Samuels Amy K, Weisrock David W, Smith Jeramiah J, France Katherine J, Walker John A, Putta Srikrishna, Voss S Randal
University of Kentucky, Department of Biology, Lexington, KY 40506, USA.
Gene. 2005 Apr 11;349:43-53. doi: 10.1016/j.gene.2004.12.037.
We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger salamander complex species also produced robustly supported trees. The D-loop, used in previous molecular phylogenetic studies of the complex, was found to contain a relatively low level of variation and we identified mitochondrial regions with higher rates of molecular evolution that are more useful in resolving relationships among species. Our results show the benefit of using complete genome mitochondrial information in studies of recently and rapidly diverged taxa.
我们报告了一项研究，该研究扩展了来自近期EST项目的线粒体转录本信息，以获取5种虎螈复合物种（美西钝口螈、东部虎螈、安德森钝口螈、加州钝口螈和杜氏钝口螈）的完整线粒体基因组序列。我们首次描述了一种代表性两栖动物中线粒体转录的各个方面，然后使用完整的线粒体序列数据在进化分歧的深层和浅层水平上研究蝾螈系统发育。美西钝口螈（N = 2481）和东部虎螈（N = 1205）的可用线粒体EST分别覆盖了线粒体基因组的92%和87%。通过使用长距离PCR和DNA测序快速获得了所有物种的完整线粒体序列。所有物种之间以及与其他远缘蝾螈相比，许多基因组结构特征（碱基对长度、碱基组成、基因数量、基因边界、密码子使用）高度相似。总体而言，钝口螈属的线粒体转录近似于在其他脊椎动物中观察到的模式。我们从EST在mtDNA上的定位推断，转录从重链和轻链启动子开始，并沿着mtDNA的全长持续进行，随后进行转录后加工。然而，观察到许多与rRNA基因对应的短转录本表明转录可能经常过早终止，从而使rRNA基因的转录产生偏差；实际上，在16S rRNA基因之后立即观察到一个rRNA转录终止信号序列。蝾螈科亲缘关系的系统发育分析始终将钝口螈科归为一个包含隐鳃鲵科和小鲵科的分支，而排除了蝾螈科。这一有力结果提出了一个新的替代假说，因为先前的研究一直将钝口螈科和蝾螈科鉴定为密切相关的类群。虎螈复合物种的系统发育分析也产生了得到有力支持的树状图。在该复合体先前的分子系统发育研究中使用的D环，发现其变异水平相对较低，并且我们鉴定出了分子进化速率较高的线粒体区域，这些区域在解析物种间关系方面更有用。我们的结果显示了在研究近期快速分化的类群时使用完整基因组线粒体信息的益处。
