mycological research 110 (2006) 1271–1289 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Molecular phylogeny of Coniochaetales Dania GARCÍAa,b, Alberto M. STCHIGELb, José CANOb, Misericordia CALDUCHb, David L. HAWKSWORTHc, Josep GUARROb,* a Instituto de Investigaciones Fundamentales en Agricultura Tropical ‘‘Alejandro de Humboldt’’, calle 1 esq.2, Santiago de las Vegas, Boyeros, C. de La Habana, Cuba b Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, C/Sant Llorenç 21, E-43201, Reus, Tarragona, Spain c MycoNova, The Yellow House, Calle Aguila 12, Colonia La Maliciosa, Mataelpino, E-28492, Madrid, Spain article info abstract Article history: Although the taxonomy of ascomycetes has changed dramatically, generic delimitation Received 23 December 2005 within the recently proposed order Coniochaetales has not been resolved. In order to clarify Received in revised form the phylogenetic relationships of genera in the Coniochaetaceae, we performed a molecular 1 June 2006 study based on the analyses of the sequences of the partial SSU and of the variable domains Accepted 7 July 2006 of the LSU rDNA genes. The phylogenetic trees obtained do not support the monophyly of Published on line 31 October 2006 the genera Coniochaeta, Coniochaetidium, Ephemeroascus, and Poroconiochaeta. A morphological Corresponding Editor: study confirmed that there were not enough differences to distinguish these genera, and the H. Thorsten Lumbsch latter three are treated as synonyms of Coniochaeta. The phialidic anamorph proved to be an informative phylogenetic character in Coniochaetales, while that the type of ascomata (cleis- Keywords: tothecial or perithecial) and the ornamentation of the ascospore walls were of little taxo- Ascomycota nomic value at the generic level. The circumscription of the genus Coniochaeta is revised. Coniocessia The genera Coniocessia and Coniolariella are proposed as new within the order Xylariales to Coniochaeta accommodate Coniochaeta nodulisporioides, and C. gamsii, respectively. The taxonomic posi- Coniolariella tion of Synaptospora and Wallrothiella subiculosa are also discussed. Xylariales ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction The family Coniochaetaceae was erected by Malloch & Cain (1971) to accommodate Coniochaeta and Coniochaetidium. The most distinctive morphological features of this family are the presence of germ-slits in the ascospores, which differentiate these fungi from Sordariaceae, and its phialidic anamorphs, which belongs to Lecythophora (Barr 1990; Weber 2002). Xylariaceae is a close family, generally, with stromatic ascomata and holoblastic conidiogenesis, which are absent in Coniochaetaceae. Coniochaetaceae has been traditionally included in Sordariales (Checa et al. 1988; Barr 1990; Lee & Hanlin 1999; Kirk et al. 2001; Eriksson 2005), but this has not been universally accepted (Weber et al. 2002) and the new order Coniochaetales has been recently proposed based on this family (Huhndorf et al. 2004). Coniochaetaceae, apart from the two mentioned genera, currently encompasses also Barrina, Coniolaria (not validly published; Eriksson & Hawksworth 1998), Ephemeroascus, Poroconiochaeta, and Synaptospora (Eriksson et al. 2004; Huhndorf et al. 2004). The genus Ascotrichella is a morphologically similar genus, characterized by non-stromatic ascomata, non-amyloid asci, ascospores with a germ-slit, and a Humicola-like anamorph; however, it has been considered to be a member of Xylariaceae (Valldosera & Guarro 1988; Whalley 1996). The genera of the Coniochaetaceae were primarily delimited by differences in their ascomata (ostiolate in Coniochaeta, and non-ostiolate in Coniochaetidium and Ephemeroascus) (Malloch & Cain 1971; van Emden 1973). The ornamentation of the * Corresponding author. E-mail address: josep.guarro@urv.net 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.07.007 1272 D. Garcı́a et al. ascospore wall was also considered a useful criterion, and the presence of pitted ascospores was used to justify the erection of Poroconiochaeta (Udagawa & Furuya 1979). The presence of phialidic, verticillate conidiogenous cells was considered a distinctive character separating Ephemeroascus from Coniochaetidium (van Emden 1973). Recent studies have demonstrated that a taxonomic classification based only on morphological characters frequently does not correlate with the phylogenetic relationships inferred from DNA sequences (Réblová & Winka 2000; Réblová & Seifert 2004). Thus, the usefulness of some of these morphological characters for delimiting supraspecific taxa has been questioned. For instance, the presence of an ostiole in the ascoma has proven to be a variable character, not useful for the delimitation of genera (von Arx 1973; Suh & Blackwell 1999). The shape and ornamentation of the ascospores have also been considered homoplastic characters with poor phylogenetic value in some groups of ascomycetes (Dettman et al. 2001; Zhang & Blackwell 2002; Solé et al. 2002; Miller 2003). In addition, the structure and morphology of the conidiogenous cells seems to be insufficient for the delimitation of taxa at the generic level (Réblová & Winka 2000). The main aim of this study was to determine the phylogenetic relationships among the genera recognized in the Coniochaetaceae. For this purpose we performed analyses of the SSU and LSU rDNA gene sequences from a set of strains of representative species of this family. Materials and methods Strains studied The fungi sequenced in this study are listed in Table 1. Additional sequences obtained from GenBank are listed in Table 2. DNA extraction, PCR amplification, sequencing, and sequence alignment The DNA was isolated as described by Cano et al. (2004). Detailed protocols for the amplification and sequencing of the SSU and LSU rDNA genes were described in Cano et al. (2002a) and Garcı́a et al. (2004), respectively. Phylogenetic analyses Phylogenetic analyses were performed using the NJ method (Saitou & Nei 1987) with the MEGA 2.1 computer program (Kumar et al. 2001). The trees were constructed using the Kimura-2-parameter distance model (Kimura 1980) with the pairwise deletion of gaps option. The robustness of branches was assessed by BS analysis with 1000 replicates. Results and discussion General analysis Fig 1 shows the phylogenetic tree of the partial SSU rDNA