Redefining Ceratocystis and allied genera

The genus Ceratocystis was established in 1890 and accommodates many important fungi. These include serious plant pathogens, significant insect symbionts and agents of timber degradation that result in substantial economic losses. Virtually since its type was described from sweet potatoes, the taxonomy of Ceratocystis has been confused and vigorously debated. In recent years, particulary during the last two decades, it has become very obvious that this genus includes a wide diversity of very different fungi. These have been roughly lumped together due to their similar morphological structures that have clearly evolved through convergent evolution linked to an insect-associated ecology. As has been true for many other groups of fungi, the emergence of DNA-based sequence data and associated phylogenetic inferences, have made it possible to robustly support very distinct boundaries defined by morphological characters and ecological differences. In this study, DNA-sequence data for three carefully selected gene regions (60S, LSU, MCM7) were generated for 79 species residing in the aggregate genus Ceratocystis sensu lato and these data were subjected to rigorous phylogenetic analyses. The results made it possible to distinguish seven major groups for which generic names have been chosen and descriptions either provided or emended. The emended genera included Ceratocystis sensu stricto, Chalaropsis, Endoconidiophora, Thielaviopsis, and Ambrosiella, while two new genera, Davidsoniella and Huntiella, were described. In total, 30 new combinations have been made. This major revision of the generic boundaries in the Ceratocystidaceae will simplify future treatments and work with an important group of fungi including distantly related species illogically aggregated under a single name.


INTRODUCTION
Ceratocystis was established in 1890 to accommodate C. fimbriata, a pathogen causing black rot of sweet potatoes in the USA (Halsted 1890). The genus now includes many important fungi including important pathogens of plants and the causal agents of sap stain in timber that are symbiotic associates of insects (Fig. 1). These fungi have ascomata with round usually dark bases that are sometimes ornamented. These bases give rise to long necks terminating in ostiolar hyphae and from which ascospores exude in slimy masses (Fig. 2). All species have ascospores surrounded by sheaths, which can be hat-shaped, ellipsoidal or obovoid and that are either evenly or unevenly distributed around the spores (Fig. 3). The asexual states of most including the type species of Ceratocystis, C. fimbriata. These studies and others (Bakshi 1951, Moreau 1952 resulted in a long-standing confusion between the two genera. This is largely because the genera have morphologically similar ascomata featuring globose bases and generally long necks from which ascospores exude in slimy masses ). According to Malloch & Blackwell (1993) the basic construction of the ascomata may be the result of an adaptation to insect-associated niches and shows the convergent evolution of fruiting structures that facilitate insect-borne transport of spores to new environments (Malloch & Blackwell 1993). Interestingly, but adding to the confusion between them, species of both Ceratocystis and Ophiostoma have evanescent asci that are seldom seen. Ascospores were confused with conidia when the genera were first discovered. The fact that both genera include species with hatshaped ascospores re-inforced debate over their relationships for many years (Van Wyk et al. 1993).
The taxonomic confusion between Ceratocystis and Ophiostoma was finally resolved once DNA sequence data became available to provide phylogenetic insights into their relatedness. Hausner et al. (1993a,b) and Spatafora & Blackwell (1994) provided the first phylogenetic trees showing that these genera are unrelated. A considerable body of evidence has contributed to the current understanding that Ophiostoma resides in the Ophiostomatales in the Sordariomycetidae and that Ceratocystis is accommodated in the Ceratocystidaceae (Microascales) in the Hypocreomycetidae (R eblov a et al. 2011, De Beer et al. 2013a. Importantly, resolution of the taxonomic confusion regarding these genera has made it possible to study them independently and thus to better understand their similarities, but also their many very different ecologies (Seifert et al. 2013).
