While mouthbrooding is not an uncommon parental care strategy in fishes, paternal mouthbrooding only occurs in eight fish families and little studied. The high cost of paternal mouthbrooding to the male implies a low risk of investment in another male’s offspring but genetic parentage patterns are poorly known for paternal mouthbrooders. Here we used single nucleotide polymorphism (SNP) genetic data to investigate parentage relationships of broods of two mouthbrooders of northern Australian rivers, mouth almighty Glossamia aprion and blue catfish Neoarius graeffei. For N. graeffei, we found that the parentage pattern was largely monogamous with the brooder male as the sire. For G. aprion, the parentage pattern was more heterogeneous including observations of monogamous broods with the brooder male as the sire (73%), polygyny (13%), cuckoldry (6%) and a brood genetically unrelated to the brooder male (6%). Findings demonstrate the potential for complex interrelationships of male care, paternity confidence, and mating behaviour in mouthbrooding fishes.

Sample collection and preparation

Samples of 115 N. graeffei (9 brooding males, 90 offspring and 16 non-brooding adults) and 261 G. aprion (18 brooding males, 180 offspring and 63 non-brooding adults) were collected across rivers of Northern Territory, Australia. G. aprion was collected in five sites: the Mary River (AHMR), Adelaide River (DRAR) and Daly River (OODR, SHFR, GJKR) and N. graeffei was collected from one site (Mary River) using electrofishing. Non-brooding males and females were included to provide basic population genetics context for our study including allele frequency estimates required for parentage analysis. Each fish was euthanized in AQUI-S (175mg/L for 20 mins) upon capture; brooding males together with their offspring were bagged individually, labelled and kept in an ice slurry until processed (see S1 – S3 for fish collection and justification of method). Fish collections were made as part of a larger study investigating trait variation in freshwater fishes, thereby utilising specimens for multiple research purposes. These species are abundant, widely distributed in the northern Australian rivers and not considered threatened species (23).

We randomly sampled 9-11 offspring from each brooding male parent. This sample size gave us a 95% chance of detecting the contribution of more than one parent (of either sex) to a brood as long as the dominant parent contributed at least 22-30% of the offspring (5,8; see S4 for explanation). Approximately 5-7 mg of adult muscle, larval tissue or whole egg was collected and stored in 70% ethanol until DNA extraction and genomic sequencing.

 SNP discovery and filtering

Samples were genotyped at single nucleotide polymorphism loci using the DArTSeq method at Diversity Arrays Technology Pty Ltd, Canberra, Australia (24). We used strict SNP filtering criteria to retain only high confidence genotypes for parentage analysis using a combination of custom R scripts, ‘adegenet’, ‘dartR’ and ‘plotly’ packages in R (25,26,27,28) (See S5 for detailed SNPs filtering and genetic diversity estimation methods).