Erratum to: Born to sing! Song development in a singing primate.

[This corrects the article DOI: 10.1093/cz/zoab018.].

Chemical composition, antimicrobial activity, and antioxidant capacity of Micromeria flagellaris Baker and M. madagascariensis Baker: Two endemic species from Madagascar as sources of essential oils.

Background: The aerial parts of Micromeria madagascariensis Baker and M. flagellaris Baker are used by the population of the Vakinankaratra and Itasy regions (Madagascar) to treat breathing difficulty, fever and/or headache, wounds, and sores. Purpose: This work aimed to characterise plant materials from M. madagascariensis and M. flagellaris to report i) chemical composition, ii) antimicrobial properties, and iii) antioxidant capacity of the essential oils extracted from the aerial parts of these species. Materials and methods: The essential oils from M. madagascariensis (MMO) and M. flagellaris (MFO) were obtained by hydrodistillation. Their chemical composition was quantified using gas chromatography coupled with mass spectrometry (GC-MS). MMO and MFO were also tested against 7 microbial strains using the disk diffusion method and their antioxidant capacity was assessed using the DPPH scavenging assay. Results: Hydrodistillation yielded 0.26% MMO and 0.29% MFO (w/w) in relation to the fresh weight. Twenty-seven compounds were identified by GC-MS in MMO extract against 36 in MFO one. The main compounds in MMO were pulegone (24.67%), trans-menthone (24.67%), eucalyptol (8.12%), ß-caryophyllene (4.98%), α-guanene (4.47), iso-menthone (3.85%), iso-pulegone (3.34%), azulene (3.28%) and 2-isopropyl-5-methylcyclohexenone (2.82%). The main compounds in the MFO were eudesma-4,11-dien-2-ol (13.88%), δ-guanene (6.62%), pulegone (6.40%), cyperone (5.56%), 4-epi-dehydrobietinol acetate (5.39%), eucalyptol (5.12%), trans-menthone (4.67%), limonene (3.77%) and sabinene (2.29%). Regarding the chemotaxonomy, M. flagellaris was very different from M. madagascariensis and both species also differed from the other Micromeria species, as confirmed by multivariate statistical analysis. Both MMO and MFO exerted activities against a large microbial spectrum; the antimicrobial activity of MMO was higher than MFO one against S. pneumoniae and C. albicans due to the presence of pulegone as the main component. MFO showed an excellent scavenging capacity with an SC50 value of 2.17 ± 0.03 µg/mL. Conclusion: The biological properties of the essential oils extracted from the selected species may explain their therapeutic value showing that Malagasy Micromeria species may be very important as new natural sources of bioactive compounds. This study may promote the effectiveness and quality of Malagasy Micromeria species, contributing to sustainable development and commercial valorisation of traditional preparations based on natural local resources.

Who you live with and what you duet for: a review of the function of primate duets in relation to their social organization.

Duets are one of the most fascinating displays in animal vocal communication, where two animals fine-tune the timing of their emissions to create a coordinated signal. Duetting behavior is widespread in the animal kingdom and is present in insects, birds, and mammals. Duets are essential to regulate activities within and between social units. Few studies assessed the functions of these vocal emissions experimentally, and for many species, there is still no consensus on what duets are used for. Here, we reviewed the literature on the function of duets in non-human primates, investigating a possible link between the social organization of the species and the function of its duetting behavior. In primates and birds, social conditions characterized by higher promiscuity might relate to the emergence of duetting behavior. We considered both quantitative and qualitative studies, which led us to hypothesize that the shift in the social organization from pair living to a mixed social organization might have led to the emergence of mate defense and mate guarding as critical functions of duetting behavior. Territory/resource ownership and defense functions are more critical in obligate pair-living species. Finally, we encourage future experimental research on this topic to allow the formulation of empirically testable predictions.

"The song remains the same": not really! Vocal flexibility in the song of the indris.

In studying communicative signals, we can think of flexibility as a necessary correlate of creativity. Flexibility enables animals to find practical solutions and appropriate behaviors in mutable situations. In this study, we aimed to quantify the degree of flexibility in the songs of indris (Indri indri), the only singing lemur, using three different metrics: Jaro Distance, normalized diversity, and entropy. We hypothesized that the degree and the co-variation of the flexibility of indris singing together would vary according to their status and sex. We found that dominant females were more flexible than dominant males when concatenating elements into strings (element concatenation). The number of different elements in a song contribution normalized by the contribution length (contribution diversity) of dominant individuals positively co-varied for seven duetting pairs. Non-dominant individuals were more variable in element concatenation than dominant individuals, and they were more diverse in phrase type than dominant females. Independently from sex and status, individual contributions did not differ in entropy (a measure of the predictability of contributions). These results corroborate previous findings regarding the dimorphism by sex and by status of individual contributions to songs. Thus, they shed light on the presence and expression of flexibility in the behavior of a non-human primate species. Indeed, they potentially show an effect of social features in shaping vocal flexibility, which underlies many communication systems, including human language. We speculate that this degree of flexibility may account for creativity.

