Dtsch. Entomol. Z. 68 (1) 2021, 101-138 | DOI 10.3897/dez.68.55732 Gy MuseuM TOR BERLIN Morphology, pollen preferences and DNA-barcoding of five Austrian species in the Colletes succinctus group (Hymenoptera, Apidae) Katharina Zenz', Herbert Zettel!, Michael Kuhlmann’, Harald W. Krenn? 1 Natural History Museum Vienna, 2” Zoological Department, Burgring 7, 1010 Vienna, Austria 2 Zoological Museum of Kiel University, Hegewischstrafe 3, 24105 Kiel, Germany 3. University of Vienna, Department of Evolutionary Biology, Integrative Zoology, UZA1, Althanstrape 14, 1090 Vienna, Austria http://zoobank. org/6A568493-F 6B0-448C-A 1CB-0B733577CEFE Corresponding author: Katharina Zenz (katharina.zenz@gmx.net) Academic editor: Michael Ohl # Received 23 June 2020 # Accepted 18 December 2020 Published 2 February 2021 Abstract Most species of the Colletes succinctus group sensu Noskiewicz, 1936 are taxonomically uncertain. This study has chosen an inte- grative approach, including pollen analysis, morphology, male genitalia, morphometry, cuticle sculpture and DNA-barcoding (CO/) to investigate the five species that were reported from Austria. It includes a detailed analysis of the male genitalia and the first de- scription 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. CO/ 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 7ripolium pannonicum. Due to pronounced intraspecific variation, it is assumed that the species of the C. swccinctus group are either species in statu nascendi or very young species. There- fore, it remains important to include ecological data in species identification. Key Words Bee, identification, integrative taxonomy, morphometrics, phylogeny, Colletes hederae, Colletes brevigena, Colletes collaris, Col- letes pannonicus, Colletes halophilus 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 cello- phane nest lining. The female produces a polyester secre- tion 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 (Prosh- chalykin 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; Kuhl- mann 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 (Noskie- wicz 1936). Especially the Colletes succinctus group is notorious for being a taxonomic challenge. It is de- fined by two synapomorphies: two deep lateral pits at sternum 6 (subgenital plate) of the males and a red- brown transparent basal margin of gaster tergum 1 (Noskiewicz 1936, Kuhlmann et al. 2007). Copyright Katharina Zenz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 102 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Col/etes In his detailed revision of Palaearctic species, Noskie- wicz (1936) assigned only two species, Colletes succinc- tus (Linnaeus, 1758) and C. collaris Dours, 1872, and two newly-described subspecies (C. succinctus aegyptiacus and C. succinctus brevigena) to the C. succinctus group. Today, this group comprises 14 species, some of them occurring in temperate Asia and North Africa and seven being native to Europe (Kuhlmann 2000, 2003; Holzler and Mazzucco 2011): Colletes succinctus, C. collaris, C. brevigena Noskiewicz, 1936 (now in the rank of spe- cies), C. halophilus Verhoeff, 1944, C. hederae Schmidt & Westrich, 1993, C. standfussi Kuhlmann, 2003 and C. pan- nonicus Holzler & Mazzucco, 2011. Although the species have different distribution areas in the Palaearctic, all ex- cept C. halophilus (from North Sea coastal habitats; West- rich 1989; Kuhlmann et al. 2007) and C. standfussi (from Greece; Kuhlmann 2003) occur sympatrically in the east- ern parts of Austria (Gusenleitner et al. 2012) (Table 1). According to collection and literature data, the Austri- an species of the C. succinctus group have one generation per year (monovoltine). Although they are all “late-sum- mer bees” (Scheuchl and Willner 2016), collection data suggest that they differ in their phenology. Colletes suc- cinctus 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; Stand- fuss 2009). The holotype of C. pannonicus was collect- ed mid-September (Holzler 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 Austri- an species of this group reportedly show different pollen preferences: the species are described as either polylec- tic, oligolectic or pseudo-oligolectic (Bischoff et al. 2005; Muller and Kuhlmann 2008; Westrich 2008; Teppner and Brosch 2015). Colletes succinctus and C. brevigena show polylectic behaviour (Michener 2007; Miller and Table 1. Distribution of the European species of the Colletes succinctus group. Austrian taxa treated in bold. Distribution Europe and Mediterranean, eastwards to Iran, southwards to North Africa, Egypt and Tunisia (Ascher and Pickering 2011-2018). Widespread in the Palaearctic, temperate Asia except for Southwest Asia; in Europe documented for Central Europe, France and Spain (Westrich 1997, Ascher and Pickering 2020). Coastal habitats of the North Sea (Westrich 1989, Kuhlmann et al. 2007, Ascher and Pickering 2011-2018). Species C. brevigena C. collaris C. halophilus C. hederae Currently spreading from the Mediterranean of Europe to Central and Western Europe, reported from Great Britain, Spain, Italy, Croatia, Greece (Schmid-Egger 1997, Rathjen 1998, Vereecken et al. 2009). C. intricans North Africa, Iberian Peninsula (Ascher and Pickering 2020). C. pannonicus| Endemic to the area around Lake Neusied1, Austria (Hélzler and Mazzucco 2011). Endemic to Thessaly, Greece (Kuhlmann 2003). Southern and central Europe, in Great Britain, in the north as far as Finland and Sweden, in western Asia as far as Kazakhstan (Westrich 1989, Kuhlmann et al. 2007, Ascher and Pickering 2020). C. standfussi C. succinctus dez.pensoft.net Kuhlmann 2008). However, it should be mentioned that C. succinctus, despite being a generalist, prefers heather (Calluna sp.) which leads to its vernacular name “heath- er bee”. Colletes collaris and C. halophilus are typical oligolectic bees, preferring pollen of Asteraceae (West- rich 1997; Kuhlmann et al. 2007; Muller 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 (Muller and Kuhlmann 2008). Colletes hederae was orig- inally 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 expan- sion (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, al- though specimens of the genus Colletes collecting pollen on ivy have been reported for a long time (Richards 1979; Janvier 1979, 1980; Westrich 1989). As the latest addition to the species group, Colletes pannonicus was described from a population using the pollen of 7ripolium pannon- icum (sea aster), supposedly being oligolectic on Astera- ceae (Holzler 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 differenc- es within the Colletes succinctus group (Kuhlmann et al. 2007, 2009; Magnacca and Brown 2012; Dellicour et al. 2014). Misinterpretations of taxa may have led to unreli- able 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 dis- cussions and investigations (e.g. Kuhlmann et al. 2007; Miller and Kuhlmann 2008; Kuhlmann et al. 2009; Magnacca and Brown 2012; Dellicour et al. 2014). The original descriptions of species described after Noskie- wicz’s (1936) revision compare the new taxa only with one previously described (usually sympatric) species, thereby neglecting similar species from other areas. How- ever, with the exceptions of C. collaris and C. standfus- si, which both can be easily recognised, the species are morphologically very difficult to distinguish (Kuhlmann 2003). Consequently, their different phenology, their pol- len 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; H6lzler 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, Dtsch. Entomol. Z. 68 (1) 2021, 101-138 an integrative approach was designed, including morphol- ogy, statistical analyses of morphometrics, pollen analy- ses, aS well as DNA-barcoding, to exclude the possibility of misidentification in previously-studied material and to provide comparison sequences for future investigations. Material and methods Specimens and preparations The study included 270 specimens (Suppl. material 1: Appendix 1) which were either newly collected or pro- vided 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). Col- lecting 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 morphologi- cal 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 atten- tion 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, pho- tography, 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. After- wards, the digital images were stacked with ZerenaStack- er 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 medi- an 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 103 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: ve- locity — 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 pro- gramme 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 spec- imens 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. succinc- tus. 