The phylogenetic position of Obolarina dryophila (Xylariales) Mycological Progress ISSN 1617-416X Volume 9 Number 4 Mycol Progress (2010) 9:501-507 DOI 10.1007/ s11557-010-0658-5 1 23 Your article is protected by copyright and all rights are held exclusively by German Mycological Society and Springer. This eoffprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Mycol Progress (2010) 9:501–507 DOI 10.1007/s11557-010-0658-5 ORIGINAL ARTICLE The phylogenetic position of Obolarina dryophila (Xylariales) Sylvie Pažoutová & Petr Šrůtka & Jaroslav Holuša & Milada Chudíčková & Miroslav Kolařík Received: 18 December 2009 / Revised: 7 January 2010 / Accepted: 11 January 2010 / Published online: 13 February 2010 # German Mycological Society and Springer 2010 Abstract The inconspicuous inner-bark parasite Obolarina dryophila is reported from wood of Quercus petraea and as an endophyte of Salix alba. In addition, viable ascospores of O. dryophila have been found in the gut of the oak bark weevil Gasterocercus depressirostris, suggesting a possible dissemination mechanism for the fungus. A phylogenetic analysis based on three genes (nrDNA, actin, β-tubulin) placed Obolarina inside the genus Biscogniauxia as a close relative of the oak pathogens B. atropunctata and B. mediterranea. Keywords Xylariales . Biscogniauxia . Camillea . RAxML analysis . Ascospore dissemination Introduction For a delimitation of taxa inside Xylariaceae, the following markers are used: perithecial ascomata embedded in more or less well-developed dark-colored stromata; cylindrical asci with an amyloid apical ring enabling discharge of S. Pažoutová (*) : M. Chudíčková : M. Kolařík Institute of Microbiology ASCR, Vídeňská 1083, 142 20 Prague 4, Czech Republic e-mail: pazouto@biomed.cas.cz P. Šrůtka Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, 165 21 Prague 6, Czech Republic J. Holuša The Forestry and Game Management Research Institute, Jíloviště-Strnady 136, 15604 Prague 5, Czech Republic ascospores; ascospores which are mainly pigmented with germ slits or pores; and predominantly holoblastic sympodial conidiation (summarized in Tang et al. 2009). However, several taxa are found that lack distinguishable apical structures: their asci are either of atypical shape or evanescent and the anamorph stage is often rare. Therefore, unequivocal taxonomic placement of these genera based on morphological markers is difficult. Stromata of all these taxa usually develop beneath or inside the bark so that active discharge of the ascospores is pointless. The systematic placement of such morphologically reduced genera Pulveria, Pyrenomyxa, and Phylacia was solved only recently. Læssøe (1994) suspected that Pulveria and Pyrenomyxa are congeneric. Stadler et al. (2005) relegated Pulveria species to the genus Pyrenomyxa based on chemical, morphological, and ultrastructural data. Phylogenetic study based on rDNA sequence comparison found that Pyrenomyxa is close to the Hypoxylon rubiginosum complex, whereas Phylacia is related to Daldinia and Entonaema (Bitzer et al. 2008). Another of these taxa, the monotypic genus Obolarina, was erected to accommodate Obolarina dryophila (Tul. & C. Tul.) Pouzar 1986. Macroscopic characters and lifestyle of Obolarina closely resemble those of Biscogniauxia. However, there are numerous differences on the micromorphological level. The amyloid apical apparatus is missing, the ascus stipes are lacking or very short, the asci are of atypical clavate shape, and the ascospores are arranged in partially biseriate and/or irregular manner, differing from the uniseriate ascospores typical for Xylariaceae (Pouzar 1986). Nordén and Sunhede (2001) studied Swedish collections of Obolarina. In some cases, they found rare stipitate asci as well as some asci with the apical thickenings similar to those observed by Candoussauand Rogers (1990) which Author's personal copy 502 may be remnants of an apical ring. The shape of asci was