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Multisource approaches in taxonomy gather different lines of evidence in order to draw strongly supported taxonomic conclusions and constitute the basis of integrative taxonomy. In the case of overlooked taxa with disjunct distributions for which sampling is more challenging, integrative approaches help to propose stable hypotheses at the species and subspecies levels. Here, based on genetic and semio-chemical traits, we performed an integrative taxonomic analysis to evaluate species delimitation hypotheses within a monophyletic group of bumblebees (Hymenoptera, Apidae, Bombus) including the formerly recognised subgenera Eversmannibombus, Laesobombus and Mucidobombus which are now included in the subgenus Thoracobombus. Our results demonstrate the conspecificity of several polytypic taxa, and we formally recognise the subspecies Bombus laesus aliceae comb. nov. Cockerell, 1931, endemic to North Africa, based on its allopatry, unique mitochondrial haplotype and divergent cephalic labial gland secretions. This highlights the need to maintain studying polytypic complexes of bumblebee taxa for which phylogenetic relationships could be still entangled and eventually implement conservation strategies for taxonomically differentiated lineages.

Social parasitic Hymenopterans have evolved morphological, chemical, and behavioral adaptations to overcome the sophisticated recognition and defense systems of their social host to invade host nests and exploit their worker force. In bumblebees, social parasitism appeared in at least 3 subgenera independently: in the subgenus Psithyrus consisting entirely of parasitic species, in the subgenus Alpinobombus with Bombus hyperboreus, and in the subgenus Thoracobombus with B. inexspectatus. Cuckoo bumblebee males utilize species-specific cephalic labial gland secretions for mating purposes that can impact their inquiline strategy. We performed cephalic labial gland secretions in B. hyperboreus, B. inexspectatus and their hosts. Males of both parasitic species exhibited high species specific levels of cephalic gland secretions, including different main compounds. Our results showed no chemical mimicry in the cephalic gland secretions between inquilines and their host and we did not identify the repellent compounds already known in other cuckoo bumblebees.

Cold-adapted species are expected to have reached their largest distribution range during a part of the Ice Ages whereas postglacial warming has led to their range contracting toward high-latitude and high-altitude areas. This has resulted in an extant allopatric distribution of populations and possibly to trait differentiations (selected or not) or even speciation. Assessing inter-refugium differentiation or speciation remains challenging for such organisms because of sampling difficulties (several allopatric populations) and disagreements on species concept. In the present study, we assessed postglacial inter-refugia differentiation and potential speciation among populations of one of the most common arcto-alpine bumblebee species in European mountains, Bombus monticola Smith, 1849. Based on mitochondrial DNA/nuclear DNA markers and eco-chemical traits, we performed integrative taxonomic analysis to evaluate alternative species delimitation hypotheses and to assess geographical differentiation between interglacial refugia and speciation in arcto-alpine species. Our results show that trait differentiations occurred between most Southern European mountains (i.e. Alps, Balkan, Pyrenees, and Apennines) and Arctic regions. We suggest that the monticola complex actually includes three species: B. konradini stat.n. status distributed in Italy (Central Apennine mountains), B. monticola with five subspecies, including B. monticola mathildis ssp.n. distributed in the North Apennine mountains; and B. lapponicus. Our results support the hypothesis that post-Ice Age periods can lead to speciation in cold-adapted species through distribution range contraction. We underline the importance of an integrative taxonomic approach for rigorous species delimitation, and for evolutionary study and conservation of taxonomically challenging taxa.

