Origins and diversity of a cosmopolitan fern genus on an island archipelago

15 Pages·2015·0.61 MB·English

Research Article
SPECIAL ISSUE: Island Plant Biology—Celebrating Carlquist’s
Legacy
Origins and diversity of a cosmopolitan fern genus
on an island archipelago
Paul G. Wolf1,2*, Carol A. Rowe1, Joshua P. Der3, Martin P. Schilling1, Clayton J. Visger4 and
John A. Thomson5
1 DepartmentofBiology,UtahStateUniversity,Logan,UT84322,USA
2 EcologyCenter,UtahStateUniversity,Logan,UT84322,USA
3 DepartmentofBiologicalScience,CaliforniaStateUniversity,Fullerton,CA92834,USA
4 DepartmentofBiology,UniversityofFlorida,Gainesville,FL32611,USA
5 NationalHerbariumofNSW,RoyalBotanicGardensandDomainTrust,MrsMacquariesRoad,Sydney,NSW2000,Australia
Received:29July2015;Accepted:9October2015;Published:20October2015
AssociateEditor:ChelseaD.Specht
Citation:WolfPG,RoweCA,DerJP,SchillingMP,VisgerCJ,ThomsonJA.2015.Originsanddiversityofacosmopolitanferngenus
onanislandarchipelago.AoBPLANTS7:plv118;doi:10.1093/aobpla/plv118
Abstract. Isolatedoceanicislandsarecharacterizedbypatternsofbiologicaldiversitydifferentfromthatonnearby
continentalmainlands.Isolationcanprovidetheopportunityforevolutionarydivergence,butalsosetthestagefor
hybridizationbetweenrelatedtaxaarrivingfromdifferentsources.Fernsdispersebyhaploidspores,whicharepro-
ducedinlargenumbersandcantravellong distancesinaircurrents,enablingtheseplantstobecomeestablished
on most oceanic islands. Here, we examine the origins and patterns of diversityof the cosmopolitan fern genus
Pteridium (Dennstaedtiaceae; bracken) on the Galapagos Islands. We use nucleotide sequences from two plastid
genes,andtwonucleargenemarkers,toexaminephylogeographyofPteridiumontheGalapagosIslands.Weincorp-
oratedatafromapreviousstudytoprovideaworldwidecontext.WealsosamplednewspecimensfromSouthand
CentralAmerica.Weusedflowcytometrytoestimategenomesizeofsomeaccessions.Wefoundthatbothplastid
andnuclearhaplotypesfallintotwodistinctclades,consistentwithatwo-diploid-speciestaxonomyofP.aquilinum
andP.esculentum.Aspredicted,theallotetraploidP.caudatumpossessesnuclearhaplotypesfrombothdiploidspe-
cies. Samples from the Galapagos include P. esculentum subsp. arachnoideum, P. caudatum and possible hybrids
betweenthem.MultiplePteridiumtaxawerealsoobservedgrowingtogetheratsomesites.Wefindevidenceformul-
tipleoriginsofPteridiumontheGalapagosIslandsandmultipleoriginsoftetraploidP.caudatumthroughoutitsrange
in Central and South America. We also posit that P. caudatum may include recent diploid hybrids, backcrossesto
P. esculentum, as well as allotetraploid plants. The Galapagos Islands are positioned close to the equator where
theycanreceivedispersingpropagulesfrombothhemispheres.Thismaypartlyexplainthehigh levelsofdiversity
foundforthiscosmopolitanfernontheseislands.
Keywords: Biogeography;bracken;ferns;Galapagos;hybridization;islands;nucleargenes;phylogeny;Pteridium.
