Morphology, pollen preferences and DNA-barcoding of five Austrian species in the Colletes succinctus group (Hymenoptera, Apidae)

Most species of the Colletes succinctus group sensu Noskiewicz, 1936 are taxonomically uncertain. This study has chosen an integrative approach, including pollen analysis, morphology, male genitalia, morphometry, cuticle sculpture and DNA-barcoding (CO1) to investigate the five species that were reported from Austria. It includes a detailed analysis of the male genitalia and the first description of the C. pannonicus male. A syntype male from the island of Crete was designated as the lectotype of Colletes succinctus brevigena Noskiewicz, 1936 to fix the species identity. New distinguishing characters were found: in females the shape of the dorsal end of the fovea facialis and, in both sexes, the structure of maxillary palpi, as well as the different puncturation on the mesopleura. Unknown structures on sterna and genitalia of the males proved to be reliable morphological characters. An identification key is provided for all studied species. Morphometry of females did not allow a clear distinction of species. CO1 sequencing confirmed previous studies that only C. collaris clearly deviates from the other species, including C. pannonicus that was analysed for the first time. Pollen analysis showed polylectic, as well as oligolectic, pollen-collecting behaviour. The collected pollen of C. pannonicus confirmed the field observations that this species is strictly oligolectic on Tripolium pannonicum. Due to pronounced intraspecific variation, it is assumed that the species of the C. succinctus group are either species in statu nascendi or very young species. Therefore, it remains important to include ecological data in species identification.


Introduction
Colletes Latreille, 1802 is a solitary bee genus belonging to the family Colletidae. Their common English name "polyester bees" is derived from a characteristic cellophane nest lining. The female produces a polyester secretion in the abdominal Dufour gland (Albans et al. 1980) and uses it to coat the nest cell walls for waterproofing with their widely split tongue (Westrich 1989).
The genus comprises 522 described species (Proshchalykin and Kuhlmann 2018; Kuhlmann and Smit 2018;Kuhlmann 2019) which are distributed all around the world, except Australia, Antarctica, Madagascar and parts of Southeast Asia (Michener 2007;Kuhlmann 2014). Only 21 species of the genus Colletes are reported from Austria (Gusenleitner et al. 2012). Based on morphological characters, the Palaearctic species were divided into 26 species groups (Noskiewicz 1936). Especially the Colletes succinctus group is notorious for being a taxonomic challenge. It is defined by two synapomorphies: two deep lateral pits at sternum 6 (subgenital plate) of the males and a redbrown transparent basal margin of gaster tergum 1 (Noskiewicz 1936, Kuhlmann et al. 2007).
According to collection and literature data, the Austrian species of the C. succinctus group have one generation per year (monovoltine). Although they are all "late-summer bees" (Scheuchl and Willner 2016), collection data suggest that they differ in their phenology. Colletes succinctus emerges first early to mid-August (Scheuchl and Willner 2016), followed by C. brevigena and C. collaris in late August (Westrich 1997;Zettel et al. 2006;Standfuss 2009). The holotype of C. pannonicus was collected mid-September (Hölzler and Mazzucco 2011) and C. hederae seems to be the species that is most adapted to the cool season. It can be found from late August until November (Kuhlmann et al. 2007).
Regarding the provisions for their offspring, the Austrian species of this group reportedly show different pollen preferences: the species are described as either polylectic, oligolectic or pseudo-oligolectic (Bischoff et al. 2005;Müller and Kuhlmann 2008;Westrich 2008;Teppner and Brosch 2015). Colletes succinctus and C. brevigena show polylectic behaviour (Michener 2007;Müller and Kuhlmann 2008). However, it should be mentioned that C. succinctus, despite being a generalist, prefers heather (Calluna sp.) which leads to its vernacular name "heather bee". Colletes collaris and C. halophilus are typical oligolectic bees, preferring pollen of Asteraceae (Westrich 1997;Kuhlmann et al. 2007;Müller and Kuhlmann 2008) and the term pseudo-oligolectic was used for C. hederae (Teppner and Brosch 2015). As its common name suggests, the "ivy bee" shows a strong preference for ivy (Hedera sp.) (Schmidt and Westrich 1993) and is widespread throughout Europe, with only a few gaps in Scandinavia (Rathjen 1998). Nonetheless, it also collects pollen from other flowers before the ivy starts blooming (Müller and Kuhlmann 2008). Colletes hederae was originally a Mediterranean species, but is currently spreading to Central and Western Europe at a rapid rate (Schmid-Egger 1997;Rathjen 1998;Vereecken et al. 2009, Saure et al. 2019. Additionally, in Austria, it shows a rapid expansion (Neumayer 2012;Zettel and Wiesbauer 2014;Ebmer et al. 2018). Due to its strong similarity to C. succinctus and C. halophilus, C. hederae was described recently, although specimens of the genus Colletes collecting pollen on ivy have been reported for a long time (Richards 1979;Janvier 1979Janvier , 1980Westrich 1989). As the latest addition to the species group, Colletes pannonicus was described from a population using the pollen of Tripolium pannonicum (sea aster), supposedly being oligolectic on Asteraceae (Hölzler and Mazzucco 2011).
Cladograms, based on molecular data, show strong agreement with Noskiewicz's (1936) morphologically based species groups (Kuhlmann et al. 2009). However, the mitochondrial gene fragment cytochrome oxidase 1 (CO1) did not show any species-specific, fixed differences within the Colletes succinctus group (Kuhlmann et al. 2007(Kuhlmann et al. , 2009; Magnacca and Brown 2012;Dellicour et al. 2014). Misinterpretations of taxa may have led to unreliable analyses and a phylogenetic analysis, including C. pannonicus, has not yet been performed.
The taxonomy and phylogeny of the species of the C. succinctus group have been the object of recent discussions and investigations (e.g. Kuhlmann et al. 2007;Müller and Kuhlmann 2008;Kuhlmann et al. 2009;Magnacca and Brown 2012;Dellicour et al. 2014). The original descriptions of species described after Noskiewicz's (1936) revision compare the new taxa only with one previously described (usually sympatric) species, thereby neglecting similar species from other areas. However, with the exceptions of C. collaris and C. standfussi, which both can be easily recognised, the species are morphologically very difficult to distinguish (Kuhlmann 2003). Consequently, their different phenology, their pollen preferences, as well as their habitat preferences, were used for differentiation (e.g. Verhoeff 1944;Schmidt and Westrich 1993;Kuhlmann 2003;Kuhlmann et al. 2007;Hölzler and Mazzucco 2011).
The aim of this study is to compare European species of the C. succinctus group, with focus on the five species that were previously reported from eastern Austria. Therefore, an integrative approach was designed, including morphology, statistical analyses of morphometrics, pollen analyses, as well as DNA-barcoding, to exclude the possibility of misidentification in previously-studied material and to provide comparison sequences for future investigations.

