Additions to the Genus Rhytidhysteron in Hysteriaceae
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Cryptogamie, Mycologie, 2016, 37 (1): 99-116 © 2016 Adac. Tous droits réservés Additions to the genus Rhytidhysteron in hysteriaceae Kasun M. THAMBUGALA a,b,c, Kevin D. HYDE b,c,d, Prapassorn D. EUNGWANICHAYAPANT c, Andrea I. ROMERO e & Zuo-Yi LIU a* aGuizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Guiyang, Guizhou 550006, People’s Republic of China bCenter of Excellence in Fungal Research, Chiang Rai 57100, Thailand cSchool of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand dKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, China ePrhideb-Conicet, Deptomento Cs. Biológicas,Facultad de Cs. Exactas y Naturales (UBA), Ciudad Universitaria,Pabellón II, 4to Piso,1428 Buenos Aires, Argentina Abstract – Rhytidhysteron (Hysteriaceae) species are widely distributed as saprobes or weak pathogens on a wide range of woody plants. In this study, several Rhytidhysteron collections were made in northern Thailand and multi-gene phylogenetic analyses were used to resolve the phylogenetic boundaries of species. Two novel species, R. thailandicum and R. neorufulum are introduced, based on morphological traits and multi-gene phylogeny. The genus is revised with a key to species. hysteriales / Phylogeny / new species / multi-gene phylogeny / Thailand INTroducTIoN Hysteriaceous fungi are primarily lignicolous or corticolous and are saprobes on wide host range in temperate and tropical regions (Boehm et al., 2009a, b; Murillo et al., 2009; Hyde et al., 2013; de Almeida et al., 2014; Liu et al., 2015; Yacharoen et al., 2015). The family Hysteriaceae was introduced by Chevallier (1826) as “Hysterineae” and this family has been treated with different genera by various authors (Zogg 1962; von Arx & Müller 1975; Kirk et al., 2001; Eriksson 2006; Lumbsch and Huhndorf 2010). Recent molecular phylogenetic analyses placed Hysteriaceae in Hysteriales, Pleosporomycetidae (Boehm et al., 2009a, b; Hyde et al., 2013; Wijayawardene et al., 2014). Hyde et al. (2013) and Wijayawardene et al. (2014) accepted 13 genera including Actidiographium Lar.N. Vassiljeva, Coniosporium Link, Gloniella Sacc., Gloniopsis De Not., Hysterium Pers., Hysterobrevium E. Boehm & C.L. Schoch, Hysterocarina H. Zogg, Hysteropycnis Hilitzer, Oedohysterium E. Boehm & C.L. Schoch, Ostreichnion Duby, Psiloglonium * Corresponding author: [email protected] doi/10.7872/crym/v37.iss1.2016.99 100 K. M. Thambugala et al. Höhn. Rhytidhysteron Speg. and Sphaeronaema Fr in the family, while de Almeida et al., (2014) introduced a new genus Hysterodifractum. Hysteriaceae is characterized by carbonaceous, immersed to erumpent to entirely superficial hysterothecia, distinctly navicular in outline, bearing a pronounced, longitudinal slit, running the length of the long axis, bitunicate asci and hyaline to pigmented, 1 to multi-septate, or muriform ascospores (Boehm et al., 2009a, b; Hyde et al., 2013; de Almeida et al., 2014). Rhytidhysteron was introduced by Spegazzini (1881) to accommodate R. brasiliense Speg. and R. viride Speg., without a type species being designated. Subsequently, Clements & Shear (1931) designated R. brasiliense as the type species (Samuels & Müller 1979; Yacharoen et al., 2015). Later, several species were added to the genus based on morphology (Spegazzini 1921; Sharma & Rawla 1985; Barr 1990; Magnes 1997) and currently there are 17 epithets listed in Index Fungorum (2016). Various authors have classifiedRhytidhysteron within the family Patellariaceae (Barr 1987; Kutorga & Hawksworth 1997; Eriksson 2006; Lumbsch and Huhndorf 2010). However, recent multi-gene phylogenetic studies have shown that Rhytidhysteron should be placed in Hysteriaceae (Boehm et al., 2009a, b, de Almeida et al., 2014; Wijayawardene et al., 2014). The genus Rhytidhysteron is characterized by closed and navicular ascomata, later opening by a longitudinal slit to become irregularly apothecioid at maturity and heavily pigmented, and with thick-walled ascospores (Boehm et al., 2009b). Aposphaeria-like’ or diplodia-like’ asexual morphs have been reported for Rhytidhysteron (Samuels & Müller 1979). In this study, we made several Rhytidhysteron collections on dead woody branches in northern Thailand. We used morphological characters and multi-gene molecular analyses to resolve species in the genus. MATErIAlS ANd METhodS Collection of samples, isolation and morphological examination Fresh specimens were collected from dead wood in northern Thailand and samples were grown on potato dextrose agar (PDA). Isolates were derived via single spore isolation following the protocols of Chomnunti et al., (2014). Germinating spores were transferred to PDA and incubated