Occurrence of Sturnira tildae De La Torre, 1959 (Chiroptera: Phyllostomidae) in the state of Maranhão, Brazil

The bat genus Sturnira is widely distributed in the Neotropical region, from northwestern Mexico to northern Argentina, and four species occur in Brazil: Sturnira lilium, Sturnira giannae, Sturnira magna, and Sturnira tildae. The present study is the first to record Sturnira tildae in the state of Maranhão, Brazil, based on morphological and molecular diagnoses. The specimen was identified based on its cranial and morphometric traits. The diagnostic traits include discreetly bilobed inner upper incisors with a broad base, lower first and second molars with lingual cusps separated by shallow grooves, and forearm longer than 45 mm. The molecular sequences of Cytochrome C Oxidase Subunit 1 (COI) and 16S rRNA genes confirmed the morphological identification and thus the occurrence of Sturnira tildae in the Amazon biome of Maranhão. This record represents an eastward extension of the known distribution of the species in the Amazonia, to Cândido Mendes, Maranhão, within an area dominated by dense rainforest and influenced by tides.


INTRODUCTION
Brazil has is the country with the third richest bat fauna in the world, with 181 species (including eight endemics) representing 68 genera and nine families (Garbino et al., 2020;Velazco 2021). Much of this diversity is found in the Amazon biome, which is home to most of the species known to occur in Brazil (Bernard et al., 2011). In this scenario, the Amazon of Maranhão, in the eastern extreme of the biome, is one of the Amazonian regions that have a fragmented distribution of bat species records, but which are gradually a number of recent studies have provided important insights into the distribution of chiropterans in this region (Bernard et al., 2011;Oliveira et al., 2011;Olímpio et al., 2016;Lima et al., 2018;Olímpio et al., 2018;Mendes et al., 2020).
Bats of the family Phyllostomidae are the most ecologically diverse group of mammals, and the second most speciose mammalian family, with 216 recognized species (Taylor & Tuttle, 2019). In recent years, a number of studies reporting the fauna of poorly-known areas associated with phylogenetic analyses, have advanced the scientific understanding of phyllostomid diversity, including the discovery of new species and the expansion of the inventories available for many genera, such as Sturnira (Miretzki et al., 2002).
Relatively few data are available on the diet or reproductive patterns of S. tildae, but this bat is assumed to be an important seed disperser, which contributes to the regeneration of forested areas (Simmons, 2005;Gardner, 2008). S. tildae shares a number of external characteristics with the other members of the genus, such as the lack of a tail, narrow and hairy interfemoral membrane, small ears, a small, broad nasal leaf, and posterior members and feet hairy, as far as the claws (Peracchi et al., 2011). The color of the pelage varies from tones of yellow to brown, with some males having tufts of orangish or dark reddish-brown hair on the shoulders (Reis et al., 2017).
The external measurements of S. tildae are similar to S. lilium, although S. lilium tends to be smaller (Gardner, 2008). Some studies point to the possibility of identification errors of identification related to the morphological similarities of these species and the overlap in their geographic distributions (Miretzki et al., 2002;Sampaio et al., 2016;Velazco & Patterson, 2017;Martins et al., 2020). In areas that are in sympatry, S. lilium differs from S. tildae only by dental and morphometric characteristics (Simmons & Voss, 1988;Martins et al., 2020). The present study uses morphological and molecular data to report on a new record of S. tildae from the state of Maranhão, which extends the known geographic distribution of the species in Brazil.

