alternate roar-barks on the same

sequence, have a differentiated function, as no animal intercalated roar-barks during the

playback, and group vocalizations were in general independent of moon illumination or

time of the night. Those issues require further investigation to clarify their underlying

cause and provide new exciting research paths in the study of maned wolf acoustic

ecology.

On very good conditions a roar-bark can reach over 3 km, but on others, less

than 160 m, which shows how drastically their environment influences their active

space. Even pointing the muzzle up and finding higher grounds to vocalize do not

guarantee better transmission. This instability was reflected in the vocal discrimination

of individuals at longer distances. The unfortunate conclusion of this work is that, at the

moment, this method is not applicable for natural recordings with the parameters used.

Yet, visual differences in roar-barks are perceivable on spectrograms, which, despite

being a subjective impression, encourages new approaches on the matter.

Nevertheless, even without vocal identification, exploring the long-range

acoustic communication of maned wolves is proving highly valuable to monitor the

species and reveal their secretive behavior.

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188

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191

Appendix I

Maned wolves do not emit more roar-barks than expected by chance during the

illuminated versus the non-illuminated portion of the night, except from new to waxing

crescent phase. In this phase only the first part of the night is illuminated, and thus the

difference may be caused by the species preference to vocalize on this time. *t=2.906,

df=33, p=0.006. Graph extracted and translated from: ÁRAUJO, D.D., FERREIRA,

L.S., ROCHA, L.H.S., & SOUSA-LIMA, R.S. 2016. Influência do ciclo lunar nas

vocalizações de lobo guará. Abstract and poster presentation at the III Conferência e

VIII Simpósio de Psicobiologia, UFRN, Natal, Rio Grande do Norte, Brazil.

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192

Appendix II

Individual variation of the time interval between the start of one maned wolf

roar-bark to the next one in the sequence. Potential for Identity Coding (PIC; as in

Robisson et al. 1993) for this parameter is 1.04. Data from 10 captive maned wolves,

24-124 roar-barks by individual, 897 roar-barks in total, recorded in 2010 by V. Sábato,

at 2 facilities in Minas Gerais, Brazil.

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193

Annex I

A sedated lactating maned wolf being examined by the Lobos da Canastra team.

This female (known as “Rose”) was captured on the night between July 12-13 2016 at

the Serra da Canastra National Park, around site F (Figure 1, Chapter 3). At this year

she was already without a VHS collar, but her data shows she lived on the present study

area since at least 2014, when she was first captured, also lactating. Photo: Rogério

Cunha de Paula. Used with permission.

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194

Annex II

Justificativa ética

A pesquisa de Luane Maria Stamatto Ferreira, CPF 049.973.494-70, RG

001850911, enquanto aluna do Programa de Pós-Graduação em Psicobiologia, nível

doutorado, e orientada pela Profa. Dra. Renata Santoro Sousa-Lima, foi autorizada pelo

Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio; licença SISBIO

número 41329-2). O ICMBio é o órgão federal com finalidade de executar e fazer

executar a política e diretrizes governamentais fixadas para o meio ambiente (lei nº

6.938/1981), sendo competência deste, através do SISBIO, conceder autorização para a

coleta de material biológico de espécimes da fauna silvestre em território brasileiro e

realização de pesquisa em unidade de conservação, como consta nas leis nº 5.197/1967,

nº 9.605/1998, nº 9.985/2000, no decreto nº 4.340/2002, lei nº 12.651/2012, e IN

ICMBio nº 03/2014. A observação e gravação de imagens e som para fins científicos

não requer autorização pelo ICMBio, como consta no IN ICMBio nº 03/2014,

entendendo-se, portanto, que sejam atividades não intrusivas e de impacto

negligenciável. Entretanto, a autorização é necessária quando as atividades de coleta são

feitas em Unidades de Conservação, como foi o caso desta pesquisa (UC: Parque

Nacional da Serra da Canastra). Logo, o projeto foi submetido e aprovado pelo ICMBio,

sob a referida licença número 41329-2, que dá autorização para Atividades com

Finalidades Científicas em UC federal (anexada a seguir).

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Ministério do Meio Ambiente - MMAInstituto Chico Mendes de Conservação da Biodiversidade - ICMBioSistema de Autorização e Informação em Biodiversidade - SISBIO

Autorização para atividades com finalidade científicaNúmero: 41329-7 Data da Emissão: 17/02/2017 09:50 Data para Revalidação*: 19/03/2018

* De acordo com o art. 28 da IN 03/2014, esta autorização tem prazo de validade equivalente ao previsto no cronograma de atividades do projeto,mas deverá ser revalidada anualmente mediante a apresentação do relatório de atividades a ser enviado por meio do Sisbio no prazo de até 30 diasa contar da data do aniversário de sua emissão.