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 dis- tal 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 late- ro-frontal view from the most dorsal point to the most ventral point of the left compound eye; Figure 1. Frontal Measuring distances for a female of the Colletes succinctus group. HL — head length, HW — head width, EL - eye length, UID — upper interocular distance, LID — lower interocular distance, MID — middle interocular distance, CL — clypeus length, CHL — cheek length. dez.pensoft.net 104 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Figure 2. Dorsal measuring distance for a female of the Co/letes succinctus group. TW — thorax width. upper interocular distance (UID) — shortest distance between the dorsal margins of the compound eyes, mea- sured in dorsal view; lower interocular distance (LID) — shortest distance between the ventral margins of the compound eyes, mea- sured in frontal view; median interocular distance (MID) — longest distance between the inner margins of the compound eyes, mea- sured 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 Holzler 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 x 100 Measurements were conducted at different magnifi- cations (24—76.8) using a calibrated LEICA MZ6 bin- ocular microscope with an ocular micrometre and lat- er converted to millimetres. The statistics programme PAST3 (Hammer et al. 2001) was used to conduct prin- cipal component analysis (PCA) and discriminant ana- lysis (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, dez.pensoft.net 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 ana- lysed 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 Muller 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 estimat- ed 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 correla- tion 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 pol- len grains were fixed and dyed with a mixture of Kaisers Glycerine-Gelatine (ROTH) and alkaline Fuchsine. A fter- wards, 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 barcod- ing (Suppl. material 4: Appendix 4). In preparation for DNA extraction, the ethanol, in which some of the ani- mals were stored, had to be washed out with PBS (phos- phate 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 suffi- cient DNA were used for further analysis. For Polymer- ase Chain Reaction (PCR) 25 ul MM Biozym Red HS Taq Master Mix, 21 ul molecular grade water (Sigma Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Aldrich) as well as 1.5 ul of each primer and the respec- tive DNA sample (1-2 ul) 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 1x TBE buffer and agarose gel (1% w/v) was used. In total, 3 ul of the samples, together with 1.5 ul 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 suc- cessfully: 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 anal- yses 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 105 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 electrophero- grams 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) (Ratnas- ingham and Hebert 2007) (Suppl. material 5: Appen- dix 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. Results 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 litera- ture (Noskiewicz 1936; Schmidt and Westrich 1993; Burger 2010; Hoélzler and Mazzucco 2011). Colletes collaris Colletes brevigena Colletes hederae shape of the dorsal end of fovea facialis dorsally extended, with oval- shaped margin at the dorsal end dorsally extended, narrower than in C. succinctus, with slightly pointed apex Colletes pannonicus widening towards dorsal end, with deep, broad and straight dorsal margin widening towards dorsal end, tapering to a medio- lateral point, lateral margin more depressed than mesal margin widening towards dorsal end, rounded dorsal margin cuticle reticulated between puncturation cuticle smooth between puncturation puncturation on frons cuticle smooth between puncturation cuticle smooth between puncturation cuticle smooth between puncturation with smooth centre, variable in size supra-clypeus with smooth centre, variable in size with smooth centre, variable in size dull, with punctures of the shiny, with larger punctures than on clypeus same size as on clypeus clypeus densely and coarsely punctured coarsely punctured, no longitudinally wrinkled, distally with inwardly distal margin of clypeus with lateral longitudinal wrinkles,| transverse furrow at the distal margin of clypeus inclined, longitudinal not exceeding mandible slightly inclining inwards before | lower end, with longitudinal | exceeding mandible base wrinkles base, longitudinal the basal end, with transverse wrinkles, inclining mesad wrinkles, latero-distally furrow at the lower end towards the end slightly inclined mesad galea shiny, microstructure-free dull and reticulated between | dull and reticulated part dull and reticulated dull and reticulated between sensilla sensilla between sensilla restricted to between sensilla between sensilla the distal half palpi mesonotum strongly punctured, with shiny coarsely punctured, with densely punctured more finely punctured than | densely punctured, with centre black-brown hairs in its in C. brevigena, with a | a shiny centre variable centre shiny centre variable in in its size its size mesopleura puncturation with distances at puncturation with shiny densely punctured, densely punctured, densely punctured, most the diameter of a puncture, | intervals that are larger than | (sporadically) punctures can | punctures merge and form | punctures can merge and usually smaller diameters of punctures merge and form wrinkles wrinkles form wrinkles propodeum hairy hairless centre of declivity hairy hairy hairy setae on terga | broad stripes of setae at posterior | narrow stripes of setae at broad stripes of setae at broad stripes of setae at | broad stripes of setae at margins posterior margins posterior margins posterior margins posterior margins tergum 1 finely and densely punctured, more deeply and coarsely | more coarsely, densely and more finely punctured more finely and with distances of punctures as long as a diameter of puncture punctured than in C. succinctus finely punctured than in C. succinctus (punctures than in other species, less densely punctured than in C. halophilus densely punctured than in C. brevigena, (sporadically) distance of punctures as long as a diameter of puncture with very short distances in between) dez.pensoft.net 106 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Figure 3. Important morphological characters for species differentiation of the Austrian species of the Colletes succinctus group: (A-C) Female C. succinctus: A. Fovea facialis; B. Terga 1 and 2; C. Proboscis; (D-F) Female: C. co//aris: D. Fovea facialis; E. Ter- ga | and 2; F. Cuticle of frons; (G—I) Female C. brevigena: G. Fovea facialis; H. Terga | and 2; I. Cuticle of mesopleura; (J—L) Female C. hederae: J. Fovea facialis; K. Terga | and 2; L. Proboscis; (M—O) Female C. pannonicus: M. Fovea facialis; N. Terga | and 2; O. Clypeus. ant — antenna, ce — complex eye, fov — fovea facialis, oc — lateral ocellus, mxp — maxillary palpus, T1 — tergum 1, T2 — tergum 2. dez.pensoft.net Dtsch. Entomol. Z. 68 (1) 2021, 101-138 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 gen- italia. 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 (Igr). 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 scle- rotised 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). Sternum 8: sub-rhomboidal. Anterior spiculum (spi) strongly elongated. Lateral processes (Ipr) bifid, bearing muscle attachments. Distal process (dpr) curved ventral- ly, with dense tuft of long setae on dorsal margin. Genital capsule: stout, most parts, including gonobase, heavily sclerotised. Gonopod (gpo) smooth and hairless, dorsolaterally with oblique depression; ventrally fused with gonostylus, dorsally separated from it by a deep fis- sure. Gonostylus (gst) curved ventrally; mesoventrally with a ridge bearing a row of setae (rs), distally with a hairy, medially curved gonostylus membrane (gme) of approximately triangular shape. Volsella (vol) strongly developed: basivolsella (bvo) short; digitus (dig) and cus- pis (cus) both plate-shaped, their opposing surfaces with numerous stout, short teeth. Penisvalva (val) with slender base; distal part curved ventrally, with several modifica- tions: a heavily-sclerotised mesodorsal ridge (mdr), a laterodorsal membrane (Idm), a basolateral groove (blg) often surrounded by stout spines, and a lateral area (lar) bearing numerous, often spine-like setae. 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 homolo- gised with the structures of other species groups. Specific characters, C. collaris: Sternum 6: Stout, lateral edges extended to posterior, appears long and broad. Little hair on the disc, blunt 107 comers weakly developed. Lateral grooves (Igr) small and spherical (Fig. 4A). — Sternum 7: Short, transverse- ly oval paired wings (wg) with densely hairy basal arm (bs), without membrane and medial process. Strongly developed shoulders (sh) (Fig. 4B). — Sternum 8: Strong- ly sclerotised median process of the anterior spiculum (spi) reaching the posterior apex, with short bifurcation in its centre. Distal process (dpr) sitting on small outwardly curved shoulders (Fig. 4C, D). — Genital capsule: Promi- nent volsella (vol) with slender digitus (Fig. 5B, D). Specific characters, C. succinctus: Sternum 6: Prominent inwardly inclined lateral grooves (Igr), long and oval shaped. Weakly pronounced blunt corners on convex hind margin and oval-shaped translu- cent area (ota), 1.5—2 times larger than lateral grooves (Igr) (Fig. 6A). — Sternum 7: Wide in appearance, with strong shoulders (sh). Basal arm (bs) of the wing (wg) with setae, especially in its proximal half. Horizontally directed distal margin of membrane (mbr) with long setae (Fig. 6B). — Sternum 8: Distal process (dpr) bas- al with wide outwardly curved shoulders. Sclerotised anterior spiculum (spi) with strong central bifurcation (Fig. 6C, D). — Genital capsule: No species-specific char- acters were detected (Fig. 7). Specific characters, C. halophilus: Sternum 6: Prominent oval-shaped translucent area with hairless centre (ota) (Fig. 8A). — Sternum 7: Hind mar- gins of membrane (mbr) of distal processes of sternum 7 (wg) almost straight (Fig. 8B). — Sternum 8: Distal process (dpr) basal with outwardly curved shoulders. Weakly pronounced bifurcation of median spiculum (spi) (Fig. 8C, D). — Genital capsule: In lateral view, membrane of gonostylus (gme) of genital arched dorsally, with dor- sobasal knob and densely hairy (Fig. 9D). Specific characters, C. hederae: Sternum 7: Basal arm (bs) narrow at base and widen- ing distally, densely hairy. Membrane (mbr) convexly curved, proximal part with dense setae, short setae at distal edge. Medial bifid elevation (mel) strongly pro- nounced (Fig. 10B). — Sterna 6, 8 and genital capsule: no species-specific characters were detected (Figs 10A, Cea py For species-specific characters of the male terminal struc- tures and genitalia of C. brevigena, see the next chapter. Lectotype designation of C. brevigena Noskiewicz (1936) described C. succinctus ssp. brevigena, based on specimens from a large distribution area: dez.pensoft.net 108 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes spi Figure 4. Male terminal structures of Colletes collaris, specimen no. 16 (LM): A. Ventral view of sternum 6; B. Ventral view of sternum 7; C. Ventral view of sternum 8; D. Lateral view of sternum 8. ba — basal arm, br — bridge, bs — base, dpr — distal process, Igr — lateral grooves, Ipr — lateral process, mbr — membrane, mel — median elevation, mp — median process, ota — oval-shaped translucent area, sh — shoulder, spi — spiculum, wg — wing. 