419

Systematic Botany (2004) 29(2) pp 419ndash431q Copyright 2004 by the American Society of Plant Taxonomists

An ITS-Based Phylogenetic Analysis of the Perennial Endemic ApiaceaeSubfamily Apioideae of Western North America

FENG-JIE SUN1 STEPHEN R DOWNIE13 and RONALD L HARTMAN2

1Department of Plant Biology University of Illinois at Urbana-Champaign Urbana Illinois 618012Department of Botany University of Wyoming PO Box 3165 Laramie Wyoming 82071

3Author for Correspondence (sdownielifeuiucedu)

Communicating Editor John V Freudenstein

ABSTRACT Phylogenetic analyses of 159 DNA sequences from the nuclear rDNA internal transcribed spacer region wereconducted to evaluate the monophyly of the herbaceous perennial genera of Apiaceae subfamily Apioideae endemic toNorth America (north of Mexico) and to determine the relationships of those elements that currently comprise Cymopteruswithin the group The results of a previous phylogenetic study were equivocal in suggesting monophyly for these perennialendemic taxa and revealed Cymopterus to be polyphyletic with its species closely linked with those of Aletes LomatiumMusineon Oreoxis Orogenia Podistera Pseudocymopterus Pteryxia and Tauschia Herein we expand sampling to include com-prehensive representation of Aletes Cymopterus Musineon Oreoxis Orogenia Podistera Pseudocymopterus and Pteryxia andgreater representation of Lomatium and Tauschia We also include all members of two genera not examined previously Glehniaand Oreonana as well as additional outgroup genera from the Angelica clade of the apioid superclade Our results indicatethat the perennial endemic apioid umbellifers of North America constitute a (weakly supported) monophyletic group withAngelica and the meso-American Arracacia clade comprising two of several possible sister groups The two subspecies ofGlehnia littoralis ally with Angelica and Peucedanum japonicum Oreonana shows afnity with several species of Cymopterus andLomatium The lack of resolution in the ITS trees precludes unambiguous hypotheses of relationship among these perennialendemic umbellifers but does show that many of these genera where resolved are not monophyletic Indeed Cymopterusand Lomatium are highly polyphyletic and permeate all major clades resolved in the molecule-derived trees Evidence frombranch lengths and low sequence divergence suggests that this group of North American umbellifers underwent rapidradiation likely during the geoclimatic events of the Late Tertiary and Quaternary

Cymopterus Raf (Apiaceae subfamily Apioideae)the spring-parsleys comprises some 35 species (Kar-tesz 1994) of herbaceous perennials distributed largelythroughout western North America (NA) Approxi-mately two-thirds of these species are restricted to theGreat Basin region roughly bounded by the Snake Riv-er of Idaho Sierra Nevada of California Colorado Riv-er of Arizona and the Rocky Mountains of Colorado(Mathias 1965) Many species are xerophytic or semi-xerophytic being conned to dry sandy or alkalinehabitats They are low in stature possess thickeneddeep taproots and have ternately cleft compound ordissected leaves The inorescence is an open com-pound umbel and the owers are mostly yellow orwhite opening from very early in the spring tothroughout the summer Most distinctive however istheir fruit for the two marginal (lateral) and usuallyone or more of the three dorsal ribs are conspicuouslywinged These wings may be membranous to spongy-corky and variously plane to wavy or corrugated In-deed the generic name is derived from the Greekkyma a wave and pteron a wing referring to the oftenundulate wings of the fruit

Much confusion exists with regard to the properdelimitation of Cymopterus and its relationship to theother indigenous umbellifers of western NA (whoseranges extend from southern Canada to northern Mex-ico) These genera include Aletes JM Coult amp RoseGlehnia F Schmidt ex Miq Harbouria JM Coult ampRose Lomatium Raf Musineon Raf Neoparrya Mathias

Oreonana Jeps Oreoxis Raf Orogenia S Watson Podis-tera S Watson Pseudocymopterus JM Coult amp RosePteryxia (Nutt ex Torr amp A Gray) JM Coult amp Roseand Shoshonea Evert amp Constance (Mathias 1930 Evertand Constance 1982 Downie et al 2002) All are pe-rennial taprooted herbs and many are similar to Cy-mopterus in their primarily low-growing and acaules-cent or subcaulescent habit Lomatium is the largest ge-nus with 78 species Glehnia Harbouria Neoparrya andShoshonea are monospecic and the remainder com-prise some 2ndash6 species each (Kartesz 1994)Consideredcollectively these plants present such a confusing in-tergradation of similar characteristics that generic de-limitation is made exceedingly difcult and in the ab-sence of mature fruits many species are essentially in-distinguishable (Jones 1908 Mathias 1930 Theobald etal 1963 Weber 1984 Cronquist 1997 Downie et al2002) Cronquist (1997) included Oreoxis Pseudocymop-terus and Pteryxia within a broadly circumscribed Cy-mopterus (Hitchcock and Cronquist 1961 Cronquist1997) Cronquist (1997) also stated that the distinctionbetween Cymopterus and Lomatium ie the conspicu-ously winged dorsal fruit ribs of the former lsquolsquois subjectto failurersquorsquo Phylogenetic studies of these endemic andlargely cordilleran genera are few and have focusedalmost exclusively upon Lomatium (Schlessman 1984Simmons 1985 Gilmartin and Simmons 1987 Soltis etal 1995 Soltis and Novak 1997 Hardig and Soltis1999)

Results from a previous phylogenetic study of 85

420 [Volume 29SYSTEMATIC BOTANY

species of NA apioid umbellifers revealed that Cymop-terus (17 examined species) and Lomatium (28 exam-ined species) are each polyphyletic with their speciesintertwined with those of Aletes Musineon Oreoxis Or-ogenia Podistera Pseudocymopterus Pteryxia and Taus-chia Schltdl (Downie et al 2002) Closely allied werethree genera of central to eastern NA distribution [Po-lytaenia DC Taenidia (Torr amp A Gray) Drude andThaspium Nutt] and Zizia WDJ Koch the latter dis-tributed widely across NA Our observations of maturefruit cross-sections as well as cladistic analysis of mor-phological characters conrmed that those character-istics of the fruit used traditionally to diagnose genera(such as wing shape and composition and the orien-tation of mericarp compression) are indeed highly var-iable thus severely limiting their usefulness in phylo-genetic estimation The results of Downie et al (2002)veried the close relationship among these genera butwere surprising in suggesting an evolutionary historyof the group much more complicated than previouslyconsidered

The restricted distribution of many endemic west-ern NA apioid genera to elevated regions of similarhabitat their similar life history and overall generalhabit and overlapping patterns of fruit variation sug-gest that this group of umbellifers may be closely re-lated An obsolete stylopodium in all genera save Pod-istera where the stylopodium is well developed as itis in most other umbellifers (Mathias and Constance1944ndash1945) is a synapomorphy uniting the group(Downie et al 2002) Further support for their mono-phyly comes from the shared presence of a protogyn-ous breeding system (and associated reproductive andecologic characteristics) atypical in a family where o-ral protandry prevails (Lindsey and Bell 1980 Lindsey1982 Barrie and Schlessman 1987 Schlessman et al1990 Schlessman and Graceffa 2002) Protogyny ispresumed derived in apioid umbellifers a response toan early owering season and unreliable pollinators(Schlessman and Graceffa 2002) However the resultsof our prior study of the group were equivocal in con-rming the monophyly of these taxa endemic to NAowing to the lack of resolution obtained at the base ofthe molecular phylogenies (Downie et al 2002)

In this paper we augment our study of Cymopterusphylogeny by expanded sampling within the genusand we consider additional material from genera thatwere poorly represented in our initial study (ie Ale-tes Lomatium Podistera Pteryxia and Tauschia) We alsoinclude representation of two genera previously un-available to us Glehnia and Oreonana but whose mor-phologies suggest afnities to Cymopterus and Tauschiarespectively (Mathias 1928 1930 Shevock and Con-stance 1979) Once more we focus our attention on theendemic perennial apioid genera of western NA andas outgroups we include additional representation

from the Angelica clade of the apioid superclade(Downie et al 2001)mdashspecically the perennial cir-cumboreal genera Seseli L Selinum L and PeucedanumL Our major objectives are to evaluate the monophylyof the perennial endemic genera of NA (north of Mex-ico) and to ascertain the phylogenetic relationships ofthose elements that currently comprise Cymopteruswithin this group We continue to utilize the nuclearrDNA internal transcribed spacer (ITS) region for phy-logenetic inference given the wealth of ITS sequencedata already accumulated for the Apiaceae for com-parative purposes their faster substitution rates com-pared to loci from the chloroplast genome (Downie etal 2001) and their utility in resolving relationshipsamong congeneric species of Apiaceae (Soltis and Ku-zoff 1993 Downie and Katz-Downie 1996 Vargas et al1999)

MATERIALS AND METHODS

Taxon Sampling Complete ITS-1 and ITS-2 sequences for 74accessions of Apiaceae subfamily Apioideae are reported here forthe rst time (Table 1) these were combined with 85 previouslypublished ITS sequences (Downie et al 2002) for a total of 159accessions

Kartesz (1994) whose checklist of Apiaceae was inuenced bythe work of Lincoln Constance recognized 35 species of Cymop-terus in NA Two species have infrageneric taxa C acaulis has vevarieties C panamintensis has two With the exception of the nar-rowly endemic and rarely collected C megacephalus ME Jones wesampled all taxa of Cymopterus We also included the recently de-scribed C constancei (Hartman 2000) and C longilobus the latterplaced in synonymy under Pteryxia hendersonii by Kartesz (1994)but maintained as a distinct species of Cymopterus by R Hartman(unpubl data) Following the checklist of Kartesz (1994) we sam-pled all seven recognized taxa of Aletes (plus the Mexican A cal-cicola Mathias and Constance 1981) both subspecies of Glehnialittoralis and all seven taxa of Pteryxia (P terebinthina var calcareais maintained as distinct from P terebinthina var albiora based onGoodrich 1986) All species of Musineon Oreonana Oreoxis Oro-genia Podistera Polytaenia and Pseudocymopterus were also includ-ed as were 30 accessions of Lomatium and ve species of Tauschia

As outgroups we included representation of the perennial cir-cumboreal genus Angelica and the meso-American Arracacia clade(ie Arracacia Prionosciadium and Rhodosciadium Downie et al2001) Mathias (1965) described Cymopterus as being derived fromlsquolsquoAngelica-like stockrsquorsquo which became adapted to drier habitats Shealso noted similarities between a fossil umbelliferous fruit from aMiocene formation in Washington State and present-day Rhodos-ciadium from Mexico (Berry 1929 as cited by Mathias 1965) Re-sults from our prior phylogenetic analysis showed that these gen-era are included alongside Cymopterus in a large polytomous clade(Downie et al 2002) We also considered members of the circum-boreal genera Seseli Selinum and Peucedanum for each of thesegenera has representatives occurring in NA The western NA ge-nus Sphenosciadium (which eventually may be transferred into An-gelica Katz-Downie et al 1999) was also included as were the NAgenera Spermolepis and Ciclospermum Aethusa cynapium was usedto root all trees based on the results of previous higher-level in-vestigations where this species was sister taxon to all aforemen-tioned taxa (Downie et al 2000c 2002) The 22 outgroup repre-sentatives included herein are all provisionally treated in the An-gelica and Arracacia clades of the apioid superclade (Plunkett andDownie 1999 Downie et al 2001)

