© Comparative Cytogenetics, 2010 . Vol. 4, No. 1, P. 79-85. ISSN 1993-0771 (Print), ISSN 1993-078X (Online)

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

A.J.B. Gomes1,3, L.R.R. Rodrigues4, J.D. Rissino1, C.Y. Nagamachi1,2, J.C. Pieczarka1,2

1Laboratório de Citogenética, ICB. Campus do Guamá, UFPa. Av. Perimetral, sn. Belém – PA – Brazil, 66075-900. 2CNPq Researcher. 3CAPES Master Scholarship on Genetics and Molecular Biology, Brazil. 4Laboratório de Genética and Biodiver- sidade. Campus de Santarém, UFOPA. AV. Marechal Rondon, sn. Bairro Caranazal. Santarém – PA - Brazil. 68.040-070. E-mails: AJBG: [email protected], LRRR: luisreginaldo.ufpa@hotmail. com, JDR: [email protected], CYN: [email protected], JCP: [email protected]

Abstract. The genus Rhinophylla Peters, 1865 (: Phyllostomidae) com- prises 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 pa- per, 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 ob- tained in the fi eld by direct extraction of bone marrow. The metaphases were ana- lyzed by conventional staining, G- and C-banding, NOR-staining and FISH with telo- meric 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 pre- vious 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 popu- lations from different geographical regions analyzed to date should be considered species of this complex, where the chromosomal rearrangements had key importance.

Key words: Rhinophylla fi scherae, Chiroptera, karyotype, cryptic species, Amazon, biodiversity.

http://pensoftonline.net/compcytogen Comparative Cytogenetics doi: 10.3897/compcytogen.v4i1.24 80 A.J.B. Gomes et al.

INTRODUCTION from Suriname (Honeycutt et al., 1980; Baker et al., 1981); and d) 2n=26, FN=48 from Bra- Phyllostomid constitute a complex as- zilian Atlantic forest (Toledo, 1973). Karyo- semblage of the Neotropical fauna with a type of R. fi scherae was described with 2n=34 long history of taxonomic controversies (Wet- and FN=56 for specimens from Colombia terer et al., 2000; Baker et al., 2003). Brazil- (Baker and Bleier, 1971; Baker et al., 1987). ian Amazonian rainforest has a rich bat fauna R. alethina was not studied to date. (Handley, 1967; Bernard et al., 2001; Sampaio, In the present paper, we report a new karyo- 2003); however, the knowledge about this re- type for Rhinophylla fi scherae from Brazilian gional fauna is far from satisfactory to under- Amazonia and discuss the biogeographical stand the complex ecological, geographic and karyotypic variation as an evidence of a spe- diversity patterns. cies complex for this taxon. There are few cytogenetic studies on bats from Brazilian Amazonia (Rodrigues et al., MATERIAL AND METHODS 2000, 2003; Neves et al., 2000; Ribeiro et al., 2003; Silva et al., 2005; Pieczarka et al., Samples 2005). The results of these studies have shown Six specimens (two males, codes LR 765 that the Phyllostomidae has a high intrafamil- and LR 855, and four females, codes LR 710, iar karyotypic variation and often new species LR 732, LR 763 and LR 818) of Rhinophylla have been detected fi rst by different karyo- fi scherae were obtained for cytogenetic analy- types. sis. The bats were collected from natural popu- The subfamily Carolliinae encompasses lations using mist nets, during the expeditions two genera: Gray, 1838 (10 species) to faunal inventory in the area of bauxite mine and Rhinophylla Peters, 1865 (3 species), which of Alcoa Inc. in Juruti, Para state, Brazil (02 are traditionally recognized as monophyletic °29´38.8˝S/56°11´27.1˝W; Fig. 1). The speci- group. The genus Rhinophylla comprises three mens were identifi ed in the fi eld with the iden- species: R. pumilio Peters, 1865 and R. fi sch- tifi cation key for bats of the Guyanna (Lim, erae Carter, 1966, broadly distributed through Engstrom, 2001). The identifi cation was con- the South America, and R. alethina Handley, fi rmed by the presence of diastema between I2 1966 restricted to west coast of Colombia and and the superior canines, as well as the hairy Peru (Handley, 1976). This genus is taxonomi- edge of interfemoral membrane (Rinehart, cally stable while Carollia has been recently Kunz, 2006). Voucher specimens were fi xed changed by the discovery of news species on in formalin 10%, preserved in ethanol 70% the past ten years (Cuartas et al., 2001; Baker and deposited in the collection of the et al., 2002; Pacheco et al., 2004; Muñoz et al., Museu Paraense Emilio Goeldi. 2004 and Solari, Baker, 2006). Chromosome preparations, cell culture Despite of the relative taxonomic stability and chromosome banding of Rhinophylla, cytogenetic studies have dem- Metaphase chromosomes were ob- onstrated karyotypic intraspecifi c variation tained in the fi eld by direct extraction of bone in R. pumilio with description of four karyo- marrow according to Baker et al. (2003). morphs: a) 2n=36, FN=62 from Colombia Chromosomal preparations and tissue biop- (Baker, 1971); b) 2n=34, FN=56 from Surina- sies were sent to the cytogenetics laboratory me (Baker, Bickham, 1980); c) 2n=34, FN=64 at the Universidade Federal do Pará in Belém,

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Evidence for new cryptic species of Rhinophylla bats 81 with different karyotypes were collected: Villavicencio Villavicencio karyotypes were collected: with different Rhinophylla fi scherae Rhinophylla fi Map of the regions were Fig. 1. (2n=34, Baker et al., 1987), Leticia (2n=34, Baker, Bleier, 1971) and Juruti (2n=38, present paper). Bleier, (2n=34, Baker et al., 1987), Leticia Baker,

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82 A.J.B. Gomes et al.

