systematic relationship of halictinae bees based on the pattern of macrocyclic lactones in the...

Insect Biochem., Vol. 12, No. 2, pp. 161 170, 1982. 0020-1790/82/020161-10503.00/0 Printed in Great Britain. © 1982 Peryamon Press Ltd.

SYSTEMATIC RELATIONSHIP OF HALICTINAE BEES BASED ON THE PATTERN OF

MACROCYCLIC LACTONES IN THE D U F O U R GLAND SECRETION

INGELA JOHANSSON*, BO G. SVENSSON~', JAN TENGO* and GUNNAR BERGSTR6M*~

*Ecological Station of Uppsala University, S-386 00 F~irjestaden, Sweden; tDepartment of Entomology, Uppsala University, S-751 22 Uppsala, Sweden and

~Department of Ecological Chemistry, GiSteborg University, S-400 33 GiSteborg, Sweden

(Received 28 May 1981)

Abstract--The volatile Dufour gland secretion from 16 species within the subfamily Halictinae has been studied by gas chromatography and mass spectrometry. Isolation and identification was performed on single individuals. From 1 to 15 individuals of each species were collected and chemically analyzed over a period of 1 to 5 yr. The secretions of each species were found to be built up by characteristic combinations of saturated and mono-unsaturated macrocyclic C16-, Cls-, C20-, C22- and C24-1actones, together with some straight chain hydrocarbons. Individuals of each species did not vary appreciably in the set-up of their secretions. Of the species studied five represent the genus Halictus and 11 represent the genus Lasioglossum. H. eurygnathus and H. rubicundus, which belong to the subgenus Halictus, have 24-tetracosanolide and 22-docosanolide as the main volatile constituents, whereas L. albipes and L. calceatus, which belong to the subgenus Evylaeus, have 16-hexadecanolide as the dominating volatile component. Based on the pattern of compounds in the secretions of the species, the systematical relationships between these species are discussed.

Key Word Index: Macrocyclic lactones, Halictinae bees, Dufour gland secretion, gas chromatography, mass spectrometry, chemical systematical characters, Halictus, Lasioglossum

I N T R O D U C T I O N

THE DUFOUR gland, which is connected to the sting apparatus of the female, reaches a considerable size during the nesting period in most ground nesting bees. This is also the case in the bee genera Halictus and Lasioglossum. Anatomical and histological inves- tigations of the Dufour gland in bees have been made by DUFOUR (1841), SEMICHON (1906), HESELHAUS (1922), PAWLOWSKY (1927) and DE LELLO (1971, 1976). Both DUFOUR (1841) and SEMICHON (1906) indicated that the secretion of the Dufour gland might be the origin of the lining or the delimiting membrane that encloses the nest cells, within which the bee larva de- velops on a stock of pollen and nectar. BATRA (1964, 1968, 1972), DE LELLO (1971) and MAY (1970) have the same opinion concerning the function of the Dufour gland in the halictine bees.

Chemical analysis of the Dufour gland secretions of bees of the subfamily Halictinae (ANDERSSON et al., 1966; BERGSTROM, 1974; HEFETZ et al., 1978; BERG- STR6M and TENG6, 1979) as well as the bee genera Colletes (BERGSTR6M, 1974; HEFETZ et al., 1979; ALBANS et al., 1980) and Hylaeus (DoFFIELD et al., 1980), in the family Colletidae, have revealed the pres- ence of several macrocyclic lactones. BERGSTR~M and TENG6 (1979) and HEFETZ et al. (1979) postulated the possible polymerisation of hydroxy acids, corresponding to the lactones, in forming a hydrophobic cell lining. HEFETZ et al. (1979) showed that the lining in

three Colletes spp. is constructed mainly from 18-hyd- roxyoctadecanoic and 20-hydroxyeicosanoic acids.

This study reports on the chemistry of the volatile part of the Dufour gland secretion of 16 species of the Halictinae genera Halictus and Lasioolossum. The relative amounts of the lactones between the species are studied and evaluated in relation to the morphological classification of Halictinae. Comments on the morphology of the Dufour gland in the species analyzed are also given.

