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

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Verkley et al.and multi-gene sequence analyses of the septoria-like generabased on numerous isolates (including S. cytisi). In their study, theyresolve the affinities and settle the nomenclature of all importantseptoria-like genera in the Dothideales and Pleosporales.Host specificity has long been a decisive criterium in speciesdelimitation in Septoria, mainly because of the paucity of usefulmorphological characters and the high level of variation therein.Traditionally, species of Septoria that were morphologically verysimilar but found on plants of different host families, were regardedas distinct taxa. Material from the same genus or from closely relatedhost genera from the same plant family that could be distinguishedby features such as conidial length and/or width and septationwere usually also considered to belong to separate species.Most taxonomists revising Septoria lacked facilities to thoroughlyinvestigate host ranges. A number of economically importantSeptoria species and species complexes have been subjected toinfection experiments on various hosts, viz. the pathogens of Apium(Cochran 1932, Sheridan 1968) and cultivated Chrysanthemum(Waddell & Weber 1963, Punithalingam & Wheeler 1965). Theresults of these studies largely seemed to confirm the general beliefthat Septoria species have host ranges that are limited to a singlegenus of plants and in relatively few cases, also include a few closelyrelated genera from the same plant family (Priest 2006). Molecularphylogenetic studies on Septoria species infecting Asteraceae(Verkley & Starink-Willemse 2004) and woody perennials (Feau etal. 2006) showed that species that are capable of infecting hostsof the same plant family do not (always) cluster in monophyleticgroups, which is indicative of disjunct evolutionary patterns of thesepathogens and their hosts. To explain these patterns, it has beenpostulated that “host jumping” occurs from typical (susceptible)hosts to “non-host” plants through asymptomatic tissue infectionand subsequent exploration of new susceptible hosts. Examplesof this were found in certain Mycosphaerella species and theirAcacia hosts (Crous et al. 2004b, Crous & Groenewald 2005), butthe mechanisms driving host jumping are not yet understood. Withour study in which we investigate the phylogenetic relationships ofspecies from a wider spectrum of host families we hope to providemore insight into the evolution of these fungal pathogens and theirhost plants and to contribute to understanding such mechanisms.Early molecular phylogenetic studies have confirmed therelationships of septoria-like fungi with sexual morphs withinMycosphaerellaceae, and that the septoria-like fungi are of poly- andparaphyletic origins (Stewart et al. 1999, Crous et al. 2001, Goodwinet al. 2001, Verkley et al. 2004a, b, Verkley & Starink-Willemse, 2004).The ITS and/or LSU nrDNA sequence data used in those studiesdid not provide sufficient phylogenetic information to discriminateclosely related species nor resolve most of the internal nodes in thetrees. Verkley et al. (2004a, b) already concluded that groups withinthe then known “Mycosphaerella clade” showed no correlation toconidiomatal structure or conidiogenesis, confirming the conclusionsdrawn by Crous et al. (2001). Feau et al. (2006) sequenced the ITS,partial β-tubulin gene, and a proportion of the mitochondrial smallsubunit ribosomal gene (mtSSU) to infer a phylogeny for Septoriaassociated with diseases of woody perennials (many of whichare here transferred to Sphaerulina). Although their inferred treesprovided improved resolution, it was clear that even more DNA lociwould be needed to fully resolve closely related species and speciescomplexes within Septoria s. str.The primary goal of our work was to improve the taxonomy ofSeptoria by adopting a polyphasic approach to taxon delimitation.To this end we studied cultures preserved in CBS, Utrecht, theNetherlands and material freshly collected in the field, did a fullcharacterisation of the morphology in planta and in vitro, andsequenced seven DNA loci, viz. nuclear ITS and (partial) LSUribosomal RNA genes, and RPB2, actin (Act), calmodulin (Cal),β-tubulin (Btub), and translation elongation factor 1-alpha (EF)genes. The obtained datasets of the seven loci were also evaluatedfor PCR amplification success rates and barcode gaps in order todetermine which individual, or combination of loci, would be bestsuited for fast and reliable species resolution and identification.Most students of Septoria have focused on material on the naturalsubstrate and did not isolate and deposit cultures in public culturecollections. Of all material we were able to successfully isolate,cultures were deposited in CBS-KNAW Fungal Biodiversity Centre(CBS) in Utrecht, The Netherlands. To assess the nomenclaturethis material was compared to type material as far as it could beobtained for study. Where useful new material and associated purecultures were designated as epitypes, to facilitate future work. Thisstudy supplements the work of Quaedvlieg et al. (2013), who attaina broader perspective and address the complicated taxonomy andpolyphyly of septoria-like fungi, proposing several new genera fortaxa that are distantly related to Septoria cytisi and allied species.MATERIAL AND METHODSCollecting, isolating and morphological comparisonInfected plant material was collected in the field and takento the laboratory. Leaves were examined directly under astereomicroscope to observe sporulating structures, or wheninsufficiently developed, incubated in a Petri-dish with wetted filterpaper for 1–2 d to enhance the development of fruiting bodies.Cirrhi of spores were removed and mounted in tapwater for themicroscopic examination of conidia. Isolates were obtained byeither transferring cirrhi directly onto 3 % malt extract agar (MEA,Oxoid) plates with 50 ppm penicillin and streptomycin, and streakedover the agar surface with an inoculation loop and some sterilewater. Sometimes conidia in water from slide preparations weretaken with a loop and streaked directly