Specimens and preparations
The study included 270 specimens (Suppl. material 1: Appendix 1) which were either newly collected or provided by the Natural History Museum of Vienna (NHM), the Upper Austrian State Museum and private collections. Pin-mounted specimens were obtained from collections. Fresh bees were collected from July to September 2017 in Vienna, Lower Austria (Retz and Ollersdorf), Upper Austria (Linz) and Burgenland around Lake Neusiedl, as well as in Poland (around the city of Sierakow). Collecting was carried out with an insect net and the bees were either euthanised in 96% ethanol to preserve DNA for DNA-barcoding or in a vial with ethyl acetate vapour.

Morphological studies
To find additional, previously-unknown morphological characters for species distinction, both females and males were examined by light microscopy and compared between species. Since a description of the males of C. pannonicus has not yet been published, special attention was paid to this species and a detailed description is given in this work. Much attention was given to the proboscis of the females and to sterna 6-8 and genitalia of the males. Therefore, these body parts were manually dissected. Morphological descriptions were chiefly based on the terminology of Michener (2007) and Boudinot (2013). However, in order to get a better overview of the male's structures of sterna 6-8 and genitalia, specific terms were additionally introduced.
For illustration of the species-specific differences, photography, as well as scanning electron microscopy, was used: Stacked digital images of the different parts of sterna 6-8 and genitalia were acquired with a Leica DFC490 camera attached to a Leica Z16 APO zoom microscope, using Leica Application Suite 4.10.0 software. Afterwards, the digital images were stacked with ZerenaStacker 64-bit and processed with Adobe Photoshop 7.0. After illustration of the entire genital capsule, further dissection became necessary to see all important structures: a median cut between the two valvulae was performed, followed by removing the valvulae from the gonostyli. These parts were also illustrated by photography.
To illustrate different structures of proboscis and head of females, a scanning electron microscope (Philips XL 30 ESEM) was used. After dissection, the samples were washed for dehydration three times in 100% ethanol and three times in 100% acetone for 15 minutes each. For drying, the critical point dryer (LEICA EM MED020) was used for around 1.56 minutes with the settings: velocity -medium; delude time -120 seconds; exchange steps -5; cycles -18; heating process -slow and speed -slow. Afterwards, the dried proboscides were glued to a copper foil with conductive silver and mounted on a carbon-taped stub. The dried heads were glued directly to the stub. For gold coating, a sputter coater (LEICA EM CPD300) was used for around 120 seconds. Images were taken with the scanning electron microscope and the programme Scandium 5.1 was used to add the scale bar.

Morphometry
In total, 103 females were used to obtain morphometric data for analysis (Suppl. material 2: Appendix 2): 10 specimens of C. halophilus, 11 specimens of C. pannonicus, 14 specimens of C. brevigena, 20 specimens of C. collaris, 22 specimens of C. hederae and 26 specimens of C. succinctus. The following distances, measured on the head and between the tegulae, were investigated (Figs 1, 2): head length (HL) -maximum head length, measured in exact frontal view along the mid-line, from vertex to distal margin of clypeus; head width (HW) -maximum head width, measured in exact frontal view from one outer edge of the compound eye to the other; eye length (EL) -length of the eye, measured in latero-frontal view from the most dorsal point to the most ventral point of the left compound eye; upper interocular distance (UID) -shortest distance between the dorsal margins of the compound eyes, measured in dorsal view; lower interocular distance (LID) -shortest distance between the ventral margins of the compound eyes, measured in frontal view; median interocular distance (MID) -longest distance between the inner margins of the compound eyes, measured in frontal view; clypeus length (CL) -maximum clypeus length, measured in frontal view from the anterior to the posterior margin; cheek length (CHL) -minimum length of the gena, measured in latero-frontal view from lower eye margin to mandible; thorax width (TW) -maximum distance between the two mesal edges of the tegulae, measured in dorsal view.
Due to their very similar morphological characters C. brevigena and C. pannonicus were examined in more detail. As already done in the species description of Hölzler and Mazzucco (2011), the relationship of head width (HW) and thorax width (TW) for C. brevigena and C. pannonicus was calculated in form of an index: Head-thorax index = TW / HW × 100 Measurements were conducted at different magnifications (24-76.8×) using a calibrated LEICA MZ6 binocular microscope with an ocular micrometre and later converted to millimetres. The statistics programme PAST3 (Hammer et al. 2001) was used to conduct principal component analysis (PCA) and discriminant analysis (LDA). The logarithmic morphometric values were used for all analyses. The PCA was based on a correlation matrix and 95% confidence intervals of variances were calculated using 1,000 Bootstrap re-samplings. For LDA, the specimens were assigned to six hypothetical groups, based on their morphological characters (C. succinctus, C. collaris, C. brevigena, C. halophilus, C. hederae and C. pannonicus) and the Jackknife method was used for re-sampling. Due to different morphological characters, the examined species C. brevigena was divided into two groups (Austrian and Mediterranean specimens) and analysed separately by LDA.

Pollen analyses
For pollen analyses, the pollen loads of 32 fresh and 41 dried female specimens (n = 73) were examined (Suppl. material 3: Appendix 3). The filling ratio of the pollen loads was determined between the grades one and five (see Müller and Kuhlmann 2008): "Five" defined fully-loaded hind legs and propodeum, so that no hair was visible and "one" meant that only one fifth of the collecting structures was loaded. Filling ratios in between (2-4) were estimated according to the researcher's personal assessment. Furthermore, these filling ratios were used to calculate the correlation between the different filling loads and the amount of pollen types. Therefore, the Pearson correlation coefficient was calculated using Past3 (Hammer et al. 2001). The pollen was removed with a fine needle and placed on a glass object slide. After prevention of clotting accumulation by adding a drop of 85% ethanol, the pollen grains were fixed and dyed with a mixture of Kaisers Glycerine-Gelatine (ROTH) and alkaline Fuchsine. Afterwards, up to 300 pollen grains per slide were counted by using a NIKON ECLIPSE E800 light microscope.
For pollen determination, literature (Beug 2004, Hesse et al. 2009), as well as the databases paldat, ponet and pollen.tstebler, were used. Illustrations were made with the programme NIS-Elements D (4.51.01) and edited with Adobe Photoshop 7.0. Furthermore, the percentage for each pollen type was calculated and all types below 5% were identified as impurities.

