The cephalic labial gland secretions of two socially parasitic bumblebees Bombus hyperboreus (Alpinobombus) and Bombus inexspectatus (Thoracobombus) question their inquiline strategy

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.     

Atypical secretions of the male cephalic labial glands in bumblebees: the case of Bombus (Rhodobombus) mesomelas Gerstaecker (Hymenoptera, Apidae)

In bumblebees, the male secretion of the cephalic labial gland is species-specific. It is highly involved in the nuptial behavior, acting as a sexual attracting pheromone. Therefore, it is also used to accurately identify the species. In contrast to this common scheme, the secretions of Bombus mesomelas are found to be strongly reduced and do not include the most volatile compounds that are present in the secretions of all the other studied bumblebee species. These secretions correspond to cuticular hydrocarbons that can be found in all bumblebee species. This was also the case for another bumblebee species from the same Rhodobombus subgenus: Bombus pomorum. This atypical composition of the male cephalic labial gland secretions seems to indicate that, at least for these two species of Rhodobombus, these secretions are not used to attract virgin females from a long distance, as it is the case for all the other bumblebee species studied.     

Reduced cephalic labial glands in the male bumblebees of the subgenus Rhodobombus Dalla Torre (Hymenoptera: Apidae: Bombus Latreille)

Until now, all males of bumblebees are known to attract conspecific females by marking places with a chemical signal secreted by their cephalic labial gland. The specific combination of patrolling flight and scentmarking is of outmost importance to bumblebees as it is their main species specific recognition system. We report here the lack of that recognition system in species of the subgenus Rhodobombus by comparing the morphology and histology of the cephalic labial glands of Bombus (Rhodobombus) mesomelas with those of a well known species, B. (Bombus) terrestris (L.) The cephalic labial glands are much smaller in B. mesomelas than in other bumblebees species and most likely non-functional. This morphology is also observed in B. pomorum and B. brodmanni which also belong to the subgenus Rhodobombus. Our morphological observations are consistent with the chemical analyses of the secretions in B. mesomelas and B. pomorum which are very limited and of most unusual composition for a bumblebee. In addition, whereas other species are thought to use their barbae mandibularis to spread their secretion onto the substrate, these structures are absent in all Rhodobombus. All these observations would mean that the males of Rhodobombus do not attract females from far away by the use of their cephalic labial gland secretions.     

Chemical reproductive traits of diploid Bombus terrestris males: Consequences on bumblebee conservation

The current bumblebee decline leads to inbreeding in populations that fosters a loss of allelic diversity and diploid male production. As diploid males are viable and their offspring are sterile, bumblebee populations can quickly fall in a vortex of extinction. In this article, we investigate for the first time a potential premating mechanism through a major chemical reproductive trait (male cephalic labial gland secretions) that could prevent monandrous virgin queens from mating with diploid males. We focus our study on the cephalic labial gland secretions of diploid and haploid males of Bombus terrestris (L.). Contrary to initial expectations, our results do not show any significant differentiation of cephalic labial gland secretions between diploid and haploid specimens. Queens seem therefore to be unable to avoid mating with diploid males based on their compositions of cephalic labial gland secretions. This suggests that the vortex of extinction of diploid males could not be stopped through premating avoidance based on the cephalic labial gland secretions but other mechanisms could avoid mating between diploid males and queens.     

First Chemical Analysis and Characterization of the Male Species‐Specific Cephalic Labial‐Gland Secretions of South American Bumblebees

The evolution of signals and reproductive traits involved in the pre‐mating recognition has been in focus of abundant research in several model species, such as bumblebees (genus Bombus). However, the most‐studied bumblebee reproductive trait, the male cephalic labial gland secretions (CLGS), remains unknown among bumblebee species from South America. In this study, the CLGS of five South American bumblebees of the subgenera Thoracobombus (Bombus excellens and B. atratus) and Cullumanobombus (B. rubicundus, B. hortulanus, and B. melaleucus) were investigated, by comparing the chemical compositions of their secretions to those of closely related European species. The results showed an obvious interspecific differentiation in both subgenera. The interspecific differentiation among the species of the Thoracobombus subgenus involved different compounds present at high contents (main compounds), while those of the Cullumanobombus subgenus shared the same main components. This suggests that among the species of the Cullumanobombus subgenus, the differentiation in minor components could lead to species discrimination.     

