Population genetic diversity and species relationships in the genus Rhinanthus L. based on microsatellite markers

The genus Rhinanthus L. is complex, containing many taxonomically unresolved taxa. In this paper we studied genetic variation and species relationships in 15 populations of six Rhinanthus species from three sections. For this purpose, we developed new microsatellite primers for R. osiliensis and used them to investigate genetic variation in two narrow endemics (R. osiliensis, R. javorkae) and in four widespread species (R. rumelicus R. wagneri, R. angustifolius and R. minor). Species‐specific private alleles were found in all species except R. osiliensis and R. angustifolius. The Bulgarian endemic R. javorkae showed the lowest genetic variation, followed by widespread R. minor and Estonian endemic R. osiliensis. Rhinanthus javorkae and R. minor were genetically most differentiated. Section Cleistolemus is weakly structured genetically, indicating close affinity between R. osiliensis, R. rumelicus, R. wagneri and R. angustifolius.     

Comparative Study of Seed Germination and Genetic Variation of Rare Endemic Rhinanthus osiliensis and Related Widespread Congener R. rumelicus (Orobanchaceae)

Genetic diversity is affected by breeding systems, life history traits and the distribution of species. Generally, inbred species with restricted geographic distribution have lower genetic variation compared to common outbreeding plant species. Rhinanthus osiliensis is a narrow endemic growing in calcareous spring fens on the island Saaremaa, Estonia. Presumably the closest congener of R. osiliensis is R. rumelicus, which is widely distributed in Eastern and Central Europe and the Balkan Peninsula. Isozymes were used to describe the mating system, levels of genetic variation and differentiation between the populations of R. osiliensis and R. rumelicus. Genetic diversity was found to be higher in widespread R. rumelicus compared to the endemic R. osiliensis. A significantly higher inbreeding coefficient with a low germination rate indicated substantial autogamy in R. osiliensis, whereas R. rumelicus displayed a mixed mating mode with a moderate inbreeding coefficient and a high germination rate. The low genetic differentiation between R. osiliensis and R. rumelicus confirmed their close affinity and the short evolutionary period of both taxa after the postglacial colonization. Considering the restricted geographic range and inbreeding in populations of R. osiliensis, this species may face a loss of genetic variation in the future.     

Rhinanthus plants found in calcareous fens on Gotland (Sweden): Are they related to Rhinanthus osiliensis from Saaremaa (Estonia)?

Rhinanthus osiliensis is a narrow endemic growing in calcareous spring fens on the island Saaremaa, Estonia. Plants very similar to R. osiliensis have been found in Sweden on the island of Gotland. Fifteen microsatellite markers were used to characterize genetic variation within R. osiliensis and the unknown taxon from Gotland. Results were compared with common Rhinanthus minor and Rhinanthus angstifolius. Our results showed that genetic diversity was remarkably lower in the Rhinanthus sp. from Gotland compared to Estonian endemic R. osiliensis. The microsatellite loci we used distinguished four studied species. In total, 46 species-specific alleles were found. The Neighbor-Joining dendrogram based on Nei's genetic distances, the Bayesian clustering analysis and the principle coordinate analysis showed that R. osiliensis and Rhinanthus sp. from Gotland are genetically differentiated and could not be the same species. In addition, endemic R. osiliensis was closer to widespread R. angustifolius. Further genetic comparison between unknown Rhinanthus sp. and local R. angustifolius and R. minor from Gotland is necessary for determine the species status and checking the possible alternative hypothesis about the origin of the unclear taxon.     

Performance of two Rhinanthus species under different hydric conditions

Rhinanthus angustifolius and Rhinanthus minor are annual hemiparasitic herbs found in open vegetations. They are closely related, hybridize frequently, and appear to have largely overlapping niches, although some floras report a preference of R. angustifolius for more humid conditions compared to R. minor. We analysed the relative fitness of both species by following their fate from germination to seed production in three different hydric treatments (wet, moist and dry) in a garden experiment. We found that R. angustifolius was either as fit as or fitter than R. minor in all conditions and for all studied parameters, with a slightly higher flower production in R. angustifolius in the wet treatment. Wet conditions had a negative effect on germination and flowering rate, but they increased flower and seed production. These observations show that environmental conditions will influence the composition of mixed Rhinanthus populations.     

