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The internal transcribed spacer 2 (ITS2) is a widely used phylogenetic marker. In the past, it has mainly been used for species level classifications. Nowadays, a wider applicability becomes apparent. Here, the conserved structure of the RNA molecule plays a vital role. We have developed the ITS2 Database (http://its2.bioapps .biozentrum.uni-wuerzburg.de) which holds information about sequence, structure and taxonomic classification of all ITS2 in GenBank. In the new version, we use Hidden Markov models (HMMs) for the identification and delineation of the ITS2 resulting in a major redesign of the annotation pipeline. This allowed the identification of more than 160 000 correct full ength and more than 50 000 partial structures. In the web interface, these can now be searched with a modified BLAST considering both sequence and structure, enabling rapid taxon sampling. Novel sequences can be annotated using the HMM based approach and modelled according to multiple template structures. Sequences can be searched for known and newly identified motifs. Together, the database and the web server build an exhaustive resource for ITS2 based phylogenetic analyses.
Background: In several studies, secondary structures of ribosomal genes have been used to improve the quality of phylogenetic reconstructions. An extensive evaluation of the benefits of secondary structure, however, is lacking. Results: This is the first study to counter this deficiency. We inspected the accuracy and robustness of phylogenetics with individual secondary structures by simulation experiments for artificial tree topologies with up to 18 taxa and for divergency levels in the range of typical phylogenetic studies. We chose the internal transcribed spacer 2 of the ribosomal cistron as an exemplary marker region. Simulation integrated the coevolution process of sequences with secondary structures. Additionally, the phylogenetic power of marker size duplication was investigated and compared with sequence and sequence-structure reconstruction methods. The results clearly show that accuracy and robustness of Neighbor Joining trees are largely improved by structural information in contrast to sequence only data, whereas a doubled marker size only accounts for robustness. Conclusions: Individual secondary structures of ribosomal RNA sequences provide a valuable gain of information content that is useful for phylogenetics. Thus, the usage of ITS2 sequence together with secondary structure for taxonomic inferences is recommended. Other reconstruction methods as maximum likelihood, bayesian inference or maximum parsimony may equally profit from secondary structure inclusion. Reviewers: This article was reviewed by Shamil Sunyaev, Andrea Tanzer (nominated by Frank Eisenhaber) and Eugene V. Koonin. Open peer review: Reviewed by Shamil Sunyaev, Andrea Tanzer (nominated by Frank Eisenhaber) and Eugene V. Koonin. For the full reviews, please go to the Reviewers’ comments section.
Biodiversity may be investigated and explored by the means of genetic sequence information and molecular phylogenetics. Yet, with ribosomal genes, information for phylogenetic studies may not only be retained from the primary sequence, but also from the secondary structure. Software that is able to cope with two dimensional data and designed to answer taxonomic questions has been recently developed and published as a new scientific pipeline. This thesis is concerned with expanding this pipeline by a tool that facialiates the annotation of a ribosomal region, namely the ITS2. We were also able to show that this states a crucial step for secondary structure phylogenetics and for data allocation of the ITS2-database. This resulting freely available tool determines high quality annotations. In a further study, the complete phylogenetic pipeline has been evaluated on a theoretical basis in a comprehensive simulation study. We were able to show that both, the accuracy and the robustness of phylogenetic trees are largely improved by the approach. The second major part of this thesis concentrates on case studies that applied this pipeline to resolve questions in taxonomy and ecology. We were able to determine several independent phylogenies within the green algae that further corroborate the idea that secondary structures improve the obtainable phylogenetic signal, but now from a biological perspective. This approach was applicable in studies on the species and genus level, but due to the conservation of the secondary structure also for investigations on the deeper level of taxonomy. An additional case study with blue butterflies indicates that this approach is not restricted to plants, but may also be used for metazoan phylogenies. The importance of high quality phylogenetic trees is indicated by two ecological studies that have been conducted. By integrating secondary structure phylogenetics, we were able to answer questions about the evolution of ant-plant interactions and of communities of bacteria residing on different plant tissues. Finally, we speculate how phylogenetic methods with RNA may be further enhanced by integration of the third dimension. This has been a speculative idea that was supplemented with a small phylogenetic example, however it shows that the great potential of structural phylogenetics has not been fully exploited yet. Altogether, this thesis comprises aspects of several different biological disciplines, which are evolutionary biology and biodiversity research, community and invasion ecology as well as molecular and structural biology. Further, it is complemented by statistical approaches and development of informatical software. All these different research areas are combined by the means of bioinformatics as the central connective link into one comprehensive thesis.
