Analysis of evolution and phylogeny of snakes of Trimeresurus genus in the southwestern islands of Japan based on structural information of venom-gland isozymes

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1. Interisland mutation of Trimeresurus flavoviridis venom phospholipase A_2 (PLA_2) isozymes.The venoms of T.flavoviridis snakes from Amami-Oshima, Tokunoshima and Okinawa islands contain basic [Asp^<49>]PLA_2s called PLA-B', PLA-B and PL-Y, respectively. They have 1-4 amino acid substitutions that are considered due to occurrence of interisland mutation under isolated environments. A neurotoxic PLA_2 isolated from Amami-Oshima T.flavoviridis venom was named PLA-N. Tokunoshima T.flavoviridis venom also contained this isozyme but Okinawa T.flavoviridis venom contains one amino acid-substituted homolog, called PLA-N(O). This is also a example of interisland mutation. It is speculated that Okiawa island first splitted from the original island and then the rest was disunited into Amami-Oshima and Tokunoshima.2. Evolution and phylogeny of snakes of Trimeresurus genus in the southwestern islands of JapanTwo novel PLA_2 isozymes with Arg at position 49 ([Arg^<49>]PLA_2) were isolated from T.elegans venom and named TeBP(R)-I and TeBP(R)-II. They are polymorphic at position 3, Val for TeBP(R)-I and lle for TeBP(R)-II. A cDNA encoding [Asp49]PLA_2 called TePLA_2 was cloned from its venom gland. A neutral [Asp49]PLA_2 was purified to homogeneity from T.tokarensis venom. However, production of its venom gland cDNA library failed. A phylogenetic tree was constructed for venom PLA_2 isozymes of Trimeresurus genus, except for T.tokarensis, in the southwestern islands of Japan. In the branch of [Lys49]PLA_2-type group, TeBP(R)-I and TeBP(R)-II of T.elegans are much closer to TmK49PLA_2 of T.mucrosquamatus than TfBPI and TfBPI of T.flavoviridis. In PLA2-type group, TePLA_2 is closer to TmPLA_2 than TfPLA2. Thus, T.elegans is the species closer to T.mucrosquamatus than T.flavoviridis. In addition, it is clearly noted that T.okinavensis and T.gramineus are the species fairly distant from T.flavoviridis, T.elegans and T.mucrosquamatus.