Fungal Diversity Multi-gene genealogies and morphological data support Diplodia cupressi sp. nov., previously recognized as D. pinea f. sp. cupressi, as a distinct species Artur Alves1*, António Correia1 and Alan J.L. Phillips2 1 Centro de Estudos do Ambiente e do Mar, Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal 2 Centro de Recursos Microbiológicos, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal Alves, A., Correia, A. and Phillips, A.J.L. (2006). Multi-gene genealogies and morphological data support Diplodia cupressi sp. nov., previously recognized as D. pinea f. sp. cupressi, as a distinct species. Fungal Diversity 23: 1-15. The causal agent of Diplodia canker of cypress (Cupressus spp.) in the Mediterranean region was originally thought to represent a sub-population of the pine pathogen Diplodia pinea and was referred to as D. pinea f. sp. cupressi. In the USA a similar fungus causing canker and dieback of Juniperus spp. was referred to as Diplodia mutila (teleomorph: Botryosphaeria stevensii). The aim of this study was to characterise the cypress pathogen in terms of morphology and sequences of the ITS region, the β-tubulin and translation elongation factor 1α genes. Phylogenetic analyses showed that the cypress canker pathogen resides in a clade together with other Diplodia species. It is, however, distinct from both D. pinea and D. mutila and more closely related to Botryosphaeria tsugae. The distinct phylogenetic position is supported by differences in conidial morphology and it is, therefore, described as Diplodia cupressi sp. nov. Key words: Botryosphaeriaceae, Cupressus, Diplodia, ITS, phylogeny, taxonomy. Introduction A Diplodia canker of Italian cypress (Cupressus sempervirens L.) was described by Solel et al. (1987) in Israel. Since then the disease has been reported from other countries including Morocco (Frisullo and Graniti, 1990), Italy (Evidente et al., 1996), South Africa (Linde et al., 1997), Greece (Xenopoulos and Tsopelas, 2000), and Tunisia (Intini et al., 2005). Isolations made from bark and outer xylem rings of cankers yielded a fungus that according to Solel et al. (1987) was very closely related to Diplodia pinea (Desm.) J. Kickx f. (syn. Sphaeropsis sapinea (Fr.:Fr.) Dyko & B. Sutton). * Corresponding author: Artur Alves; e-mail: aalves@bio.ua.pt 1 They considered the cypress pathogen to represent a distinct subpopulation of the plurivorous species D. pinea, and named it D. pinea f. sp. cupressi. It differed from D. pinea by the smaller conidia, and the lack of ornamentation on the inner surface of the conidium wall. Also, unlike D. pinea, which is a common shoot blight and canker pathogen of Pinus species, the cypress isolates were unable to cause any disease symptoms in artificial inoculations of pine trees (Solel et al., 1987; Linde et al., 1997; Xenopoulos and Tsopelas, 2000). Swart et al. (1993) challenged the close relationship of D. pinea and D. pinea f. sp. cupressi and showed that both fungi are considerably different in terms of morphological characters like conidium size and shape, growth rates on various culture media, and isozyme profiles. This led them to suggest that, in order to avoid further confusion, the cypress pathogen should be referred to as Sphaeropsis sp. until its taxonomy could be further elucidated. The observations of Swart et al. (1993) are further supported by molecular data, namely ITS and mt rDNA nucleotide sequence data (Zhou and Stanosz, 2001a, b) and ISSR fingerprinting (Zhou et al., 2001). A fungus identified as Botryosphaeria stevensii Shoem. (anamorph D. mutila Fr.) was reported as the cause of a canker disease and dieback of Juniperus species in the United States (Tisserat et al., 1988; Flynn and Gleason, 1993; Stanosz and Moorman, 1997). Tisserat et al. (1988) observed the teleomorph and anamorph states of the fungus on J. scopulorum Sarg. in Kansas, and confirmed its pathogenicity by artificial inoculations of J. scopulorum, J. virginiana L., and J. chinensis L. Flynn and Gleason (1993) as well as Stanosz and Moorman (1997) reported only the anamorph in Pennsylvania and Iowa, respectively. On the basis of morphological and pathological data, as well