Biogeographical karyotypic variation of Rhinophylla fi scherae ( Chiroptera : Phyllostomidae ) suggests the occurrence of cryptic species

The genus Rhinophylla Peters, 1865 (Carolliinae: Phyllostomidae) comprises three species: R. pumilio Peters, 1865, R. fi scherae Carter, 1966 and R. alethina Handley, 1966. Only the fi rst two species have been cytogenetically studied to date. Previous studies on specimens of Rhinophylla fi scherae from two populations from East of Andes (Colombia) showed the karyotype with 2n=34 and FN=56. In this paper, we report the results of cytogenetic analysis of six specimens of Rhinophylla fi scherae from Brazil. Probably chromosomal differences can be found among the populations because of the geographic distance. Metaphase chromosomes were obtained in the fi eld by direct extraction of bone marrow. The metaphases were analyzed by conventional staining, Gand C-banding, NOR-staining and FISH with telomeric probes. Rhinophylla fi scherae has 2n=38 and Fundamental Number FN=68, with small amounts of constitutive heterochromatin in the centromeric regions of the chromosomes and the long arm of pair 16. Fluorescence in situ hybridization using telomeric probes did not show any interstitial sequences. Hybridization with human 18S and 28S rDNA probes and silver staining revealed the presence of Nucleolar Organizer Regions at the long arms of pairs 16 and 18. The pattern of G-banding showed that this population had a huge chromosome variation compared with previous studies on specimens of Rhinophylla fi scherae. The chromosomal differences among populations that have been morphologically classifi ed as R. fi scherae suggest that this species should be considered a cryptic species complex, and that the populations from different geographical regions analyzed to date should be considered species of this complex, where the chromosomal rearrangements had key importance.


INTRODUCTION
Phyllostomid bats constitute a complex assemblage of the Neotropical bat fauna with a long history of taxonomic controversies (Wetterer et al., 2000;Baker et al., 2003).Brazilian Amazonian rainforest has a rich bat fauna (Handley, 1967;Bernard et al., 2001;Sampaio, 2003); however, the knowledge about this regional fauna is far from satisfactory to understand the complex ecological, geographic and diversity patterns.
There are few cytogenetic studies on bats from Brazilian Amazonia (Rodrigues et al., 2000(Rodrigues et al., , 2003;;Neves et al., 2000;Ribeiro et al., 2003;Silva et al., 2005;Pieczarka et al., 2005).The results of these studies have shown that the Phyllostomidae has a high intrafamiliar karyotypic variation and often new species have been detected fi rst by different karyotypes.
In the present paper, we report a new karyotype for Rhinophylla fi scherae from Brazilian Amazonia and discuss the biogeographical karyotypic variation as an evidence of a species complex for this taxon.

Samples
Six specimens (two males, codes LR 765 and LR 855, and four females, codes LR 710, LR 732, LR 763 and LR 818) of Rhinophylla fi scherae were obtained for cytogenetic analysis.The bats were collected from natural populations using mist nets, during the expeditions to faunal inventory in the area of bauxite mine of Alcoa Inc. in Juruti, Para state, Brazil (02 °29´38.8˝S/56°11´27.1˝W;Fig. 1).The specimens were identifi ed in the fi eld with the identifi cation key for bats of the Guyanna (Lim, Engstrom, 2001).The identifi cation was confi rmed by the presence of diastema between I2 and the superior canines, as well as the hairy edge of interfemoral membrane (Rinehart, Kunz, 2006).Voucher specimens were fi xed in formalin 10%, preserved in ethanol 70% and deposited in the mammal collection of the Museu Paraense Emilio Goeldi.

Chromosome preparations, cell culture and chromosome banding
Metaphase chromosomes were obtained in the fi eld by direct extraction of bone marrow according to Baker et al. (2003).Chromosomal preparations and tissue biopsies were sent to the cytogenetics laboratory at the Universidade Federal do Pará in Belém, Comp.Cytogenet., 2010 4(1)

Fluorescence in situ hybridization (FISH)
FISH with digoxigenin-labeled telomeric probes (All human Telomere probes, Oncor) was performed according to the manufacturer's protocol.To confi rm the NOR labelled sites Biotin-dUTP was incorporated into the human 18S and 28S rDNA probes using nick translation.Briefl y, the slides were incubated in RNAse and pepsin solutions following the procedure described by Martins, Galetti

RESULTS
All six specimens of Rhinophylla fi scherae from Brazilian Amazonia (Fig. 1) have 2n=38 and FN=68 (Fig. 2, a), of which 12 chromosomes pairs are metacentric/submetacentric, four pairs are subtelocentric and two pairs are acrocentric.The X chromosome is submetacentric, while the Y is small and acrocentric.Cbanding detected constitutive heterochromatin at the centromeric regions of all chromosomes (Fig. 2, b), and in the long arm of pair 16.Both Ag-NOR staining (not shown) and FISH with 28S and 18S rDNA probes (Fig. 2, c) revealed Nucleolar Organizer Regions (NORs) in the proximal region of the long arm of pairs 16 and 18. FISH with telomeric probe hybridized only at the tips of the chromosomes (Fig. 2,  d), without any interstitial telomeric sequence (ITS).

DISCUSSION
Several studies have confi rmed the occurrence of Rhinophylla fi scherae in Pará State (Eastern Amazonian region), and the previous reports were consistent with the diagnostic traits for this species (Bernard et al., 2001;Bernard, Fenton, 2007).Rhinophylla fi scherae is clearly distinguished from R. pumilio by dental and external characters (Rinehart, Kunz, 2006) but, apparently, there is no evident external morphological variation within R. fi scherae populations.
The extensive chromosomal divergence between the R. fi scherae from different geographical regions suggests that these two cytotypes probably are not part of the same species.This would be an additional cryptic species situation, as already observed in Carollia brevicauda Schinz, 1821 and C. sowelli Baker, Solari et Hoffmann, 2002(Baker et al., 2002) and Carollia castanea H. Allen, 1890 and C. benkeithi Solari et Baker, 2006(Solari, Baker, 2006).Molecular data would be helpful to reinforce this hypothesis, but they are not available at the moment.Wright et al. (1999) used data from the Cytochrome-B gene to study the phylogenetic relationships between the genera Carollia and Rhinophylla.Their results suggest that R. pumilio has been separated from R. fi scherae for a relatively long time (8-10 million years).In the most parsimonious tree the branch leading to R. pumilio and R. fi scherae was supported by low bootstrap values and Bremer decay, which was interpreted as a result of intense divergence intra-and inter-species, and may suggest that the nominal taxa R. pumilio and/or R. fi scherae may encompasses more than one species.Our karyotypic results are consistent with these interpretations for R. fi scherae.
Based on data here presented the population from Juruti (PA, Brazil) will be named herein as Rhinophylla fi scherae.Further, detailed studies using G-banding and chromosome painting, as well as molecular and morphological analyses of all the geographic

Fig. 2
Fig. 2, a-d.Rhinophylla fi scherae metaphases.a -G-banded.b -C-banded.c -FISH with rDNA 28 and 18S probes.d -FISH with telomeric probes.Bar = 5µm.(The chromosomal pair 16 from another specimen in the box shows a more intense heterochromatin block in the long arm).