Chromosome comparison between two species of Phyllostomus ( Chiroptera-Phyllostomidae ) from Eastern Amazonia , with some phylogenetic insights

The neotropical bat genus Phyllostomus (Chiroptera Phyllostomidae) traditionally includes four species: P. hastatus, P. discolor, P. elongatus and P. latifolius (Valdez, 1970). The genera Phyllostomus and Phylloderma, solely comprising the species Phylloderma stenops, are closely related by morphology (Williams and Genoways, 1980), karyotypes (Baker, 1973, 1979) and albumin data (Honeycutt and Sarich, 1987). Baker et al. (1988) suggested the inclusion of Phylloderma stenops in Phyllostomus. Evidence of strong affinity between Phyllostomus and Phylloderma was reinforced by cytochrome-b sequence analysis (Van den Bussche and Baker, 1993). In this paper, by adding Phyllostomus stenops (Baker et al., 1988), we increased to five the number of species in the genus Phyllostomus. Widely distributed throughout in the Central and South America, P. hastatus occurs from Honduras to Southeastern Brazil; P. discolor occurs from Mexico to Argentina while P. elongatus is restricted to South America in Colombia, Venezuela, the Guianas, Brazil, Ecuador, Peru and Bolivia; P. latifolius was registered in the Kanuko Mountains, of Guyana and in Suriname whereas P. stenops is found from Mexico to Northern Bolivia and Southeastern Brazil (Valdez, 1970; Jones and Carter, 1976; Honeycutt et al., 1980; Wilson and Reeder, 1993). Karyotypic studies in Phyllostomus have demonstrated that all species possess 2n = 32; P. discolor has FN = 60 while P. hastatus, P. elongatus, P. stenops and P. latifolius have FN = 58 (Baker, 1973, 1979; Honeycutt et al., 1980). Chromosome comparisons by G-banding between P. discolor and P. hastatus from Central America (Patton and Baker, 1978) have demonstrated that only the smallest autosome (pair 15), a chromosome metacentric in P. discolor and acrocentric in P. hastatus, underwent rearrangement after divergence of the two species. A similar result was found in specimens from Southeastern Brazil (Varella-Garcia et al., 1989). C-banding studies revealed constitutive heterochromatin only at centromeric regions in P. hastatus, P. discolor and P. elongatus (Baker, 1979; Varella-Garcia et al., 1989). Ag-NOR staining identified NOR site, located only on chromosome 15 in P. hastatus and P. discolor (Morielle and Varella-Garcia, 1988). According to Patton and Baker (1978), the karyotype of Macrotus waterhousii (2n = 46, FN = 60) is considered primitive to the family Phyllostomidae. Phyllostomus hastatus, P. discolor, Mimon crenulatum and Tonatia minuta are associated by five synapomorphic chromosome rearrangements: four Robertsonian fusions (18/3, 8/9, 17/ 12 and 29/27) and one inversion (4/5 inv.). Phyllostomus and Mimon share three additional synapomorphic fusions (22/13, 14a/21 and 28/30). The monophyly of Phyllostomus was established but the interspecific relationships remain unresolved (Honeycutt and Sarich, 1987; Baker et al., 1988; Van Den Bussche and Baker, 1993). The Amazonian Region bat fauna is the world’s richest (Findley, 1993). Despite this, few studies have been made of bats from that region, at least from Brazilian Amazonia. This study presents a chromosome comparison between Phyllostomus discolor and P. hastatus, through G-bandChromosome comparison between two species of Phyllostomus (Chiroptera Phyllostomidae) from Eastern Amazonia, with some phylogenetic insights

Widely distributed throughout in the Central and South America, P. hastatus occurs from Honduras to Southeastern Brazil; P. discolor occurs from Mexico to Argentina while P. elongatus is restricted to South America in Colombia, Venezuela, the Guianas, Brazil, Ecuador, Peru and Bolivia; P. latifolius was registered in the Kanuko Mountains, of Guyana and in Suriname whereas P. stenops is found from Mexico to Northern Bolivia and Southeastern Brazil (Valdez, 1970;Jones and Carter, 1976;Honeycutt et al., 1980;Wilson and Reeder, 1993).
The Amazonian Region bat fauna is the world's richest (Findley, 1993).Despite this, few studies have been made of bats from that region, at least from Brazilian Amazonia.This study presents a chromosome comparison between Phyllostomus discolor and P. hastatus, through G-band-ing, C-banding, sequential G/C-banding and NOR staining patterns, in specimens exclusively from Brazilian Amazonia and discusses the phylogeny of genus Phyllostomus from the chromosomal standpoint.