*Correspondingauthor’se-mailaddress:[email protected]
PublishedbyOxfordUniversityPressonbehalfoftheAnnalsofBotanyCompany.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/
licenses/by/4.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 1
Wolfetal.—OriginsofGalapagosPteridium
Introduction morphologicalcharacters,includingthosethatareused
for taxonomic treatments. Furthermore, regional and
Oceanic islands provide an ideal biological setting for
localtreatmentsoftendonotincorporatethecontextof
evolutionarychangeandthusforthestudyofevolution-
variationthatisseenattheglobalscale.Afewauthors
aryprocesses.Onislandsthataredistantlyisolatedfrom
have examined Pteridium in a worldwide context. For
continents, there can be an increased opportunity for
example,Ching(1940)consideredthegenustocomprise
organismsto diverge genetically from those in the ori-
6 species, whereas a year later, Tryon (1941) treated
ginal source populations. Furthermore, remote islands
Pteridium as a single species with 2 subspecies and 12
can act as a sink for individuals of the same species
varieties. Page (1976) reviewed information on geo-
(orcloselyrelatedspecies)arrivingfrommorethanone
graphic variation and concluded that there is probably
originalsource,therebysettingthestageforhybridiza-
morethanonespecies,buthemadenoformaltaxonomic
tion, increased genetic diversity, or both. Angiosperms
changes.Morerecently,inaseriesofarticles(Thomson
and gymnosperms colonize islands most often via
2000,2012;Thomson etal.2008),Thomson hasrecog-
seeds,whichcontaindiploidembryos.Thisiscontrasted
nizedtwomaindiploidspecies:P.aquilinum(correspond-
in ferns (monilophytes) and lycophytes, which are dis-
ingtoTryon’ssubspeciesaquilinum)fromEurope,North
persedbyhaploidspores.Inmanycases,spore-bearing
America,AsiaandAfricaandP.esculentum(correspond-
plantsrequiretwospores,andsubsequentlytwogameto-
ing approximately to Tryon’s subspecies caudatum).
phytes, for successful establishment (Soltis and Soltis
Pteridiumesculentumistreatedbysomeauthorsastwo
1987;Soltisetal.1988).Moreover,spore-dispersedplants
species:P.esculentuminAustraliaandNewZealandand
appeartohavehighlevelsofgeneflowrelativetoseed
P. arachnoideum in South America (see, for example,
plants (Soltis and Soltis 1987), presumably because
Schwartsburdetal.2014),whereasotherstreatesculen-
spores are smaller and more easily transported in the
tum and arachnoideum as subspecies of P. esculentum
air than most seeds. An increased dispersal potential
(Thomson 2012; Zhou et al. 2014), a system we follow
can result in colonization of new areas from multiple
here. Regardless of rank assignment, evidence for two
sporesources,andmayinvolveanincreasedopportunity maincladesofPteridiumincludesanalysesofplastidDNA
forhybridizationrelativetoseedplants.Here,weexplore variation(Deretal.2009;Zhouetal.2014).Further,several
thispossibilityasweassesstheoriginsanddiversityofthe hybridsandallotetraploidshavebeenexamined(Thomson
cosmopolitan fern genus Pteridium on the Galapagos andAlonso-Amelot2002;Zhouetal.2014).Deretal.(2009)
Islands. noted that development of nuclear genomic markers
TheGalapagosIslandsareagroupof(cid:2)14mainislands wouldbecriticalforestablishingtheoriginsofhybridtaxa
and (cid:2)100 rocks or islets, (cid:2)1000km west of mainland andforothersystematicstudiesofPteridium.
Ecuador(Snelletal.1996).Theislandsvaryinageranging SouthAmericaishometotwomainPteridiumtaxa:dip-
from (cid:2)0.7to (cid:2)4.2millionyearssinceemergenceabove loidP.esculentumsubsp.arachnoideumandallotetraploid
sea level (White et al. 1993). However, additional evi- P. caudatum, the lattera hybrid between P. esculentum
dence points to a much older archipelago existing at fromSouthAmericaandP.aquilinumfromNorthAmerica
the same spot prior tothe current emergence(Christie (Thomson and Alonso-Amelot 2002). Tetraploidy was
etal.1992).Rassmann(1997)citedthisideatoexplain inferred on the basis of Feulgen cytometry (Tan and
why estimates of some lineage ages are more than Thomson 1990) and spore size and guard cell length
5millionyears old. The relative isolation of the islands (ThomsonandAlonso-Amelot2002).Thehybridoriginof
from the nearest mainland coasts (Ecuador and Costa P.caudatumisfurthersupportedbytheadditivepattern
Rica) has resulted in a high level of endemism. There of DNA markers from P. aquilinum and P. esculentum
are estimated to be 236 endemic plant species on the (Thomson2000;ThomsonandAlonso-Amelot2002).Add-
islands (Tye and Francisco-Ortega 2011). Origins of the itional characters that can be used to distinguish
Galapagos flora are now thought to be quite diverse P.caudatumfromP.esculentumsubsp.arachnoideumin
and include northern and southern Andes and other SouthAmericaincludethepresenceofgnarledtrichomes
partsof SouthAmerica,aswellasCentralAmericaand between veins abaxially (Thomson and Martin 1996)
the Caribbean (Tye and Francisco-Ortega 2011). The and free laminar lobes on P. esculentum. An additional
widerangeofelevationsandrainfallpatternsresultsin taxon was recently described from north eastern Brazil
adiversityofecologicalhabitats(Itow2003). (Schwartsburdetal.2014).