sequences of 29 strains, belonging to 11 species of Coniochaetaceae, some representative members of the class Sordariomycetes, and Aphanoascus mephitalis as out-group. Three major clades were formed, which corresponded to the three subclasses of Sordariomycetes, i.e. Hypocreomycetidae (98 % BS value), Sordariomycetidae (86 % BS value), and Xylariomycetidae (73 % BS value). The recently erected order Coniochaetales (Huhndorf et al. 2004) formed a well-supported subclade (81 % BS value) within the Sordariomycetidae. In this analysis the genus Coniochaeta, as currently circumscribed, was found to be polyphyletic. C. nodulisporioides was placed within the Xylariomycetidae very far from the other species of the genus, which were mixed with species of the genera Coniochaetidium, Ephemeroascus, and Poroconiochaeta. Since in pyrenomycetes domains of the LSU rDNA gene have been found to evolve more rapidly than the SSU rDNA gene (Zhang & Blackwell 2002), partial sequences of the former were used to study the relationships between Coniochaeta and allied genera and to investigate more precisely the placement of C. nodulisporioides. Fig 2 shows the phylogenetic tree obtained from the analysis of 44 strains, belonging to different species of the class Sordariomycetes. In this analysis we have Table 1 – Source of specimens examined and sequenced in this study Species Coniochaeta cruciata C. hansenii C. nodulisporioides C. tetraspora Coniochaetidium boothii C. ostreum Coniolaria murandii Ephemeroascus verticillatus Poroconiochaeta discoidea P. punctulata P. savoryi (syn. Thielavia savoryi) P. tetraspora Strains FMR 7409 CBS 885.68 CBS 281.77T FMR 8167 CBS 381.74T CBS 507.70T LCP 04.5004 CBS 816.71T CBS 158.80T FMR 7408 CBS 725.74T FMR 7415T Source Soil, Nigeria Dung of rabbit, The Netherlands Soil, Jordan Soil, Spain Soil, India Twig of Larrea sp. Soil, USA Soil, The Netherlands Soil, Japan Soil, Argentina Wood of Juniperus scopulorum, UK Soil, Russia EMBL no. SSU LSU AJ875176 AJ875182 AJ875185 AJ875178 AJ875181 AJ875184 AJ875222 AJ875223 AJ875224 AJ875225 AJ875226 AJ875227 AJ875233 AJ875232 AJ875230 AJ875231 AJ875229 AJ875228 AJ875183 AJ875179 AJ875175 AJ875180 AJ875177 CBS, Centraalbureau voor Schimmelcultures (Utrecht); FMR, Culture Collection, Facultad de Medicina (Reus); LCP, Laboratoire de Cryptogamie, Muséum National d’Histoire Naturelle (Paris). T, type or ex-type culture. Molecular phylogeny of Coniochaetales 1273 Table 2 – Sequences obtained from GenBank and used in the present study Species EMBL no. SSU LSU Achaetomium globosum A. luteum Albertiniella polyporicola Aphanoascus mephitalis Apioclypea sp. Apiospora setosa A. sinensis Aporothielavia leptoderma Appendicospora sp. Arthrinium phaeospermum Ascotricha chartarum A. guamensis A. lusitanica A. xylina Astrocystis cocoës Aureobasidium pullulans Barrina polyspora Camarops microspora Cephalotheca sulfurea Cercophora septentrionalis Chaetomium globosum Clypeosphaeria uniseptata Coniochaeta lignaria – – – AB015779 – – – – – – AB048283 AB048281 AB048282 AB048789 – – – – – – – – – C. malacotricha – C. pulveracea – C. subcorticalis C. tetraspora C. velutina Coniochaeta sp. Cryptendoxyla hypophloia Cryptosphaeria eunomia Daldinia concentrica Diaporthe decedens D. detrusa Diatrype disciformis Dicyma olivacea Discula destructiva D. umbrinella Dothidea sambuci Eutypa sp. – AF048813 – AF048799 – – U32402 – – U32403 AB048284 – – – – Fasciatispora petrakii Graphostroma platystoma Halorosellinia oceanica Hypocrea gelatinosa H. lutea H. schweinitzii Hypomyces polyporinus Hyponectria buxi Hypoxylon fragiforme H. haematostroma Lasiosphaeria ovina Lecythophora decumbens L. fasciculata L. hoffmanii L. lignicola L. luteoviridis – – – – – L36986 U32410 – AY083810 AF346543 – – – – – – AJ312097 AJ312105 AF096185 – AY083836 AY346259 AY083831 AF096186 AY083833 AY083832 – – – – AY083823 AF050239 AY346258 AY083821 AF096188 U47823 U47825 AY083830 AF353583 AF353584 AF353586 AF353588 AF353589 AF353590 AF353591 AF353592 AF353593 – AF353594 – AF096190 AY083826 U47828 AF408348 AF408349 U47829 – AF408359 AF408359 AF382387 AY083825 AY346280 AY083828 AY083827 AY083822 U00737 U00739 – – AY083834 AY083829 – AF064643 AF353597 AF353598 AF353599 AF353601 AF353603 (continued on next page) Table 2 (continued) Species EMBL no. SSU L. mutabilis Nectria cinnabarina N. pseudotricha Neocosmospora vasinfecta Oxydothis frondicola Rosellinia necatrix Sordaria fimicola S. humana Wallrothiella subiculosa Xylaria curta X. hypoxylon – U32412 – U32414 – – – AF48811 – U32417 U20378 LSU AF353605 U00748 U17410 – AY083835 AY083824 AF132330 – U17428 U47840 U47841 included also the genera Barrina, recently related to Coniochaetales (Huhndorf et al. 2004), Coniolaria, a genus very similar to Coniochaeta but with a holoblastic anamorph (Seigle-Murandi et al. 1995); and moreover Wallrothiella subiculosa, which showed a confused taxonomic position within Sordariales (Réblová & Winka 2000). The topology of the tree was similar to that derived from SSU sequence data. The order Coniochaetales received a high BS support (96 %), the species of Coniochaeta again were revealed as polyphyletic, and the placement of C. nodulisporioides within the subclass Xylariomycetidae was also confirmed. Similarly to Coniochaeta, the genera Coniochaetidium, Ephemeroascus, and Poroconiochaeta did not form natural groups. The species of these genera were distributed in different branches, interspersed with species of other genera. Interestingly, W. subiculosa appeared within the Coniochaetales clade, and Coniolaria murandii was in the subclass Xylariomycetidae, related to two species of Xylaria (Xylariaceae) included in the study (73 % BS value). Fig 3 shows the phylogenetic tree derived from the analysis of LSU rDNA sequences but restricted only to members of Coniochaetales. In this study Aporothielavia leptoderma, Lasiosphaeria ovina, and Sordaria fimicola (Sordariales) were used as out-group. All the strains of Coniochaetales formed an ingroup with 98 % of BS support. They were distributed among three major clades and a basal group, although, in general, only terminal branches received a significant BS support. Coniochaeta was revealed as paraphyletic and its species appeared distributed into the different clades interspersed with members of the other genera included in the study. Barrina