Phylogeny of Horsetails (Equisetum) Based on the Chloroplast Rps4

Home , Equisetum giganteum, Equisetum variegatum

Phylogeny ofHorsetails ( Equisetum)based on the Chloroplast rps4 Gene and Adjacent Noncoding Sequences

JEAN-MICHEL GUILLON LaboratoireEcologie ,Syste´matique et Evolution. UMR 8079CNRS. Ba ˆtiment 360,U niversite´Paris-Sud,91405 Orsay-cedex,France (email: [email protected])

CommunicatingEditor: P aulS .Manos

ABSTRACT. Equisetum isa genusof 15 extant species thatare the sole surviving representatives ofthe class Sphenopsida. ChloroplastD NAsequence data were used toexamine the monophylyof the tw oaccepted subgenera ( Equisetum and Hippochaete )and thep utative basal position of Equisetumgiganteum .Aplastid DNAregionthat includes rps4 was sequenced forall species of Equisetum.Phylogenetic analyses usingparsimony ,likelihoodor posterior probability criteria, all support the followinginferences: (1) Equisetumbogotense isbasal withinthe genus; (2)all otherspecies group intw omajorsister clades: (i)the restof subgenus Equisetum (7 species) and (ii)subgenus Hippochaete (7 species). Onthe basis ofthe present phylogeny,bisexuality would notbe theancestral state inthe Equisetum genus, but characters shared amongspecies ofthe subgenus Equisetum,suchas supercial stomata and protrudingantheridia, could be ancestral inthe genus.

Horsetails ( Equisetum L.)are the only survivors of gametophytes, alsoshow aclear-cutdistin ction be- the formerly more diverse Sphenopsida,free-sporing tween the twosubgenera (Table1; Duck ett 1973, plants characterized byarticulate stems bearing 1979a). whorls oflea ves ateach node .According tothe fossil Most living species ofhorsetails have very broad record, primitiveforms possibly assignable toSphen- circumborealdistribution s, their latitude ranging be- opsida appearedin the lateDevonian (Stewartand tween 408 and 608 north (Hauke1963, 1978). The ex- Rothwell 1993;T aylor and Taylor 1993).Sphenops ida ceptions are E. bogotense , E. giganteum , and E. myri- then reached maximum diversity during the Carbon- ochaetum,from Central and SouthAmerica, E. laeviga- iferous. Afterwards,major extinction episodes took tum,which is restricted toN orth America, E. diffusum, placeduring the early Permian and the lateJurassic. endemicofthe Himalayas,and E. ramosissimum , which Since the beginning ofthe Cenozoic,all known Sphen- ranges from Europe,Africa,and Asia tosome Oceanic opsida havebeen herbaceousforms thatare indistin- Islands. On accountof overlappin gdistributions, guishable from living horsetails (Brown 1975;McIver manyinterspecic hybrids, involving allspecies except and Basinger 1989;Stew artand Rothwell 1993).F ossils E. bogotense ,are found in the wild. These hybrids are attributedto the genus Equisetites thatclosely resemble consideredtobe sterile (butsee Krahulec et al.1996) modern Equisetum date from the Permian (Boureau and torely on vegetativereproduction forpersistenc e 1964;Stew artand Rothwell 1993),and possibly even and growth. Most ofthe hybrids have alsobeen ex- from the Carboniferous (Emberger 1968;T aylor and perimentally synthesized bycontrolled crosses (Duck- Taylor 1993).On accountof the similarity between ett 1979b).N otably,the pattern ofhybridiza tion in the Equisetites fossils and living Equisetum species, the dis- genus Equisetum canbe used todistinguish twodis- tinction made between these twogenera has been tinctgroups ofspecies. Within eachgroup, species are questioned tothe point thatArnold (1947)proposed connected byh ybridization, whereas hybridization that Equisetum might beregarded asthe oldest surviv- cannotoccur between groups. This division based on ing vascularplant genus in the world. hybrid occurrenceperfectly