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Labial gland marking secretions of male Bombus lucorum bumblebees from Europe and China reveal two separate species: B. lucorum (Linnaeus 1761) and Bombus minshanicola (Bischoff 1936)
Biochemical Systematics and Ecology 39 (2011) 587–593
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Labial gland marking secretions of male Bombus lucorum bumblebees from Europe and China reveal two separate species: B. lucorum (Linnaeus 1761) and Bombus minshanicola (Bischoff 1936) Andreas Bertsch a, *, Horst Schweer b a b
Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Straße, D-35032 Marburg, Germany Department of Pediatrics, Philipps-University Marburg, Baldingerstraße, D-35043 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2011 Accepted 29 April 2011 Available online 24 May 2011
Differences in male bumblebee labial gland secretions can be used to separate species pairs. Cephalic labial gland secretions from male Bombus lucorum Linnaeus bumblebees from Europe, and male ‘B. lucorum’ bumblebees from China, were analyzed by gas chromatography/mass spectrometry (GC/MS). In the European B. lucorum L., ethyl tetradec9-enoate was identified as the major compound (58% peak area), along with a complex mixture of straight-chain alkenols, acetates, hydrocarbons, and wax type esters. In contrast, the main component of the labial gland secretions of the Chinese ‘B. lucorum’, ethyl dodecanoate (31%), did not dominate the secretion, which additionally contained large amounts of 3,7,11-trimethyldodeca-6,10-dien-1-ol (¼2,3-dihydrofarnesol, 24% peak area), ethyl octadec-9-enoate (15% peak area) and a mixture of acyclic diterpenes (alcohols, aldehydes, and acetates). Furthermore, in B. lucorum, 3,7,11-trimethyldodeca-6,10-dien-1ol was only detected in trace amounts (0.05%) and ethyl octadec-9-enoate corresponded to only 0.8% of the mixture. These results indicate that ‘B. lucorum’ from China is a separate taxon from B. lucorum L. Further analysis revealed that ‘B. lucorum’ from China has been previously described as Bombus minshanicola Bischoff 1936 from Gansu/China. Differences in the chemical composition of male bumblebee labial gland secretions are discussed in comparison with other known bumblebee species pairs. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Hymenoptera Bombus lucorum Bombus minshanicola Cephalic labial gland Gas chromatography Mass spectrometry
1. Introduction For a long time, so-called cryptic species have presented problems for taxonomy. Since taxa were defined by morphological features, the lack of separating morphological characteristics was a significant impediment. With the advent of DNA markers and other tools for biochemical systematics, things changed fundamentally. A good example could be the bumblebee species Bombus lucorum (Linnaeus 1761), which was tentatively separated from Bombus terrestris in the beginning of the 20th century, and established as a separate species on the basis of its morphology (Krüger, 1920, 1951), male labial gland secretions (Calam, 1969; Bertsch, 1997; Urbanova et al., 2001) and mitochondrial DNA markers (Bertsch et al., 2005; Bertsch, 2009). In fact, Scholl and Obrecht (1983) coined the term “B. lucorum-complex” to point out that we have to handle a complex of three taxa which are difficult to separate by morphological characteristics alone. With the use of DNA markers and male labial gland secretions, these taxonomical problems could be solved (Bertsch, 2009). Genetically, the B. lucorum-complex does not exist,
* Corresponding author. E-mail addresses: [email protected] (A. Bertsch), [email protected] (H. Schweer). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.04.010
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and the main substance identified in the male labial gland secretions (ethyl tetradec-9-enoate) of B. lucorum is unique, clearly separating this species from all the other taxa involved. Interestingly, Cameron et al. (2007) reported bumblebee DNA sequences from Sichuan/China that they also refer to as ‘B. lucorum’, but which differ essentially from the DNA sequences of B. lucorum from Europe, a result confirmed by the unpublished COI sequence of ‘B. lucorum’ from North China (GenBank GU085202); proving that this Chinese ‘B. lucorum’ is genetically very different from European B. lucorum. This ‘B. lucorum/China’ is not identical with the taxon B. lucorum. To better understand this interesting bumblebee taxon ‘B. lucorum’ from China, we investigated male cephalic labial gland secretions and included GC analysis of the European B. lucorum species for direct comparison. 2. Materials and methods 2.1. Materials Male ‘B. lucorum’ bumblebees were collected at Cirsium leo, approximately 50 km west of Yongdeng, Gansu, China (36 41.260 N, 102 42.770 E). For comparison, male B. lucorum Linnaeus bumblebees were also collected from Kirkwall, Orkney Islands, Mainland, UK (58 59.050 N, 2 57.560 W). Because safe identification using morphological characteristics alone is difficult and problematic, all specimens have been unambiguously identified using DNA markers (Bertsch, 2009, Table 1 specimen LUC-01 and Bertsch, 2010, Table 1 specimen MIN-03). Old males were recognized by their frayed wings and discarded. The wing borders of all males used for labial gland preparations were smooth, indicative of active males. The cephalic part of the labial gland was dissected from the head while frozen, and placed in a vial (glands from five males where pooled in each vial) containing 0.2 ml pentane. Because the aim of this investigation was to elucidate the chemical complexity and composition of male bumblebee labial glands, and not to study gland content variability, we pooled glands from five males into a single vial. This method allowed for detection of even minor compounds present in the glands.