Once Ceratocystis was clearly recognised as unrelated to Ophiostoma, an increasingly clear picture emerged of a genus that included species that were morphologically and ecologically very distinct from one another. These differences have been substantially amplified by the discovery of many new and often cryptic species, revealed through DNA-sequence comparisons (Wingfield et al. 1996, Witthuhn et al. 1998. For example, perhaps the two best-known species names within Ceratocystis, C. fimbriata and C. moniliformis, are now known to represent complexes of many different species . Recognition of these complexes has made it possible to interpret their very clear differences. Wingfield et al. (2013) provided the first intensive, phylogenetically based reconsideration of the taxonomy of Ceratocystis. This study included all available sequence data up to 2006 when the study was completed, and it clearly exposed five very different taxonomic groups. These included the species of the C. fimbriata complex, the C. moniliformis complex, and the C. coerulescens complex, as well the Thielaviopsis and Ambrosiella complexes, known only by their asexual states. Importantly, species in these complexes could easily be separated by their morphological and ecological differences. The DNA sequence data used merely reaffirmed the circumscription of the groups. Wingfield et al. (2013) provided substantial evidence that species in Ceratocystis s. l. should be assigned to discrete genera. They argued that this would substantially reduce taxonomic confusion among these very different groups of fungi and importantly, also enhance understanding of their different ecologies. Wingfield et al. (2013) were not able to place all species of Ceratocystis s. l. in discrete complexes. Some, such as C. paradoxa, C. adiposa and C. fagacearum fell away from all clearly defined species groups. In retrospect, it appears that this problem stemmed from a lack of sampling and was resolved by the discovery of additional species that could define complexes based on these isolated phylogenetic branches. Such a pattern has become clearly evident from a recent study of a large collection of isolates that would previously have been identified as C. paradoxa (Mbenoun et al. 2014a). These isolates have now been shown to represent a number of very different but related species that are now recognised as comprising the C. paradoxa complex. It is, therefore, very likely that other complexes will emerge in Ceratocystis s. l., as new species are collected and treated in the future.
Ceratocystis s. l., as it is currently defined includes many ecologically important fungi (Fig. 1). For example, most species in the C. fimbriata complex are important and in some cases devastating plant pathogens (Kile 1993. These include C. albifundus, a virulent pathogen of Acacia mearnsii in Africa (Roux & Wingfield 2013), C. cacaofunesta, a pathogen of cacao in South America (Engelbrecht et al. 2007), C. platani, an invasive alien pathogen of Platanus trees in Europe (Gibbs 1981, Ocasio-Morales et al. 2007), and C. manginecans that has devasted mango (Mangifera indica) and Acacia mangium trees in the Middle East and south-east Asia respectively (Van Wyk et al. 2007, Tarigan et al. 2011. Species in the C. coerulescens complex include associates of bark beetles (Coleoptera: Scolytinae) as well as important causal agents of sap-stain in timber (Seifert 1993. The Thielaviopsis complex includes plant pathogens, while the Ambrosiella complex comprise obligate associates of ambrosia beetles (Coleoptera: Scolytinae) (Batra 1967, Kile 1993. Species in the C. moniliformis complex are mostly wound-inhabiting saprobes or mild pathogens, often causing sap stain in timber (Hedgcock 1906, Seifert 1993. The members of the C. paradoxa complex are all pathogens of monocotyledonous plants, including pineapples and palms (Mitchell 1937, Alvarez et al. 2012, Mbenoun et al. 2014a).
All available evidence shows that Ceratocystis s. l. represents a suite of morphologically, phylogenetically and ecologically different fungi. There is no reasonable argument for retaining them in a unitary genus, and indeed, doing so would result only in confusion arising from a diminished lack of appreciation of their dramatic differences. Placing them in discrete genera will enhance the perception of opportunities to understand these organisms and, where applicable, to manage or conserve them. It will provide an improved interpretive framework for analysing Fig. 2. Morphological features of the ascomata of species of Ceratocystis s.l. A, B. Ascomata of C. albifundus and C. fimbriata respectively, on woody substrates with masses of ascospores emerging from their necks. C-E. Ascomata showing different morphological features such as light-coloured bases of C. albifundus (CMW4059), pear-shaped ascomatal bases characteristic of C. pirilliformis (CMW6579), ornamented bases and divergent necks of C. cerberus (now T. cerberus) (CMW 36668). F, G. Apices of ascomata showing a range of forms of ostiolar hyphae such as long, divergent ostiolar hyphae of C. ethacetica (CMW 36671) (now T. ethacetica) and short, convergent ostiolar hyphae of C. inquinans (now H. inquinans) (CMW 21106). H, I. Hat-shaped ascospores being released from ostiolar hyphae in C. sumatrana (now H. sumatrana) (CMW 21113) and C. pirilliformis (CMW 6670). J. Bases of ascomata in the C. moniliformis s.l. complex (now Huntiella) with distinct plates at the bases of the ascomatal necks, and (K, L) spine-like ornamentations of H. microbasis (CMW 21117) and H. oblonga (CMW 23803) respectively. M. Digitate ornamentations on the ascomatal bases in species residing in C. paradoxa s.l. (now Thielaviopsis) (CMW 36642).  Kile et al. 1996], hat-shaped (e.g. C. fimbriata, CMW 15049), oblong (e.g. C. paradoxa, now T. paradoxa, CMW 36642) and obovoid (e.g. C. laricicola, now E. laricicola, CMW 20928). E-H. Simple tubular conidiophores commonly tapering to their apicies, and found in most species of Ceratocystis s.l. E. Flasked-shaped phialidic conidiophores of T. paradoxa (CMW 36642) releasing obovoid secondary conidia. F. Phialide releasing cylindrical conidia of C. pirilliformis (CMW 6670). G. Chlamydospore of T. basicola (CMW 7068) and H. C. pirilliformis (CMW 6670). I-L. Darkly pigmented, thick-walled aleurioconidia of (I) T. paradoxa ( REDEFINING CERATOCYSTIS AND ALLIED GENERA www.studiesinmycology.org the ecological differences among the species, such as differences in pathogenicity and insect associations, particularly when complete genome sequences become available for these fungi, as they have recently done for C. fimbriata s. str., C. moniliformis s. str. and C. manginecans (Wilken et al. 2013, Van der Nest et al. 2014).
