1 Supporting Information for Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use Edina Nemesházi, Zoltán Gál, Nikolett Ujhegyi, Viktória Verebélyi, Zsanett Mikó, Bálint Üveges, Kinga Katalin Lefler, Daniel Lee Jeffries, Orsolya Ivett Hoffmann, Veronika Bókony Table of contents: I. Identification of sex reversal and assessing its relationships with human land use ................. 2 Tables............................................................................................................................ 2 Table S1. Sampling locations and land-use variables Table S2. Putative sex-linked PCR targets successfully sequenced in agile frogs with primers designed based on common frog sequences Table S3. PCR programs used for sequencing and molecular sexing Table S4. Loadings of land-use variables in the principal components Table S5. Genotype-phenotype combinations found in each clutch Figures........................................................................................................................... 8 Figure S1. Molecular sexing with SNP-specific PCR primers designed for Rds1 and Rds2 Figure S2. HRM-based genotyping on Rds3 Figure S3. Gonads in juvenile agile frogs Figure S4. Distribution of the breeding ponds along the „urban PC” and the „agricultural PC” Figure S5. Geographical distribution of our capture sites on both sides of the river Danube in Hungary II. Developmental abnormalities .............................................................................................. 12 Tables............................................................................................................................ 14 Table S6. Parameter estimates of the statistical models comparing sex-reversed and normal froglets Figures........................................................................................................................... 16 Figure S5. Larval growth and development speed, and juvenile spleen size and pigmentation in lab-raised froglets Figure S6. Froglets' body mass (without gut mass) at dissection Figure S7. Froglets' testis size References .............................................................................................................................. 19