Pteridiumisaworldwidegenusthathasbeentreated MostchromosomecountsofPteridiumshow2n¼104
from as few as one species to .20. There are several (Page1976;Sheffieldetal.1989;Thomson2000;Tindale
taxonomicchallengesinthegenus,oneofwhichisthe andRoy2002; Bainard et al. 2011),withothercomple-
high level of variability and phenotypic plasticity for ments,suchastriploidy(Sheffieldetal.1993),assumed
2 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Wolfetal.—OriginsofGalapagosPteridium
to be rare. One count of 2n¼52 from Spain (Lo¨ve and (2) To determine how many Pteridium taxa are on the
Kjellqvist 1972) has not been corroborated despite GalapagosIslands.
resampling from the same area (Sheffield et al. 1989). (3) ToexaminewhetherdifferentPteridiumtaxaoccupy
Jarrettetal.(1968)reportedthefirstobservationofcyto- different islands, or elevations on the Galapagos
logicalvariationinthegenus,withacountof 2n¼208 Islands.
(tetraploid)foronesporophyteofPteridiumfromtheGal- (4) ToexaminethepossiblemainlandoriginsofGalapa-
apagosIslands.Thisreporthasbeenthemotivationfor gosPteridium.
previousaswell asthecurrentfocusonPteridiumfrom
these islands. Klekowski (1973) used gametophytes
grownfromsporescollectedfromtheislandstoexamine Methods
theabilitytoself-fertilizeandcrosswithPteridiumfrom
We sampled 17 Pteridium from three of the Galapagos
other sources. The results demonstrated that bracken
Islands(Fig.1;Table1):SantaCruz,IsabelaandSanCris-
fromHawaii(P.aquilinum)andsamplesfromtheGalapa-
tobal.We alsoscouted on Floriana,butwereunable to
gosIslandswereinterfertilewithPteridiumfromCentral
locateanyPteridiumonthatisland.Ateachsite,wecol-
and South America.However,Hawaiian and Galapagos
lectedexpandingfrondsegmentsontosilicagel,andcol-
Pteridiumwereintersterilewitheachother.
lectedintactfrondsforherbariumspecimens,deposited
Recentexaminationof Pteridiumcollectionsfromthe
attheherbariumoftheCharlesDarwinResearchStation
Galapagosislandssuggeststhatmorethanonetaxonis
(CDS).Inaddition,weincludedaselectionofDNAsam-
presentonGalapagos.WesetouttoexamineGalapagos
ples from the plastid gene studyof Der et al. (2009) to
Pteridiumwiththefollowingobjectives:
obtainnucleargenesequences.Weselectedrepresenta-
(1) ToexaminetheoriginsofP.caudatumonbothislands tives of each of the major plastid clades of Der et al.
andmainland. (2009). We also included additional Pteridium samples
Figure1. MapofAmericashowinglocationsofPteridiumsampledforthisstudy.InsetshowsdetailsofGalapagosIslands.Approximatetaxon
boundariesarebasedonTryon(1941),TryonandTryon(1982)andMickelandSmith(2004).
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 3
Wolfetal.—OriginsofGalapagosPteridium
Table1. Voucherandlocalityinformationforsamplesusedinthisstudy.Code(asusedintreefigures)indicatescollectorandnumber,withfull
nameinparentheseswhenabbreviatedincode.‘Possiblehybrids’arelikelytobebetweenP.esculentumsubsp.arachnoideumandP.caudatum.
Code Herbarium Taxon Country Island/province/state Latitude(88888) Longitude(88888) Elevation(m)
............................................................................................................................................................................
