Four nests of Hoplitis tuberculata in three different trap nests were found. Five further nests were found in tree trunks and stumps in the vicinity of the trap nests (Figs 4, 5). The maximal diameter of the nine burrows selected by the bees as nesting sites was 4 mm (n=1), 4.5 mm (n=6), 5 mm (n=1) and 6 mm (n=1).

All four nests built in the bamboo sticks of the trap nests had a similar structure and consisted of i) a basal wall that sealed the nest against the rear end, ii) a varying number of brood cells each delimited towards the nest entrance by a cell partition, iii) a (facultative) vestibule in front of the last cell and iv) a nest plug that closed the nest at the front end. The distance from the basal wall to the outermost wall of the nest plug was 6.5 cm, 7.6 cm, 8.3 cm and 10.9 cm.

The basal walls consisted of masticated leaves (“leaf pulp”) and had a width of about 1 mm. In two nests, a free space between pith and basal wall with a length of 0.4 cm and 3.5 cm, respectively, was present, which was loosely filled with small particles, such as pebbles, wood and leaf fragments, seeds or earth crumbs (Fig. 10). In the other two nests, the basal wall was constructed directly adjacent to the pith that filled the rear of the bamboo sticks (Fig. 14). Some of these particles, which were most probably transported into the nest by the female bees, partly adhered to the inside of the basal wall, suggesting that the females incorporated them into the leaf pulp matrix during the first steps of basal wall construction.

Figures 10–14. 

Nest architecture of Hoplitis tuberculata: 10 Opened nest in a hollow bamboo stick with – from left to right – i) short space filled with small particles followed by the basal wall, ii) two brood cells, which are delimited towards the nest entrance by a three-layered cell partition, iii) vestibule loosely filled with small particles, iv) nest plug consisting of one wall each at the rear and the front end enclosing a space that is filled with small particles and divided up by three additional walls (the low amount of particles in the space between the third and the fourth wall, the presence of only traces of the fourth and the outermost wall and the lack of particles between the two outermost walls is due to the loss of particles and walls during the splitting of the stick) 11 Three-layered cell partition composed of two walls enclosing an interlayer that is densely filled with particles 12 Three-layered cell partition being flush with the nest plug, which consists of several walls with densely packed small particles in between 13 Vestibule between outermost cell partition and innermost part of the nest plug loosely filled with small particles 14 Opened nest in a hollow bamboo stick with i) basal wall built directly adjacent to the pith in the rear of the stick, ii) four brood cells each delimited by a three-layered cell partition directly followed by iii) the nest plug.

The four nests contained one (n=1), two (n=2) and four (n=1) linearly arranged brood cells, which had a length of 8.5-12.5 mm (Fig. 10, 14). In two nests, which were opened in late fall, the brood cells harboured postdefecating larvae spun in a semitransparent, brownish-white cocoon, indicating that Hoplitis tuberculata overwinters as prepupa. The cell partitions had a width of 3-5 mm and invariably consisted of three layers (Fig. 11): two walls each of 0.5-1 mm width, which had been constructed from leaf pulp, enclosed an interspace of 2-4 mm length, which was densely packed with pebbles, earth crumbs, seeds, wood chips or fragments of leaves and needles. While the outer sides of both walls of the cell partitions were very carefully worked forming a plane surface, the inner sides were more irregular. Some of the particles of the interlayer were partly incorporated into the leaf pulp matrix of the inside of the outer wall.

Three of the four nests contained a vestibule between the outermost cell partition and the nest plug measuring 2.2 cm, 8.1 cm and 8.9 cm in length (Fig. 10). The vestibule was loosely filled with a small amount of 20 to 30 particles, such as pebbles, earth crumbs, wood chips or fragments of leaves and needles (Fig. 13). In one nest, no vestibule was developed; instead, the outermost cell partition and the nest plug bordered directly at each other (Fig. 12, 14).

The nest plugs measured 1.2 cm, 1.3 cm, 1.4 cm and 1.9 cm in length. They consisted of one wall each at the rear and the front end, which enclosed a space that was divided up by one (n=1) or three (n=3) additional walls (Fig. 10, 12, 14). All interspaces between the walls were usually densely packed with small pebbles, earth crumbs, wood chips or fragments of leaves and needles. The walls of the nest plug had a width of 0.5-1 mm, were built from leaf pulp and partly contained foreign particles on their inside, which had been glued to the leaf pulp matrix during wall construction. In two nests, single pebbles and/or earth crumbs were embedded in the leaf pulp matrix of the outside of the front wall (Fig. 6, 7), whereas in the other two nests the front wall consisted exclusively of leaf pulp. This variability in the presence or absence of small particles incorporated into the front wall was also apparent in the five nests found in the vicinity of the trap nests: the front wall of two nests contained small particles on its outside (Fig. 8), whereas that of the other three nests was built of leaf pulp only (Fig. 9).

