Random Sperm Use and Genetic Effects on Worker Caste Fate in Atta Colombica Leaf-Cutting Ants
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Molecular Ecology (2011) 20, 5092–5102 doi: 10.1111/j.1365-294X.2011.05338.x Random sperm use and genetic effects on worker caste fate in Atta colombica leaf-cutting ants LUKE HOLMAN,*1,2 MARLENE STU¨ RUP,*1 KALEVI TRONTTI† and JACOBUS J. BOOMSMA* *Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark, †Department of Biosciences, PO Box 65, Viikinkaari 1, University of Helsinki, Helsinki 00014, Finland Abstract Sperm competition can produce fascinating adaptations with far-reaching evolutionary consequences. Social taxa make particularly interesting models, because the outcome of sexual selection determines the genetic composition of groups, with attendant sociobio- logical consequences. Here, we use molecular tools to uncover some of the mechanisms and consequences of sperm competition in the leaf-cutting ant Atta colombica, a species with extreme worker size polymorphism. Competitive PCR allowed quantification of the relative numbers of sperm stored by queens from different males, and offspring genotyping revealed how sperm number translated into paternity of eggs and adult workers. We demonstrate that fertilization success is directly related to sperm numbers, that stored sperm are well-mixed and that egg paternity is constant over time. Moreover, worker size was found to have a considerable genetic component, despite expectations that genetic effects on caste fate should be minor in species with a low degree of polyandry. Our data suggest that sexual conflict over paternity is largely resolved by the lifetime commitment between mates generated by long-term sperm storage, and show that genetic variation for caste can persist in societies with comparatively high relatedness. Keywords: competitive PCR, cryptic female choice, fair raffle, genetic caste determination, sperm competition Received 23 June 2011; revision received 14 September 2011; revision accepted 16 September 2011 den Boer et al. 2010; Manier et al. 2010). ‘Fair raffle’ Introduction sperm competition is a form of null model in which the Sperm competition (Parker 1970) is a widespread phe- fertilization success of each competing male is equal to nomenon with manifold evolutionary and ecological the proportion of sperm belonging to that male in the consequences (e.g. Miller & Pitnick 2002; Boomsma fertilization set (e.g. Parker 1990). Deviations from the et al. 2005; Holman & Snook 2008; Price et al. 2008; den fair raffle show that some males’ sperm have more Boer et al. 2010; Manier et al. 2010). Sperm competition chance of achieving fertilization than those of other occurs whenever ejaculates of different males compete males, for example because they differ in traits that for a common set of eggs, and selects for traits that affect fertilization success (e.g. insemination order, afford males a greater share of paternity in polyandrous motility or survival; Snook 2005), or because females females. Males frequently produce more sperm to bias paternity towards particular males (‘cryptic female numerically out-compete those of rivals, and may pos- choice’; Eberhard 1996). sess adaptations that reduce the number of competing The eusocial Hymenoptera are unique among insects rival sperm (Birkhead & Møller 1998; Simmons 2001; in many respects, including their mating biology. Reproductive activity is typically concentrated into a Correspondence: Luke Holman, Fax: 61255573; single brief ‘mating flight’, after which queens store E-mail: [email protected] sperm for unusually long periods (up to 20–30 years) 1 These authors contributed equally to the work. and never mate again (Boomsma et al. 2005). This 2Present address: Ecology, Evolution & Genetics, Research mating system imposes unique selective pressures on School of Biology, Australian National University, Canberra, ACT 0200, Australia. males, e.g. to produce sperm that remain viable Ó 2011 Blackwell Publishing Ltd SPERM USE AND CASTE FATE IN ATTA 5093 and competitive for many years. Additionally, genetic variation in caste fate, because only genetically hymenopteran males typically only receive fitness diverse colonies can contain multiple genotypes with returns from daughters that develop into queens, which different biases towards particular female morphs. Con- tend to be produced months or years after the mating trary to this intuitive expectation, the degree of genetic flight. This mating system reduces interlocus sexual caste determination across species is not clearly corre- conflict, because a male’s fitness depends on the long- lated with polyandry, suggesting a more complex pic- term survival of his mate and her colony, constraining ture (Schwander et al. 2005, 2010). A complete the evolution