Wolf1001 CDS P.esculentumsubsp. Ecuador SantaCruz 20.63 290.38 592
arachnoideum
Wolf1002 CDS P.caudatum Ecuador SantaCruz 20.66 290.40 420
Wolf1003 CDS P.esculentumsubsp. Ecuador SantaCruz 20.64 290.33 874
arachnoideum
Wolf1004 CDS P.esculentumsubsp. Ecuador SantaCruz 20.65 290.33 732
Arachnoideum
Wolf1005a CDS Possiblehybrid Ecuador SantaCruz 20.66 290.33 580
Wolf1005c CDS P.caudatum Ecuador SantaCruz 20.66 290.33 580
Wolf1006 CDS P.caudatum Ecuador SantaCruz 20.67 290.32 476
Wolf1007 CDS P.esculentumsubsp. Ecuador Isabela 20.81 291.09 1009
arachnoideum
Wolf1008 CDS P.esculentumsubsp. Ecuador Isabela 20.83 291.09 1006
arachnoideum
Wolf1009 CDS P.esculentumsubsp. Ecuador Isabela 20.84 291.09 822
arachnoideum
Wolf1010 CDS P.esculentumsubsp. Ecuador Isabela 20.84 291.07 627
arachnoideum
Wolf1011 CDS Possiblehybrid Ecuador Isabela 20.85 291.04 405
Wolf1012 CDS P.esculentumsubsp. Ecuador SanCristobal 20.91 289.55 381
arachnoideum
Wolf1013 CDS P.esculentumsubsp. Ecuador SanCristobal 20.90 289.48 683
arachnoideum
Wolf1014 CDS Possiblehybrid Ecuador SanCristobal 20.90 289.48 676
Wolf1015 CDS P.esculentumsubsp. Ecuador SanCristobal 20.90 289.52 739
arachnoideum
Wolf1016 CDS P.esculentumsubsp. Ecuador SanCristobal 20.90 289.53 544
arachnoideum
Wolf1017 CDS Possiblehybrid Ecuador SanCristobal 20.90 289.53 544
Wolf1018 UTC P.aquilinumsubsp. USA Hawaii 19.43 2155.28 1247
decompositum
Wolf1019 UTC P.aquilinumsubsp. USA Florida 29.63 281.92 42
pseudocaudatum
AL147 DUKE P.aquilinumsubsp.feei Mexico Oaxaca 17.17 296.60 2660
(A.Larsson)
IJ786 LPB,UC P.esculentumsubsp. Bolivia FranzTamayo 214.62 268.95 2350
(Jime´nez) arachnoideum
IJ1245 LPB,UC P.esculentumsubsp. Bolivia Ayopaya 216.65 266.62 2750
(Jime´nez) arachnoideum
IJ2048 LPB,UC Pteridiumsp. Bolivia FedericoRoma´n 210.48 265.57 140
(Jime´nez)
Continued
4 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Wolfetal.—OriginsofGalapagosPteridium
Table1. Continued
Code Herbarium Taxon Country Island/province/state Latitude(88888) Longitude(88888) Elevation(m)
............................................................................................................................................................................
Wolf1020 UTC P.caudatum CostaRica SanJose 9.56 283.80 2270
Wood15788 HAW P.aquilinumsubsp. USA Hawaii 22.15 2159.65 1280
decompositum
Wolf1023 HAW P.aquilinumsubsp. USA Hawaii 21.40 2157.89 419
decompositum
Worthington DUKE Pteridiumsp. Puerto Ponce 18.13 266.68 792
35231 Rico
JJdG14388 NSW P.esculentumsubsp. French Saint-Laurent-du-Maroni 4.70 253.97 480
(deGranville) 729390 arachnoideum Guiana
Matos231 NY P.esculentumsubsp. Brazil Bahia 214.71 239.60 700
01198119 arachnoideum
Ortiz497 NY P.esculentumsubsp. Ecuador Esmeraldas 0.40 278.80 1925
00089157 arachnoideum
Delprete10293 NY P.esculentumsubsp. Brazil Goias 217.80 248.82 1150
01019119 arachnoideum
Prado2351 SP P.esculentumsubsp. Brazil Parana` 225.14 250.03 1000
arachnoideum
Prado2337 SP P.esculentumsubsp. Brazil Sa˜oPaulo 222.77 245.53 1888
arachnoideum
Wolf795 UC1622577 H.incisa
Wolf387 UTC P.scaberula
Wolf376 UTC B.pubescens LaRe´union
from the mainland of Central and South America, and used(Thomson2000,2012;ThomsonandAlonso-Amelot
outgroupsHistiopteris,BlotiellaandPaesia(Table1). 2002;Thomsonetal.2008).
