Distance- based approaches
The Kimura two-parameter (K2P) and p-distance model are used to
calculate the pairwise genetic distance and construct the
neighbor-joining tree (NJ) on the MEGA7.0. The haplotype diversity and
nucleotide diversity of COI sequences were calculated using
DnaSP. Then ML tree analysis was implemented using RaxmlGUI. All
analysis used the default parameters and 1,000 bootstraps. In all trees,
bootstrap values below 70% are not shown
(Shen et al., 2016 ).
According
to the phylogenetic trees of the original sequencing sequences and
morphological characteristics, no less than 3 DNA sequences of each
category were selected for further species confirmation by the
IDENTIFICATION of BOLD (Barcode of Life Data System) and the BLAST of
NCBI (National Biotechnology Information Center), to evaluate the
accuracy of the morphological identification results and obtain
reference sequences with high relative similarity. In the selection of
similarity sequences, we have defined 97% as a relatively loose
standard to indicate potential species identification
(E. H. K. Wong & Hanner, 2008 ).
In this study, a total 42 COI sequence with high similarity is obtained
by aligning from GenBank. Gammarus pisinnus (GenBank accession
number: KF824592) selected as the outer group. All DNA sequences
obtained in this study were submitted to GenBank and BOLD; their
accession numbers are provided in the electronic appendix (Table S2,
Table S3), which can be found on the website of the National Center for
Biotechnology Information (NCBI).
In addition to, Automatic Barcode Gap Discovery (ABGD) analysis was
implemented on the website
(http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html), using
K80, relative gap width (X = 1.0) and set the rest of the parameters as
default values.