Fungal Diversity The genus Oxydothis: new palmicolous taxa and phylogenetic relationships within the Xylariales Iman Hidayat1,2,∗, Rajesh Jeewon3, Chaiwat To-anun1, and Kevin D. Hyde3∗ 1 Department of Plant Pathology, Chiang Mai University, Chiang Mai, Thailand Mushroom Research Centre, Moo3 Ban Phadeng, Pa Pae, Chiang Mai, 50150 Thailand 3 Centre for Research in Fungal Diversity, Department of Ecological & Biodiversity, The University of Hong Kong, Hong Kong SAR, PR China 2 Hidayat, I., Jeewon, R., To-anun, C. and Hyde, K.D. (2006). The genus Oxydothis: New palmicolous taxa and phylogenetic relationships within Xylariales. Fungal Diversity 23: 159179. Oxydothis (Xylariales) is an ascomycete genus, commonly encountered on decaying monocotyledons, such as palms. During our study on diversity of palmicolous fungi in northern Thailand, we encountered three new species of Oxydothis: O. cyrtostachicola, O. inaequalis and O. wallichianensis, and these are described and illustrated in this paper. The three novel species differ from other morphologically similar Oxydothis species in ascomata shape and ostiole position, ascal ring, and ascospore morphology. Phylogenetic affiliations of the new taxa with members of related ascomycete families within the Xylariales are discussed based on morphology and nrDNA sequence data. The phylogenetic relationships of Oxydothis and its familial placement remain obscure based on the 28S nrDNA sequence analyses. Large sub unit nrDNA (28S) gene sequences do not provide significant phylogenetic information concerning the evolutionary relationships of these xylariaceous fungi. ITS nrDNA sequence analyses, however, indicate that Oxydothis is more closely related to members of the Amphisphaeriaceae than Diatrypaceae or Xylariaceae. Key words: Arecaceae, new species, palm fungi, ribosomal DNA, Xylariales Introduction Oxydothis Penz. & Sacc. (Xylariales) includes more than 70 species, which are saprobic, endophytic or parasitic on members of the Gramineae, Liliaceae, Palmae and Pandanaceae (Penzig and Saccardo, 1897; Hyde et al., 2000). The genus is typified by Oxydothis grisea Penz. & Sacc. (Penzig and Saccardo, 1897) and delimited based on morphological features such as long ∗ Corresponding authors, Iman Hidayat, e-mail: hidayatiman@yahoo.com; Kevin D. Hyde, email: kdhyde@hkucc.hku.hk 159 cylindrical asci with a J+ subapical apparatus; and long fusiform to filiform, hyaline, bicelled ascospores, which taper from the centre to spine–like ends, pointed or rounded processes (Penzig and Saccardo, 1897; Hyde, 1994). The familial placement of Oxydothis, however, is still tentative. There is uncertainty as to which morphological characters should be used to delimit the genus, as its taxonomic and phylogenetic relationships with other members of the Xylariales are still not resolved. Based on morphological similarities with Leiosphaerella Höhn., and presence of horizontal and clypeate ascomata, Oxydothis was placed within the Amphisphaeriaceae (Muller and Arx, 1962; Muller and Arx, 1973; Wehmeyer 1976; Samuels and Rossman, 1987). The genus was, however, referred to the Physosporellaceae (Phyllachorales) by Barr (1976) and later transferred to the Hyponectriaceae based on the perithecial ascomata with papillate ostiole, and cylindrical asci (Hawksworth et al., 1995). A scanning Electron Microscopy (SEM) study of the ultrastructure of the Oxydothis ascus could not resolve the generic and familial affiliations of Oxydothis (Wong and Hyde, 1999). Wang and Hyde (1999) excluded Oxydothis from the Hyponectriaceae based on the arrangement of the ascomata, structure of the asci and ascospores, which are unlike Hyponectria buxi. Phylogenetic analyses of 5.8S nrDNA and internal transcribed spacer (ITS2) sequence data have revealed that Oxydothis has evolutionary affiliations with members of the Clypeosphaeriaceae (Kang et al., 1998). Although Clypeosphaeriaceae was found to be heterogeneous, Kang et al. (1999b, 2002) placed Oxydothis within the family. Phylogenies analyses of 28S and 18S nrDNA revealed closer affiliations of Hyponectria with Oxydothis and Appendicospora, although these relationships had poor statistical support (Smith et al., 2003). During our study on diversity