Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla

Abstract

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.