Experimental Strategy Details of the DNA extractions PCRamplications and DNA sequencing are provided elsewhere(Downie and Katz-Downie 1996 Downie et al 2000a) Total ge-

2004] 421SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

nomic DNA was obtained from ca 20 mg of dried leaf tissueusing the DNeasy Plant Mini Kit (Qiagen Inc Valencia CA) andPCR-amplied using primers lsquolsquoITS4rsquorsquo and lsquolsquoITS5rsquorsquo (White et al1990) Cycle sequencing reactions using these same primers wereperformed on the puried PCR products using AmpliTaq DNApolymerase and uorescent dye-labeled terminators (ABI PrismBigDye Terminator vers 30 Ready Reaction Cycle Sequencing KitApplied Biosystems Foster City CA) Sequencing products wereresolved by electrophoresis using Applied Biosystemrsquos 373A DNAsequencer Both ITS spacer regions were sequenced in their entire-ty on both DNA strands All ITS-1 and ITS-2 data have been de-posited in GenBank (Table 1) and the data matrix is available inTreeBASE (study accession number S1010 matrix accession num-ber M1706)

Data Analysis DNA sequences fromboth spacer regions werealigned using CLUSTAL X (Jeanmougin et al 1998) and adjustedmanually as necessary Sequence data from the invariant 58S sub-unit were excluded as they were unavailable for several previouslypublished taxa owing to the manual sequencing methods used toobtain these earlier data Uncorrected pairwise nucleotide differ-ences (lsquolsquoprsquorsquo) were determined using PAUP vers 40 (Swofford1998) as they are commonly provided in other angiosperm ITSstudies (Baldwin et al 1995) To facilitate analysis taxa bearingidentical sequences were combined (ie two accessions of Cymop-terus constancei two accessions of Oreoxis trotteri three accessionsof Pteryxia terebinthina var albiora three accessions of P terebin-thina var calcarea two of ve accessions of Aletes macdougalii subspbreviradiatus and the three remaining accessions of A macdougaliisubsp breviradiatus) for a nal tally of 150 terminals for the cla-distic analyses Fewer than 03 of all data matrix cells werescored as missing data the sequences with missing data were ob-tained from GenBank and submitted by others

The resulting data matrix was rst analyzed using equally-weighted maximum parsimony (MP) with gap states treated asmissing data Alignment gaps were not scored as separate pres-ence-absence characters because of ambiguous length mutations inseveral previously submitted Lomatium sequences as a result ofcompressions MP trees were sought using the heuristic searchstrategies of PAUP and the inverse-constraint approach describedin Downie et al (1998) The maximum number of MP trees wasset at 20000 and these trees were permitted to swap to comple-tion Bootstrap values (Felsenstein 1985) were calculated from100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-addition of taxaonly those values compatible with the 50 majority-rule consen-sus tree were recorded Estimates of support obtained using thismethod are generally similar to those obtained from bootstrapanalyses with branch swapping particularly for well-supportednodes (Mort et al 2000) The number of additional steps requiredto force particular taxa into a monophyletic group was examinedusing the constraint option of PAUP The g1 statistic of skewedtree-length distribution was calculated from 10000 random par-simony trees generated by PAUP and was used to assess theamount of nonrandom structure in the data (Hillis and Huelsen-beck 1992)

A maximum likelihood (ML) analysis of these ITS data was thenperformed Prior to this analysis the program Modeltest vers 306(Posada and Crandall 1998) was used to select an evolutionarymodel of nucleotide substitution that best ts these data Usingthe hierarchical likelihood ratio test statistic Modeltest selected theTrN1G (Tamura and Nei 1993) model (base frequencies 02725A 02154 C 02312 G 02809 T estimates of substitution ratesAlaquoC 1 AlaquoG 22734 AlaquoT 1 ClaquoG 1 ClaquoT 45457 GlaquoT 1proportion of invariable sites 5 0 gamma distribution shape pa-rameter 5 05930) These settings were entered into PAUP and aheuristic search carried out using ten random addition sequencereplicates and TBR branch-swapping under ML optimization Abootstrap analysis was conducted using the neighbor-joiningmethod and the ML parameters estimated by Modeltest Bootstrapvalues were calculated from 100 replicate analyses

Owing to its computational efciency with large data sets aphylogenetic analysis was also performed using MrBayes vers201 (Huelsenbeck and Ronquist 2001) a Bayesian inference pro-

gram Starting trees were chosen at random and one million gen-erations were run with sampling occurring every 100 generationsTree topologies were based on the general time reversible (GTR)model of maximum likelihood (with the shape parameter of thegamma distribution inferred by Modeltest) and four simultaneousMarkov Chain Monte Carlo chains to model nucleotide rate het-erogeneity Two-thousand trees were discarded as lsquolsquoburn-inrsquorsquo priorto calculating the 50 majority-rule tree Numbers on internalbranches of the tree indicate the percent of time the clade occursamong the sampled trees (ie the posterior probability of thatclade existing Huelsenbeck and Ronquist 2001)

RESULTS

Sequence Analysis Alignment of 150 ITS-1 andITS-2 sequences (representing 159 accessions) resultedin a matrix of 458 positions with none excluded be-cause of alignment ambiguity Characteristics of thesealigned sequences as separate spacer regions or com-bined are presented in Table 2 Thirty-seven gapsranging between 1 and 6 bp in size were introducedto facilitate alignment 33 of these were 1-bp in lengthtwo were 2-bp in length one was 4-bp in length andone was 6-bp in length Uncorrected pairwise sequencedivergence values across both spacers and all taxaranged from identity to 121 of nucleotides Pairwisesequence divergence values for the 126 ingroup ter-minal taxa ranged from identity to 94 of nucleotides(the latter between Cymopterus basalticus and Tauschiatexana) Pairwise distances within Cymopterus (n541)ranged from identity to 73 distances within Loma-tium (n530) ranged from identity to approximately80 Percent G 1 C content over both spacers rangedbetween 542 and 599 (mean 5 571) The g1 sta-tistic for 10000 random trees was20220 This value issignicantly more skewed (ie more negative) thanrandom data (g1 5 2009 for 250 variable positionsand 25 or more taxa P 001) indicating that theseITS data contain signicant amounts of phylogeneticsignal (Hillis and Huelsenbeck 1992) No evidence ofobvious ITS length variants representative of multiplerDNA repeat types was observed

Phylogenetic Analysis MP analysis of 150 ITS-1and ITS-2 sequences (159 accessions) resulted in thepreset limit of 20000 minimal length trees each of 881steps (consistency indices of 04654 and 03762 withand without uninformative characters respectivelyand a retention index of 06405) The strict consensusof these trees is presented in Fig 1 Bootstrap esti-mates were generally low with few values exceeding85

The consensus tree revealed a monophyletic assem-blage of endemic perennial NA genera although theclade was very weakly supported (with 50 boot-strap value) Included in this group are 126 terminals(reecting 135 accessions) represented by 18 generaAletes Cymopterus Harbouria Lomatium Musineon Neo-parrya Oreonana Oreoxis Orogenia Podistera PolytaeniaPseudocymopterus Pteryxia Shoshonea Taenidia Tauschia

422 [Volume 29SYSTEMATIC BOTANY

TABLE 1 Voucher information and GenBank accession numbers for those accessions of Apiaceae subfamily Apioideae examined fornuclear rDNA ITS sequence variation Equivalent information for those taxa considered in a previous phylogenetic study is presentedin Downie et al (2002)

Aletes calcicola Mathias amp ConstancemdashMexico Coahuila Sierra de San Marcos opposite Los Fresnos 4ndash5 Apr 1969 Pinkavaet al 6117 (UC) ITS-1 AY146818 ITS-2 AY146884 A lifolius Mathias Constance amp WL TheobmdashUSA Texas Culberson CoHunter Peak at Pine Top Guadalupe Mountains National Park 6 Sep 1987 Higgins 17453 (BRY) ITS-1 AY146819 ITS-2AY146885 A macdougalii JM Coult amp Rose WL subsp breviradiatus Theob amp CC TsengmdashUSA Utah San Juan Co Manti-LaSal National Forest Notch Canyon jct of Cottonwood Wash 5 Jun 1985 Atwood 11143 (BRY) ITS-1 AY146820 ITS-2 AY146886USA New Mexico San Juan Co Chaco Culture Nat Hist Park top of Chacra Mesa 2 May 1993 Heil 7374 (UNM) ITS-1AY146821 ITS-2 AY146887 USA Utah Gareld Co Dixie National Forest Death Hollow 28 May 1989 Franklin 6499 (BRY)ITS-1 AY146822 ITS-2 AY146888 USA New Mexico San Juan Co NM 575 015 mi NNW of US 64 NW of Blanco 10 Jul1999 Sun amp Hartman 649 (ILL) ITS-1 AY146823 ITS-2 AY146889 A macdougalii subsp macdougaliimdashUSA Arizona CoconinoCo Grand Canyon Kaibab Trail to Roaring Springs 23 Jun 1933 Eastwood amp Howell 986 (UC) ITS-1 AY146824 ITS-2 AY146890

Angelica grayi (JM Coult amp Rose) JM Coult amp RosemdashUSA Colorado Gareld Co Blair Mountain 17 mi NNW ofGlenwood Springs 24 Aug 1991 Vanderhorst amp Palaci 4490 (RM) ITS-1 AY146825 ITS-2 AY146891