Behavioural Correlates of Lemur Scent-Marking in Wild Diademed Sifakas (Propithecus diadema) in the Maromizaha Forest (Madagascar).

Scent-marking through odours from excreta and glandular secretions is widespread in mammals. Among primates, diurnal group-living lemurs show different deployment modalities as part of their strategy to increase signal detection. We studied the diademed sifaka (Propithecus diadema) in the Maromizaha New Protected Area, Eastern Madagascar. We tested whether the scent-marking deposition occurred using a sequential rubbing of different body parts. We also tested if glands (i.e., deposition of glandular secretions) were more frequently rubbed than genital orifices (i.e., deposition of excreta) by comparing different kinds of rubbing behaviour. We then investigated if the depositor's rank and sex affected the sequence of rubbing behaviour, the height at which the scent-marking happened, and the tree part targeted. We found that glandular secretions were often deposited with urine, especially in dominant individuals. The probability of anogenital and chest marking was highest, but chest rubbing most frequently occurred in dominant males. Markings were deposited at similar heights across age and sex, and tree trunks were the most used substrate. Males exhibited long and more complex scent-marking sequences than females. Our results indirectly support the idea that diademed sifakas deploy a sex-dimorphic mixture of glandular secretions and excreta to increase the probability of signal detection by conspecifics.

Singing more, singing harsher: occurrence of nonlinear phenomena in a primate' song.

Nonlinear phenomena (NLP) in animal vocalizations arise from irregularities in the oscillation of the vocal folds. Various non-mutually exclusive hypotheses have been put forward to explain the occurrence of NLP, from adaptive to physiological ones. Non-human primates often display NLP in their vocalizations, yet the communicative role of these features, if any, is still unclear. We here investigate the occurrence of NLP in the song of a singing primate, the indri (Indri indri), testing for the effect of sex, age, season, and duration of the vocal display on their emission. Our results show that NLP occurrence in indri depends on phonation, i.e., the cumulative duration of all the units emitted by an individual, and that NLP have higher probability to be emitted in the later stages of the song, probably due to the fatigue indris may experience while singing. Furthermore, NLP happen earlier in the vocal display of adult females than in that of the adult males, and this is probably due to the fact that fatigue occurs earlier in the former because of a greater contribution within the song. Our findings suggest, therefore, that indris may be subjected to physiological constraints during the singing process which may impair the production of harmonic sounds. However, indris may still benefit from emitting NLP by strengthening the loudness of their signals for better advertising their presence to the neighboring conspecific groups.

Evidence for acoustic discrimination in lemurs: A playback study on wild indris Indri indri.

Indris Indri indri are group-living lemurs that occupy stable territories over several years and perform remarkable long-distance vocal displays. Vocal exchanges between long-term territory neighbors may contribute to assessing reciprocal resource-holding potentials, thus adaptively reducing the costs of territorial defense by limiting aggressive escalation. Previous work showed that indris' songs show distinctive acoustic features at individual and group level. However, the possibility that indris use such cues for individual or group-level recognition has never been investigated experimentally. We conducted a playback experiment to test whether indris discriminate between familiar and nonfamiliar songs. Our rationale lies in the hypothesis of the dear enemy phenomenon, which predicts that territorial animals will show reduced aggression levels toward familiar neighbors compared with novel rivals. We played back stimulus recordings to wild indris from their territory boundaries and examined their responses in terms of vocal and behavioral indicators of willingness to engage in a fight. In line with our predictions, focal animals responded more rapidly and approached more often the speaker in response to playback stimuli of nonfamiliar individuals than to stimuli of neighboring groups. These results indicate that indris can discriminate between different classes of intruders based on distinctive acoustic features of their song choruses. We suggest that increased aggression directed toward unfamiliar intruders may be explained by higher threat levels associated with dispersal and group formation dynamics. We further discuss the relevance of these findings in a strepsirrhine primate model for comparative studies of vocal communication and sociality.

