Second capture of Promops centralis (Chiroptera) in French Guiana after 28 years of mist-netting and description of its echolocation and distress calls

The Amazonian basin harbours some of the most bat-diverse ecosystems worldwide. Yet, information on elusive, high-flying bat species such as Molossidae is scarce or virtually missing in the literature, which hampers conservation efforts both locally and globally. The recent advent of new technologies specifically designed to survey bats, such as passive ultrasound detectors and acoustic lures, has significantly increased understanding of bat ecology and distribution, and has allowed researchers to gather new and valuable information which was impossible to collect in the past. We undertook a rapid bat diversity assessment in French Guiana using acoustic lures to aid in capturing high-flying insectivorous bat species. Here we report the second and third capture record of Promops centralis (Chiroptera, Molossidae) for French Guiana, captured after 28 years since the first and only captures so far in the county. One individual was a post-lactating female and represents the first record of breeding P. centralis in French Guiana. We provide (i) morphometric and acoustic data (including the species’ distress calls) as well as detail photography to aid in species identification; and (ii) COI and CytB sequences of the two individuals (first mitochondrial sequences for French Guiana).


INTRODUCTION
Harbouring a rich diversity of habitats, including the tropical Amazonian forest, French Guiana represents a hotspot of bat diversity. Currently, 106 bat species from nine families are listed in this county (Catzeflis et al. 2013;Moratelli et al. 2015;Rufray 2015). The advent of new techniques for surveying bats such as the use of bioacoustics is currently pushing forward our understanding of bat ecology and distribution (Britzke et al. 2013;Gibb et al. 2019), allowing the discovery of new species and opening ecological research questions and fields that were entirely inaccessible in the past.
The most common survey technique in bat studies used in the tropics is the ground mist-netting, followed by canopy netting, and direct roost search (MacSwiney et al. 2008). Nevertheless, the use of acoustic studies is increasing in the Neotropics (Barataud et al. 2013) due to the technological advances in new affordable detectors and automatic classifiers (Hill et al. 2019). While mist-netting has been proved to be highly efficient for phyllostomid bats, bioacoustics seem to be more appropriate for aerial insectivorous bats such as molossids or emballonurids (MacSwiney et al. 2008). More recently, the development of ultrasonic acoustic lures to attract bats to the mist-nets has gained momentum amongst bat researchers and naturalists (Quackenbush et al. 2016;Samoray et al. 2019), especially for targeting elusive insectivorous species. Although their effectiveness has been proven in different contexts and for several species (Hill and Greenaway 2005;Lintott et al. 2013;Quackenbush et al. 2016), acoustic lures have been rarely used in the tropics (Chaverri et al. 2018).
Amongst all bat families that occur in the Neotropics, Molossidae is one of the least known, probably due to the difficulty of capturing molossids using traditional sampling methods (Kalko et al. 2008). These species roost in relatively inaccessible crevices or cavities and forage at high altitudes above forest canopies, in open areas and over aquatic habitats, where mist-netting is either impossible or inefficient Torrent et al. 2018). As a result, natural history data for these species are generally scarce or virtually missing in the literature. Among molossids, the big crested mastiff bat, Promops centralis is one of the most elusive and less studied species (Hintze et al. 2019). As many other molossids, P. centralis seems to be a habitat-opportunist, having been reported in a wide range of environments such as forests, wetlands, deserts, and urban areas (Gregorin and Taddei 2000;Lim and Engstrom 2001;Kalko 2010, 2011;Fischer et al. 2015;González-Terrazas et al. 2016;Hintze et al. 2019). In terms of echolocation, P. centralis is easily recognizable, with alternate concave-convex pairs of pulses produced at ~30 and ~35 kHz, respectively (López-Baucells et al. 2016;Arias-Aguilar et al. 2018).
We undertook a rapid bat diversity assessment in French Guiana using mist nets and acoustic lures, with the main aim of improving the natural history understanding of molossid bats. Here we report the second and third capture records of P. centralis for French Guiana, captured after 28 years since the first and only captures in the county so far by Simmons and Voss (1998). We present data on the morphology, echolocation (including distress calls), and mitochondrial cytochrome oxidase I (COI) and cytochrome b (CytB) gene sequences of P. centralis from French Guiana.

Sampling design
We performed a total of 14 consecutive nights of bat trapping with mist nets between the 8 and 21 November 2019 at 14 sites ( Figure 1; see details in the Supplementary Material, Table S1). At each site, we installed between two and five nets of 12 m length and 2.5 m height (Ecotone, Gdynia, Poland), and trapping took place from sunset for four hours. Each survey night, three mist nets were equipped with an acoustic lure (Bat Lure, Apodemus, Wageningen, The Netherlands) broadcasting social and distress calls (see Chaverri et al. 2018) of bats from four different families: Phyllostomidae, Vespertillionidae, Molossidae and Emballonuridae.