as RAPD marker analysis, Stanosz et al. (1998) concluded that the cypress pathogen referred to as D. pinea f. sp. cupressi in Israel was the same species as the juniper pathogen identified as B. stevensii in the United States. A close affinity between D. mutila and D. pinea f. sp. cupressi was noted by Swart et al. (1993), who showed that conidial dimensions of the two taxa were similar. However, Tisserat et al. (1988) reported differences between the juniper isolates and an isolate of B. stevensii obtained from Malus pumila Mill., as well as published descriptions of B. stevensii. Differences were noted in colony morphology, pigment production, radial growth rate and ability to rot apple fruit. Phylogenetic analysis of ITS and mt rDNA regions (Zhou and Stanosz, 2001a,b) and ISSR fingerprinting analysis (Zhou et al., 2001) showed that B. stevensii (D. mutila) isolates from juniper are closely related to the 2 Fungal Diversity cypress pathogen D. pinea f. sp. cupressi but considerably different from B. stevensii isolates from Malus pumila. The purpose of this study was to clarify the taxonomy of the fungus causing disease on cypress and juniper. For this we studied the type specimen and authentic cultures of D. pinea f. sp. cupressi in terms of morphological characteristics and nucleotide sequence data of ITS, EF1-α, and β-tubulin regions. Materials and methods Isolates Single-conidial or ascospore isolates were prepared according to the methods described by Alves et al. (2004). Additional isolates were obtained from the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. Isolates were maintained on ½ strength Difco PDA. Morphology Morphological characters were determined from isolates sporulating on 2% water agar bearing autoclaved poplar twigs and incubated at 25°C under fluorescent light to induce sporulation. Structures were mounted in 100% lactic acid and digital images were recorded with a Leica DFC 320 camera on a Leica DMR HC microscope. Measurements were made with the Leica IM500 measurement module. From 50 measurements of each type of structure the mean, standard deviation and 95% confidence intervals were calculated. Dimensions are given as the range of dimensions with minimum and maximum dimensions in parentheses followed by mean and 95% confidence limits. DNA isolation, PCR amplification and sequencing The procedures described by Alves et al. (2004) were used to extract genomic DNA from fungal mycelium and to amplify part of the nuclear rRNA cluster using the primers ITS1 and ITS4 (White et al., 1990). The primers EF1728F and EF1-986R (Carbone and Kohn, 1999) and Bt2a and Bt2b (Glass and Donaldson, 1995) were used to amplify part of the translation elongation factor 1-alpha (EF1-α) gene and part of the β-tubulin gene respectively. PCR reactions were carried out with Taq polymerase, nucleotides and buffers supplied by MBI Fermentas (Vilnius, Lithuania) and PCR reaction mixtures were prepared according to Alves et al. (2004) and Phillips et al. (2006). The 3 amplification conditions for EF1- α and β -tubulin regions were as follows: initial denaturation of 5 min at 95ºC, followed by 30 cycles of 30 seconds at 94ºC, 45 seconds at 55ºC, and 90 seconds at 72ºC, and a final extension period of 10 min at 72ºC. In some cases where amplification of the EF1- α region was not accomplished, a second PCR was performed using 1 µL of the first PCR amplification as template. The amplified PCR fragments were purified with the JETQUICK PCR Purification Spin Kit (GENOMED, Löhne, Germany). Both strands of the PCR products were sequenced with the ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTAQ DNA Polymerase (PE Applied Biosystems, Foster City, California, USA) in a Bio-Rad iCycler Thermal Cycler. Cycle sequencing procedure was described by Alves et al. (2004). The sequences were obtained with the ABI PRISM® 310 Genetic Analyzer (PE Applied Biosystems, Foster City, California, USA) and were read and edited with Chromas 1.45 (http://www.technelysium.com.au/chromas. html). All sequences were checked manually and nucleotide arrangements at ambiguous positions were clarified using both primer direction sequences. Sequences were deposited