Original articleFinding hidden females in a crowd: Mate recognition in fig wasps
Introduction
Mate recognition is often the first and most important step in reproduction. For those organisms that mate in large aggregations, mate recognition must occur in crowded conditions. Such aggregations can either consist of conspecifics as in garter snakes (Shine and Mason, 2001) and bark beetles (Byers and Wood, 1980) or of multiple species as in mycophagous Drosophila (Jaenike et al., 1992), fiddler crabs (Detto et al., 2006), Anopheles mosquitoes (Diabaté et al., 2006) and fig wasps (Janzen, 1979; Weiblen, 2002; Herre et al., 2008; Ghara and Borges, 2010). Mate recognition signals function not only as barriers to hybridisation in mixed-species aggregations, but are also important for reproductive isolation in sympatric species. The evolution of mate recognition signals can often be important in the radiation and maintenance of species isolation in species that are sympatric and syntopic sharing not only geographic ranges, but also habitats within these ranges (Symonds and Elgar, 2004; Mullen et al., 2007; Smadja and Butlin, 2009; Nanda and Singh, 2011). Species specificity of premating signals in sympatric species is higher than those between allopatric species (Butlin, 1987; Coyne and Orr, 1989; Noor, 1999). Such signal specificity is also high in sympatric species under syntopic conditions (Shine et al., 2002).
In crowded and dark spaces, chemical (Partan and Marler, 2005) and/or vibratory signals (Hebets and Papaj, 2005) could conceivably be more important than visual signals in mate recognition. In crowded situations where several species are simultaneously sexually mature and are ready to mate within the same micro-environment, i.e. in syntopy, the ability of males to accurately find a conspecific female becomes crucial. Such systems afford valuable insights into mechanisms by which mate recognition could occur under signalling constraints. Since sex pheromones could function over longer or shorter distances depending on their volatility, it is expected that heavier relatively non-volatile cuticular hydrocarbons could serve as contact sex pheromones (Singer, 1998; Ginzel, 2010), while compounds with low volatility could serve as mate recognition signals over short distances without contact having to occur (Yoshida, 1978; Simser and Coppel, 1980). In species in which females are hidden by physical barriers, males have to find such hidden females using signals that can either penetrate or coat such barriers such as the retreat silk of ant-mimicking spiders (Borges et al., 2007) and the cocoons of hymenopteran parasitoids (Howard, 1993), or employ proxies for female signals as in male gall wasps which use altered host plant volatiles to find females hidden within plant stems (Tooker et al., 2002; Tooker and Hanks, 2004), or male Heliconius butterflies that use host plant volatiles and the presence of immature larvae to find females developing within pupal cases which they proceed to guard until female eclosion (Estrada and Gilbert, 2010; Estrada et al., 2010).
Mate recognition by male wasps in the nursery pollination mutualism between fig and fig wasps has to function under the constraints of crowding, darkness, syntopy and simultaneous sexual maturation of multiple fig wasp species as well as the possible requirement for proxies for the location of hidden females. In this mutualism, individual fig wasps develop in galled flowers within an enclosed, globular, thick-walled inflorescence called the fig syconium, while many of the uniovulate flowers also develop seeds after being pollinated by mutualistic fig wasps. Wingless male fig wasps emerge first from their galls, and either release females from their galls before mating or insert genitalia into female-containing galls to mate with virgin females (Weiblen, 2002; Cook and Rasplus, 2003; Cook and Segar, 2010). Depending on the fig species and syconium size, syconia may contain hundreds to thousands of flowers (Janzen, 1979; Verkerke, 1989; Kjellberg et al., 2001; Cook and Rasplus, 2003), with hundreds of developing wasps and seeds. Besides the mutualistic fig wasp species (belonging to Agaonidae), most species of figs are parasitised by other galler, inquiline or parasitoid fig wasp species (belonging to other subfamilies in the Chalcidoidea), all of which also develop within individual galls within the enclosed syconium. Between 1 and 30 species of syntopic fig wasps could occupy the syconia of a single fig species (Cook and Rasplus, 2003; Cook and Segar, 2010). In some fig species, two species of congeneric pollinating wasps may also develop within the same syconium (Michaloud et al., 1996). Therefore, if males are able to detect the pre-mating chemical signals of different fig wasp species, they can be expected to experience a chemical equivalent of the cocktail party problem encountered in acoustic communication in a noisy environment (Bee and Micheyl, 2008), i.e., the need to perceive specific mate recognition signals in a noisy chemical environment. Furthermore, since in some fig species such as Ficus racemosa, females cannot free themselves from their galls and rely upon males to do so (A. Krishnan, pers. observ.), males need to either use a species-specific chemical or vibratory signal that is emitted by females themselves and that can pass through the gall wall, or rely on proxy cues that coat the gall wall and indicate the presence of conspecific females within galls. Furthermore, since male wasps (especially the pollinator males) have a short lifespan of 24–48 h (Kjellberg et al., 1988; Ghara and Borges, 2010) within which they have to mate as well as cut an opening in the syconium wall to release pollinator females, male pollinator wasps should also be under intense selection pressure to identify galls containing conspecific females quickly and accurately.
