Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences

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Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences
  Phylogenetic relationships of   Nemania plumbea  sp. nov. andrelated taxa based on ribosomal ITS and RPB2 sequences  Alvin M. C. TANG*, Rajesh JEEWON, Kevin D. HYDE Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong,Pokfulam Road, Hong Kong, China a r t i c l e i n f o Article history: Received 4 August 2006Received in revised form2 December 2006Accepted 10 January 2007Published online 24 January 2007 Corresponding Editor: H. Thorsten Lumbsch Keywords: Molecular systematics SordariomycetesXylariaceae a b s t r a c t During a survey fungal diversity of xylariaceous fungi in Thailand, a new  Nemania  species, N. plumbea , was identified.  Nemania plumbea  is characterized by soft-textured grey stromataon a persistent mat of white hyphae, pale brown ascospores with a short germ-slit on themore convex side. It also produces a  Geniculosporium -like anamorph in culture. In order toevaluate its phylogenetic relationships among related species and genera, ITS-5.8S rDNAand RPB2 were analysed separately and simultaneously. Results from the phylogeneticanalyses indicate that there is close phylogenetic association between  N. plumbea  and N. aenea . A preliminary account into the natural grouping of   Xylariaceae  based on ITS-5.8S rDNA and RPB2 sequences is also discussed. ª  2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Nemania  is a plurivorousgenus containing about37species( Ju&Rogers2002).Speciesmainlyoccuronrottingwoodofangio-sperms (e.g.  Betulaceae ,  Fabaceae ,  Fagaceae ,  Salicaceae ). Taxo-nomic delineation of this genus has been obscure in the past,and investigators often classified species under  Hypoxylon (Miller1961;Martin1967;Whalley etal. 1983).However,Pouzar(1985a,1985b)andlaterPetrini&Rogers(1986)gave Nemania ge-nericstatus. Nemania ischaracterizedbymoreorlesscarbona-ceous, dark brown to black stromata that do not releasecoloured pigments in 10 % potassium hydroxide (KOH) ( Ju &Rogers 2002). A whitish soft tissue is present between or be-neath the perithecia. Ascospores are frequently pale brown,mostlywithaninconspicuousgerm-slitandperisporeindehis-cence in 10 % KOH. The anamorphs, if known, are  Geniculospo-rium  or at least  Geniculosporium -like ( Ju & Rogers 2002). Comprehensive reviews of   Nemania  have been provided byGranmo  et al.  (1999) and Ju & Rogers (2002). However, previous accounts and treatments of this genus are few (see van derGucht 1995; Thienhirun 1997; Ju & Rogers 1999). The generic concepts were recently confirmed by a phylogenetic studybased on ribosomal ITS sequences showing that  Nemania  isphylogenetically distinct from  Biscogniauxia ,  Camillea ,  Creo-sphaeria ,  Hypoxylon  and  Kretzschmaria  (Sa´nchez-Ballesteros et al.  2000).  Nemania  and  Xylaria  have long been suggested tobe similar as they have immature ascospores that have an ev-anescent cellular appendage, and they have endophytic * Corresponding author.E-mail address: available at www.sciencedirect.comjournal homepage: mycological research 111 (2007) 392–402 0953-7562/$ – see front matter  ª  2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2007.01.009  lifestyles(Rogers 1979; Ju & Rogers 2002), but this hasnot been addressed properly in molecular studies. Recently, phyloge-netic relationships of several xylariaceous