polyspora, C. cruciata, C. hansenii, and W. subiculosa, formed a first basal group, although they were genetically distant from each other. Three strains of C. malacotricha, the type species of Coniochaetidium (C. ostreum), and E. verticillatus formed clade I, although not well-supported. Clade II included a branch with the single species Lecythophora hoffmannii, and a well-supported group (85 % BS value) constituted by three strains of Coniochaeta ligniaria, one of C. velutina, and one each of the following four species of Lecythophora: L. fasciculata, L. lignicola, L. mutabilis, and L. luteoviridis. Clade III was divided into two branches. The basal branch was only formed by the type species of genus Poroconiochaeta (P. discoidea), that appeared very far from the other species 1274 D. Garcı́a et al. Dicyma olivacea Ascotricha guamensis Subclass Xylariomycetidae Ascotricha chartarum 97 Ascotricha lusitanica Ascotricha xylina Daldinia concentrica 75 Hypoxylon fragiforme Hypoxylon haematostroma Coniocessia nodulisporioides 73 76 Xylaria curta Xylaria hypoxylon Diatrype disciformis Sordaria fimicola Subclass Sordariomycetidae Coniochaeta tetraspora Coniochaeta tetraspora 86 Coniochaeta sp. Coniochaetidium boothii Ephemeroascus verticillatus 81 Poroconiochaeta discoidea Poroconiochaeta punctulata Poroconiochaeta savoryi Coniochaeta hansenii Coniochaeta cruciata 100 98 Nectria cinnabarina Neocosmospora vasinfecta 100 Hypocrea schweinitzii Hypomyces polyporinus Subclass Hypocreomycetidae Poroconiochaeta tetraspora Aphanoascus mephitalis 0.01 Fig 1 – NJ tree based on nucleotide sequences of the SSU rDNA gene. Branch lengths are proportional to genetic distance, indicated by a bar. BS values of 70% or greater are indicated above the internodes. of this genus included in the study. The second branch encompassed three well-supported groups. The first comprised Coniochaetidium boothii, Coniochaeta subcorticalis, and C. pulveracea (100 % of BS value); the second one comprised C. tetraspora and L. decumbens (97 % of BS support); and the third grouped three species of Poroconiochaeta, P. punctulata, P. savoryi, and P. tetraspora (96 % BS support). Fig 4 shows the phylogenetic tree obtained from the analysis of LSU sequences of 27 strains of six families of Xylariales (Apiosporaceae, Clypeosporaceae, Diatrypaceae, Graphostomataceae, Hyponectriaceae, and Xylariaceae), and the aim of this analysis was to determine more precisely the phylogenetic relationships of C. nodulisporioides and C. murandii, which previous threes excluded from Coniochaetales, with members of such families. Some members of Sordariales, i.e. Aporothielavia leptoderma, Lasiosphaeria ovina, and Sordaria fimicola, were used as out groups. Although few members of each family were included in the analysis, only Diatrypaceae (98 % BS value) and Apiosporaceae (100 % BS value) were phylogenetically supported. Xylariaceae was revealed as polyphyletic. Hypoxylon fragiforme and Daldinia concentrica were grouped in the basal clade (98 % BS support), which was very distant from the other species of Xylariaceae such as Astrocystis cocoës, Fasciatispora petrakii, Halorosellinia oceanica, Rosellinia necatrix, and X. hypoxylon. Coniochaeta nodulisporioides showed a certain, although distant relationship with Oxydothis frondicola, and members of Hyponectriaceae (Appendicospora sp. and Hyponectria buxi). Coniolaria murandii appeared to be related with A. cocoës and R. necatrix, members of Xylariaceae (78 % BS value). In general, the analyses from the LSU rDNA sequences correlated poorly with the morphological criteria used for delimiting genera in Coniochaetales, suggesting that the current taxonomic classification is artificial. Our analysis seems to demonstrate that Xylarioideae and Hypoxyloideae are not closely related as traditionally accepted. This could be due to insufficient taxon sampling and to the use of poorly characterized reference sequences. These results are in contrast to the recent analysis of Triebel et al. (2005), based on 5.8 S/ITS rDNA sequences, which, although resulted in a low resolution at the infrageneric level, recognized the two core groups (Hypoxyloideae/Xylarioideae) as monophyletic with Diatrypaceae as a sister group. In another recent study the rDNA sequences were regarded as difficult to align and interpret and then discarded to infer the phylogeny of Molecular phylogeny of Coniochaetales 1275 Coniochaeta hansenii Barrina polyspora 72 Coniochaeta cruciata Wallrothiella subiculosa Poroconiochaeta discoidea Coniochaeta ligniaria Coniochaeta velutina 92 Coniochaeta tetraspora Poroconiochaeta tetraspora 96 96 Order Coniochaetales Ephemeroascus verticillatus Coniochaeta malacotricha Coniochaetidium ostreum 100 91 Coniochaetidium boothii Coniochaeta pulveracea Coniochaeta subcorticalis 100 99 Diaporthe decedens Diaporthe detrusa 100 Discula destructiva Discula umbrinella Order Diaporthales Cryptendoxyla hypophloia Cephalotheca sulfurea Albertiniella polyporicola 98 Family Cephalothecaceae Poroconiochaeta punctulata Poroconiochaeta savoryi Lasiosphaeria ovina 89 Sordaria fimicola 96 Aporothielavia leptoderma Chaetomium globosum 100 100 88 73 Hypocrea gelatinosa Hypocrea lutea Nectria cinnabarina Nectria pseudotrichia Xylaria curta Xylaria hypoxylon Coniolariella gamsii Diatrype disciformis Coniocessia nodulisporioides 99 Order Xylariales 91 Order Hypocreales Achaetomium globosum Achaetomium luteum Order Sordariales Cercophora septentrionalis Hypoxylon fragiforme Daldinia concentrica 100 Dothidea sambuci Aureobasidium pullulans 0.05 Fig 2 – NJ tree based on nucleotide sequences of the LSU rDNA gene. Branch lengths are proportional to distance. BS values of 70% or greater are indicated above the internodes. Hypoxyloideae. However, from analysis of actin and b-tubulin sequences the major groups were reflected as inferred from morphological and chemical traits (Hsieh et al. 2005). However, in a study on Diatrypaceae also based on rDNA sequences, Acero et al. (2004) were unable to recognize the classical boundaries among this fungal group. It is possible that rDNA may have evolved independently from morphological traits to some extent in many groups of fungi, and if further molecular studies confirm this, it would be reasonable to use different concepts for Coniochaetales and Xylariales. Coniochaeta