Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

Acoustic characterization of the threatened bat Neoeptesicus innoxius

(Chiroptera: Vespertilionidae) and two sympatric species in Western Ecuador

Carlos Restrepo-Giraldo1; https://orcid.org/0000-0003-4111-2669

Andrea Au Hing-Cujilán2; https://orcid.org/0000-0002-9938-8716

Tania Paz-Ramírez2; https://orcid.org/0000-0002-5381-9808

Jaime A. Salas3,4; https://orcid.org/0000-0003-3468-5178

Natalia Molina-Moreira2*; https://orcid.org/0000-0002-8197-1137

1. Laboratorio de Paisajes Antrópicos Sostenibles, División de Ciencias Ambientales, Instituto Potosino de Investigación

Científica y Tecnológica, San Luis Potosí, México; carlos.restrepo@ipicyt.edu.mx

2. Escuela de Ciencias Ambientales, Facultad de Ingeniería, Universidad Espíritu Santo, Samborondón, Guayas, Ecuador;

natimolina@uees.edu.ec (*Correspondence), andrea.auhing@outlook.com, pazramirezt@gmail.com

3. Carrera de Biología, Facultad de Ciencias Naturales, Universidad de Guayaquil, Guayaquil, Guayas, Ecuador; jaime.

salasz@ug.edu.ec

4. Instituto Nacional de Biodiversidad (INABIO), Quito-Ecuador.

Received 04-II-2025. Corrected 31-III-2025. Accepted 02-IX-2025.

ABSTRACT

Introduction: Bioacoustics allows the study of the ecology and behavior of bats through the analysis of echoloca-

tion signals. In insectivorous bats, foraging strategies, prey preference, and habitat use are closely related to the

emission patterns of echolocation signals. Identifying significant bat habitats and improving conservation efforts

can benefit from understanding these relationships.

Objectives: To describe the echolocation signals of an endangered species Neoeptesicus innoxius, and other com-

monly detected species in the study area, Myotis nigricans and Molossus molossus, to contribute to the construc-

tion of a bat echolocation call reference library in Western Ecuador.

Methods: Mist nets were used to capture bats, and reference recordings were subsequently obtained using the

Anabat Swift ultrasonic detector. Echolocation pulse selection for each species was carried out using Kaleidoscope

Pro 5.6.8 and BatSound 4.2.1, measuring the following spectral and temporal parameters of the echolocation sig-

nals in the search phase: initial, final, and maximum energy frequency, pulse duration, and interpulse interval.

Results: N. innoxius presented the echolocation signals with the greatest variability of the spectral and temporal

parameters, emitting pulses with high modulation speed (with one prominent FM component), and of low mod-

ulation speed (with one prominent QCF component); M. nigricans emissions were characterized by broadband

signals of very short duration; and M. molossus presented alternation in its echolocation signals, where both high

and low pulses had very little variability.

Conclusions: The acoustic description of an endangered species like N. innoxius alongside the descriptions of

other species frequently detected at the same study site, contributes to the construction of a bat echolocation

call reference library. This input is the basis of future research of the ecology and behavior of the insectivorous

bats that inhabit the Western Ecuador, which in turn is very valuable for designing tools and strategies for bat

conservation.

Keywords: insectivorous bats; echolocation; Isla Santay; Ecuador; Molossidae; Vespertilionidae.

https://doi.org/10.15517/jm7kr371

VERTEBRATE BIOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

INTRODUCTION

Traditionally, the study of the ecology of

bats (order Chiroptera) has been based on their

capture with mist nets or direct roost observa-

tion. However, not all species are susceptible

to being captured or can be easily observed,

such as the case of insectivorous bats, since

they are extremely agile and small, and their

high-frequency echolocation emissions make it

difficult to observe their behavior and monitor

their movements (Jones & Rayner, 1989). Insec-

tivorous bats use echolocation as the primary

sensory modality, which aids in orientation,

object classification, and search for potential

resources (Schnitzler & Kalko, 2001). This trait

enables their study with bioacoustics using

ultrasonic detectors, which usually record spe-

cies of bats that fly close to the ground and at

high altitudes, therefore the complementary use

of mist nets and ultrasonic detectors increases

the probability of species detection, helping to

better estimate bat species richness in each area

(Orozco-Lugo et al., 2013).

