利用核糖体DNA限制性酶切谱(ARDRA)分子技术和小亚基单位核糖体RNA(SSrRNA)基因序列分析对五种双眉虫(原生动物,纤毛门,游仆目)种内及种间关系的探讨

RECONSIDERATION ON THE INTER- AND INTRA-SPECIES RELATIONSHIPS OF DIOPHRYS-COMPLEX (PROTOZOA,CILIOPHORA,HYPOTRICHIA) BASED ON ARDRA RIBOPRINTING METHOD AND ANALYSES OF SSrRNA SEQUENCES

从12种限制性内切酶中筛选出6种可获得种间多样性的内切酶:Alu Ⅰ,Taq Ⅰ,Hae Ⅲ,Hinf Ⅰ,Msp Ⅰ,Xba Ⅰ,对广义双眉虫Diophrys-complex5个种(伪寡毛双眉虫Diophrys apoligothrix、悬游双眉虫D.appendiculata、盾圆双眉虫D.scutum、秀丽拟双眉虫Paradiophrys irmgard、针毛类双眉虫Diophryopsis hystrix)共7个种群的核糖体基因(18S小亚基、部分23S大亚基及其内转录间隔区域)进行多位点酶切。结果显示,种间差异明显大于种内差异。利用RAPDistance1.04软件构建的邻接树表明,盾圆双眉虫的3个种群表现出高度的同源性;3个近缘属(双眉虫属、拟双眉虫属、类双眉虫属)可以被明确区分,并支持类双眉虫属与拟双眉虫属的独立性。从GenBank/EMBL数据库中获得广义双眉虫及相近种的小亚基单位核糖体RNA(SSrRNA)基因序列,利用邻接法(NJ),贝叶斯法(Bayesian)和最大简约法(MP)构建的系统发生树具有基本一致的拓扑结构,结果显示:狭义双眉虫属Diophrys为单源发生系,并与类双眉虫属Diophryopsis组成姐妹群;尽管拟双眉虫属Paradiophrys具有广义双眉虫典型的形态学特征,但与尾刺虫属Uronychia有着较近的亲缘关系。本工作同时表明,核糖体DNA限制性酶切(ARDRA)技术可靠地区分纤毛虫的形态相似种,并在双眉虫属间水平的系统关系推定中存在一定的适用性。

Diophrys-complex species are hard to be identified and separated properly because of the overlapping and variable cirri-pattern features. In this work, the SSrRNA, ITS1-5.8S-ITS2 and partial LSrRNA gene for 7 populations of 5 Diophrys-complex species, viz. Diophrys appendiculata, D. scutum, D.apoligothrix, Diophryopsis hystrix and Paradiophrys irmgard, were amplified and digested using a number of restriction enzymes. The amplified ribosomal DNA restriction analysis （ARDRA） riboprinting can significantly separate all of 5 D/ophrys-complex species, while different populations of D. scutum showed nearly identical patterns. Phylogenetic analysis inferred from the ARDRA riboprinting patterns indicates that Diophrys and Diophryopsis showed relatively dose relationship, while Paradiophrys usually clustered at the basal of the root. Inferred from the SSrRNA gene sequences, phylogenetic relationships of these 5 Diophrys-complex species were reconstructed using multiple algorithms. The result indicates that Diophrys and Diophryopsis formed a sister clade, while Paradiophrys is more closely related to Uronychia, suggesting that the Diophrys-complex species evolved as a paraphyletic group.