Revising Ceratocystis s. l. and providing genera to accommodate the well-defined groups in this aggregate genus must be done in conformity with the principles of the new International Code for algae, fungi and plants (Melbourne Code) adopted at the 18 th International Botanical Congress (McNeill et al. 2012). Importantly, this must reflect the One Fungus One Name (1F1N) principles that originally emerged from the Amsterdam Declaration  ) and subsequent discussions (Hawksworth 2011, Norvell 2011. In this regard, De Beer et al. (2013b) listed six genus names as possible synonyms of Ceratocystis s. l. One of these names belongs to a sexual genus Endoconidiophora, originally described for E. coerulescens (Münch 1907). The five other names were all considered to denote asexual genera under the dual nomenclature system: they included Thielaviopsis (Went 1893, type species T. ethacetica), Chalaropsis (Peyronel 1916, type species Ch. thielavioides), Hughesiella (Batista & Vital 1956, type species Hu. euricoi), Ambrosiella (Von Arx & Hennebert 1965, type species A. xylebori), and Phialophoropsis (Batra 1967, type species Ph. trypodendri). These names are available for new generic circumscriptions accommodating groups currently residing in Ceratocystis s. l.
The major aim of this study was to revise the generic boundaries for species currently accommodated in Ceratocystis s. l. This task involved obtaining material from as many species as possible and applying 1F1N principles. Generating the full genome sequences for 19 species including representatives of all the phylogenetic groups in Ceratocystis s. l. provided the opportunity to screen multiple gene regions to address genuslevel questions. In addition, gene regions from the AFTOL project (Lutzoni et al. 2004, Hibbett et al. 2007, the ITS barcoding initiative (Schoch et al. 2012), as well as additional barcoding genes from an ongoing project at CBS (Stielow et al. 2014) were used to design Microascales-specific primers and to select the most appropriate gene regions to clearly resolve generic boundaries for Ceratocystis s. l.

Cultures
All cultures used in this study were obtained from the Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa (CMW) and Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands (CBS). Single spore or single hyphal-tip cultures were prepared and maintained on 2 % Malt Extract Agar (MEA). A list of isolates used in this study is presented in Table 1.

DNA extraction
Single spore/single hyphal-tip cultures were inoculated in YM broth (2 % malt extract, 0.2 % yeast extract) and incubated at 25°C with shaking for 2-5 d. Mycelium was harvested and freeze-dried in 2 mL Eppendorf tubes. The freeze-dried mycelium was submerged in liquid nitrogen, followed by pulverising the mycelium with a pipette tip. About 10 mg of mycelial "powder" was used for DNA extraction using PrepMan Ultra Sample Preparation reagent (Applied Biosystems, Foster City, California) as described in Duong et al. (2012).

