DOI: 10.5061/dryad.905qfttmn
title: Diverse parentage relationships in paternal mouth-brooders

description: 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.
description: <em>Sample collection and preparation</em>
 Samples of 115 <em>N. graeffei </em>(9 brooding
 males, 90 offspring and 16 non-brooding adults) and 261 <em>G.
 aprion</em> (18 brooding males, 180 offspring and 63 non-brooding
 adults) were collected across rivers of Northern Territory, Australia.
 <em>G. aprion </em>was collected in five sites: the Mary River
 (AHMR), Adelaide River (DRAR) and Daly River (OODR, SHFR, GJKR) and
 <em>N. graeffei</em> 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.
 <em> SNP discovery and filtering</em>
 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).