defined as clavate when young and oblong to elliptical later. Thickness of the Obolarina ascal wall remains constant during the ascus development, whereas in the Biscogniauxia species, it changes from thin to thick to medium. Ascospores of Obolarina are markedly inequilateral with a sigmoid germ slit. In Biscogniauxia, the upper part of a two-layered stroma is dehisced together with the bark, and at the exposed surface, the anamorphic stage appears first, preceding perithecial development. On the other hand, the stromata of Obolarina probably remain enclosed inside the bark during their whole development. Pouzar (1986) only found the dead stromata exposed. Cultivation of Obolarina from the ascospores was first achieved using stromata excised from the bark (Candoussau and Rogers 1990). These authors also observed an anamorph of Obolarina in the culture on acidified cornmeal agar, with terminal holoblastic conidiation and simple unbranched sporophores resembling poorly developed Rhinocladiella. The taxonomic position of Obolarina is unclear. Pouzar (1986) noted the macroscopic similarity to Biscogniauxia, but based on differences in micromorphology, he did not exclude an association with yet another group of stromatic Pyrenomycetes. Eriksson and Hawksworth (1986) speculated that Obolarina may be congeneric with Helicogermslita, due to the markedly spiral germ slit on their ascospores; however, scanning electron micrograph has shown that the germ slit of Obolarina was shallow and differed from that of Helicogermslita. Candoussau and Rogers (1990), considered Obolarina to be closer to Biscogniauxia, sharing the bipartite type of stroma and association with the host bark. The only published molecular data for Obolarina are those of SSU nrDNA that suggest its affinity to Xylaria (Andersson et al. 1995). The genus Biscogniauxia appears paraphyletic (SánchezBallesteros et al. 2000; Peláez et al. 2008) with species of the closely related genus Camillea Fr. clustering inside. Camillea species are almost entirely found in the Neotropics with only a few Paleotropical species (Læssøe et al. 1989; van der Gucht 1992; Hastrup and Læssøe 2009), whereas Biscogniauxia is ubiquitous. Camillea differs from Biscogniauxia by form-genera of anamorphs (predominantly Xylocladium vs Nodulisporium), ascospore color (hyaline vs colored), and richly ornamented ascospores lacking germ slit. However, Rogers et al. (2002) described C. labiatirima which has a germ slit and smooth spores. Apical rings of Camillea asci are high, angular, with a strong iodine reaction, whereas Biscogniauxia rings are flat. Camillea stromata are often erect, which does not occur in Biscogniauxia. Ju and Rogers (1996) classified both genera into Hypoxyloideae; they have Nodulisporium-like anamorphs, but differ from Hypoxyloideae in having bipartite stromata Mycol Progress (2010) 9:501–507 and lack of KOH-extractable stromatal pigments (Ju et al. 1998) During our study of wood insects in bark of broadleaved trees, cultures of O. dryophila have been isolated from ascospores originating from excised fungal stromata and from gut of the bark weevil Gasterocercus depressirostris Fabricius, 1793 (Coleoptera, Curculionidae) as well as from a symptomless inner bark layer of willow. Nuclear rDNA, actin, and β-tubulin gene sequences were analyzed phylogenetically to investigate ordinal and familial placement of Obolarina. The taxonomic position of this monotypic genus among the Xylariaceae was determined, and the results are presented here to add to the rather scarce data about the biology of this specialized fungus. Materials and methods Isolates and cultivations Mature stromata of Obolarina dryophila were excised from oak bark, and pure cultures were obtained as single-spore isolates from ascospores plated in a sufficient dilution on 2% MEA. Adults of G. depressirostris were reared from oak wood logs. The logs (0.8 m long, 0.1–0.3 m diam.) were cut in January