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β-tubulin (benA, tub-2) and calmodulin (caM) are crucial genes in the taxonomy of Aspergillus section Nigri. Widely used β-tubulin primers are not specific for the benA gene for some taxa and preferentially amplify the tubC paralogue. Sequences of the tubC paralogue are widely combined with benA sequences in recent taxonomical works as well as other works, resulting in incongruent trees. In this study we newly provide benA sequences for several ex-type strains, which were characterised using the tubC gene only. We designed a highly specific forward primer to benA designated Ben2f for use in Aspergillus section Nigri, and tested specificity of numerous primer combinations to β-tubulin paralogs. The primer pairs with the highest specificity to the benA gene and functional across species in section Nigri includes Ben2f/Bt2b, Ben2f/T22 and T10/T22. We also provide tools based on codon usage bias analysis that reliably distinguish both paralogues. Exon/intron arrangement is the next distinctive characteristic, although this tool is not valid outside section Nigri. The species identity of taxa from the A. aculeatus clade used in previous molecular studies was revised using combined molecular data (ITS, benA, caM). These data together with two different PCR-fingerprinting methods indicated that A. japonicus should be treated as a synonym of A. violaceofuscus. Similarly, A. fijiensis is reduced to synonymy with A. brunneoviolaceus.

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β-tubulin paralogue &lt;I&gt;tubC&lt;/I&gt; is frequently misidentified as the &lt;I&gt;benA&lt;/I&gt; gene in &lt;I&gt;Aspergillus&lt;/I&gt; section &lt;I&gt;Nigri&lt;/I&gt; taxonomy: primer specificity testing and taxonomic consequences