Background: Females have often been shown to exhibit preferences for certain male traits. However, little is known about behavioural rules females use when searching for mates in their natural habitat. We investigated mate sampling tactics and related costs in the territorial strawberry poison frog (Oophaga pumilio) possessing a lek-like mating system, where both sequential and simultaneous sampling might occur. We continuously monitored the sampling pattern and behaviour of females during the complete period between two successive matings.
Results: We found no evidence that females compared males by visiting them. Instead females mated with the closest calling male irrespective of his acoustic and physical traits, and territory size. Playback experiments in the natural home ranges of receptive females revealed that tested females preferred the nearest speaker and did not discriminate between low and high call rates or dominant frequencies.
Conclusions: Our results suggest that females of O. pumilio prefer the closest calling male in the studied population. We hypothesize that the sampling tactic in this population is affected by 1) a strongly female biased sex ratio and 2) a low variance in traits of available males due to strong male-male competition, preventing low quality males from defending a territory and mating.
Background
Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing.
Results
We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase).
Conclusions
This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.
Microbial activity is known to have profound impact on bee ecology and physiology, both by beneficial and pathogenic effects. Most information about such associations is available for colony-building organisms, and especially the honey bee. There, active manipulations through worker bees result in a restricted diversity of microbes present within the colony environment. Microbial diversity in solitary bee nests remains unstudied, although their larvae face a very different situation compared with social bees by growing up in isolated compartments. Here, we assessed the microbiota present in nests and pre-adults of Osmia bicornis, the red mason bee, by culture-independent pyrosequencing. We found high bacterial diversity not comparable with honey bee colonies. We identified a variety of bacteria potentially with positive or negative interactions for bee larvae. However, most of the other diverse bacteria present in the nests seem to originate from environmental sources through incorporated nest building material and stored pollen. This diversity of microorganisms may cause severe larval mortality and require specific physiological or symbiotic adaptations against microbial threats. They may however also profit from such a diverse environment through gain of mutualistic partners. We conclude that further studies of microbiota interaction in solitary bees will improve the understanding of fitness components and populations dynamics.
Background
VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1.
Results
In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme.
Conclusions
The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.
Background:
In previous studies, the gram-positive firmicute genus Paenibacillus was found with significant abundances in nests of wild solitary bees. Paenibacillus larvae is well-known for beekeepers as a severe pathogen causing the fatal honey bee disease American foulbrood, and other members of the genus are either secondary invaders of European foulbrood or considered a threat to honey bees. We thus investigated whether Paenibacillus is a common bacterium associated with various wild bees and hence poses a latent threat to honey bees visiting the same flowers.
Results:
We collected 202 samples from 82 individuals or nests of 13 bee species at the same location and screened each for Paenibacillus using high-throughput sequencing-based 16S metabarcoding. We then isolated the identified strain Paenibacillus MBD-MB06 from a solitary bee nest and sequenced its genome. We did find conserved toxin genes and such encoding for chitin-binding proteins, yet none specifically related to foulbrood virulence or chitinases. Phylogenomic analysis revealed a closer relationship to strains of root-associated Paenibacillus rather than strains causing foulbrood or other accompanying diseases. We found anti-microbial evidence within the genome, confirmed by experimental bioassays with strong growth inhibition of selected fungi as well as gram-positive and gram-negative bacteria.
Conclusions:
The isolated wild bee associate Paenibacillus MBD-MB06 is a common, but irregularly occurring part of wild bee microbiomes, present on adult body surfaces and guts and within nests especially in megachilids. It was phylogenetically and functionally distinct from harmful members causing honey bee colony diseases, although it shared few conserved proteins putatively toxic to insects that might indicate ancestral predisposition for the evolution of insect pathogens within the group. By contrast, our strain showed anti-microbial capabilities and the genome further indicates abilities for chitin-binding and biofilm-forming, suggesting it is likely a useful associate to avoid fungal penetration of the bee cuticula and a beneficial inhabitant of nests to repress fungal threats in humid and nutrient-rich environments of wild bee nests.
Solitary bees build their nests by modifying the interior of natural cavities, and they provision them with food by importing collected pollen. As a result, the microbiota of the solitary bee nests may be highly dependent on introduced materials. In order to investigate how the collected pollen is associated with the nest microbiota, we used metabarcoding of the ITS2 rDNA and the 16S rDNA to simultaneously characterize the pollen composition and the bacterial communities of 100 solitary bee nest chambers belonging to seven megachilid species. We found a weak correlation between bacterial and pollen alpha diversity and significant associations between the composition of pollen and that of the nest microbiota, contributing to the understanding of the link between foraging and bacteria acquisition for solitary bees. Since solitary bees cannot establish bacterial transmission routes through eusociality, this link could be essential for obtaining bacterial symbionts for this group of valuable pollinators.
Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments have demonstrated collective responses of the whole Gr-set. We here disentangle the contributions of all three honeybee sugar receptors (AmGr1-3), combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. We show an expanded sugar spectrum of the AmGr1 receptor. Mutants lacking AmGr1 have a reduced response to sucrose and glucose but not to fructose. AmGr2 solely acts as co-receptor of AmGr1 but not of AmGr3, as we show by electrophysiology and using bimolecular fluorescence complementation. Our results show for the first time that AmGr2 is indeed a functional receptor on its own. Intriguingly, AmGr2 mutants still display a wildtype-like sugar taste. AmGr3 is a specific fructose receptor and is not modulated by a co-receptor. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. Our comprehensive study on the functions of AmGr1, AmGr2 and AmGr3 in honeybees is the first to combine investigations on sugar perception at the receptor level and simultaneously in vivo. We show that honeybees rely on two gustatory receptors to sense all relevant sugars.
The composition and richness of herbivore and plant assemblages change along climatic gradients, but knowledge about associated shifts in specialization is scarce and lacks controlling for the abundance and phylogeny of interaction partners. Thus, we aimed to test whether the specialization of phytophagous insects in insect‐plant interaction networks decreases toward cold habitats as predicted by the ‘altitude niche‐breadth hypothesis’ to forecast possible consequences of interaction rewiring under climate change. We used a non‐invasive, standardized metabarcoding approach to reconstruct dietary relationships of Orthoptera species as a major insect herbivore taxon along a broad temperature gradient (~12°C) in Southern Germany. Based on Orthoptera surveys, feeding observations, collection of fecal pellets from >3,000 individuals of 54 species, and parallel vegetation surveys on 41 grassland sites, we quantified plant resource availability and its use by herbivores. Herbivore assemblages were richer in species and individuals at sites with high summer temperatures, while plant richness peaked at intermediate temperatures. Corresponding interaction networks were most specialized in warm habitats. Considering phylogenetic relationships of plant resources, however, the specialization pattern was not linear but peaked at intermediate temperatures, mediated by herbivores feeding on a narrow range of phylogenetically related resources. Our study provides empirical evidence of resource specialization of insect herbivores along a climatic gradient, demonstrating that resource phylogeny, availability, and temperature interactively shape the specialization of herbivore assemblages. Instead of low specialization levels only in cold, harsh habitats, our results suggest increased generalist feeding due to intraspecific changes and compositional differences at both ends of the microclimatic gradient. We conclude that this nonlinear change of phylogeny‐based resource specialization questions predictions derived from the ‘altitude‐niche breadth hypothesis’ and highlights the currently limited understanding of how plant‐herbivore interactions will change under future climatic conditions.
Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall–specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.
Background
Brood parasites can exert strong selection pressure on their hosts. Many brood parasites escape their detection by mimicking sensory cues of their hosts. However, there is little evidence whether or not the hosts are able to escape the parasites’ mimicry by changing these cues. We addressed this question by analyzing cuticular hydrocarbon (CHC) profiles of Cerceris and Philanthus wasps and their brood parasites, cuckoo wasps mimicking the CHC profiles of their hosts. Some of these hosts use hydrocarbons to preserve their prey against fungal infestation and thus, they cannot significantly change their CHC composition in response to chemical mimicry by Hedychrum brood parasites.
Results
We found that the CHC overlap between brood parasites and their hosts was lower in case of host wasps not preserving their prey than in case of prey-preserving host wasps, whose CHC evolution is constrained. Furthermore, the CHC profiles in non-preserving host wasps is more strongly diversified in females than in males, thus in the sex that is chemically mimicked by brood parasites.
Conclusion
Our results provide evidence for a chemical arms race between those hosts that are liberated from stabilizing selection on their chemical template and their parasites.