SISBIODados do titular

Nome: RENATA SANTORO DE SOUSA LIMA MOBLEY CPF: 705.712.446-53

Título do Projeto: Ecologia acústica do lobo-guará (Chrysocyon brachyurus) no Parque Nacional da Serra da Canastra ? MG

Nome da Instituição : UFRN - UNIVERSIDADE FEDERAL DO RIO G. NORTE CNPJ: 24.365.710/0001-83

Cronograma de atividades# Descrição da atividade Início (mês/ano) Fim (mês/ano)

1 Piloto no campo 12/2013 12/20132 Coleta de dados 01/2014 10/20173 Levantamento bibliográfico 01/2014 12/20174 Análise de dados 02/2014 12/20175 Elaboração de documentos cientificos 06/2014 12/2017

Observações e ressalvas

1As atividades de campo exercidas por pessoa natural ou jurídica estrangeira, em todo o território nacional, que impliquem o deslocamento de recursos humanos emateriais, tendo por objeto coletar dados, materiais, espécimes biológicos e minerais, peças integrantes da cultura nativa e cultura popular, presente e passada,obtidos por meio de recursos e técnicas que se destinem ao estudo, à difusão ou à pesquisa, estão sujeitas a autorização do Ministério de Ciência e Tecnologia.

2

Esta autorização NÃO exime o pesquisador titular e os membros de sua equipe da necessidade de obter as anuências previstas em outros instrumentos legais, bemcomo do consentimento do responsável pela área, pública ou privada, onde será realizada a atividade, inclusive do órgão gestor de terra indígena (FUNAI), daunidade de conservação estadual, distrital ou municipal, ou do proprietário, arrendatário, posseiro ou morador de área dentro dos limites de unidade de conservaçãofederal cujo processo de regularização fundiária encontra-se em curso.

3Este documento somente poderá ser utilizado para os fins previstos na Instrução Normativa ICMBio n° 03/2014 ou na Instrução Normativa ICMBio n° 10/2010, no queespecifica esta Autorização, não podendo ser utilizado para fins comerciais, industriais ou esportivos. O material biológico coletado deverá ser utilizado para atividadescientíficas ou didáticas no âmbito do ensino superior.

4O titular de licença ou autorização e os membros da sua equipe deverão optar por métodos de coleta e instrumentos de captura direcionados, sempre que possível,ao grupo taxonômico de interesse, evitando a morte ou dano significativo a outros grupos; e empregar esforço de coleta ou captura que não comprometa a viabilidadede populações do grupo taxonômico de interesse em condição in situ.

5O titular de autorização ou de licença permanente, assim como os membros de sua equipe, quando da violação da legislação vigente, ou quando da inadequação,omissão ou falsa descrição de informações relevantes que subsidiaram a expedição do ato, poderá, mediante decisão motivada, ter a autorização ou licençasuspensa ou revogada pelo ICMBio, nos termos da legislação brasileira em vigor.

6Este documento não dispensa o cumprimento da legislação que dispõe sobre acesso a componente do patrimônio genético existente no território nacional, naplataforma continental e na zona econômica exclusiva, ou ao conhecimento tradicional associado ao patrimônio genético, para fins de pesquisa científica,bioprospecção e desenvolvimento tecnológico. Veja maiores informações em www.mma.gov.br/cgen.

7Em caso de pesquisa em UNIDADE DE CONSERVAÇÃO, o pesquisador titular desta autorização deverá contactar a administração da unidade a fim de CONFIRMARAS DATAS das expedições, as condições para realização das coletas e de uso da infra-estrutura da unidade.

Outras ressalvas

1 Não pode haver novas capturas além das previstas no projeto citado no parecer ora emitido. Todas as citações referentes ao PNSC devem tercomo base o Plano de Manejo de 2005.

Equipe# Nome Função CPF Doc. Identidade Nacionalidade1 Jean Pierre Santos assistente de campo 013.190.686-00 M7928211 SSP-MG Brasileira2 FLAVIO HENRIQUE GUIMARÃES RODRIGUES Colaborador 536.695.171-20 1028375 SSP-DF Brasileira3 Victor Sábato Rocha Colaborador 081.762.096-60 MG13000879 SSP-MG Brasileira4 Luciana Helena Silva Rocha Responsável de campo 047.443.344-74 1800413 ITEP-RN Brasileira5 Julio Baumgarten Colaborador 473.370.951-04 - Brasileira6 Marcello Montagno do Valle assistente de campo 115.682.788-45 6895163 SSP-SP Brasileira7 Luane Maria Stamatto Ferreira Responsável de campo 049.973.494-70 001850911 ITEP-RN Brasileira8 Julia Simões Damo assistente de campo 119.633.486-21 19157461 PCMG-MG Brasileira

SISBIOEste documento (Autorização para atividades com finalidade científica) foi expedido com base na Instrução Normativa nº 03/2014. Através do código

de autenticação abaixo, qualquer cidadão poderá verificar a autenticidade ou regularidade deste documento, por meio da página do Sisbio/ICMBio na

Internet (www.icmbio.gov.br/sisbio).

