Methods for species delimitation in bumblebees (Hymenoptera,Apidae, Bombus): towards an integrative approach

Delimitation of closely related species is often hindered by the lack of discrete diagnostic morphological characters. This is exemplified in bumblebees (genus Bombus). There have been many attempts to clarify bumblebee taxonomy by using alternative features to discrete morphological characters such as wing shape, DNA, or eco‐chemical traits. Nevertheless each approach has its own limitations. Recent studies have used a multisource approach to gather different lines of speciation evidence in order to draw a strongly supported taxonomic hypothesis in bumblebees. Yet, the resulting taxonomic status is not independent of selected evidence and of consensus methodology (i.e. unanimous procedure, majority, different weighting of evidence). In this article, we compare taxonomic conclusions for a group of taxonomically doubtful species (the Bombus lapidarius‐group) obtained from the four commonly used lines of evidence for species delimitation in bumblebees (geometric morphometric of wing shape, genetic differentiation assessment, sequence‐based species delimitation methods and differentiation of cephalic labial gland secretions). We ultimately aim to assess the usefulness of these lines of evidence as components of an integrative decision framework to delimit bumblebee species. Our results show that analyses based on wing shape do not delineate any obvious cluster. In contrast, nuclear/mitochondrial, sequence‐based species delimitation methods, and analyses based on cephalic labial gland secretions are congruent with each other. This allows setting up an integrative decision framework to establish strongly supported species and subspecies status within bumblebees.     

Too strict or too loose? Integrative taxonomic assessment of Bombus lapidarius complex (Hymenoptera: Apidae)

The latest progress of the taxonomy is the use of integrative approach for species delimitation based on a multisource dataset. However, the taxonomic decision that should be made when convergence between the different lines of evidence is not observed remains debated. Here, we investigate the consequences of the application of an ‘integration by cumulation’ approach on the taxonomic statuses within the Bombus lapidarius complex when using an integrative taxonomic framework (i.e. nuclear and mitochondrial markers along with reproductive traits) compared with a strict ‘integration by congruence’ method. Our results show similar taxonomic conclusions whatever the decision-making approach used except for one taxon. According to the differentiation observed in our integrative taxonomic framework, recent divergence time and other field observations for this taxon, we assume that a too strict decision-making method could fail to detect recently diverged species. This is exemplified by the new species Bombus bisiculus sp. n. occurring in South Italy and Sicily.     

Evidence for hilltopping in bumblebees?

1. Male bumblebees are known to exhibit a range of mate‐location behaviours, including perching on prominent objects and darting at passing queens, patrolling of scent‐marked flight routes, and waiting outside nest entrances for virgin queens to emerge. Here we provide evidence for a fourth strategy, known as hilltopping. This behaviour is widely known from a range of invertebrates, but has not previously been described in bumblebees. 2. We studied the distribution of bumblebees along transects ascending four hills in Scotland and demonstrate that, relative to workers, males of four bumblebee species or species groups (Bombus lapidarius, B. monticola, B. pascuorum, and B. lucorum/magnus/cryptarum/terrestris) tend to congregate at or near the tops of hills. This is, to our knowledge, the first evidence for hilltopping in bumblebees and the first record of any putative mate‐locating behaviour for male B. pascuorum, a very common species in Europe. 3. We note that, in common with most previous studies of mate‐locating behaviour in bumblebees, attraction of virgin queens and mating were not observed.     

Influence of urbanisation on the prevalence of protozoan parasites of bumblebees

1. Increasing urbanisation is often cited as a cause of declining biodiversity, but for bumblebees there is evidence that urban populations of some species such as Bombus terrestris L. may be more dense than those found in agricultural landscapes, perhaps because gardens provide plentiful floral resources and nesting opportunities. 2. Here we examine the influence of urbanisation on the prevalence of the main protozoan parasites of bumblebees in west central Scotland. We would expect transmission rates and prevalence of parasites to be higher in high density host populations, all else being equal. 3. Workers of two bee species, B. terrestris and B. pascuorum, were sampled over a 45‐day period in mid to late summer, and parasites were detected in faeces and via dissection. A comparison of the two methods suggests that faecal sampling is considerably less sensitive than dissection, failing to detect infection in 27.8%, 55.1%, and 80% of cases of infection with the parasites Crithidia bombi, Nosema bombi, and Apicystis bombi, respectively. 4. For all three parasites, broad patterns of prevalence were similar, with prevalence tending to increase with urbanisation in B. terrestris but not in B. pascuorum. The different patterns of seasonal prevalence in the two bee species suggest that intraspecific transmission is more important that interspecific transmission. 5. Our observation of greater parasite prevalence among B. terrestris in urban compared with rural areas suggests that urban habitats may present greater opportunities for parasite transmission. Greater bee densities in urban areas may be the driving factor; however, further study is still needed. For example, differences in disease prevalence between habitats could be driven by differences in the types and abundance of flowers that are available, or in exposure to environmental stressors.     