Shared pollinators and pollen transfer dynamics in two hybridizing species, Rhinanthus minor and R. angustifolius

Gene flow between hybridizing plant species depends strongly on pollinator behaviour, which affects pollen transfer among floral types and reproductive isolation. We examined bumblebee behaviour and pollen transfer between two hybridizing Rhinanthus species that are very similar in ecology and floral traits. The two species, Rhinanthus minor and R. angustifolius, shared similar pollinator guilds and assemblages, but pollinator recruitment and flower visitation rates were higher in R. angustifolius sites, probably because of its higher reward levels and better visibility. When presented with Rhinanthus flowers, bumblebees that previously foraged on R. angustifolius were less prone to visit R. minor inflorescences, while R. minor foragers accepted both species in similar proportions. Although Rhinanthus has been cited as a case of mechanical isolation resulting from interactions between bee behaviour and differences in stigma and anther placement, we found no support for efficient mechanical reproductive isolation. Bumblebees that foraged on R. minor flowers carried more pollen, but pollen placement on their bodies was similar to that of bees that visited R. angustifolius, and cross-specific stigmatic pollen deposition was similar in both directions. However, the asymmetry in pollinator handling time between the two species, due to dissimilar pollen rewards, may have lowered relative heterospecific pollen receipt on R. angustifolius, suggesting that net gene flow resulting from pollen transfer dynamics is more likely towards R. minor, although this effect remains weak and will be most likely counterbalanced by context-based labile pollinator preference.     

Outcrossing rates in two self‐compatible,hybridising Rhinanthus species: implications for hybrid formation

The congeners Rhinanthus angustifolius and Rhinanthus minor, two annual hemiparasites pollinated by bumblebees, are known to hybridise in the wild. Both species are self‐compatible, but the capacity for autonomous selfing is higher in R. minor. This suggests a difference in realized outcrossing rates, which have not been determined before in these species. Using microsatellites, both species turned out to have mixed mating systems, but with a much lower multilocus outcrossing rate (0.13) for R. minor compared to R. angustifolius (0.76). We hypothesised that a higher outcrossing rate should lead to a higher chance of heterospecific pollination, and we therefore determined the rate of hybrid formation on each species in an artificial mixed population. Hybrid seeds were produced at low frequency (4.5%), and no significant difference was found between the species. It is therefore likely that post‐pollination processes influence hybrid seed formation to counteract the expected difference in heterospecific pollen deposition. We checked fruit set, seed set and the rate of autonomous selfing in controlled crosses in the greenhouse in 2 years, and found that fruit set (2003) or seed set (2010) were lower in R. angustifolius × R. minor crosses relative to the reciprocal cross. Hybrid seeds produced on R. angustifolius also had a much lower germination rate, so most of the established F₁ hybrid plants have the R. minor cytoplasm. The formation of advanced hybrids depends on pollinator preference, which is biased towards R. angustifolius if present in sufficient numbers, because it offers more rewards.     

Closely related parasitic plants have similar host requirements and related effects on hosts

The performance of root hemiparasites depends strongly on host species identity, but it remains unknown whether there exist general patterns in the quality of species as hosts for hemiparasites and in their sensitivity to parasitism. In a comparative approach, the model root hemiparasites Rhinanthus minor and R. alectorolophus were grown with 25 host species (grasses, forbs, and legumes) at two nutrient levels. Hosts grown without parasites served as a control. Host species identity strongly influenced parasite biomass and other traits, and both parasites grew better with legumes and grasses than with forbs. The biomass of R. alectorolophus was much higher than that of R. minor with all host plants and R. alectorolophus responded much more strongly to higher nutrient availability than R. minor. The performance of the two species of Rhinanthus with individual hosts was strongly correlated, and it was also correlated with that of R. alectorolophus and the related Odontites vulgaris in previous experiments with many of the same hosts, but only weakly with that of the less closely related Melampyrum arvense. The negative effect of R. minor on host biomass was less strong than that of R. alectorolophus, but stronger relative to its own biomass, suggesting that it is more parasitic. The impact of the two parasites on individual hosts did not depend on nutrient level and was correlated. Several legumes and grasses were tolerant of parasitism. While R. minor slightly reduced mean overall productivity, R. alectorolophus increased it with several species, indicating that the loss of host biomass was more than compensated by that of the parasite. The results show that closely related parasites have similar host requirements and correlated negative effects on individual hosts, but that there are also specific interactions between pairs of parasitic plants and their hosts.     