Concerning Coniochaeta, the different analyses demonstrated that C. hansenii and C. cruciata were the most divergent species. Both were included in a basal clade together with Barrina and Wallrothiella (Fig 3). C. hansenii has only been reported from dung (Mahoney & LaFavre 1981; Checa et al. 1988), and as other coprophilic species (e.g. C. multispora, C. philocoproides, C. polymegasperma, C. polysperma) this species is characterized by multispored asci. C. cruciata was described based on only 1276 D. Garcı́a et al. Poroconiochaeta punctulata Poroconiochaeta savoryi 96 Poroconiochaeta tetraspora Coniochaeta tetraspora Lecythophora decumbens 100 Coniochaetidium boothii 92 Clade III 97 Coniochaeta subcorticalis Coniochaeta pulveracea 86 Coniochaeta pulveracea Poroconiochaeta discoidea Lecythophora hoffmannii Coniochaeta ligniaria 97 Coniochaeta ligniaria Clade II Coniochaeta ligniaria Lecythophora mutabilis 85 Lecythophora fasciculata Coniochaeta velutina Lecythophora lignicola Lecythophora luteoviridis Ephemeroascus verticillatus 98 100 Clade I Coniochaetidium ostreum Coniochaeta malacotricha Coniochaeta malacotricha Coniochaeta malacotricha Wallrothiella subiculosa Coniochaeta hansenii Coniochaeta cruciata Barrina polyspora Lasiosphaeria ovina 87 Sordaria fimicola Aporothielavia leptoderma 0.01 Fig 3 – NJ tree based on nucleotide sequences of the LSU rDNA gene from different taxa related with Coniochaeta. Branch lengths are proportional to distance. BS values of 70% or greater are indicated above the internodes. one strain isolated from Quercus ilex (Checa et al. 1988). This species is easily differentiated by its cruciform ascospores (in optical section), which resemble those of the soil-borne C. extramundana. Recent studies in Sordariales and Xylariales demonstrated that the number of ascospores per ascus and the ascospore morphology are homoplastic characters, which have appeared several times in the course of evolution, and are without phylogenetic value at generic level (Miller 2003; Acero et al. 2004). On the basis of their peculiar characteristics and the relative genetic distance from the rest of the species of Coniochaeta, it could be acceptable to erect two new genera to accommodate C. cruciata and C. hansenii. However, at the moment we have included both species in Coniochaeta because they have the most distinctive morphological features of this genus. This distant phylogenetic position from the rest of Coniochaeta could be a result of a limited number of species being included in the analyses. The close genetic relationship between C. tetrasperma and Lecythophora decumbens indicates that probably they are two states of the same species; the latter had not previously been connected to any teleomorph (Weber 2002; Weber et al. 2002), but the conidial state of C. tetrasperma described by Cain (1961) was very similar to L. decumbens. Both form reduced adelophialides, and the shape and size of conidia are also similar (Cain 1961; Weber 2002). The different colour of the colonies, dark in L. decumbes and pinkish in C. tetrasperma, does not seem to be of phylogenetic value, because the well-supported clade III formed by C. ligniaria, C. velutina, and some species of Lecythophora (Fig 3) encompasses species with pinkish to salmon–orange colonies, as well as species with darker ones. The present study also showed that C. nodulisporioides, which possesses an anamorph with holoblastic conidiogenesis (Hawksworth 1978), is close to Xylariales (Figs 1–2, 4). This agrees with previous authors (Barr 1990; Laessøe 1994; Hawksworth & Rogers, in Barr & Cannon 1994), who considered that Coniochaeta should be restricted to the species with Molecular phylogeny of Coniochaetales 1277 Astrocystis cocoës 78 Coniolariella gamsii Rosellinia necatrix Xylaria curta Xylaria hypoxylon Halorosellinia oceanica Graphostroma platystoma Cryptosphaeria eunomia 99 Eutypa sp. Diatrype disciformis 74 Eutypa sp. Fasciatispora petrakii Apioclypea sp. Clypeosphaeria uniseptata Apiospora setosa 99 100 Apiospora sinensis Arthrinium phaeospermum Coniocessia nodulisporioides 100 Oxydothis frondicola Appendicospora sp. Hyponectria buxi 98 Hypoxylon fragiforme Daldinia concentrica Camarops microspora Lasiosphaeria ovina Sordaria fimicola Aporothielavia leptoderma 0.02 Fig 4 – NJ tree based on nucleotide sequences of the LSU rDNA gene from different genera of Xylariales, Coniolaria murandii, and Coniochaeta nodulisporioides. Branch lengths are proportional to distance. BS values of 70% or greater are indicated above the internodes. phialidic anamorphs and that those species with a Nodulisporium- or Geniculosporium-like anamorph (e.g. C. nodulisporioides, C. emodensis) should be excluded from the genus. In Xylariales, molecular studies have demonstrated that the type of anamorph is of taxonomic value (Sánchez-Ballesteros et al. 2000; Sánchez-Ballesteros 2001). The hyaline anamorph of C. nodulisporioides is unique in Xylariales, and in spite of the members of Nodulisporium being pigmented, it has been considered Nodulisporium-like (Hawksworth 1978). Based on molecular and morphological data, we propose the new genus Coniocessia to accommodate C. nodulisporioides, although its inclusion in any of the known families of Xylariales needs further investigation. The morphological features of Coniocessia are similar to those of Xylariaceae, but this study seems to demonstrate that this species is phylogenetically closer to Hyponectriaceae than to Xylariaceae (Fig 4). However, these data have to be taken with caution considering the BS support is very low and that the latter family needs redefinition. Members of Hyponectriaceae, as well as members of the other families of Xylariales, with the exception of Xylariaceae, can be differentiated from Coniocessia by their hyaline, yellow to pinkish or light brown ascospores, and by the absence of an elongate germ-slit (Barr 1990; Hyde et al. 1998). In this study, Oxydothis frondicola was the species genetically closest to Coniocessia (Fig 4). However, species of Oxydothis are characterized by long, cylindrical asci with an amyloid sub-apical apparatus, and hyaline to light brown, long to filiform, bicellular ascospores lacking an elongate germ-slit, and in addition, they have a Selenosporella-like anamorph (Samuels & Rossman 1987; Barr 1990; Fröhlich & Hyde 1994). Udagawa & Horie (1982) proposed Coniochaeta emodensis (for a single isolate from soil in Nepal (Fig 5). It is characterized by non-stromatic ascomata, non-amyloid asci, dark brown ascospores with a longitudinal germ-slit, and an anamorph with holoblastic, sympodial conidiogenesis, and denticulate conidiogenous cells. Unfortunately, material of this species was not available for study. However, the authors provided a detailed description and very informative illustrations of the species, which allow its recognition as a member of Xylariales. Some authors have considered C. emodensis and C. nodulisporioides to be closely related species (Udagawa & Horie 1982; Barr 1990; Laessøe 1994), but we think that they are not 1278 D. Garcı́a et al. Fig 5 – Coniochaeta emodensis. A. Asci with ascospores. B. Ascospores. C. Conidiophores, conidiogenous cells, and conidia. Bars [ 10 mm. congeneric. Their anamorphs are very different: C. nodulisporioides has hyaline conidiophores and conidia, and swollen conidiogenous cells with persistent conspicuous denticles, whereas C. emodensis possesses pale yellowish brown conidiophores and conidiogenous cells with very short and fragile denticles. The anamorph of C. emodensis, as well as those of some members of Xylariaceae (Collodiscula, Entoleuca, Euepixylon, Leprieuria, Nemania, Podosordaria, Rosellinia, and Whalleya) (Whalley 1996; Rogers et al. 2002) has been referred to as Geniculosporium or Geniculosporium-like. However, all these species are morphologically very different from C. emodensis. They are characterised by well-developed stromatic tissues, which in some genera are also multiperitheciate (Entoleuca, Euepixylon, Leprieuria, Nemania, Podosordaria, and Whalleya), bipartite (Whalleya) or stipitate (Leprieuria); and by asci with a distinct, and often amyloid apical apparatus. Moreover, ascospores in Euepixylon have a poroid germ-slit and an inconspicuous, hyaline appendage in immature stages (Rogers et al. 2002; Fournier & Magni 2004), and Collodiscula, has two-celled ascospores which lack a distinct germination site (Samuels et al. 1987). Although the circumscription of Xylariaceae and the delimitation of their genera remain unclear (Whalley 1996; Sánchez-Ballesteros 2001; Smith et al. 2003), we think that C. emodensis belongs to an undescribed genus of that family. However, as no molecular data are available to confirm this at this moment a new genus for C. emodensis is not proposed. Coniochaetidium The genus Coniochaetidium is only distinguished from Coniochaeta by the presence of cleistothecial ascomata. It encompasses four species, C. boothii, C. mirabile, C. nuciforme, and C. ostreum (Malloch & Cain 1971; von Arx 1975; Udagawa & Tsubouchi 1986; Guarro et al. 1997; Garcı́a et al. 2003). Molecular phylogeny of Coniochaetales However, our phylogenetic analyses confirmed that this character was of poor taxonomic value. The two species included in the study, C. ostreum and C. boothii, were nested in different groups, interspersed with species of Coniochaeta and Ephemeroascus. Coniochaetidium is therefore treated as a synonym of Coniochaeta. As mentioned above, the nature of the anamorph has been proved to be a phylogenetic informative character in Coniochaetales. However, the conidial state in C. ostreum was simply described as producing ‘aleuriospores’ (Malloch & Cain 1971), an obsolete and confused term (Hughes 1953; Kendrick 1971; Kirk et al. 2001). Von Arx (1975) referred to the presence of blastoconidia, although we were not able to see such conidia in an ex-type culture. Instead, we observed only abundant chlamydospores in chains or arising terminally. A holoblastic anamorph with a retrogressive conidiogenesis was described for C. mirabile, and identified as a member of Cladobotryum (Udagawa & Tsubouchi 1986). Retrogressive conidiogenesis is quite rare in conidial fungi (Kendrick 1985), but has been described for some anamorphic states of Hypomyces such as Cladobotryum and Trichothecium (Rogerson & Samuels 1993; Gams et al. 1998). However, phylogenetic studies in Hypomyces suggested that retrogressive conidiogenesis does not have taxonomic value at the suprageneric level. Species of Hypomyces with retrogressive conidiogenesis formed a clade, which also included other species with phialidic conidiogenesis (Verticillium and Gliocladium) (Põldmaa et al. 1999). Therefore, C. mirabile is accepted here as member of the re-circumscribed genus Coniochaeta. 1279 Ephemeroascus van Emden (1973) erected the genus Ephemeroascus for a single species isolated from soil in The Netherlands, characterized by cleistothecial ascomata and ascospores with an elongate germ-slit. That author separated E. verticillatus from Coniochaetidium on the presence of phialoconidia, and excluded it from Coniochaetaceae because of the absence of pinkish orange colonies, because Malloch & Cain (1971) had referred to this feature as one of the most distinctive characters in the family. However, C. velutina and some species of Lecythophora are characterized by the production of more darkly pigmented colonies (Weber 2002). The anamorph of Ephemeroascus was described having erect conidiophores and discrete phialides in a verticillate arrangement (van Emden 1973), which was later defined as Verticillium-like (von Arx 1975; Mahoney & LaFavre 1981; Kirk et al. 2001). A similar anamorph was observed in C. cypraeaspora (van der Linde 1991) and was referred to as Paecilomyces-like. Future molecular studies are needed to evaluate when the presence of a verticillate anamorph has phylogenetic value within Coniochaeta, but our results suggest that this character or the presence of cleistothecial ascomata are insufficient for excluding Ephemeroascus from Coniochaeta. Both genera are therefore treated here as synonyms. Poroconiochaeta The distinctive character used to segregate Poroconiochaeta from Coniochaeta was the presence of pitted ascospores in Fig 6 – Coniochaeta species with ornamented ascospores (SEM). A. C. discoidea. B–C. C. ornata. D–E. C. punctulata. F. C. savoryi. Bars [ 10 mm. 1280 the former (Udagawa & Furuya 1979). The ornamentation of the ascospore wall had been traditionally considered an important taxonomic character to define genera in many groups of ascomycetes. However, in recent years numerous studies have demonstrated that