in the GenBank public database. Nucleotide sequences for all three DNA regions of additional Botryosphaeria species were taken from GenBank (Table 1). Phylogenetic analyses The ITS, EF1- α and β -tubulin sequences were aligned with ClustalX version 1.83 (Thompson et al., 1997), using pairwise alignment parameters of gap opening = 10, gap extension = 0.1, and multiple alignment parameters of gap opening = 10, gap extension = 0.2, transition weight = 0.5, delay divergent sequences = 25%. Alignments were checked and manual adjustments were made where necessary. Phylogenetic information contained in indels (insertions/deletions) was incorporated into the phylogenetic analyses using simple indel coding as implemented by GapCoder (Young and Healy, 2003). Phylogenetic analyses of sequence data were done using PAUP*v. 4.0b10 (Swofford, 2003) for Maximum-parsimony (MP) and Neighbourjoining (NJ) analyses and Mr Bayes v.3.0b4 (Ronquist and Huelsenbeck, 2003) for Bayesian analyses. The outgroup taxa selected for rooting the trees were B. lutea and B. ribis. Trees were visualized with TreeView (Page, 1996). The kimura-2-parameter nucleotide substitution model (Kimura 1980) was used for distance analysis. All characters were unordered and of equal weight. Bootstrap values were obtained from 1000 NJ bootstrap replicates. 4 Fungal Diversity Maximum-parsimony analyses were performed using the heuristic search option with 1000 random taxa additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight and alignment gaps were treated as missing data. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated by 1000 bootstrap replications (Hillis and Bull, 1993). Other measures used were tree length (TL) consistency index (CI), retention index (RI) and homoplasy index (HI). Bayesian analyses employing a Markov Chain Monte Carlo method were performed with Mr Bayes v.3.0b4 (Ronquist and Huelsenbeck, 2003). The general time-reversible model of evolution (Rodriguez et al., 1990), including estimation of invariable sites and assuming a discrete gamma distribution with six rate categories (GTR+Γ+G) was used. Four MCMC chains were run simultaneously, starting from random trees, for 1,000,000 generations. Trees were sampled every 100th generation for a total of 10,000 trees. The first 1000 trees were discarded as the burn-in phase of each analysis. Posterior probabilities (Rannala and Yang, 1996) were determined from a majority-rule consensus tree generated with the remaining 9,000 trees. This analysis was done three times starting from different random trees to ensure that trees from the same tree space were being sampled during each analysis. The validity of the incongruence length difference (ILD) test (= partition homogeneity test in PAUP*) for determining whether multiple data sets should be combined has been questioned (Cunningham, 1997; Barker and Lutzoni, 2002). In this study we adopted the method of assessing combinability of data sets by comparing highly supported clades among trees generated from different data sets to detect conflict. High support typically refers to bootstrap support values of ≥ 70% and Bayesian posterior probabilities ≥ 95% (Alfaro et al., 2003). If no conflict exists between the highly supported clades in trees generated from these different data sets, this suggests the genes share similar phylogenetic histories, and phylogenetic resolution and support could ultimately be increased by combining the data sets. Results Phylogenetic analyses Approximately 550, 400 and 300 bases were determined for the ITS, βtubulin and EF1-α genes, respectively, of the isolates (Table 1). New sequences were deposited in GenBank (Table 1) and the alignments in TreeBase 5 Table 1. Isolates studied. Identity Host Locality Collector ITS2 β-tubulin Isolate EF1-α 1 number Vitis vinifera Oeiras, Portugal A.J.L. Phillips AY259091 AY573217 DQ458848 CBS 110299 B. lutea Ribes sp. New York, USA B. Slippers AY236935 AY236877 AY236906 CBS 115475 B. ribis CBS 230.30 B. stevensii Phoenix dactylifera California, U.S.A. L.L. Huillier DQ458886 DQ458869 DQ458849 CBS 112553 B. stevensii Vitis vinifera Montemor-o-Novo, Portugal A.J.L. Phillips AY259093 AY573219 DQ458850 