The value of twinned pollinator-pollen metabarcoding: bumblebee pollination service is weakly partitioned within a UK grassland community.

Predicting ecological impact of declining bumblebee (Bombus) populations requires better understanding of interactions between pollinator partitioning of floral resources and plant partitioning of pollinator resources. Here, we combine Cytochrome Oxidase 1 (CO1) barcoding for bumblebee identification and rbcL metabarcoding of pollen carried by bees in three species-rich UK pastures. CO1 barcoding assigned 272 bees to eight species, with 33 individuals belonging to the cryptic Bombus lucorum complex (16 B. lucorum and 17 B. cryptarum). Seasonal bias in capture rates varied by species, with B. pratorum found exclusively in June/July and B. pascuorum more abundant in August. Pollen metabarcoding coupled with PERMANOVA and NMDS analyses revealed all bees carried several local pollen species and evidence of pollen resource partitioning between some species pairings, with Bombus pratorum carrying the most divergent pollen load. There was no evidence of resource partitioning between the two cryptic species present, but significantly divergent capture rates concorded with previous suggestions of separation on the basis of foraging behaviour being shaped by local/temporal differences in climatic conditions. Considering the bee carriage profile of pollen species revealed no significant difference between the nine most widely carried plant species. However, there was a sharp, tipping point change in community pollen carriage across all three sites that occurred during the transition between late July and early August. This transition resulted in a strong divergence in community pollen carriage between the two seasonal periods in both years. We conclude that the combined use of pollen and bee barcoding offers several benefits for further study of plant-pollinator interactions at the landscape scale.

Dual-locus DNA metabarcoding reveals southern hairy-nosed wombats (Lasiorhinus latifrons Owen) have a summer diet dominated by toxic invasive plants.

Habitat degradation and summer droughts severely restrict feeding options for the endangered southern hairy-nosed wombat (SHNW; Lasiorhinus latifrons). We reconstructed SHNW summer diets by DNA metabarcoding from feces. We initially validated rbcL and ndhJ diet reconstructions using autopsied and captive animals. Subsequent diet reconstructions of wild wombats broadly reflected vegetative ground cover, implying local rather than long-range foraging. Diets were all dominated by alien invasives. Chemical analysis of alien food revealed Carrichtera annua contains high levels of glucosinolates. Clinical examination (7 animals) and autopsy (12 animals) revealed that the most degraded site also contained most individuals showing signs of glucosinolate poisoning. We infer that dietary poisoning through the ingestion of alien invasives may have contributed to the recent population crashes in the region. In floristically diverse sites, individuals appear to be able to manage glucosinolate intake by avoidance or episodic feeding but this strategy is less tractable in the most degraded sites. We conclude that recovery of the most affected populations may require effective Carrichtera management and interim supplementary feeding. More generally, we argue that protection against population decline by poisoning in territorial herbivores requires knowledge of their diet and of those food plants containing toxic principles.

Replacing Sanger with Next Generation Sequencing to improve coverage and quality of reference DNA barcodes for plants.

We estimate the global BOLD Systems database holds core DNA barcodes (rbcL + matK) for about 15% of land plant species and that comprehensive species coverage is still many decades away. Interim performance of the resource is compromised by variable sequence overlap and modest information content within each barcode. Our model predicts that the proportion of species-unique barcodes reduces as the database grows and that 'false' species-unique barcodes remain >5% until the database is almost complete. We conclude the current rbcL + matK barcode is unfit for purpose. Genome skimming and supplementary barcodes could improve diagnostic power but would slow new barcode acquisition. We therefore present two novel Next Generation Sequencing protocols (with freeware) capable of accurate, massively parallel de novo assembly of high quality DNA barcodes of >1400 bp. We explore how these capabilities could enhance species diagnosis in the coming decades.

Assessing the value of imperfect biocontainment nationally: rapeseed in the United Kingdom as an exemplar.

