A new osmiine bee with a spectacular geographic disjunction: Hoplitis ( Hoplitis ) onosmaevae sp. nov. (Hymenoptera, Anthophila, Megachilidae)

A new osmiine bee species, Hoplitis ( Hoplitis ) onosmaevae sp. nov. (Megachilidae), is described. So far, this species is exclusively known from the Mercantour National Park in the southwestern French Alps and from mountainous ranges in Turkey and northern Iraq, two areas separated by at least 2000 km. Phylogenetic analyses based on mitochondrial and nuclear genes revealed that H. on-osmaevae is closely related to H. adunca (Panzer, 1798), H. benoisti (Alfken, 1935) and H. manicata (Morice, 1901). Hoplitis onos-maevae is presumably narrowly oligolectic and harvests pollen only on flowers of Onosma L. (Boraginaceae). It has a particularly long proboscis, which is probably an adaptation to collect nectar from the long-tubed flowers of this plant genus. The females collect pollen by buzzing the Onosma flowers, a rare behavior in megachilid bees. The species nests in insect burrows in dead wood, similar to H. adunca and H. manicata but unlike other closely related representatives of the subgenus Hoplitis , suggesting a single origin of nesting in dead

Species of the subgenus Hoplitis are mostly oligolectic or mesolectic and collect the pollen either only on Boraginaceae or Fabaceae or on both of them.The pattern of frequent transitions between the exploitation of these two plant families among related species and the frequent exploitation of both families by the same species has been referred to as the "Boraginaceae-Fabaceae paradox" (Sedivy et al. 2013a).Within the Boraginaceae, the genus Echium L. is exploited by many species, but some species also collect the pollen from other genera, such as Onosma L., Lithodora Grisebach or Heliotropium L. Numerous species have particular morphological adaptations for pollen collection, such as hooked bristles on the proboscis or on the fore legs (Müller 2012;Sedivy et al. 2013a).Regarding nesting biology, different behaviours are found within this subgenus.Probably all species of the annulata and monstrabilis groups nest in the ground in self-excavated burrows (Rozen et al. 2009;Sedivy et al. 2013b;Le Goff 2017;Fateryga et al. 2023;Müller 2023b).Most species of the adunca group build their nests exposed in shallow depressions of rocks and stones or hidden within rock cavities and use sand as nesting material, often combined with small pebbles and hardened with salivary secretions, e.g.Hoplitis anthocopoides (Schenck, 1853) or H. loti (Morawitz, 1867) (Sedivy et al. 2013b).Two species of the adunca group nest in insect borings in dead wood or in hollow stems, in addition to other preexisting cavities, and use sand to separate their brood cells: H. adunca and H. manicata (Morice, 1901) (Müller 2023b;Sedivy et al. 2013b).
We describe here a new Hoplitis species of the adunca group.This new species was detected during faunistic inventories of the Mercantour National Park in the Alps in southern France.Upon morphological comparison, the French specimens were found to exactly correspond to specimens of an undescribed species from southern Turkey.In the present publication, this new species is morphologically diagnosed, its phylogenetic position within the subgenus Hoplitis is explored using genetic analyses, and its nesting biology and floral association are described based on field observations in southern France and microscopical analysis of pollen contained in the female scopae.

Material studied
The following material was examined for this study: 1. males and females of the undescribed species from Mercantour National Park, France, from southern and eastern Turkey and northern Iraq; 2. males and females of Hoplitis holmboei (Mavromoustakis, 1948), H. homalocera Zanden, 1991, and H. linguaria (Morawitz, 1875), which are Boraginaceae specialists and also possess a very long proboscis (Sedivy et al. 2013a), that served for comparison.As no specimens of a fourth Hoplitis species with an extraordinarily long proboscis, H. semilinguaria Tkalců, 1992 (known only in the female sex), could be located neither in NMPC nor in OLML (Tkalců collection), where the types should be deposited according to the original description, the comparison was performed based on the original description.

Terminology
Morphological and anatomical terminology is based on Michener (2007).

