spumarius EU672977 Peru:

Iquitos region Atelopus tricolor EU672978 Bolivia: Yungas de La Paz Atelopus varius U52779 Panama Atelopus varius AY325996 Costa Rica: near Las Alturas Atelopus zeteki DQ283252 Panama: Las Filipinas Atelopus oxapampae EU672979 Peru: Oxapampa region Atelopus sp. ‘cusco’ EU672980 Peru: near Puente Fortaleza Atelopus sp. ‘cocha’ AF375509 Colombia: Laguna Cocha Rhinella marina DQ283062 Peru Dendrophryniscus brevipollicatus AF375515 Brazil Osornophryne puruanta EU672982 Ecuador Osornophryne antisana EU6729823 Ecuador Osornophryne sp. 1 EU672981 Ecuador Osornophryne sp. 2 EU6729824 Ecuador Eleutherodactylus cf. johnstonei AF124123 Unknown DNA was extracted AZD6738 datasheet selleck products from toe clips. Tissue samples (stored in 99% ethanol) were digested using proteinase K (final concentration 1 mg/mL), homogenised and subsequently purified following a high-salt extraction protocol (Bruford et al. 1992). Polymerase chain reaction (PCR) primers for the fragment of the 16S rRNA gene were 16SA-L and 16SB-H of Palumbi et al.

(1991), used as in Van der Meijden et al. (2007). PCR products were purified via spin columns (Qiagen). Sequencing was performed directly using the corresponding

PCR primers. New sequences were combined with existing sequences taken from GenBank in the final dataset containing 27 taxa including bufonid and non-bufonid outgroups (Table 1). Sequences were aligned using ClustalW Proteases inhibitor (Selleckchem 3 Methyladenine Thompson et al. 1994) and subsequently edited by hand. The final alignment contained a total of 570 positions of which 219 were variable and 136 were parsimony-informative. Phylogeny reconstruction was performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Gaps were treated as unknown characters. The best fitting models of sequence evolution were determined by the AIC criterion as implemented in Modeltest 3.06 (Posada and Crandall 1998). ML tree searches were performed using PhyML, version 2.4.4 (Guindon and Gascuel 2003). Bootstrap branch support values were calculated with 200 replicates. The Bayesian analyses of the combined and separate datasets was conducted with MrBayes 2.