2.2. Gas chromatography/mass spectrometry A Finnigan MAT TSQ700 gas chromatograph/tandem mass spectrometer was employed. Gas chromatography was carried out using a Hewlett Packard Ultra 1 column (50 m, 0.2 mm internal diameter, 0.11 mm film thickness) in splitless mode, with helium as the carrier gas and at an inlet pressure of 300 kPa. The split valve was opened for 1 min. An initial temperature of 120 C was held for 1 min, then increased at a rate of 8 /min to 280 C, followed by an increase at a rate of 3 /min to 310 C, and finally by 1 /min to 320 C. This final temperature was held for 10 min. Mass spectrometer conditions were as follows: the interface temperature was 300 C, source temperature 130 C, electron energy 70 eV, emission current 0.2 mA, and electron multiplier 1400 V. When using the mass spectrometer in positive ion chemical ionization (CI) mode, the ammonia CI gas pressure was 70 Pa. Compounds were identified by comparison with compounds in the NIST 2002 Library (National Institute of Standards and Technology, USA), along with retention times and molecular ions from the CI spectra. To determine the positions of double bonds, derivatization with dimethyl disulfide was used, as described by Buser et al. (1983). 3. Results Typical chromatograms of male cephalic labial gland secretions of ‘B. lucorum’ from China (¼ Bombus minshanicola), and B. lucorum from Europe are shown in Fig. 1. Compounds identified are summarized in Table 1. The labial glands contained a mixture of acyclic diterpenes (alcohols, aldehydes, and acetates) and various straight-chain fatty acid derivatives (alcohols, aldehydes, esters and both saturated and unsaturated hydrocarbons with C21–C29). In the male labial gland of ‘B. lucorum’ from China, the major compound identified was ethyl dodecanoate (31% of the total peak area, peak 5) (see Fig. 1). In addition, large amounts of 3,7,11-trimethyldodeca-6,10-dien-1-ol (peak 10, 24% of total peak area) and ethyl octadec-9-enoate (peak 49, 15% of total peak area) were detected. Furthermore, minor amounts (1%–5%) of dodecanoic acid (peak 2), tetradecanal (peak 6), 3,7,11-trimethyldodeca-6,10-dienal (peak 7), two alcohols, octadeca-9,12, 15-trien-1-ol (peak 37), and icos-15-en-1-ol (peak 59), and one ester, 3,7,11-trimethyldodeca-6,10-dien-1-yl dodecanoate (peak 85) were detected. An additional alcohol, octadeca-9,12-dien-1-ol (peak 36), two more aldehydes, 3,7,11-trimethyldodeca-2,6,10-trienal (peaks 11 & 12), five more ethyl esters, ethyl tetradecanoate (peak 15), ethyl hexadecanoate (peak 32), ethyl octadecatrienoate (peak 48), and ethyl octadec-11-enoate and ethyl octadec-13-enoate (peaks 50 and 52) and three acetates, 3,7,11-trimethyldodeca-6,10-dien-1-yl acetate (peak 17), octadecatrienyl acetate (peak 51), octadecyl acetate (peak 53) and an additional acid, octadec-9-enoic acid (peak 45), were also detected in small amounts (between 0.2% and 1%). In the male labial glands of B. lucorum from Europe, the major compound identified was ethyl tetradec-9-enoate (58% of the total peak area, peak 14) (see Fig. 1). No other compound was detected in large amounts (>5%). However, minor amounts (1%–5%) of a series of alcohols, hexadecan-1-ol (peak 21), octadeca-9,12-dien-1-ol (peak 36), and octadeca-9,12,15-trien-1ol (peak 37) were detected. An additional alcohol, icosan-1-ol (peak 60), and an aldehyde, octadecenal (peak 31), two ethyl esters, ethyl hexadec-11-enoate (peak 29) and ethyl octadec-9-enoate (peak 49), were also detected in small amounts (between 0.2% and 1%).
A. Bertsch, H. Schweer / Biochemical Systematics and Ecology 39 (2011) 587–593
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B. minshanicola 5
100%
-Ethyl dodecanoate 10 49
75%
-Ethyl octadec-9-enoate -3,7,11-Trimethyl-dodeca-6,10-dien-1-ol
50%
6
25%
37 85
7
Octadeca-9,12,15-trien-1-ol-
59
-Icos-15-en-1-ol
2
74
10:00
100%
B. lucorum
15:00
20:00
25:00
20:00
25:00
14
-Ethyl tetradec-9-enoate 75%
15
50% 5
28 37
-Ethyl dodecanoate 21
25%
-Octadeca-9,12,15-trien-1-ol 48
Hexadecan-1-ol-
60
-Icosan-1-ol
10:00
15:00
RT (min:sec)
Fig. 1. Total ion chromatograms of cephalic labial gland pentane extracts from ‘Bombus lucorum’ China (¼ B. minshanicola) and Bombus lucorum from Europe. Numbers correspond to the numbers in the peak list (Table 1). Some compounds listed in Table 1 were present in quantities too low to be visible in these chromatograms.
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Table 1 Compounds of the cephalic labial glands of ‘B. lucorum’ from China (¼B. minshanicola) and B. lucorum from Europe, peak areas more than 10% in bold. No.
Compound
RT [min:sec]
RI
1 2 3 4 5 6 7 8 9 10 11
