Mycol. Res. 107 (5): 557–566 (May 2003). f The British Mycological Society 557 DOI: 10.1017/S0953756203007706 Printed in the United Kingdom. Multiple gene genealogies and microsatellite markers reflect relationships between morphotypes of Sphaeropsis sapinea and distinguish a new species of Diplodia Juanita de WET1, Treena BURGESS3#, Bernard SLIPPERS1, Oliver PREISIG3, Brenda D. WINGFIELD2 and Michael J. WINGFIELD3* 1 Department of Microbiology and Plant Pathology, Forestry Agricultural Biotechnology Institute (FABI ), Tree Pathology Co-operative Programme (TPCP), University of Pretoria, South Africa. 2 Department of Genetics, Forestry Agricultural Biotechnology Institute (FABI ), Tree Pathology Co-operative Programme (TPCP), University of Pretoria, South Africa. 3 Forestry Agricultural Biotechnology Institute (FABI ), Tree Pathology Co-operative Programme (TPCP), University of Pretoria, South Africa. E-mail : mike.wingfield@fabi.up.ac.za Received 9 October 2002; accepted 5 March 2003. Sphaeropsis sapinea is an opportunistic pathogen causing serious damage to conifers, pre-disposed by adverse environmental conditions or mechanical damage. Three different morphological forms of the fungus have been described and are commonly referred to as the A, B and C morphotypes. Isolates of the different morphotypes have also been separated based on differences in pathogenicity and molecular characteristics. These differences, however, overlap and have not been considered sufficiently robust to justify the description of separate taxa. The aim of this study was to consider relationships between isolates representing different S. sapinea morphotypes, using multiple gene genealogies inferred from partial sequences of six protein-coding genes and six microsatellite loci. Genealogies generated for the protein-coding genes and microsatellite loci were not congruent but both consistently grouped isolates representing the A and C morphotypes in separate but closely related clades. In contrast, isolates of the B morphotype grouped together in a clade that was equally different to the A and C morphotypes as it was to the clade encompassing isolates of Botryosphaeria obtusa. These results provide strong evidence to show that the B morphotype isolates are distantly related to S. sapinea and represent a discrete taxon, which we describe here as Diplodia scrobiculata sp. nov. INTRODUCTION Sphaeropsis sapinea (syn. Diplodia pinea) is a latent, opportunistic pathogen of conifers occurring world-wide (Eldridge 1961, Swart & Wingfield 1991). It can have devastating effects on trees when it is associated with stress-inducing factors such as drought, hail, adverse temperatures or mechanical wounding (Purnell 1957, Chou 1987). S. sapinea causes extensive losses in commercial plantation forestry, especially where susceptible Pinus spp. are intensively propagated (Zwolinski, Swart & Wingfield 1990). Three distinct morphotypes (A, B and C) have been described for S. sapinea. The A morphotype is characterised by fluffy mycelium and smooth conidial walls, while the B morphotype has mycelium appressed to the surface of the agar and pitted conidial walls (Wang et al. 1985, Wang, Blanchette & Palmer 1986, Palmer, * Corresponding author. # Present address: School of Biological Sciences and Biotechnology, Murdoch University, Perth, Western Australia. Stewart & Wingfield 1987). C morphotype isolates have fluffy mycelium and smooth conidial walls similar to the A morphotype, but the conidia are significantly longer in the C morphotype (de Wet et al. 2000). Isolates of the C morphotype are also considerably more pathogenic than those of the A morphotype (de Wet et al. 2002). An I morphotype of S. sapinea was described as being intermediate between the A and B morphotypes (Hausner et al. 1999), but subsequent studies based on SSR markers (Burgess, Wingfield & Wingfield 2001a) showed that this fungus represents the anamorph state of Botryosphaeria obtusa. The authenticity of the morphotypes of S. sapinea has been