generic distinction based only on this character does not have phylogenetic support (SánchezBallesteros 2001; Dettman et al. 2001; Cano et al. 2002b; Solé et al. 2002; Sugiyama et al. 2002; Zhang & Blackwell 2002; Huhndorf et al. 2004; Garcı́a et al. 2004). This has been confirmed by our results. The species of Poroconiochaeta did not form a monophyletic group, but they were interspersed with species of Coniochaeta and Coniochaetidium (Figs 2–3). A detailed examination of all the species accepted in the genus Poroconiochaeta revealed that the most representative features of the D. Garcı́a et al. colonies, ascomata, asci, and even the anamorph of P. discoidea (Udagawa & Furuya 1979) were similar to those described for the genus Coniochaeta. Therefore, Poroconiochaeta is also considered a synonym of Coniochaeta. Other genera Wallrothiella is a large and confused genus, which has been considered to be a member of Trichosphaeriaceae (Trichosphaeriales) (von Arx & Müller 1954), Niessliaceae (Hypocreales) (Barr 1990) or related to Chaetosphaeriaceae (Chaetosphaeriales) (Samuels & Barr 1997; Réblová & Winka 2000). Phylogenetic studies based on LSU sequences excluded it from the cited families, but did not resolve its taxonomic position (Zhang & Fig 7 – Some representative types of Coniochaeta ascospores. A. C. angustispora. B. C. arxii. C. C. boothii. D. C. cephalothecoides. E. C. cruciata. F. C. discoidea. G. C. malacotricha. H. C. mirabile. I. C. nuciforme. J. C. Ornata. Bars [ 10 mm. Molecular phylogeny of Coniochaetales Blackwell 2002; Réblová & Winka 2000; Réblová & Seifert 2004). Wallrothiella is characterized by non-stromatic perithecia, asci with a non-amyloid apical ring, hyaline, globose ascospores with no germ pore or slit, and a phialidic anamorph described for W. subiculosa (syn. Pseudogliomastix protea) (Gams & Boekhout 1985) characterized by branched, pigmented conidiophores and somewhat darker conidia. Our LSU rDNA sequences analyses showed that W. subiculosa belonged to the Coniochaetales. Barrina is a monotypic genus characterized by immersed ascomata, multispored asci, and one-celled, hyaline ascopores, which germinate within the ascus producing a phialidic anamorph (ascoconidia) (Ramaley 1997). Ramaley (1997) and Gams (2000) considered this genus to be related to Lasiosphaeriaceae and indicated a close resemblance of that anamorph with Lecythophora. Our results agree with Huhndorf et al. 1281 (2004), who demonstrated a close phylogenetic relationship between Barrina and Coniochaeta. The placement of Wallrothiella and Barrina in Coniochaetales revealed here (Figs 2–3) was unexpected because the ascospores of these genera are hyaline and do not have germ-slits. However, according to Rogers (1994), this feature is of poor phylogenetic value at the suprageneric level because the presence of germ-slit has showed a convergent evolution and appeared many times in different groups of fungi such as Hypocreales (Hypocreomycetidae) and Xylariales (Xylariomycetidae). Our results suggest that the presence of a phialidic anamorph in Coniochaetales is more phylogenetically informative than the presence of germ-slits, and that Barrina and Wallrothiella must be included in Coniochaetales. Synaptospora is another genus included in Coniochaetaceae (Eriksson & Hawksworth 1993) that has ascospores without Fig 8 – Some representative types of Coniochaeta ascospores. A. C. ostrea. B. C. ovata. C. C. punctulata D. C. saccardoi. E. C. savoryi. F. C. scatigena. G. C. verticillata. Bars [ 10 mm. 1282 a germ-slit. Its ascospores are one-celled, brown, and globose, and tend to fuse in groups (Cain 1957). Although some authors considered that its relationships with members of Coniochaetaceae are unclear (Huhndorf et al. 1999; Réblová 2002), our studies demonstrate that the absence of germ-slit is not an excluding character for pertaining to Coniochaetales. The type of perithecia and the absence of a germ-slit relate this genus to W. subiculosa. Seigle-Murandi et al. (1995) proposed the genus Coniolaria for a single isolate with some features typical of Coniochaeta D. Garcı́a et al. but with a holoblastic Dactylaria-like anamorph. Recently, Eriksson et al. (2004) accepted Coniolaria as a member of Coniochaetaceae. Our results demonstrated that the accommodation of this genus in Xylariales, as proposed by other authors (in http://www.indexfungorum.org), is more appropriate. Genera of Xylariaceae that do not have stromatic tissues are Ascotricha, Ascotrichella, and Calceomyces. Coniolaria can be differentiated from the genera mentioned by the type of anamorph, which have been described as Dicyma, Humicola-like, and Nodulisporium-like respectively (Hawksworth 1971; Fig 9 – Some representative types of Coniochaetaceae anamorphs. A. Barrina polyspora (Lecythophora-like). B. Coniochaeta boothii (chlamydospores). C. C. cypraespora (Paecilomyces-like). D. C. ligniaria (Lecythophora sp.). E. C. mirabile (Cladobotryumlike). F. C. ostrea (clamydospores). G. C. vetulina (Lecythophora sp.). H. C. verticillata (Verticillium-like). I. Wallrothiella subiculosa (Pseudogliomastix protea). Bars [ 10 mm. Molecular phylogeny of Coniochaetales Udagawa & Ueda 1988; Valldosera & Guarro 1988; Whalley 1996; Rogers et al. 2002; Fournier & Magni 2004). Moreover, Calceomyces has reticulate ascospores and asci with an amyloid apical ring (Udagawa & Ueda 1988). In the original description of C. murandii, a Dactylaria anamorph was described and illustrated (Seigle-Murandi et al. 1995). However, we have examined an ex-type culture of this species and noticed some characters not typical of that genus. Dactylaria forms hyaline conidia from cylindrical denticulate conidiogenous cells on sympodially proliferating erect conidiophores (de Hoog 1985; Goh & Hyde 1997). However, C. murandii (i.e. C. gamsii) has branched conidiophores from which emerge pigmented conidia that often give rise to one or several short chains of secondary conidia. These characters do not fit with the description of Dactylaria, but they are distinctive of Rhinocladiella (de Hoog 1977), which has some resemblance to Geniculosporium and Nodulisporium (Barron 1983). Rhinocladiella has been connected with some xylariaceous genera such as Hypoxylon and Obolarina (Petrini & Müller 1986; Candoussau & Rogers 1990; Laessøe & Spooner 1994; Whalley 