Nesting site

At three study sites – all open larch forests or larch-meadow forests (Figs 2–4) –, a total of 19 nests of O. nigriventris was found, 13 of which were abandoned and at least one year old, whereas six contained bee immatures upon discovery (Tab. 1). Some of the old nests were used as nesting sites by other aculeate hymenopterans in the year of discovery, such as the osmiine bee Osmia leaiana and the eumenine wasp Euodynerus quadrifasciatus (Fabricius) (Figs 22–24). All 19 nests were in bark pieces of Larix decidua, which lay in grassy vegetation on sun-exposed ground (Figs 1, 7, 17, 19). The bark pieces measured 13–73 cm in length, 8.5–34 cm in width and 3–8 cm in thickness (Tab. 1). Eight bark pieces contained one nest, four pieces two nests and one piece three nests. No nests were found in the thick bark of coniferous tree stumps or still living trees. The three study sites where no nests were found were all windfalls lacking thick-barked conifers, such as Larix or Pinus (Figs 5, 6).

Figures 7–16. 

Nest architecture of Osmia nigriventris. 7–9) Nest seals of nests 19, 16 and 18. 10) Five-layered nest plug of nest 18. 11) Single-layered nest plug of nest 17. 12) Ring-shaped threshold. 13, 14) Three-layered cell partitions. 15) Brood cells with larval provisions. 16) Brood cells with larval cocoons.

Figures 17–24. 

Nests of Osmia nigriventris. 17, 18) Nest 16 X-rayed (left) and dissected (right). 19, 20) Nest 18 X-rayed (left) and dissected (right). 21) Old nest 8 with short blind burrow on the right. 22–23) Old nests 2 and 6 later used by Euodynerus quadrifasciatus as nesting site. 24) Old nest 1 with brood cells of Osmia leaiana in the upper half and remnants of the old nest of O. nigriventris in the lower half.

Nest architecture

The 19 nests were all tunneled out by the female bees with no indication that preexisting burrows e.g. of cerambycid beetle larvae were used. The nesting burrows were carefully carved out and measured 6–7 mm in diameter. They were neither lined with glandular secretions nor with leaf pulp except for several small areas of few mm² covered with a thin layer of leaf pulp, which was probably applied to smooth out irregularities or to fill small cracks. The nest entrances, which were circular in shape (Fig. 8) and had a diameter of 6.5–7 mm, were either on the lateral side of the bark piece (n = 12; Fig. 9), on the upper side, i.e. facing upwards (n = 6; Fig. 7), or on the underside, i.e. facing to the ground (n = 1; Fig. 8, Tab. 1). In the latter case, the space between the ground and the nest entrance was relatively large, granting easy access for the nesting bee. The entrances of those seven nests, which did not start on the lateral side of the bark piece, were on the scaly exterior surface of the bark (n = 4; Fig. 7) or on the smooth interior surface (n = 3; Fig. 8).

The nests consisted either of a single linear to curved burrow (n = 9; Figs 17, 18, 21), or of a burrow system comprising 2–4 mostly linear fingers (n = 10; Figs 19, 20, 22–24, Tab. 1), which were usually excavated in roughly the same plane and often ran more or less parallel to each other. Except for the 9–14 mm long and almost vertical entrance burrow of those nests that did not start on the lateral side of the bark piece, the nesting burrows ran more or less parallel to the upper and under surface of the bark. The total length of the excavated burrows per nest was correlated with the number of brood cells and ranged from 3.4–29.8 cm (Tab. 1). The nests contained 1–12 brood cells, which were either constructed singly in short burrows (Figs 19, 20) or in a linear series of 2–4 in longer burrows (Figs 15–24, Tab. 1). The brood cells had a length of 10–14 mm and were widest in the middle (6.5–9 mm) and slightly constricted at both ends (6–7 mm). Nest 3 was an exception in that its burrow system had a total length of 67.5 cm and comprised 26 brood cells distributed in nine fingers, which were tunneled out in three different planes within the bark and contained up to five linearly arranged cells (Tab. 1). In contrast to all the other nests, which were most probably constructed by a single female only, the high number of brood cells suggests that nest 3 was likely built by several females, either in the same or in consecutive years.