MATERIAL AND METHODS
Bats were collected in June 2016 in a fragment of forest located within the Amazon biome of the Brazilian state of Maranhão, in the municipality of Cândido Mendes. Specimen collection followed the procotol of Pacheco (2004) and permits were duly provided by the Brazilian federal agency for the environment (IBAMA/ SISBIO permit number 42670-3). The vegetation of the study region is dense rainforest typical of the Amazon biome, and it has a humid equatorial climate, as well as being influenced by the tides of the Maracaçumé River, which crosses the town of Cândido Mendes (Bandeira, 2013).
The specimen described here was collected using a 3 m high and 12 m long mist-net, with a 25 mm mesh. The age of the specimen was determined in the field based on the ossification of the phalangeal epiphyses, and its sex and reproductive status were also recorded (Brunet & Austad, 2004). The specimen was photographed, euthanized, labeled, and stored on ice for transportation to the Genetics and Molecular Biology Laboratory (GENBIMOL) of Universidade Estadual do Maranhão (UEMA) in Caxias, Maranhão, where it was weighed and measured, and a sample of a muscle tissue was extracted, which was stored in ethanol 70° for the molecular analyses.
The skull was extracted through the buccal aperture. Once clean, the skull was clarified with 10% peroxide and dried in a stove at 30℃, after which, it was labeled and stored in a clean recipient. The specimen was fixed in formaldehyde and preserved in ethanol at UEMA's Laboratory of Genetics and Molecular Biology, on the campus of Caxias, and then transferred to the mammal collection of the Federal University of Paraíba, in João Pessoa, Brazil, where is it deposited. The specimen was identified based on its external traits and craniometric measurements, following Vizzoto & Taddei (1973), Simmons & Voss (1998), and Reis et al. (2013Reis et al. ( , 2017. Measurements taken include the length of right and left forearms, ear, tragus, foot, greatest length of the skull, basal length and condylobasal length, width of the brain case and the mastoid, zygomatic width, the length of the upper tooth row, and the width across the molars (Table 1). Body mass, after the specimen had been dead for hours, was determined using a precision balance after euthanasia of the specimen.
The total DNA was extracted from the muscle tissue using Promega's Wizard Genomic DNA Purification kit, following the default protocol. Two mitochondrial genes, Cytochrome C Oxidase Subunit 1 (COI) and rRNA 16S, were amplified by Polymerase Chain Reaction (PCR) using the primers LCO-1490 and HCO-2198 for the COI gene (Folmer et al., 1994) and L1987 and H2609 for the rRNA 16S (Palumbi et al., 2002). The samples were sequenced by the dideoxyterminal method of Sanger et al. (1977) in an ABI Prism™ 3500 (Applied Biosystems, USA) automatic DNA sequencer, using the Big Dye kit. The sequences were edited and aligned in BIOEDIT 7.0 (Hall, 1999), with the genetic divergence matrices being complied in MEGA X (Kumar et al., 2018) using the Kimura 2-parameter algorithm. The sequences were plotted in the BOLD Systems v4 (www.boldsystems.org) and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) platforms to confirm their identification and determine their degree of similarity with sequences of other Sturnira species.

RESULTS
We collected an adult female S. tildae (field number: RRM 117, voucher: UFPB 11757) in June 2016 from a farm located within the urban perimeter of the town of Cândido Mendes (01°27′21″S, 45°43′32″W), in the Amazon biome, within an area containing remnants of rainforest. The length of the right forearm was 45.77 mm, while its left forearm was 46.34 mm. The length of ear of the specimen was 12.95 mm, the tragus 5.46 mm, and the foot 12.45 mm. The specimen was a non-lactating which had ectoparasites, orange-brown pelage, with the venter paler than the dorsum, and dark hairs around the eyes (Fig. 1).
Sturnira tildae was also identified based on the following set of morphological cranial traits: small brain case and broad, high rostrum, slightly bilobed upper incisors with a wide base and narrower points, and first and second lower molars with lingual cusps separated by shallow grooves with no vertical border ( Fig. 2A-D and Table 1).
The present study provides the first record of S. tildae from the Brazilian state of Maranhão, and only the fifth for the Brazilian Northeast geographic region (two from Bahia state - Faria & Baumgarten, 2007;Faria, 2006, one from Pernambuco;Martins et al., 2020 and one from Ceará - Novaes & Laurindo, 2014). The geographically closest locality to Cândido Mendes with records of S. tildae is Santa Barbara, in the neighboring state of Pará, which is ca. 280 km due west (Fonseca, 2006). There is also a record from the municipality of Muaná, in Pará (Marques-Aguiar et al., 2002), which is 402 km west of Cândido Mendes. Both these localities are within the Amazon biome. South of Maranhão, the nearest locality is in the municipality of Goiatins, in Tocantins state (Maas et al., 2018), which is 724 km to the south, in the Cerrado savanna biome, while the nearest record to the east is from the municipality of Barbalha, in Ceará state, at a distance of 967 km, in the Caatinga biome (Novaes & Laurindo, 2014) (Fig. 3). The present study extends the known distribution of S. tildae to the eastern extreme of the Amazon biome.
The sequence of the rRNA 16S gene obtained from the specimen analyzed in the present study diverged by 0.4% from those of S. tildae from French Guiana and by 4.8% from S. lilium. The BLAST search identified a genetic similarity of 100% with S. tildae from French Guiana. In the case of the COI gene sequences, intraspecific genetic divergence between the study specimen and sequences from French Guiana, Surinam, Ecuador, and Guyana was 0.92-1.58%, while it was 11.8% in relation to S. lilium. In the BOLD Systems platform, the sequence returned a similarity of 99.09% with S. tildae from French Guiana (Table 2). These results are consistent with the morphological and craniometric identification of the specimen, and confirm the occurrence of S. tildae in the Amazon biome of Maranhão state.

