DNA Barcoding, Prey Spectrum Analysis, and Vegetative Propagation of Nepenthes mirabilis × rafflesiana, A Rarely Sighted Pitcher Plant Hybrid from Peninsular Malaysia

A solitary female Nepenthes hybrid (Nepenthes mirabilis × rafflesiana), bearing 20-cm tall reddish pitchers, was discovered in the last remaining peat swamp forest in Johor, Peninsular Malaysia. The nothospecies (natural hybrid) is considered a very rare occurrence and has been designated as a taxon of high ecological importance, underscoring the urgent need for in-depth study and targeted conservation initiatives. To confirm its genetic identity, DNA sequencing was conducted, and the phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) indicated a strong genetic relationship between the hybrid (accession no. OR741796) and N. rafflesiana. Consistent with this, the ITS1 secondary structure of the hybrid exhibited a conserved folding structure similar to that of N. rafflesiana. It was recorded that the nothospecies primarily preys on hymenopterans from the family Formicidae, including at least seven genera. Furthermore, mosquito larvae from the genus Toxorhynchites were observed in both upper and lower pitchers, highlighting the pitcher plant's importance as a crucial breeding site for the predatory elephant mosquito. Given that only one living specimen of the nothospecies was discovered, we optimised a shoot-based vegetative propagation method that allowed several clones to be produced, which will be vital for the success of future reintroduction efforts. This is the first report to simultaneously address the molecular, ecological, and horticultural aspects of a Nepenthes natural hybrid in Peninsular Malaysia.