Three types of partitions were recorded in the nests of O. nigriventris: i) partitions that separate the brood cells from each other (“inner cell partitions”); ii) partitions that seal the outermost brood cell towards the nest entrance or a finger towards its mouth (“outer cell partitions”), and iii) partitions that close the nest at the entrance (“nest plug”). The inner cell partitions, which ranged in length from 4–12 mm, were invariably three-layered (n = 24; Figs 13–16), consisting of a central layer of densely packed small bark particles (2–9 mm long) sandwiched between two layers of leaf pulp (marginally 1–2 mm thick). As revealed by two thresholds of leaf pulp at the entrance of two still empty brood cells (Fig. 12), the females mark the position of the later cell partition before they begin to provision a brood cell; these two thresholds were ring-shaped, which seems to be typical also for other Osmia species of the subgenus Melanosmia, such as O. parietina (A. Müller, unpublished data). The construction of the outer cell partitions, which ranged in length from 2–10 mm, was slightly more variable than that of the inner cell partitions: most of them were three-layered as the inner cell partitions and had similar dimensions (n = 9), whereas some partitions at the mouth of a finger were either five-layered with three layers of leaf pulp (marginally 1–2 mm thick) enclosing two layers of small bark particles (1–2 mm long; n = 3) or consisted of a single leaf pulp layer only (marginally 2 mm thick; n = 3). The construction of the nest plugs, which were usually flush with the nest opening, considerably varied between the different nests (Figs 10, 11): the plugs ranged from 3–11 mm in length and consisted either of a single layer of leaf pulp (marginally 3–5 mm thick; n = 3), of two layers of leaf pulp (marginally 1.5–3.75 mm thick) enclosing a 1 mm long layer of small bark particles (n = 1), of three layers of leaf pulp (marginally 2–3 mm thick) enclosing two slightly less than 1 mm long layers of bark particles (n = 1), or of five layers of leaf pulp (marginally 1.5–2 mm thick) enclosing four 1–3 mm long layers of bark particles (n = 1). The length of the vestibule, i.e. the distance between the interior side of the nest plug and the outermost layer of the outer cell partition, ranged from 0.5–2.8 cm.

DNA metabarcoding revealed that the plugs of nests 14–19 were constructed from masticated leaves of Potentilla spec. (Rosaceae) and Helianthemum nummularium (Cistaceae) (Tab. 2). In addition, a low proportion of reads corresponded to Lotus corniculatus (Fabaceae). Although the percentage of sequence reads obtained by next-generation sequencing is only a rough indication for the relative amount of a plant taxon in the extracted DNA, this low proportion might be due to a contamination e.g. with pollen of Lotus, which is the most important pollen host of O. nigriventris in the Alps (see below).

Female mandibular strength

The assessment of the mandibular strength revealed a low intraspecific variability indicating that the shape and the force of the female mandibles are species-specific traits (Suppl. material 2). Among the nine O. (Melanosmia) species examined, O. nigriventris and O. pilicornis, which both excavate their nests in wooden substrate, possess by far the strongest mandibles (Fig. 25, Suppl. material 2). In contrast, the mandibles of those species, which either nest in preexisting cavities (O. inermis, O. parietina, O. uncinata) or in loose soil and litter (O. alticola, O. maritima, O. xanthomelana), are distinctly weaker. Compared with these species, the mandibles of O. nigriventris are relatively shorter, have less diverging margins and are apically only slightly curved inwards resulting in a compact and chisel-like shape that appears to be well suited to tunnel out burrows in bark or dead wood (Figs 28, 29).

Figure 25. 