CBS 112554 B. stevensii Pyrus communis Monte da Caparica, Portugal A.J.L. Phillips AY259095 DQ458870 DQ458851 JL 375 B. stevensii Fraxinus excelsior Llanars, Catalonia, Spain J. Luque DQ458887 DQ458871 DQ458852 CMW 7060 B. stevensii Fraxinus excelsior Maarseveen, Netherlands H.A. van der Aa AY236955 AY236904 AY236933 Quercus suber Aveiro, Portugal A. Alves AY259100 AY573227 DQ458853 CBS 112549 B. corticola CBS 112547 B. corticola Quercus ilex Córdoba, Spain M.E. Sanchez AY259110 DQ458872 DQ458854 CBS 418.64 B. tsugae Tsuga heterophylla British Columbia, Canada A. Funk DQ458888 DQ458873 DQ458855 CBS 112555 B. obtusa Vitis vinifera Montemor-o-Novo, Portugal A.J.L. Phillips AY259094 AY573220 DQ458856 CBS 119049 B. obtusa Vitis sp. Italy L. Mugnai DQ458889 DQ458874 DQ458857 CBS 124.13 B. rhodina Unknown U.S.A. J.J. Taubenhaus DQ458890 DQ458875 DQ458858 CAA 006 B. rhodina Vitis vinifera California, USA T.J. Michailides DQ458891 DQ458876 DQ458859 Eastern Cape, South Africa D. Pavlic DQ458892 DQ458877 DQ458860 CBS 115812 L. gonubiensis Syzygium cordatum Cupressus sempervirens Bet Dagan, Israel Z. Solel DQ458893 DQ458878 DQ458861 CBS 168.87 D. cupressi CBS 261.85 D. cupressi Cupressus sempervirens Bet Dagan, Israel Z. Solel DQ458894 DQ458879 DQ458862 CBS 393.84 D. pinea Pinus nigra Putten, Netherlands H.A. van der Aa DQ458895 DQ458880 DQ458863 CBS 109725 D. pinea Pinus patula Habinsaran, South Africa M.J. Wingfield DQ458896 DQ458881 DQ458864 CBS 109727 D. pinea Pinus radiata Stellenbosch, South Africa W.J. Swart DQ458897 DQ458882 DQ458865 CBS 109943 D. pinea Pinus patula Indonesia M.J. Wingfield DQ458898 DQ458883 DQ458866 CBS 109944 D. scrobiculata Pinus greggii Mexico M.J. Wingfield DQ458899 DQ458884 DQ458867 CBS 113424 D. scrobiculata Pinus greggii Mexico M.J. Wingfield DQ458900 DQ458885 DQ458868 1 Acronyms of culture collections: CAA – A. Alves, Universidade de Aveiro, Portugal; CBS – Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CMW – M.J. Wingfield, FABI, University of Pretoria, South Africa; JL – J. Luque, IRTA, Spain. Isolates in bold are cultures ex-type. 2 Sequence numbers in italics were retrieved from GenBank. All others were determined in the present study. 6 Fungal Diversity (S1542). Sequences of the three genes were aligned and analysed separately by maximum parsimony and Bayesian analysis, and the resulting trees were compared. No major conflicts were detected between single gene phylogenies indicating that the genes could be combined, thus resulting in increased phylogenetic resolution. The sequence alignment of 23 isolates (Table 1), including the two outgroup species, consisted of 579 characters for the ITS region, 331 for the EF1-α gene and 413 for the β -tubulin gene, including alignment gaps. Indels were coded separately and added to the end of the alignment as characters 1326-1397. In the analyses, alignment gaps were treated as missing data. The combined dataset consisted of 1397 characters, of which 1043 were constant and 83 variable characters were parsimony-uninformative. Maximum parsimony analysis of the remaining 271 parsimony-informative characters resulted in a single most parsimonious trees (TL = 518 steps, CI = 0.8205, HI = 0.1795, RI = 0.8818, RC = 0.7235). Bayesian and NJ analyses produced trees with the same topology as the MP tree. The Bayesian analysis was done three times and the resulting trees in each run were identical. The MP tree is shown in Fig. 1 with MP bootstrap supports above and posterior probabilities below the branches. Phylogenetic analyses clearly separated the ingroup taxa into several clades which correspond to known species as well as two D. pinea morphotypes. The two species with Lasiodiplodia anamorphs (B. rhodina (Berk. & M.A. Curtis) Arx and L. gonubiensis Pavlic, Slippers & M.J. Wingf.) appear as a highly supported and distinct clade at the base of the ingroup. In phylogenetic terms D. cupressi is most closely related to B. tsugae, with both species forming a larger clade together with several isolates identified as B. stevensii. Morphology The two isolates received as D. pinea f. sp. cupressi (CBS 261.85 and CBS 168.87) sporulated well in culture. Conidia were of the type associated with Diplodia mutila. Thus, they were hyaline, thick-walled and aseptate with both ends rounded, or with a truncate base. Morphology in culture corresponded with the characteristics of a specimen lodged by Z. Solel in IMI 303475. These specimens and cultures differed from D. pinea in which the conidia are brown and larger. Conidia of the isolates from Cupressus are somewhat wider than typical for D. mutila. Since the isolates from Cupressus are phylogenetically and morphologically distinct, it is described here as a new species in Diplodia. 