confirmed using DNA-based techniques, such as randomly amplified polymorphic DNA (RAPDs) (Smith & Stanosz 1995, de Wet et al. 2000), restriction fragment length polymorphisms (RFLPs) (Hausner et al. 1999) and DNA sequences of the rRNA operon (de Wet et al. 2000). More recently, inter simple or short sequence repeat (ISSR) fingerprinting and simple sequence repeat (SSR) markers have also been used to A new species of Diplodia 558 Table 1. Isolates used in this study. Other collection numbersc Reference Isolatesa Typeb Origin Host Collector CMW8225 CMW190 CMW4885 CMW4876 CMW5870 (PREM57463) CMW8228 (PREM57464) CMW4898 (PREM57465) CMW4900 (PREM57466) CMW189 (PREM57461) *CMW4334 (PREM57462) ^ CMW8753 CMW8230 CMW8231 CMW8232 *CMW8233 ^ CMW4891 A A C C B Australia USA Indonesia Indonesia CA, USA P. radiata P. resinosa P. patula P. patula P. radiata T. Burgess M. A. Palmer M. J. Wingfield M. J. Wingfield T. Gordon B CA, USA P. radiata T. Gordon B Mexico P. greggii M. J. Wingfield B Mexico P. greggii M. J. Wingfield B USA P. banksiana M. A. Palmer 124 Palmer et al. (1987) B USA P. resinosa G. R. Stanosz 474 Blodgett & Stanosz (1999)* B B. obtusa B. obtusa B. obtusa B. obtusa Lasiodiplodia theobromae Italy Canada Canada South Africa South Africa Indonesia Pinus. sp. Picea glauca P. banksiana Malus domestica M. domestica P. patula L. Sparapano J. Reid J. Reid W. A. Smit W. A. Smit M. J. Wingfield 97-73 920729 810704 Stanosz et al. (1999) Hausner et al. (1999) Hausner et al. (1999) a Isolates marked (*) were included only in the microsatellite genealogy and those marked (^) were included only in the protein-coding gene genealogy. b Morphotype designation is based on descriptions provided by Palmer et al. (1987) and de Wet et al. (2000). c Isolation numbers used in previous studies for which references are provided in the last column. provide increased resolution to the differentiation between these morphotypes (Zhou, Smith & Stanosz 2001, Burgess et al. 2001a). These techniques alone, however, are not always informative when comparing closely related species or elements of the same species. This weakness can be resolved by using genealogies inferred from multiple protein-coding genes (Taylor et al. 2000) combined with highly polymorphic microsatellite loci (Geiser, Pitt & Taylor 1998, Fisher et al. 2000, Koufopanou et al. 2001, Steenkamp et al. 2002). In this study, our aim was to construct multiple gene genealogies from partial sequences of six protein-coding genes (Bt2 of b-tubulin, chitin synthase (CHS), elongation factor 1a (EF-1a), actin (ACT), calmodulin (CAL) and glutaraldehyde-6-phosphate (GPD)), and six microsatellite loci (SS5, SS7, SS8, SS9, SS10 and SS11) to elucidate the phylogenetic relationships between isolates of S. sapinea representing the different morphotypes. MATERIALS AND METHODS Fungal isolates Eleven Sphaeropsis sapinea isolates (Table 1) from the USA, Australia, Mexico, California, Italy, and Indonesia were used in this study. These isolates represented all three morphotypes described for S. sapinea. Four isolates of the closely-related Botryosphaeria obtusa, were included for comparison, and Lasiodiplodia theobromae (syn. B. rhodina) was used as an outgroup taxon (Table 1). The S. sapinea isoalte from south-western Australia was obtained by direct isolation from the pith tissue of P. radiata cones, and those from Mexico from P. greggii cones. The Indonesian and Californian isolates were obtained from pycnidia on P. patula or P. radiata shoots, with die-back symptoms. Single conidial cultures were generated for all the isolates and cultured on 2 % Malt Extract Agar (MEA) (2 % m/v Biolab malt extract ; 2% m/v Biolab agar) in Petri dishes at 25 xC. All the single conidial cultures were transferred to 2 % MEA slants in McCartney bottles and stored at 4 x. All isolates are maintained in the Culture Collection of the Tree Pathology Co-operative Programme (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa. Representative isolates have also been deposited