Evolution of the suctorial proboscis in pollen wasps (Masarinae, Vespidae)

Arthropod Structure & Development 31 (2002) 103–120 www.elsevier.com/locate/asd

Evolution of the suctorial proboscis in pollen wasps (Masarinae, Vespidae) Harald W. Krenna,*, Volker Maussb, John Planta b

a Institut fu¨r Zoologie, Universita¨t Wien, Althanstraße 14, A-1090, Vienna, Austria Staatliches Museum fu¨r Naturkunde, Abt. Entomologie, Rosenstein 1, D-70191 Stuttgart, Germany

Received 7 May 2002; accepted 17 July 2002

Abstract The morphology and functional anatomy of the mouthparts of pollen wasps (Masarinae, Hymenoptera) are examined by dissection, light microscopy and scanning electron microscopy, supplemented by field observations of flower visiting behavior. This paper focuses on the evolution of the long suctorial proboscis in pollen wasps, which is formed by the glossa, in context with nectar feeding from narrow and deep corolla of flowers. Morphological innovations are described for flower visiting insects, in particular for Masarinae, that are crucial for the production of a long proboscis such as the formation of a closed, air-tight food tube, specializations in the apical intake region, modification of the basal articulation of the glossa, and novel means of retraction, extension and storage of the elongated parts. A cladistic analysis provides a framework to reconstruct the general pathways of proboscis evolution in pollen wasps. The elongation of the proboscis in context with nectar and pollen feeding is discussed for aculeate Hymenoptera. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mouthparts; Flower visiting; Functional anatomy; Morphological innovation; Evolution; Cladistics; Hymenoptera

1. Introduction Evolution of elongate suctorial mouthparts have occurred separately in several lineages of Hymenoptera in association with uptake of floral nectar. They can be found, for example, in various ‘symphytans’ (Jervis and Vilhelmsen, 2000), parasitoid Apocrita (Jervis, 1998), sphecids (Ulrich, 1924), Scoliidae, Sapygidae, Tiphiidae (Osten, 1982, 1991) and in many bees (Michener, 1944, 2000). In Vespidae, despite the fact that the adults of both sexes obtain at least some nourishment from floral nectar (Kugler, 1970; Proctor et al., 1996), a very long elongate suctorial proboscis is not common, except in Eumeninae (Osten, 1982) and Masarinae. The Masarinae, or pollen wasps, are unique among the vespids for their bee-like habits of provisioning each larval brood cell with pollen and nectar. Female pollen wasps use their mouthparts to gather pollen and nectar from flowers and for nest construction (Gess and Gess, 1992; Gess, 1996, 2001; Mauss, 1996, 2000; Mauss and Mu¨ller, 2000). * Corresponding author. Tel.: þ43-1-4277-54497; fax: þ 43-1-42779544. E-mail addresses: [email protected] (H.W. Krenn), volker. [email protected] (V. Mauss).

Some have very long proboscides; however, in contrast to bees, the proboscis is formed only by the glossa and, in some species, it is looped back into the prementum when in repose (Bradley, 1922; Schremmer, 1961; Richards, 1962; Osten, 1982; Carpenter, 1996/1997; Gess, 1998). The traditional classification of the Masarinae, dating back to Saussure (1854), was based on the misunderstanding that the glossa of one group (based on Paragia ) cannot be retracted at all and the glossa of the other group (based on Masaris ) can be retracted into the prementum. Carpenter’s (1996/1997) study of the Paragiina clarified the morphological misunderstanding and demonstrated that the glossa in all groups is retractable. The separation of the Masarinae into two main lineages, the Paragiina and Masarina, however, was upheld in that study by other features. Currently the Masarinae contains 14 genera with about 300 species (Carpenter 1982, 2001) and is divided into the Gayellini and Masarini. The latter tribe consists of Paragiina (Australian region only), Masarina (widespread except Australia) and Priscomasarina, which was established to accommodate a newly discovered species from Namibia (Gess, 1998, Fig. 1). The evolution of an elongate proboscis occurred at least twice in the Masarinae. Elongation of the proximal part of

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Fig. 1. Dendrogram showing hypothesized phylogeny of Masarinae, combined from Carpenter (1982, 1989, 1993, 1996) and Gess (1998). Taxa in bold type are investigated in this study.

the glossa or of the distal part thus defines two lineages, the subtribe Masarina and Metaparagia (Paragiina) (Carpenter, 1996/1997). As a relatively small group of flower visiting Hymenoptera, the Masarinae offer the possibility to examine the pathways of mouthpart evolution in the context of nectar feeding. We focus on a comparative functional anatomy of the glossa in Masarini since in some genera it is relatively short yet retractable while in others it is extremely long. We delineate several morphological innovations which are important for the formation and functioning of a suctorial proboscis, in addition to discussing further evolutionary aspects of the proboscis in Hymenoptera.

2. Material and methods

braunsi Schulthess, Celonites peliostomi Gess and Quartinioides sp. (classification after Carpenter (2001), who regards Quartinioides as a subgenus of Quartinia ). Fresh specimens were fixed in 70% ethanol or DuboscqBrasil solution (Romeis, 1989). Whole mount preparations of the mouthparts were made from dissected heads. They were soaked in diluted lactic acid at 40 –50 8C for 1 –2 days, washed in distilled water, and embedded in polyvinyl lactophenol without dehydration on glass slides. The preparations were covered with glass slips and dried at 50 8C. Serial semithin-section technique was used to examine mouthpart anatomy with light microscopy and to reconstruct the possible functional mechanisms of glossal movements. The isolated heads were dehydrated with acidified DMP (2,2-dimethoxypropane) and acetone, then embedded in ERL-4206 epoxy resin under vacuum impregnation. Semithin sections were cut using diamond knives. They were stained with a mixture of 1% azure II and 1% methylene blue in an aqueous 1% borax solution for approximately 1 min at 80 8C. Series of sagittal semithin sections were prepared for all the above listed species of Masarinae. Preparations were made of P. decipiens and C. hispanicus individuals with retracted and extended proboscides. The mechanism of glossal movements was studied in thawed specimens of freeze-killed C. hispanicus and in freshly collected C. lusitanicus, C. hispanicus and C. fonscolombei. For viewing in the scanning electron microscope (SEM), fixed samples of P. decipiens, C. hispanicus, C. peliostomi and Quartinioides sp. were dehydrated in ethanol and submerged in hexamethyldisilazane prior to air drying (Bock, 1987). A graphite adhesive tape was used to mount them on SEM viewing stubs. The samples were sputtercoated with gold and viewed in a Jeol JSM-35 CF SEM.

