Species identification and numbers

The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) has been applied in the genus Diaporthe to define the species boundaries in recent studies (Udayanga et al. 2012b; Gomes et al. 2013; Tan et al. 2012). Therefore species delimitation is currently based on DNA sequence data and comparison of morphological characters (Santos and Phillips 2009; Santos et al. 2010; Diogo et al. 2010; Udayanga et al. 2014a, b). Although the genus Diaporthe has received much attention, few phylogenetic studies have thus far been conducted; hence the taxonomy of some of the species in this genus is still uncertain including many of the common plant pathogens. Index Fungorum lists 892 Diaporthe names and 983 Phomopsis names whereas MycoBank (2014) lists 919 Diaporthe names and 1,040 Phomopsis names. However, the names available in the literature are mostly applied based on host association and morphology except fewer species described in the last two decades based on DNA sequence data. Ex-type cultures are available for less than 100 species known despite a large number of species listed in databases and literature. The delimitation of species within the genus Diaporthe improved once DNA sequence data were incorporated (Castlebury and Mengistu 2006; Van Rensburg et al. 2006; Santos et al. 2010; Udayanga et al. 2012b, 2014a, b) since this facilitates obtaining detailed insight into complex evolutionary relationships.

Molecular phylogeny

Since the first molecular phylogenetic study in Diaporthe (Rehner and Uecker 1994), rDNA ITS, partial sequences  of translation elongation factor 1-α (TEF) and  mating  type genes (MAT 1-1-1/1-2-1) have commonly been used in molecular phylogenetic studies in this genus (Van Niekerk et al. 2005;  Van  Rensburg  et  al.  2006;  Santos et al. 2010; Udayanga et al. 2011; Sun et al. 2012). Udayanga et al. (2012a) used ITS, TEF, β- tubulin and CAL genes with a selected set of ex-type cultures and additional isolates to infer the phylogeny of the genus. In a parallel study, a multi-marker phylogeny was effectively used to describe novel species in Diaporthe based on fresh collections from Thailand (Udayanga et al. 2012b). Gomes et al. (2013) used a Brazilian collection of isolates and existing ex-type cultures for a combined phylogenetic analysis of five genetic markers which included ITS, TEF, β- tubulin, CAL and HIS. They introduced several novel taxa from Brazilian collections from medicinal plants with one epitype for Diaporthe anarcardi from Kenya. Udayanga et al. (2014a, b) revisited the Diaporthe species associated with Citrus worldwide with a comprehensive assessment of the genes including ITS, TEF, β- tubulin, CAL and ACT. The study revisited several important phytopathogens including  D.  citri,  D.  cytosporella,  D.  forniculina  and D. rudis, with the epitypes designated with modern The clarification of  D. foeniculaina and D. rudis revealed the potential extensive host association of some species.

Udayanga et al. (2014a) further emphasized that ITS alone can cause much confusion in defining closely related taxa, which has also been noted by several previous researchers regarding closely related species in Diaporthe (Farr et al. 2002a, b; Murali et al. 2006; Santos et al. 2010). The variation of ITS sequences can result in superfluous, multiple terminal branches in combined analyses, even when other gene regions do not support these distinctions (Udayanga et al. 2014a, b). The TEF gene is informative when it comes to clarifying species limits in Diaporthe (Table, Fig.).

Table  Diaporthe. Details of the isolates used in the phylogenetic tree

Fig.  Phylogram generated from parsimony analysis based on„ combined ITS, EF1-α, β- tubulin, and CAL sequenced data of Diaporthe. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Diaporthella corylina CBS 121124.

Recommendations

ITS and TEF are recommended for preliminary identification of the species (Castlebury et al. 2001; Castlebury 2005; Santos and Phillips 2009; Santos et al. 2010). ITS, TEF, β- tubulin, CAL, HIS and ACT should be used in the combined analysis (selection of 4–5 genes), with recommended primers in relevant publications (Udayanga et al. 2012b, 2014a, b; Gomes et al. 2013).