genera were evalu-atedusingprotein-codinggenes,suchas b -tubulinand a -actin(Hsieh  et al.  2005). This was found to be particularly useful inxylariaceous fungi as limited success has been achieved indelineating genera and resolving generic relationships byribosomal DNA genes (Sa´nchez-Ballesteros  et al.  2000; Smith et al.  2003; Triebel  et al.  2005). The combination of genesfrom different loci is needed to clarify the taxonomic confu-sion surrounding   Annulohypoxylon, Biscogniauxia ,  Camillea , Daldinia ,  Entoleuca ,  Hypoxylon ,  Nemania  and  Xylaria .Inthisstudy,DNAsequencesfromITS-5.8SrDNAandRPB2were analysed separately and in combination to establish thephylogenetic relationships of the new species  Nemania plum-bea  and related species. A total of 32 novel DNA sequencesfrom 21 taxa were generated. This study also provides a pre-liminary account into the natural groupings of genera within Xylariaceae  based on ITS-5.8S rDNA and RPB2 sequences. Materials and methods Collection, observation, and cultures Corticated wood samples were collected in the forest of theMushroom Research Centre, Chiang Mai Province, Thailand,duringtherainyseasoninJune2005.Microscopicobservationsweremadeinwater.Melzer’sreagentwasusedtotesttheamy-loidityofapicalring;aqueousCongored,blue–blackWatermanink or phloxine for measurements of ascal stipes, and 10 %potassium hydroxide (KOH) for testing the dehiscence of theperispore. Measurements were taken at   1000 magnificationonsamplesof30ascospores,at  400on20asciandtenperithe-cia.Singlesporecultureswereobtainedfromasinglesporeiso-lation method (Choi  et al.  1999). Cultures were grown on maltextract agar (MEA). The external stromatal colours wererecorded and coded according to Rayner (1970). Herbariumspecimens and living cultures were deposited in the Mush-room Research Centre Herbarium and the University of Hong Kong Culture Collection (HKUCC), respectively. DNA extraction and PCR GenomicDNAwas extracted from myceliagrownon MEA cul-ture following a protocol as outlined by Jeewon  et al.  (2002,2004) and Cai  et al.  (2005, 2006); otherwise DNA was extracted directlyfromascomatausingDNAextractionkit(E.Z.N.A.  Fo-rensic DNA kit, Omega Bio-Tek, Doraville, GA). Target regionsof the ITS-5.8S rDNA regions and RPB2 regions were amplifiedsymmetrically using ITS4 and ITS5 (White  et al.  1990) andfRPB2-5F and fRPB2-7Cr, respectively (Liu  et al.  1999). PCR pro-files used are outlined in Promputtha  et al.  (2005). PCR prod-ucts were purified with a DNA and Gel Band Purification Kit(Amersham Biosciences, Piscataway, NJ). Sequencing reac-tions were carried out using the same primers as mentionedabove in a 3730 DNA analyser (Applied Biosystems, FosterCity, CA) at the Genome Research Centre (University of Hong Kong). DNA sequences have been deposited in GenBank andare listed in Table 1. Phylogenetic analysis DNAsequenceswerealigned(Table1)usingBioEdit(Hall1999) and ClustalX 1.83 (Thompson  et al.  1997). Manual gapadjustments were made to improve the alignment. Phyloge-netic analyses were conducted using PAUP version 4.0b10(Swofford 2002) and MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001). Alignmentgapsweretreated as missingdata. Weightedparsimony analyses were performed using a symmetric stepmatrix generated with the program STMatrix version 2.2(Francois Lutzoni and Stefan Zoller, Department of Biology,Duke University), by which the relative frequencies of nucleo-tide