Initial Characterization of Freshwater Sponge Species by PCR Using Random and Specific Primers

Abstract

A nongemmulating species of freshwater sponge, Discospongilla cooperi, was recently discovered in western Montana. Because traditional classification of freshwater sponges is based upon morphological characteristics of gemmules, freshwater sponges that do not produce gemmules cannot be reliably classified using morphological criteria. This project was designed as a preliminary step to differentiate D. cooperi from three other freshwater sponges that are known to exist in western Montana — Ephydatia muelleri, Spongilla lacustris, and Eunapiusfragilis - and determine the phylogenetic relationship ofthese freshwater sponges using molecular criteria. DNA was purified using proteinase K digestions or microwave disruption followed by phenol-chloroform extraction and then amplified with the polymerase chain reaction (PCR). Sizes of PCR products were used to characterize multiple individuals from each of the four sponge species studied. Random primers (RAPD) and primers specific for a receptor tyrosine kinase gene of the marine sponge Geodia cydonium were used for the PCR reaction. Reproducible PCR products were obtained from E. muelleri and D. cooperi. These PCR products provided enough information to differentiate E. muelleri and D. cooperi. Some molecular variability was observed between individuals within each of these species as well. This variability was attributed to differences in PCR conditions, DNA degradation, ineffective DNA purification, and DNA polymorphisms. S. lacustris and E. fragilis did not produce consistent PCR products. Consequently, the phylogenetic relationship between these four species of freshwater sponge could not be determined.