Selection of gene regions and primers
Ten different gene regions [the nuclear ribosomal DNA large subunit (LSU), the nuclear ribosomal DNA small subunit (SSU), nuclear ribosomal DNA internal transcribed spacer regions (ITS), the 60S ribosomal protein RPL10 (60S), beta-tubulin (BT), translation elongation factor 1-alpha (EF1), translation elongation factor 3-alpha (EF3), mini-chromosome maintenance complex component 7 (MCM7), the RNA polymerase II largest subunit (RPB1), and the RNA polymerase II second largest subunit (RPB2)] were extracted from 19 Ceratocystis draft genome sequences that included species from all the major clades. The genome sequences, of which three have been published (Wilken et al. 2013, Van der Nest et al. 2014, are available at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria. Phylogenetic analyses were conducted with all ten gene regions (data not shown). LSU, 60S, and MCM7 were selected as candidate genes for further investigation including all the isolates in the study, based on their level of support at the basal nodes, the ease of amplification and sequencing, and the popularity of their use in studies of other fungal lineages.
The ITS region has been widely used in phylogenetic studies to distinguish between species in Ceratocystis. However, due to the recent discovery of multiple ITS forms in certain species of Ceratocystis (Al Adawi et al. 2013, Naidoo et al. 2013, and the fact that gene regions were chosen that were slightly more conserved to resolve the genus level questions, the ITS was intentionally not used in the present study. Primers LR0R and LR5 (Vilgalys & Hester, 1990) were used in PCR amplification and sequencing of LSU. Primers Algr52_412-433_f1 and Algr52_1102_1084_r1 (Stielow et al. 2014) were used for PCR amplification and sequencing of 60S. Based on the sequences obtained from genomes, new primers Cer-MCM7F (ACICGIGTITCIGAYGTNAAGCC) and Cer-MCM7R (TTRGCAACACCAGGRTCACCCAT) were designed and used in PCR amplification and sequencing of MCM7.

PCR and sequencing
All PCR reactions were done in a total volume of 25 μL. The reaction mixture consisted of 2.5 μL of 10X PCR reaction buffer, 2.5 mM MgCl 2 , 200 μM of each dNTP, 0.2 μM of each of the forward and reverse primers for LSU (1 μM of each primer in case of degenerate primers for 60S and MCM7), 1 U FastStart Taq DNA Polymerase (Roche) and 2 μL of genomic DNA solution. The PCR thermal conditions included an initial denaturation at 96°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 s, and 72°C for 60 s, and ended with a final extension at 72°C for 8 min. The annealing temperature was set at 55°C for all gene regions and all isolates at first. In some cases where the PCR failed or non-specific amplification was observed, we experimented with different annealing  temperatures (between 52°C and 60°C) until successful amplification was obtained. Direct sequencing of PCR products was done using BigDye ® Terminator v. 3.1 Cycle Sequencing kit (Applied Biosystems) with a 1/16 reaction and at 55°C annealing temperature for all primers. Sequencing PCR products were precipitated using the sodium acetate and ethanol precipitation protocol, followed by fragment separation using an ABI PRISM ® 3100 Genetic Analyzer (Applied Biosystems).

Phylogenetic analyses
Sequences from different gene regions were aligned using an online version of MAFFT v. 7 (Katoh & Standley 2013). The three gene regions (LSU, 60S and MCM7) were combined and analysed as a single dataset. Each of the gene regions was also analysed separately and results were compared with those of the combined analyses. Maximum parsimony (MP) analyses were performed in MEGA6 (Tamura et al. 2013) with 1000 bootstrap replications. The subtree-pruning-regrafting (SPR) algorithm was selected, and alignment gaps and missing data included. Maximum likelihood (ML) analyses were done using raxmlGUI (Silvestro & Michalak 2012) with the GTR+G+I substitution model selected. Ten parallel runs with four threads and 1000 bootstrap replications were conducted. Bayesian inference (BI) analyses were performed using MrBayes v. 3.2 (Ronquist et al. 2012) employing the GTR+G+I substitution model. Ten parallel runs, each with four chains, were conducted. Trees were sampled at every 100 th generation for 5 M generations. After sampling, 25 % of trees were discarded as a burn-in phase and posterior probabilities were calculated from all the remaining trees.

Morphology
Morphological descriptions from the protologues of all species treated in this study were carefully considered when genera were redefined. Based on these species descriptions, the most common characters of all species in a genus were selected and incorporated in the emended and new genus descriptions. Over time, different authors often used different terminology describing similar characters. We aligned the generic descriptions of the different genera with each other using similar terminology.

RESULTS
Maximum likelihood, BI and MP trees obtained from analyses of the individual gene regions (Figs 4-6) and the combined datasets (Fig. 7) of the LSU, 60S and MCM7 sequences, consistently resulted in nine well-supported major lineages. Although trees derived from individual datasets had different topologies (Figs 4-6), they were not significantly incongruent with the trees obtained from the combined analyses (Fig. 7). This was indicated by the fact that most major lineages found in the combined analyses were present in trees resulting from individual datasets.