Genomic data reveal deep genetic structure but no support for current taxonomic designation in a grasshopper species complex

Taxonomy has traditionally relied on morphological and ecological traits to interpret and classify biological diversity. Over the last decade, technological advances and conceptual developments in the field of molecular ecology and systematics have eased the generation of genomic data and changed the paradigm of biodiversity analysis. Here we illustrate how traditional taxonomy has led to species designations that are supported neither by high throughput sequencing data nor by the quantitative integration of genomic information with other sources of evidence. Specifically, we focus on Omocestus antigai and Omocestus navasi, two montane grasshoppers from the Pyrenean region that were originally described based on quantitative phenotypic differences and distinct habitat associations (alpine vs. Mediterranean‐montane habitats). To validate current taxonomic designations, test species boundaries, and understand the factors that have contributed to genetic divergence, we obtained phenotypic (geometric morphometrics) and genome‐wide SNP data (ddRADSeq) from populations covering the entire known distribution of the two taxa. Coalescent‐based phylogenetic reconstructions, integrative Bayesian model‐based species delimitation, and landscape genetic analyses revealed that populations assigned to the two taxa show a spatial distribution of genetic variation that do not match with current taxonomic designations and is incompatible with ecological/environmental speciation. Our results support little phenotypic variation among populations and a marked genetic structure that is mostly explained by geographic distances and limited population connectivity across the abrupt landscapes characterizing the study region. Overall, this study highlights the importance of integrative approaches to identify taxonomic units and elucidate the evolutionary history of species.     

When taxonomy meets genomics: lessons from a common songbird

Taxonomy is being increasingly informed by genomics. Traditionally, taxonomy has relied extensively on phenotypic traits for the identification and delimitation of species, though with a growing influence from molecular phylogenetics in recent decades. Now, genomics opens up new and more powerful tools for analysing the evolutionary history and relatedness among species, as well as understanding the genetic basis for phenotypic traits and their role in reproductive isolation. New insights gained from genomics will therefore have major effects on taxonomic classifications and species delimitation. How a genomics approach can inform a flawed taxonomy is nicely exemplified by Mason & Taylor ( 2015 ) in this issue of Molecular Ecology. They studied redpolls, which comprise a genus (Acanthis) of fringillid finches with a wide distribution in the Holarctic region, and whose species taxonomy has been a matter of much controversy for decades (Fig.  1 ). Current authoritative checklists classify them into one, two or three species, and five or six subspecies, based largely on geographical differences in phenotypic traits. Previous studies, including a recent one of the subspecies on Iceland (Amouret et al. 2015 ), have found no evidence of differentiation between these taxa in conventional molecular markers. The lack of genetic structure has been interpreted as incomplete lineage sorting among rapidly evolving lineages. Now Mason & Taylor ( 2015 ), using a large data set of genomewide SNPs, verify that they all belong to a single gene pool with a common evolutionary history, and with little or no geographical structuring. They also show that phenotypic traits used in taxonomic classifications (plumage and bill morphology) are closely associated with polygenic patterns of gene expression, presumably driven by ecological selection on a few regulatory genes. Several lessons can be learned from this study. Perhaps the most important one for taxonomy is the risk of taxonomic inflation resulting from overemphasizing phenotypic traits under local adaptation and ignoring a lack of phylogenetic signal in molecular markers.     

Following the cold: geographical differentiation between interglacial refugia and speciation in the arcto‐alpine species complex Bombus monticola (Hymenoptera: Apidae)

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.     