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 His- tory, 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 3 succinctus L. ssp. brevigena Nosk. det. Noskiewicz.”, dez.pensoft.net which were studied by Noskiewicz in the course of pre- paring his monograph and, therefore, are putative syn- types. For the reason of taxonomic stability, we select a male (no. 270) from Crete (Greece) as the lectotype (see Figs 12-14). Arguments for selecting this speci- men were (1) its almost perfect condition and (2) avoid- ance of a type locality in the southern Caucasus which would make future molecular research on this taxon more difficult. Lectotype (4, present designation, Natural History Museum Vienna): “Oertzen Creta 1884.” [= Crete Island, Greece; printed], “Colletes 3 succinctus L. ssp. brevige- na Nosk. det. Noskiewicz.” [mostly handwritten], “270” [handwritten], “Colletes brevigena Noskiewicz, 1936 det. Dtsch. Entomol. Z. 68 (1) 2021, 101-138 109 A val B val gst rs gpo 0.5mm Figure 5. Male genitalia of Co//etes collaris, specimen no. 16 (LM): A. Dorsal view of genital capsule; B. Ventral view of genital cap- sule; C. Lateral view of genital capsule; D. Mediolateral view of gonopod and gonostylus with volsella; E. Lateral view of penis val- va. blg — basolateral groove, bvo — basivolsella, us — cuspis, dig — digitus, gme — gonostylus membrane, gpo — gonopod, gst — gono- stylus, lar — lateral area, ldm — laterodorsal membrane, mdr — mesodorsal ridge, rs — row of setae, val — penis valva, vol — volsella. dez.pensoft.net Ane, Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes = S 2 rm) Figure 6. Male terminal structures of Colletes succinctus, specimen no. 243 (LM): A. Ventral view of sternum 6; B. Ventral view of sternum 7; C. Ventral view of sternum 8; D. Lateral view of sternum 8. ba — basal arm, br — bridge, bs — base, dpr — distal process, Igr — lateral grooves, Ipr — lateral process, mbr — membrane, mel — median elevation, mp — median process, ota — oval-shaped translucent area, sh — shoulder, spi — spiculum, wg — wing. K. Zenz 2018* [printed], “Lectotype Colletes succinctus brevigena Noskiewicz, 1936 des. Katharina Zenz et al. 2020” [printed on red paper]. Paralectotypes deposited in the Natural History Museum Vienna: 1 4 (legs partly broken) labelled as the lectotype; 1 & (legs and antennae partly broken) labelled “Pola Schlett.” [= Pula, today in Croatia, leg. Schletterer; printed]; 8 3d (in various conditions) labelled “Transkauk. Helenendorf 1886.” [= Goygol in Azerbaijan; printed “6” on some labels handwritten]; all specimens with Noskiewicz’s and Zenz’s identification label as the lectotype and a type label “Para- lectotype Colletes succinctus brevigena Noskiewicz, 1936 labelled by K. Zenz 2020” [printed on red paper]. dez.pensoft.net Specific characters of terminal structures and genita- lia of the male C. brevigena: Sternum 7: Very pronounced shoulders (sh) with broad basal arms (ba), densely hairy. Concavely curved dis- tal margin of membrane (mbr) bearing few hairs (Fig. 13B). — Sternum 8: Spiculum (sp) with a broad, sclerotised bifurcation (Fig. 13C, D). — Sternum 6 and genital capsule: no species-specific characters were de- tected (Figs 13A, 14). For species-specific characters of the male termi- nal structures and genitalia of C. pannonicus, see the next chapter. Dtsch. Entomol. Z. 68 (1) 2021, 101-138 val 0.5 mm Figure 7. Male genitalia of Colletes succinctus, specimen no. 243 (LM): A. Dorsal view of genital capsule; B. Ventral view of genital capsule; C. Lateral view of genital capsule; D. Mediolateral view of gonopod and gonostylus with volsella; E. Lateral view of penis valva. blg — basolateral groove, bvo — basivolsella, cus — cuspis, dig — digitus, gme — gonostylus membrane, gpo — gonopod, gst — gonostylus, lar — lateral area, ldm — laterodorsal membrane, mdr — mesodorsal ridge, rs — row of setae, val — penis valva, vol — volsella. dez.pensoft.net 112 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Cc 0.5mm spi Figure 8. Male terminal structures of Colletes halophilus, specimen no. 180 (LM): A. Ventral view of sternum 6; B. Ventral view of sternum 7; C. Ventral view of sternum 8; D. Lateral view of sternum 8. ba — basal arm, br — bridge, bs — base, dpr — distal process, Igr — lateral grooves, Ipr — lateral process, mbr — membrane, mel — median elevation, mp — median process, ota — oval-shaped translucent area, sh — shoulder, spi — spiculum, wg — wing. First description of the male of C. pannonicus Colletes pannonicus 1s a recently described species that has been solely found in the Seewinkel near Lake Ne- usied| (Holzler 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 fe- males in Seewinkel on 7ripolium pannonicum (sea aster). Examined material. Austria, Burgenland: 1 <4, Po- dersdorf, 4.9.1991, leg. M. Madl, coll. H. Zettel (spec. no. 174); 1 3, Illmitz, Hélle; 4.9.2006, leg. & coll. H. dez.pensoft.net Wiesbauer (spec.no. 166), 1 3, Podersdorf, 9.9.2012, leg. & coll. H. Wiesbauer (spec.no. 175); 1 4, Podersdorf; 29. 8.2014, leg. & coll. H. Wiesbauer (spec.no. 176); 2 64, IlImitz, Holle, 29-30.8.2014, leg. & coll. H. Wiesbauer (spec.no. 167-168); 1 3, Neusiedl am See, Kalvarien- berg, 47°56'33.69"N, 16°51'39.23"E, 8. 9.2016, leg. & coll. L.W. Gunczy (spec.no. 171). Description. Face with long yellow-whitish hair. Galea reticulated between sensilla, segments of max- illary 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 Dtsch. Entomol. Z. 68 (1) 2021, 101-138 aks A gst gst vol gpo E Z E 2 9 o i=) Cc E = pl —) Figure 9. Male genitalia of Colletes halophilus, specimen no. 180 (LM): A. Dorsal view of genital capsule; B. Ventral view of genital capsule; C. Lateral view of genital capsule; D. Mediolateral view of gonopod and gonostylus with volsella; E. Lateral view of penis valva. blg — basolateral groove, bvo — basivolsella, cus — cuspis, dig — digitus, gme — gonostylus membrane, gpo — gonopod, gst — gonostylus, lar — lateral area, lIdm — laterodorsal membrane, mdr — mesodorsal ridge, rs — row of setae, val — penis valva, vol — volsella. dez.pensoft.net 114 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Cc dpr 0.5mm Figure 10. Male terminal structures of Colletes hederae, specimen no. 197 (LM): A. Ventral view of sternum 6; B. Ventral view of sternum 7; C. Ventral view of sternum 8; D. Lateral view of sternum 8. ba — basal arm, br — bridge, bs — base, dpr — distal process, Igr — lateral grooves, Ipr — 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 colour- ed. Stripes of setae on terga yellow-whitish. Punctures on tergum | large, but smaller than on mesonotum. Ter- gum | 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 yel- low hair. Tergum 2 densely punctured, sporadically with intervals of the size of one puncture diameter; cuticle shiny (Fig. 15D). Specific characters of terminal structures and genita- lia of the male C. pannonicus: Sternum 6: Lateral grooves (Igr) oval and large (Fig. 16A). — Sternum 7: Slender wings (wg), with setae on their dez.pensoft.net proximal half. Hairless membrane (mbr), sigmoid-curved hind margin bearing short hairs (Fig. 16B). — Sternum 8: Distal process (dpr) basal with outwardly curved shoul- ders, with sclerotised, short and discontinuous median bifurcation (Fig. 16C). — Genital capsule: no species-spe- cific characters were detected (Fig. 17). 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 sub- jected to a detailed examination and determined according to the following identification keys for females and males: Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Wi A B gst mdr 0.5mm gme 0.5mm Figure 11. Male genitalia of Colletes hederae, specimen no. 197 (LM): A. Dorsal view of genital capsule; B. Ventral view of gen- ital capsule; C. Lateral view of genital capsule; D. Mediolateral view of gonopod and gonostylus with volsella; E. Lateral view of penis valva. blg — basolateral groove, bvo — basivolsella, cus — cuspis, dig — digitus, gme — gonostylus membrane, gpo — gonopod, gst — gonostylus, lar — lateral area, lIdm — laterodorsal membrane, mdr — mesodorsal ridge, rs — row of setae, val — penis valva, vol — volsella. dez.pensoft.net KS: Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Figure 12. Male of Colletes brevigena, lectotype: A. Lateral view; B. Dorsal view; C. Frontal view; D. Terga 1 and 2. T1 — tergum 1, T2 — tergum 2. 