Insights into the genus Diaporthe: phylogenetic species delimitation in the D. eres species complex

Dhanushka Udayanga Lisa A. Castlebury Amy Y. Rossman Ekachai Chukeatirote Kevin D. Hyde 0 ) Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming 650201, People's Republic of China The genus Diaporthe comprises pathogenic, endophytic and saprobic species with both temperate and tropical distributions. Cryptic diversification, phenotypic plasticity and extensive host associations have long complicated accurate identifications of species in this genus. The delimitation of the generic type species Diaporthe eres has been uncertain due to the lack of ex-type cultures. Species limits of D. eres and closely related species were evaluated using molecular phylogenetic analysis of eight genes including nuclear ribosomal internal transcribed spacer (ITS), partial sequences of actin Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer. - (ACT), DNA-lyase (Apn2), translation elongation factor 1- (EF1-), beta-tubulin (TUB), calmodulin (CAL), 60s ribosomal protein L37 (FG1093) and histone-3 (HIS). The occurrence of sequence heterogeneity of ITS within D. eres is observed, which complicates the analysis and may lead to overestimation of the species diversity. The strict criteria of Genealogical Concordance Phylogenetic Species Recognition (GCPSR) were applied to resolve species boundaries based on individual and combined analyses of other seven genes except the ITS. We accept nine distinct phylogenetic species including Diaporthe alleghaniensis, D. alnea, D. bicincta, D. celastrina, D. eres, D. helicis, D. neilliae, D. pulla and D. vaccinii. Epitypes are designated for D. alnea, D. bicincta, D. celastrina, D. eres, D. helicis and D. pulla. Modern descriptions and illustrations are provided for these species. Newly designed primers are introduced to amplify and sequence the Apn2 (DNA- lyase) gene in Diaporthe. Based on phylogenetic informativeness profiles, EF1-, Apn2 and HIS genes are recognised as the best markers for defining species in the D. eres complex. In the last two decades much progress has been made in the ability to define fungal species through the use of molecular data (Hibbett and Taylor 2013; Hyde et al. 2013). Circumscribing species within cryptic species complexes that have complicated life histories is essential for determining patterns of speciation and potential hyperdiversity within a genus (Bickford et al. 2007; Silva et al. 2012a; Fekete et al. 2012; ODonnell et al. 2013). Genealogical Concordance Phylogenetic Species Recognition (GCPSR) as an approach for defining fungal species was proposed by Taylor et al. (2000), based on Avise and Balls (1990) genealogical concordance species concept requiring the analysis of several unlinked genes. This approach is often used as an alternative to morphological and biological species recognition (Dettman et al. 2003a). However, there have been relatively a few evaluations of the utility of genes to delineate closely related species in genera with broad host ranges and wide geographic distributions (Giraud et al. 2008; Dupis et al. 2012; Groenewald et al. 2013; Wikee et al. 2013; Salgado-Salazar et al. 2013). The principles of GCPSR are based on the assumption that recombination within a lineage is likely to be the reason for conflict within gene trees, with the transition from conflict to congruence representing the species boundaries (Taylor et al. 2000). Selecting multiple genes with strong phylogenetic signals, the absence of significant incongruence and the application of standard criteria to determine the species boundaries are important factors in the best practices of resolving species complexes (ODonnell et al. 2000; Bischoff et al. 2009; Watanabe et al. 2011; Salichos and Rokas 2013; Damm et al. 2013; Quaedvlieg et al. 2014). Dettman et al. (2003a) further upgraded the operational criteria of GCPSR with the implementation of a two-step process to resolve complex species level phylogenies in fungi. Independent evolutionary lineages are recognised by genealogical concordance and nondiscordance, and subsequently these lineages are subjected to the ranking based on genetic differentiation and exhaustive subdivision process to determine the species limits (Dettman et al. 2003a, b). These methods have been implemented in species complexes including the model ascomycete Neurospora (Dettman et al. 2003b, 2006) and some important plant pathogenic fungal genera (ODonnell et al. 2004; Taylor et al. 2006; Cai et al. 2011; Laurence et al. 2014). The genus Diaporthe comprises pathogenic, endophytic and saprobic species with both temperate and tropical geographic distributions (Rehner and Uecker 1994; Rossman et al. 2007; Udayanga et al. 2011; Huang et al. 2013). Species recognition criteria in Diapo (...truncated)