Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3-22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed >= 2.3% sequence divergence between taxa and 200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.

Morphological and molecular characters reveal differentiation in a Neotropical social bee, Melipona beecheii (Apidae : Meliponini)

Morphometrics and DNA microsatellites were used to analyse the genetic structure of populations of the stingless bee M. beecheii from two extremes of its geographic range. The results showed that populations from Costa Rica and Yucatan exhibit substantial phenotypic and molecular differentiation. Bees from Yucatan were smaller and paler than those from Costa Rica. The value of multilocus F-ST = 0.280 (P <0.001) confirmed that there were significant molecular genetic differences between the two populations. Populations showed significant deviation from Hardy Weinberg equilibrium and the values of FIS (the inbreeding coefficient) were positive for Costa Rica = 0.416 and the Yucatan Peninsula = 0.193, indicating a lack of heterozygotes in both populations possibly due to inbreeding. The DNA sequence of 678 bp of the mitochondrial gene COI differed between populations by 1.2%. The results of this study should be considered in conservation programmes, particularly with regard to the movement of colonies between regions.

An optimized method for the generation of AFLP markers in a stingless bee (Melipona quadrifasciata) reveals a high degree of intracolonial genetic polymorphism

This study describes an optimized protocol for the generation of Amplified Fragment Length Polymorphism (AFLP) markers in a stingless bee. Essential modifications to standard protocols are a restriction enzyme digestion (EcoRI and Tru1I) in a two-step procedure, combined with a touchdown program in the selective PCR amplification step and product labelling by incorporation of alpha[P-33]dATP. In an analysis of 75 workers collected from three colonies of Melipona quadrifasciata we obtained 719 markers. Analysis of genetic variability revealed that on average 32% of the markers were polymorphic within a colony. Compared to the overall percentage of polymorphism (44% of the markers detected in our bee samples), the observed rates of within-colony polymorphism are remarkably high, considering that the workers of each colony were all of spring of a singly mated queen.

Specific and sensitive detection of Nosema bombi (Microsporidia : Nosematidae) in bumble bees (Bombus spp.; Hymenoptera : Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323 bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 107 down to 10 spores diluted in 100 mu l of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of similar to 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to b more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy. (c) 2005 Elsevier Inc. All rights reserved.

Satellite flies (Leucophora personata, Diptera : Anthomyiidae) and other dipteran parasites of the communal bee Andrena agilissima (Hymenoptera : Andrenidae) on the island of Elba, Italy

Solitary and presocial aculueate Hymenoptera are parasitized by a range of dipteran species in the families Axithomyiidae, Bombyliidae, Conopidae, Phoridae, and Sarcophagidae that are likely to impact on their hosts. We undertook a study over several years of a univoltine and communal bee, Andrena agilissima, and its main dipteran parasites, in particular the satellite fly Leucophora personata (Diptera: Anthomyiidae). Behavioural and ecological data were collected from one nesting aggregation of the host bee on the island of Elba, Italy, from 1993 to 2003, and from a foraging site of the bee, ca 5 km from the nesting aggregation. Other Diptera associated with A. agilissmia at the field site were the bee fly Bombylius fimbriatus (Bombyliidae), the conopid fly Zodion cinereum (Conopidae), and the scuttle fly Megaselia andrenae (Phoridae). The phenology of the Diptera broadly overlapped with that of their host across the season of activity (end of April and all of May). Diurnal activity patterns differed slightly; L. personata in particular was active at the host's nesting site before A. agilissima. Female satellite flies also showed a range of behaviours in gaining entry to a host nest. We summarize published data on this and other Leucophora species that parasitize Andrena host bees. Host bees returning to their nests occasionally undertook zig-zag flight manoeuvres if followed by a satellite fly that were generally successful in evading the fly. Satellite flies that entered a nest, presumably to oviposit, were less likely to remain therein if another host bee entered the same nest, suggesting that one advantage to communal nesting for this host is a reduction in brood cell parasitism by L. personata. We provide the first clear evidence for parasitism by a Zodion of any Andrena host. Both L. personata and M. andrenae concentrated their parasitic activities in the zone of the host nesting aggregation with highest nest densities. Three of the Diptera, L. personata, B. fimbriatus, and Z. cinereum, seemed to have extremely low rates of parasitism whilst that of M. andrenae appeared low. Though they have refined parasitic behaviour that allows them to gain entry into host nests (L. personata, B. fimbriatus, and M. andrenae) or to parasitize adults (Z. cinercum), these parasites seem not to impact upon the dynamics of the host A. agilissima at the nesting aggregation, and the host possesses traits to reduce parasitism.