MATERIALS A N D M E T H O D S

Biological material

All the species analyzed were collected on the island of ()land in the Baltic Sea, except Lasioglossum boreale, which emanates from Abisko, northern Sweden. Two specimens of Halictus rubicundus collected in Kansas, U.S.A., were also available (Table 1). The nomenclature and the system- atics of Halictinae follow EBMER (1976) and for L. boreale, cf. SVENSSON et al. (1977). The specimens analyzed are de- posited in the BGS collections (Department of Entomo- logy, Uppsala University), and the chromatograms/mass spectra are at the Ecological Station on Oland.

The bees were collected during their nesting periods for the chemical analyses. At that time the gland is completely filled. The prehibernating females, in contrast, had a thin wrinkled gland (Fig. 1). After collection, the bees were kept in a refrigerator at +8°C. The Dufour gland (Fig. 1) was usually dissected the day of collection. The preparation of the glands, described earlier by BERGSTROM and TENGO

161

162 INGELA JOHANSSON et al.

Table 1. Number of specimens of each species analysed daring 1973-1980. A full account of the taxonomy of the species studied is given in Table 3

1973 1974 1975 1977 1978 1979 1980 n

H. (H.) eurygnathus 3 2 I 2 8 H. (H.) rubicundus 2 1 3 H. (S.) tumulorum 4 2 3 9 H. IS.) leucahaeneus 8 2 1 11 H. (S.) confusus 1 1 L, [L.) leucozonium 1 6 3 2 3 15 L, (L.) quadrinotatum 2 1 3 L. (E.) aeratum 1 3 4 L, (E.) leucopum 1 I 2 5 9 L. (E.) morio 1 9 1 11 L. (E.) semilucens 3 3 L. (E.) villosulum 1 1 2 L. ( E.) Jidvicorne 6 2 3 11 L. (E.) boreale 1 1 L. (E.) albipes 1 3 1 5 10 L. (E.) calceatum 1 1 2 4 8

(1974), was made under insect saline solution: NaCI 9.0, KCI 0.42, CaCI2 0.25, NaHCO3 0.20, glucose 2.5 g/l dis- tilled water. The gland was either extracted in hexane or put directly in a precolumn tube of the gas chromatograph.

Chemical techniques

The volatile part of the Dufour gland content was subjected to combined gas chromatographic/mass spectro- metric analyses (GCMS), using a LKB 2091 GCMS instrument at the Ecological Station. Separate gas chromatographic analyses (GC) were also made with a Perkin- Elmer 100 GC. In both cases glass capillary columns were used. The stationary phases were OV-17/FFAP (5:6 w/w) (own preparation) and WG-I1 (Werner Giinther Analyse- technik, W. Germany).

Typically, the gas chromatographic analyses were started at + I50°C and then programmed for an 8°C/rain rise to +230°C, thereafter being isothermal for approx. 30min. Gas chromatographic retention values and mass spectra of components in the secretion were compared with those of reference compounds. Extracts were made in 0.2 ml of hexane and in most analyses a 2/A portion of this extract was used for the GCMS analysis, Some compounds, mainly esters of low volatility and of medium polarity, comparable to the components of the highest mass in the secretion, were used as references to ensure that the gas chromatographic variables (type of column, temperature program- ming, gas flow, etc.) accurately represented the true proportions between the components. In Table 2, the proportions of the different lactones present in the species are based on gas chromatograms of hexane extracts of single glands.

RESULTS

During dissection the morphology of the Dufour gland was noted for each species. PAWLOWSKY (1927) and DE LELLO (1971) presented diagrams of the gland of some species. The gland in Halic tus (H.) rubicundus and in H. (H.) sexcinctus (PAWLOWSKY, 1827; DE LELLO, 1971) are very alike in their general appearance, that is, they have one appendix emerging near the base of the gland (Fig. 1). In Lasioglossum (E.) punctat iss imum (PAwLOWSKY, 1927) there is an ad- ditional very short appendix bulging out opposite to the larger one. BORDAS (1895) also found two appen- dices to the gland in Lasioolossum (L.) leucozonium. In the species now studied there was no short appendix

present in the Halic tus members, whereas a short appendix was found in the Lasioglossum species (Fig. 1). The gland is much smaller in the Halic tus species studied than in the Lasioglossum species, in relation to the size of the abdomen. The generic difference in the shape of the Dufour gland between Hal ic tus and Lasioglossum has-not been stressed earlier. Prelimi- nary studies of the Dufour gland in some species of Sphecodes and Dufourea also revealed generic characters.

