Individual-level specialisation and interspecific resource partitioning in bees revealed by pollen DNA metabarcoding.

It is increasingly recognised that intraspecific variation in traits, such as morphology, behaviour, or diet is both ubiquitous and ecologically important. While many species of predators and herbivores are known to display high levels of between-individual diet variation, there is a lack of studies on pollinators. It is important to fill in this gap because individual-level specialisation of flower-visiting insects is expected to affect their efficiency as pollinators with consequences for plant reproduction. Accordingly, the aim of our study was to quantify the level of individual-level specialisation and foraging preferences, as well as interspecific resource partitioning, in three co-occurring species of bees of the genus Ceratina (Hymenoptera: Apidae: Xylocopinae), C. chalybea, C. nigrolabiata, and C. cucurbitina. We conducted a field experiment where we provided artificial nesting opportunities for the bees and combined a short-term mark-recapture study with the dissection of the bees' nests to obtain repeated samples from individual foraging females and complete pollen provisions from their nests. We used DNA metabarcoding based on the ITS2 locus to identify the composition of the pollen samples. We found that the composition of pollen carried on the bodies of female bees and stored in the brood provisions in their nests significantly differed among the three co-occurring species. At the intraspecific level, individual females consistently differed in their level of specialisation and in the composition of pollen carried on their bodies and stored in their nests. We also demonstrate that higher generalisation at the species level stemmed from larger among-individual variation in diets, as observed in other types of consumers, such as predators. Our study thus reveals how specialisation and foraging preferences of bees change from the scale of individual foraging bouts to complete pollen provisions accumulated in their nests over many days. Such a multi-scale view of foraging behaviour is necessary to improve our understanding of the functioning of plant-flower visitor communities.

Vertical stratification of plant-pollinator interactions in a temperate grassland.

Visitation of plants by different pollinators depends on individual plant traits, spatial context, and other factors. A neglected aspect of small-scale variation of plant-pollinator interactions is the role of vertical position of flowers. We conducted a series of experiments to study vertical stratification of plant-pollinator interactions in a dry grassland. We observed flower visitors on cut inflorescences of Centaurea scabiosa and Inula salicina placed at different heights above ground in two types of surrounding vegetation: short and tall. Even at such a small-scale, we detected significant shift in total visitation rate of inflorescences in response to their vertical position. In short vegetation, inflorescences close to the ground were visited more frequently, while in tall vegetation, inflorescences placed higher received more visits. Moreover, we found major differences in the composition of the pollinator community on flowers at different heights. In a second experiment, we measured flower visitation rate in inflorescences of Salvia verticillata of variable height. Total flower visitation rate increased markedly with inflorescence height in this case. Data on seed set of individual plants provide evidence for a corresponding positive pollinator-mediated selection on increased inflorescence height. Overall, our results demonstrate strong vertical stratification of plant-pollinator interactions at the scale of mere decimetres. This may have important ecological as well as evolutionary implications.

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation.

Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.

Non-coding RNA may be associated with cytoplasmic male sterility in Silene vulgaris.

Cytoplasmic male sterility (CMS) is a widespread phenomenon in flowering plants caused by mitochondrial (mt) genes. CMS genes typically encode novel proteins that interfere with mt functions and can be silenced by nuclear fertility-restorer genes. Although the molecular basis of CMS is well established in a number of crop systems, our understanding of it in natural populations is far more limited. To identify CMS genes in a gynodioecious plant, Silene vulgaris, we constructed mt transcriptomes and compared transcript levels and RNA editing patterns in floral bud tissue from female and hermaphrodite full siblings. The transcriptomes from female and hermaphrodite individuals were very similar overall with respect to variation in levels of transcript abundance across the genome, the extent of RNA editing, and the order in which RNA editing and intron splicing events occurred. We found only a single genomic region that was highly overexpressed and differentially edited in females relative to hermaphrodites. This region is not located near any other transcribed elements and lacks an open-reading frame (ORF) of even moderate size. To our knowledge, this transcript would represent the first non-coding mt RNA associated with CMS in plants and is, therefore, an important target for future functional validation studies.

The Evolution of the FT/TFL1 Genes in Amaranthaceae and Their Expression Patterns in the Course of Vegetative Growth and Flowering in Chenopodium rubrum.

The FT/TFL1 gene family controls important aspects of plant development: MFT-like genes affect germination, TFL1-like genes act as floral inhibitors, and FT-like genes are floral activators. Gene duplications produced paralogs with modified functions required by the specific lifestyles of various angiosperm species. We constructed the transcriptome of the weedy annual plant Chenopodium rubrum and used it for the comprehensive search for the FT/TFL1 genes. We analyzed their phylogenetic relationships across Amaranthaceae and all angiosperms. We discovered a very ancient phylogenetic clade of FT genes represented by the CrFTL3 gene of C. rubrum Another paralog CrFTL2 showed an unusual structural rearrangement which might have contributed to the functional shift. We examined the transcription patterns of the FT/TFL1 genes during the vegetative growth and floral transition in C. rubrum to get clues about their possible functions. All the genes except for the constitutively expressed CrFTL2 gene, and the CrFTL3 gene, which was transcribed only in seeds, exhibited organ-specific expression influenced by the specific light regime. The CrFTL1 gene was confirmed as a single floral activator from the FT/TFL1 family in C. rubrum Its floral promoting activity may be counteracted by CrTFL1 C. rubrum emerges as an easily manipulated model for the study of floral induction in weedy fast-cycling plants lacking a juvenile phase.