Multiple gene genealogies and phenotypic data reveal cryptic species of the Botryosphaeriaceae: A case study on the Neofusicoccum parvum/N. ribis complex

Neofusicoccum parvum and N. ribis (Botryosphaeriaceae, Ascomycetes) are closely related, plant pathogenic fungi with a world-wide distribution on a wide range of woody hosts. Species boundaries in the N. parvum/N. ribis complex have eluded definition, despite the application of various tools for characterisation. In this study, we test the hypothesis that only one species exists amongst isolates from the N. parvum/N. ribis complex, identified from Syzygiumcordatum trees across their native distribution in South Africa. Genealogical concordance phylogenetic species recognition (GCPSR) was applied based on concordance of genealogies obtained from DNA sequence data for five nuclear loci. These data showed that the single species hypothesis must be rejected. Rather, all analyses support the existence of three previously unrecognised, cryptic species within the N. parvum/N.ribis complex from S. cordatum, in addition to N. parvum and N. ribis. The three lineages reflecting these cryptic taxa are sympatric across their geographical range, indicating barriers to gene flow other than geographic isolation. Phenotypic characters failed to detect all the species uncovered by the GCPSR. Sequence data of the Internal Transcribed Spacer (ITS) of the ribosomal DNA locus, which is thought to be useful for barcoding in fungi, did not distinguish all the species with confidence. RNA polymerase II subunit (RPB2) was the most informative to distinguish all the species a posteriori to the application of GCPSR. The results reflect the critical importance of using multiple gene genealogies and adequate sampling to identify cryptic species and to characterise the true diversity within the Botryosphaeriaceae.