2.1. Field observation Flower visiting behavior and water uptake were observed in Ceramius fonscolombei Latreille, C. hispanicus Dusmet, C. lusitanicus Klug in Spain (Arago´n, Province Teruel: Barranco de Zorita, 19 – 26 June 1998; north of Almohaja, 16 –18 June 1998; east of Los Iban˜ez, 7 – 12 June 2000; Rambla de Rio Seco, west of Valdecebro, 9– 16 June 2000) and in C. tuberculifer Saussure in France (Alpes-de-HauteProvence: Peyresq 19– 28 July 1994; Montagne de Boules 26 –29 July 1994) in part with the aid of close-up binoculars and documented by macro-photography (scale 1:1). 2.2. Morphology The mouthparts of females were examined using light microscopy in Priscomasaris namibiensis Gess (Priscomasarina), Paragia decipiens Shuckard (Paragiina), Ceramius hispanicus and C. fonscolombei which are considered to be basal representatives of Masarina, and several higher Masarina, i.e. Masarina familiaris Richards, Jugurtia

3. Results 3.1. Flower visiting behavior The behavioral pattern exhibited by Ceramius on flowers differed according to the shape of the flower and whether pollen or nectar was collected. On flowers with exposed anthers [Helianthemum spec. (Cistaceae) (C. lusitanicus and C. hispanicus ); Reseda spec. (Resedaceae) (C. fonscolombei )] females primarily harvested pollen directly (Fig. 2), their mandibles clasped and nibbled the anthers; their maxillae were visibly active during ingestion of the loosened pollen. The proboscis was never extended. Pollen uptake at zygomorphic flowers with hidden anthers (Lotus corniculatus L. (Fabaceae) (C. hispanicus ); Dorycnium hirsutum (L.) Ser. (Fabaceae) (C. lusitanicus ); Teucrium montanum L. (Lamiaceae) (C. tuberculifer )) was indirect; pollen was brushed from the anthers or from parts of the

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Figs. 2 and 3. Fig. 2: Ceramius lusitanicus female collecting pollen with mandibles and maxillae at a flower of Helianthemum organifolium (Lam.) Pers. Fig. 3: C. hispanicus female imbibing water from moist soil with extended glossa (arrow).

body and brought between the mouthparts by movements of the forelegs, the distal parts of which form pollen brushes. Although nectar uptake is difficult to verify, it can be supposed to occur at several zygomorphic flowers with a deep tubular corolla (Marrubium supinum L., Nepeta nepetella L. (both Lamiaceae) (C. hispanicus ); Teucrium montanum (C. tuberculifer ); Echium vulgare L. (Boraginaceae), Dorycnium hirsutum (C. lusitanicus )). The glossa was never extended before the wasp had put its head into the corolla of these flowers, but on some occasions it could be observed that the glossa was still somewhat extended when the wasp pulled its head back. The glossa was always completely retracted shortly thereafter and wasps never flew off with extended mouthparts. Ceramius fonscolombei was observed to visit the easily accessible flowers of Reseda, presumably for nectar uptake, since its short proboscis could be seen extended toward the nectar-bearing dorsal enlargement on the disc of the flower, with the mandibles slightly opened. Ceramius uses water to moisten the soil during particular stages of nest construction (Gess and Gess, 1992; Gess, 1996, 2001; Mauss and Mu¨ller, 2000). To collect water the females of C. hispanicus (Fig. 3), C. lusitanicus and C. fonscolombei landed at the edge of a water site or on damp soil. They opened their mandibles and extended the glossa. The extension process was very rapid. During the following period of water uptake only the distal tip of the glossa reached the wet surface. Normally the proboscis was slightly bend ventrad. The distal bifurcated section of the glossa was straight and parallel. On a few occasions the proboscis was bend slightly dorsally in C. lusitanicus with the distal tip lying on the ground. The posture of the wasp depends on the length of its glossa. Females of C. fonscolombei with a short proboscis lowered their heads close to the water surface, while individuals of C. hispanicus and C. lusitanicus with a long proboscis raised

their heads above the main body axis (Fig. 3). When imbibing water the outer surface of the glossa appears to be covered with adherent water which resulted in shiny reflections. 3.2. Mouthpart morphology The gross morphology of the head, mandibles and maxillae is briefly summarized for the investigated Masarinae. The surface of the head and exposed areas of the mouthparts are covered with long unbranched bristles. Viewed frontally, the clypeus projects over the labrum. Long bristles of the labrum protrude from under the clypeus (Figs. 4 and 8). When the mandibles are closed, they obscure the frontal view of the maxillae and labium except for the tips of the glossa and palpi. The labium and maxillae are visible from the posterior view of the head (Fig. 7). The basal parts of the maxilla, i.e. the cardo and stipes, lie between the labium and the head. The stipes is arched and tilted at a slight angle against the labium. Proximally it is attached to the apex of the cardo and distally it bears the lacinia, galea, and maxillary palpus which has six segments in P. namibiensis and five in P. decipiens. The lacinia is a large, flat lobe overlapping the anterior part of the galea. The distal portion of the galea is composed of several plates, one of which bears on the inner surface a longitudinal row of bristles. Pollen grains are commonly found on this galeal comb. The inner surface of the galea is basally continuous with the preoral cavity, which is formed by the epipharynx, the underside of the labrum and the large muscular hypopharynx. The hypopharynx contains the voluminous infrabuccal pouch, which in some specimens was filled with pollen (Figs. 6 and 13). Parts of the lacinia and galea, which are positioned near the infrabuccal pouch are responsible for pushing pollen grains into the mouth (Fig. 6). The short-tongued mouthparts of P. namibiensis and P.

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Figs. 4–6. Fig. 4: Head of P. decipiens (Paragiina); mandibles (md) are open and the glossa (gl) is extended. Clypeus (cl) partly covers the labrum (lr). Fig. 5: Bifurcate glossa (gl) of P. decipiens (Paragiina) in dorsal view; paraglossae (pgl) lie laterally at the basis of the glossa; dorsal side of glossa bears transverse cuticular lamellae which enclose the food canal of the bifid distal region. Fig. 6: Longitudinal section through head of P. namibiensis (Priscomasarina). Glossa (gl) folded under the preoral cavity (poc). Infrabuccal pouch (ibp) filled with pollen grains; m. intralabialis posterior (mip) folds the posterior lingual plate (plp) against the prementum (pr); glossa rod (glr) is bent in posterior direction. Extension of the glossa is achieved by contraction of m. intralabialis anterior (mia) which permits the anterior lingual plate (alp) to revert back to its extended position parallel to the prementum.

decipiens correspond in many features to the plesiomorphic condition for vespids, e.g. Euparagiinae (Bradley, 1922), Eumeninae (Richards, 1962; Osten, 1982) and Vespinae (Kirmayer, 1909; Brocher, 1922; Duncan, 1939). The labial palpus is 4-segmented, the glossa is bifid and has a length of approximately 1.5 mm in P. decipiens (Figs. 4 and 5). The glossa is short compared to the prementum, whereas the paraglossae are relatively large and conspicuous (Fig. 5). The prementum is elongate and u-shaped with large median arches adjoining the hypopharynx on its lateral edges. The glossa emerges from the distal end of the prementum and is flanked by the paraglossa, which arise from the paraglossal sclerite. Intermediate the glossa and the prementum on the posterior side is the large and strongly flexible ‘posterior lingual plate’ (Duncan, 1939) which arises out of the apical prementum and leads into the short glossal rod; intermediate on the anterior side is the ‘anterior lingual plate’ (Duncan, 1939) which is characterized by its lateral arms. While the mandibles and maxillae are similar in form and function in all investigated Masarinae major differences

occur in the morphology of the glossa which forms the principle organ of fluid uptake. The plesiomorphic glossa of vespids and basal pollen wasps can be morphologically divided into a proximal section and a distal, often bilobed or bifurcated section with the acroglossal buttons. The anterior surface of the glossa bears transverse rows of flattened hairlike cuticular structures, however, in the Masarini these are modified into lamella-shaped plates. The lamellae in Priscomasaris transverse the entire glossal surface, while in Paragia, they are divided medially into two rows extending from the glossal base to the tips of the deeply bifid glossa (Fig. 5). The food canal of the proximal section of the glossa is a deep longitudinal pocket set between the lateral rows of lamellae. On the anterior surface of each glossal lobe, the lamellae arch toward the hair-like cuticular structures emerging from the posterior surface and together they form a narrow food canal (Fig. 5). An acroglossal button with associated sensilla is located on the posterior apex of each glossal lobe. The paraglossa are elongate, extending beyond the proximal section of the glossa, and

