Karyotype of three Lonchophylla species (Chiroptera, Phyllostomidae) from Southeastern Brazil

Abstract Lonchophylla Thomas, 1903 is a Neotropical bat genus that comprises 12 species, with little cytogenetic information available. Here we present the description of the karyotype of three species collected in Southeastern Brazil. Lonchophylla bokermanni Sazima, Vizotto & Taddei, 1978, Lonchophylla dekeyseri Taddei, Vizotto & Sazima, 1983, and Lonchophylla peracchii Dias, Moratelli & Esberard, 2013 showed the same diploid number 2n = 28 and the same autosomal fundamental number FNa = 50, in both Lonchophylla bokermanni and Lonchophylla peracchii. We observed that the karyotypes were also cytogenetically similar when we compared the studied species with other species within the same genus. It is therefore not possible to differentiate the species using only karyotypes with conventional staining. However, this information increases the knowledge of the genus and can be one more important character for a better phylogenetic comprehension of this taxon.


Introduction
In recent years, new species and a genus of the subfamily Lonchophyllinae were described: Lonchophylla peracchii Dias, Moratelli & Esberard, 2013, L. inexpectata Moratelli & Dias, 2015, and Hsunycteris Parlos, Timm, Swier, Zeballos & Baker, 2014(Dias et al. 2013, Parlos et al. 2014, Moratelli and Dias 2015. For the description of bat species, morphological and morphometric characteristics are usually employed, but the use of other tools such as cytogenetic analysis can provide essential information for evolutionary relationships of bats (Varella- Taddei 1989, Garcia andPêssoa 2010), as already seen for rodents, for example (Romanenko and Volobouev 2012). Although there are few cytogenetic data for Lonchophyllinae, they were nevertheless informative for systematic rearrangements of this taxon (see Parlos et al. 2014).
In Brazil, there are records for five species of this genus: Lonchophylla bokermanni Sazima, Vizotto & Taddei, 1978, Lonchophylla dekeyseri Taddei, Vizotto & Sazima, 1983, Lonchophylla inexpectata, Lonchophylla mordax Thomas, 1903 and the new species, Lonchophylla peracchii mentioned above. There are karyotype data available until now for the two congeneric taxa from outside the country, Lonchophylla robusta Miller, 1912and Lonchophylla concava Goldman, 1914(Parlos et al. 2014), but no cytogenetic data were available for Brazilian species. Therefore, this study is the first to describe the karyotype of L. bokermanni, L. dekeyseri and L. peracchii. using 12 to 16 mist-nets (9 × 3 m, 35 mm mesh) that remained open in the first six hours after the sunset for seven consecutive nights.
Chromosomes in metaphases were obtained through in vitro bone marrow culture grown in Dulbecco´s MEM with 10% fetal bovine serum and colchicine for 2 hours, following by an incubation in KCl 0.075M solution at 37 °C by 30 minutes, centrifuged, fixed in Carnoy solution (methanol: acetic acid, 3:1). The fixation step was repeated three times. Preparation was done by dropping one drop by distance onto clean microscope slides and air-dried. Conventional staining with Giemsa 5% was used to observe diploid number (2n) and Fundamental Number of autosomal arms (FNa) and chromosome morphology variation. This analysis was carried out using an optic photomicroscope (Nikon Eclipse 50i), in a 1,000 increase -lenses of 100 plus 10 ocular lenses.

Results and discussion
All three species showed the same diploid number 2n = 28 and an autosomal fundamental number FNa = 50 was observed (Figure 2). The autosomal complement of males L. bokermanni and L. peracchii consists of 12 pairs of meta/submetacentrics varying from large to small, and a pair of small acrocentric chromosomes (FNa = 50). Two size classes of autosomal chromosomes can be observed -the eight first are all large chromosomes, and in the second row (Figure 2A-C), with smaller ones, including four metacentric and the smallest chromosome of the karyotype, the only acrocentric ones. The X chromosome is a medium sized metacentric and the Y is a minute acrocentric, smaller than the last pair of autosomal complement. Similarly, the karyotype of L. dekeyseri can be characterized but the identification of the sex chromosome pair was impossible in the sole collected specimen which was a female.
Karyotype comparison is considered as an important tool to establish phylogenetic relationships and as a taxonomic tool to confirm some species identities (Baker 1970, Silva et al. 2006, Urdampilleta et al. 2013). However, the resolution power of the cytogenetic method is not the same for all groups. Sometimes, it is necessary to analyze as many as possible the species' karyotypes (Garcia and Pessoa 2010). In bats, the few available published karyotype data from South America (Moratelli and Morielle-Versute 2007) make it difficult to propose, using such kind of information, new or different taxonomic arrangements and a better comprehension of the systematics of Neotropical bats (Garcia and Pessoa 2010).
The karyotype conservatism in Microchiroptera has been observed in other studies (Varella- Garcia et al. 1989, Sousa andAraújo 1990) which well corroborate with our results. Even if species distinction is not evident for representatives of the Lonchophylla genus through the conventional chromosome characteristics, the generic separation of Lonchophylla -Hsunycteris is supported by their different karyotypes.

Acknowledgments
Julia L Luz, Maíra SM Godoy, Luciana M Costa, Luciana G Pereira, Edvandro A Ribeiro and André C Siqueira helped in the fieldwork; Luana Azamor helped in laboratory work; to FAPERJ for IC Grant toBrunna Almeida and for scholarship for Renan F. Souza; and CNPq for financial support. Lena Geise has CNPq and Uerj/Prociencia fellowship.