In summary, the females used two different materials for nest construction: i) leaf pulp to build all the walls within the nest (Fig. 2) and ii) small particles, which were amassed in interspaces between walls and occasionally also incorporated into the leaf pulp matrix of certain walls. The origin of the leaf pulp is not known in detail; one female, however, was repeatedly observed to chew sepals of Potentilla erecta to collect leaf pulp (Fig. 3). The small particles were most probably all collected from the ground as judged by the observation that several females picked up small pebbles and earth crumbs from a small unpaved path.

The microscopical analysis of 87 female pollen loads revealed that Hoplitis tuberculata is polylectic harvesting pollen from the flowers of at least eight plant families (Tab. 1). However, pollen of Fabaceae strongly dominated constituting 84.5% of the total pollen grain volume, followed by pollen of Cistaceae represented by 8.9%. Pollen of all other plant families was recorded in small percentages only. The strong preference for Fabaceae pollen is also evident from the finding that all 87 pollen loads contained pollen of this plant family, 54 of which were pure Fabaceae pollen loads. Among the Fabaceae, Lotus was by far the most important pollen host (Tab. 1); its pollen represented 63.9% of the total pollen grain volume and was recorded in 84 out of 87 pollen loads, 35 of which were pure Lotus pollen loads. The second most important Fabaceae pollen host was Hippocrepis; its pollen represented 11.0% of the total pollen grain volume and was recorded in 31 out of 87 loads.

Pollen of Lotus was also the most important pollen type recorded in six brood cell provisions of two nests collected at the study site (Tab. 2). All cells contained large to very large amounts of Lotus pollen. The provisions of several cells, however, also contained considerable amounts of non-Fabaceae pollen, such as pollen of Vaccinium and Rubus.

As shown in the present study, Hoplitis tuberculata uses leaf pulp to construct the walls of both brood cells and nest plug. The erroneous assumption of mud as being the exclusive or predominant nest building material (Westrich 1989, Amiet et al. 2004) is probably based on the observation of females that collected earth crumbs or pebbles on the ground to amass them later in interspaces within their nests. In fact, among Hoplitis species of the large subgenus Alcidamea only two species have been recorded so far to use mud for nest construction, i.e. the stem-nesting Nearctic H. grinnelli, which constructs its cell partitions with clay while the nest plug consists of alternating layers of clay and pith (Davidson 1896, as H. producta), and the Palaearctic H. fulva, which seals its nest in preexisting cavities of loess scarps with a plug of mud whereas its brood cells are constructed from leaf pulp alone (Marikovskaya 1968).

Peculiar characteristics of the nest architecture of Hoplitis tuberculata include i) the three-layered cell partitions, ii) the presence of a vestibule loosely filled with small particles and iii) a nest plug that consists of densely packed layers of small particles sandwiched between at least three walls.

The construction of three-layered cell partitions composed of two walls with an interlayer of densely packed small particles in between seems to be unique among Palaearctic osmiine bees, which usually partition linearly arranged brood cells by single walls only (Müller 2015 and references therein). However, such three-layered cell partitions are known from several North American Hoplitis species, which are all members of the subgenus Alcidamea but differ in their preferred nesting sites. Species, which use preexisting cavities, such as H. albifrons, H. fulgida and H. spoliata, transport small particles such as pebbles, wood chips, earth crumbs or fragments of conifer needles from outside into their nest to include them into the cell partitions (Fye 1965, Medler 1967, Clement and Rust 1976). Interestingly, these three species are closely related to H. tuberculata and also belong to the Hoplitis tuberculata species group (see Introduction). Among them, the construction of three-layered cell partitions seems to be the rule in H. fulgida and is most common in H. albifrons, while H. spoliata frequently omits the interlayer resulting in single leaf pulp walls between the brood cells (Fye 1965, Medler 1967, Clement and Rust 1976). In contrast, species, which excavate their own burrows in pithy stems, such as H. hypocrita, H. pilosifrons or H. sambuci, tightly pack particles of pith taken from the burrow walls in between the two leaf pulp layers (Michener 1955, Clement and Rust 1976). Although the latter three species are not members of the Hoplitis tuberculata species group, their shared habit of constructing three-layered cell partitions suggests a close relatedness to that group as shown by Sedivy et al. (2013).

