phylogenetic relationships in ephedra (gnetales): evidence ... · phylogenetic relationships in...

834

Systematic Botany (2004) 29(4) pp 834ndash849q Copyright 2004 by the American Society of Plant Taxonomists

Phylogenetic Relationships in Ephedra (Gnetales) Evidence from Nuclearand Chloroplast DNA Sequence Data

STEFANIE M ICKERT-BOND1 and MARTIN F WOJCIECHOWSKI

School of Life Sciences Arizona State University Tempe Arizona 85287-45011Author for correspondence Current Address Department of Botany The Field Museum of Natural History

1400 South Lake Shore Drive Chicago Illinois 60605-2496 (sbondfieldmuseumorg)

Communicating Editor Gregory M Plunkett

ABSTRACT Sequences from the nuclear ribosomal internal transcribed spacer region 1 (nrDNA ITS1) and the plastidrps4 gene from the genus Ephedra (Ephedraceae Gnetales) were obtained in order to infer phylogenetic relationships char-acter evolution and historical biogeography in the genus Within Ephedra the length of the nrDNA ITS1 varied from 1081to 1143 basepairs (bp) in contrast to dramatically shorter lengths in the outgroups (Gnetum Welwitschia and Pinus) Therps4 locus varied in length from 645 to 661 bp in the same set of taxa Both parsimony and maximum likelihood analysesof these sequences resulted in a well-resolved phylogeny that supports the monophyly of Ephedra but not its subdivisioninto the traditional sections Ephedra Asarca and Alatae The resulting phylogeny also indicates a derivation of the New Worldclade from among the Old World taxa Among the Old World species three highly-supported monophyletic groups arerecognized that are highly concordant with morphological evidence The New World clade includes two main subclades ofNorth and South American species that are strongly supported while the position of two mostly Mexican species E pedun-culata and E compacta remains unresolved Character reconstruction of ovulate strobilus types in Ephedra indicates that fleshybracts are ancestral with shifts to dry winged bracts having occurred multiple times Low levels of sequence divergencewithin the North American clade suggest either recent and rapid ecological radiation or highly conservative ribosomal DNAevolution within the clade

Ephedra L the single genus of the family Ephed-raceae (Gnetales) is a well-delimited genus of ca 60species that are distributed in deserts and other aridregions in both the Old and the New Worlds Itsunique combination of morphological and biochemicalcharacters includes small decussate or whorledephemeral leaves on photosynthetic stems (Kubitzki1990) vessels and dimorphic tracheary elements (Carl-quist 1996) ribbed pollen (Steeves and Barghoorn1959) ovules with a single integument subtended bya pair of fleshy or wing-like bracts and the presenceof ephedrine and other alkaloids (Stapf 1889 Martens1971 Takaso 1984 Caveney et al 2001) The genus hasgenerally been considered monophyletic (Price 1996Huang 2000 Rydin et al 2002) although this has notbeen tested rigorously Chromosome numbers indicatea base number of x 5 7 with evidence of polyploidyand interspecific hybridization in both Old World andNew World forms (Hunziker 1953 Pachomova 1971Ickert-Bond 1999)

Ephedra is one of the few gymnosperms adapted toextreme aridity and as such is highly reduced vege-tatively Both the small number of characters and theconvergent evolution of species occurring in xeric hab-itats have limited taxonomic study Early taxonomicwork on Ephedra was based mostly on features of theovule-bearing strobilus Meyer (1846) divided the ge-nus into two informal groups section I Plagiostoma CA Mey and section II Discostoma C A Mey on thebasis of characters of the ovule and the extension ofthe micropyle called the tubillus Later Stapf (1889)proposed three formal subgroups based on ovule mor-

phology Section Pseudobaccatae is the most species-richand includes those with fleshy succulent bracts whilesection Alatae is comprised of those with dry wingedbracts from both the Old World (E alata Decne E prze-walskii Stapf E lomatolepis Schrenk E strobilacea Bun-ge) and New World (E trifurca and E torreyana) Sec-tion Asarca has two New World species E californicaand E aspera which are neither winged nor fleshy andhave ovulate strobili with bracts having scarious mar-gins Other investigators have attempted to test theserelationships using additional morphological charac-ters including pollen (Steeves and Barghoorn 1959)cuticle micromorphology (Pant and Verma 1974) andwood anatomy (Carlquist 1989 1992 1996) None ofthese studies supported the subdivision of Ephedraproposed by Stapf (1889)

In a group of plants where morphologically-basedtaxonomy has been so difficult the use of molecularsequence data provides an essential tool for resolvingrelationships To date however little molecular infor-mation is available for Ephedra The genus was includedin a survey of rbcL sequences of the Gnetales (Hasebeet al 1992) with only a few of the more than 50 speciesof Ephedra included (Price 1996) or as exemplars inhigher-level analyses of land plants using rbcL atpB26S and 18S ribosomal DNA (Magallon and Sander-son 2001 Rydin et al 2002 Soltis et al 2002) Infra-generic relationships were studied by Huang (2000) us-ing the matK gene and nrDNA ITS locus but limitedsampling and a taxon bias toward New World speciesobscured inferences about the genus as a whole andmany clades were not well supported

2004] 835ICKERT-BOND AND WOJCIECHOWSKI EPHEDRA PHYLOGENY

The present study seeks to increase our knowledgeof diversity and evolution in the genus Ephedra by pre-senting hypotheses for infrageneric relationships usingDNA sequence data from both the chloroplast and nu-clear genomes This study endeavors to 1) test themonophyly of Ephedra 2) provide a preliminary test ofthe validity of the traditional taxonomic divisions intosections Alatae Asarca and Ephedra (Stapf 1889 Mus-sayev 1978 Freitag and Maier-Stolte 1994) 3) enhanceour understanding of infrageneric relationships amongspecies of Ephedra and 4) provide a preliminary testof the biogeographical diversification of the genusCharacter evolution is discussed as it relates to the re-sulting phylogeny

MATERIALS AND METHODS

Taxon Sampling A total of 71 taxa were sampled in this study(Appendix 1) including 51 species from all lineages of Ephedra asdefined by Stapf (1889) two recently described South Americanspecies (Hunziker 1994 Matthei 1995) and two recently describedOld World species (Assadi 1996 Freitag and Maier-Stolte 2003)Multiple accessions of some species were used to confirm ques-tionable identifications Sampling of the New World species in-cluded all known taxa except for Ephedra trifurcata Zoellner Thisspecies is only known from the type specimen collected in 1978and could not be relocated by the first author while visiting thetype locality (Marga Marga Chile) in 2001 Selection of Old Worldspecies was based on consultation with M Maier-Stolte and HFreitag University of Kassel Germany who are currently revisingthose taxa (Freitag and Maier-Stolte 1992 1994 2003) Outgrouptaxa include Gnetum Welwitschia Hooker f and the more distantlyrelated Pinus L All new sequences have been deposited in Gen-bank (httpwwwncbinlmnihgov) and the final data matricesof both the nrDNA ITS1 and the plastid rps4 gene have been de-posited in TreeBASE (study accession number S1144 matrix ac-cession numbers M1966ndashM1967)

DNA Extraction and PCR Amplification Fresh or silica-driedmaterial was available for most taxa but herbarium material wasused for a few samples Genomic DNAs were isolated using DNAextraction kits (QIAGEN Corporation Alameda California) DNAsequences were amplified by polymerase chain reaction (Palumbi1996) on a DNA engine Dyad (MJ Research Waltham Massachu-setts) with the following conditions initial denaturation (928C 2min) followed by 40 cycles of denaturation (928C 45 sec) an-nealing (558C 30 sec) and extension (728C 30 sec) and concludingwith a final extension (728C 7 min) The PCR amplifications wereperformed in 25 ml reactions containing 10ndash100 ng genomic DNA02 mM deoxyribonucleotide triphosphates (equimolar) 05 unitsPlatinum Taq polymerase (Invitrogen) oligonucleotide primers at05 mM and Mg21 at 15 mM Glycerol was substituted with 5DMSO to relax the secondary structure of ITS (Liston et al 1996)

PCR amplification of the internal transcribed spacer region ofthe nuclear ribosomal DNA (nrDNA ITS1) used the primer ITS5and 58SR (Liston et al 1996) and a pair of internal primers de-signed specifically for Ephedra (ITS1-Ep1S 59-GGACGGTCTTT-GACCAGTTTATA-39 ITS1-Ep2R 59- GCGACGTAGGAAAGGAAATAG-39 modified from Huang 2000) Amplification yielded asingle product of ca 1200 nucleotides for Ephedra whereas se-quences from outgroup taxa varied greatly in length ranging from400 to 900 bp Amplification of the ca 700 bp plastid gene rps4used the primers trnSR2 (59-GCTTACCGGGGTTCGAATC-39) andrps5F (59-ATGTTCCCGTTATCGAGGACCT-39) designed by RCranfill University of California Berkeley This sequence includesca 30 bp of the intergenic spacer between the 39 end of the rps4gene and the trnS gene Most taxa were amplified according toreaction conditions given as above but an annealing temperatureof 558C and 40 amplification cycles were used

Sequencing Purified PCR products (QIAquicky PCR purifi-cation kit QIAGEN) were sequenced for complimentary strandsSequencing reactions employed the same primers as PCR for theplastid rps4 gene Because the nrDNA ITS1 region in Ephedra spansca 1200 bp both the external PCR primers and the additionalpair of internal primers (ITS1-Ep1S and ITS1-Ep2R) were used togenerate sequences across the full length of nrDNA ITS1 in bothdirections PCR products were cycle-sequenced using fluorescentdye-labeled chemistry (Big Dyes Perkin-Elmer Foster City Cali-fornia) Separation and identification of cycle-sequence productswas conducted on an ABI automated 377XL sequencer at the Ar-izona State University DNA Laboratory

Alignment and Analysis Sequences were assembled into con-tigs using Sequencher version 41 (Gene Codes Corporation AnnArbor Michigan) Sequence alignments were initially made inClustalX (Jeanmougin et al 1998) and edited manually The plas-tid gene rps4 codes for a protein product and is easily alignableby eye The nrDNA ITS1 sequences are somewhat more difficultto align due to dramatic length variation (ranging from 400 to1200 bp) and high sequence divergence between Ephedra and allknown outgroups

PAUP 40 beta10 (Swofford 2003) was used to reconstruct phy-logenetic relationships All characters were unordered and weight-ed equally Parsimony analyses were conducted using heuristicsearch methods and employed all addition sequence options (SIM-PLE CLOSEST RANDOM addition sequences) in combinationwith tree-bisection reconnection (TBR) branch-swapping and theMULTREES option which saves all minimal trees with MAX-TREES set to 5000 Fourteen indels (insertionsdeletions) in thenrDNA ITS1 data set (varying from one to 25 base positions inlength for a total of 72 characters) and were routinely excludedfrom all analyses However the indels were also coded as separatebinary characters and added at the end of the data set and ana-lyzed to determine whether they were phylogenetically informa-tive For all data sets support for individual tree branches wasestimated using non-parametric bootstrap methods (Felsenstein1985) Bootstrap proportions (BP) were obtained from 100 repli-cates of heuristic searches as described above (1000 random ad-dition sequences TBR branch swapping and MULTREES select-ed)