Only few exceptions were observed in the cases of 60S and LSU datasets. In one exceptional case, the 60S dataset (Fig. 5) showed Lineage 6 as split into two clades. In another case, the LSU tree (Fig. 4)     REDEFINING CERATOCYSTIS AND ALLIED GENERA www.studiesinmycology.org close to each other. Neither of these placements, however, was supported by phylogenetic statistics. Among the three gene regions used, MCM7 proved to be the most informative and resulted in trees with topologies similar to those obtained from the combined dataset. The first of the nine lineages (Figs 4-7), representing the largest number of species, included C. fimbriata (type species of Ceratocystis) and 31 other species previously included in the C. fimbriata complex. The second lineage included CMW 22736, representing T. thielavioides (type species for Chalaropsis), T. ovoidea, and two isolates from the USA and Belgium, previously described as T. thielavioides, but clearly distinct from CMW 22736. These two isolates are thus referred to as Chalaropsis sp. 1. The third lineage included C. coerulescens, type species for Endoconidiophora, and seven species previously considered part of the C. coerulescens complex. Isolates representing C. virescens, C. eucalypti, T. australis and T. neocaledoniae represented the fourth lineage, which did not include a type species of a previously described genus. Lineage 5 was previously referred to as the C. paradoxa complex, and included C. ethacetica (type species of Thielaviopsis), C. euricoi, C. musarum, C. radicicola and the recently described species, C. cerberus. The sixth lineage was the second largest and included C. moniliformis s. str. and 17 other species, but contained no type species representing a previously described genus. Two new species that are currently being described (Mbenoun et al., unpubl. data) grouped in this lineage, and were labelled according to provisional species names provided by M. Mbenoun (unpublished), namely Huntiella chlamydospora nom. prov. and H. pycnanthi nom. prov. Isolates of Ambrosiella xylebori (type species for Ambrosiella), A. hartigii and A. beaveri formed a distinct lineage. The last two lineages comprised Knoxdaviesia and Graphium species used as outgroups in all analyses.
Five of the 79 species in Ceratocystis s. l. were not accommodated in any of the nine major lineages discussed above (Figs 4-7). Ceratocystis adiposa and C. major had identical sequences in ITS (data not shown), LSU and 60S, and formed a distinct clade that was most closely related to lineage 7 (representing Ambrosiella). Ceratocystis fagacearum and A. ferruginea, although significantly different from each other, formed a clade of their own separating them from other Ceratocystis and Ambrosiella lineages. The fifth species, T. basicola, formed a unique lineage distinct from, but related to species in lineage 2 as its closest relatives.

GENERIC DESCRIPTIONS AND NOMENCLATOR
Phylogenetic data generated in this study revealed seven wellsupported lineages in Ceratocystis s. l. The distinction between these lineages is also supported by morphological and ecological data for the species in these groups. These lineages are, therefore, treated as distinct genera. Five of the lineages incorporate the type species of earlier described genera, and we thus emend the descriptions of Ambrosiella, Ceratocystis s. str., Chalaropsis, Endoconidiophora, and Thielaviopsis, based on the types and other species accommodated in the lineages. Two lineages for which existing names are not available are treated as novel genera, described here as Davidsoniella and Huntiella. Where necessary, new combinations are provided for the names of species in these genera. Species previously treated in Ceratocystis, but excluded from the newly defined genera in the Ceratocystidaceae (Tables 2 and 3), invalidly described species (Table 4), and homonyms from kingdoms other than the Fungi (Table 5), are not treated in the nomenclator, but listed in the tables as indicated. Sexual state not known. Conidiophores phialidic, single to aggregated in sporodochia, hyaline, unbranched or sparingly branched, one-celled to septate. Conidia formed in chains or as terminal aleurioconidia.