Cymopterus aboriginum ME JonesmdashUSA California Mono Co ca 5 mi E of Benton along Hwy 6 21 Jun 1990 Atwood13838 (BRY) ITS-1 AY146826 ITS-2 AY146892 C acaulis (Pursh) Raf var greeleyorum JW Grimes amp PL PackardmdashUSAOregon Malheur Co McBride Creek Road 24 May 1989 Smithman et al LS-2045 (CIC) ITS-1 AY146827 ITS-2 AY146893 Cacaulis var higginsii (SL Welsh) S GoodrichmdashUSA Utah Kane Co E of Coyote Wash 8 Apr 1993 Chapman sn (BRY) ITS-1 AY146828 ITS-2 AY146894 C acaulis var parvus S GoodrichmdashUSA Utah Tooele Co Indian Rice Grass Duneeld NWof English village 7 Jun 1993 Johnson 351 (BRY) ITS-1 AY146829 ITS-2 AY146895 C beckii SL Welsh amp S GoodrichmdashUSAUtah San Juan Co SW of Abajo Mountains S of the Causeway 10 Jul 1992 Franklin 7564 (BRY) ITS-1 AY146830 ITS-2AY146896 C cinerarius A GraymdashUSA California Mono Co Sweetwater Canyon 15 Jul 1944 Alexander amp Kellogg 3893 (UTC)ITS-1 AY146831 ITS-2 AY146897 C constancei RL HartmmdashUSA Colorado Montrose Co San Miguel and lower DoloresRiver drainages Simbad Valley 30 May 1995 Hartman 50943 (RM) ITS-1 AY146832 ITS-2 AY146898 USA Wyoming SubletteCo Upper Green River Plain Cretaceous Mountain 19 May 1993 Hartman 37182 (RM) ITS-1 AY146833 ITS-2 AY146899 Ccorrugatus ME JonesmdashUSA Nevada Mineral Co Walker River Indian Reservation Rawhide Flats 15 May 1988 Tiehm 11640(BRY) ITS-1 AY146834 ITS-2 AY146900 C coulteri (ME Jones) MathiasmdashUSA Utah Sevier Co Triangle Mt Soldiers Canyon15 May 1990 Thorne amp Zupan 7377 (BRY) ITS-1 AY146835 ITS-2 AY146901 C davisii RL HartmmdashUSA Idaho Cassia CoN slope of Mt Harrison 30 Jul 1983 Cronquist 11840 (UTC) ITS-1 AY146836 ITS-2 AY146902 C deserticola BrandegeendashUSACalifornia San Bernardino Co 1 mi S of Kramer 21 Mar 1958 Raven 11891 (UTC) ITS-1 AY146837 ITS-2 AY146903 Cdouglassii RL Hartm amp ConstancemdashUSA Idaho Custer Co 2 mi N of Horseheaven Pass and just W of Spring Hill VABMPahsimeroi Mountains Challis National Forest 26 Jun 1984 Atwood 10302 (BRY) ITS-1 AY146838 ITS-2 AY146904 C gilmaniiC MortonmdashUSA Nevada Clark Co Desert Range SE base Desert National Wildlife Refuge 73 mi from Sheep Peak summit17 May 1987 Moreeld et al 4463 (BRY) ITS-1 AY146839 ITS-2 AY146905 C glaucus NuttmdashUSA Idaho Valley Co ridgebetween Fisher Creek Saddle and Black Tip along IdahoValley Co line 26 Jul 1989 Ertter et al 8742 (RM) ITS-1 AY146840ITS-2 AY146906 C goodrichii SL Welsh amp NeesemdashUSA Nevada Lander Co Toiyabe Range 2 mi N of Bunker Hill 16 Jul1981 Neese et al 10737 (BRY) ITS-1 AY146841 ITS-2 AY146907 C lapidosus (ME Jones) ME JonesmdashUSA Wyoming UintaCo Uinta Co Road 290 37 air mi W of Lonetree 7 Jul 1983 Hartman 15767 (UTC) ITS-1 AY146842 ITS-2 AY146908 Clongilobus (Rydb) WA WebermdashUSA Wyoming Sublette Co Wyoming Range 15 Aug 1993 Hartman 43986 (RM) ITS-1AF358482 ITS-2 AF358549 C macrorhizus BuckleymdashUSA Texas Tom Green Co Grandview campground on W side of FisherLake 19 Mar 1983 Ertter amp Strachan 4690 (RM) ITS-1 AY146843 ITS-2 AY146909 C minimus (Mathias) MathiasmdashUSA UtahIron Co Cedar Breaks National Monument just below N rim where rst reached by Rattlesnake Trail 25 Jul 1993 Stone 1656(BRY) ITS-1 AY146844 ITS-2 AY146910 C newberryi (S Watson) ME JonesmdashUSA Utah San Juan Co 45 mi downstreamfrom Bluff 20 May 1983 Barneby 17896 (BRY) ITS-1 AY146845 ITS-2 AY146911 C panamintensis JM Coult amp Rose varacutifolius (JM Coult amp Rose) MunzmdashUSA California San Bernardino Co south side of Ord Mtn north of Lucerne Valley8 Apr 1988 Boyd et al 1776 (OSC) ITS-1 AY146846 ITS-2 AY146912 C ripleyi BarnebymdashUSA Nevada Nye Co Stone CabinValley 24 May 1995 Holmgren amp Holmgren 12299 (BRY) ITS-1 AY146847 ITS-2 AY146913 C rosei (ME Jones ex JM Coult ampRose) ME JonesmdashUSA Utah Sanpete Co Hills W of Ninemile Reservoir 8 May 1987 Franklin 4604 (BRY) ITS-1 AY146848ITS-2 AY146914

Glehnia littoralis F Schmidt ex Miq subsp littoralismdashTaiwan Taitung Hsien Lanyu (Orchid Island) Liao et al 1235 (MO)ITS-1 AY146849 ITS-2 AY146915 G littoralis subsp leiocarpa (Mathias) HultenmdashUSA Oregon Tillamook Co by the beachon the south side of Cape Kiwanda 10 Aug 1975 Halse 1228 (OSC) ITS-1 AY146850 ITS-2 AY146916

Lomatium ambiguum (Nutt) JM Coult amp RosemdashUSA Montana Madison Co ca 15 mi from roadrsquos end on Bear TrapCanyon Trail 30 May 1979 Lowry 1867 (ILL) ITS-1 AY146851 ITS-2 AY146917 L bradshawii (Rose ex Mathias) Mathias ampConstancemdashUSA Oregon Benton Co NE Corvallis W of Canterbury Circle area around Frazier Creek 8 Jun 1980 Halse 2215(ILL) ITS-1 AY146852 ITS-2 AY146918 L brandegei (JM Coult amp Rose) JF MacbrmdashUSA Washington Chelan Co 5 mi SWof Leavenworth on Snow Creek Trail 29 May 1977 Lowry 621 (ILL) ITS-1 AY146853 ITS-2 AY146919 L grayi (JM Coult ampRose) JM Coult amp Rose var grayimdashUSA Wyoming Lincoln Co Tunp Range Preacher Hollow 16 mi NNE of Cokeville 22May 1993 Hartman 37444 (RM) ITS-1 AY146854 ITS-2 AY146920 L grayi var depauperatum (ME Jones) MathiasmdashUSA UtahJuab Co Gilson Mountains 7 mi from Lynndyl 7 May 1981 Goodrich 15335 (RM) ITS-1 AY146855 ITS-2 AY146921

Musineon lineare (Rydb) MathiasmdashUSA Utah Cache Co Toney Grove Trailhead Trail to Naomi Peak 9 Jul 1989 Hartman24364 (RM) ITS-1 AY146856 ITS-2 AY146922

Oreonana clementis (ME Jones) JepsmdashUSA California Tulare Co Mt Guyot 22 Jul 1949 Howell 25604 (UTC) ITS-1AY146857 ITS-2 AY146923 O purpurascens Shevock amp ConstancemdashUSA California Tulare Co Sequoia National Forest SlateMountain along USFS trail 31E14 26 Apr 1977 Shevock 5428 (BRY) ITS-1 AY146858 ITS-2 AY146924 O vestita (S Watson)JepsmdashUSA California San Bernardino Co Angeles National Forest San Gabriel Mtns between Mt Harwood and Mt SanAntonio Peak 4 May 1977 Shevock 5433 (BRY) ITS-1 AY146859 ITS-2 AY146925

2004] 423SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 1 Continued

Oreoxis trotteri SL Welsh amp S GoodrichmdashUSA Utah Grand Co E of Courthouse Rock 20 May 1985 Welsh 23427 (BRY)ITS-1 AY146860 ITS-2 AY146926 USA Utah Grand Co 135 mi NNW of Moab 4 Jun 1985 Neese amp Welsh 16984 (BRY) ITS-1 AY146861 ITS-2 AY146927

Orogenia fusiformis S WatsonmdashUSA Oregon Josephine Co ats near S boundary with Siskiyou NF Oregon Caves NationalMonument 24 May 1996 Zika 12864 (OSC) ITS-1 AY146862 ITS-2 AY146928

Peucedanum japonicum Thunb ex A Murraymdashcult Botanical Conservatory Mulhouse France (no 97138) 8 Dec 1999 Reduronsn (Herb Reduron) ITS-1 AF495826 ITS-2 AF495827 P ostruthium (L) WDJ Koch (5Imperatoria ostruthium L)mdashDownie etal (1998) ITS-1 U78403 ITS-2 U78463

Podistera macounii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Alaska ridge W of Sheep Creek 30 Jun 1977 Reed etal 6047 (BRY) ITS-1 AY146863 ITS-2 AY146929 USA Alaska Mountains between Essie Creek and the headwaters of Seven-tymile Drainage 21 Jun 2002 Larsen et al sn (ALA) ITS-1 AY146864 ITS-2 AY146930 P nevadensis (A Gray) S WatsonmdashUSA California El Dorado Co Lake Tahoe Basin Management Unit Freel Peak 27 Jul 2002 Matson 634 (ILL) ITS-1 AY146865ITS-2 AY146931 P yukonensis Mathias amp ConstancemdashUSA Alaska Kathul Mountain along the Yukon River roughly halfwaybetween Eagle and Circle 11 Jun 2002 Larsen amp Batten 02-1263 (ALA) ITS-1 AY146866 ITS-2 AY146932

Polytaenia texana (JM Coult amp Rose) Mathias amp ConstancemdashUSA Texas Burnet Co E of Briggs 25 May 1985 Barrie 1403(RM) ITS-1 AY146867 ITS-2 AY146933

Pseudocymopterus longiradiatus Mathias Constance amp WL TheobmdashUSA New Mexico Otero Co Sacramento Mts ca 3mi SSW of Bent SW side of Domingo Peak 7 Aug 1990 Sivinski 1546 (UNM) ITS-1 AY146868 ITS-2 AY146934

Pteryxia davidsonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA New Mexico Grant Co Bear Mountain near SilverCity 17 Jun 1903 Metcalfe 165 (RM) ITS-1 AY146869 ITS-2 AY146935 P hendersonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Utah Duchesne Co 075 mi SE of Davis Lake Uinta Mtns 23 Aug 1996 Huber 3502 (BRY) ITS-1 AY146870 ITS-2AY146936 P petraea (ME Jones) JM Coult amp RosemdashUSA Nevada Nye Co Toiyabe National Forest Toquima Range IronSprings 5 Jun 1978 Goodrich 11292 (UTC) ITS-1 AY146871 ITS-2 AY146937 P terebinthina (Hook) JM Coult amp Rose varterebinthinamdashUSA Washington Benton Co Pit 30 between the 200 area 17 May 1993 McKinnon amp Sackschewsky 327 (BRY)ITS-1 AY146872 ITS-2 AY146938 P terebinthina var albiora (Nutt ex Torr amp A Gray) MathiasmdashUSA Wyoming SubletteCo Upper Green River Basin ca 95 air mi NNW of La Barge 20 May 1993 Hartman 37374 (RM) ITS-1 AY146873 ITS-2AY146939 USA Utah Daggett Co Flaming Gorge National Recreation area 5 Jun 1995 Refsdal amp Goodrich 3591 (RM) ITS-1AY146874 ITS-2 AY146940 P terebinthina var calcarea (ME Jones) MathiasmdashUSA Wyoming Uinta Co 275 mi N of Lone-tree 2 Jul 1999 Goodrich 26060 (BRY) ITS-1 AY146875 ITS-2 AY146941 USA Utah Daggett Co Ashley National Forest FlamingGorge National Recreation area E side of Flaming Gorge Reservoir 26 May 1992 Goodrich 24045 (BRY) ITS-1 AY146876 ITS-2 AY146942 USA Wyoming Big Horn Co Big Horn Mountains Cold Spring Road 26 May 1980 Hartman amp Dueholm 11224(RM) ITS-1 AY146877 ITS-2 AY146943 USA Wyoming Carbon Co ca 7 air mi NE of Savery 14 Jun 1979 Hartman amp Coffey8961 (RM) ITS-1 AY146878 ITS-2 AY146944 P terebinthina var californica (JM Coult amp Rose) MathiasmdashUSA CaliforniaMadera Co near Whiskey Creek 1 mi NW of Ellis Meadow Sierra Nevada 1 Jul 1938 Constance 2390 (RM) ITS-1 AY146879ITS-2 AY146945 P terebinthina var foeniculacea (Nutt ex Torr amp A Gray) MathiasmdashUSA Idaho Custer Co Morgan Creek10 mi N of Challis 26 Jun 1984 Atwood 10320 (RM) ITS-1 AY146880 ITS-2 AY146946