The monophyly of previously proposed taxonomic groupings(sects Ephedra Alatae and Asarca) was investigated by comparingtrees consistent with topological constraints to trees obtained fromunconstrained analyses Trees with topological constraints wereconstructed using MacClade 403 (Maddison and Maddison 2000)and loaded into PAUP Heuristic searches were then conductedto find the shortest trees consistent with each imposed constraint

Maximum likelihood (ML) analyses were conducted with thenrDNA ITS1 dataset using PAUP to compare with results fromparsimony analyses A single tree derived from parsimony orneighbor joining analysis was used as the starting tree for a heu-ristic search (addition sequence lsquolsquoASISrsquorsquo) with TBR branch swap-ping and MULTREES in effect Base frequencies were empiricallydetermined and the transition transversion (K) ratio and gammashape parameter (G) were estimated on tree number 6 (of 198 mostparsimonious trees) from parsimony analysis (3283000 and00535576 respectively) and the tree derived from neighbor join-ing (348352 and 0073199 respectively) under the HKY85 modelof sequence evolution chosen using results from ModelTest ver-sion 306 (Posada and Crandall 1998) Rate heterogeneity acrosssites followed a gamma distribution with four categories Pairwisedistances were calculated across all sites as lsquouncorrected prsquo valuesusing PAUP

Rooting followed the outgroup method for the rps4 data butwas problematic within the nrDNA ITS1 sequences due to dra-matic length differences between sequences of the outgroup andingroup Multiple analyses were undertaken both with and with-out inclusion of outgroups One approach to deal with large lengthvariation and sequence divergence between the outgroup and theingroup was proposed by Simmons and Freudenstein (2003) thatutilizes ClustalX (Jeanmougin et al 1998) and was employed hereIn ClustalX blocks of 30 invariant bases were added to the se-

836 [Volume 29SYSTEMATIC BOTANY

TABLE 1 Comparison of rate constancy of nrDNA ITS sequence evolution in Ephedra based on likelihood ratio tests with placementof the root in all possible positions The log likelihood values with assumption of molecular clock (2lnL0) and without (2lnL1) valuesfor molecular clock assumption are consistently 316824174 Molecular clock in these data is rejected based on chi-square values (df 546 P 5 001 CV 5 71201) Taxa abbreviations EA1 5 Ephedra antisyphilitica 900 EAM 5 Ephedra americana Argentina EAP1 5 Ephedraaphylla Fr 14a 01 EAP2 5 Ephedra aphylla F30181 EAS 5 Ephedra aspera EBO 5 Ephedra boelkei EBR 5 Ephedra breana EC1 5 Ephedracoryi SIB 953 EC2 5 Ephedra coryi SIB 952 ECA 5 Ephedra californica ECH 5 Ephedra chilensis ECO 5 Ephedra compacta ECU 5 Ephedracutleri EDI 5 Ephedra distachya EFA 5 Ephedra fasciculata EFE 5 Ephedra fedtschenkoae EFL 5 Ephedra frustillata EFN 5 Ephedra funereaEFR 5 Ephedra fragilis EFU 5 Ephedra funerea SIB 964 EGR 5 Ephedra gracilis EIN 5 Ephedra intermedia ELA 5 Ephedra laristanica EMO5 Ephedra monosperma EMU 5 Ephedra multiflora ENE 5 Ephedra nevadensis EOC 5 Ephedra ochreata EPE 5 Ephedra pedunculata ERA5 Ephedra rupestris Arg ERE 5 Ephedra regeliana ERU 5 Ephedra rupestris ESA 5 Ephedra saxatilis ESI 5 Ephedra sinica var pumila ESO5 Ephedra somalensis EST 5 Ephedra strobilacea ETA 5 Ephedra triandra ETI 5 Ephedra transitoria ETO 5 Ephedra torreyana CrimminsETP 5 Ephedra torreyana powelliorum ETR 5 Ephedra trifurca ETS 5 Ephedra torreyana AZ ETW 5 Ephedra tweediana EVI 5 Ephedraviridis

Root placed along the branch betweentaxa listed and the remaining taxa

Molecular clockassumption 2lnL0

Without the molecular clockassumption 2lnL1 Likelihood ratio 22nLR

ELA 320269271609 316824174 68901955EPEEVIESOEDIEMO ESA

321448556536323276675754323555968010324299145664325154758526

92487065712905004134635891494994416661169

ECAEAP2 EFRECHEST ETIECO4 EAM ERU

326065509676326536480176326873771349327193190747327317757967

1848267219424613200991952073803420987168

ESI EINEFE ERE EC1 EAP1ETW ETR ETAEBRECO1 EC2

328140952837328532565718328990698269329251888925329612217738

226335582341678424333049248554325576088

ETOETPEGREMU EBOEA1ERA EOC

331193671356331414447345332035824580332421645634332917170745333683603110

287389952918054730423302311949443218599433718859

ETS EFNEASEFUENE ECU EFA

333902081343334221571065335544379305335860790784

34155815347947943744041138073234

quences at the 59 and 39 ends of nrDNA ITS1 These blocks keptthe 59 and 39 ends of the Ephedra sequences consistently alignedwith outgroup sequences reducing overall sequence divergenceIn the program alignment parameters for pairwise alignmentswere set to lsquolsquoslow-accuratersquorsquo with a lsquolsquogap opening costrsquorsquo of 10 anda lsquolsquogap extension costrsquorsquo of 5 for both pairwise and multiple align-ments In multiple alignment mode lsquolsquodelay divergent sequencesrsquorsquowas set to 40 and lsquolsquoDNA transition weightrsquorsquo was set to 050 Theseinvariant sequence blocks were then excluded from all phyloge-netic analyses

Two alternative methods of rooting were explored The first wasmidpoint rooting which assigns the root to the midpoint of thelongest path between two terminal taxa (ie between the two mostdivergent lineages in the ingroup Swofford et al 1996) Secondreconstruction of a tree under the assumption of a molecular clockwas used to infer the root of the tree Analyses were conductedto determine likelihood scores for a tree of ingroup taxa generatedby the neighbor-joining method both with and without enforce-ment of a molecular clock where the root was forced to attach toeach of the possible branches on the same ingroup topology (seeSchultheis and Baldwin 1999) The number of ingroup sequenceswas reduced to represent only one accession per taxon to mini-mize the number of necessary calculations (Table 1) Estimatednucleotide frequencies and the substitution model parameter val-ues consistent with the General Time Reversal model [GTR1I1G]

under the Akaike Information Criterion were determined usingModelTest version 306 (Posada and Crandall 1998) Rate hetero-geneity across all lineages was then assessed using a likelihoodratio test (Felsenstein 1988 Huelsenbeck and Rannala 1997)

RESULTS

Nuclear Locus Results from likelihood ratio anal-yses of nrDNA ITS1 data with and without enforce-ment of a molecular clock (Table 1) for the ingrouptaxa show that clock-like evolution can be rejected forall root placements within Ephedra except along the lin-eage between the Old World species E laristanica andthe remaining taxa In all other rootings examinedbranch lengths were not consistent with equal rates ofnucleotide change Midpoint rooting also resulted inplacement of the root between E laristanica and all re-maining taxa Results from analyses of the dataset inwhich invariant blocks were included at both the 39and 59 ends of the nrDNA ITS1 of both the ingroup


FIG 1 Phylogenetic relationships in Ephedra based on parsimony analysis of nrDNA ITS1 sequence data Tree is strictconsensus of 198 equally parsimonious trees of length 234 steps (CI 5 07179 RI 5 09282) derived from heuristic analyses(random addition sequence TBR branch swapping MULTREES in effect) Bootstrap values are given above branches of cladesthat were supported in both the strict consensus and the bootstrap consensus trees Named groups following classifications byStapf (1889) except as noted M 5 Mussayev (1978) FMS 5 Freitag and Maier-Stolte (1994) and their geographic distribution(NW New World NA North America SA South America OW Old World) appear in margin at right

and the outgroup are compatible with results fromboth midpoint rooting of the ingroup taxa alone andfrom root placement based on a molecular clock as-sumption (ie between E laristanica and the remainingtaxa) For subsequent analyses and presentation of re-sults trees were rooted with E laristanica

The aligned length of the nrDNA ITS1 region for 55ingroup taxa was 1158 bp There were 14 indels thatwere also added to the data set as binary charactersbut the inclusion of these extra characters had no effecton the overall tree topology The nrDNA ITS1 data setincluded 112 parsimony-informative characters andyielded 198 most parsimonious trees (234 steps) Pair-wise distances ranged from 0ndash55 within Ephedra

The strict consensus of these trees (Fig 1) when

rooted with Ephedra laristanica shows the New Worldspecies of Ephedra comprising a well-supported cladewhile the Old World taxa form a paraphyletic grade ofthree well-supported clades at the base of the treeWithin the New World clade there is strong support(BP595) for a clade comprising all species from NorthAmerica except for two primarily Mexican species Ecompacta and E pedunculata Within the main NorthAmerican clade E viridis is sister to the remainingspecies Species of section Asarca do not form a mono-phyletic group within the main North American cladewhereas two species of section Alatae subsection Tri-furcae E torreyana and E funerea form a moderately-supported clade (BP570) within this main clade Al-though all South American species belong to one high-


ly-supported clade (BP598) relationships among thesespecies are not well resolved except the clade includ-ing E tweediana E triandra and E chilensis

Among the Old World taxa there is strong supportfor several clades Ephedra monosperma E pachyclada varsinaica E saxatilis and the newly described E somalen-sis comprise a very well-supported clade (BP599) thatis congruent with Ephedra section Ephedra group Dis-tachyae subgroup Leptocladae In this clade E monos-perma and E saxatilis are sister taxa and this clade issister to a clade of E somalensis and E pachyclada varsinaica Further Ephedra section Ephedra group Distach-yae subgroup Distachyae which includes E sinica varpumila E intermedia E fedtschenkoae and E regelianais strongly supported (BP593) as monophyletic With-in this clade E fedtschenkoae and E regeliana are weakly(BP563) supported as sister species Ephedra strobilaceaof section Alatae comprises a clade with E transitoriaand E sarcocarpa with E strobilacea sister to E transi-toria and E sarcocarpa of group Sarcocarpae Finally thespecies representing Ephedra section Ephedra groupFragilis are well resolved as monophyletic

