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Additional methods, additional Table S1. showing the data matrix of the analyzed morphological characters, additional Table S2. showing the data matrix used for statistical analyses, additional Table S3. giving the Eigenvalues of the morphological characters from the categorical principal components analysis, additional Figure S1. explaining the morphology of the PPG, additional Figure S2. showing sagittal section of the head capsule of Philanthus rugosus, additional results of the hierarchical cluster analysis based on PPG and MG morphology, including additional Figure S3. showing the dendrogram resulting from the hierarchical cluster analysis, additional Figure S4. showing the results of the ancestral state reconstruction of presence vs. absence of the MG, and additional Figure S5 showing the results of the ancestral state reconstruction of presence vs. absence of secretory cells of the PPG. (PDF 1617Â kb)

Background Sexual selection is thought to promote evolutionary changes and diversification. However, the impact of sexual selection in relation to other selective forces is difficult to evaluate. Male digger wasps of the tribe Philanthini (Hymenoptera, Philanthinae) scent mark territories to attract receptive females. Consequently, the organs for production and storage of the marking secretion, the mandibular gland (MG) and the postpharyngeal gland (PPG), are subject to sexual selection. In female Philanthini, these glands are most likely solely subject to natural selection and show very little morphological diversity. According to the hypothesis that sexual selection drives interspecific diversity, we predicted that the MG and PPG show higher interspecific variation in males than in females. Using histological methods, 3D-reconstructions, and multivariate statistical analysis of morphological characters, we conducted a comparative analysis of the MG and the PPG in males of 30 species of Philanthini and three species of the Cercerini and Aphilanthopsini, two related tribes within the Philanthinae. Results We found substantial interspecific diversity in gland morphology with regard to gland incidence, size, shape and the type of associated secretory cells. Overall there was a phylogenetic trend: Ensuing from the large MGs and small PPGs of male Cercerini and Aphilanthopsini, the size and complexity of the MG was reduced in male Philanthini, while their PPG became considerably enlarged, substantially more complex, and associated with an apparently novel type of secretory cells. In some clades of the Philanthini the MG was even lost and entirely replaced by the PPG. However, several species showed reversals of and exceptions from this trend. Head gland morphology was significantly more diverse among male than among female Philanthinae. Conclusion Our results show considerable variation in male head glands including the loss of an entire gland system and the evolution of a novel kind of secretory cells, confirming the prediction that interspecific diversity in head gland morphology is higher in male than in female Philanthini. We discuss possible causes for the remarkable evolutionary changes in males and we conclude that this high diversity has been caused by sexual selection. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0963-6) contains supplementary material, which is available to authorized users.

Additional methods, additional table S1 listing the bee species included in the chemical analyses, additional table S2 showing the data matrix used for the cluster analysis, additional table S3 depicting the chemical composition of the cuticular extracts of the bees and wasps, and additional figure S1 showing dendrogram of cluster analysis based on postpharyngeal and mandibular gland morphology. (PDF 600 kb)

Microbes pose severe threats to animals as competitors or pathogens and strongly affect the evolution of life history traits like parental care. Females of the European beewolf Philanthus triangulum, a solitary digger wasp, provision their offspring with paralyzed honeybees and embalm them with the secretion from large postpharyngeal glands (PPG) that contain mainly unsaturated hydrocarbons. This coating changes the physico-chemical properties of the prey surface, causes a reduction of water condensation and retards growth of mold fungi. Here we examined the closely related South American genus Trachypus, which shows a life-history similar to Philanthus. We investigated whether Trachypus spp. also possess PPGs and embalm larval provisions. Using histological methods and 3D reconstructions we show that Trachypus boharti and T. elongatus possess PPGs that are similar to P. triangulum but somewhat smaller. The ultrastructure of the gland epithelium suggests that the gland content is at least partly sequestered from the hemolymph. Chemical analyses using gas chromatography / mass spectrometry revealed that both the cuticle and PPGs of Trachypus contain mainly unsaturated long-chain hydrocarbons. The gland of T. boharti additionally contains long-chain ketones. The hydrocarbons from the PPG of T. elongatus occurred on prey bees excavated from nests in the field but not on conspecific control bees. While the embalming only slightly elevated the amount of hydrocarbons on prey bees, the proportion of unsaturated hydrocarbons, which is crucial for the antifungal effect, was significantly increased. The Trachypus species under study possess PPGs that are very similar to the PPG of P. triangulum with regard to morphology, ultrastructure and chemistry. Moreover, we provide clear evidence that T. elongatus females embalm their prey, presumably as a means of prey preservation. The observed differences among Trachypus and Philanthus in gland size and prey embalming may have evolved in response to divergent ecological conditions.