DNA-barcoding
The legs of 46 specimens were used for DNA barcoding (Suppl. material 4: Appendix 4). In preparation for DNA extraction, the ethanol, in which some of the animals were stored, had to be washed out with PBS (phosphate buffered saline). Afterwards, the samples were shock-frozen in a mixture of dry ice and ethanol and crushed. The following DNA extraction was performed using Qiagen's "DNeasy Blood and Tissue Kit" and the protocol "Purification of Total DNA from Animal Tissues (Spin-Column Protocol)".
After extraction, the samples were analysed with a nanodrop (Nanodrop 200/2000c Spectrophotometer) for DNA quantification and only samples containing sufficient DNA were used for further analysis. For Polymerase Chain Reaction (PCR) 25 μl MM Biozym Red HS Taq Master Mix, 21 μl molecular grade water (Sigma Aldrich) as well as 1.5 μl of each primer and the respective DNA sample (1-2 μl) were mixed. The PCR machine "TProfessional Thermocycler" (Biometra) was used to amplify the gene using the primer-pair LCO1490 and HCO2198 (Folmer et al. 1994) in the following settings: the starting denaturation cycle at 95 °C for 3 minutes was followed by 33 cycles for 30 seconds each, 33 alignment cycles at 47 °C for 30 seconds each, 33 elongation cycles at 72 °C for 45 seconds each and one final cycle at 72 °C for 10 minutes.
For the subsequent gel electrophoresis, a mixture of 1× TBE buffer and agarose gel (1% w/v) was used. In total, 3 μl of the samples, together with 1.5 μl Orange DNA Loading Dye (Thermo Science) and the Sizemarker GeneRuler 1kb -DNA Ladder (Thermo Science), were loaded on to the gel.
The purification was carried out with Qiagen's "QI-Aquick PCR Purification Kit" and the DNA sequencing was executed by Microsynth AG.
Unfortunately, only 21 samples were sequenced successfully: 20 specimens of the European C. succinctus group and one specimen of C. creticus, which was used as an outgroup (Suppl. material 4: Appendix 4). The remaining samples could not be used for further analyses due to either contamination or unsuccessful DNA extraction. Furthermore, some sequences could not be used because of infestation with Wolbachia (a genus of gram-negative bacteria common in sexual organs of arthropods) and very poor electropherograms which could not be evaluated. In general, electropherograms were not ideal, which is probably due to an artefact band (200 bp). Therefore, base-calling in all electropherograms had to be checked carefully.
The obtained electropherograms were proof-read, aligned and cut to the same length by removing the primer sequences with Bioedit 7.2.6. and FinchTV 1.4.0 (Geospiza, Inc). POPArt (Bandelt et al. 1999) was used to create a median-joining network from the sequences obtained, as well as from reference data (47 sequences) from the Barcode of Life Database (BOLD) (Ratnasingham and Hebert 2007) (Suppl. material 5: Appendix 5). In addition, genetic distances were calculated and a neighbour-joining tree was generated with MEGA 7.0.26 (Kumar et al. 2016) before it was illustrated with FigTree 1.4.3.

Morphology of the species
In addition to already-known morphological features, new characters were discovered to better distinguish between the Austrian species of the Colletes succinctus group (Table 2, Fig. 3). Table 2. Expression of the distinctive characters (females and males) of the investigated species in the C. succinctus group. Newly found characters for species differentiation, identified in this study in bold font and already known characters extracted from literature (Noskiewicz 1936;Schmidt and Westrich 1993;Burger 2010;Hölzler and Mazzucco 2011  Morphological characters of male sterna 6-8 and genitalia In addition to external morphological characters, the male specimens can also be distinguished by shape and pubescence of sterna 6-8 and the morphology of the genitalia. It was possible to find differences in their shape and pubescence.

General description, Colletes succinctus group:
Sternum 6: large, lacking lateral tubercles; the convex hind margin medially protruded and with variably-developed blunt corners at each side. Before hind margin with two deep lateral grooves (lgr). An oval-shaped translucent area (ota) of variable size in the middle. Sternum 7: base (bs) curved, narrow, connected with distal structures via a bridge (br) with a narrow sclerotised medial stalk. Distal part strongly modified: a central, diamond-shaped, distally bifid medial elevation (mel) leads to strongly sclerotised basal shoulders (sh), which bear the paired wings (wg). Except for C. collaris (see description of C. collaris), each wing consisting of a sclerotised, densely pilose medial processes (mp) and a weakly sclerotised, flexible lateral part with a hairy basal arm (ba) and a distal, almost bold membrane (mbr).
Compared to other species groups, the distal part of sternum 7 is smaller and distinctly shorter in the C. succinctus group (Noskiewicz 1936). The specific structures, described above, cannot be clearly homologised with the structures of other species groups.
For species-specific characters of the male terminal structures and genitalia of C. brevigena, see the next chapter.
Lectotype designation of C. brevigena Noskiewicz (1936) described C. succinctus ssp. bre vi gena, based on specimens from a large distribution area: Macedonia, Dalmatia, Cyprus, Crete, North Persia and the Caucasus; however, he questioned the synsubspecificity of a male from Austria near Neusiedlersee (note that this is the type area of C. pannonicus). Noskiewicz (1936) did not designate a holotype; a lectotype was not selected by subsequent authors. The depositories of the syntype series were not published by Noskiewicz (1936), but later it was reported that no types of C. brevigena are represented in the Noskiewicz collection in the Museum of Natural History, University of Wroclaw, Poland (Wanat et al. 2014;and Marek Wanat, pers. comm. to Herbert Zettel).
The Natural History Museum Vienna keeps eleven male specimens, all identically labelled "Colletes ♂ succinctus L. ssp. brevigena Nosk. det. Noskiewicz.", which were studied by Noskiewicz in the course of preparing his monograph and, therefore, are putative syntypes. For the reason of taxonomic stability, we select a male (no. 270) from Crete (Greece) as the lectotype (see . Arguments for selecting this specimen were (1) its almost perfect condition and (2) avoidance of a type locality in the southern Caucasus which would make future molecular research on this taxon more difficult.

