STUDIES IN MYCOLOGY 75: 213–305. A new approach to species delimitation in Septoria G.J.M. Verkley1*, W. Quaedvlieg1,2, H.-D. Shin3, and P.W. Crous1,2,4 1CBS-KNAW Fungal Biodiversity Centre, Upssalalaan 8, 3584 CT, Utrecht, the Netherlands; 2Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; 3Division of Environmental Science and Ecological Engineering, Korea University, Seoul 136-701, Korea; 4Wageningen University and Research Centre (WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands *Correspondence: G.J.M. Verkley, [email protected] Abstract: Septoria is a large genus of asexual morphs of Ascomycota causing leaf spot diseases of many cultivated and wild plants. Host specificity has long been a decisive criterium in species delimitation in Septoria, mainly because of the paucity of useful morphological characters and the high level of variation therein. This study aimed at improving the species delimitation of Septoria by adopting a polyphasic approach, including multilocus DNA sequencing and morphological analyses on the natural substrate and in culture. To this end 365 cultures preserved in CBS, Utrecht, The Netherlands, among which many new isolates obtained from fresh field specimens were sequenced. Herbarium material including many types was also studied. Full descriptions of the morphology in planta and in vitro are provided for 57 species. DNA sequences were generated for seven loci, viz. nuclear ITS and (partial) LSU ribosomal RNA genes, RPB2, actin, calmodulin, Btub, and EF. The robust phylogeny inferred showed that the septoria-like fungi are distributed over three main clades, establishing the genera Septoria s. str., Sphaerulina, and Caryophylloseptoria gen. nov. Nine new combinations and one species, Sphaerulina tirolensis sp. nov. were proposed. It is demonstrated that some species have wider host ranges than expected, including hosts from more than one family. Septoria protearum, previously only associated with Proteaceae was found to be also associated with host plants from six additional families of phanerogams and cryptogams. To our knowledge this is the first study to provide DNA-based evidence that multiple family-associations occur for a single species in Septoria. The distribution of host families over the phylogenetic tree showed a highly dispersed pattern for 10 host plant families, providing new insight into the evolution of these fungi. It is concluded that trans-family host jumping is a major force driving the evolution of Septoria and Sphaerulina. Key words: Evolution, host jumping, host specificity, Multilocus Sequence Typing (MLST), Mycosphaerella, Mycosphaerellaceae, new genus, new species, Pleosporales, Phloeospora, Septoria, Sphaerulina, taxonomy, systematics.. Taxonomic novelties: New genus – Caryophylloseptoria Verkley, Quaedvlieg & Crous; New species – Sphaerulina tirolensis Verkley, Quaedvlieg & Crous; New combinations – Caryophylloseptoria lychnidis (Desm.) Verkley, Quaedvlieg & Crous, Caryophylloseptoria silenes (Westend.) Verkley, Quaedvlieg & Crous, Caryophylloseptoria spergulae (Westend.) Verkley, Quaedvlieg & Crous, Sphaerulina aceris (Lib.) Verkley, Quaedvlieg & Crous, Sphaerulina cornicola (DC. : Fr.) Verkley, Quaedvlieg & Crous, Sphaerulina gei (Roberge ex Desm.) Verkley, Quaedvlieg & Crous, Sphaerulina hyperici (Roberge ex Desm.) Verkley, Quaedvlieg & Crous, Sphaerulina frondicola (Fr.) Verkley, Quaedvlieg & Crous, Sphaerulina socia (Pass.) Quaedvlieg, Verkley & Crous; Epitypifications (basionyms)– Ascochyta lysimachiae Lib., Septoria astragali Roberge ex Desm., Septoria cerastii Roberge ex Desm., Septoria clematidis Roberge ex Desm., Septoria cruciatae Roberge ex Desm., Septoria spergulae Westend., Septoria epilobii Westend., Septoria galeopsidis Westend., Septoria gei Roberge ex Desm., Septoria hyperici Roberge ex Desm., Septoria rubi Westend., Septoria senecionis Westend., Septoria urticae Roberge ex Desm. doi:10.3114/sim0018. Hard copy: June 2013. INTRODUCTION to extract DNA and successfully amplify and sequence nuclear ribosomal RNA genes to determine its position in a comprehensive Fungi classified in the genus Septoria Sacc. are asexual morphs phylogeny inferred for Mycosphaerellaceae. of Ascomycota causing leaf spot diseases on many cultivated and Most taxonomists adopted a generic concept of Septoria that wild plants. Some 3000 Septoria names