Notes:
We followed the emended generic description for Ambrosiella by Harrington et al. (2010), who restricted the genus to those species belonging to the Microascales. DNA sequence data is not available for A. trypodendri, type species of Phialophoropsis, which means the synonymy of the latter genus with Ambrosiella cannot be confirmed for the present. All known Ambrosiella species are associates of ambrosia beetles. Notes: Sexual state unknown. Ambrosiella trypodendri is the type species of Phialophoropsis (Batra 1967). No cultures are available for this species. However, Harrington et al. (2010) argued that it is morphologically similar to Ambrosiella and provided a new combination for it. Seifert has examined the type, and made a drawing from it that was used to represent this species in The Genera of Hyphomycetes   Emended generic diagnosis. Ascomatal bases globose, brown to black, unornamented or with undifferentiated ornamental hyphae. Ascomatal necks long, tapering to apex, straight, dark-brown to black, hyaline at apex. Ostiolar hyphae divergent, non-septate, tapered, light brown to hyaline. Asci dehiscent. Ascospores one-celled, hat-shaped, hyaline, accumulating in cream-coloured masses at tips of necks. Primary conidiophores phialidic, flask-shaped. Secondary conidiophores flaring or wide-mouthed. Primary conidia cylindrical, hyaline. Secondary conidia barrel to subglobose shaped, hyaline to light brown. Aleurioconidia globose, ovoid to pyriform, singly or in chains, pale-brown to brown.

Notes:
The most characteristic features of this genus are the ascomatal bases lacking distinct ornamentations and hat-shaped ascospores. The possibility that Ro. coffeae might not be a synonym of C. fimbriata is discussed under the latter species, below. However, even if the two species are distinct, the species from coffee would probably still group in Ceratocystis s. str., which means Rostrella will remain a synonym of Ceratocystis. Tarigan et al. 2013). The name C. fimbriata should be restricted to the fungus from sweet potato and to other isolates belonging to the same phylogenetic species. Pontis (1951) listed Rostella coffeae as a synonym of C. fimbriata, but mentioned biological differences between isolates from the coffee tree and sweet potato. Several recent studies, based on DNA sequence comparisons for multiple gene regions, have distinguished host-specific and geographically-separated populations, including populations from coffee, in the C. fimbriata species complex , Barnes et al. 2001, Baker Engelbrecht et al. 2003, Marin et al. 2003, Johnson et al. 2005. Van Wyk et al. (2010) described two of these host-specific groups from coffee in Colombia as new species, but did not consider the possibility that one of them might represent R. coffeae, probably because the latter was originally described from coffee in Java (Indonesia). For the present we treat R. coffeae as a synonym of C. fimbriata until future studies with fresh isolates from coffee in Java provide further insights into this question. Description: Kiffer & Delon (1983: 166-170, figs 1-2).
Notes: The synonymy of St. dimorpha with C. paradoxa was suggested by Mbenoun et al. (2014a). These authors also discussed and explained the treatment of the names and authorities of the previously considered sexual and asexual states, as suggested by Hawksworth et al. (2013). Descriptions : Hunt (1956: 11, 17, 20); Nag Raj & Kendrick (1975: 106, 142, fig. 38);Upadhyay (1981: 69, figs 205-213); Mbenoun et al. (2014a). Notes: Paulin-Mahady et al. (2002) and Mbenoun et al. (2014a) confirmed the synonymy of T. punctulata and C. radicicola based on similar sequences. Based on the Melbourne Code (McNeill et al. 2012) the older epithet must take preference, implying that this species will in future be treated as T. punctulata, and not as the better known C. radicicola, unless conservation of the later name against the earlier is proposed and accepted.

Ceratocystis incertae sedis
Four species could not be consistently accommodated in any of the seven major clades for which genera have been provided. We believe that they represent discrete genera but we have not provided generic names for these lineages. With increased sampling and further study, additional species are likely to be found that will populate these clades. At that time, genera can be provided for them. For the present they have been retained in their existing genera. We also list C. erinaceus and C. norvegica here for which isolates could not be obtained, and for which the generic placements remains uncertain.
Notes , Moreau (1952), Griffin (1968), Olchowecki & Reid (1974), and Nag , all treated C. major and C. adiposa as distinct.  suggested the synonymy of C. major with C. adiposa. Identical SSU sequences for the two species by Hausner et al. (1993b) suggested that the synonymy is sound, and this was confirmed in the present study where the two species had identical sequences in ITS, LSU and 60S. Description: Nag Raj & Kendrick (1975: 108-113, figs 39-40 as Ca. elegans).
