Fig. 1 Construction of pollination network and comparison of analysis methods of mixed pollen composition. The contents of the blue and the green boxes refer to morphological and molecular methods, respectively. The content in the orange box is the PCR-free genome-skimming (metagenomics) approach proposed in this paper. The black network model at the bottom represents the real pollination network, and the blue, green and orange network models represent network structures constructed by the corresponding methods. Due to the limitations of various methods, the constructed networks are potentially deviated from the real network. Metabarcoding and metagenomic techniques can alleviate issues caused by intra-specific morphological variations; but some closely related species remain difficult to differentiate. Compared to metabarcoding, the metagenomic technology can reduce species bias caused by PCR and improve the accuracy in relative abundance.

Fig. 2 The pipeline for P-distance distribution map of mitochondrial and chloroplast genomes

Fig. 3 The P-distance map among species and genera of the mitochondrial genomes and the chloroplast genomes. (a) The P-distance map of 13 protein-coding genes in the mitochondrial genomes among six species in Apis; (b) The P-distance map of 13 protein-coding genes in the mitochondrial genomes among 20 species in Apidae; (c) The P-distance map of 67 protein-coding genes in the chloroplast genomes among 31 species in Dendrobium; (d) The P-distance map of 67 protein-coding genes in the chloroplast genomes among 84 species in Orchidaceae. In panels a & b, the orange area is the location of the COI barcode, and the orange dotted circle represents the median value of the COI barcode’s P-distance. In panels c & d, the orange area is the location of the matK barcode, the purple area is the location of the rbcL barcode, and the orange dotted circle represents the median value of the matK barcode’s P-distance, the purple dotted circle represents the median value of the rbcL barcode’s P-distance.