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Fig. 7. Schematic drawing of head of P. decipiens (Paragiina). Striped muscles indicate those responsible for glossal retraction (A, B), and glossal extension (C, D). (A) Posterior view, glossa retracted. (B) Longitudinal section of head; glossa retracted by contraction of m. intralabialis posterior (mip) and m. craniolabialis anterior (mca). (C) Posterior view, glossa extended. (D) Longitudinal section of head, glossa extended by contraction of m. intralabialis anterior (mia) and m. craniolabialis posterior (mcp). Occipital foramen (of); cardo (c); stipes (st); maxillary palpus (mxp); mandible (md); prementum (pr); paraglossa (pgl); glossa (gl), labial palpus (lp).

their concave median surfaces laterally embrace the base of the glossa (Fig. 5). In both species, glossa and paraglossae fold together in repose (Fig. 7). The plesiomorphic resting position of the labium is a zshaped fold (Figs. 6 and 7). When folded, the glossal base frontally closes the preoral cavity (Figs. 6 and 7). In this position the glossa is bent toward the hypopharynx at a right angle to the prementum. The posterior lingual plate is flexed against the prementum and the short glossal rod bends the distal bifurcated section of the glossa in the opposite direction (Fig. 6). The musculature of the labium which is considered responsible for direct movements of the glossa is diagramed in Fig. 7. The muscles are labeled according to origin and attachment sites and numbered after Matsuda (1965) with regard to probable homology within the Hymenoptera. Comparison of serial head sections with the glossa in retracted and extended positions enabled us to draw conclusions on the functional mechanism of glossal movements. The glossa is folded primarily by contraction of musculus intralabialis posterior (M42), which folds back the posterior lingual plate, and by contraction of m. craniolabialis anterior (M34), which draws back the anterior lingual plate (Fig. 7B). Extension of the glossa is achieved by m. intralabialis anterior (M43) which permits the anterior lingual plate to revert back to its extended position parallel to the prementum, and by m. craniolabialis posterior (M35), which originates on the clypeus and extends at a right angle to the prementum. Its contraction pulls the proximal prementum toward the proboscidial fossa of the head capsule and probably thus contributes to initial extension of the glossa (Fig. 7D). Proboscis of Ceramius species. The major modification

in the labium of Ceramius species, as compared to P. decipiens, regards glossal length, formation of a closed food tube, increased flexibility at the articulation between the basal glossa and prementum, and the resting position of the glossa. We investigated two species of Ceramius, the relatively short-tongued C. fonscolombei (glossal length 2 mm) and the long-tongued C. hispanicus with a glossal length of 5.6 mm (^ 0.2; n ¼ 10). In both species, the cuticular structures of the glossa build an enclosed median food tube along its entire length and it can be retracted into the prementum. Despite variation in glossal length, the functional mechanisms presumed to be responsible for retraction and protraction appear identical, at least with regard to internal anatomy. The elongate suctorial glossa of Ceramius and most other higher Masarina can be functionally and morphologically divided into three sections: a short proximal section, a long middle section, and a distal, usually bifurcated, section (Fig. 10). The proximal section of the glossa encompasses the posterior articulation to the prementum (Fig. 9). The distal prementum connects via the ‘hinge plate’ (Duncan, 1939) to the well-sclerotized posterior lingual plate which is continuous with the glossal rod. The internal elastic glossal rod extends the entire length of the glossa to the bifurcated section. The anterior side of the proximal glossa is connected to the anterior lingual plate by a thin and flexible cuticle which allows the glossa to telescope under the anterior lingual plate. Distally, the anterior lingual plate is forked to embrace the lateral base of the glossa which itself is adjoined to the paraglossal sclerite as well as to the lateral prementum processes. The posterior and lateral sides of the glossa are characterized by an elastic cuticular membrane up to the middle of the glossa (Fig. 16).

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The food tube of the middle section is formed by two longitudinal and adjacent rows of lamellae on the anterior surface. The arching lamellae of each row overlap the preceding ones and the two rows come together to form a completely closed median food tube that extends the entire length of the glossa (Figs. 11 and 16). The broad surfaces of the plates are finely sculptured, a feature that may help to ensure a tight closure between the plates yet permit flexibility (Fig. 11). In the proximal section of the glossa, the food canal widens, the lamellae are larger and the two rows do not overlap as tightly as in the middle section. The proximal widening opens into the preoral cavity which is covered by the labrum and distal parts of the maxilla. At the bifurcated section of the glossa, the food tube splits and continues along each glossal lobe (Figs. 12 and 16). Each food canal in this section is formed by the strongly curved and overlapping lamellae on the anterior side, while the posterior side is formed by additional cuticular structures that curve upward from the underside of the glossa, together enclosing a narrow canal along the inner margin of each glossal lobe (Fig. 16). They have small spines, possibly to increase surface area. Fluids are probably taken up through the slits between the lamellae and between the hair-like structures (Fig. 12). The acroglossal buttons are reduced in size and bear numerous short conical sensilla each with a single terminal pore. In the retracted position, the glossa is almost entirely withdrawn into the prementum and lodged underneath the anterior lingual plate (Figs. 8, 9, 14 and 16). The glossa rod is connected to the prementum by the intervening hinge plate and posterior lingual plate which permits two 908 flexions of the glossa (Fig. 9). First is the flexion of the hinge plate on the prementum, and second the flexion between the hinge plate and posterior lingual plate, together they result in a reversal of the direction of the glossa (Figs. 13 and 14). At about one third of its length the retracted glossa bends about 1508 forward so that its anterior surface lies directly under the anterior lingual plate, the tips of the glossal lobes lie between the maxillae and mandibles. The membranous cuticle of the proximal glossa half is pulled into the prementum and forms a cavity (Fig. 14). In cross-section, the prementum is strongly u-shaped to provide space for the loop of the retracted glossal rod. The anterior side of the glossa, which is connected to the median area of the anterior lingual plate, retracts telescopically through the forked arms of the anterior lingual plate. The flexible sleeve-like anterior surface of the glossa