in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, and the National Collection of Fungi (PREM), Pretoria. DNA extractions The single conidial isolates (Table 1) were grown in liquid ME medium in 1.5 ml Eppendorf tubes for one week at 25 x. After centrifugation, the mycelium pellet was freeze dried and DNA was extracted using the technique described by Raeder & Broda (1985). The DNA concentrations of the samples were determined against a standard molecular marker and diluted to 5 ng mlx1 for further studies. J. de Wet and others Amplification of partial protein-coding genes and microsatellite loci The Bt2 regions of the b-tubulin gene (Glass & Donaldson 1995), parts of five other protein-coding genes (Carbone & Kohn 1999) and six microsatellite loci (Burgess et al. 2001a) were amplified from 14 isolates (Table 1). The 25 ml reaction mixture consisted of 2.5 ml Expand PCR buffer (2 mM Tris-HCl, pH 7.5 ; 1.5 mM MgCl2 ; 10 mM KCl), 100 mM of each dNTP, 300 nM of each primer, 2 ng DNA template and 0.25 U Expand HighTM Fidelity Taq polymerase (Roche Biochemicals). The following temperature profile was followed : 2 min at 94 x, 10 cycles of 30 s at 94 x, 45 s at 60 x and 1 min at 72 x, the last three temperature intervals were repeated for another 30 cycles with a 5 s increase per cycle for the elongation step at 72 x. Sequencing PCR products were visualised on a 1 % agarose gel containing ethidium bromide using UV illumination. The PCR products were purified using the Roche High Pure PCR product purification kit (Roche Diagnostics). Both DNA strands were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit and an ABI PrismTM 377 DNA sequencer (Applied Biosystems, Warrington). All the reactions were done using protocols recommended by the manufacturers. Sequence data for all the isolates (Table 1) were processed using Sequence Navigator version 1.0.1 (Perkin Elmer) and manually aligned. Phylogenetic analyses Parsimony and distance analyses were performed on the individual data sets, as well as the combined data sets after partition homogeneity tests were performed on the individual protein-coding gene and microsatellite sequences using PAUP (Swofford 2002). A partition homogeneity test was also performed to test whether the protein-coding and microsatellite genealogies could be combined (Farris et al. 1994, Huelsenbeck, Bull & Cunningham 1996). In all cases, parsimony analyses were based on a strict heuristic search with a treebisection reconnection (TBR) branch swapping algorithm, stepwise addition and collapse of branches if maximum lengths was zero. Bootstrap values were determined after 103 replications and only groups with frequencies >50 % were retained. Distances were determined using ‘neighbour-joining ’ with an uncorrected ‘p ’ parameter. RESULTS Amplification and sequencing of protein-coding genes and microsatellite loci Portions of six protein-coding genes and six microsatellite loci were successfully amplified from Sphaeropsis 559 sapinea and Botryosphaeria obtusa isolates, while only protein-coding gene regions could be amplified from B. rhodina isolates. Sequences generated from the amplification products ranged from 170 to 565 bp in length. Introns occurring in the partial gene sequences of Bt2 of b-tubulin, EF-1a, ACT, CAL and GPD and the sequences flanking the microsatellites were included in the phylogenetic analyses. Phylogenetic analyses Neighbour-joining distance phylograms were generated for each of the six protein-coding genes with bootstrap values (Fig. 1). The partition homogeneity test showed that no significant conflict exists between the phylogenies of the individual protein-coding genes (P=0.01). The individual sequences were consequently combined into one data set containing 2272 characters, of which 62 variable characters were parsimony informative, 238 were parsimony uninformative and the remainder were constant. Neighbour-joining distance phylograms were also generated for each of the six microsatellite loci (Fig. 2). The partition