Comparison of results from parsimony and maxi-mum likelihood analyses (Figs 2 5) are generally con-gruent with respect to overall topology and estimatedbranch lengths For example a highly-supported cladeof New World taxa is nested within Old World groupsand both reconstruction methods are also consistent inshowing taxa with the largest number of inferred nu-cleotide substitutions to be E laristanica E aphylla Efragilis and E foeminea

Chloroplast Locus The aligned length of the rps4gene and the partial sequence of the intergenic spacerregion between rps4 and trnS ranged from 673 to 685bp Pairwise distance ranged from 0ndash19 within Ephe-dra Results of parsimony analyses using Pinus Wel-witschia and Gnetum as outgroups strongly supportsthe monophyly of the genus Ephedra (Fig 3) Althoughthese data provide very little resolution within Ephedra(13 parsimony informative characters within the in-group not including indels) they do suggest the NewWorld Ephedra are nested within Old World lineages(Fig 3) Ephedra somalensis is sister to the rest of Ephe-dra with relationships of the remaining taxa unre-solved except for a few weakly-supported clades Con-sistent with results from the nrDNA ITS1 data (Fig 1)the New World species are nested within Old Worldgroups although bootstrap support is weak Branchlengths estimated by parsimony (Fig 4) show very fewcharacter changes in Ephedra compared to Gnetum andWelwitschia and long branches separate the genera

DISCUSSION

Molecular Evolution Based on results of compar-ison of uncorrected pairwise distances the nrDNAITS1 region in Ephedra evolves approximately three

times faster than the protein-coding region of plastidrps4 and the adjacent intergenic spacer region Al-though this region is only twice as long as the rps4region it has ten times as many the number of infor-mative characters It is therefore evident that thenrDNA ITS1 region is more phylogenetically infor-mative for deducing relationships in Ephedra The ITS1region has been used for phylogeny reconstruction inseveral gymnosperm studies (Liston et al 1996 Ger-nandt et al 2001 Li et al 2001 Sinclair et al 2002) andextensively in angiosperms (Baldwin et al 1995 Al-varez and Wendel 2003) However it should be notedthat the rps4 gene has shown phylogenetic utility instudies of pteridophytes (Pryer et al 2001 Schneideret al 2004) and monocots (Nadot et al 1994)

Low levels of sequence divergence in nrDNA ITS1(as in rps4) in the New World clade (Fig 2) as com-pared to that observed within Old World groups sug-gests that either Ephedra underwent a more recent orrapid radiation in the New World or that evolution ofthe nrDNA region has been more conservative in NewWorld Ephedra A similar pattern has recently beenshown in the New World thistle Cirsium Mill wherethe western North American lineage exhibits a lowersequence divergence compared to other New Worldgroups (Kelch and Baldwin 2003) Molecular phylo-genetic analysis of the conifer genus Torreya Arn alsoshows well-supported Old and New World clades butlow levels of sequence divergence within each of thempointing to a recent origin of extant taxa (Li et al2001) a pattern similar to the results for Ephedra Otherexamples of a long stem lineage and low divergencewithin resulting crown groups have been noted re-cently in Myristicaceae (Sauquet et al 2003) and Chlor-anthaceae (Zhang and Renner 2003) For example inthe Chloranthaceae the fossil record dates back to theEarly Cretaceous (Doyle et al 2003) but age estimatesbased on molecular data (Zhang and Renner 2003)date the divergence time of the crown group as con-siderably more recent in the Tertiary (14ndash45 MYA)

Monophyly of Ephedra Previous studies have pro-vided morphological and molecular evidence consis-tent with the monophyly of Ephedra within the broadercontext of seed plant relationships (Price 1996 Huang2000 Rydin et al 2002 Huang and Price 2003 Wonand Renner 2003) In this study we used a more com-prehensive sampling of Ephedra as well as outgrouptaxa to address this issue Molecular sequence datafrom the plastid rps4 gene strongly support the mono-phyly of Ephedra (Figs 3 4) as well as the monophylyof those species sampled from Gnetum

Phylogenetic Relationships Within Ephedra Re-sults from both data sets are consistent in showing thatEphedra comprises a basal paraphyletic grade of OldWorld lineages within which a highly-supported NewWorld clade is nested (Figs 1ndash5) Results presented


FIG 2 Maximum likelihood tree of nrDNA ITS1 data showing estimated branch lengths Relationships among major cladesof Ephedra are similar to those inferred from parsimony analyses of the same data except that Ephedra compacta is now sisterto main North American clade Phylogram shown is one of four trees (2InL 5 304849402) derived from maximum likelihoodanalyses under a HKY851G model of nucleotide substitution Geographic distribution of taxa (NW New World NA NorthAmerica SA South America OW Old World) appear in margin at right

here suggest that neither the subdivision of Ephedrainto the three traditional sections Alatae Asarca andPseudobaccatae (5 Ephedra L) as circumscribed by Stapf(1889) nor the slightly modified system by Mussayev(1978) who recognized five sections (Alatae AsarcaEphedra Scandentes (Stapf) Mussayev Monospermae

Pachom) is supported All the sections based on mor-phology by Stapf (1889) and Mussayev (1978) are dis-tributed worldwide except for the strictly North Amer-ican section Asarca Within the New World clade foursubclades are highly-supported one composed ofmost of the North American taxa and one of strictly


FIG 3 Phylogeny of Ephedra based on parsimony analysis of plastid rps4 (including rps4 gene and rps4- trnS intergenicspacer) sequence data Tree is strict consensus of 1000 (MAXTREES) equally parsimonious trees of length 465 steps (CI 5 0892RI 5 0962) derived from heuristic analyses (random addition sequence TBR branch swapping MULTREES in effect) Bootstrapvalues are given above branches for clades resolved in strict consensus Geographic distribution of taxa indicated in margin atright (NW New World OW Old World)


FIG 4 General morphology of the ovulate bract in Ephedra A Ephedra torreyana of section Alatae subsection Trifurcaeshowing dry wing-bracted ovulate strobili (3 35) B Detail of individual bract note stalk at base and margin elaborated intowing (3 6) C Ephedra antisyphilitica of section Ephedra subsection Antisyphiliticae showing fleshy bracts of the ovulate strobili(3 39) D Detail of bracts showing fusion of more than 50 of the entire lengths of the bract note absence of stalk at base(3 76) E Ephedra nevadensis of section Asarca showing bracts that are dry not papery or fleshy (3 35) F Detail of bracts fusedbasally but well below midpoint (3 64) G Detail of single bract with margin entire not elaborated into wing (3 65)


South American species The wing-bracted section Ala-tae as circumscribed by Stapf (1889) and Mussayev(1978) is not monophyletic in this analysis (Fig 1) Toachieve monophyly trees derived from the MP analy-sis would have to be 55 steps (ca 22) longer Ephedrastrobilacea is the only species with dry winged bractsfrom the Old World section Alatae Tropidolepides Stapf(5 subsection Alatae (Stapf) Mussayev) included in thisanalysis It is strongly supported as sister to a cladeof two species (E sarcocarpa and E transitoria) withfleshy bracts (BP599) In Stapfrsquos classification E sar-cocarpa was placed in section Pseudobaccatae Stapf sub-section Pachycladae Stapf among other fleshy-bractedephedras of the Old World while E transitoria was notdescribed until after Stapfrsquos treatment (Riedl 1961)Freitag and Maier-Stolte (1994) provide an alternativeclassification including E sarcocarpa E transitoria anda third species E lomatolepis in their group SarcocarpaeFreitag and Maier-Stolte which is monophyletic in thisanalysis (Figs 1 2 Sarcocarpae clade) although withweak support (BP557) Our molecular results are con-sistent with the distribution of similar morphologicalcharacters such as wide hyaline margins of the oth-erwise fleshy bracts (Fig 5A B) distinctly stalked mi-crosporangia (Fig 5C) and pollen morphology as firstnoted by Freitag and Maier-Stolte (1994)

The New World subsection of section Alatae Habro-lepides Stapf (5 subsection Trifurcae Mussayev) is notmonophyletic in this analysis although two species Efunerea and E torreyana are closest relatives (BP570)in the main North American clade It appears that dry-winged bracts originated multiple times in the NewWorld Other members of Stapfrsquos original subsectionHaprolepides which includes the North American E tri-furca and the South American E multiflora and E boel-kei do not cluster with the other wing-bracted taxafrom the New World although E boelkei a species withwinged bracts recently described from Argentina(Roig 1984) forms a clade with E multiflora (Figs 25) in the South American clade These two species arevery closely related and at least one specialist (J Hun-ziker pers comm Instituto Darwinion San Isidro Ar-gentina 2000) considers E boelkei to be a polyploidderivative of E multiflora that has subsequently becomemorphologically distinct This hypothesis needs to beinvestigated using both molecular and cytologicalmethods

The second traditionally-described section AsarcaStapf which includes E californica E fasciculata and Easpera (Mussayev 1978) is not supported as monophy-letic in our analysis For these taxa to form a cladetrees derived from parsimony analysis would require17 more steps than the globally most parsimonioustrees ( 7 longer) It is interesting to note that Ecalifornica a species with dry bracts that are marginallywinged that has commonly been placed in section

Asarca (Stapf 1889 Mussayev 1978 Price 1996) is high-ly supported as the closest relative of E trifurca of sec-tion Alatae subsection Trifurcae (BP598) which is de-lineated by winged bracts of the ovule-bearing strobili(Fig 5A B)

Results from analyses presented here also suggestthat the third section Pseudobaccatae (5 Ephedra) thefleshy-bracted ephedras is polyphyletic Monophyly ofthese taxa would require trees with 40 additional steps( 16 longer) However within section Pseudobacca-tae several relationships are highly supported Mostspecies of subsection Scandentes Stapf (5 section Scan-dentes (Stapf) Mussayev subsection Scandentes) (5group Fragilis Freitag amp Maier-Stolte) form a highly-supported monophyletic group (Figs 1 2 Fragilisclade) This grouping is consistent with putativeshared development of lsquolsquolargersquorsquo leaves (up to four cmin E foliata Boiss) sessile microsporangia (Fig 5C)Fragilis-type pollen and a general climbing habit (Fig5D Freitag amp Maier-Stolte 1994) The newly describedE laristanica from Iran (Assadi 1996) was used as theoutgroup to the rest of Ephedra in this analysis but fitswell within subsection Scandentes Mussayev (5groupFragilis) morphologically (Freitag and Maier-Stoltepers comm University of Kassel Germany 2003)