onto a plate. After 1–3 dat room temperature, germinated conidia were transferred on tofresh media without antibiotics. New isolates were deposited in theCBS. Cultures taken from the CBS Collection were activated fromlyophilised or cryopreserved material and inoculated on oatmeal(OA) and MEA plates. A complete overview of the material used inthis study is presented in Table 1.For the morphological study in planta hand sections weremade from infected leaves, mounted in water and examinedunder an Olympus BX 50 microscope equipped with brightfield and differential interference contrast (DIC) objectives, andphotographed using a mounted Nikon Digital Sight DS-5M camera.Conidial masses were mounted in water and 30 spores measured.For culture studies, 7–14-d-old cultures were transferred to freshOA, MEA and cherry decoction agar (CHA) plates and placed in anincubator under n-UV light (12 h light, 12 h dark) at 15 ºC to promotesporulation (if otherwise, this is indicated in the descriptions). Mediawere prepared according to Crous et al. (2009). Colony colourswere described according to Rayner (1970). Sporulating structuresobtained from cultures were used for the morphological descriptionin vitro. Photographs of culture plates were taken after 2–3 wkon a photo stand with daylight tubes with a Pentax K110 D digitalcamera. Cultures were incubated up to 40 d to observe sporulationand other features.214
A new approach to species delimitation in SeptoriaDNA isolation, PCR and sequencingGenomic DNA was extracted from fungal mycelium growing onMEA, using the UltraClean® Microbial DNA Isolation Kit (MoBio Laboratories, Inc., Solana Beach, CA, USA). Strains (Table1) were sequenced for seven loci: Actin (Act), calmodulin (Cal),β-tubulin (Btub), internal transcribed spacer (ITS), Translationelongation factor 1-alpha (EF) 28S nrDNA (LSU) and RNApolymerase II second largest subunit (RPB2); the primer setslisted in Table 2 were used. The PCR amplifications wereperformed in a total volume of 12.5 µL solution containing10–20 ng of template DNA, 1 × PCR buffer, 0.7 µL DMSO(99.9 %), 2 mM MgCl 2, 0.4 µM of each primer, 25 µM of eachdNTP and 1.0 U Taq DNA polymerase (GoTaq, Promega).PCR amplification conditions were set as follows: an initialdenaturation temperature of 96 °C for 2 min, followed by 40cycles at the denaturation temperature of 96 °C for 45 s, primerannealing at the temperature stipulated in Table 2, primerextension at 72 °C for 90 s and a final extension step at 72 °C for2 min. The resulting fragments were sequenced using the PCRprimers together with a BigDye Terminator Cycle SequencingKit v. 3.1 (Applied Biosystems, Foster City, CA). Sequencingreactions were performed as described by Cheewangkoon et al.(2008). All novel sequences were deposited in NCBI’s GenBankdatabase and alignments and phylogenetic trees in TreeBASE.Sequence alignement and phylogenetic analysesA basic alignment of the obtained sequence data was first doneusing MAFFT v. 7 (http://mafft.cbrc.jp/alignment /server/index. html;Katoh et al. 2002) and if necessary, manually improved in BioEditv. 7.0.5.2 (Hall 1999). To check the congruency of the multigenedataset, a 70 % neighbour-joining (NJ) reciprocal bootstrapmethod with maximum likelihood distance was performed (Mason-Gamer & Kellogg 1996, Lombard et al. 2010). Bayesian analyses(critical value for the topological convergence diagnostic set to0.01) were performed on the concatenated loci using MrBayes v.3.2.1 (Huelsenbeck & Ronquist 2001) as described by Crous etal. (2006a) using nucleotide substitution models that were selectedusing MrModeltest (Table 3) (Nylander 2004).Kimura-2-parameter valuesThe inter-and intraspecific distances for each individual datasetwere calculated using MEGA v. 4.0 (Tamura et al. 2007) with theKimura-2-parameter (pairwise deletion) model.RESULTSIdentification of the best DNA barcode loci forSeptoria speciesAmplification successThe PCR amplification success rates were very high for all sevenloci, varying from 97 % for RPB2 to 100 % for ITS and LSU (Table3). Good amplification reactions of RPB2 required a 2–3 timeshigher DNA input then the other loci and this locus is therefore lessfavorable for easy identification. The other six loci amplified withoutproblems.Kimura-2-parameter valuesThe Kimura-2-parameter (K2P) distribution graphs are depicted inFig. 1. They visualise the inter- and intraspecific distances per locus(barcoding gap). A good barcoding locus should have no overlapbetween the inter- and intraspecific K2P distances and should havean average interspecific distance that is at least 10 times as high asthe average intraspecific distance of that locus (Hebert et al. 2003).The seven loci show a rather constant degree of intraspecific variationof 0.01 in their K2P distribution graphs, however their interspecificvariations shows considerable differences. The average interspecificvariation in both ITS and LSU datasets is very low (0.015) comparedto their intraspecific variation (0.01), leading to a very low inter- tointraspecific variation ratios of 1.5 : 1 for these two loci (Fig. 1).These low ratios are far below the required 10 : 1 ratio, indicating ageneral lack of natural variation within these two loci, making them illsuitedfor effective identification of the individual species used in thisdataset. These low K2P results for ITS and LSU are consistent withFrequencyFrequencyFrequency120010008006004002000140012001000800600400200070006000500040003000200010000DistanceDistanceDistanceInter RPB2Inter TubInter EFIntra RPB2Intra TubIntra EFInter ActInter CalIntra ActIntra CalInter ITSInter LSUIntra ITSIntra LSUFig. 1. Frequency distributions of the Kimura-2-parameter distances (barcodinggaps) for the seven PCR loci.www.studiesinmycology.org215
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Verkley et al.Fig. 23. Septoria lac
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Verkley et al.much more profound di
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Verkley et al.Fig. 30. Septoria nap
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Verkley et al.Fig. 38. Septoria sis
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