PhylogeographicPhylogeographic analysesanalyses ofof geneticgenetic variationvariation withinwithin ArmillariaArmillaria ostoyaeostoyae fromfrom thethe westernwestern UnitedUnited StatesStates

Photo: Raini Rippy

J.W. HANNA (1,2), N.B. Klopfenstein (1), M.-S. Kim (1), G.I. McDonald (1), J.A. Moore (2) (1) Department of Forest Resources, University of Idaho, Moscow, Idaho (2) USDA Forest Service, Rocky Mountain Research Station, Moscow, Idaho A shift in tree species composition has occurred in many forests of the western USA Hanna et al. 2004

Fire Suppression Photo: National Park Service

White Pine Blister Rust Selective Harvest and Planting Photo: John W. Hanna Photo: Master Garden Products ChangingChanging forestforest compositioncomposition

ForestForest compositioncomposition hashas shiftedshifted awayaway fromfrom seralseral speciesspecies (e.g.,(e.g., pine,pine, larch)larch) towardtoward climaxclimax speciesspecies (e.g.,(e.g., truetrue fir,fir, DouglasDouglas--fir)fir)

AsAs aa result,result, ArmillariaArmillaria diseasedisease hashas becomebecome moremore prevalentprevalent

Hanna et al. 2004 ArmillariaArmillaria speciesspecies andand relativerelative pathogenicitypathogenicity

NABSa Species Pathogenicity Primary Host

I A. ostoyae High Conifers VI A. mellea High Hardwoods A. tabescens High? Hardwoods II A. gemina Moderate? Hardwoods IX A. nabsnona Moderate? Hardwoods III A. calvescens Low? Mixed V A. sinapina Low? Mixed VII A. gallica Low? Mixed XI A. cepistipes Low? Mixed X Unnamed Rarely pathogenic Mixed

aNABS = North American Biological Species Korhonenn, 1978, Anderson and Ullich, 1979, Berube and Dessureault, 1988, Berube and Dessureault 1989, Volk et al., 1996 Hanna et al. 2004 ArmillariaArmillaria ostoyaeostoyae:: PrimaryPrimary causecause ofof ArmillariaArmillaria

diseasedisease inin westernwestern conifersconifers Hanna et al. 2004

USDA Forest Service, Missoula Archives Hanna et al. 2004 ImpactImpact ofof ArmillariaArmillaria rootroot diseasedisease onon treetree growthgrowth

Volume loss has been shown as high as 40% over 4-8 years in eighteen-year-old Douglas-fir.

Cruickshank et al., 2000 PreviousPrevious studiesstudies onon A.A. ostoyaeostoyae • Distinct differences in A. ostoyae epidemiology have been noted among coastal and interior regions of western North America (McDonald 1990, Goheen and Otrosina 1998, Morrison and Pellow 2002).

• Genets of A. ostoyae can show varying levels of pathogenicity and virulence (Omdal et al. 1995, Morrison and Pellow 2002).

• Genetic variability within A. ostoyae has been demonstrated among genets (Anderson and Stasovski 1992, Piercey-Normore et al. 1997, Schulze et al. 1997, Chillali et al. 1998, Terashima et al. 1998, White et al. 1998, Sicoli et al. 2003).

• Little is known about intraspecific genetic variation within A. ostoyae and its relationship to phylogeny and ecological behavior. Hanna et al. 2004 ObjectivesObjectives

AssessAssess geneticgenetic diversitydiversity amongamong genetsgenets ofof A.A. ostoyaeostoyae toto examineexamine intraintra-- andand interspecificinterspecific phylogeographicphylogeographic relationships.relationships. InvestigationInvestigation ofof geneticgenetic diversitydiversity maymay bebe importantimportant toto understand:understand:

1)1) varyingvarying levelslevels ofof pathogenicitypathogenicity andand virulencevirulence withinwithin A.A. ostoyaeostoyae

2)2) phylogeographicphylogeographic relationshipsrelationships amongamong A.A. ostoyaeostoyae genetsgenets andand genetsgenets ofof otherother ArmillariaArmillaria species,species, andand

3)3) adaptationadaptation toto diversediverse environmentalenvironmental factorsfactors