matches with the taxonom- Morphologicalvariability within living species and icdivision ofth egenus into the twosubgenera Equi- extensive hybridizationamong them has long ob- setum and Hippochaete (Duckett1979b). scured the taxonomy ofthe genus Equisetum. Since the The sectional classication of the subgenera Equi- workofH auke(1963,1978), which signicantly clari- setum and Hippochaete is more controversial. Different ed the picture,15species of Equisetum have been divisions ofthe subgenus Equisetum have been pro- widely accepted.The taxonomy ofthe genus Equisetum posed, depending on the charactersprioritized fortax- recognizes twosubgenera ( Equisetum and Hippochaete ; onomy. These charactersinclude (i)stem dimorphism Milde 1861),on the basisof stomatal position: super- (Braun 1839),(ii) en dodermal patterns (Ptzer 1867), cial in Equisetum (E. arvense, E. bogotense , E. diffusum, (iii) surfacemorpholo gy ofsilica deposits (Page 1972), E. uviatile , E. palustre, E. pratense, E. sylvaticum , and E. and (iv)antheridiu mmorphology (Duckett1973). Al- telmateia),butsunk en belowthe epidermal surfacein though there wassome correlationbetween Duckett’s Hippochaete (E. giganteum , E. hyemale, E. laevigatum , E. observations concerning antheridiummorphology and myriochaetum , E. ramosissimum , E. scirpoides , and E. var- the classication proposed byP age (1972),Ha uke iegatum).Other characters,such asthe morphology of (1974)considere dthatthe availableinformati on was

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TABLE 1. Characters differingbetw een the twosubgenera Eq- and Nickrent 1999),or (ii)both n uclearand mitochon- 1 2 uisetum and Hippochaete. except E. bogotense and E. diffusum. drial small-subunit rDNAsequences(Renzaglia et al. except E. laevigatum, and E. ramosissimum incolder climates. 3 except E. ramosissimum,E. myriochaetum and E. giganteum. 4 except E. 2000),or (iii) plastid atpB, rbcL, and rps4 and nuclear myriochaetum and E. laevigatum. Data fromDuckett (1973, 1979a), small-subunit ribosomal DNAsequences(Pryer et al. Hauke (1963, 1968, 1969, 1974, 1978) and Page (1972). 2001)also concluded thathorsetails and ferns are a monophyletic group and the closest living relatives to Characters Equisetum Hippochaete seed plants.The exactposition of Equisetum with re- Sporophyte spectto ferns varies among the studies, some placing stem Equisetum asa sister cladeto leptosporan giate ferns persistence annual1 perennial2 morphology branched unbranched3 (Duffand Nickrent 1999;Renzaglia et al.2000), some cone apex rounded pointed4 weakly supporting the grouping of Equisetum with silica deposits ornamented amorphous Marattiopsida(Pry er et al.2001), and some placing Eq- position ofstomata supercial sunken uisetum asthe sister group toall ferns (Kenrick and Gametophyte Crane 1997). lamellae morphology unistratose column In contrast,phylogen etic relationships within the antheridia genus Equisetum have remained largely unexplored. position protruding sunken Evolutionin Equisetum wasdiscussed bySchaffner cover cell number usually .2 usually 2 (1925,1930) and Hauke(1963),although acladistic Chromosomesize small large analysis ofm orphologicalcharacters has not yet been undertaken in horsetails. Considering that E. giganteum has large stems and persistent sheath teeth, asdo most insufcient toform ulatea sectional classication of the fossil members ofthis group,and becauseit has reg- subgenus Equisetum.Later,Hauke(1978)constructed a ularwhorls ofbranches and stomatain manylines, as phenetic dendrogrambased on pairwise comparisons found throughoutthe subgenus Equisetum, both au- ofspecies forman ycharacters.A ccording toHauke ’s thors concluded that E. giganteum wasth