Reconstructing the mating system of Santa Cruz Galapagos giant tortoises with implications for the conservation of a critically endangered species

Mating systems, the process of how males and females choose their mates, influence reproductive strategies, social structure, and genetic diversity within populations. Knowledge of mating systems is also important for informing conservation strategies. The two species of Galapagos giant tortoise on Santa Cruz Island (Chelonoidis donfaustoi and C. porteri) are keystone species in their respective habitats. To assist in conservation efforts, more knowledge of their mating systems in wild populations is necessary, including whether multiple paternity is present. To enable investigation of reproductive behavior in Santa Cruz giant tortoises, we developed a genotyping-in-thousands by sequencing (GT-seq) panel that can identify species and individuals and estimate relatedness. Given the conservation status of these species, we designed and optimized the panel to genotype cloacal swab samples using a minimally invasive approach. The developed panel includes 325 SNPs; 38 targeted for species identification and 287 for individual identification and relatedness estimation. Genotypic data collected from 220 samples from 42 nests and attending mothers were used to reconstruct the mating system to support nesting data and to determine the presence of multiple paternity within the population. In total, 5 of the 14 females sampled had nests with different fathers in the same nesting season. Four of these females had nests with two fathers, indicating multiple paternity. Overall, the findings of this research contribute to the existing literature on Galapagos giant tortoise mating systems and can help inform future management decisions within the system.