of palmicolous fungi in northern Thailand, we collected four species of Oxydothis. There have been a number of novel ascomycetous taxa collected from Thailand (Chatmala et al., 2004; Pinruan et al., 2004; Somrithipol and Jones, 2005; Hunter et al., 2006), but this is the first time we identified three new Oxydothis species. These are Oxydothis cyrtostachicola sp. nov. is described from decaying fronds of Cyrtostachys renda (Arecaceae), while Oxydothis wallichianensis sp. nov. and O. inaequalis sp. nov. are described from decaying leaves and fronds of Wallichia siamensis (Arecaceae), respectively. The new species are described and illustrated in this paper. In addition we performed phylogenetic analyses of partial 28S and ITS + 5.8S nrDNA sequence data to elucidate possible familial placement of Oxydothis within the Xylariales. 160 Fungal Diversity Materials and methods Microscopic examination Decaying fronds of Wallichia siamensis and Cyrtostachys renda were collected in northern Thailand during July and October 2005. The microscopic observation of Oxydothis spp. was performed as outlined in Shenoy et al. (2005) and Photita et al. (2005). DNA extraction, amplification and sequencing The total genomic DNA from the novel fungal species was extracted directly from the ascomata growing on the palm substrate, following a modified protocol of Hirata and Takamatsu (1996). Ascomata were picked up using fine forceps, and suspended in 500 µl of 5% Chelex solution (Bio-Rad, Richmond, Calif.). Ascomata were disrupted by means of a micropestle and content vortexed thoroughly for 1 min. Tube was incubated at a temperature of 100˚C for 15 mins and vortexed again for 1 min to allow maximum disruption. The contents were centrifuged at a speed of 14000g for 30 seconds and the supernatant was transferred to a new tube. This sample was directly used as templates for PCR. DNA amplification was performed by polymerase chain reaction (PCR). For partial 28S nrDNA amplification, LROR and LR5 primers (Vilgalys and Hester, 1990) were used, while ITS5 and ITS4 primers (White et al., 1990) were used for ITS nrDNA amplification. The amplification procedure for ITS followed the protocol outlined in Jeewon et al. (2004) and Wang et al. (2005), while details for PCR conditions for 28S are outlined in Cai et al. (2005). Total reaction volume used was 25 µl. The PCR products spanning approximately 850 bp (partial 28S nrDNA) and 550 bp (ITS nrDNA), were checked on 1% agarose electrophoresis gels stained with ethidium bromide. The amplified PCR products were purified using minicolumns, purification resin and buffer according to the manufacturer’s protocol (Amersham Biosciences, Catalog no. 27–9602–01). DNA sequencing was carried out using the above-mentioned primers in an Applied Biosystems 3730 DNA Analyzer at the Genome Research Centre, The University of Hong Kong. Sequence alignment and phylogenetic analyses For each species of Oxydothis, sequences obtained from the respective primers (LROR and LR5; ITS5 and ITS4) were aligned in Clustal X (Thomson Table 1. Sequences used in the analyses. 161 Genbank accession no. ITS nrDNA Oxydothis cyrtostachicola DQ 660334 Oxydothis daemonoropsicola DQ 660335 Oxydothis inaequalis DQ 660336 Amphisphaeria sp. AF375998 Arecophila bambusae Arecophila sp. Asordaria arctica AY681175 Bartalinia bischofiae Bartalinia laurina AF405302 Bartalinia robillardoides AF405301 Bertia moriformis Bionectria ochroleuca Bionectria pityrodes Cainia graminis Camarops tubulina Camarops ustulinoides Chaetomium globosum Clypeosphaeria mamillana AF009808 Cryptosphaeria pullmanensis AJ302419 Cryptosphaeria subcutanea AJ302420 Diatrype bullata DQ006946 Diatrype disciformis Diatrype sp. DQ006957 Diatrype stigma AF192323 Discostroma fuscellum AF377284 Discostroma sp. Discostroma tosta AF009814 Discostroma tricellulare AF377285 Dothidea insculpta Dothidea sambuci Eutypa armeniacae DQ006948 Eutypa lata DQ006944 Eutypa sp. Eutypella vitis DQ006943 Halorosellinia oceanica Kretzschmaria clavus AJ390434 Lepteutypa cupressi Monographella albescens AJ132506 Species 162 28S nrDNA DQ 660337 DQ 660338 DQ 660339 AF452038 AF452039 AF382367 AF382369 AY695261 AY489716 AY489728 AF431949 AY346266 AY346267 AF286403 U47829 