Hanna et al. 2004 Materials and Methods

Hanna et al. 2004 Summary of Armillaria species and genets used in phylogeographic analysis Species Origin Number Collector(s) or Reference of isolatesa AArmillaria. calvescens Michig aostoyaen, USA 2 M.T. Banik Quebec, Canada 1 J.A. Bérubé AOrigin. cepistipes British Columbia, Canad#a isolates2 D.J. MoCollector(s)rrison or Reference Washington, USA 1 H.H. Burdsall Jr. A.Chihuahua, gallica Britis hMexico Columbia, Canada 11 D.J. MoCrris.G.on Shaw III Michigan, USA 1 M.T. Banik Eastern, WFinlandisconsin, USA 11 M.T. BaUnknownnik A. gemina New York, USA 2 J.J. Worrall c Idaho, USAWest Virginia, USA 231 M.E. MiIeFTNClke , G.I. McDonald A. mellea New Hampshire, USA 1 T.C. Harrington Montana,Vi rUSAginia, USA 81 G.F. BillsB.A. Ferguson et al. 2003 Wisconsin, USA 1 M.T. Banik A. nabsnona Alaska, USA 1 C.G. ShaG.I.w III McDonald British Columbia, Canada 1 D.J. Morrison New Hampshire,Idaho, USA USA 11 G.I. McDTona.C.ld Harrington A. ostoyae Chihuahua, Mexico 1 C.G. Shaw III New Mexico,Eastern, FUSAinland 141 UnknowGn .I. McDonald, Omdal et al. 1995 Idaho, USA 23 IFTNCc, G.I. McDonald Oregon, MUSAontana, USA 68 B.A. FerFergusonguson, G.I. McD etona al.ld 2003, New Hampshire, USA 1 T.C. Harrington New Mexico, USA 14 G.I. McDG.I.onald ,McDonald Omdal et al. 1995 Oregon, USA 6 Ferguson et al. 2003, G.I. McDonald Primorye,Prim Russiaorye, Russia 11 G.M. FilipG.M. Filip Utah, USA 3 G.I. McDonald Utah, USAWashington, USA 319 J.F. AmmG.I.irati, IMcDonaldFTNCc, M-S. Kim, G.I. McDonald A. sinapina British Columbia, Canada 1 D.J. Morrison c Washington,Michiga nUSA, USA 191 M.T. BaJni.F.k Ammirati, IFTNC , Washington, USA 1 J.F. Ammirati A. tabescens Georgia, USA 2 G. SchnaM-S.bel Kim, G.I. McDonald South Carolina, USA 1 G. Schnabel NABS Xb Idaho, USA 3 G.I. McDonald a Each isolate represents a distinct genet b NABS = North American Biological Species Hanna et al. 2004 c Intermountain Forest Tree Nutrition Cooperative, Department of Forest Resources, University of Idaho, Moscow, ID 83844 ArmillariaArmillaria inspectionsinspections andand collectionscollections

Hanna et al. 2004 From G.I. McDonald BarkBark fanfan collectioncollection typetype FruitingFruiting bodiesbodies

RhizomorphRhizomorph Hanna et al. 2004 collectioncollection typetype WoodWood collectioncollection typetype ArmillariaArmillaria spp.spp. collectionscollections

•• ArmillariaArmillaria spp.spp. isolatesisolates werewere reducedreduced toto uniqueunique genetsgenets byby somaticsomatic pairingpairing

Hanna et al. 2004 Armillaria species were identified

Somatic pairing Mating tests

IGS RFLP

Hanna et al. 2004 MaterialsMaterials andand MethodsMethods (Continued)

Using a direct-PCR method, sequences were obtained for nuclear ribosomal DNA

Large subunit (LSU),

Internal transcribed spacer and 5.8S (ITS), and

Intergenic spacer one (IGS) from 77 genets of Armillaria ostoyae from western North America (and 3 individuals each from 10 North American Armillaria species). Hanna et al. 2004 PrimerPrimer setssets usedused forfor amplification/sequencingamplification/sequencing

Region Primer Use Reference . LSU: 5.8SR Amplification Vilgalys 2004 LR7 Amplification Vilgalys 2004 LR0R Sequencing Vilgalys 2004 LR5 Sequencing Vilgalys 2004 LR15 Sequencing Vilgalys 2004

ITS: ITS-1F Amp & Seq Gardes and Bruns 1993 ITS4 Amp & Seq White et al. 1990

IGS: LR12R Amp & Seq Veldman et al. 1981 O-1 Amp & Seq Duchesne and Anderson 1990 A5SR1 Amp & Seq Hanna et al. 2004

Hanna et al. 2004 AA directdirect PCRPCR (polymerase(polymerase chainchain reaction)reaction) methodmethod waswas usedused toto amplifyamplify segmentssegments ofof nuclearnuclear ribosomalribosomal DNA.DNA.