Life history variation between species of the relictual genus Borderea (Dioscoreaceae): phylogeography, genetic diversity, and population genetic structure assessed by RAPD markers

The genus Borderea consists of two species, B. pyrenaica and B. chouardii, taxa which have been previously considered as conspecific due to their overall close morphology. These two sole species of the rare genus of Dioscoreaceae are endemic to the Pyrenees (Spain, France). This mountain range likely operated as a refugium for these plants during the last glaciations. B. chouardii is only known from a single population in the Spanish Prepyrenees and has been classified as at risk of extinction in the Red List of Endangered Species (IUCN); B. pyrenaica shows a narrow distribution range in the central Pyrenees and Prepyrenees. We analysed genetic variation, population structure and differentiation in these two taxa using RAPD markers. Our study was conducted on the same seven populations for which very low levels of genetic differentiation were detected previously through allozyme analysis. By contrast, high levels of genetic variability were detected through the RAPD hypervariable markers. Twelve RAPD primers produced 112 distinct bands in the 397 surveyed individuals, totalling 395 different RAPD phenotypes. Only four bands were monomorphic across all samples of Borderea, whereas 21 of the polymorphic bands were species‐specific (20 for B. chouardii, and one for B. pyrenaica). The largest genetic distances were those between the B. chouardii and the B. pyrenaica phenotypes. An analysis of molecular variance showed greater variance between groups (B. chouardii vs. B. pyrenaica, 76.08%) than within groups (3.60%). RAPD band specificity, phenotypic distances, and the partitioning of variance all support the taxonomic separation of the two species. Statistical evaluation of within‐ and among‐population RAPD genetic variability in B. pyrenaica showed that genetic variability was higher within populations (>80%) than among them. No clear pattern of RAPD differentiation could be observed among the six studied populations of this taxon though slight differences in genetic diversity could be observed in the more isolated Prepyrenean populations compared with the more widespread Pyrenean ones. These results suggest a recent postglacial origin of the present B. pyrenaica populations. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 483–498.     

The first record of the rare oligolectic bumblebee Bombus gerstaeckeri Morawitz (Hymenoptera: Apidae: Bombini) lrom Ukraine

The oligolectic bumblebee Bombus gerstaeckeri Morawitz has been recorded in the Ukrainian Carpathians for the first time during field research from 2002 to 2007. Queens and workers of the species have been observed foraging on two Aconitum spp. within 540 m and 1400 m altitude. The additional forage resource of this species, that is Salvia glutinosa L., has been recorded at low height. Three competitor bumblebees, not confined only to Aconitum spp. have been observed foraging on monkshoods: B. hortorum (L.), B. pascuorum (Scopoli) and B. wurflenii Radoszkowski.     

Systematic revision of the living species of Bullidae (Mollusca: Gastropoda: Cephalaspidea), with a molecular phylogenetic analysis

Bullidae are a worldwide family of marine shelled cephalaspidean gastropods with a mainly tropical distribution, but also with some representatives in temperate waters. The taxonomy of the group has in the past been based only on shell characters, and the few anatomical accounts available have not addressed more than one to three species, so there has been no agreement about the number of valid species. Seventy‐two specific names and 16 varietal names have been proposed worldwide. The systematics of the family Bullidae are revised, based not only on shells but also on anatomy of all extant species and on DNA sequence data. Twelve species are recognized worldwide, including one new species here described, and all are assigned to the genus Bulla. Two species occur in the eastern Atlantic, B. striata and B. mabillei; two in the western Atlantic, B. occidentalis and B. solida; two in the eastern Pacific, B. gouldiana and B. punctulata; and six in the Indo‐West Pacific, B. ampulla, B. arabica sp. nov. , B. orientalis, B. peasiana, B. quoyii and B. vernicosa. Full synonymies and taxonomic histories are provided for each species. In order to promote taxonomic stability, neotypes are designated for B. striata, B. solida, B. nebulosa (valid name B. gouldiana) and B. vernicosa, and lectotypes for B. occidentalis, B. mabillei, B. punctulata, B. ampulla and B. quoyii. The type locality of B. ampulla is restricted to Mauritius. Bullidae show a general morphological stasis, with anatomy being very similar between species. However, there are high levels of intraspecific variability in the shell, radula and male genital system. In some cases species could only be separated based on molecular data . After defining the characters and geographical range of each species it became clear that sympatric species (a maximum of three) show distinctive shells and reproductive structures, which makes identification straightforward. This study employs an integrative approach, combining information on shells, anatomy, DNA and geographical distribution, in order to resolve the systematics of a difficult taxonomic group. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 153 , 453–543.     

