Cross%lineage,hybridization,in,the,genus,Arctostaphylos,(Ericaceae),,

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1 Crosslineage,hybridization,in,the,genus,Arctostaphylos,(Ericaceae),, EricVanDyke,ReedCollegeBio332(May2017) Abstract, TheevolutionaryhistoryofthegenusArctostaphylosissplitbetweentwodeeplineages.Many Arctostaphylosspeciesarehypothesizedtoresultfromhybridization,butcrossJlineagehybridsare uncommon.iidentifiedsixspecieswithnuclearribosomaldnasequencesthatcontainnucleotide combinationsfrombothlineages.inthesecrossjlineagehybrids,polymorphicnucleotidesarecommon atthesevenbasepositionsthatseparatethetwolineages.incrossjlineagehybridspecieswhere individualsfrommultiplepopulationsareavailable,nucleotidevariationbetweenpopulationsispresent atthesesevenpositions.atleastfourofthesecrossjlineagehybridsarepolyploids,suggestingthat wholegenomeduplicationenableshybridizationbetweenlineages.sequencevariationbetween populationssuggeststhattheymayresultfrommultipleindependentpolyploidy.isearchedfor potentialparentalspeciesforthesecrossjlineagehybridsbycomparingtheirsequenceswiththoseofall otherextanttaxaandevaluatingnucleotideadditivity.theresultsofferinsightsintoarctostaphylos biogeographyandpaleoendemismaswellashybridizationandpolyploidy. Introduction, ThegenusArctostaphyloscomprises67species(Kauffmannetal.2015,FNA2009).Onespecies,A.-uva0 ursi,isgloballydistributed.theremainderarenativetothewesternunitedstates,northernmexico, andsouthernbritishcolumbia.nearlythreequartersofarctostaphylosarelocalendemics;manyof t"em are confined to i/land/ of unu/ual and generall4 poor /oil. Fort4 are of con/ervation concern. ElevenArctostaphylosspeciesarepolyploids. ManzanitaParkinnorthMontereyCounty,California,containsmorethan150haofcoastalchaparralon sandysoil.thisshrublandhabitatisdominatedbythreemanzanitaspecies:twonarrowendemics,- Arctostaphylos-pajaroensisandA.-hookeri,andaregionalendemic,-A.-crustacea.Thethreetaxaare easilydistinguishable.a.-pajaroensisisatall,uprightshrubwithtriangularleavesandgrayshreddy bark,a.-hookeriisalow,matjformingshrubwithshinygreenleavesandsmoothredbark,anda.- crustaceaisintermediateinstatureandhasaprominentbasalburl.duringmorethanadecadeof frequenthikesatthepark,ichallengedmyselftofindindividualswiththesecharactersinunusual combinations.theseapparenthybridsdooccur,andtheyinvariablycombinethetriangularleavesofa.- pajaroensiswiththeburlofa.-crustacea.iwasmotivatedinthissearchbytheknowledgethatlocal nurseriessellanaturalhybridnameda.- Sun/et t"at combine/charactersfroma.-hookeriwith charactersfroma.-crustacea.butineverfoundoneinthewild.