In Ecuador, bat bioacoustics studies have

been around for more than a decade, and sur-

prisingly the number of publications remain

scarce. Nonetheless, one of the most relevant

efforts for building Ecuador’s first reference

library of bat echolocation calls took place in the

Amazon, at the Yasuní National Park, grouping

the acoustic description of 28 species of bats

recorded by active (Petterson 240 x ultrasound

detector) and passive detection (BioAcous-

tic Technology AR125 ultrasound detector)

(Rivera-Parra & Burneo, 2013). The first tech-

nique is performed on a mobile transect, better

suited for detecting bats using understory flight

RESUMEN

Caracterización acústica del murciélago amenazado Neoeptesicus innoxius

(Chiroptera: Vespertilionidae) y dos especies simpátricas en el occidente de Ecuador

Introducción: La bioacústica permite estudiar la ecología y el comportamiento de los murciélagos mediante el

análisis de las señales de ecolocalización. En los murciélagos insectívoros, las estrategias de alimentación, la pre-

ferencia de presas y el uso del hábitat están estrechamente relacionados con los patrones de emisión de las señales

de ecolocalización. La comprensión de estas relaciones puede ayudar a identificar los hábitats importantes de los

murciélagos y mejorar los esfuerzos de conservación.

Objetivos: Describir las señales de ecolocalización de la especie en categoría Vulnerable Neoeptesicus innoxius, en

conjunto con las de otras dos especies detectadas frecuentemente en el mismo sitio de estudio, Myotis nigricans y

Molossus molossus, para contribuir a la construcción de una biblioteca de referencias ultrasónicas de murciélagos

insectívoros en el Occidente del Ecuador.

Métodos: Para la captura de murciélagos se utilizaron redes de niebla y posteriormente se obtuvieron graba-

ciones de referencia con el detector ultrasónico Anabat Swift. Mediante los programas Kaleidoscope Pro 5.6.8 y

BatSound 4.2.1 se seleccionaron los mejores pulsos grabados de cada especie y se midieron los siguientes paráme-

tros espectrales y temporales de las señales de ecolocalización en fase de búsqueda: frecuencia inicial, frecuencia

final, frecuencia de máxima energía, duración del pulso e intervalo entre pulsos.

Resultados: N. innoxius presentó señales con mayor variabilidad en sus parámetros espectrales y temporales,

emitiendo señales de alta velocidad de modulación con un componente FM predominante, y de baja velocidad

de modulación con un componente QCF predominante; las emisiones de M. nigricans se caracterizan por ser de

banda ancha y de muy corta duración; y M. molossus presentó alternancia en sus señales de ecolocalización, donde

tanto las señales altas como las bajas presentan muy poca variabilidad.

Conclusiones: La descripción acústica de una especie en categoría Vulnerable como N. innoxius al igual que las

de otras especies que se detectan y capturan frecuentemente en el mismo sitio de estudio, contribuyen a la con-

strucción de una biblioteca de referencia de los sonidos que emiten estos animales para hacer uso del hábitat. Este

aporte es la base de futuras investigaciones para entender mejor la ecología y el comportamiento de los murcié-

lagos insectívoros que habitan en el Occidente Ecuatoriano, lo que a su vez es crucial para diseñar instrumentos

de conservación adecuados.

Palabras clave: murciélagos insectívoros; ecolocalización; Isla Santay; Ecuador; Molossidae; Vespertilionidae.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

paths or forest edges; and the latter is based

on the deployment of detectors for extended

periods over fixed points at variable heights,

better suited for detecting bats flying over the

canopy. This highlights the importance of com-

bining different methodological approaches for

working in the Amazon rainforest with its high

diversity of habitats and species.

Less than a decade ago a second effort

for describing bat echolocation signals was

carried out in Western Ecuador, specifically

in Cerro Blanco Protected Forest, a remnant

of dry forest close to the city of Guayaquil,

reporting a total of 17 phonotypes belonging to

several families: Emballonuridae, Molossidae,

Mormoopidae, Noctilionidae, Phyllostomidae

and Vespertilionidae, with the latter being the

most abundant and diverse family (Tinajero,

2017). This proves bioacoustics methods valu-

able for enhancing our understanding on bat

ecology and behavior over a wide range of

habitats, ecosystems and perturbation gradients

(Stahlschmidt & Brühl, 2012).

Insectivorous bats occupy different habi-

tats such as dry forests and mangroves, along

the coastal profile and in marine-coastal areas

in Ecuador (Paz-Ramírez et al., 2018; Paz-

Ramírez & Salas, 2019; Salas, 2019). Both eco-

systems are known for their species diversity,

regional endemism, and presence of threat-

ened species (Burneo et al., 2015), making

it necessary to implement study techniques

and tools that would allow identifying suit-

able habitats to improve conservation efforts

based on a more comprehensive understanding

of diversity, ecology, and behavior of species

(Torres-Domínguez et al., 2022). The use of

bioacoustics would help to partially solve this

problem, especially in ecosystems such as man-

groves, where the height of the canopy and the

difficulty of access to firm ground do not allow

the use of classic techniques such as mist net-

ting (Estrada-Villegas et al., 2018).

The use of complementary methods to

strengthen the ability to study and understand

bats is currently recognized (Mancini et al.,

2024). A holistic view of the bat commu-

nity can be achieved by combining different

recording and capture techniques, especially

in Neotropics where the insectivorous species

that are very difficult to capture with mist nets

converge with other species that are better rep-

resented with this capture method, such as the

frugivorous and nectivorous bats of the Phyl-

lostomidae family (Pech-Canche et al., 2011).

