Bat acoustic libraries are important tools that assemble echolocation calls to allow the comparison and discrimination to confirm species identifications.  The Sonozotz project represents the first nation-wide library of bat echolocation calls for a megadiverse country.  It was assembled following a standardized recording protocol that aimed to cover different recording habitats, recording techniques, and call variation inherent to individuals.  The Sonozotz project included 69 species of echolocating bats, a high species richness that represents 50% of bat species found in the country.  We include recommendations on how the database can be used and how the sampling methods can be potentially replicated in countries with similar environmental and geographic conditions.  To our knowledge, this represents the most exhaustive effort to date to document and compile the diversity of bat echolocation calls for a megadiverse country.  This database will be useful to address a range of ecological questions including the effects of anthropogenic activities on bat communities through the analysis of bat sound.

Geographic and environmental coverage

We selected multiple localities scattered across Mexico to maximize the number of species included in the database.  We divided the Mexican territory into eight study regions based on topography, environmental complexity and the collaboration of bat experts working in each region: 1) Californian region (Baja California, Baja California Sur and Sonora); 2) Northeast region (Durango, Sinaloa and Chihuahua); 3) West region (Colima, Nayarit and Jalisco); 4) East region (Puebla, Tlaxcala and Veracruz): 5) North center region (Aguascalientes, Guanajuato, San Luis Potosí, Nuevo León and Zacatecas); 6) South center region (Estado de México, Morelos, Hidalgo and Querétaro); 7) Southeast region (Campeche, Quintana Roo and Yucatán); and 8) Southwest region (Chiapas, Oaxaca and Tabasco). Number of individuals recorded per species in each region is presented in Table 1. Based on this organization, we sampled in 27/32 (84%) of the Mexican states and six out of the seven ecoregions (defined as geographically distinctive areas containing a group of natural communities that share most of their species, environmental conditions and ecological dynamics- Challenger & Soberón, 2008) that have been defined for the Mexican territory by the National Commission for the Knowledge and Use of Biodiversity (CONABIO) (Figure 2, INEGI, CONABIO & INE, 2008).  The localities sampled covered an altitudinal gradient ranging from sea level to 3600 m.a.s.l., and a great variety of ecosystems ranging from the northern xerophytic shrub lands to the southeastern tropical forests.  

Capture, handling and sampling of bats

For each individual, we recorded sex, age (juvenile or adult), reproductive status (females: inactive, pregnant, or lactating; males: abdominal, inguinal or scrotal testes), and standard morphometric measurements (forearm length, head and body length, tail length, and body weight).  Individual bats were photographed in standard formats and angles to provide support for posterior taxonomic identification and to create a photographic library of Mexican insectivorous bats.  In addition, we obtained a small wing biopsy (diameter = 2 mm) stored in 96% ethanol to also serve as genetic reference material for future studies, in the case that intraspecific acoustic variation can hint to the presence of cryptic species. 

When identification certainty was < 80% (based on the judgment of the most experienced collector), a voucher specimen was collected to confirm its identity based on cranial and postcranial characters and measurements.  All handling and sampling procedures followed ethical recommendations provided by Sikes and the Animal Care and Use Committee of the American Society of Mammalogists (2016).  This project had collection permits (SGPA/DGVS/05867/16, SGPA/DGVS/07291/17) issued by the Secretaría de Medio Ambiente y Recursos Naturales to M. Briones-Salas.

Individual data and biopsy samples were labeled with unique consecutive numbers which kept information on the region, site, and locality and will be freely available to researchers together with the acoustic material.  Collected tissues were deposited in the Regional Collection of Durango (Mammalia), at CIDIIR-Durango, and voucher specimens were deposited at the Mammalogy Collection, CIB at Centro de Investigaciones Biológicas del Noroeste (CIBNOR).

Recording of bat echolocation calls

The ultimate purpose of the Sonozotz project was to build a reference call library that could be used to identify free-flying bats while foraging or commuting under natural conditions. Therefore, we aimed to record search calls under the conditions most commonly encountered by the species depending on their traits, habits, and behavior.  Species-specific recommendations included recording mode: 1) hand release at ground level: bats were released from 1.5-2 m from the ground; 2) hand release at heights > 5 m; 3) flight cage: rooms or enclosures that allowed the bat to fly, we used this technique for species low intensity calls (e.g. Lampronycteris brachyotis, Lophostoma brasiliensis); 4) zip-lining: bats are attached to a 2-m length of small elastic cord by a loose-fitting loop of the cord pulled over the bat’s foot, the other end of the elastic cord is attached via a small snap swivel to 30–50 m of taut monofilament line about 1 m above the ground; 5) inside the bag; 6) take-off flight from perch, distance to microphone (0.5 m, 0.5-1 m, 5 m, or > 10 m). We also described the recording environment (stationary inside bag, closed, edge, or open).  Bat echolocation calls were recorded immediately after processing individuals, which were afterwards released on site.  All bats were recorded in real time with broad-band bat detectors (Avisoft UltraSoundGate 116H; Avisoft Bioacoustics, Glienicke, Germany), coupled with a sensitive condenser microphone (CM16/CMPA; Avisoft Bioacoustics, Glienicke, Germany) through a XLR-5 cable, and a laptop Dell Inspiron 7348 (Dell Inc.) running the software Avisoft-RECORDER (Avisoft Bioacoustics, Glienicke, Germany) through a USB cable.  Recording settings were fixed on the software using standard parameters.  We recorded in channel 1 without going over 15-sec per recording sequence employing a sampling rate of 300 kHz, a sample resolution of 16 bits, and a high-pass filtering of 4 kHz.  We named files directly on the software window typing the unique codes previously assigned that preserved information on the region, site, locality, date, time, and individual.  Complementary information was recorded on channel 2 (voice notes) to store information on environmental conditions, recording mode, and any other significant information for the recording output.

