Supplementary Material for: Sexing Frogs by Real-Time PCR: Using Aromatase <b><i>(cyp19) </i></b>as an Early Ovarian Differentiation Marker

Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, <i>Lithobates sylvaticus</i> and <i>Silurana tropicalis</i> tadpoles were sampled at different developmental stages covering the entire process of sex differentiation. Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase <i>(cyp19)</i>, forkhead box L2 <i>(foxl2)</i> and the cytochrome 17-alpha-hydroxylase/17,20 lyase <i>(cyp17)</i>. Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of <i>cyp17,</i><i>cyp19</i> and <i>foxl2</i> in <i>L. sylvaticus</i> adult gonads and gonad-mesonephros complex (GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of <i>cyp19</i> alone can be used as a robust predictor of phenotypic sex in<i> L. sylvaticus </i>tadpoles. In addition, we validated this method measuring <i>cyp19</i> mRNA levels in <i>S. tropicalis </i>GMCs. We propose measuring <i>cyp19</i> as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future.