to February and incubated in cage nets and polyethylene bags at 2–4°C. In April, they were transferred to 20–26°C and sprinkled bi-weekly with sterile water. The emerged weevils were killed by ethyl acetate vapor and dissected. Their gut content was observed microscopically in water, and pieces containing ascospores plated on 2% MEA. All cultivations were done at 24°C in the dark. Endophytic isolates were obtained from slivers of surface-sterilized inner bark of a branch of Salix alba using ethanol–sodium hypochlorite sterilization (Sieber and Hugentobler 1987). Cultures of all isolates were stored on slants of 2% MEA at 4°C. The isolates were deposited in the Culture Collection of Fungi (CCF, Faculty of Sciences, Charles University, Prague, CZ), the specimens of Obolarina from the Kuntínov hill and Mořina were deposited in the PRM herbarium (National Museum, Prague, CZ) (Table 1). Microscopy Spores were mounted in Melzer’s reagent and photographed and measured using an Olympus BX51 microscope equipped with a digital camera CAMEDIA and image-processing software QuickPHOTO Camera 2.2. Measurements were made from 50 ascospores. The statistical treatment of spore size data was done using Kyplot 2.0 beta 15 (Yoshioka 2002) available at http://www.priceless warehome.org/WoundedMoon/files/kyp2b15.exe. Author's personal copy Mycol Progress (2010) 9:501–507 503 Table 1 Isolates, their origin and respective GenBank accession numbers Species Locality Collector Obolarina dryophila Czech Republic, Kuntínov hill Šrůtka P. Obolarina dryophila Obolarina dryophila Obolarina dryophila Obolarina dryophila Biscogniauxia nummularia Czech Republic, Czech Karst, Mořina Czech Republic, Kuntínov hill France, Ariège, Rimont, Las Muros Slovakia, Jurský Šúr Šrůtka P. (PRM 915934b) Šrůtka P. (PRM 915933) Fournier J. (JF07198) Holuša J. Slovakia, Jurský Šúr Holuša J. Original substrate Quercus petraea (gut of Gasterocercus depressirostris) Quercus petraea (stromata) Quercus petraea (stromata) Quercus ilex (stromata) Salix alba (endophyte) Salix alba (endophyte) Culture accession no. Accession no. β-Tubulin Actin rDNA CCF 3914a GQ428319 GQ428307 GQ428313 CCF 3915 GQ428320 GQ428308 GQ428314 CCF 3916 GQ428321 GQ428309 GQ428315 CCF 3917 GQ428322 GQ428310 GQ428316 CCF 3918 GQ428323 GQ428311 GQ428317 CCF 3919 GQ428324 GQ428312 GQ428318 a CCF Culture Collection of Fungi, Department of Botany, Faculty of Sciences, Charles University, Benátská 2, 128 01 Prague, Czech Republic b PRM PRM Herbarium, Mycological Department, National Museum, Václavské nám. 68. 115 79 Prague, Czech Republic DNA extraction and PCR amplification DNA was prepared from young cultures using UltraClean Microbial DNA Isolation Kit (Mo-Bio Laboratories, Solana Beach, CA) according to the manufacturer’s manual. Nuclear rDNA containing ITS1, 5.8 S, ITS2, and D1D2 regions of 28 S) was amplified with the primers ITS5 or ITS1F combined with NL4 (White et al. 1990; Gardes and Bruns 1993) in a Mastercycler Gradient (Eppendorf, Hamburg) as follows: one cycle of 3 min at 95°C, 30 s at 55°C, and 1 min at 72°C; 30 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C and a closing cycle 30 s at 95°C, 30 s at 55°C, and 10 min at 72°C. The reaction mix consisted of PCR buffer (Finnzymes, Oy, Finnland), 0.2 mM deoxynucleotides, 2 pmol of each primer, and 1 U DynaZyme (Finnzymes) and 5–50 ng DNA in 25 µl of total volume. Part of the β-tubulin gene was amplified with the primers T1 and T22 (O'Donnell and Cigelnik 1997) and a fragment of the actin gene was obtained using primers ACT512F and ACT783R (Carbone and Kohn 1999). The reaction mixture for these amplifications was as above, except for addition of MasterAmp PCR enhancer from Epicentre Biotechnologies (Madison, WI); the annealing temperature of the PCR run was lowered to 52°C. Amplicons were custom-purified and sequenced at Macrogen (Seoul, Korea); the sequences were deposited in the NCBI database (Table 1). Sequence