2 ItisnotunusualtofindtwoorthreeArctostaphylosspeciesgrowingtogetheratthesamesite, occasionallymore.sometimestherearehybrids,sometimest"ere aren t. T"i/ /tud4 i/ an attempt to applymoleculardataandthetoolsofphylogeneticanalysistoanswerthequestion:why? Methods, IdownloadedallArctostaphylosnucleotidesequencesfromGenBankthatincludetheITS,5.8S,or26S regionsofnuclearribosomaldna.thesesequencesresultfromseveralstudies,particularlymarkoset al.(1998),boykinetal.(2005),andwahlertetal.(2009).ialsodownloadedarctous-rubrasequences thatincludethesameregionsasanoutgrouptopolarizemyresultingphylogeny.ichosearctous-rubra, anarctic/tundrashrub,basedonitspositionasclosesistertoarctostaphyloswithintheericaceae(kron etal.2002).iupgradedtaxonomicnomenclatureasnecessarytoagreewiththecurrenttreatment(fna 2009)andassignedabriefidentifier(sixJcharacteracronym,optionalsingleJcharactersubspecies identifier,anddifferentiatingnumber)toeachsequence.sequenceidentifiersarelistedinappendix1 alongwithcorrespondingtaxonname,genbankaccessionnumber,ploidy,studyauthor,andcollection locationandvoucheridentificationifavailable.theits+5.8sdatasetconsistsofsequencesfromallbut fourarctostaphylosspecies(a.-bolensis,a.-incognita,a.-moranii,anda.-nortensis)andincludesmultiple sequencesformanyspecies(differentsubspeciesandcollectionlocations).the26sdatasetconsistsof asmallersample(18species). IassembledandmanuallyalignedtheITS1+5.8S+ITS2and26Ssequencesinseparatenucleotide charactermatrices.whereits1andits2wereseparateaccessions,icombinedthesebeforealignment. Manualalignmentwasstraightforwardduetoarelativelysmallnumberofvariablecharactersand indels.afteralignment,itrimmedallsequencestouniformlength(584bpforits1+5.8s+its2and264 bpfor26s). Thesealignedcharactermatricesprovideddatanecessarytoinfertheevolutionaryhistoryof ArctostaphylosthoughphylogeneticinferenceusingthePAUP*software.Inaddition,theyenabled directobservationofnucleotidevariabilityandpolymorphismwithinandbetweennucleotide sequences,providingameansfortestinghypothesizeddiploidandpolyploidhybridizationbetween species. Results,, Phylogenetic,analysis, SequencesfromtheITS,5.8S,and26SregionsinArctostaphyloscontainnucleotidesubstitutionsand polymorphismsatrelativelyfewbasepositions,sodirectobservationandrecognitionofbetweenj speciespatterns(apomorphies)andspeciesjspecificpatterns(autapomorphies)wasrelatively straightforward.ifirstremovedallbasepositionswithnophylogeneticallyinformativecharacters(no statechanges,orchangesonlywithinasingletaxon);thisdramaticallyreducedthesizeofthedatasets. Ifurtherreduceddatasetsizebycombiningallidenticalsequencesfromasinglespecies,including

3 Figure 1. Reduced, rearranged, and colored ITS+5.8S character matrix (nucleotides) with cross-l i neage sequences r emoved. CLADE2a_3 includes four taxa with identical sequences. CLADE2b_52 includes 52 taxa with identical sequences.

cross-lineage sequences removed. Figure 3. Reduced, rearranged, and colored ITS+5.

4 Figure 2. Reduced, rearranged, and colored 26S character matrix (nucleotides) with cross-lineage sequences removed. Figure 3. Reduced, rearranged, and colored ITS+5.8S cross-lineage sequences. All but one taxon are polyploid (red identifiers). Figure 4. Reduced, rearranged, and colored 26S cross-lineage sequences. All taxa are polyploid.

5 Figure 5. ITS+5.8S majority-rule consensus tree with cross-lineage sequences removed. Orange bar is the genus Arctostaphylos. Red bars are the patula and columbiana clades. Brown bar is the ohloneana clade.