Paternity frequency and maternity of males in some stingless bee species

In monogynous hymenopteran societies, the number of mates of a queen strongly influences the potential for conflict between workers and queens over the maternity of males. Queens always 'prefer' their own sons to sons of workers, regardless of queen mating frequency. When a queen mates once, workers are more closely related to, and therefore are expected to prefer, their own sons and then sons of sisters to sons of the queen. However, if effective paternity frequency exceeds 2, workers on average should prefer queen-produced males to males produced by their sisters. We studied the queen mating frequency of seven stingless bee species: the Mexican species Scaptotrigona mexicana , S. pectoralis and the Australian species Austroplebeia symei , Trigona clypearis , T. hockingsi , T. mellipes and T. sapiens . We then determined whether males arise from eggs laid by workers or queens in A. symei , T. clypearis , T. hockingsi and T. mellipes . We show that all seven species investigated are most likely singly mated and that the queen dominates reproduction. This indicates that the queen's mating frequency alone does not determine whether workers or the queen produces the males.

Landscape effects on extremely fragmented populations of a rare solitary bee, Colletes floralis

Globally there is concern over the decline of bees, an ecologically important group of pollinating insects. Genetic studies provide insights into population structure that are crucial for conservation management but that would be impossible to obtain by conventional ecological methods. Yet conservation genetic studies of bees have primarily focussed on social species rather than the more species-rich solitary bees. Here we investigate the population structure of Colletes floralis, a rare and threatened solitary mining bee, in Ireland and Scotland using nine microsatellite loci. Genetic diversity was surprisingly as high in Scottish (Hebridean island) populations at the extreme northwestern edge of the species range as in mainland Irish populations further south. Extremely high genetic differentiation among populations was detected; multilocus FST was up to 0.53, and G’ST and Dest were even higher (maximum: 0.85 and 1.00 respectively). A pattern of isolation by distance was evident for sites separated by land. Water appears to act as a substantial barrier to gene flow yet sites separated by sea did not exhibit isolation by distance. Colletes floralis populations are extremely isolated and probably not in regional migration-drift equilibrium. GIS-based landscape genetic analysis reveals urban areas as a potential and substantial barrier to gene flow. Our results highlight the need for urgent site-specific management action to halt the decline of this and potentially other rare solitary bees.

Genetic differentiation across the social transition in a socially polymorphic sweat bee, Halictus rubicundus

Eusociality is widely considered a major evolutionary transition. The socially polymorphic sweat bee Halictus rubicundus, solitary in cooler regions of its holarctic range and eusocial in warmer parts, is an excellent model organism to address this transition, and specifically the question of whether sociality is associated with a strong barrier to gene flow between phenotypically divergent populations. Mitochondrial DNA (COI) from specimens collected across the British Isles, where both solitary and social phenotypes are represented, displayed limited variation, but placed all specimens in the same European lineage; haplotype network analysis failed to differentiate solitary and social lineages. Microsatellite genetic variability was high and enabled us to quantify genetic differentiation among populations and social phenotypes across Great Britain and Ireland. Results from conceptually different analyses consistently showed greater genetic differentiation between geographically distant populations, independently of their social phenotype, suggesting that the two social forms are not reproductively isolated. A landscape genetic approach revealed significant isolation by distance (Mantel test r = 0.622, p

Quantifying forage specialisation in polyphagic insects: the polylectic and rare solitary bee, Colletes floralis (Hymenoptera: Colletidae)

Many insect species vary in their degree of foraging specialisation, with many bee species considered polyphagic (polylectic). Wild, non-managed bee species vary in their conservation status, and species-specific biological traits such as foraging specialisation may play an important role in determining variance in population declines. Current agri-environment schemes (AESs) prescribe the introduction of flower seed mixes for agricultural systems to aid the conservation of wild bees. However, the extent to which flower combinations adequately meet bee foraging requirements is poorly known. We quantitatively assessed pollen use and selectivity using two statistical approaches: Bailey's Intervals and Compositional Analysis, in an examplar species, a purportedly polylectic and rare bee, Colletes floralis, across 7 sites through detailed analysis of bee scopal pollen loads and flower abundance. Both approaches provided good congruence, but Compositional Analysis was more robust to small sample sizes. We advocate its use for the quantitative determination of foraging behaviour and dietary preference. Although C. floralis is polylectic, it showed a clear dietary preference for plants within the family Apiaceae. Where Apiaceae was uncommon, the species exploited alternative resources. Other plant families, such as the Apiaceae, could be included, or have their proportion increased in AES seed mixes, to aid the management of C. floralis and potentially other wild solitary bees of conservation concern. © 2011 The Authors. Insect Conservation and Diversity © 2011 The Royal Entomological Society.