5.8S Nuclear IGS-2 Nuclear Small rDNA ITS-1 ITS-2 Nuclear Large rDNA IGS-1 5S IGS-2 Small rDNA rDNA

PCR product was then sequenced and nuclear ribosomal DNA regions investigated for phylogenetic signal. These segments of DNA repeat several hundred times within the genome of an individual. Hanna et al. 2004 PhylogeneticPhylogenetic analysisanalysis • Careful attention was given to both sequence editing and alignment.

• Only sequences having an non-ambiguous alignment were compared.

For example: A. mellea and A. tabescens were only compared to other North American Armillaria species in the LSU region. ITS and IGS regions contain numerous indels making alignment impractical.

Hanna et al. 2004 PhylogeneticPhylogenetic analysisanalysis (continued)

• Repetitive sequences were eliminated from the sequence alignments so that only unique genotypes would be compared.

• Neighbor-joining, Parsimony, and Bayesian methods of phylogenetic inference were used for phylogenetic inference.

Hanna et al. 2004 DirectDirect PCRPCR revealsreveals heterogeneousheterogeneous sequencessequences

DirectDirect PCRPCR hashas beenbeen previouslypreviously shownshown toto detectdetect 90%90% ofof thethe heterogeneousheterogeneous rDNArDNA productsproducts inin anan individualindividual andand thethe relativerelative peakpeak heightheight seemsseems toto reflectreflect relativerelative copycopy numbernumber (Rauscher(Rauscher etet al.al. 2002).2002).

Hanna et al. 2004 DirectDirect PCRPCR andand heterogeneousheterogeneous sequencessequences

• Sequence chromatograms from direct PCR showed that heterogeneity was common within Armillaria individuals for all rDNA regions analyzed.

• Heterogeneity within rDNA regions show evidence of intra-individual variation (intra- or interspecific hybridization).

Hanna et al. 2004 DirectDirect PCRPCR andand heterogeneousheterogeneous rDNArDNA

• Phylogenetic signal is reduced when heterogeneous rDNA is characterized by ambiguous characters at polymorphic nucleotide sites.

For example, sequences with 4 dimorphic sites …ATTRGCCAYTTGCGKCCGTAMGGCA… may represent 16 possible genotypes.

– When possible, chromatograms showing heterogeneous PCR product were split into homogenous sequence representations before phylogenetic analysis with three different methods.

– Only individuals with well-defined rDNA sequences were used for phylogenetic analyses.

Hanna et al. 2004 Three methods for editing chromatograms

1a. Editing of a “frame-shift”

1b. Application of specific primers on heterogeneous PCR product

1c. Splitting a single SNP (single nucleotide polymorphism)

(R = A or G) Hanna et al. 2004 Editing of a “frame-shift” Hanna et al. 2004 Downstream (toward 5S) IGS-1 rDNA section of isolate PC514

Edited (predicted) sequences and representative chromatograms

Primer site CTTTGAACGGCAAC

Primer site GTTTGAACGGCAAAC Application of specific primers on heterogeneous PCR product Primers designed from the predicted sequences from previous slide are applied to the product Hanna et al. 2004 CTTTGAACGGCAAC and GTTTGAACGGCAAAC (Note: actual primers are reverse compliment) Upstream (toward LSU) IGS-1 rDNA section of isolate PC514

Resulting sequences and chromatograms from reverse direction sequencing “unzipping” Splitting a single SNP (single nucleotide polymorphism) nLSU rDNA section of isolate SSF4 Hanna et al. 2004

Resulting sequence edits and representative chromatograms Results

• Neighbor-joining, Parsimony, and Bayesian methods showed congruent results

Hanna et al. 2004 LSUGA4

LSUOS4 LSUOS5

nLSU* LSUOS3 LSUNA2 LSUNA1 LSUGA3 LSUOS6 LSUOS7 LSUOS8 99 97 LSUOS9 LSUOS10 LSUOS11 98 LSUTA2 93 92 LSUGA2 92

94 LSUGA1, 100 LSUCA2, LSUX4 LSUCE2, LSUTA1 100 LSUOS1, LSUSI2 LSUCA1, LSUCA3 LSUCE1, LSUX3 LSUGE1, LSUX2 LSUSI1, LSUGE2 LSUX1 LSUOS2 NORTHWEST Armillaria ostoyae LSUME1 ROCKIES A. ostoyae