This, in turn, provides less biased information

on their ecology, life history, and other aspects

relevant to conservation (Carvalho et al., 2023;

Fenton et al., 1992). Additionally, the advantage

of using acoustic methods lies in the fact that

they are non-invasive and allow obtaining more

precise information on the relative activity and

richness of species (Barlow, 1999).

Nevertheless, the use of acoustic methods

applied to bat ecology and conservation poses

significant challenges for researchers given the

need for standardization of different proto-

cols and procedures for obtaining valuable

information, from capturing and handling the

specimens, to managing, systematizing and

analyzing the data on acoustic parameters from

the echolocation signal recordings. Nonethe-

less, the advancement of technologies for the

processing and analysis of acoustic data and the

automated identification of species (Cao et al.,

2023; Mora et al., 2002; Yoh et al., 2022), togeth-

er with the apparition of low-cost ultrasonic

detection devices, have allowed more affordable

and better quality recordings of bat echoloca-

tion signals, which in turn led to an increase in

the number of reference libraries and descrip-

tions of emission patterns of different bat

species in America, Europe, and Asia (Arévalo-

Cortés et al., 2024; Görföl et al., 2022; Rydell et

al., 2017; Zamora-Gutierrez et al., 2020).

Changes in echolocation signal emission

patterns and habitat use estimated from ultra-

sonic detection can be used to design and

implement conservation actions, since changes

in persistence, activity patterns, distribution, or

habitat use, and selection of insectivorous bats

can be estimated (López-Bosch et al., 2021).

However, to achieve it, it is essential to gener-

ate sound reference libraries for each species,

since there is an intrinsic variation in acous-

tic parameters due to sexual, developmental,

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

geographical or individual differences, among

others (Barclay & Brighman, 2004; Frick, 2013;

Jacobs et al., 2007; Jones, 1997; Jones & Hold-

eried, 2007).

In this work, the echolocation signals of

three species of insectivorous bats in Western

Ecuador are described: Neoeptesicus innoxius,

an endemic species shared between Western

Ecuador and Northwestern Peru (Cláudio et al.,

2023; Salas et al., 2023), and two species with a

wide geographical distribution, and with whom

N. innoxius frequently occurs on Santay Island:

Myotis nigricans and Molossus molossus. With

these descriptions, we seek to contribute to a

reference library of bat echolocation calls on

the Ecuadorian coast, that serves as a baseline

for future work that could prescind of direct bat

capture, while enabling researchers to identify

threatened and sensitive species in free flight.

MATERIALS AND METHODS

Study area: The field work was carried

out under the research permit MAAE-DBI-

CM-2022-0234 from the Ministry of the Envi-

ronment of Ecuador, in the Isla Santay and

Isla Gallo National Recreation Area, com-

monly called “Isla Santay”, created in 2010

and located between two highly populated

urban centers: Guayaquil and Durán, prov-

ince of Guayas, Western Ecuador. This pro-

tected area has an area of approximately 2 215

hectares, with an elevation that ranges from

zero to ten m.a.s.l., presenting a variety of

habitats: mangroves, dry forest, and grasslands.

The mangrove habitat is composed primar-

ily by species like red mangrove (Rhizophora

mangle); red crawling mangrove (Rhizophora

racemosa); red knight mangrove (Rhizophora

harrisonii); jelí mangrove (Conocarpus erec-

tus); black mangrove (Avicennia germinans)

and white mangrove (Laguncularia racemosa)

(Cruz-Cordovez, 2019).

Bat Capture: The capture of bats was

carried out during 30 nights between Sep-

tember 2019 and March 2022 (14 nights for

September-December 2019; five nights for

January-February 2020; four nights for Octo-

ber-December 2020; four nights for January-

February 2021; three nights for January-March

2022), sampling simultaneously each night at

four sites (Guayaquil-Durán route, Huaquillas

Trail, Guayaquil-Comuna Santay route, and

Ecoaldea) located along the mangrove edges

adjacent to open areas mostly comprised by

grasslands and wetlands, considering the pres-

ence of known hollowed trees that serve as

roosts. These sites were selected based on pre-

vious capture success at the general study area,

as they are almost entirely edge habitats laying

between highly cluttered spaces and open spac-

es, hence allowing for a better chance to capture

species that fly fast in open spaces, and species

with more maneuverable flights exploiting the

mangrove interior. At each sampling site, four

nylon mist nets (six m long x 2.6 m wide and

with a 38 mm mesh opening) were deployed

per night, remaining active from approximately

18:00 to 00:00, located at ground and sub-

canopy levels and separated from each other

at least by 200 m, in places near water sources

over the trails with open spaces along the man-

grove edges. Environmental variables such as

air temperature, wind speed, moon phase and

relative humidity were collected once per night

(data not provided). Once the individuals were

captured, the basic morphometric measure-

ments of each specimen were obtained for in

situ identification (Tirira, 2017), along with sex

and reproductive status.