invaginates at the distal end of anterior lingual plate (Figs. 13 and 14), extending back beneath the plate near to salivarium where it turns forward. The anterior lingual plate extends as a long and narrow sclerite to the proximal end of the prementum (Figs. 13 and 14). The paraglossae are short and can be only partially retracted. The pronounced difference in labial musculature between P. decipiens and the Ceramius species concerns the course of the m. intralabialis anterior (M43). This muscle extends between the inner premental margin and the anterior lingual plate. In Ceramius it is fan-shaped due to the strongly u-shaped prementum and the elongation of the anterior lingual plate. One part of this muscle extends from the proximal end of the prementum to the anterior lingual plate at a right angle to the course of the prementum (Figs. 13 and 14). Another part extends from the lateral margin of the prementum to the anterior lingual plate at an oblique angle. Further portions of this muscle extend between the premental processes and the lateral arms of the anterior lingual plate. Together with the shape of the prementum, the thin fan-shaped muscles form a deep cavity or pouch in which the glossa retracts (Fig. 14). A functional model for the mechanism of extension and retraction of the glossa (Fig. 17) was derived from dissections and comparison of the sectional series in specimens with the proboscis in retracted and extended positions (Figs. 14 and 15). In Ceramius the contraction of the fan-shaped m. intralabialis anterior (M43) constricts the space between anterior lingual plate and the prementum and squeezes the premental pouch which envelopes the glossa (Fig. 15). In this manner, the glossa rod is moved forward out of the pouch. Contraction of the anterior part of these muscles forces the entire anterior lingual plate forward, and the anterior side of the glossa turns inside out. Due to its elastic properties the glossa immediately projects forward to its full extent, as determined in freeze-killed and thawed specimens. The role of the m. craniolabialis posterior (M35) is not entirely clear. Its contraction may pull the prementum deeper into the head cavity which would contribute to the compression of the space between prementum and anterior lingual plate (Fig. 17). Opening of the mandibles is a likely precondition for glossal extension. According to the field observations the mandibles were always observed to be open when the glossa was extended (Fig. 3). During the initial phase of retraction of the glossa, the posterior lingual plate is folded back into the prementum by

Figs. 8–12. Fig. 8: Head of C. hispanicus (Masarina) in frontal view. Mandibles (md) closed; glossa retracted into prementum. Clypeus (cl) covers the labrum. Fig. 9: C. hispanicus (Masarina); distal portion of the labium in lateral view; left mandible and maxilla removed. Glossa retracted into prementum (pr), only glossal tips (gl) visible; posterior lingual plate (plp) at a right angle to hinge plate (hp) which is at a right angle to prementum. Clypeus (cl), mandible (md), labial palpus (lp), maxillary palpus (mxp). Fig. 10: Head of C. hispanicus (Masarina) in lateral view. Glossa (gl) extended; hinge plate (hp) and posterior lingual plate (plp) are extended outward forming the articulation of the glossa (gl) and prementum (pr); glossa tip (glt) is bifurcated; paraglossa (pgl) is short. Fig. 11: Cross cut through the middle section of the glossa. Overlapping cuticle lamellae form the food tube (ft) along the anterior side; the glossa rod (glr) provides stability to the glossa. Fig. 12: Bifurcate glossal tip in C. hispanicus. Each glossal half has a separate food canal formed by spiny cuticular structures; tip bears acroglossal button (ab).

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Figs. 13–16. Fig. 13: Ceramius fonscolombei (Masarina), longitudinal section through head. Relatively short glossa (gl) is held in resting position. Posterior lingual plate (plp) is folded and glossal rod (glr) is retracted into the prementum (pr). Anterior lingual plate (alp) is longer than retracted glossa. Paraglossa (pgl), clypeus (cl) and labrum (lr) form frontal closure of the preoral cavity (poc); distal plates of maxillae (mx) transport pollen into the infrabuccal pouch (ibp). Fig. 14: C. hispanicus (Masarina), longitudinal sections through the prementum (pr) with retracted glossa (gl). Glossal rod (glr) articulates with prementum via posterior lingual plate (plp) and hinge plate (hp); glossal tip (glt) at same level as paraglossa (pgl); long anterior lingual plate (alp) give

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Fig. 17. Model of the functional mechanism of glossal movement in Ceramius. (A) Glossa retracts by contraction of m. intralabialis posterior (mip) and m. craniolabialis anterior (mca).(B) Glossa unfolds by contraction of m. intralabialis anterior (mia) and m. craniolabialis posterior (mcp). Areas of articulation between prementum (pr) and hinge plate (hp) and between hinge plate and posterior lingual plate (plp) are extended. Arrows indicate movements of mouthparts.

contraction of m. intralabialis posterior (M42) (Fig. 17). At a particular point the elastic properties of the glossal rod force the glossa to suddenly slip into the premental pouch. The membranous cuticle of the anterior side invaginates under the anterior lingual plate. The posterior side turns into the prementum by the double flexion of the glossa (Fig. 17). Contraction of m. craniolabialis anterior (M34) pulls back the anterior lingual plates and the lateral glossal base (Fig. 17). Proboscis of higher Masarina. In most of the higher Masarine taxa, the glossa is longer relative to body length than in the previously discussed species. In J. braunsi and M. familiaris the glossa has a length of 3.0 –3.3 mm which is equal to one third body length. In Quartinioides sp. it is about 4.9 – 5.0 mm long which is about as long as the body.

The principle morphology of the glossae and the basic mechanism of retraction in all investigated higher Masarina is the same as described for Ceramius. The glossa is retracted between the prementum and the anterior lingual plate, but due to its great length the looped glossa extends beyond the proximal end of the prementum to a varying degree in the different species. A sac formed by membranous cuticle (‘glossal sac’, Richards, 1962) is visible on the posterior side of the head as a lightly colored sac behind the more darkly sclerotized prementum. In J. braunsi, as in Ceramius, the glossa lies in one great loop within the prementum and protrudes beyond the proximal end of the prementum and cardines (Fig. 18). The musculature of the labium does not envelope the sides of the glossal pouch. The m. intralabialis anterior (M43) is

attachment site of m. intralabialis anterior (mia); m. intralabialis posterior (mip) attaches at posterior lingual plate. Fig. 15: C. hispanicus (Masarina), longitudinal sections through prementum (pr), glossa (gl) extended. The two articulations between prementum and hinge plate (hp) and between hinge plate and posterior lingual plate (plp) are extended. Anterior lingual plate (alp) pressed against prementum due to contraction of m. intralabialis anterior (mia) and m. craniolabialis posterior (mcp); m. craniolabialis anterior (mca) attaches at anterior lingual plate. Fig. 16: C. hispanicus (Masarina), cross-sections through the glossa in (A) the proximal half, (B) the distal half, and (C) the tip region. Cuticular structures of the lateral glossal wall form the food tube (ft) on the anterior side of the glossa. The glossal rod (glr) stiffens the glossa on the posterior side. The lumen of the glossa (gll) is voluminous in the proximal half and narrow distally; the bifid tip region has a double food tube formed by curved cuticular structures from both sides of the glossa.