6 subspecies,underanewcommonidentifierwithanappendednumber.also,intwocases,icombined multipletaxawithidenticalsequencesunderacommonidentifier(clade2a_3andclade2b_52). MembershipofthesecombinedsequencegroupsarelistedinAppendix2.Allsequenceswithcharacter differences,includingpolymorphisms,wereretained.finally,irearrangedtheorderofthecharacters (nucleotides)andcoloredgroupsofderivedcharacters(apomorphiesandautapomorphies)toaidvisual identificationofanypatterns.portionsofthereduced,rearranged,andcoloredcharactermatricesare showninfigure1(its+5.8s)andfigure2(26s). Severalgroupsofderivedcharactersareapparentwithinthesenucleotidesequences.First,15 characterpositions(orange) sevenintheits1region,fourinits2,andfourin26s differnearly uniformlyfromtheancestralstate(theoutgroup,rubra_1).thesepositionsareobviouslyresponsible forthesynapomorphythatdefinesthegenusarctastaphylos.also,sevencharacterpositions(red) twoinits1,twoinits2,andthreein26s assumecomplementarycharacterstatesasagroup.these positionsareclearlyresponsibleforasynapomorphythatsplitsthegenusintotwodeeplineages.this twojcladephylogenywasfirstobservedbydenford(1981)usingflavonoidsasmarkers.while investigatingthemonophylyoffivearctostaphylossubspecies,markosetal.(1998)recoveredthesetwo cladesfromnuclearribosomaldnausingitsand26ssequencesandrflpmarkers.boykinetal.(2005) andwahlertetal.(2009)demonstratedthatthetwojcladestructurepersistedastheyincreasedthe numberofarctostaphylostaxasampled(appendix3).afewoneandtwojcharacterapomorphies(blue andgreen)arealsoevidentwithinthenucleotidesequences,aswellasonelongerderivedgroupthat encompassessevencharacterpositions(brown).thissevenjcharacterapomorphywillbediscussed below. OfcoursethedatasetsarenotquiteasuniformasFigures1and2imply.Withinthegroupofseven characterpositionsthatdividesarctostaphylosintotwolineages(fourinits+5.8sandthreein26s), variousassortmentsofindividualnucleotidesfrombothclades,andpolymorphismsthatcombine nucleotidesfrombothclades,appearin15sequencesfromsixspecies(figures3and4). Nevertheless,afterremovingthose15crossJcladesequencesfromtheITS+5.8Smatrix,maximum parsimonyinpaup*generatedamajorityjruleconsensustreeforthegenusarctostaphylosthat correspondscloselywiththepredictedphylogeny(comparethecoloredbarsinfigure5withlikej coloredcellsinfigure1)andaddsadditionaltaxatothephylogeniespublishedbymarkosetal.(1998), Boykinetal.(2005),andWahlertetal.(2009). Crosslineage,hybrid,parentage, CrossJlineagesequencescauseproblemsforphylogenyinference,buttheyprovidevaluableinsightsinto hybridizationbetweenspecies.hybridizationinarctostaphyloshasbeenanactivetopicofresearchand conjectureformanydecades.manyarctostaphylosspeciesarehypothesizedtohavearisenthrough diploidhybridizationorpolyploidy,butreportsofcrossjlineagehybridizationareuncommon(wahlertet al.2006,parker2007).

7 IidentifiedsixhybridspecieswithnuclearribosomalDNAsequencesthatcontainnucleotide combinationsfrombothlineages(figures3and4).inthesecrossjlineagehybrids,polymorphic nucleotidesarecommonatthesevenbasepositionsthatseparatethetwolineages.incrossjlineage hybridswhereindividualsfrommultiplesequencesareavailable,nucleotidevariationbetween populationsispresentatthesesevenpositions.theprevalenceoffourjcharacterjstateambiguityatthe positionsthatsplitthegenusinseveral26ssequencessuggestspolyploidyintheoriginofthesespecies (Figure4).Notsurprisingly,theliteratureconfirmsthatfourofthecrossJcladespeciesarepolyploids, andtheploidyofanotherisuncertain. ItestedwhethertheparentsofknownorhypothesizedcrossJlineagehybridscouldbeidentifiedby performingpairwisesequencecomparisonsbetweenallotherextanttaxaandevaluatingnucleotide additivityinthetargettaxon.similarattemptstoevaluatepossiblehybridizationandpolyploidywithits sequenceadditivityhavebeenreported(e.g.whittall2000,hardigetal.2002). Figure 6. A. patula and A. viscida are not additive for A. mewukka; seven nucleotides (brown) are not available from either parent (ITS+5.8S). Figure 7. A. ohloneana is additive for A. mewukka in hybrids with any of various taxa from the same lineage (ITS+5.8S). Figure 8. A. ohloneana is additive for all selections of A. manzanita in hybrids with any of various taxa from the opposite lineage (ITS+5.8S). Figure 9. A. hookeri (and several other possible parents) is additive for all A. pungens sequences in hybrids with any of various taxa from the opposite lineage (ITS+5.8S).