Patterns of Genetic and Reproductive Traits Differentiation in Mainland vs. Corsican Populations of Bumblebees

Populations on islands often exhibit lower levels of genetic variation and ecomorphological divergence compared to their mainland relatives. While phenotypic differentiation in characters, such as size or shape among insular organisms, has been well studied, insular differentiation in quantitative reproductive traits involved in chemical communication has received very little attention to date. Here, we investigated the impact of insularity on two syntopic bumblebee species pairs: one including species that are phylogenetically related (Bombus terrestris and B. lucorum), and the other including species that interact ecologically (B. terrestris and its specific nest inquiline B. vestalis). For each bumblebee species, we characterized the patterns of variation and differentiation of insular (Corsican) vs. mainland (European) populations (i) with four genes (nuclear and mitochondrial, 3781 bp) and (ii) in the chemical composition of male marking secretions (MMS), a key trait for mate attraction in bumblebees, by gas chromatography-mass spectrometry (GC-MS). Our results provide evidence for genetic differentiation in Corsican bumblebees and show that, contrary to theoretical expectations, island populations of bumblebees exhibit levels of genetic variation similar to the mainland populations. Likewise, our comparative chemical analyses of MMS indicate that Corsican populations of bumblebees are significantly differentiated from the mainland yet they hold comparative levels of within-population MMS variability compared to the mainland. Therefore, insularity has led Corsican populations to diverge both genetically and chemically from their mainland relatives, presumably through genetic drift, but without a decrease of genetic diversity in island populations. We hypothesize that MMS divergence in Corsican bumblebees was driven by a persistent lack of gene flow with mainland populations and reinforced by the preference of Corsican females for sympatric (Corsican) MMS. The impoverished Corsican bumblebee fauna has not led to relaxation of stabilizing selection on MMS but to consistent differentiation chemical reproductive traits on the island.     

Towards understanding the dynamics of hybridization and apomixis in the evolution of the genus Boechera (Brassicaceae)

We have reviewed past and recent research on the radiation of the North American genus Boechera (Brassicaceae), and discuss future prospects for our understanding of the evolutionary processes and patterns of differentiation in this highly polymorphic genus. Boechera comprises about 80 biennial to perennial species of exclusively North American and Greenlandic distribution. Hybridization and apomixis, in conjunction with polyploidy, were found to play a major role in the origin and maintenance of intraspecific and interspecific polymorphism. However, taxonomic classification within the genus has mainly been based on morphological characters, and diagnostic features discriminating between taxa are often limited. Molecular markers are promising tools for: the assessment of infrageneric phylogen‐etic relationships, the isolation of basal sexual taxa, the detection of hybridization and estimation of its frequency among lineages in time and space, the identification of centres of genetic and species diversity, and the comparison of various reproductive modes. We present and discuss the major achievements obtained since the introduction of molecular methods to Boechera research with respect to these aims, and point out possible shortcomings of specific marker systems. The distribution and transmission of apomixis, polyphyletic origin of genotypes and cytotypes, variation in reproductive system, and the lack of consensus between current taxonomic concepts and evolutionary evidence are the major topics discussed here in the context of hybridization.     

Reuse of voucher specimens provides insights into the genomic associations and taxonomic value of wing colour and genitalic differences in a pest group (Lepidoptera: Tortricidae: Choristoneura)

Subtle morphological differences can be essential to diagnosing closely related species, and an understanding of the genetic basis of these characters can contribute to understanding their divergences. We used voucher specimens from previous genetic analyses of population structure to subsequently analyse genome-wide associations linking morphology to genetic variation in spruce budworms, a group of economically important and morphologically similar forest pests. In particular, we assessed the taxonomic value and genetic architecture of two morphological traits (wing pattern and genitalic spicule abundance) that have been reported to differ among spruce budworm species. Our results suggest that phallic spicule number has greater taxonomic utility than wing pattern for distinguishing Choristoneura fumiferana (Clemens) from Choristoneura occidentalis occidentalis Freeman and Choristoneura occidentalis biennis Freeman. However, there was considerable overlap among taxa for all phenotypic characters analysed. In a genome-wide association study, wing pattern variation was significantly associated with four single nucleotide polymorphism (SNP) loci, including two adjacent SNPs. One SNP was flanked by sequence resembling RNA-directed DNA polymerase from mobile element jockey-like. This locus is a promising candidate for the study of wing pattern development in spruce budworms, as jockey-like transposable elements and polymerases have documented roles in wing spot production in other Lepidoptera. Our study links classical taxonomic characters and genomic data to provide insights into the potential genetic architecture of species differences. It also demonstrates previously untapped morphological and taxonomic value in voucher specimens from earlier molecular genetic analyses.     