LSUME3 CIRCUMBOREAL A. ostoyae LSUME2 Non-A. ostoyae Armillaria spp. * Ambiguous sequences from 14 heterogeneous isolates not included Radial 90% majority rule consensus tree of Armillaria spp. based on 24,001 trees from Bayesian inference analysis of the nuclear large ribosomal subunit (nLSU). Numbers between clades indicate estimated posterior probability. Hanna et al. 2004 RUSSIA FINLAND CANADA

NEW HAMPSHIRE WA MT

NORTHWEST OR ROCKIES ID ROCKIES x CIRCUMBOREAL WY CIRCUMBOREAL

Phylogeographic distribution of NV Armillaria ostoyae genets based UT CO on major clades from a 90% CA majority rule consensus tree produced from sequences of the nuclear large ribosomal subunit AZ (nLSU) using Bayesian inference NM analysis.

nLSU MEXICO

Hanna et al. 2004 CircumbCircumborealoreal GroupGroup DistributionDistribution

Finland

Russian Far East Utah New Hampshire

Hanna et al. 2004 Observed trends in LSU sequence analyses raise the possibility that A. ostoyae distribution is related to paleogeographic events

CoetzeeCoetzee etet al.al. (2003)(2003) postulatepostulate thatthat relationshipsrelationships amongamong SouthernSouthern HemisphereHemisphere ArmillariaArmillaria speciesspecies isis relatedrelated toto continentalcontinental driftdrift andand thethe breakupbreakup ofof Gonwanaland.Gonwanaland.

Dr. Ron Blakey – Northern Arizona University

Hanna et al. 2004 ITSOS24

ITSOS25

ITSOS19 ITSOS20 ITSOS18 ITSOS21 ITS* ITSOS17

ITSOS16

ITSOS15 ITSOS22

ITSOS14 99

ITSOS13 90 ITSOS23

ITSOS12 100 ITSOS11 ITSGE1 ITSOS10

ITSOS9 ITSOS8 ITSOS7 ITSOS1 NORTHWEST A. ostoyae ITSOS6 ROCKIES A. ostoyae

A. gemina ITSOS5 ITSOS2 * Ambiguous sequences from 31 heterogeneous isolates not included ITSOS4 ITSOS3

Radial 90% majority rule consensus tree of Armillaria spp. based on 24,001 trees from Bayesian inference analysis of the internal transcribed spacer and 5.8S rDNA (ITS). Numbers between clades indicate estimated posterior probability. Hanna et al. 2004 CANADA

NEW HAMPSHIRE WA MT

NORTHWEST OR ROCKIES & NEW HAMPSHIRE ID WY Phylogeographic distribution of Armillaria ostoyae genets based NV on major clades from a 90% UT CO majority rule consensus tree CA produced from sequences of the internal transcribed spacer and 5.8S rDNA (ITS) using Bayesian inference analysis. AZ NM

ITS

MEXICO

Hanna et al. 2004 IGSGE3

IGS* IGSGE1 IGSGE2

IGSGE5** IGSGE4

IGSOS23

IGSOS22 93

IGSOS21 IGSGE6** IGSOS1 IGSOS20 100 IGSOS2 IGSOS19 100 IGSOS3

IGSOS4 IGSOS18 96 IGSOS5

IGSOS6 99 IGSOS7 IGSOS17 IGSOS8 IGSOS14 IGSOS9 IGSOS13 IGSOS10 IGSOS12 IGSOS11 IGSOS15 NORTHWEST & NEW HAMPSHIRE A. ostoyae ROCKIES A. ostoyae A. gemina * Ambiguous sequences from 10 heterogeneous isolates not included ** A.ostoyae x A. gemina interspecific hybrid (IGSGE5 x IGSGE4) IGSOS16 Radial 90% majority rule consensus tree of Armillaria spp. based on 24,001 trees from Bayesian inference analysis of the intergenic spacer one (IGS). Numbers between clades indicate estimated posterior probability. Hanna et al. 2004 CANADA

NEW HAMPSHIRE WA MT

NORTHWEST & NEW HAMPSHIRE OR ROCKIES ID NORTHWEST x ROCKIES WY Phylogeographic distribution of Armillaria ostoyae genets based NV UT CO on major clades from a 90% CA majority rule consensus tree produced from sequences of the intergenic spacer one (IGS) using Bayesian inference AZ NM analysis.