Another well-supported clade within the Old Worldsection Ephedra corresponds to Freitag and Maier-Stol-tersquos subgroup Leptocladae which includes E monospermaand E saxatilis (from the Himalayas and Mongolia)and E somalensis and E pachyclada var sinaica (fromSomalia and Israel) (BP 5 99) These taxa share themorphological characters of typically 1-seeded ovulatestrobili sessile microsporangia (Fig 5C) and Distachya-type pollen (Freitag and Maier-Stolte 1994 2003) Thedisjunct distribution of these four species betweeneast-central Asia and east Africa may reflect a combi-nation of vicariance and extinction events an expla-nation suggested for a similar distribution of taxa inthe family Nyctaginaceae (Thulin 1994) Ephedra so-malensis grows in mesophytic habitats which may in-dicate that it is a relictual species of a possible oncemore widespread Tertiary laurophyllous vegetation innortheast Africa (Axelrod 1979 Freitag and Maier-Stol-te 2003) Species of subgroup Leptocladae were tradi-tionally placed in subsection Leptocladae Stapf or byMussayev (1978) in section Monospermae Pachomovasubsection Monospermae To render subsection Monos-permae monophyletic would require 15 additional stepsrelative to the most parsimonious trees ( 6 longer)

Freitag and Maier-Stoltersquos group Distachyae is notsupported as monophyletic in this analysis but E dis-tachya and E distachya subsp helvetica form a weakly-supported clade (BP566) that itself is weakly-sup-ported (BP554) as the sister group to a highly-sup-ported clade (BP599) of E strobilacea from section Ala-tae and group Sarcocarpae (Figs 1 2 5) The other


FIG 5 Parsimony reconstruction of character state evolution of several unordered morphological key characters optimizedon one of 198 equally parsimonious trees of length 234 steps (shown in Fig 1) using MacClade A Ovulate bract charactersNamed sections follow classifications by Stapf (1889) B Wing margin of ovulate bracts FIG 5 Continued C Synangia char-acters of staminate strobili D Plant habit

members of the group Distachyae form a highly-sup-ported clade (BP593) but relationships within thisclade are not well resolved

New World species of section Ephedra (5Pseudobac-catae Stapf) are not resolved as monophyletic but arescattered within the New World among North Amer-

ican and South American groups Monophyly of thisclade in the New World would require the addition of30 steps ( 12 longer than the most parsimonioustrees) It is interesting to note that all of the SouthAmerican species of section Pseudobaccatae subsectionAmericanae Mussayev form a highly-supported clade


(BP598) while the two remaining members of sub-section Americanae E pedunculata and E compacta re-main unresolved within the New World clade

Character Evolution Traditional delimitations ofinfrageneric relationships in Ephedra as proposed byStapf (1889) and Mussayev (1978) based on a few char-acters of the ovulate bracts are not supported by mo-lecular data (Fig 5) None of the three sections thefleshy-bracted ephedras of section Ephedra (5 Pseudo-baccatae Stapf) the intermediate ephedras of sectionAsarca Stapf or the wing-bracted ephedras of sectionAlatae Stapf are supported herein as clades Based onour phylogenetic results it appears that fleshy bractsare the ancestral condition and that dry winged bractsoriginated multiple times (Fig 5) The question of or-igin(s) of the intermediate condition (section Asarca)cannot be fully addressed here because relationshipswithin the main North American clade are not well-resolved It is noteworthy that bracts characterizingsection Asarca are partially connate as in section Ephe-dra but distinct in section Alatae Furthermore thebracts of members of section Asarca particularly in Enevadensis swell considerably during the growing sea-son (S Ickert-Bond pers obs) but subsequent devel-opment into fleshy bracts does not occur

While the results from analysis of molecular data donot confirm classifications based on ovulate bract char-acters the larger number of species possessing fleshy-bracted ovules is intriguing and might be correlatedwith greater dispersal ability or different rates of spe-ciation The occurrence of red fleshy bracts in sectionEphedra is generally considered indicative of endozo-ochory (Stapf 1889 Freitag and Maier-Stolte 1994Danin 1996 Hodar et al 1996) In contrast the drywing-bracted diaspores of section Alatae are a goodexample of anemochory (Stapf 1889 Danin 1996) Theseeds of section Asarca often accumulate at the stembase and are carried off by packrats other small ro-dents and possibly lizards (S Ickert-Bond pers obs)

While differing dispersal mechansims have been in-ferred to explain the high species diversity of angio-sperms compared to non-fruiting plants (Tiffney andMazer 1995 Smith 2001) recent studies have shownthat similar effects may occur in the pollination anddispersal mechanisms of some gymnosperms as well(Norstog 1990 Schneider et al 2002) Animal dispersalof angiosperm diaspores has been thought to be re-sponsible for greater specialization and thus increasedspeciation but the situation may be more complex ingymnosperms than previously recognized It is not themere fact of biotic versus abiotic dispersal but a com-bination of traits including dispersal type ecologicalconditions and growth form that may explain thegreater species diversity in certain plant groups (Her-rera 1989 Eriksson and Bremer 1991 Tiffney and Ma-zer 1995 Smith 2001) The coincidence of higher spe-

cies number with fleshy diaspores in Ephedra seems tosuggest that fleshiness may have affected the rate ofspeciation or of extinction In contrast anemochoroustaxa in Ephedra inhabit highly specialized niches suchas hyperarid deserts sometimes in dry salt lakes oftendevoid of animal life (Danin 1996) Thus it may behabitat availability and not dispersal ability that hasprevented increased diversity within this group a phe-nomenon that has been suggested to account for thediversity of herbaceous members of Rubiaceae (Eriks-son and Bremer 1991)

Habit diversity in Ephedra is another interesting mor-phological character Most species grow as much-branched shrubs but a few climbing or small tree-likespecies are known as well (Stapf 1889 Pearson 1929Carlquist 1996 Price 1996) A lianoid habit (Fig 5D)is reconstructed as having been derived in three dif-ferent clades in the Old World Fragilis group in theNorth American E pedunculata and in the South Amer-ican clade (E tweediana E triandra)

Characters of the staminate strobilus in Ephedrashow another interesting pattern (Fig 5C) Distinctlystalked synangia (fused sporangia) sensu Hufford(1996) are reconstructed as derived from sessile onesThis character had been used to imply the distinctive-ness of species in section Alatae both in the Old as wellas the New World however this section is not mono-phyletic in our analyses (Figs 1 5A) Results do sug-gest a close relationships of the wing-bracted taxa ofsection Alatae subsection Alatae with Freitag andMaier-Stoltersquos group Sarcocarpae in the Old Worldwhich is distinguished by fleshy bracts with a widehyaline margin

Other morphological characters that have not beenused for species identification may indeed have someutility at broader infrageneric levels For exampleSteeves and Barghoorn (1959) and El-Ghazaly andRowley (1997) considered pollen morphology to be toovariable for species identification or inferring infrage-neric relationships in Ephedra yet general pollen-typeswere used in combination with other characters to de-lineate clades by Freitag and Maier-Stolte (1994) Withfurther inspection of the results of phylogenetic anal-yses additional morphological characters of value maybe discovered

Polyploidy in Ephedra is present but generally onlyat the tetraploid level (Choudry 1984 Hunziker 19531955) with the exception of a single hexaploid countin Ephedra funerea (Ickert-Bond 2003) No obvious geo-graphic or taxonomic relationship between the distri-bution of diploid and polyploid taxa exists since poly-ploidy occurs worldwide and in all sections of the ge-nus (Choudry 1984)

Biogeographical Implications Three hypothesesconcerning the center of origin for Ephedra have beenput forth 1) the genus originated in Central Asia [hy-


pothesis 1 of Pachomova (1969)] 2) it originated inSouth America [hypothesis 2 of Pachomova (1969)] 3)it originated in the mountainous littorals of the Med-iterranean [hypothesis 3 of Soskov (1968) and Mussay-ev (1978)] To investigate biogeographical patterns inEphedra evidence derived from plate tectonics fossilsand phylogenetic analysis is brought to bear on thesecompeting hypotheses

Unequivocal gnetalean megafossils known fromEarly Cretaceous strata include Drewria potomacensisCrane and Upchurch of Aptian age from the PotomacGroup of Virginia USA and Cratonia cotyledon RydinMohr and Friis from the late Aptian-early Albian CratoFormation of Brazil (Crane and Upchurch 1987 Rydinet al 2003) Both of these genera show a mosaic ofmorphological characters seen among extant genera ofthe Gnetales suggesting that they represent examplesof a group that was considerably more diverse duringthe Cretaceous when they were also more widely dis-tributed geographically Characters currently uniqueto one or another extant genus such as the unusualcotyledon morphology of the narrowly-endemic genusWelwitschia were more broadly distributed in thesetwo genera suggesting later Cretaceous or early Ter-tiary extinction of genera with intermediate morphol-ogies

There are additional megafossils with strong gne-talean affinities (Crane 1996) that await more detailedstudy including samples from Early Cretaceous local-ities in China (Guo and Wu 2000) Russia (Krassilov1986) and Portugal (Crane et al 1995 Rydin et al2004) The habit of these fossils ranges from large-leaved taxa as in the Gnetum-Welwitschia lineage (Kras-silov 1986 Crane and Upchurch 1987 Rydin et al2003) to small-leaved forms as in the Ephedra lineage(Crane and Maisey 1991 Mohr et al 2003 2004 Rydinet al 2004) The age and geographic distribution ofthese gnetalean megafossils thus suggests that thecrown group of Gnetales once ranged from Eurasiaacross the North Atlantic land bridge to North Amer-ica and was present in eastern South America (at leastthe Welwitschia and Ephedra lineages) Given the pre-sent day western African distribution of Welwitschia itcan be hypothesized that this group had a broadernorthern Gondwanan distribution (Rydin et al 2003)during the Cretaceous

Dispersed fossil pollen grains of questionable gne-talean affinity are abundant from the Triassic to theRecent The form genus Equisetosporites Daugherty(Ephedripites Bolkhovitina) comprises grains that aresimilar to those of modern Ephedra but also includeother forms which exceed the modern pollen diversityof the genus including grains having psilate end platestwisted muri etc (Osborn et al 1993 Hesse et al2000) Fossil grains assigned to Ephedra are knownfrom a variety of localities since the Paleocene includ-

ing sites in southeastern and western North Americaand Australia (Cookson 1956 Leopold and Clay-Poole2001) Ephedra pollen has been reported from the mid-dle Eocene Green River Formation of Wyoming whereit was thought to occur on dry well-drained slopes(Wodehouse 1933) the middle Eocene Claiborne For-mation in western Kentucky where it presumablygrew in sandy xeric coastal habitats (Graham 1999)and in the late Eocene Florissant Colorado florawhere it may have occurred on dry ridges supportinga semiarid vegetation (Leopold and Clay-Poole 2001)Ephedra is also present in the Tertiary of Australiawhere it is now extinct (Cookson 1956) The Paleocene-Eocene period (ca 60 MYA) might thus indicate thetime when crown group Ephedra originated