There You Are! Automated Detection of Indris' Songs on Features Extracted from Passive Acoustic Recordings.

The growing concern for the ongoing biodiversity loss drives researchers towards practical and large-scale automated systems to monitor wild animal populations. Primates, with most species threatened by extinction, face substantial risks. We focused on the vocal activity of the indri (Indri indri) recorded in Maromizaha Forest (Madagascar) from 2019 to 2021 via passive acoustics, a method increasingly used for monitoring activities in different environments. We first used indris' songs, loud distinctive vocal sequences, to detect the species' presence. We processed the raw data (66,443 10-min recordings) and extracted acoustic features based on the third-octave band system. We then analysed the features extracted from three datasets, divided according to sampling year, site, and recorder type, with a convolutional neural network that was able to generalise to recording sites and previously unsampled periods via data augmentation and transfer learning. For the three datasets, our network detected the song presence with high accuracy (>90%) and recall (>80%) values. Once provided the model with the time and day of recording, the high-performance values ensured that the classification process could accurately depict both daily and annual habits of indris' singing pattern, critical information to optimise field data collection. Overall, using this easy-to-implement species-specific detection workflow as a preprocessing method allows researchers to reduce the time dedicated to manual classification.

Fungal Patterns from Soils in Madagascar: an Insight from Maromizaha Forest (Evergreen Humid Forest) to Outside (Deciduous Forest).

Soil fungal diversity was studied by next-generation sequencing and compared in two different Malagasy ecosystems, the first a New Protected Area (Maromizaha NAP) that is a rich humid evergreen forest and the second a degraded and declined deciduous forest (Andaravina) whose area has been also eroded. Both areas, however, have comparable annual rainfalls and soil pH values. So it was of interest to examine the soil fungal diversity in each system and compare them. We detected 1,817,658 reads representing Ascomycota, which were dominant in both habitats (55.9%), followed by unidentified fungi (21.5%), Basidiomycota (12.7%) and Mortierellomycota (6.7%), with Mucoromycota, Chytridiomycota, Glomeromycota and other phyla accounting for less than 5% in total. In detail, 1,142 OTUs out of 1,368 constitute the common core shared by both sampling areas, which are characterized by tropical climate, whereas 185 are Maromizaha specific and 41 Andaravina specific. The most represented guilds involve fungi related to saprotrophic behaviour, with a greater tendency towards pathotrophic mode. A significant variability in terms of richness and abundance is present within Maromizaha, which is a heterogeneous environment for fungi but also for plant composition, as it emerged from the vegetational survey of the investigated sites. A few fungal sequences match taxa from Madagascar, highlighting the scarce representativeness of fungi from this island in the fungal databases and their still low knowledge. Enlarging studies in Madagascar will help not only to unravel its largely unknown fungal biodiversity but also to give a contribution for studies on the reconstruction of the diversity of soil fungi worldwide.

Born to sing! Song development in a singing primate.

In animal vocal communication, the development of adult-like vocalization is fundamental to interact appropriately with conspecifics. However, the factors that guide ontogenetic changes in the acoustic features remain poorly understood. In contrast with a historical view of nonhuman primate vocal production as substantially innate, recent research suggests that inheritance and physiological modification can only explain some of the developmental changes in call structure during growth. A particular case of acoustic communication is the indris' singing behavior, a peculiar case among Strepsirrhine primates. Thanks to a decade of intense data collection, this work provides the first long-term quantitative analysis on song development in a singing primate. To understand the ontogeny of such a complex vocal output, we investigated juvenile and sub-adult indris' vocal behavior, and we found that young individuals started participating in the chorus years earlier than previously reported. Our results indicated that spectro-temporal song parameters underwent essential changes during growth. In particular, the age and sex of the emitter influenced the indris' vocal activity. We found that frequency parameters showed consistent changes across the sexes, but the temporal features showed different developmental trajectories for males and females. Given the low level of morphological sexual dimorphism and the marked differences in vocal behavior, we hypothesize that factors like social influences and auditory feedback may affect songs' features, resulting in high vocal flexibility in juvenile indris. This trait may be pivotal in a species that engages in choruses with rapid vocal turn-taking.

Categorical rhythms in a singing primate.