homogeneity test on these data also showed that no significant conflict exists between the individual microsatellite phylogenies (P=0.01). They were thus combined into one data set containing 1783 characters, of which 146 variable characters were parsimony informative, 263 were parsimony uninformative and the remainder were constant. The partition homogeneity test showed that significant conflict exists between the combined microsatellite and the combined protein-coding gene phylogenies (P=0.26) and that they could not be combined. Three distinct clades with bootstrap values higher than 50 % emerged from the combined neighbour-joining distance phylogram, generated from the protein-coding gene sequences, as well as the microsatellite sequences (Fig. 3). One clade included all the A and C morphotype isolates of S. sapinea. These isolates were closely related but clearly distinguishable from each other. A second clade contained all of the B morphotype isolates. A third clade contained B. obtusa isolates together with isolates (CMW8230 and CMW8231), previously described as the I morphotype of S. sapinea (Hausner et al. 1999) and now known to represent B. obtusa (Burgess et al. 2001a). The clade containing the B morphotype isolates was equally distant from the clade encompassing the A and C morphotype isolates as it was from that including isolates of B. obtusa. High levels of sequence similarity were observed for S. sapinea isolates representing the A and C morphotypes and no correlation to geographical distribution was observed for them. Isolates of the B morphotype encompassed a high degree of genetic diversity. Although a more thorough study with more isolates from a wider geographical distribution needs to be done, groupings of isolates according to geographical origin were starting to emerge. Based on the combined A new species of Diplodia (a) 560 CMW8225 CMW190 1 of 24 MP trees CI = 0.95 RI = 0.92 RC = 0.88 1 CMW189 CMW8753 (b) CMW190 CMW4885 1 of 100 MP trees CI = 0.82 RI = 0.85 RC = 0.70 1 CMW4876 CMW8225 CMW8228 CMW8753 2 CMW5870 CMW8230 CMW4898 3 CMW8231 CMW4900 CMW8232 CMW8232 CMW189 3 CMW8230 CMW4900 CMW8231 CMW4885 2 CMW5870 CMW8228 1 CMW4876 CMW4898 CMW4891 CMW4891 0.005 changes (c) 0.01 changes 1 of 1 MP trees CI = 0.98 RI = 0.95 RC = 0.94 CMW8225 CMW190 1 CMW4885 (d) 1 of 17 MP trees CI = 0.94 RI = 0.84 RC = 0.79 1 CMW4885 CMW4876 CMW190 CMW8230 CMW8231 CMW8225 CMW4876 CMW5870 3 CMW4898 CMW8232 2 CMW189 CMW189 CMW8753 CMW8753 CMW4900 CMW5870 2 CMW8230 CMW8232 CMW8228 CMW4898 3 CMW8231 CMW4900 CMW4891 CMW4891 0.01 changes 0.01 changes (e) CMW8228 CMW8753 2 CMW4898 1 of 8 MP trees CI = 0.94 RI = 0.81 RC = 0.76 CMW4876 CMW4885 CMW190 CMW8225 1 CMW8225 CMW8232 3 CMW8230 CMW8231 CMW4900 3 CMW5870 CMW189 CMW189 2 CMW4898 2 CMW8228 CMW4900 CMW4891 0.005 changes CMW4885 1 CMW8231 CMW5870 1 of 100 MP trees CI = 0.88 RI = 0.56 RC = 0.49 CMW190 CMW4876 CMW8230 CMW8232 (f ) CMW8753 CMW4891 0.005 changes Fig. 1. Phenograms constructed for partial sequences of six protein coding genes, (a) Bt2 of the b-tubulin gene, (b) ACT, (c) EF-1a, (d) CAL, (e) CHS, (f) GPD, using neighbour-joining distance analysis with an uncorrected ‘ p ’ parameter and parsimony based on a strict heuristic search to determine bootstrap values. Bootstrap values were determined after 103 replications and only groups with frequencies >50 % were retained. Clade 1, Sphaeropsis sapinea (A and C morphotypes) ; Clade 2, Diplodia scrobiculata ; and Clade 3, Botryosphaeria obtusa. MP, most parsimonious ; CI, consistency index; RI, retension index; and RC, reconstructed consistency index. J. de Wet and others 561 (a) (b) 1 of 2 MP trees CI = 1 RI = 1 RC = 0 3 CMW8231 CMW823 CMW8228 CMW4900 CMW4898 CMW5870 CMW8232 CMW8233 1 of 9 MP trees CI = 0.96 RI = 0.97 RC = 0.04 2 CMW190 CMW4900 CMW4334 1 CMW4334 CMW8225 1 CMW189 CMW4876 CMW4898 CMW4876 CMW8225 2 CMW4885 CMW8231 CMW190 CMW4885 CMW8230 CMW5870 3 CMW8228 CMW8232 CMW8233 CMW189 (d) (c) 2 1 of 4 MP trees CI = 0.97 RI = 0.98 RC = 0.03 CMW5870 CMW8228 CMW4900 