Recording echolocation signals: An

Anabat Swift ultrasonic detector (Titley Sci-

entific, E.U.) was used to record the echolo-

cation signals from bats on hand release and

free flight, which were then analyzed with the

software BatSound 4.2.1 (Pettersson Elektronik

AB, 2013). The detector was configured to

record in continuous mode and Full Spectrum,

saving the files in WAV format, at a sampling

rate of 250 kHz, with the omnidirectional

ultrasonic microphone oriented at a position

of approximately > 90° with respect to the

ground and aiming towards the bats flight

path, with 5 ms minimum trigger time, 16 dB

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

sensitivity level, and with a recording trigger

configured at 10 kHz minimum frequency.

Once the recordings were obtained, individu-

als were released close to their capture site

(Martínez-Medina et al., 2021).

Data analysis; description of echoloca-

tion signals: Recordings were filtered with

SeaWave 2.0 (Centro Interdisciplinare di

Bioacustica e Ricerche Ambientali, 2013) to

separate recorded files containing noise from

files containing pulses or echolocation sig-

nals. The files containing the bat echolocation

signals to be described were then selected

based on its signal-to-noise ratio (S/N), only

keeping files whose signals had an overall

amplitude difference of at least 25 dB regarding

the background noise of each file. BatSound

4.2.1 was used to reconstruct the spectro-

grams based on a Fast Fourier Transforma-

tion (FFT) of 1 024 samples, allowing a better

resolution in the frequency domain, given the

specific need of separating two species with

overlapping bandwidths, reconstructing also

the power spectra which corresponds to the

energy profile of the component frequencies

of a given sound, yielding information on what

frequencies are concentrating the most energy

(Martínez-Medina et al., 2021).

Some search phase signals were selected

from the echolocation sequences obtained for the

three species, to measure the following param-

eters: Initial Frequency (FINI), Final Frequency

(FFIN) and Maximum Energy Frequency or

Peak Frequency (FME), and Bandwidth (AB,

subtracting the final frequency from the initial)

measured in kilohertz (kHz) and Pulse Duration

(DUR) and Interpulse Interval (IPI), measured

in milliseconds (ms) (Martínez-Medina et al.,

2021). The average value and its standard devia-

tion were calculated for all these parameters,

except for the IPI, for which the median was

calculated. The calculation of the median in this

case responds to the need to avoid biases in the

central distribution of the duration of the IPI,

since this can be overestimated when ignoring

the fact that sometimes aerial insectivorous bats

can omit the emission of a pulse while travel-

ing between sites, making the intervals between

emissions twice as long in such cases (Adams,

2017; Stidsholt et al., 2021) (Fig. 1).

Fig. 1. Acoustic parameters for the description of echolocation signals, adapted from “Standards for recording echolocation

signals and building bat reference libraries in Colombia” (Martínez-Medina et al., 2021).

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

Additionally, N. innoxius and M. nigricans

modulation rates were calculated for their two

main components: Modulated Frequency (FM)

and Quasi-Constant Frequency (QCF) by divid-

ing the bandwidth of each component by its

duration; where each component was separated

at the inflection point of the signal so that the

measurement of the FM structure was obtained

from the beginning of the signal to its inflection

point and the measurement of the QCF com-

ponent was taken from the inflection point to

the end of the signal. The inflection point fre-

quency is also compared for both species. The

modulation rate refers to the speed at which

the component frequencies of an echolocation

signal are changing, with high change speeds

associated with the emission of FM pulses, and

low change speeds associated with the emis-

sion of QCF pulses. Furthermore, when bats

combine both types of emissions (FM-QCF)

there is a point of transition between both com-

ponent structures, called the inflection point,

where the modulation rate changes abruptly

(Martínez-Medina et al., 2021).

Subsequently, a modulation speed propor-

tion was estimated between both components

to support qualitative descriptions of the pulse

shapes, where high modulation speed pulses

will have a preponderance of deep and very

fast modulation FM structures, being the most

prominent components of the signal. While

pulses with relatively lower modulation rates

will tend towards curvilinear shapes, and the

QCF components will be predominant within

each signal. Although the use of frequency

rate change measurements in echolocation sig-

nals is rare, understanding how the modula-

tion of these and their components change

is especially useful to classify their structure

(Redgwell et al., 2009), discriminate species in

different flight contexts (Limpens, 2004) and

understand the sensory capabilities and limita-

tions of the two species that, in this case, have

been referenced under the same conditions,

that is, subjected to the same structural com-

plexity of the flight space (edge of mangrove)

(Jones & Teeling, 2006).