emost prim- results, only onegroup is worth recognizing; this in- itive member ofthe genus.On the basisof the sup- cludes E. arvense, E. pratense, and E. sylvaticum . In con- posed primitive statusof E. giganteum ,the most ad- trast,from the pattern ofh ybridizationwithin the sub- vanced members ofthe subgenus Hippochaete were genus Equisetum,Duckett (1979b)suggested that E. proposed tobe E. laevigatum and E. scirpoides (Schaffner pratense and E. sylvaticum would form aunit separate 1925;Hauke 1963). from the remaining species. Besides the evolutionofsporophy ticcharacters, the Aclassication of the subgenus Hippochaete has also phylogeneticstatusof E. giganteum has implications for been proposed byHa uke(1963).Section Incunabula the evolution ofsexuality in Equisetum gametophytes. contains the regularly branched E. giganteum , section Gametophytes ofhorsetails are usually unisexual: ga- Hippochaete contains species with evergreen, un- metophytes rst develop asmale or female,with fe- branched aerial stems ( E. hyemale, E. variegatum , and E. male gametophytes later producing antheridia,when scirpoides),and the section Ambigua contains those spe- not fertilized (Duckett 1970,1972). Interestingl y,ga- cies with intermediate morphology ( E. myriochaetum , metophytes of E. giganteum are functionally bisexual E. ramosissimum , and E. laevigatum ).This division is (i.e.,producing antheridia and archegoniasimulta- partly supported byexperimen taldata on hybridiza- neously), aphenologythatis uniquein the genus tion (Duckett 1979b),but the morphology of Hippo- (Hauke1969, 1985). Because he considered E. gigan- chaete gametophytes shows no cleardiscontin uities teum tobe primitive ,Haukehypothesizedthatbisex- that tthe sectional division (Duckett 1979a).M ore- uality couldhave been the primitive condition in Eq- over, the frequent occurrenceof hybrids within sub- uisetum.In contrast, E. bogotense gametophytes seem to genus Hippochaete suggests thatthe taxaare more bestrictly unisexual (i.e.,never showing sex reversal closely interrelatedthan in subgenus Equisetum. from archegoniato antheridia production),aphenol- The phylogeneticposition ofhorsetails has been in- ogy proposed tobe the most advanced in the genus vestigated in several studies. Examinationofearly fos- (Hauke1968, 1969). sil record and morphologicalcharacters of land plants Wehere report amolecular phylogenyofthe genus favorsthe grouping ofhorsetails with ferns in amono- Equisetum using rps4 chloroplastsequences. The objec- phyletic clade(K enrick and Crane 1997).The same tive ofthis study is toin vestigate the relationships conclusion wasreached with the study ofcharacters among species ofhorsetails, and in particularto ex- derived from spermatogenesis, development, and mor- amine(i)the monophyly ofthe twosubgenera and (ii) phology (Renzaglia et al.2000). Recently ,molecular the basalposition of E. giganteum ,in order toexplore phylogenies ofextant vascularplants using either (i) the evolution ofcharacters in this remarkablegroup of mitochondrial small-subunit rDNAsequences (Duff plants. 2004] GUILLON:PHYLOGENY OF EQUISETUMBASED ON RPS4 253

TABLE 2. Voucher and EMBL/GenBank informationfor speci- one fernsequence was found inthe data base,and thiscould not mensused inthis study. be aligned with Equisetum sequences. Thus, only Equisetum sequences were included inthe alignment. These noncoding sequences were divided intoconserved regionstha tcould be aligned Equisetum L. E. arvense L.(Guillon 5, P-00271032; AJ583677). amongall species (shown on awhite backgroundinFig. 1), and E.bogotense Kunth (McAlpins.n., UC-1733471; AJ583678). E. blocksof v ariable regions(DN Ainsertions,deletions, repetitions, diffusum D. Don (Gross1, P-00271037; AJ583679). E.uviatile and stretchesof polyT orpolyA) tha twere present orabsent de- L.