Proposed divergence times of the crown group Gne-tales based on estimates derived from analyses ofDNA sequence data range widely from about 80 to 218MYA (Sanderson and Doyle 2001 Ickert-Bond and Wo-jciechowski 2002 Sanderson 2002 Soltis et al 2002)This variation in estimated divergence times is due inpart to the analysis of different genes different parti-tions of the same gene or different assumptions onseed plant topology Using a molecular clock-basedanalysis of rbcL data Huang and Price (2003) estimat-ed an age for crown group Ephedra ranging from 8 to32 MYA This date is considerably younger than thewell-established pollen record of Ephedra that begins inthe Paleocene (Graham 1999)

Results from the analyses presented here appear tosupport the idea of an origin and early diversificationof Ephedra in the Old World with a subsequent diver-sification to the New World (Figs 1ndash5) This clearlyrefutes the second hypothesis of Pachomova (1969) fora South American origin of Ephedra Our results alsosuggest diversification of the New World clade withderivation of the South American taxa from those ofNorth America but the relationships are not adequate-ly resolved to answer this definitively

The close relationship among the four New Worldclades is supported by high bootstrap values (Fig 1)Similar close relationships of taxa occurring in desertsof North America and South America can be found inthe genera Larrea Cav (Cortes and Hunziker 1997 Liaet al 2001) Prosopis L (Thorne 1986) and FagoniaTourn ex L (Porter 1974) One explanation for this re-curring pattern is that these plant taxa may be of re-cent origin and their distributions are the result oflong-distance dispersal (Axelrod 1979 Graham 1999)After North and South America separated in the Cre-taceous a large marine barrier extended across muchof Central America until ca 36 MYA and it was notuntil ca 24 MYA that land connections (the Panamaland bridge) existed that would allow exchange oflarge terrestrial animals and plants lacking means forlong-distance dispersal (Graham 1992 Burnham and


Graham 1999) It has generally been hypothesized thatherbaceous genera evolved in North America and weredispersed south to temperate South America relativelyrecently (Raven 1963 Porter 1974 Thorne 1986 Wen etal 2002) while woody species are considered to havea South American ancestry (Thorne 1973 1986) Re-sults from analyses of molecular sequence data pre-sented here (Figs 1ndash5) argue that the migration in NewWorld Ephedra was not from South America to NorthAmerica (Figs 1ndash5) but instead proceeded from NorthAmerica including Mexico to South America Rela-tionships of the four lines in the New World clade areunresolved in the strict consensus however Ephedra pe-dunculata a species mainly Mexican in distributiongroups with the North American clade in some of ourmost parsimonious trees this topology either shows Ecompacta (strictly Mexican in distribution) as sister tothe core North American clade while in other mostparsimonious trees these two taxa remain unresolvedThis is evidence against the hypothesis that the firstNew World species of Ephedra split into a North Amer-ican and a South American clade by an initial vicari-ance event

ACKNOWLEDGMENTS We would like to thank the Royal Botan-ic Gardens Edinburgh the Jardin Botanique de Montreal BLeuenberger (B) R Puente-Martinez (ASU) G Walters (MO) andH Won (MO) for providing plant materials We are particularlygrateful for numerous Ephedra samples provided by M Maier-Stol-te and H Freitag (KAS) who guided species selection for the OldWorld and also provided extensive discussion on the manuscriptWe also thank the Navajo Nation Kofa National Wildlife RefugeUS Fish and Wildlife Service Death Valley National Park BigBend National Park the Ministerio del Ambiente Ecuador for per-mission to collect materials A Fiero-Fierre (MO) D Neil (MO)M Munoz-Schick (SI) and L Novarra (MCNS) for field support inSouth America and M Lavin K B Pigg and D J Pinkava forcritically reading earlier versions of this manuscript and J ADoyle for helpful discussion during this study The manuscriptwas greatly improved by the many helpful comments made by JA Doyle an anonymous reviewer and the editors The researchwas supported by an American Dissertation Fellowship from theAAUW Educational Foundation an American Society of Plant Tax-onomists Graduate Research Award a Botanical Society of Amer-ica John S Karling Graduate Research Award a Phelps DodgeScholarship (Phelps Dodge Foundation) the Gertrude ClaypoolFund (National Council of State Garden Clubs) and support fromthe (former) Department of Plant Biology and the Graduate Col-lege at Arizona State University to SI-B and MFW This paperrepresents a partial fulfillment of the requirements for the degreeof Doctor of Philosophy (to SI-B) at Arizona State University

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ASSADI M 1996 A new species of Ephedra L and reports of twonew or interesting grasses from Iran Iranian Journal of Botany7 1ndash5

AXELROD D I 1979 Desert vegetation its age and origin Pp 1ndash72 in Arid land plant resources eds J R Goodin and D K

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BALDWIN B G M J SANDERSON J M PORTER M F WOJCIE-CHOWSKI C S CAMPBELL and M J DONOGHUE 1995 TheITS region of the nuclear ribosomal DNA a valuable sourceof evidence on angiosperm phylogeny Annals of the MissouriBotanical Garden 82 247ndash277

BURNHAM R J and A GRAHAM 1999 The history of neotropicalvegetation new developments and status Annals of the Mis-souri Botanical Garden 86 546ndash589

CARLQUIST S 1989 Wood bark and pith anatomy of New Worldspecies of Ephedra Aliso 12 103ndash118

mdashmdashmdash 1992 Wood bark and pith anatomy of Old World speciesof Ephedra and a summary for the genus Aliso 13 255ndash295

mdashmdashmdash 1996 Wood bark and stem anatomy of Gnetales a sum-mary International Journal of Plant Sciences (Supplement) 157S58ndashS76

CAVENEY S D A CHARLET H FREITAG M MAIER-STOLTE andA N STARRATT 2001 New observations of the secondarychemistry of World Ephedra (Ephedraceae) American Journalof Botany 88 1199ndash1208

CHOUDRY A S 1984 Karyomorphological and cytogenetical stud-ies in Ephedra Journal of Science Hiroshima University Series BDivision 2 19 57ndash109

COOKSON I C 1956 Pollen grains of the Ephedra-type in Austra-lian Tertiary deposits Nature 177 47ndash48

CORTES M C and J H HUNZIKER 1997 Isozymes in Larrea di-varicata and Larrea tridentata (Zygophyllaceae) a study of twoamphitropical viacariants and autopolyploidy Genetica 101115ndash124

CRANE P R 1996 The fossil history of the Gnetales InternationalJournal of Plant Sciences (Suppplement) 157 S50ndashS57

mdashmdashmdash and G R UPCHURCH 1987 Drewria potomacensis gen et spnov an Early Cretaceous member of the Gnetales from thePotomac Group of Virginia American Journal of Botany 741722ndash1736

mdashmdashmdash and J G MAISEY 1991 Fossil plants Pp 414ndash419 in Santanafossils an illustrated atlas ed J G Maisey Neptune City TFHpublications

mdashmdashmdash E M FRIIS and K R PEDERSEN 1995 The origin and earlydiversification of angiosperms Nature 374 27ndash33

DANIN A 1996 Plants of desert dunes Berlin New York SpringerVerlag

DOYLE J A H EKLUND and P S HERENDEEN 2003 Floral evo-lution in Chloranthaceae implications of a morphologicalphylogenetic analysis International Journal of Plant Sciences(Supplement) 164 S365ndashS382

EL-GHAZALY G and J ROWLEY 1997 Pollen wall of Ephedra foliataReview of Palaeobotany and Palynology 21 7ndash18

ERIKSSON O and B BREMER 1991 Fruit characteristics life formsand species richness in the plant family Rubiaceae AmericanNaturalist 138 881ndash900

FELSENSTEIN J 1985 Confidence limits on phylogenies an ap-proach using the bootstrap Evolution 39 783ndash791

mdashmdashmdash 1988 Phylogenies from molecular sequences inferenceand reliability Annual Review of Genetics 22 521ndash566

FREITAG H and M MAIER-STOLTE 1992 A new species and a newcombination in the genus Ephedra from Arabia EdinburghJournal of Botany 49 89ndash93

mdashmdashmdash and mdashmdashmdash 1994 Chorology of trees and shrubs in southwestAsia and adjacent regions Vol X Kornik Polish Academy ofSciences Institute of Dendrology

mdashmdashmdash and mdashmdashmdash 2003 The genus Ephedra in NE Tropical AfricaKew Bulletin 58 415ndash426

GERNANDT D S A LISTON and D PINERO 2001 Variation in thenrDNA ITS of Pinus subsection Cembroides implications formolecular systematic studies of pine species complexes Mo-lecular Phylogenetics and Evolution 21 449ndash467


GRAHAM A 1992 Utilization of the isthmian land bridge duringthe Cenozoic-paleobotanical evidence for timing and the se-lective influence of altitudes and climate Review of Palaeobo-tany and Palynology 72 119ndash128

mdashmdashmdash 1999 Late Cretaceous and Cenozoic history of North Americanvegetation New York and Oxford Oxford University Press

GUO S-X and X-W WO 2000 Ephedripites from latest JurassicYixian Formation in western Liaoning Northeast China ActaPalaeontologica Sinica 39 81ndash91

HASEBE M M ITO R KOFUJI K IWATSUKI and K UEDA 1992Phylogenetic relationships in Gnetophyta deduced from rbcLgene sequences Botanical Magazine Tokyo 105 385ndash391

HERRERA C M 1989 Seed dispersal by animals a role in angio-sperm diversification American Naturalist 133 309ndash322

HESSE M M WEBER and H HALBRITTER 2000 A comparativestudy of the polyplicate pollen types in Arales Laurales Zin-giberales and Gnetales Pp 227ndash239 in Pollen and spores mor-phology and biology eds M M Harley C M Morton and SBlackmore Kew Royal Botanic Gardens

HODAR J A F CAMPOS and B A ROSALES 1996 Trophic ecologyof the ocellated lizard Lacerta lepida in an arid zone of south-ern Spain relationships with availability and daily activity ofprey Journal of Arid Environments 33 95ndash107

HOLMGREN P K N H HOLMGREN and L C BARNETT 1990Index Herbariorum Edition 8 Part I The Herbaria of the WorldBronx New York Botanical Garden Press

HUANG J 2000 Molecular systematics and evolution of the genusEphedra PhD dissertation The University of Georgia Ath-ens Georgia USA

mdashmdashmdash and R A PRICE 2003 Estimation of the age of extant Ephe-dra using rbcL sequence data Molecular Biology and Evolution20 435ndash440

HUELSENBECK J P and B RANNALA 1997 Phylogenetic methodscome of age testing hypotheses in an evolutionary contextScience 276 227ndash232

HUFFORD L 1996 The morphology and evolution of male repro-ductive structures of Gnetales International Journal of PlantSciences (Supplement) 157 S95ndashS112

HUNZIKER J H 1953 Numero de cromosomas de varias especiessudamericanas de Ephedra Revista Argentina de Agronomıa 20141ndash143

mdashmdashmdash 1955 Morphologıa cromosomica de nueve especies argen-tinas del genero Ephedra Revista de Investigaciones Agrıcolas 9201ndash209

mdashmdashmdash 1994 Ephedraceae Pp 15ndash23 in Flora Fanerogamica Argen-tina Cordoba Programa Proflora (Conicet)