Integration of conflict into integrative taxonomy: fitting hybridization in species delimitation of Mesocarabus (Coleoptera: Carabidae)

In species differentiation, characters may not diverge synchronously, and there are also processes that shuffle character states in lineages descendant from a common ancestor. Species are thus expected to show some degree of incongruence among characters; therefore, taxonomic delimitation can benefit from integrative approaches and objective strategies that account for character conflict. We illustrate the potential of exploiting conflict for species delimitation in a study case of ground beetles of the subgenus Carabus (Mesocarabus), where traditional taxonomy does not accurately delimit species. The molecular phylogenies of four mitochondrial and three nuclear genes, cladistic analysis of the aedeagus, ecological niche divergence and morphometry of pronotal shape in more than 500 specimens of Mesocarabus show that these characters are not fully congruent. For these data, a three‐step operational strategy is proposed for species delimitation by (i) delineating candidate species based on the integration of incongruence among conclusive lines of evidence, (ii) corroborating candidate species with inconclusive lines of evidence and (iii) refining a final species proposal based on an integrated characterization of candidate species based on the evolutionary analysis of incongruence. This procedure provided a general understanding of the reticulate process of hybridization and introgression acting on Mesocarabus and generated the hypothesis of seven Mesocarabus species, including two putative hybrid lineages. Our work emphasizes the importance of incorporating critical analyses of character and phylogenetic conflict to infer both the evolutionary history and species boundaries through an integrative taxonomic approach.     

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)

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 tetradec-9-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 (=2,3-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-1-ol 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.     

Distribution and flower visitation records of bumblebees in Lebanon (Hymenoptera: Apidae)

Summary

West Palearctic bumblebees are common wildflowers and crop pollinators that are well studied in their central and northern distribution ranges, but fewer information is available on their southern distribution areas. Lebanon falls on the southern limit of their distribution and no published information is available on the local bumblebees. Our study aims to produce a data baseline of the local bumblebee species. In order to do so we grouped available old records of bumblebees in Lebanon with recent author collections and produced preliminary distribution maps. We listed four species: Bombus terrestris, B. argillaceus, B. niveatus vorticosus and B. melanurus. Preliminary distribution shows that Bombus terrestris and B. argillaceus are widespread and have a large foraging range, whereas B. niveatus vorticosus and B. melanurus have a restricted distribution to altitudes above 1800 m with a smaller foraging range. The male cephalic labial gland secretions analysis of local Bombus terrestris specimens provides preliminary evidence that the local subspecies could be Bombus terrestris calabricus. Therefore, we highlight the importance of regulating foreign Bombus terrestris subspecies importation for agriculture purposes, as well as monitoring B. niveatus vorticosus and B. melanurus that are rendered vulnerable by their isolated populations.     

Integrative taxonomy and preliminary assessment of species limits in the Liolaemus walkeri complex (Squamata,Liolaemidae) with descriptions of?three?new species from Peru

Species delimitation studies based on integrative taxonomic approaches have received considerable attention in the last few years, and have provided the strongest hypotheses of species boundaries. We used three lines of evidence (molecular, morphological, and niche envelopes) to test for species boundaries in Peruvian populations of the Liolaemus walkeri complex. Our results show that different lines of evidence and analyses are congruent in different combinations, for unambiguous delimitation of three lineages that were “hidden” within known species, and now deserve species status. Our phylogenetic analysis shows that L. walkeri, L. tacnae and the three new species are strongly separated from other species assigned to the alticolor-bibronii group. Few conventional morphological characters distinguish the new species from closely related taxa and this highlights the need to integrate other sources of data to erect strong hypothesis of species limits. A taxonomic key for known Peruvian species of the subgenus Lioalemus is provided.     

Molecular and chemical characters to evaluate species status of two cuckoo bumblebees: Bombus barbutellus and Bombus maxillosus (Hymenoptera,Apidae, Bombini)

Many methods, based on morphological, molecular or chemical characters, have been used to address the question of species taxonomic status. Integrative taxonomy aims to define stronger supported taxonomic hypotheses by considering complementary datasets from different characters. By following an integrative approach, the present study includes molecular, chemical and morphological criteria to establish the taxonomic status of two rare and doubtful cuckoo bumblebee taxa: Bombus (Psithyrus) barbutellus and Bombus (Psithyrus) maxillosus. These two sympatric taxa are discriminated by few morphological criteria (mainly wing darkness and hair length). We used these morphological character diagnoses to establish an a priori status of our samples (23 specimens). We developed a combined molecular dataset from one nuclear gene, elongation factor 1α (EF‐1α), and one mitochondrial gene, cytochrome c oxidase subunit I (COI), spanning 1623 bp, and a chemical dataset of sexual marking pheromones (73 compounds). The molecular data were subjected to maximum‐likelihood and Bayesian phylogenetic inference under partitioned model and maximum parsimony. The chemical data were analysed by clustering and the two‐group k‐means method to test divergences between the two species. The resulting phylogenetic trees show no consistent divergence between the two taxa. Moreover, we found no divergence in the sexual marking pheromones in the clustering and two‐group k‐means analyses. These converging results support the conspecificity of both taxa. Nonetheless, our determinations using the traditional morphological criteria separated our samples into two taxa. We conclude that the morphological criteria seem to relate to intraspecific variations: B. maxillosus is regarded as a syn.n. of B. barbutellus.     

