Department of Natural History Science, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.
Mol Phylogenet Evol. 2013 Mar;66(3):889-97. doi: 10.1016/j.ympev.2012.11.015. Epub 2012 Dec 2.
To investigate the phylogenetic position of Blakiston's fish owl (Bubo blakistoni), we sequenced the mitochondrial (mt) DNA control region and cytochrome b (cyt b) for nine Bubo species. Maximum-likelihood analyses of combined control region and cyt b sequences, and cyt b sequences alone, showed that species formerly placed in genus Ketupa comprise a monophyletic group. Unexpectedly, we discovered a long cluster of 20-25 tandem repeat units 77 or 78bp long in the third control region domain in four of the nine Bubo species for which the control region was sequenced (B. blakistoni, B. flavipes, and B. ketupu in the Ketupa clade; B. lacteus), leading to overall control region lengths of 3.0-3.8kpb estimated from agarose gel electrophoresis. The control region in B. lacteus is the longest (3.8kbp) reported to date in vertebrates. Sequencing of eight repeat units at each end of the cluster in 20 B. blakistoni individuals detected several types of repeat units 77 or 78bp long, and six patterns in the order of unit types. The occurrence of a repeat cluster in all three species examined in the Ketupa clade suggests their common ancestor also had a cluster, whereas a maximum parsimony tree showed repeat-unit types grouping by species, rather than by paralog groups, suggesting independent origins of the clusters. We reconcile these results with a turnover model, in which the range in cluster-length variation and unit types at the 5' end are hypothetically functionally constrained by the protein-binding function of the control region, but otherwise there is a continual turnover of units in evolutionary time, with new unit types arising through mutations, proliferating by duplication of single and double repeat blocks, and being lost through deletion. Estimated free energies for reconstructed secondary structures of single and especially pairs of repeat units were higher than for homologous single-unit blocks in species lacking a repeat cluster, supporting slipped-strand mispairing as the mechanism of cluster turnover.
为了研究 Blakiston's 鱼鹰（Bubo blakistoni）的系统发育位置，我们对 9 种 Bubo 物种的线粒体（mt）DNA 控制区和细胞色素 b（cyt b）进行了测序。联合控制区和 cyt b 序列以及 cyt b 序列的最大似然分析表明，以前归入 Ketupa 属的物种构成了一个单系群。出乎意料的是，我们在 9 种测序的 Bubo 物种中的 4 种（Ketupa 分支中的 B. blakistoni、B. flavipes 和 B. ketupu；B. lacteus）的第三个控制区结构域中发现了一个长的 20-25 个串联重复单元，每个单元长 77 或 78bp，导致琼脂糖凝胶电泳估计的总控制区长度为 3.0-3.8kpb。B. lacteus 的控制区是迄今为止在脊椎动物中报道的最长的（3.8kbp）。在 20 个 B. blakistoni 个体的重复簇的每一端测序 8 个重复单元，检测到几种 77 或 78bp 长的重复单元类型，以及六种单元类型的顺序模式。在 Ketupa 分支中检查的所有 3 个物种中都出现重复簇，表明它们的共同祖先也有一个簇，而最大简约树显示重复单元类型按物种分组，而不是按同源基因分组，表明簇的起源是独立的。我们将这些结果与一个更替模型相协调，在该模型中，5' 端的簇长度变化和单元类型的范围在理论上受到控制区蛋白结合功能的功能限制，但在进化时间内，单元不断更替，新的单元类型通过突变产生，通过单个和双重复块的复制而增殖，并通过缺失而丢失。重建的单重复单元和特别是对重复单元对的二级结构的估计自由能高于缺乏重复簇的物种中同源单单元块的自由能，支持滑动链错配作为簇更替的机制。