First description of the male of C. pannonicus
Colletes pannonicus is a recently described species that has been solely found in the Seewinkel near Lake Neusiedl (Hölzler and Mazzucco 2011). The "preliminary description" was based on a female specimen (holotype) and since the males of C. pannonicus have not yet been described, this is done on the following, based on dry specimens collected sympatrically with C. pannonicus females in Seewinkel on Tripolium pannonicum (sea aster).
Examined material. Description. Face with long yellow-whitish hair. Galea reticulated between sensilla, segments of maxillary palpi long and narrow. Mesonotum coarsely and densely punctured, in some specimens with a shiny centre of varying size, with long orange-brownish hair. Mesopleura densely punctured, (sporadically) punctures  Lateral view of sternum 8. ba -basal arm, br -bridge, bs -base, dpr -distal process, lgr -lateral grooves, lpr -lateral process, mbr -membrane, mel -median elevation, mp -median process, ota -oval-shaped translucent area, sh -shoulder, spi -spiculum, wg -wing. merge and form wrinkles, with yellow-whitish hair. Hair on propodeum yellowish-white to yellow-orange coloured. Stripes of setae on terga yellow-whitish. Punctures on tergum 1 large, but smaller than on mesonotum. Tergum 1 deeply and coarsely punctured, on disc dense, to the sides with interspaces of the size of 0.5-1 puncture diameter; cuticle shiny; basal declivity with long yellow hair. Tergum 2 densely punctured, sporadically with intervals of the size of one puncture diameter; cuticle shiny ( Fig. 15D).

Identification keys
Using a range of external morphological characters (Table  1), as well as the identified differences in male genitalia, it was possible to establish identification keys for both females and males. All investigated specimens were subjected to a detailed examination and determined according to the following identification keys for females and males:   Lateral view of sternum 8. ba -basal arm, br -bridge, bs -base, dpr -distal process, lgr -lateral grooves, lpr -lateral process, mbr -membrane, mel -median elevation, mp -median process, ota -oval-shaped translucent area, sh -shoulder, spi -spiculum, wg -wing.   Lateral view of sternum 8. ba -basal arm, br -bridge, bs -base, dpr -distal process, lgr -lateral grooves, lpr -lateral process, mbr -membrane, mel -median elevation, mp -median process, ota -oval-shaped translucent area, sh -shoulder, spi -spiculum, wg -wing. Identification key to females of the Austrian species of the C. succinctus group 1 Apical tergal hair bands narrow over entire width and more narrowed towards the middle (Fig. 3E). Propodeum hairless in centre of declivity. Mesonotum continuously punctured with scattered black-brown hair in the middle. Puncture at mesopleura separated by intervals that are two or three times larger than diameter of punctures. Cuticle of frons reticulated and punctured (Fig. 3F). Clypeus with longitudinal wrinkles that (sporadically) incline inwards towards the end (Fig. 18A). Galea with microstructure between sensilla (Fig. 14C, D). Narrow, elongated fovea facialis with slightly pointed apex (Figs 3D, 18B)  Clypeus with mesally curved longitudinal wrinkles, the most lateral 1-2 wrinkles of each side meeting each other in the middle behind fore-margin (Fig. 19A). Tergum 1 densely and finely punctured, punctures becoming abruptly smaller towards hind margin. Tergum 2 with slit-shaped structure, slits fine and loosely standing next to each other (Fig. 3K).

Non-assignable specimens
All studied specimens were subjected to a control of their species affiliation. The specimens were re-identified with the help of the new key presented herein. A total of 21 (19 females and two males) of the 270 specimens were assigned to another species of the C. succinctus group than previously. Three males were excluded from the Colletes succinctus   group due to the absence of lateral pits on sternum 6 (Suppl. material 1: Appendix 1). In an additional 29 specimens, it was not possible to assign them to a species with certainty, due to their character combination (Suppl. material 6: Appendix 6). These aberrant specimens showed either a mix of species-specific characters of C. succinctus and C. brevigena or of C. succinctus and C. hederae. One specimen even showed characters of all three species. One specific male specimen from Velden in Carinthia (no. 124) could be assigned to the C. succinctus group due to its deep lateral pits on sternum 6. However, it differs from all other Austrian species by peculiar characters: the mesopleura are so densely punctured that the punctures merge into each other (as is only known from C. halophilus) and tergum 1 shows a very sparse puncturation with distances on disc about twice as long as a diameter of puncture. Tergum 2 is only superficially punctured.

Variation within species
Identified females of all species showed a pronounced intraspecific variation (Table 3). Especially, the head width (HW) seems to be very variable in all species. The intraspecific variation is most pronounced in C. succinctus. Here, the females differ very strongly in all measuring distances with the exception of the cheek length (CHL). Colletes collaris and C. halophilus, on the other hand, show a pronounced intraspecific variability in their head length (HL) and width (HW) and C. brevigena varies both in head width (HW) and thorax width (TW).
Colletes hederae differ mostly in head width (HW) and upper interocular distance (UID). Colletes pannonicus, on the other hand, exhibits the lowest interspecific polymorphism, although the specimens differed to a large extent in their head width (HW). On average, the females of C. hederae are the largest specimens (Table 3). They have by far the largest head, the longest clypeus, the longest lower interocular distance and the longest cheeks. In addition, they have the longest eyes, closely followed by C. brevigena. In turn, the largest average upper interocular distance is shown in females of C. halophilus, as well as the longest middle interocular distance, followed by C. hederae. The specimens of C. brevigena show the widest thorax on average, closely followed by C. halophilus and C. hederae.
Overall, the females of the species C. succinctus have the smallest mean head size (Table 2). They show the lowest values for head width, clypeus length, upper ocular distance, lower ocular distance and middle ocular distance. In terms of head length, they present the smallest mean value together with C. pannonicus.

Principal components analysis of all species (PCA)
All measurements were analysed using a principal component analysis (PCA). Based on 1,000 bootstrap re-samplings, the first principal component (PC1) explains just under 71% of the total sample variance, the second explains over 11% and the third explains about 7% of the variance.

C. succinctus (n = 26) C. collaris (n = 20) C. brevigena (n = 14) C. halophilus (n = 10) C. hederae (n = 22) C. pannonicus (n = 11) Mean (HL)
2  (Table 4). Therefore, it is primarily a measure for body size. Principal component 2 dependents strongly on CHL. For example, when plotting PC1 and PC2, specimens of C. hederae are defined by high loadings on both axes, which means the majority have large bodies and long cheeks (Fig. 23). PC3 is strongly positively influenced by TW and negatively influenced by HL (Table 4). Thus, specimens with a wider thorax tend to have a shorter head. Fig. 23 shows principal components 1 and 2, based on the measured values of the investigated species. Overall, the morphometric data of all species overlap with each other to some extent. By comparing only two species, it is possible to distinguish between some of them. The specimens of C. hederae and C. succinctus overlap only minimally. The same can be seen when comparing the data of C. pannonicus with C. hederae or C. collaris. However, measurements of all other species overlap greatly.