Limited mitochondrial DNA variation within South Africa's black rhino (Diceros bicornis minor) population and implications for management

The taxonomy of African black rhinoceros (Diceros bicornis) remains unresolved. Maintaining levels of genetic diversity and species rescue by reintroduction and restocking requires its resolution. We compared the sequences of the mitochondrial DNA (mtDNA) control region for a total of 101 D. bicornis from three subspecies: D. b. minor, D. b. michaeli and D. b. bicornis. A single unique haplotype was found within the 65 D. b. minor samples from KwaZulu‐Natal (KZN) Province, South Africa, 55 of which came from Hluhluwe‐iMfolozi Game Park (HiP) and Mkuzi Game Reserve (MGR) source populations. However, six different haplotypes were represented in eleven D. b. minor samples from Zimbabwe. Similarly, published autosomal microsatellite data indicate low levels of diversity within the KZN D. b. minor populations. The low levels of mtDNA diversity within the KZN metapopulation point to the possible need for genetic supplementation. However, there is a need to determine whether the low levels of genetic variation within KZN D. b. minor are a result of the recent bottleneck or whether KZN historically always had low diversity.     

Dormancy and germination of sixRhinanthus species in relation to climate

The genusRhinanthus (Orobanchaceae) consists of annual hemiparasites that occur in a wide range of climates. Patterns of dormancy and germination were studied for six species sampled in areas ranging from the Pyrenees to Northern Scandinavia, and from sea level up to about 2500 m altitude in the Alpine region. Dormancy was broken by a 2 to 6 months period of cold stratification. Optimal temperature and length of the stratification period appeared to vary between and within species. Two patterns of dormancy and germination were distinguished. Seeds of the first group, including the widely distributedR. minor andR. angustifolius, further referred to as the LW group, require a relatively long period of cold stratification. Moreover, their germination is accelerated if they are subjected to a widening range of higher temperatures in the last weeks of the stratification period. In the other species (R. alectorolophus, R. glacialis, R. mediterraneus and perhapsR. antiquus) the release of dormancy is completed in a rather short period. Higher temperatures in the last weeks of the stratification period hardly affect the germination process of this SN-group, with short dormancy and no accelerated germination at higher temperatures. In both groups, temperatures above the low values during stratification reduce germination percentages and induce secondary dormancy in non-germinated seeds. The effects strongly vary with timing and temperature. The patterns have a genetic basis and seem to be species-specific. They do not vary with climate conditions, since samplesR. minor, whether collected in sub-arctic or sub-alpine areas or at sea level, generally react according to the LW pattern, and samples ofR. alectorolophus from areas at sea level up to montane regions according to the SN one. The ecological significance of the various stratification requirements and effects of higher temperatures on germination is discussed in relation to the local climate of the species and the evolutionary history of the genus.     

Genetic diversity of the endangered coastal violet <Emphasis Type="Italic">Viola grayi</Emphasis> Franchet et Savatier (Violaceae) and its genetic relationship to the species in subsection <Emphasis Type="Italic">Rostratae</Emphasis>

Viola grayi, a coastal violet, is found on sandy seashores in limited regions in Japan, but not on rocky sea cliffs or in inland areas. This stemmed violet is classified in the subsection Rostratae. Because of recent human activity on sandy seashores, this species is now recorded as endangered. The present status of this species was surveyed and its genetic diversity was analyzed using 17 newly developed simple sequence repeat markers. The results are compared with those for two commonly found inland violet species in the same subsection, V. grypoceras and V. kusanoana. The coastal violet populations showed markedly lower genetic diversity than those of the common species, V. grypoceras, whereas V. kusanoana showed intermediate genetic diversity. About a half of the total genetic variation of V. grayi is found among populations in analysis of molecular variance. In contrast, only less than 25% of the total variation is found among populations for the two common violets. These three species showed a remarkably high inbreeding coefficient, which indicates high inbreeding under natural mating conditions, although these species have both chasmogamous and cleistogamous flowers. In a population-based dendrogram, V. grayi was clustered in a single and concise group, whereas V. grypoceras and V. kusanoana were found in somewhat discrete positions. In the Bayesian analysis of genetic structure of the coastal violet, delta K was the highest at K = 7, and the seven clusters almost correspond to seven populations studied here. Implications of the conservation of this endangered coastal violet are presented.     