AF382380 DQ247802 AY544681 AY346280 AY083822 AF382379 Fungal Diversity Table 1 continued. Sequences used in the analyses. Species Monographella albescens Muscodor albus Muscodor albus Nitschkia grevillei Oxydothis frondicola Pestalotiopsis versicolor Pestalotiopsis westerdijkii Seiridium cardinale Seiridium unicorne Sordaria fimicola Truncatella angustata Ustulina deusta Xylaria acuta Xylaria arbuscula Xylaria hypoxylon Xylaria mali Xylaria polymorpha Xylaria sp. Genbank accession no. ITS nrDNA AJ132509 AY244622 AY527044 AF009803 DQ334862 DQ137856 AF377298 AF377299 AY681188 DQ093715 AF201718 28S nrDNA AY346294 AY083835 AF382357 AF382377 AY780079 AY544676 AY183369 AY327478 AF163040 AF163042 AY315404 AY544648 et al., 1997) and Bioedit (Hall, 1999). In total, two datasets were analysed: Dataset based on 28 nrDNA sequences (Dataset I), and Dataset based on 5.8S nrDNA (Dataset II). Genbank accession numbers and taxa used are listed in Table 1. Phylogenetic analyses were performed in PAUP* (Swofford, 2002). Ambiguously aligned sites were excluded from the all analyses. Unweighted parsimony (UP) and weighted parsimony (WP) analyses were performed. Gaps were treated as missing data. WP analyses were also performed using a symmetric step matrix generated with the program STMatrix version 2.2 (François Lutzoni and Stefan Zoller, Department of Biology, Duke University, Durhan, NC). Trees were inferred using the heuristic search option with 1000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics (tree length [TL]. consistency index [CI], retention index [RI], related consistency index [RC], homoplasy index [HI], and log likelihood [-ln L]) were calculated for trees generated under different optimality criteria. Clade stability was assessed in bootstrap analyses with 1000 replicates, each with 10 replicates of random stepwise addition of taxa. Random sequence addition was used in the bootstrap analyses. Kishino-Hasegawa tests (Kishino and Hasegawa, 1989) were performed in order to determine whether trees were 163 significantly different. Trees were figured in Treeview (Page 1996). Other details are outlined in Arenal et al. (2005) and Promputta et al. (2005). The best-fit model of evolution was determined by MrModeltest2.2 (Posada and Crandall, 1998). Posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (BMCMC) in MrBayes 3.0b4 (Huelsenbeck and Ronquist, 2001), using above estimated model of evolution. Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100th generations (resulting 10,000 total trees). The first 1,000 trees that represented the burn-in phase of the analyses were discarded and the remaining 9,000 were used for calculating posterior probabilities (PP) in the majority rule consensus rule tree. The analyses were repeated five times starting from different random trees to ensure trees from the same tree space were being sampled during each analyses. Posterior probabilities equal to and above 95% were regarded as significant. Results Taxonomy Oxydothis cyrtostachicola Hidayat, To-anun & K.D. Hyde, sp. nov. Mycobank 510055 (Figs 1-4) Etymology: In reference to the host genus, Cyrtostachys. Ascomata 130–155 µm diam, 20–34 μm alta, immersa, subglobosa, ostiolata. Asci 102– 120 × 12–13 µm, 8-spori, pedunculati, aparatu apicale J–, praediti Ascosporae 48–52 × 5–6 µm, hyalinae, fusiformis, bicellulares. Ascomata forming under slightly raised, ellipsoidal regions on the host surface, black border, solitary or in groups 2–3; in section immersed, subglobose, ostiole eccentric, long axis horizontal to that of the host surface with neck at one end, forming ca 130–155 µm diam. × 20–34 µm high. Peridium comprised of 2–3 layers outer layers of oblong, dark-brown cells and with an additional inner layer of oblong, hyaline cells. Paraphyses deliquesce early. Asci 102–120 × 12–13 µm, 8-spored, unitunicate, clavate, short pedicellate, J-, refractive subapical ring, has a canal leading to the apex. Ascospores 48–52 × 5–6 µm, fusiform, 1-septate, hyaline, tapering gradually from the central septum to pointed processes, without spine-like form. Anamorph: Unknown. Habitat: Saprobic on Cyrtostachys renda fronds. Known distribution: Northern Thailand. Material examined: THAILAND: Chiang Mai, Chiang Mai University garden (latitude 18.84.00; longitude 98.97.00), on petioles of Cyrtostachys renda, 30 October 2005, FIH 151 (MRC 0007; holotype designated here). 