Morphology DNAsequencing
Preliminary morphological analysis of samples was DNAwasextractedfromfresh,desiccatedorherbarium
undertakenindependentlyofmolecularstudies.Photo- tissueusingtheDNeasyPlantMinikit(QIAGEN,Valencia,
graphs of fresh unpressed pinnae supplemented by CA,USA),followingthemanufacturer’sprotocol.
macrophotographs of the abaxial surface of individual The plastid markers trnS–rpS4 (spacer+gene) and
ultimate segments were used for the examination of rpL16 intron wereamplified in 25mL polymerase chain
gross features of laminal dissection, presence/absence reactions(PCRs)usingthefern-specificprimerspublished
of free laminal lobes on pinna and pinnule axes, and in Small et al. (2005). However, the complete plastid
abaxiallaminalindumentum. genome sequence of P. aquilinum (GenBank accession
Weusedsmallsubsamplesofeachaccessioncompris- NC_014348) was used to redesign the rpl16 reverse
ing one or two dried pinnules for moredetailed micro- and the trnS primers (names now with ptaq suffix; see
scopic study following Thomson and Martin (1996). Table2).
Weexaminedtheindumentofabaxialpinnuletandseg- NuclearprimerswerebasedonthoseofRothfelsetal.
mentmidveins,determinedpresenceversusabsenceof (2013).WechosetwonucleargenesSQD1(Region1)and
gnarled intervein trichomes, measured false-indusial ApPEFP_C(Region2),redesigning(nowwithsuffix_ptaq)
width, estimated the number of cells per millimetre theforwardApPEFP_CandSQD1reverse,basedonPteri-
alongtheoutermarginofthefalseindusiumandmea- dium sequences from a study of the transcriptome
suredstomatalguardcelllength.Taxonomicdesignation (Der et al. 2011). SQD1 encodes sulfoquinovosyldiacyl-
wasbasedonpreviousdescriptionsofthecharacterswe glyerol 1 involved in the biosynthesis of sulfolipids and
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 5
Wolfetal.—OriginsofGalapagosPteridium
Table2. PrimersequencesforPCRandDNAsequencing.Suffix‘ptaq’ of standard, Vicia faba (26.9pg; Dolezˇel et al. 1998),
denotesprimersdesignedinthisstudy. wereco-choppedonachilledsurfaceusingafreshrazor
bladein500mLofice-coldextractionbuffer(0.1Mcitric
Primername Primersequence,5′–3′
.................................................................................. acid, 0.5% v/v Triton X-100) (Hanson et al. 2005), with
rpl16_r_ptaq TCCTCTATGTTGCTTACGATAT 1%w/vPVP-40(Yokoyaetal.2000).Tissuewaschopped
trns_gga_ptaq CTACCGAGGGTTCAAATCCCTC intoasemi-fineslurry,andthe suspensionwasswirled
byhanduntiltheliquidreachedalightgreentinge.The
SQD_r2_ptaq CCTTTGCCATAAACTGTAAGGGGGTG
suspensionwaspouredthroughacellstrainer(BDFalcon;
EMSQD1E1F6 GCAAGGGTACHAAGGTHATGATCATAGG
Becton,DickinsonandCompany,FranklinLakes,NJ,USA).
ApPEFP_f25_ptaq AATGCTCTAAGTCATTGTTACCGATC RNaseA (1mgmL21) and 350mL of propidium iodide
ApPEFP_C4218_r7 TTGTAAATCTCTGTRTCRGATGYYGT staining solution (0.4M NaPO , 10mM sodium citrate,
4
rps4_int_f1 CAGATTACTGAAAAACTAGC 25mM sodium sulfate, 50mgmL21 propidium iodide)
were added to140mLof filtrate,incubatedat 258C for
rps4_int_r1 AGAAGAGCGAAAGGGTTC
30min,followedbyupto2honice.Thestainedsolutions
rpl16_int_f1 GCGAAGCTGAAAACGATGCC
wereanalysed with an Accuri C6usinga 488nm laser,
rpl16_int_r1 GTTCCATTTCTAAATAGCGG and10000eventswerecapturedpersample.Therelative
genomesizewascalculatedusingtheratioofthemean
fluorescentpeakofthesampletotheinternalstandard
ApPEFP_Cencodesanappr-1-pprocessingenzymefamily
multipliedbythegenomesizeofthestandard.