Selinum candollei DCmdashDownie and Katz-Downie (1996) ITS-1 U30564 ITS-2 U30565 S carvifolia (L) LmdashFrance Bas-Rhin between Herbsheim and Boofzheim 14 Aug 2001 Reduron sn (Herb Reduron) ITS-1 AY179028 ITS-2 AY179028

Seseli gracile Waldst amp KitmdashKatz-Downie et al (1999) ITS-1 AF008605 ITS-2 AF009084 S libanotis (L) WDJ KochmdashKatz-Downie et al (1999) ITS-1 AF008603 ITS-2 AF009082 S tortuosum LmdashPortugal Lisboa Sintra Praja das Macas cult BotanicalConservatory Mulhouse France (no 98042) 2 Aug 2001 Hildenbrand et al sn (Herb Reduron) ITS-1 AY179031 ITS-2 AY179031

Tauschia arguta (Torr amp A Gray) JF MacbrmdashUSA California San Diego Co Corte Madera Ranch near Pine Valley 22May 1983 Van der Werff amp Clark 4571 (MO) ITS-1 AY146881 ITS-2 AY146947 T glauca (JM Coult amp Rose) Mathias ampConstancemdashUSA California Trinity Co SE of Burnt Ranch 11 July 1990 Spellenberg 10254 (RM) ITS-1 AY146882 ITS-2AY146948 T kelloggii (A Gray) JF MacbrmdashUSA California Butte Co SW of intersection of Black Bart Road and ForbestownRoad about 35 air mi SW of Forgestown 16 Jun 1993 Ahart 6972 (UC) ITS-1 AY146883 ITS-2 AY146949

TABLE 2 Sequence characteristics of the two internal transcribed spacer (ITS) regions separately and combined for 150 terminals(159 accessions) of Apiaceae subfamily Apioideae

Sequence characteristic ITS-1 ITS-2Combined

(ITS-1 amp ITS-2)

Length variationNo of aligned positionsNo of aligned positions constantNo of aligned positions parsimony-informativeNo of aligned positions autapomorphicNo of unambiguous alignment gapsNo of unambiguous gaps parsimony-informativeMaximum pairwise sequence divergence ()

206ndash2182259189452111125

220ndash226233937961165

131

427ndash4414581841681063716121

424 [Volume 29SYSTEMATIC BOTANY

FIG 1 Strict consensus of 20000 minimal length 881-steptrees derived from equally-weighted maximum parsimonyanalysis of aligned ITS-1 and ITS-2 sequences from 150 ter-minals (159 accessions) of Apiaceae subfamily ApioideaeConsistency indices 5 04654 and 03762 with and without

not

uninformative characters respectively retention index506405 Numbers on branches represent bootstrap estimatesfor 100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-additionvalues 50 are not indicated All Cymopterus accessions areboldfaced Numbers in parentheses refer to numbers of ac-cessions bearing identical DNA sequences that were combinedto facilitate analysis

Thaspium and Zizia A large basal polytomy obscuresrelationships among these umbellifers yet four majorclades containing six or more taxa each are apparentThe rst and most distal of these clades comprises 37taxa representing 13 accessions of Cymopterus 16 ac-cessions of Lomatium 3 accessions each of Oreonanaand Pteryxia and 2 accessions of Orogenia A secondclade comprises all ve accessions of Cymopterus acau-lis plus C bulbosus C newberryi and Lomatium concin-num A third clade comprises some but not all infra-specic accessions of Pteryxia terebinthina plus Pteryxiapetraea Cymopterus corrugatus C coulteri and C ibapen-sis The fourth major clade contains Cymopterus con-stancei C macrorhizus C montanus C multinervatus Cpurpurascens and Aletes calcicola Several additionalclades of four to ve taxa each exist and are also ofheterogeneous generic composition

The results of the MP analysis revealed that Cymop-terus (boldfaced in all tree gures presented herein)and Lomatium the two largest genera of NA Apiaceaeare highly polyphyletic and that their species are in-extricably linked with those of the other endemic pe-rennial genera of the region In addition the generaAletes Musineon Oreonana Oreoxis Orogenia PodisteraPseudocymopterus Pteryxia and Tauschia are also notmonophyletic Constraining the 41 examined taxa ofCymopterus to monophyly and rerunning the MP anal-ysis resulted in trees 35 steps longer than those mostparsimonious (Table 3) Lomatium is monophyletic intrees 45 steps longer than those most parsimoniousSimilar analyses were performed by constraining eachof the other aforementioned genera with Aletes Glehn-ia Oreoxis Pteryxia and Tauschia each monophyletic intrees 10 or more steps greater than those most parsi-monious (Table 3) In contrast Polytaenia Thaspiumand Zizia are all monophyletic in unconstrainedsearches

The remaining 24 accessions examined formed a po-lytomy basal to this clade and included a monophy-letic Angelica with the inclusion of Sphenosciadium andPacic NA Glehnia littoralis subsp leiocarpa the cladeof Selinum candollei Seseli Peucedanum japonicum andPacic Asian Glehnia littoralis subsp littoralis the lattertwo taxa forming a strongly-supported sister group(97 bootstrap value) the Arracacia clade (ArracaciaPrionosciadium and Rhodosciadium) the clade of Ciclo-spermum 1 Spermolepis and Peucedanum ostruthium Se-

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

420 [Volume 29SYSTEMATIC BOTANY

species of NA apioid umbellifers revealed that Cymop-terus (17 examined species) and Lomatium (28 exam-ined species) are each polyphyletic with their speciesintertwined with those of Aletes Musineon Oreoxis Or-ogenia Podistera Pseudocymopterus Pteryxia and Taus-chia Schltdl (Downie et al 2002) Closely allied werethree genera of central to eastern NA distribution [Po-lytaenia DC Taenidia (Torr amp A Gray) Drude andThaspium Nutt] and Zizia WDJ Koch the latter dis-tributed widely across NA Our observations of maturefruit cross-sections as well as cladistic analysis of mor-phological characters conrmed that those character-istics of the fruit used traditionally to diagnose genera(such as wing shape and composition and the orien-tation of mericarp compression) are indeed highly var-iable thus severely limiting their usefulness in phylo-genetic estimation The results of Downie et al (2002)veried the close relationship among these genera butwere surprising in suggesting an evolutionary historyof the group much more complicated than previouslyconsidered

The restricted distribution of many endemic west-ern NA apioid genera to elevated regions of similarhabitat their similar life history and overall generalhabit and overlapping patterns of fruit variation sug-gest that this group of umbellifers may be closely re-lated An obsolete stylopodium in all genera save Pod-istera where the stylopodium is well developed as itis in most other umbellifers (Mathias and Constance1944ndash1945) is a synapomorphy uniting the group(Downie et al 2002) Further support for their mono-phyly comes from the shared presence of a protogyn-ous breeding system (and associated reproductive andecologic characteristics) atypical in a family where o-ral protandry prevails (Lindsey and Bell 1980 Lindsey1982 Barrie and Schlessman 1987 Schlessman et al1990 Schlessman and Graceffa 2002) Protogyny ispresumed derived in apioid umbellifers a response toan early owering season and unreliable pollinators(Schlessman and Graceffa 2002) However the resultsof our prior study of the group were equivocal in con-rming the monophyly of these taxa endemic to NAowing to the lack of resolution obtained at the base ofthe molecular phylogenies (Downie et al 2002)

In this paper we augment our study of Cymopterusphylogeny by expanded sampling within the genusand we consider additional material from genera thatwere poorly represented in our initial study (ie Ale-tes Lomatium Podistera Pteryxia and Tauschia) We alsoinclude representation of two genera previously un-available to us Glehnia and Oreonana but whose mor-phologies suggest afnities to Cymopterus and Tauschiarespectively (Mathias 1928 1930 Shevock and Con-stance 1979) Once more we focus our attention on theendemic perennial apioid genera of western NA andas outgroups we include additional representation

from the Angelica clade of the apioid superclade(Downie et al 2001)mdashspecically the perennial cir-cumboreal genera Seseli L Selinum L and PeucedanumL Our major objectives are to evaluate the monophylyof the perennial endemic genera of NA (north of Mex-ico) and to ascertain the phylogenetic relationships ofthose elements that currently comprise Cymopteruswithin this group We continue to utilize the nuclearrDNA internal transcribed spacer (ITS) region for phy-logenetic inference given the wealth of ITS sequencedata already accumulated for the Apiaceae for com-parative purposes their faster substitution rates com-pared to loci from the chloroplast genome (Downie etal 2001) and their utility in resolving relationshipsamong congeneric species of Apiaceae (Soltis and Ku-zoff 1993 Downie and Katz-Downie 1996 Vargas et al1999)

MATERIALS AND METHODS

Taxon Sampling Complete ITS-1 and ITS-2 sequences for 74accessions of Apiaceae subfamily Apioideae are reported here forthe rst time (Table 1) these were combined with 85 previouslypublished ITS sequences (Downie et al 2002) for a total of 159accessions

Kartesz (1994) whose checklist of Apiaceae was inuenced bythe work of Lincoln Constance recognized 35 species of Cymop-terus in NA Two species have infrageneric taxa C acaulis has vevarieties C panamintensis has two With the exception of the nar-rowly endemic and rarely collected C megacephalus ME Jones wesampled all taxa of Cymopterus We also included the recently de-scribed C constancei (Hartman 2000) and C longilobus the latterplaced in synonymy under Pteryxia hendersonii by Kartesz (1994)but maintained as a distinct species of Cymopterus by R Hartman(unpubl data) Following the checklist of Kartesz (1994) we sam-pled all seven recognized taxa of Aletes (plus the Mexican A cal-cicola Mathias and Constance 1981) both subspecies of Glehnialittoralis and all seven taxa of Pteryxia (P terebinthina var calcareais maintained as distinct from P terebinthina var albiora based onGoodrich 1986) All species of Musineon Oreonana Oreoxis Oro-genia Podistera Polytaenia and Pseudocymopterus were also includ-ed as were 30 accessions of Lomatium and ve species of Tauschia

As outgroups we included representation of the perennial cir-cumboreal genus Angelica and the meso-American Arracacia clade(ie Arracacia Prionosciadium and Rhodosciadium Downie et al2001) Mathias (1965) described Cymopterus as being derived fromlsquolsquoAngelica-like stockrsquorsquo which became adapted to drier habitats Shealso noted similarities between a fossil umbelliferous fruit from aMiocene formation in Washington State and present-day Rhodos-ciadium from Mexico (Berry 1929 as cited by Mathias 1965) Re-sults from our prior phylogenetic analysis showed that these gen-era are included alongside Cymopterus in a large polytomous clade(Downie et al 2002) We also considered members of the circum-boreal genera Seseli Selinum and Peucedanum for each of thesegenera has representatives occurring in NA The western NA ge-nus Sphenosciadium (which eventually may be transferred into An-gelica Katz-Downie et al 1999) was also included as were the NAgenera Spermolepis and Ciclospermum Aethusa cynapium was usedto root all trees based on the results of previous higher-level in-vestigations where this species was sister taxon to all aforemen-tioned taxa (Downie et al 2000c 2002) The 22 outgroup repre-sentatives included herein are all provisionally treated in the An-gelica and Arracacia clades of the apioid superclade (Plunkett andDownie 1999 Downie et al 2001)

Experimental Strategy Details of the DNA extractions PCRamplications and DNA sequencing are provided elsewhere(Downie and Katz-Downie 1996 Downie et al 2000a) Total ge-