Equipment
Morphological examination of specimens was done with a Perfex Sc 6.38 binocular (7× to 45× magnification).Measurements were realized with a micrometric eyepiece.All pictures of collected and prepared specimens were taken with a Keyence VHX 1000 digital microscope.A reflex body Nikon D7200 combined with a macro lens Nikkor 60 mm were used for field photographs.

Geographical informations
French collecting sites were georeferenced using a field GPS.Turkish and Iraqi specimens were collected by different entomologists between 1968 and 2022 (see list of paratypes below and Suppl.material 1 for details) and the label information regarding the collecting sites is heterogenous.In case the coordinates were not indicated on the label, they were extrapolated from the locality data.All coordinates are given in WGS 84.The distribution map was generated using QGIS Desktop 3.28.3 and Au-toNavi Satellite layer.

Microscopical pollen analysis
Pollen was removed from the metasomal scopa of five females from France (one specimen) and three localities in Turkey (four specimens) as well as from two brood cells of a nest discovered in France, embedded in glycerol gelatine on a slide and compared with reference slides containing pollen of different Boraginaceae species and genera under a microscope at 400× magnification.

Genetic analyses
For two individuals of the new species collected in Saint-Dalmas-le-Selvage, France, in 2020 (see list of paratypes below), we sequenced four gene fragments included in the phylogenetic study of Sedivy et al. (2013c): the mitochondrial gene cytochrome oxidase I (COI) and three nuclear genes conserved ATPase domain (CAD), elongation factor 1-alpha (EF) and long-wavelength rhodopsin (Opsin), using standard DNA sequencing protocols and primers developed for bees or for Megachilidae (Praz et al. 2008;Litman et al. 2011;Sedivy et al. 2013c).Genetic distances were computed using the Kimura 2-parameter (K2P) distance model in a test version of Paup 4.0 (Swofford 2002) kindly provided by D. Swofford.The new sequences have been deposited on Genbank (accession numbers PP389027-PP389032) and BOLD (accession numbers HYMAA896-24 and HYMAA897-24).Single gene analyses were performed using maximum Likelihood in RAxML 8.2 (Stamatakis 2014), using a single partition and a GTR + G model; the four genes were then concatenated and analysed using RAxML.We implemented two distinct partitioning schemes: first, with four partitions, one per gene; and second, with seven partitions, three for the three nucleotide positions of COI, three for the three nucleotide positions of the nuclear genes, and one partition for the introns; in both cases, a GTR + G model was fitted to each partition.

