Information on genetic relationships among individuals is essential to many studies of the behaviour and ecology of wild organisms. Parentage and relatedness assays based on large numbers of single nucleotide polymorphism (SNP) loci hold substantial advantages over the microsatellite markers traditionally used for these purposes. We present a double-digest restriction site-associated DNA sequencing (ddRAD-seq) analysis pipeline that, as such, simultaneously achieves the SNP discovery and genotyping steps and which is optimized to return a statistically powerful set of SNP markers (typically 150-600 after stringent filtering) from large numbers of individuals (up to 240 per run). We explore the trade-offs inherent in this approach through a set of experiments in a species with a complex social system, the variegated fairy-wren (Malurus lamberti) and further validate it in a phylogenetically broad set of other bird species. Through direct comparisons with a parallel data set from a robust panel of highly variable microsatellite markers, we show that this ddRAD-seq approach results in substantially improved power to discriminate among potential relatives and considerably more precise estimates of relatedness coefficients. The pipeline is designed to be universally applicable to all bird species (and with minor modifications to many other taxa), to be cost- and time-efficient, and to be replicable across independent runs such that genotype data from different study periods can be combined and analysed as field samples are accumulated.

Extra-pair paternity (EPP), where offspring are sired by a male other than the social male, varies enormously both within and among species. Trying to explain this variation has proved difficult because the majority of the interspecific variation is phylogenetically based. Ideally, variation in EPP should be investigated in closely related species, but clades with sufficient variation are rare. We present a comprehensive multifactorial test to explain variation in EPP among individuals in 20 populations of nine species over 89 years from a single bird family (Maluridae). Females had higher EPP in the presence of more helpers, more neighbours or if paired incestuously. Furthermore, higher EPP occurred in years with many incestuous pairs, populations with many helpers and species with high male density or in which males provide less care. Altogether, these variables accounted for 48% of the total and 89% of the interspecific and interpopulation variation in EPP. These findings indicate why consistent patterns in EPP have been so challenging to detect and suggest that a single predictor is unlikely to account for the enormous variation in EPP across levels of analysis. Nevertheless, it also shows that existing hypotheses can explain the variation in EPP well and that the density of males in particular is a good predictor to explain variation in EPP among species when a large part of the confounding effect of phylogeny is excluded.

44Information on genetic relationships among individuals is essential to many 45 studies of the behavior and ecology of wild organisms. Parentage and relatedness 46 assays based on large numbers of SNP loci hold substantial advantages over the 47 microsatellite markers traditionally used for these purposes. We present a double-digest 48 restriction site-associated DNA sequencing (ddRAD-seq) analysis pipeline that, as such, 49 simultaneously achieves the SNP discovery and genotyping steps and which is 50 optimized to return a statistically powerful set of SNP markers (typically 150-600 after 51 stringent filtering) from large numbers of individuals (up to 240 per run). We explore the 52 tradeoffs inherent in this approach through a set of experiments in a species with a 53 complex social system, the variegated fairy-wren (Malurus lamberti), and further validate 54 it in a phylogenetically broad set of other bird species. Through direct comparisons with 55 a parallel dataset from a robust panel of highly variable microsatellite markers, we show 56 that this ddRAD-seq approach results in substantially improved power to discriminate 57 among potential relatives and considerably more precise estimates of relatedness 58

Cooperatively breeding species vary widely in degree of social complexity, and disentangling relationships among group members can reveal the costs and benefits of cooperation. Here, we explore the social system of a relatively unstudied cooperatively breeding bird, the Variegated Fairywren (Malurus lamberti), and explore how social complexity and group dynamics may affect cooperation and conflict. We used a combination of field-based population monitoring and detailed social association observations to determine group membership annually across four breeding seasons (2014 – 2017), and used a ddRAD-seq genotyping method to determine genetic relationships within social groups. Social groups ranged in size from 2 – 8 individuals and nearly half of all social groups had multiple adult individuals of both sexes. Approximately two-thirds of those groups exhibited plural breeding, in which multiple females within the same social group nested individually on the same territory. Genetic relationships were diverse across social groups, and many consisted of a combination of relatives and non-relatives of each sex. Notably, although related females often were present within a social group, co-breeding females in the same social group were never closely related to each other. Given extensive variation in relatedness among group members, cooperation in the Variegated Fairywren is likely maintained by a combination of direct and indirect fitness benefits.