Discussion

Our chemical analyses of samples directly taken from the PPG of male P. triangulum revealed a blend of 55 substances including 11 compounds previously described as components of the marking pheromone in P. triangulum males (Schmidt et al. 1990; Schmitt et al. 2003). These results, together with our observation that (as described for other Philanthus species (Schmidt et al. 1985)) the compounds isolated from heads or PPGs of male P. triangulum can also be found on freshly marked territories of beewolf males (Herzner, Kaltenpoth, Kroiss, Schmitt, and Strohm, unpublished data), support our hypothesis that the PPG of male European beewolves functions as the reservoir of the marking pheromone.

The 44 substances that had not been described by (Schmitt et al. 2003) were included here due to different reasons. In the earlier study most of the minor hydrocarbons were omitted, because they were also found in cuticle extracts and we concluded that they might not be part of the marking pheromone (see also Schmidt et al. 1990). Here we show that the hydrocarbons are in fact present in the PPG. Therefore, we consider them as a part of the marking secretion. Most importantly, for the current study we pooled the gland extracts of three large males, concentrated the resulting sample considerably and injected it by hand.

This procedure allowed for the detection of very minor substances in the pheromone blend.

One of the compounds that is new to the cephalic secretion of male European beewolves is (Z)-9-octadecen-1-ol. It has been previously isolated from glands and cuticle of several apid species (see e.g. Andersen et al. 1988; Bergström 1985) and from jojoba (Simmondsia chinensis) seeds (Tobares et al. 2003). 1-Eicosanol has been described as a component of the marking secretion of males of a cuckoo bumblebee (Kullenberg et al. 1970) and is present in the female sex pheromone of the European grapevine moth Lobesia botrana (Arn et al.

1988). It can also be found on cocoons and on the cuticles of different developmental stages of the honeybee Apis mellifera (Donzé et al. 1998). The ketone ∆-16-pentacosen-8-one is also present in the PPG of P. triangulum females (Strohm et al., unpublished data). ∆-18-peptacosen-10-one has been found to be a pheromone component of the white-spotted longicorn beetle Anoplophora malasiaca (Yasui et al. 2003).

Based on descriptions in the literature (Borg-Karlson & Tengö 1980; Evans & O´Neill 1988;

Gwynne 1978; McDaniel et al. 1992; Schmidt et al. 1990; Schmidt et al. 1985) we had initially assumed that the marking pheromone in P. triangulum is secreted from the mandibular glands. Owing to its delicate structure (Herzner et al. 2007) the PPG of male beewolves is

destroyed when dissecting heads to remove and extract the mandibular glands. When the PPG is injured, its content instantly spills out and disperses in the head capsule. As a consequence the mandibular gland gets contaminated with the PPG content. This was possibly the case in some previous studies on beewolf male pheromones (Borg-Karlson &

Tengö 1980; Schmidt et al. 1990; Schmidt et al. 1985; Schmitt et al. 2003).

When taking extreme care during dissection it is possible to remove the mandibular glands without harming the PPG. When we extract these mandibular glands that are not contaminated with the content of the PPG we mostly detect only very minor amounts of some pheromone components in the GC-MS (Herzner & Strohm, unpublished data). Therefore, in total head extracts, the vast majority of the detected compounds seems to originate from the PPG and not from the mandibular glands.

In our analyses all components of the PPG were also present in total head extracts and vice versa. Furthermore, the proportions of all compounds were very similar between the two types of extracts. The only component that showed a marked discrepancy in its relative abundance between the PPG and the head extracts was (S)-2,3-dihydrofarnesoic acid, which was always more abundant in the head extracts. The reason for this difference is yet unclear.

Owing to the extraordinary size of the PPG (Herzner et al. 2007), its content dominates head extracts in a way that the contribution of other tissues or the cuticle to the extract can be neglected. The high congruence between direct samples of the PPG and total head extracts of male European beewolves shows that the latter yield valid samples of the marking secretion.

Therefore, it seems justified and far more practical to use total-head extracts for studies on the male beewolf marking pheromone. In particular, when doing quantitative analyses the risk of loosing some secretion due to non perfect dissection can be circumvented.