substitutions were calculated and converted into costs of changes. Unweighted MP trees were found using 1K heuristicsearch and including parsimony-informative charactersin stepwise (random) addition and tree bisection and recon-struction (TBR) as branch swapping algorithm. Max-treeswere set to 5K, branches of zero length were collapsed andall parsimonious trees were saved. Branch support for allparsimony analyses was estimated by performing 1K BSreplicates(Felsenstein1985)withaheuristicsearchconsisting of ten random-addition replicates for each BS replicate.Other details are outlined in Kodsueb  et al.  (2006) and Shenoy et al.  (2006).ML best-fit models were estimated using MrModelTest 2.2(Posada & Crandall 1998; Nylander 2004). Independent substi- tution models were obtained from the test for Bayesian anal-yses in MrBayes 3.0b4. The Bayesian analyses were conductedwiththeMarkovchainsrunfor1Mgenerationsandtreesweresampled every 100th generation resulting in 10K trees. Thefirst 1K trees, which represented the burn-in phase of theanalysis, were discarded, and the remaining 9K trees wereused for calculating PPs in the consensus tree. Descriptivetree statistics tree length (TL), CI, RI, RC, and HI were calcu-latedforalltreesgeneratedunderdifferentoptimalitycriteria.Trees were figured in Treeview (Page 1996). Results ITS based phylogenies TheITS-5.8SrDNAdatasetcontained44taxaof663characters(TreeBASE accession no. SN2885). Two hundred and ninety-one base pairs of ambiguously aligned regions were excludedfrom parsimony, weighted parsimony and Bayesian analyses.Of the remaining 372 unambiguously aligned characters, 170were constant, 21 parsimony uninformative and 178 parsi-mony informative. For the same dataset, with the use of step-matrix (A 4 C ¼ 2.37, A 4 G ¼ 2.32, A 4 T ¼ 2.36, C 4 T ¼ 2.19, C 4 G ¼ 2.34, G 4 T ¼ 2.34, A 4 gap ¼ 2.31, C 4 gap ¼ 2.26,G 4 gap ¼ 2.30, T 4 gap ¼ 2.25) in weighted parsimony analy-ses, the number of parsimony uninformative and informativecharacterswere20and179,respectively. Gapsweretreatedasthe fifth character for the analyses. Unweighted parsimonyanalysis (with gaps treated as missing characters) generated13 equally parsimonious trees and for the same analysis(withgaptreatedasfifthcharacter)generated12equallyparsi-monious trees. Weighted parsimony analysis resulted in onePhylogenetic relationships of   Nemania plumbea  sp. nov. and related taxa 393  Table 1 – Taxa used in the phylogenetic analyses and their accession numbers Taxon names Specimen voucher Origin GenBank accession No.ITS RPB2 Order  BolinialesBoliniaceae Camarops amorpha  SMH 1450 Puerto Rico – AY780156 C. tubulina  SMH 4614(a) Denmark – AY780157 Order  ConiochaetalesConiochaetaceae Coniochaeta discoidea  SANK12878 Japan – AY780191 Coniochaetidium savoryi  TRTC 51980 Malawi – AY780174 Coniochaeta ligniaria  CBS 178.75 Netherlands – DQ631958 C. velutina  IFO 9439 Unknown – DQ631959 Order  DiaporthalesDiaporthaceae Diaporthe phaseolorum  NRRL 13736 Unknown – AY641036 Gnomoniaceae Gnomonia ribicola  CBS 115443 Hong Kong – DQ368642 Plagiostoma euphorbiae  CBS 340.78 Netherlands – DQ368643 Valsaceae Amphiporthe castanea  CBS 392.93 Italy – DQ368644 Diaporthales Insertae sedis Cryphonectria parasitica  CP 155 Unknown – AY485619 Order  Halosphaeriales : Halosphaeriaceae Corollospora maritima  CBS 264.59 Germany – DQ368632 Neptunella longirostris  HK AT-2061 Hong Kong – DQ368633 Order  Hypocreales : Ceratostomataceae Melanospora