Morphological, acoustic and genetic analyses
Mist nets were checked every 20 min, and bats were removed from the nets as soon as possible. Species were identified using identification keys from López-Baucells et al. (2016) and Charles-Dominique et al. (2001). Information on sex, age (juvenile or adult), reproductive status (not reproductively active, reproductively active, lactating female, or pregnant female), body mass, and forearm length were taken for all captured individuals. Sex and reproductive status were assessed by inspecting genitalia, while age was determined by trans-retro-illuminating wing joints (Anthony 1988). Bat capture and handling were conducted following guidelines approved by the American Society of Mammalogists (Sikes and Gannon 2011). To confirm species identification of individuals with ambiguous morphological criteria, one to two wing punches were taken per individual and were subsequently processed and analysed to obtain mitochondrial genetic classification (hereafter referred as DNA barcoding). When the acoustic repertoire of a species was poorly described in the literature, we recorded echolocation calls while releasing individuals, either during the night if the acoustic activity of free-flying bats was very low, or at sunrise of the same night to avoid the simultaneous presence of other individuals. Different ultrasound microphones were used for the recordings: M500-384 (Pettersson, Uppsala, Sweden) and Echo Meter Touch Pro (Wildlife Acoustics, Maynard, USA). Releases were performed in open environments (forest clearings or wide roads). We also recorded distress calls when an individual was handled. Lactating females were always released at night. VOL. 50(4) 2020: 327 -334 ACTA AMAZONICA Echolocation and distress calls of the two P. centralis individuals captured were analysed using Kaleidoscope (Wildlife Acoustics, USA) with a 512-Hanning FFT window. For each individual, we measured the frequency of maximum energy of ten good-quality echolocation calls. The first series of pulses recorded during the release were discarded as they were not truly representative calls. Distress calls were only recorded from the male while handled. Measurements such as call duration and frequency of maximum energy of harmonics were conducted on 19 good-quality calls.
Genetic analyses were conducted at the Department of Microbiology and Immunology at KU Leuven (Leuven, Belgium). Mitochondrial DNA from both individuals was extracted from muscular tissue (wing punches) using total nucleic acid extraction with RNAeasy mini kit (Cat No. 74104, Qiagen, USA), following specification from the manufacturer. Both COI and CytB sequences were amplified using One-Step RT-PCR kit (Cat No. 210210, Qiagen, Germany), following specifications from the manufacturer. Primers Molcit-F AATGACATGAAAAATCACCGTTGT and MVZ-16 AAATAGGAARTATCAYTCTGGTTTRAT were used to amplify CytB (Ibáñez et al. 2006) and UTyr 5'-ACCYCTGTCYTTAGATTTACAGTC-3' and C1L705 5'-ACTTCDGGGTGNCCRAARAATCA-3' COI (Hassanin et al. 2012). Thermocycling was conducted as follows: 15 min at 94°C, 30 sec at 94°C, 30 sec at 55 °C, 1 min at 72 °C and 10 min at 72°C. Molecules were amplified for 40 cycles (a cycle corresponding to a repetition of the underlined temperatures and times). Amplicons for both individuals were purified using ExoSAP-IT (Cat No. 78201.1, Thermo Fisher Scientific, USA) and sequenced by Sanger sequencing (Macrogen Europe, Meibergdreef, Amsterdam-Zuidoost). Chromatograms were assembled with UGENE v1.31.0 and manually corrected. Sequences are deposited in GenBank as

RESULTS
We captured 450 individuals belonging to 40 bat species (overall results of the survey are available in www.fauneguyane.fr), including two individuals of Promops centralis. The identification was primarily based on morphological features, and was subsequently confirmed with acoustic and genetic analyses. The first individual was captured on 8 November at 18:25 in a net placed along a swimming pool located in a private garden of Remire-Montjoly (Figure 1). It was a nonreproductively active male with a forearm length of 51.8 mm, for which no other morphological features were measured. It was released at the same location. We captured the second individual on 21 November around 20:20, 131 km away from the first location, in a net placed over a shallow water body located near a secondary road westward of Iracoubo (Figure 1). This individual was captured in front of a bat lure playing distress and social calls of several molossid species, including distress calls recorded from the first P. centralis we captured. The individual was a post-lactating female and therefore represents the first report of breeding P. centralis in French Guiana (Figure 2). All the external morphological measurements have been included in Table 1.
Regarding their echolocation, both individuals presented the typical acoustic features of P. centralis, with alternate concave-convex pairs of modulated pulses. When hand-   Simmons and Voss (1998) Figure 3; available at https://sonotheque.mnhn.fr/sounds/ MNHN/SO/2020-192 and https://sonotheque.mnhn.fr/ sounds/mnhn/so/2020-174). The most common distress calls of P. centralis consisted of long frequency-modulated calls (mean ± SD: 114.5 ± 52.5 ms; 19 calls), with several harmonics with mean frequency of maximum energy at 15.8 (SD 2.9), 30.7 (SD 5.5), 44.3 (SD 4.4) and 53.6 (SD 6.2) kHz ( Figure 3; sample MNHN-SO-2020-193 available at https:// sonotheque.mnhn.fr/sounds/MNHN/SO/2020-193). These structures are described by Nagel (2006) as tonal calls, more specifically trills (> 3 frequency modulations, with irregular patterns), and could appear noisy. Reconstruction of both COI and CytB sequences of the two individuals from DNA barcoding yielded in two diagnostic sequences of 655 and 784 nucleotides, respectively. All four sequences were analysed with BLAST (Mega-Blast) (Camacho et al. 2009) using the complete NCBI nucleotide database (accessed on April 2020). All sequences were affiliated to Promops centralis with an average nucleotide identity > 99% (Table 2). Controversially, the COI sequence from the female individual (GenBank accession number MT350797) had Promops davisoni as one of its closest references (GenBank accession number MH185193.1). Detailed inspection of secondary hits of MT350797 against NCBI Blast database showed comparable identities with Promops centralis. However, the CytB sequence from the same individual (GenBank accession number MT350796) was classified as Promops centralis (closest reference as GenBank accession number MG029508.1). Based on similarity results from CytB and COI sequences, it can be assumed that both individuals belong to the Promops centralis, concording with our previous assessment (Table 2).

