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,2016,36(6):1245-1249
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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犖犲狑犚犲犮狅狉犱狅犳狋犺犲犔犻犮犺犲狀犌犲狀狌狊犘犲犾狋犻犵犲狉犪犳狉狅犿犆犺犻狀犪
NIUDongling1,ZHUQiang2,WANGZhongzhong1,SHIJing1,
BAIMingsheng1,ZHENGXueping1
(1LifeScienceSchool,NingxiaUniversity,Yinchuan750021,China;2.NingxiaForestResearchInstitution,Yinchuan
750000,China)
犃犫狊狋狉犪犮狋:Anewrecordspecies犘犲犾狋犻犵犲狉犪犿狅狀狋犻犮狅犾犪 Vitik.wasidentifiedfromthenorthwestofChina,
basedonmorphologicalandchemicalanalysis,andaphylgeneticanalysisofnrDNAITSsequencewasalso
carriedout.Itwasdistinguishedbyitsnumerouslaminalphylidia,oftenpruinose,alongthemarginor
cracks,marginalpartstomentose,tuftedrhizomeswithpalenearmarginsanddarkenedtowardscenter.
Detaileddescriptionanddiscussionwithaliedspecieswereprovided.
犓犲狔狑狅狉犱狊:northwestofChina;犘犲犾狋犻犵犲狉犪;ITSsequences;newrecordspecies
Thegenus犘犲犾狋犻犵犲狉犪iscosmopolitanmono
phyleticgenuswithmorethan91species,which
typicalyhavemuscicolousandterricolousfoliose
thalus[13].Mostof犘犲犾狋犻犵犲狉犪specieshaveacya
nobacterialphotobointthatcarriesoutnitrogen
fixationandplaysamajorecologicalroleinnitro
gencyclingofecosystem.Thisgenusisacommon
lichengrouprepresentedby28taxainChina[45],
distributedinalgeographicaldistributionsofChi
na.However,untilnowthisgenusisnotstudied
thoroughlyinChina,inwhichthepotentialfor
findingnew speciesishigh.Duringourrecent
fieldworkinLiupanshanandHelanshanMountains
locatedinthenorthwestofChina,wefindonead
ditionaltaxa(犘犲犾狋犻犵犲狉犪犿狅狀狋犻犮狅犾犪Vitik.)belong
ingtothisgenusbasedonmorphologyandmolecu
larcharacters.
1 Materialsandmethods
1.1 犕狅狉狆犺狅犾狅犵犻犮犪犾犪狀犪犾狔狊犻狊
Specimensforthisstudywerecolectedfrom
LiupanshanandHelanshanMountains,locatedin
thenorthwestofChina.Themorphologicalde
scriptionsweremadefromdrymaterials.Morpho
logicalandanatomicalcharacterswereobserved
andphotographedusingaXTS20 (FukaiScience
and Technology DevelopmentCo.,China)and
LeicaDM2500(LeicainstrumentCo.,Germany).
Thevoucherspecimensforthisstudywerekeptin
theherbariumofColegeofLifeScienceofNingxia
University(NXAC,Yinchuan,Ningxia,China).
Besidesourowncolectionweexaminedthespeci
mensofthesamespeciesdepositedinWiliamLou
is& ChicitaF.CulbertsonLichenHerbarium &
Library(DukeUniversity,Durham,NC,USA)
1.2 犆犺犲犿犻犮犪犾犪狀犪犾狔狊犻狊
Chemicalanalyseswerecarriedoutusingspot
test[6].Theseinvolveapplyingtinyamountsofa
reagenttolichenandobservinganyresultingcolor
change.Fourreagentswereusedinthesespot
tests,K (10%aqueouspotassiumhydroxide),C
(sodiumhypochloritesolution),KC(10% Kfol
lowedbyCsolution),P (5%alcoholicPpheny
lenediamine).