analysis For a preliminary ordinal and familial placement, each sequence was used as a query for BLASTN searches against the NCBI nonredundant nucleotide databases (Altschul et al. 1990), where Biscogniauxia and Camillea appeared as the closest relatives. Sequences of the species belonging to the closest related genera were then downloaded from the GenBank, aligned by MUSCLE web interface (http://www.ebi.ac.uk/Tools/muscle/index.html) (Edgar 2004), and manually corrected in the BioEdit program (Hall 1999) Phylogenetic analysis was done on two datasets with Annulohypoxylon squamulosum used as an outgroup. The nrDNA alignment (Rib) was constructed from 5.8 S/ITS nrDNA sequences and the calculations were based on the positions 34–90, 154–198, and 210–544 according to the outgroup (EF026139) including gaps in these intervals. The alignment consisted of 451 positions, 123 of them parsimonyinformative. In the alignments of actin and β-tubulin, all columns containing gaps were omitted from further analysis. The Biscogniauxia sequences in these two datasets originated from the study of Hsieh et al. (2005). As the Hsieh’s dataset did not contain sequences of B. nummularia—a type species of the genus Biscogniauxia—they were obtained in the present study. A test based on maximum agreement subtrees (de Vienne et al. 2007) confirmed the congruence of actin and β-tubulin trees (Icong =2.681, p value=1.02E−10); therefore, the alignments were pooled to a single dataset (TubAct) with 387 parsimony-informative positions out of 1,609. Phylogenetic analyses were carried out using RAxML version 7.0.4 with a rapid bootstrapping (1,000 replicates) (Stamatakis et al. 2008) and a bootstopping (Pattengale et al. 2009) running at the CIPRES web portal (Miller et al. 2009). For the Rib dataset, the analysis was performed under the GTR+G+I model of rate heterogeneity and ML estimate of parameter α. Subsequent thorough ML search was stopped after 300 bootstrap replicates by bootstopping Author's personal copy 504 criterion. Empirical base frequencies were: pi(A): 0.267536 pi(C): 0.244303 pi(G): 0.218232 pi(T): 0.269930. Model information: parameter α: 0.511761; invariant sites: 0.262819; substitution rates A–C: 1.928, A–G: 5.983, A– T: 1.927, C–G: 1.673, C–T: 11.096, G–T: 1.00; tree-length: 1.129963; ML optimization likelihood: −2974.657. For the TubAct dataset, the model was GTR with ML estimate of parameter α. Subsequent thorough ML search was stopped after 200 bootstrap replicates by bootstopping criterion. Empirical base frequencies were: pi(A): 0.196984 pi(C): 0.307859 pi(G): 0.247111 pi(T): 0.248046. Model information: parameter α: 0.286065; substitution rates A–C: 0.880, A–G: 3.443, A–T: 1.449, C–G: 10.821, C–T: 5.769, G–T: 1.00; tree-length: 1.129963; tree-length: 0.669142; ML optimization likelihood: −9133.234. In both trees bootstrap support values were drawn on best-scoring ML tree. Trees were drawn using MEGA4 (Tamura et al. 2007). Results Natural occurrences of Obolarina The oak isolates were obtained in the course of a study of xylophagous insects. From the samples collected at Kuntínov hill (Pannonian wood), apart from woodwasps and their parasites, various species of bark beetles were reared; among them were also 20–30 adults of G. depressirostris. In the guts of the dissected weevils, aggregates consisting of a mixture of wood fibers and remnants of dark fungal stromata were observed (Fig. 1). In these remnants, brown 1-guttulate inequilateral ascospores with sigmoid germ slits were found. The gut content of Gasterocercus weevils was spread on MEA and after 1–2 days some of the ascospores germinated into colonies. Fig. 1 Remnants of Obolarina dryophila stromata from the gut of Gasterocercus depressirostris. Note the intact ascospores. Scale bar 20 µm Mycol Progress (2010) 9:501–507 Fig. 2 Mature ascospores of Obolarina dryophila. Scale bar 20 µm The oak specimens from Kuntínov were searched for fruiting bodies. Only