A cost-effective protocol for total DNA isolation from animal tissue

Guanidine hydrochloride (GHCl) is frequently used for the isolation of RNA from eukaryotic cells. However, it appears that its use for the rapid isolation of chromosomal DNA has been little appreciated. Intact and readily digestible DNA was prepared from a range of murine tissues and cell lines which had been dispersed in GHCl and ethanol precipitated, and then rinsed in 70% ethanol and resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. This simple technique is ideally suited to the preparation of DNA from large numbers of tissue samples.

Geographic barriers and elevational gradients have long been recognized as important in species diversification. Here, we illustrate an example where both mechanisms have shaped the genetic structure of the Neotropical rainfrog, Pristimantis ornatissimus, which has also resulted in speciation. This species was thought to be a single evolutionary lineage distributed throughout the Ecuadorian Chocó and the adjacent foothills of the Andes. Based on recent sampling of P. ornatissimus sensu lato, we provide molecular and morphological evidence that support the validity of a new species, which we name Pristimantis ecuadorensis sp. nov. The sister species are elevational replacements of each other; the distribution of Pristimantis ornatissimus sensu stricto is limited to the Ecuadorian Chocó ecoregion (< 1100 m), whereas the new species has only been found at Andean localities between 1450–1480 m. Given the results of the Multiple Matrix Regression with Randomization analysis, the genetic difference between P. ecuadorensis and P. ornatissimus is not explained by geographic distance nor environment, although environmental variables at a finer scale need to be tested. Therefore this speciation event might be the byproduct of stochastic historic extinction of connected populations or biogeographic events caused by barriers to dispersal such as rivers. Within P. ornatissimus sensu stricto, morphological patterns and genetic structure seem to be related to geographic isolation (e.g., rivers). Finally, we provide an updated phylogeny for the genus, including the new species, as well as other Ecuadorian Pristimantis.