1.3 犕狅犾犲犮狌犾犪狉犪狀犪犾狔狊犻狊
DNAwasobtainedfromdrysamples.Extrac
tion folowed a modification of DOYLE and
DOYLEs(1987)method[78].rDNAinternaltran
scribedspacers(ITS12)wereamplifiedviapoly
merasechainreaction(PCR)usingfolowingprim
ers:ITS1F[9]andITS4[10].PCRmixtures(25μL)
consistedof25μgBSA,1U犜犪狇DNApolymer
ase,dNTPs(0.2mmol/L),primers(0.5μmol/L
犜犪犫犾犲1 犞狅狌犮犺犲狉犻狀犳狅狉犿犪狋犻狅狀狅犳犘犲犾狋犻犵犲狉犪犿狅狀狋犻犮狅犾犪犞犻狋犻犽.狊犪犿狆犾犲狊狑犻狋犺犌犲狀犅犪狀犽犪犮犮犲狊狊犻狅狀狀狌犿犫犲狉犪狀犱
狊狆犲犮犻犿犲狀狊犳狉狅犿犌犲狀犅犪狀犽犐犜犛狊犲狇狌犲狀犮犲狊狅犫狋犪犻狀犲犱
Species Vouncher GenBankaccession
NP39 犘.犿狅狀狋犻犮狅犾犪 HelanshanNingxia,130002 KT781508
NP36 犘.犿狅狀狋犻犮狅犾a HelanshanNingxia,130003 KT781509
NP41 犘.犿狅狀狋犻犮狅犾犪 LiupanshanNingxia,120007(1) KT781510
NP19 犘.犿狅狀狋犻犮狅犾犪 Liupanshan,Ningxia,120016 KT781512
NP20 犘.犿狅狀狋犻犮狅犾犪 Helanshan,Ningxia,130005 KT781513
NP21 犘.犿狅狀狋犻犮狅犾犪 Helanshan,Ningxia,130004 KT781514
AY257877 犘.犿狅狀狋犻犮狅犾犪 Switzerland,Vust452188(G) AY257877
AY257881 犘.犿狅狀狋犻犮狅犾犪 Switzerland,Vust452187(G) AY257881
AY257886 犘.狊犮狅狋狋犲狉犻 Canada,Goward811289a(UBC) AY257886
AY257874 犘.犿狅狀狋犻犮狅犾犪 Poland,Toborowicz13.08.1976(KTC) AY257874
AY257872 犘.犿狅狀狋犻犮狅犾犪 Poland,Faltynowicz5239(UGDAL) AY257872
KF957622 犘.狆狅狀狅犼犲狀狊犻狊 Italy,3523(HBTO) KF957622
AY257875 犘.犿狅狀狋犻犮狅犾犪 Switzerland,Vust452186(G) AY257875
AY257883 犘.狆狅狀狅犼犲狀狊犻狊 Canada,Goward821233(CANL) AY257883
KJ413221 犘.犿狅狀狋犻犮狅犾犪 SSM353(UI) KJ413221
KC139753 犘.犿狅狀狋犻犮狅犾犪 SSM125(UI) KC139753
AY257873 犘.犿狅狀狋犻犮狅犾犪 Yugoslavia,Vitikainen7196(H) AY257873
FJ709039 犘.狆狅狀狅犼犲狀狊犻狊 HOB0207086213 FJ709039
AY257884 犘.狆狅狀狅犼犲狀狊犻狊 Poland,Bielczyk2116(KRAML) AY257884
AY257885 犘.狆狅狀狅犼犲狀狊犻狊 Poland,Kiszka2.09.1988(KRAPL) AY257885
AY257961 犘.犲犾犻狊犪犫犲狋犺犪犲 Poland,Bielcayk42135(KRAML) AY257961
Specimennamefolowedbyanasteriskrepresentsoutgroupsequence.
6421 ! " # $ % & 36O
each),andPCR buffer,adjusted with H2Oto
25μL.Thefolowingthermalcyclerprofilewas
employedforPCRreactions:aninitialdenatur
ationof95 ℃for5min,folowby35cyclesof
95℃for45s,52℃for90s,and72℃for90s,
withafinalextensionof72℃for10min.Amplifi
cationproductswerecleanedwithExoAPandse
quencedusingBigDyechemistrywithanABI3730
automatedsequencer (PE Applied Biosystems,
USA).SequenceswereassembledusingSequench
er4.2(GeneCodes,USA).Alnewlyobtained
sequencesweresubjectedtoBlastsearchestoveri
fytheiridentityandsubmittedtoGenBank.
1.4 犘犺狔犾狅犵犲狀犲狋犻犮犪狀犪犾狔狊犻狊
TheentireITSsequencesof6samplesexamined
and14representativesselected(Table1)werealigned
byMuscleimplementedin犕犈犌犃version6[11].The
alignmentswereanalyzedusingneighborjoining(NJ),
thesoftwarebased Kimura2parameter modelwas
used,andgapswereexcludedinthepairwisedistance
estimation,thesupportvaluesweretestedby1000
bootstrapreplications.
2 Resultsanddiscussion
2.1 犘犺狔犾狅犵犲狀犲狋犻犮犪狀犪犾狔狊犲狊
SequencesofITSaloneweresufficienttoiden
tifyalspeciesfromthe犘犲犾狋犻犵犲狉犪犮犪狀犻狀犪species
complex.The犘.狆狅狀狅犼犲狀狊犻狊groupwaswelde
finedasamonophyleticclade(Fig.1),whichhas
beenidentifiedbefore[12].ITSsequencesofrepre
sentativecolectionsshowedthatalstudied犘犲犾狋犻
犵犲狉犪 species from Liupanshan and Helanshan
Mountains,belongedtothespecies犘.犿狅狀狋犻犮狅犾犪
clade,supportforthemonophylyofthistaxonwas
strong(BS=78%).Thespecimensindifferentge
ographicalregionwereindifferentlineages,the
specimensfrom Liupanshan wereinalineage,
thosefrom Helanshaninanotherlineage.‘犘.
