Electrophoretic and Immunological Studies on the Relationship of the Brachyphyllinae and the Glossophaginae

-Electrophoretic and albumin immunological data indicate that the Brachyphyllinae as currently conceived is a natural assemblage, with Erophylla sezekorni and Phyllonycteris aphylla being more closely related to each other than either is to Brachyphylla cavernarum. In both data sets, values that distinguish Erophylla from Phyllonycteris are in the general range of values that characterize congeneric species of mammals. Immunological distance values for the species Glossophaga soricina, Monophyllus redmani, Anoura caudifer, Leptonycteris sanborni, Choeroniscus minor, and Hylonycteris underuoodi indicate that these taxa are approximately equidistant from the Brachyphyllinae. Immunological comparisons of Glossophaga and Monophyllus to Anoura, Leptonycteris, Choeroniscus, and Hylonycteris indicate that the four latter genera are more closely related to Glossophaga and Monophyllus than Glossophaga and Monophyllus are to the genera of the Brachyphyllinae. Values from Lonchophylla thomasi and Lionycteris spurrelli suggest a more distant relationship of these glossophagine genera to the Brachyphyllinae than the other glossophagine genera examined. Our data suggest that a 2N = 32, FN = 60 karyotype like that characteristic of Glossophaga is primitive for a clade including the Brachyphyllinae and the glossophagine genera Anoura, Glossophaga, Monophyllus, Leptonycteris, Hylonycteris, and Choeroniscus. These data suggest that the polyphyletic origin of certain glossophagine genera, as proposed by Baker (1967), is unlikely. This study was designed to provide data on the evolution and systematics of the only endemic subfamily of Antillean bats, the Brachyphyllinae (Phyllostomidae). The evolutionary affinities and number of genera in the Brachyphyllinae (=Phyllonycterinae; see Baker, 1979) has been a persistent source of debate (Silva-Taboada and Pine, 1969; Baker and Bass, 1979). One of the three genera, Brachyphylla, currently placed in this subfamily has been placed in four different subfamilies (Baker and Bass, 1979). Data from G-band chromosomes show that relative to the proposed primitive karyotype for the family Phyllostomidae (Patton and Baker, 1978), Brachyphylla shares a derived karyotype with Erophylla and Phyllonycteris (the other two genera recognized in the Brachyphyllinae). However, this derived karyotype is also shared with two genera (Glossophaga and Monophyl lus) of the subfamily Glossophaginae. Therefore, these chromosomal data cannot conclusively document that Brachyphylla is more closely related to Erophylla and Phyllonycteris than to the glossophagine genera Monophyllus and Glossophaga. We address this systematic question using biochemical data from both starch-gel electrophoresis and albumin immunology. The current chromosomal data for Glossophaga, Monophyl lus , and the brachyphyllines can be interpreted in at least two ways. First, the karyotype of the brachyphyllines and Glossophaga and Monophyllus is pleisiomorphic for all glossophagines 1. Mamm., 62(4):665-672, 1981 665 666 JOURNAL O F MAMMALOGY Vol. 62, No. 4 as well as the brachyphyllines, or second, this karyotype is the result of a group of synapomorphic character states that document that some glossophagine genera (Monophyllus, Glossophaga, and possibly others) and the Brachyphyllinae shared a common ancestor after diverging from other glossophagines (as proposed in fig. 8 of Gardner, 1977). The systematic implications of the two alternatives are radically different. In the first case, recognition of the Brachyphyllinae would be justifiable from a phylogenetic standpoint, with the Glossophaginae as a sister taxon. However, the second case would imply that despite the classical anatomical and dental differences that serve as the basis for recognition of the two subfamilies, there would be no phylogenetic basis for recognition of the Brachyphyllinae as a sister taxon to the Glossophaginae (Baker and Bass, 1979). The immunological technique of microcomplement fixation was used to examine the above alternative evolutionary hypotheses. MATERIALS AND METHODS Electrophoretic analysis.