164 Fungal Diversity Figs 1-4. Micrographs of Oxydothis cyrtostachicola (from holotype). 1. Vertical section through the ascoma. 2. Asci and ascal ring. 3-4. Ascospores. Bars: 1 = 20 µm; 3-4 = 5 µm. Isotype: THAILAND: Chiang Mai, Chiang Mai University garden (latitude 18.84.00; longitude 98.97.00), on petioles of Cyrtostachys renda, October 30, 2005, FIH 151 (HKU (M) 17170). Oxydothis daemonoropsicola J. Fröhl. & K.D. Hyde Material examined: THAILAND: Chiang Mai, Doi Suthep (latitude 18.48.00; longitude 98.56.00), decaying rachis of Wallichia siamensis, 21 July, 2005, Iman Hidayat FIH 019 (MRC 0005, 0006). Oxydothis inaequalis Hidayat, To-anun & K.D. Hyde, sp. nov. Mycobank 510054 (Figs 5-10) Etymology: From the Latin inaequalis meaning “unequal, various” in reference to the ascospore processes that appear slightly unequal in length. 165 Figs 5-10. Micrographs of Oxydothis inaequalis (from holotype). 5. Vertical section through the ascoma. 6. Ascomata on the host surface. 7-8. Ascospores. 9. Ascus. 10. Ascal ring. Bars: 5 = 110 µm; 6 = 10 mm; 7 = 7 µm; 8 = 7.5 µm; 9 = 12 µm; 10 = 4 µm. 166 Fungal Diversity Ascomata 110–120 µm diam, 22–30 µm alta, immersa, ellipsoida, ostiolata. Asci 200– 285 × 11.25–13.75 µm, 8-spori, unitunicati, pedunculati, aparatu apicale J+, 3.5–4 µm alti, 3– 3.5 µm diam. praediti Ascosporae 78–100 × 5–6.25 µm, 1-2 seiate, fusiformis, hyalinae, bicellulares. Stromata 5–40 mm long × 5–10 mm wide, visible as blackened ellipsoidal regions on the host surface, lacking borders. Ascomata 110–120 µm diam. × 22–30 µm high, forming slightly raised domes, singly or clustered in groups up to 10 (mostly 2–3); in section immersed, ellipsoid, long axis horizontal to that of the host surface, papilla at one end curving upwards to the host surface. Stromatic tissue surrounds the ascomata within the host hypodermis. Peridium 10–12.5 µm thick, comprised of 2–3 layers; outer layers of oblong, dark-brown cells and sometimes with an additional inner layer of oblong, hyaline cells. Paraphyses deliquescent early, septate, ca 2.5 µm in diam. Asci 200–285 × 11.25–12.5 µm, 8-spored, unitunicate, cylindrical, short pedicellate, J+, 4–6(–7) µm high, 3–4 µm diam., wedge-shaped, subapical ring, apically truncate. Ascospores (75–) 78–87.5 (–100) × 5–7.5 µm, 1–2 seriate, fusiform, 1-septate, hyaline, tapering gradually to form long pointed processes. The ascospores processes are sometimes uneven which may make the septum appear slightly eccentric. Anamorph: Unknown. Habitat: Saprobic on the Wallichia siamensis fronds. Known distribution: Northern Thailand. Material examined: THAILAND, Chiang Mai, Doi Suthep (latitude 18.48.00; longitude 98.56.00), decaying rachis of Wallichia siamensis, 21 July 2005, Iman Hidayat FIH 018 (MRC 0004; holotype designated here), Isotype: THAILAND, Chiang Mai, Doi Suthep (latitude 18.48.00; longitude 98.56.00), decaying rachis of Wallichia siamensis, 21 July 2005, Iman Hidayat FIH 018 (HKU (M) 17169). Oxydothis wallichianensis Hidayat, To-anun & K.D. Hyde, sp. nov. Mycobank No. 510053 (Figs 11-16) Etymology: In reference to the host genus, Wallichia. Ascomata 70–150 µm diam, 55–100 µm alta, subglobosa, ostiolata. Asci 87.5–125 × 10–15 µm, 8-spori, unitunicati, pedunculati, aparatu apicale J+, 2–2.5 µm alti, 3–3.5 µm diam. praediti. Ascosporae 32.5–55 × 6.25–7.5 µm, 1-2 seriate, fusiformis, hyaline, bicellulares. Stromata 20–30 mm long × 15–25 mm wide, surrounded by ellipsoidal, brown borders. Ascomata 70–150 µm diam. × 55–100 µm high, stromata domes on the host surface, mostly clustered in groups of up to 18; in section immersed to erumpent, subglobose, papilla at one end curving upwards to the host surface. Peridium 5–8.75 µm thick comprised of 2–3 layers outer layers of oblong, dark–brown cells. Asci 87.5–125 × 10–15 µm, 8-spored, unitunicate, cylindrical, pedicellate, with a J+, 2–2.5 µm high × 3–3.5 µm diam., wedgeshaped, subapical ring. Ascospores 32.5–55 × 6.25–7.5 µm, 1–2-seriate, 167 Figs 11–16. Micrographs of Oxydothis wallichianensis (from the holotype). 11-12. Ascomata on the host surface. 