Sequence analysis was then conducted with reference to the analysis
pipeline detailed by (Meier et al., 2016). The reads from the Paired-end
were merged by the software PEAR 0.9.6 (Zhang et al. , 2014).. The reads
of each PCR products were then matched to their specific template
specimen which was achieved due to primer pair combination that were
uniquely labelled. A python script by (Srivathsan, unpublished) was used
to 1) demultiplex data, 2) tally the reads for each sample, 3) identify
and cluster identical reads into groups, 4) identify dominant groups of
reads and combine with variants that were otherwise of identical length
and lastly 5) tally the reads found in the group showing highest
identity and compare with the group showing the next highest identity
(Meier et al., 2016). Quality control was carried out by a set of
criteria namely more than 50x read count, more than 10x barcode count
and for the number of dominant reads to be five times or more than
second most dominant reads (Meier et al., 2016). This was to ensure that
coverage attributed to each barcode was sufficient and not from
confounding sequences such as contaminant DNA fragments. In addition,
quality control rejects dominant sequences that may have arisen out of
amplification error in the PCR step. Next, the sequences that passed the
quality control were entered into the search query in Basic Local
Alignment Search Tool (BLAST) to search for sequences that match
>97% to non-Onthophagus taxa, which were contaminant
sequences and thus eliminated from analysis. After quality control,
MEGA7, an online software, was used to align the sequences to ensure
that there were no stop codons. Then, a new Python script (Srivathsan,
unpublished) was used to construct c and a threshold of 3% which is
widely used to distinguish between species in literatures on insects
(Hebert et al., 2003;
Srivathsan
& Meier, 2012).
Figure S4. Haplotype map based on 434 specimens across all sampling
sites, CCNR (n=64), Ubin (n=122), Perak (n=5), Kenyir (n=8), Gombak
(n=40), Langkawi (n=195).