zamiae  CBS 421.87 Spain – DQ368634 Hypocreaceae Hypocrea pallida  GJS 89-83 Unknown – AY015636 H. schweinitzii  CBS 243.63 New Zealand – DQ368635 Hypomyces polyporinus  CBS 168.89 Germany – DQ368636 Hydropisphaera erubescens  ATCC 36093 Unknown – AY545731 Nectriaceae Gibberella moniliformis  ATCC 38932 Unknown – AY533830 Order  MicroascalesChadefaudiellaceae Faurelina elongata  CBS 126.78 India – DQ368639 Microascaceae Petriella setifera  CBS 110344 Spain – DQ368640 Microascales  inc. sed. Ceratocystis fimbriata  CBS 374.83 Spain – DQ368641 Order  SordarialesChaetomiaceae Chaetomium elatum  ATCC 42780 Unknown – AF107791 Zopfiella ebriosa  CBS 111.75 Netherlands – AY780200 Chaetosphaeriaceae Melanochaeta hemipsila  SMH 2125 Puerto Rico – AY780184 Chaetosphaeria ovoidea  SMH 2605 Michigan – AY780173 Lasiosphaeriaceae Bombardia bombarda  SMH 4821 France – AY780154 Cercophora caudata  CBS 606.72 Netherlands – DQ368646 Lasiosphaeria ovina  SMH 3286 North Carolina – AY600292 Podospora fimbriata  CBS 144.54 Unknown – AY780189 Schizothecium curvisporum  ATCC 36709 Kenya – AY780192 Sordariaceae Gelasinospora tetrasperma  ATCC 96230 Canada – AY780177 Neurospora crassa  FGSC 2489 USA – AF107789 Sordaria fimicola  CBS 723.96 Papua New Guinea AY681188 DQ368647 S. macrospora  Buck s.n. Canada – AY780195 394 A. M. C. Tang   et al.  Table 1 (  continued  ) Taxon names Specimen voucher Origin GenBank accession No.ITS RPB2 Order  Xylariales Amphisphaeriaceae Bartalinia robillardoides  BRIP 14180 Unknown AF405301 DQ368653 Discostroma botan  HHUF 4642 Japan – DQ368648 Pestalotiopsis versicolor  BRIP 14534 Unknown AF409993 DQ368654 Seiridium cardinale  ICMP 7323 New Zealand AF409995 DQ368655 S. eucalypti  CBS 343.97 Australia – DQ368656 Diatrypaceae Diatrype disciformis  F-091,971 Spain AJ390410 –  Apiosporaceae Apiospora tintinnabula  ICMP 6889 New Zealand – DQ368649 A. setosa  ICMP 4207 New Zealand – DQ368650 Xylariaceae Annulohypoxylon annulatum  GB 5659 New Jersey AJ390395 – A. atroroseum  ATCC 76081 Ile de le Reunion AJ390397 – A. stygium  JF TH-28-01 Thailand DQ631935 DQ631960 Annulohypoxylon  sp. CM AT-010 Thailand AJ390409 DQ631962 Biscogniauxia atropunctata  ATCC 13359A Florida AJ390411 – B. capnodes  CM AT-015 Thailand DQ631933 – B. marginata  ATCC 62608 Pennyslvania AJ390417 – B. mediterranea  CBS 280.61 USA AJ390413 – B. nummularia  CBS 969.70 UK AJ390415 – B. repanda  ATCC 62606 Unknown AJ390418 – B.  sp. JF 06-05 Thailand DQ631932 – Creosphaeria sassafras  CM AT-018 Thailand DQ631934 DQ631964 Daldinia grandis  BJ Coppins 8630 UK AF176982 – D. concentrica  M-0066225 UK AY616681 DQ368651 D. concentrica  CBS 139.73 Netherlands AF163021 – D. fissa  Lind & Andreasen s.n. Denmark AF176976 – D. loculata  HJ108 Latvia AF176959 – D. petriniae  H Knudsen s.n. Denmark AF176970 – D. vernicosa  CBS 157.32 USA AF163022 – Hypoxylon fendleri  F-108, 405 Mexico AJ390400 – H. monticulosum  CM AT-04 Thailand DQ631938 – H. monticulosum  HK AT-PTC015 Hong Kong  DQ631939 DQ631950 H. monticulosum  GZ AT-M050 Guizhou DQ631936 DQ631955 Kretzschmaria clavus  JP 3113 Puerto Rico AJ390434 – K. deusta  CBS 826.72 Belgium AJ390435 – Muscodor albus  Unknown USA AF324336 – M. vitigenus  Unknown USA AY100022 – Nemania aenea  ATCC 68818 Czech/France AJ390426 DQ631951 N. aenea  CBS 680.86 Switzerland AJ390427 – N. aenea  var.  aureolutea  ATCC 60819 Switzerland AJ390428 – N. aenea  var.  macrospora  ATCC 60822 California AJ390433 – N. bipapillata  