Intraspecific support for the polygyny-vs.-polyandry hypothesis in the bulldog ant Myrmecia brevinoda ZENG-QIANG QIAN,* HELGE SCHLU ¨ NS,*† 1 BIRGIT C. SCHLICK-STEINER,‡ 1 FLORIAN M. STEINER,‡ 1 SIMON K. A. ROBSON,* 1 ELLEN A. SCHLU ¨ NS*† 1 and ROSS H. CROZIER* 2 *School of Marine and Tropical Biology, James Cook University, Townsville, QLD 4811, Australia, †Department of Apiculture, University of Agricultural Sciences and Veterinary Medicine, 400372 Cluj-Napoca, Romania, ‡Molecular Ecology Group, Institute of Ecology, University of Innsbruck, 6020 Innsbruck, Austria Abstract The number of queens per colony and the number of matings per queen are the most important determinants of the genetic structure of ant colonies, and understanding their interrelationship is essential to the study of social evolution. The polygyny-vs.-polyandry hypothesis argues that polygyny and polyandry should be negatively associated because both can result in increased intracolonial genetic variability and have costs. However, evidence for this long-debated hypothesis has been lacking at the intraspecific level. Here, we investigated the colony genetic structure in the Australian bulldog ant Myrmecia brevinoda. The numbers of queens per colony varied from 1 to 6. Nestmate queens within polygynous colonies were on average related (r qq = 0.171 ± 0.019), but the overall relatedness between queens and their mates was indistinguishable from zero (r qm = 0.037 ± 0.030). Queens were inferred to mate with 1–10 males. A lack of genetic isolation by distance among nests indicated the prevalence of independent colony foundation. In accordance with the polygyny-vs.-polyandry hypothesis, the number of queens per colony was significantly negatively associated with the estimated number of matings (Spearman rank correlation R = )0.490, P = 0.028). This study thus provides the rare intraspecific evidence for the polygyny-vs.-polyandry hypothesis. We suggest that the high costs of multiple matings and the strong effect of multiple mating on intracolonial genetic diversity may be essential to the negative association between polygyny and polyandry and that any attempt to empirically test this hypothesis should place emphasis upon these two key underlying aspects. Keywords: genetic relatedness, Hymenoptera, microsatellites, Myrmeciinae, polyandry, polygyny Received 12 March 2011; revision received 4 June 2011; accepted 14 June 2011 Introduction Eusocial insect colonies with the simplest relatedness structure are headed by a single, once-mated queen, which is considered ancestral in the evolution of insect eusociality (Hughes et al. 2008a). However, multiple queens per colony (polygyny) and multiple paternity (polyandry) are not rare at all, especially in ants (reviewed by Keller & Reeve 1994; Crozier & Pamilo 1996; Schmid-Hempel & Crozier 1999; Baer 2011). As estimated from the data set of Hughes et al. (2008b), 41.54% and 44.62% of formicid species show some level of polygyny and polyandry, respectively. The number of queens per colony has received significant attention (e.g. Ho ¨ lldobler & Wilson 1977; Keller 1993, 1995), because it affects the relatedness among colony members and thus the indirect benefits workers gain by helping to raise the brood (Heinze & Keller 2000). It is to a queen’s disad- vantage to share the colony with (especially unrelated) queens owing to a decrease in individual reproductive output (i.e. individual fitness) and to the workers’ disad- vantage to raise queens that are unrelated (or distantly related) to them (Craig & Crozier 1979). Additional dis- advantages proposed for polygyny include an increased Correspondence: Zeng-Qiang Qian, Fax: +61 7 4725 1570; E-mail: zengqiang.qian@yahoo.com 1 These authors contributed equally to the paper. 2 Prof Ross H. Crozier deceased on 12 November 2009. Ó 2011 Blackwell Publishing Ltd Molecular Ecology (2011) 20, 3681–3691 doi: 10.1111/j.1365-294X.2011.05195.x