CMW8228 1 of 12 MP trees CI = 0.90 RI = 0.95 RC = 0.10 CMW189 CMW4334 CMW5870 CMW4898 CMW4900 1 CMW4898 1 CMW4334 2 CMW4885 3 CMW4876 CMW189 CMW8231 CMW190 CMW4885 CMW8225 3 CMW190 CMW8232 CMW8230 CMW4876 CMW8225 CMW8231 CMW8233 CMW8233 CMW8231 (e) 1 of 100 MP trees CI = 0.95 RI = 0.94 (f ) RC = 0.05 CMW8228 CMW5870 3 CMW8232 1 of 2 MP trees CI = 0.94 RI = 0.97 RC = 0.06 2 CMW5870 CMW8228 CMW189 CMW4900 3 CMW189 CMW4900 CMW8232 CMW4334 CMW4334 CMW8232 CMW4898 CMW8231 CMW4898 CMW8231 1 1 CMW8230 CMW4876 CMW4885 CMW8230 CMW8233 CMW4885 CMW8233 CMW190 CMW4876 CMW8225 CMW190 CMW8225 Fig. 2. Phenograms constructed for sequence data of six SSR loci, (a) SS5, (b) SS7, (c) SS8, (d) SS9, (e) SS10, (f) SS11, using neighbour-joining distance analysis with an uncorrected ‘ p ’ parameter and parsimony based on a strict heuristic search to determine bootstrap values. Bootstrap values were determined after 103 replications and only groups with frequencies >50 % were retained. Clade 1, Sphaeropsis sapinea (A and C morphotypes) ; Clade 2, Diplodia scrobiculata ; and Clade 3, Botryosphaeria obtusa. MP, most parsimonious ; CI, consistency index ; RI, retension index; and RC, reconstructed consistency index. A new species of Diplodia 562 (a) (b) CMW190 CMW8225 93 1 of 9 MP trees CI = 0.89 RI = 0.76 RC = 0.67 1 CMW4876 98 63 CMW4876 CMW8230 3 CMW8231 3 100 88 100 99 100 CMW8233 CMW189 CMW4900 81 CMW5870 CMW4898 CMW8228 CMW5870 2 2 CMW4900 66 CMW8231 CMW8232 100 CMW8232 85 52 CMW4885 CMW8230 66 CMW8225 1 CMW4885 65 76 CMW190 1 of 3 MP trees CI = 0.93 RI = 0.91 RC = 0.85 100 100 CMW8228 CMW189 CMW4898 88 100 CMW4334 CMW8753 CMW4891 10 changes 0.005 substitutions/site Fig. 3. (a) Phenogram constructed for combined sequences of the six protein-coding genes, (b) phenogram constructed for combined sequence data of six SSR loci using neighbour-joining distance analysis with an uncorrected ‘p ’ parameter and parsimony based on a strict heuristic search to determine bootstrap values. Bootstrap values were determined after 103 replications and only groups with frequencies >50 % were retained. Clade 1, Sphaeropsis sapinea (A and C morphotypes) ; Clade 2, Diplodia scrobiculata ; and Clade 3, Botryosphaeria obtusa. MP, most parsimonious ; CI, consistency index; RI, retension index; and RC, reconstructed consistency index. protein-coding gene genealogy (Fig. 3), the B morphotype isolates from the universal United States (Central, West and Mexico) (CMW189, CMW5870, CMW8228, CMW4898, CMW4900) grouped separately from the single European B morphotype isolate (CMW8753). Furthermore, the combined microsatellite genealogy could differentiate the B morphotype isolates from the universal United States into three sub-clades, one from the Central US (CMW189, CMW4334), one from California (CMW5870, CMW8228) and one from Mexico (CMW4898, CMW4900). TAXONOMY The results of the phylogenetic comparisons presented in this study provide robust evidence to justify treating isolates of the B morphotype of S. sapinea as a discrete taxon. We, therefore, provide the following description for the fungus : Diplodia scrobiculata J. de Wet, B. Slippers & M. J. Wingfield, sp. nov. (Figs 4–10) Etym. : Latin, scrobiculata=minutely pitted, in reference to the texture of the conidial walls. Culturae colonias supra submurinas vel murinas, infra atromurinas, marginibus sinuatis faciunt. Coloniae creverunt optime ad 25 xC, et superficiem medii in 8 diebus velabant. Mycelium atratum septatum ad agarum appressum. Conidiomata in foliis pinorum pycnidialia, mycelio obtecta. Pycnidia atro-vinaceo-brunnea in foliis pinorum vel in agaro immersa, 150 mm diam Cellulae conidiogenae holoblasticae, proliferatione percurrenti limitata, ut videtur annellationibus paucis, 10 mm diam Conidia clavata vel truncata, 1–3 septata, parietibus crassis, scrobiculatis, atrovinacea vel atrobrunnea, 39.5r14 mm. Typus: USA : Wisconsin : Jackson County, Pinus banksiana. 