(Husby5, P-00271028; AJ583680). E.giganteum L. (Husby 7, pending on species (shownon agreyor black backgroundinFig. P-00271026; AJ583681). E. hyemale L.(Ducketts.n., CGG- 1). Conserved noncodingD NAsequences were juxtaposed, small 19660161; AJ583682). E.laevigatum A.Braun (Husby2, P- indelsbeing coded as asingleevent, toyield the second matrix 00271030; AJ583683). E.myriochaetum Cham. &Schlecht. (Ar- (308 characters,1.4% ofcells scored as missing;TreeB ASEmatrix mond93, E-15013; AJ583684). E. palustre L.(Guillon 4, P- M1596). Variable regionsw ere treated separatelybecause theyre- 00271033; AJ583685). E. pratense Ehrh. (Page 1256, E-15005; sult fromlarge repetition, insertionor deletion events thatare very AJ583686). E.ramosissimum Desf.subsp. debile (Roxb.)Hauke differentfrom single nucleotid esubstitutions. Therefore,athird (Walker 7714, E-15011; AJ583687). E.scirpoides Michx. (Dun- matrix(15 characters, no missingda ta; TreeBASEmatrix M1597) and s.n., G-452418; AJ583688). E.sylvaticum L.(Page 7889, E- included blocksof v ariable regions,considered as multistate char- 15001; AJ583689). E.telmateia Ehrh. subsp. braunii (Milde) acters (i.e., coded as 1, 2or3, according tothe color code inFigure Hauke (Doran22, CONN-122648; AJ583690). E.variegatum 1, all possible transitionsbeing equally weighted). Schleicher (Guillon 3, P-00271034; AJ583691). Phylogenetic Analyses. Formaximum parsimony (MP) analy- Outgroups (sequenceswere obtained fromGenBank/ sesof the rstand second matrices, branch and bound searches EMBL). Angiopteris evecta (J.R. Forst.)Hoffmann (AF313591). were conducted usingP AUP* 4.0b (Swofford1998), saving all most parsimonious trees. Internalsupport forrelationsh ips was as- Danaea elliptica Sm.(AF313589). Ophioglossum reticulatum L. sessedusing bootstrap analyses with1,000 replicates. ForMP (AF313594). Pterisplumula Desv.(AF321694). Salvinia molesta analysis ofthe third matrix, a heuristic search was conducted us- Mitchell(AF313600). ingP AUP* 4.0b (Swofford1998), startingfrom a treeconstructed bystepwise addition, and branch swapping withtree-bisectio n- reconnection(TBR).A 1,000 replicate randomstepwise addition sequence was specied, and all mostparsimonio us treesw ere MATERIALS AND METHODS saved. Maximumlikelihood (ML) analysis was conducted on the rst Sampling. Allwidely recognized extant species of Equisetum matrixusing P AUP*4.0b (Swofford1998). Ageneral timereversibl e were represented inthis analysis. The voucher and EMBL/ modelof substitution with site-speci c ratesfor rst,second and GenBank accession details forspecimens used inthis study are thirdcodon positions(GTR 1SS)w as chosen, based on Akaike In- listedin T able 2. formationCriterion. The MPtree with the greatest -ln score was DNAExtraction, PCR,and N ucleotideSequenc ing. Total ge- used toestimate the model parameters .Heuristic search was then nomicDN Awas extracted fromfresh or silica geldried plant ma- conducted,startingfrom a treeconstructed bystepwise addition, terial usingthe DNAeasy y Plant MiniKit (Qiagen).The chloro- and branch swapping withTBR. Toavoid deleteriouslyrestricting plast regionincluding the rps4 gene and noncoding ankingse- thesearch toa singleisland oftrees, arandomstepwise addition quences (approximately1100 base pairs) was amplied usingthe sequence was specied for100 replicates. Resampling ofcodons universal primersS and T,as described byDemesure et al. (1995). was performed1,000 timesusing the Bootstrap Codon program Amplication mixtures(25 ml)contained 50 mMK Cl, 10 mMTris- fromJohn H uelsenbeck’s laboratory.ML analyses were thencon- HCl (pH 9.0 at25 8C),50 mMK Cl, 0.1% Triton t X-100, 2.5 mM ducted as above,recalculatingthe modelparameters foreach of

