DNA barcoding facilitates many evolutionary and ecological studies, including the examination of the dietary diversity of herbivores. In this study, we present a survey of ecological associations between herbivorous beetles and host plants from seriously threatened European steppic grasslands. We determined host plants for the majority (65%) of steppic leaf beetles (55 species) and weevils (59) known from central Europe using two barcodes (trnL and rbcL) and two sequencing strategies (Sanger for mono/oligophagous species and Illumina for polyphagous taxa). To better understand the ecological associations between steppic beetles and their host plants, we tested the hypothesis that leaf beetles and weevils differ in food selection as a result of their phylogenetic relations (within genera and between families) and interactions with host plants. We found 224 links between the beetles and the plants. Beetles belonging to seven genera feed on the same or related plants. Their preferences were probably inherited from common ancestors and/or resulted from the host plant's chemistry. Beetles from four genera feed on different plants, possibly reducing intrageneric competition and possibly due to an adaptation to different plant chemical defences. We found significant correlations between the numbers of leaf beetle and weevil species feeding on particular plants for polyphagous taxa, but not for nonpolyphagous beetles. Finally, we found that the previous identifications of host plants based on direct observations are generally concordant with host plant barcoding from insect gut. Our results expand basic knowledge about the trophic relations of steppic beetles and plants and are immediately useful for conservation purposes.

beetle_sampling_sitesList of sampling sites (with GPS coordinates) with beetle species sampled in these sites (separately for weevils and leaf beetles). Additionally, all sites are presented in google maps .kmz fileCO1 SangerSequences of Cytochrome Oxidase subunit I mitochondrial gene generated from examined weevil and leaf beetle species. Alignments prepared for phylogenetic trees reconstructions and Maximum Likelihood trees reconstructed with use of PhyML software.CO1.ziprbcL&trnL SangerSequences of trnL and rbcL plant barcodes generated with use of Sanger technique from steppic plants and from DNA isolates obtained from steppic beetles. RbcL alignment use for phylogenetic tree reconstruction and rbcL Maximum Likelihood tree reconstructed with use of PhyML software.rbcL&trnL.ziptrnL&rbcL IlluminaIllumina MiSeq trnL and rbcL plant barcode sequences (forward and reverse) generated from DNA isolates obtained from 15 selected beetles and additional sample which contain sequences generated from 8 selected steppic plants (Eryngium planum, Inula ensifolia, Onobrychis viciifolia, Adonis vernalis, Salvia pratensis, Rosa canina, Arenaria serpyllifolia, and Elymus repens, prepared for method validation purposes)Illumina trnL&rbcL.zip