Sperling F A, Hickey D A
Department of Biology, University of Ottawa, Ontario, Canada.
Mol Biol Evol. 1994 Jul;11(4):656-65. doi: 10.1093/oxfordjournals.molbev.a040144.
A combination of polymerase-chain-reaction amplification and automated DNA sequencing was used to survey variation in a species complex of pest insects, the spruce budworms (Choristoneura fumiferana species group), and an outgroup species, C. rosaceana. We sequenced an mtDNA region of 1,573 bp that extends from the middle of cytochrome oxidase subunit I (COI) through tRNA leucine (UUR) to the end of cytochrome oxidase subunit II. In addition, we examined levels of intraspecific variation within a 470-bp region of the COI gene. Choristoneura fumiferana clearly represented the oldest lineage within its species group, with 2.7%-2.9% sequence divergence from the other species. In contrast, the four remaining species (C. pinus, C. biennis, C. occidentalis, and C. orae) had closely related or identical mtDNA, with < 1% divergence among most of their haplotypes. Despite its older lineage and widespread geographic distribution, C. fumiferana showed significantly lower intraspecific genetic diversity than did C. occidentalis. Choristoneura orae shared haplotypes with C. occidentalis and C. biennis, and species-level separation of these three species was not supported. Two divergent, uncommon haplotypes were also found in C. occidentalis and C. biennis. The divergent haplotype in C. biennis had an unusually high number of inferred amino acid replacements, suggesting selective differences between mitochondrial DNA haplotypes. Transition:transversion ratios in Choristoneura paralleled those found in Drosophila; transition:transversion ratios were highest in closely related sequences but decreased with increasing sequence divergence. Nucleotide composition showed an A+T bias that was near the high end of the range known for insects. This work illustrates the potential utility of direct DNA sequencing in assessing population structures, species limits, and phylogenetic relationships among organisms that have not previously been subjected to DNA analysis.
采用聚合酶链反应扩增和自动DNA测序相结合的方法，对害虫云杉芽虫（Choristoneura fumiferana物种组）的一个物种复合体以及一个外群物种玫瑰色云杉芽虫（C. rosaceana）的变异情况进行了调查。我们对一段1573 bp的线粒体DNA区域进行了测序，该区域从细胞色素氧化酶亚基I（COI）中部延伸至亮氨酸转运RNA（UUR），再到细胞色素氧化酶亚基II末端。此外，我们还检测了COI基因470 bp区域内的种内变异水平。云杉芽虫（Choristoneura fumiferana）显然代表了其物种组内最古老的谱系，与其他物种的序列差异为2.7%-2.9%。相比之下，其余四个物种（C. pinus、C. biennis、C. occidentalis和C. orae）的线粒体DNA关系密切或相同，其大多数单倍型之间的差异小于1%。尽管云杉芽虫（Choristoneura fumiferana）谱系更古老且地理分布广泛，但其种内遗传多样性却显著低于西方云杉芽虫（C. occidentalis）。玫瑰色云杉芽虫（C. orae）与西方云杉芽虫（C. occidentalis）和两年生云杉芽虫（C. biennis）共享单倍型，不支持这三个物种在物种水平上的分离。在西方云杉芽虫（C. occidentalis）和两年生云杉芽虫（C. biennis）中还发现了两种不同的、罕见的单倍型。两年生云杉芽虫（C. biennis）中不同的单倍型具有异常高数量的推断氨基酸替换，表明线粒体DNA单倍型之间存在选择性差异。云杉芽虫（Choristoneura）的转换：颠换比率与果蝇中的情况相似；转换：颠换比率在亲缘关系密切的序列中最高，但随着序列差异的增加而降低。核苷酸组成显示出A+T偏好，接近昆虫已知范围的高端。这项工作说明了直接DNA测序在评估种群结构、物种界限以及以前未进行过DNA分析的生物体之间的系统发育关系方面的潜在用途。