New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae)

Rock-dwelling gastropods are usually patchily distributed in limestone habitats, presumably have low active and passive dispersal ability and often represent narrow-ranged endemic taxa. Their current taxonomy is predominantly shell morphology based, and it remains unknown whether the morphologically differentiated and geographically separated populations represent phylogenetic clades. In this study, we analysed the hyperdiverse, terrestrial door snail genus Montenegrina. Based on the current taxonomy defined by shell morphology, it contains 29 species and 106 subspecies distributed in the Balkan region. The constructed phylogenetic tree using three mitochondrial markers was used to test whether it agrees with the current taxonomy. In this comprehensive tree, about half of the species and subspecies are monophyletic. Some of the paraphylies could be reasonably resolved by taxonomic changes; that is, some subspecies should be reassigned or raised to species level. Other incongruencies probably arose due to introgression even between distant clades. The histone genes turned out to be unsuitable for elucidating the phylogeny of Montenegrina. In the species-delimitation tests, considerably more molecular operational taxonomic units were delimited than the number of presently described species. The present data indicate that (a) shell morphology-based taxonomy and taxon recognition can be problematic in such a large and morphologically highly variable genus; (b) the potential error due to incomplete sampling presents a problem in a genus as variable as Montenegrina; (c) multi-locus analyses should be conducted to arrive at a better basis for species delimitation; and (d) integrative approaches including genetic as well as morphological/anatomical data from a comprehensive geographic sample are necessary.     

Targeted agri‐environment schemes significantly improve the population size of common farmland bumblebee species

Changes in agricultural practice across Europe and North America have been associated with range contractions and local extinction of bumblebees (Bombus spp.). A number of agri‐environment schemes have been implemented to halt and reverse these declines, predominantly revolving around the provision of additional forage plants. Although it has been demonstrated that these schemes can attract substantial numbers of foraging bumblebees, it remains unclear to what extent they actually increase bumblebee populations. We used standardized transect walks and molecular techniques to compare the size of bumblebee populations between Higher Level Stewardship (HLS) farms implementing pollinator‐friendly schemes and Entry Level Stewardship (ELS) control farms. Bumblebee abundance on the transect walks was significantly higher on HLS farms than ELS farms. Molecular analysis suggested maximum foraging ranges of 566 m for Bombus hortorum, 714 m for B. lapidarius, 363 m for B. pascuorum and 799 m for B. terrestris. Substantial differences in maximum foraging range were found within bumblebee species between farm types. Accounting for foraging range differences, B. hortorum (47 vs 13 nests/km²) and B. lapidarius (45 vs 22 nests/km²) were found to nest at significantly greater densities on HLS farms than ELS farms. There were no significant differences between farm type for B. terrestris (88 vs 38 nests/km²) and B. pascuorum (32 vs 39 nests/km²). Across all bumblebee species, HLS management had a significantly positive effect on bumblebee nest density. These results show that targeted agri‐environment schemes that increase the availability of suitable forage can significantly increase the size of wild bumblebee populations.     

Use of genetic markers to quantify bumblebee foraging range and nest density

Bumblebees (Hymenoptera: Apidae) are important pollinators of crops and wildflowers, but many species have suffered dramatic declines in recent decades. Strategies for their conservation require knowledge of their foraging range and nesting density, both of which are poorly understood. Previous studies have mainly focussed on the cosmopolitan bumblebee species Bombus terrestris , and implicitly assume this to be representative of other species. Here we use a landscape-scale microsatellite study to estimate the foraging range and nesting density of two ecologically dissimilar species, B. terrestris and B. pascuorum . Workers were sampled along a 10 km linear transect and 8–9 polymorphic microsatellite markers used to identify putative sisters. We provide the first published estimates of the number of colonies using a circle of radius 50 m in an agricultural landscape: 20.4 for B. terrestris and 54.7 for B. pascuorum . Estimates of nest density differed significantly between the two species: 13 km⁻² for B. terrestris and 193 km⁻² for B. pascuorum . Foraging ranges also differed substantially, with B. pascuorum foraging over distances less than 312 m and B. terrestris less than 625 m. Clearly bumblebee species differ greatly in fundamental aspects of their ecology. This has significant implications for the development of conservation strategies for rare bumblebees and isolated plant populations, for the management of bumblebees as pollinators, and for predicting patterns of gene flow from genetically modified plants.     