0.5 mm Figure 13. Male terminal structures of Colletes brevigena, specimen no. 270 (LM): A. Ventral view of sternum 6; B. Ventral view of sternum 7; C. Ventral view of sternum 8; D. Lateral view of sternum 8. ba — basal arm, br — bridge, bs — base, dpr — distal process, Igr — lateral grooves, Ipr — lateral process, mbr — membrane, mel — median elevation, mp — median process, ota — oval-shaped translucent area, sh — shoulder, spi — spiculum, wg — wing. dez.pensoft.net Dtsch. Entomol. Z. 68 (1) 2021, 101-138 abel gst vol Figure 14. Male genitalia of Colletes brevigena, specimen no. 270 (LM): A. Dorsal view of genital capsule; B. Ventral view of genital capsule; C. Lateral view of genital capsule; D. Mediolateral view of gonopod and gonostylus with volsella; E. Lateral view of penis valva. lg — basolateral groove, bvo — basivolsella, cus — cuspis, dig — digitus, gzme — gonostylus membrane, gpo — gonopod, gst — gonostylus, lar — lateral area, lIdm — laterodorsal membrane, mdr — mesodorsal ridge, rs — row of setae, val — penis valva, vol — volsella. dez.pensoft.net 118 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Figure 15. Male of Colletes pannonicus: A. Lateral view; B. Dorsal view; C. Frontal view; D. Terga 1 and 2. T1 — tergum 1, T2 — tergum 2. 0.5mm Figure 16. Male terminal structures of Colletes pannonicus, specimen no. 171 (LM): A. Ventral view of sternum 6; B. Ventral view of sternum 7; C. Ventral view of sternum 8; D. Lateral view of sternum 8. ba — basal arm, br — bridge, bs — base, dpr — distal pro- cess, Igr — lateral grooves, Ipr — lateral process, mbr — membrane, mel — median elevation, mp — median process, ota — oval-shaped translucent area, sh — shoulder, spi — spiculum, wg — wing. dez.pensoft.net Dtsch. Entomol. Z. 68 (1) 2021, 101-138 ES gst 0.5mm 0.5mm Figure 17. Male genitalia of Colletes pannonicus, specimen no. 171 (LM): A. Dorsal view of genital capsule; B. Ventral view of genital capsule; C. Lateral view of genital capsule; D. Mediolateral view of gonopod and gonostylus with volsella; E. Lateral view of penis valva. blg — basolateral groove, bvo — basivolsella, cus — cuspis, dig — digitus, gme — gonostylus membrane, gpo — gono- pod, gst — gonostylus, lar — lateral area, ldm — laterodorsal membrane, mdr — mesodorsal ridge, rs — row of setae, val — penis valva, vol — volsella. dez.pensoft.net 120 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Col/etes 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 hair- less 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 pointediapex Criss. SDs LEB) Jen tars Pee ae nee Ae ee Ne a Be OUR Se ee a RO Ltr eee aon C. collaris — stripes. or-hair-at.the: posteriorumaregins of easter. terga: Widest. 216s RRA pices pM Epic dee sls SEW Lael gnecics 2 2 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). Mesonotum densely punctured with shiny centre, variable in its size. Mesopleura densely punctured, punctures can merge and form wrinkles. Galea with microstructure between sensilla (Fig. 19C). Upper end of fovea facialis rounded, slighty kicineyeshapecs@hiosess: (RO): ..mees hn sensed eserente ue tne nn tas sel gavert ody AR co A Ral ee ee ONRE Yul Aorta jPod oan? C. hederae — Wrinkles on the clypeus straight, longitudinal or slightly inclined mesally near apex, never meeting each other in middle...3 =) Galea between sensilla shiny, without microreticulation. Segments of the maxillary palpi short and stout (Figs 3C, 20C, D). Mesonotum densely punctured with shiny area in its centre. Mesopleura densely punctured with scattered intervals of the diameter of puncture. Tergum 1 sparsely punctured and slowly shrinking towards the distal end, distance of puncture on disc as long as diameter of punctures. Tergum 2 coarse and densely, oval punctured (Fig. 3B). Clypeus with longitudinal wrinkles, at most slightly inclined mesally at distal end; with transverse, long furrow at anterior margin (Fig. 20A). Fovea facialis elongated, narrow, with oval-shaped margin at the upper end (Figs 3A, 20A)....... C. succinctus — Galea with microreticulation between sensilla. Segments of maxillary palpi strongly elongated .................cccccseeeeeeeeees 4 sil Figure 18. Head of a female of Colletes collaris, no. 190 (SEM): A. Latero-frontal view of head; B. Upper part of fovea facialis in dorsal view, right antenna on the left; C. Detailed view of setae and microstructure of galea; D. Latero-dorsal view of proboscis. ant — antenna, ce — complex eye, cl — clypeus, fov — fovea facialis, ga — galea, gl — glossa, lap — labial palp, mxp — maxillary palp, pgl — paraglossa, scl — supra-clypeus, sen — sensilla. dez.pensoft.net Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Al Ba Whit rn as al 200 ym Figure 19. Head of a female of Colletes hederae, no. 202 (SEM): A. Latero-frontal view of head; B. Upper part of fovea facialis in dorsal view, lateral ocellus on the left; C. Detailed view of setae and microstructure of galea; D. Latero-dorsal view of proboscis. ce — complex eye, cl — clypeus, fov — fovea facialis, ga — galea, gl — glossa, mxp — maxillary palp, oc — lateral ocellus, pgl — para- glossa, scl — supra-clypeus, sen — sensilla. Fovea facialis widening towards upper end, with deep, broad and straight margin (Figs. 3M and 21B). Mesonotum densely punctured, distance of punctures on disc up to twice as long as diameter of punctures, the sparsely punctured area Can vary in its size. Mesopleura densely punctured. Tergum 1 with dense, large punctures, (sporadically) distance of puncture as long as diameter of punctures. Tergum 2 very densely punctured (Fig. 3N). Clypeus with longitudinal wrinkles, distal wrinkles slightly inclined to the middle (Figs. 30 and 21A).............ccccccccceccece ec eeeeeeeteeseeaes C, pannonicus Fovea facialis widening towards the upper end and tapering to a medio-lateral point, outer margin more depressed than inner margin (Figs 3G, 22B). Mesonotum and mesopleura densely punctured (Fig. 31). Tergum 1 densely punctured, (sporadically) with distances of punctures as long as diameter of punctures. Tergum 2 punctured (Fig. 3H). Clypeus longiieinal hy whinkedtCele sie Ay ns: ee sagen cy att eons reas Sa Oey teres GIST RCE Beret HSRC SR AAALee JA tN, C. brevigena Identification key to males of the Central European species of the C. succinctus group 1 Narrow stripes of hair at the posterior margin of the terga. Propodeum hairless in centre of declivity. Distal processes of sternum 7 short (wg), transverse-oval, medially not extended, but broadly separated from each other (mel), completely densely haired (Fig. 4B). Mesonotum with dense puncturation. Mesopleura sporadically punctured, with intervals twice the size of a diameter. Galea with microstructure between sensilla. Stripes of hair on terga narrowed towards the middle. Ter- gum 1 densely punctured, partly with diameter-sized intervals in-between punctures. Tergum 2 with coarse puncturation, intervals about the size of 1-2 diameters. Tergum 3 coarsely punctured with over diameter-sized intervals ......... C. collaris Broadtsiripessot half al (hes poOSlenonminiaroinOte teeter iat §, Secs ae once clic teath oe eee AAP e Se odoinn d Bee cies cc cmntud veh Solna, eee o Galea between sensilla shiny without microreticulation. Segments of the maxillary palpi short and stout. Hind margins of membrane (mbr) of distal processes of sternum 7 (wg) almost straight and with long setae. Wings (wg) very broad (Fig. 6B). Mesonotum coarsely punctured, with smooth area in its centre. Mesopleura coarsely and densely punctured, with diameter-sized distances of punctures. Tergum 1 densely punctured, with intervals of a puncture diameter. Punc- turation of tergum 2 roundish, smaller than on tergum 1. Tergum 3 puncturation with intervals of double or triple CAR CLCS Haifa. ner sin ceaaatnoun ces ddecbichh ae aauertehhay eelshathe ee hahaa ney sdeliane ce odectbhuae es iid hare Apap wae del dah ens dude aide Loh aes hihi C. succinctus Cuticle of galea between sensilla reticulated. Segments of the maxillary palpi long and slender. Hind margins of the wing-shaped processes of sternum 7 (wg) concave and with short setae. WINGS (WG) NALOW ...........cc cee ee eee eea ee eeeeaeeeenes 3 dez.pensoft.net 122 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes In lateral view membrane of gonostylus (gme) of genital arched dorsally, with dorsobasal knob and densely haired (Fig. 9D). Mesonotum coarsely punctured, with smooth area in its centre, varying in its size. Mesopleura very densely punctured, punctures merge into each other, wrinkled. Galea with reticulate structure between sensilla. Tergum 1 dense- ly punctured, intervals about the size of a diameter. Puncturation of tergum 2 round and dense, of tergum 3 slit-shaped NESE OAS Oo tte! feet lng’ iobtsoe seem oe vigaadie sxe mension nahiot ere eck cate ppltgansetae eet dcahts Stee tak raha & ihc t eal aver aches tills Mesahinles C. halophilus In lateral view, gonostylus .base-cf genital straightiand shainy: :8.0.8 Liss se dB an cdanlcjehins aha M een cline (larcgalels decent OS categinclee ahaa J 4 Base of basal arms (ba) of wings only half as wide as apex. Membrane of wings (mbr) concave, proximally densely hairy (Fig. LOB). Mesonotum densely punctured, sometimes with a smooth area in its centre. Mesopleura densely punctured, slightly wrinkled. Galea with reticulate structure between sensilla. Tergum 1 densely and finely punctured. Puncturation on tergum 2 dense and oval-shaped, finer than on tergum 1, of tergum 3 densely and slit-like structured...... C. hederae Base-of. 