Species taxonomy of bumblebees (Bombus Latreille, 1802) is well known to be problematic due to a potentially high intra-specific variability of morphological traits while different species can converge locally to the same color pattern (cryptic species). Assessing species delimitation remains challenging because it requires to arbitrarily select variable traits whose accuracy continues to be debated. Integrative taxonomic approach seems to be very useful for this group as different independent traits are assessed to propose a rational taxonomic hypothesis. Among operational criteria to assess specific status, the reproductive traits involved in the pre-mating recognition (i.e., the male cephalic labial gland secretions, CLGS) have been premium information. Since these secretions are supposed to be species-specific, these chemical traits can bring essential information where species delimitation is debated. Here, we describe and compare the CLGS of 161 male specimens of nine Alpinobombus taxa: alpinus, balteatus, helleri, hyperboreus, kirbiellus, natvigi, neoboreus, polaris, and pyrrhopygus. We aim also to test the congruence between this new information (reproductive traits) and published genetic dataset. Our results emphasized six distinct groups with diagnostic major compounds: (a) alpinus + helleri with hexadec-9-en-1-ol; (b) polaris + pyrrhopygus with two major compounds hexadec-9-en-1-ol and hexadec-9-enal; (c) balteatus with tetradecyl acetate; (d) kirbiellus with geranyl geranyl acetate; (e) hyperboreus + natvigi with octadec-11-en-1-ol; (f) neoboreus with octadec-9-en-1-ol. Based on this new information, we can confirm the species status of B. alpinus, B. balteatus, B. hyperboreus, B. kirbiellus, B. neoboreus, and B. polaris. We also confirm the synonymy of helleri (Alps) with alpinus (Sweden). However, the specific status of natvigi (Alaska) and pyrrhopygus (Sweden) is questionable and these taxa do not have specific CLGS composition.

Volatiles released by seedlings of Norway spruce infested with the aphid Cinara pilicornis were analyzed using SPME–GC–MS. Among the stress-induced compounds released by the host plant, citronellol, cis–trans-nepetalactone and cis–trans-nepetalactol was found. These compounds originated from the aphids and they were assumed to be pheromone components for this aphid species. To determine the relative stereochemistry of the nepetalactone, a diagnostic method was developed. The method was based on multivariate analysis of tabulated relative intensities of mass fragments of the four nepetalactone diastereomers. In the practical method described, a few pairs of fragments in the mass spectra were compared and, in combination with the Kovat's index, were used to unambiguously identify the relative stereochemistry of the nepetalactone.

The absolute configurations of citronellol, 2,3-dihydrofamesol, and 2,3-dihydrofarnesal in male marking pheromones of seven species of bumblebees and cuckoo bumblebees were determined by enantioselective gas chromatography on a capillary column coated with 60% heptakis(2,3-di-O-acetyl-6-O-TBDMS)-beta-cyclodextrin in polysiloxane PS 268. Pure (-)-S-enantiomers of all three terpenes were found in the labial glands of all investigated specimens of the following species: Bombus (Bombus) terrestris, B. (Bombus) lucorum, B. (Pyrobombus) pratorum, B. (Pyrobombus) pyrenaeus, B. (Pyrobombus) jonellus, B. (Pyrobombus) impatiens, and the cuckoo bumblebee B. (Ashtonipsithyrus) bohemicus. Within species, specimens were collected at different localities and in different years. Except for 2,3-dihydrofamesol in B. terrestris, this is the first report on the absolute configuration of terpenes in marking pheromones of bumblebees., QC 20100525 QC 20111020

Enantiomerically pure (S)-(+)- linalool was the main constituent in the extracts of the cephalic secretions of virgin females, mated females, freshly emerged males, and patrolling males of the solitary bee Colletes cunicularius. After copulation, the content of (S)-(+)- linalool emitted by the female was strongly reduced. Electrophysiological experiments revealed that both enantiomers of linalool elicited responses from the antennae of the males. Field tests using the pure enantiomers and the racemate of linalool showed that the number of male bees attracted was highest for (S)-(+)- linalool. The search flight activity in the mating flight area increased dramatically when patrolling males were presented with (S)-(+)- linalool vs (R)-(+)- linalool. Taken together, these data indicate a mate attractant pheromone function of (S)-(+)- linalool., QC 20100525

The compositions of 23 monoterpene hydrocarbons of six pine species (Pinus sylvestris, P. yunnanensis ll,ris, P. armandii, P. tropicalis, P. cubensis and P. caribaca) were compared, using multivariate data analysis. Four of the six species were clearly different from the other species in a Principal Components Analysis (PCA) model, based on the relative amounts (selective normalization) of the monoterpenes. The correlation coefficients between constituents were determined separately for each species and the strongest correlations were found between (+)-alpha -pinene and (+)-camphene and between the corresponding (-)-enantiomers, in all species. This pattern, i.e. a good correlation in all species, was neither shown by the correlation of the structurally more similar (+)-alpha/beta -pinenes, nor by the (-)-alpha/beta -pinenes or within the enantiomeric pairs of alpha -pinene and beta -pinene. For these pairs of monoterpenes, good correlations were found in some species. None of the species showed good correlations in all the investigated monoterpene pairs presented here. Correlations between monoterpenes in insect-attacked trees (P. cubensis and P. caribaea, attacked by Dioryctria horneana, and P. yunnanensis, attacked by Tomicus piniperda) were also determined. The results are discussed from chemotaxonomic and biosynthetic points of view., QC 20100525

