Colletotrichum is one of the most important plant pathogenic genera that is responsible for numerous diseases which can have a profound impact on the agricultural sector. Species delineation is difficult due to a lack of distinctive phenotypic variation. Therefore, in this study three different genomic approaches based on phylogenetic, evolutionary and coalescent-based methods are applied to establish robust species boundaries. The reliability of five different DNA barcodes was also assessed to provide further insights into species delineation. The ITS region can resolve the placement of taxa up to the species complex level. The GAPDH and TUB2 markers are determined to be the most informative for most complexes. However, no single marker could discriminate between species in all complexes, therefore different molecular approaches based on multi-locus datasets are recommended. This is the first study to provide an estimated divergence time for all species complexes in Colletotrichum. The estimated divergence time for species complexes ranged between 4.8 to 32.2 MYA. Based on the high level of congruent results obtained from the different molecular approaches, a new species complex, the Colletotrichum agaves complex is introduced. This complex consists of five taxa which are characterised by the presence of straight or slightly curved conidia with obtuse apices. This study shows that coalescent approaches and multi-locus phylogeny are crucial to establish species boundaries in Colletotrichum. The taxonomic placement of three singleton taxa Colletotrichum axonopodi, C. cariniferi and C. parallelophorum is revised. We accept 248 species and provide recommendations regarding species boundaries in the graminicola–caudatum complex.

炭疽菌是最重要的植物致病属之一，可导致多种对农业部门产生深远影响的疾病。由于缺乏独特的表型变异，很难对物种进行描述。因此，在这项研究中，基于系统发育，进化和基于聚结的方法的三种不同的基因组方法被用于建立稳固的物种边界。还评估了五个不同DNA条形码的可靠性，以提供对物种划分的进一步了解。ITS区域可以将分类单元的位置解析到物种复杂程度。该GAPDH和TUB2标记被确定为对大多数复合物最有用的信息。但是，没有一个单一的标记物可以区分所有复合物中的物种，因此建议使用基于多位点数据集的不同分子方法。这是第一个提供估计炭疽菌中所有物种复合物发散时间的研究。估计的物种复合物的发散时间介于4.8至32.2 MYA之间。基于从不同分子方法获得的高水平一致性结果，一种新的物种复合物，炭疽菌（Colletotrichum）龙舌兰复合体介绍。该复合体由五个类群组成，其特征是存在具有钝顶的笔直或稍弯曲的分生孢子。这项研究表明，聚结方法和多位点系统发育对于在炭疽菌中建立物种边界至关重要。三个单的类群的分类学位置炭疽病axonopodi，C. cariniferi和C. parallelophorum被修改。我们接受248种，并就graminicola-caudatum复合体中的物种边界提供建议。