pPhospholipases Asub2/sub (PLAsub2/subs) were purified from the iTrimeresurus stejnegeri/i venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLAsub2/sub were determined. In addition to the six previously documented PLAsub2/sub isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLAsub2/subs. The molecular masses and partial sequences of most of the purified PLAsub2/subs matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLAsub2/subs of crotalid venoms, a novel type of PLAsub2/sub with Asn-49 substitution at the Casup2+/sup-binding site was discovered. This type of PLAsub2/sub is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that iT. stejnegeri/i displayed high geographic variations of the PLAsub2/subs within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLAsub2/subs of this species and other related Asian vipers reveals that iT. stejnegeri/i contains venom genes related to those from several sympatric pit vipers, including the genera iTropedolaemus/i and iGloydius/i besides the iTrimeresurus/i itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species./p
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