Notes: Sexual state unknown. Delon & Kiffer (1978) synonymised T. basicola with Ca. elegans, at the time treated in Chalara (Nag . Paulin-Mahady et al. (2002) showed the species is best treated in Thielaviopsis, thus reversing the synonymy and bringing the name to its current state. Although our data have shown that T. basicola does not form part of Thielaviopsis as defined in the present study, the species is best treated in this genus until an epitype is designated that is linked to the holotype specimen. On that basis a final generic placement can be ascertained. Notes: The asexual state of this causal agent of oak wilt was described first as Ca. quercina (Henry 1944), while the sexual state was later discovered and described as E. fagacearum (Bretz 1952 Notes: A culture for this species could not be obtained. The sequences generated by Reid et al. (2010) suggest that this species falls outside the C. coerulescens complex (now Endoconidiophora), in which it would otherwise fit based on morphology and its conifer host. An accurate generic placement will only be possible once a culture can be obtained from which the appropriate sequences can be generated. Until such time it is best treated in Ceratocystis s.l.

DISCUSSION
Ceratocystis s. str. as it is defined in the present study is typified by the well-known species C. fimbriata. The genus currently includes 32 species, all of which were included in the analyses making up this study. The genus includes many important plant pathogens of angiosperm trees, but also of root crops (Kile 1993, Roux & Wingfield 2013. These fungi all have ascomata with smooth non-ornamented bases and hat-shaped ascospores; two morphological features that distinguish them from species now in the genera Thielaviopsis (previously C. paradoxa s.l.) and Huntiella (previously C. moniliformis s. l.). Both the latter genera have ornamented ascomatal bases, although the morphology of the ornamentations is different in the two genera. In some cases, species boundaries for Ceratocystis s. str. are very clear, for example in the cases of the tree pathogens C. platani, C. cacaofunesta, and C. albifundus (Wingfield et al. 1996. In others, distinction at the species level has been debated (Fourie et al. 2014, Harrington et al. 2014a). Problems have for example arisen where the ITS region has suggested the existence of species boundaries but where it is now recognised that there are often two or more ITS forms within a single isolate (Al Adawi et al. 2013, Naidoo et al. 2013, Harrington et al. 2014a). Revisions of these species boundaries are likely to emerge when additional tools, especially those taken from whole genome sequences (Wilken et al. 2013), become available to discriminate more clearly between species. Another problem, already recognised for this group, is that hybridisation has occurred between species ), a factor that will also confuse the recognition of discrete taxa. What is clear, however, is that there are many species already known in this group and many more will likely be found in the future.
The asexual genus Chalaropsis has been emended to accommodate three species that are found on woody substrates. Two of these three were included in the analyses, along with information from a fourth undescribed species discovered in a culture collection. None of these fungi are known to have any economic or critical important ecological significance.
The genus Endoconidiophora was emended to accommodate an important group of eight species that occur mostly on conifers and many of which are symbionts of conifer-infesting bark beetles. These fungi have previously been referred to as "the Gymnosperm section" in the C. coerulescens s. l. group (Harrington 2009) and they include a number of important pathogenic species such as E. polonica, E. laricicola, E. laricis and E. rufipennis (Redfern et al. 1987, Christiansen & Solheim 1990, Solheim & Safranyik 1997. Other species are mostly agents of sap stain in conifer timber. Unlike species in Ceratocystis s. s., Huntiella and Thielaviopsis as circumscribed here, these fungi have ascospores that are not hat-shaped but rather are obovoid, with distinct sheaths (Fig. 3). Ceratocystis norvegica, a species from conifers in Norway (Reid et al. 2010) that seems to fit the description of Endoconidiophora, but for which material was not available, should be considered in future treatments of this genus.
Davidsoniella is described as a new genus to accommodate members of what has previously been referred to as "the Angiosperm section" of C. coerulescens s. l. (Harrington 2009. The group includes four species, of which two, D. virescens and D. eucalypti, have known sexual states. The fusiform ascospores with evenly distributed hyaline sheaths are similar to but distinct from those of species now accommodated in Endoconidiophora. The remaining two species (D. australis and D. neocaledoniae) are known only by their asexual morphs, with the "chalaraor thielaviopsis-like" morphology typical of all species in Ceratocystis s. l. other than Ambrosiella. Interestingly, three of these fungi (D. virescens, D. neocaledoniae and D. australis) are important tree pathogens (Hepting 1944, Dadant 1950, Kile & Walker 1987 while one (D. eucalypti) is not known to be a pathogen (Kile et al. 1996). Three species (D. eucalypti, D. neocaledoniae and D. australis) are known exclusively from Australasia, while D. virescens occurs in North America. One more species fits the description of Davidsoniella, namely C. erinaceus from oak in Europe (Boh ar 1996). No sequence data exist for this species but it should be considered in future treatments of this genus.
