Comparative cytogenetic analysis between Lonchorhina aurita and Trachops cirrhosus (Chiroptera, Phyllostomidae)

Phyllostomidae comprises the most diverse family of neotropical bats, its wide range of morphological features leading to uncertainty regarding phylogenetic relationships. Seeing that cytogenetics is one of the fields capable of providing support for currently adopted classifications through the use of several markers, a comparative analysis between two Phyllostomidae species was undertaken in the present study, with a view to supplying datasets for the further establishment of Phyllostomidae evolutionary relationships. Karyotypes of Lonchorhina aurita (2n = 32; FN = 60) and Trachops cirrhosus (2n = 30; FN = 56) were analyzed by G- and C-banding, silver nitrate staining (Ag-NOR) and base-specific fluorochromes. Chromosomal data obtained for both species are in agreement with those previously described, except for X chromosome morphology in T. cirrhosus, hence indicating chromosomal geographical variation in this species. A comparison of G-banding permitted the identification of homeologies in nearly all the chromosomes. Furthermore, C-banding and Ag-NOR patterns were comparable to what has already been observed in the family. In both species CMA3 /DA/DAPI staining revealed an R-banding-like pattern with CMA 3 , whereas DAPI showed uniform staining in all the chromosomes. Fluorochrome staining patterns for pericentromeric constitutive heterochromatin (CH) regions, as well as for nucleolar organizing regions (NORs), indicated heterogeneity regarding these sequences among Phyllostomidae species.


Introduction
The family Phyllostomidae, which comprises the New World leaf-nosed bats, is considered the third largest of the order Chiroptera. This family is the most diverse group among Neotropical bats, with approximately 56 genera and 141 species (Baker et al., 2003;Simmons, 2005). Phyllostomidae bats exhibit wide variation in morphological features, and are adapted to a extensive range of ecological niches, with dietary specialization which includes fruit, nectar, pollen, insects, vertebrates and blood. This great diversity has been problematic for systematics, and concurs to hindering efforts to reconstruct the phylogenetic history of the family (Wetterer et al., 2000;Jones, 2002;Baker et al., 2003).
The subfamily Phyllostominae is one of the groups which has been questioned by researchers, but without consensus. Several authors agree that this subfamily is not a monophyletic group, although only recently has a new pro-posal been made as to its subdivision. Baker et al. (2003), on analyzing mtDNA sequence data, grouped this information together with previous phylogenies based on the RAG2 gene (Baker et al., 2000), morphology (Wetterer et al., 2000) and karyotypes (Baker et al., 1973(Baker et al., , 1989Baker and Bass, 1979), to suggest a classification with 56 genera in 11 subfamilies for the Phyllostomidae. In this classification, members were distributed among five subfamilies: Macrotinae, Micronycterinae, Lonchorhininae, Phyllostominae and Glyphonycterinae. Lonchorhininae, which is comprised of a single genus (Lonchorhina), diverged before the radiation of Phyllostominae and nectarivorous bats, appearing as a basal branch relative to Phyllostominae.
Cytogenetic studies constitute an important approach for understanding phylogenetic relationships among bats. By comparing banding patterns and the localization and constitution of different markers, it has been possible to characterize several taxa and develop hypotheses on evolutionary relationships, as well as models of chromosomal evolution (Baker et al., 1989;Baker, 2006).
Thus, in this work, chromosomal features of Lonchorhina aurita (Lonchorhininae) and Trachops cirrhosus (Phyllostominae) were studied by conventional analysis, G-and C-banding, staining with silver nitrate and base-specific fluorochromes (CMA 3 and DAPI) in order to establish mutual cytogenetic differences. These data will be helpful in understanding the chromosome structure and evolution of the family Phyllostomidae, as well as systematic aspects and phylogenetic relationships among members.