13. Vertical section through the ascoma. 14. Peridium. 15. Ascospores. 16. Asci and ascal ring. Bars: 11, 12 = 2 mm; 13, 16 = 50 μm; 14 = 5 μm; 15 = 10 μm. fusiform, 1-septate, hyaline, tapering abruptly near the ends to form long spinelike processes. Anamorph: Unknown. Habitat: Saprobic on Wallichia siamensis leaves. Known distribution: Northern Thailand. 168 Fungal Diversity Material examined: THAILAND: Chiang Mai, Doi Suthep (latitude 18.48.00; longitude 98.56.00), on decaying leaves of Wallichia siamensis, 21 July 2005, Iman Hidayat FIH 010 (MRC 0002; holotype designated here). Isotype: THAILAND: Chiang Mai, Doi Suthep (latitude 18.48.00; longitude 98.56.00), on decaying leaves of Wallichia siamensis, 21 July 2005, Iman Hidayat FIH 010 (HKU (M) 17174). 28S nrDNA phylogenies The 28S nrDNA dataset consisted of 26 taxa. The other reference taxa included members of known ascomycete families of the Sordariomycetes. Dothidea sambuci and D. insculpta were the designated outgroups. The final dataset comprised 913 characters. The dataset was deposited in TreeBASE (http://www.treebase.org/) under the study accession no. S1603. Likelihoodratio test in MrModeltest2.2 suggested that the best fit model of evolution for this dataset was SYM+I+G. Thirty-nine characters (ambiguous regions) were excluded in the analyses. In parsimony analyses, when gaps were treated as missing data, there were 505 constant characters, 95 parsimony-uninformative characters, and 274 parsimony–informative characters for both UP and WP. Two trees were obtained from UP and WP analyses. Based on K-H test (P* ≥ 0.05), these four trees were not significantly different (details not shown). The single parsimonious tree (TL = 899, CI = 0.600, RI = 0.688, RC = 0.412, HI = 0.400, -ln L = 5890.26251) generated from WP analyses with Dothidea sambuci and D. insculpta as outgroups and treating gaps as missing data is shown in Fig. 17. Bootstrap values (equal to or above 50%) based on 1000 replicates are shown on the upper branches, while values of the posterior probabilities (PP) resulted from BMBMC analyses are represented on the lower branches. ITS nrDNA phylogenies This dataset consisted of 37 taxa and analyses covered the ITS and 5.8S region. Sordaria fimicola and Asordaria arctica were the designated outgroups. The final dataset comprised 612 characters. The dataset was deposited in TreeBASE (http://www.treebase.org/) under the study accession no. S1602. Likelihood-ratio test in MrModeltest2.2 suggested that the best-fit model of evolution for this dataset was GTR+I+G. One hundred and six characters (ambiguous regions) were excluded in the analyses. In parsimony analyses, when gaps were treated as missing data, there were 199 constant characters, 36 parsimony-uninformative characters, and 271 parsimonyinformative characters for both UP and WP. Four trees were generated from 169 Xylaria acuta * Xylaria hypoxylon 89 Xylariaceae Halorosellinia oceanica * Arecophila bambusae 100 81 Arecophila sp. Cainiaceae * Cainia graminis Oxydothis daemonoropsicola 95 Oxydothis frondicola Xylariales Oxydothis cyrtostachicola * Xylariomycetidae * Oxydothis inaequalis * Bartalinia laurina 86 Discostroma sp. 99 Amphisphaeriaceae 80 99 Lepteutypa cupressi Seiridium cardinale Diatrype disciformis 99 Diatrypaceae Eutypa sp. 100 Bionectria ochroleuca Bionectria pityrodes 97 Bertia moriformis 100 Nitschkia grevillei 73 Camarops tubulina Camarops ustulinoides 99 98 Chaetomium globosum Sordaria fimicola Sordariomycetidae 100 Hypocreomycetidae 99 Dothidea sambuci Out group Dothidea insculpta 10 Fig. 17. Phylogenetic tree based on partial 28S nrDNA sequence data showing placement of members of the Oxydothis within class Sordariomycetes. Bootstrap values ≥ 50% from maximum parsimony analyses are shown above internodes and thickened branches indicate Bayesian analyses when posterior probabilities values ≥ 95%. Bootstrap values which are less than 50% shown by asterisk. 