1996), but in 1283 recent revisions of Xylariaceae its anamorphs have been assigned to Nodulisporium- or Virgariella-like genera (Ju et al. 1998; Rogers et al. 2002). In addition, some molecular studies have connected Rhinocladiella with Capronia (Herpotrichiellaceae, Chaetothyriales) (Spatafora et al. 1995; Haase et al. 1999). Further studies are required to ascertain a more concrete position of the anamorph of C. gamsii among the hyphomycetes genera. Coniolaria has some morphological resemblance to Obolarina. Both genera have non-amyloid short stipitate asci without a distinct apical apparatus (Candoussau & Rogers 1990; SeigleMurandi et al. 1995; Rogers et al. 2002; Fournier & Magni 2004). However, Obolarina has bipartite, multiperitheciate stromata, an anamorph characterized by unbranched coniodiophores, and inconspicuous conidial secession scars, ramoconidia being absent (Candoussau & Rogers 1990). The generic name Coniolaria was not published validly so we propose the new name Coniolariella here. Recently, the new species Coniochaeta gamsii identical to the previously not validly published name C. murandii has been described from barley leaves in Iran (Asgari & Zare 2006). Fig 10 – Coniocessia nodulisporioides. A. Detail of the peridium. B–D. Asci. E–G. Ascospores. H. Ascospores (SEM). I–M. Anamorph (SEM). Bars [ 10 mm. 1284 Taxonomy Coniochaeta (Sacc.) Cooke, Grevillea 16: 16 (1887). (Figs 8, 9). Coniochaetidium Malloch & Cain, Can. J. Bot. 49: 878 (1971). Ephemeroascus Emden, Trans. Br. mycol. Soc. 61: 599 (1973). Poroconiochaeta Udagawa & Furuya, Trans. mycol. Soc. Japan 20: 5 (1979). Ascomata superficial or semi-immersed in the natural substrate, superficial or sometimes immersed in culture, pyriform and ostiolate or globose and non-ostiolate; setose, hairy or glabrous; dark or slightly pigmented. Peridium membranaceous to pseudoparenchymatous, rarely coriaceous; textura angularis, intrincata, or less frequently cephalothecoid. Asci cylindrical, clavate, sub-globose or near globose, short stipitate, with a truncate to rounded apex, usually with a conspicuous to indistinct non-amyloid apical ring, 4-, 8- to multispored, thin-walled, evanescent. Hamathecium paraphysate or absent. Paraphyses when present numerous, hyaline, filiform, simple, septate, and evanescent. Ascospores narrowly ellipsoid to fusoid, broadly ellipsoidal to globose, lenticular or cruciform, with rounded to apiculate ends, flattened on one or both sides; one-celled, smooth or pitted; brown to dark brown, olive-greenish to dark olivaceous or black; germ-slit straight, longitudinal, conspicuous or indistinct, rarely go all the way around the spore; sheath hyaline, gelatinous, present in some species. Anamorphs belonging to Lecythophora, Verticillium, Paecilomyces, and Cladobotryum. Type species: Coniochaeta ligniaria (Grev.) Massee, Grevillea 16: 37 (1887). Note: According the on-line version of the Index Fungorum (http://www.speciesfungorum.org/Names/Names.asp), the genus Coniochaeta includes around 70 species and six synonyms. Although that was not monographed, three articles included keys for species of Coniochaeta known in pure culture (Hawksworth & Yip 1981), and isolated from Argentina (Romero et al. 1999) and Spain (Checa et al. 1988). The larger synopsis of Coniochaeta by Mahoney & LaFavre (1981), and includes comparative tables for 33 species. Coniochaeta boothii (Manohar. & P. Rama Rao) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 7C and 9B) Basionym: Thielavia boothii Manohar. & P. Rama Rao, Trans. Br. mycol. Soc. 61: 196 (1973). Coniochaetidium boothii (Manohar. & P. Rama Rao) Arx, Stud. Mycol. 8: 26 (1975). Strain examined: CBS 381.74T D. Garcı́a et al. Basionym: Coniochaetidium mirabile Udagawa & Tsub., Mycotaxon 27: 63 (1986). Coniochaeta nuciforme (Guarro, Gené, Al-Bader & Abdullah) D. Garcı́a, Stchigel & Guarro, comb. nov. (Fig. 7I) Basionym: Coniochaetidium nuciforme Guarro, Gené, Al-Bader & Abdullah, Mycoscience 38: 123 (1997). Strain examined: FMR 5776 Coniochaeta ornata D. Garcı́a, Stchigel & Guarro, nom. nov. (Figs 6B–C, and 7J) Basionym: Poroconiochaeta tetraspora D. Garcı́a, Stchigel & Guarro, Mycologia 95: 525 (2003). Strain examined: FMR 7408T Non C. tetraspora Cain 1961. Coniochaeta ornata has ostiolate and nonostiolate ascomata, and four-spored asci. It can be easily differentiated from C. tetraspora by its pitted ascospores. Coniochaeta ostrea (Malloch & Cain) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 8A and 9F) Basionym: Coniochaetidium ostreum Malloch & Cain, Can. J. Bot. 49: 879 (1971). Strain examined: CBS 507.70T Coniochaeta punctulata (Udawaga & Furuya) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 6D-E, and 8C) Basionym: Poroconiochaeta punctulata Udagawa & Furuya, Trans. mycol. Soc. Japan 20: 8 (1979). Strain examined: FMR 7408 Coniochaeta savoryi (C. Booth) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 6F and 8E) Basionym: Thielavia savoryi C. Booth, Mycol. Pap. 83: 5 (1961). Synonyms: Coniochaetidium savoryi (C. Booth) Malloch & Cain, Can. J. Bot. 49: 880 (1971). Germslitospora savoryi (C. Booth) Lodha, in Subramanian, Tax. Fungi 1: 250 (1978). Poroconiochaeta savoryi (C. Booth) D. Garcı́a, Stchigel & Guarro, Mycologia 95: 528 (2003). Strain examined: CBS 725.74T Coniochaeta verticillata (Emden) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 8G and 9H) Basionym: Ephemeroascus verticillatus Emden, Trans. Br. mycol. Soc. 61: 601 (1973). Strain examined: CBS 816.71T Coniochaeta discoidea (Udagawa & Furuya) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 6A and 7F) Basionym: Poroconiochaeta discoidea Udagawa & Furuya, Trans. mycol. Soc. Japan 20: 6 (1979). Strain examined: CBS 158.80T Coniocessia D. Garcı́a, Stchigel, D. Hawksw. & Guarro, gen. nov. Etym.: cessio (L)-distant, moved away, referred to the exclusion of C. nodulisporiodes from Coniochaeta, the type species of the new genus. Coniochaeta mirabile (Udagawa & Tsub.) D. Garcı́a, Stchigel & Guarro, comb. nov. (Fig. 7H). Ascomata subglobosa vel pyriformia, ostiolata. Asci cylindrici vel subcylindrici, structura apicalis nula. Ascosporae unicellulares, Molecular phylogeny of Coniochaetales 1285 Ascomata globosa, ostiolata, glabra. Asci cylindrici, structura apicalis nula. Ascosporae unicellulares, ellipsoideae, brunneae vel nigrae, cum sulco