2.1.3 Molecular sorting and investigating genetic variation
DNA was extracted from 739 specimens from Singapore populations
(CCNR=129 and Pulau Ubin=167) and Malaysian populations (Central
Peninsular MY=109 and Langkawi=334). The cytochrome oxidase I gene (COI)
fragment has been widely used as to distinguish and identify species for
most animals as the mutation rate of COI gene is approximately parallel
to speciation time scale, thus able to differentiate between species
that are closely related (Fraija-Fernández et al., 2018; Hebert et al.,
2003; Waugh, 2007). Furthermore, the COI gene is an ideal species marker
for insects due to the simple sequence alignment, primer sites that are
robust and easily available and low likelihood of recombination and
possession of introns (Foottit & Adler, 2009). A 3% threshold for
uncorrected pairwise distances was used as it is commonly used in
literature to differentiate species (Hebert et al., 2003; Meiklejohn,
Wallman, & Dowton, 2011; Srivathsan & Meier, 2012).
For all specimens, the right mid femur was dissected into 7 μl of
QuickExtract solution and the DNA was extracted by following Lucigen’s
(the manufacturer’s) protocol (Lucigen, 2018). After DNA extraction, COI
amplification was conducted on extracted DNA. The reaction volumes for
COI amplification for each sample was 5μl of CWBIO 2xTaq MasterMix
(Dye), 1 μl of extracted DNA, 1 μl of sterilised Millipore water, 1 μl
of forward primer, “mlCOIintF”: 5’- GGW ACW GGW TGA ACW GTW TAY CCY
CC-3’ (Leray et al., 2013), 1 μl of reverse primer, “jgHCO2198”:
5’-TAN ACY TCN GGR TGN CCR AAR AAY CA-3’ (Geller et al., 2013) and 1 μl
of 0.001 mg Bovine Serum Albumin (BSA). Polymerase Chain Reaction (PCR)
was performed using the Eppendorf Mastercycler nexus gradient using a
step-up cycling protocol. The following protocol was used: initial
denaturation (94°C, 5 minutes) followed by 35 cycles of denaturation
(94°C, 1 minute), annealing (47°C, 2 minutes) and extension (72°C, 1
minute) and lastly, final extension (72°C, 5 minutes). In addition,
primers were labelled with a sequence tag of 7-9bp such that all
specimens will have a unique combination of labelled forward and reverse
primers. The successfully amplified PCR products were pooled and sent
for NGS sequencing, and sequences were analysed by constructing a
cluster fusion diagram using uncorrected pairwise distances. The full
sequencing and bioinformatics pipeline is appended in Appendix2.
In order to estimate gene flow between populations, a haplotype map was
constructed for the COI sequences of approximately 306 base pairs, with
434 specimens of O . babirussa from Singapore and Malaysia
populations. A minimum spanning network with the software PopART was
used to examine haplotypes and their geographical distribution (Leigh &
Bryant, 2015). The haplotype map is appended in Appendix2 (Figure S4).
The results of the molecular investigation showed that morphologically
sorted O . babirussa were genetically distinct from other
similar looking morphospecies, lending support to our morphological
sorting. The haplotype map also showed that populations across all
sampling sites were panmictic, justifying our groupings of mainland
peninsular Malaysia sites.