Commonly used dynamic sorption techniques for collecting biologically active volatile compounds have been compared. Solid phase microextraction (SPME) using two types of fibers (polydimethylsiloxane, PDMS, 100 mu m, and carbowax/divinylbenzene, CW/DVB, 65 mu m) was compared to purge and trap methods (Porapak Q, Tenax TA and charcoal) and a technique based on absorption in methanol in a cooling bath. Sampling was done in a stream of purified air (20 ml/min) in a closed and temperature-regulated (27 degrees C) glass tube, passing over a capillary tube containing a hexane solution of tridecane, heptadecane, 1-octen-3-ol, 1-hexadecanol, ethyl tetradecanoate, alpha-pinene, linalool, terpinen-4-ol, cis-verbenol, verbenone, beta-caryophyllene, E,E-farnesol, and geranylgeraniol. With all of the methods, the sampling was performed for a period of 30 min before extraction and analysis was done on a GC-FID system. In general, SPME gave a higher response for all compounds except for alpha-pinene, which was only extracted by the CW/DVB fiber. Purge and trap methods and methanol absorption gave the same response for all substances extracted. None of the methods extracted hexadecanol and geranylgeraniol under the conditions used. However, the SPME equipped with the PDMS coating extracted heptadecane, E,E-farnesol and ethyl tetradecanoate. Our results show that SPME, when selecting the fibers to fit the polarity and volatility of the compounds, is an outstanding extraction method compared to purge and trap and methanol absorption, especially for a qualitative analysis. The best conditions for storing fibers exposed to compounds of high volatility were at low temperatures (6 degrees C) in sealed vials, while the worst way was to leave the exposed fiber unprotected at room temperature (22 degrees C). The dynamic sampling system was effectively tested on a fruiting body of a polypore fungus (Ganoderma applanatum) emitting 1-octen-3-ol, and again SPME showed to be the most sensitive te, QC 20100525

The floral fragrance of the shrub Daphne mezereum in central Sweden was collected by means of the head-space technique and investigated by GC-MS and multi-dimensional GC. (S)-(+)-Linalool was the main constituent (95%) of the flower fragrance and its enan, Addresses: BorgKarlson AK, ROYAL INST TECHNOL, DEPT CHEM ORGAN CHEM, S-10044 STOCKHOLM, SWEDEN. ACAD SCI CZECH REPUBL, INST ORGAN CHEM & BIOCHEM, CR-16610 PRAGUE, CZECH REPUBLIC. UPPSALA UNIV, DEPT SYSTEMAT BOT, S-75236 UPPSALA, SWEDEN.

The floral fragrance of the shrub Daphne mezereum in central Sweden was collected by means of the head-space technique and investigated by GC-MS and multi-dimensional GC. (S)-(+)-Linalool was the main constituent (95%) of the flower fragrance and its enantiomeric purity exceeded 99% in the samples. The (2S, 5S)- and (2R, 5S)-furanoid and the (3R, 6S)- and (3S, 6S)-pyranoid linalool oxide isomers constituted 2-5% of the fragrance. The elution order of these compounds on a permethylated beta-cyclodextrin column is reported. A fragrance sample of D. mezereum as well as (S)-(+)-linalool attracted males of the vernal solitary bee species Colletes cunicalarius and Andrena cinerea. A racemic mixture of the two enantiomeric pairs of known furanoid linalool oxides was only weakly attractive to the bees. The role of the fragrance in the pollination specialization of the plant is discussed.

Volatiles from the essential oil of the liverwort Conocephalum conicum were analysed. The chirality of the monoterpene hydrocarbons was studied by two-dimensional ps chromatography. All compounds except beta-phellandrene showed high optical purity. For the identification of enantiomers, (+)-beta-phellandrene and (-)-alpha-thujene were prepared from (+)-limonene and (+)-sabinene, respectively.