Ethyl decanoate Dodecanoic acid Ethyl dodecenoate (isomer I) Ethyl dodecenoate (isomer II) Ethyl dodecanoate Tetradecanal 3,7,11-Trimethyldodeca-6,10-dienal 1-Methylethyl dodecanoate Tetradecan-1-ol 3,7,11-Trimethyldodeca-6,10-dien-1-ol 3,7,11-Trimethyldodeca-2,6,10-trienal (isomer I) 3,7,11-Trimethyldodeca-2,6,10-trienal (isomer II) Tetradecanoic acid Ethyl tetradec-9-enoate Ethyl tetradecanoate Hexadecenal 3,7,11-Trimethyldodeca-6,10-dien-1-yl acetate 3,7,11-Trimethyldodeca-2,6,10-trienoic acid Hexadecanal Ethyl tetradecadienoate Hexadecan-1-ol Nonadecane Hexadecenoic acid Hexadecanoic acid Ethyl hexadecadienoate Icosadiene Ethyl hexadec-7-enoate Ethyl hexadec-9-enoate Ethyl hexadec-11-enoate Octadecatrienal Octadecenal (isomer I) Ethyl hexadecanoate Octadecadienal Ethyl hexadecadienoate Octadecenal (isomer II) Octadeca-9,12-dien-1-ol Octadeca-9,12,15-trien-1-ol Octadec-9-en-1-ol Octadecan-1-ol Henicosene Henicosane Octadeca-9,12-dienoic acid Octadeca-9,12,15-trienoic acid Octadecenoic acid (isomer I) Octadec-9-enoic acid (isomer II) Icosa-5,8,11,14-tetraenoic acid Ethyl octadecadienoate Ethyl octadecatrienoate Ethyl octadec-9-enoate Ethyl octadec-11-enoate Octadecatrienyl acetate Ethyl octadec-13-enoate Octadecyl acetate Ethyl octadecanoate Icosenal (isomer I) Icosenal (isomer II) Docosane Icosen-1-ol (isomer I) Icos-15-en-1-ol (isomer II) Icosan-1-ol Tricos-9-ene Tricos-7-ene Tricos-5-ene 3,7-Dimethyloct-6-en-1-yl dodecanoate Tricosane
6:00 8:27 8:36 8:41 8:45 8:54 8:57 9:07 9:47 10:02 10:13
1378 1562 1569 1572 1579 1586 1591 1612 1657 1676 1689
– – 30.71 1.65 1.59 0.18 0.19 23.61 0.36
10:32
1711
0.55
10:53 11:10 11:21 11:25 11:30 11:34 11:36 11:39 12:29 12:57 13:20 13:33 13:35 13:37 13:40 13:42 13:45 13:49 13:52 13:55 13:57 13:59 14:03 14:36 14:38 14:52 14:58 15:03 15:25 15:30 15:32 15:36 15:41 15:47 15:52 15:56 16:04 16:06 16:09 16:12 16:20 16:23 16:25 16:30 16:35 17:07 17:14 17:21 17:22 17:25 17:34 17:38 17:42
1735 1746 1762 1768 1775 1782 1785 1789 1864 1900 1946 1960 1962 1965 1967 1970 1973 1977 1980 1983 1986 1989 1995 2042 2045 2058 2061 2068 2100 2107 2110 2116 2123 2132 2139 2143 2152 2155 2159 2163 2173 2177 2182 2193 2200 2247 2258 2263 2270 2274 2288 2294 2300
0.04 0.02 0.31 0.05 0.37 0.04 0.08 – 0.08 0.01 0.01 0.01 – – – – 0.08 0.10 0.02 0.20 – – 0.01 0.24 3.27 0.11 – 0.02 0.62 – – 0.06 0.53 – – 0.23 14.52 0.56 0.22 0.36 0.44 – – 0.10 0.05 0.04 1.37 – 0.53 – – 0.05 1.53
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
B. minshanicola [% peak area] 0.09 1.93
B. lucorum [% peak area]
Mþ [m/z]
0.01 0.04 0.03 0.04 9.56 0.14 0.14 – – 0.03 –
200 200 226 226 228 212 222 242 214 224 220
–
220
0.03 58.4 4.64 – – – 0.02 0.02 3.54 – 0.02 – 0.08 0.34 0.18 3.55 0.31 0.07 0.42 0.03 0.06 0.03 – 1.64 4.66 0.02 0.16 – 0.15 0.01 0.02 – 0.05 0.08 0.03 1.27 0.82 0.04 – – – 0.04 0.01 0.02 0.03 0.08 0.10 0.94 0.28 0.03 0.01 – 2.92
228 254 256 238 266 254 240 252 242 268 254 256 280 278 282 282 282 262 266 284 264 282 266 266 264 268 270 294 296 280 278 282 282 304 308 306 310 310 306 310 312 312 294 294 310 296 296 298 322 322 322 338 324
DMDS adducts
18:51 {117, 231; Mþ 348}
20:38 {173, 203; Mþ 376} 20:42 {145, 231; Mþ 376} 20:51 {117, 259; Mþ 376}
24:42 {173, 189; Mþ 362}
22:23 {173, 203; Mþ 376}
22:39 {173, 231; Mþ 404} 22:42 {145, 259; Mþ 404} 22:48 {117, 287; Mþ 404}
23:37 {117, 273; Mþ 390} 23:30 {173, 243; Mþ 416} 23:35 {145, 271; Mþ 416}
A. Bertsch, H. Schweer / Biochemical Systematics and Ecology 39 (2011) 587–593
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Table 1 (continued ) B. lucorum [% peak area]
Mþ [m/z]
0.10
–
350
0.06 0.06 0.07 0.08 0.07 0.02 0.63 0.29 – 0.22 0.01
– – – – – 0.02 0.06 0.13 1.68 0.13 1.45 –
338 338 338 340 338 336 338 324 350 350 352 378
2550 2568 2600 2677 2683 2700 2760
0.06 0.04 – 0.37 – – 4.83
– 0.01 0.01 0.92 0.20 0.09 –
368 364 366 378 378 380 406
2766 2773 2778 2891
0.55 – 0.02 0.04
– 0.01 – 0.12
396 392 404 406
No.
Compound
RT [min:sec]
RI
66
3,7,11-Trimethyldodeca-6,10-dien-1-yl octanoate Icos-15-en-1-yl acetate (isomer I) Icosen-1-yl acetate (isomer II) Icosen-1-yl acetate (isomer III) Ethyl icosanoate Icosen-1-yl acetate (isomer IV) Tetracosene Tetracosane Docos-15-en-1-ol Pentacos-9-ene Pentacos-7-ene Pentacosane 3,7,11-Trimethyldodeca-6,10-dien-1-yl decanoate Dodecyl dodecanoate Hexacosene Hexacosane Heptacos-9-ene Heptacos-7-ene Heptacosane 3,7,11-Trimethyldodeca-6,10-dien-1-yl dodecanoate Tetradecyl dodecanoate Octacosene Nonacosadiene Nonacos-9-ene
18:13
2341
18:14 18:18 18:25 18:32 18:34 18:45 18:58 19:19 19:30 19:35 19:47 20:15
2342 2347 2356 2366 2368 2371 2400 2450 2476 2491 2500 2542
20:20 20:31 20:47 21:30 21:36 21:44 22:16 22:19 22:23 23:26 23:33
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
þ
RT ¼ retention time; RI ¼ retention index; M double bond could not be assigned.