170 Fungal Diversity 99 Xylaria arbuscula 74 Xylaria hypoxylon Xylaria mali 54 Xylaria sp Kretzschmaria clavus 86 65 Xylariaceae Xylaria polymorpha 94 97 Ustulina deusta 100 Muscodor albus Muscodor vitigenus 52 Diatrype sp. 99 Diatrype bullata * 99 Eutypa lata Eutypa 99 * 99 Diatrypaceae Diatrype stigma 68 armeniacae Cryptosphaeria subcutanea Cryptosphaeria pullmonensis 91 Monographella albescens Clypeosphaeria mamillana 99 Seiridium cardinale 58 100 Seiridium unicorne Discostroma tosta 90 Discostroma fuscellum Amphisphaeria sp. 95 Discostroma tricellulare 99 Bartalinia robillardoides Bartalinia laurina 99 Truncatella angustata * 74 Amphisphaeriaceae * 96 Clypeosphaeriaceae Eutypella vitis 100 Monographella albescens 100 Pestalotiopsis versicolor Pestalotiopsis westerdijkii 98 Oxydothis frondicola Oxydothis daemonoropsicola 59 83 Oxydothis cyrtostachicola Oxydothis inaequalis Asordaria arctica Sordaria fimicola Out group 0.1 Fig. 18. Phylogenetic tree based on ITS nrDNA sequence data showing placement of members of the Oxydothis within the Xylariales. Bootstrap values ≥ 50% from maximum parsimony analyses are shown above internodes and thickened branches indicate Bayesian analyses when posterior probabilities values ≥ 95%. Bootstrap values, which are less than 50% shown by an asterisk. 171 = 0.318, HI = 0.543, -ln L = 6699.36613) generated from WP analyses and treating gaps as missing data is shown in Fig. 18. Bootstrap values (equal to or above 50%) based on 1000 replicates are shown on the upper branches, while values of the posterior probabilities (PP) resulted from BMBMC analyses are represented on the lower branches. Discussion Oxydothis is a common ascomycete genus, with 72 species which have been reported from monocotyledons and especially on palms (Hyde, 1994; Shenoy et al., 2005). Fröhlich and Hyde (2000) reported that Oxydothis was the most common ascomycete genus occurring on palms. The basic taxonomic features of Oxydothis were discussed by Hyde (1994), who emphasized on several morphological features to delimit the species, i.e., ascoma orientation, ascal ring size and shape, and ascospore apex structure. Table 2. Comparison of Oxydothis wallichianensis with similar Oxydothis species (measurements from Hyde, 1994c and Fröhlich and Hyde, 2000) Taxa O. wallichianensis O. batuapoensis Ascus Length (μm) 87.5–125 83.5–117.5 O. dispariapicis 224.2–277 O. licualicola 155–205 O. livistonicola O. parvula O. perangusta O. elaeidis O. sabalensis ca 260 110–130 96–130 95–130 95–130 Width (μm) 10–15 (4.9–) 5.3–7.2 14.1–17 (10.5–) 12.5–15 ca 10 8–10 6.4–8 14–16 14–16 Ascal ring Height (μm) 2–2.5 0.2–0.4 Ascospores Length (μm) 32.5–55 33.9–48.8 2–3.4 Width (μm) 3–3.5 1.3–1.55 (–2) 2.4–3 1.75–2.5 3 Width (μm) 6.25–7.5 2.9–3.4 (–4.1) (98–) (6–) 115–132.5 7.3–8.8 60–80 5–7.5 1.2–1.6 1–1.6 0.96–1.45 1.6 0.8–1 2.4–3.2 2–2.8 1.1–1.45 2.4–3.2 2.6–4 74–96 49–62 48–64 44–56 44–56 6–8 4–6 3–3.8 6–8 4–6 The three novel species differ from other morphologically similar Oxydothis species in ascomata shape and ostiole position, ascal ring, and ascospore morphology. The ascospores of Oxydothis wallichianensis are similar to those of eight other species and these taxa are compared in Table 2. Oxydothis elaeidis and O. sabalensis are the most similar species. The character that distinguishes O. wallichianensis from O. elaeidis is the ascal ring size, which is larger in the former (2–2.5 × 3–3.5 µm vs 1.6 × 2.4–3.2 µm). The grouping of ascomata (up to 20) under brown regions are also specific to 172 Fungal Diversity O. wallichianensis and distinguishes it from other similar Oxydothis species, such as O. parvula, O. sabalensis, O. batuapoensis where ascomata are mostly solitary. Oxydothis asymmetrica is the only Oxydothis species with similar ascospores to those of Oxydothis inaequalis, both in size and shape. The significant characters that distinguish it from O. inaequalis are ascus size (200– 285 × 11.25 µm vs. (6.8–) 8.1–11.9 (–12) µm) and ascal ring size (3.5–4 × 3– 3.5 µm vs. 0.8–1.55 × 2.1–2.5 µm) (Table 3). Oxydothis asymmetrica also differs in having lenticular ascomata in section with a central pore, while O. inaequalis has ellipsoidal ascomata with an eccentric orientation. Table 3. Comparison of Oxydothis inaequalis with O. asymmetrica (measurements from Hyde, 1994c; Fröhlich and Hyde, 2000) Ascus Length (μm) O. inaequalis 200-285 O. asymmetrica 157.1-208.8 Taxa Width (μm) 11.25–13.75 (6.8-)8.1-11.9(-12) Ascal ring Height (μm) 3.5-4 0.8-1.55(-7) Width (μm) 3–3.5 2.1-2.5 