How many fathers? Study design implications when inferring multiple paternity in crocodilians.

Up to ten males have been reported to sire clutches of crocodilian eggs but review of the underlying study designs raised questions of potential upward bias of inferred sire numbers. To test this premise, different scenarios were explored using a published dataset of 16 known single-sire saltwater crocodile pairs and their offspring which were originally confirmed using an 11 loci microsatellite panel in CERVUS. Varying the number of microsatellites, omitting one or both parental genotypes and using different parentage analysis techniques revealed that total allele number, rather than number of loci, determined inferred sire accuracy in two opposing ways. Using the single-locus minimum method and GERUD, which both require prior knowledge of family groupings (ie nests), fewer alleles (and loci) accurately inferred only one father. In contrast, CERVUS and COLONY required all 11 loci (65 alleles) and both parental genotypes to (a) assign correct family groups and (b) infer the correct sire number, except in one family where two sires were equally assigned based on their number of homozygous loci. When less genotype information was provided, CERVUS and COLONY inferred up to six and seven sires, respectively. Consequently, crocodilian maternal genotypes should at least be obtained either directly (blood, tissue) or indirectly (eggshells, eDNA) in addition to a representative population sample of adult genotypes before embarking on multiple paternity determination. Running different scenarios using different analytical methods and reporting on the lowest number of inferred sires is encouraged remembering these sires are hypothetical until proven by true genotype.