Results
Field    2350 m; 25.5.1989;Klaus Warncke leg.; ETHZ.See Suppl.material 1 for a complete list of records as well as Fig. 2 for a distribution map.
Diagnosis.In most species of Hoplitis (Hoplitis), the length of the proboscis is at most one-third as long as the body.Only H. linguaria, H. holmboei, H. homalocera and H. semilinguaria have a longer proboscis, which reaches about half of the body length.Hoplitis onosmaevae possesses an even longer proboscis, which is approximately as long as the body (Fig. 3).In both sexes of H. holmboei and H. homalocera, the vertex is comparatively short (ocelloccipital distance less than two ocellar diameters) and, when seen in front view, not elevated behind ocelli, but regularly rounded across its width; in H. onosmaevae the vertex is longer (ocelloccipital distance about three ocellar diameters) and is strongly elevated behind ocelli (Figs 3,4).Hoplitis onosmaevae differs from H. linguaria in both sexes by the color of the tegulae, which are reddish-orange in H. linguaria and brown (although often reddish brown along the external margin) in H. onosmaevae.In the female, H. onosmaevae can be distinguished from H. linguaria by the lateral margins of S6, which have a thickened rim, and by the apical margin of S6, which ends medially in a sharp spine as in H. adunca (Fig. 7); in H. linguaria, S6 lacks thickened rims or a medioapical spine.Moreover, the punctation of the supraclypeal area in H. onosmaevae is comparatively fine and dense with only small interspaces especially in the middle, whereas it is sparse in H. linguaria in the middle.In the male, H. onosmaevae can be distinguished from H. linguaria by the shape of the membranous appendage at the apical margin of S6 and the form of the gonostylus.In H. onosmaevae, the appendage of S6 is longitudinally raised medially, wider at the base than at the apex, tapers into a narrowed tip and is covered with short light orange hairs in the middle (Figs 12,14).The gonostylus is threadlike and slightly clubbed at the apex.In H. linguaria, the membraneous appendage of S6 is flat, short and distinctly bilobed and the gonostylus is much thicker and narrowed at the apex.Compared to H. semilinguaria, which is known only in the female, the inner margins of the compound eyes are diverging towards the clypeus in H. onosmaevae, whereas they are parallel in H. semilinguaria.In addition, S6 of H. linguaria lacks thickened marginal rims as is probably also the case in H. semilinguaria.Tkalců (1992) compared these two species and did not provide any information on the structure of S6.
Hoplitis onosmaevae can be separated from the common species H. adunca, which is similar in size and also has thickened marginal rims and a medioapical spine on female S6, by the yellowish-brown hind tibial spurs (Fig. 9) (tibial spur black in H. adunca), in addition to the longer proboscis.
Integument color.Cuticula generally black, except where indicated hereafter.Proboscis brown, glossa or-  ange.Cuticula reddish-brown laterally along apical margin of clypeus and more or less along margin of sternites and tergites, on internal surface of femora, apical part of leg segments, especially last tarsi and claws.All tibial spurs yellowish-brown to orange.Tegulae dark brown, external margin often reddish brown.Wing venation dark brown.Eyes grey-brown in the field.Vestiture color.Generally white, including scopa, except where in-dicated hereafter.Hairs slightly darker, greyish brown, on scutum.Some hairs along apical margin of clypeus and on tergal discs yellowish white.Hairs on internal surface of tibiae and tarsi, on basal part of femora and apical half of trochanters yellowish-orange.
Head.Vertex strongly elevated and tilted forward when seen in front or 3/4 view.Ocelloccipital distance approximately equal to three ocellar diameters (Fig. 4).Inner margin of compound eyes slightly diverging below (ratio between upper and lower interocular distance approximately 0,92).Clypeus strongly convex, protruding, apical margin with approximately nine teeth, lateral teeth triangular, median teeth nearly cylindrical (Figs 4,5).Mandibles tridentate, apical tooth elongate, acute (Fig. 5).Proboscis equal to body length when fully extended, galea half as long as body, with short bristles basally.Labrum longer that wide.Head overall shiny, shagreened only on parts of clypeus (see below), densely punctate; interspaces absent or narrow, except on vertex and genae where interspaces can be as wide as one puncture diameter, on frons laterally and close to ocellar area where the interspaces can reach two puncture diameters.Frons along frontal line and supraclypeal area more densely and finely punctate (supraclypeal area sometimes comprises some narrow interspaces centrally).Clypeus more strongly punctate than elsewhere on head, interspaces up to 1 or 2 puncture diameters, punctation becoming denser and finer apically; clypeus in basal half with median impunctate longitudinal line (Fig. 5), line sometimes reaching apical margin.Labrum unpunctured and shiny in basal third, mat and shagreened apically, here more or less punctate.Proboscis surface unpunctured, smooth and shiny on the front, mat and shagreened laterally and dorsally.Antennae.FL2 slightly longer than FL3 and FL4 together; FL (5-6-)7-10 brown-orange below (Fig. 5).
Mesosoma.Pronotum weakly shagreened with shallow punctation, but shiny.Mesepisternum (except ventral part), scutum and scutellum shiny, densely and relatively strongly punctate, interspaces well visible, up to 2 to 3 punctures diameters on central part of scutum.Mesepisternal concavity shiny with sparse punctation.Axillae shagreened, densely and finely punctate.Metanotum shiny with distinct punctation.Metepisternum weakly shagreened but shiny.Propodeal triangle nearly entirely shagreened, shiny in its lower part; propodeal posteriorly nearly impunctate around propodeal pit, more densely punctate laterally; sides of propodeum densely and finely punctate (Fig. 8).Mesepisternum ventrally impressed along longitudinal axis, this concavity open posteriorly between mid-legs insertions, here shiny and sparsely punctate.Mesosoma entirely hairy except on propodeal triangle and propodeal pit (Fig. 8).Hairs on scutellum, lateral part of mesepisternum and on propodeum as long as on vertex, shorter on scutum and even more so on ventral parts of mesepisternum.Legs.External extensions on apex of first and mid tibiae relatively thin, elongated and curved.Spur of front tibiae modified, internal margin lamellar, external margin thick and curved, apex of spur pointed, internally with a minute fringe of hairs.Front metatarsi basally with an internal notch.Inner spurs of hind tibiae straight and regularly tapering, only slightly curved apically (Fig. 9).
Male (habitus: Figs 16,17).Body length 10-12 mm.Integument color.As in ♀ except when mentioned below.Eyes grey-green in the field.Vestiture color.Predominantly brown-orange in fresh specimens, fading to yellowish to greyish white.