ICKERT-BOND S M 1999 Morphology and microgametogenesis inArizona native Joint-fir Ephedra (Ephedraceae) Journal of theArizona-Nevada Academy of Science (Proceedings Supplement) 3410

mdashmdashmdash 2003 Systematics of New World Ephedra L (Ephedraceae)integrating morphological and molecular data PhD disser-tation Arizona State University Tempe Arizona USA

mdashmdashmdash and M F WOJCIECHOWSKI 2002 Timing the evolutionarydivergence of crown group Gnetales integration of molecularand fossil data International symposium (Abstracts) Plantspecies-level systematics patterns processes and new appli-cations Leiden National Herbarium Nederland

JEANMOUGIN F J D THOMPSON M GOUY D G HIGGINS and TJ GIBSON 1998 Multiple sequence alignment with ClustalXTrends in Biochemical Science (TIBS) 23 403ndash405

KELCH D G and B G BALDWIN 2003 Phylogeny and ecologicalradiation of New World thistles (Cirsium Cardueae- Com-positae) based on ITS and ETS rDNA sequence data Molec-ular Ecology 12 141ndash151

KRASSILOV V A 1986 New floral structures from the Lower Cre-taceous of Lake Baikal area Review of Palaeobotany and Paly-nology 47 9ndash16

KUBITZKI K 1990 Ephedraceae Pp 379ndash385 in The families andgenera of vascular plants vol 1 eds K V Kramer and P SGreen Berlin and New York Springer Verlag

LEOPOLD E B and S T CLAY-POOLE 2001 Florissant leaf andpollen floras of Colorado compared climatic implications Pp17ndash69 in Fossil flora and stratigraphy of the Florissant FormationColorado eds E Evanoff K M Gregory-Wodzicki and K RJohnson Denver Proceedings of the Denver Museum of Na-ture and Science Series 4

LI J C C DAVIS M J DONOGHUE S KELLEY and P DEL TREDICI2001 Phylogenetic relationships of Torreya (Taxaceae) inferredfrom sequences of nuclear ribosomal DNA ITS region Har-vard Papers in Botany 6 275ndash281

LIA V V V A CONFALONIERI C I COMAS and J H HUNZIKER2001 Molecular phylogeny of Larrea and its allies (Zygo-phyllaceae) reticulate evolution and the probable time of cre-osote bush arrival to North America Molecular Phylogeneticsand Evolution 21 309ndash320

LISTON A W A ROBINSON J M OLIPHANT and E R ALVAREZ-BUYLLA 1996 Length variation in the nuclear ribosomalDNA internal transcribed spacer region of non-floweringseed plants Systematic Botany 21 109ndash120

MADDISON D R and W P MADDISON 2000 MacClade analysis ofphylogeny and character evolution Sunderland Sinauer Associ-ates

MAGALLON S and M J SANDERSON 2001 Absolute diversifica-tion rates in angiosperm clades Evolution 55 1762ndash1780

MARTENS P 1971 Les gnetophytes Handbuch der Pflanzenanato-mie Band 12 Teil 2 Berlin Stuttgart Gebruder Borntraeger

MATTHEI O 1995 Ephedraceae Pp 328ndash337 in Flora of Chile Vol1 eds C Marticorena and R Rodriguez Concepcion Univ-ersidad de Concepcion

MEYER C A VON 1846 Versuch einer Monographie der GattungEphedra durch Abbildungen erlautert Memoirs de lrsquoacademieimperiale des sciences de Saint-Petersbourg Sixieme serie Sci-ences naturelles 5 225ndash298

MOHR B A R C RYDIN and E M FRIIS 2003 Gnetalean diver-sity during the Early Cretaceous of Brazil Botany 2003httpwww2003botanyconferenceorg Accessed Sept2004

mdashmdashmdash M E BERNARDES-DE-OLIVEIRA A M F BARRETO and MC CASTRO-FERNANDES 2004 Gnetophyte preservation anddiversity in the Early Cretaceous Crato Formation (Brazil)VII International Organization of Paleobotany ConferenceBariloche Argentina Abstract

MUSSAYEV I F 1978 On geography and phylogeny of some rep-resentatives of the genus Ephedra L Botanicheskii Zhurnal 63523ndash543

NADOT S R BAJON and B LEJEUNE 1994 The chloroplast generps4 as a tool for the study of Poaceae Plant Systematics andEvolution 191 27ndash38

NORSTOG K J 1990 Studies of cycad reproduction at FairchildTropical Garden Memoirs of the New York Botanical Garden 5763ndash81

OSBORN J M T N TAYLOR and M R DE LIMA 1993 The ultra-structure of fossil ephedroid pollen with gnetalean affinitiesfrom the Lower Cretaceous of Brazil Review of Palaeobotanyand Palynology 77 171ndash184

PACHOMOVA M G 1969 On the taxonomy of the genus Ephedra(Some comments on the works of U D Soskow and V ANikitin) Botanicheskii Zhurnal 54 697ndash705

mdashmdashmdash 1971 Ephedraceae Pp 25ndash33 in Plants of Central Asia plantcollections from China and Mongolia (Rasteniia tsentralnoi Azii)ed V I Grubov Enfield Science Publishers

PALUMBI S R 1996 Nucleic acids II The polymerase chain reac-tion Pp 205ndash247 in Molecular systematics D M Hillis C Mo-ritz and B K Mable (eds) Sunderland Sinauer Associates

PANT D D and B K VERMA 1974 Taxonomy of the genus Ephe-


dra significance of stem and leaf epidermis and cuticle Bo-tanical Journal of the Linnean Society 69 287ndash308

PEARSON H H W 1929 Gnetales Cambridge Cambridge Uni-versity Press

PORTER D M 1974 Disjunct distribution in the New World Zyg-ophyllaceae Taxon 23 339ndash346

POSADA D and K A CRANDALL 1998 MODELTEST testing themodel of DNA substitution Bioinformatics 14 817ndash818

PRICE R A 1996 Systematics of the Gnetales a review of mor-phological and molecular evidence International Journal ofPlant Sciences (Supplement) 157 S40ndashS49

PRYER K M H SCHNEIDER A R SMITH R CRANFILL P GWOLF J S HUNT and S D SIPES 2001 Horsetails and fernsare a monophyletic group and the closest living relative toseed plants Nature 409 618ndash622

RAVEN P R 1963 Amphitropical relationships in the floras ofNorth and South America The Quarterly Review of Biology 38151ndash177

RIEDL H 1961 Notizen zur Orient-Flora Anzeiger der Osterrei-chischen Akademie der Wissenschaften Mathematisch-naturwis-senschafliche Klasse 98 22ndash28

ROIG F A 1984 Ephedra boelkei (Ephedraceae) nueva especie su-damericana de la seccion Alatae Stapf Parodiana 3 11ndash19

RYDIN C M KALLERSJO and E M FRIIS 2002 Seed plant phy-logenetic relationships and the systematic position of Gne-tales based on nuclear and chloroplast DNA conflicting datarooting problems and the monophyly of conifers Internation-al Journal of Plant Sciences 163 197ndash214

mdashmdashmdash B MOHR and E M FRIIS 2003 Cratonia cotyledon gen etsp nov a unique seedling related to Welwitschia Biology Let-ters The Royal Society of London Supplement 1 S29

mdashmdashmdash P K RAUNSGAARD and E M FRIIS 2004 The evolutionaryhistory of Ephedra evidence from fossils and molecular data(abstract) Botany 2004 Salt Lake City Utah httpwwwbotanyconferenceorgenginesearchindexphp AccessedSept 2004

SANDERSON M J 2002 Estimating absolute rates of molecular evo-lution and divergence times a penalized likelihood ap-proach Molecular Biology and Evolution 19 101ndash109

mdashmdashmdash and J A DOYLE 2001 Sources of error and confidenceintervals in estimating the age of angiosperms from rbcL and18S rDNA data American Journal of Botany 88 1499ndash1516

SAUQUET H J A DOYLE T SCHARASCHKIN T BORSCH K WHILU L W CHATROU and A LE THOMAS 2003 Phylogeneticanalysis of Magnoliales and Myristicaceae based on multipledata sets implications for character evolution Botanical Jour-nal of the Linnean Society 142 125ndash186

SCHULTHEIS L M and B G BALDWIN 1999 Molecular phyloge-netics of Fouquieriaceae evidence from nuclear rDNA ITSstudies American Journal of Botany 86 578ndash589

SCHNEIDER D M WINK F SPORER and P LOUNIBOS 2002 Cy-cads their evolution toxins herbivores and insect pollina-tors Naturwissenschaften 89 281ndash294

SCHNEIDER H E SCHUETTPELZ K M PRYER R CRANFILL S MA-GALLON and R LUPIA 2004 Ferns diversified in the shadowof angiosperms Nature 428 553ndash557

SINCLAIR W T R R MILL M F GARDNER P WOLTZ T JAFFREJ PRESTON M L HOLLINGSWORTH A PONGE and M MOLL-ER 2002 Evolutionary relationships of the New Caledonianheterotrophic conifer Parasitaxus usta (Podocarpaceae) in-ferred from chloroplast trnL-F intronspacer and nuclearrDNA ITS2 sequences Plant Sytematics and Evolution 233 79ndash104

SIMMONS M P and J V FREUDENSTEIN 2003 The effects of in-creasing genetic distance on alignment of and tree construc-tion from rDNA internal transcribed spacer sequences Mo-lecular Phylogenetics and Evolution 26 444ndash451

SMITH J F 2001 High species diversity in fleshy-fruited tropicalunderstory plants The American Naturalist 157 646ndash653

SOLTIS P S D E SOLTIS V SAVOLAINEN P R CRANE and T GBARRACLOUGH 2002 Rate heterogeneity among lineages oftracheophytes integration of molecular and fossil data andevidence for living fossils Proceedings of the National Academyof Sciences USA 99 4430ndash4435

SOSKOV U D 1968 Three lines of development within the sectionEphedra of the genus Ephedra L in the flora of the USSRBotanicheskii Zhurnal 53 85ndash91 (In Russian)

STAPF O 1889 Die Arten der Gattung Ephedra Denkschrift der Kais-erlichen Akademie der Wissensschaften Mathematisch-Naturwis-senschaftliche Klasse 56 1ndash112

STEEVES M W and E S BARGHOORN 1959 The pollen of EphedraJournal of the Arnold Arboretum 11 221ndash255

SWOFFORD D L G J OLSEN P J WADDELL and D M HILLIS1996 Phylogenetic inference Pp 407ndash514 in Molecular system-atics eds Hillis D M C Moritz and B K Mable Sunder-land Sinauer Associates

mdashmdashmdash 2003 PAUP Phylogenetic analysis using parsimony (andother methods) Version 4 Sunderland Sinauer Associates