This report is based on analyses made during 1973-1980 (Table 1). Each analysis was made on single individuals. The number of individual specimens of each species analyzed varies, To the extent that we have analyzed several single individuals of the same species (see Table 2) we can say that the pattern of quantitative proportions between components does not vary greatly. This is exemplified by the three gas chromatograms given in Fig. 8, below [L. (E.) albipes]. An investigation of the intraspecific variation in the components of the Dufour gland secretion in halictinae bees is in progress (BRIAN H. SMrrH, Eco- logical Station of Uppsala University). Totally, 16 species were studied representing two subgenera of Halictus and two of Lasioglossum with two, three, two and nine species per subgenus.

The individual compounds in the secretions were identified through their gas chromatographic retention values and by their mass spectra. As an example, the mass spectrum of the largest macrocyclic lactone of the secretion from H. (H.) eurygnathus is given in Fig. 2. It shows the typical fragmentation pattern of a saturated macrocyclic lactone. The molecular peak (M = 366) is quite large with a relative intensity of 34~o. The fragment corresponding to loss of water (M - 18 = 348) gives a prominent peak with 27% relative intensity. Other characteristic fragments in the mass spectrum are M - 60 = 306 and M/e = 60. The hydrocarbon backbone of the macrocyclic lactone gives a series of strong fragments M/e = 43, 55, 69, 83, 97, 111, etc., with M/e = 55 being the base peak. The same pattern holds for all the even-num- bered macrocyclic lactones of hydrocarbon chain lengths C16 to C24.

Lactones of halictinae bees 163

Table 2. Macrocyclic lactones in the Dufour gland secretion of 5 Halictus and 11 Lasioglossum species

Fig n C16 C18 C20 C22 C24 $81 urtsat sat u~Isa! sal unsat, sat unsat sal unsat.

OOOOQO OQO

. . . . . O0 QGQO000

- , , . o ....

OOGOQOQO00

The symbols comprise means of all sampled individuals of a given species and represent the relative percentage (composition) of the lactones present in the secretion (lined portion indicate the pl'esence of a second isomer). Quantitative estimates are based on the peak areas in the chromatograms.

C16, Cis, C2o, C22 and C24 means that there are 16, 18, 20 22 and 24 carbon atoms, respectively, in the macrocyclic lactone chain. • = lactone dominates secretion, relative amount not calculated. • = lactone present, relative amount not calculated, sat. = saturated, unsat. = unsaturated.

l e u c a h e n e u s mor io l e u c o z o n ium

I m m

Fig. 1. General appearance of the Dufour gland in Halictus (Seladonia) leucaheneus, Lasioelossum (LasiooIossum) leucozonium and L. (Evylaeus) morio. The gland is completely filled during the nesting period, however, the prehibernating females have a thin wrinkled gland (shown for leucaheneus). The glands were straightened and loosened from other tissue to show their shape. Note the double appendix

in the Lasioolossum species.

164

t00

INGELA JOHANSSON et al.

~ e o

~ 6 0

.~ &O' m

~ 20'

0 ~ 0

SS 80 23~ 16

1.3

97 M

M-18 366

II] IL J ,,, , - !

20 &O 60 80 I00 120 t/.O 160 180 200 220 2&O 260 280 300 320 340 360 mle

Fig. 2. Mass spectrum of the largest macrocyclic lactone component in the Dufour gland secretion of H.(H.) eurygnathus, identified as 24-tetracosanolide.