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Darwin’s theory of sexual selection explains the evolution of flamboyant male traits through female choice. It does not, however, address the question why males typically court and females choose. This asymmetry is now thought to be the result of the dichotomy in reproductive expenditures: Females invest primarily in parental care and males invest predominantly in mate attraction or competition. Based on this view, several hypotheses for the origin and maintenance of female preferences have been proposed. They include the classical sexual selection models, i.e. female choice for direct and indirect benefits as well as the more recent concepts of female choice for genetic compatibility and receiver bias models. The complementary choice scenario assumes that females choose mates with regard to genetic compatibility. The receiver bias concept views male traits and female preferences within the framework of communication theory and encompasses various more or less distinct models, two of which are sensory exploitation and sensory trap. Both models postulate that male signals evolved in response to pre-existing perceptual biases of females. The sensory trap hypothesis additionally emphasizes that pre-existing female preferences for certain cues evolved in non-sexual contexts, like e.g. foraging. Males that mimic these cues and elicit a favourable out-of-context response by females may increase their reproductive success. This thesis examines the evolution of the pheromone communication in the European Beewolf Philanthus triangulum. Beewolf females are specialized hunters of honeybees and provision their progeny with paralyzed prey. Male beewolves establish and scent mark territories with a pheromone from a head gland to court females. The concordant occurrence of the otherwise rare alcohol (Z)-11-eicosen-1-ol in the male pheromone and in the alarm pheromone of honeybees, the exclusive prey of the females, suggests a sensory trap process as an explanation for the evolution of the male pheromone in P. triangulum. According to this hypothesis, we tested three predictions: First, foraging honeybees should emit eicosenol. Via chemical analysis we could show that honeybee workers in fact smell of eicosenol during foraging. The occurrence of eicosenol on the cuticle and in the headspace of honeybees is a new finding. Second, beewolf females should use eicosenol as a cue for prey detection or identification. Using behavioural assays, we demonstrated that prey recognition in beewolf females is accomplished by olfactory cues and that eicosenol is an essential cue in this process. The sensory sensitivity of beewolf females to eicosenol must be extremely high, since they perceive the trace amounts present in the head space of honeybees. This sensitivity may be due to specialized olfactory receptors on the antennae of beewolf females. An inventory of the flagellar sensilla of both sexes showed that females carry one type of sensillum that is missing in males, the large sensillum basiconicum. This chemo-sensitive sensillum most likely plays a role in prey recognition. The third prediction is that beewolf males incorporate bee-like substances, including eicosenol, into their pheromone, and possibly catch females in a sensory trap. A reanalysis of the male pheromone revealed, among others, eicosenol and several alkanes and alkenes as pheromonal compounds. Our own analyses of the chemical profiles of honeybee workers and beewolf pheromone disclosed a surprisingly strong resemblance between the two. Eight of the eleven substances of the male pheromone are also present on the cuticle and in the headspace of honeybees. Notwithstanding this similarity, the male pheromone does not function as a sensory trap for females. Nevertheless, the extensive congruence between the odour bouquets of the females’ prey and the male pheromone strongly suggests that the male signal evolved to exploit a pre-existing female sensory bias towards bee odour, and, thus represents a case of sensory