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Figs. 18 and 19. Fig. 18: Longitudinal section through head of J. braunsi (Masarina). Glossal rod (glr) is retracted into a loop which bulges beyond the prementum (pr) and cardo (c). Posterior lingual plate (plp) is folded backward by m. intralabialis posterior (mip); hinge plate (hp) is bent against the prementum. Micrograph is composite of photos of two sections from the same series. Fig. 19: Longitudinal section through head of Quartinioides sp. (Masarina). The glossa (gl) is retracted in several loops into the prementum (pr); glossal tip (glt) frontally covered by distal plates of the maxillae (mx). m.intralabialis posterior (mip).

smaller and extends only into the proximal third between the anterior lingual plate and the prementum. This muscle is composed of two portions, one runs obliquely in the posterior direction to the proximal/median region of the prementum, the other portion extends in a lateral direction and inserts on the lateral margin of the prementum. In M. familiaris the glossa sac is remarkably enlarged and arches over the hypostomal bridge. Due to the transparency of the cuticle, the loop of the glossal rod is visible from outside. The stipites have processes directed toward the median sides behind the proximal end of the prementum. In Celonites peliostomi the glossal sac is large and extends well beyond the head. In Quartinioides sp. the prementum is rather flat, broad and rounded on the posterior side and extends with two slender arms over the lateral sides. No glossal sac is present. In comparison to the short body length, the glossa of Quartinioides sp. is extremely long and very thin, being about ten times as long as the prementum. The bifurcate section makes up about 85% of total glossal length. Longitudinal sections through the head reveal that the glossa retracts into several longitudinal and transversal

loops within the prementum (Fig. 19). In this species, as in the examined Jugurtia and Masarina, the m. intralabialis anterior (M43) is weak and does not envelope the glossal pouch. 3.3. Cladistics The Masarinae have been subjected to previous cladistic analyses. In Carpenter’s (1982) phylogenetic study, which was based on 50 characters and numerous vespid taxa including the pollen wasps, the superfamily Vespoidea was reduced to the single family Vespidae with the following arrangement: Euparagiinaeþ (Masarinae þ (Eumeninae þ (Stenogastrinae þ (Polistinae þ (Vespinae))))). The Euparagiinae were removed from the masarids leaving two tribes of pollen wasps, the Gayellini and Masarini. The Gayellini were analyzed by Carpenter (1989). Carpenter (1993) presented a dendrogram of the Masarinae, based on about 50 unpublished characters in which Paragia þ Metaparagia were the sister-group to the remainder of the Masarini. In an analysis of the Australian species of pollen wasps, Carpenter (1996/1997) separated the Masarini into

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Table 1 List of characters and character coding used in cladistic analysis of Fig. 20 Head 1. Clypeal dorsal margin: (0) straight; (1) bisinuate 2.Wing-shaped clypeus: (0) absent; (1) present 3. Eye emargination: (0) present; (1) absent 4. Number of male antennal articles: (0) thirteen; (1) twelve 5. Female mandibles: (0) quadridentate; (1) tridentate; (2) bidentate. Polarity as in Gess (1998) Mouthparts 6. Paraglossa: (0) about as long as or longer than proximal section of glossa; (1) shorter; (2) reduced or absent 7. Prementum: (0) longer or about as long as proximal section of glossa; (1) shorter than proximal section of glossa 8. Glossa: (0) shorter than head length; (1) longer than head length; (2) about as long or longer than body 9. Glossa retractable into prementum: (0) partially; (1) almost fully with one loop; (2) almost fully and coiled into several loops 10. Glossal sac: (0) absent; (1) moderate in size; (2) large extending beyond cardo. Ceramius was coded with state one; however, state two may be present in some species 11. Glossal anterior surface with: (0) transverse rows of hairs; (1) transverse rows of lamellae; (2) median food canal formed by non-overlapping lamellae; (3) median food tube formed by overlapping lamellae. Additive 12. Glossal lobe: (0) without processes; (1) with two rows of flattened processes forming a sponge-like extension; (2) flattened processes overlap and curve together to form a tube. Additive 13. Anterior lingual plate: (0) short; (1) long and narrow sclerite to the proximal end of the prementum 14. Acroglossal buttons: (0) present; (1) absent 15. Maxillary palpi: (0) six-segmented; (1) three-segmented; (2) two-segmented; (3) one-segmented. Additive. Character is variable in Paragiina and Ceramius Mesosoma 16. Pretegular carina: (0) present; (1) absent. Polarity as in Carpenter (1996/1997) and Gess, 1998) 17. Propodeal spiracle: (0) lateral; (1) more or less dorsal 18. Male foretrochanter: (0) without process; (1) with process Forewing 19. Marginal cell: (0) not narrower basally than apically; (1) 2r-rs curving basal to insertion of RS so that it is narrower 20. Submarginal cell number: (0) three; (1) two 21. CuA2 and A: (0) angled where meeting; (1) rounded together 22. First discal cell: (0) shorter than subbasal cell; (1) as long or longer than subbasal cell 23. CuA: (0) diverging from M þ CuA; (1) distal to insertion of cu-a; (2) based to insertion of cu-a 24. Cu-a: (0) transverse; (1) inserted on CuA and aligned with A 25. Longitudinal folding: (0) absent; (1) present Hindwing 26. Free apical section of A: (0) present; (1) absent 27. Jugal lobe: (0) present; (1) reduced Biology 28. Larvae feed on: (0) insect prey; (1) pollen and nectar

two subtribes, Paragiina (containing Paragia and Metaparagia ) and Masarina. The analysis of Gess (1998) with consideration of 17 characters split the Masarini into three subtribes with Priscomasaris as only member of a new subtribe, Priscomasarina, which formed a sister group relation to remaining subtribes, Paragiina þ Masarina. The present analysis utilizes 28 characters (Table 1) many of which are adopted from Gess (1998) and Carpenter (1982, 1996/1997). Three multistate characters (11, 12, 15) representing transformation series were coded as additive. Euparagia (Euparagiinae) was selected as the outgroup. Computer analysis on the data matrix of Table 2 using NONA (Goloboff, 1993) yields one cladogram (Fig. 20) with a step length of 49, consistency index of 0.79 and retention index 0.81. Cladograms were examined and characters plotted using WinClada (Nixon, 2000). The cladogram in Fig. 20 confirms the tribal and

subtribal arrangement of taxa as presented in Gess (1998). The clade Paragiina þ Masarina is supported by two synapomorphies, both features of the glossa, i.e. food canal of proximal glossa formed by lamellae (character 11, state 2), and the presence of a food canal on the glossal lobes (character 12, state 2). A processed male foretrochanter (character 18) was regarded as another potential synapomorphy in the analysis of Gess (1998), however, the character plotting is equivocal in this study, since it is present in Paragia and Ceramius but not the other investigated Masarina. The cladistic analysis shows that the trend toward elongation of the proboscis is accompanied by morphological innovations, such as the presence of lamellae on the anterior glossa (character 11, state 1) leading to the formation of a median food canal between the lamellae (character 11, state 2). Both states are necessary preconditions for the formation

114

Table 2 Distribution of 28 characters (Table 1) used in cladistic analysis (Fig. 20). Character numbers in bold type Head Clypeal dorsal margin, 1