protein,ADP-ribose-1-monophosphatase(Appr-1-pase),
aubiquitouscellularprocessingenzyme.ThePCRprimer
sequences(Table 2)usedwereasfollows:SQD_r2_ptaq Results
combinedwithEMSQD1E1F6,andApPEFP_f25_ptaqcom-
In general, we found Galapagos Pteridium to be highly
binedwithApPEFP_C4218_r7.Polymerasechainreaction
variable for both morphological and molecularcharac-
conditionsfollowedDeretal.(2009),annealingat56.58C
ters.WefindevidenceoftwoPteridiumtaxapluspossible
for the plastid and nuclear genes. For sequencing, we
hybrids,andmultiplecolonizationeventsfromdifferent
usedallPCRprimersplusnewinternalprimers(Table2)
mainlandsources.
for the two plastid genes: rps4_int_f1, rps4_int_r1,
rpl16_int_f1andrpl16_int_r1.Inmanysamples,thetwo
Morphology
nucleargeneampliconscontainedmultiplehaplotypes.
Pteridium caudatum and P. esculentum can be distin-
To sequence each haplotype separately, we cloned the
guishedmorphologicallybyacombinationofcharacters
PCRproductsusingtheStrataClonePCRCloningKit(Agi-
(Thomson 2000; Thomson and Alonso-Amelot 2002;
lentTechnologies,SantaClara,CA,USA).DNAsequences
Table3).Weinferredthat11ofoursampleswereclearly
wereassembledandeditedusingSequencher4.6(Gene
P.e.subsp.arachnoideum,2wereclearlyP.caudatumand
CodesCorporation,AnnArbor,MI,USA).
4weredifficulttodetermineandinferredtobepossible
Phylogeneticanalysis hybrids. The two P. caudatum samples were found at
SequenceswerealignedwithMAFFTversion7.215using thetwolowestsitesonSantaCruz.Thepossiblehybrids
the L-INS-i algorithm for accurate alignments. Newly werefoundatthelowestsitesonIsabelaandSanCristo-
generated sequences for the plastid genes rps4 and balandmid-elevationsitesonSantaCruzandSanCristo-
rpl16 were combined with data from Der et al. (2009; bal.WefounddifferentPteridiumtaxagrowingwithina
GenBank accession numbers FJ177158–FJ177206 for kilometreofeachotheronSantaCruzandSanCristobal.
the trnS–rps4 spacer+gene and FJ177239–FJ177287 Samples from one site included one frond that was
fortherpL16intron)andconcatenatedforphylogenetic P. e. arachnoideum (Wolf 1005a) and another frond
analysis.Maximumlikelihood(ML)phylogeneticinference (collected within 1m of the other) was P. caudatum
wasperformedseparatelyforeachnucleargeneandthe (Wolf1005c)orahybrid.
plastid datawith RAxMLversion 8.1.17 using100 rapid Most of our Galapagos samples of Pteridium fell into
bootstrapreplicatesfollowed byaMLsearch under the one of the two distinct categories for stomatal guard
GTRGAMMA model of evolution.Trees were rootedwith cell length:thosewithameanbelow40mmand those
thethreeoutgroups. above 40mm. Wolf 1002 and Wolf 1006 fall within the
range expected for tetraploid P. caudatum (Thomson
Flowcytometry
and Alonso-Amelot 2002) and close to the guard cell
Genome size was determined using flow cytometry. length (46.5mm, Thomson 2000) for the Galapagos
Approximately0.75cm2offreshleaftissueand0.5cm2 plant showing 4n¼208 (K Sheet H2146/97/1, Jarrett
6 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Wolfetal.—OriginsofGalapagosPteridium
Table3. TypicalmorphologiesforP.caudatum,P.esculentumsubsp.arachnoideum,andpossiblehybrids(orintrogressants)betweenthem.
InformationbasedonTryon(1941)andThomsonandAlonso-Amelot(2002).
Determination WolfID# Freelobes False False Stomatal Abaxial Abaxialsurface:
onsegment indusium: indusium: guardcell surface veinindumentum
axes width(mm) cells/mm length(mm) betweenveins:
lengthalong gnarled
margin trichomes
............................................................................................................................................................................