IGS MEXICO

Hanna et al. 2004 ResultsResults (continued) rDNArDNA heterogeneityheterogeneity (intra(intra--individualindividual variation)variation) waswas commoncommon withinwithin thethe 7777 A.A. ostoyaeostoyae individualsindividuals LSU – 37 (48%) ITS – 45 (58%) IGS – 46 (60%)

OnlyOnly 16%16% ofof individualsindividuals werewere homogenoushomogenous inin allall threethree rDNArDNA regionsregions Hanna et al. 2004 Armillaria ostoyae intraspecific hybridization - IGS-1 rDNA

NABS I – Northwest Group Hanna et al. 2004

NABSNABS I Group I - Northwest B x Rockies Hybrid NABS I Group A

NABS I – Rockies Group • The Rockies group appears to have evolutionary ties to Armillaria ostoyae in Japan.

• From this we can hypothesize movement of this group to or from Asia.

• 70-100 million years ago a land bridge may have been present to facilitate this movement

• At the time Alaska and northern Asia had a temperate climate

Hanna et al. 2004 www.scotese.com

Hanna et al. 2004 FactorsFactors associatedassociated withwith geneticgenetic racesraces andand intraspecificintraspecific hybridizationhybridization withinwithin A.A. ostoyaeostoyae

•• AdaptationAdaptation toto aa varietyvariety ofof environmentsenvironments

•• MayMay bebe responsibleresponsible forfor differingdiffering levelslevels ofof pathogenicitypathogenicity andand virulencevirulence

•• “Hybrid“Hybrid vigor”vigor” maymay contributecontribute toto enhancedenhanced growth,growth, survival,survival, and/orand/or pathogenicitypathogenicity

•• HybridsHybrids maymay adaptadapt betterbetter toto environmentalenvironmental changechange

Hanna et al. 2004 ConclusionsConclusions

• Armillaria ostoyae is genetically diverse.

• Phylogenetic results from the LSU region show three distinct groups (circumboreal, Rockies, and Northwest) of A. ostoyae present in western North America.

• Genetically diverse groups of Armillaria ostoyae show various evolutionary histories and spatial distributions.

• There is phylogeographic congruence of Rockies and Northwest groups among three different rDNA regions.

Hanna et al. 2004 AcknowledgementsAcknowledgements

IntermountainIntermountain ForestForest TreeTree NutritionNutrition CoCo--opop

UniversityUniversity ofof IdahoIdaho

BOISEBOISE Corp.Corp.

USDAUSDA ForestForest ServiceService

Hanna et al. 2004 DuringDuring thethe lastlast centurycentury manymany forestsforests withinwithin thethe westernwestern UnitedUnited StatesStates havehave seenseen aa shiftshift inin treetree speciesspecies compositioncomposition duedue to:to:

•• FireFire SuppressionSuppression

•• WhiteWhite PinePine BlisterBlister RustRust

•• SelectiveSelective HarvestingHarvesting andand PlantingPlanting PracticesPractices

Hanna et al. 2004 Western Montana 1909

Gruell et al., 1982 Western Montana 1948

Gruell et al., 1982 Hanna et al. 2004 WhiteWhite PinePine BlisterBlister RustRust

• By 1940 Cronartium ribicola the fungus responsible for white pine blister rust had reached epidemic proportions on the remaining white pine forests .

• Today less than 10% of the original 5 million acres of white pine cover type exists.

Fins et al., 2001 Photo: John Hanna SelectiveSelective HarvestingHarvesting andand PlantingPlanting PracticesPractices Hanna et al. 2004 AK (Armillaria Killed)

Very few stands of white pine remain. Those that do contain genes resistant to blister rust. With each generation natural selection produces trees that are more resistant. Unfortunately as much as 90% of the potential genetic resistance within white pine was lost due to selective logging of the best trees and later in

blister rust “salvage” operations. Fins et al., 2001

Photo: BC Ministry of Forests Photo: John Hanna Photo: Raini Rippy Symptoms of Armillaria Root Disease

Chris Schnepf ResinosisResinosis CrownCrown thinningthinning and/orand/or stressstress conescones Hanna et al. 2004 ArmillariaArmillaria ostoyaeostoyae (NABS(NABS I)I) •• SeriousSerious plantplant pathogenpathogen foundfound throughoutthroughout thethe NorthernNorthern HemisphereHemisphere thatthat causescauses rootroot andand buttbutt rotrot onon diversediverse woodywoody plantplant hosts.hosts.