germinalis longitudinale. Conidiophora ramosa; cellulae conidiogenae integratae, denticulatae; conidia solitaria vel in series breves ad ramoconidia. Typus: Coniolariella gamsii (Agari & Zare) D. Garcı́a, Stchigel & Guarro 2006. Ascomata globose, ostiolate, glabrous, surrounded at the base by brown, thick hyphae. Stroma absent. Peridium pseudoparenchymatous to coriaceous, textura intricata to textura angularis. Hamathecium paraphysate. Asci cylindrical, 8-spored, without apical structures, non-amyloid. Ascospores one-celled, ellipsoidal, brown, dark brown to black, smooth, without sheath, germ-slit longitudinal. Conidiophores branched, slightly pigmented. Conidiogenous cells integrate, sub-hyaline to pale olive–brown, elongating sympodially, conidia-bearing denticles with pigmented scars. Conidia sub-hyaline to pale olivaceous, solitary or in small chains on ramoconidia, with pigmented scars at the base. Coniolariella gamsii (Asgari & Zare) D. Garcı́a, Stchigel & Guarro, comb. nov. (Figs 12–13) Basionym: Coniochaeta gamsii Asgari & Zare, Nova Hedwigia 82: 228 (2006). Synonym: Coniolaria murandii Seigle-Mur., Guiraud, Steiman & Sage, Cryptogamic Botany 5: 347 (1995); nom. inval. (Art. 36). Fig 11 – Coniocessia nodulisporioides. A. Asci with ascospores. B. Ascospores. C. Conidiophore, conidiogenous cells, and conidia. Bars [ 10 mm. atrobrunneae, cum sulco germinalis longitudinale. Conidiophora ramosa; cellulae conidiogenae integratae, polyblasticae et denticulatae; conidia laevia vel leniter verruculosa, subglobosa vel pyriformia. Typus: Coniocessia nodulisporioides. Ascomata superficial, subglobose to pyriform, ostiolate, glabrous or pilose. Stroma absent. Peridium pseudoparenchymatous to coriaceous; outer layer with textura intricata. Asci cylindrical to subcylindrical, without an apical ring. Hamathecium paraphysate. Ascospores one-celled, ellipsoidal, dark brown, smooth-walled, with a longitudinal germ-slit. Conidiophores branched; conidiogenous cells integrate, terminal, discrete, polyblastic, denticulate; conidia smooth-walled or slightly verruculose, subglobose to pyriform, apiculate at the base. Coniocessia nodulisporioides (D. Hawksw.) D. Garcı́a, Stchigel, D. Hawksw. & Guarro, comb. nov. (Figs 10–11) Basionym: Coniochaeta nodulisporioides D. Hawksw., Norw. J. Bot. 25: 15 (1978). Strain examined: CBS 281.77T Coniolariella D. Garcı́a, Stchigel & Guarro, gen. nov. Etym.: derived from the original name of the type, Coniolaria. Ascomata globosa, ostiolata. Paraphysis numerosa. Asci cylindrici, structura apicalis nula, stipitati. Ascosporae unicellulares, ellipsoideae vel citriformis, brunneae vel nigrae, cum sulco germinalis longitudinale. Conidiophora ramosa; cellulae conidiogenae integratae, sympodice producere; conidia solitaria vel in series breves, ellipsoidea, cilindrica, ramoconidia obclavata. Ascomata superficial, solitary or in small groups, globose, ostiolate, glabrous, dark brown to black, 500–1500 mm diam, surrounded by brown to dark brown, thick-walled smooth hyphae, 2–5 mm diam. Peridium pseudoparenchymatous to coriaceous, two-layered. Outer layer of textura intricata to textura angularis, brown to dark brown. Inner layer formed by several layers of thick walled, pale brown to brown, angular to sub-globose, 4–6 mm diam cells, of textura angularis to textura epidermoidea. Paraphyses numerous, filiform, septate, hyaline, 135–200  1.5–2 mm. Asci 8-spored, cylindrical, with rounded tip and without apical structures, non-amyloid, 100–130  11–16 mm, stipitate, stipe 14–20 mm long. Ascospores one-celled, obliquely uniseriate, broadly ellipsoidal to citriform, with apiculate ends, at first hyaline, becoming brown to black, thick-walled, smooth, 16–20  6–12 mm, without gelatinous sheath, germ-slit longitudinal, straight to slightly sinuous at one end. Conidial state growing on the ascomata or independently. Conidiophores macronematous, branched, up to 200 mm long, 2–3 mm wide, pale olive brown, smooth and septate. Conidiogenous cells integrated, subhyaline to pale olive-brown, smooth, cylindrical, polyblastic, elongating sympodially during conidiogenesis, with conspicuous conidia-bearing denticles, denticles plane to slightly prominent, less than 1 mm long, with pigmented scars, often apical branches secedes and functions as conidia (ramoconidia). Ramoconidia pale olivaceous, smooth, 1286 D. Garcı́a et al. Molecular phylogeny of Coniochaetales 1287 Fig 13 – Coniolariella gamsii. A. Conidiophores, conidiogenous cells, and conidia. B. Asci with ascospores. C. Ascospores. Bars [ 10 mm. thin-walled, one- or two-celled, obclavate, apiculate at the base with pigmented seceded scar, apices with small, pigmented, plane denticles, giving rise to secondary conidia, 14–19(–21)  2–4 mm. Conidia sub-hyaline to pale olivaceous, one-celled, cylindrical, rounded apices, with a distinct, small, brown spot; apiculate at the base with pigmented scar, 9–11  2–3 mm, solitary or in small chains on ramoconidia. Fig 12 – Coniolariella gamsii. A. Ascomata. B. Detail of the outer layer of the peridium. C. Detail of the inner layer of the peridium. D. Asci with ascospores. E. Detail of the ascus. F-H. Ascospores (LM). I–K. Ascospores (SEM). L–N. Conidiophores, conidiogenous cells, and conidia. O–Q. Detail of the formation of secondary conidia (ramoconidia). R–U. Conidiophores, conidiogenous cells, and conidia (SEM). Bars: A [ 100 mm, B–R [ 10 mm. 1288 Acknowledgements We are indebted to the curators of the Centraalbureau voor Schimmelcultures (Utrecht, CBS) and Fungal Strain Collection, Laboratoire Cryptogamie, Museum National d’Historie Naturalle (Paris, LCP) for providing strains, and to Nuria Pilas and Felix Gilgado for their technical assistance. D. G. is grateful for a fellowship grants from the Universitat Rovira i Virgili, and D. L. H. for support from the Ramón y Cajal programme of the Ministry of Science and Technology of Spain. This study was supported by the Ministry of Science and Technology of Spain, grant CGL 2004-00425/BOS. references Acero FJ, González V, Sánchez-Ballesteros J, Rubio V, Checa J, Bills GF, Salazar O, Platas G, Peláez F, 2004. Molecular phylogenetic studies on the Diatrypaceae based on rDNA-ITS sequences. Mycologia 96: 249–259. von Arx JA, 1973. Ostiolate and nonostiolate pyrenomycetes. 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