Species delimitation methods reveal cryptic diversity in the Hypnea cornuta complex (Cystocloniaceae,Rhodophyta)

The simple and convergent morphologies of many red algae make these species difficult to identify using traditional morphological characters. Many cryptic species have been described in recent years based on molecular datasets, and this has led to the application of an integrative taxonomy approach in species delimitation. Here, we performed several species delimitation methods (mBGD, ABGD, SPN, PTP, GMYCs and GMYCm) based on two different loci (COI-5P and rbcL) in species of the Hypnea cornuta complex. These methods were combined with morphological and phylogenetic data, extensive sampling, analysis of topotype material, and historically relevant herbarium samples. Our findings demonstrate that the groups morphologically assigned to H. cornuta and H. stellulifera consist of five different cryptic species. H. cornuta is a polyphyletic taxon composed of three well-separated lineages, thus requiring sequencing of type or topotype specimens to determine which one is Hypnea cornuta sensu stricto. We have revealed that the distribution of H. stellulifera is limited to Asia, while the Brazilian specimens initially assigned to this species were clarified as a new endemic species: Hypnea cryptica sp. nov. Our results indicated that only an integrative approach combining several lines of evidence, including morphology, nomenclature history, molecular data, biogeography and ecology can correctly solve the taxonomic status of widely distributed cryptic species.     

Integration of molecular, ecological, morphological and endosymbiont data for species delimitation within the Pnigalio soemius complex (Hymenoptera: Eulophidae)

Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis‐inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host–plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London     

Cryptic diversity in Brevipalpus mites (Tenuipalpidae)

Defining the taxonomic identity of organisms is a prerequisite for their study, and in the case of economically important species, misidentification may lead to the application of inappropriate prevention and control strategies. Flat mites of the Brevipalpus genus include several crop pests and the systematics of these mites represents a challenge for acarologists. Many of the most economically important Brevipalpus species have repeatedly been inaccurately identified. Such problematic classification has been attributed to the likely occurrence of cryptic species in the genus. In this study, we used an integrative approach that combined molecular analyses, including sequence‐based species delimitation, with detailed morphological identification using traits that have recently showed to be taxonomically informative. Sequences of mitochondrial cytochrome c oxidase subunit I (COI) were obtained from individuals collected from host plants belonging to 14 genera and 13 families across 29 locations in the Americas (Brazil, Chile, USA). The phylogenetic analyses included previously published Brevipalpus sequences from GenBank, and the final data set was classified into 44 haplotypes. Six putative species were recognised by COI‐based species delimitation analysis, and morphological evidence supported each of these species. The integrative approach revealed the occurrence of cryptic species in the Brevipalpus genus and contributed to the clarification of previously noted incongruences. The results presented here allow for the evaluation of taxonomic characteristics in a phylogenetic context and indicate new characters for the differentiation of Brevipalpus species. In addition, Brevipalpus incognitus n. sp. Ferragut & Navia, a cryptic species detected in this study, is described based on morphological and molecular traits. Implications of the advances in Brevipalpus systematics presented herein with respect to pest management are briefly discussed.     

Exocrine gland secretions of virgin queens of five bumblebee species (Hymenoptera: Apidae, Bombini)

Secretions of three different glands (mandibular gland, labial gland, and Dufour's gland) of virgin queens of five bumblebee species (Bombus lucorum, B. lapidarius, B. hypnorum, B. pascuorum, and B. terrestris) were analysed. Around 200 compounds were identified in the secretions. The compositions of the secretions of labial and mandibular glands were species-specific. Dufour's gland of all species produced mainly hydrocarbons, both saturated and unsaturated, the proportions of which differed quantitatively between the species studied.     

Analysis of the labial gland secretions of the male bumblebee Bombus griseocollis (Hymenoptera: Apidae)

The labial gland secretions from males of the bumblebee Bombus (Separatobombus) griseocollis De Geer, a bumblebee exhibiting perching behaviour, were analysed by gas chromatography/mass spectrometry (GC/MS) in the electron impact and positive ion chemical ionization mode. The major compound of the complex mixture of alkenols, acetates, hydrocarbons, wax type esters and steroids is tetradecyl acetate, considerable amounts of hexadecyl, geranyllinaloyl, geranylgeranyl, docosyl, tetracosenyl and hexacosenyl acetate were also found. 1,3-Tetradecanediol diacetate, detected as a minor component, has not yet been identified in male bumblebee labial gland secretions. Besides small amounts of primary alcohols (tetradecanol and hexadecanol) the tertiary alcohol geranyllinalool (3,7,11,15-tetramethyl-hexadeca-1,6,10,14-tetraene-3-ol) was also present. The primary alcohols were also present as esters of butanoic, dodecanoic, tetradecanoic, and hexadecanoic acid. Besides the usual mixture of un- and mono-unsaturated straight chain hydrocarbons, the labial gland contains the isoprenoid hydrocarbons beta-springene [(6E, 10E)-7,11,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene] and two isomers of a-springene [(3Z,6E,10E)- and (3E,6E,10E)-3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene]. The close relationship in chemical composition in male bumblebees with perching and flight pass behaviour is discussed.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.