7 D. pinea C CBS109725 98 1.00 D. pinea C CBS109943 59 0.96 D. pinea A CBS393.84 90 0.98 D. pinea A CBS109727 D. scrobiculata CBS109944 100 100 1.00 D. scrobiculata CBS113423 1.00 B. obtusa CBS112555 98 1.00 B. obtusa CBS119049 B. stevensii CBS112553 100 1.00 60 0.50 88 B. stevensii CBS230.30 0.98 86 1.00 100 1.00 B. stevensii JL375 B. stevensii CMW7060 B. stevensii CBS112554 100 71 1.00 0.98 D. cupressi CBS168.87 100 1.00 90 D. cupressi CBS261.85 1.00 B. tsugae CBS418.64 100 B. corticola CBS112547 1.00 100 1.00 B. corticola CBS112549 B. rhodina CAA006 100 1.00 100 B. rhodina CBS124.13 1.00 L. gonubiensis CBS115812 B. lutea CBS110299 B. ribis CBS115475 10 Fig. 1. Single most parsimonious tree resulting from combined ITS, EF1-α and β-tubulin sequence data. Bootstrap support values from 1000 replications are shown above the nodes with pooled posterior probabilities from three independent Bayesian analyses below the nodes. The tree was rooted to B. lutea and B. ribis. The bar represents 10 changes. 8 Fungal Diversity Diplodia cupressi A.J.L. Phillips & A. Alves, sp. nov. Mycobank: MB510137 (Figs 2-10) Etymology: Named for the host genus it was first reported on, namely Cupressus. Conidiomata in contextu hospitis inclusa, solitaria, stromatiformia, globosa, usque 300 µm diametro. Cellulae conidiogenae 12.5-20 × 4-4.5 µm, holoblasticae, hyalinae, subcylindricae, percurrenter cum 1-4 proliferationibus prolificentes, vel in plano eodem periclinaliter incrassatae. Conidia 23.5-28.5 × 13.5-15.0 µm, hyalinae, unicellulares, parietibus crassis, ovoidea, apicibus obtuse rotundato, in fundo obtuse rotundato, cum aetas colorescentia uno cum septo. Microconidiophorae cylindricae, hyalinae usque 10 µm longae, 2.5-3 µm latae. Cellulae microconidiogenae 10-14 × 2-2.5 µm, cylindricae, hyalinae, holoblasticae, phialidibus typicus periclinater spissescentibus. Microconidia hyalinae, unicellulares, 4-5 × 1.5 µm. Conidiomata up to 300 µm diam., solitary, separate, uniloculate, dark brown to black, globose, ostiolate, wall composed of thick-walled textura angularis, becoming thin-walled and hyaline toward the inner region. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 12.5-20 × 4-4.5 µm, hyaline, smooth, holoblastic forming conidia at their tips, proliferating internally giving rise to periclinal thickenings or proliferating percurrently with 1-4 close or widely spaced annellations, formed from the inner wall of the pycnidium. Conidia (21.5–)23.5–28.5(–30.5) × (12.0–)13.5– 15.0(–16.0) µm, 95% confidence limits = 24.4–25.4 × 13.9–14.5 µm, ( x ± S.D. of 50 = 24.9 ± 1.9 × 14.2 ± 0.9 µm, L/W = 1.76 ± 0.18) thick-walled, wall up to 2 µm wide, ovoid with both ends rounded, aseptate, hyaline and remaining so for a long time, becoming brown and one-septate after discharge from the pycnidia. Microconidiophores hyaline, smooth, cylindrical, up to 10 µm long, 2.5-3 µm wide. Microconidiogenous cells discrete or integrated, hyaline, smooth, cylindrical, holoblastic or proliferating via determinate phialides with periclinal thickening, 10–14 × 2–2.5 µm. Microconidia hyaline, smooth, aseptate, rod-shaped with rounded ends, 4–5 × 1.5 µm. Teleomorph: An unknown Botryosphaeria sp. Habitat: On cankered stems of Cupressus and Juniperus species. Known distribution: Greece, Israel, Italy, Morocco, South Africa, Tunisia, USA. Material examined: ISRAEL, Bet Dagan, dried culture from cankered stems of Cupressus sempervirens, 1986, Z. Solel (HERB IMI 303475; holotype, culture ex-type CBS 168.87). Discussion The taxonomy of the fungal pathogen causing canker of cypress and juniper has been confused. Solel et al. (1987) considered it to be a forma specialis of the plurivorous pine pathogen D. pinea (= S. sapinea) and named it 9 Figs 2-10. Diplodia cupressi (from cultures ex-holotype). 