B. minshanicola [% peak area]
–
¼ molecular ion; DMDS adducts ¼ RT min:sec {xxx, xxx; M
þ
DMDS adducts
24:53 {117, 315; Mþ 432}
25:44 {145, 273; Mþ 418} 25:50 {173, 271; Mþ 444}
28:20 {173, 299; Mþ 472}
31:36 {173, 327; Mþ 500}
xxx}, in some minor compounds, position of the
4. Discussion 4.1. Flight path activity and male labial gland secretions Flight path activity and scent marking has long been observed for many bumblebee species, and male cephalic labial gland secretions have been investigated in great detail (Bergström et al., 1981; Valterova and Urbanova, 1997). In early studies, Sladen (1912) stated that the male scent ‘attracts not only males but the queens’, and following the first chemical investigations, Kullenberg et al. (1973) formulated the hypothesis that these compounds are actually sex pheromones used to attract virgin females, a hypothesis not yet confirmed by empirical evidence. Flight path activity and scent marking by ‘B. lucorum’ from China is completely unknown, and even in Northern Europe, where B. lucorum is one of the most abundant bumblebees, bumblebee flight path activities in the field have not been investigated in detail; and only casual observations have been reported (Bringer, 1973). In fact, quantitative flight path activity and scent marking data for European B. lucorum males are only available from climatic test chambers (Bertsch, 1984). The most useful application of male bumblebee labial gland secretions to date has been the separation of critical taxa, to establish taxonomical rank. For example, separating specimens of B. terrestris and B. lucorum in some parts of Europe by morphological characteristics alone is so difficult that both species have for a long time been treated as a single species. The detection of large differences in the chemical composition of compounds from male labial gland secretions finally established both taxa as separate species (Calam, 1969; Kullenberg et al.,1970, 1973). The same problem was encountered with Bombus vestalis and Bombus bohemicus, two parasitic bumblebees of the subgenus Psithyrus which are morphologically very similar and often difficult to separate; but analysis of their male labial gland secretions separated them very clearly (Bergman et al., 1996; Urbanova et al., 2004). B. lapponicus auct. is another good example, where all efforts to separate taxa by morphological characteristics alone had failed (Pittioni, 1942); finally, investigation of male labial gland secretions revealed two separate species, Bombus monticola Smith and Bombus lapponicus Fabricius (Bergström and Svensson, 1973; Svensson, 1973, 1980).
4.2. Differences in metabolic pathways Compounds of male labial gland secretions may be variable. Individual gland contents may change quantitatively and qualitatively (a) over the lifetime (see Zacek et al., 2009 for B. terrestris) and (b) during the day (secretions are used for scent marking in the morning, so glands may be depleted later in the day; see Bergman, 1997 for Bombus lapidarius) and (c) geographically between well established subspecies (Coppée et al., 2008 for B. terrestris). But this variability does not alter the typical pattern of substances it is always possible to identify the pattern of substances typical for a certain species. The
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differences found in labial gland secretions between all of the above species pairs were not quantitative, but qualitative, changing the pattern of substances. Male bumblebee labial gland secretions can be roughly divided according to the biochemical pathways which synthesize the compounds. Using this classification system, two types of secretions have been identified: PP type secretions, containing fatty acid derivatives only (Polyketide Pathway: PP); and PP þ MAP type secretions, which, in addition to fatty acid derivatives, contain mevalonic acid derivatives (Mevalonic Acid Pathway: MAP) in the form of acyclic diterpenes (Bergman et al., 1996). The species pairs B. lucorum – B. terrestris, B. bohemicus – B. vestalis and B. monticola – B. lapponicus belong to this class; the first members of these species pairs contain PP type labial gland secretions and the second members contain PP þ MAP type secretions. The species pair analyzed in this investigation, B. lucorum from Europe and ‘B. lucorum’ from China, also fits well with this classification system. B. lucorum from Europe contains PP type labial gland secretions and ‘B. lucorum’ from China contains PP þ MAP type labial gland secretions. It is another example of a species pair which is difficult to separate by morphological characteristics alone, but has essentially different male labial gland secretions. Because B. lucorum from ‘Europe’ and ‘B. lucorum’ from China use different species recognition signals, they are separate taxa. Furthermore, ‘B. lucorum’ from China most probably is a taxon in the rank of a species. 4.3. Taxonomic consequences As the subgenus Bombus sensu stricto has been thoroughly studied, and has already delivered a huge number of color variants and names (Krüger, 1951, 1954, 1956, 1958), it seemed improbable to us that ‘B. lucorum’ from China did not already have a name. Because B. lucorum lanchouensis Vogt 1908 was proposed to be synonymous with Bombus patagiatus, as B. patagiatus ssp. lanchouensis (Tkalcu, 1967), only one taxon remains from the area (Northern Sichuan & Gansu/China). This taxon was collected in 1930 by Dr. D. Hummel during a Sven Hedin expedition, and was described as B. terrestris ssp. minshanicola (Bischoff, 1936) from Gansu/China, and later transferred to B. lucorum by Krüger (1951). Recent genetic investigations established this taxon as a separate species, B. minshanicola Bischoff 1936, genetically closer to the North American Bombus affinis and Bombus franklini than to the European B. lucorum (Bertsch, 2010). Thus, this investigation of male labial gland secretions of ‘B. lucorum’ from China confirms these genetic analysis results. Acknowledgments An unknown collaborator from the Gansu Institute of Apiculture, Tianshui/China and Dr. Jiandong An, Institute of Apiculture, Chinese Academy of Agricultural Sciences, Beijing/China supplied the ‘B. lucorum’material from Gansu. We would like to thank them for their valuable help. References Bergman, P., Bergström, G., Appelgren, M., 1996. Labial gland marking secretion in males of two Scandinavian cuckoo bumblebee species (genus Psithyrus). Chemoecology 7, 140–145. Bergström, G., Svensson, B.G., 1973. Studies on natural odoriferous compounds VIII. Characteristic marking secretions of two forms lapponicus and scandinavicus of B. lapponicus Fabr. (Hymenoptera, Apidae). Chem. Scripta 4, 231–239. Bergman, P., 1997. Chemical Communication in Bumblebee Premating Behaviour. PhD thesis, Göteborg University, Sweden. Bergström, G., Svensson, B.G., Appelgren, M., Groth, I., 1981. Complexity of bumble bee marking pheromones: biochemical, ecological and systematical interpretations. 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Kullenberg, B., Bergström, G., Stallberg-Stenhagen, S., 1970. Volatile components of the cephalic marking secretion of male bumble bees. Acta Chem. Scand. 24, 1481–1483. Kullenberg, B., Bergström, G., Bringer, B., Carlberg, B., Cederberg, B., 1973. Observations on scent marking by Bombus Latr. and Psithyrus Lep. Males (Hymn, Apidae) and localization of site of production of the secretion. Zoon Suppl. 