Only echolocation signals with a high sig-

nal-to-noise ratio that allowed the signals of

interest to be separated from background noise

were included in the analyses; to do this, the

power spectrum of each recording fragment

with echolocation signals was examined, con-

firming that there was a difference of at least 25

dB between the power level of the background

noise and the power level of the signals to be

measured. As an exclusion criterion, recording

segments that did not exceed 25 dB differ-

ence between power profiles of the noise and

the signal of interest were omitted since the

measurement of characteristics such as inten-

sity or maximum frequencies may be affected

(Martínez-Medina et al., 2021).

Statistical analysis: In this study, the pres-

ence of N. innoxius and M. nigricans is reported

in the same acoustic monitoring and mist net

capture sites, so we consider it convenient to

make a comparison between the signals of these

species to set the precedent on the spectral

and temporal variables that characterize and

differentiate them, given that in some cases

variables such as the final frequency, the inter-

pulse interval and the frequency of maximum

energy of N. innoxius usually have values that

are distributed in the same range as those of the

same variables in M. nigricans. The statistical

computing language R (R Core Team, 2024)

was used to construct two-sample t-tests and

compare the different spectral and temporal

variables measured for N. innoxius and M.

nigricans, testing the null hypothesis that there

was no true difference between the means of

IPI, DUR, FINI, FFIN, AB, FME, frequency at

the inflection point of the signal, and modula-

tion rates of the FM and QCF components of

both species with a 95 % confidence interval.

In the same way, the hypothesis that there was

no true difference between the means of DUR

and the IPI of the high and low pulses of M.

molossus, as well as with the means of the AB

of both types of pulses, was tested through a

test Wilcoxon rank sum given the non-normal

distribution of the data.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

Additionally, given that M. molossus can

emit alternatively between low or high pulses,

we explored more about the variability that

each of these scenarios of alternation in the

emission of pulses can imprint on some vari-

ables such as the duration of these and their

intervals, testing whether the distribution of the

variances of the duration of the high and low

pulses and their interpulse intervals come from

the same distribution with an alpha of 0.05.

Finally, a post hoc Tukey HSD multiple means

comparison test was run at a 95 % confidence

level to determine which data groups differed

from each other.

RESULTS

Description of Neoeptesicus innoxius

echolocation signals: For this species, 163

signals from 11 search phase echolocation

sequences were measured. This species emit-

ted signals with prominent FM components

accompanied by a small QCF component at the

end of each pulse with nearly linear modulation

rates between 0.8-2.5 kHz/ms, and relatively

short durations varying from 5.9-7.7 ms. Its

FINI ranged between 77-61.6 kHz, and its FFIN

ranged between 51.8-46.5 kHz, resulting in

signals with bandwidths from 11.79-28.47 kHz.

The average MEF of N. innoxius echoloca-

tion signals was between 53.3-50.6 kHz, which

corresponds to the frequency range between

10.1-3.6 kHz above the inflection point fre-

quency, when the change in signal emission

from a FM structure to a QCF structure occurs;

although it also accumulated energy around the

inflection point in signals with slower modula-

tion. The IPI of the echolocation signals of N.

innoxius had a median duration of 93.5 ms,

being the longest of the two species of the Ves-

pertilionidae family described in the present

work (Table 1).

The average modulation speed of the FM

structure of N. innoxius echolocation signals

Table 1

Spectral and temporal parameters (mean ± standard deviation) extracted from the search phase echolocation sequences of

the three species of insectivorous bats studied on Isla Santay, Ecuador.

Family Species FINI

(kHz)

FFIN

(kHz)

FME

(kHz)

ABAND

(kHz)

DUR

(ms)

IPI

(ms) SPEED (kHz/ms)

Vespertilionidae M. nigricans

N = 6; n = 109

96.10 ±

8.68

48.31 ± 2.14 a) 53.55

± 2.67

b) 67.63

± 5.95

47.78

± 9.24

4.89

± 0.70

84.3

± 11.79

FM 14.08

± 2.95

QCF 6.25 ± 2.37

N. innoxius

N = 11; n = 163

69.33 ±

7.73

49.20 ± 2.64 51.99

± 1.30

20.13

± 8.34

6.82

± 0.88

93.5

± 12.78

FM 6.02

± 2.97

QCF 1.71 ± 0.84

Molossidae M. molossus

N = 7; n = 76

H: 43.70 ±

3.24

L: 39.02 ±

3.00

H: 36.81 ±

3.10

L: 35.56

± 2.20

40.69

± 3.38

37.38

± 2.87

6.89

± 1.90

6.46

± 1.90

9.82

± 2.32

0.38

± 1.97

H-H: 112

± 30.98

H-L: 136.6

± 34.38

L-H: 84.65

± 31.46

L-L: 81.6

± 36.52

FINI: initial frequency; FFIN: final frequency; FME: maximum energy frequency; DUR: pulse duration; ABAND:

bandwidth; IPI: interpulse interval; N: number of recordings/sequences analyzed; n: number of signals analyzed (hand

released and free flight). For M.nigricans, two maximum amplitude frequency values are reported, where a) corresponds to

the maximum amplitude frequency of the QCF component, and b) corresponds to the maximum amplitude frequency of the

