Unlocking Demography: Developing an e <scp>DNA</scp> ‐Based Toolkit to Measure Sex Ratios From Populations

Demographic information, such as sex ratios, is essential for understanding population dynamics and informing conservation strategies. Yet obtaining sex ratios in natural populations can be challenging due to logistical, ethical and legal constraints. Environmental DNA (eDNA) has revolutionised non-invasive biodiversity monitoring, but its potential for assessing demographic parameters remains largely unexplored. Here we present an eDNA-based method to monitor sex ratios of populations by quantifying sex-specific SNP alleles. Using RADseq data from Balkan crested newts (Triturus ivanbureschi), we identified a male-specific allele that was consistently present in all males and absent in females. We then developed a Droplet Digital PCR (ddPCR) assay to quantify allele ratios and validated it on mock (DNA extract mixtures) and eDNA samples with known sex ratios. Our sex-specific SNP assay successfully distinguished male- and female-biassed ratios in mock samples and showed a strong positive relationship between the proportion of males and the male-specific allele. While resolution was lower in eDNA samples, sex ratio estimates reflected population composition, particularly when corrected for biomass. Performance was mainly influenced by inter-individual variation in male allele copy numbers, but this effect diminished as the number of males increased, reflecting natural populations better. For effective field application, maximising nuclear eDNA recovery, validating marker specificity and accounting for species-specific life history traits when sampling will be crucial. With further field validation, our eDNA-based method could support large-scale, non-invasive sex ratio monitoring, offering valuable insights into species phenology and population dynamics to guide conservation efforts.