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Weir et al.Table 2. Primers used in this study, with sequences and sources.Gene Product name Primer Direction Sequence (5’–3’) ReferenceACT Actin ACT-512F Foward ATG TGC AAG GCC GGT TTC GC Carbone & Kohn 1999ACT-783R Reverse TAC GAG TCC TTC TGG CCC AT Carbone & Kohn 1999CAL Calmodulin CL1 Foward GAR TWC AAG GAG GCC TTC TC O’Donnell et al. 2000CL2A Reverse TTT TTG CAT CAT GAG TTG GAC O’Donnell et al. 2000CL1C Foward GAA TTC AAG GAG GCC TTC TC This studyCL2C Reverse CTT CTG CAT CAT GAG CTG GAC This studyCHS-1 Chitin synthase CHS-79F Foward TGG GGC AAG GAT GCT TGG AAG AAG Carbone & Kohn 1999CHS-345R Reverse TGG AAG AAC CAT CTG TGA GAG TTG Carbone & Kohn 1999GAPDH Glyceraldehyde-3-phosphate dehydrogenase GDF Foward GCC GTC AAC GAC CCC TTC ATT GA Templeton et al. 1992GDR Reverse GGG TGG AGT CGT ACT TGA GCA TGT Templeton et al. 1992GS Glutamine synthetase GSF1 Foward ATG GCC GAG TAC ATC TGG Stephenson et al. 1997GSF3 Foward GCC GGT GGA GGA ACC GTC G This studyGSR1 Reverse GAA CCG TCG AAG TTC CAG Stephenson et al. 1997GSR2 Reverse GAA CCG TCG AAG TTC CAC This studyITS Internal transcribed spacer ITS-1F Foward CTT GGT CAT TTA GAG GAA GTA A Gardes & Bruns 1993ITS-4 Reverse TCC TCC GCT TAT TGA TAT GC White et al. 1990SOD2 Manganese-superoxide dismutase SODglo2-F Foward CAG ATC ATG GAG CTG CAC CA Moriwaki & Tsukiboshi 2009SODglo2-R Reverse TAG TAC GCG TGC TCG GAC AT Moriwaki & Tsukiboshi 2009TUB2 β-Tubulin 2 T1 Foward AAC ATG CGT GAG ATT GTA AGT O’Donnell & Cigelnik 1997T2 Reverse TAG TGA CCC TTG GCC CAGT TG O’Donnell & Cigelnik 1997Bt2b Reverse ACC CTC AGT GTA GTG ACC CTT GGC Glass & Donaldson 1995Table 3. Nucleotide substitution models used in phylogeneticanalyses.Gene All taxa Musae clade Kahawae cladeITS TrNef+G TrNef+G TrNefGAPDH HKY+G TPM1uf+G TrNCAL TIM1+G TIM1+G TrN+GACT HKY+G TrN JCCHS-1 TrNef+G TrNef+G K80GS TIM2+G TIM3+GSOD2 HKY+G GTR+I+GTUB2 TrN+G HKY+GConidia were measured and described using conidia taken fromthe conidial ooze on acervuli and mounted in lactic acid, at least24 conidia were measured for each isolate, range measurementsare provided in the form (lower extreme–) 25 % quartile – 75 %quartile (–upper extreme), all ranges were rounded to the nearest0.5 µm. Cultures were examined periodically for the developmentof perithecia. Ascospores were measured and described fromperithecia crushed in lactic acid.Appressoria were producing using a slide culture technique. Asmall square of agar was inoculated on one side with conidia andimmediately covered with a sterile cover slip. After 14 d the coverslip was removed and placed in a drop of lactic acid on a glassslide.All morphological character measurements were analysedwith the statistical programme “R” v. 2.14.0 (R Development CoreTeam 2011). The R package ggplot2 (Wickham 2009) was used forgraphical plots. The box plots show the median, upper and lowerquartiles, and the ‘whisker’ extends to the outlying data, or to amaximum of 1.5× the interquartile range, individual outliers outsidethis range are shown as dots.Taxa treated in the taxonomic sectionSpecies, subspecific taxa, and formae speciales within the C.gloeosporioides species complex are treated alphabetically byepithet. The names of formae speciales are not governed by theInternational Code of Botanical Nomenclature (ICBN) (McNeillet al. 2006, Art. 4, Note 4), and are hence enclosed in quotationmarks to indicate their invalid status. Other invalid names thatare governed by the ICBN are also enclosed in quotation marks.Accepted names are marked with an asterisk (*). The breadth ofthe taxonomic names treated includes:all taxonomic names with DNA sequence data in GenBankthat place them in the C. gloeosporioides complex as it has beendefined here on the basis of the ITS gene tree. The sense that thenames were used in GenBank may have been misapplied;names that