FM component. The values of the alternating high (H) and low (L) signals of M. molossus are reported, as well as the specific

interpulse intervals for when the interval is produced between a pair of high pulses (H-H), a high pulse and a low pulse (H-L),

a low pulse and a high pulse (L-H), and two low pulses (L-L). Only the IPI values correspond to the median and standard

deviation. SPEED: modulation speed of an FM or QCF element within the same signal.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

ranged between 9-3 kHz/ms, while the aver-

age modulation speed of its QCF component

had a linear trend, giving a more curvilinear

and concave appearance to their echolocation

signals compared to those of M. nigricans,

which turned out to be more vertical than

curvilinear (Fig. 2).

Description of Myotis nigricans echolo-

cation signals: From this species, 109 search

phase echolocation signals were measured from

6 echolocation sequences that may or may not

contain approach and terminal phases. The M.

nigricans signals were formed by a nearly verti-

cal FM component that becomes curvilinear

after the second third of the signal, or the final

part of the signal that follows the inflection

point where the modulation speed is slowed.

Its FINI ranged between 87-104 kHz, and the

FFIN ranged between 46-50 kHz, resulting in a

usually broadband signal with a bandwidth of

38.5-57 kHz, and shorter durations (4.2-5.5 ms)

compared to N. innoxius (Fig. 3).

The average MEF of the slowest modulated

terminal part of the FM structure was between

50.8-56.2 kHz, while the average MEF of the

FM component ranged between 61.6-73.5 kHz.

In this way, M. nigricans accumulates the great-

est amount of its energy in the last three kHz of

the lower limit of the FM structure and towards

the inflection point between the two types of

structure. Furthermore, the greatest amount of

energy accumulated in the final third of the M.

nigricans signals was located between four-16

kHz after the signal inflection point. The IPI of

M. nigricans have a median duration of 84.3 ms,

which turns out to be the shortest of the two

species of the Vespertilionidae family described

here (Table 1).

The average modulation speed (kHz

change per unit time) of the FM component

of M. nigricans signals ranged between 17-11.1

kHz/ms, while the average modulation rate

of its QCF component varied between 8.6-3.8

kHz/ms, indicating that although there is a

two to three times reduction in the modulation

speed compared to the FM component, the

Fig. 2. Echolocation signals of N. innoxius from Isla Santay, Ecuador, which were reconstructed using Kaleidoscope Pro 5.6.8

(Wildlife Acoustics Inc., 2024) in a spectrogram of 1 024 points of FFT window size, with a Hanning window type and 85

% sample overlap. X-axis in milliseconds (ms); Y-axis in kiloheartz (kHz). The oscillogram is shown in red in the upper box

and the spectrogram in the lower box, with intensity (dB) on a color scale from green to orange (less intense to more intense

respectively). Values of intervals between pulses, durations and frequencies are shown as recorded in nature.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

modulation of the QCF component is still fast

enough with modulation speeds above linear

modulation (one kHz/ms), making its appear-

ance more vertical than diagonal.

Description of Molossus molossus echo-

location signals: For this species, 76 echoloca-

tion signals were measured from seven search

phase sequences, where approach and termi-

nal sequences were also present. This species

presents two types of signals: high and low,

both consisting of a prominent QCF element

that was sometimes complemented by a very

short FM element at the beginning or end of

the signals; The FINI of the high signal varied

between 46.9-40.4 kHz, and its FFIN ranged

between 39.9-33.7 kHz. The FINI and FFIN

of low signals were in a range between 42-36

kHz, and 34.7-30.3 kHz, respectively. Most of

the energy of M. molossus echolocation sig-

nals is held between 34-44 kHz, depending on

whether it is a high pulse or a low pulse. Both

types of signals also have a similar bandwidth,

ranging between four-eight kHz, with high

signals normally having relatively wider bands.

It should be noted that high and low signals can

overlap by two or three kHz in their bandwidth,

although their bandwidths do not differ from

each other (Wilcoxon rank sum test with con-

tinuity correction W = 789, p-value = 0.4593)

(Fig. 4).