Genetic diversity of Philaenus spumarius and P. tesselatus (Hemiptera,Aphrophoridae): implications for evolution and taxonomy

The meadow spittlebugs Philaenus spumarius and P. tesselatus are closely related taxa with uncertain taxonomic position in the light of previous morphological, ecological, cytological and molecular research. Despite morphological homogeneity of P. spumarius (with the exception of high colour and pattern polymorphism) across its wide Holarctic range, it is possible that additional taxa (species or subspecies) exist. Philaenus spumarius is a potentially important pest in parts of its range where it was introduced. We used DNA markers to describe the genetic diversity of P. spumarius and P. tesselatus and to verify the taxonomic status of P. tesselatus and remote populations of the former species. The mitochondrial (cytochrome B) data showed that there are two main groups encompassing a northeastern (Asia and north‐central Europe) and a southwestern (Mediterranean area and western Europe, including North American specimens) clade. According to the elongation factor‐1α gene, there are three main clades: northeastern (Eurasiatic clade, E1), southeastern (east Mediterranean – Caucasus clade, E2) and southwestern (Iberian clade, E3). These two or three mitochondrial and nuclear clades could be considered as separate taxonomic units. On the other hand, all studied individuals of both species possessed the same internal transcribed spacer 2 haplotype. American specimens most probably originated from some western European populations. All studied specimens of P. tesselatus belong to the southwestern clade and western Mediterranean cluster. Therefore, together with cytological data, its species status is doubtful. To definitively solve the taxonomic status of P. tesselatus and populations of P. spumarius, further research using more samples and more genetic markers are needed.     

Diploid male production in a rare and locally distributed bumblebee, Bombus florilegus (Hymenoptera, Apidae)

The European bumblebee B. terrestris was recently introduced in Japan for agricultural purposes and has now become naturalized. The naturalization of this exotic species may have great detrimental effects on closely related native Japanese bumblebees. The Japanese bumblebee Bombus florilegus is a rare and locally distributed species found in the Nemuro Peninsula of Hokkaido, Japan. In order to assess its population genetics, we estimated the genetic structure of B. florilegus in 16 breeding colonies (queen, workers, and males) and 20 foraging queens by analyzing microsatellite DNA markers. Of the 36 queens analyzed by genotyping and dissection, 32 had been inseminated by a male. The remaining 4 had not been inseminated at all. Of the 4 nonmated queens, one was triploid. Diploid males were found in 4 breeding colonies. Based on the microsatellite data, it appears that B. florilegus has low reproductive success. Since matched mating and nonmating within local populations are high, the extinction risk is correspondingly higher. Our results suggest that conservation of the Japanese B. florilegus is required in order to protect it from both habitat destruction and the naturalization of alien species. Received 19 July 2007; revised 13 October 2007; accepted 15 October 2007.     

Molecular and morphological variation of rare endemic oncocyclus irises (Iridaceae) of Lebanon

Oncocyclus irises endemic to Lebanon form a complex of three closely related taxa replacing each other over short geographical distances in a linear habitat. In order to characterize the appropriate taxonomic levels and to assess their conservation status, we investigated patterns of phenotypic variability and the partitioning of genetic variation within and among populations using random amplified polymorphic DNA (RAPD) markers. Multivariate analysis (principal components analysis and multiple correspondence analysis), based on 16 quantitative and six qualitative characters, revealed no separation between populations or taxa. Moreover, no morphological character could be used to define clear boundaries between populations/taxa. The genetic characterization revealed high levels of polymorphism and diversity (H_s). Principal components analysis showed population delimitations, but no groupings reflecting the currently defined taxa could be identified. Both morphological and genetic data showed that Lebanese oncocyclus irises could not be fitted into clear taxonomic boundaries. Consequences for conservation are discussed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 123–135.