after stripping the bark, were the mature stromata containing similar ascospores found and which were identified as Obolarina dryophila by Prof. Jack D. Rogers (Washington State University) (Fig. 2). Fruiting bodies of the same fungus were also obtained from other locations and the cultures established. Ascospores from the Kuntínov specimen were longer ð13:5 < 16 17 < 19:2  4:5 < 5:5 6:5 < 7:7mmÞ, than those from Mořina ð12:5 < 13:5 16 < 18:5  5:0 < 5:5 6:5 < 7:0mmÞ. Another culture with the same morphology (young culture lanose, whitish, becoming yellow-green to dark green with age, non-sporulating) has been isolated as an endophyte from the inner bark of an otherwise symptomless willow branch. Conidiation was not observed in any of these cultures. Molecular results The sequences of rDNA and actin were identical for all O. dryophila isolates. In the β-tubulin sequence, there were two differences found between the endophytic isolate from willow and the isolates originating from oak and gut of the oak bark weevil. BLAST search (Altschul et al. 1990) of the NCBI database has shown that the sequences closest to those of O. dryophila were from Biscogniauxia species, and for the rDNA, also from species of Camillea. The highest similarities (with a query coverage 100%) found were as follows: rDNA (ITS1–5.8 S-ITS2 region) 93%, actin 96%, β-tubulin 89%. The topology of trees obtained from Rib and TubAct datasets was similar (Figs. 3 and 4). Two clades with a high support were found in both phylogenies: a rather heterogeneous clade A (containing O. dryophila and, in the Rib tree, also Camillea species) and Clade B (consisting of B. Author's personal copy Mycol Progress (2010) 9:501–507 505 Fig. 3 Phylogenetic tree generated from RAxML analysis based on ITS rDNA sequences. Bootstrap values ≥50% are shown above or below branches. The tree is rooted with Annulohypoxylon squamulosum. The associated GenBank accession numbers are given after the species name Obolarina dryophila Biscogniauxia mediterranea EF026134 100 Biscogniauxia mediterranea AJ246224 62 Biscogniauxia atropunctata AF201705 100 Biscogniauxia atropunctata AJ390412 Biscogniauxia atropunctata AJ390412 78 Biscogniauxia latirima EF026135 Biscogniauxia philippinensis var. microspora EF026136 71 81 Clade A 99 Endophyte (from Juniperus virginiana) EF419906 100 Biscogniauxia sp.(endophyte of Coffea arabica) EU009960 Biscogniauxia sp. SUT290 DQ322095 85 Camillea obularia AJ390423 Camillea tinctor AJ390421 66 Camillea tinctor DQ322082 94 Biscogniauxia cylindrispora EF026133 85 Biscogniauxia nummularia AJ246231 100 Biscogniauxia sp. (endophyte of Hevea brasiliensis) FJ884075 58 86 Biscogniauxia capnodes EF026131 96 Clade B Biscogniauxia nummularia AJ390415 73 56 Biscogniauxia anceps EF026132 Biscogniauxia marginata AJ390417 Biscogniauxia bartholomaei AF201719 Biscogniauxia repanda AJ390418 73 Biscogniauxia simplicior EF026130 Biscogniauxia arima EF026150 Annulohypoxylon squamulosum EF026139 0.05 Fig. 4 Phylogenetic tree generated from RAxML analysis based on combined actin and β-tubulin sequences. Bootstrap values ≥50% are shown above or below branches. The tree is rooted with Annulohypoxylon squamulosum. The sequences originate from the study of Hsieh et al. (2005) except for those of O. dryophila and B. nummularia which were obtained in this study 100 Obolarina dryophila (from Quercus) Obolarina dryophila (from Salix) 100 99 Biscogniauxia atropunctata Biscogniauxia philippinensis var. microspora 100 74 100 Biscogniauxia latirima Clade A Biscogniauxia mediterranea 64 Biscogniauxia latirima 97 Biscogniauxia uniapiculata Biscogniauxia formosana 96 100 Biscogniauxia cylindrispora 86 Biscogniauxia anceps 100 Biscogniauxia capnodes var. rumpens 89 100 Biscogniauxia capnodes Biscogniauxia granmoi Biscogniauxia simplicior Biscogniauxia arima Biscogniauxia citriformis Annulohypoxylon squamulosum 0.05 Clade B Biscogniauxia nummularia Author's personal copy 506 