TAKASO T 1984 Structural changes in the apex of the female stro-bilus and the initiation of the female reproductive organ(ovule) in Ephedra distachya L and E equisetina Bge Acta Bo-tanica Neerlandica 33 257ndash266

THORNE R A 1973 Major disjunctions in the geographic rangesof seed plants The Quarterly Review of Biology 47 365ndash411

mdashmdashmdash 1986 A historical sketch of the vegetation of the Mojaveand Colorado deserts of the American Southwest Annals ofthe Missouri Botanical Garden 73 642ndash651

THULIN M 1994 Aspects of disjunct distributions and endemismin the arid parts of the horn of Africa particularly SomaliaPp 1105ndash1119 in Proceedings of the XIIIth Plenary Meeting As-sociation pour lrsquoEtude taxonomique de la Flore drsquoAfrique Tropicale(AETFAT) eds J H Seyani and A C Chikuni ZombaNational Herbarium and Botanic Gardens of Malawi

TIFFNEY B H and S J MAZER 1995 Angiosperm growth habitdispersal and diversification reconsidered Evolutionary Ecol-ogy 9 93ndash117

WEN J P P LOWRY J L WALCK and K O YOO 2002 Phyloge-netic and biogeographic diversification in Osmorhiza (Api-aceae) Annals of the Missouri Botanical Garden 3 414ndash428

WODEHOUSE R P 1933 The Tertiary pollen-II the oil shales ofthe Green River formation Bulletin of the Torrey Botanical Club60 479ndash535

WON H and S RENNER 2003 Horizontal gene transfer from flow-ering plants to Gnetum Proceedings of the National Academy ofSciences USA 100 10824ndash10829

ZHANG L-B and S RENNER 2003 The deepest splits in the Clor-anthaceae as resolved by chloroplast sequences InternationalJournal of Plant Sciences (Supplement) 164 S383ndashS392

APPENDIX 1List of taxa and their voucher information for nrDNA ITS1 and

plastid rps4 sequences Letters in parentheses refer to different ac-cessions used in the molecular analysis All vouchers are depos-ited in the ASU herbarium unless otherwise noted in parenthesesacronyms follow Index Herbariorum (Holmgren et al 1990) c 5cultivated Infrageneric groups follow Stapf (1889) Mussayev(1978) and Freitag and Maier-Stolte (1994)

Sect Alatae subsect Alatae (Stapf) Mussayev E przewalskii(Stapf) Andrz Mongolia Gobi Altai Hurka amp Neuffer 12228 (KAS)rps4 AY591484 no ITS1 Pakistan Eberhardt 98ndash743 (KAS) rps4AY591483 no ITS1 E strobilacea Bunge Iran Rechinger 27161(US) rps4 AY591448 ITS1 AY599162

Sect Alatae subsect Trifurcae Mussayev E boelkei F A RoigArgentina Mendoza Ickert-Bond 1252 rps4 AY591473 ITS1AY599175 E funerea Coville and C V Morton (1) USA Arizona


Ickert-Bond 473 rps4 AY591454 ITS1 AY599168 (2) USA Califor-nia Ickert-Bond 964 no rps4 ITS1 AY599170 E multiflora Phil exStapf Chile Atacama Desert Ickert-Bond 1211 rps4 AY591471 ITS1AY599173 E torreyana S Watson (1) USA Arizona Ickert-Bond941 no rps4 ITS1 AY599155 (2) USA Arizona Crimmins sn norps4 ITS1 AY599166 E torreyana S Watson var powelliorum TWendt USA Texas Big Bend Ickert-Bond 1126 rps4 AY591453ITS1 AY599147 E trifurca Torr ex S Watson USA Arizona Ick-ert-Bond 753 no rps4 ITS1 AY599164

Sect Ephedra subsect Americanae Mussayev E americanaHumb amp Bonpl ex Willd (1) Ecuador Bolivar Ickert-Bond 1105rps4 AY591464 ITS1 AY599143 (2) Argentina Salta Ickert-Bond1219 no rps4 ITS1 AY599178 E breana Phil Chile El Loa Ickert-Bond 1234 rps4 AY591472 E chilensis Miers Chile Valparaiso Ick-ert-Bond 1240 rps4 AY591470 ITS1 AY599142 E compacta Rose(1) Mexico Nuevo Leon Puente 2238 no rps4 ITS1 AY599163 (2)Mexico Puebla-Veracruz Puente 2243B no rps4 ITS1 AY599161(3) Mexico San Luis Potosi Puente 1901 rps4 AY591474 ITS1AY599157 E coryi E L Reed (1) USA Texas Ickert-Bond 952rps4 AY591461 ITS1 AY599153 (2) USA Texas Ickert-Bond 953no rps4 ITS1 AY599151 E frustillata Miers Chile MagallanesIckert-Bond 1247 rps4 AY591462 ITS1 AY599174 E gracilis PhilChile Valparaiso Ickert-Bond 1201 rps4 AY591465 ITS1 AY599150E ochreata Miers Argentina Mendoza B 380819 (B) rps4AY591463 ITS1 AY599176 E pedunculata Engelm ex S WatsonUSA Texas Ickert-Bond 920 rps4 AY591460 ITS1 AY599144 Erupestris Benth (1) Ecuador Cotopaxi Ickert-Bond 1100 rps4AY591467 ITS1 AY599167 (2) Argentina Salta Ickert- Bond1220rps4 AY591466 ITS1 AY599171 E triandra Tul emend J H HunzArgentina Salta Ickert-Bond 1227 rps4 AY591468 ITS1 AY599165E tweediana Fisch and CA Mey emend J H Hunz ArgentinaSalta Ickert-Bond 1225 rps4 AY591469 ITS1 AY599159

Sect Ephedra subsect Antisyphilticae Mussayev E antisyphil-itica S Watson USA Texas Puente 2314 no rps4 ITS1 AY599152USA Texas Ickert-Bond 900 rps4 AY591452 ITS1 AY599148

Group Distachyae subgroup Distachyae s str Freitag andMaier-Stolte E distachya L (1) Syria NE of Damascus Freitag30144 (KAS) rps4 AY591441 ITS1 AY599133 (2) Rumania Buca-rest Diaconescu 30205 (B) rps4 AY591481 no ITS1 (3) S Kazakhs-tan coast of Aral lake Wucherer 453 (KAS) no rps4 ITS1AY599135 (4) Turkey Ronya Freitag 28772 (KAS) no rps4 ITS1AY599134 E distachya L subsp helvetica (CA Mey) Aschersonamp Graebner (1) Switzerland Freitag 20332 (KAS) no rps4 ITS1AY599136 (2) Switzerland B37921 (B) rps4 AY591480 no ITS1 Efedtschenkoae Pauls Arrowhead Alpines Nursery (c) Ickert-Bondsn rps4 AY591442 ITS1 AY59915 E intermedia C A Mey Ka-zakhstan Mosqua B 37886 (B) no rps4 ITS1 AY599179 E rege-liana Florin China SW Xinjiang U Wundisch 956 (KAS) rps4AY591449 ITS1 AY599160

Group Distachyae subgroup Leptocladae Freitag and Maier-Stolte E equisetina Bunge Kazakhstan Freitag sn (KAS) no rps4no ITS1 E gerardiana Wall Pakistan Kaghan Valley Miehe 4717(KAS) rps4 AY591482 no ITS1 E major Host subsp major Tur-key WSW Eregli Freitag amp Adiguzel 28780 (KAS) rps4 AY591489ITS1 E major Host subsp procera (Fischer amp C A Mey) Bornm(1) Turkey Van-Golo Freitag 202 (KAS) rps4 AY591487 no ITS1(2) Iran Baghrot Valley Gamp S Miehe 4526 (KAS) rps4 AY591488no ITS1 E monosperma C A Mey (2) Mongolia Gobi Altai Hur-ka amp Neuffer 12182 (KAS) rps4 AY591443 ITS1 AY599139 E sax-atilis (Stapf) Florin China SE Tibet Miehe 95ndash18ndash11 (KAS) rps4AY591445 ITS1 AY599140 China Xizang S Tibet Miehe 98ndash00505

(KAS) rps4 AY591491 no ITS1 E saxatilis (Stapf) Florin var sik-kimensis (Stapf) Florin China Xizang S Tibet Dichore 6535 (KAS)rps4 AY591490 no ITS1 E somalensis Freitag and Maier-StolteSomalia Sanaag Region Thulin 10944 (KAS) rps4 AY591444 ITS1AY599141

Group Sarcocarpae Freitag and Maier-Stolte E transitoria As-sadi Jordan Shaumari Wildlife Res Freitag 30123 (KAS) rps4AY591450 ITS1 AY599172 E sarcocarpa Aitch and Hemsl IranKavir desert near Mobarakiyeh Freitag 13966 (KAS) no rps4 ITS1AY599137

Group Fragilis Freitag and Maier-Stolte E altissima Desf varalgerica Stapf Montreal Botanical Garden (c) JBM2830ndash41 rps4AY591479 no ITS1 E aphylla Forssk (1) Jordan Wadi Musa Frei-tag 30181 (KAS) rps4 AY591438 ITS1 AY599128 (2) Italy Sicilynear Manfredia Freitag F14a 01 (KAS) rps4 AY591439 ITS1AY599127 E foeminea Forssk Greece Delphi Freitag 190801(KAS) rps4 AY591478 ITS1 AY599131 E fragilis Desf (1) ItalySicily Freitag 18001 (KAS) rps4 AY591477 no ITS1 (2) JordanWadi Musa Freitag 30180 (KAS) rps4 AY591440 ITS1 AY599129(3) Italy Sicily near Manfredia Freitag F14 01 (KAS) rps4AY591476 no ITS1 (4) Morocco Walters 675 (KAS) rps4 AY591475no ITS1 (5) SE Spain Freitag 27237 (KAS) no rps4 ITS1 AY599130E laristanica Assadi Iran Fars Assadi amp Sardabi 41781 (KAS) rps4AY591437 ITS1 AY599126

Subsect Pachycladae E pachyclada Boiss var sinaica (Riedl)H Freitag and M Maier-Stolte Sinai S Musa Freitag 19960 (KAS)no rps4 ITS1 AY599138 E sinica Florin Mongolia Gobi Altai Hur-ka amp Neuffer 11937 (KAS) rps4 AY591446 no ITS1 Companionplants (c) Ickert-Bond sn (ASU) rps4 AY591486 no ITS1 E sinicaFlorin var pumila Mongolia Hovd Aymag Hurka amp Neuffer 10242(KAS) rps4 AY591447 ITS1 AY599169