•• AdverselyAdversely impactsimpacts commercialcommercial timbertimber productionproduction byby causingcausing treetree mortalitymortality andand growthgrowth reduction.reduction.

J.W.Hanna 2004 Photo: Raini Rippy DirectDirect PCRPCR andand heterogeneousheterogeneous productproduct • When possible, chromatograms showing heterogeneous PCR product were split into homogenous sequence representations before phylogenetic analysis with three different methods

Hanna et al. 2004 CharacteristicsCharacteristics ofof ArmillariaArmillaria

•• ArmillariaArmillaria rootroot diseasedisease waswas firstfirst describeddescribed byby RobertRobert HartigHartig inin 18731873 •• FungusFungus // BasidiomyceteBasidiomycete •• ConstructsConstructs rhizomorphsrhizomorphs fromfrom vegetativevegetative hyphaehyphae •• FruitingFruiting bodiesbodies areare mushroomsmushrooms •• SomeSome ArmillariaArmillaria myceliummycelium isis luminescentluminescent

Photo: Raini Rippy Hanna et al. 2004 Hanna et al. 2004 ExampleExample ofof ArmillariaArmillaria ostoyaeostoyae MortalityMortality CentersCenters Hanna et al. 2004 AsAs aa result…result…

• A high percentage of forest stands within the Inland Northwest are no longer dominated by fire dependent seral tree species such as western larch, western white pine, and Ponderosa pine.

• Shade tolerant species such as grand fir and Douglas-fir now make up the majority of our forest stands. These stands are often young and dense.

• Remaining old growth stands are in danger due to the abundance of “ladder fuels”. This creates an increased risk of stand replacing crown fires. Langston, 1995 Photo: Moosefest.com • Douglas-fir and grand fir Hanna et al. 2004 are highly susceptible to Armillaria ostoyae.

• Plantations of Ponderosa pine not locally adapted to a site can also be highly susceptible.

•Locally adapted seral species such as western larch, western white pine, and Ponderosa pine show the greatest resistance to Armillaria root rot. Shaw and Kile, 1991 Hanna et al. 2004 ArmillariaArmillaria SpeciesSpecies andand GenetGenet IdentificationIdentification

Black line Colorless antagonisms = different species = same species

Photo: Mee-Sook Kim Microscopic observation of Armillaria mating for species identification

ClampClamp connectionsconnections

Hanna et al. 2004 From G.I. McDonald Inferring phylogenies from genes containing a small number of informative sites becomes highly ambiguous if hybrid individuals are polymorphic at those sites

Polymorphic site 1 R = G or A

Polymorphic site 2 Y = C or T

Polymorphic site 3 K = G or T

Polymorphic site 4 M = A or C

2 polymorphic sites = 4 possible genotypes 3 polymorphic sites = 8 possible genotypes

4 polymorphic sites = 16 possible genotypes Hanna et al. 2004 Primer sets applied to heterogeneous PCR product

A triplicate of reverse primers were created and applied to product shown to contain a SNP at base pair position 683 of the IGS-1 region

Region Primer .

IGS: AOHR1T (5’-TGCCGTTCAAAA-3’)

AOHR1G (5’-TGCCGTTCAAAC-3’)

AOHR1C (5’-TGCCGTTCAAAG-3’)

Hanna et al. 2004 PhylogeneticPhylogenetic approachesapproaches

• Techniques for reducing heterogeneous individuals into homogenous parental representation were not always possible.

• Remaining individuals containing two or more nucleotide sites showing intra-individual variation were excluded from phylogenetic analysis to eliminate ambiguity of phylogenic signal.

Hanna et al. 2004 77 A. ostoyae individuals were sequenced in each of three regions

Chromatograms showing heterogeneity (intra-individual variation)

LSU – 37 (48%) ITS – 45 (58%) IGS – 46 (60%)

Reduced to two homogenous sequences with editing techniques

LSU – 23 (30%) ITS – 14 (18%) IGS -36 (47%) Remaining heterogeneous individuals excluded from analysis

LSU – 14 (18%) ITS – 31 (40%) IGS – 10 (13%)

Only 16% of individuals were homogenous in all three regions

Hanna et al. 2004 Hanna et al. 2004 ArmillariaArmillaria MortalityMortality CentersCenters

USDA Forest Service, Missoula Archives