Linear discriminant analysis of all species (LDA)
Based on their morphological characters, the species could not be separated efficiently. Even with a Jackknife re-sampling, only 54.13% of all measured specimens were classified as their previously-assigned species (hypothetical group). Thus, around half of all specimens were assigned to different species by the LDA (Suppl. material 7: Appendix 7). A detailed listing of assignments for each measured specimen, by the author as well as by the LDA, with or without Jackknife re-sampling, is given in Suppl. material 8: Appendix 8.  Table 5. Result of the linear discriminant analysis of the species Colletes brevigena, which is divided into the hypothetical groups C. brevigena MED (with Mediterranean origin) and C. brevigena A (Austrian specimens), including Jackknife re-sampling (1,000). Species in bold letters were classified differently from the hypothetical assignment.

Point Given group
Classification

Linear discriminant analysis of C. brevigena
For a more detailed analysis of the species C. brevigena, all specimens (n = 18) were divided into two hypothetical groups: thirteen specimens from the Mediterranean region were grouped as "C. brevigena MED" and the remaining five females from Austria were grouped as "C. brevigena A". Based on the morphometric data, the discriminant analysis calculated that the specimens belonged to their previously-assigned hypothetical species in 100% of cases. Only after a Jackknife re-sampling, the affiliation of five Mediterranean C. brevigena and two Austrian C. brevigena was reversed, resulting in different assignments in about 40% (Table 5) of the specimens.

The taxonomically-challenging species C. brevigena and C. pannonicus
Head-thorax index: By comparing the head-thorax index of C. brevigena and C. pannonicus, the species cannot be differentiated. In relation to the thorax width, the examined females of C. brevigena show broader heads than the specimens of C. pannonicus; however, the two species overlap in their minimum to maximum head-thorax index range to a great extent (C. brevigena 71.7 to 87.5 vs. C. pannonicus 70.7 to 82.2).

Principal component analyses (PCA):
Principal component 1 explains around 70% of the total sample variance and is defined by high positive loadings of all variables, except for variable CHL (cheek length) and TW (thorax width). Principal component 2, however, explains only 13% of the variance and shows high positive loadings of CHL and high negative loadings of TW.
Therefore, PC1 is interpreted as a measure for body size, whereas PC2 is mainly a measure for sizes of CHL and TW ( Table 6).
The examined specimens of C. pannonicus and C. brevigena overlap in their measurements to a great extent. On average, the specimens of C. brevigena are larger, but like in the PCA of all investigated species, the intraspecific variation is larger than the interspecific differences (Fig. 24).

Pollen determination
Based on their morphological characters, the pollen grains found on the studied specimens were assigned to  Asteraceae (liguliflorae and tubuliflorae), Araliaceae, Ericaceae, Resedaceae and Rutaceae (Fig. 25). In addition, tricolporate/reticulate pollen grains were also found in the pollen load of one specimen, but it was not possible to determine them more precisely. In some cases, it was possible to determine the pollen grains to genus level (Citrus sp., Reseda sp.) or even species level (Tripolium pannonicum, Calluna vulgaris and Hedera helix). The pollen of the family Asteraceae has a spiny (echinate) surface structure. They are either entirely spiked (tubuliflorae) or have spineless windows on their surface (liguliflorae). Ivy (Hedera helix) belongs to the Araliaceae family. Its pollen grains show a reticulate surface and are tricolporate: it has three colpi as well as three pores from which the pollen tubes emerge. Heather (Calluna vulgaris) belongs to the family Ericaceae and is arranged as tetradae: four spheroidal-shaped pollen grains are associated with each other. They possess pores and a scabrate to verrucate structure. The pollen of Citrus sp. (Rutaceae) is tetracolporate and with a reticulate surface.
Colletes hederae: Colletes hederae is the most represented species in this pollen analysis (n = 27). It shows a polylectic pollen-collecting behaviour with a strong preference for Hedera helix of the Araliaceae family (79.5%). In addition, 20.3% of the collected pollen comes from Citrus sp. of the family Rutaceae. There is only one specimen that shows contamination (0.2%) by Citrus sp. All females from Stammersdorf (no. 198 Colletes brevigena: The only specimen of C. brevigena (no. 190) represented in this pollen analysis was caught at the same time as a specimen of C. collaris (no. 195) in Ollersdorf on the flowers of Reseda sp. This specimen collected Reseda pollen in large quantities (~ 98%) and only a small proportion of the pollen load is due to contamination (2.3%).
Colletes pannonicus: All specimens of C. pannonicus (n = 5) were captured near Lake Neusiedl and show an oligolectic behaviour, collecting pollen on Asteraceae tubuliflorae (99.6%), whereas only 0.4% of the load is due to contamination. Their individual pollen packages contain 98-100% pollen of this Asteraceae type, which can be assigned to Tripolium pannonicum and the contamination rate is ~ 2% (Suppl. material 3: Appendix 3).
Specimens without assignment to a species: Due to unclear morphological characters, seven specimens, which were used for pollen analyses, could not be clearly assigned to a specific species. Thus, they are marked with "???" in Suppl. material 3: Appendix 3. Most of these females were captured on the Bisamberg (n = 5) and chiefly collected Reseda sp. pollen. Two of them (nos. 62 and 64) additionally collected a small amount of pollen from Asteraceae tubuliflorae (13-19%). The other three (nos. 61, 63 and 65) had pollen loads with over 90% Reseda sp. and some amounts of contamination (0.3-2.0%). A female from Spitzerberg near Hainburg (no. 66) also preferred Reseda sp. (99.3%) and a single female (no. 148), for which both the date and location of capture are unknown, had a pure pollen package with Calluna vulgaris pollen (100%).

