Genetic verification of multiple paternity in two free-ranging isolated populations of African wild dogs (Lycaon pictus)

Abstract

The African wild dog, second most endangered carnivore in Africa, has a well-developed, highly cooperative pack system. The usual structure of a pack consists of a dominant breeding pair, the alpha male and female, several subordinates, non-breeding adults and dependent offspring. Domestic dog microsatellites were used to study the parentage in three packs and confirm that more than one dog, including the subordinate males, can sire pups within a litter as previously suggested. The study was performed on two isolated populations of wild dogs in the North West Province of South Africa. In Madikwe Game Reserve, skin samples from 47 dogs were obtained by means of biopsy darts (adults) and skin slivers taken from the ear (subadults) and stored in absolute ethanol. In Pilanesberg National Park, blood samples from 18 captured dogs were collected in EDTA blood tubes. The wild dogs were photographed and individually identified according to coat patterns. Behavioural data to determine ranking were collected from all three packs. DNA was extracted from collected samples using proteinase-K digestion followed by isolation of DNA with phenol/chloroform/isoamyl alcohol. A total of 16 microsatellite loci that consistently amplified and appeared to be polymorphic in wild dogs, were used. Polymerase Chain Reaction (PCR) was performed using two panels of microsatellite loci in multiplex reactions. An amount of 1 µl of PCR product was loaded on to the 3130 XL Genetic Analyser with Genescan 500 LIZ (Applied Biosystems) size standard and analysed using STRand (Board of Regents, University of California) software program. CERVUS 2.0 software was used to calculate allele frequencies, expected and observed heterozygosity, frequency of null alleles, polymorphic information content and exclusion probabilities for parentage assignment. Parentage verification was also performed manually. The parentage analysis revealed that at least one pup was not sired by the alpha male in each of the five litters studied. Although previous studies suggested that the alpha male sires the majority of offspring in the pack, our results confirm that subordinate males commonly sire pups with the alpha female if and when the opportunity arises. This is possibly a mechanism to decrease the effects of inbreeding.