Bat Distribution Size or Shape as Determinant of Viral Richness in African Bats

Maganga, Gaël D.; Bourgarel, Mathieu; Vallo, Peter; Dallo, Thierno D.; Ngoagouni, Carine; Drexler, Jan Felix; Drosten, Christian; Nakouné, Emmanuel R.; Leroy, Eric M.; Morand, Serge

In order to improve the quality of the comparative analysis, a phylogenetic tree was built using 14 new molecular sequences of the bat mitochondrial cytochrome b gene ( Table 2 ). Total genomic DNA was extracted from ethanol-preserved tissue samples (muscle, liver or spleen) with Genomic DNA Tissue Mini Kit (Geneaid Biotech) according to the manufacturer's protocol. We amplified the mitochondrial gene for cytochrome b (cytb) using primer pairs F1 (modified; 59-CCACGACCAATGACAY-GAAAA-39) and R1 from Sakai et al. [26] in most microbats, L14724 and H15915 from Irwin et al. [27] in hipposiderids and fruit bats, LGL765F and LGL766R from Bickham et al. [28, 29] in long-fingered bats (Miniopterus inflatus). The volume of PCR reaction was 25 ml, it contained 12.5 ml Combi PPP Master Mix (Top-Bio, Prague, Czech Republic), 200 mM of forward and reverse primers respectively, and 2.5 ml of extracted DNA. PCR protocol consisted in an initial denaturation at 94uC for 3 min, 35 cycles of denaturation for 40 s at 94uC, annealing for 40 s at 50uC, and extension for 90 s at 65uC, and a final extension at 65uC for 5 min. Resulting PCR products were inspected on 1.5% agarose gel and purified with Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech). If multiple bands appeared, the one of appropriate length was excised and purified from gel using the same purification kit. Purified PCR products were sequenced commercially (Macrogen, Seoul, Korea) with the respective forward primer using BigDye Terminator sequencing chemistry (Applied Biosystems, Foster City, CA, USA) on ABI 3730xl sequencer. Sequences were edited in Sequencher 4.6 (Gene Codes, Ann Arbor, MI, USA), manually checked for correct base reading and protein coding frame, and aligned by eye in BioEdit 7.0 [30] . Sequences of two artiodactyl taxa, Bos taurus (D34635) and Ovis ammon (AJ867276) were added to the alignment as outgroup taxa for rooting the bat phylogeny. Phylogenetic tree including branch lengths was inferred from aligned nucleotide sequences in PAUP*4.0b (Sinauer Associates, Sunderland, Massachusetts, USA) under maximum likelihood (ML) criterion and general time-reversible model of evolution with a portion of invariable sites and gamma distributed variation rates (GTR+I+C), which was suggested as the best evolutionary model and whose parameters were estimated in Modeltest 3.7. Topological constraints were set before computation of the ML tree, as corresponding to acknowledged phylogenetic relationships among genera, families and higher taxonomic ranks of bats as referred by Teeling et al. [31] and Almeida et al. [32] . Due to a priori definition of the tree topology, analysis of nodal support was not performed. The constrained ML tree was, however, compared to unconstrained ML tree using a Shimodaira-Hasegawa (SH) test, in order to assess possible significant difference, which might indicate unreliability of the constrained tree. Sequences generated in this study were