A new approach to species delimitation in Septoria - CBS - KNAW

Verkley et al.therefore be proposed for it. Sphaerulina rubi Demaree & Wilcoxis already in use for another fungus with a Cylindrosporium sexualstate (C. rubi Ellis & Morgan, conidia 40–55 × 2.5 µm cf. Saccardo),so Sphaer. westendorpii is proposed here as nomen novum.Sphaerulina rehmiana has been associated with Septoria rosaeCBS 355.58, which has been identified as S. rosae, is geneticallydistinct from Sphaer. westendorpii (Quaedvlieg et al. 2013).Insufficiently known speciesFor the following species no host material was available and thesehave only been studied in culture, mostly based on older isolates,for which details are not described when the strain is regarded asdegenerate.Septoria hippocastani Berk. & Broome, Ann. Mag. nat.Hist., Ser. 2, 5: 379. 1850.Material examined: Germany, Pfälzer Wald, on Aesculus hippocastanum, Sep1961, deposited Nov 1961, W. Gerlach, living culture CBS 411.61 (= BBA 9619).Note: CBS 411.61 is degenerated and sterile, but based onmultilocus sequence analysis it can be concluded that it is aSeptoria s. str. (Fig. 2).Septoria limonum Pass., Atti Soc. crittog. ital., 2: 23. 1879.Description in vitro (18 ºC, near UV): Colonies on OA 20–29 mmdiam in 3 wk, with an even, colourless margin; colonies plane,spreading, immersed mycelium in the centre flesh, surrounded by abroad zone of dark vinaceous to brown-vinaceous, aerial myceliumabsent, or scarce, with few tufts of pure white aerial hyphae; reverseconcolorous. No sporulation observed. Colonies on MEA 25–32 mmdiam in 3 wk, with an even to somewhat ruffled, buff to colourlessmargin; colonies spreading, somewhat elevated in the centre,immersed mycelium appearing grayish, the colony surface almostentirely covered by a dense mat of white to grey, woolly-floccoseaerial mycelium; reverse in the centre rust, surrounded by a broadzone of olivaceous-grey to greenish grey, which is sharply borderedby the narrow buff to luteous margin. No sporulation observed.Material examined: Italy, Citrus limonium, isolated Mar. 1951, deposited by G.Goidanich, living culture CBS 419.51.Notes: In the multilocus sequence analysis (Fig. 2) this straingroups with CBS 356.36 (S. citricola) and few other strains in aweakly supported clade close to the plurivorous Septoria protearumand isolates of Septoria citri. Due to the lack of morphologicalinformation linked to this strain, its identity remains uncertain.DISCUSSIONThe type species of the genus Septoria, S. cytisi, could not beincluded in the multilocus analysis due to the fact that only LSU andITS sequences were available for this species. However, as shownby Quaedvlieg et al. (2011), the position of this taxon is beyonddoubt central to the clade indicated here as the main Septoriaclade. Several “typical” Septoria species infecting herbaceousplants proved genetically distant from S. cytisi and its relatives, andcan best be classified in separate genera, Sphaerulina (Quaedvlieget al. 2013) and Caryophylloseptoria.The identification of Septoria has thus far mainly relied on hosttaxonomy and morphological characters of the shape, size, andseptation of conidia (Jørstad 1965, Teterevnikova-Babayan 1987,Andrianova 1987, Vanev et al. 1997, Muthumary 1999, Shin &Sameva 2004, Priest 2006). Taxonomists have noted that conidialwidth is generally a more reliable character for species identificationthan conidial length, which is more variable. Some also noticed thatSeptoria material collected from the same location and host species,but under different environmental conditions or at different times inthe same season, can differ considerably in average conidial sizes,particularly length (Jørstad 1965). These findings are also confirmedin our study. Reliable identification based on morphologicalcomparison alone is not possible for many Septoria species, andreference sequences will have to be produced for many more taxain future. This will require critical studies of type specimens andalso require the recollection of fresh material. It is crucial that thetypes of the oldest names available for Septoria on certain hostswill need to be studied as part of such work, and where necessaryepitypes designated to fix the genetic application of these names.Although hardly practised thus far by taxonomists, isolation and studyin culture is a valuable and indispensable tool for Septoria speciesdelimitation and identification. We noted that the shape of conidiaon OA generally agree best with those in the source material onthe natural substrate. Under standardised incubation conditions onstandard media cultures originating from deviant voucher material,for example because it developed under adverse conditions, showagain their “normal” phenotypes which is better for comparisonpurposes. Extracting DNA from axenic cultures is straight-forwardand less prone to errors caused by contaminants, a problem oftenencountered when extracting DNA from plant tissue.The K2P results show that the five protein coding genes usedduring this research should all theoretically be able to distinquishevery species in this dataset as their average inter- to intraspecificdistance ration is over 10:1. The problem is that these are averagenumbers, not absolute numbers. For example, the Btub K2P graphin Fig. 1 starts at 0 and not at at 0.29, meaning that there actuallyare a few species in our dataset that are not distinguishable by Btubalone (although obviously by far most species in fact are). To avoidthis, we recommend using at least two of the protein coding lociused in this study for identification of Septoria and allied genera.Because EF and Btub both have very high PCR success ratesand have the highest species resolution percentage of all the lociused in this study, we recommend using these two loci for speciesidentification purposes. It is advisable, however, to first sequencethe ITS and LSU for a preliminary genus identification by blasting inGenBank and other useful databases.The multilocus sequence dataset generally provided goodresolution, with maximum to high bootstrap support for almost allterminal and most of the deeper nodes of the phylogenetic tree. Theintraspecific variation in the genes investigated is limited for mosttaxa, even if specimens originate from such distant geographicorigins as New Zealand, Korea and Europe (S. convolvuli, S.leucanthemi, S. polygonorum). Strains assigned to Septoria citripossibly represent a species complex, one of few groups withinthe main Septoria clade that was not resolved. One case of crypticspeciation is revealed in the S. chrysanthemella complex, whereat least two genetically discrete entities can be found that arephenotypically difficult to distinguish.Our results confirm that most species of Septoria have narrowhost ranges, being limited to a single genus or a few genera of thesame plant family. There were a few notable exceptions, however.We demonstrated that the supposed single-family host ranges302