have been described in included fungi forming pycnidial conidiomata with holoblastic, literature (Verkley et al. 2004a, b). Sexual morphs are unknown hyaline, smooth-walled conidiogenous cells with sympodial and/or for most taxa, but those reported were mostly classified in percurrent proliferation and hyaline, smooth, filiform to cylindrical Mycosphaerella and Sphaerulina (Von Arx 1983, Sutton & multi-septate conidia (Sutton 1980, Constantinescu 1984, Sutton & Hennebert 1994, Crous et al. 2000, Verkley & Priest 2000, Crous et Pascoe 1987, 1989, Farr 1991, 1992). Similar fungi forming acervular al. 2001, Aptroot 2006). Several overviews of the taxonomic work conidiomata were classified inPhloeospora , with Phloeospora ulmi done on these fungi have been provided in the literature (Shin & as the type species, yet some researchers adopted a broader Sameva 2004, Priest 2006, Quaedvlieg et al. 2013). Priest (2006) concept to include Phloeospora in Septoria (Jørstad 1965, Von Arx discussed the complex nomenclatural history of Septoria. The type 1983, Andrianova 1987, Braun 1995). Recent DNA-sequencing species of Septoria, S. cytisi, is a fungus occurring on the woody studies have shown that the morphological characters that were legume Cytisus laburnum (= Laburnum anagyroides) and several used to delimit coelomycete genera in the past, in particular those other, mostly herbaceous Fabaceae (Farr 1992, Muthumary 1999). pertaining to conidiomatal structure and conidiogenesis, did not The phylogenetic position of this species for which no cultures correlate well with the sequence-inferred phylogenies (Crous et al. are available has for long been uncertain. However, using well- 2001, Verkley et al. 2004a, b). Quaedvlieg et al. (2013) present in identified herbarium material, Quaedvlieg et al. (2011) were able their broad-scope study the results of an in-depth morphological Copyright CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. Studies in Mycology For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 213 VERKLEY ET AL. and multi-gene sequence analyses of the septoria-like genera characterisation of the morphology in planta and in vitro, and based on numerous isolates (including S. cytisi). In their study, they sequenced seven DNA loci, viz. nuclear ITS and (partial) LSU resolve the affinities and settle the nomenclature of all important ribosomal RNA genes, and RPB2, actin (Act), calmodulin (Cal), septoria-like genera in the Dothideales and Pleosporales. β-tubulin (Btub), and translation elongation factor 1-alpha (EF) Host specificity has long been a decisive criterium in species genes. The obtained datasets of the seven loci were also evaluated delimitation in Septoria, mainly because of the paucity of useful for PCR amplification success rates and barcode gaps in order to morphological characters and the high level of variation therein. determine which individual, or combination of loci, would be best Traditionally, species of Septoria that were morphologically very suited for fast and reliable species resolution and identification. similar but found on plants of different host families, were regarded Most students of Septoria have focused on material on the natural as distinct taxa. Material from the same genus or from closely related substrate and did not isolate and deposit cultures in public culture host genera from the same plant family that could be distinguished collections. Of all material we were able to successfully isolate, by features such as conidial length and/or width and septation cultures were deposited in CBS-KNAW Fungal Biodiversity Centre were usually also considered to belong to separate species. (CBS) in Utrecht, The Netherlands. To assess the nomenclature Most taxonomists revising Septoria lacked facilities to thoroughly this material was compared to type material as far as it could be investigate host ranges. A number of economically important obtained for study. Where useful new material and associated pure Septoria species and species complexes have been subjected to cultures were designated as epitypes, to facilitate future work. This infection experiments on various hosts, viz. the pathogens of Apium study supplements the work of Quaedvlieg et al. (2013), who attain (Cochran 1932, Sheridan 1968) and cultivated Chrysanthemum a broader perspective and address the complicated taxonomy