2004] 421SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

nomic DNA was obtained from ca 20 mg of dried leaf tissueusing the DNeasy Plant Mini Kit (Qiagen Inc Valencia CA) andPCR-amplied using primers lsquolsquoITS4rsquorsquo and lsquolsquoITS5rsquorsquo (White et al1990) Cycle sequencing reactions using these same primers wereperformed on the puried PCR products using AmpliTaq DNApolymerase and uorescent dye-labeled terminators (ABI PrismBigDye Terminator vers 30 Ready Reaction Cycle Sequencing KitApplied Biosystems Foster City CA) Sequencing products wereresolved by electrophoresis using Applied Biosystemrsquos 373A DNAsequencer Both ITS spacer regions were sequenced in their entire-ty on both DNA strands All ITS-1 and ITS-2 data have been de-posited in GenBank (Table 1) and the data matrix is available inTreeBASE (study accession number S1010 matrix accession num-ber M1706)

Data Analysis DNA sequences fromboth spacer regions werealigned using CLUSTAL X (Jeanmougin et al 1998) and adjustedmanually as necessary Sequence data from the invariant 58S sub-unit were excluded as they were unavailable for several previouslypublished taxa owing to the manual sequencing methods used toobtain these earlier data Uncorrected pairwise nucleotide differ-ences (lsquolsquoprsquorsquo) were determined using PAUP vers 40 (Swofford1998) as they are commonly provided in other angiosperm ITSstudies (Baldwin et al 1995) To facilitate analysis taxa bearingidentical sequences were combined (ie two accessions of Cymop-terus constancei two accessions of Oreoxis trotteri three accessionsof Pteryxia terebinthina var albiora three accessions of P terebin-thina var calcarea two of ve accessions of Aletes macdougalii subspbreviradiatus and the three remaining accessions of A macdougaliisubsp breviradiatus) for a nal tally of 150 terminals for the cla-distic analyses Fewer than 03 of all data matrix cells werescored as missing data the sequences with missing data were ob-tained from GenBank and submitted by others

The resulting data matrix was rst analyzed using equally-weighted maximum parsimony (MP) with gap states treated asmissing data Alignment gaps were not scored as separate pres-ence-absence characters because of ambiguous length mutations inseveral previously submitted Lomatium sequences as a result ofcompressions MP trees were sought using the heuristic searchstrategies of PAUP and the inverse-constraint approach describedin Downie et al (1998) The maximum number of MP trees wasset at 20000 and these trees were permitted to swap to comple-tion Bootstrap values (Felsenstein 1985) were calculated from100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-addition of taxaonly those values compatible with the 50 majority-rule consen-sus tree were recorded Estimates of support obtained using thismethod are generally similar to those obtained from bootstrapanalyses with branch swapping particularly for well-supportednodes (Mort et al 2000) The number of additional steps requiredto force particular taxa into a monophyletic group was examinedusing the constraint option of PAUP The g1 statistic of skewedtree-length distribution was calculated from 10000 random par-simony trees generated by PAUP and was used to assess theamount of nonrandom structure in the data (Hillis and Huelsen-beck 1992)

A maximum likelihood (ML) analysis of these ITS data was thenperformed Prior to this analysis the program Modeltest vers 306(Posada and Crandall 1998) was used to select an evolutionarymodel of nucleotide substitution that best ts these data Usingthe hierarchical likelihood ratio test statistic Modeltest selected theTrN1G (Tamura and Nei 1993) model (base frequencies 02725A 02154 C 02312 G 02809 T estimates of substitution ratesAlaquoC 1 AlaquoG 22734 AlaquoT 1 ClaquoG 1 ClaquoT 45457 GlaquoT 1proportion of invariable sites 5 0 gamma distribution shape pa-rameter 5 05930) These settings were entered into PAUP and aheuristic search carried out using ten random addition sequencereplicates and TBR branch-swapping under ML optimization Abootstrap analysis was conducted using the neighbor-joiningmethod and the ML parameters estimated by Modeltest Bootstrapvalues were calculated from 100 replicate analyses

Owing to its computational efciency with large data sets aphylogenetic analysis was also performed using MrBayes vers201 (Huelsenbeck and Ronquist 2001) a Bayesian inference pro-

gram Starting trees were chosen at random and one million gen-erations were run with sampling occurring every 100 generationsTree topologies were based on the general time reversible (GTR)model of maximum likelihood (with the shape parameter of thegamma distribution inferred by Modeltest) and four simultaneousMarkov Chain Monte Carlo chains to model nucleotide rate het-erogeneity Two-thousand trees were discarded as lsquolsquoburn-inrsquorsquo priorto calculating the 50 majority-rule tree Numbers on internalbranches of the tree indicate the percent of time the clade occursamong the sampled trees (ie the posterior probability of thatclade existing Huelsenbeck and Ronquist 2001)

RESULTS

Sequence Analysis Alignment of 150 ITS-1 andITS-2 sequences (representing 159 accessions) resultedin a matrix of 458 positions with none excluded be-cause of alignment ambiguity Characteristics of thesealigned sequences as separate spacer regions or com-bined are presented in Table 2 Thirty-seven gapsranging between 1 and 6 bp in size were introducedto facilitate alignment 33 of these were 1-bp in lengthtwo were 2-bp in length one was 4-bp in length andone was 6-bp in length Uncorrected pairwise sequencedivergence values across both spacers and all taxaranged from identity to 121 of nucleotides Pairwisesequence divergence values for the 126 ingroup ter-minal taxa ranged from identity to 94 of nucleotides(the latter between Cymopterus basalticus and Tauschiatexana) Pairwise distances within Cymopterus (n541)ranged from identity to 73 distances within Loma-tium (n530) ranged from identity to approximately80 Percent G 1 C content over both spacers rangedbetween 542 and 599 (mean 5 571) The g1 sta-tistic for 10000 random trees was20220 This value issignicantly more skewed (ie more negative) thanrandom data (g1 5 2009 for 250 variable positionsand 25 or more taxa P 001) indicating that theseITS data contain signicant amounts of phylogeneticsignal (Hillis and Huelsenbeck 1992) No evidence ofobvious ITS length variants representative of multiplerDNA repeat types was observed

Phylogenetic Analysis MP analysis of 150 ITS-1and ITS-2 sequences (159 accessions) resulted in thepreset limit of 20000 minimal length trees each of 881steps (consistency indices of 04654 and 03762 withand without uninformative characters respectivelyand a retention index of 06405) The strict consensusof these trees is presented in Fig 1 Bootstrap esti-mates were generally low with few values exceeding85

The consensus tree revealed a monophyletic assem-blage of endemic perennial NA genera although theclade was very weakly supported (with 50 boot-strap value) Included in this group are 126 terminals(reecting 135 accessions) represented by 18 generaAletes Cymopterus Harbouria Lomatium Musineon Neo-parrya Oreonana Oreoxis Orogenia Podistera PolytaeniaPseudocymopterus Pteryxia Shoshonea Taenidia Tauschia

422 [Volume 29SYSTEMATIC BOTANY

TABLE 1 Voucher information and GenBank accession numbers for those accessions of Apiaceae subfamily Apioideae examined fornuclear rDNA ITS sequence variation Equivalent information for those taxa considered in a previous phylogenetic study is presentedin Downie et al (2002)

Aletes calcicola Mathias amp ConstancemdashMexico Coahuila Sierra de San Marcos opposite Los Fresnos 4ndash5 Apr 1969 Pinkavaet al 6117 (UC) ITS-1 AY146818 ITS-2 AY146884 A lifolius Mathias Constance amp WL TheobmdashUSA Texas Culberson CoHunter Peak at Pine Top Guadalupe Mountains National Park 6 Sep 1987 Higgins 17453 (BRY) ITS-1 AY146819 ITS-2AY146885 A macdougalii JM Coult amp Rose WL subsp breviradiatus Theob amp CC TsengmdashUSA Utah San Juan Co Manti-LaSal National Forest Notch Canyon jct of Cottonwood Wash 5 Jun 1985 Atwood 11143 (BRY) ITS-1 AY146820 ITS-2 AY146886USA New Mexico San Juan Co Chaco Culture Nat Hist Park top of Chacra Mesa 2 May 1993 Heil 7374 (UNM) ITS-1AY146821 ITS-2 AY146887 USA Utah Gareld Co Dixie National Forest Death Hollow 28 May 1989 Franklin 6499 (BRY)ITS-1 AY146822 ITS-2 AY146888 USA New Mexico San Juan Co NM 575 015 mi NNW of US 64 NW of Blanco 10 Jul1999 Sun amp Hartman 649 (ILL) ITS-1 AY146823 ITS-2 AY146889 A macdougalii subsp macdougaliimdashUSA Arizona CoconinoCo Grand Canyon Kaibab Trail to Roaring Springs 23 Jun 1933 Eastwood amp Howell 986 (UC) ITS-1 AY146824 ITS-2 AY146890

Angelica grayi (JM Coult amp Rose) JM Coult amp RosemdashUSA Colorado Gareld Co Blair Mountain 17 mi NNW ofGlenwood Springs 24 Aug 1991 Vanderhorst amp Palaci 4490 (RM) ITS-1 AY146825 ITS-2 AY146891