'basal-arms -(ba)-of wings: almost’ as DrOad! AS APOK:e eee cise ss canes tates cesas nnv'eivnd hemes Helves wen ee niles hogron KebSlbawenmeeneeded dete & 5 Basal arms (ba) completely densely hairy, membrane (mbr) sparsely hairy (Fig. 13B). Mesonotum coarsely punctured, sometimes with a smooth area of varying size in its middle. Mesopleura very densely punctured. Galea with reticulate structure between sensilla. Tergum 1 with coarse and large punctures. Puncturation of tergum 2 dense and roundish, SUSAN Cer SUL AESU Ne. a el 21 24 U1 gy Alban enn nnes uc Aner Tt ek anes NL CeRrrtnes Lente A, oe ins chen I + re Pee C. brevigena Basal arms (ba) haired in lower half, membrane (mbr) hairless (Fig. 16B). Puncturation on mesonotum dense and coarse, sometimes with shiny centre. Mesopleura densely punctured, sometimes with wrinkles. Galea with reticulate microstructure between sensilla. Tergum 1 coarse and large punctured. Tergum 2 coarse and densely punctured, round ANG ASIMAler LAM OMS U I: Le a ee Pes ed a tao Or at chara eter ae mason Ritaevae Aromat etd naan Scale astaalt atart Pacem Saguey C, pannonicus Non-assignable specimens the help of the new key presented herein. A total of 21 (19 females and two males) of the 270 specimens were assigned All studied specimens were subjected to a control of their — to another species of the C. succinctus group than previous- species affiliation. The specimens were re-identified with — ly. Three males were excluded from the Colletes succinctus 50 pm pgl 200 um Figure 20. Head of a female of Colletes succinctus, no. 237 (SEM): A. Latero-frontal view of head; B. Upper part of fovea facialis in dorsal view, lateral ocellus on the left; C. Detailed view of setae and microstructure of galea; D. Latero-dorsal view of proboscis. ant — antenna, ce — complex eye, cl — clypeus, fov — fovea facialis, ga — galea, gl — glossa, mxp — maxillary palp, oc — ocellus, pgl — paraglossa, sen — sensilla, scl — supra-clypeus. dez.pensoft.net Dtsch. Entomol. Z. 68 (1) 2021, 101-138 123 Figure 21. Head of a female of Co/letes pannonicus, no. 232 (SEM): A. Latero-frontal view of head; B. Upper part of fovea facialis in dorsal view, lateral ocellus on the left; C. Detailed view of setae and microstructure of galea; D. Latero-dorsal view of proboscis. cl — clypeus, ce — complex eye, fov — fovea facialis, ga — galea, gl — glossa, lap — labial palp, mxp — maxillary palp, oc — lateral ocellus, pgl — paraglossa, sen — sensilla, scl — supra-clypeus. Figure 22. Head of a female of Colletes brevigena, no. 190 (SEM): A. Latero-frontal view of head; B. Upper part of fovea facialis in dorsal view, lateral ocellus and antenna on the left; C. Detailed view of setae and microstructure of galea; D. Latero-dorsal view of proboscis. ant — antenna, ce — complex eye, cl — clypeus, fov — fovea facialis, oc — lateral ocellus, sen — sensilla, scl — supra-clypeus. dez.pensoft.net 124 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Table 3. Mean, maximum and minimum values of all measured distances (in millimetres) for six European species of the Colletes suc- cinctus group (females; n = 103). Measured distances: (HL) head length, (HW) head width, (EL) eye length, (CL) clypeus length, (UID) upper interocular distance, (LID) lower interocular distance, (MID) middle interocular distance, (CHL) cheek length, (TW) thorax width. C. succinctus (n= 26) — C. collaris (n = 20) Mean (HL) 2.60 2.69 2.73 Min. (HL) 2.35 2.47 2.51 Max. (HL) 2.76 3.00 2.92 Mean (HW) 3.39 3.42 3.60 Min. (HW) 3.06 3.19 3.28 Max. (HW) 3.70 3.83 3.9 Mean (EL) 2.00 2.00 2.17 Min. (EL) 1.88 1.85 1.98 Max. (EL) 2.24 2.24 231 Mean (CL) 1.07 1.10 1.13 Min. (CL) 0.83 0.95 1.03 Max. (CL) 1.21 1.24 131 Mean (UID) 1.99 2.03 2.12 Min. (UID) 1.55 1.86 1.91 Max. (UID) oAy, 2.27 2.27 Mean (LID) 1.80 1.81 1.50 Min. (LID) 1.34 1.70 1.70 Max. (LID) 2.04 1.99 2.11 Mean (MID) 2.36 2.38 2.52 Min. (MID) 1.80 D199 DED Max. (MID) 2.63 2.68 2.73 Mean (CHL) 0.14 0.15 0.13 Min. (CHL) 0.10 0.11 0.08 Max. (CHL) 0.16 0.20 0.29 Mean (TW) 2.61 2.54 2.82 Min. (TW) 2.25 2.19 2.58 Max. (TW) 2.89 2.99 3.20 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: Ap- pendix 6). These aberrant specimens showed either a mix of species-specific characters of C. succinctus and C. bre- vigena 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 Aus- trian 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. Morphometric measurements (females) Variation within species Identified females of all species showed a pronounced in- traspecific variation (Table 3). Especially, the head width (HW) seems to be very variable in all species. The in- traspecific variation is most pronounced in C. succinc- tus. Here, the females differ very strongly in all mea- suring distances with the exception of the cheek length (CHL). Colletes collaris and C. halophilus, on the oth- er hand, show a pronounced intraspecific variability in their head length (HL) and width (HW) and C. brevigena dez.pensoft.net C. brevigena (n = 14) C. halophilus (n=10) ~~ C. hederae (n= 22) — C. pannonicus (n = 11) 2.76 2.90 2.60 2.59 2.59 2.47 3.05 3.09 2.76 3.64 32/2 3.3 3.44 3.47 3.28 3.83 3.99 3.64 2.13 2.19 2.05 1.98 2.05 1.92 2:27 2.31 2.14 1.17 1.23 1.10 1.06 1.13 1.03 1.31 1.34 1.16 2.22 2.15 2.08 2.06 1.99 1.96 2.32 2.35 2.17 2.00 2.04 1.89 1.83 1.93 1.80 2.17 2.19 1.96 2.62 2.6 2.45 2.42 2.47 2.29 2.81 2.84 255 0.15 0.16 0.14 0.13 0.11 0.11 0.16 0.20 0.15 2.8 2.73 2.64 253 2.47 2.53 2.99 2.99 2.99 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 poly- morphism, although the specimens differed to a large ex- tent 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 dis- tance 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 speci- mens 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 ocu- lar distance, lower ocular distance and middle ocular dis- tance. 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 com- ponent analysis (PCA). Based on 1,000 bootstrap re-sam- plings, 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. The loadings of the nine measuring distances show that PC] (principal component 1) correlates with all variables Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Table 4. Loadings of principal component 1 (PC1), PC2 and PC3 for each measured distance of the six European members of the Colletes succinctus group. Measured distances: (HL) head length, (HW) head width, (EL) eye length, (CL) clypeus length, (UID) upper interocular distance, (LID) lower interoc- ular distance, (MID) middle interocular distance, (CHL) cheek length, (TW) thorax width. PCI PC2 PC3 HL (log) 0.313 0.071 -0.716 HW (log 0.375 -0,102 -0.140 EL (log) 0.359 -0.105 -0,345 CL (log) 0.348 0.170 0.082 UID (log) 0.360 0.001 0.334 LID (log) 0.378 -0,028 0.123 MID (log) 0.376 0.022 0.197 CHL (log) 0.074 0.941 0.090 TW (log) 0.303 -0.241 0.411 except for variable CHL (cheek length) (Table 4). There- fore, 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 nega- tively influenced by HL (Table 4). Thus, specimens with a wider thorax tend to have a shorter head. Fig. 23 shows principal components | 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 speci- mens of C. hederae and C. succinctus overlap only mini- mally. 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. 125 Table 5. Result of the linear discriminant analysis of the spe- cies Colletes brevigena, which 1s divided into the hypothetical groups C. brevigena MED (with Mediterranean origin) and C. brevigena A (Austrian specimens), including Jackknife re-sam- pling (1,000). Species in bold letters were classified differently from the hypothetical assignment. Point Given group Classification Jackknifed 42 C. brevigena MED C. brevigena MED C. brevigena A 44 C. brevigena MED C. brevigena MED C. brevigena MED 45 C. brevigena MED C. brevigena MED C. brevigena A 53 C. brevigena MED C. brevigena MED C. brevigena MED 70 C. brevigena MED C. brevigena MED C. brevigena MED 71 C. brevigena MED C. brevigena MED C. brevigena MED 74 C. brevigena MED C. brevigena MED C. brevigena A 75 C. brevigena MED C. brevigena MED C. brevigena A 76 C. brevigena MED C. brevigena MED C. brevigena MED 77 C. brevigena MED C. brevigena MED C. brevigena MED 78 C. brevigena MED C. brevigena MED C. brevigena MED 79 C. brevigena MED C. brevigena MED C. brevigena MED 161 C. brevigena MED C. brevigena MED C. brevigena A 190 C. brevigena A C. brevigena A C. brevigena MED 264 C. brevigena A C. brevigena A C. brevigena A 265 C. brevigena A C. brevigena A C. brevigena A 266 C. brevigena A C. brevigena A C. brevigena MED 267 C. brevigena A C. brevigena A C. brevigena A 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 (hy- pothetical 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. e @ e eo e e i 2 e oS ty 6 e “ * e 6 oa 7 7 iy We = @ -9.0 07.5 -6.0 -4.5 « -3.0 o6 sa 4.5 a bad - ee = -0.75} ro) 6 ° LZ r -1.504 Colletes succinctus ° ey Colletes collaris -2.25 Colletes brevigena e -3.00 Colletes halophilus Colletes hederae Colletes pannonicus Component 1 Figure 23. Scatter plot of PC 1 and PC 2, based on nine morphometric values of six European members of the Colletes succinctus group. dez.pensoft.net 126 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Linear discriminant analysis of C. brevigena For a more detailed analysis of the species C. brevigena, all specimens (n = 18) were divided into two hypothet- ical 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 be- longed 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 exam- ined females of C. brevigena show broader heads than the specimens of C. pannonicus; however, the two spe- cies 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 | 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 posi- tive loadings of CHL and high negative loadings of TW. Component 2 C. pannonicus C. brevigena 3.0 Table 6. Loadings of principal component 1 (PC1) and