8 SearchingforpotentialhybridparentsoftaxawithITS+5.8SsequencesthatcontainthesevenJcharacter apomorphymentionedabove(browninfigures1,3,and5)producedsurprisingresults.arctostaphylosmewukka,aspecieslonghypothesizedtobeofhybridorigin,hasbeencarefullystudied(epling1947, Dobzhansky1953,Roof1967,Schmid1968).A.-mewukkawasfirstdescribedasadiploidhybrid betweenarctostaphylos-patulaanda.-viscida,twomanzanitaswithoverlappingranges(mcminn1939). Schierenbecketal.(1992)performeddetailedmorphologicalandcytologicalstudiesanddeclaredthe speciesandsubspeciestobetheresultofmultipleindependent(polyphyletic)allopolyploidywitha.- patulaanda.-viscidaasparents.yetpairwisesequencecomparisonshowsthatthecombinationofa.- patulaanda.-viscidaisnotadditivefora.-mewukka(figure6).onlyoneextantspeciessatisfiesthe additivityrequirement:a.-ohloneana(figure7).furthermore,whilea.-patulaanda.-viscidaarefrom differentlineages,a.-mewukkacanresultfromhybridizationbetweena.-ohloneanaandvarioustaxain thesamelineage.a.-mewukkai/ fairl4 wide/pread at middle elevation/ in California / Sierra Nevada. A.- ohloneanaisarare,recentlydescribedspeciesfromjustonecoastalsantacruzcountylocation well overonehundredmilestothewest(kauffmann2015). T"ere / more. Arctostaphylos-manzanitaanditssubspeciesareagroupofmorphologicallyand geographicallydiversepolyploidtaxa.thisvariabilityisalsoevidentinthevarietyofnucleotide combinationsinitssequences.threea.-manzanitasequencescontainsome(butnotall)ofthesame sevenjcharacterapomorphy,eitherasvariablecharactersorpolymorphisms(figure8).thisvariability suggestsa.-manzanita / numeroussubspeciesandvarietiesmayhaveoriginatedthroughmultiple independentpolyploidy.onceagain,pairwisecomparisonfounda.-ohloneanaastheonlypossibilityfor oneofa.-manzanita / hybridparents(figure8).accordingtovaseyandparker(2008),a.-ohloneana superficiallyresemblesa.manzanita.a.-manzanitaisfoundinavarietyoflocationsandhabitats, includingasmallsiteabout30mileseastofa.-ohloeana / coa/tal location(kauffmann2015). LikeArctostaphylos-manzanita,Arctostaphylos-pungens-hasavarietyofnucleotidecombinationsand polymorphismsinitssequences.asina.-manzanita,thisvariabilitysuggestsmultipleindependent polyploidy.againisearchedforpotentialhybridparentsfora.-pungensbycomparingsequencesand evaluatingnucleotideadditivity.inthiscase,morethanonespeciessatisfiestheadditivityrequirement; A.-Hookeri,forexample(Figure9).Arctostaphylos-pungensisgenerallyconsidereddiploid,butRoof (1976) write/g Hit" a /ingle known exceptionk in /out"ern MexicoK t"e A.-pungensofMexicoand /out"ern California "a/ been determined to be a tetraploidk wit" a gamete number of 26.IfRoofis correct,a.-pungensisanothercrossjlineagepolyploid.thishelpsexplainwhykeeley(1976)observed unexpectedmorphologicalcharactersemergefromhybridizationbetween(tetraploid?)a.-pungens.and diploida.-glauca.believinga.-pungenstoalsobediploid,keeleydeclaredthepopulationahybrid swarmwithbackcrossestobothparentsratherthanvariableoffspringfromapolyploidparent. Discussion,, Two,lineages,(patula,and,columbiana,clades), SeveralauthorshaveproposedbiogeographicalexplanationsfortherapidpostJglacialradiationof Arctostaphylos.RavenandAxelrod(1978)describetheXerothermicPeriod,atimeofwarmingand