Mandibular strength of nine European Osmia species of the subgenus Melanosmia. For each species, the force of the right mandible of three females was averaged except for O. alticola, O. maritima and O. steinmanni, for which only one or two specimens were available. Orange = nesting in self-excavated burrows in wooden substrate, green = nesting in preexisting cavities, blue = nesting in loose soil or litter, grey = nesting unknown (after Müller 2019 and references therein).

Brood parasites

The obtained sequences of the barcoding gene Cytochrome Oxidase I were all clean and readable. Comparison with BOLD databases indicate that 13 out of the 17 overwintering prepupae belong to O. nigriventris, whereas two prepupae each from the nests 15 and 19 were brood parasites, i.e. three specimens of the bombyliid fly Anthrax anthrax (Schrank) and one specimen of the sapygid wasp Sapyga similis (Fabricius). From the latter species, a dead imago was also found in a brood cell of nest 1. The percentage of match between the barcoding sequences of the 17 prepupae and the BOLD reference sequences exceeded 99% in all cases.

Osmia nigriventris collected the pollen almost exclusively from flowers of Fabaceae, Ericaceae and Cistaceae (Tab. 3, Figs 26, 30). Pollen of these three plant families constituted 96.6% of the total pollen grain volume. Among the Fabaceae, Lotus was by far the most important pollen host. Its pollen was represented by 55.8% of the total pollen grain volume and was recorded in 42 out of 50 pollen loads. Among the Ericaceae, both Rhododendron and Vaccinium were exploited. Their pollen contributed 18.1% and 8.5%, respectively, to the total pollen grain volume and was found in 10 and 7 loads, respectively, rendering these two genera the second and third most important host taxon of O. nigriventris. Helianthemum (Cistaceae) was the fourth most important host taxon. Its pollen constituted 5.6% of the total pollen grain volume and was recorded in 5 loads.

Figures 26–29. 

26, 27) Females of Osmia nigriventris collecting pollen on Rhododendron ferrugineum (left, photo H. Wiesbauer) and sucking nectar on Rubus (right, photo S. Kühne). 28) Female mandible of Osmia nigriventris. 29) Female mandible of Osmia inermis.

Figure 30. 

Pollen host spectrum of Osmia nigriventris based on the microscopical analysis of 50 scopal loads from 41 different localities in the Swiss and French Alps.

The larval provisions of four brood cells originating from three nests contained only pollen of Fabaceae and Ericaceae (Tab. 4). One provision exclusively consisted of Fabaceae pollen, whereas the other three provisions were mixtures of the pollen of both families. Lotus was the most important pollen host in each of the four provisions.

In Central Europe, the imaginal period of O. nigriventris extends from May to August (Fig. 31). At the lowest extra-alpine occurrences at elevations below 900 m a.s.l., the flight activity might start as early as the beginning of May and does not extend beyond the first decade of June, whereas near the timberline in the subalpine zone of the Alps the imagines do not emerge before the end of May and fly till the end of August.

Figure 31. 

Phenology of Osmia nigriventris in Central Europe. For each pentade, the number of female and male records per elevation is given. For a given locality and date, only one record per sex was considered.

X-raying of the nests 14–19 performed in November 2018 revealed that 13 out of the 19 brood cells contained prepupae of O. nigriventris spun in a cocoon (Figs 16–20), suggesting that O. nigriventris passes at least its first winter in the prepupal stage (see Discussion). In two cells, the eggs or the freshly eclosed larvae had died leaving behind the entire larval provisions (Fig. 15), and the remaining four cells contained prepupae of Anthrax anthrax and Sapyga similis (see above).

In one brood cell each of three abandoned nests, the adult bee had left its cocoon in spite of the fact that the cell partitions were still intact, suggesting that the imagines occasionally leave their natal nest through a new burrow gnawed across the bark rather than through the nest entrance.