have been used in the literature in recent yearsfor which a possible relationship to C. gloeosporioides has beensuggested;all subspecific taxa and formae speciales within C.gloeosporioides and Glomerella cingulata.We have not considered the full set of species in Colletotrichum,Gloeosporium and Glomerella that were placed in synonymy withC. gloeosporioides or Glomerella cingulata by von Arx & Müller(1954) or von Arx (1957, 1970).For each accepted species, comments are provided regardingthe limitations of ITS, the official barcoding gene for fungi, todistinguish that species from others within the C. gloeosporioidescomplex.124
The Colletotrichum gloeosporioides species complexRESULTSPhylogeneticsDNA sequences of five genes were obtained from all 158 isolatesincluded in the study and concatenated to form a supermatrix of2294 bp. The gene boundaries in the alignment were: ACT: 1–316,CAL: 317–1072, CHS-1: 1073–1371, GAPDH: 1372–1679, ITS:1680–2294. A BI analysis of the concatenated dataset is presentedin Fig.1. This tree is annotated with the species boundaries of thetaxa that we accept in the C. gloeosporioides complex, and theclades representing these taxa formed the basis for investigatingthe morphological and biological diversity of our species. Ex-typeand authentic isolates are highlighted in bold and labelled with thenames under which they were originally described. The posteriorprobability (PP) support for the grouping of most species rangesfrom 1 to 0.96, however support for deeper nodes is often lower,e.g. 0.53 for the root of C. ti and C. aotearoa, indicating that thebranching may be uncertain for the root of these species. Branchlengths and node PP are typically lower within a species thanbetween species.The large number of taxa in Fig. 1 makes it difficult to visualisethe interspecific genetic distance between the recognised species.The unrooted tree in Fig. 2 represents the results of a BI analysisbased on a concatenation of all eight genes, but restricted to theex-type or authentic cultures from each of the accepted taxa. Theanalysis was done without out-group taxa and clearly shows twoclusters of closely related species that we informally label theMusae clade, and the Kahawae clade.To better resolve relationships within the Musae and Kahawaeclades a further set of BI analyses included eight genes and,wherever possible, several representative isolates of each of theaccepted species. All eight gene sequences were concatenatedto form a supermatrix for each clade. For the Musae clade of32 isolates the alignment was 4199 bp and the gene boundarieswere: ACT: 1–292, TUB2: 293–1008, CAL: 1009–1746, CHS-1:1747–2045, GAPDH: 2046–2331, GS: 2332–3238, ITS: 3239–3823, SOD2: 3824–4199. For the Kahawae clade of 30 isolatesthe alignment was 4107 bp and the gene boundaries were: ACT:1–281, TUB2: 282–988, CAL: 989–1728, CHS-1: 1729–2027,GAPDH: 2028–2311, GS: 2312–3179, ITS: 3198–3733, SOD2:3734–4107. The additional genes sequenced provided additionalsupport for our initial species delimitations with better resolutionfor some closely related species. For example, the highlypathogenic coffee berry isolates (referred to here as C. kahawaesubsp. kahawae) were distinguished from other C. kahawaeisolates.Analyses based on concatenated data sets can maskincongruence between individual gene trees. The low levels ofsupport within some parts of the species-tree analysis (Fig. 3),in part reflects incongruence between gene trees. The levels ofsupport for the Kahawae clade and for the Musae clade are strong(PP=1) but the species that we accept within these clades havelower levels of support than is shown between the other speciesoutside of the clades. The scale bar in Fig. 3 represents a time scale,calibrated at zero for the present day, and at 1 for the last commonancestor (LCA) of the C. gloeosporioides and C. boninense speciescomplexes. The