Sound analyses

We used BATSOUND PRO v.4.21 (Pettersson Elektronik AB, Uppsala, Sweden) to visually inspect all recorded sequences and remove those recordings that had: a) non-search-phase calls, b) calls not belonging to the targeted species, and c) low signal-to-noise ratio.  We distinguished search phase calls from approach-phase and social calls by their duration, frequency and pattern of change over time. 

The file consists in a spreadsheet (Metadata) that contains detailed information of each recording. Field content are described as follows:  

Individual Id: ID number of the individual recorded

Recording ID: ID number of the echolocation file

Call Library Short name: Indicates the acronym of the name of the Acoustic Library 

Call Library Full Name: Indicates the full name of the Acoustic Library

Call Library City: Indicates the city where the Acoustic library is hosted

Call Library Country: Indicates the country where the Acoustic library is hosted

Call Library Institution Short name: Indicates the acronym of the Institution where the Acoustic library is hosted

Call Library Institution Full Name: Indicates the full name of the Institution where the Acoustic library is hosted

Observers: Names of the people involved in the recording of the bats 

Field trip group: Names of the people involved in the field trip session 

Date: Day of recording

Month: Month of recording

Year: Year of recording

Country: Country where the recording was made

State: State where the recording was made

Municipality: Municipality where the recording was made

Locality: Locality where the recording was made

Datum: Datum of the recording locality 

UTM Zone: UTM zone of the recording locality

UTMX (m): UTMX of the recording locality

UTMY (m): UTMY of the recording locality

Geo Method: Georeference method used

GPS Model: GPS model use to fix the recording locality

Accuracy (m): Accuracy of the fixing of recording locality

Altitude (m): Altitude of the recording locality provided in meters above sea level

Vegetation Type (in Spanish): Type of vegetation where the recording was made Description is in Spanish

Ecoregion (In Spanish): CONABIO’s ecoregion where the recording was made Description is in Spanish

Family: Family of the individual recorded

Genus: Genus of the individual recorded

Species: Species of the individual recorded

Sex: Sex of the individual recorded 

Age: Age of the individual recorded 

Forearm length (mm): Forearm length of the individual recorded 

Weight (gr): Weight of the individual recorded

Reproductive status: Reproductive status of the individual recorded

ID Certainty: Identification certainty based on the judgment of the most experienced collector, value is in percentage

ID Photograph: Identification code of the photograph of the individual recorded. Letters indicate the author of the photograph.

Tissue's collection name: Name of the collection where the tissue is deposited

Tissue's collection number: Number of the collected tissues

Voucher's collection name:  Name of the collection where the voucher is deposited

Voucher's collection number: Number of the collected voucher

Recording technique: Technique used to record the individual. This was decided based on literature information and personal experience taking in consideration the flight and echolocation attributes.

Recording environment: Characteristics of the environment where the individual was recorded

Flight height (m): Flight height of the bat when recorded. Value 9999 stands for 0 m, when individuals were recorded inside a bag

Ultrasound Conversion Technique: Ultrasound Conversion Technique used to record the individual

Sampling rate (kHz): Sampling rate used to record the individual. For this project we employed a sampling rate of 300 kHz

Sampling resolution (bits): Sampling resolution used to record the individual. For this project we employed a sampling resolution of 16 bits 

High-pass filtering: Filter resolution used to record the individual.  For this project we employed a high-pass filtering of 4 kHz

Expansion factor: Expansion factor used to record the individual. For this project we employed a factor of 1 (real time)

Detector Brand: Brand of the bat detector

Detector Model: Model of the bat detector

Type of microphone: Type of microphone used to record the individual

Brand and model sound card: Brand and model sound card used to record the individual

Recording unit: Recording unit used to record the individual

Storage: Type of storage used to save the recording of the individual

Habitat description (in Spanish): Detailed description of the characteristics of the locality where the individual was captured and recorded. Information is provided in Spanish 

The metadata will be also hosted by CONABIO through a public web app (selia.conabio.gob.mx) to be launched by mid 2020. This app will provide access to recordings and their metadata for consultation and download. It will also provide tools for visualization and annotation of acoustic data as well as access to other open ultrasonic and audible materials.