1987, M. A. Palmer CMW189 (PREM 57461 – holotypus). Cultures (Fig. 4) pale mouse grey (15kkkkkd) to mouse grey (15kkkkk) viewed from the top of the Petri dish, dark mouse grey (15kkkkkk) to fuscous black (13kkkkkm) viewed from the bottom of the Petri dish, colonies with sinuate edges ; optimal growth at 25 x, covering the medium surface (9 cm Petri dishes) in 8 d. Mycelium dark, septate, appressed to the agar surface. Conidiomata (Fig. 5) pycnidial, covered in mycelium, dark, immersed in pine needles or in the agar, (100–) 150 (–250) mm diam, single, papillate ostiole. Conidiogenous cells (Figs 6–7) discrete, dark, smooth, 10 mm in diameter, holoblastic with limited percurrent proliferation seen as small numbers of annellations. Conidia (Figs 8–9) clavate to truncate, dark mouse grey (15kkkkkk), (37.5–) 39.5 (–41.5) mm (13–) 14 (–15.5) mm, 1–3 septa, thick, pitted walls (Wang et al. 1985, Wang et al. 1986). Substratum : Needles of Pinus banksiana, P. resinosa, and P. greggii. Distribution : USA (Wisconsin, Minnesota, California), Mexico, and Europe (France, Italy). Other specimens examined: USA : Minnesota : Wadena County, Pinus resinosa, 1987, G. R. Stanosz CMW4334. California : Pinus radiata, 2000, T. Gordon CMW5870, CMW8228. Mexico : Pinus greggii, 1998, M. J. Wingfield CMW4898, CMW4900. [All in PREM57462, PREM57463, PREM57464, PREM57465, PREM57466.] J. de Wet and others Figs 4–9. Diplodia scrobiculata (holotype). Fig. 4. Colony characteristics on malt extract agar. Fig. 5. Section through pycnidium with conidia. Figs 6–8. Conidiophores with conidiogenous cells. Fig. 9. Conidia with up to three septa. 563 A new species of Diplodia 564 (c) (a) (b) Fig. 10. Diplodia scrobiculata (holotype). (a) Pycnidium ; (b) conidiogenous cells ; and (c) conidia. Bars (a)=100 mm ; (b)–(c)=20 mm. DISCUSSION Multiple gene genealogies constructed from six proteincoding gene regions and six microsatellite-rich loci provide robust evidence that the B morphotype isolates of Sphaeropsis sapinea represent a distinct species. We have thus provided the name Diplodia scrobiculata for this fungus. Our results also reinforce those of Zhou et al. (2001) using dominant ISSR markers, and Burgess et al. (2001a) using co-dominant SSR markers, suggesting the A and B morphotypes of S. sapinea represent distinct taxa. The construction of multiple gene genealogies, supported by distinct morphological differences, has enabled us to infer reliable and consistent phylogenetic relationships between the morphotypes of S. sapinea s. lat. We found that isolates of the A and C morphotypes are much more closely related to each other than they are to D. scrobiculata. D. scrobiculata isolates were equally distant from those of the A and C morphotypes of S. sapinea as they were from isolates of Botryosphaeria obtusa. Phylogenetic relationships inferred from these gene genealogies corroborate results obtained using SSR markers, based on sizes (Burgess et al. 2001a). Therefore, in this case SSR markers alone would have been adequate to infer species level relationships, even though initial empirical studies have suggested otherwise (Fisher et al. 2000). Botryosphaeria spp. are very similar based on their teleomorph characteristics but their anamorphs characteristics are very diverse and reside in a number of anamorphs genera. Recent morphological and molecular data suggested that these anamorphs can be divided into two main groups (Jacobs & Rehner 1988, Crous & Palm 1999, Denman et al. 2000, Zhou & Stanosz 2001). The first group have ellipsoid, thick-walled conidia that septate regularly and darken with age, and are characterised by the genus Diplodia. The second group have fusoid, thin-walled conidia that septate and darken less often with age, and are characterised by the genus Fusicoccum. The new species described here is concurrent with Diplodia group described by the above authors, and is therefore described in this genus. Based on the above evidence, we agree with the argument of Denman et al. (2000), that percurrent proliferation and time of septation of S. sapinea, that is considered characteristic, is not