Shape of clypeus, 2

Eye margination, 3

Number of antennal articles, 4

Female mandibles, 5

Paraglossa length, 6

Prementum length, 7

Glossa length, 8

Glossa retracted into prementum, 9

Glossal sac, 10

Lamellae on glossa, 11

Glossal lobe with food tube, 12

Anterior lingual plate, 13

Acroglossal buttons, 14

Maxillary palpi segment number, 15

0 1 0 0 0 0 0 0 0 0

0 0 0 0 1 0 0 0 0 0

0 0 1 1 0 0 0 0 0 0

0 0 1 1 1 1 1 1 1 1

2 0 1 1 1 2 1 1 2 2

0 0 0 0 1 2 1 1 2 2

0 0 0 0 1 1 1 1 1 1

0 0 0 0 1 1 1 1 2 1

0 0 0 0 1 1 1 1 2 1

0 0 0 0 1 2 2 2 0 1

0 0 1 2 3 3 3 3 3 3

0 0 1 2 2 2 2 2 2 2

0 0 0 0 1 1 1 1 1 1

0 0 0 0 0 1 0 0 0 0

0 0 0 0 0 1 2 2 3 3

Mesosoma

Euparagia Gayella Priscomasaris Paragia Ceramius Celonites Masarina Jugurtia Quartinioides Quartinia

Forewing

Pretegular carina, 16

Propodeal spiracle, 17

Male foretrochanter, 18

Marginal cell, 19

0 1 1 0 0 0 0 0 0 0

0 0 0 1 0 1 0 0 0 0

0 0 0 1 1 0 0 0 0 0

0 0 0 1 0 0 0 0 0 0

Hindwing Submarginal cell number, 20 0 0 1 1 1 1 1 1 1 1

CuA2 and A, 21

First discal cell, 22

CuA, 23

Cu-a, 24

Longitudinal folding, 25

0 0 0 1 0 0 0 0 0 0

1 0 1 1 1 1 1 1 1 1

0 1 2 2 2 2 0 0 0 0

0 0 1 1 1 1 1 1 1 1

0 0 0 0 0 1 0 0 1 1

Free apical section of A, 26 0 0 1 1 1 1 1 1 1 1

Biology Jugal lobe, 27

Larval food, 28

0 1 1 1 1 1 1 1 1 1

0 1 1 1 1 1 1 1 1 1

H.W. Krenn et al. / Arthropod Structure & Development 31 (2002) 103–120

Euparagia Gayella Priscomasaris Paragia Ceramius Celonites Masarina Jugurtia Quartinioides Quartinia

Mouthparts

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115

Fig. 20. Cladogram of Masarinae based on data in Table 2. Subtribes indicated on right margin. Character numbers are given above line and character states below. The outgroup is represented by Euparagia. Morphological innovations associated with the production of a suctorial proboscis are formation of a food canal, a looped glossa, and a closed food tube. Glossa retracted in several loops is an autapomorphy in Quartinioides.

of the closed food tube of the elongated glossa (character 11, state 3) in Masarina. Furthermore, the lengthening of the anterior lingual plate (character 13, state 1) seems to be crucial for the development of novel mechanisms enabling the extension of the glossa out of the glossal sac. Elongation of the glossa in Masarina is also associated with shortening of the paraglossa (character 6, states 1 and 2). The presence of a moderate-sized protruding glossal sac (character 10, state 1) is interpreted by the analysis as a synapomorphy of the Masarina; however, it is absent in Quartinioides. A large protruding sac (character 10, state 2) is regarded as convergent in Celonites and the clade Jugurtia þ Masarina, however, it could be a synapomorphy of the higher Masarina with a reversion in Quartinia and a loss in Quartinioides.

4. Discussion 4.1. Morphological innovations in the suctorial proboscis of pollen wasps Flower visiting behavior in insects is connected with a host of modifications in the mouthparts. Many of these are adaptations for pollen collection and ingestion as well as nectar consumption. Radical transformations of the mouthparts are evident in various forms of elongation that are associated with nectar feeding from flowers with a deep corolla (Schremmer, 1961; Jervis, 1998; Jervis and Vilhelmsen, 2000). The evolution of an elongate suctorial glossa from a short homologous condition is exemplified in the pollen wasps. The basal taxa of the pollen wasps, i.e. Gayella, Priscomasaris, Paragia possess a relatively short

glossa which has cuticular structures that allow uptake of nectar and water, presumably, in large part by adhesion. The functional morphology which enables a passive uptake of liquids, at least until the vicinity of the preoral cavity where pharyngeal suction takes over, is regarded as plesiomorphic for the Masarinae since it appears to differ little from that of other wasps in Euparagiinae (Bradley, 1922), Eumeninae (Richards, 1962; Osten, 1982) or Vespinae (Kirmayer, 1909; Brocher, 1922; Duncan, 1939). The higher Masarina possesses an elongate suctorial proboscis with morphological innovations of the labium, i.e. the lamellar structures of the glossa forming a food tube, the specialized apex and basiglossal articulation, as well as the shape and muscles of the prementum. Morphological innovations enabling mouthpart elongation are often novel solutions to biomechanical problems, such as formation of suction tubes, mechanisms of movement and new resting positions for the long proboscis. Some of these will be referred to below. Suction. The elongate proboscis in Lepidoptera operates like a drinking-soda straw, in that fluid is sucked along an air-tight tube due to pressure created by the muscular pharyngeal pump (Kingsolver and Daniel, 1995). The same analogy applies to the glossa of higher pollen wasps, where the lamellar cuticle structures of the glossa, which must be homologous to the rows of hair structures on the glossa of other Vespidae, form the long and air-tight median food tube. Other mechanisms for ensuring the air-tightness of a food canal include the coming together or the interlocking of different parts, either temporarily, like in bees, or permanently. Permanent linkage of the two halves of the proboscis is achieved in Lepidoptera by a series of hooks

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and overlapping cuticle plates (Hepburn, 1971; Krenn and Kristensen, 2000). Intake region. Closed suctorial proboscides require specialized regions at the apex of the food canal for fluid uptake. In pollen wasps, this takes place through the slit-like openings in the food canal of the glossal lobes. In other insects the apical regions of the food canal are outfitted with specialized sensilla, for example in Lepidoptera (Krenn, 1998; Krenn and Kristensen, 2000; Krenn et al., 2001) and Diptera (Szucsich and Krenn, 2000, 2002). However, in pollen wasps, the acroglossal button and its sensillae are not strongly modified even in the most derived species. The long tongue of bees has different requirements. The glossa is enclosed inside the food tube and independently performs licking movements extending beyond the ensheathing tube (Snodgrass, 1956; Harder, 1982; Plant, personal observation). The apical food tube must first be loaded with nectar by means of the glossal movements and capillary action, before it is drawn through the food tube to the mouth by suction action (Kingsolver and Daniel, 1995). A presuction nectar-loading stage is not necessary in butterflies and the higher pollen wasps, since suction begins with immersion of the apical uptake region in the nectar. Mechanisms of protraction and retraction. The labiomaxillary complex of aculeate Hymenoptera permits a slight extension and retraction. The mechanisms for this have been described for Vespula (Duncan, 1939), sphecids (Ulrich, 1924), scoliids (Osten, 1982, 1988), and the shorttongued bee Andrena (Harder, 1983). It involves at least three major steps, the movement of the cardines which swing the proboscis in or out of the proboscidial fossa, the z-shaped fold between prementum and glossa, and the folding or unfolding of the galea. When a significant elongation of apical parts of the proboscis takes place, new steps of extension and retraction are added onto the preexisting ones. Storage of glossa. The length of the proboscis is contingent on its required storage space as well as the retraction method; in the pollen wasps, e.g. Ceramius, the space available inside the prementum is a limiting factor for the length of the glossa. One solution taken by the higher Masarina is to store the glossa outside the prementum by creating an ‘opening’ in the cuticular membrane between the cardines through which the glossa invaginates into a large sac. The basic mechanism, however, remains the same as in Ceramius, in that the glossa is retracted into one loop even if it is so large as to protrude out of the prementum. Quartinioides has taken another direction, its glossa lies entirely within the prementum but in several irregular crisscrossing loops. The mechanism of extension in Quartinioides, however, remains puzzling. It may be significant that its glossa, although very long, is also extremely thin, at least in the species examined. Richards (1962) suggested that hemolymph pressure was important for extension. It is likewise not known how the glossa of Celonites and Jugurtia is projected out from its fully retracted position