of competitive male traits that reduce the understanding of the origin and maintenance of eusoci- survival of either (Boomsma et al. 2005). Males are also ality and genetic caste determination therefore requires expected to have comparatively little power to influence an appreciation of reproductive biology, including paternity after the mating flight, because the majority of sperm competition when queens mate multiply. fertilizations occur long after the male has died and the Here, we investigate sperm use and genetic caste nonsperm components of the ejaculate (e.g. seminal determination in the leaf-cutting ant Atta colombica. Col- proteins; Baer et al. 2009) have broken down (den Boer onies of this species contain a single queen and up to a et al. 2010). million workers (Ho¨lldobler & Wilson 1990), which are Despite these evolutionary constraints, specialized classified into three nondiscrete size classes termed male adaptations to sperm competition have been minors, media and majors (2–15 mm in length). Smal- reported, including chemically complex seminal fluid ler workers preferentially tend the brood and fungus (Baer et al. 2009) that lowers the survival of rival male garden, while larger workers specialize in foraging and sperm while preserving self-sperm (den Boer et al. 2010; colony defence (Wilson 1980). Queens store sperm from King et al. 2011). Other putative male adaptations to a small number of males (typically 2–3; Fjerdingstad & sexual selection include mating plugs (Baer et al. 2000; Boomsma 1998; Baer & Boomsma 2006; Helmkampf Mikheyev 2003; Baer 2011) and detachable male genita- et al. 2008) during a single mating flight, and males lia (Wilhelm et al. 2011). Large variation in paternity produce seminal fluid that increases the survival of skew (the difference in proportion of offspring fathered their own sperm while hindering that of rivals’ sperm by each male; Boomsma & Ratnieks 1996) has been (den Boer et al. 2010). However, antagonistic interac- reported both within and across species (e.g. Boomsma tions among ejaculates are apparently halted after stor- & Sundstrom 1998; Boomsma & Van der Have 1998), age when male seminal fluid is replaced by secretions although the proximate and ultimate causes of this vari- from the queen’s spermatheca (the sperm storage ation remain virtually unknown. The intracellular bacte- organ) (den Boer et al. 2009, 2010), suggesting that overt rium Wolbachia, which can influence paternity skew by forms of sperm competition do not continue after per- rendering some mating types infertile (cytoplasmic manent storage. Sperm may be released from the sper- incompatibility; Werren 1997), is also present in many matheca at random, which would lead to fair raffle social Hymenoptera (e.g. Van Borm et al. 2001), but the competition, or there could be a bias in sperm use consequences of infection are largely unknown. towards particular males. Such a bias could be caused Sperm competition in social insects is of particular by differences in the fertilization ability of sperm from interest because of the sociobiological consequences of different ejaculates, cryptic female choice, physical strat- mating systems. The degree of polyandry and the out- ification of stored sperm leading to temporal variation come of postcopulatory sexual selection together deter- in sperm use (‘sperm clumping’; Boomsma & Ratnieks mine relatedness among the queen’s offspring. 1996; Wiernasz & Cole 2010) or incompatibility between Relatedness is a key parameter in the origin and mainte- male and female nuclear or cytoplasmic genotypes nance of eusociality, because altruistic traits such as (Wolbachia has been reported to infect this species, worker sterility are only selectively favoured when they although it is unknown whether it affects compatibility; provide enough benefit to sufficiently close relatives Van Borm et al. 2001). Moreover, evidence for genetic (Hamilton 1964; Boomsma 2007, 2009). Within-colony effects on worker caste fate has been found in the sister relatedness also affects the degree of queen-worker con- genus Acromyrmex (Hughes et al. 2003; Hughes & flict over male production and the sex ratio (Sundstro¨m Boomsma 2008), although its presence in Atta is & Boomsma 2000), as well as the degree of conflict unknown. All else being equal, we would expect between worker full-sibling lineages (patrilines) over the genetic variation for caste to be substantially reduced in paternity of new queens (Wenseleers 2007). Moreover, Atta because queen mating frequency is lower than in genetic factors are now known to influence the develop- Acromyrmex (10.6 ± 1; Sumner et al. 2004), suggesting ment of females into queens or different worker morphs that genes that cause a strong predisposition towards (reviewed in Anderson et al. 2008; Schwander