1, 23–30. Pittioni, B., 1942. Die boreoalpinen Hummeln und Schmarotzerhummeln. I. Teil: Einleitung. Mitteilungen Königl. Naturwissenschaftl. Instituten Sofia XIV, 155–212. Sladen, F.W.L., 1912. The Humble-bee, Its Life-history and How to Domesticate It. Macmillan & Co., London. Reprint Logaston Press 1989. Scholl, A., Obrecht, E., 1983. Enzymelektrophoretische Untersuchungen zur Artabgrenzung im Bombus-lucorum-Komplex. Apidologie 14, 65–78. Svensson, B.G., 1973. Morphological studies on two Scandinavian Subspecies of B. lapponicus Fabr. Entomologisk Tidskrift 94, 140–147. Svensson, B.G., 1980. Species-isolating Mechanisms in Male Bumble Bees (Hymenoptera, Apidae). Ph D thesis, University of Uppsala, Sweden. Tkalcu, B., 1967. Sur deux espèces de Bourdons décrites par William Nylander. Bulletin de la Société Entomologique de Mulhouse 1967, 41–58. Urbanova, K., Valterova, I., Hovorka, O., Kindl, J., 2001. Chemotaxonomical characterization of males of Bombus lucorum (Hymenoptera: Apidae) collected in the Czech Republic. Eur. J. Entomol. 98, 111–115. Urbanova, K., Halik, J., Hovorka, O., Kindl, J., Valterova, I., 2004. Marking pheromones of the cuckoo bumblebee males (Hymenoptera, Apoidea, Bombus Latreille): composition of labial gland secretions of six species found in the Czech Republic. Biochem. Systematics Ecol. 32, 1025–1045. Valterova, I., Urbanova, K., 1997. Chemické signálu Cmeláku. ([Chemical signals of bumble bees]). Chemické Listy 91, 846–857 (in Czech). Zacek, P., Kalinova, B., Sobotnik, J., Hovorka, O., Ptacek, V., Coppée, A., Verheggen, F., Valterova, I., 2009. Comparison of age-dependent quantitative changes in the mal labial gland secretions of Bombus terrestris and Bombus lucorum. J. Chem. Ecol. 35, 698–705.
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Labial gland marking secretions of male Bombus lucorum bumblebees from Europe and China reveal two separate species: B. lucorum (Linnaeus 1761) and Bombus minshanicola (Bischoff 1936) Andreas Bertsch a, *, Horst Schweer b a b
Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Straße, D-35032 Marburg, Germany Department of Pediatrics, Philipps-University Marburg, Baldingerstraße, D-35043 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2011 Accepted 29 April 2011 Available online 24 May 2011
Differences in male bumblebee labial gland secretions can be used to separate species pairs. Cephalic labial gland secretions from male Bombus lucorum Linnaeus bumblebees from Europe, and male ‘B. lucorum’ bumblebees from China, were analyzed by gas chromatography/mass spectrometry (GC/MS). In the European B. lucorum L., ethyl tetradec9-enoate was identified as the major compound (58% peak area), along with a complex mixture of straight-chain alkenols, acetates, hydrocarbons, and wax type esters. In contrast, the main component of the labial gland secretions of the Chinese ‘B. lucorum’, ethyl dodecanoate (31%), did not dominate the secretion, which additionally contained large amounts of 3,7,11-trimethyldodeca-6,10-dien-1-ol (¼2,3-dihydrofarnesol, 24% peak area), ethyl octadec-9-enoate (15% peak area) and a mixture of acyclic diterpenes (alcohols, aldehydes, and acetates). Furthermore, in B. lucorum, 3,7,11-trimethyldodeca-6,10-dien-1ol was only detected in trace amounts (0.05%) and ethyl octadec-9-enoate corresponded to only 0.8% of the mixture. These results indicate that ‘B. lucorum’ from China is a separate taxon from B. lucorum L. Further analysis revealed that ‘B. lucorum’ from China has been previously described as Bombus minshanicola Bischoff 1936 from Gansu/China. Differences in the chemical composition of male bumblebee labial gland secretions are discussed in comparison with other known bumblebee species pairs. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Hymenoptera Bombus lucorum Bombus minshanicola Cephalic labial gland Gas chromatography Mass spectrometry
1. Introduction For a long time, so-called cryptic species have presented problems for taxonomy. Since taxa were defined by morphological features, the lack of separating morphological characteristics was a significant impediment. With the advent of DNA markers and other tools for biochemical systematics, things changed fundamentally. A good example could be the bumblebee species Bombus lucorum (Linnaeus 1761), which was tentatively separated from Bombus terrestris in the beginning of the 20th century, and established as a separate species on the basis of its morphology (Krüger, 1920, 1951), male labial gland secretions (Calam, 1969; Bertsch, 1997; Urbanova et al., 2001) and mitochondrial DNA markers (Bertsch et al., 2005; Bertsch, 2009). In fact, Scholl and Obrecht (1983) coined the term “B. lucorum-complex” to point out that we have to handle a complex of three taxa which are difficult to separate by morphological characteristics alone. With the use of DNA markers and male labial gland secretions, these taxonomical problems could be solved (Bertsch, 2009). Genetically, the B. lucorum-complex does not exist,
* Corresponding author. E-mail addresses: [email protected] (A. Bertsch), [email protected] (H. Schweer). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.04.010
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and the main substance identified in the male labial gland secretions (ethyl tetradec-9-enoate) of B. lucorum is unique, clearly separating this species from all the other taxa involved. Interestingly, Cameron et al. (2007) reported bumblebee DNA sequences from Sichuan/China that they also refer to as ‘B. lucorum’, but which differ essentially from the DNA sequences of B. lucorum from Europe, a result confirmed by the unpublished COI sequence of ‘B. lucorum’ from North China (GenBank GU085202); proving that this Chinese ‘B. lucorum’ is genetically very different from European B. lucorum. This ‘B. lucorum/China’ is not identical with the taxon B. lucorum. To better understand this interesting bumblebee taxon ‘B. lucorum’ from China, we investigated male cephalic labial gland secretions and included GC analysis of the European B. lucorum species for direct comparison. 2. Materials and methods 2.1. Materials Male ‘B. lucorum’ bumblebees were collected at Cirsium leo, approximately 50 km west of Yongdeng, Gansu, China (36 41.260 N, 102 42.770 E). For comparison, male B. lucorum Linnaeus bumblebees were also collected from Kirkwall, Orkney Islands, Mainland, UK (58 59.050 N, 2 57.560 W). Because safe identification using morphological characteristics alone is difficult and problematic, all specimens have been unambiguously identified using DNA markers (Bertsch, 2009, Table 1 specimen LUC-01 and Bertsch, 2010, Table 1 specimen MIN-03). Old males were recognized by their frayed wings and discarded. The wing borders of all males used for labial gland preparations were smooth, indicative of active males. The cephalic part of the labial gland was dissected from the head while frozen, and placed in a vial (glands from five males where pooled in each vial) containing 0.2 ml pentane. Because the aim of this investigation was to elucidate the chemical complexity and composition of male bumblebee labial glands, and not to study gland content variability, we pooled glands from five males into a single vial. This method allowed for detection of even minor compounds present in the glands.