Cymopterus aboriginum ME JonesmdashUSA California Mono Co ca 5 mi E of Benton along Hwy 6 21 Jun 1990 Atwood13838 (BRY) ITS-1 AY146826 ITS-2 AY146892 C acaulis (Pursh) Raf var greeleyorum JW Grimes amp PL PackardmdashUSAOregon Malheur Co McBride Creek Road 24 May 1989 Smithman et al LS-2045 (CIC) ITS-1 AY146827 ITS-2 AY146893 Cacaulis var higginsii (SL Welsh) S GoodrichmdashUSA Utah Kane Co E of Coyote Wash 8 Apr 1993 Chapman sn (BRY) ITS-1 AY146828 ITS-2 AY146894 C acaulis var parvus S GoodrichmdashUSA Utah Tooele Co Indian Rice Grass Duneeld NWof English village 7 Jun 1993 Johnson 351 (BRY) ITS-1 AY146829 ITS-2 AY146895 C beckii SL Welsh amp S GoodrichmdashUSAUtah San Juan Co SW of Abajo Mountains S of the Causeway 10 Jul 1992 Franklin 7564 (BRY) ITS-1 AY146830 ITS-2AY146896 C cinerarius A GraymdashUSA California Mono Co Sweetwater Canyon 15 Jul 1944 Alexander amp Kellogg 3893 (UTC)ITS-1 AY146831 ITS-2 AY146897 C constancei RL HartmmdashUSA Colorado Montrose Co San Miguel and lower DoloresRiver drainages Simbad Valley 30 May 1995 Hartman 50943 (RM) ITS-1 AY146832 ITS-2 AY146898 USA Wyoming SubletteCo Upper Green River Plain Cretaceous Mountain 19 May 1993 Hartman 37182 (RM) ITS-1 AY146833 ITS-2 AY146899 Ccorrugatus ME JonesmdashUSA Nevada Mineral Co Walker River Indian Reservation Rawhide Flats 15 May 1988 Tiehm 11640(BRY) ITS-1 AY146834 ITS-2 AY146900 C coulteri (ME Jones) MathiasmdashUSA Utah Sevier Co Triangle Mt Soldiers Canyon15 May 1990 Thorne amp Zupan 7377 (BRY) ITS-1 AY146835 ITS-2 AY146901 C davisii RL HartmmdashUSA Idaho Cassia CoN slope of Mt Harrison 30 Jul 1983 Cronquist 11840 (UTC) ITS-1 AY146836 ITS-2 AY146902 C deserticola BrandegeendashUSACalifornia San Bernardino Co 1 mi S of Kramer 21 Mar 1958 Raven 11891 (UTC) ITS-1 AY146837 ITS-2 AY146903 Cdouglassii RL Hartm amp ConstancemdashUSA Idaho Custer Co 2 mi N of Horseheaven Pass and just W of Spring Hill VABMPahsimeroi Mountains Challis National Forest 26 Jun 1984 Atwood 10302 (BRY) ITS-1 AY146838 ITS-2 AY146904 C gilmaniiC MortonmdashUSA Nevada Clark Co Desert Range SE base Desert National Wildlife Refuge 73 mi from Sheep Peak summit17 May 1987 Moreeld et al 4463 (BRY) ITS-1 AY146839 ITS-2 AY146905 C glaucus NuttmdashUSA Idaho Valley Co ridgebetween Fisher Creek Saddle and Black Tip along IdahoValley Co line 26 Jul 1989 Ertter et al 8742 (RM) ITS-1 AY146840ITS-2 AY146906 C goodrichii SL Welsh amp NeesemdashUSA Nevada Lander Co Toiyabe Range 2 mi N of Bunker Hill 16 Jul1981 Neese et al 10737 (BRY) ITS-1 AY146841 ITS-2 AY146907 C lapidosus (ME Jones) ME JonesmdashUSA Wyoming UintaCo Uinta Co Road 290 37 air mi W of Lonetree 7 Jul 1983 Hartman 15767 (UTC) ITS-1 AY146842 ITS-2 AY146908 Clongilobus (Rydb) WA WebermdashUSA Wyoming Sublette Co Wyoming Range 15 Aug 1993 Hartman 43986 (RM) ITS-1AF358482 ITS-2 AF358549 C macrorhizus BuckleymdashUSA Texas Tom Green Co Grandview campground on W side of FisherLake 19 Mar 1983 Ertter amp Strachan 4690 (RM) ITS-1 AY146843 ITS-2 AY146909 C minimus (Mathias) MathiasmdashUSA UtahIron Co Cedar Breaks National Monument just below N rim where rst reached by Rattlesnake Trail 25 Jul 1993 Stone 1656(BRY) ITS-1 AY146844 ITS-2 AY146910 C newberryi (S Watson) ME JonesmdashUSA Utah San Juan Co 45 mi downstreamfrom Bluff 20 May 1983 Barneby 17896 (BRY) ITS-1 AY146845 ITS-2 AY146911 C panamintensis JM Coult amp Rose varacutifolius (JM Coult amp Rose) MunzmdashUSA California San Bernardino Co south side of Ord Mtn north of Lucerne Valley8 Apr 1988 Boyd et al 1776 (OSC) ITS-1 AY146846 ITS-2 AY146912 C ripleyi BarnebymdashUSA Nevada Nye Co Stone CabinValley 24 May 1995 Holmgren amp Holmgren 12299 (BRY) ITS-1 AY146847 ITS-2 AY146913 C rosei (ME Jones ex JM Coult ampRose) ME JonesmdashUSA Utah Sanpete Co Hills W of Ninemile Reservoir 8 May 1987 Franklin 4604 (BRY) ITS-1 AY146848ITS-2 AY146914

Glehnia littoralis F Schmidt ex Miq subsp littoralismdashTaiwan Taitung Hsien Lanyu (Orchid Island) Liao et al 1235 (MO)ITS-1 AY146849 ITS-2 AY146915 G littoralis subsp leiocarpa (Mathias) HultenmdashUSA Oregon Tillamook Co by the beachon the south side of Cape Kiwanda 10 Aug 1975 Halse 1228 (OSC) ITS-1 AY146850 ITS-2 AY146916

Lomatium ambiguum (Nutt) JM Coult amp RosemdashUSA Montana Madison Co ca 15 mi from roadrsquos end on Bear TrapCanyon Trail 30 May 1979 Lowry 1867 (ILL) ITS-1 AY146851 ITS-2 AY146917 L bradshawii (Rose ex Mathias) Mathias ampConstancemdashUSA Oregon Benton Co NE Corvallis W of Canterbury Circle area around Frazier Creek 8 Jun 1980 Halse 2215(ILL) ITS-1 AY146852 ITS-2 AY146918 L brandegei (JM Coult amp Rose) JF MacbrmdashUSA Washington Chelan Co 5 mi SWof Leavenworth on Snow Creek Trail 29 May 1977 Lowry 621 (ILL) ITS-1 AY146853 ITS-2 AY146919 L grayi (JM Coult ampRose) JM Coult amp Rose var grayimdashUSA Wyoming Lincoln Co Tunp Range Preacher Hollow 16 mi NNE of Cokeville 22May 1993 Hartman 37444 (RM) ITS-1 AY146854 ITS-2 AY146920 L grayi var depauperatum (ME Jones) MathiasmdashUSA UtahJuab Co Gilson Mountains 7 mi from Lynndyl 7 May 1981 Goodrich 15335 (RM) ITS-1 AY146855 ITS-2 AY146921

Musineon lineare (Rydb) MathiasmdashUSA Utah Cache Co Toney Grove Trailhead Trail to Naomi Peak 9 Jul 1989 Hartman24364 (RM) ITS-1 AY146856 ITS-2 AY146922

Oreonana clementis (ME Jones) JepsmdashUSA California Tulare Co Mt Guyot 22 Jul 1949 Howell 25604 (UTC) ITS-1AY146857 ITS-2 AY146923 O purpurascens Shevock amp ConstancemdashUSA California Tulare Co Sequoia National Forest SlateMountain along USFS trail 31E14 26 Apr 1977 Shevock 5428 (BRY) ITS-1 AY146858 ITS-2 AY146924 O vestita (S Watson)JepsmdashUSA California San Bernardino Co Angeles National Forest San Gabriel Mtns between Mt Harwood and Mt SanAntonio Peak 4 May 1977 Shevock 5433 (BRY) ITS-1 AY146859 ITS-2 AY146925

2004] 423SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 1 Continued

Oreoxis trotteri SL Welsh amp S GoodrichmdashUSA Utah Grand Co E of Courthouse Rock 20 May 1985 Welsh 23427 (BRY)ITS-1 AY146860 ITS-2 AY146926 USA Utah Grand Co 135 mi NNW of Moab 4 Jun 1985 Neese amp Welsh 16984 (BRY) ITS-1 AY146861 ITS-2 AY146927

Orogenia fusiformis S WatsonmdashUSA Oregon Josephine Co ats near S boundary with Siskiyou NF Oregon Caves NationalMonument 24 May 1996 Zika 12864 (OSC) ITS-1 AY146862 ITS-2 AY146928

Peucedanum japonicum Thunb ex A Murraymdashcult Botanical Conservatory Mulhouse France (no 97138) 8 Dec 1999 Reduronsn (Herb Reduron) ITS-1 AF495826 ITS-2 AF495827 P ostruthium (L) WDJ Koch (5Imperatoria ostruthium L)mdashDownie etal (1998) ITS-1 U78403 ITS-2 U78463

Podistera macounii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Alaska ridge W of Sheep Creek 30 Jun 1977 Reed etal 6047 (BRY) ITS-1 AY146863 ITS-2 AY146929 USA Alaska Mountains between Essie Creek and the headwaters of Seven-tymile Drainage 21 Jun 2002 Larsen et al sn (ALA) ITS-1 AY146864 ITS-2 AY146930 P nevadensis (A Gray) S WatsonmdashUSA California El Dorado Co Lake Tahoe Basin Management Unit Freel Peak 27 Jul 2002 Matson 634 (ILL) ITS-1 AY146865ITS-2 AY146931 P yukonensis Mathias amp ConstancemdashUSA Alaska Kathul Mountain along the Yukon River roughly halfwaybetween Eagle and Circle 11 Jun 2002 Larsen amp Batten 02-1263 (ALA) ITS-1 AY146866 ITS-2 AY146932

Polytaenia texana (JM Coult amp Rose) Mathias amp ConstancemdashUSA Texas Burnet Co E of Briggs 25 May 1985 Barrie 1403(RM) ITS-1 AY146867 ITS-2 AY146933

Pseudocymopterus longiradiatus Mathias Constance amp WL TheobmdashUSA New Mexico Otero Co Sacramento Mts ca 3mi SSW of Bent SW side of Domingo Peak 7 Aug 1990 Sivinski 1546 (UNM) ITS-1 AY146868 ITS-2 AY146934

Pteryxia davidsonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA New Mexico Grant Co Bear Mountain near SilverCity 17 Jun 1903 Metcalfe 165 (RM) ITS-1 AY146869 ITS-2 AY146935 P hendersonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Utah Duchesne Co 075 mi SE of Davis Lake Uinta Mtns 23 Aug 1996 Huber 3502 (BRY) ITS-1 AY146870 ITS-2AY146936 P petraea (ME Jones) JM Coult amp RosemdashUSA Nevada Nye Co Toiyabe National Forest Toquima Range IronSprings 5 Jun 1978 Goodrich 11292 (UTC) ITS-1 AY146871 ITS-2 AY146937 P terebinthina (Hook) JM Coult amp Rose varterebinthinamdashUSA Washington Benton Co Pit 30 between the 200 area 17 May 1993 McKinnon amp Sackschewsky 327 (BRY)ITS-1 AY146872 ITS-2 AY146938 P terebinthina var albiora (Nutt ex Torr amp A Gray) MathiasmdashUSA Wyoming SubletteCo Upper Green River Basin ca 95 air mi NNW of La Barge 20 May 1993 Hartman 37374 (RM) ITS-1 AY146873 ITS-2AY146939 USA Utah Daggett Co Flaming Gorge National Recreation area 5 Jun 1995 Refsdal amp Goodrich 3591 (RM) ITS-1AY146874 ITS-2 AY146940 P terebinthina var calcarea (ME Jones) MathiasmdashUSA Wyoming Uinta Co 275 mi N of Lone-tree 2 Jul 1999 Goodrich 26060 (BRY) ITS-1 AY146875 ITS-2 AY146941 USA Utah Daggett Co Ashley National Forest FlamingGorge National Recreation area E side of Flaming Gorge Reservoir 26 May 1992 Goodrich 24045 (BRY) ITS-1 AY146876 ITS-2 AY146942 USA Wyoming Big Horn Co Big Horn Mountains Cold Spring Road 26 May 1980 Hartman amp Dueholm 11224(RM) ITS-1 AY146877 ITS-2 AY146943 USA Wyoming Carbon Co ca 7 air mi NE of Savery 14 Jun 1979 Hartman amp Coffey8961 (RM) ITS-1 AY146878 ITS-2 AY146944 P terebinthina var californica (JM Coult amp Rose) MathiasmdashUSA CaliforniaMadera Co near Whiskey Creek 1 mi NW of Ellis Meadow Sierra Nevada 1 Jul 1938 Constance 2390 (RM) ITS-1 AY146879ITS-2 AY146945 P terebinthina var foeniculacea (Nutt ex Torr amp A Gray) MathiasmdashUSA Idaho Custer Co Morgan Creek10 mi N of Challis 26 Jun 1984 Atwood 10320 (RM) ITS-1 AY146880 ITS-2 AY146946

Selinum candollei DCmdashDownie and Katz-Downie (1996) ITS-1 U30564 ITS-2 U30565 S carvifolia (L) LmdashFrance Bas-Rhin between Herbsheim and Boofzheim 14 Aug 2001 Reduron sn (Herb Reduron) ITS-1 AY179028 ITS-2 AY179028