Silver staining and G-and C-banding procedures were undertaken through routine cytogenetic techniques, according to Howell and Black (1980), Seabright (1971) and Sumner (1972), respectively. Triple staining CMA 3 /DA/DAPI was carried out according to Schweizer (1980) with various modifications (Santos and Souza, 1998a). For sequential staining (AgNO 3 /CMA 3 /DAPI), the slides stained with silver nitrate were distained after photographing (Dos Santos Guerra, 1991) and re-stained with CMA 3 /DA/DAPI. Photomicrographs were taken by means of a Leica DMLB photomicroscope for conventional, silver staining and fluorescence staining. G-and C-banding were captured by a CytoVision image capture system.
The G-banding pattern disclosed the precise identification of all chromosome pairs. Comparative banding analysis inferred homeologies between the two species in pairs 1 to 3 and 5 to 8 (Figures 1a and 1b). Furthermore, in L. aurita the chromosome pairs 9, 10, 11 and 15 appeared to correspond to pairs 13, 12, 10 and 14 in T. cirrhosus, respectively. C-banding revealed constitutive heterochromatin (CH) in the pericentromeric regions of all the autosomes and the X chromosome, whereas the Y chromosome was almost completely heterochromatic in both species (Figures 2a and 2b).
Triple staining CMA 3 /DA/DAPI in these species revealed an R-banding-like pattern with the CMA 3 dye (GCrich regions) (Figures 3a and 3c), and uniform staining of all chromosomes with DAPI (Figures 3b and 3d). In addition, CMA 3 -positive blocks were observed in the pericentromeric region of some chromosomes, thereby indicating the GC-richness of CH.
Staining with silver nitrate (AgNO 3 ) revealed a single pair of NORs located at the secondary constriction in both species: in the short arm of pair 13 in L. aurita (Figure 4a) and in the long arm of pair 11 in T. cirrhosus (Figure 4c). The signals resulting from sequential staining AgNO 3 /CMA 3 /DAPI, revealed CMA 3 positive NORs in L. aurita (Fig. 4b), whereas these regions were CMA 3 neutral in T. cirrhosus (Figure 4d).

Discussion
Our data regarding diploid number, chromosome morphology and sex determination system obtained for both L. aurita and T. cirrhosus are in agreement with those previously described, except for the X chromosome in T. Barros et al. 749 cirrhosus. We observed an acrocentric X in specimens from Pernambuco, Brazil, although this has been described as subtelocentric in individuals from Mexico and Trinidad (Baker, 1967;Hsu et al. 1968;Baker and Hsu, 1970).
The majority of Phyllostomidae species have a biarmed X chromosome (metacentric, submetacentric or subtelocentric) this condition being considered basal for the family (Rodrigues et al., 2003). Acrocentric morphology of the X chromosome has been described in only three other Phyllostomidae species, Micronycteris hirsuta (Micronycterinae), Mesophylla macconnelli and Vampyressa pusilla (Stenodermatinae) (Baker and Hsu, 1970;Baker et al., 1973;Gardner, 1977). However, despite having encountered the same morphology, we suggest that the acrocentric morphology of the X chromosome in T. cirrhosus (Phyllostominae) is an apomorphic character that has evolved independent of the condition observed in the three aforementioned species, as they are distantly related. The most probable event involved in the morphological change of the X chromosome in T. cirrhosus could be pericentric inversion occurring in an ancestral metacentric or submetacentric X.