2.2. Gas chromatography/mass spectrometry A Finnigan MAT TSQ700 gas chromatograph/tandem mass spectrometer was employed. Gas chromatography was carried out using a Hewlett Packard Ultra 1 column (50 m, 0.2 mm internal diameter, 0.11 mm film thickness) in splitless mode, with helium as the carrier gas and at an inlet pressure of 300 kPa. The split valve was opened for 1 min. An initial temperature of 120 C was held for 1 min, then increased at a rate of 8 /min to 280 C, followed by an increase at a rate of 3 /min to 310 C, and finally by 1 /min to 320 C. This final temperature was held for 10 min. Mass spectrometer conditions were as follows: the interface temperature was 300 C, source temperature 130 C, electron energy 70 eV, emission current 0.2 mA, and electron multiplier 1400 V. When using the mass spectrometer in positive ion chemical ionization (CI) mode, the ammonia CI gas pressure was 70 Pa. Compounds were identified by comparison with compounds in the NIST 2002 Library (National Institute of Standards and Technology, USA), along with retention times and molecular ions from the CI spectra. To determine the positions of double bonds, derivatization with dimethyl disulfide was used, as described by Buser et al. (1983). 3. Results Typical chromatograms of male cephalic labial gland secretions of ‘B. lucorum’ from China (¼ Bombus minshanicola), and B. lucorum from Europe are shown in Fig. 1. Compounds identified are summarized in Table 1. The labial glands contained a mixture of acyclic diterpenes (alcohols, aldehydes, and acetates) and various straight-chain fatty acid derivatives (alcohols, aldehydes, esters and both saturated and unsaturated hydrocarbons with C21–C29). In the male labial gland of ‘B. lucorum’ from China, the major compound identified was ethyl dodecanoate (31% of the total peak area, peak 5) (see Fig. 1). In addition, large amounts of 3,7,11-trimethyldodeca-6,10-dien-1-ol (peak 10, 24% of total peak area) and ethyl octadec-9-enoate (peak 49, 15% of total peak area) were detected. Furthermore, minor amounts (1%–5%) of dodecanoic acid (peak 2), tetradecanal (peak 6), 3,7,11-trimethyldodeca-6,10-dienal (peak 7), two alcohols, octadeca-9,12, 15-trien-1-ol (peak 37), and icos-15-en-1-ol (peak 59), and one ester, 3,7,11-trimethyldodeca-6,10-dien-1-yl dodecanoate (peak 85) were detected. An additional alcohol, octadeca-9,12-dien-1-ol (peak 36), two more aldehydes, 3,7,11-trimethyldodeca-2,6,10-trienal (peaks 11 & 12), five more ethyl esters, ethyl tetradecanoate (peak 15), ethyl hexadecanoate (peak 32), ethyl octadecatrienoate (peak 48), and ethyl octadec-11-enoate and ethyl octadec-13-enoate (peaks 50 and 52) and three acetates, 3,7,11-trimethyldodeca-6,10-dien-1-yl acetate (peak 17), octadecatrienyl acetate (peak 51), octadecyl acetate (peak 53) and an additional acid, octadec-9-enoic acid (peak 45), were also detected in small amounts (between 0.2% and 1%). In the male labial glands of B. lucorum from Europe, the major compound identified was ethyl tetradec-9-enoate (58% of the total peak area, peak 14) (see Fig. 1). No other compound was detected in large amounts (>5%). However, minor amounts (1%–5%) of a series of alcohols, hexadecan-1-ol (peak 21), octadeca-9,12-dien-1-ol (peak 36), and octadeca-9,12,15-trien-1ol (peak 37) were detected. An additional alcohol, icosan-1-ol (peak 60), and an aldehyde, octadecenal (peak 31), two ethyl esters, ethyl hexadec-11-enoate (peak 29) and ethyl octadec-9-enoate (peak 49), were also detected in small amounts (between 0.2% and 1%).
A. Bertsch, H. Schweer / Biochemical Systematics and Ecology 39 (2011) 587–593
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B. minshanicola 5
100%
-Ethyl dodecanoate 10 49
75%
-Ethyl octadec-9-enoate -3,7,11-Trimethyl-dodeca-6,10-dien-1-ol
50%
6
25%
37 85
7
Octadeca-9,12,15-trien-1-ol-
59
-Icos-15-en-1-ol
2
74
10:00
100%
B. lucorum
15:00
20:00
25:00
20:00
25:00
14
-Ethyl tetradec-9-enoate 75%
15
50% 5
28 37
-Ethyl dodecanoate 21
25%
-Octadeca-9,12,15-trien-1-ol 48
Hexadecan-1-ol-
60
-Icosan-1-ol
10:00
15:00
RT (min:sec)
Fig. 1. Total ion chromatograms of cephalic labial gland pentane extracts from ‘Bombus lucorum’ China (¼ B. minshanicola) and Bombus lucorum from Europe. Numbers correspond to the numbers in the peak list (Table 1). Some compounds listed in Table 1 were present in quantities too low to be visible in these chromatograms.
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Table 1 Compounds of the cephalic labial glands of ‘B. lucorum’ from China (¼B. minshanicola) and B. lucorum from Europe, peak areas more than 10% in bold. No.