Phylogeny
The obtained sequences were aligned for comparison (Suppl. material 9: Appendix 9). In the CO1 sequences of the specimens of C. collaris, 27 single base-pair differences were found, which clearly separate them from the other species. These, in turn, show little to no fixed substitutions in their alignment and therefore cannot be separate from each other.
The 21 obtained sequences, as well as 47 reference sequences received from the DNA-Barcode of Life Database (BOLD) (Suppl. material 5: Appendix 5), were used for further analysis by the neighbour-joining tree (NJT) method (Fig. 26). In the retrieved phylogenetic tree, the species C. collaris clusters in two clades strongly supported with Bootstrap values of 99% each and thus appears paraphyletic (as later found out, by a misidentification): clade 1 contains all the specimens from the present study (n = 4), as well as data from BOLD (n = 16), whereas clade 2 consists of two BOLD sequences (KC469653 and KC469654). The other four species (C. brevigena, C. succinctus, C. hederae and C. pannonicus) collapse into one large clade, which is supported by a moderate Bootstrap value of 88% (Fig. 26). They appear as the sister group to one specimen of C. brevigena (no. DQ085546), with a Bootstrap value of 99%.
For a better illustration of the relationships amongst these clustering species, a median joining network was created (Fig. 27). Most of the closely-related specimens share one common haplotype. As in the NJT, the two C. collaris clades are highly distinct. Clade 2 of C. collaris (KC469653 and KC469654), later identified as C. luzhouensis Kuhlmann, 2007, is separated from the main haplotype (C. succinctus, C. brevigena, C. hederae and C. pannonicus) by fifty substitution steps. One specimen of C. succinctus (no. BCZSM-HYM02017) and one specimen of C. brevigena (no. DQ085546) differ from the group by three substitution steps and two hypothetical haplotypes that could not be found in the sample.

Genetic distances
The interspecific distances between the investigated species are lowest between C. brevigena and C. hederae and highest between C. collaris and all the other species with the exception of the outgroup (Colletes creticus) ( Table 7). The intraspecific distance, in turn, is slightly higher than the interspecific distance in C. brevigena, C. succinctus, C. pannonicus and C. hederae. It is the lowest amongst the C. collaris examined in this study (Table 8). Some of the investigated specimens of C. brevigena, C. succinctus and C. hederae have identical sequences and show Figure 26. Neighbour-joining tree of the sequences of the species of the Colletes succinctus group obtained in this study (n = 21, marked in red) and reference data (n = 47) from BOLD with outgroup and Bootstrap values (1,000 re-samples). The specimens KC469653 and KC469654 in BOLD (marked in green), originally assigned to C. collaris, were later identified as a different species, C. luzhouensis Kuhlmann, 2007, that does not belong to the C. succinctus group. The scale bar represents 0.01 substitutions per site. no genetic distance (Table 7). A pairwise genetic distance matrix can be found in Suppl. material 10: Appendix 10.
Including sequence data from BOLD shows no differences. In addition, in this combined dataset, C. collaris is the most differentiated species (Tables 7 and 8). However, two sequences from BOLD, labelled as C. collaris, were highly differentiated resulting in a higher intraspecific distance than the interspecific distances to the other ingroup species and even higher than distances between ingroup and outgroup (Table 7).

Morphology
New features were found for species differentiation in the Austrian C. succinctus group. Nevertheless, there is a pronounced intraspecific variation (between populations of different collecting sites) in all species. This also concerns previously-described diagnostic characters for females (Noskiewicz 1936;Schmidt and Westrich 1993;Burger 2010;Hölzler and Mazzucco 2011), like the wrinkles on the clypeus, the puncturation on the mesonotum Table 7. Genetic mean, minimum (min.) and maximum (max.) p distances (%) between the European species of the Colletes succinctus group, based on the specimens investigated (A) as well as on reference data (B) from the Barcode of Life Databank (BOLD).  and the presence/absence of the reticulate micro-sculpture on the galea. Due to these sometimes very confusing character combinations, it was not possible to clearly assign all specimens of this study to a species. A few female specimens showed combined traits of the species C. succinctus and C. hederae or C. succinctus and C. brevigena, one even a mixture of characters of all three species. This can best be explained by the hypothesis that, in these young species, not all diagnostic characters are yet fixed in all populations. By combining the new features with the already-known characters from literature (Noskiewicz 1936;Verhoeff 1944;Schmidt and Westrich 1993;Hölzler and Mazzucco 2011;Amiet et al. 2014), it was possible to establish identification keys for females from Austria and males from Central Europe. With these identification keys, the species C. succinctus, C. collaris and C. hederae can be reliably distinguished in both sexes. Colletes halophilus and C. pannonicus live in very similar habitats, but were never compared to each other. The initial hypothesis that they could be synonyms was not corroborated: the dorsobasal knob on the membrane of the gonostylus distinguishes C. halophilus from all other Central European species. Colletes brevigena and C. pannonicus remain to be difficult to determine as they are very similar in appearance. Only slight differences of the dorsal shape of the female's fovea facialis, as well as the male's genitalia, provide indications for the two-species hypothesis. The cuticle of the fovea facialis bears many secretory cells and is more strongly developed in females than in males (Schuberth and Schönitzer 1993). The differently shaped dorsal margins of the fovea are often used as a character for species identification in other genera, especially Andrena (Schmid-Egger and Scheuchl 1997).

Morphometry
The determination of females by use of the new identification key is not supported by the discriminant analysis (LDA). Based on their morphometric data, the LDA assigned only about half of all specimens to the same species as previously determined by the authors. For this method, specimens of C. collaris were used as reference species. Colletes collaris possesses several morphological characters (pilosity of propodeum, narrow band of setae on terga etc., compare Amiet et al. 2014) which do not show intraspecific polymorphism. As C. collaris was observed to have been mis-assigned by the discriminant analysis, the character sets, which were used for the LDA, are not suitable to differentiate the species.
In general, the morphometric analysis of the selected head characters and thorax width alone did not prove to be informative enough to distinguish females of the examined species of the C. succinctus group, as, for example, suggested for C. pannonicus by Hölzler and Mazzucco (2011). No morphometric studies have yet been published for the studied species, but other species of the genus Colletes have already been investigated: because of their similar appearance, especially in their puncturation on terga, C. inexpectatus Noskiewicz, 1936 andC. daviesanus Smith, 1846 were regarded as synonyms (Warncke 1978). Prǐdal (1999) was able to verify that both represent independent species, amongst other characters, by the measurements of the male's hind tarsus. Therefore, the present results call for subsequent examinations. Maybe it is possible to gain more information about species differentiation by measuring legs or antennae. Additionally, males should be added to morphometric studies.