The CH in Phyllostomidae is generally located in the pericentromeric regions of chromosomes (Varella-Garcia et al., 1989), as observed in L. aurita and T. cirrhosus. However, additional CH blocks have been found in interstitial and telomeric regions in several species, notably Carollia perspicillata, Choeroniscus minor, Glossophaga soricina, Artibeus lituratus, A. planirostris,A. jamaicencis,A. cinereus,Sturnira lilium,Platyrrhinus lineatus,Uroderma magnirostrum,U. bilobatum,Diaemus youngi,750 Cytogenetics of Phyllostomidae bats   Desmodus rotundus and Diphylla ecaudata (Varella-Garcia et al., 1989;Souza and Araújo, 1990;Souza, 1998a, 1998b;Neves et al., 2001;Santos et al., 2001;Silva et al., 2005). The Y chromosomes of L. aurita and T. cirrhosus were almost entirely heterochromatic, which is a common pattern in Phyllostomidae species (Varella-Garcia et al., 1989;Souza and Araújo, 1990). The occurrence of one pair of NORs located in secondary constrictions of chromosomes in L. aurita and T. cirrhosus seems to be an ancestral condition among phyllostomid bats (Morielle and Varella-Garcia, 1988;Santos et al., 2002). NOR staining by GC-specific fluorochromes, as observed in L. aurita, has also been discerned in Artibeus lituratus, A. jamaicencis, Desmodus rotundus and Diphylla ecaudata, although these regions were CMA 3 neutral in Carollia perspicillata, Phyllostomus discolor and T. cirrhosus. This indicates heterogeneity regarding the base composition of intergenic regions related to NORs among species of the family Phyllostomidae Souza, 1998a, 1998b;Santos et al., 2001. In L. aurita and T. cirrhosus karyotypes, CMA 3 staining resulted in a pattern similar to R-banding, although, a G-band-like pattern was not observed with DAPI staining. In both species, the pericentromeric CH regions of some chromosomes presented positive staining with CMA 3 (CMA 3 + ). In certain species, such as Carollia perspicillata, the presence of euchromatic bands (R-and G-bands) and heterochromatin heterogeneity (CMA 3 -positive, DAPI-positive and CMA 3 /DAPI-neutral) after CMA 3 /DA/DAPI staining has been observed (Santos and Souza, 1998a). On the other hand, the CH in three species of Artibeus (A. lituratus, A. jamaicencis and A. cinereus), as well as Desmodus rotundus and Diphylla ecaudata, indicated no AT-or CG-richness after staining with these dyes (Santos and Souza, 1998b;Santos et al., 2001). Such a differential response to GC-and AT-specific fluorochromes in several Phyllostomidae species is a result of variability in heterochromatin composition within the family (Santos et al., 2001).
In the family Phyllostomidae, it is common to use the karyotype of Macrotus waterhousii as a reference for the numbering system, since it is believed to represent the ancestral karyotype for the family (Baker, 2006). From the present work, it is obvious that the two species analyzed share considerable homeologies, with 11 identical chromosome pairs. Their karyotypes are highly derived when compared to the ancestral state (see Baker, 1979 for M. waterhousii standard reference G-banded karyotype). However, several chromosomal arms in M. waterhousii can be recognized as being homeologous to arms in the karyotypes of the two studied species. This gives support to the inference that the evolutionary trend in Phyllostomidae appears to lead to a reduction in diploid number by centric fusion events, with retention of the linkage groups. Furthermore, these 11 chromosomes were probably present in the ancestor before the radiation of Lonchorhina, the common ancestor of Phyllostominae (sensu Baker et al. 2003) and nectarivorous bats.
There are three biarmed elements, recognizable in the M. waterhousii karyotype, which remained unchanged in L. aurita and T. cirrhosus. These chromosomes correspond to M. waterhousii arms 6/7, 25/26, and 15/16 (Pairs 8, 9 and 11 of L. aurita and 8, 13 and 10 of T. cirrhosus). They have been described as unchanged in members of different subfamilies (i.e. Desmodontinae, Baker et al. 1988;Glossophaginae, Baker and Bass, 1979; and other Phyllostominae species, Patton and Baker, 1979), thereby indicating that they were already present in the common ancestor of all phyllostomid bats.
We have been unable to detect homeologies among some chromosomes for the studied species, and the banding pattern of distinct arms of these chromosomes is such as not to allow us to be certain of their correspondence. It is likely that most of these arms have undergone inversion prior to translocation, thereby hindering the identification of the rearrangements involved in karyotypic changes between these bats.
Members of the Phyllostomidae family have conserved karyotypes but show intergeneric variability, making a comparative analysis using classical banding difficult (Baker, 1979;Pieczarka et al., 2005). However, further comparative chromosomal studies with molecular cytogenetic techniques based on fluorescence in situ hybridization (FISH) are expected to provide a better understanding of the karyotypic changes that have occurred during the evolution of this family, as well as the phylogenetic relationships among the members of this complex group of bats.