Seseli gracile Waldst amp KitmdashKatz-Downie et al (1999) ITS-1 AF008605 ITS-2 AF009084 S libanotis (L) WDJ KochmdashKatz-Downie et al (1999) ITS-1 AF008603 ITS-2 AF009082 S tortuosum LmdashPortugal Lisboa Sintra Praja das Macas cult BotanicalConservatory Mulhouse France (no 98042) 2 Aug 2001 Hildenbrand et al sn (Herb Reduron) ITS-1 AY179031 ITS-2 AY179031

Tauschia arguta (Torr amp A Gray) JF MacbrmdashUSA California San Diego Co Corte Madera Ranch near Pine Valley 22May 1983 Van der Werff amp Clark 4571 (MO) ITS-1 AY146881 ITS-2 AY146947 T glauca (JM Coult amp Rose) Mathias ampConstancemdashUSA California Trinity Co SE of Burnt Ranch 11 July 1990 Spellenberg 10254 (RM) ITS-1 AY146882 ITS-2AY146948 T kelloggii (A Gray) JF MacbrmdashUSA California Butte Co SW of intersection of Black Bart Road and ForbestownRoad about 35 air mi SW of Forgestown 16 Jun 1993 Ahart 6972 (UC) ITS-1 AY146883 ITS-2 AY146949

TABLE 2 Sequence characteristics of the two internal transcribed spacer (ITS) regions separately and combined for 150 terminals(159 accessions) of Apiaceae subfamily Apioideae

Sequence characteristic ITS-1 ITS-2Combined

(ITS-1 amp ITS-2)

Length variationNo of aligned positionsNo of aligned positions constantNo of aligned positions parsimony-informativeNo of aligned positions autapomorphicNo of unambiguous alignment gapsNo of unambiguous gaps parsimony-informativeMaximum pairwise sequence divergence ()

206ndash2182259189452111125

220ndash226233937961165

131

427ndash4414581841681063716121

424 [Volume 29SYSTEMATIC BOTANY

FIG 1 Strict consensus of 20000 minimal length 881-steptrees derived from equally-weighted maximum parsimonyanalysis of aligned ITS-1 and ITS-2 sequences from 150 ter-minals (159 accessions) of Apiaceae subfamily ApioideaeConsistency indices 5 04654 and 03762 with and without

not

uninformative characters respectively retention index506405 Numbers on branches represent bootstrap estimatesfor 100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-additionvalues 50 are not indicated All Cymopterus accessions areboldfaced Numbers in parentheses refer to numbers of ac-cessions bearing identical DNA sequences that were combinedto facilitate analysis

Thaspium and Zizia A large basal polytomy obscuresrelationships among these umbellifers yet four majorclades containing six or more taxa each are apparentThe rst and most distal of these clades comprises 37taxa representing 13 accessions of Cymopterus 16 ac-cessions of Lomatium 3 accessions each of Oreonanaand Pteryxia and 2 accessions of Orogenia A secondclade comprises all ve accessions of Cymopterus acau-lis plus C bulbosus C newberryi and Lomatium concin-num A third clade comprises some but not all infra-specic accessions of Pteryxia terebinthina plus Pteryxiapetraea Cymopterus corrugatus C coulteri and C ibapen-sis The fourth major clade contains Cymopterus con-stancei C macrorhizus C montanus C multinervatus Cpurpurascens and Aletes calcicola Several additionalclades of four to ve taxa each exist and are also ofheterogeneous generic composition

The results of the MP analysis revealed that Cymop-terus (boldfaced in all tree gures presented herein)and Lomatium the two largest genera of NA Apiaceaeare highly polyphyletic and that their species are in-extricably linked with those of the other endemic pe-rennial genera of the region In addition the generaAletes Musineon Oreonana Oreoxis Orogenia PodisteraPseudocymopterus Pteryxia and Tauschia are also notmonophyletic Constraining the 41 examined taxa ofCymopterus to monophyly and rerunning the MP anal-ysis resulted in trees 35 steps longer than those mostparsimonious (Table 3) Lomatium is monophyletic intrees 45 steps longer than those most parsimoniousSimilar analyses were performed by constraining eachof the other aforementioned genera with Aletes Glehn-ia Oreoxis Pteryxia and Tauschia each monophyletic intrees 10 or more steps greater than those most parsi-monious (Table 3) In contrast Polytaenia Thaspiumand Zizia are all monophyletic in unconstrainedsearches

The remaining 24 accessions examined formed a po-lytomy basal to this clade and included a monophy-letic Angelica with the inclusion of Sphenosciadium andPacic NA Glehnia littoralis subsp leiocarpa the cladeof Selinum candollei Seseli Peucedanum japonicum andPacic Asian Glehnia littoralis subsp littoralis the lattertwo taxa forming a strongly-supported sister group(97 bootstrap value) the Arracacia clade (ArracaciaPrionosciadium and Rhodosciadium) the clade of Ciclo-spermum 1 Spermolepis and Peucedanum ostruthium Se-

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

2004] 421SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

nomic DNA was obtained from ca 20 mg of dried leaf tissueusing the DNeasy Plant Mini Kit (Qiagen Inc Valencia CA) andPCR-amplied using primers lsquolsquoITS4rsquorsquo and lsquolsquoITS5rsquorsquo (White et al1990) Cycle sequencing reactions using these same primers wereperformed on the puried PCR products using AmpliTaq DNApolymerase and uorescent dye-labeled terminators (ABI PrismBigDye Terminator vers 30 Ready Reaction Cycle Sequencing KitApplied Biosystems Foster City CA) Sequencing products wereresolved by electrophoresis using Applied Biosystemrsquos 373A DNAsequencer Both ITS spacer regions were sequenced in their entire-ty on both DNA strands All ITS-1 and ITS-2 data have been de-posited in GenBank (Table 1) and the data matrix is available inTreeBASE (study accession number S1010 matrix accession num-ber M1706)

Data Analysis DNA sequences fromboth spacer regions werealigned using CLUSTAL X (Jeanmougin et al 1998) and adjustedmanually as necessary Sequence data from the invariant 58S sub-unit were excluded as they were unavailable for several previouslypublished taxa owing to the manual sequencing methods used toobtain these earlier data Uncorrected pairwise nucleotide differ-ences (lsquolsquoprsquorsquo) were determined using PAUP vers 40 (Swofford1998) as they are commonly provided in other angiosperm ITSstudies (Baldwin et al 1995) To facilitate analysis taxa bearingidentical sequences were combined (ie two accessions of Cymop-terus constancei two accessions of Oreoxis trotteri three accessionsof Pteryxia terebinthina var albiora three accessions of P terebin-thina var calcarea two of ve accessions of Aletes macdougalii subspbreviradiatus and the three remaining accessions of A macdougaliisubsp breviradiatus) for a nal tally of 150 terminals for the cla-distic analyses Fewer than 03 of all data matrix cells werescored as missing data the sequences with missing data were ob-tained from GenBank and submitted by others

The resulting data matrix was rst analyzed using equally-weighted maximum parsimony (MP) with gap states treated asmissing data Alignment gaps were not scored as separate pres-ence-absence characters because of ambiguous length mutations inseveral previously submitted Lomatium sequences as a result ofcompressions MP trees were sought using the heuristic searchstrategies of PAUP and the inverse-constraint approach describedin Downie et al (1998) The maximum number of MP trees wasset at 20000 and these trees were permitted to swap to comple-tion Bootstrap values (Felsenstein 1985) were calculated from100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-addition of taxaonly those values compatible with the 50 majority-rule consen-sus tree were recorded Estimates of support obtained using thismethod are generally similar to those obtained from bootstrapanalyses with branch swapping particularly for well-supportednodes (Mort et al 2000) The number of additional steps requiredto force particular taxa into a monophyletic group was examinedusing the constraint option of PAUP The g1 statistic of skewedtree-length distribution was calculated from 10000 random par-simony trees generated by PAUP and was used to assess theamount of nonrandom structure in the data (Hillis and Huelsen-beck 1992)

A maximum likelihood (ML) analysis of these ITS data was thenperformed Prior to this analysis the program Modeltest vers 306(Posada and Crandall 1998) was used to select an evolutionarymodel of nucleotide substitution that best ts these data Usingthe hierarchical likelihood ratio test statistic Modeltest selected theTrN1G (Tamura and Nei 1993) model (base frequencies 02725A 02154 C 02312 G 02809 T estimates of substitution ratesAlaquoC 1 AlaquoG 22734 AlaquoT 1 ClaquoG 1 ClaquoT 45457 GlaquoT 1proportion of invariable sites 5 0 gamma distribution shape pa-rameter 5 05930) These settings were entered into PAUP and aheuristic search carried out using ten random addition sequencereplicates and TBR branch-swapping under ML optimization Abootstrap analysis was conducted using the neighbor-joiningmethod and the ML parameters estimated by Modeltest Bootstrapvalues were calculated from 100 replicate analyses

Owing to its computational efciency with large data sets aphylogenetic analysis was also performed using MrBayes vers201 (Huelsenbeck and Ronquist 2001) a Bayesian inference pro-

gram Starting trees were chosen at random and one million gen-erations were run with sampling occurring every 100 generationsTree topologies were based on the general time reversible (GTR)model of maximum likelihood (with the shape parameter of thegamma distribution inferred by Modeltest) and four simultaneousMarkov Chain Monte Carlo chains to model nucleotide rate het-erogeneity Two-thousand trees were discarded as lsquolsquoburn-inrsquorsquo priorto calculating the 50 majority-rule tree Numbers on internalbranches of the tree indicate the percent of time the clade occursamong the sampled trees (ie the posterior probability of thatclade existing Huelsenbeck and Ronquist 2001)

RESULTS

Sequence Analysis Alignment of 150 ITS-1 andITS-2 sequences (representing 159 accessions) resultedin a matrix of 458 positions with none excluded be-cause of alignment ambiguity Characteristics of thesealigned sequences as separate spacer regions or com-bined are presented in Table 2 Thirty-seven gapsranging between 1 and 6 bp in size were introducedto facilitate alignment 33 of these were 1-bp in lengthtwo were 2-bp in length one was 4-bp in length andone was 6-bp in length Uncorrected pairwise sequencedivergence values across both spacers and all taxaranged from identity to 121 of nucleotides Pairwisesequence divergence values for the 126 ingroup ter-minal taxa ranged from identity to 94 of nucleotides(the latter between Cymopterus basalticus and Tauschiatexana) Pairwise distances within Cymopterus (n541)ranged from identity to 73 distances within Loma-tium (n530) ranged from identity to approximately80 Percent G 1 C content over both spacers rangedbetween 542 and 599 (mean 5 571) The g1 sta-tistic for 10000 random trees was20220 This value issignicantly more skewed (ie more negative) thanrandom data (g1 5 2009 for 250 variable positionsand 25 or more taxa P 001) indicating that theseITS data contain signicant amounts of phylogeneticsignal (Hillis and Huelsenbeck 1992) No evidence ofobvious ITS length variants representative of multiplerDNA repeat types was observed

Phylogenetic Analysis MP analysis of 150 ITS-1and ITS-2 sequences (159 accessions) resulted in thepreset limit of 20000 minimal length trees each of 881steps (consistency indices of 04654 and 03762 withand without uninformative characters respectivelyand a retention index of 06405) The strict consensusof these trees is presented in Fig 1 Bootstrap esti-mates were generally low with few values exceeding85

