Narrow flower specialization in two European bee species of the genus Colletes ( Hymenoptera : Apoidea : Colletidae )

Colletes anchusae Noskiewicz, 1924 and C. wolfi Kuhlmann, 1999 (Colletidae) are closely related bee species with vicariant distributions, the former occurring in east and southeast Europe and Turkey, the latter restricted to the Italian peninsula. Microscopical analysis of scopal pollen revealed that in Europe both species are monolectic collecting pollen exclusively from flowers of Cynoglottis barrelieri (All.) Vural & Kit Tan (Boraginaceae). In Turkey, C. anchusae possibly visits also Cynoglottis chetikiana Vural & Kit Tan. The distributions of the two bee species and of Cynoglottis coincide. The females of both Colletes spe­ cies are equipped with stout, curved bristles on their foretarsi used for scraping pollen out of the narrow flower tube of Cynoglottis. Compared to other European species of Colletes the foretarsi of C. anchusae and C. wolfi are shortened, presumably an adaptation to the short corolla tube of their host plant.

There is little doubt that the females of C. anchusae and C. wolfi use the tarsal bristles to extract pollen from flowers with hidden anthers.Indeed, in Hungary and the Ukraine C. anchusae was repeatedly observed on flowers of Anchusa bar relieri (All.)Vitman, a member of the Boraginaceae with narrow flower tubes (Noskiewicz, 1924(Noskiewicz, , 1936;;Moczar, 1961).A. barrelieri differs from other Anchusa species in having dis tinctly smaller pollen grains and a short corolla tube (Vural & Kit Tan, 1983).Because of these differences Vural & Kit Tan (1983) put A. barrelieri in its own genus, Cynoglottis.Recently, a second Cynoglottis species, C. chetikiana Vural & Kit Tan, was described from Turkey (Vural & Kit Tan, 1983).
The aim of this study was to examine the host plant spectrum of C. anchusae and C. wolfi, to compare the distributions of these two bee species with that of their host plants and to ana lyze potential adaptations of bee morphology to flower structure.

Pollen sources
To assess the pollen plants of C. anchusae and C. wolfi we analyzed the scopal contents of females from museum and pri vate collections by light microscopy using the method outlined by Westrich & Schmidt (1986).We identified the pollen grains at a magnification of 400* and 1000* with the aid of Clarke (1980), the literature cited in Westrich & Schmidt (1986) and a reference collection containing the pollen of nearly all European genera of the Boraginaceae, including Cynoglottis.
Information on the distribution and flight period of C. anchusae and C. wolfi were extracted from Noskiewicz (1924Noskiewicz ( , 1936) ) and the Colletes data bank of M. Kuhlmann (unpub lished).

Bee morphology and flower structure
To examine the structure of the flowers of C. barrelieri her barium specimens were soaked in water.
To estimate the fit between leg length and corolla depth the relative length of the foreleg of C. anchusae and C. wolfi was compared with that of 16 other European and North African Colletes species, each belonging to another species group (see Kuhlmann, 2000), by calculating the ratio of leg length to forewing length.Forewing length was used as a measure of body size.The lengths of femur, tibia and tarsus (up to the inser tion of the claws), and forewing (along the anterior margin) were measured under a dissecting microscope to the nearest 1/20 mm.

Bee morphology and flower structure
The corolla of C. barrelieri is blue to bluish-violet.The limb is almost flat, 6-9 mm across, with rounded lobes (Fig. 2).The narrowly campanulate tube is short, measuring 1.0 to 1.5 mm in depth and 2.0 mm in diameter.At its apex the tube is narrowed by five whitish faucal scales, which protrude slightly from the tube.The anthers are hidden under the hairy scales and reach the base of the scales.The style is included and about half as long as the tube.
The forelegs of both C. anchusae and C. wolfi are modified in two ways.The slightly curved tibia is on its outer surface dis tinctly concave and hairless (Fig. 3) and the basitarsus is beset   (Noskiewicz, 1924(Noskiewicz, , 1936)).In Hungary, C. anchusae is also believed to be a specialist of this plant species (Moczar, 1961).The close match in the distribu tions of the bees and Cynoglottis is a further indication of this intimate relationship as is the coincidence of the flowering time of Cynoglottis with the flight period of the bees.