No significant differences were found

(Welch Two Sample t-test t = -1.1153, df =

74, p-value = 0.2683) between the mean dura-

tions of high and low echolocation signals of

M. molossus, showing similar ranges. (7.5 ms

to 12.1 ms, or 8.4 ms to 12.3 ms respectively),

although the duration of low signals tends to

extend almost 1 ms longer compared to the

duration of high signals. The IPI of the M.

molossus signals differ (Wilcoxon rank sum

test with continuity correction W = 1 028.5,

p-value < 0.01), and can be shorter or longer

depending on the measured pair of pulses: a

high and a low pulse, vice versa, two high pulses

or two low pulses. The longest intervals have

been observed from high to low signals (maxi-

mum 170.9 ms), while the shortest intervals

Fig. 3. Echolocation signals of M. nigricans from Isla Santay, Ecuador, which were reconstructed using Kaleidoscope Pro

5.6.8 in a spectrogram of 1 024 points of FFT window size, with a Hanning window type and 85 % sample overlap. X-axis in

milliseconds (ms) and Y-axis in kilohertz (kHz). The oscillogram is shown in red in the upper box and the spectrogram in

the lower box, with intensity (dB) on a color scale from green to orange (less intense to more intense respectively). Values of

intervals between pulses, durations and frequencies are shown as recorded in nature.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

are observed between two low pulses (mini-

mum 45 ms), or from a low pulse to a high

pulse (p < 0.01).

Although the paired test based on the type

of pulse (high and low) did not show significant

differences in the average duration of the puls-

es, the post hoc test showed that the duration

of a high pulse when followed by another high

pulse is significantly shorter than the duration

of the same high pulse when it is followed by

a low pulse (p < 0.005). Similarly, the band-

width of the low pulses when followed by other

low pulses was significantly narrower than the

bandwidth of the high pulses that are followed

by other high pulses (p < 0.05).

Differences between N. innoxius and M.

nigricans echolocation signals: After compar-

ing the set of values of each variable describing

the echolocation behavior of N. innoxius and

M. nigricans, we found that IPI (t = 5.6833,

df = 243.74, p-value = < 0.001), duration (t =

19.873, df = 261.21, p-value < 0.001) and FFIN

(t = 4.7223, df = 155.29, p-value < 0.001) of N.

innoxius echolocation signals were significantly

higher in comparison to those of M. nigricans

signals. In contrast, the bandwidth (t = -25.026,

df = 215.01, p-value < 0.001), FINI (t = -25.905,

df = 212.63, p-value < 0.001), MEF (t = -5.6356,

df = 142.82, p-value = < 0.001), and the modu-

lation rates of the FM (t = -21.882, df = 232.68,

p-value < 0.001) and the QCF (t = -19.043, df

= 126.42, p-value < 0.001) components of M.

nigricans were significantly higher compared to

those of N. innoxius (Fig. 5).

DISCUSSION

The present study constitutes the most

recent contribution towards the construction

of a regional and national reference library of

echolocation signals, adding three more species

to the list of insectivorous bats inhabiting the

lowlands of Western Ecuador. But more impor-

tantly, it provides the first acoustic description

of N. innoxius, an endemic species distributed

in the region comprised between Southwest

Ecuador and Northwest Peru, which is listed as

Fig. 4. Echolocation signals of M. molossus from Isla Santay, Ecuador, which were reconstructed using Kaleidoscope Pro

5.6.8 in a spectrogram of 1 024 points of FFT window size, with a Hanning window type and 85 % sample overlap. X-axis in

milliseconds (ms) and Y-axis in kilohertz (kHz). The oscillogram is shown in red in the upper box and the spectrogram in

the lower box, with intensity (dB) on a color scale from green to orange (less intense to more intense respectively). Values of

intervals between pulses, durations and frequencies are shown as recorded in nature.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

Vulnerable (VU) in the Red List of Mammals of

Ecuador (Tirira, 2021), and as Near Threatened

(NT) in the IUCN red list of species (Velazco

& Aguirre, 2020), being rarely captured in the

study area where this research is carried out, the

coastal lowlands.

The species studied here have been report-

ed sharing roosts or have been captured simul-

taneously in the same mist nets in the Guayas

region, Western Ecuador (Linares & Zabala,

2018; Salas et al., 2014; Salas et al., 2023) and

are part of the same soundscape of Isla Santay.

Hence, we consider it relevant to include a

detailed description of their emission patterns,

even though M. nigricans and M. molossus are

commonly recorded in Neotropical bioacous-

tics studies (Arévalo-Cortés et al., 2024; Barbo-

sa et al., 2022). This is in response to the need of

having enough diverse references for estimating

how different sources of variation shape the

echolocation signal designs that each insectivo-

rous bats species employs, with the geographi-

cal variations and local spatial context being

two of the most relevant factors altering their

emission patterns (Barclay & Brighman, 2004;

Schnitzler & Kalko, 2001).

As observed here, some spectral and tem-

poral features in the signals of N. innoxius and

M. nigricans tend to overlap, hence it would

be useful to understand the differences that

separate the signal designs of the two species

to avoid potential identification errors and

the subsequent generation of unreliable data,

which can have serious consequences for spe-

cies conservation (Russo & Voigt, 2016). Both

N. innoxius and M. nigricans exploit the same

habitat, specializing in forest edges, and exhib-

iting a convergent echolocation signal design,

based mostly on FM signals, suited for render-

ing high spatial resolution at short distances

(Jones & Holderied, 2007; Siemers et al., 2001;

Schnitzler & Kalko, 2001), therefore the differ-

ences on the modulation rates of the FM and

QCF components of these signals could be

useful for separating both species, also having

the potential to shed light on the resource par-

titioning experienced by these species along the

mangrove edge at Isla Santay.