Phylogeography of Chinese cherry (Prunus pseudocerasus Lindl.) inferred from chloroplast and nuclear DNA: insights into evolutionary patterns and demographic history

Chinese cherry (Prunus pseudocerasus Lindl.) is a commercially valuable fruit crop in China. In order to obtain new insights into its evolutionary history and provide valuable recommendations for resource conservation, phylogeographic patterns of 26 natural populations (305 total individuals) from six geographic regions were analyzed using chloroplast and nuclear DNA fragments. Low levels of haplotype and nucleotide diversity were found in these populations, especially in landrace populations. It is likely that a combined effect of botanical characteristics impact the effective population size, such as inbreeding mating system, long life span, as well as vegetative reproduction. In addition, strong bottleneck effect caused by domestication, together with founder effect after dispersal and subsequent demographic expansion, might also accelerate the reduction of the genetic variation in landrace populations. Interestingly, populations from Longmen Mountain (LMM) and Daliangshan Mountain (DLSM) exhibited relatively higher levels of genetic diversity, inferring the two historical genetic diversity centers of the species. Moreover, moderate population subdivision was also detected by both chloroplast DNA (G_ST = 0.215; N_ST = 0.256) and nuclear DNA (G_ST = 0.146; N_ST = 0.342), respectively. We inferred that the episodes of efficient gene flow through seed dispersal, together with features of long generation cycle and inbreeding mating system, were likely the main contributors causing the observed phylogeographic patterns. Finally, factors that led to the present demographic patterns of populations from these regions and taxonomic varieties were also discussed.     

Hybridization in Annual Plants: Patterns and DynamicsDuring a Four-Year Study in Mixed Rhinanthus Populations

Hybridization in annual plants is rare, but their short life cycle provides an excellent opportunity to study the dynamics of hybridization. Hybridization occurs between the annual hemiparasites Rhinanthus minor and Rhinanthus angustifolius (Orobanchaceae). Using flower morphology, Kwak (1980) found a prevalence of hybrids close to R. angustifolius in a single population. We aim to find whether this pattern is also found using genetic markers, whether it is generally occurring in mixed populations, and whether these populations are stable over time. We used species-specific genetic markers to determine the number of individuals in a range of hybrid classes in three mixed populations of different ages during four consecutive years. In the young population, F₁ hybrids were found in the first year and mostly hybrids between R. minor and these F₁s in the second year, but in the years after that, hybrids close to R. angustifolius became more abundant. We also found this in the two older populations, where hybrids close to R. angustifolius always occurred in higher frequencies than hybrids close to R. minor. Over time, R. angustifolius strongly increased in frequency in two populations. Patterns of marker presence and absence suggested that advanced-generation hybrids are mainly formed by backcrossing with one of the parents, predominantly R. angustifolius whenever its frequency in the population is higher than 15%. The dynamics of mixed populations depend on the ecological conditions that regulate the presence of the two parental species, and introgression into R. angustifolius seems prevalent.     

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (H_E) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.     