Head.Vertex as in ♀.Inner orbital edges slightly diverging below (less so than in ♀).Clypeus protruding, apical margin denticulate, but less regularly and strongly than in ♀; teeth partly hidden by dense apical fringe of hairs.Mandibles bidentate, upper tooth short, apical tooth sharp.Proboscis, galea and labrum proportions as in ♀.Vestiture very dense on frons, paraocular and supraclypeal areas and clypeus.Punctation overall more homogeneous than in ♀, finer and denser on average; clypeus entirely punctate except a narrow impunctate area along lateral margins, punctation fine and dense, difficult to see due to dense vestiture.Labrum medially shiny, unpunctured or with sparse punctation, shagreened and more densely punctate laterally.Antennae.Scape densely punctate and hairy, 3 times as long as wide at apex; width at apex twice basal width.FL2 triangular, at most 1,5 as long as wide at apex, as long as FL3 and FL4 together.FL3 and FL4 twice as wide as long.FL8-10 nearly square, FL11 approximately 1,5 times as long as wide.Flagella flattened dorso-ventrally; only internal surface of FL2 (distinctly) and FL3 (slightly) convex.FL5-or FL6-11 orange ventrally (Figs 10,11).
Mesosoma and legs.As in ♀ except where indicated hereafter.Punctation denser and finer on scutum and lateral parts of mesepisternum, and denser on area around propodeal pit.
Metasoma.Tergal punctation generally as in ♀ but punctures less impressed and area with fine punctation on tergal margins wider.T7 finely punctate and shagreened basally, smooth and shiny medially, punctation becoming rugose and sparse apically.T2-3 depressed basally.T6 with a strong tooth laterally, apical margin irregular, slightly sinuate medially.In dorsal view, lateral margin of T7 slightly concave medially, apical margin rounded (Fig. 13).Metasomal vestiture as in ♀ except where indicated hereafter.Lateral fasciae of T1 less pronounced and more elongated, T2-5 with shorter hairs, T6 with continuous fasciae on apical margin, fasciae narrower in the middle but not interrupted, T7 with long hairs laterally but fewer hairs medially.S1 thicker than S2-4, with lamellar margin.S2-5 with a transverse premarginal gibbosity (inconspicuous on S5), S5 with longitudinal central groove.Margins of S1-4 slightly sinuate, S5 straight medially, rounded laterally.S6 basally with short and wide translucent lamella, median extension trapezoidal, with sinuate margins (Figs 12, 14; see also Diagnosis).S1 with strong and dense punctation on thickened part.S2-4 with dense punctation, shagreened and with fine punctation basally and premarginally, transverse gibbosities smooth and shiny, with sparse punctation medially.Median extension of S6 mostly shagreened (Fig. 14).Apical margin of S1-3 with sparse fringe of relatively long hairs, apical fringe shorter on S4, almost absent on S5.S6 baso-laterally with dense and long fringe of hairs, hairs emerging behind translucent lamella; surface of median extension of S6 with short yellowish-orange hairs.Gonostyli thread-like, slightly clubbed apically.(Antalya and Mersin province), from the western Pontic Mountains in northwestern Turkey (Bolu Province) and from the Tinée Valley in the French southern Alps (Provence-Alpes-Côte-d'Azur region) (Fig. 2).In France, the species appears to be extremely rare and localized.So far it has only been found at two sites separated by less than 5 km; at each site, only few individuals were observed (the estimated number of individuals was 3 and 10-20, respectively).The search for the species was unsuccessful in several other localities in the French southern Alps with of Onosma tricerosperma subsp.fastigiata.These localities were situated 10 km to 18 km to the east, the north and the southwest of the two known sites of H. onosmaevae.Localities surveyed were the Bachelard valley above Fours-Saint-Laurent (Alpes-de-Haute-Provence, 44.32°N,6.69°E) between 1750 m and 1900 m on 11.7.2019 and 27.6.2020,the Ubaye upper-valley, around la Barge and Maljasset (Alpes-de-Hautes-Provence, Saint-Paul-sur-Ubaye, 44.59°N, 6.83°E) between 1700 m and 2150 m on 11.7.2019 and 25.6.2020, the Ubayette valley, around Larche (Alpes-de-Haute-Provence, Larche, 44.45°N, 6.45°E) between 1650 m and 2000 m on 13.7.2019,above Saint-Ours (Alpes-de-Haute-Provence, Val d' Oronaye,44.48°N,6.81°E) between 1800 m and 1950 m on 26.6.2020, and the Barlatte upper-valley (Alpes-Maritimes, Châteauneuf-d'Entraunes, 44.18°N, 6.83°E) between 1700 m and 1850 m (MNP core area) on 25.6.2022.However, some of these unsuccessful visits were not performed under ideal conditions (e.g.end of blooming time or low abundance of blooming host plants), so that the occurrence of H. onosmaevae at all these sites cannot be excluded with certainty.
Field observations.Field observations were conducted at the "Vallon du torrent de Jalorgues" (Figs 15, 22) on 23.6.2020,when Hoplitis onosmaevae was found for the first time, and on 28.6.2020.The first visit took place in good weather conditions and lasted from 10:00 to 15:30.Several females and males were seen, most observations and pictures were realized on that day and a nesting site was found.During the second visit, fewer individuals were observed, possibly due to the less favorable weather conditions and we focused on a closed nest found five days before, around which a single female was seen.
Behaviour at Onosma stands.Several females were observed collecting pollen and nectar on flowers of Onosma tricerosperma subsp.fastigiata (Figs 18,20,21).After landing on the corolla, they entered the flower headfirst, clung to the style and then vibrated the flower by buzzing to extract the pollen.This behavior was observed several times.Each visited flower was sonicated usually twice during a few seconds.The females then collected nectar on the same flower and repeated the same sequence on the next flower.The bees landed frequently on the ground or on a stone between two flower visits to concentrate the nectar by widely spreading the mandibles and the labrum and slightly unfolding the proboscis (Fig. 19).Males were observed patrolling Onosma patches in their search for females, resting frequently nearby on the ground or on a stone (Figs 16,17).They sometimes interrupted their patrolling flights to ingest nectar from Onosma flowers.
Pollen hosts.The pollen contained in the female scopae and the brood cells was morphologically identical to reference pollen samples of Onosma; while the French samples certainly belong to Onosma, the unambiguous identification of Onosma pollen was not possible for the Turkish samples, as several closely related Boraginaceae genera with similar pollen morphology occur in Turkey.
Nesting biology.A nesting site was found on 23.6.2020 in the "Vallon du torrent de Jalorgues" by observing the flight direction of a female leaving the main patch of host plants.The nesting site was situated at a distance of approximately 35-40 m from the Onosma patch, where three dead trunks of larch (Larix decidua Miller, 1768) were present, one lying and two still standing (Fig. 22).Two nests in preexisting insect burrows were discovered, one in the lying trunk and the other in a standing trunk.One nest was left untouched, while the second was partially opened five days later.This nest consisted of an unknown number of brood cells, of which the two outermost were excavated.These cells were in line and parallel to the external surface of the tree trunk.The following nest architecture was observed (Fig. 25): 1. a nest plug composed of small pebbles glued together with fine sand, which was probably mixed with secretions (Fig. 23); 2. an empty chamber; 3. a partition constructed with sand, but without pebbles; 4. the outermost cell containing a solid provision mass (egg or larva not visible); 5. a partition built with sand; 6. the penultimate cell containing a solid provision mass (egg or larva not visible); 7. another partition built with sand.