Compound
RT [min:sec]
RI
1 2 3 4 5 6 7 8 9 10 11
Ethyl decanoate Dodecanoic acid Ethyl dodecenoate (isomer I) Ethyl dodecenoate (isomer II) Ethyl dodecanoate Tetradecanal 3,7,11-Trimethyldodeca-6,10-dienal 1-Methylethyl dodecanoate Tetradecan-1-ol 3,7,11-Trimethyldodeca-6,10-dien-1-ol 3,7,11-Trimethyldodeca-2,6,10-trienal (isomer I) 3,7,11-Trimethyldodeca-2,6,10-trienal (isomer II) Tetradecanoic acid Ethyl tetradec-9-enoate Ethyl tetradecanoate Hexadecenal 3,7,11-Trimethyldodeca-6,10-dien-1-yl acetate 3,7,11-Trimethyldodeca-2,6,10-trienoic acid Hexadecanal Ethyl tetradecadienoate Hexadecan-1-ol Nonadecane Hexadecenoic acid Hexadecanoic acid Ethyl hexadecadienoate Icosadiene Ethyl hexadec-7-enoate Ethyl hexadec-9-enoate Ethyl hexadec-11-enoate Octadecatrienal Octadecenal (isomer I) Ethyl hexadecanoate Octadecadienal Ethyl hexadecadienoate Octadecenal (isomer II) Octadeca-9,12-dien-1-ol Octadeca-9,12,15-trien-1-ol Octadec-9-en-1-ol Octadecan-1-ol Henicosene Henicosane Octadeca-9,12-dienoic acid Octadeca-9,12,15-trienoic acid Octadecenoic acid (isomer I) Octadec-9-enoic acid (isomer II) Icosa-5,8,11,14-tetraenoic acid Ethyl octadecadienoate Ethyl octadecatrienoate Ethyl octadec-9-enoate Ethyl octadec-11-enoate Octadecatrienyl acetate Ethyl octadec-13-enoate Octadecyl acetate Ethyl octadecanoate Icosenal (isomer I) Icosenal (isomer II) Docosane Icosen-1-ol (isomer I) Icos-15-en-1-ol (isomer II) Icosan-1-ol Tricos-9-ene Tricos-7-ene Tricos-5-ene 3,7-Dimethyloct-6-en-1-yl dodecanoate Tricosane
6:00 8:27 8:36 8:41 8:45 8:54 8:57 9:07 9:47 10:02 10:13
1378 1562 1569 1572 1579 1586 1591 1612 1657 1676 1689
– – 30.71 1.65 1.59 0.18 0.19 23.61 0.36
10:32
1711
0.55
10:53 11:10 11:21 11:25 11:30 11:34 11:36 11:39 12:29 12:57 13:20 13:33 13:35 13:37 13:40 13:42 13:45 13:49 13:52 13:55 13:57 13:59 14:03 14:36 14:38 14:52 14:58 15:03 15:25 15:30 15:32 15:36 15:41 15:47 15:52 15:56 16:04 16:06 16:09 16:12 16:20 16:23 16:25 16:30 16:35 17:07 17:14 17:21 17:22 17:25 17:34 17:38 17:42
1735 1746 1762 1768 1775 1782 1785 1789 1864 1900 1946 1960 1962 1965 1967 1970 1973 1977 1980 1983 1986 1989 1995 2042 2045 2058 2061 2068 2100 2107 2110 2116 2123 2132 2139 2143 2152 2155 2159 2163 2173 2177 2182 2193 2200 2247 2258 2263 2270 2274 2288 2294 2300
0.04 0.02 0.31 0.05 0.37 0.04 0.08 – 0.08 0.01 0.01 0.01 – – – – 0.08 0.10 0.02 0.20 – – 0.01 0.24 3.27 0.11 – 0.02 0.62 – – 0.06 0.53 – – 0.23 14.52 0.56 0.22 0.36 0.44 – – 0.10 0.05 0.04 1.37 – 0.53 – – 0.05 1.53
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
B. minshanicola [% peak area] 0.09 1.93
B. lucorum [% peak area]
Mþ [m/z]
0.01 0.04 0.03 0.04 9.56 0.14 0.14 – – 0.03 –
200 200 226 226 228 212 222 242 214 224 220
–
220
0.03 58.4 4.64 – – – 0.02 0.02 3.54 – 0.02 – 0.08 0.34 0.18 3.55 0.31 0.07 0.42 0.03 0.06 0.03 – 1.64 4.66 0.02 0.16 – 0.15 0.01 0.02 – 0.05 0.08 0.03 1.27 0.82 0.04 – – – 0.04 0.01 0.02 0.03 0.08 0.10 0.94 0.28 0.03 0.01 – 2.92
228 254 256 238 266 254 240 252 242 268 254 256 280 278 282 282 282 262 266 284 264 282 266 266 264 268 270 294 296 280 278 282 282 304 308 306 310 310 306 310 312 312 294 294 310 296 296 298 322 322 322 338 324
DMDS adducts
18:51 {117, 231; Mþ 348}
20:38 {173, 203; Mþ 376} 20:42 {145, 231; Mþ 376} 20:51 {117, 259; Mþ 376}
24:42 {173, 189; Mþ 362}
22:23 {173, 203; Mþ 376}
22:39 {173, 231; Mþ 404} 22:42 {145, 259; Mþ 404} 22:48 {117, 287; Mþ 404}
23:37 {117, 273; Mþ 390} 23:30 {173, 243; Mþ 416} 23:35 {145, 271; Mþ 416}
A. Bertsch, H. Schweer / Biochemical Systematics and Ecology 39 (2011) 587–593
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Table 1 (continued ) B. lucorum [% peak area]
Mþ [m/z]
0.10
–
350
0.06 0.06 0.07 0.08 0.07 0.02 0.63 0.29 – 0.22 0.01
– – – – – 0.02 0.06 0.13 1.68 0.13 1.45 –
338 338 338 340 338 336 338 324 350 350 352 378
2550 2568 2600 2677 2683 2700 2760
0.06 0.04 – 0.37 – – 4.83
– 0.01 0.01 0.92 0.20 0.09 –
368 364 366 378 378 380 406
2766 2773 2778 2891
0.55 – 0.02 0.04
– 0.01 – 0.12
396 392 404 406
No.
