Mycosphere Doi 10.5943/mycosphere/3/5/8 Identification and comparison of Xylaria curta and Xylaria sp. from Western Ghats-Courtallum Hills, India Ramesh V1, Thalavaipandian A2, Karunakaran C3 and Rajendran A4* Department of Botany, Biomedical Research Laboratory, VHNSN College, Virudhunagar – 626 001, Tamilnadu, India 3 Department of Chemistry, Biomedical Research Laboratory, VHNSN College, Virudhunagar – 626 001, Tamilnadu, India 1,2,4 Ramesh V, Thalavaipandian A, Karunakaran C, Rajendran A 2012 – Identification and Comparison of Xylaria curta and Xylaria sp. from Western Ghats-Courtallum Hills, India. Mycosphere 3(5), 607–615, Doi 10.5943 /mycosphere/3/5/8 Xylaria curta and Xylaria sp., originating from evergreen forests of Courtallum Hills, Western Ghats Tamil Nadu, India were identified based on 18S rRNA gene sequence comparisons and morphological characteristics. These two species nested within a subclade that also contained X. curta from Thailand and X. longipes from Spain. Key words – Ascomycetes – MEGA 5 – Molecular phylogeny – Neigbour-joining method – Nuclear small subunit 18s RNA Article Information Received 23 August 2012 Accepted 14 September 2012 Published online 25 September 2012 *Corresponding author: Rajendran A – e-mail – arvhnsnbotany@yahoo.co.in Introduction Traditional classification and identification of fungi has relied upon microscopic features, colony characteristics on artificial media and biochemical reactions (Sutton & Cundell 2004). Such methods have served in the past but they have major drawbacks as they cannot be applied to non-cultivatable organisms and occasionally biochemical characteristic of some organisms do not fit into the patterns of any known genus and species. Amplification and sequencing of target regions within the ribosomal DNA gene complex has emerged as a useful adjunctive tool for the identification of fungi and does not depend on fungus sporulation for identification (Buzina et al. 2001, Iwen et al. 2002, Rakeman et al. 2005, Schwarz et al. 2006). The internal transcribed spacer (ITS) regions 1 and 2 located between the highly conserved small (18S) and large (28S) ribosomal subunit genes in the rRNA operon are known to have sufficient sequence variability to allow identification to the species level for many fungi (Brandt et al. 2005, White et al. 1990). Nucleic acid sequences from small subunit ribosomal RNAs (18s rRNAs) have proved useful for phylogenetic analysis in eukaryotes. Because of their ubiquity and evolutionary conservation these molecules are useful for inferring distant phylogenetic relationships providing a means of assessing relationship between organisms which lack any informative homologous morphological or developmental traits (Sogin et al. 1977, Woese 1987, Field et al. 1988). The Xylariaceae is a large and relatively well-known ascomycete family found in most countries (Whalley 1996), and it contains 35 genera (Eriksson & Hawksworth 1993). It is characterized by perithecial ascocarps bearing paraphyses and periphyses 607 Mycosphere Doi 10.5943/mycosphere/3/5/8 that are embedded in a stroma. The asci of most species bear a ring at the apex that appears as a characteristic amyloid ascal plug when stained with iodine. Many species actively decay wood of living or dead angiosperms and are known to be saprobic in most cases (Rogers 1979). Xylaria is a large and the first described genus of the Xylariaceae (Martin 1970). Xylaria species are saprobic or sometimes weakly to strongly parasitic on woody plants and usually have erect elongated stromata. Although they are found mostly on wood, some species are found on sawdust, leaf, dung or soil. Species of Xylaria are difficult to identify and classify especially as the stromata of a given species often vary greatly in colour, size and sometimes in general shape (Whalley 1996). Until now, identification of fungal species has been mainly done on the basis of morphological and microscopically characteristics but this is not suitable for differentiating closely related species of Xylaria. In the present study, Xylaria curta and Xylaria sp.R005 are described for the first time