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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 four loci tree as almost equivalent to the five 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.
Comparison of MP trees generated from the same alignment of 5 genes, when changing the position ofthe TEF sequences in that alignment
The numbers represent the bootstrap value for the branching in each tree. Red – MP tree with TEF in the first position of the alignment. Mauve – MP tree with TEF in the second position of the alignment. Green – MP tree with TEF in the third position of the alignment. Blue – MP tree with TEF in the fourth position of the alignment. Black – MP tree with TEF in the fifth position of the alignment. The only small effect of shifting the position of TEF is observed in the TEF-ITS-TUB-HIS-CAL (red) and ITS-TEF-TUB-HIS-CAL (mauve) trees. In TEF-ITS-TUB-HIS-CAL case, flipping is observed in a small number of terminal branches. That flipping does not affect the overall topology of the tree, which is identical to that of the remaining trees. These results suggest that changing the order of the genes in the alignment will have a negligible effect on the topology of the phylogenetic trees.
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