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Identification of free-living Oophila amblystomatis (Chlorophyceae) from Yellow Spotted Salamander and Wood Frog breeding habitat

Abstract: Aquatic egg masses of several North American amphibians associate symbiotically with the unicellular green alga Oophila amblystomatis (Chlorophyceae). Studies conducted on egg masses of the yellow spotted salamander prior to the molecular era concluded that O. amblystomatis invaded egg masses from the surrounding water but no definitive identification of this taxon from known breeding habitat has been reported. A recent report provided evidence for intergenerational transfer of symbionts, thereby heightening the need to generate more definitive evidence of their provenance. To that end we developed a collection and filtration method, and combined with PCR and sequencing of 18S ribosomal DNA, show that pond water in which the yellow spotted salamander and the wood frog deposit egg masses contains algae that nest within the recently reported Oophila clade. More specifically, some of the algal sequences obtained through this method nest within the Oophila subclade that contains algae that exclusively associate with the yellow spotted salamander. Other sequences nest with the Oophila subclade that associate with wood frogs, supporting the possibility of host specialization. This study supports, and provides the means to further test, the hypothesis that algae from breeding habitat are a source of algae that form symbiotic associations with amphibian egg masses.