2. Conidiomata formed in culture on an autoclaved pine needle. 3-6. Conidiogenous cells. 7. Microconidiogenous cells. 8. Microconidia. 9. Hyaline, aseptate, thick-walled conidia. 10. Brown, one-septate conidia. Bars: 2 = 500 µm; 3-10 = 10 µm. D. pinea f. sp. cupressi, while in the United States it was identified as B. stevensii or its anamorph D. mutila. In this paper we studied the type specimen and authentic cultures of D. pinea f. sp. cupressi in terms of morphological characteristics and nucleotide sequences of ITS, EF1-α, and β-tubulin regions. On the basis of the morphological and multigene sequence data we recognize the cypress and juniper canker pathogen as a new species and describe it as D. cupressi sp. nov. Diplodia cupressi is clearly differentiated from D. pinea and D. mutila on the basis of morphological, cultural, and molecular characters. Conidia of D. cupressi are thick-walled, smooth, initially hyaline and aseptate and remain so for a long time, ultimately becoming one-septate and dark-walled after discharge from the pycnidia. In contrast, conidia of D. pinea become dark at an early stage of development and whilst within the pycnidial cavity. Diplodia cupressi differs from D. pinea not only in the stage at which the conidia 10 Fungal Diversity become pigmented, there are some other characters that differentiate the two species. Conidia of D. pinea become septate just before germination, while in D. cupressi septation does not seem to be associated with germination. Furthermore, conidia of D. pinea are larger than those of D. cupressi with average lengths exceeding 40 µm. As discussed previously by Swart et al. (1993) and Stanosz et al. (1998) and confirmed by the data presented in this study D. cupressi differs markedly from D. pinea and cannot be considered merely as a host specialized sub-population within this species. The thick-walled conidia that remain hyaline for a long time are characteristic of Diplodia as typified by D. mutila (Alves et al., 2004, Phillips et al., 2005). In this respect, D. cupressi closely resembles D. mutila. However, the conidia of D. cupressi are wider than typical of D. mutila (Alves et al., 2004) and correspond well with those described by Solel et al. (1987) in the original description of this fungus. They also correspond to the description of the fungus Tisserat et al. (1988) referred to as B. stevensii. Since they are larger than in the type of D. mutila and in the anamorph associated with the type of B. stevensii (Alves et al., 2004) we conclude that this fungus is not B. stevensii (D. mutila). This is supported by previous observations from Tisserat et al. (1988) who reported that several differences existed between D. cupressi and isolates obtained from apple and identified as B. stevensii. Phylogenetic analyses of separate and combined nucleotide sequence data from three genes (ITS, EF1-α, and β-tubulin) place D. cupressi within the Clade 1 of the Botryosphaeriaceae (Crous et al., 2006), but clearly separate it from all other Diplodia species. In phylogenetic terms D. cupressi is most closely related to B. tsugae, a species occurring on Tsuga heterophylla and known only from British Columbia (Funk, 1964), than to B. stevensii. Morphologically D. cupressi is easily separated from the anamorph of B. tsugae, which has much larger conidia (see Table 2). Although no teleomorph has been described for this species it apparently does exist and is clearly a member of the genus Botryosphaeria. The teleomorph was reported by Tisserat et al. (1988) on dead branches of J. scopulorum and at the time identified as B. stevensii. However, no specimens of the teleomorph could be traced (Tisserat pers. comm.). Tisserat et al. (1988) established the connection between the teleomorphic and anamorphic states by culture of ascospores. According to these authors pseudothecia were immersed in the host, asci measured 120 × 16 µm; ascospores were hyaline, smooth, thick walled, elliptical to ovate, and 32-40(37) × 12-16(14) µm. Although dimensions of