Ascospores Length Width (μm) (μm) 78-100 5-6.25 67.5-81.5 4.7-.5(-7) The third Oxydothis species from Wallichia siamensis is Oxydothis daemonoropsicola. The characteristics of ellipsoidal ascomata, long cylindrical asci, J+ ascal ring, and fusiform ascospores are typical of O daemonoropsicola. The size of asci, ascal ring, and ascospores are also close to those of the type as reported by Fröhlich and Hyde (2000) (Table 4). Table 4. Comparison of Oxydothis sp. FIH 019 with similar Oxydothis species (measurements from Hyde, 1994c; Fröhlich and Hyde, 2000). Taxa Ascus Length (μm) FIH 225–255 Width (μm) Oxydothis sp. 12.5– 019 13.75 O. daemonoropsicola 222.2–282.8 12.1–18.1 O. megalospora Ascal ring Height Width (μm) (μm) 2–2.25 2–3 Ascospores Length Width (μm) (μm) 95–105 5–6.25 2.55–3.8 2.55–3.8 91.8–112.2 5.1–7.7 3–5 103.7–126.9 5.6–7.6 224.5–292.8 11.7–16.6 3–7.5 Only four species of Oxydothis are characterized by a J– subapical ring: O. ianei (Taylor and Hyde, 2003), O. livistonae, O. nonamyloidea and O. nontincta (Fröhlich and Hyde, 2000). Species with J- subapical rings are distinguished by ascospore shape, size, and in having or lacking mucilage at 173 the ends. Oxydothis cyrtostachicola is characterized by fusiform ascospores, which taper gradually from the central septum to pointed processes, (not spine– like), and without mucilaginous drops at the ends. O. cyrtostachicola is closer to O. ianei and O. nonamyloidea, however, the asci and ascospores in O. cyrtostachicola are distinct from O. ianei and O. nonamyloidea in size. These taxa are compared in Table 5. Table 5. Comparison of Oxydothis cyrtostachicola with J– Oxydothis species (measurements from Hyde, 1994e; Fröhlich and Hyde, 2000; Taylor and Hyde, 2003) Taxa O. cyrtostachicola O. ianei O. nonamyloidea O. nontincta O. livistonae Ascus Length (μm) 102–120 108–174 205–260 (136–)146–200 Ca 300 Width (μm) 12–13 7.2–9.6 18–22 11–14 11–14 Ascospores Length (μm) 48–52 56–68 94–115 68–96 150–170 Width (μm) 5–6 2.6–3.8 3.5–4.5 5–6.4(–7) 4–5.5 Barr (1990) placed Oxydothis in the Hyponectriaceae based on its similarity with Pemphidium. Oxydothis is very similar to Pemphidium Mont. in ascomata, asci, and ascospore morphology. The latter, however, differs from Oxydothis in having a non-amyloid subapical ring and 1-celled, cylindrical ascospores with unusual polar appendages (Hyde et al., 2000). Wehmeyer (1975) placed Pemphidium in the Amphisphaeriaceae and this placement was accepted by Eriksson and Hawksworth (1991), who emphasized more on ascus and paraphyses morphology for it inclusion in the family (non–amyloid apical ring in asci is not typical of the Amphisphaeriaceae). Wang and Hyde (1999) later excluded Oxydothis from the Hyponectriaceae based on arrangement of ascomata, structure of asci, and ascospores. They also excluded Pemphidium from the Hyponectriaceae due to few similarities with Hyponectria. The non–amyloid subapical ring is not rare in Oxydothis since four reported Oxydothis species, O. ianei (Taylor and Hyde, 2003), O. livistonae, O. nonamyloidea and O. nontincta (Fröhlich and Hyde, 2000) have non-amyloid subapical rings. Samuels and Rossman (1987) reported a Selenosporella anamorph for Oxydothis selenosporellae; however Selenosporella is also a synanamorph of Iodosphaeria (Lasiosphaeriaceae; Sordariales) (Samuels et al., 1987). This may probably explain the distinctive evolutionary history of Oxydothis in relation to the members of the Amphisphaeriaceae, which are known to produce Pestalotiopsis-like anamorphs (Nag Raj, 1977; Kang et al., 1999a; 174 Fungal Diversity Jeewon et al., 2002; 2003). An ultrastructure of asci and ascospores of Oxydothis also suggested that Oxydothis is closer to the Diatrypaceae than the Amphisphaeriaceae (Wong and Hyde, 1999). We used 28S nrDNA sequence data to confirm the placement of Oxydothis within the Xylariales. Molecular phylogenies in previous studies (Kang et al., 1998, 1999b, 2002; Smith et al., 2003) supported the placement of Oxydothis within the Xylariales (Sordariomycetes). Morphological characters such as presence of pseudostroma, ascoma perithecial, papillate ostiole, periphysate, cylindrical asci with J+ ring, and transversely septate ascospores also supports its placement