MgCl2, 200 mMofeach ofthe four dNTPs, 0.4 mMofeach primer, the1,000 bootstrap replicates, with10 random-additionreplicates and 1unit ofT aq DNApolymerase(Promega).The PCR products per heuristic search. were checked on agarose gelwith ethidium bromide and puried Bayesian phylogenetic analyses were conducted on the rstma- byPEG precipitation(Rosenthal et al. 1993). Puried PCR prod- trixusing MrBayes v ersion2.0 (Huelsenbeck and Ronquist2002). ucts were sequencedusingMWG Biotech customsequencin gser- We used a GTR1SSmodel, startingwith a randomtree .Four Mar- vice. kov chains were run for220,000 generationsand sampled every Outgroups, SequenceAlignment, andIndel Coding. Outgroups 100 generations.Sta tionarityw as reached atapproximatelygen- were chosen withinferns beca use recent phylogenetic studies eration20,000, sothe rst200 treesor ‘ ‘burnin ’’ofthe chain were agree on groupinghorsetails and fernsin a monophyletic clade discarded, and phylogenetic inferencesare based on thosetrees sisterto seed plants (Kenrickand Crane 1997; Duffand Nickrent sampled aftergeneratio n20,000. 1999; Renzaglia et al. 2000; Pryeret al. 2001). Outgroupsw ere representative ofmajor groups offerns for which rps4 sequence data are available: Filicopsida ( Pterisplum ula and Salvinia molesta ), RESULTS Marattiopsida ( Danaea elliptica and Angiopteris evecta )and Ophiog- lossopsida ( Ophioglossumreticulatum ).Analysesalso including (i) Among the 624characters present in the rst matrix seed plant sequences or(ii) L ycopodiopsida and Isoetopsida sequences, inaddition tofern sequence s, gaveidentical results. (rps4 sequences),291sites (47%)were variableand 187 Automated alignmentof sequences was performedusing the (30%)were phylogeneticallyinformativ e.MPanalysis ClustalX program(Thompson et al. 1994), and followed byre ne- ofthis dataset yielded veequally parsimonious trees, mentby eye .Sequence matricesare available fromTreeBASE (studyaccession numberS964, matrixaccession numberM1595- which only differed in the positions of E. palustre, E. M1597). pratense, and E. telmateia (Fig. 2). Equisetumbogotense is Alignmentof the complete rps4 coding sequences was straight- sister tothe rest ofthe genus (90%BS, DI 5 5), divided forward among Equisetum species, but onlypartial rps4 sequences into twoclades: (i)the subgenus Hippochaete (91% BS, were available foroutgroup sand tworegionsprov ed difcult to align. Onlyunambiguo uslyaligned sequences ofoutgroup swere DI 5 4)and (ii)the subgenus Equisetum without E. used inthe matrix,bases ofuncertain alignmentbeing coded as bogotense (86% BS, DI 5 3).Support is lower for missing.Thus, thenumber of characters was 624 and theper- branches within eachgroup, except for the following centage ofcells scored as missingdata was 4% inthe rps4 data set(TreeB ASEma trixM1595). clades: (E. arvense, E. diffusum, E. uviatile ) (98% BS, DI Forupstream and downstreamnoncoding DNAregions,only 5 5), and (E. giganteum , E. hyemale, E. laevigatum , E. 254 SYSTEMATIC BOTANY [Volume 29 e x i 5 h r t t d a n n i m u o d d r e r s g i o k h t c p a a e t b h x t y u j e n r , i s g s e t i r c e h t e g c i p l a s r , a 2 m h u c 5 t e e s t d i a n u t q s u i o E t l r l g u l a k m c g a s n b a o y d e m e r a d g o d , c e 1 , n s g 5 e i i l c d a e n e p u s b o r n n g a o k c c g t a n a i b h d t k n c s e a e l p c b e n : d e s u t q w n e e o l s s l : b o a f d n r s a o u o t d r n e g e d k s c o e a c r b p d e s n t l i a e h d s n u W i o . g d 4 o s n l p a o r h m o t t o g h 9 n 3 e s l a d e n l d a b e r a 9 i e 5 r d a i s v s e c n f o n o c e s u e q A r e e r s w o g s d n i T n d f u o o o c r s n g e o k h c n c a t e b e h r t r t s o f l : o o d c n n e o u i t o m a r a z g s i k r a c o a g n b e o t a y s c e r n o d g i n r g a o e r t . k , n a c t e n a a o l m i d t B n i . s g g i x o n l i i r p s A t s a i n e . m m v 1 i d . g 5 n G I a ? o F c . t ) e a s ( 3 2004] GUILLON:PHYLOGENY OF EQUISETUMBASED ON RPS4 255