Representative capillary gas chromatograms of volatile compounds from individual extracts of Dufour gland secretions of six different species are given in Figs. 3-8. Of these, two species belong to the genus Halictus and four to the genus Lasioglossum. The first example (Fig. 3), shows H. (H.) eurygnathus. In this species macrocyclic lactones with a long hydrocarbon chain dominate the secretion. Thus, the two main components of this species are 22-docosanolide and 24-tetracosanolide. Tricosane gives a large peak in this chromatogram, however, the intensity of the straight chain hydrocarbons varies. In the other Halictus species shown, H. (Seladonia) leucaheneus (Fig. 4), 18-octadecanolide, 20-eicosanolide and 22-docosanolide are the very dominant components with smaller amounts of the corresponding monounsaturated macrocyclic lactones and 24-tetracosanolide. Lasioglossum (L.) leucozonium, L. (Evylaeus) fulvi-

corne and L. (E.) semilucens (see gas chromatograms in Figs. 5, 6 and 7, respectively), represent species which all have 18-octadecanolide as the main component and smaller amounts of 20-eicosanolide and even smaller amounts of 22-docosanolide. These species are quite similar to each other. However, L. (L.) leucozonium has a monounsaturated 20-eicosanolide as the second largest component, and L. (E.)fulvicone stands out by having a double set of two unsaturated lactones in both the 20-eicosanolide and the 22-docosanolide area. L. leucozonium differs also from most of the Lasioglossurn species, together with L. (E.) albipes and L. (E.) calceatum, by having very small amounts of 22-docosanolide and no detectable 24-tetracosanolide.

L. (E.) albipes and L. (E.) calceaturr~ which have been reported on before by our group (ANDERSSON et al., 1966), represent the other extreme [see example

8

_I 5

Ct~~ C2s

I I 30 20

C2s 80:250

J I

I IO A ~sothermal +230 ° C /

M in

0

Proor + 150 °C

Fig. 3. Capillary gas chromatogram of a portion of an extract of the Dufour gland secretion from one H.(H.) euryonathus Analytical conditions are given in the text. The numbers of the peaks represent the following lactones: 1. 16-hexadecanolide; 2. 16-hexadccenolide; 3. 18-octadecanolide; 4. 18-octadece- nolid¢; 5. 20-eicosanolide; 6. 20-eicosenolid¢; 7. 22-docosanolide; 8. 22-docosenolide; 9. 24-tetracosa-

nolide; 10. 24-tetracosenolide.


7 5

I I 30 20

, C2

LJ

3 8 0 : 2 4 7

I i I0

isol"hermol t 2 3 0 *C

Min

_1° Progr +150 *C

Fig. 4. Capillary gas chromatogram of a portion of an extract of the Dufour gland secretion from one H.(S.) leucaheneus. The numbers of the peaks are explained in the legends to Fig. 3.

given by L. (E.) albipes in Fig. 8]. These species have a secretion characterized by 16-hexadecanolide (trivial name dihydroambrettolide) as the main component. Both species also have 18-octadecanolide and a corresponding monounsaturated lactone, in about twice the amount of the saturated one, as secondary compounds.

DISCUSSION

Halictus s.l. is still accepted by some authors at the generic level. However, several external morphologi-

cal characteristics justify a segregation into two separate genera, viz. Halictus and Lasioglossum (EBMER, 1969; MICHENER, 1978a). WARNCKE (1975) briefly pro- posed several subgenera within the subgenus. For the north European species the most commonly adopted subgeneric classification is a division of the genera Halictus and Lasioglossum into Halictus/Seladonia and Lasioglossum/Evylaeus, respectively. The subgenus Dialictus recognized by some authors (e.g. FITTON et al., 1978) is a part of the species of the large subgenus Evylaeus. The systematic points of view on Halictus s.l., applied to the species used in this study, are summarized in Table 3.

LB. 12,2 c

I 3O

8 7

t 20

3 80:258

4

I iso,~ . . . . , * 2 ; % ,0 j Io

M i n Progr ÷ 150 *C

Fig. 5. Capillary gas chromatogram of a portion of an extract of the Dufour gland secretion from one L.(L.) leucozonium. The number of the peaks are explained in the legend to Fig. 3.