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).

Pará, Brazil. G-banding patterns were obtained (1998). The slides were then dehydrated in an by saline solution (2xSSC) incubation and ethanol series (70, 90 and 100%), aged in a Wright’s staining following Verma and Babu 65°C incubator for one hour, and denaturated (1995). C-banding was processed according to in 70% formamide/2xSSC at 65°C for one Sumner (1972), and Ag-NOR staining follow- minute. Fourteen microliters of the hybridiza- ing Howell and Black (1980). The karyotypes tion mixture (50% formamide, 1xSSC, 10% were organized according to Baker, Bleier dextran sulfate, 5 mg salmon sperm DNA, (1971). and 2 mg mouse Cot-1 DNA) and 1 µl of la- Fluorescence in situ hybridization belled rDNA probe were denatured at 65°C (FISH) and dropped onto the denatured chromosome FISH with digoxigenin-labeled telomeric preparations, which were then mounted with probes (All human Telomere probes, Oncor) 24x24 mm cover slips. In situ hybridization was performed according to the manufactur- experiments were incubated for 48-72 h at er’s protocol. To confi rm the NOR labelled 37°C. The hybridization signal was detect- sites Biotin-dUTP was incorporated into the ed with avidin-Cy3 as described previously human 18S and 28S rDNA probes using nick (Yang et al., 1995; Pieczarka et al., 2005). translation. Briefl y, the slides were incubat- The images were captured with an Axiocam ed in RNAse and pepsin solutions following Mrm CCD camera, which was controlled by the procedure described by Martins, Galetti the Zeiss Axiovision 3.0 software. The chro-

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Evidence for new cryptic species of Rhinophylla bats 83 mosomes were identifi ed by their morphology very small pair of chromosomes) previously and DAPI (4’,6-diamidino-2-phenylindole) described by conventional analysis (sample inverted banding pattern. from Leticia) and G-banding (sample from Vil- lavicencio, Baker et al., 1987). Comparative RESULTS G-banding analysis between the karyotypes All six specimens of Rhinophylla fi scherae of R. fi scherae from Villavicencio (Colombia) from Brazilian Amazonia (Fig. 1) have 2n=38 and from Juruti (Brazil) shows an extensive and FN=68 (Fig. 2, a), of which 12 chromo- chromosomal reorganization remaining few somes pairs are metacentric/submetacentric, chromosomes shared without rearrangement. four pairs are subtelocentric and two pairs are The extensive chromosomal divergence acrocentric. The X chromosome is submeta- between the R. fi scherae from different geo- centric, while the Y is small and acrocentric. C- graphical regions suggests that these two banding detected constitutive heterochromatin cytotypes probably are not part of the same at the centromeric regions of all chromosomes species. This would be an additional cryptic (Fig. 2, b), and in the long arm of pair 16. Both species situation, as already observed in Car- Ag-NOR staining (not shown) and FISH with ollia brevicauda Schinz, 1821 and C. sowelli 28S and 18S rDNA probes (Fig. 2, c) revealed Baker, Solari et Hoffmann, 2002 (Baker et al., Nucleolar Organizer Regions (NORs) in the 2002) and Carollia castanea H. Allen, 1890 proximal region of the long arm of pairs 16 and C. benkeithi Solari et Baker, 2006 (Solari, and 18. FISH with telomeric probe hybridized Baker, 2006). Molecular data would be help- only at the tips of the chromosomes (Fig. 2, ful to reinforce this hypothesis, but they are d), without any interstitial telomeric sequence not available at the moment. (ITS). Wright et al. (1999) used data from the Cytochrome-B gene to study the phylogenetic relationships between the genera Carollia and DISCUSSION Rhinophylla. Their results suggest that R. pu- Several studies have confi rmed the occur- milio has been separated from R. fi scherae for rence of Rhinophylla fi scherae in Pará State a relatively long time (8-10 million years). In (Eastern Amazonian region), and the previ- the most parsimonious tree the branch leading ous reports were consistent with the diagnos- to R. pumilio and R. fi scherae was supported tic traits for this species (Bernard et al., 2001; by low bootstrap values and Bremer decay, Bernard, Fenton, 2007). Rhinophylla fi sch- which was interpreted as a result of intense erae is clearly distinguished from R. pumilio divergence intra- and inter-species, and may by dental and external characters (Rinehart, suggest that the nominal taxa R. pumilio and/or Kunz, 2006) but, apparently, there is no evi- R. fi scherae may encompasses more than one dent external morphological variation within species. Our karyotypic results are consistent R. fi scherae populations. with these interpretations for R. fi scherae. The karyotype with 2n=38 (24M/ Based on data here presented the popula- SM+8ST+4A) found in R. fi scherae from tion from Juruti (PA, Brazil) will be named Juruti, Pará state, Brazil (this study – Fig. 1) herein as Rhinophylla fi scherae. Further, is clearly different of the R. fi scherae from detailed studies using G-banding and chro- Colômbia (Baker, Bleier, 1971; Baker et al., mosome painting, as well as molecular and 1987), with 2n=34 (20M/SM+4ST+6A with a morphological analyses of all the geographic

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