from southern Tamil Nadu, India based on molecular analysis as well as morphological characteristics. Materials and methods Fungal isolates Fruiting bodies of the two Xylaria strains were isolated from the soil and decomposing wood bark in tropical evergreen forest of Southern Western Ghats of Courtallum Hills, Tamil Nadu. The fruiting bodies were cleaned, air dried and stored at room temperature in paper bags. The studied specimens were deposited at the Department of Botany, VHNSN College, Virudhungar, Tamil Nadu. Cultures were initiated from perithecial contents or mycelial plugs of freshly collected stromata, propagated and studied as described by Stadler et al. (2005) on potato dextrose agar (PDA) medium at 30˚C. Pure cultures were maintained on PDA slants at -20˚C in culture collection. Cultures were grown in 500-ml Erlenmeyer flasks containing 200 ml of culture media (PDA). Radial growth rate of the isolates were measured in petri plates containing PDA after 15, 20, 25 and 30 days at 30˚C. Each specimen was examined for morphological characteristics of asci, ascospores, paraphyses and other structures of taxonomic value. Spore dimensions were determined for 50 spores. Lactophenol cotton blue and distilled water were used as mounting media for microscopy. Dried materials were rehydrated in 3% aqueous KOH. Photography was carried out with a light microscope and binocular microscope (COSLAP) with computer attached. DNA extraction, amplification and sequencing Fungal isolates were incubated 2–3 weeks at 30˚C on PDA. The mycelium was harvested and transferred into 2 ml plastic tubes using a spatula and lyophilized for DNA isolation. Total genomic DNA was extracted using the method of Doyle & Doyle (1987). The ITS1 5.8s and ITS2 regions of nrDNA were amplified using the primers ITS1 and ITS2 (White et al. 1990) and the 18S region was amplified using the (5'TCCGTAGGTGAACCTGCGG-3' and 5'TCCTCCGCTTATTGATATGC-3’) primers (White et al. 1990). Polymerase chain reactions (PCR) were performed using a Perkin Elmer 480 with 35 cycles of 94°C denaturation for 1 min, 50°C annealing for 30 s, and 72°C extension for 1 min with an additional 7 min extension at 72°C after cycling. The PCR amplicons were purified using Qiaquick spincolumns according to manufacturer protocols. All the PCR products were sequenced at Applied Biosystems, Foster City, California, USA and an additional internal 18S primer NS 1.5, NS 2, NS 4 (White et al. 1990) and BMBBR (Lane et al. 1985) were used to improve sequencing results. Isolates of 18s rRNA fungal sequences obtained were submitted to GenBank (NCBI, USA) (accession numbers: JF795289 and JF795290). All the studies of DNA extraction and isolation were done by Synergy Scientific Services, Chennai. Phylogenetic analysis Phylogenetic analysis was conducted in MEGA 5 software (Tamura et al. 2007). Sequenced ITS1-5.8S-ITS2 regions were aligned initially using the alignment algorithm Clustal W (Thompson et al. 1997) with the gap 608 Mycosphere Doi 10.5943/mycosphere/3/5/8 Fig. 1 – Macro and microscopic features of Xylaria curta. 1, 2. Natural habitat of fruiting bodies. 3. Cultural morphology on PDA plates (diameter 9 cm) after 2 weeks of incubation. 4. Ascal apical rings and ascospores in water (×200). 5. Cross section showing prethecia and ascospores 6. A partial rosette of maturing asci. 609 Mycosphere Doi 10.5943/mycosphere/3/5/8 open penalty 7.0 and gap extension penalty 4.0. Due to some variation in areas of ITS1 and ITS2 regions, an alignment was then improved manually. The evolutionary history was inferred using the neighbor- joining method (Saitou & Nei 1987). All positions containing gaps with missing data were eliminated from the dataset. Strengths of internal branches of resulting trees were statistically tested by the bootstrap analysis of 1000 replications (Felsenstein 1985). Additional sequences were retrieved from GenBank (Table 1). Results Identification of fungal strain The two fungal strains were identified as Xylaria curta and Xylaria sp. based on the nuclear ribosomal ITS1-5.8S-ITS2 sequence analysis. The ITS sequence analysis revealed that the fungal