O. nigriventris has a vast Holarctic distribution (Figs 32–34, Suppl. material 3). In the Nearctic, O. nigriventris occurs in the western and northern United States (Colorado, Wyoming, Idaho, Montana, Oregon, Washington, Alaska, Minnesota, Michigan) and Canada (Alberta, British Columbia, Yukon, Northwest Territories, Manitoba, Ontario, Quebec, Newfoundland and Labrador) within a belt that extends from 40° to 69° northern latitude (see also Rightmyer et al. 2010 and Ascher and Pickering 2019). In the Palaearctic, its range is split into three main parts: i) Alps (France, Switzerland, Italy, Germany, Austria) from 1200 m a.s.l. up to the timberline at 2300 m a.s.l., ii) Caucasus and iii) the boreal zone from Scandinavia (Norway, Sweden, Finland), the Baltic (Lithuania, Latvia, Estonia) and European Russia eastwards over Siberia, northwestern China and Mongolia to northeasternmost China and the Russian Far East between 41° and 70° northern latitude. Widely scattered outposts of its distribution exist in the lowlands of northern Germany and Poland as well as in some low mountain ranges in Germany, the Czech Republic and Slovakia, where the species is regarded as a glacial relict (Figs 32, 34). Among the 53 records of O. nigriventris north of the Alpine arc, which range in altitude from 55 m to 730 m a.s.l., 43 (81.1%) date from before 1995, suggesting that numerous former Central European occurrences outside the Alps no longer exist.

Figures 32–34. 

Distribution of Osmia nigriventris in 32) the Palaearctic, 33) the Nearctic and 34) Central Europe. For Central Europe, records before 2000 are given in black, those from 2000 to the present in yellow. See Suppl. material 2 for a complete list of all distributional data. Made with Natural Earth (www.naturalearthdata.com).

At three different localities in the Grisons and the Valais, numerous nests of Osmia nigriventris were discovered, which were all constructed in thick bark pieces of Larix decidua lying on sun-exposed ground of open subalpine forests. Nests of O. nigriventris were also detected by Frey-Gessner (1881) and F. Amiet (personal communication) in pieces of Pinus sylvestris bark at the Alp Ponchet above Chandolin/Valais and near Samedan/Grisons, respectively, and by Stöckhert (1933) in the thick bark of a Pinus stump in Franken (Germany). Furthermore, a female of O. nigriventris was observed to fly off from its putative nest in a bark piece lying at the base of a Pinus sylvestris stump in Brandenburg/Germany (Saure 2017, personal communication). Lastly, L. Gyllenhall, a Swedish entomologist living from 1752 to 1840, found O. nigriventris nesting in the bark of a pine in Vastergötland (Sweden), which later prompted Gerstaecker (1869) to give this species the name O. corticalis (cortex = bark), now regarded as a junior synonym of O. nigriventris (Nilsson, 2009). These records of bark nests at five different localities in the Swiss Alps and at one locality each in central Germany, northern Germany and southern Sweden indicate that the construction of nests in the bark of thick-barked conifers is a typical trait of O. nigriventris. As numerous conifers including Larix and Pinus seal injuries of the bark with resin (Krokene 2015 and references therein), O. nigriventris is expected to nest only in bark which adheres to dead stumps or is detached from the stems and lies on the ground. However, O. nigriventris is not strictly specialized to bark as nesting substrate, as is revealed by its occurrence at localities, where no thick-barked conifers occur, such as the three windfalls investigated in the present study. Here, the species is supposed to excavate its nests in dead wood of Picea abies stumps. In fact, Giraud (1861) observed several females of O. nigriventris nesting in self-excavated burrows in a thick board of Abies alba, which formed part of a pasture fence. This shows that while O. nigriventris might have a pronounced preference for nesting in bark, it is capable of constructing its nests also in dead wood.

