Perivitelline Membrane‐Bound Sperm as a Source of Paternal Genomic <scp>DNA</scp> to Inform Breeding Male Marine Turtle Genetics and Demographics

Sex in marine turtles is determined by incubation conditions, raising concerns of population feminization and loss of genetic diversity due to warming temperatures. Demographic data on breeding males are limited due to their relative inaccessibility. Hatchling sampling can inform multiple paternity (MP) and breeding sex ratios (BSR) but is logistically intensive, limiting the number of nests and populations analyzed. Here, we present a novel approach to characterize successfully breeding males by genotyping sperm trapped in the perivitelline membrane (PVM) surrounding the yolk of a single egg per clutch. We compared maternal genotypes via eggshells with PVM extract genotypes from 27 loggerhead turtle (<i>Caretta caretta</i>) eggs and 13 green turtle (<i>Chelonia mydas</i>) eggs from Melbourne Beach, Florida, USA, at 16 and 13 microsatellite loci, respectively. Sampled offspring genotypes (620 loggerhead and 1117 green turtle) provided ground-truthing of PVM sperm allele detections. Paternity analyses resolved 38 loggerhead and 29 green turtle sires, all but two of which were correctly inferred via PVM allele calls. All paternal alleles representing single paternity clutches and primary contributors of multiply sired clutches were detected in the PVM genotypes except in one case of maternal DNA swamping of the PVM. Seven of eight sires that contributed ≤ 11% of offspring were detected via PVM genotyping, comparable to inferences based on sampling 20 hatchlings per nest. PVM-inferred paternal genotypes from single-paternity clutches were > 99% concordant with reconstructed sire genotypes in both species. Although destructive to a single egg per clutch, this method is non-invasive to nesting females and hatchlings. Its scalability across space and time enables long-term monitoring of MP and BSR, key demographic metrics for assessing population viability under climate change. Male genotypes generated from the PVM in single paternity nests may also inform population assignment, assessments of genetic connectivity, and relatedness analyses.