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.