within the Xylariales. Although Oxydothis species are nested in the Xylariales with 80% bootstrap support, there is insufficient evidence to place Oxydothis within any known families of the Xylariales. We note that molecular analyses does not support Oxydothis bears a monophyletic group and in addition O. frondicola did not cluster with other Oxydothis as would be expected based on morphological evidence. Table 6. Morphological comparison of Oxydothis with Amphisphaeriaceae and Hyponectriaceae Morphological characters Stromata Oxydothis Amphisphaeriaceae Hyponectriaceae Pseudostroma Ascomata Crustose often clypeate Globose Subglobose or often ellipsoidal and long axis parallel to the host surface Long cylindrical Cylindrical Reduced (clypeus absent) Subglobose Asci shape Apical apparatus Ascospores colour Ascospores shape Ascospores ornamentation J+ or J– subapical ring, Usually J+, small variable in shapes (discoid, wedge shape, and cylindrical) and sizes Hyaline Hyaline to brown Cylindric–clavate to clavate Small J+ or J–, apical ring Hyaline to pale brown Long fusiform or filiform, Ellipsoidal to fusiform Various gradually tapering from the centre to pointed processes, which may be spine–like, or with round ends Often with small amounts Rarely ornamented; Lacking ornaments of mucilage without germ however often with pores germ pores 175 The association of Oxydothis with other known xylariaceous taxa receives very weak statistical support and most clades are not phylogenetically resolved. Therefore we refrain from making any systematic conclusions based on our 28S nrDNA sequence dataset. Another major observation in this study was the lack of statistical support for all the major nodes within the Xylariales. Even addition of more taxa with broader taxon sampling from all families failed to resolve some of the major clades. Similar results were obtained from previously published nrDNA phylogenies (Smith et al., 2003; Duong, et al., 2004; Bahl et al., 2005).Parsimony analyses of the ITS dataset supported the monophyly of Oxydothis with 59% bootstrap support. Oxydothis frondicola and O. daemonoropsicola are closely related to each other, while O. cyrtostachicola and O. inaequalis share close phylogenetic affinities and both of these relationships are strongly supported. This is in accordance with their ascospore morphology. Oxydothis frondicola and O. daemonoropsicola produce filiform ascospores, while O. cyrtostachicola and O. inaequalis posess fusiform ascospores which tapering gradually from the central septum to pointed processes. Phylogenetic results also indicate that Oxydothis share close phylogenetic affinities to the Amphisphaeriaceae (74% bootstrap support). It is therefore highly plausible that Oxydothis is more closely related to members of the Amphisphaeriaceae, but whether it should be accomodated within this family as have been postulated by Muller and Arx, (1962; 1973), Wehmeyer (1976), Samuels and Rossman (1987), and Samuels et al., (1987) based on morphology is still doubtful. On the other hand, whether Oxydothis should be accommodated in a new family will definitely need further detailed study with more species (including those from Pemphidium, Leiosphaerella, and Iodosphaeria) and sequence analyses based on protein-coding genes. Single gene phylogeny based on nrDNA is insufficient and inefficient in resolving phylogenetic relationships within the Xylariales (Smith et al., 2003; Bahl et al., 2005). We made several attempts to promote the formation of the anamorph of Oxydothis in culture and amplify partial RPB2 gene from a number of specimens but were unsuccessful. Although the phylogeny of the Hyponectriaceae is still obscure, we propose that Oxydothis should be excluded from the Hyponectriaceae and Amphisphaeriaceae. Morphological characters such as ellipsoidal ascomata with long axis parallel to the host surface, long cylindrical asci with canal leads to the apex, variability of apical apparatus shape (discoid, wedge shape, and cylindrical) and size, ascospores with long fusiform or filiform with gradually tapering from the centre to pointed processes, support the exclusion from those families (Table 6). 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