Biogeography
In the past twelve years, numerous new bee species have been discovered in mainland France.These new discoveries mainly concern species known from adjacent or other European countries (Vereecken and Barbier 2009;Aubert et al. 2010;Dufrêne et al. 2014;Aubert 2016;Genoud and Dufrêne 2016;Gabiot and Dufrêne 2018;Le Divelec 2020;Le Divelec and Aubert 2020).Alternatively, some of the newly discovered taxa are cryptic species that were previously confused with closely related taxa (Ghisbain et al. 2018;Praz et al. 2019Praz et al. , 2022;;Kasparek 2020;Litman et al. 2021).The discovery of overlooked, newly immigrated or cryptic species may continue or even accelerate in the future as interest in bees increases and identifications and species delimitation are more and more complemented by DNA analyses.Much rarer, however, is the discovery of morphologically well characterized bee species that are new to science, such as H. onosmaevae.
This discovery is particularly remarkable as this species shows a highly disjunct distribution, occurring in southern France and at least 2000 km away in Turkey and Iraq.The morphology of both females and males of the European and the Asian populations is completely identical including the shape of the male antenna and the form of the membraneous appendage of male sternum 6, which are highly diagnostic characters in the subgenus Hoplitis.
Thus, these disjunct populations are treated here as conspecific despite the lack of genetic data for the eastern populations.The bumblebee Bombus brodmannicus Vogt, 1909 shows an even more disjunct distribution occurring in the southern Alps the eastern Pontic Mountains, the Armenian highlands and the Caucasus (Rasmont and Iserbyt 2014).Another putative Onosma specialist was discovered in France just a few years ago, Eucera breviceps (Friese, 1911), which was previously known only from Turkey and Syria (Aubert et al. 2024).It recently turned out, however, that this species is also present in Greece, Bulgaria and Italy.It cannot be excluded that H. onosmaevae also occurs in mountainous areas between France and Turkey, e.g. in Italy or the Balkans, where the bee fauna has not yet been thoroughly studied.In fact, the occurrence of H. onosmaevae in Bolu province close to the European part of Turkey renders the new species' occurrence in Greek mountain ranges quite probable.