DISCUSSION
Using an integrative approach combining morphological, acoustic and genetic data, we confirmed the presence of Promops centralis in French Guiana, adding the second and third capture records for this county after more than 28 years from the first captures of this species (Simmons and Voss 1998). We captured one male and one post-lactating female in two sites located 131 km apart and situated > 80 and > 45 km away, respectively, from the site where the first capture of P. centralis was made. Our capture records contribute to improve current knowledge of P. centralis distribution in French Guiana. At a larger scale, our records fall into the potential but poorly known distribution range of P. centralis in South America recently modelled by Hintze et al. (2019).
Mist-net surveys have been intensively conducted in French Guiana since the 1990s, especially by Charles-Dominique et al. (2001) and through numerous research projects (e.g. Thoisy et al. 2014;Moratelli et al. 2015;Catzeflis et al. 2016;Lavergne et al. 2016;Filippi-Codaccioni et al. 2018). Yet it was only from 2011, with the advent of  bioacoustics, that P. centralis was registered again using bat detectors (e.g. Barataud et al. 2013). The 25 acoustic records so far suggest that P. centralis is well distributed in French Guiana (Figure 1; www.faune-guyane.fr) and present in many habitats, from tropical rainforest to marshes and lowland wetlands. Nevertheless, despite the relatively high capture effort and, more recently, the use of new capture techniques (e.g. canopy nets) the enigmatic and poorly studied P. centralis has remained uncaught in French Guiana for nearly three decades.
Our results are of great interest considering the rarity of the mentions of this species in French Guiana and the difficulties of capturing high-flying foragers. It is likely, albeit not evidence-based, that the second P. centralis was attracted to the net due to the distress calls of the first individual, that were broadcasted by the acoustic lure. We provide the first recordings of distress calls for this species and make them available on open access, so other researchers can use them in the field. Comprehensive bat call libraries are essential to improve bat sampling efficacy and will probably drive the future of both acoustic and mist-netting bat surveys worldwide (Waters and Gannon 2004;López-Baucells et al. 2019). Further work is needed to confirm whether distress calls of P. centralis indeed attract conspecifics, as observed in other species (Chaverri et al. 2018).
Promops centralis may be more common than previously expected in Central and South America (Hintze et al. 2019), and several factors can explain the lack of records in the literature. Firstly, this species is difficult to capture using ground-nets due to its foraging behaviour. Optimizing net configuration and location may, however, help in increasing the capture rate (Trevelin et al. 2017). In this study, the two individuals of P. centralis were captured in nets placed across a shallow water body in open spaces. While high-flying species are adapted to foraging above canopy levels, they descend to drink, and setting mist-nets across water bodies located in open space represents a better option than setting-up nets above the canopy. Besides, few acoustic surveys are conducted in the tropics, especially in the Amazon (MacSwiney et al. 2008;López-Baucells et al. 2016). More information about the species distribution range could easily be gained by deploying more detectors in suitable habitats. Moreover, it is likely that the species is commonly confused by bat researchers and naturalists with other widespread molossid species that present similar morphology, such as Molossus rufus (Simmons and Voss 1998). It is therefore of utmost importance to generate high-quality taxonomic affiliation of unique captures, as was achieved in this study.

CONCLUSIONS
Our reports of P. centralis constitute the most updated information for the species in French Guiana. We present morphological, acoustic, and genetic baseline information that will hopefully optimize the output of future bat research in the Neotropics -including genetic mitochondrial sequences and raw acoustic data -all of them usually scarce in the literature. The lack of genetic and acoustic references still represents an important obstacle that hinders bat research and conservation in the tropics. Further research is needed to understand the potential of acoustic lures to attract and capture elusive, highflying bat species.