Combining multiple analytical approaches for the identification of population structure and genetic delineation of two subspecies of the endemic Arabian burnet moth Reissita simonyi (Zygaenidae; Lepidoptera)

Habitat fragmentation and landscape topology may influence the genetic structure and connectivity between natural populations. Six microsatellite loci were used to infer the population structure of 35 populations (N = 788) of the alpine Arabian burnet moth Reissita simonyi (Lepidoptera, Zygaenidae) in Yemen and Oman. Due to the patchy distribution of larval food plants, R. simonyi is not continuously distributed throughout the studied area and the two recognized subspecies of this endemic species (Reissita s. simonyi/R. s. yemenicola) are apparently discretely distributed. All microsatellites showed prevalence of null alleles and therefore a thorough investigation of the impact of null alleles on different population genetic parameters (F _ST, inbreeding coefficients, and Population Graph topologies) is given. In general, null alleles reduced genetic covariance and independence of allele frequencies resulting in a more connected genetic topology in Population Graphs and an overestimation of pairwise F _ST values and inbreeding coefficients. Despite the presence of null alleles, Population Graphs also showed a much higher genetic connectivity within subspecies (and lower genetic differentiation (via F _ST)) than between; supporting existing taxonomic distinction. Partial Mantel tests showed that both geographical distance and altitude were highly correlated with the observed distribution of genetic structure within R. simonyi. In conclusion, we identified geographical and altitudinal distances in R. simonyi as well as an intervening desert area to be the main factors for spatial genetic structure in this species and show that the taxonomic division into two subspecies is confirmed by genetic analysis.     

The impact of Rhinanthus minor in newly established meadows on a productive site

Question: What is the impact of the presence of Rhinanthus minor on forb abundance in newly established swards? Location: Wetherby, West Yorkshire, UK (53°55’N, 1°22’W). Method: A standard meadow mix containing six forbs and six grasses was sown on an ex‐arable field and immediately over‐sown using a randomised plot design with three densities of Rhinanthus minor (0, 600, and 1000 seeds per m²). Above‐ground biomass was analysed over a period of three years, while detailed assessments of sward composition were performed during the first two years. Results: Values of grass biomass were reduced in the presence of Rhinanthus, especially at the higher sowing density. The ratio of grass: forb biomass was also lower in association with Rhinanthus, but only at the higher sowing density. The presence of Rhinanthus had no effect on species number or diversity, which decreased between years regardless of treatment. Conclusions: Although not tested in a multi‐site experiment, the benefit of introducing Rhinanthus into newly established swards to promote forb abundance was determined. The efficacy of Rhinanthus presence is likely to depend on whether species not susceptible to the effects of parasitism are present.     

Little to no inbreeding depression in a tapeworm with mixed mating

Meta‐studies on hermaphrodites have found a negative relationship between primary selfing rates and levels of inbreeding depression (ID) and, thus, generally support purging in inbred systems. However, in plants, high among‐taxa variance in ID results in no difference in the mean ID between outcrossing and mixed‐mating taxa. Selective interference likely explains high ID among mixed‐mating taxa, whereas low levels of ID among mixed‐mating taxa are not as stressed. Among animal hermaphrodites, primarily molluscs, there are little data on mixed‐mating systems. To fill a taxonomic and mating system gap, we tested for ID in a mixed‐mating tapeworm, Oochoristica javaensis. We provide a direct estimate of ID across infection of an intermediate host by comparing selfing rates at two life history stages. We found little to no evidence for ID, and the level of ID falls in line with what is reported for highly selfing species even though O. javaensis has mixed mating. We discuss this result within the context of kin mating in O. javaensis. Our results emphasize that primary selfing rates alone may be insufficient to classify the inbreeding history in all species when testing for a relationship to ID. Mixed‐mating taxa, and possibly some outcrossing taxa, may exhibit low levels of ID if biparental inbreeding is also driving purging. We advocate that ID studies report estimates of inbreeding history (e.g. F_IS or identity disequilibrium) from nature‐derived adult samples to provide context rather than relying on primary selfing rates alone.     

Rhinanthus minor population genetic structure and subspecies: Potential seed sources of a keystone species in grassland restoration projects