166 INGF.LA JOHANSSON #l al,

d c21 9 B

3 80:245

LL: T

30 20 Isot~errnQI~23G *(2 IO 0

~A(n Pro( F, +150 °C

Fig. 6, Capillary gas chromatogram of a portion of an extract of the Dufour gland secretion from one L.(E.) fulvicorne. The numbers of the peaks are explained in the legend to Fig. 3.

H. (H3 rubicundus and H, (H.) eurygnathus belong, from a morphological viewpoint to different groups within the subgenus (MtcHENER, 1978a). This is also the case for the two L. (Lasioglossum) species. Regard- ing the three Seladonia species studied, H. (S.) tumulorum and H. (S.) confusus are very closely related (Enr,~R, 1979). Concerning the Evylaeus species analyzed the systematic affinity is most apparent for the "species pairs" aeratum-leucopum and albipes-calcea- turn. The others belong to separate species groups, However, L. {E.)fulvicorne and L. (E,) boreale are together with L, (E,) albipes and L. (g.) calceatum, included in the large calceatum-group [Emvm~, 1976~.

If the systematic positions of the species analyzed {Table 3) are considered in relation to the patterns o[ volatile compounds found in the Dufour gland secretions (Table 2) some chemotaxonomical evalu- ations can be done. In H. (Haffctus) the 24-tetracosanolide and the 22-docosanolide are dominant, whereas in H. (Seladonia) the 20-eicosanolide and the 18-octadecanolide are most apparent. The 18-octadecanolide is dominant in the L, (Lasioglossum) species. The L, ~Evylaeus) species exhibit a mixed picture. In L, (E.) albipes and L. (E.) calceatum the 16-hexadecanolide toether with the 18-octadecanolide are very apparent. Those 'two species have a lactone compo-

3 BO : 246

30

LJ 20 Z$othlrmal ÷230 .C ~0 L I I I O

mgr÷~50 °C Min

Fig. 7. Capillary gas chromatogram.of a porlior~ of an extract of the Dufour gland secretion from one L.(E,) semilucens, The number of the peaks are explained in the legend to Fig. 3.


sition that is distinctly different when compared to the H. (Halictus) species, where the lactones with higher molecular weights dominate. The other L. (Evylaeus) species exhibit the same major components as the (H.) Seladonia species. However, in the latter species the 22-docosanolide is a more prominent component (cf. Fig. 9).

All species exhibit a species-specific blend. The results of this investigation suggest that there may be generic and subgeneric trends in the composition of macrocyclic lactones in the Dufour gland secretion. There are no absolute chemical limits between the subgenera Halictus, Seladonia, Lasioglossum and Evy- laeus, although this subgeneric classification seems to

be the best fit with the chemical patterns found. There are no chemical grounds for a segregation of the Dia- lictus species from Evylaeus.

HEFETZ et al. (1978) analyzed North American specimens of L. (L.) leucozonium. Their results regard- ing this species differ from ours in that the 20-eicosanolide constitutes a large portion of the volatile secretion in their analyses. They analyzed another eight species belonging to the genera Lasioglossum, Agapos- temon (both belonging to tribe Halictini) and Aucdoch- Iorella (tribe Augochlorini), but found neither 16-hexadecanolide nor 24-tetracosanolide in the Dufour gland secretion. Both differences in handling of the biological material and differences in analytical tech-

(a)

I I 30 20

80:235

I I0 Isothermal +230 °C

MIn

J 0 )

Proof +150 =C

(b)

I I 3O 20

¢ 3

JJ I s o ? h e r m o l +230 °C I0

Min

I 80:253

J MIn

L Figs 8(a) and (b).

I0

Progr.+ 150 °C

(c) 3

7

INGELA JOHANSSON el al. 168

80:56

1 I I M i n . I ! 30 20 I$oThermol -t-230 '~ I0 ~ O

Procr. I- 150 *C Min

Fig. 8. Three capillary gas chromatograms of portions of extracts of the Dufour gland secretion from individual L.(E.) albipes. The numbers of the peaks are explained in the legend to Fig. 3.

niques can account for this. Exchange of biological material may be needed to resolve details in the distri- bution pattern of macrocyclic lactones. It is worth noting that we have so far not found any species with a secretion that spans the whole series of even-num- bered macrocyclic lactones from Ct6 to C24 (see Table 2).