The PPG has long been considered to be idiosyncratic to ants. In ant workers it typically contains a blend of straight and methyl-branched hydrocarbons (Cabrera et al. 2004; Lucas et al. 2004; Soroker et al. 1995) that mainly plays a role in nestmate recognition (Crozier &

Dix 1979; Lenoir et al. 1999; Soroker et al. 1998; Vienne et al. 1995). Female P. triangulum also possess large PPGs (Strohm et al. 2007) that contain a blend of mainly straight unsaturated and saturated as well as minor proportions of methyl-branched long-chain hydrocarbons and long-chain unsaturated ketones (Strohm et al., unpublished data). Many of the hydrocarbons as well as the ketones ∆-16-pentacosen-8-one and ∆-18-heptacosen-10-one present in the male PPG are also found in the female PPG. The female PPG is morphologically different from the male gland and contains, besides the ketones, no substances with functional groups. The PPG secretion of female beewolves is used for prey

preservation (Strohm & Linsenmair 2001) and thus has a completely different function from the PPG content in beewolf males.

Thus, in beewolf females the PPG is subject to natural selection to increase the protection against fungal infestations of their honeybee prey and larvae (Strohm & Linsenmair 2001).

The PPG of males, by contrast, is subject to sexual selection (Herzner 2004; Herzner et al.

2006; Herzner et al. 2005; Kaltenpoth & Strohm 2006). Such a change in function and sexual dimorphism regarding glands and their contents is not unusual (e.g. the methathoric and dorsal abdominal glands in some Heteroptera (Aldrich 1988; Aldrich et al. 1996; Ho et al. 2003; Zhang & Aldrich 2003)). The significance of this sexual dimorphism remains mostly unclear, however. Even in ants, the morphology and function of the PPG seem to exhibit inter- and intraspecific variability (Eelen et al. 2006; Schoeters & Billen 1997).

In addition to the morphological and chemical differences of the PPG between the sexes there is a dimorphism with regard to the composition of the gland content within each sex of P.

triangulum. The majority of males (87%) had pentacosene as the by far most abundant hydrocarbon (C25-type). Some males (13%), however, possessed almost equal proportions of pentacosene and heptacosene (C25/C27-type) with a bimodal and non overlapping distribution of the two types with regard to the proportion of heptacosene. The chemical dimorphism in the PPG content of beewolf females is much more pronounced than in males. In females, the main compound, accounting for 70-80% of the blend, is either pentacosene or (Z)-9-heptacosene and there are no intermediate types with similar amounts of both compounds like the C25/C27 type in males (Strohm et al., unpublished data). The proportions of the pentacosene-morph and the heptacosene-morph in females are 80% and 20% and thus similar to the proportions of the C25-type and the C25/C27-type in males (87% vs. 13%). The meaning of this chemical dimorphism is not yet clear. Noteworthy, the dimorphism is only exhibited by hydrocarbons without functional groups. Moreover, if the dimorphism had a genetic basis, recognition of relatives - in particular with regard to the avoidance of inbreeding (Herzner et al. 2006) – would be facilitated.

Our findings might have important implications for the evolution of the PPG within the Hymenoptera. If the PPG existed only in ants, it would be tempting to assume that it had evolved in response to the requirements of the social communication system (Eelen et al.

2006; Hölldobler & Wilson 1990; Lenoir et al. 1999). The existence of a PPG in beewolf females suggested that this gland might have evolved in some early Aculeates for the purpose of prey preservation (Strohm & Linsenmair 2001, Strohm et al., unpublished data). Now we know that the PPG in male beewolves stores the marking pheromone. This beewolf marking pheromone most probably represents the male sex pheromone, since females are obviously attracted to the territories by the windborne pheromone and most matings occur in the

males’ territory or in the nearby vegetation (Alcock 1975; Evans & O´Neill 1988; Gwynne 1978; Herzner et al. 2006; Herzner et al. 2005; O’Neill 1979; O’Neill 1983; Schmitt et al.

2003; Simon Thomas & Poorter 1972; Strohm 1995; Strohm & Lechner 2000). Therefore, the pheromone is subject to sexual selection with regard to both its composition and amount (Herzner 2004; Herzner et al. 2007; Herzner et al. 2006; Herzner et al. 2005; Kaltenpoth &

Strohm 2006; Herzner et al., unpublished data). Since sexual selection can be a very strong evolutionary force (Higashi et al. 1999; Kaneshiro & Boake 1987; Lande 1981) the evolution of the gland might have been influenced by the requirements of males to store large amounts of pheromones to attract females. Regardless of the evolutionary origin of the PPG our findings show that this gland, that was assumed to have a ‘social’ function in ants, serves a selfish function as a reservoir for a male sex pheromone in a solitary wasp.