sufficient to separate it from the genus Diplodia. We, therefore, suggest reverting to the older name D. pinea for this taxon. No sexual state is known for any form of S. sapinea, although, together with D. scrobiculata, molecular evidence (Burgess et al. 2001a) shows that it clearly represents an anamorph of Botryosphaeria. Burgess, Wingfield & Wingfield (2001b), have also shown that the A morphotype isolates representing S. sapinea sensu stricto are overwhelmingly clonal. D. scrobiculata J. de Wet and others isolates occasionally produce spermatia-like spores (Palmer et al. 1987), suggesting the presence of a sexual state in this fungus. Recent studies using SSR markers have shown a considerably higher degree of genetic diversity amongst isolates of D. scrobiculata than those of the A and C morphotypes of S. sapinea (Burgess, unpubl.). D. scrobiculata could represent a recently derived lineage of Botryosphaeria, which has only recently lost its ability to reproduce sexually. Alternatively, sexual reproduction in this fungus may possibly be suppressed by unfavourable conditions such as those in culture and sexual structures may yet be found in nature. In contrast, we believe the A and C morphotypes of S. sapinea represent ancient lineages that have stabilised over time and have acquired a virtually clonal existence. S. sapinea, as reflected by the A and C morphotypes of this fungus, appears to be native to and widely distributed across the natural range of Pinus species. The two morphotypes that represent this species differ in their distribution, host specificity and virulence. The A morphotype is common and has a wide distribution in Southern hemisphere countries including South Africa, Australia and New Zealand, where it was probably introduced together with pine seed imports (Swart et al. 1991, Burgess & Wingfield 2001). The C morphotype of S. sapinea has, thus far, been found only on Pinus spp. in Indonesia and isolates are significantly more virulent than those of the A morphotype (de Wet et al. 2002). D. scrobiculata has a much more restricted distribution. The fungus was initially known only on Pinus banksiana and Pinus resinosa in the north central United States (Wang et al. 1985, Palmer et al. 1987), but has recently been reported from other conifers in Europe (Stanosz, Swart & Smith 1999). There is no conclusive evidence to show that is has been introduced into pinegrowing areas of the southern hemisphere. The wide array of phylogenetic comparisons presented in this study provide robust evidence to support the recognition of D. scrobiculata (formerly S. sapinea B morphotype) as a distinct species. This is also supported by the results of other molecular genetic comparisons (Zhou et al. 2001, Burgess et al. 2001a), as well as useful morphological and ecological data previously published (Wang et al. 1985, Palmer et al. 1987, de Wet et al. 2002). Isolates of D. scrobiculata are characterized by dark, septate mycelium appressed to the surface of the agar. This is consistently different to S. sapinea isolates that have fluffy, aerial mycelium. Conidia of D. scrobiculata are dark brown with thick, pitted walls and 1–3 septa (Wang et al. 1985, Wang et al. 1986, Palmer et al. 1987). Conidiogenous cells are holoblastic with annelidic proliferations and based on this characteristic, D. scrobiculata and S. sapinea are apparently indistinguishable. S. sapinea was one of the earliest fungi to be recognised as a common inhabitant of Pinus spp. (Fisher 1912). It is also one of the best-known pathogens of Pinus spp. grown as exotics in the tropics and southern 565 hemisphere (Burgess & Wingfield 2001). Thus, the discovery of taxonomically and ecologically meaningful differences in isolates of S. sapinea in the north central United States in the late 1980’s, was relatively recent. During the past 15 years, substantial evidence has accumulated to show that these differences reflect both inter- and intraspecies variation. 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