exploitation. In addition to the above described scenario concerning mostly the ‘design’ of the male pheromone, we addressed possible indirect benefits female beewolves may gain by basing their mating decisions on signal ‘content’. We show that the pheromone of male beewolves varies between families and may, thus, contain information about the degree of relatedness between the female and a potential mate. Females could use this information to choose genetically complementary males to avoid inbreeding and the production of infertile diploid sons. Collectively, our results provide strong evidence for a receiver bias process in the evolution of the male pheromone of P. triangulum. They further indicate that the pheromone composition may subsequently have been influenced by other natural or sexual selection pressures, like e.g. complementary female choice., Darwins Theorie der Sexuellen Selektion deutet die Evolution übersteigerter Männchenmerkmale als Ergebnis der Weibchenwahl. Sie erklärt jedoch nicht, warum Männchen in der Regel um Weibchen werben und Weibchen unter den werbenden Männchen wählen. Man glaubt heute, daß dies auf eine Asymmetrie im reproduktiven Aufwand zwischen den Geschlechtern zurückzuführen ist: Weibchen investieren überwiegend in elterliche Fürsorge, Männchen v.a. in Balzsignale. Hierauf basierend wurden mehrere Hypothesen zur Evolution von Präferenzen bei weiblicher Partnerwahl vorgeschlagen. Dazu gehören die klassischen Modelle der Sexuellen Selektion, wie ‚direct’ und ‚indirect benefit’ sowie die neueren Konzepte der Weibchenwahl aufgrund genetischer Kompatibilität und die 'Receiver Bias' Modelle. Letztere betrachten Männchenmerkmale und Weibchenpräferenzen im Rahmen der Kommunikationstheorie und umfassen mehrere ähnliche Modelle wie z.B. 'Sensory Exploitation' und 'Sensory Trap'. Sie postulieren, daß das Design von Männchensignalen in Anpassung an existierende Präferenzen der Weibchen entsteht. Die 'Sensory Trap' Hypothese betont zudem, daß diese sensorischen Präferenzen der Weibchen für bestimmte Signale in einem der natürlichen Selektion unterliegenden (nicht sexuellen) Kontext entstanden, so z.B. zum Auffinden von Nahrung. Männchen imitieren diese Signale um vorteilhafte Reaktionen der Weibchen auszulösen und so ihren Reproduktionserfolg zu erhöhen. Die vorliegende Dissertation untersucht die Evolution der Pheromonkommunikation des Europäischen Bienenwolfs. Bienenwolfweibchen sind spezialisierte Jäger der Honigbiene und versorgen ihre Nachkommen mit gelähmter Beute. Bienenwolfmännchen etablieren Reviere und markieren diese mit einem Pheromon um Weibchen anzulocken. Das übereinstimmende Vorkommen des sonst sehr seltenen Alkohols (Z)-11-Eicosen-1-ol sowohl im Pheromon der Männchen als auch im Alarmpheromon der Honigbienen, der ausschließlichen Beute der Weibchen, deutete darauf hin, daß es sich bei dem Pheromon um eine 'Sensory Trap' für Weibchen handeln könnte. Entsprechend dieser Hypothese testeten wir drei Vorhersagen: Erstens, furagierende Honigbienen sollten nach Eicosenol riechen. Mit Hilfe chemischer Analysen konnten wir erstmals zeigen, daß Honigbienensammlerinnen nach Eicosenol riechen. Zweitens sollten Bienenwolfweibchen Eicosenol für die Identifikation ihrer Beute nutzen. Wie wir in unseren Verhaltenstests zeigen konnten, verlassen sich die Weibchen für die Beuteerkennung auf Duftsignale. Hierbei ist Eicosenol eine notwendige Komponente für die Identifizierung der Honigbienen. Die sensorische Empfindlichkeit der Weibchen für Eicosenol scheint extrem hoch zu sein, da sie diese nur in Spuren im Luftraum um Honigbienen vorhandene Substanz, wahrnehmen können. Die hohe sensorische Empfindlichkeit der Weibchen könnte durch spezialisierte olfaktorische Rezeptoren bedingt sein. Die Analyse der antennalen Sensillen zeigte, daß Weibchen einen Sensillentyp besitzen, der bei Männchen nicht vorkommt: die großen Sensilla basiconica. Diese chemosensitiven Sensillen könnten eine entscheidende Rolle bei der Beuteerkennung spielen. Die dritte Vorhersage ist, daß Bienenwolfmännchen die für