Compound
RT [min:sec]
RI
66
3,7,11-Trimethyldodeca-6,10-dien-1-yl octanoate Icos-15-en-1-yl acetate (isomer I) Icosen-1-yl acetate (isomer II) Icosen-1-yl acetate (isomer III) Ethyl icosanoate Icosen-1-yl acetate (isomer IV) Tetracosene Tetracosane Docos-15-en-1-ol Pentacos-9-ene Pentacos-7-ene Pentacosane 3,7,11-Trimethyldodeca-6,10-dien-1-yl decanoate Dodecyl dodecanoate Hexacosene Hexacosane Heptacos-9-ene Heptacos-7-ene Heptacosane 3,7,11-Trimethyldodeca-6,10-dien-1-yl dodecanoate Tetradecyl dodecanoate Octacosene Nonacosadiene Nonacos-9-ene
18:13
2341
18:14 18:18 18:25 18:32 18:34 18:45 18:58 19:19 19:30 19:35 19:47 20:15
2342 2347 2356 2366 2368 2371 2400 2450 2476 2491 2500 2542
20:20 20:31 20:47 21:30 21:36 21:44 22:16 22:19 22:23 23:26 23:33
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
þ
RT ¼ retention time; RI ¼ retention index; M double bond could not be assigned.
B. minshanicola [% peak area]
–
¼ molecular ion; DMDS adducts ¼ RT min:sec {xxx, xxx; M
þ
DMDS adducts
24:53 {117, 315; Mþ 432}
25:44 {145, 273; Mþ 418} 25:50 {173, 271; Mþ 444}
28:20 {173, 299; Mþ 472}
31:36 {173, 327; Mþ 500}
xxx}, in some minor compounds, position of the
4. Discussion 4.1. Flight path activity and male labial gland secretions Flight path activity and scent marking has long been observed for many bumblebee species, and male cephalic labial gland secretions have been investigated in great detail (Bergström et al., 1981; Valterova and Urbanova, 1997). In early studies, Sladen (1912) stated that the male scent ‘attracts not only males but the queens’, and following the first chemical investigations, Kullenberg et al. (1973) formulated the hypothesis that these compounds are actually sex pheromones used to attract virgin females, a hypothesis not yet confirmed by empirical evidence. Flight path activity and scent marking by ‘B. lucorum’ from China is completely unknown, and even in Northern Europe, where B. lucorum is one of the most abundant bumblebees, bumblebee flight path activities in the field have not been investigated in detail; and only casual observations have been reported (Bringer, 1973). In fact, quantitative flight path activity and scent marking data for European B. lucorum males are only available from climatic test chambers (Bertsch, 1984). The most useful application of male bumblebee labial gland secretions to date has been the separation of critical taxa, to establish taxonomical rank. For example, separating specimens of B. terrestris and B. lucorum in some parts of Europe by morphological characteristics alone is so difficult that both species have for a long time been treated as a single species. The detection of large differences in the chemical composition of compounds from male labial gland secretions finally established both taxa as separate species (Calam, 1969; Kullenberg et al.,1970, 1973). The same problem was encountered with Bombus vestalis and Bombus bohemicus, two parasitic bumblebees of the subgenus Psithyrus which are morphologically very similar and often difficult to separate; but analysis of their male labial gland secretions separated them very clearly (Bergman et al., 1996; Urbanova et al., 2004). B. lapponicus auct. is another good example, where all efforts to separate taxa by morphological characteristics alone had failed (Pittioni, 1942); finally, investigation of male labial gland secretions revealed two separate species, Bombus monticola Smith and Bombus lapponicus Fabricius (Bergström and Svensson, 1973; Svensson, 1973, 1980).
4.2. Differences in metabolic pathways Compounds of male labial gland secretions may be variable. Individual gland contents may change quantitatively and qualitatively (a) over the lifetime (see Zacek et al., 2009 for B. terrestris) and (b) during the day (secretions are used for scent marking in the morning, so glands may be depleted later in the day; see Bergman, 1997 for Bombus lapidarius) and (c) geographically between well established subspecies (Coppée et al., 2008 for B. terrestris). But this variability does not alter the typical pattern of substances it is always possible to identify the pattern of substances typical for a certain species. The
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differences found in labial gland secretions between all of the above species pairs were not quantitative, but qualitative, changing the pattern of substances. Male bumblebee labial gland secretions can be roughly divided according to the biochemical pathways which synthesize the compounds. Using this classification system, two types of secretions have been identified: PP type secretions, containing fatty acid derivatives only (Polyketide Pathway: PP); and PP þ MAP type secretions, which, in addition to fatty acid derivatives, contain mevalonic acid derivatives (Mevalonic Acid Pathway: MAP) in the form of acyclic diterpenes (Bergman et al., 1996). The species pairs B. lucorum – B. terrestris, B. bohemicus – B. vestalis and B. monticola – B. lapponicus belong to this class; the first members of these species pairs contain PP type labial gland secretions and the second members contain PP þ MAP type secretions. The species pair analyzed in this investigation, B. lucorum from Europe and ‘B. lucorum’ from China, also fits well with this classification system. B. lucorum from Europe contains PP type labial gland secretions and ‘B. lucorum’ from China contains PP þ MAP type labial gland secretions. It is another example of a species pair which is difficult to separate by morphological characteristics alone, but has essentially different male labial gland secretions. Because B. lucorum from ‘Europe’ and ‘B. lucorum’ from China use different species recognition signals, they are separate taxa. Furthermore, ‘B. lucorum’ from China most probably is a taxon in the rank of a species. 4.3. Taxonomic consequences As the subgenus Bombus sensu stricto has been thoroughly studied, and has already delivered a huge number of color variants and names (Krüger, 1951, 1954, 1956, 1958), it seemed improbable to us that ‘B. lucorum’ from China did not already have a name. Because B. lucorum lanchouensis Vogt 1908 was proposed to be synonymous with Bombus patagiatus, as B. patagiatus ssp. lanchouensis (Tkalcu, 1967), only one taxon remains from the area (Northern Sichuan & Gansu/China). This taxon was collected in 1930 by Dr. D. Hummel during a Sven Hedin expedition, and was described as B. terrestris ssp. minshanicola (Bischoff, 1936) from Gansu/China, and later transferred to B. lucorum by Krüger (1951). Recent genetic investigations established this taxon as a separate species, B. minshanicola Bischoff 1936, genetically closer to the North American Bombus affinis and Bombus franklini than to the European B. lucorum (Bertsch, 2010). Thus, this investigation of male labial gland secretions of ‘B. lucorum’ from China confirms these genetic analysis results. 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