Sect Asarca E aspera Engelm ex S Watson (2) USA TexasIckert-Bond 1136a rps4 AY591455 ITS1 AY599146 E californica SWatson USA California Ickert-Bond 968 rps4 AY591458 ITS1AY599145 E cutleri Peebles USA Arizona Ickert-Bond 1006 rps4AY591456 ITS1 AY599156 E fasciculata A Nelson USA Ari-zona Ickert-Bond 513 rps4 AY591457 ITS1 AY599180 E nevadensisS Watson USA Nevada Ickert-Bond 959 rps4 AY591451 ITS1AY599154 E viridis Coville USA Arizona Ickert-Bond 941 rps4AY591459 ITS1 AY599149

No sectional affiliation E sp nov lsquolsquoBhutanicarsquorsquo Bhutan TsochenChen Miehe 00ndash124ndash02 (KAS) rps4 AY591492 no ITS1

Gnetum L G cuspidatum Bl Malaysia Won 551 (MO) rps4AY5914 no ITS1 G gnemon L Montreal Botanical Garden (c)JBM2240ndash50 rps4 AY591428 no ITS1 G gnemonoides Brongn Ma-laysia Won 553 (MO) rps4 AY591429 ITS1 AY624586 G indicumMerrill Royal Botanical Garden Edinburgh (c) E19550226 (E) rps4AY591434 ITS1 AY624585 G leptostachyum Bl Malaysia San157112 (MO) rps4 AY591435 no ITS1 G leyboldii Tul Costa RicaLa Selva Landrum sn rps4 AY591432 no ITS1 G lufoenseCYCheng Singapore Won 600 (MO) rps4 AY591436 ITS1AY624584 G montanum Mgf Royal Botanical Garden Edinburgh(c) E19791010 (E) rps4 AY591433 no ITS1 G parvifolium (Warb)Cheng University of Tokyo Botanical Garden (c) Kato sn rps4AY591431 no ITS1 G ula Brogn University of California Botan-ical Garden (c) Ickert-Bond sn rps4 AY591427 no ITS1

Pinus thunbergii Parl No locality or voucher rps4 D17510 ITS1AF037025

Welwitschia mirabilis Hooker f Hunt Botanical Garden (c)Cranfill sn rps4 AY188246 no ITS1 California State UniversityFullerton (c) no voucher no rps4 ITS1 U50740


The present study seeks to increase our knowledgeof diversity and evolution in the genus Ephedra by pre-senting hypotheses for infrageneric relationships usingDNA sequence data from both the chloroplast and nu-clear genomes This study endeavors to 1) test themonophyly of Ephedra 2) provide a preliminary test ofthe validity of the traditional taxonomic divisions intosections Alatae Asarca and Ephedra (Stapf 1889 Mus-sayev 1978 Freitag and Maier-Stolte 1994) 3) enhanceour understanding of infrageneric relationships amongspecies of Ephedra and 4) provide a preliminary testof the biogeographical diversification of the genusCharacter evolution is discussed as it relates to the re-sulting phylogeny

MATERIALS AND METHODS

Taxon Sampling A total of 71 taxa were sampled in this study(Appendix 1) including 51 species from all lineages of Ephedra asdefined by Stapf (1889) two recently described South Americanspecies (Hunziker 1994 Matthei 1995) and two recently describedOld World species (Assadi 1996 Freitag and Maier-Stolte 2003)Multiple accessions of some species were used to confirm ques-tionable identifications Sampling of the New World species in-cluded all known taxa except for Ephedra trifurcata Zoellner Thisspecies is only known from the type specimen collected in 1978and could not be relocated by the first author while visiting thetype locality (Marga Marga Chile) in 2001 Selection of Old Worldspecies was based on consultation with M Maier-Stolte and HFreitag University of Kassel Germany who are currently revisingthose taxa (Freitag and Maier-Stolte 1992 1994 2003) Outgrouptaxa include Gnetum Welwitschia Hooker f and the more distantlyrelated Pinus L All new sequences have been deposited in Gen-bank (httpwwwncbinlmnihgov) and the final data matricesof both the nrDNA ITS1 and the plastid rps4 gene have been de-posited in TreeBASE (study accession number S1144 matrix ac-cession numbers M1966ndashM1967)

DNA Extraction and PCR Amplification Fresh or silica-driedmaterial was available for most taxa but herbarium material wasused for a few samples Genomic DNAs were isolated using DNAextraction kits (QIAGEN Corporation Alameda California) DNAsequences were amplified by polymerase chain reaction (Palumbi1996) on a DNA engine Dyad (MJ Research Waltham Massachu-setts) with the following conditions initial denaturation (928C 2min) followed by 40 cycles of denaturation (928C 45 sec) an-nealing (558C 30 sec) and extension (728C 30 sec) and concludingwith a final extension (728C 7 min) The PCR amplifications wereperformed in 25 ml reactions containing 10ndash100 ng genomic DNA02 mM deoxyribonucleotide triphosphates (equimolar) 05 unitsPlatinum Taq polymerase (Invitrogen) oligonucleotide primers at05 mM and Mg21 at 15 mM Glycerol was substituted with 5DMSO to relax the secondary structure of ITS (Liston et al 1996)

PCR amplification of the internal transcribed spacer region ofthe nuclear ribosomal DNA (nrDNA ITS1) used the primer ITS5and 58SR (Liston et al 1996) and a pair of internal primers de-signed specifically for Ephedra (ITS1-Ep1S 59-GGACGGTCTTT-GACCAGTTTATA-39 ITS1-Ep2R 59- GCGACGTAGGAAAGGAAATAG-39 modified from Huang 2000) Amplification yielded asingle product of ca 1200 nucleotides for Ephedra whereas se-quences from outgroup taxa varied greatly in length ranging from400 to 900 bp Amplification of the ca 700 bp plastid gene rps4used the primers trnSR2 (59-GCTTACCGGGGTTCGAATC-39) andrps5F (59-ATGTTCCCGTTATCGAGGACCT-39) designed by RCranfill University of California Berkeley This sequence includesca 30 bp of the intergenic spacer between the 39 end of the rps4gene and the trnS gene Most taxa were amplified according toreaction conditions given as above but an annealing temperatureof 558C and 40 amplification cycles were used

Sequencing Purified PCR products (QIAquicky PCR purifi-cation kit QIAGEN) were sequenced for complimentary strandsSequencing reactions employed the same primers as PCR for theplastid rps4 gene Because the nrDNA ITS1 region in Ephedra spansca 1200 bp both the external PCR primers and the additionalpair of internal primers (ITS1-Ep1S and ITS1-Ep2R) were used togenerate sequences across the full length of nrDNA ITS1 in bothdirections PCR products were cycle-sequenced using fluorescentdye-labeled chemistry (Big Dyes Perkin-Elmer Foster City Cali-fornia) Separation and identification of cycle-sequence productswas conducted on an ABI automated 377XL sequencer at the Ar-izona State University DNA Laboratory

Alignment and Analysis Sequences were assembled into con-tigs using Sequencher version 41 (Gene Codes Corporation AnnArbor Michigan) Sequence alignments were initially made inClustalX (Jeanmougin et al 1998) and edited manually The plas-tid gene rps4 codes for a protein product and is easily alignableby eye The nrDNA ITS1 sequences are somewhat more difficultto align due to dramatic length variation (ranging from 400 to1200 bp) and high sequence divergence between Ephedra and allknown outgroups

PAUP 40 beta10 (Swofford 2003) was used to reconstruct phy-logenetic relationships All characters were unordered and weight-ed equally Parsimony analyses were conducted using heuristicsearch methods and employed all addition sequence options (SIM-PLE CLOSEST RANDOM addition sequences) in combinationwith tree-bisection reconnection (TBR) branch-swapping and theMULTREES option which saves all minimal trees with MAX-TREES set to 5000 Fourteen indels (insertionsdeletions) in thenrDNA ITS1 data set (varying from one to 25 base positions inlength for a total of 72 characters) and were routinely excludedfrom all analyses However the indels were also coded as separatebinary characters and added at the end of the data set and ana-lyzed to determine whether they were phylogenetically informa-tive For all data sets support for individual tree branches wasestimated using non-parametric bootstrap methods (Felsenstein1985) Bootstrap proportions (BP) were obtained from 100 repli-cates of heuristic searches as described above (1000 random ad-dition sequences TBR branch swapping and MULTREES select-ed)

The monophyly of previously proposed taxonomic groupings(sects Ephedra Alatae and Asarca) was investigated by comparingtrees consistent with topological constraints to trees obtained fromunconstrained analyses Trees with topological constraints wereconstructed using MacClade 403 (Maddison and Maddison 2000)and loaded into PAUP Heuristic searches were then conductedto find the shortest trees consistent with each imposed constraint

Maximum likelihood (ML) analyses were conducted with thenrDNA ITS1 dataset using PAUP to compare with results fromparsimony analyses A single tree derived from parsimony orneighbor joining analysis was used as the starting tree for a heu-ristic search (addition sequence lsquolsquoASISrsquorsquo) with TBR branch swap-ping and MULTREES in effect Base frequencies were empiricallydetermined and the transition transversion (K) ratio and gammashape parameter (G) were estimated on tree number 6 (of 198 mostparsimonious trees) from parsimony analysis (3283000 and00535576 respectively) and the tree derived from neighbor join-ing (348352 and 0073199 respectively) under the HKY85 modelof sequence evolution chosen using results from ModelTest ver-sion 306 (Posada and Crandall 1998) Rate heterogeneity acrosssites followed a gamma distribution with four categories Pairwisedistances were calculated across all sites as lsquouncorrected prsquo valuesusing PAUP

Rooting followed the outgroup method for the rps4 data butwas problematic within the nrDNA ITS1 sequences due to dra-matic length differences between sequences of the outgroup andingroup Multiple analyses were undertaken both with and with-out inclusion of outgroups One approach to deal with large lengthvariation and sequence divergence between the outgroup and theingroup was proposed by Simmons and Freudenstein (2003) thatutilizes ClustalX (Jeanmougin et al 1998) and was employed hereIn ClustalX blocks of 30 invariant bases were added to the se-







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1848267219424613200991952073803420987168


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34155815347947943744041138073234




RESULTS













DISCUSSION


















































LITERATURE CITED




































































































































321448556536323276675754323555968010324299145664325154758526

92487065712905004134635891494994416661169


326065509676326536480176326873771349327193190747327317757967

1848267219424613200991952073803420987168


328140952837328532565718328990698269329251888925329612217738

226335582341678424333049248554325576088


331193671356331414447345332035824580332421645634332917170745333683603110

287389952918054730423302311949443218599433718859


333902081343334221571065335544379305335860790784

34155815347947943744041138073234




RESULTS













DISCUSSION


















































LITERATURE CITED









































































































































DISCUSSION


















































LITERATURE CITED










































































































































































LITERATURE CITED






























































































































phylogenetic relationships in ephedra (gnetales): evidence ... · phylogenetic relationships in...

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