FIG.2. Oneof ve shortesttrees obtained ina parsimonyanalysis of rps4 sequence data (rst matrix). Numbers above branches are branch lengths.Bremer decay indices (DI),followed bybootstrap percentages (based on 1,000 replicates), are given below branches. Anull decay index means that the branch collapses inthe strictconsensus tree. Lines withno bootstrap values below denote nodes supported in ,50% ofthe replications. Tree length 5 508 steps; CI 5 0.76; RI 5 0.78. myriochaetum , E. ramosissimum ) (95% BS, DI 5 3). Eq- logenetically informative.MPanalysis ofthis dataset uisetumscirpoides is sister toa cladeformed bythe rest yielded six equally parsimonious trees, which only dif- ofthe subgenus Hippochaete (96% BS, DI 5 4). fered in the positions of E. palustre, E. telmateia , and E. For ML analysis ofthe rst matrix, model param- giganteum (Fig. 5).Strong bootstrapsupport is ob- eter values were estimated from the MPtree with the tained forthe samebranches aswith MPanalysis of greatest -ln score (-ln likelihood 5 2913.36106).All 100 coding sequences,though with the caveatthat the pre- replicates ofthe heuristic search using the ML opti- sent trees are not rooted. mality criterion resulted in the sametree (Fig. 3).Both Among the 15characters present in the third matrix MPand ML trees are congruent, with abasalposition (blocksof v ariablenoncoding regions, shown on agrey of E. bogotense ,and twomajor clades corresponding to or blackbackgroun din Fig. 1),14 w ere variableand 7 the subgenera Equisetum (without E. bogotense ) and were phylogenetically informative. MPanalysis ofthis Hippochaete .Bootstrapv alues are similar in bothanal- dataset yielded eight equally parsimonious trees yses yet with ahigher supportin ML for(i) ( E. arvense, (length 5 18steps; consistency index 5 1.0;retention E. diffusum, E. uviatile , E. sylvaticum (84%BS), and (ii) index 5 1.0),which only differed in the positions of E. (E. laevigatum , E. myriochaetum ) (82% BS). palustre, E. pratense, E. telmateia , and E. sylvaticum (re- The Bayesian analysis ofthe rst matrix yielded a sult not shown). In these eight trees, subgenera Equi- consensus tree (Fig. 4)com parableto those obtained setum and Hippochaete are separated by ve steps (DI with MPand identical tothat obtained with ML. Two 5 5), and E. scirpoides is sister tothe rest ofthe sub- clades weakly supported in MPand ML analyses are genus Hippochaete (DI 5 2). here recovered with 100%freq uency: ( E. hyemale, E. laevigatum, E. myriochaetum , E. ramosissimum ) and (E. hye- DISCUSSION male, E. ramosissimum ). Among the 308characters present in the second Trees resulting from the contrasting algorithmic matrix (noncoding sequencesconserved among all Eq- analyses of rps4 and adjacentnoncoding 5 9 and 39 se- uisetum species, shown on awhite background in Fig. quences are in good agreement.Througho utthis dis- 1),99 sites (32%)w ere variableand 53(17%) w ere phy- cussion, only those clades thatw ere supported in all 256 SYSTEMATIC BOTANY [Volume 29

FIG.3. Tree obtained inmaximum likelihood analysis of rps4 sequence data (rst matrix), using a GTR modelof substitution withsite-speci c rates foreach ofthe threecodon positions (-lnlikelihood 5 2911.14035). Numbersabove branches are mu- tational rates per siteand thosebelow branches are bootstrap values fornodes supported in .50% ofthe replications.

