Romanovskaia V A, Rokitko P V
Mikrobiol Z. 2011 Mar-Apr;73(2):3-12.
To determine a possibility of application of phylogenetic criteria for estimating the taxa rank, the intra- and interspecies, as well as intergeneric relatedness of methanotrophs on the basis of 16S rRNA gene sequences was estimated. We used sequences of 16S rRNA genes of the studied isolates of obligate methanotrophs which have been deposited in UCM (Ukrainian Collection of Microorganisms), and of type strains of other obligate methanotrophs species (from GenBank database). It is shown, that the levels of interspecies and intergeneric relatedness in different families of methanotrophs are not identical, and therefore they can be used for differentiation of taxa only within one family. The carried out analysis has shown, that it is necessary to reconsider taxonomic position: (1) of two phenotypically similar species of Methylomonas (M. aurantiaca and M. fodinarum), similarity of 16S rRNA genes which is 99.4%, similarity of their total DNA--up to 80% that rather testifies to strain differences, than to species differences; (2) of species Methylomicrobium agile and M album which are phylogenetically more related to genus Methylobacter (97% of affinity), than Methylomicrobium (94% of affinity); (3) of genera of the family Beijerinckiaceae (Methylocella and Methylocapsa), and also genera of the family Methylocystaceae (Methylosinus and Methylocystis), whereas high level of relatedness (97% and more) of these bacteria with other methanotrophic genera (within one family) practically corresponds to a range of relatedness of species (within some genera) in the family Methylococcaceae. When determining phylogenetic criteria which can characterize the ranks of taxa, it was revealed, that the levels of interspecies relatedness of methanotrophic genera of the families Methylocystaceae and Beijerinckiaceae (97.8-99.1% and 97.8%, accordingly) considerably exceed the level of genera formation in the family Methylococcaceae (94.0-98.2%) and, moreover, approach the value of intraspecies relatedness of the family Methylococcaceae (97.5-99.3%). Coefficients of intraspecies relatedness of methanotrophs of the families Methylocystaceae and Beijerinckiaceae are sometimes equal to interspecies relatedness. Hence, taxa of various rank can have the identical level of genes divergence. Thus, methanotrophic taxons of the families Methylocystaceae and Beijerinckiaceae have not demonstrated precise phylogenetic criteria which could correspond both to the species rank, or the genus rank. At the same time, the criteria, being adequate to the rank of certain taxa, are revealed in methanotrophs of the family Methylococcaceae. The level of genotypic relatedness of strains of the same species is in the range of 97.5-99.3%, species of the same genus--94-98%, the highest levels of relatedness between genera of this family are 90-96%.
为了确定应用系统发育标准来估计分类单元等级的可能性，基于16S rRNA基因序列对甲烷营养菌的种内、种间以及属间亲缘关系进行了评估。我们使用了保存在乌克兰微生物菌种保藏中心（UCM）的专性甲烷营养菌研究菌株的16S rRNA基因序列，以及其他专性甲烷营养菌物种的模式菌株序列（来自GenBank数据库）。结果表明，甲烷营养菌不同科的种间和属间亲缘关系水平并不相同，因此它们仅可用于同一科内分类单元的区分。所进行的分析表明，有必要重新考虑分类地位：（1）甲基单胞菌属的两个表型相似物种（橙色甲基单胞菌和食泉甲基单胞菌），其16S rRNA基因相似度为99.4%，总DNA相似度高达80%，这更证明了菌株差异而非物种差异；（2）敏捷甲基微菌和白色甲基微菌物种，它们在系统发育上与甲基杆菌属的亲缘关系更近（亲和力为97%），而非与甲基微菌属（亲和力为94%）；（3）拜耶林克氏菌科的属（甲基孢囊菌属和甲基帽菌属），以及甲基孢囊菌科的属（甲基弯曲菌属和甲基孢囊菌属），而这些细菌与其他甲烷营养菌属（在同一科内）的高度亲缘关系（97%及以上）实际上与甲基球菌科内某些属的物种亲缘关系范围相对应。在确定能够表征分类单元等级的系统发育标准时发现，甲基孢囊菌科和拜耶林克氏菌科甲烷营养菌属的种间亲缘关系水平（分别为97.8 - 99.1%和97.8%）大大超过甲基球菌科的属形成水平（94.0 - 98.2%），而且接近甲基球菌科的种内亲缘关系值（97.5 - 99.3%）。甲基孢囊菌科和拜耶林克氏菌科甲烷营养菌的种内亲缘关系系数有时等于种间亲缘关系。因此，不同等级的分类单元可能具有相同的基因分歧水平。这样，甲基孢囊菌科和拜耶林克氏菌科的甲烷营养分类单元并未表现出既符合物种等级也符合属等级的精确系统发育标准。同时，在甲基球菌科甲烷营养菌中发现了与特定分类单元等级相适应的标准。同一物种菌株的基因型亲缘关系水平在97.5 - 99.3%范围内，同一属的物种为94 - 98%，该科属间的最高亲缘关系水平为90 - 96%。