What are the origins of musical rhythm? One approach to the biology and evolution of music consists in finding common musical traits across species. These similarities allow biomusicologists to infer when and how musical traits appeared in our species1. A parallel approach to the biology and evolution of music focuses on finding statistical universals in human music2. These include rhythmic features that appear above chance across musical cultures. One such universal is the production of categorical rhythms3, defined as those where temporal intervals between note onsets are distributed categorically rather than uniformly2,4,5. Prominent rhythm categories include those with intervals related by small integer ratios, such as 1:1 (isochrony) and 1:2, which translates as some notes being twice as long as their adjacent ones. In humans, universals are often defined in relation to the beat, a top-down cognitive process of inferring a temporal regularity from a complex musical scene1. Without assuming the presence of the beat in other animals, one can still investigate its downstream products, namely rhythmic categories with small integer ratios detected in recorded signals. Here we combine the comparative and statistical universals approaches, testing the hypothesis that rhythmic categories and small integer ratios should appear in species showing coordinated group singing3. We find that a lemur species displays, in its coordinated songs, the isochronous and 1:2 rhythm categories seen in human music, showing that such categories are not, among mammals, unique to humans3.

Disentangling the Possible Drivers of Indri indri Microbiome: A Threatened Lemur Species of Madagascar.

Research on the gut microbiome may help with increasing our understanding of primate health with species' ecology, evolution, and behavior. In particular, microbiome-related information has the potential to clarify ecology issues, providing knowledge in support of wild primates conservation and their associated habitats. Indri (Indri indri) is the largest extant living lemur of Madagascar. This species is classified as "critically endangered" by the IUCN Red List of Threatened Species, representing one of the world's 25 most endangered primates. Indris diet is mainly folivorous, but these primates frequently and voluntarily engage in geophagy. Indris have never been successfully bred under human care, suggesting that some behavioral and/or ecological factors are still not considered from the ex situ conservation protocols. Here, we explored gut microbiome composition of 18 indris belonging to 5 different family groups. The most represented phyla were Proteobacteria 40.1 ± 9.5%, Bacteroidetes 28.7 ± 2.8%, Synergistetes 16.7 ± 4.5%, and Firmicutes 11.1 ± 1.9%. Further, our results revealed that bacterial alpha and beta diversity were influenced by indri family group and sex. In addition, we investigated the chemical composition of geophagic soil to explore the possible ecological value of soil as a nutrient supply. The quite acidic pH and high levels of secondary oxide-hydroxides of the soils could play a role in the folivorous diet's gut detoxification activity. In addition, the high contents of iron and manganese found the soils could act as micronutrients in the indris' diet. Nevertheless, the concentration of a few elements (i.e., calcium, sulfur, boron, nickel, sodium, and chromium) was higher in non-geophagic than in geophagic soils. In conclusion, the data presented herein provide a baseline for outlining some possible drivers responsible for the gut microbiome diversity in indris, thus laying the foundations for developing further strategies involved in indris' conservation.

Linguistic laws of brevity: conformity in Indri indri.

Vocal and gestural sequences of several primates have been found to conform to two general principles of information compression: the compensation between the duration of a construct and that of its components (Menzerath-Altmann law) and an inverse relationship between signal duration and its occurrence (Zipf's law of abbreviation). Even though Zipf's law of brevity has been proposed as a universal in animal communication, evidence on non-human primate vocal behavior conformity to linguistic laws is still debated, and information on strepsirrhine primates is lacking. We analyzed the vocal behavior of the unique singing lemur species (Indri indri) to assess whether the song of the species shows evidence for compression. As roars have a chaotic structure that impedes the recognition of each individual utterance, and long notes are usually given by males, we focused on the core part of the song (i.e., the descending phrases, composed of two-six units). Our results indicate that indris' songs conform to Zipf's and Menzerath-Altmann linguistic laws. Indeed, shorter phrases are more likely to be included in the song, and units' duration decrease at the increase of the size of the phrases. We also found that, despite a sexual dimorphism in the duration of both units and phrases, these laws characterize sequences of both males and females. Overall, we provide the first evidence for a trade-off between signal duration and occurrence in the vocal behavior of a strepsirrhine species, suggesting that selective pressures for vocal compression are more ancestral than previously assumed within primates.

I Like the Way You Eat It: Lemur (Indri indri) Gut Mycobiome and Geophagy.