JP 3034 Puerto Rico AJ390429 – N. chestersii  ATCC 38988/JF 04024 Wales/France AJ390430 DQ631949 N. diffusa  FR AT-113 France DQ658238 DQ631947 N. diffusa  GZ AT-F006 Guizhou – DQ631957 N. maritima  JF04055 France DQ631941 DQ631946 N. plumbea  JF TH-04-01 Thailand DQ641634 DQ631952 N. serpens  ATCC 16078 Canada AJ390431 – N. serpens  FR AT-114 France DQ631942 DQ631948 Stilbohypoxylon quisquiliarum  JF TH-06-04 Thailand DQ631943 DQ631954 Xylaria  sp. CM AT-016 Thailand DQ631937 – X. grammica  XT09003 Thailand DQ631945 DQ631953 X. cornu damae  XT09009 Thailand DQ631944 DQ631956 X. hypoxylon  CBS 724.69 Canada AF163031 – X. mali  Unknown UK AJ309350 DQ368652 Xylariaceous  sp. 1 CBS 385.35 Unknown AF163040 – Sordariomycetes  incertae sedis Thyridiaceae Sinosphaeria bambusicola  SMH1999 Puerto Rico – AY780193( continued on next page ) Phylogenetic relationships of   Nemania plumbea  sp. nov. and related taxa 395  tree. The best-fit model selected for Bayesian analysis byMrModeltest was GTR þ I þ G. Fig 1 shows the tree generatedfrom weighted parsimony (TL ¼ 2428, CI ¼ 0.448, RI ¼ 0.670,RC ¼ 0.300, HI ¼ 0.552).Ten  Nemania  species constitute a well-supported (both BSand Bayesian) monophyletic clade and appeared to bephylogenetically distinct from other genera such as  Annulohy- poxylon ,  Biscogniauxia ,  Daldinia ,  Hypoxylon ,  Muscodor  and Xylaria  (Fig 1). Within the  Nemania  clade, most of the internalsubclades did not receive reliable branch support. The newspecies  N. plumbea  grouped with  N. bipapillata  AJ390429 and N. aenea  AJ390426, but with poor statistical confidence. In 10  Nemania aenea AJ390427  Nemania aenea var. aureolutea AJ390428   Nemania aenea var. macrospora AJ390433  Nemania serpens AJ390431  Nemania chestersii AJ390430  Nemania bipapillata AJ390429  Nemania plumbea DQ641634  Nemania aenea AJ390426  Nemania maritima DQ631941   Nemania serpens DQ631942  Biscogniauxia marginata AJ390417  Biscogniauxia repanda AJ390418  Biscogniauxia mediterranea AJ390413  Biscogniauxia sp. DQ631932  Biscogniauxia atropunctata AJ390411  Biscogniauxia nummularia AJ390415  Biscogniauxia capnodes DQ631933  Xylaria grammica DQ631944  Xylaria hypoxylon AJ309350  Xylaria mali AF163040  Xylaria cornu damae AF163031 Kretzschmaria deusta AJ390435 Kretzschmaria clavus AJ390434 Stilbohypoxylon quisquiliarum DQ631937  Muscodor albus AF324336  Muscodor vitigenus AY100022  Annulohypoxylon atroroseum AJ390397  Annulohypoxylon stygium AJ390409Xylariaceous sp. 1 DQ631943  Annulohypoxylon annulatum AJ390395  Hypoxylon monticulosum DQ631938  Hypoxylon monticulosum DQ631939  Hypoxylon monticulosum DQ631936  Hypoxylon fendleri AJ390400  Annulohypoxylon sp.   DQ631935  Daldinia fissa  AF176976  Daldinia concentrica  AF163021  Daldinia vernicosa  AF163022  Daldinia petriniae  AF176970  Daldinia grandis  AF176982  Daldinia loculata  AF176959  Daldinia concentrica  AY616681 Creosphaeria sassafras  DQ631934  Diatrype disciformis  AJ390410706253918871991001001009784748110089978977809864 Fig 1 – Phylogeny obtained from weighted parsimony analysis based on ITS rDNA dataset. BS values  50 % are shown abovethe branches and Bayesian PPs   95 % are indicated as thickened branches.   Table 1 (  continued  ) Taxon names Specimen voucher Origin GenBank accession No.ITS RPB2 Order  PleosporalesPleosporaceae Pleospora herbarum  EGS04-188C Unknown – AF107804 396 A. M. C. Tang   et al.
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