PC2 for each measured character in members of C. pannonicus and C. brevigena. Measurements: (HL) head length, (HW) head width, (EL) eye length, (CL) clypeus length, (UID) upper in- terocular distance, (LID) lower interocular distance, (MID) middle interocular distance, (CHL) cheek length, (TW) tho- rax width. PCI PC2 HL (log) 0.351 0.050 HW (log) 0.378 -0.123 EL (log) 0.366 -0.116 CL (log) 0.323 0.177 UID (log) 0.368 0.062 LID (log) 0.374 0.016 MID (log) 0.379 0.062 CHL (log) 0.100 0.837 TW (log) 0.255 -0.478 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. bre- vigena 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 var- iation is larger than the interspecific differences (Fig. 24). Pollen analysis Pollen determination Based on their morphological characters, the pollen grains found on the studied specimens were assigned to Component 1 Figure 24. Scatter plot of PC 1 and PC 2, based on nine morphometric values of two European members of the Colletes succinctus group: (pink) C. pannonicus, (dark green) C. brevigena. dez.pensoft.net Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Asteraceae (liguliflorae and tubuliflorae), Araliaceae, Eri- caceae, 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 pos- sible to determine the pollen grains to genus level (Citrus sp., Reseda sp.) or even species level (7ripolium panno- nicum, 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 tricolpo- rate: it has three colpi as well as three pores from which d27, the pollen tubes emerge. Heather (Ca/luna vulgaris) be- longs 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) 1s tetracol- porate and with a reticulate surface. Relationship between filling ratio and number of pol- len types Females with smaller pollen packages and therefore a lower filling ratio, collected fewer different pollen types than females with larger pollen loads and a high filling ratio (r = 0.343, p = 0.003). Figure 25. Selection of pollen grains collected by the females of the Austrian species of the Colletes succinctus group, scale bar 10 um: A. Asteraceae tubuliflorae; B. Asteraceae tubuliflorae (7ripolium pannonicum), C. Asteraceae liguliflorae; D. tricolporate/ reticulate; E. Resedaceae (Reseda sp.); F. Ericaceae (Calluna vulgaris); G. Araliaceae (Hedera helix), H. Rutaceae (Citrus sp.). dez.pensoft.net 128 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes Pollen preferences of the Austrian species of the C. succinctus group Colletes succinctus: Pollen analysis indicated that C. succinctus is polylectic. More than half (57%) of their average pollen load consisted of Reseda sp., close- ly followed by 40% Ericaceae pollen, which could be assigned to Calluna vulgaris. Only 1% of Asteraceae liguliflorae and 2% of Asteraceae tubuliflorae could be identified in the package and the rest was interpreted as contamination (Asteraceae, Ericaceae and undeter- mined). Fourteen of the 21 investigated C. succinctus females were collected in Retz, nine of them on Calluna vulgaris (Ericaceae) and five on Reseda sp. (Reseda- ceae): only seven of the nine specimens collected on C. vulgaris showed pure pollen packages consisting of that specific pollen. One female (no. 88) also preferred C. vulgaris (86.3%), but additionally collected a small proportion of Asteraceae liguliflorae (13.7%). Only in one specimen (no. 87), which was caught on C. vul- garis, no pollen of the same plant could be found. Its pollen package consisted of 100% Reseda sp. The five captured specimens on Reseda sp. possessed over 90% pollen from this plant in their collecting devices. One specimen (no. 92) additionally collected C. vulgaris in smaller quantities (8.3%) (Suppl. material 3: Appendix 3). Two C. succinctus females captured in Ollersdorf possessed pollen packages with over 99% Reseda sp. All specimens from Oberweiden (no. 160) and Bisam- berg (n = 4) collected preferably on Reseda sp. Of those, only one female (no. 98) additionally collected 28.7% of its pollen package on Asteraceae tubuliflorae (Suppl. material 3: Appendix 3). Colletes collaris: In summary, the 13 investigated fe- males of C. collaris show a polylectic pollen-collecting behaviour with a strong preference for Asteraceae (66%). This is closely followed by Reseda sp. (31%). Only 2% are due to pollen of the type tricolporate/reticulate and 1% remained indeterminable. Flower consistency of specimens was rarely observed. A single female (no. 1), captured at Bisamberg, had a 100% pure pollen package of Reseda sp. and a further specimen (no. 3) also from Bisamberg collected 100% of Asteraceae of the type tu- buliflorae. Resedaceae and Asteraceae tubuliflorae are also very popular with the remaining females from Bis- amberg (nos. 4—7). One of them (no. 4) collected approx- imately equal parts of Reseda sp. (36.7%), Asteraceae tubuliflorae (33.3%) and tricolporate/reticulate pollen (30%). Another one (no. 6) preferred Reseda sp. (46%), closely followed by Asteraceae tubuliflorae (32%) and Asteraceae liguliflorae (16%) and a further 6% of the pol- len load is attributed to contamination (Suppl. material 3: Appendix 3). A female (no. 9) from Langenlois preferred Asteraceae tubuliflorae (78.3%) to Reseda sp. (21.7%) as did the specimen from Engabrunner Haide (no. 10). The latter collected 76.7% of Asteraceae tubuliflorae and 23.3% of Reseda sp. (Suppl. material 3: Appendix 3). A female from Ollersdorf (no. 11) again preferred dez.pensoft.net Asteraceae liguliflorae (56.7%) to Asteraceae tubuliflo- rae (43.3%). Another female from Ollersdorf (no. 195), caught on Reseda sp., collected mainly on this plant (77.3%), followed by Asteraceae tubuliflorae (21.3%). It shows a small amount of 1.3% that is presumably con- tamination-related (Suppl. material 3: Appendix 3). The three studied females from Lake Neusiedl (nos. 191-193) preferred Asteraceae tubuliflorae, which can be assigned to Tripolium pannonicum. Only no. 192 additionally col- lected 18.3% of its pollen load on Asteraceae liguliflorae (Suppl. material 3: Appendix 3). Colletes hederae: Colletes hederae is the most repre- sented species in this pollen analysis (n = 27). It shows a polylectic pollen-collecting behaviour with a strong pref- erence 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 spec- imen that shows contamination (0.2%) by Citrus sp. All females from Stammersdorf (no. 198), Hainburg (nos. 29-30), Linz (no. 221) and different parts of Vienna (nos. 221, 225 and 228—230) collected pure pollen packages of H. helix flowers. The specimens captured at Donaupark on H. helix (nos. 211—212 and 219) in turn possess not only pollen of H. helix, but also pollen of Citrus sp., some in smaller and some in larger quantities. One female (no. 217) collected 50% H. helix and 50% Citrus sp. (Suppl. material 3: Appendix 3). Colletes brevigena: The only specimen of C. brevi- gena (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 spec- imen 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 ol- igolectic behaviour, collecting pollen on Asteraceae tubu- liflorae (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 1s ~ 2% (Suppl. material 3: Appendix 3). Specimens without assignment to a species: Due to un- clear morphological characters, seven specimens, which were used for pollen analyses, could not be clearly as- signed to a specific species. Thus, they are marked with “22?” in Suppl. material 3: Appendix 3. Most of these fe- males 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 pre- ferred Reseda sp. (99.3%) and a single female (no. 148), for which both the date and location of capture are un- known, had a pure pollen package with Calluna vulgaris pollen (100%). Dtsch. Entomol. Z. 68 (1) 2021, 101-138 DNA-barcoding 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 dif- ferences 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 se- quences 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) meth- od (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 paraphylet- ic (as later found out, by a misidentification): clade 1 con- tains 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. bre- vigena (no. DQ085546), with a Bootstrap value of 99%. 129 For a better illustration of the relationships amongst these clustering species, a median joining network was created (Fig. 27). Most of the closely-related speci- mens 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 identi- fied as C. /uzhouensis 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 spe- cies are lowest between C. brevigena and C. hederae and highest between C. col/aris 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. succinc- tus and C. hederae have identical sequences and show C. succinctus BOLD ( Pepe TH IRL C. succinctus BOLD (TCDB-T510) IRL C. succinctus BOLD C. succinctus BOLD Ge succinctus BOLD Ranaeeo4) IRL DQ085523) IR TCDB-T508) IRL 0DQ085579) IR C 3 it ZSM HYM eee C. succinctus BOLD ( BMNHYE) *970601 C. hederae BOLD | C. pannonicus ee C. hederae (P3 C. hederae (a y C. hederae (BK2 88 | C. succinctus ese C. succinctus (B | K 4 99 C. succinctus ee C. succinctus C. succinctus (73: *970408) GB BoD (BC ZSM HYM 02017) GER C ZSM HYM 08377) GER HYM 02007) GER seprece B 3 h ‘97062 C. hederae BOLD Be 6CP NHMW-HYM hy Mra 1961) A 4 C. brevigena BOLD (DQ085546) 96 99 C. creticus (7348) 0.01 64 08554: 2 : i 4000261) JPN C. collaris BOLD (DNA000260) JPN C. collaris BOLD (KC469646 C. collaris BOLD (esese40) wae 46.9644) C. céllaris BOLD (KC4696 49) C. collaris Bia C. collaris (BK. C. collaris fe C. collaris BOLD (KC469642) 99 : ha C, collaris BOLD (KC469653) C. collaris BOLD (KC469654) Figure 26. Neighbour-joining tree of the sequences of the species of the Co/letes 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. swccinctus group. The scale bar represents 0.01 substitutions per site. dez.pensoft.net 130 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes BCZSMHYMO2017 0Q085S46 7348 KC 469653 KC469654 . brevigena . collaris group 1 . collaris group 2 . hederae . succinctus . pannonicus . creticus @c @c @c @c @c @c @c KC469642 KC469645 KC469649 KC469644 KC469641 KC469643 KC469651 Figure 27. Median-joining network of the analysed species of the Colletes succinctus group (n = 20) and reference data (n = 47) from BOLD (Barcode of Life Database). (tick marks) substitution steps, (black dots) hypothetical haplotype that cannot be found. 