Here, we investigated the possible linkages among geophagy, soil characteristics, and gut mycobiome of indri (Indri indri), an endangered lemur species able to survive only in wild conditions. The soil eaten by indri resulted in enriched secondary oxide-hydroxides and clays, together with a high concentration of specific essential micronutrients. This could partially explain the role of the soil in detoxification and as a nutrient supply. Besides, we found that soil subject to geophagy and indris' faeces shared about 8.9% of the fungal OTUs. Also, several genera (e.g. Fusarium, Aspergillus and Penicillium) commonly associated with soil and plant material were found in both geophagic soil and indri samples. On the contrary, some taxa with pathogenic potentials, such as Cryptococcus, were only found in indri samples. Further, many saprotrophs and plant-associated fungal taxa were detected in the indri faeces. These fungal species may be involved in the digestion processes of leaves and could have a beneficial role in their health. In conclusion, we found an intimate connection between gut mycobiome and soil, highlighting, once again, the potential consequent impacts on the wider habitat.

Sexually dimorphic phrase organization in the song of the indris (Indri indri).

Animal acoustic communication often takes the form of complex sequences, composed of multiple distinct acoustic units, which can vary in their degree of stereotypy. Studies of sequence variation may contribute to our understanding of the structural flexibility of primates' songs, which can provide essential ecological and behavioral information about variability at the individual, population, and specific level and provide insights into the mechanisms and drivers responsible for the evolutionary change of communicative traits. Several methods have been used for investigating different levels of structural information and sequence similarity in acoustic displays. We studied intra and interindividual variation in the song structuring of a singing primate, the indri (Indri indri), which inhabits the montane rain forests of Madagascar. Indri groups emit duets and choruses in which they combine long notes, short single units, and phrases consisting of a variable number of units (from two to six) with slightly descending frequency. Males' and females' contributions to the song differ in the temporal and frequency structure of song units and repertoire size. We calculated the similarity of phrase organization across different individual contributions using the Levenshtein distance, a logic distance that expressed the minimum cost to convert a sequence into another and can measure differences between two sequences of data. We then analyzed the degree of similarity within and between individuals and found that: (a) the phrase structure of songs varied between reproductive males and females: female structuring of the song showed a higher number of phrases if compared to males; (b) male contributions to the song were overall more similar to those of other males than were female contributions to the song of other females; (c) male contributions were more stereotyped than female contributions, which showed greater individual flexibility. The picture emerging from phrase combinatorics in the indris is in agreement with previous findings of rhythmic features and song repertoire size of the indris, which also suggested that female songs are potentially less stereotyped than those of males.

Evidence of genetic monogamy in the lemur Indri (Indri indri).

Monogamy is a rare strategy among mammals but relatively common among primates. The study of the evolution of monogamy in mammals and primates is lacking empirical studies that assess the relationship between a pair-living social organization and genetic monogamy. Sexual or genetic monogamy can only be assessed by performing molecular analyses and investigating rates of extra-pair paternity (EPP). Studying the occurrence of EPP can provide valuable insights into reproductive strategies and their adaptive value. The indri is a pair-living primate that lives in stable groups. Their social units are composed of the reproductive pair and up to four more individuals, but extra-pair copulation (EPC) can occur. This raises the question of whether this event may or may not lead to EPP. Here, we investigated whether a pair-living social organization corresponds to genetic monogamy in indris (Indri indri). We analyzed the paternity of 12 offspring from seven pairs using a set of six microsatellite loci on fecal samples (mean number of alleles 11.7 ± 1.8 (mean ± standard deviation). We found that in 92% of cases the genetic profile of the offspring matched the paired male of the group for all the loci considered. In the only case of paternity mismatch, the paternity assignment remained inconclusive. Our results show that I. indri genetic monogamy is the norm and supports the hypothesis that pair-living social organization is associated with low EPP rate. Also, our results are in contrast with the hypothesis of infertility as a reason to engage in EPC for this species.

Finding Meanings in Low Dimensional Structures: Stochastic Neighbor Embedding Applied to the Analysis of Indri indri Vocal Repertoire.

Although there is a growing number of researches focusing on acoustic communication, the lack of shared analytic approaches leads to inconsistency among studies. Here, we introduced a computational method used to examine 3360 calls recorded from wild indris (Indri indri) from 2005-2018. We split each sound into ten portions of equal length and, from each portion we extracted spectral coefficients, considering frequency values up to 15,000 Hz. We submitted the set of acoustic features first to a t-distributed stochastic neighbor embedding algorithm, then to a hard-clustering procedure using a k-means algorithm. The t-distributed stochastic neighbor embedding (t-SNE) mapping indicated the presence of eight different groups, consistent with the acoustic structure of the a priori identification of calls, while the cluster analysis revealed that an overlay between distinct call types might exist. Our results indicated that the t-distributed stochastic neighbor embedding (t-SNE), successfully been employed in several studies, showed a good performance also in the analysis of indris' repertoire and may open new perspectives towards the achievement of shared methodical techniques for the comparison of animal vocal repertoires.