The function of the three-layered cell partitions is counterintuitive at first sight as the thick nest plug built by Hoplitis tuberculata and its relatives (see below) is expected to already provide enough protection against the intrusion of nest predators. We hypothesize that these strong cell partitions might impede mobile larvae of predators, which already infested a brood cell before the nest was sealed, from invading adjacent cells. In fact, larvae of some Trichodes beetle species (Cleridae), which are antagonists of above-ground nesting megachilid bees, attack several brood cells in sequence by breaking through the cell walls (Carré 1980).

The presence of a vestibule filled with small particles seems to be another typical trait common to the members of the Hoplitis tuberculata species group except for H. spoliata, where the vestibule is empty (Fye 1965, Medler 1967, Clement and Rust 1976). As in H. tuberculata, vestibules may occasionally be absent in nests of H. albifrons and H. fulgida with the last provisioned cell being flush with the nest plug (Fye 1965, Clement and Rust 1976). In contrast to the three-layered cell partitions, which seem to be a unique character of only a few species of the subgenus Alcidamea, vestibules with amassed small particles are rather widespread among osmiine bees. They are known from some Hoplitis species of the subgenera Anthocopa and Alcidamea other than the Hoplitis tuberculata species group, from several Osmia species of the subgenera Erythrosmia, Neosmia and Pyrosmia as well as from Wainia elizabethae (Gess and Gess 1988, Müller 2015 and references therein).

The architecture of the nest plug varies both within and among the North American members of the Hoplitis tuberculata species group. The nest plug of H. spoliata usually consists of a single layer of leaf pulp, rarely of three or four layers with short empty spaces in between (Medler 1967). In contrast, the nest plug of H. albifrons and H. fulgida is usually three-layered with two walls enclosing an interspace tightly packed with small particles (Fye 1965, Clement and Rust 1976). In the latter species, one or two additional walls are occasionally present in the particle-filled space between the rear and the front wall of the nest plug, mirroring the situation found for H. tuberculata in the present study. As in H. tuberculata, small pebbles are often but not always cemented into the leaf pulp matrix of the front wall in H. fulgida and H. spoliata (Hicks 1926, Fye 1965, Medler 1967).

In summary, although the nest architecture of Hoplitis tuberculata is unique among Palaearctic osmiine bees, it corresponds to that of its closest North American relatives, indicating that nesting site, nest building material and nest structure are conserved traits within the Hoplitis tuberculata species group.

The present study shows that Hoplitis tuberculata is polylectic and collects pollen from the flowers of at least eight different plant families, among which Fabaceae clearly dominate. Fabaceae pollen was recorded in each pollen load and constituted almost 85% of the total pollen grain volume. In contrast, several brood cell provisions contained considerable amounts of non-Fabaceae pollen suggesting that pollen hosts other than Fabaceae may locally also play an important role for larval nourishment. Indeed, the significance of Fabaceae pollen as deduced from the analysis of pollen loads of females, which most probably all had been collected during flower visits, might have been overestimated. The probability that specimens of H. tuberculata are collected at flowers of Lotus or Hippocrepis rather than at flowers of e.g. Vaccinium or Rubus is likely higher because the conspicuously yellow Fabaceae flowers act as true magnet for each bee researcher due to the fact that they attract a multitude of different bee species. Considering this possible bias, the host plant spectrum of H. tuberculata recorded in the present study is similar to that found in Finland (Käpylä 1978). Here, pollen of seven different plant families represented by at least 3% per load was recorded in 16 pollen loads of females collected both at flowers and nesting sites. As in the present study, Fabaceae (Caragana, Lathyrus, Lotus), Ericaceae (Vaccinium), Rosaceae (Geum, Potentilla, Rubus), Ranunculaceae (Anemone, Ranunculus) and Asteraceae (Leontodon) were among the plant families exploited for pollen. In addition, flowers of Asparagaceae (Convallaria, Polygonatum) and Violaceae (Viola) also served as pollen hosts, the former being the most important pollen sources followed by flowers of the Fabaceae.

In summary, the pollen hosts of Hoplitis tuberculata known so far belong to ten different plant families, among which Fabaceae predominate but probably not to that large degree as might be expected from the analysis of the female pollen loads from the Alps alone.