In the last few decades unimproved semi-natural grasslands have been affected by intensification of land use and habitat fragmentation. Because of their biodiversity these species-rich grasslands are of high conservation importance and efforts are under way to restore such habitats. Detailed knowledge of within species diversity will aid deciding on the optimal seed source for such restoration projects, e.g. local genotypes or ecotypes. Rhinanthus minor is a species that is typically found in semi-natural grasslands and is commonly used in grassland restoration projects. This is because R. minor is a hemiparasitic plant that takes minerals and nutrients from its host, which in turn decreases the host's biomass and leads to opportunities for less competitive species in the vegetation. Here, we investigate genetic diversity within and between R. minor populations. This allowed us to test whether the six different subspecies of R. minor that have been described in the UK, based on their morphology, flowering time, and habitat, can be differentiated using molecular markers. We identified moderate levels of genetic differentiation between R. minor populations within the UK. In addition, R. minor individuals from the UK appear to be distinct from R. minor and Rhinanthus angustifolius individuals from other European countries based on microsatellite genotyping and DNA sequencing of cpDNA and rDNA ITS. The molecular markers used in the current study did not separate populations of R. minor based on either their subspecies or habitat. The implication for the use of R. minor in grassland restoration projects seems to be that it is not necessary to use local seeds or seeds from the same subspecies.     

Long‐distance pollen and seed dispersal and inbreeding depression in Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) in the Brazilian savannah

Hymenaea stigonocarpa is a neotropical tree that is economically important due to its high‐quality wood; however, because it has been exploited extensively, it is currently considered threatened. Microsatellite loci were used to investigate the pollen and seed dispersal, mating patterns, spatial genetic structure (SGS), genetic diversity, and inbreeding depression in H. stigonocarpa adults, juveniles, and open‐pollinated seeds, which were sampled from isolated trees in a pasture and trees within a forest fragment in the Brazilian savannah. We found that the species presented a mixed mating system, with population and individual variations in the outcrossing rate (0.53–1.0). The studied populations were not genetically isolated due to pollen and seed flow between the studied populations and between the populations and individuals located outside of the study area. Pollen and seed dispersal occurred over long distances (>8 km); however, the dispersal patterns were isolated by distance, with a high frequency of mating occurring between near‐neighbor trees and seeds dispersed near the parent trees. The correlated mating for individual seed trees was higher within than among fruits, indicating that fruits present a high proportion of full‐sibs. Genetic diversity and SGS were similar among the populations, but offspring showed evidence of inbreeding, mainly originating from mating among related trees, which suggests inbreeding depression between the seed and adult stages. Selfing resulted in a higher inbreeding depression than mating among relatives, as assessed through survival and height. As the populations are not genetically isolated, both are important targets for in situ conservation to maintain their genetic diversity; for ex situ conservation, seeds can be collected from at least 78 trees in both populations separated by at least 250 m.     

Detecting hybridization in mixed populations ofRhinanthus minor andRhinanthus angustifolius

Rhinanthus minor andRhinanthus angustifolius are known to hybridize in mixed populations in nature. These hybridization events can have important evolutionary consequences. The development and use of species-specific RAPD and ISSR markers allowed the detection of hybrid individuals not always distinguishable with morphological characters. Two mixed populations of different ages were studied. In a young mixed 2-year-old population, both individuals of the two parental species and F₁ hybrids were found using genetic analysis, showing that hybridization occurred rapidly. Flower morphology of F₁ hybrids was too variable to distinguish all these hybrids from the parental species. This morphological variability of F₁ hybrids was also confirmed in artificial crosses in the greenhouse. In an old and no longer mixed 30-year-old population, onlyR. angustifolius plants and a few genetically introgressed individuals close toR. angustifolius were present. Genetic markers showed traces of past hybridization and introgression. Unidirectional introgression ofR. minor intoR. angustifolius with the complete disappearance ofR. minor from this population was observed.     

Impacts of inbreeding on bumblebee colony fitness under field conditions

Background  

Inbreeding and the loss of genetic diversity are known to be significant threats to small, isolated populations. Hymenoptera represent a special case regarding the impact of inbreeding. Haplodiploidy may permit purging of deleterious recessive alleles in haploid males, meaning inbreeding depression is reduced relative to diploid species. In contrast, the impact of inbreeding may be exacerbated in Hymenopteran species that have a single-locus complementary sex determination system, due to the production of sterile or inviable diploid males. We investigated the costs of brother-sister mating in the bumblebee Bombus terrestris. We compared inbred colonies that produced diploid males and inbred colonies that did not produce diploid males with outbred colonies. Mating, hibernation and colony founding took place in the laboratory. Once colonies had produced 15 offspring they were placed in the field and left to forage under natural conditions.