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Old World fruitbats were divided into the cynopterine, epomophorine, rousettine, eonycterine, and notopterine sections by Knud Andersen (1912). Among these, the eonycterine and notopterine sections together comprise the subfamily Macroglossinae, which includes forms with specializations for nectarivory. Single-copy DNA hybridization data argue against the monophyly of four of Andersen's sections and further suggest paraphyly or polyphyly of the Macroglossinae. DNA hybridization data provide support for an endemic African clade that includes Megaloglossus (an eonycterine), Epomophorus (an epomophorine), and Lissonycteris (a rousettine). Analyses of mitochondrial 12S rRNA-tRNA valine gene sequences corroborate the African clade but provide less resolution than hybridization data for most branches on the pteropodid tree. Here, we report 11 new 16S rRNA sequences and analyze a mitochondrial data set that includes 12S rRNA, tRNA valine, and 16S rRNA for 18 pteropodid genera. Parsimony, minimum evolution, and maximum likelihood were all employed in phylogenetic analyses. The addition of 16S rRNA sequences to the mitochondrial data set resulted in increased support for several clades, including Macroglossus + Syconycteris, Cynopterus + Thoopterus, Rousettus + the endemic African clade, and Eonycteris + Rousettus + the endemic African clade. Statistical tests suggest that another endemic African genus, Eidolon, is dissociated from the African clade and represents an independent invasion into Africa. We constructed a molecular phylogenetic framework that incorporated clades that were strongly supported by both single-copy DNA hybridization and 12S rRNA-tRNA valine-16S rRNA sequences. Using this framework as a backbone phylogenetic constraint, we then analyzed a morphological data matrix for 34 pteropodid genera with parsimony. Results of this analysis suggest that other epomophorines and Myonycteris (a cynopterine) are also part of the endemic African clade.