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 differ- ences. In addition, in this combined dataset, C. collaris is the most differentiated species (Tables 7 and 8). How- ever, two sequences from BOLD, labelled as C. collaris, were highly differentiated resulting in a higher intraspe- cific distance than the interspecific distances to the other ingroup species and even higher than distances between ingroup and outgroup (Table 7). Discussion Morphology New features were found for species differentiation in the Austrian C. succinctus group. Nevertheless, there is a pronounced intraspecific variation (between popula- tions of different collecting sites) in all species. This also concerns previously-described diagnostic characters for females (Noskiewicz 1936; Schmidt and Westrich 1993; dez.pensoft.net Table 7. Genetic mean, minimum (min.) and maximum (max.) p distances (%) between the European species of the Colletes suc- cinctus group, based on the specimens investigated (A) as well as on reference data (B) from the Barcode of Life Databank (BOLD). Species 1 Species 2 Mean Mean Min. Min. Max. Max. distance distance A B A B A B C. hederae C. succinctus 0.50 0.03 0.00 0.00 1.00 0.60 C. hederae C. collaris 5.20 4.84 4.90 0.00 5.30 10.38 C. succinctus — C. collaris 5.10 4.87 4.90 3.99 5.40 10.38 C. hederae C. pannonicus 0.50 0.00 0.30 3.99 0.80 0.00 C. succinctus C. pannonicus 0.50 0.03 0.30 0.00 0.60 0.60 C. pannonicus 4.80 4.84 460 3.99 4.90 C. brevigena 0.40 0.07 0.00 0.00 1.00 0.60 C. brevigena 0.50 0.10 0.00 0.00 1.00 0.60 C. collaris C. brevigena 5.20 487 490 3.99 5.30 C. pannonicus C. brevigena 0.50 0.07 0.20 0.00 0.80 0.60 C. collaris C. hederae C. succinctus C. hederae C. creticus 6.80 6.19 6.80 6.19 6.80 6.19 C. succinctus — C. creticus 6.90 6.22 6.80 6.19 7.00 6.19 C. collaris C. creticus 7.90 8.02 7.80 7.39 8.00 11.58 C. pannonicus C. creticus 6.80 6.19 6.70 6.19 6.80 6.19 C. brevigena C. creticus 6.80 6.19 6.70 6.19 6.80 6.19 Burger 2010; Hoélzler and Mazzucco 2011), like the wrin- kles on the clypeus, the puncturation on the mesonotum Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Table 8. Genetic mean p distance within European species of the Colletes succinctus group, based on investigated specimens (A), as well as merged with reference data (B) from Barcode of Life Database (BOLD). Mean distance A Mean distance B C. hederae 0.6 0.0 C. succinctus 0.5 0.1 C. collaris 0.2 2.5 C. pannonicus 0.5 0.0 C. brevigena 0.4 0.1 and the presence/absence of the reticulate micro-sculp- ture on the galea. Due to these sometimes very confusing character combinations, it was not possible to clearly as- sign all specimens of this study to a species. A few female specimens showed combined traits of the species C. suc- cinctus 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; Holzler and Mazzuc- co 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 dor- sobasal knob on the membrane of the gonostylus distin- guishes 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 ap- pearance. 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 Schonitzer 1993). The differently shaped dorsal margins of the fovea are often used as a charac- ter for species identification in other genera, especially Andrena (Schmid-Egger and Scheuchl 1997). Morphometry The determination of females by use of the new identi- fication key is not supported by the discriminant analy- sis (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 morpholog- ical 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 ac al 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 exam- ined species of the C. succinctus group, as, for example, suggested for C. pannonicus by Holzler and Mazzucco (2011). No morphometric studies have yet been pub- lished for the studied species, but other species of the ge- nus Colletes have already been investigated: because of their similar appearance, especially in their puncturation on terga, C. inexpectatus Noskiewicz, 1936 and C. davie- sanus Smith, 1846 were regarded as synonyms (Warncke 1978). Pridal (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 dif- ferentiation 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. succinc- tus, C. collaris and C. hederae showed a polylectic pol- len-collecting behaviour, C. pannonicus appeared to be oligolectic, but examination was based on a single popu- lation. 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 Miller 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 expect- ed 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 pol- len load of pure Reseda sp.; this species is described as polylectic by Muller 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 dez.pensoft.net 132 their pollen packages. This plant genus is recognised for the first time as a pollen source for C. hederae. The other females of C. hederae were captured in Linz and several sites in Vienna, directly on H. helix. During the rather late flight period of C. hederae (September—November; Kuhl- mann et al. 2009), there is no particularly large selection of flowering plants available. Ivy flowers offer an easily accessible and nutrient-rich pollen resource. Nonetheless, it was observed that, if other nutritious pollen sources are available, C. hederae disregards Hedera sp. and starts collecting pollen from the other sources. Therefore, the polylectic pollen-collecting behaviour of C. hederae is sometimes referred to as pseudo-oligolectic; this means that they are sometimes only oligolectic by lack of choice (Teppner and Brosch 2015). Little is known about the pollen-collecting behaviour of C. pannonicus. Field observations led to the assump- tion that C. pannonicus 1s strictly oligolectic on Tripolium pannonicum (sea aster) (Holzler 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 7’ 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 accord- ance with findings by Muller and Kuhlmann (2008), they also collected pollen of Reseda sp. Asteraceae are omnipresent, provide large amounts of pollen and nec- tar 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 impor- tant 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 detoxifi- cation and for an easier extraction of important nutrients from the pollen plasma (Miller and Kuhlmann 2008). Asteraceae are the perfect nutrient supplier for adapt- ed bees, being available almost without competition. In Colletes, Asteraceae are preferentially collected by oligo- lectic 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. hederae (Miller 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 monophy- lum 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 se- quences from BOLD, C. collaris separates into two clades dez.pensoft.net Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes and forms a paraphylum. This can be explained by check- ing 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 CO/ 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, ex- cept 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 CO/ sequence, which is in accordance with previous studies (Kuhlmann et al. 2007, 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 appear- ance, it is difficult to distinguish them strictly by mor- phology. 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 (Holzler & Mazzucco, 2011), whereas the previously-stated morphometric dif- ferences 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 diftfer- ent morphological character states than specimens from the Mediterranean. Therefore, both species were investi- gated 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 7ripolium pannonicum (sea aster). That all known specimens were caught around Lake Neusied! is surprising, as there are no geographical barriers which would prevent a wider distri- bution. Specialisation to a distinct habitat, salt meadows, seems the most likely and hitherto accepted explanation that C. pannonicus could not be found elsewhere in Aus- tria. The proposed (H6lzler and Mazzucco 2011) and here confirmed apparent dependence on 7. pannonicum could be explained by the fact that hardly any other Asterace- ae are blooming at these sites in late autumn. 7ripolium pannonicum is a widespread plant found in Europe and in temperate regions of Asia (Eurot+tMed 2006) and it can be Dtsch. Entomol. Z. 68 (1) 2021, 101-138 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 distribution area spanning from the Balkans, to Crete, Persia and the Caucasus, but many specimens are untraceable. The selection of a lectotype was necessary to define this prob- lematic species. The type locality is Crete (Greece). A clear morphological distinction could be detect- ed between Austrian specimens (from Bisamberg and Ollersdorf, Lower Austria) of C. brevigena and spec- imens 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 ana- lysis (LDA) of morphometric data resulted in clear sepa- ration 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 ex- plained 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 punc- turation of C. hederae, but as typical for females of C. brevigena, they have longitudinal wrinkles on their clypeus. Therefore, the authors classified them with re- servation as C. brevigena (Zettel et al. 2006). Concluding the findings for C. brevigena, this study raises doubts about the close relationship between spec- imens/populations of C. brevigena occurring in Austria and specimens from the Mediterranean region. In addi- tion 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 Novem- ber). 