Kin discriminators in the eusocial sweat bee Lasioglossum malachurum: the reliability of cuticular and Dufour's gland odours

The ability to discriminate degrees of relatedness may be expected to evolve if it allows unreciprocated altruism to be preferentially directed towards kin (Hamilton in J Theor Biol 7:1-16, 1964). We explored the possibility of kin recognition in the primitively eusocial halictid bee Lasioglossum malachurum by investigating the reliability of worker odour cues that can be perceived by workers to act as indicators of either nest membership or kinship. Cuticular and Dufour's gland compounds varied significantly among colonies of L. malachurum, providing the potential for nestmate discrimination. A significant, though weak, negative correlation between chemical distance and genetic relatedness (r = -0.055, p

Female choice in the red mason bee, Osmia rufa (L.) (Megachilidae)

Females are often thought to use several cues and more than one modality in selection of a mate, possibly because they offer complementary information on a mate's suitability. In the red mason bee, Osmia rufa, we investigated the criteria a female uses to choose a mating partner. We hypothesized that the female uses male thorax vibrations and size as signs of male viability and male odor for kin discrimination and assessment of genetic relatedness. We therefore compared males that had been accepted by a female for copulation with those rejected, in terms of their size, their immediate precopulatory vibrations (using laser vibrometry), the genetic relatedness of unmated and mated pairs (using microsatellite markers) and emitted volatiles (using chemical analyses). Females showed a preference for intermediate-sized males that were slightly larger than the modal male size. Furthermore, male precopulatory vibration burst duration was significantly longer in males accepted for copulation compared with rejected males. Vibrations may indicate vigor and assure that males selected by females are metabolically active and healthy. Females preferentially copulated with males that were genetically more closely related, possibly to avoid outbreeding depression. Volatiles of the cuticular surface differed significantly between accepted and rejected males in the relative amounts of certain hydrocarbons, although the relationship between male odor and female preference was complex. Females may therefore also use differences in odor bouquet to select among males. Our investigations show that O. rufa females appear to use multiple cues in selecting a male. Future investigations are needed to demonstrate whether odor plays a role in kin recognition and how the multiple cues are integrated in mate choice by females.

Complex sociogenetic organization and the origin of unrelated workers in a eusocial sweat bee, Lasioglossum malachurum

Sweat bees (Halictidae) exhibit great interspecific and intraspecific diversity in their social organisation, yet there is remarkably little information on the sociogenetic organisation of any species. Lasioglossum malachurum is a eusocial sweat bee with an annual lifecycle that exhibits considerable variation in its social organisation across its wide geographic range from northern to southern Europe. We collected all adults from 31 L. malachurum nests at Eichkogl, Austria, near the latitudinal centre of its distribution, and genotyped 148 workers using 5 highly variable microsatellite loci developed for this species. Nests were often queenless (48% of nests) during the second phase of worker activity, when colonies were provisioning the sexual brood. Pedigree reconstruction and estimates of nestmate genetic relatedness demonstrated that nests often (32% of nests) contained alien workers, probably as a result of worker drifting from their natal to a foreign nest. Queen effective mating frequency was variable (harmonic mean m(e) = 1.24), but sometimes high (maximum 2.7). These data demonstrate that nests of L. malachurum do not have a classical eusocial sociogenetic organisation (monogyny, monandry) and thereby pose a challenge to exclusively relatedness based arguments for the evolution of eusociality in the taxon.

Characterization of 14 polymorphic microsatellite loci for the facultatively eusocial sweat bee Halictus rubicundus (Hymenoptera, Halictidae) and their variability in related species

Sweat bees display considerable variation in social organization and a few species, such as Halictus rubicundus, are even facultatively eusocial. Fourteen polymorphic, unlinked microsatellite loci were isolated from H. rubicundus and characterized in 45 females. The number of alleles per locus ranged from two to 18 (mean 10.1), observed heterozygosity ranged from 0.24 to 0.98 (mean 0.71) and expected heterozygosity ranged from 0.24 to 0.98 (mean 0.70). Six or more loci cross-amplified in four other sweat bees. These loci will be useful for the study of social evolution and population genetic structure in H. rubicundus and many other sweat bees.