The emended asexual genus Thielaviopsis includes species previously placed in C. paradoxa s. l., some of which have known sexual states. Until recently all species in this group were aggregated in the single species, C. paradoxa, but Mbenoun et al. (2014b)'s sequencing and mating studies disclosed six species in what they referred to as the C. paradoxa complex. They showed that the type species of Thielaviopsis, T. ethacetica, though previously treated as anamorph of C. paradoxa (Nag , is a distinct species. In one species, T. euricoi, no sexual state has been observed, but the others all produce hat-shaped ascospores. The outstanding characteristic of this genus, however, is the presence of prominent, digitate appendages on the ascomatal bases (Fig. 2). Most of these fungi occur on monocotyledenous plants including palms, pineapple and banana and some are important plant pathogens (Mitchell 1937, Bliss 1941, Abdullah et al. 2009).
Huntiella was established in this study to accommodate a well-recognised and large group of species that have previously been referred to as residing in C. moniliformis s. l. Nineteen species are recognised in Huntiella of which 18 were included in the analyses. Two of these species are in the process of being described (Mbenoun unpubl. data). Species of Huntiella have hat-shaped ascospore (Fig. 3) similar to those found in Ceratocystis s. str. but they have very distinct ascomata. The latter feature necks with basal plates that easily disconnect from the ascomatal bases, which are also ornamented with spines (Fig. 2). Huntiella spp. are very commonly encountered on tree REDEFINING CERATOCYSTIS AND ALLIED GENERA www.studiesinmycology.org wounds and they are typically non-pathogenic (Roux et al. 2004. The genus Ambrosiella is perhaps the most unusual in Ceratocystis s. l. The five species accommodated in this genus (three of which were included in the analyses) are all symbionts of wood-boring "ambrosia" beetles and they lack a known sexual state. They are the only species in Ceratocystis s. l. that do not have typical "chalara-like" conidiogenous cells. Instead they have tubular tapering conidiophores and rectangular conidia formed in chains.
Four species in Ceratocystis s. l. did not reside in any of the six major phylogenetic clades arising from this study. These species included C. adiposa, C. fagacearum, Thielaviopsis basicola and Ambrosiella ferruginea. These clearly represent discrete genera, which as collections increase in the future, will most likely accommodate additional species. This would be the same situation that has arisen for other genera now recognised in Ceratocystis s. l. and that previously included very few obvious species. For the present, we have chosen not to provide generic descriptions for these species. We believe that they are likely to be more clearly defined in the future, particularly since three of the four require additional work to obtain living material that can be unambiguously reconciled with their typification. Three of these four fungi (C. fagacearum, C. adiposa and T. basicola) are well-recognised plant pathogens (Butler 1906, Yarwood 1981, Juzwik et al. 2008) and we recognise that name changes could cause some confusion. It will thus be important to make it clear in studies that these fungi are phylogenetically unrelated to the genera in which they are currently treated.
Phylogenetic analyses based on three carefully selected gene regions in this study have provided robust data to be able to distinguish more effectively between a large number of important and very different fungi that have, for many years, been unfortunately lumped in a single genus. The improved resolution has emerged through intensive collecting initiatives in new areas and through the application of new technologies that have improved our ability to recognise cryptic taxa. As global collecting initiatives expand for fungi residing in the Ceratocystidaceae, the taxa accommodated in the genera established in this study will surely increase and the boundaries of the few remaining monotypic lineages will also be elucidated.
The new and rapidly stabilising nomenclatural code for fungi (McNeill et al. 2012) underpins a natural classification and a single name for all fungal taxa. This is a major and positive change that will ultimately promote a more effective taxonomy for fungi and it will ensure easier relationships with important associated disciplines such as plant pathology (Hawksworth 2011. A one fungus one name scheme has already been presented for the so-called ophiostomatoid fungi including the Ceratocystidaceae (De Beer et al. 2013b). In the present study, we have followed this approach rigorously. As far as possible, available generic names, in all cases those associated with asexual morphs have been used. In two instances entirely new generic names have been established and these honour two important early pioneers of the taxonomy of the ophiostomatoid fungi. They are John Hunt who produced the first comprehensive monograph of Ceratocystis ) and Ross W. Davidson who dedicated his career to collecting, identifying and describing species of ophiostomatoid fungi including several Ceratocystis spp.