Honigbienen typischen Substanzen, darunter auch Eicosenol, in ihr Pheromon integrieren, um Weibchen in einer 'Sensory Trap' zu fangen. Eine Neuanalyse des Männchenpheromons zeigte, u.a. Eicosenol und einige Alkane und Alkene als Pheromonbestandteile. Unsere Analysen zeigten eine überraschende Übereinstimmung der chemischen Profile von Honigbienen und Bienenwolfmännchen. Acht der elf Substanzen des Pheromons finden sich auch im Duft der Honigbiene. Trotz dieser erstaunlichen Ähnlichkeit fungiert das Männchenpheromon nicht als 'Sensory Trap' für Weibchen. Die ausgeprägte Kongruenz der Düfte von Weibchenbeute und Männchenpheromon deutet aber darauf hin, daß bei der Evolution des Männchenpheromons vermutlich eine bereits existierende sensorische Präferenz der Weibchen für Bienenduft ausgenutzt wurde, d.h. daß es sich dabei um 'Sensory Exploitation' handelt. Das oben beschriebene Szenario für die Evolution des Männchenpheromons betrifft hauptsächlich das Design des Männchensignals. Darüber hinaus haben wir den möglichen Informationsgehalt des Pheromons untersucht. Das Pheromonmuster variiert beim Europäischen Bienewolf mit der Familienzugehörigkeit. Das Pheromon könnte somit Information über den Verwandtschaftsgrad zwischen einem Weibchen und einem potentiellen Paarungspartner enthalten. Weibchen könnten diese Information nutzen um sich nur mit genetisch kompatiblen Männchen zu paaren und auf diese Weise Inzucht zu vermeiden. Die vorliegenden Ergebnisse liefern starke Evidenzen für einen 'Receiver Bias' Prozess bei der Evolution des Pheromons von P. triangulum. Sie deuten außerdem darauf hin, daß die Zusammensetzung des Pheromons in der Folge durch weitere Selektionsdrücke, wie z.B. Weibchenwahl für genetische Kompatibilität, beeinflusst wurde.

Background Hymenoptera that mass-provision their offspring have evolved elaborate antimicrobial strategies to ward off fungal infestation of the highly nutritive larval food. Females of the Afro-European Philanthus triangulum and the South American Trachypus elongatus (Crabronidae, Philanthinae) embalm their prey, paralyzed bees, with a secretion from a complex postpharyngeal gland (PPG). This coating consists of mainly unsaturated hydrocarbons and reduces water accumulation on the prey’s surface, thus rendering it unfavorable for fungal growth. Here we (1) investigated whether a North American Philanthus species also employs prey embalming and (2) assessed the occurrence and morphology of a PPG among females of the subfamily Philanthinae in order to elucidate the evolution of prey embalming as an antimicrobial strategy. Results We provide clear evidence that females of the North American Philanthus gibbosus possess large PPGs and embalm their prey. The comparative analyses of 26 species from six genera of the Philanthinae, using histological methods and 3D-reconstructions, revealed pronounced differences in gland morphology within the subfamily. A formal statistical analysis based on defined characters of the glands confirmed that while all members of the derived tribe Philanthini have large and complex PPGs, species of the two more basal tribes, Cercerini and Aphilanthopsini, possess simple and comparatively small glands. According to an ancestral state reconstruction, the complex PPG most likely evolved in the last common ancestor of the Philanthini, thus representing an autapomorphy of this tribe. Conclusion Prey embalming, as described for P. triangulum and T. elongatus, and now also for P. gibbosus, most probably requires a complex PPG. Hence, the morphology and size of the PPG may allow for inferences about the origin and distribution of the prey embalming behavior within the Philanthinae. Based on our results, we suggest that prey embalming has evolved as an antimicrobial strategy in and is restricted to the tribe Philanthini, which seems to face exceptional threats with regard to fungal infestations of their larval provisions. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0565-0) contains supplementary material, which is available to authorized users.