Pollen Analysis
In this study, the species of the Colletes succinctus group occurring sympatrically in Austria were both polylectic and oligolectic: the investigated females of C. succinctus, C. collaris and C. hederae showed a polylectic pollen-collecting behaviour, C. pannonicus appeared to be oligolectic, but examination was based on a single population. In addition, a correlation between the filling ratio of the pollen packages and the number of different pollen types could be determined: the larger the pollen load, the more different pollen types could be found.
The present study showed a preference of the "heather bee" C. succinctus for Reseda sp. (hitherto unknown as a pollen source), closely followed by Calluna vulgaris and Asteraceae, whereas Müller and Kuhlmann (2008) found that the species collected pollen on Ericaceae, Araliaceae, Asteraceae and Apiaceae. Interestingly, some specimens of the C. succinctus, collected in Retz, had the expected pollen of Calluna vulgaris, but some others used the pollen of Reseda sp. thriving in close vicinity, although there was no obvious shortage of Calluna flowers. That the females had either pure Calluna vulgaris or Reseda sp. pollen loads can be explained by flower consistency (cf. Waser 1986).
The single analysed female of C. brevigena had a pollen load of pure Reseda sp.; this species is described as polylectic by Müller and Kuhlmann (2008), collecting pollen on a variety of different families. These authors also described C. hederae as polylectic, whereas Bischoff et al. (2005) described the species as oligolectic due to the fact that examined nest cells and pollen loads only showed pollen of Hedera helix. In this study, most of the females collected pure pollen loads from Hedera helix flowers. Only females from Donaupark in Vienna showed polylectic behaviour by adding pollen of Citrus sp. to  (Teppner and Brosch 2015).
Little is known about the pollen-collecting behaviour of C. pannonicus. Field observations led to the assumption that C. pannonicus is strictly oligolectic on Tripolium pannonicum (sea aster) (Hölzler and Mazzucco 2011). The first pollen analysis, conducted within this study, confirmed that C. pannonicus from the same population is oligolectic on Asteraceae. However, as all investigated females were collected on T. pannonicum, the results can either be explained by strict oligolectic behaviour or by flower consistency.
Although the examined females of C. collaris showed a strong preference for Asteraceae, which is in accordance with findings by Müller and Kuhlmann (2008), they also collected pollen of Reseda sp. Asteraceae are omnipresent, provide large amounts of pollen and nectar and flower, depending on the species, from spring to autumn, but they have a low protein and amino acid content (Somerville and Nicol 2006) and a possibly toxic pollen kit (Williams 2003) and the extraction of important nutrients from the pollen plasma is difficult (Peng et al. 1985). Therefore, expensive physiological adaptations of the bees to this pollen type are necessary: for coping with a low protein and amino acid content, for detoxification and for an easier extraction of important nutrients from the pollen plasma (Müller and Kuhlmann 2008). Asteraceae are the perfect nutrient supplier for adapted bees, being available almost without competition. In Colletes, Asteraceae are preferentially collected by oligolectic species, but are largely avoided by the majority of polylectic species; exceptions are foremost found of the Colletes succinctus group: C. succinctus, C. brevigena and C. hede rae (Müller and Kuhlmann 2008).

DNA-barcoding
Only specimens of C. collaris can be clearly separated from the other species. By analysing only sequences of the material of this study, C. collaris forms a monophylum and the sister group to the other analysed species, which collapse into one large clade and show little to no genetic distance to each other. However, after adding sequences from BOLD, C. collaris separates into two clades and forms a paraphylum. This can be explained by checking the two C. collaris sequences that appear secluded in the tree: an additional BOLD blast showed that both specimens belong to Colletes luzhouensis Kuhlmann, 2007, a species native to China, which explains not only the high intraspecies genetic distance of C. collaris, but also the high genetic distances to the other species. Thus, C. collaris forms a monophylum and is the sister group to a clade comprising the remaining studied species of the C. succinctus group. A previous study of Kuhlmann et al. (2009), albeit including only one specimen of C. collaris, analysed the gene fragments CO1 and 28S and achieved the same result: C. collaris represented the sister group to C. intricans, C. succinctus, C. hederae, C. brevigena and C. halophilus. In this study, C. pannonicus was examined for the first time by using DNA barcoding. However, it cannot be distinguished from the other investigated species, except C. collaris. Since the species collapse into one large clade/haplogroup (except C. collaris), it is not possible to assign specimens to species using the CO1 sequence, which is in accordance with previous studies (Kuhlmann et al. 2007(Kuhlmann et al. , 2009. For future investigations of the group, it would be recommended to investigate other genes.

