从12S rRNA基因序列探讨中国10种壁虎的系统关系

对肌肉样品采用SDS／蛋白酶K裂解，酚－氯仿抽提和乙醇沉淀提取总DNA，用线粒体12SrDNA通用引物扩增，用银染测序和ABI Prism 310型遗传分析仪测定了Gekko japonicus,G.swinhonis,G.hokouensis,G.gecko,Cyrtopodion elongatus,C.russowi,Teratoscincus roborowskii,Hemidactylus bowringii.,H.frenatus和Gehyra mutilata 10种壁虎的线粒体12SrRNA基因片段的序列，对位排列后的序列长421bp,含201个变异位点，属间核苷酸变异范围为0．228－0．282，属内是0．005－0．263。重建的分子系统树将10种壁虎聚为三支：第1支是大壁虎和截趾虎，第2支由壁虎属其余的3个种构成一个单系；第三支由基余3个属的5个种组成，研究结果表明：截趾虎与壁虎属4物种亲缘关系较近；吐鲁番沙虎和弯脚虎属2物种，晰虎属2物种之间的亲缘关系较近，支持Russell(1976)关于弯脚虎属与晰虎属间进化关系的研究结果而不支持Kluge(1987)将沙虎属另立亚科的系统发生假说；铅山壁虎与无蹼壁虎最先聚为姐妹群，再与多疣壁相聚组成一个单系。

PHYLOGENY OF TEN SPECIES OF CHINESE GEKKONID LIZARDS (GEKKONIDAE: LACERTILIA) INFERRED FROM 12 S rDAN DNA SEQUENCES

The studies on phylogenetic relationships of gekkonid lizards have been largely based on morphological characters before, including the structure of digits and internal anatomy. In China, the phylogeny among some gekkonid lizards based on karyotype, nucleolar organizer and LDH (lactic dehydrogenase) isozyme was r e ported. However, nucleotide sequence data have not yet been employed to study th e phylogeny of gekkonid lizards. In the present study, phylogenetic relationship among 10 Chinese gekkonid lizard species was inferred based on sequences of 1 2S rRNA gene fragment.  1. Material and method 　　Total DNA was extracted from about 50 mg of the muscle tissue from 10 gekkonid lizards (Gekko japonicus, G. swinhonis, G. hokouensis, G. gecko, Cyrtopodion elongatus, C. russowi, Teratoscincus roborowskii, Hemidactylus bowringii, H. frenatus and Gehyra mutilata) collected in China. The samples were either frozen (－20℃), or ethanol or formalin preserved (Table 1). Two primers L1091 and H1478 (Kocher et al., 1989) were used for amplification and sequencing. Sequencing were performed manually using silver-staining technique or automatically in an ABI Prism 310 Genetic Analyzer. 　　All sequences were aligned with the program of Clustal w (Thompson et al., 1994). The DNA sequences were analyzed with the MEGA program (1.02 edition). For distance estimation, we deleted sites with gaps in each pairwise comparison (Table 2). The phylogenetic trees of NJ, UPGMA and MP were reconstructed with the same program based upon Juke-Cantor distance, with bootstrap test (1 000 replications) (Fig.1). 2. Result and discussion 　　The sequences have been deposited in GenBank (accession numbers: AF236819 and AF323510～323518). Aligned sequences of 421 available base pairs (including 201 variant sites) were obtained. The percentage of sequence divergence (sequence difference / (sequence size) among the five genera was 0.228～0.282, within the genera 0.005～0.263. Tricotomous clusters were obtained by those methods in all three phylogenetic trees: The first cluster contained the sister group of G. mutilata and G. gecko; The second cluster contained the monophyletic lineage of other three species of Gekko; The third cluster contained the remaining five species. 　　All three phylogenetic trees reconstructed indicate that G. mutilata is closely related to the genus Gekko, especially G. gecko. This result is in agreement with the traditional systematic classification. 　　The feet of the Cyrtopodion (=Cyrtodactylus) geckos are of creep moving type, which adapts to the desert habitat. Which have With dilated toes and hyperextended digits, the feet are adapted to arboreal life. Though the two genera differ not only in morphology but also in habitat use, they cluster in all three phylogenetic trees. This outcome supports the result of Russell (1976), who considered the muscle, bone and tendon systems of the feet and digits developed along with the scansors as well as setae and pointed out that the antepenultimate phalanxes of both Cyrtodactylus and Hemidactylus were very small and erect and the extremely reduced nature of this particular phalanx was unique to the cyrtodactyl-hemidactyl linege. 　　The results of cladistic study of the Gekkonoidea (Kluge, 1987) indicated that Teratoscincus was the sister-lineage to the remaining taxa of Gekkonidae, and a new subfamily, Teratoscincinae, should be established. Our result did not support Kluge's hypothesis. The genetic distances between T. roborowskii and other gekkonid lizards based upon the percentage divergence of the DNA sequences was similar to those among the other geckos. T. roborowskii was a sister group to the clade constituted by the two species of Cyrtopodion in UPGMA tree, or clustered with Cyrtopodion and Hemidactylus in a monophyletic group in MP tree. 　　There were studies on karytype, nucleolar organizer and LDH isozyme of G. japonicus, G. swinhonis and G. hokouensis (Chen et al., 1986; Guo et al., 1995; Shen et al., 1996). However, the phylogenetic relationships of these Gekko speices have not yet been resolved. Our molecular data indicated that G. hokouensis and G. swinhonis clustered as the sister group of G. japonicus. The molecular data also supported the validity of G. hokouensis. 　　Although G. gecko was included in the clade constituted by the other three species of Gekko in MP tree, the sequence divergences of 12S rRNA gene fragment between G. gecko and the other Gekko species (P=0.259～0.263) were higher than those among different genera of gekkonid lizards. Additionally, it clustered with G. mutilata in the NJ and UPGMA trees. Therefore, the phylogenetic position of G. gecko remains unresolved.