9 dryingclimate,whenboundariesbetweennorthernforestjdominatedvegetation(thearctojtertiary Geoflora)andsouthernscrubJdominatedvegetation(MadroJTertiaryGeoflora)werechangingrapidly. TheyattributemuchoftheexplosivespeciationinspecieslikeArctostaphylostothesechanges.Thetwo deeplineageswithinthearctostaphylosphylogenylikelyoriginatedwiththesetwovegetationtypes. BecauseArctostaphylosdiversityandtaxonomyareheavilyinfluencedbythesplitbetweentwo lineages,theselineagesdeservemoreimaginativenamesthan Group One/GroupTwo (Marko/etal. 1998)or Clade 1/Clade2 (Boykinetal.2005,Wahlertetal.2009).Irecommendthatthemost representativeorwidespreaddiploidspeciesineachlineageshouldlenditsname,andprovisionally suggest patulaclade for t"o/e /pecie/ wit" Great Ba/inRRock4 Mountain (MadroJTertiary)affinities, and columbianaclade for t"o/e /pecie/ wit" northern(arctojtertiary)affinities., The1ohloneana1clade1 ThesmallcladethatsharesadistinctivesevenJnucleotideapomorphyisalsodeservingofaname.Its rare,recentlyjdescribeddiploidmember,arctostaphylos-ohloneana,maybethepaleoendemicremnant fromaformerlywidejrangingspecies(vaseyandparker2008)thatparticipatedinmultipleindependent polyploidytoproduceanassortmentofa.-manzanitaanda.-mewukkasubspecies.isuggest ohloneana clade for t"i/ group thatisprovidingvaluableinsightsintodiversificationandbiogeographyin Arctostaphylos. Too,many,manzanitas?, Twoverydifferentpatternsofdiversificationareevidentin-Arctostaphylos.SameJlineagespecies conformrigidlytoanucleotide /ignature K thehandfulofapomorphies,eachinvolvingjustafewbase positions,thatcollectivelydefinesitsplaceinthephylogeny(coloredcellsinfigures1and2andcolored barsinfigure5).ninetypercentofarctostaphylostaxaexhibitthisconsistency.onesignatureis commonto34species,halfthegenus.incontrast,eachcrossjlineagepopulationacquiresitsown uniquearrangementofnucleotidesandpolymorphisms,especiallyatthesevenbasepositionsthat separatethelineages,ratherthanrigidlyconformingtoacommon,speciesjwidesignature(figures3and 4).Theirphylogenyisareticulatednetworkratherthanasimpletree.Ineverycasebutone,these hybridspeciesarepolyploids,suggestingthatwholegenomeduplicationenableshybridizationbetween lineages(schierenbecketal.1992). Arctostaphylostaxonomists"ave been ent"u/ia/tic /plitter/ atleastfromtheperspectiveofnuclear ribosomaldna.raven(1969)characterizedthesituation: A variety of workers have continued to present new combinations and new taxa without ever approaching the overall view of the group necessary to achieve taxonomic synthesis. A useful taxonomic system for a complex group such as Arctostaphylos will never be built up on such blocks, and indeed, the overall pattern of variation tends to become more and more obscure as the new taxa are proliferated. Roof(1976)K following Raven / leadk took a freshapproach tothegenusandattemptedacomplete revisionbasedonjustsixbasicspecies.alltherestheconsideredhybridstobereclassifiedas subspeciesandvarieties(inconsistentlyappliedtermsforregionalorlocal coherentevolutionary

10 subsets,hamiltonandreichard1992).forsamejlineagespecies,onenucleotidesignatureistypically sharedbyavarietyofpopulations,whetherspecies,subspecies,orsamejlineagehybridsthathave undergoneconcertedevolution.forthesetaxa,roof / radical lumping approachseemsappropriate, notwithstandingconservationconcerns(willendangeredpopulationscontinuetoreceiveprotectionif t"e4 re no longercalledspecies?).butnotsoforcrossjlineagepolyploidsandhybrids.thebest documentedexampleofcrossjlineagediversificationinthisstudyisarctostaphylos-manzanita,with individual/ from nine population/. Roof de/cribe/ combination/ too numerou/ and deviou/ to recite ina.-manzanitaand worrie/ t"at t"ere i/ danger of attac"ing to it more /ub/pecie/ t"an it can po//ibl4 bear. Suc"taxademandarethinkingofthespeciesconcept.Whatisaspeciesifeverypopulationhas itsownuniquearrangementofnucleotides?shouldeverypopulationconstituteaseparatespecies?if so,doeseveryspeciesdeserveendangeredspeciesprotection? Figure 10. The three Arctostaphylos at Manzanita Park (ITS+5.8S). A diploid in the patula clade, a diploid in the columbiana clade, and a polyploid. What would a polyploid A. Sunset l ook l i ke? Arctostaphylos, Sunset, HybridizationhasbeenreportedtoberelativelyfrequentbetweensomepairsofArctostaphylosspecies. Forotherpairs,hybridsappearinfrequently,ifever.Reproductivebarriersarepresumedtobestronger betweenthetwolineagesthanwithin(parker2007).instudieswherehybridizationwasfrequent,the parentsweretypicallymembersofthesamelineage;wherehybridizationwasuncommon,theywere fromoppositelineages(vaseyandparker2014).asimilarsituationexistsinthegenusceanothus:two deeplineagesareseparatedbystrongreproductivebarriers,whicharebelievedtobegeographicaland edaphicratherthanintrinsic(hardigetal.2002). Manzanitasfromthetwolineagesarefrequentlyfoundgrowingtogetherinthewild.Oftentheyare accompaniedbyapolyploid.thisisthesituationatmanzanitapark.arctostaphylos-hookeriisadiploid inthepatulaclade,a.pajaroensis-isadiploidinthecolumbianaclade,anda.-crustaceaispolyploid. Arctostaphylos Sun/et i/ acrossjlineagehybrid.iswouldbeinterestingtolearnifitispolyploid. Needs, TheexistenceoftwodeeplineagesintheinferredArctostaphylosphylogeny,thepatulaandcolumbiana clades,isonlymoderatelysupportedbybootstrapresampling(markosetal.1998,boykinetal.2005, Wahlertetal.2009).Withtheexceptionoftheohloneanaclade,cladesdeeperinthephylogenyreceive evenlowersupport.themostobviouspathtoincreasingstatisticalsupport,andfordetermining