Distribution
C. wolf i and C. anchusae differ morphologically only slightly (Kuhlmann, 1999) indicating a recent separation of these two species.This is compatible with the hypothesis that populations of the ancestor of the two species survived the ice age in at least two refuges, one in Italy and another in southeast Europe or Turkey, from where they spread northwards after the end of the last glacial period.The gap in the distribution area of their host plant might then have prevented the two populations from coming into contact again.
Both C. anchusae and C. wolfi are considered to be very rare.However, their flower specialization might account for why these bees are so rarely collected in the field.We expect them to be much more widespread occurring wherever Cynoglottis grows.

Bee morphology and flower structure
Thirteen Central European species of bees belonging to four families and five genera are known to have evolved stout, curved bristles either on the forelegs or on the proboscis (Müller, 1995).In all these species the specialized bristles serve to extract pollen from the flowers of plants whose anthers are hidden in narrow tubes.Similar morphological specializations are also known in bees from outside Europe (Shinn, 1967;Thorp, 1979Thorp, , 2000;;Houston, 1991).Here too, the modified hairs are a tool for removing pollen from narrow flower tubes.Though field observations are still lacking both C. anchusae and C. wolfi are thought to use the specialized bristles on their fore tarsi to harvest pollen from Cynoglottis.Females of C. nasutus are known to insert both forelegs simultaneously into a flower tube of Anchusa officinalis before extracting pollen from the hidden anthers with repeated upward and downward movements of their forelegs (Müller, 1995).Females of C. anchusae and C. wolfi probably collect pollen in much the same way.
The function of the modified female tibia in C. anchusae and C. wolfi is obscure.The tibial curvature might enable the bees to bring the tarsi of their forelegs closer together and in a parallel position before they are simultaneously inserted into the narrow entrance of a flower.
In both C. anchusae and C. wolfi the development of hooked bristles on the foretarsus is accompanied by a shortening of the foretarsus.The tarsal length is similar to the tube length of C. barrelieri.Consequently, the shortening of the foretarsus might be an adaptation to the short corolla tube of the host plant allowing the bees to use the total length of their tarsi to scrape pollen out of the flower.In contrast to C. anchusae and C. wolfi forelegs, head and proboscis of C. nasutus are distinctly length ened compared to other European Colletes species, doubtless an adaptation for collecting pollen and nectar from the deep tubular flowers of Anchusa officinalis, which are 7 to10 mm long (Mül ler, 1995).
For this nine females of C. anchusae, ten females of C. wolfi and two females each of C. albomaculatus (Lucas, 1849), C. cunicularius (Linnaeus, 1761), C. daviesanus Smith, 1846, C. fiormosus Perez, 1895, C. gallicus Radoszkowski, 1891, C. hederae Schmidt & Westrich, 1993, C. hylaeifiormis Eversmann, 1852, C. impunctatus Nylander, 1852, C. lacunatus Dours, 1872, C. maidli Noskiewicz, 1936, C. marginatus Smith, 1846, C. meyeri Noskiewicz, 1936, C. mlokossewiczi Radosz kowski, 1891, C. moricei Saunders, 1904, C. nasutus Smith, 1853 and C. nigricans Gistel, 1857 were selected.RESULTS Pollen sources The pollen samples from both C. anchusae and C. wolfi con sisted of exactly the same pollen type.At a magnification of 1000*, the pollen grains in the 26 samples could not be distin guished from a reference pollen sample of C. barrelieri.Distribution C. barrelieri occurs from Italy eastwards to Turkey and Syria, and northwards to the Ukraine and Russia (Fig. 1).The distribu tion is split into two parts, a western part covering Italy and an eastern part ranging from Slovenia eastwards.C. chetikiana, the second species of the genus, is endemic to north, central and south Anatolia.In Turkey, the distributions of the two Cyno glottis species partially overlap.Both species grow on dry rocky slopes, stony pastures, fallow fields and open coniferous forest, mostly on limestone.Depending on the altitude the flowering time is May to July.C. wolfi and C. anchusae show a vicariant distribution (Fig. 1).C. wolfi is confined to Italy whereas C. anchusae is found in east and southeast Europe and Turkey.All known localities of the two Colletes species fall within the distribution area of Cynoglottis.Collection records date from 7 May to 28 July (n = 24) for C. anchusae and from 27 May to 19 July (n = 25) for C. wolfi.