Even when they can overlap in the edge

area for which both species emit signals of rela-

tively broad band, the faster modulation of M.

nigricans signals enables it to hunt for insects

Fig. 5. Comparison of spectral and temporal parameters between N. innoxius (grey) and M. nigricans (orange). Boxes

represent Q1 and Q3, the thick line represents median, whiskers represent maximum and minimum values, diamonds inside

the boxes represent the mean, and black dots represent randomly jittered data points, allowing a better view of overlapping

points distribution.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

closer to the clutter of the mangrove (~ 70 cm

for a signal with a minimum duration of 4.19

ms, Table 1) compared to what N. innoxius can

achieve with an echolocation signal of slower

modulation and longer duration (~1.02 m for

a signal with a minimum duration of 5.94 ms,

Table 1) without experiencing backward mask-

ing, which happens when the echoes from the

background clutter engulf those of the prey that

sits on or fly near it, rendering it impossible

for the bat to separate the relevant informa-

tion from the background noise (Schnitzler &

Kalko, 2001).

On the other hand, the signals of N. innox-

ius present a QCF structure which modulation

speed ranges from 0.8 to 2.5 kHz/ms resulting

in an elongated component that concentrates

the vast majority of signal’s energy in a narrow

band of frequencies, thus having the potential

to travel further in open spaces adjacent to

border habitats were the faint signal of M. nigri-

cans would decay in intensity rendering poorer

information with greater distances, enabling N.

innoxius to exploit open grasslands and wet-

lands more efficiently (Stilz & Schnitzler, 2012).

The emission pattern described here for

M. nigricans mostly agrees with those reported

in previous studies, although with variations

in relevant features such as signal shape and

final frequency. These differences could result

from geographical, individual or flight context

variations, since M. Nigricans is an edge spe-

cialist that can move between open areas and

cluttered sites, changing the modulation rate of

the FM and QCF components of its echoloca-

tion signals as seen in this study, being able to

modulate the QCF component speed between

3-8 kHz/ms (Arias-Aguilar et al., 2018; Kraker-

Castañeda et al., 2018; Surlykke & Kalko, 2008).

Although the spectral and temporal values

described here for M. molossus agree with oth-

ers reported for the same species, the compari-

son between its alternating pulses has not been

approached until this study. Even though, this is

a widely referenced species, and the alternation

pattern of its echolocation signals is already

well known (Arias-Aguilar et al., 2018; Jung et

al., 2014; Kössl et al., 1999; Mora et al., 2004),

there is still a lack of clarity about the varia-

tion of temporal features between high and

low pulses. Current literature does not address

different signal emission patterns between dif-

ferent types of pulse pairs (high-low, low-

high, high-high, low-low) and only reports two

interval values, one for low pulses and one for

high pulses (Arévalo-Cortés et al., 2024). As

illustrated here, some traits vary between the

different combinations of high and low pulses

that could help better describe and identify

this species.

Finally, Isla Santay is recognized as an

Important Area for Bat Conservation or

AICOM (Salas, 2022), so the records collected

will be used as a reference library, which will

be a key tool for the conservation of this group

by facilitating the operation of monitoring pro-

grams. For instance, we now acknowledge that

N. innoxius can exploit open areas besides the

mangrove interior and edges, hence a monitor-

ing program dedicated to this species should

include all the potential habitats for foraging

and commuting like grasslands and wetlands,

in addition to only focusing monitoring efforts

over mangroves (Zamora-Gutierrez et al., 2016).

Nonetheless, while the references for N. innox-

ius presented in this work were acquired over a

spatial context dominated by mangrove edges,

we urge that more references in other spatial

contexts need to be added to the repertoire of

this species, since the combination of the FM

and QCF components of its signals reflect a

higher vocal complexity that will not be enough

well represented by references from edge habi-

tats only (Martínez-Medina et al., 2021).

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025704, enero-diciembre 2025 (Publicado Set. 18, 2025)

ACKNOWLEDGMENTS

The authors would like to thank the Santay

Island National Recreation Area, especially the

administrator, park rangers and community

for their support and accompaniment during

the field phase. They would also like to thank

Gabriela Gonzalez Olimón for her collabora-

tion in the translation and Luciana Carrera for

her collaboration. Funding: This research is

part of the “Programa Biodiversidad Sostenible

del Manglar al Coral 2021-2050”, funded by the

Universidad de Especialidades Espíritu Santo,

through its Research Center. Under the con-

tract code: MAAE-DBI-CM-2022-0234.

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