11 whethertheconclusionsreportedinthisstudyarerobust,istoacquiresequencedatabeyondthe nuclearribosomalrepeats includinglowjcopycodingregionsandplastidgenomes. Theassumptionhasbeenthat,becauseofconcertedevolution,onlyasingleindividualfromasingle populationisnecessarytoobtainrepresentativesequencedataforarctostaphylos(boykinetal.2005). ForsameJlineagespecies,thisislikelythecase.ButforcrossJlineagehybrids,thefewavailable sequencesfromdifferentpopulationsinasinglespecies(arctostaphylos-manzanita,a.-pungens,anda.- uva0ursi)clearlycontradicttheassumption.thereforeagreaterbreadthofsamplingamongthesix crossjlineagespeciesisalsoneeded.thesesequencesshouldcapturethegeographicrangeofeach species,withemphasisonunusualedaphics(e.g.serpentine)andregionsthatoverlapwithother Arctostaphylostaxa. Finally,moredefinitivechromosomecountsareneededforafewcrossJlineagespecies.Istherearange ofploidyinarctostaphylosmewukkaanda.pungens?chromosomalvariationslikelyinfluencethe trajectoryofspeciesevolutionandhybridization. References, Boykin,L.M.etal.2005.TwolineagesofArctostaphylos(Ericaceae)identifiedusingtheinternal transcribedspacer(its)regionofthenucleargenome.madroño52(3). Dobzhansky,T.1953.NaturalhybridsoftwospeciesofArctostaphylosintheYosemiteregionof California.Heredity7. Epling,C.1947.Actualandpotentialgeneflowinnaturalpopulations.TheAmericanNaturalist81(797). FNAEditorialCommittee,eds.2009.FloraofNorthAmerica,vol.8.TheFloraofNorthAmerica Association,NewYorkandOxford. Hamilton,C.W.andReichard,S.H.1992.CurrentPracticeintheuseofsubspecies,variety,andformain theclassificationofwildplants.taxon41(3). Hardig,T.M.etal.2002.Morphologicalandmolecularanalysisofputativehybridspeciationin Ceanothus(Rhamnaceae).SystematicBotany27(4). Kauffmann,M.E.etal.2015.FieldGuidetoManzanitas:California,NorthAmerica,andMexico. BackcountryPress,Kneeland,CA. Keeley,J.E.1976.MorphologicalevidenceofhybridizationbetweenArctostaphylos-glaucaand-A.- pungens(ericaceae).madroño23(8).