FIG.4. The 50% majority-ruleconsensus treeobtained ina Bayesian analysis of rps4 sequence data (rst matrix). Numbers above branches are mean branch lengths.Numbers below branches are the frequencyof recovery ofeach clade. 2004] GUILLON:PHYLOGENY OF EQUISETUMBASED ON RPS4 257

FIG.5. Oneof 6 shortesttrees obtained inaparsimonyanalysis ofnoncoding ankingsequences that can be aligned among all Equisetum species (shownon awhite background inFig. 1; second matrix).Numbers above branches are branch lengths. Bremerdecay indices, followed bybootstrap percentages (based on 1,000 replicates) are given below branches. Lines withno bootstrap values below denote nodes supported in ,50% ofthe replications. Tree length 5 134 steps; CI 5 0.90; RI 5 0.94. analyses are considered. Overall, the results supporta sexuality provide anydenitive datato support the basalposition forthe E. bogotense lineage, and then the theory that Hippochaete contains the most primitiveex- divergence oftw oclades corresponding tosubgen us tanthorsetails.. ..it is difcult to attribute ,with any Equisetum (without E. bogotense )and subgenus Hippo- condence ,specic morphologicalcharacters and fea- chaete.Within the subgenus Equisetum, only E. arvense, tures ofthe coning behavior toeither advanced or E. diffusum, and E. uviatile group in aconsistently primitive status.’’ The present results substantiatethis supported clade.Relationships are better resolved view,contradictingthe widely held opinion thatbisex- within the subgenus Hippochaete ,with the successive uality,asfound in E. giganteum ,is the ancestral state divergences ofthe E. scirpoides and E. variegatum line- in the genus Equisetum. ages, and aconsistent sister group relationship be- The basalposition of E. bogotense has other inter- tween E. laevigatum and E. myriochaetum . From now, I esting implications forthe evolution ofcharacters with- shall designate Euequisetum the cladethat includes E. in the genus.Indeed, somedistinctivefeatures of E. arvense, E. diffusum, E. uviatile , E. palustre, E. pratense, bogotense,such as lamentous and strictly unisexual E. sylvaticum , and E. telmateia (i.e.,subgen us Equisetum gametophytes with highly protruding archegonial without E. bogotense ). necks and antheridia (Hauke1968, 1969), may reect The basalposition of E. bogotense contrasts with pre- anearly divergence from other Equisetum lineages. On vious opinions concerning the most ancestral lineage the other hand, charactersshared among Euequisetum in Equisetum; E. giganteum wasconsidere dtobe the species and E. bogotense may bean cestral forhorsetails. most primitive member ofthe genus (Schaffner1925, Among such charactersare: (i) branched stems, (ii)a 1930;Hauk e1963),whereas E. bogotense gametophyte bluntcone ,(iii) anouter endodermis common toall charactersw ere thought tore ect an adv anced posi- vascularbundles ofthe stem, (iv)super cial stomata, tion (Hauke1968,1969). Indeed, Hauke (1963)consid- (v)unistratose lamellae in gametophytes, and (vi)pro- ered thatlarge size,evergreen stems and bisexualga- truding antheridia. This pattern ofev olution is in keep- metophytes, asshown by E. giganteum ,tobe primitiv e ing with the observationthat Equisetites fossils from states. Duckett (1979a,p .179and p.201)already the Mesozoicshow bluntcones and generally regular stressed that‘ ‘neither gametophyte morphology nor branching (Boureau1964). Furtherm ore,it is congruent 258 SYSTEMATIC BOTANY [Volume 29 with the timing ofearliest appearancesin the fossil branched stems, bisexualgametoph ytes, stomataar- record; the most ancient reported Hippochaete species ranged in many lines). Within subgenus Equisetum, no dates from the early Cenozoic(Brown 1975),whereas previous classication proposed the grouping of E. ar- E. bryanii,assigned tothe subgenus Equisetum, dates vense, E. diffusum, and E. uviatile ,which is highly sup- from the middle Jurassic (Gould1968). ported in ouranalyses. Consistent with this cladeare During completionofthe present work, Des Marais the observations that E. arvense hybridizes in the wild et al.