MATERIAL AND METHODS
The sample included 3 specimens of Phyllostomus discolor (1M, 2F) from Belém (1 o 11' S, 48 o 29' W), and 4 P. hastatus (1M, 3F) from Peixe-Boi (1 o 11' S, 47 o 19' W), both in Pará State, Brazil.All specimens were taken from natural populations and collected with mist nets; voucher specimens are found in the mammal collections of the Emílio Goeldi Museum in Pará.Access numbers are: 26328, 26329 and 26330 for P. discolor and 26331, 26332, 26333 and 26334 for P. hastatus.Metaphase spreads were got with fibroblast cultures from ear biopsies and bone marrow preparations (Baker and Qumsiyeh, 1988).Slides were prepared using air-drying method and G-bands obtained by trypsin treatment (Scheres, 1972), C-bands were performed according to Sumner (1972), sequential G/C-banding followed both techniques (some metaphases were G-banded, photographed and later submitted to a C-banding process), and Ag-NOR staining followed Howell and Black (1980).

RESULTS
The karyotype of Phyllostomus discolor possesses 2n = 32 and FN = 60; the autosome complement is composed of 15 bi-armed pairs, while that P. hastatus has 2n = 32, FN = 58, with 14 bi-armed plus one acrocentric pair.In both species, the X chromosome is medium submetacen-tric and the Y is minute acrocentric.Figures 1 and 2 show, respectively, the G-and C-bands representative of P. hastatus and similar those of P. discolor.C-banding revealed constitutive heterochromatin restricted to the pericentromeric region of all chromosomes.
Comparative analysis of G-banding pattern between P. discolor and P. hastatus shows that both species conserved all chromosomes without rearrangements, except for chromosome 15, altered by pericentric inversion (Figure 3).Both species presented Ag-NOR labelling at a distal region of pair 15 (Figure 3d).

DISCUSSION
Karyotypes of P. discolor and P. hastatus from Eastern Amazonia are identical to those described for Central American specimens (Patton and Baker, 1978), and Southeastern Brazil (Morielle and Varella-Garcia, 1988;Varella-Garcia et al., 1989).Our results corroborate the hypothesis that Phyllostomus has a conservative rate of chromosomal evolution, in contrast to other widespread bat genera, e.g., Tonatia and Micronycteris that present a high rate of chromosomal evolution (Baker and Bickham, 1980).
P. discolor and P. hastatus differ cytogenetically by a single pericentric inversion in chromosome 15, probably derived by fusion of acrocentrics 28 and 30 from Macrotus waterhousii (Patton and Baker, 1978).This chromosome, a small metacentric in P. discolor, is acrocentric in P. hastatus, P. elongatus, P. stenops and P. latifolius (Baker, 1973(Baker, , 1979;;Honeycutt et al., 1980).Considering that P. discolor is primitive to genus Phyllostomus (Valdez, 1970;Honeycutt and Sarich, 1987;Baker et al., 1988), and shares  with Mimon crenulatum the metacentric chromosome 15 (Patton and Baker, 1978), the apparently derived acrocentric chromosome 15 is shared by P. hastatus, P. elongatus, P. stenops and P. latifolius.Chromosome 15 of P. discolor presents the NOR on the distal region of one arm, while it appears on the distal region of a short arm in the 15 of P. hastatus.Therefore, the chromosome portion involved in pericentric inversion seems unlikely to contain DNAr sequences, because of the distal position of the NOR site.In general, few species of the family Phyllostomidae have been studied by Ag-NOR staining (Baker, 1979).In the specific case of Phyllostomus, NOR studies are needed to verify if: 1) P. elongatus and P. stenops present NORs at distal regions of the short arm of chromosome 15 as in P. hastatus, 2) Mimon crenulatum has NOR sites on chromosome 15 as in P. discolor, and 3) Macrotus waterhousii presents NORs in the acrocentric 28 or 30.This information will increase understanding of chromosomal evolution in the genus Phyllostomus.
Interspecific relationships in the genus Phyllostomus have been difficult to clarify.Using morphological characters, Valdez (1970) proposed a close relationship between P. elongatus and P. latifolius.In contrast, albumin distance analysis (Honeycutt and Sarich, 1987) indicated a closer connection between P. hastatus and P. elongatus, joined by P. stenops in one group while P. discolor and P. latifolius emerged at the cladogram base.The allozymic data from Baker et al. (1988), while not adequately resolving interspecific relationships in Phyllostomus, suggested that the five species diverged at approximately the same time.Phylogenetic analysis with cytochrome-B sequence (Van Den Bussche and Baker, 1993) confirmed the hastatuselongatus association suggested by Honeycutt and Sarich (1987), and suggested that P. discolor is sister taxa to hastatus-elongatus, with P. latifolius joining this group and P. stenops at the root of the tree.
Cytogenetic data are compatible with the phylogeny based on albumin data, proposed by Honeycutt and Sarich (1987).On the other hand, from the cytogenetic standpoint, the hypothesis based on cytochrome-B (Van Den Bussche and Baker, 1993) is less parsimonious because if the biarmed form of chromosome 15 is primitive, then the derived form (acrocentric) would have twice evolved: once at lineage P. stenops, again at lineage P. latifolius and the finally at lineage hastatus-elongatus.
Additional studies are need for clarifying the interspecific relationships in the genus Phyllostomus.Special attention should be given to Ag-NOR staining or DNAr hybridization studies in Mimon crenulatum, Phyllostomus stenops and P. elongatus to ascertain the supposed homeology of chromosome 15.