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Background  Species identification is essential for controlling disease, understanding epidemiology, and to guide the implementation of phytosanitary measures against fungi from the genus  Diaporthe . Accurate  Diaporthe  species separation requires using multi-loci phylogenies. However, defining the optimal set of loci that can be used for species identification is still an open problem.    Methods  Here we addressed that problem by identifying five loci that have been sequenced in 142  Diaporthe  isolates representing 96 species:  TEF1 ,  TUB ,  CAL ,  HIS  and ITS. We then used every possible combination of those loci to build, analyse, and compare phylogenetic trees.    Results  As expected, species separation is better when all five loci are simultaneously used to build the phylogeny of the isolates. However, removing the ITS locus has little effect on reconstructed phylogenies, identifying the  TEF1 - TUB - CAL - HIS  4-loci tree as almost equivalent to the 5-loci tree. We further identify the best 3-loci, 2-loci, and 1-locus trees that should be used for species separation in the genus.    Discussion  Our results question the current use of the ITS locus for DNA barcoding in the genus  Diaporthe  and suggest that  TEF1  might be a better choice if one locus barcoding needs to be done.

Evaluating multi-locus phylogenies for species boundaries determination in the genusDiaporthe