Host plants
Hoplitis species of the subgenus Hoplitis show a tight association with two plant families, the Boraginaceae and the Fabacaeae (Sedivy et al. 2013a).Although our results are based on few samples only, analyses of pollen contained in the female scopae and the brood cells as well as field observations strongly suggest that H. onosmaevae sp.nov. is narrowly oligolectic on Onosma.The long mouthparts and the ability to buzz the flowers are clearly adaptations for the collection of nectar and pollen from Onosma.Pollen collection on Boraginaceae genera with narrow-tubed flowers requires morphological or behavioral adaptations known to occur in numerous species of diverse bee groups including Hoplitis species of the subgenus Hoplitis (Müller 1995).Among the Hoplitis species of the adunca group, H. flabellifera (Morice, 1901) possesses curved bristles on the front tarsi used to extract pollen from flowers of the genus Anchusa L. (Sedivy et al. 2013a).The recently discovered H. lithodorae Müller, 2012 has specialized hooked bristles on the galeae of the proboscis, which serve to scratch pollen out of the narrow corolla of the flowers of Lithodora, its exclusive pollen host (Müller 2012;Aubert 2016).Among the Hoplitis species of the annulata group, H. aqabaensis (Warncke, 1991), oligolectic on Echiochilon Desfontaine, has long and stiff pollen-collecting bristles on the ventral side of the first segment of the labial palpi and the base of the second segment (Sedivy et al. 2013a).In the persica species group, Hoplitis persica (Warncke, 1991), which is presumably oligolectic on Heliotropium, has apically curved and capitate bristles on the second segment of the labial palpi most probably used to extract pollen from the narrow-tubed Heliotropium flowers (Sedivy et al. 2013a).
In contrast to the Boraginaceae genera mentioned above, pollen collection on flowers of Onosma does not require morphological specializations on proboscis or front legs.
In this genus, the pollen is shed into a cone that is formed by the five adjacent anthers and can be extracted either by buzzing or with mandibles and fore legs (Teppner 1995(Teppner , 2011;;Trunz et al. 2020).Beside H. onosmaevae, four additional species of the subgenus Hoplitis are known to apply buzzing to extract pollen from cone-shaped flowers of the Boraginaceae (Sedivy et al. 2013a): Hoplitis holmboei, which is oligolectic on several genera of the Boraginaceae, such as Onosma, Echium and Lithodora, H. homalocera, which exclusively collects pollen on Podonosma Boiss., a close relative of Onosma, and two undescribed species of Hoplitis (Hoplitis sp. nov. 2 and H. sp. nov. 3 in Sedivy et al. 2013a), which buzz the flowers of Onosma and Trichodesma R. Brown, respectively.At least two species that belong to other families than Megachilidae are also known to buzz the flowers of Onosma: Eucera breviceps, oligolectic on Onosma (Aubert et al. 2024), and the short-tongued bee Andrena symphyti Schmiedeknecht, 1883, oligolectic on Boraginaceae and known to collect pollen also on Symphytum L. and Cerinthe L. (Teppner 2011).The females of Osmia apicata Smith, 1853, a Boraginaceae oligolege with a strong preference for Onosma (Haider et al. 2013), do not buzz the Onosma flowers but scrape the pollen out of the cone with their fore legs after intruding their head into the corolla (Gogala and Surina 2011).The front legs of H. onosmaevae are probably not involved in pollen collection, although the females insert the head and the front part of the mesosoma into the corolla.Instead, by placing the ventral side of the mesosoma against the style (Fig. 20), the vibration of the thorax is transmitted to the cone through the style during buzzing (Buchmann 1983;Teppner 2018).The extraordinarily elongated proboscis of H. onosmaevae, which is probably not used for pollen extraction, allows males and females to access the nectar, which accumulates at the base of the Onosma corolla tube.
The phylogenetic position of H. onosmaevae strongly suggests that specialization on Onosma has occurred at least twice among species of the adunca group, once in H. onosmaevae and once in the clade containing H. holmboei and Hoplitis sp.nov.2, whereas H. holmboei, and possibly Hoplitis sp.nov.2, also collect pollen from other genera of Boraginaceae.The phylogenetic position of H. linguaria, which is most probably oligolectic on Onosma (Müller 2023b), and of H. semilinguaria, which might also be an Onosma oligolege due to its long proboscis, is unknown.However, morphology suggests that they are not closely related to H. onosmaevae, which differs from these two species by the presence of a marginal rim and a medioapical spine on S6, two characters that H. onosmaevae shares with its closest relatives H. adunca, H. benoisti and H. manicata.Whether H. linguaria and H. semilinguaria are closely related to each other or to H. holmboei will require further investigation; depending on their phylogenetic position, specialization on Onosma may have indepently evolved two, three or even four times in the subgenus Hoplitis.