at 25°C in the dark. Cultural characteristics, such as mycelium colour, shape, texture and growth rate were determined. Type and voucher specimens are deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, and New Zealand Fungal & Plant Disease Collection (PDD), while cultures are deposited at the Mae Fah Luang University Culture Collection (MFLUCC) with duplicates in International Collection of Microorganisms from Plants (ICMP) Landcare Research, New Zealand. Specimens were observed and examined with a Motic SMZ 168 stereomicroscope. Micro-morphological characters of the taxon were examined with a Nikon ECLIPSE 80i compound microscope and images were captured using a Nikon ECLIPSE 80i compound microscope with a Canon EOS 550D digital camera. Observations and photographs were made from material mounted in water and Indian ink was added to water mounts to show the presence of gelatinous sheaths around the ascospores. Measurements were made with the Tarosoft (R) Image Frame Work and images used for figures were processed with Adobe Photoshop CS3 Additions to the genus Rhytidhysteron in Hysteriaceae 101 Extended version 10.0 software. Faces of fungi numbers and Index Fungorum numbers are provided as detailed in Jayasiri et al. (2015) and Index Fungorum (2016). DNA extraction, PCR amplification and sequencing Fresh fungal mycelium was grown on PDA at 25°C for 21 days. Extraction of genomic DNA from mycelia and sequencing of PCR products were carried out following the method of Thambugala et al. (2015). Four partial gene portions were amplified in this study including LROR and LR5 (Vilgalys and Hester, 1990) for the nuclear ribosomal large subunit (LSU), ITS4 and ITS5 (White et al., 1990) for the internal transcribed spacer (ITS), EF1-983F and EF1-2218R (Carbone and Kohn 1999) for translation elongation factor 1-alpha (EF1-α) and NS1 and NS4 (White et al., 1990) for the nuclear ribosomal small sub unit (SSU). The amplifications were performed in 25 μL of PCR mixtures containing 9.5 μL ddH2O, 12.5 μL 2×PCR Master Mix (TIANGEN Co., China), 1 μL of DNA template, 1 μL of each primer (10 μM). The amplification reactions were performed and analyzed as described by Thambugala et al., (2015). Phylogenetic analyses The phylogenies of the new strains were determined using two analyses: The first phylogenetic analysis was carried out based on a combined data set of LSU, SSU and EF1-α sequence data of 80 isolates belonging to Hysteriaceae (Table 1) in order to show the placement of the genus Rhytidhysteron in the family, with Delitschia winteri (CBS 225.62) as the outgroup taxon. The second phylogenetic analysis based on a combined data set of LSU, SSU, EF1-α and ITS sequence data were done only for the species in the genus Rhytidhysteron, using 19 isolates and Gloniopsis praelonga (CBS 112415) as the outgroup taxon. All newly generated sequences are deposited in GenBank. Isolates and GenBank accession numbers used in these analyses are listed in Table 1. SeqMan v. 7.0.0 (DNASTAR, Madison, WI) was used to compute consensus sequences. The sequence data were aligned and combined using Bioedit (Hall 1999) and MEGA 5.0 (Tamura et al., 2011). Alignments were checked and manual improved manually where necessary. The maximum likelihood analysis was performed using RAxML v 7.4.2 Black Box software (Stamatakis et al., 2008). The general time reversible model (GTR) including estimation of invariable sites was applied with a discrete gamma distribution with four rate classes. The best scoring trees were selected for the each analysis and illustrated with MEGA 5.0 and with Adobe Illustrator CS3. rESulTS Phylogenetic analyses The first analysis was based on a combined data set of LSU, SSU and EF1-α sequence data belonging to Hysteriaceae and the best scoring RAxML tree is shown in Fig. 1. The outgroup taxon, Delitschia winteri (CBS 225.62) is clearly 102 K. M. Thambugala et al. Table 1. GenBank and culture collection accession numbers of species included in the phylogenetic study. Newly generated sequences are shown in bold GenBank Accession no. Species Isolate no.* LSU SSU EF ITS Delitschia winteri CBS 225.62 DQ678077 DQ678026 DQ677922 Gloniopsis arciformis GKM L166A GU323211 GU323180 _ _ Gloniopsis praelonga CBS 112415 FJ161173 FJ161134 FJ161090 _ CMW 19983 FJ161193 FJ161152 _ _ CBS 123337 FJ161195 FJ161154 FJ161103 _ Gloniopsis sp. MFLUCC 14-0581 KU377563 KU377568 KU497491 _ Gloniopsis subrugosa GKM 1214 GQ221895 _ GU397336 _ CBS 123346 FJ161210 FJ161170 _ _ GKM 1010 GQ221891 _ _ _ SMH 557 GQ221896 _ GU397337 _ Graphyllium caracolinensis HUEFS 42838 KF914914 _ _ _ Hysterium angustatum GKM5211 GQ221906 _ _ _ CMW 20409 FJ161194 FJ161153 _ _ SMH 5216 GQ221908 _ GQ221933 _ GKM 243A GQ221899 _ _ _ CBS 123334 FJ161207