| Summary: | Diversity of species within <i>Aspergillus niger</i> clade, currently represented by <i>A. niger sensu stricto</i> and <i>A. welwitshiae</i>, was investigated combining three-locus gene sequences, <i>Random Amplified Polymorphic DNA</i>, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as <i>A. niger</i> (<i>n</i> = 247) and 17 as <i>A. welwitschiae</i> (<i>n</i> = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other <i>loci</i>, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species <i>A. welwitschiae</i> and <i>A. niger</i>. This species is illustrated and described as <i>Aspergillus vinaceus</i> sp. nov. In contrast to <i>A. niger</i> and <i>A. welwitschiae</i>, <i>A. vinaceus</i> strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of <i>A. niger</i> and <i>A. welwitschiae,</i> and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in <i>A. vinaceus.</i> The strain type of <i>A. vinaceus</i> sp. nov. is ITAL 47,456 (T) (=IBT 35556).
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