In bees having a large Dufour gland, its secretion obviously has a function in forming the brood cell lining as indicated in several recent papers (DE LELLO, 1971; BATRA, 1972; MAY, 1974; BERGSTR6M and TENG6, 1974, 1979; DUFFIELD et at., 1980). HEFETZ et al. (1979) and ALBANS et al. (1980) showed that the hydrophobic cell lining of Colletes bees is built up through polymerization of hydroxy acids. This prob- ably also occurs in the Dufour gland secretion of the bees studied here. The secretion might well have other functions as hypothesized by e.g. BERGSTR6M and TENG6 (1979) and DUFFIELD et al. (1980). The lactones may serve a semiochemical function. F rom the point of view of relative volatility of the macrocyclic lactones there is a considerable difference in vapor press- ures between 16-hexadecanolide and 24-tetracosanolide. It remains to be seen if this has a biological meaning.

The Halictinae genus Sphecodes comprises species which are cleptoparasitic mainly on Halictus (s.l.) species (MlC~m'r~g, 1978b). The Sphecodes species vaguely exhibit species-specific host choice. Often a group of e.g. Lasioolossum (L.) species is parasitized by one Sphecodes species. The volatiles in the secretion of the host may be important cues in the host- finding of Sphecodes as well as in other host-parasite relationships of this kind. Odour correspondence between host and cleptoparasite has been shown in the relation between Andrena and Nomada bees (TENC,6 and BERGSTR6M, 1977)

For the series H. (Halictus)-H. (Seladonia)-L. ( Ev ylaeus)/ Aoapost emon/ Augochlorella-L. ( Lasioglos- sum)-L. (E.) albipes/calceatum there are decreasing amounts of lactones of lower volatility and increasing amounts of lactones of higher volatility (see Table 2). The results of this study support in broad outline the classification based on morphological characteristics.

Note

After submission of this paper an article by DUFFIELD et al. was published in J. chem. Ecol. (1981) 7, 319-331, which describes the chemical analyses of the Dufour gland secretion in 18 Nearctic halictine bee species. The authors found macrocyclic lactones ranging from Cla to C26 and, in smaller amounts, branched Cs-alkenoi esters. Since four of the subgenera and three of the species have been investi- gated by both groups and since we both attempt to draw some systematic conclusions, a few comments may be in order.

(1) DUFFIELO et al. state that there is "no evidence of unique, species-specific blends". Considering our results reported in this paper and the following facts, we maintain that there is a species specificity, which is of systematic value.

(a) The Dufour glands were analysed individually. (b) In 9 of the species 8-15 specimens were analysed. (c) The specimens were collected during several years

and from different localities. (d) There is a high constancy in the proportions of the

lactones between the different analyses (cf. Fig. 8).

(2) DUFFIELO et al. state that "the higher taxa are also not chemically unique, although trends are apparent, that

~may have phylogenetic implications". This is close to our opinion, as can be seen above.

(3) There are three species analysed by both DUFFIELD et al. and ourselves. In these cases there are some differences in the results, as shown in Table 4 below (using the nota- tion of the American group to show the proportions of the

Lactones of halictinae bees

Table 3. Classification of the species analysed according to MICHENER (1978a), EBMER t1976) and FITTON et al. (1978)

169

Genus Subgenus Group Species

Halictus Latreille

Lasioglossum Curtis

Halictus s.str. s.str. Monilapis

Cockerell

Seladonia Robertson

Lasioglossum s.str.