the asci fall within the range of the type of B. stevensii, the ascospores are much larger than were reported for the type of B. stevensii, which average 31.5 11 Table 2. Botryosphaeria and Diplodia species associated with gymnosperms. Teleomorph Anamorph B. laricis A form of Macrophoma (Wehm.) Arx sapinea & E. Müll. B. tsugae A. Diplodia sp. Funk Botryosphaeria D. cupressi A.J.L. Phillips sp. & A. Alves “B. stevensii” “D. mutila” Unknown D. pinea (Desm.) J. Kickx f. Unknown D. scrobiculata J. de Wet et al. Unknown D. cyparissa Cooke & Harkn. Unknown D. juniperi Westend. Unknown D. kansensis Ellis & Everh. Unknown D. thujae Sacc. Unknown D. thujae G.H. Otth Unknown D. thujae Westend. Unknown D. thujana Peck & Clinton Unknown D. thyoidea Cooke & Ellis Unknown D. virginiana Cooke & Ravenel Unknown D. megalospora Berk. & M.A. Curtis Unknown D. conigena Desm. Conidia Colour/septation Size (µm) dark, aseptate 17.5-44.1 × 11.2-21 hyaline, aseptate hyaline, aseptate hyaline, aseptate dark, aseptate dark, 1-3-septate hyaline, aseptate dark, 1-septate dark, 1-septate Unknown dark, 1-septate dark, 1-septate dark dark dark, 1-septate Unknown dark, 1-septate References Smerlis (1970) 36-41 × 18-22 Funk (1964) this work 23.5-28.5 × 13.5-15 23-32 × 12-15 Tisserat et al. (1988) 30-45 × 10-16 Punithalingam and Waterston (1970) de Wet et al. (2003) 37.5-41.5 × 13-15.5 Saccardo (1884) 20-22 × 9 18-20 × 8-10 Saccardo (1884) 20-27 × 12-15 Saccardo (1895) 20-25 × 10 20 × 9 18-20 × 9-10 18-23 25-28 × 12-13 20-25 × 10 37 × 12 Saccardo (1884) Saccardo (1895) Saccardo (1899) Saccardo (1884) Saccardo (1884) Saccardo (1884) Saccardo (1884) 26-30 × 12-15 Saccardo (1884) ± 2.3 × 11.4 ± 0.9 µm (Alves et al., 2004). Also, ascospores of B. stevensii are fusiform, widest in the middle, both ends obtuse, hyaline, thin-walled, smooth, aseptate, rarely becoming pale brown and 1- or 2-septate with age. It is thus apparent that the ascomycete reported by Tisserat et al. (1988) represents a previously undescribed species for which no name is provided due to the lack of a suitable specimen. It can be distinguished from its closest phylogenetic relative, B. tsugae, whose asci and ascospores are larger. Many Botryosphaeria and Diplodia species have been associated with Gymnosperms (Table 2). Cultures are lacking for most of these species thus making appropriate comparisons impossible. However, it can be seen from the data retrieved from the literature and summarized in Table 2 that none of the available names is suitable for D. cupressi. 12 Fungal Diversity Due to the close morphological resemblance D. cupressi has in the past been identified as D. mutila (Tisserat et al., 1988; Flynn and Gleason, 1993; Stanosz and Moorman, 1997). For this reason we included in this study several isolates obtained from different hosts and whose morphological features support their identification as B. stevensii or its anamorph D. mutila. These cultures differed phylogenetically from D. cupressi and grouped in a highly supported clade. Although closely related phylogenetically, some variation can be seen between these cultures in terms of nucleotide sequence from the three genes analysed (ITS, EF1-α, and β-tubulin). This variability falls within the range that can be found between different species in the genus Botryosphaeria and so these isolates probably represent a complex of cryptic species that needs to be resolved. Acknowledgements This work was financed by Fundação para a Ciência e a Tecnologia (FCT) under project Project POCTI/AGR/56140/2004 Artur Alves was supported by grant number SFRH/BD/10389/2002 from FCT. The curator of IMI is thanked for the loan of IMI 303475. References Alfaro, M.E., Zoller, S. and Lutzoni, F. (2003). Bayes or Bootstrap? A simulation study comparing the performance of Bayesian Markov Chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Molecular Biology and Evolution 20: 255–266. Alves, A., Correia, A., Luque, J. and Phillips, A.J.L. (2004). Botryosphaeria corticola sp. nov. on Quercus species, with notes and description of Botryosphaeria stevensii and its anamorph Diplodia mutila. Mycologia 96: 598-613. 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