Nesting biology
The nesting biology is unknown for more than two thirds of the Hoplitis species of the adunca group (Müller 2023b).Most species for which the nesting habits are known build their nests exposed in depressions of rocks or hidden rock cavities and all use sand as nesting material, often combined with small pebbles (Sedivy et al. 2013b).Exceptions are H. fertoni (Pérez, 1890), which uses snail shells as nesting site, as well as H. adunca and H. manicata, which nest in insect burrows in dead wood and hollow stems beside other types of preexisting cavities.Interestingly, Hoplitis onosmaevae, which is closely related to the latter two species based on our phylogenetic analysis, was found to build its nests also in insect burrows in dead wood, suggesting that nesting in existing cavities in wood or stems has probably evolved only once in the subgenus Hoplitis.Whether Hoplitis benoisti, which builds its nests in depressions and cavities in rocks or in insect burrows in the soil (Sedivy et al. 2013b), has reverted to the ancestral nesting behaviour of the adunca group or whether it is sister to the other three species suggesting the origin of nesting in wood and stems in the ancestor of H. adunca, H. manicata and H. onosmaevae is unclear, since the phylogenetic relationships of these four species are not resolved.

Conservation
The extreme rarity of Hoplitis onosmaevae in France, where it is currently known only from two restricted and little distant localities, does not seem to be fully accounted for by the rarity of its host plant, since large Onosma tricerosperma subsp.fastigiata populations are known outside the known range of H. onosmaevae, for example in the southern part of the Massif Central and at lower altitudes in the southern Alps.Hoplitis onosmaevae was only found at elevations between 1900 m and 2000 m in France and between 1075 m and 2350 m in Turkey and Iraq.This suggests that H. onosmaevae may have a particularly narrow climatic niche in France and does not find suitable climatic conditions at lower elevations, despite the presence of the host plant.This situation is reminiscent of that of Bombus brodmannicus, which is also much more narrowly distributed in the Alps than its host plant, Cerinthe (Boraginaceae).Possibly, these two bee species are associated with climatic conditions that are only found at certain elevations in restricted areas of the southern Alps, for example humid and cold winters and particularly hot and dry summers.Other factors may also limit the distribution of H. onosmaevae, particularly the presence of dead wood as nesting substrate, which represents a scarce resource in the steppe-like grasslands, where Onosma plants preferentially grow.The local history of forest use by humans may also partly explain the rarity of H. onosmaevae; massive deforestation in the southern Alps from approximately 2000 BC (Beaulieu 1977) may have reduced the area of habitat suitable for the species by reducing the number of its potential nidification sites.
The strongly disjunct distribution of H. onosmaevae and the bee's extremely local occurrence in limited areas of the southern French Alps have important implications for conservation.The species has most probably a very narrow ecological niche, making it highly susceptible to future changes in its habitats, for example due to changes in agricultural practices or to climate change.Even if the two known sites of H. onosmaevae are located in the core area of the Mercantour National Park, human activities can threaten its populations.Pastoralism, for example, is a significant economic activity in the Park and considered as an important heritage, playing a role in maintaining open habitats (Parcs nationaux de France 2012).However, excessive pastoral pressure or overgrazing can easily impact the populations of the host plants, either by direct consumption of the plants or by repeated trampling.The negative impact of excessive grazing could even be aggravated by unfavorable climatic conditions, such as prolonged droughts, which are expected to reduce the availability of flowers and further increase pressure on the vegetation.The presence of dead wood in close neighbourhood to the Onosma stands is a further essential prerequisite for the occurrence of H. onosmaevae, rendering the preservation and promotion of a sufficient supply of dead wood at the French sites occupied by H. onosmaevae essential.

