Beyond DNA sequencing : integrative approaches to resolving selected higher and lower taxonomic problems in Afrotropical Chiroptera.
Of the approximate 300 currently recognised bat species known from the Afrotropics, very few have been studied in sufficient detail to a) provide accurate species and distributional limits for extant taxa, b) identify possible cryptic species, and c) ascertain the closest sister lineage of numerous taxonomic groups. For those species where DNA-based phylogenies are available, the use of additional taxonomic markers and methods has provided further insights into the evolutionary history of certain extant chiropteran groups. This work comprises a series of systematic studies of African and Malagasy Chiroptera aimed at investigating sequence-based evolutionary hypotheses of higher and lower level taxa using comparative molecular cytogenetic and morphometric techniques. Efforts were directed at resolving taxonomic inconsistencies of chiropteran taxa from the African subregion and/or Madagascar, for which there is a general paucity of comprehensive and/or resolved phylogenies. Taxa belonging to the families Pteropodidae, Hipposideridae, Myzopodidae, and Molossidae were chosen for study because molecular-based have failed to provide consensus regarding evolutionary relationships amongst the above-mentioned taxonomic groups, or are in stark contrast to phylogenies based on morphological data. In addition, molecular cytogenetics and geometric morphometric approaches were used because they have had been applied in few evolutionary studies of Afrotropical bats. With the exception of a few karyotypic descriptions, scant data are available that details the chromosomal diversity and karyotypic evolution of bats from Madagascar in relation to their conspecifics or congenerics on other continents. To understand better the mechanisms that may have structured the karyotypes of extant Malagasy Chiroptera and the utility of chromosomal characters in retracing their evolutionary history, eight species from seven families were analysed using G- and C-banding and chromosome painting. Robertsonian (Rb) fusions and fissions were the dominant mode of genome restructuring amongst taxa and, for the most part, proved useful characters for investigations of phylogenomic relationships amongst families and genera. Chromosomal data generated from painting studies employing Myotis myotis (MMY) chromosomal probes, produced phylogenetically important characters that supported two conflicting hypotheses regarding the evolutionary affinities of the Myzopodidae, a family of bats endemic to Madagascar. The Rb fusion MMY 9+11 detected in Myzopodidae, also common to Phyllostomidae, could suggest a close association of Myzopoda aurita with the superfamily Noctilionoidea. However, the Rb fusion MMY 3+4 that is also present in vesper bats, suggests closer evolutionary ties between M. aurita and the Vespertilionoidea. A sex-autosome translocation, a cytogenetic character previously confined to phyllostomid and vespertilionid bats, was also detected in M. aurita casting further uncertainties on the evolutionary origins of this deep-branching species. This study highlighted the need for more refined cytogenetic investigations based on human-derived chromosomal paints and the application of highresolution bacterial artificial chromosomal (BACs) probes to map intrachromosomal breakpoints and/or subchromosomal rearrangements in the genome of Myzopoda. Heterochromatic polymorphisms and inversions appear to be important mechanisms of karyotypic evolution amongst pteropodid genera. Painting data revealed that at least five structural arrangements might be linked to the evolutionary divergence of pteropodine and rousettine fruit bats. A cryptic pericentric inversion was detected in the genome of Pteropus rufus corresponding to the homologue of MMY 4+19 (equivalent to HSA3+21); an ancestral syntenic character proposed for eutherian mammals. Proposed synapomorphies of the rousettine clade, as defined by molecular DNA studies, include the derived state of the MMY 4+19 homologue and the non-centric fusion of MMY 16/17+24 homologue. Integration of painting data on Hipposideros commersoni with published comparative maps of other hipposiderids enabled a brief revision of the postulated ancestral hipposiderid chromosomal complement. These data disputed previously proposed chromosomal synapomorphies of Hipposideridae and supported the basal position of H. commersoni within the genus. The inclusion of other hipposiderid genera, in particular Malagasy Paratriaenops and southern African Cloeotis, in chromosome painting studies may allow for further inferences regarding the evolutionary history of this diverse family. Morphometric approaches were employed to resolve uncertainties concerning species-level relationships within Afrotropical Otomops. Multivariate analyses delineated three well-supported morphological groups that corresponded to recently described genetic lineages and revealed several species-specific morphological traits for taxonomic diagnoses. Otomops from Djibouti, Ethiopia, Kenya, and Yemen constitute an undescribed morphologically and genetically cohesive group that requires a formal taxonomic description. Understanding the ecological and possible physiological adaptive value of morphological variation can provide valuable insights into the evolutionary history of this Afrotropical species complex. This work has provided further insights into the systematics of certain Afrotropical Chiroptera through the use of molecular cytogenetic and geometric morphometric techniques. Specifically, it has facilitated the interpretation of ancestral, independent and convergent chromosomal characters in the evolution of Afrotropical taxa belonging to the families Pteropodidae, Hipposideridae, and Myzopodidae, and has also elucidated lineage-specific morphological attributes in members of the genus Otomops thereby advancing our understanding of chiropteran diversity within the region.