Solitary bees are subject to a variety of pressures that cause severe population declines. Currently, habitat loss, temperature shifts, agrochemical exposure, and new parasites are identified as major threats. However, knowledge about detrimental bacteria is scarce, although they may disturb natural microbiomes, disturb nest environments, or harm the larvae directly. To address this gap, we investigated 12 Osmia bicornis nests with deceased larvae and 31 nests with healthy larvae from the same localities in a 16S ribosomal RNA (rRNA) gene metabarcoding study. We sampled larvae, pollen provisions, and nest material and then contrasted bacterial community composition and diversity in healthy and deceased nests. Microbiomes of pollen provisions and larvae showed similarities for healthy larvae, whilst this was not the case for deceased individuals. We identified three bacterial taxa assigned to Paenibacillus sp. (closely related to P. pabuli/amylolyticus/xylanexedens), Sporosarcina sp., and Bacillus sp. as indicative for bacterial communities of deceased larvae, as well as Lactobacillus for corresponding pollen provisions. Furthermore, we performed a provisioning experiment, where we fed larvae with untreated and sterilized pollens, as well as sterilized pollens inoculated with a Bacillus sp. isolate from a deceased larva. Untreated larval microbiomes were consistent with that of the pollen provided. Sterilized pollen alone did not lead to acute mortality, while no microbiome was recoverable from the larvae. In the inoculation treatment, we observed that larval microbiomes were dominated by the seeded bacterium, which resulted in enhanced mortality. These results support that larval microbiomes are strongly determined by the pollen provisions. Further, they underline the need for further investigation of the impact of detrimental bacterial acquired via pollens and potential buffering by a diverse pollen provision microbiome in solitary bees.
Cryptic species and hidden ecological interactions of halictine bees along an elevational gradient
(2021)
Changes of abiotic and biotic conditions along elevational gradients represent serious challenges to organisms which may promote the turnover of species, traits and biotic interaction partners. Here, we used molecular methods to study cuticular hydrocarbon (CHC) profiles, biotic interactions and phylogenetic relationships of halictid bees of the genus Lasioglossum along a 2,900 m elevational gradient at Mt. Kilimanjaro, Tanzania. We detected a strong species turnover of morphologically indistinguishable taxa with phylogenetically clustered cryptic species at high elevations, changes in CHC profiles, pollen resource diversity, and a turnover in the gut and body surface microbiome of bees. At high elevations, increased proportions of saturated compounds in CHC profiles indicate physiological adaptations to prevent desiccation. More specialized diets with higher proportions of low‐quality Asteraceae pollen imply constraints in the availability of food resources. Interactive effects of climatic conditions on gut and surface microbiomes, CHC profiles, and pollen diet suggest complex feedbacks among abiotic conditions, ecological interactions, physiological adaptations, and phylogenetic constraints as drivers of halictid bee communities at Mt. Kilimanjaro.
Among the Microbacteriaceae the species of Subtercola and Agreia form closely associated clusters. Phylogenetic analysis demonstrated three major phylogenetic branches of these species. One of these branches contains the two psychrophilic species Subtercola frigoramans and Subtercola vilae, together with a larger number of isolates from various cold environments. Genomic evidence supports the separation of Agreia and Subtercola species. In order to gain insight into the ability of S. vilae to adapt to life in this extreme environment, we analyzed the genome with a particular focus on properties related to possible adaptation to a cold environment. General properties of the genome are presented, including carbon and energy metabolism, as well as secondary metabolite production. The repertoire of genes in the genome of S. vilae DB165\(^T\) linked to adaptations to the harsh conditions found in Llullaillaco Volcano Lake includes several mechanisms to transcribe proteins under low temperatures, such as a high number of tRNAs and cold shock proteins. In addition, S. vilae DB165\(^T\) is capable of producing a number of proteins to cope with oxidative stress, which is of particular relevance at low temperature environments, in which reactive oxygen species are more abundant. Most important, it obtains capacities to produce cryo-protectants, and to combat against ice crystal formation, it produces ice-binding proteins. Two new ice-binding proteins were identified which are unique to S. vilae DB165\(^T\). These results indicate that S. vilae has the capacity to employ different mechanisms to live under the extreme and cold conditions prevalent in Llullaillaco Volcano Lake.
Societal Impact Statement
Pollen relates to many aspects of human and environmental health, which protection and improvement are endorsed by the United Nations Sustainable Development Goals. By highlighting these connections in the frame of current challenges in monitoring and research, we discuss the need of more integrative and multidisciplinary pollen research related to societal needs, improving health of humans and our ecosystems for a sustainable future.
