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%.