Female indris determine the rhythmic structure of the song and sustain a higher cost when the chorus size increases.

Among the behavioral traits shared by some nonhuman primate species and humans there is singing. Unfortunately, our understanding of animals' rhythmic abilities is still in its infancy. Indris are the only lemurs who sing and live in monogamous pairs, usually forming a group with their offspring. All adult members of a group usually participate in choruses that are emitted regularly and play a role in advertising territorial occupancy and intergroup spacing. Males and females emit phrases that have similar frequency ranges but may differ in their temporal structure. We examined whether the individuals' contribution to the song may change according to chorus size, the total duration of the song or the duration of the individual contribution using the inter-onset intervals within a phrase and between phrases. We found that the rhythmic structure of indri's songs depends on factors that are different for males and females. We showed that females have significantly higher variation in the rhythm of their contribution to the song and that, changes according to chorus size. Our findings indicate that female indris sustain a higher cost of singing than males when the number of singers increases. These results suggest that cross-species investigations will be crucial to understanding the evolutionary frame in which such sexually dimorphic traits occurred.

First ethnobotanical inventory and phytochemical analysis of plant species used by indigenous people living in the Maromizaha forest, Madagascar.

ETHNOPHARMACOLOGICAL RELEVANCE: Characterized by one of the highest rates of endemism and biodiversity in the world, Madagascar provides a wide variety of medicinal plants, that could represent a potential source of new drugs. The main aim of this study was to investigate the potential medicinal properties of the plant species used by indigenous people in Maromizaha forest and to provide the first ethnobotanical inventory of the area. MATERIALS AND METHODS: Data were collected through open semi-structured interviews with local informants, the reported plants were collected and identified to create a specimen herbarium. Informant Consensus Factor (ICF) was calculated for each ailment category mentioned in the use-reports. A selection of seven medicinal plants was submitted to phytochemical and antimicrobial analysis. The results were discussed and compared with those described in ethnobotanical and pharmacological literature. RESULTS: One hundred and three villagers were interviewed and a total of 509 use-reports were recorded. Information on 117 plant species belonging to 57 botanical families were provided. 12 categories of indigenous uses were recognized, among them the higher ICF values were recorded for cardiovascular complaints (0.75), general and unspecific diseases (0.74), digestive disorders (0.69), and diseases of the skin (0.55). The traditional medicinal uses of 18 species (15 endemic) were described for the first time. In total, 22 different bioactive compounds were identified; polyphenols, monoterpenes, organic acids, and vitamin C were observed in the chemical composition of all the analyzed samples. Macaranga perrieri showed the highest values of both total polyphenolic compounds and antioxidant activity. Antimicrobial activity was observed in leaf and bark extracts of Dilobeia thouarsii. CONCLUSION: These results confirmed the importance of investigating the traditional use of plant species, suggesting the crucial role of ethnobotanical studies for rural development, biodiversity conservation, and the sustainable use of plant resources in the studied area.

Call and be counted! Can we reliably estimate the number of callers in the indri's (Indri indri) song?

Estimating the number of animals participating in a choral display may contribute reliable information on animal population estimates, particularly when environmental or behavioral factors restrict the possibility of visual surveys. Difficulties in providing a reliable estimate of the number of singers in a chorus are many (e.g., background noise masking, overlap). In this work, we contributed data on the vocal chorusing of the indri lemurs (Indri indri), which emit howling cries, known as songs, uttered by two to five individuals. We examined whether we could estimate the number of emitters in a chorus by screening the fundamental frequency in the spectrograms and the total duration of the songs, and the reliability of those methods when compared to the real chorus size. The spectrographic investigation appears to provide reliable information on the number of animals participating in the chorusing only when this number is limited to two or three singers. We also found that the Acoustic Complexity Index positively correlated with the real chorus size, showing that an automated analysis of the chorus may provide information about the number of singers. We can state that song duration shows a correlation with the number of emitters but also shows a remarkable variation that remains unexplained. The accuracy of the estimates can reflect the high variability in chorus size, which could be affected by group composition, season and context. In future research, a greater focus on analyzing frequency change occurring during these collective vocal displays should improve our ability to detect individuals and allow a finer tuning of the acoustic methods that may serve for monitoring chorusing mammals.