nummularia, B. anceps, and B. capnodes). O. dryophila grouped in both trees with B. atropunctata and B. mediterranea; in the Rib tree, this grouping has a weaker support than in the TubAct tree. Discussion Stromata of O. dryophila have been found so far only on oaks (Q. robur, Q. petraea; in France, also Q. pedunculata and Q. ilex) (Pouzar 1986; Candoussau and Rogers 1990; Nordén and Sunhede 2001). In the present study, O. dryophila was detected as an endophyte in the inner bark of an untypical host, Salix alba, where no signs of a stromatal formation have been observed. Similarly, the Biscogniauxia species closest to O. dryophila (B. atropunctata and B. mediterranea) produce fruiting bodies on oaks, causing cankers in water-stressed trees (Nugent et al. 2005), but they were revealed as endophytes in less typical niches, like a symptomless oak wood (Luchi et al. 2005), oak leaves (Hoffman et al. 2008), and even conifers (Hoffman and Arnold 2008). Our finding of viable ascospores in the gut of Gasterocercus depressirostris might represent an alternative mechanism of spreading for Obolarina. As its fruiting bodies remain hidden inside the host bark during the whole development, anamorphic spores are probably not formed and ascospores are not actively ejected, so the possibility for an ascospore dissemination might lie in bark insects. Gasterocercus beetles occur in older oak forests, and their larvae develop in sapwood of trunks and branches of drying oaks, which is also an ideal niche for Obolarina development. Consumption of B. atropunctata var. atropunctata stromata on Quercus virginiana by no less than 15 species of Coleoptera (both adults and larvae) was observed by Lawrence (1977) in Georgia (USA); some of the species preferred conidia, the other ones fed on the stromatic tissue. Lawrence also included a short list of European beetles recorded as feeding on Hypoxylon and Daldinia species, however, none of the species mentioned belonged to the family Curculionidae. With Obolarina and Camillea nested inside, the genus Biscogniauxia is paraphyletic. Only recently, paraphyly was also found in the genus Daldinia, where Entonaema liquescens and a new species and genus Ruwenzoria pseudoannulata, both with distinctly different morphological markers, were found wedged between two clades of Daldinia species (Stadler et al. 2009a). Moreover, one of the Daldinia clades is phylogenetically closer to the genera with such a drastically different morphology as Thamnomyces (Stadler et al. 2009b) and Phylacia (Bitzer et al. 2008), than to species from the other Daldinia clade. Despite considerable morphological heterogeneity, all these Mycol Progress (2010) 9:501–507 genera have similar metabolite profiles in both the cultures and stromata, sharing at least seven pathways of the polyketide biosynthesis (Stadler et al. 2004, 2009b). A similar situation seems to be present among the genera Biscogniauxia, Camillea, and Obolarina. Obolarina lacks a iodine-positive apical ring; however, thin ring-like iodinenegative structures observed by Nordén and Sunhede (2001) in some of the asci may represent the reduced discoid rings of Biscogniauxia. Only a bipartite stroma and short-stiped to sessile asci remain as the morphological characteristics common to these three genera, together with the more distantly related Whalleya and Theissenia (Ju et al. 2007). No morphological markers were found that would be typical for taxa residing in the clades A or B, but the species on the more ancestral positions of the phylograms tended to reduced or missing carbonaceous tissue enclosing ostioles (B. arima, B. marginata, B. repanda, B. simplicior, and B. bartholomaei) (Ju et al. 1998). Acknowledgements This work was supported by the Czech Institutional Research Concept No. AV0Z5020903 and Czech Science Foundation grant 206/07/0283. Thanks are due to J.D. Rogers, Z. Pouzar and M. 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