Summary
Pollen is at once intimately part of the reproductive cycle of seed plants and simultaneously highly relevant for the environment (pollinators, vector for nutrients, or organisms), people (food safety and health), and climate (cloud condensation nuclei and climate reconstruction). We provide an interdisciplinary perspective on the many and connected roles of pollen to foster a better integration of the currently disparate fields of pollen research, which would benefit from the sharing of general knowledge, technical advancements, or data processing solutions. We propose a more interdisciplinary and holistic research approach that encompasses total environmental pollen diversity (ePD) (wind and animal and occasionally water distributed pollen) at multiple levels of diversity (genotypic, phenotypic, physiological, chemical, and functional) across space and time. This interdisciplinary approach holds the potential to contribute to pressing human issues, including addressing United Nations Sustainable Development Goals, fostering social and political awareness of these tiny yet important and fascinating particles.
Biodiversity loss, as often found in intensively managed agricultural landscapes, correlates with reduced ecosystem functioning, for example, pollination by insects, and with altered plant composition, diversity, and abundance. But how does this change in floral resource diversity and composition relate to occurrence and resource use patterns of trap-nesting solitary bees? To better understand the impact of land-use intensification on communities of trap-nesting solitary bees in managed grasslands, we investigated their pollen foraging, reproductive fitness, and the nutritional quality of larval food along a land-use intensity gradient in Germany. We found bee species diversity to decrease with increasing land-use intensity irrespective of region-specific community compositions and interaction networks. Land use also strongly affected the diversity and composition of pollen collected by bees. Lack of suitable pollen sources likely explains the absence of several bee species at sites of high land-use intensity. The only species present throughout, Osmia bicornis (red mason bee), foraged on largely different pollen sources across sites. In doing so, it maintained a relatively stable, albeit variable nutritional quality of larval diets (i.e., protein to lipid (P:L) ratio). The observed changes in bee–plant pollen interaction patterns indicate that only the flexible generalists, such as O. bicornis, may be able to compensate the strong alterations in floral resource landscapes and to obtain food of sufficient quality through readily shifting to alternative plant sources. In contrast, other, less flexible, bee species disappear.
Osmotic adaptation and accumulation of compatible solutes is a key process for life at high osmotic pressure and elevated salt concentrations. Most important solutes that can protect cell structures and metabolic processes at high salt concentrations are glycine betaine and ectoine. The genome analysis of more than 130 phototrophic bacteria shows that biosynthesis of glycine betaine is common among marine and halophilic phototrophic Proteobacteria and their chemotrophic relatives, as well as in representatives of Pirellulaceae and Actinobacteria, but are also found in halophilic Cyanobacteria and Chloroherpeton thalassium. This ability correlates well with the successful toleration of extreme salt concentrations. Freshwater bacteria in general lack the possibilities to synthesize and often also to take up these compounds. The biosynthesis of ectoine is found in the phylogenetic lines of phototrophic Alpha- and Gammaproteobacteria, most prominent in the Halorhodospira species and a number of Rhodobacteraceae. It is also common among Streptomycetes and Bacilli. The phylogeny of glycine-sarcosine methyltransferase (GMT) and diaminobutyrate-pyruvate aminotransferase (EctB) sequences correlate well with otherwise established phylogenetic groups. Most significantly, GMT sequences of cyanobacteria form two major phylogenetic branches and the branch of Halorhodospira species is distinct from all other Ectothiorhodospiraceae. A variety of transport systems for osmolytes are present in the studied bacteria.
Bees need food of appropriate nutritional quality to maintain their metabolic functions. They largely obtain all required nutrients from floral resources, i.e., pollen and nectar. However, the diversity, composition and nutritional quality of floral resources varies with the surrounding environment and can be strongly altered in human-impacted habitats. We investigated whether differences in plant species richness as found in the surrounding environment correlated with variation in the floral diversity and nutritional quality of larval provisions (i.e., mixtures of pollen, nectar and salivary secretions) composed by the mass-provisioning stingless bee Tetragonula carbonaria (Apidae: Meliponini). We found that the floral diversity of larval provisions increased with increasing plant species richness. The sucrose and fat (total fatty acid) content and the proportion and concentration of the omega-6 fatty acid linoleic acid decreased, whereas the proportion of the omega-3 fatty acid linolenic acid increased with increasing plant species richness. Protein (total amino acid) content and amino acid composition did not change. The protein to fat (P:F) ratio, known to affect bee foraging, increased on average by more than 40% from plantations to forests and gardens, while the omega-6:3 ratio, known to negatively affect cognitive performance, decreased with increasing plant species richness. Our results suggest that plant species richness may support T. carbonaria colonies by providing not only a continuous resource supply (as shown in a previous study), but also floral resources of high nutritional quality.