protruding beyond the basal part of the prementum. Compressing the sides of the prementum together is probably not sufficient to eject the looped glossa. It is astonishing that the labial musculature in the examined species of the subtribe Masarina, e.g. Ceramius, is only slightly modified from the plesiomorphic condition in pollen wasps; all muscles can be readily homologized with those in other Vespinae and in general with other Hymenoptera (Duncan, 1939; Matsuda, 1965). Compared to the short-tongued Masarinae, e.g. Paragia, only one muscle has modified its course. Due to the elongation of attachment sites on the anterior lingual plate and the particularly arched prementum, one part of this muscle is positioned up to 908 differently from the plesiomorphic condition. In the derived condition the contraction of this muscle compresses the glossal pouch inward and seems to be the major force in initiating extension of the glossa, at least in Ceramius. In the plesiomorphic condition, the same muscle functions for extension as well, thus no new neural motor pattern is necessary for the control of the glossa movements. 4.2. Comparative remarks on proboscis evolution in Hymenoptera Convergent evolution of an elongated proboscis associated with flower visiting behavior is apparent in many groups of Hymenoptera. Even within the Masarinae a second clade, the Australian Metaparagia independently evolved an elongated glossa for probing flowers with deep corollas (especially Goodeniaceae; Gess et al., 1995; Gess, 1996). However, in this taxon the proximal section of the glossa is greatly elongated and the paraglossae reach to the bifurcated section of the glossa; in addition the proboscis is also retractable into the prementum (Carpenter, 1996/1997) but the associated morphological changes and the mechanism of retraction are undetermined. Examples of long or moderately long mouthparts are numerous in other aculeate Hymenoptera, i.e. Eumeninae (Vespidae), e.g. species of Eumenes, Pterocheilus, Raphiglossa, Labochilus (Schremmer, 1961; Richards, 1962; Bohart and Stange, 1965; Giordani Soika, 1974; Haeseler, 1975; Osten, 1982; Mauss, personal observation), many Sphecinae including Ammophila, Sphex, some Bembicinae (Ulrich, 1924; Bohart and Menke, 1976; Osten, 1982), certain Tiphiidae, Sapygidae, and Scoliinae (Osten, 1982, 1991) and some Chrysididae and Pompilidae (Jervis, 1998). Most of these derived elongate mouthparts differ from that in Masarinae in that the food canal is not formed exclusively by the glossa but includes other parts of the labium and maxillae. For example, in the long-tongued bees (Apidae and Megachilidae) the elongate and flattened labial palpi together with the galea form a stationary sheath-like tube within which the glossa operates (Snodgrass, 1956). Retraction of the glossa into the prementum as in the Masarina is not unique in Hymenoptera. It has been described for Scolia (Scoliidae) (Ko¨nigsmann, 1976;

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Table 3 Composition of food canal produced by elongation of apical mouthparts in various aculeate Hymenoptera. Type of mouthpart specializations (CNEA, see text) follows Jervis (1998) Glossa

Masarinae Eumeninae Eumeninae Scoliidae Chrysididae Sphecinae Sphecinae Long-tongued bees Andrenidae

þ þ þ þ þ þ þ þ

Paraglossa

þ þ

Galea

Labial palpi

þ þ

?

þ þ þ

þ

þ

þ

þ

Andrenidae Halictidae Halictidae Colletidae Colletidae Colletidae

þ þ

þ

þ þ

Colletidae Andrenidae

þ þ

Melittidae

þ

a

þ

Maxill. palpi

þ þ

þ þ þ þ

Metaparagia and subtribe Masarina Raphiglossa (CNEA type 1) Eumenes (Osten, 1982) Scolia (Osten, 1982) Parnopes (Linsenmaier, 1997) Ammophila (Ulrich, 1924) (CNEA type 1) Bembix Megachilidae, Apidae (CNEA type 4) Protomeliturgini, Melitturgini, Perditini (e.g. Perdita ), Calliopsini (e.g. Callonychium ), Andrena (Callandrena ) micheneriana (LaBerge, 1978) (CNEA type 4) Neffapis longilingua (Panurginae) (Rozen and Ruz, 1995) (CNEA type 6) Various Rophitinae Ariphanarthra palpalis (Eickwort, 1969) (CNEA type 5) Leioproctus (Filiglossa ) filamentosus (Michener 2000) Niltonia (Colletinae) (Laroca et al., 1989) Chilimelissaa, Xeromelissa (Xeromelissinae), species of Hylaeus (Pseudhylaeus ), H. (Prosopisteroides ) (Hylaeinae), Euhesma, and Euryglossa tubulifera (Euryglossinae) (Michener, 1965; Houston, 1983) (CNEA type 5) Palaeorhiza papuana (males only) (Michener, 1965) Oxaeidae, Andrena (Iomelissa ) violae (Michener, 1944), A. (Charitandrena ) hattorfiana, A. (Taenandrena ) lathyri Pseudophilanthus tsavoensis (Michener, 1981, as Agemmonia )

Incorrectly given as labial palpi in Laroca et al. (1989).

Micha, 1927; Osten, 1982, 1988) and Epomidiopteron (Tiphiidae) (Osten, 1991). However, the proboscis at least in Scolia is not a thin suctorial tube as both the glossa and paraglossae are enlarged and fold back into the prementum (Osten, 1982). In long-tongued bees, the elongated apical parts of the proboscis fold back beneath the prementum. Depending on the total glossal length the folded proboscis may exceed the thorax and abdomen. Some short-tongued bees have independently evolved an elongate proboscis of very similar construction to that in Apidae and Megachilidae, for example, especially in Rophitinae (Halictidae) and Panurginae (Andrenidae) (Michener, 1944, 2000), and also in one species of Andrena (Andreninae) (LaBerge, 1978). Individual species of shorttongued bees have also achieved other forms of elongation, presumably with formation of a food canal, by production of the maxillary palpi, or the labial palpi, or both together. Elongation of the glossa alone is also occasionally found in short-tongued bees, however, it is not apparent how or if a special food tube is formed. The structures which constitute the elongated section of the apical food canal are listed for various bees and other aculeate Hymenoptera in Table 3. It is reasonable to assume that the structures of the maxillae and the labium underlie different selection pressures and evolutionary constraints arising from their role in foreleg cleaning, nectar feeding, pollen ingestion, nest and brood cell construction and other functions of the proboscis. A change in one of these behaviors may free