狊犮狅狋狋犲狉犻’isanonmonophyleticputativespecies,
andoneofthespecimenshasbeenprovedsisterto
犘.犿狅狀犻犮狅犾犪,so‘犘.狊犮狅狋狋犲狉犻’isnotvalidlypub
lished[13].Thespecimenof ‘犘.狊犮狅狋狋犲狉犻’from
Canade(Table1)belongedtoHelanshanlineage,
whichmeansthis‘犘.狊犮狅狋狋犲狉犻’shouldbetreatedas
amemberof犘.犿狅狀狋犻犮狅犾犪.Thespecimensfrom
LiupanshanweresistertothespecimensfromPo
landwithhighsupport(BS=99%),thespecimens
fromHelanshanandSwitzerlandweredelimitedas
amonophyleticentity(BS=97%).Sequencedata
werealsocomparedusingBlastanalysisofITS12
regionsdepositedinNCBI.Theywere100%iden
ticalto犘犲犾狋犻犵犲狉犪犿狅狀狋犻犮狅犾犪.
2.2 犜犺犲狊狆犲犮犻犲狊
犘犲犾狋犻犵犲狉犪犿狅狀狋犻犮狅犾犪Vitik.,ActaBot.Fennica
152:64(1994,Fig.2)
Thalus:foliose,smaltomediumsized,3-9
cmindiam.,adnate;lobes:flattenedandelon
gate,0.5-3cmlongand1-3mmwide,withup
turned,curled,withnumerousflatphylidia;up
persurface:gray,partialybrownwhendry,to
mentosemarginaly,dul marginaly,smoothfor
wardscentral,sometimespruinoseonthesurface;
phylidia:flat squamulose along margins and
cracks,oftenpruinose;medula:whitewithinter
wovenhyphae;photobiont:nostoc;lowersurface:
whitemarginwithwhiteraisedveins,brownto
wardscenter;rhizines:tufted,palenearmargins,
Supportisindicatedforbranchesbybootstrapfrequencies
exceeding50%undertheNeighborJoining
Fig.1 Phylogenetictreeof犘.犿狅狀狋犻犮狅犾犪and
selectedspeciesof犘.狆狅狀狅犼犲狀狊犻狊group
basedonITSnrDNA
74216© ./0,ª:NOP1ehijk(«¬)
A.generalhabit;B.marginalphylidiawithpruinose;C.pruinoseonthesurface;D.tuftedrhizines
Fig.2犘犲犾狋犻犵犲狉犪犿狅狀狋犻犮狅犾犪.
darkenedtowardscenter;apothecia:notseen,said
tobeflatorsaddleshaped,upto5mmindiam.;
pycnidia:notseen.
Chemistry:spottestsonthecortexandme
dulaK,C,KC,P.
EcologyandDistribution:犘.犿狅狀狋犻犮狅犾犪main
lygrowsamongmossesoversoil.Itsworlddistri
butionincludesNorth America,Europeand A
sia[14].Atpresentthisspeciesismainlyrecorded
fromLiupanshanwhichislocatedinNingxiaand
HelanshanwhichislocatedinNingxiaandInner
MongoliafromtheNorthwestofChina.Further
more,itismainlycolectedfromforestzonesin
NatureReserveofNingxiaandInnerMongolia.
Notes:ITSresultsindicatethatthistaxon
fromthenorthwestofChinaiscomprisedoftwo
geographicalydistinctlineages,butthereisnoob
viousmorphologicaldifferencebetweenthem,so
twogeographicalpopulationsof犘.犿狅狀狋犻犮狅犾犪are
treatedhereasataxon.Thepopulationfromthe
northwestofChinaissistertothespecimensfrom
Europe(PolandandSwitzerland)withhighsup
port.Wespeculateitisduetotheirsimilarclimat
icconditions.犘.犿狅狀狋犻犮狅犾犪isidentifiedbyitslam
inalphylidiaalongthemarginorcracks,lobeends
withgreywhitishtomentose,tuftedrhizines.The
speciesiscloseto犘.狆狅狀狅犼犲狀犻狊basingonGenBank
Blast,butthelatterisdistinguishedbyshortof
phylidiaandpaleunbranchedtofibrous,discrete
rhizines.
Specimensexamined:China,Prov.Ningxia,
Guyuan,Liupanshan,Qiuqianjia,1718 m,on
moss,2012,120016;Liupanshan,Erlonghe,2
039 m,on moss,2012,120017;Helanshan,
8421 ! " # $ % & 36O
Suyukou,2086m,onmoss,2013,130005;13
0004;Prov.InnerMongolia:Helanshan,South
Temple,2000m,onmoss,2013,130002;13
0003.- (specimensaredepositedin NXAC).
France:Lorraine,LaMadeleine,Fusboesch,345
m,onsoil,2008,07.27.02/8.- (specimensare
depositedinDukeUniversityHerbarium).
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