Most often, fig wasp males mate with females found within their own syconium since males usually die within their natal syconium (Galil and Eisikowitch, 1968; Herre et al., 2008; Ghara and Borges, 2010). However, in some species, fig wasp males may leave their natal syconium and mate with females from other syconia (Greeff, 2002; Greeff et al., 2009, Greeff et al., 2003). This may be to avoid inbreeding (Greeff et al., 2009), although it is believed that fig wasps with their haplodiploid sex determination system can tolerate high levels of inbreeding (McKey, 1989); often only a single or few foundress female wasps may lay eggs within individual syconia (Kathuria et al., 1999; Zavodna et al., 2007). Consequently, sib matings are likely to be quite frequent (Zavodna et al., 2007). However, whether male fig wasps prefer female signals from their natal syconia or from non-natal syconia is not clear, though studies on the pollinator Pegoscapus assuetus seem to indicate that males prefer females from natal syconia (Frank, 1985). The cues employed by P. assuetus males to differentiate between females from natal and non-natal syconia were not investigated.
With this background, we have used F. racemosa to understand how male pollinator fig wasps solve the problem of finding hidden females. We determined if male Ceratosolen fusciceps can distinguish between galls of conspecific females from those of non-pollinator females (which can co-occur with pollinators within the same syconia) on the basis of (1) whole galls (with the gall occupant within the gall); (2) empty galls (gall occupant removed to remove potential vibratory signals); (3) volatile signals from empty galls and (4) surface hydrocarbon signals of empty galls. We also conducted choice assays to test if males prefer galls containing females from non-natal fig syconia (which are definitely non-sibs) to females from natal fig syconia (which have higher probabilities of being sibs).
Section snippets
Species biology and study site
The syconia of F. racemosa are pollinated by the mutualistic agaonid wasp Ceratosolen fusciceps Mayr and are also host to six other species of non-pollinating fig wasps in the subfamilies Sycophaginae and Sycoryctinae respectively (gallers – Apocryptophagus stratheni Joseph, Apocryptophagus testacea Mayr, Apocryptophagus fusca Girault and the parasitoids – Apocryptophagus agraensis Joseph, Apocrypta westwoodi Grandi and Apocrypta sp. 2) (Ghara and Borges, 2010) that develop within them.
Results
Most pollinator males made choices within one minute of starting the assay. In the whole gall choice assays, most males that chose female pollinator galls began chewing open the galls and attempted to mate with the females inside. Pollinator males did not attempt to open non-pollinator galls. In the choice assays with empty galls and volatiles, most males choosing female pollinator galls exhibited searching behaviour near the galls at the end of 5 min and a few tried to chew on the opened
Discussion
Mating aggregations can occur at oviposition sites (Jaenike et al., 1992), overwintering sites (Shine and Mason, 2001), feeding sites (Byers and Wood, 1980), leks (Diabaté et al., 2006) and adult emergence sites (Thornhill, 1976). Mating aggregations in fig wasps occur at adult emergence sites which are the enclosed interiors of fig syconia (Janzen, 1979; Weiblen, 2002; Herre et al., 2008). The interior of an F. racemosa syconium in wasp pre-dispersal phase is a crowded and dark arena which may
Acknowledgements
We thank the Ministry of Environment and Forests, Government of India, for supporting this research. We also thank Gautam Pramanik, Santhosh Revadi, Mahua Ghara, Yuvaraj Ranganathan, Pratibha Yadav, Vignesh Vishvanathan and Yathiraj Ganesh for help with collecting fig syconia. We are also grateful to Devaveena Dey and Sravanth Hindupur for supplying us with six-well cell culture plates. This paper stems from the work of many summer research fellows and students doing lab rotation projects.
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