-Bats were collected with mistnets from natural populations (species, localities, and sample sizes given under specimens examined). Immediately after sacrifice, liver, kidney, and heart samples were removed and frozen in liquid nitrogen. Techniques for tissue preparation, electrophoresis, and biochemical staining are essentially those described by Selander et al. (1971). Seventeen isozymes were assayed. These presumed loci were Isocitrate dehydrogenase-l,2 (Idh-l,2), Malate dehydrogenase-l,2 (Mdh-l,2), Lactate dehydrogenase-l,2 (Ldh-l,2), Leucine aminopeptidase (Lap), Albumin (Alb), Glutamate oxalate transaminase-1,2 (Got-l,2), a-Glycerophosphate dehydrogenase (a-Gpd), 6-Phosphogluconate dehydrogenase (6-Pgd), Hemoglobin (Hb), Indophenol oxidase-2 (Ipo-2), Phosphoglucomutase-2 (Pgm-2), Sorbitol dehydrogenase (Sdh), and Peptidase (Pep). The substrate used to resolve Pep was Glycyl-L-l'eucine. The most common allele for a given locus was designated 100 if cathodal, 100 if anodal. All other variants are described in percentages of this common allozyme. Loci were designated numerically, with the most anodal isozyme being "1" and more cathodal loci being given progressively higher numbers. By using the allozyme data, Rogers' D values (Rogers, 1972) were calculated between the species studied. Immunology.-Albumins were purified from serum (Glossophaga) and pooled tissues (Brachyphylla, Phyllonycteris, L%fonophyllus) remaining from the electrophoretic studies. For each albumin, 5 g of tissue were homogenized in 15 ml isotris buffer, dialyzed against 50 mM Tris (pH = 8.0), and centrifuged. To the supernatant was added 20 mg Rivanol (2-ethoxy 6, 9 diaminoacridine lactate) in 1 ml of the above buffer and the resulting precipitate was separated by centrifugation. The precipitate (mainly a Rivanol-albumin complex) was then treated with 5 ml of 0.5 M Trizma-HC1 until the fine yellow particles of free Rivanol were regenerated. The Rivanol was then separated by centrifugation and the supernatant dialyzed against 0.2 M Trissulfate (pH = 8.9) and vacuum-dialyzed to a volume of 0.7 ml. Fifty percent glycerol (0.2 ml) was then added and the albumin isolated by preparative polyacrylamide electrophoresis using the above-mentioned Tris-sulfate buffer for the gel and a Tris-borate electrode buffer (3.2 g Tris, 0.45 g boric acid per liter). The acrylamide concentration was 7.5%. The albumin band was identified using ANS (8-Anilino-I-Napthalenesulfonic Acid Magnesium Salt), cut out of the gel, and eluted in 12 ml of isotris buffer. Albumin from Glossophaga was prepared in the same way except that the original dialysis was done on a six-fold diluted serum sample. Antisera to the albumins of Brachyphylla cavernarum, Phyllonycteris aphylla, Monophyllus plethodon, and Glossophaga soricina were prepared in rabbits (three to four Dutch belted rabbits per albumin) according to the schedule of Sarich (1969). In each case, the antisera to a particular bat species were titered using the microcomplement fixation (MC'F) procedure and pooled in reciprocal proportion to their titers (Sarich and Wilson, 1966). These pooled antisera were used for all subsequent analyses. Albumins of certain bat species were compared and their corresponding immunological differences calculated using quantitative microcomplement fixation. Immunological differences were reported in albumin immunological distance units (AID) with one unit being approximately equivalent to one amino acid substitution (Prager and Wilson, 1971; Maxson and Wilson, 1974). Data analysis.-The Fitch and Margoliash (1967) method was used to construct phylogenetic Nooember 1981 BAKER E T AL.-BRACHYPHYLLINAE AKD GLOSSOPHAGIKAE 667 Ad o ? I g %so o 0 0 2 s I ,y I I