Among the osmiine bees, bark appears to be only exceptionally used as nesting substrate. Apart from O. nigriventris, reports for nesting in bark exist only for the Palaearctic species O. uncinata Gerstaecker and the three Nearctic species O. atriventris Cresson, O. densa Cresson and O. cahuilla Cooper, all belonging to the subgenus Melanosmia. The first three species differ from O. nigriventris in that they colonized already existing beetle burrows in bark and also use preexisting linear cavities in other substrates, such as dead wood or stems (Graenicher 1906, Stöckhert 1933, Linsley and MacSwain 1951, Cane et al. 2007, Else and Edwards 2018). This suggests that the selection of bark for nesting was rather accidental in these three species. In O. cahuilla, 21 individuals emerged from a single nest within the bark of a pine stump (Cooper 1993). As O. cahuilla is hypothesized to be related to the Palaearctic O. pilicornis (Cooper 1993), which nests in self-excavated burrows in dead wood (Prosi et al. 2016), the bark nest might possibly have been gnawed out by the nesting female as in O. nigriventris.

The inner cell partitions, which consist of an interlayer of bark particles sandwiched between two thin layers of leaf pulp, are diagnostic for the nests of O. nigriventris. Doubtlessly, the interlayer is constructed by amassing bark particles that accumulated within the burrow during the process of nest excavation. Among the osmiine bees, which usually separate linearly arranged brood cells by one-layered walls, such three-layered cell partitions are otherwise known only from the Palaearctic Hoplitis tuberculata Nylander and its North American relatives (Müller 2015). In these Hoplitis species, the interlayer consists of pebbles, wood chips, earth crumbs, fragments of conifer needles or particles of pith. These strong cell partitions are hypothesized to impede mobile larvae of predators, which already infested a brood cell before the nest was closed, from invading adjacent cells, such as larvae of Trichodes beetles (Cleridae). The same function might also apply to the massive cell partitions of O. nigriventris.

The mandibles of O. nigriventris and O. pilicornis turned out to be the strongest among the European O. (Melanosmia) species. This finding corresponds well to the habit of both species to tunnel out their nests in hard wooden substrate and supports the view that both species are specialized excavators and do not colonize preexisting cavities for nesting, such as insect borings in dead wood. Such a specialization also explains why the repeated attempts to establish these two species in trap nests composed of drilled borings in dead wood all have failed (A. Müller and R. Prosi, unpublished data).

Anthrax anthrax (Bombyliidae) and Sapyga similis (Sapygidae) were recorded as brood parasites of O. nigriventris in the present study. Most species of Anthrax are ectoparasitoids of above-ground nesting aculeate Hymenoptera attacking the full-grown larvae of their hosts (Yeates 1997). A. anthrax was found to parasitize the crabronid wasp Pemphredon lugens Dahlbom, the megachilid bees Megachile nigriventris Schenck, Hoplitis adunca (Panzer), Osmia bicornis (Linnaeus) and O. cornuta (Latreille) as well as the apid bee Anthophora retusa (Linnaeus), revealing a wide host range (Brechtel 1986, Litt 1996, Krunić et al. 2005, Peeters et al. 2012, Westrich 2018). In contrast to A. anthrax, S. similis was already known to be an antagonist of O. nigriventris (Stöckhert 1933, Amiet 2008). It develops in the nests of other O. (Melanosmia) species, such as O. inermis (Zetterstedt), O. parietina Curtis, O. uncinata and probably also O. maritima Friese and O. xanthomelana (Kirby) (Stöckhert 1933, Zanden 1982, Westrich 2002, 2018), suggesting a preference for Osmia species of the subgenus Melanosmia as hosts. However, S. similis was also reared from nests of Chelostoma grande (Nylander) (Westrich 2002) and might thus be expected to parasitize further osmiine bee species occurring in the Alps and the Jura Mountains, where S. similis has its main distribution in Switzerland (Amiet 2008). Chrysura hirsuta (Gerstaecker) (Chrysididae) is most probably a further brood parasite of O. nigriventris (Berland and Bernard 1938, Trautmann 1927, Stöckhert 1933, Paukkunen et al. 2014), although no proof for its development within the nests of O. nigriventris exists so far. As in S. similis, Osmia species of the subgenus Melanosmia appear to be the main hosts of C. hirsuta (Prosi et al. 2016, Westrich 2018).

