Molecular Identification of Dendropsophus Species

Samples of genomic DNA were obtained from Dendropsophusseniculus (ZUEC 17225), Dendropsophussoaresi (ZUEC 16867) and Dendropsophusnovaisi (ZUEC 17858) following the procedure reported by Medeiros et al. (2013) (link). A fragment of approximately 1300 bp of the 16S ribosomal RNA gene was PCR-amplified using the primers 12L13(L) (Feller and Hedges 1998 (link)) and 16Sbr(H) (Palumbi et al. 1991 ). The amplified products were purified with the GFX PCR and Gel Band DNA purification Kit (GE Healthcare) and directly sequenced in an automatic DNA ABI/Prism sequencer (Applied Biosystems) using BigDye Terminator kits (Applied Biosystems) and the primers 12L13 (Feller and Hedges 1998 (link)), TitusI(H) (Titus 1992), Hedges16L2a (Hedges 1994 ), Hedges16H10 (Hedges 1994 ), 16Sar(L) (Palumbi et al. 1991 ) and 16Sbr(H) (Palumbi et al. 1991 ). DNA sequences were aligned using ClustalW option implemented in BioEdit v. 7.2.5 (Hall 1999 ) and compared to each other and to the 16S rDNA sequence of Dendropsophusseniculus available at GenBank (AY843666).