Conclusions
Given the extreme rarity of Hoplitis onosmaevae in France and the species' high vulnerability to habitat changes due to its strong dependence on Onosma and dead wood, a detailed conservation plan should be worked out in the southwestern Alps, including the following measures: 1. additional fieldwork to identify further populations and to determine the current distribution of H. onosmaevae, including in the Italian Alps, where the species could also be present; 2. the active conservation of the known populations of H. onosmaevae, for example by adopting an appropriate management plan, ensuring that the habitat characteristics do not change and providing an adequate supply of plant and nesting ressources for the bee, with the aim of increasing the overall size of the population; 3. adaptation of the eco-pastoral management plans in the regions of the Mercantour National Park where the species occurs, to preserve and increase the abundance of the Onosma stands.
especially Marie-France Leccia and the staff from the Tinée and Var areas for facilitating the logistics; Jan Macek (Praha Museum), Martin Schwarz and Esther Öckermüller (Biologiezentrum Linz) for help in the search for the types of H. Lucas Ferron for the drawing of the nest; and Herbert Zettel and Max Kasparek as well as the subject editor Stefan Dötterl for reviewing the manuscript.
Field work in Mercantour National Park in 2018 was co-financed by the Government of Monaco and the Prince Albert II of Monaco Foundation.

Figure 1 .
Figure1.Phylogenetic tree based on maximum likelihood analyses of sequence data of the mitochondrial gene COI and of the three nuclear genes conserved ATPase domain (CAD), elongation factor 1-alpha (EF) and long-wavelength rhodopsin (Opsin).Numbers above branches: statistical support based on 1000 bootstrap replicates (values below 50 are omitted) in the analysis with 4 partitions (by gene); number below branches: support in analysis with 7 partitions (by codon position; a hyphen under a node indicates that this node was not recovered in the analysis); the topology is from the analysis with 4 partitions.