Some 'ant'swers: Application of a layered barcode approach to problems in ant taxonomy.

DNA barcoding has emerged as a routine tool in modern taxonomy. Although straightforward, this approach faces new challenges, when applied to difficult situation such as defining cryptic biodiversity. Ants are prime examples for high degrees of cryptic biodiversity due to complex population differentiation, hybridization and speciation processes. Here, we test the DNA barcoding region, cytochrome c oxidase 1 and two supplementary markers, 28S ribosomal DNA and long-wavelength rhodopsin, commonly used in ant taxonomy, for their potential in a layered, character-based barcoding approach across different taxonomic levels. Furthermore, we assess performance of the character-based barcoding approach to determine cryptic species diversity in ants. We found (i) that the barcode potential of a specific genetic marker varied widely among taxonomic levels in ants; (ii) that application of a layered, character-based barcode for identification of specimens can be a solution to taxonomical challenging groups; (iii) that the character-based barcoding approach allows us to differentiate specimens even within locations based on pure characters. In summary, (layered) character-based barcoding offers a reliable alternative for problematic species identification in ants and can be used as a fast and cost-efficient approach to estimate presence, absence or frequency of cryptic species.