(2003) also investiga ted the phylogeneticrelation- with both E. diffusum (E. x wallichianum ) and E. uvi- ships among extant horsetails, using rbcL and trnL-F atile (E. x litorale), and that E. x litorale is the most com- chloroplast DNAsequence data.Their MPanalyses mon hybrid in the subgenus Equisetum (Hauke1978; supportthe samebasalposition for E. bogotense , but Duckett 1979b). their ML analysis placesit assister tosubgen us Hip- Sincethe sequences used in this study occuronly pochaete.In either case,becausesubgen us Hippochaete in plastids, and since maternal transmissionofplastids would benested within subgenus Equisetum, my con- has been reported in the genus Equisetum (Guillon and clusions regarding the evolutionofcharacters in horse- Raquin 2000),the present phylogenyis in theory con- tails are equally valid. Future research focusedon E. cerned only with maternal lineages. The gene tree may bogotense would beuseful in order tocon rm its po- not exactlyre ect species relationships ifh ybridization sition. Indeed, E. bogotense and E. diffusum have been between differentiated Equisetum species has taken the least studied horsetail species. Specically ,it would placein the past.Although hybrids between extant beinteresting toperform hybridizationexperiments species are generally considered tobe sterile ,Krahulec using E. bogotense .Naturalh ybrids of E. bogotense are et al.(1996) observed the development ofgam eto- unknown,butthis may merely reect the absenceof phytes from E. x moorei (ramosissimum x variegatum) interfertile sympatricspecies. In addition, cytological and E. x meridionalis (hyemale x ramosissimum ). Unfor- studies are needed todetermine whether E. bogotense tunately,the authors did not report whether antheridia is alsodiploid and has the samechromosome number or archegoniawere expressed in these hybrid game- (n 5 108)as all kary ologically studied members ofthe tophytes. However, it is important tokeep in mind genus (Hauke1974). Page’sstatement (1972,p .367):‘ ‘the lackof clear rela- In the light ofthe present phylogeny,variousmor- tionships between the living [horsetail] species and phological charactersappear to show somedegree of their reticulate pattern oflinkage seem consistent with homoplasy. Consider forexam ple the shape ofthe cone the view thath ybridizationhas played asignicant if apex;the most parsimonious scenario is the one as- not dominant role in their evolution.’’Future studies suming that(i) a bluntcone wasthe ancestral statefor of Equisetum phylogenyshould address this point by extant horsetails, (ii)a pointed apexlater appearedin sequencing nuclear genes tocompare with the existing anancestor ofsubgenus Hippochaete ,and (iii) the apex plastid data. reversed toa rounded statein anancestor ofthe ( E. ACKNOWLEDGEMENTS .Ithank Sophie Nadot and Bernard Le- laevigatum, E. myriochaetum )clade.Other examples of jeune foruseful advice and discussion, and critical reading ofthis homoplasictraits are:(i) the coning behavior (hetero- manuscript.OdileJonot, P aola Bertolino, and Catherine Colom- phyadic stems found in E. arvense and E. telmateia ), (ii) beauare acknowledged fortheir help inD NAextractions and am- the stem branching pattern (branched throughoutthe plica tions. Chad Husbyprovided plants fromhis personal col- lections and commentedon the manuscript. Chad Husbyand subgenus Equisetum and in E. ramosissimum , E. myri- Franz Tod kindlycollected manywild plants forme .Iam also ochaetum, and E. giganteum ),(iii) the endodermal pat- gratefulto many people who sentme plants and, insome cases, tern (with anindividual endodermis found in both E. voucheredthem for me: PeterA tkinsonat CambridgeU niversity uviatile and E. giganteum ),and (iv)the shape ofan- Botanic Garden; HollyForbes at Universityof California Botanic Garden; MikeGross a tGeorgian Court College; Philip O.Ashby, theridial opercularcells and archegonialn ecks (most Fiona Inchesand Adele Smitha tRoyal Botanic Garden, Edin- elongated in E. bogotense and E. uviatile ;Hauke1963, burgh;Sophie Dunand-Martinand Philippe Clerc atConservatoire 1968,1978; Duckett 1973,1979a). et JardinBotanique de la Villede Gene`ve; ClintonM orseand An- Previous taxonomicdivisions within bothsubgen- drew Doran at Universityof Connecticu t; Nathalie Machon atCon- era are not supported bythe present phylogeny.With- servatoireN ational Botanique du Bassin Parisien. Ithank Jean- Noe¨lLabat, Dario De Franceschi,Se´verine Dramet, and Juliette in Hippochaete ,the only concordanceis the grouping of Chatard attheMuseum N ational d’Histoire N aturelle de Paris. E. laevigatum with E. myriochaetum ,which wasalready Nicolas Boileauand ChristopheDe ´ola assisted mewith the phy- present in Hauke’sclassication (1963). In contrast,the logenetic analyses. Tatiana Giraud, Claire Lavigne,Roxana Yock- phylogenysupportsa basalposition within Hippochae- tengand OdylleCudelou are also acknowledged fortheir help . Finally,Iwould liketo thank Alan R. Smithfor sharing with me te for E. scirpoides ,which wasformerly thought topos- unpublished results. sess advanced characters(small size,unbranched stems, outer common endodermis in bothstems and LITERATURE CITED rhizome,stomataarranged in single lines), instead of ARNOLD, C. A. 1947. Anintrod uction topaleobotan y. 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