DISCUSSION
In the present study, the cranial and morphological measurements of the specimen, are typical of S. tildae, as defined by De La Torre (1959) Martins et al. (2020). The cranial and dental measurements of the specimen (skull, basal, and condylobasal lengths, the length of the upper tooth row, and the postorbital and zygomatic widths, the width of the braincase, and the width across the molars) are all very similar to those of the S. tildae specimens collected in Recife, Pernambuco (Martins et al., 2020) and Morretes, Paraná (Miretzki et al., 2002). This indicates that differences due to the geographic or sexual variation are negligible.
The species S. tildae has great morphological similarities with S. lilium, making identification difficult. (Simmons & Voss, 1998). A number of diagnostic traits can nevertheless be used to distinguished the two taxa, including the length of the forearm, which exceeds 45 mm in S. tildae, but never exceeds this value in S. lilium (Simmons & Voss, 1998 (Fazzolari-Correa, 1995;Davis, 1980). All these differences were also observed in the S. tildae specimen analyzed in the present study.
The dentition provides the most reliable evidence for the differentiation of the two species, given the presence of bilobed internal superior incisives and wider in S. tildae, than observed in S. lilium (Eisenberg, 1989), as well as the lingual cusps of the first and second molars of the mandible, S. lilium has high lingual cusps, separated by a deep vertical notch between the first and second molars and in S. tildae these cusps are low and separated by shallow notches (Miretzki et al., 2002). These authors considered the differences in the configuration of the cusps of the mandibular molars to be completely reliable for the distinction of the specimens of S. lilium and S. tildae from Paracou, in French Guiana, and this conclusion was further confirmed in the present study.
The analyses of the 16S rRNA and COI genes revealed a high degree of similarity, in both cases, between the specimen presented here and S. tildae from French Guiana, with a genetic divergence of less than 2% in comparison with the specimens from of different localities, which is consistent with the 2% intraspecific threshold of the COI marker established for bats by Clare et al. (2011). Despite the fact that S. lilium and S. tildae present major morphological similarities, the analysis of the rRNA 16S sequences indicated a divergence of 4.8% between the S. tildae specimen presented here and S. lilium, increasing to 11.8% for the COI gene. The genetic data thus support conclusively the morphological and craniometric analyses, and confirm the occurrence of S. tildae in Maranhão.
In Brazil, S. tildae has been recorded in the Amazon, Atlantic Forest, Cerrado, and Caatinga biomes. In the Amazon biome (IBGE, 2019), there are records from the Brazilian states of Amazonas, Acre, Rondônia, Pará, Amapá, Roraima, and Mato Grosso (Tavares et al., 2008;Reis et al., 2013;Novaes & Laurindo, 2014;Reis et al., 2017), and the closest locality to the present study site is 280 km west, in Pará. This is the fifth record of the occurrence of S. tildae in the Brazilian Northeast. The previous records included one from the Caatinga biome, in Barbalha, Ceará state (Novaes & Laurindo, 2014), and three from the Atlantic Forest, in Ilhéus (Faria & Baumgarten, 2007) and Una (Faria, 2006), both in Bahia, and Recife in Pernambuco state (Martins et al., 2020). Barbalha is very close -about 20 km -to Floresta Nacional do Araripe. The area is considered to be a relict of Atlantic Forest in the middle of the Caatinga (Silveira et al., 2019).

CONCLUSIONS
The present study recorded the occurrence of S. tildae in the Amazon biome of the Brazilian state of Maranhão, based on both morphological and molecular evidence. This is the first record of the species for the state, and the fifth for the Brazilian Northeast, and extends the known distribution of the species to the municipality of Cândido Mendes, 280 km east of the nearest locality in the Amazon biome. The findings of the study reinforce the need for attention when identifying the species of the genus Sturnira, in particular in areas where S. lilium and S. tildae are likely to be captured together and then released, given that the considerable morphological similarities of these sympatric taxa may result in errors of identification. As S. tildae occurs more frequently in moist forests, new records are expected in other Amazonian areas in Maranhão, as well as in Brejos de Altitude habitats within the Caatinga biome (see Tabarelli & Santos, 2004).

ACKNOWLEDGMENTS
ACSL and FHSC were supported by postgraduate fellowships and BATPC was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) postdoctoral fellowships. CLSC was supported by a postgraduate grant from the Universidade Estadual do Maranhão (UEMA). This study was financed in part by CAPES -Financial Code 001 and the Foundation for Research and Scientific and Technological Development of Maranhão (FAPEMA).