The challenging species Colletes brevigena and C. pannonicus
Colletes brevigena and C. pannonicus proved to be the most challenging species. Due to their similar appearance, it is difficult to distinguish them strictly by morphology. There is a small difference in the female's fovea facialis that was not mentioned in the original description of the holotype of C. pannonicus (Hölzler & Mazzucco, 2011), whereas the previously-stated morphometric differences could not be approved in larger material. The previously undescribed male of C. pannonicus shows only discrete differences in its genitalia. Furthermore, some Austrian specimens of C. brevigena showed different morphological character states than specimens from the Mediterranean. Therefore, both species were investigated more closely.
All examined specimens of C. pannonicus were found solely near to the type locality in the Seewinkel where they can be observed flying around the flowers of Tripolium pannonicum (sea aster). That all known specimens were caught around Lake Neusiedl is surprising, as there are no geographical barriers which would prevent a wider distribution. Specialisation to a distinct habitat, salt meadows, seems the most likely and hitherto accepted explanation that C. pannonicus could not be found elsewhere in Austria. The proposed (Hölzler and Mazzucco 2011) and here confirmed apparent dependence on T. pannonicum could be explained by the fact that hardly any other Asteraceae are blooming at these sites in late autumn. Tripolium pannonicum is a widespread plant found in Europe and in temperate regions of Asia (Euro+Med 2006) and it can be expected that C. pannonicus has a wider distribution than presently known. The seeming endemism is either caused by under-collecting in other suitable areas or, possibly, by confusion with similar species, mainly C. brevigena. Noskiewicz (1936) described C. succinctus ssp. brevigena, based on specimens from a large dis tri bution area spanning from the Balkans, to Crete, Persia and the Cau casus, but many specimens are untraceable. The selec tion of a lectotype was necessary to define this problematic species. The type locality is Crete (Greece).
A clear morphological distinction could be detected between Austrian specimens (from Bisamberg and Ollersdorf, Lower Austria) of C. brevigena and specimens from the Mediterranean region. The Austrian females are unusually large and show a puncturation on terga, mesonotum and mesopleura that is similar to C. succinctus and/or C. hederae. Only some specimens from Spitzerberg (Lower Austria) are more similar to Mediterranean C. brevigena. A linear discriminant analysis (LDA) of morphometric data resulted in clear separation of Austrian and Mediterranean specimens (100%). Subsequently, in a Jackknife re-sampling, only 60% of all specimens could be assigned to their original group (Austrian or Mediterranean). This lower value may be explained by the small number of Austrian specimens (n = 5) in comparison to the higher number of Mediterranean specimens (n = 13). To determine whether Austrian and Mediterranean specimens differ in their measurements or are more similar than assumed in this study, a larger number of samples would be needed for measurement. Unfortunately, due to the rarity of this species in Austria, this was not possible during this study. Zettel et al. (2006) also listed questionable females of C. brevigena in Ollersdorf. These specimens showed a puncturation on their terga that is similar to the puncturation of C. hederae, but as typical for females of C. brevigena, they have longitudinal wrinkles on their clypeus. Therefore, the authors classified them with reservation as C. brevigena (Zettel et al. 2006).
Concluding the findings for C. brevigena, this study raises doubts about the close relationship between specimens/populations of C. brevigena occurring in Austria and specimens from the Mediterranean region. In addition to the morphological differences, also the phenology differs: in the Mediterranean, C. brevigena is bivoltine (Kuhlmann 2003;Standfuss 2009): the first generation flies in May and the second generation later in the year (according to collection data from September to November). In Austria, however, there is only one generation active from August to September (Zettel et al. 2006). It can be assumed that C. brevigena is a Mediterranean species that post-glacially migrated northwards, where it must have adapted to different environmental conditions, most importantly to a shorter warm season. In this case, it would be expected that -like in some other bee species (e.g. Andrena pontica Warncke, 1972; Scheuchl and Willner 2016) -the second generation is omitted because of the longer-lasting development. In the peculiar case of C. brevigena, however, the spring generation is omitted. As most specimens of the genus Colletes in Central Europe spend their diapause (hibernation) as a pre-pupa (in the last larval stage) (Westrich 1989), this delayed development into an imago should be genetically fixed and, therefore, must be a trait that was passed on by its ancestor. A similar case is also known from the species complex of Andrena argentata Smith, 1844. This bivoltine species shows a trans-Palaearctic distribution. In England and Sweden, it either has no spring generation or the summer generation is richer in specimens. Additionally, in this case, it is assumed that the two generations do not belong to the same species (Scheuchl and Willner 2016).
Thus, it would be quite possible that the Austrian specimens in question are of a different species that is very similar to C. brevigena in the Mediterranean. However, it is also possible that C. brevigena shows a highly pronounced geographic variation. The studied populations in Austria and the Mediterranean region are geographically far apart and differ to such an extent that they can be regarded as conspecific only with difficulty. It would be advisable to study C. brevigena populations from intermediate areas, for example, from Hungary or the northern Balkans. Thereby, it may be possible to find transitional morphs that could corroborate the conspecificity of the two morphologically different groups. Both possibilities would merit further investigation.
Integrative approach to separate C. brevigena and C. pannonicus Colletes brevigena and C. pannonicus share a very similar morphological character set. Genetic data (mitochondrial gene CO1) are not useful to differentiate between them. It is difficult to distinguish the two species by the discriminating characters described by Hölzler and Mazzucco (2011), as morphometric data greatly overlap. For example, Hölzler and Mazzucco (2011) stated that the female of C. pannonicus possesses a wider head in relation to the thorax width, when compared to C. brevigena. This was not confirmed by this study. The values of both species greatly overlap, with C. brevigena even showing mean values for a slightly wider head than C. pannonicus. These findings can support the assumption from the previous chapter, that C. pannonicus can easily be misidentified as C. brevigena. In the salt meadows of Seewinkel, however, the two species do not occur sympatrically, but C. pannonicus shares the habitat with C. collaris. Colletes brevigena, on the other hand, is only found in steppe biotopes (Zettel et al. 2006). Due to the few subtle differences, it is quite possible that specimens of C. pannonicus were misidentified as C. brevigena in other areas of south-eastern Europe. Additionally, the oligolectic collecting behaviour on Tripolium pannonicum in Seewinkel could simply be based on the lack of alternative resources. Therefore, it is important to include pollen preferences, as well as habitat preferences, to differentiate between both species.
Specimens without assignment to a species For 21 specimens, it was not possible to assign them to a species, based on their morphological characters alone. They showed mixed characters of several species (C. succinctus, C. brevigena and C. hederae), especially, regarding the puncturation on terga, mesopleura and mesonotum, as well as structures on clypeus and galea.
The intraspecific variation of the species of the C. succinctus group has always been an issue for taxonomists (Noskiewicz 1936;Verhoeff 1944;Schmidt and Westrich 1993;Hölzler and Mazzucco 2011). Some specimens show mixed characters and cannot be clearly identified. Therefore, the ecology (pollen preferences of females and phenology) has been included in species differentiation (Kuhlmann et al. 2007). For example, female no. 148 shows C. succinctus-typical terga, mesopleura and clypeus, but does not have a shiny centre on the mesonotum. Since this was already mentioned by Noskiewicz (1936) as a typical C. succinctus characteristic, further aspects have to be investigated. When examining the pollen package of the specimen, it consists purely of Calluna vulgaris. Thus, the probability is very high that this specimen is a specimen of C. succinctus which, however, did not develop all characters typical for this taxon. In addition, the females (nos. 61-66), which were collected at Bisamberg (Vienna), show combined characters of C. succinctus, C. brevigena and C. hederae. These specimens were collected mid-September on Reseda sp. (pollen loads studied). Although Reseda sp. is only known as a pollen source for C. brevigena and C. collaris in literature (Müller and Kuhlmann 2008) and for C. succinctus in the present study, it is not impossible that C. hederae also collects pollen on this flower. Colletes hederae is a polylectic species, which shows preferences for Hedera helix, but also collects pollen on other plants, should their preferred plant not yet be in bloom (Müller and Kuhlmann 2008;Westrich 2008;Teppner and Brosch 2015). Moreover, flower visits of C. hederae on Reseda sp. have been observed after this study was carried out (H. Zettel, unpubl.).

Conclusion
Based on the results presented here, it can be assumed that the species of the C. succinctus group are either species in statu nascendi or evolutionary of very recent origin. In any case, incomplete lineage sorting, as well as gene flow, might explain the close genetic relationships. This study was able to find further helpful characters for a morphological identification of the Austrian species of the C. succinctus group. The main result is that the species complex C. succinctus-brevigena-hederae-pannonicus is more complicated than assumed by all previous taxonomists (Noskiewicz 1936, Verhoeff 1944, Schmidt and Westrich 1993, Hölzler and Mazzucco 2011, as there is high variation in morphological and ecological characters. For some specimens, it is still difficult to identify them by studying their morphology. Thus, the ecology of the specimens continues to be an important tool for species differentiation. Wanat A, Jaloszyński P, Wanat M (2014)