P.caudatum 1002,1006 Absent 0.3–0.5 (cid:2)31 .40 Absent Glabrous
P.esculentumsubsp. 1001,1003,1004, Present 0.1–0.3 (cid:2)48 ,40 Present Densefine
arachnoideum 1007,1008,1009, acicularwhite
1010,1012,1013, hairs,some
1015,1016 twisted;fine
white
arachnoidhairs
Indeterminate: 1005a Absent 0.2 64 32.3 Present Veinhairsless
possibly densethanfor
introgressant typicalsubsp.
arachnoideum
1011 Absent 0.4 56 39.1 Present Asfor1005a
1014 Absent 0.15 48 39.3 Present Asfor1005a
1017 Absent 0.15–0.2 40 34.3 Absent Asfor1005a
Table4. GenestatisticsandGenBankaccessioninformation.
Gene Numberof Variable Parsimony-informative Differencesbetweenaquilinum GenBankaccession
characters characters characters andesculentumhaplotypes numbers
............................................................................................................................................................................
ApPEFP_C 785 51 28 17 KT345729–KT345821
SQD1 752 36 26 15 KT345856–KT345898
rps4 1036 33 25 11+1indel KT345822–KT345855
rpl16 792 27 21 9+2indels KT345899–KT345934
Total 3365 147 100 55
etal.1968),corroboratingourmorphology-baseddeter- informative,and55distinguishedtheP.aquilinumclade
mination of these samples(Table 3).Ploidy level of the fromtheP.esculentumclade(Table4).Phylogeneticana-
otherGalapagossamplesstudiedcannotbedetermined lysesofthetwoplastidgeneswerecongruent,asfound
due to the extended, apparently continuous, series of previously(Deretal.2009).Thus,alignmentsofthetwo
guardcelllengthsrepresented(Table3).Thewiderange plastidgeneswereconcatenatedforacombinedanalysis
of lengths observed suggests that both diploid and (Fig.2).Inthethreeanalyses(twoplastidgenesconcate-
triploid levels might be represented. On the basis of nated,SQD1andApPEFP_C),theaquilinumandesculen-
morphology,11ofoursamplesareP.esculentumsubsp. tumcladesofPteridiumweresistertoeachother.Table4
arachnoideum and 4 may be hybrids carrying genomic providesthe ranges of GenBankaccessionnumbers for
elements from outside arachnoideum, and may be each gene and Supporting Information—File S1 lists
triploid(Table3). the GenBank accession number for each sequence.
All trees and associated nucleotide alignments are
DNA deposited in TreeBASE (http://purl.org/phylo/treebase/
Overall,nucleotidedatafrom thefourgenescontained phylows/study/TB2:S18018). We include outgroups for
3365 characters of which 100 were phylogenetically phylogenetic analysis of the nuclear genes. However,
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 7
Wolfetal.—OriginsofGalapagosPteridium
Figure2. Phylogenetictreebasedonthecombinedplastidgenedataset.ThetreewasrootedwithBlotiellapubescens,Paesiascaberulaand
Histiopterisincisa.
8 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Wolfetal.—OriginsofGalapagosPteridium
inclusionofoutgroupsintheplastidtreesresultedinvery haplotypes and4sampleshad onlyesculentum haplo-
shortingroupbranchlengths,andisthereforenotshown. types.AllGalapagosspecimenshaveesculentumnuclear
Thetreetopologywithoutoutgroupsisthesameasthat haplotypes,11withanesculentumhaplotypeonly,10of
withoutgroups. whichwereP.e.arachnoideum.FiveGalapagossamples
Wedetectedatotalof19aquilinumand31esculentum have aquilinum and esculentum haplotypes, two of
plastidhaplotypes.InsamplesfromtheGalapagosIslands, which were P. caudatum, two appeared to be hybrids
wedetected12plastidhaplotypes,oneofwhichhasbeen and one was P. e. arachnoideum. One specimen (Wolf
sampledpreviouslyinP.e.arachnoideumfromVenezuela 1004)appearedtohaveaquilinumandesculentumhap-
and Mexico (Der et al. 2009). The remaining Galapagos lotypes for SQD1, but only aquilinum haplotypes for
plastid haplotypes were nested within a clade that ApPEFP_C. All mainland samples of P. caudatum have
included haplotypes from Mexico and South America both aquilinum and esculentum haplotypes. Diploid
(Fig.2).AllsamplesofP.aquilinumhadtheexpectedplastid individualsshouldhavenomorethantwohaplotypesat
haplotype,andallnewsamplesfromSouthAmericanPter- a locus, whereas tetraploids are expected to have no
idium, including those from the Galapagos Islands, had morethanfourhaplotypes.However,8ofthe13hetero-
esculentum haplotypes. A specimen from Costa Rica zygousindividualshad extrahaplotypes:3P.caudatum
(Wolf1020),whichappearstobeP.caudatum,hadanaqui- samples with 5, 5 and 7 haplotypes, 1 P. esculentum
linumplastidhaplotype,asdidthesingleMexicansample with6haplotypesand4P.aquilinumsampleswith3,3,
ofP.a.feei(A.Larsson147),ataxonnotpreviouslyincluded 3and5haplotypes.Inmostcases,extrahaplotypesdif-
inmolecularstudies.Previousstudies(Thomsonetal.2008; fered from others from the same individual by one
Deretal.2009)havenotedtwo5-bppolymorphicrepeats nucleotide,andatthemostthreenucleotides.Allextra
inthetrnS–rps4spacer.Together,thesepolymorphisms haplotypesappear tobethe result of single nucleotide
account for three haplotypes: haplotype C in outgroups autapomorphies,andwecannotaccountforextrahaplo-
andtheP.esculentumclade,haplotypeAinP.aquilinum typesbyrecombination,whetherinplantcells,PCRtubes
andhaplotypeBinonlyEuropeanandAfricanP.aquilinum. orduringcloning.