strains had more than 90% sequence similarity with those strains obtained from GenBank. Morphology and cultural characteristics Xylaria curta Fruiting bodies 6–15 cm in length, 0.5– 1 cm in broad (Fig. 1), growing either singly or in groups which are typically seen emerging from soil, dark brown, becoming darker at maturity. Stromata with fertile part is cylindrical-clavate, with rounded, fertile apices, unbranched, single or clustered, shortstipitate, stipe smooth and black. External surface is blackish with golden brown scales, roughened with small wrinkles, internally white and occasionally becoming hollow. Perithecia completely immersed, up to 0.5 mm diam., ostioles black, papillate. Asci 8-spored, uniseriate, cylindrical, stipitate, Ascospores are 17.3–17.8 µm broad, 33.1–33.8 µm in length. ellipsoid-inequilateral to broad ellipsoidinequilateral, dark brown, unicellular, smooth, germ slit conspicuous, straight, running fulllength of spore (Fig. 1). Substrate is undetermined decaying wood. Growth rate is high, 5.8–7.5 cm/week, covering petri dish in 6–8 days. Mycelial mat is white at early stage, later it is brown to black coloured. Hyphae are thin-walled and branched. The fruiting bodies arise in the spring. They may persist for several months or even years and can release spores continuously during this time. Xylaria sp.R005 Fruiting bodies 2–8 cm in length, 1–3 cm in diameter (Fig. 2), often growing in groups of three clustered into “finger-like or hand-shaped” forms which emerge around stumps or decaying trees, dark grey to brown, becoming dark black at maturity, subcylindric at first, becoming flattened; upper branches appear powdered white, finally tipped black when mature, stalk black and hairy. Stromata cylindrical, dichotomously branched several times or infrequently unbranched, surface with conspicuous perithecial mounds, wrinkled. Asci 8-spored, uniseriate, cylindrical, Ascospores are 15.1–16.5 µm broad, 30.1–33.5 µm long, brown to dark brown, unicellular, short with narrowly rounded ends, smooth. Growth rate is slow, colonies not reaching the edge of petri dish (9 cm diameter) in 1 month. The mycelial mat white at first, later black, mostly submerged, with irregular margins. Phylogenetic analysis Phylogenetic relationships inferred from ITS1-5.8S-ITS2 region sequences of Xylaria species are shown in Fig. 3. The tree is divided into two main clusters (A and B), each divided into two sub-clusters (A1 and B1). In sub-cluster A1 xylariaceae sp, X. longipes and Xylaria sp. were grouped together. X. curta, Xylaria sp. and X. longipes were grouped together in sub-cluster A2. In sub-cluster B1 Xylaria sp., X. juruensis and X. polymorpha were grouped together at a bootstrap value of 55%. X. levis, X. plebeja, X. polymorpha, X. curta and Xylaria sp. were grouped together at a bootstrap value of 72% in sub-cluster B2. Based on the sequence data the X. curta and Xylaria sp. were connected to each other in the same group and nested in a cluster consisting of X. longipes. The tree showed no phylogenetically close relationship between the two fungi and certain genera of Xylariaceae. Discussion This study provides molecular evidence for identification of Xylaria curta and Xylaria 610 Mycosphere Doi 10.5943/mycosphere/3/5/8 Table 1 ITS sequence data used in this study. Species Geographic origin GenBank No Xylaria polymorpha Xylaria sp.SOF11 Xylaria curta* Xylaria sp.XF10 Xylaria sp.CH2* Xylaria polymorpha Xylaria longipes Xylaria longipes Xylaria curta Xylaria curta Xylaria juruensis Xylaria laevis Xylaria plebeja Xylaria curta Xylaria sp.E10500C Xylariaceae sp.vega244 Xylariaceae sp.vega348 Xylariaceae sp.vega457 China China India India India Japan Netherlands Spain Taiwan Taiwan Taiwan Taiwan Taiwan Thailand USA USA USA USA AB274817 JF703668 JF795289 HQ435666 JF795290 AB512310 AF163038 AY909016 GU322443 GU322444 GU322439 GU324747 GU324740 DQ322144 JN572047 EU010004 EU009959 EU010003 Asterisks indicate the sequences obtained from the present study sp. in southern Tamil Nadu, India. Kshirasagar et al. (2009) reported that ten species of Xylaria from Western Ghats of Maharashtra