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 spe- cies (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 133 C. brevigena, however, the spring generation is omitted. As most specimens of the genus Colletes in Central Eu- rope spend their diapause (hibernation) as a pre-pupa (in the last larval stage) (Westrich 1989), this delayed devel- opment into an imago should be genetically fixed and, therefore, must be a trait that was passed on by its ances- tor. A similar case is also known from the species com- plex of Andrena argentata Smith, 1844. This bivoltine species shows a trans-Palaearctic distribution. In Eng- land 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 spec- imens 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 pro- nounced geographic variation. The studied populations in Austria and the Mediterranean region are geographical- ly 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 inter- mediate 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 CO/) are not useful to differentiate between them. It is difficult to distinguish the two species by the discrim- inating characters described by Holzler and Mazzucco (2011), as morphometric data greatly overlap. For exam- ple, Hélzler 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, howev- er, the two species do not occur sympatrically, but C. pan- nonicus shares the habitat with C. collaris. Colletes brevi- gena, 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 mis- identified 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. dez.pensoft.net 134 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 me- sonotum, 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 tax- onomists (Noskiewicz 1936; Verhoeff 1944; Schmidt and Westrich 1993; Holzler 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 dif- ferentiation (Kuhlmann et al. 2007). For example, female no. 148 shows C. succinctus-typical terga, mesopleu- ra and clypeus, but does not have a shiny centre on the mesonotum. Since this was already mentioned by Nosk- iewicz (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, how- ever, did not develop all characters typical for this taxon. In addition, the females (nos. 61—66), which were collect- ed at Bisamberg (Vienna), show combined characters of C. succinctus, C. brevigena and C. hederae. These speci- mens were collected mid-September on Reseda sp. (pol- len loads studied). Although Reseda sp. is only known as a pollen source for C. brevigena and C. collaris in \iterature (Muller and Kuhlmann 2008) and for C. succinctus in the present study, it is not impossible that C. hederae also col- lects 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 (Muller and Kuhlmann 2008; Westrich 2008; Teppner and Brosch 2015). Moreover, flower visits of C. hederae on Reseda sp. have been ob- served 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 spe- cies in Statu nascendi or evolutionary of very recent or- igin. 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 spe- cies complex C. succinctus-brevigena-hederae-pannon- icus is more complicated than assumed by all previous taxonomists (Noskiewicz 1936, Verhoeff 1944, Schmidt and Westrich 1993, Hoélzler and Mazzucco 2011), as there is high variation in morphological and ecological char- acters. For some specimens, it is still difficult to identify them by studying their morphology. Thus, the ecology of dez.pensoft.net Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Colletes the specimens continues to be an important tool for spe- cies differentiation. Acknowledgements We are very grateful to Elisabeth Haring (Natural History Museum Vienna), Dominique Zimmermann (Natural Histo- ry Museum Vienna) and Christian Baranyi (University of Vi- enna) for the great expertise, the persistent patience and the always open door. Our gratitude goes out to Alexander Dos- tal, Andreas Link, Dominique Zimmermann, Esther Ocker- miller, Franz Seyfert, Heinz Wiesbauer, Karl Mazzucco, Lorenz Wido Gunzy, Sabine Schoder and Volker Haeseler as well as the Natural History Museum Vienna and the Upper Austrian State Museum Linz for providing specimens for this study. We thank Alice Laciny for her repeatedly skills as the “best proof-reader of all time” and Eva Frischherz, Josef Frischherz and Stefan Ehrengruber for their support, patience and the occasional delivery of soul food. 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Westrich P (2008) Flexibles Pollensammelverhalten der ansonsten streng oligolektischen Seidenbiene Colletes hederae Schmidt and Westrich (Hymenoptera: Apidae). Eucera 2: 17-29. Williams NM (2003) Use of novel pollen species by specialist and gen- eralist solitary bees (Hymenoptera: Megachilidae). Oecologia 134: 228-237. https://doi.org/10.1007/s00442-002-1104-4 Zettel H, Ebmer AW, Wiesbauer H (2006) Zur Kenntnis der Wildbienen (Hymenoptera: Apidae) in Wien, Niederésterreich und dem Burgen- land (Osterreich). Beitrage zur Entomofaunistik 7: 49-62. Zettel H, Wiesbauer H (2014) Zur Kenntnis der Wildbienen (Hymeno- ptera: Apidae) in Wien, Niederosterreich und dem Burgenland (Osterreich) — 6. Beitrage zur Entomofaunistik 15: 113-133. Supplementary material 1 Appendix 1 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: Specimen data Explanation note: Examined specimens of the Austrian species of the Colletes succinctus group, sorted by ID number, including genus, species (currently assigned and previously assigned), sex, collecting site and date, owner of specimen as well as all applied approaches. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.68.55732.suppl1 dez.pensoft.net Supplementary material 2 Appendix 2 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: Morphometrical data Explanation note: List of all morphometrically measured individuals, listed by ID number, including species and measured values in millimetres. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.55732.suppl2 Supplementary material 3 Appendix 3 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: Specimen data Explanation note: List of all individuals of the Austrian species of the Colletes succinctus group used for pol- len analysis, listed by species. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.55732.suppl3 Supplementary material 4 Appendix 4 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: Specimen data Explanation note: List of specimens used for DNA bar- coding, including ID number, genus, species, sex, se- quencing number and status of sequencing. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.55732.suppl4 Dtsch. Entomol. Z. 68 (1) 2021, 101-138 Supplementary material 5 Appendix 5 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: CO/ sequences Explanation note: List of CO/ sequences of specimens of the Colletes succinctus group, which were obtained from the Barcode of Life Database (BOLD). Including sample ID number, genus, species as well as sex and origin (if specified in database). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.68.55732.suppl5 Supplementary material 6 Appendix 6 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: List of individuals Explanation note: List of individuals for which no exact species affiliation could be determined. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.68.55732.suppl6 Supplementary material 7 Appendix 7 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: Species assignment Explanation note: Species assignment before and after the LDA, including Jackknife resampling (1,000). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.55732.suppl7 137 Supplementary material 8 Appendix 8 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: Species assignment Explanation note: Species assignment before and after the LDA, including Jackknife resampling (1,000). Species in bold letters were classified differently than the hypothetical assignment. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.68.55732.suppl8 Supplementary material 9 Appendix 9 Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- mann, Harald W. Krenn Data type: CO/ sequences Explanation note: Alignment of the self-obtained CO] sequences showing distinguishing substitutions of the Austrian species of the Colletes succinctus group and outgroup taxa (C. creticus). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.55732.suppl9 dez.pensoft.net 138 Katharina Zenz et al.: Morphology, pollen preferences and DNA-barcoding of Austrian Coll/etes Supplementary material 10 Copyright notice: This dataset is made available under the Open Database License (http://opendatacom- Appendix 10 mons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended Authors: Katharina Zenz, Herbert Zettel, Michael Kuhl- to allow users to freely share, modify, and use this mann, Harald W. Krenn Dataset while maintaining this same freedom for Data type: Pairwise genetic mean p distances others, provided that the original source and au- Explanation note: Pairwise genetic mean p distances (%) thor(s) are credited. between the examined individuals of the European — Link: https://doi.org/10.3897/dez.68.55732.suppl10 species of the Colletes succinctus group, based on the individuals investigated. dez.pensoft.net