Dialictus Robertson

Evylaeus Robertson

tumulorum

aeratum I

morio--"Gruppe im weiteren Sinn"

minutissimum

villosulum

calceatum--"Gruppe im weiteren Sinn"

rubicundus (Christ, 1791)

eurygnathus BliJthgen, 1930

Corlfttsus Smith, 1853

tumulorum (Linnaeus, 1758)

leucaheneus Ebmer, 1972

leucozonium (Schrank, 1781)

quadrinotatum (Kirby, 1802)

aeratum (Kirby, 1802)

leucopum (Kirby, 1802)

morio (Fabricius, 1793)

semilucens (AIfken, 1914)

cillosulum (Kirby, 1802)

fulvicorne (Kirby, 1802)

boreale Svensson. Ebmer & Sakagami 1977

albipes (Fabricius, 1781)

calceatum (Scopoli, 1763)

"/e 50

40

30

2O

10

o

)/' "'" "i ,/ '~, o "',,

o

el6 C18 C20 C22 C24

0 L. IL.)~yucozoniu,!

L.~v~laeus~. b specie=

L. (E.) alblpes ~d caleeatum

Fig. 9. Proportion of the macrocyclic lactones in the Dufour gland secretion of four halictine subgenera.

170 INGELA JOHANSSON et at.

Table 4.

Ct 8 C20 Cz 2 C2, Source sat. unsat, sat. unsat, sat. unsat, sat.. unsat.

x x x x x x D H. (H.) rubicundus

X X X X J

X X X D H. (S.) confusus x x x Xx x J

× × D L. (L.) leucozonium

x x x x x x x J

D = DUFFIELD et al. J = JOHANSSON et al. ( the present paper)

lactones). There may be differences between the American and the European populations*, however, as we have pointed out in the text, both handling of the biological material as well as differences in analytical techniques can influence the results. An exchange of biological material might help to clarify the differences in results.

Acknowledgements--This study has been financially sup- ported by the Swedish Natural Science Research Council and the Faculty of Mathematics and Sciences, Uppsala University. We thank Mr. BRXAN H. SMrm for helpful comments on the manuscript. Mr. THOMAS JONSSON, Mrs. INGA GRO'rH and Miss MONICA APPLEGREN have assisted in parts of the work.

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ANDERSSON C.-O., BERGSTROM G., KULLENBERG B. and STk, LLBERG-STENHAGEN S. (1966) Identification macrocy- lic lactones as odouriferous components of the scent of the solitary bees Halictus calceatus Scop. and Halictus albipes F. Ark. Kemi 26, 191-198.

BATRA S. W. T. (1964) Behaviour of the social bee, Lasiog- Iossum :ephyrum, within the nest (Hymenoptera: Halicti- dae). Insectes soc. II, 159-186.

BATRA S. W. T. (1968) Behaviour of some social and solitary halictine bees within their nests: a comparative study (Hymenoptera: Halictidae). J. Kans. ent. Soc. 41, 120-133.

BATRA S. W. T. (1972) Some properties of the nest-building secretions of Nomia, Anthrophora, Hylaeus and other bees. J. Kans. ent. Soc. 45, 208-218.

BEROS'rR6M G. (1974) Macrocyclic lactones in the Dufour gland secretion of the solitary bees Colletes cunicularius L. and Halictus calceatus Scop. (Hymenoptera, Apidae). Chem. Scr. 5, 39-46.

BERGSTR6M G. and TENG~ J. (1974) Farnesyl and geranyl esters as main volatile constituents of the secretion from Dufour's gland in 6 species of Andrena (Hymenoptera, Apidae). Chem. Scr. 5, 28-38.

BERGS'IX6M G. and TENG6 J. (1979) C24-, C22-, C20- and Cta-macrocyclic lactones in halictide bees. Acta chem. scand. B33, 390.

BOROAS L. (1895) Appareil glandulaire des Hymenopt6res. Annls. Sci. nat. Zool. 7, S6r. T 19, 1-362.

DUFFIELD R. M., FERNADES A., McKAY S., WHEELER J. W. and SHELLING R. R. (1980) Chemistry of the exocrine secretions of Hylaeus modestus (Hymenoptera: Colleti- dae). Comp. Biochem. Physiol. 67B, 159-162.

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systematic relationship of halictinae bees based on the pattern of macrocyclic lactones in the...

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