Código de autenticação: 48814279Página 1/3

Page 196: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Ministério do Meio Ambiente - MMAInstituto Chico Mendes de Conservação da Biodiversidade - ICMBioSistema de Autorização e Informação em Biodiversidade - SISBIO

Autorização para atividades com finalidade científicaNúmero: 41329-7 Data da Emissão: 17/02/2017 09:50 Data para Revalidação*: 19/03/2018

* De acordo com o art. 28 da IN 03/2014, esta autorização tem prazo de validade equivalente ao previsto no cronograma de atividades do projeto,mas deverá ser revalidada anualmente mediante a apresentação do relatório de atividades a ser enviado por meio do Sisbio no prazo de até 30 diasa contar da data do aniversário de sua emissão.

SISBIODados do titular

Nome: RENATA SANTORO DE SOUSA LIMA MOBLEY CPF: 705.712.446-53

Título do Projeto: Ecologia acústica do lobo-guará (Chrysocyon brachyurus) no Parque Nacional da Serra da Canastra ? MG

Nome da Instituição : UFRN - UNIVERSIDADE FEDERAL DO RIO G. NORTE CNPJ: 24.365.710/0001-83

Locais onde as atividades de campo serão executadas# Município UF Descrição do local Tipo

1 MG PARQUE NACIONAL DA SERRA DA CANASTRA UC Federal

Atividades X Táxons# Atividade Táxons

1Observação e gravação de imagem ou som de taxon em UCfederal

Canidae

Material e métodos1 Método de captura/coleta (Carnívoros) Bioacústica

Destino do material biológico coletado# Nome local destino Tipo Destino

1 UFRN - UNIVERSIDADE FEDERAL DO RIO G. NORTE

SISBIOEste documento (Autorização para atividades com finalidade científica) foi expedido com base na Instrução Normativa nº 03/2014. Através do código

de autenticação abaixo, qualquer cidadão poderá verificar a autenticidade ou regularidade deste documento, por meio da página do Sisbio/ICMBio na

Internet (www.icmbio.gov.br/sisbio).

Código de autenticação: 48814279Página 2/3

Page 197: UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · domo arigato gozaimashita ao meu sensei James! Por fim, agradeço aos lobos-guará do Parque Nacional da Serra da Canastra que,

Ministério do Meio Ambiente - MMAInstituto Chico Mendes de Conservação da Biodiversidade - ICMBioSistema de Autorização e Informação em Biodiversidade - SISBIO

Autorização para atividades com finalidade científicaNúmero: 41329-7 Data da Emissão: 17/02/2017 09:50 Data para Revalidação*: 19/03/2018

* De acordo com o art. 28 da IN 03/2014, esta autorização tem prazo de validade equivalente ao previsto no cronograma de atividades do projeto,mas deverá ser revalidada anualmente mediante a apresentação do relatório de atividades a ser enviado por meio do Sisbio no prazo de até 30 diasa contar da data do aniversário de sua emissão.

SISBIODados do titular

Nome: RENATA SANTORO DE SOUSA LIMA MOBLEY CPF: 705.712.446-53

Título do Projeto: Ecologia acústica do lobo-guará (Chrysocyon brachyurus) no Parque Nacional da Serra da Canastra ? MG

Nome da Instituição : UFRN - UNIVERSIDADE FEDERAL DO RIO G. NORTE CNPJ: 24.365.710/0001-83

Registro de coleta imprevista de material biológicoDe acordo com a Instrução Normativa nº 03/2014, a coleta imprevista de material biológico ou de substrato nãocontemplado na autorização ou na licença permanente deverá ser anotada na mesma, em campo específico, porocasião da coleta, devendo esta coleta imprevista ser comunicada por meio do relatório de atividades. O transporte domaterial biológico ou do substrato deverá ser acompanhado da autorização ou da licença permanente com a devidaanotação. O material biológico coletado de forma imprevista, deverá ser destinado à instituição científica e, depositado,preferencialmente, em coleção biológica científica registrada no Cadastro Nacional de Coleções Biológicas (CCBIO).

Táxon* Qtde. Tipo de amostra Qtde. Data

* Identificar o espécime no nível taxonômico possível.

SISBIOEste documento (Autorização para atividades com finalidade científica) foi expedido com base na Instrução Normativa nº 03/2014. Através do código

de autenticação abaixo, qualquer cidadão poderá verificar a autenticidade ou regularidade deste documento, por meio da página do Sisbio/ICMBio na

Internet (www.icmbio.gov.br/sisbio).

Código de autenticação: 48814279Página 3/3