separate species-tree analyses for the Musae andKahawae clades provide a finer resolution of evolutionary historywithin each clade, the time scale based on the same calibrationas Fig. 3.The UPGMA-based ITS gene tree (Fig 6). shows that C.theobromicola, C. horii, C. gloeosporioides, G. cingulata “f. sp.camelliae”, C. asianum, C. musae, C. alatae, C. xanthorrhoeae allform monophyletic clades and may be distinguished with ITS, butmany species are unable to be discriminated using this gene alone.Note that C. cordylinicola and C. psidii are represented by a singleisolate, meaning that variation within ITS sequences across thesespecies has not been tested.Morphology and biologyBrief morphological descriptions, based on all specimens examined,are provided for only those species with no recently publisheddescription. Conidial sizes for all accepted species are summarisedin Fig. 7. Within a species, conidial sizes are reasonably consistentacross isolates, independent of geographic origin or host. However,differences between species are often slight and size ranges oftenoverlap (Fig. 7). The shape of appressoria is generally consistentwithin a species, some being simple in outline, others complex andhighly lobed.Several species are characterised in part by the developmentof perithecia in culture. These include four species in the Musaeclade (C. alienum, C. fructicola, C. queenslandicum, and C.salsolae) and three in the Kahawae clade (C. clidemiae, C.kahawae subsp. ciggaro, and C. ti). Ascospore size and shapecan be a useful species-level diagnostic feature (Fig. 8). In mostspecies the ascospores are strongly curved and typically taperingtowards the ends, but in C. clidemiae and C. ti, they are more orless elliptic with broadly rounded ends and not, or only slightly,curved. Individual isolates within any of these species may lose theability to form perithecia, perhaps associated with cultural changesduring storage.Large, dark-walled stromatic structures are present in thecultures of some species not known to form perithecia. Oftenembedded in agar, less commonly on the surface or amongstthe aerial mycelium, these structures differ from perithecia incomprising a compact tissue of tightly tangled hyphae rather thanthe pseudoparenchymatous, angular cells typical of perithecialwalls. They have a soft, leathery texture compared to the morebrittle perithecia. These stromatic structures sometimes developa conidiogenous layer internally, and following the productionof conidia they may split open irregularly, folding back to form astromatic, acervulus-like structure. These kind of structures arealso formed by some species in the C. boninense species complex(Damm et al. 2012b, this issue).The macroscopic appearance of the cultures is often highlydivergent within a species (e.g. C. fructicola and C. theobromicola),in most cases probably reflecting different storage histories of theisolates examined. Prolonged storage, especially with repeatedsubculturing, results in staling of the cultures, the aerial myceliumoften becoming dense and uniform in appearance and colour, anda loss of conidial and perithecial production, and variable in growthrate (Fig. 9). In some species, individual single ascospore or singleconidial isolates show two markedly different cultural types, seenotes under C. kahawae subsp. ciggaro.Some species appear to be host specialised, e.g. C. horii,C. kahawae subsp. kahawae, C. nupharicola, C. salsolae, C. ti,and C. xanthorrhoeae, but those most commonly isolated havebroad host and geographic ranges, e.g. C. fructicola, C. kahawaesubsp. ciggaro, C. siamense, and C. theobromicola. Colletotrichumgloeosporioides s. str. is commonly isolated from Citrus in manywww.studiesinmycology.org125
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Studies in Mycology (ISSN 0166-0616
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