structures for evolutionary recruitment. In some cases it may be possible to indicate which structures are preoccupied, for example, in pollen wasps the galea is connected with pollen eating and therefore presumably not available for elongation. However, in general it is difficult to determine why one particular structure or one set of structures undergoes modification and not another. To summarize, at least three morphological – functional groups of mouthparts can be distinguished which may be related to feeding habits of adult Hymenoptera. (1) The unspecialized small labiomaxillary complex. (2) Apomorphic ‘short-tongued’ and (3) Apomorphic elongate or ‘long-tongued’. The first group is presumably plesiomorphic for Hymenoptera (Jervis, 1998). The main body of the mouthparts usually does not extend beyond the reach of the open mandibles. The labial and maxillary palpi, however, are very long and active in performing tactile sensory movements. The mouthparts are used to lick and suck nectar, honeydew or prey body-fluid, examples are Syspasis (Ichneumonidae) (Richards, 1977), Ampulex (Ulrich, 1924) and Psenulus (Sphecidae). Modifications of the plesiomorphic condition have led to development of short and long-tongued conditions which are associated with nectar feeding. The short-tongued proboscis as in many bees and wasps can extend somewhat beyond the reach of the open mandibles since it has undergone a general increase in size or length of its major

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parts. These parts of the labiomaxillary complex involve the basal section (cardo, hypopharynx, labrum), the mid-section (stipes, subgalea, laciniae, prementum) and not merely the apical section (glossa, paraglossa, labial and maxillary palpi, postpalpal galea). The short-tongued mouthparts are thus well developed compared to the plesiomorphic small proboscis. The principles of proboscis extension, retraction and formation of the food canal are similar to those of the plesiomorphic small proboscis, however, morphological differentiation may occur in the postmental region (Plant and Paulus, 1987), shape of the glossa, articulation of the base of the glossa, and a reduction in the relative length and function of the maxillary and labial palpi. An elongate proboscis may be defined, as in the Megachilidae and Apidae, when the glossa exceeds the length of the prementum (Harder, 1983). An apical elongation can occur by other structures as well. Essential for the discussion here is when these lengthenings necessitate the addition of new construction designs and morphological innovations with respect to food canal formation, storage of elongated parts, mechanisms of extension and retraction, etc. Jervis (1998) and Jervis and Vilhelmsen (2000) documented eight types of mouthpart elongations in Hymenoptera for the uptake of nectar from flowers with long, narrow, tubular corollas and referred to them as CNEA (concealed nectar extraction apparatus). Briefly stated, these are: (1) glossa and galea elongate, (2) glossa elongate and galea only moderately elongate, (3) glossa, galea and maxillary palpi elongate, (4) glossa, galea and labial palpi elongate, (5) maxillary palpi elongate, (6) glossa and labial palpi elongate, (7) maxillary and labial palpi elongate, (8) prementum and stipes elongate. These types were intended to account for the surprising variation found in various groups of symphytans and parasitoid Apocrita. We list in Table 3 those structures which partake in the elongation of the apical food canal for various bees and other aculeate Hymenoptera. Some of these examples correspond to CNEA types of Jervis (1998) and are indicated in the table, others would constitute new types of CNEA, e.g. the mouthpart elongation of the higher Masarinae, since it is achieved only by the glossa. It can be seen that major aspects of the functional morphology between the plesiomorphic small mouthparts and the apomorphic short-tongued condition are similar. We seek to underscore the functional –morphological differences between short-tongued and elongated proboscides and to point out the morphological consequences of elongation, rather than to emphasize the lengths of the different parts. Thus we would not include in Jervis’ (1998) CNEA type 1 most of those flower visiting bees and wasps with mouthparts that have undergone a slight or moderate or short elongation. These forms correspond to our group 2, apomorphic short. A short-tongued condition probably represents the evolutionary starting point for further modification by elongation.

Furthermore, the mouthpart condition found in typical short-tongued bees such as Hylaeus, Colletes, Andrena and Melitta, does not generally permit these insects to utilize concealed nectar sources. The basal taxa of the Masarinae with short mouthparts are likewise restricted to plant species with easily accessible, actinomorphic flowers, e.g. Priscomasaris on Molluginaceae and Aizoaceae (Gess 2001), Paragia on Myrtaceae, Proteaceae, Mimosaceae and Bromeliaceae (Houston, 1984, 1986; Snelling, 1986; Gess, 1996). Our field observations on flower visiting behavior confirm that species of Ceramius with elongate mouthparts are able to utilize derived flower types with concealed nectaries (e.g. Fabaceae, Lamiaceae, Pontederiaceae; reviewed by Gess and Gess (1989), Gess (1996), Mauss (1996), Mauss and Mu¨ller (2000) and Garcete-Barrett and Carpenter (2000)) while the relatively short-tongued Ceramius fonscolombei visits flowers with readily accessible nectar. The morphology of the proboscis and its mechanisms of extension in Ceramius permit a rapid exploitation of flowers with very narrow corolla tubes. Pollen wasps can extend their proboscis into a narrow corolla tube after landing on the flower since the glossa is propelled forward from the looped resting position. In contrast, the long proboscis of bees requires more space to swivel out and unfold into the feeding position. Many long-tongued bees must unfold their proboscis before insertion into flowers and those with a particularly long proboscis, such as euglossids and Anthophora, hover in front of blossoms and approach flowers with an extended proboscis. Based on the proposed phylogeny and biogeographic pattern of the pollen wasps it is possible to roughly estimate when the evolution from licking/sucking mouthparts to a pure suctorial proboscis should have occurred (Fig. 20). The basal subfamilies of the Vespidae, including the Masarinae, appear to have become established in the early to middle Cretaceous (Grimaldi, 1999). The basal-most group of pollen wasps, Gayellini, is limited to the Neotropics. The Masarini, however, represent a typical disjunct Gondwanan distribution with the Paragiina endemic in the Australian region and Masarina restricted to the remaining areas (Gess, 1992; Carpenter, 1993). In addition, most genera of Masarinae are highly endemic to continental areas. The diversification of pollen wasps probably thus took place after the middle of the Cretaceous and coincided with the diversification of angiosperms (Crane, 1993; Grimaldi, 1999). The independent evolution of an elongated proboscis in the stem-groups of the subtribe Masarina and Metaparagia (Paragiina) must have occurred after separation of the Australian land mass in the middle Cretaceous, about 100 million years ago (Fukarek, 1995).

Acknowledgements We are especially grateful to J. Carpenter (New York)

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and F. Gess and S. Gess (Grahamstown) who kindly collected some of the investigated material for us and to L. Castro (Teruel) for his extraordinary hospitality and indispensable support of V. Mauss during the field studies. M. Lo´pez (Diputacion General de Aragon) kindly issued the required collection permits. We thank U. Hannappel, C. Wirkner and A. Pernstich for technical assistance, T. Osten for valuable comments on the manuscript, and the Obero¨sterreichische Landesmuseum—Biologiezentrum Linz for making available papers of the Fritz Schremmer collection. Parts of the work were supported by the Austrian Science Fund (Project 13944 Bio).

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