12 Kron,K.A.etal.2002.PhylogeneticClassificationofEricaceae:MolecularandMorphologicalEvidence. TheBotanicalReview68(3). Markos,S.E.etal.1998.PhylogenyoftheArctostaphylos-hookericomplex(Ericaceae)basedonnrDNA sequencedatafromtheitsregion.madroño45(3). McMinn,H.E.1939.AnIllustratedManualofCaliforniaShrubs.UniversityofCaliforniaPress,Berkeley, CA. Parker,V.T.2007.DiversityandevolutionofArctostaphylosandCeanothus.Fremontia35(4). Raven,P.H.1969.Review:SupplementtoACaliforniaFlorabyPhilipA.Munz.Madroño20(4). Raven,P.H.andAxelrod,D.I.1978.OriginandRelationshipsoftheCaliforniaFlora.Universityof CaliforniaPress,BerkeleyCA. Roof,J.1967.Arctostaphylos-mewukka:ahybrid.TheFourSeasons2(2). Roof,J.1976.AfreshapproachtothegenusArctostaphylosinCalifornia.TheFourSeasons5(2). Schierenbeck,K.A.etal.1992.Morphologicalandcytologicalevidenceforpolyphyleticallopolyploidyin Arctostaphylos-mewukka(Ericacacese).PlantSystematicsandEvolution179. Schmid,R.etal.1968.BiosystematicevidenceforhybridizationbetweenArctostaphylos-nissenanaand A.-viscida.Brittonia20. Vasey,M.C.andParker,V.T.2008.AnewlydescribedspeciesofArctostaphylos(Ericaceae)fromthe centralcaliforniacoast.madroño55(3). Vasey,M.C.andParker,V.T.2014.Driversofdiversityinwoodyplantlineagesexperiencingcanopyfire regimesinmediterraneantypeclimates.in:rajakaruna,n.etal.,eds.plantecologyandevolutionin harshenvironments.novapublishers,newyork. Wahlert,G.A.etal.2006.TheArctostaphylos-bakerispeciescomplexfromSonomaCounty,California. TheFourSeasons12(4). Wahlert,G.A.etal.2009.AphylogenyofArctostaphylos(Ericaceae)inferredfromnuclearribosomalITS sequences.journalofthebotanicalresearchinstituteoftexas3(2). Whittall,J.etal.2000.DetectingnucleotideadditivityfromdirectsequencesisaSNAP:Anexample fromsidalcea(malvaceae).plantbiology2.

13 Appendices, Appendix 1: ITS+5.8S sequence identifiers, taxon names, GenBank accession numbers, ploidy, study authors, collection locations, and voucher identification.

14 Appendix 1 (cont.): ITS+5.8S

15 Appendix 1 (cont.): ITS+5.8S

Appendix 1: 26S sequence identifiers, taxon names, GenBank accession numbers, ploidy, study authors, collection locations, and voucher identification.

16 Appendix 1: 26S sequence identifiers, taxon names, GenBank accession numbers, ploidy, study authors, collection locations, and voucher identification. CLADE2a_3: NEVADA1,CRUSTA1,CRUSTAs1,GLANDU2 CLADE2b_52: MANZAN1,MANZAN3,MANZAN4,MANZANe1,MANZANg1,TOMENTd1,CRUSTAi1,CRUSTAr1, ANDERS1,AURICU1,BAKERI1,BAKERIs1,CANESC0,CANESC1,CANESCs1,CATALI0,CATALI1,COLUMB1, CONFER1,CRUZEN0,CRUZEN1,FRANCI1,GABILA1,GLANDU0,GLANDUc1,GLANDUm1,GLAUCA1, GLUTIN1,HOOVER1,IMBRIC1,INSULA1,LUCIAN1,MONTAN1,MONTAR1,MONTER1,MORROE1, OBISPO1,OSOENS1,PAJARO0,PAJARO1,PALLID1,PILOSU0,PILOSU1,PILOSU2,PURISS1,REFUGI0, REGISM1,SILVIC1,VIRGAT1,XMANST1,XMEDIA1,XREPEN1 Appendix 2: Identical sequences combined under a common identifier (CLADE2a_3 and CLADE2b_52).

17 Appendix 3: Assignment of taxa to patula and columbiana clades by Denford (1981, flavonoids), Markos (1998; RFLP, ITS, and 26S), Boykin (2005, ITS), Wahlert (2009, ITS), and this study (ITS). Note the confusion when attempting to classify crosslineage taxa.

18 Appendix 3 (cont.): Assignment of taxa to patula and columbiana clades.

Modern Evolutionary Classification. Lesson Overview. Lesson Overview Modern Evolutionary Classification

Lesson Overview 18.2 Modern Evolutionary Classification THINK ABOUT IT Darwin s ideas about a tree of life suggested a new way to classify organisms not just based on similarities and differences, but