Developing New Phylogenetic Markers with Enhanced Phylogenetic Resolution Using Genome Sequence Information

Taxonomic revisions of fungi have traditionally relied on molecular phylogenies using universal markers such as nuclear and mitochondrial rRNA, cytochrome oxidase, RNA polymerase II, minichromosome maintenance complex component 7, and elongation factor 1. However, these markers often fail to resolve the phylogenetic relationships of closely related fungal species, and further development of fungal universal markers for classification is limited. To address this, the study attempted to develop semi-universal markers targeting specific fungal families rather than the entire kingdom, assessing their potential for taxonomic purposes. Cladoniaceae and Parmeliaceae were selected due to their high species diversity and significant taxonomic challenges. High-resolution phylogenetic markers for single-copy genes with high substitution rates were developed for these families. In this study, primer sequences for nine newly developed phylogenetic markers are presented: cell division control protein 12 (CDC12), histone acetyltransferase esa1 (ESA1), GPI-anchor transamidase (GPITA), the intergenic spacer between hypothetical protein and elongation factor 3 (HYP2-EF3), isocitrate dehydrogenase (ICDH), mitochondrial-processing peptidase-like protein subunit beta (MPP2), SNF7 family protein (SNF7), zf-C2HC5-domain-containing protein (ZFCP), and V-type ATP synthase subunit B (VATPS). These markers exhibited substitution rates at least three times and up to seven times higher than the ITS domain. The phylogeny of Cladonia gracilis (Cladoniaceae), reconstructed using concatenated sequences of ten markers (ITS, CDC12, ESA1, GPITA, HYP2-EF3, ICDH, MPP2, SNF7, VATPS, and ZFCP), displayed well-resolved branches with high bootstrap support. These markers are expected to provide sufficient resolution for the phylogenetic analysis of closely related species in Cladoniaceae and Parmeliaceae. Experimental conditions and bioinformatic tools for multiplex PCR with barcode primers and sequencing on the MiSeq platform were established, ensuring labor- and cost-effective sequencing performance. This approach demonstrates that semi-universal phylogenetic markers targeting the family level can be used to draw taxonomic conclusions for problematic taxa and potentially be applied to other taxonomic groups.

真菌的分类学修订传统上依赖于采用通用标记的分子系统发育分析，这类通用标记涵盖核与线粒体核糖体RNA（rRNA）、细胞色素氧化酶、RNA聚合酶II、微型染色体维持复合体组件7（minichromosome maintenance complex component 7）以及延伸因子1。然而，此类标记往往难以解析亲缘关系相近的真菌物种间的系统发育关系，且面向真菌分类的通用标记开发仍存在瓶颈。为解决这一局限，本研究尝试开发针对特定真菌科而非整个真菌界的半通用标记，并评估其用于分类学研究的潜力。研究选取了物种多样性极高且分类学难题突出的石蕊科（Cladoniaceae）与梅衣科（Parmeliaceae）作为研究对象，针对这两个科开发了高替换率的单拷贝基因高分辨率系统发育标记。本研究报道了9个新开发的系统发育标记的引物序列：细胞分裂控制蛋白12（cell division control protein 12, CDC12）、组蛋白乙酰转移酶ESA1（histone acetyltransferase esa1, ESA1）、GPI锚定转酰胺酶（GPI-anchor transamidase, GPITA）、假想蛋白与延伸因子3之间的基因间隔区（intergenic spacer between hypothetical protein and elongation factor 3, HYP2-EF3）、异柠檬酸脱氢酶（isocitrate dehydrogenase, ICDH）、线粒体加工肽酶样蛋白β亚基（mitochondrial-processing peptidase-like protein subunit beta, MPP2）、SNF7家族蛋白（SNF7 family protein, SNF7）、含zf-C2HC5结构域的蛋白（zf-C2HC5-domain-containing protein, ZFCP）以及V型ATP合酶B亚基（V-type ATP synthase subunit B, VATPS）。上述标记的替换率至少为ITS区域的3倍，最高可达7倍。利用包含10个标记（ITS、CDC12、ESA1、GPITA、HYP2-EF3、ICDH、MPP2、SNF7、VATPS及ZFCP）的串联序列重建的纤细石蕊（Cladonia gracilis, Cladoniaceae）系统发育树，展现出解析度良好且自展支持率优异的分支结构。这些标记有望为石蕊科与梅衣科内亲缘关系相近物种的系统发育分析提供充足的解析能力。本研究还建立了基于条形码引物的多重PCR实验条件以及在MiSeq平台上的测序流程，可实现高效且低成本的测序作业。该研究证实，针对科级分类单元的半通用系统发育标记可用于解决疑难类群的分类学问题，且具备推广应用至其他分类类群的潜力。