3.2 Genetic distance
The level of genetic divergence in the COI genes is summarized in Table S1. Intraspecific K2P distances ranged from 0 to 15.07% with an average of 0.78%, whereas interspecific K2P distance within one genus ranged from 0.62% to 33.36% (average 16.37%). Interspecific K2P distance within one genus was 21-fold higher than those of intraspecific K2P distances. Although the maximum intraspecific distance and the distance to the nearest neighbour overlapped partially (Fig. 2), the averages of the nearest neighbor distances were 28-fold higher than those of the maximum intraspecific distances. Based on the Barcode Gap analysis, the minimum interspecific distance for 187 species was larger than the maximum intraspecific distance (98.9%; Fig. 3). For the speciesP. laetum and P. bullum , the maximum intraspecific variations overlapped with the NN distance, leading to the absence of a barcode gap. For the species Chironomus heterodentatus and Chironomus sp. XJ, the distances to the NN were less than 2% of sequence divergence, but more than the maximum intraspecific value. We compared the means of intraspecific K2P distance for nine macroinvertebrate taxonomic groups (Table S1). In particular, Mollusea exhibited the largest mean intraspecific distance (1.93%), whereas Odonata yielded the lowest value of 0.28%.
3.3 Phylogenetic tree-based identification and cluster analysis
Through the NJ method, phylogenetic trees of nine taxonomic groups show that all individuals grouped into species-specific clusters with high bootstrap values expect P. laetum and P. bullum (Fig. 4). For those species with two or more representatives, the conspecifics in monophyletic clades was associated with high support (100% bootstrap in the NJ tree). Despite very low interspecific variations (0.62%), speciesChironomus sp. XJ and Chironomus heterodentatus can be well distinguished by the phylogenetic analyses. One abnormal cluster occurred in an individual ofP. bullum , where it clustered with five individuals ofP. laetum with a 0.63% mean interspecific distance (ranged 0.21%-1.06%). SpeciesDicranota guerini , Ameletus montanus, Glyptotendipes sp. XJ, Euryhapsis sp, Cricotopus ornatus, Atherix sp. XJ, Epeorus sp5 and Radix auriculariaexhibited large internal splits in their monophyletic clusters with >90% support values and, simultaneously, large K2P distances between the internal clusters (Fig. S1). In addition, the clades ofSigara striata andChironomus pallidivittatus formed with some subclusters with relatively large mean K2P genetic distance (Fig. S2).
The cluster analysis of 265 individual sequences from the BOLD data show that the species clustered well with the homologous specimens (Fig. 4B-4J). Firstly, specimens of the same species in our study clustered within the same cluster, and then clustered with those from other areas (Germany, United States, Mexico, Canada, Norway, Italy, Finland, Japan, Iran etc). Based on the BLAST analysis, the query cover and identity of COI sequences for the same species from the present study and other reports in BOLD database did not reach 100%.