could not be distinguished phylogenetically. Similarly, X. escharoidea and X. nigripes have been characterized by Rogers et al. (2005). The genus Xylaria shows great variation in morphology, but few phylogentic studies have been conducted to infer the relationships of taxa within the genus. Nuclear small subunit ribosomal RNA gene regions are usually used as a molecular tool to analyze fungal taxa at a family or order level and ITS regions are commonly used to examine phylogenetic positions or relationship at a species or intraspecies level. Morphological and anatomical data clearly differentiated these two species from each other. X. curta was found in soil whereas Xylaria sp.R005 was found in decaying wood. The morphological characters of these two Xylaria species are identical with X. angulosa (AB274814) found from soil (Rogers et al. 1987). On the other hand, many species of Xylaria are actively found in decaying wood of angiosperms and are known to be saprobic (Rogers 1979). In this study X. curta and Xylaria sp.R005 were differentiated from X. polymorpha and other species of Xylaria on the basis of variation in internal transcribed spacers ITS1 and ITS2. Our fungal strains formed a segregated clade with X. curta and Xylaria sp., supported by low bootstrap values of 68 and 72%, respectively (Fig. 3). Phylogenetic relationship of some Xylaria species was also studied by Lee et al. (2000) in which Xylaria species were classified into three groups based on morphological and molecular similarity, viz. X. apiculata, X. arbuscula, X. muli in group A, X. acuta, X. castorea, X. cornu-damae, X. enteroleuca, X. fioriana and X. longipes in group B, X. hypoxylon and X. polymorpha in group C. A few characters of ascospores, perithecia and stromata support the grouping of Xylaria inferred from molecular data, but there seems to be no character of universal significance that can justify the phylogenetic results. It may indicate that convergent evolution of characters occurred many times within Xylaria. Such possible changes in convergent evolution, along with variations associated with developmental stages of fruit bodies might have caused confusions in identifying and classifying Xylaria species. Phylogenetic analyses based on molecular data such as ITS 611 Mycosphere Doi 10.5943/mycosphere/3/5/8 Fig. 2 – Macro and microscopic features of Xylaria sp. 1. Natural habitat of fruiting body. 2. Morphological variability of fruiting body. 3. Cultural morphology on liquid medium (PDB) in 500 ml conical flask. 4, Cultural morphology on agar plates (diameter 9 cm) after 4 weeks of incubation. 5, 6. Ascal apical rings and ascospores in water (×200). 612 Mycosphere Doi 10.5943/mycosphere/3/5/8 Fig. 3 – Phylogenetic relationship between Xylaria species, inferred from ITS nucleotide sequence data. Bootstrap values are shown for those branches that had >30% support in a bootstrap analysis of 1000 replicates. The numbers of nucleotide changes among taxa are represented by branch length and scale bar equals the number of nucleotide substitutions per site. Asterisks indicate the sequence obtained from the present study. A and B indicates major clusters and 1 and 2 indicates sub-clusters referred to in the text. sequences of the present study proved to be very practical for taxonomic investigations at specific or generic levels in identification or classification of fungi with highly variable morphology like Xylaria. 613 Mycosphere Doi 10.5943/mycosphere/3/5/8 Increased taxon sampling from other parts of India and other continents are needed to elucidate the genetic diversity of Xylaria species complex. In future the phylogenetic structure will be increased through additional gene sequences. Acknowledgments Authors thank the Managing Board of Virudhunagar Hindu Nadar’s Senthikumara Nadar College, Virudhunagar-626 001, Tamil Nadu, India for providing research facilities. References Brandt ME, Gaunt D, Iqbal N, McClinton S, Hambleton S, Sigler L. 2005 - Falsepositive Histoplasma capsulatum GenProbe chemiluminescent test result caused by a Chrysosporium species. 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