Paternal biocontainment methods (PBMs) act by preventing pollen-mediated transgene flow. They are compromised by transgene escape via the crop-maternal line. We therefore assess the efficacy of PBMs for transgenic rapeseed (Brassica napus) biocontainment across the United Kingdom by estimating crop-maternal hybridization with its two progenitor species. We used remote sensing, field surveys, agricultural statistics, and meta-analysis to determine the extent of sympatry between the crop and populations of riparian and weedy B. rapa and B. oleracea. We then estimated the incidence of crop-maternal hybridization across all settings to predict the efficacy of PBMs. Evidence of crop chloroplast capture by the progenitors was expanded to a national scale, revealing that crop-maternal gene flow occurs at widely variable rates and is dependent on both the recipient and setting. We use these data to explore the value that this kind of biocontainment can bring to genetic modification (GM) risk management in terms of reducing the impact that hybrids have on the environment rather than preventing or reducing hybrid abundance per se.

Confocal observations of late-acting self-incompatibility in Theobroma cacao L.

Cocoa (Theobroma cacao) has an idiosyncratic form of late-acting self-incompatibility that operates through the non-fusion of incompatible gametes. Here, we used high-resolution confocal microscopy to define fine level changes to the embryo sac of the strongly self-incompatible cocoa genotype SCA 24 in the absence of pollination, and following compatible and incompatible pollination. All sperm nuclei had fused with the female nuclei by 48 h following compatible pollinations. However, following incompatible pollinations, we observed divergence in the behaviour of sperm nuclei following release into the embryo sac. Incomplete sperm nucleus migration occurred in approximately half of the embryo sacs, where the sperm nuclei had so far failed to reach the female gamete nuclei. Sperm nuclei reached but did not fuse with the female gamete nuclei in the residual cases. We argue that the cellular mechanisms governing sperm nucleus migration to the egg nucleus and those controlling subsequent nuclear fusion are likely to differ and should be considered independently. Accordingly, we recommend that future efforts to characterise the genetic basis of LSI in cocoa should take care to differentiate between these two events, both of which contribute to failed karyogamy. Implications of these results for continuing efforts to gain better understanding of the genetic control of LSI in cocoa are discussed.

Production of haploids and doubled haploids in oil palm.

BACKGROUND: Oil palm is the world's most productive oil-food crop despite yielding well below its theoretical maximum. This maximum could be approached with the introduction of elite F1 varieties. The development of such elite lines has thus far been prevented by difficulties in generating homozygous parental types for F1 generation. RESULTS: Here we present the first high-throughput screen to identify spontaneously-formed haploid (H) and doubled haploid (DH) palms. We secured over 1,000 Hs and one DH from genetically diverse material and derived further DH/mixoploid palms from Hs using colchicine. We demonstrated viability of pollen from H plants and expect to generate 100% homogeneous F1 seed from intercrosses between DH/mixoploids once they develop female inflorescences. CONCLUSIONS: This study has generated genetically diverse H/DH palms from which parental clones can be selected in sufficient numbers to enable the commercial-scale breeding of F1 varieties. The anticipated step increase in productivity may help to relieve pressure to extend palm cultivation, and limit further expansion into biodiverse rainforest.

Spontaneous gene flow from rapeseed (Brassica napus) to wild Brassica oleracea.

Research on the environmental risks of gene flow from genetically modified (GM) crops to wild relatives has traditionally emphasized recipients yielding most hybrids. For GM rapeseed (Brassica napus), interest has centred on the 'frequently hybridizing' Brassica rapa over relatives such as Brassica oleracea, where spontaneous hybrids are unreported in the wild. In two sites, where rapeseed and wild B. oleracea grow together, we used flow cytometry and crop-specific microsatellite markers to identify one triploid F1 hybrid, together with nine diploid and two near triploid introgressants. Given the newly discovered capacity for spontaneous introgression into B. oleracea, we then surveyed associated flora and fauna to evaluate the capacity of both recipients to harm cohabitant species with acknowledged conservational importance. Only B. oleracea occupies rich communities containing species afforded legislative protection; these include one rare micromoth species that feeds on B. oleracea and warrants further assessment. We conclude that increased attention should now focus on B. oleracea and similar species that yield few crop-hybrids, but possess scope to affect rare or endangered associates.