The two nuclear genes showed a similar pattern of
differentiation as the plastid genes: a set of distinct Flowcytometry
nucleotidedifferences(15forSQD1and17forApPEFP_C) We were able to estimate c-values for fresh Pteridium
distinguishedaquilinumfromesculentumhaplotypes.For frondson aconsistentbasis,but wewere unable to do
SQD1,samplesofP.caudatumwereheterozygousforthe soforanydrysamples,includingthoseinsilicageland
above nucleotide positions indicating that they were herbarium specimens.We estimatehaploid genomesize
additiveforP.aquilinumandP.esculentumhaplotypes. (mean+coefficient of variation) of 15.88 (+0.67)pg
However, although we were able to sequence a few for P. a. decompositum from Hawaii (Wolf 1018), 16.13
distinct haplotypes from heterozygous plants, this was (+0.67)pg for P. a. pseudocaudatum from Florida (Wolf
largely unsuccessful. Bacterial cells carrying the SQD1 1019)and29.2(+2.1)pgforP.caudatumfromCostaRica
PCRproductappearedtobeclumpingsothatsinglecol- (Wolf1020).Theseareconsistentwithpreviousestimates
onies were usually not single clones and therefore fordiploidandtetraploidPteridium,respectively(Tanand
remainedheterozygous,despitere-streakingofcolonies. Thomson1990;Bainardetal.2011).
We suspect that clumping was a function of partial
expression of the gene. Conversely, we were able to
Discussion
cloneseveralhaplotypesofApPEFP_Cfromheterozygous
individualsandwefoundthatP.caudatumplantsindeed Inthisarticle,weexaminedmorphologicalandmolecular
possessedbothaquilinumand esculentum haplotypes. variation inPteridiumfromGalapagos Islands.Tomake
Wedetectedatotalof12aquilinumand13esculentum inferences about taxonomic variation and possible ori-
haplotypes for SQD1 (Fig. 3), and 32 aquilinum and 30 ginsontheislands,wefirstprovidedcontextwithother
esculentumhaplotypesforApPEFP_C(Fig.3).Inthesam- mainlandandworldwidesamples, includingsequences
ples from the Galapagos Islands,we detected 7aquili- fromapreviousstudy(Deretal.2009).Wefirstdiscuss
num and 14 esculentum ApPEFP_C haplotypes, and 1 variation for morphological and molecular characters,
esculentum and 9 aquilinum haplotypes for SQD1. Of followedbytheimplicationsfortheoriginsofGalapagos
the six samples that were heterozygous for SQD1, we Pteridium.
wereabletosequencefivehaplotypesfromthreeindivi-
duals. Of the 17 samples that were heterozygous for Moleculardata
ApPEFP_C,8hadbothaquilinumandesculentumhaplo- A growingbodyof researchhasexamined variation for
types, 5 heterozygous samples had only aquilinum nuclear-encoded genes within species of ferns (for
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 9
Wolfetal.—OriginsofGalapagosPteridium
Figure3. PhylogenetictreebasedonthenucleargenesApPEFP_C(A)andSQD1(B).TreeswererootedwithB.pubescens,P.scaberulaand
H.incisa.
10 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015