Microscopic analysis of both female pollen loads and larval provisions revealed that Osmia nigriventris is mesolectic, restricting pollen harvesting almost exclusively to flowers of Fabaceae, Ericaceae and to a lesser extent Helianthemum (Cistaceae). Known pollen host species are Lotus corniculatus L. and Hippocrepis comosa L. among the Fabaceae, Rhododendron ferrugineum L., R. hirsutum L., Vaccinium myrtillus L. and V. vitis-idaea L. among the Ericaceae and Helianthemum nummularium (L.) Mill. among the Cistaceae (Frey-Gessner 1899–1907, Stöckhert 1933, Westrich 2018, Dylewska 1993, this study). In contrast to the literature, no preference for Vaccinium over Fabaceae was evident in the present study as assumed by Stöckhert (1933) and Westrich (2018), and pollen of Potentilla (Rosaceae), which is listed as an additional pollen source by Westrich (2018), was never recorded in any pollen load.

The pollen host spectrum of O. nigriventris is similar to that of other predominantly alpine Osmia (Melanosmia) species, for which pollen of Fabaceae also plays a crucial role for larval development (Müller 2018). Compared with these O. (Melanosmia) species, which comprise narrow Fabaceae specialists collecting pollen only on two closely related genera as well as generalists harvesting pollen on up to eleven different plant families, O. nigriventris occupies an intermediate position with respect to its degree of host plant specialization. The combined exploitation of Fabaceae and Ericaceae found to be typical for O. nigriventris in the Alps has also been observed in other Palaearctic O. (Melanosmia) species inhabiting Ericaceae-rich alpine and boreal habitats, such as O. disjuncta Tkalců, O. inermis (Zetterstedt) and O. steinmanni Müller (Hicks 2009, Müller 2018, 2019) and might possibly also apply to O. laticeps Thomson currently considered to be oligolectic on Vaccinium (Nilsson 2009).

Similar to the other European Osmia species of the subgenus Melanosmia, which are early flying bees active in spring and early summer (Westrich 2018, SwissBeeTeam 2019), O. nigriventris already emerges in phenological spring, i.e. between the beginning of May at low altitudes and the first decade of June at high altitudes. As the nests of O. nigriventris from Blatten/Valais (1880 m a.s.l.) contained prepupae upon examination in winter 2018, it seems improbable that the time span between the end of winter and the onset of the early flight period is sufficiently long to allow for the entire metamorphosis to the imaginal stage. We therefore hypothesize that O. nigriventris needs two years for its development in the subalpine zone of the Alps and that it overwinters here as prepupa in the first and as imago in the second winter. This hypothesis is supported by observations made on other O. (Melanosmia) species: O. inermis needs at least two years for its development on the German Swabian Alb and in Scotland, where the first winter is passed as prepupa and the second as diapausing adult (Else and Edwards 1996; Westrich 2002), and O. parietina was found to emerge not until two years after egg deposition in the Valais at 1430–1800 m a.s.l. (Westrich 2002).

O. nigriventris belongs to the few bee species that have an autochthonous Holarctic distribution (Ebmer 2011). Although the species is not threatened at the European scale due to its wide distribution and large population size in Scandinavia (Nieto et al. 2014), it is scarce and locally distributed throughout the Alps and appears to have disappeared from many of its former Central European extra-alpine glacial refugia at low altitudes. Though this decline at low altitudes might have been caused by habitat deterioriation, it might also be a first indication that climate warming is increasingly negatively affecting population survival of this cold-adapted species, which raises concern about the species’ future in the upper montane and lower subalpine zone of the Alps.