The consensus tree revealed a monophyletic assem-blage of endemic perennial NA genera although theclade was very weakly supported (with 50 boot-strap value) Included in this group are 126 terminals(reecting 135 accessions) represented by 18 generaAletes Cymopterus Harbouria Lomatium Musineon Neo-parrya Oreonana Oreoxis Orogenia Podistera PolytaeniaPseudocymopterus Pteryxia Shoshonea Taenidia Tauschia

422 [Volume 29SYSTEMATIC BOTANY

TABLE 1 Voucher information and GenBank accession numbers for those accessions of Apiaceae subfamily Apioideae examined fornuclear rDNA ITS sequence variation Equivalent information for those taxa considered in a previous phylogenetic study is presentedin Downie et al (2002)

Aletes calcicola Mathias amp ConstancemdashMexico Coahuila Sierra de San Marcos opposite Los Fresnos 4ndash5 Apr 1969 Pinkavaet al 6117 (UC) ITS-1 AY146818 ITS-2 AY146884 A lifolius Mathias Constance amp WL TheobmdashUSA Texas Culberson CoHunter Peak at Pine Top Guadalupe Mountains National Park 6 Sep 1987 Higgins 17453 (BRY) ITS-1 AY146819 ITS-2AY146885 A macdougalii JM Coult amp Rose WL subsp breviradiatus Theob amp CC TsengmdashUSA Utah San Juan Co Manti-LaSal National Forest Notch Canyon jct of Cottonwood Wash 5 Jun 1985 Atwood 11143 (BRY) ITS-1 AY146820 ITS-2 AY146886USA New Mexico San Juan Co Chaco Culture Nat Hist Park top of Chacra Mesa 2 May 1993 Heil 7374 (UNM) ITS-1AY146821 ITS-2 AY146887 USA Utah Gareld Co Dixie National Forest Death Hollow 28 May 1989 Franklin 6499 (BRY)ITS-1 AY146822 ITS-2 AY146888 USA New Mexico San Juan Co NM 575 015 mi NNW of US 64 NW of Blanco 10 Jul1999 Sun amp Hartman 649 (ILL) ITS-1 AY146823 ITS-2 AY146889 A macdougalii subsp macdougaliimdashUSA Arizona CoconinoCo Grand Canyon Kaibab Trail to Roaring Springs 23 Jun 1933 Eastwood amp Howell 986 (UC) ITS-1 AY146824 ITS-2 AY146890

Angelica grayi (JM Coult amp Rose) JM Coult amp RosemdashUSA Colorado Gareld Co Blair Mountain 17 mi NNW ofGlenwood Springs 24 Aug 1991 Vanderhorst amp Palaci 4490 (RM) ITS-1 AY146825 ITS-2 AY146891

Cymopterus aboriginum ME JonesmdashUSA California Mono Co ca 5 mi E of Benton along Hwy 6 21 Jun 1990 Atwood13838 (BRY) ITS-1 AY146826 ITS-2 AY146892 C acaulis (Pursh) Raf var greeleyorum JW Grimes amp PL PackardmdashUSAOregon Malheur Co McBride Creek Road 24 May 1989 Smithman et al LS-2045 (CIC) ITS-1 AY146827 ITS-2 AY146893 Cacaulis var higginsii (SL Welsh) S GoodrichmdashUSA Utah Kane Co E of Coyote Wash 8 Apr 1993 Chapman sn (BRY) ITS-1 AY146828 ITS-2 AY146894 C acaulis var parvus S GoodrichmdashUSA Utah Tooele Co Indian Rice Grass Duneeld NWof English village 7 Jun 1993 Johnson 351 (BRY) ITS-1 AY146829 ITS-2 AY146895 C beckii SL Welsh amp S GoodrichmdashUSAUtah San Juan Co SW of Abajo Mountains S of the Causeway 10 Jul 1992 Franklin 7564 (BRY) ITS-1 AY146830 ITS-2AY146896 C cinerarius A GraymdashUSA California Mono Co Sweetwater Canyon 15 Jul 1944 Alexander amp Kellogg 3893 (UTC)ITS-1 AY146831 ITS-2 AY146897 C constancei RL HartmmdashUSA Colorado Montrose Co San Miguel and lower DoloresRiver drainages Simbad Valley 30 May 1995 Hartman 50943 (RM) ITS-1 AY146832 ITS-2 AY146898 USA Wyoming SubletteCo Upper Green River Plain Cretaceous Mountain 19 May 1993 Hartman 37182 (RM) ITS-1 AY146833 ITS-2 AY146899 Ccorrugatus ME JonesmdashUSA Nevada Mineral Co Walker River Indian Reservation Rawhide Flats 15 May 1988 Tiehm 11640(BRY) ITS-1 AY146834 ITS-2 AY146900 C coulteri (ME Jones) MathiasmdashUSA Utah Sevier Co Triangle Mt Soldiers Canyon15 May 1990 Thorne amp Zupan 7377 (BRY) ITS-1 AY146835 ITS-2 AY146901 C davisii RL HartmmdashUSA Idaho Cassia CoN slope of Mt Harrison 30 Jul 1983 Cronquist 11840 (UTC) ITS-1 AY146836 ITS-2 AY146902 C deserticola BrandegeendashUSACalifornia San Bernardino Co 1 mi S of Kramer 21 Mar 1958 Raven 11891 (UTC) ITS-1 AY146837 ITS-2 AY146903 Cdouglassii RL Hartm amp ConstancemdashUSA Idaho Custer Co 2 mi N of Horseheaven Pass and just W of Spring Hill VABMPahsimeroi Mountains Challis National Forest 26 Jun 1984 Atwood 10302 (BRY) ITS-1 AY146838 ITS-2 AY146904 C gilmaniiC MortonmdashUSA Nevada Clark Co Desert Range SE base Desert National Wildlife Refuge 73 mi from Sheep Peak summit17 May 1987 Moreeld et al 4463 (BRY) ITS-1 AY146839 ITS-2 AY146905 C glaucus NuttmdashUSA Idaho Valley Co ridgebetween Fisher Creek Saddle and Black Tip along IdahoValley Co line 26 Jul 1989 Ertter et al 8742 (RM) ITS-1 AY146840ITS-2 AY146906 C goodrichii SL Welsh amp NeesemdashUSA Nevada Lander Co Toiyabe Range 2 mi N of Bunker Hill 16 Jul1981 Neese et al 10737 (BRY) ITS-1 AY146841 ITS-2 AY146907 C lapidosus (ME Jones) ME JonesmdashUSA Wyoming UintaCo Uinta Co Road 290 37 air mi W of Lonetree 7 Jul 1983 Hartman 15767 (UTC) ITS-1 AY146842 ITS-2 AY146908 Clongilobus (Rydb) WA WebermdashUSA Wyoming Sublette Co Wyoming Range 15 Aug 1993 Hartman 43986 (RM) ITS-1AF358482 ITS-2 AF358549 C macrorhizus BuckleymdashUSA Texas Tom Green Co Grandview campground on W side of FisherLake 19 Mar 1983 Ertter amp Strachan 4690 (RM) ITS-1 AY146843 ITS-2 AY146909 C minimus (Mathias) MathiasmdashUSA UtahIron Co Cedar Breaks National Monument just below N rim where rst reached by Rattlesnake Trail 25 Jul 1993 Stone 1656(BRY) ITS-1 AY146844 ITS-2 AY146910 C newberryi (S Watson) ME JonesmdashUSA Utah San Juan Co 45 mi downstreamfrom Bluff 20 May 1983 Barneby 17896 (BRY) ITS-1 AY146845 ITS-2 AY146911 C panamintensis JM Coult amp Rose varacutifolius (JM Coult amp Rose) MunzmdashUSA California San Bernardino Co south side of Ord Mtn north of Lucerne Valley8 Apr 1988 Boyd et al 1776 (OSC) ITS-1 AY146846 ITS-2 AY146912 C ripleyi BarnebymdashUSA Nevada Nye Co Stone CabinValley 24 May 1995 Holmgren amp Holmgren 12299 (BRY) ITS-1 AY146847 ITS-2 AY146913 C rosei (ME Jones ex JM Coult ampRose) ME JonesmdashUSA Utah Sanpete Co Hills W of Ninemile Reservoir 8 May 1987 Franklin 4604 (BRY) ITS-1 AY146848ITS-2 AY146914

Glehnia littoralis F Schmidt ex Miq subsp littoralismdashTaiwan Taitung Hsien Lanyu (Orchid Island) Liao et al 1235 (MO)ITS-1 AY146849 ITS-2 AY146915 G littoralis subsp leiocarpa (Mathias) HultenmdashUSA Oregon Tillamook Co by the beachon the south side of Cape Kiwanda 10 Aug 1975 Halse 1228 (OSC) ITS-1 AY146850 ITS-2 AY146916

Lomatium ambiguum (Nutt) JM Coult amp RosemdashUSA Montana Madison Co ca 15 mi from roadrsquos end on Bear TrapCanyon Trail 30 May 1979 Lowry 1867 (ILL) ITS-1 AY146851 ITS-2 AY146917 L bradshawii (Rose ex Mathias) Mathias ampConstancemdashUSA Oregon Benton Co NE Corvallis W of Canterbury Circle area around Frazier Creek 8 Jun 1980 Halse 2215(ILL) ITS-1 AY146852 ITS-2 AY146918 L brandegei (JM Coult amp Rose) JF MacbrmdashUSA Washington Chelan Co 5 mi SWof Leavenworth on Snow Creek Trail 29 May 1977 Lowry 621 (ILL) ITS-1 AY146853 ITS-2 AY146919 L grayi (JM Coult ampRose) JM Coult amp Rose var grayimdashUSA Wyoming Lincoln Co Tunp Range Preacher Hollow 16 mi NNE of Cokeville 22May 1993 Hartman 37444 (RM) ITS-1 AY146854 ITS-2 AY146920 L grayi var depauperatum (ME Jones) MathiasmdashUSA UtahJuab Co Gilson Mountains 7 mi from Lynndyl 7 May 1981 Goodrich 15335 (RM) ITS-1 AY146855 ITS-2 AY146921

Musineon lineare (Rydb) MathiasmdashUSA Utah Cache Co Toney Grove Trailhead Trail to Naomi Peak 9 Jul 1989 Hartman24364 (RM) ITS-1 AY146856 ITS-2 AY146922

Oreonana clementis (ME Jones) JepsmdashUSA California Tulare Co Mt Guyot 22 Jul 1949 Howell 25604 (UTC) ITS-1AY146857 ITS-2 AY146923 O purpurascens Shevock amp ConstancemdashUSA California Tulare Co Sequoia National Forest SlateMountain along USFS trail 31E14 26 Apr 1977 Shevock 5428 (BRY) ITS-1 AY146858 ITS-2 AY146924 O vestita (S Watson)JepsmdashUSA California San Bernardino Co Angeles National Forest San Gabriel Mtns between Mt Harwood and Mt SanAntonio Peak 4 May 1977 Shevock 5433 (BRY) ITS-1 AY146859 ITS-2 AY146925

2004] 423SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 1 Continued

Oreoxis trotteri SL Welsh amp S GoodrichmdashUSA Utah Grand Co E of Courthouse Rock 20 May 1985 Welsh 23427 (BRY)ITS-1 AY146860 ITS-2 AY146926 USA Utah Grand Co 135 mi NNW of Moab 4 Jun 1985 Neese amp Welsh 16984 (BRY) ITS-1 AY146861 ITS-2 AY146927

Orogenia fusiformis S WatsonmdashUSA Oregon Josephine Co ats near S boundary with Siskiyou NF Oregon Caves NationalMonument 24 May 1996 Zika 12864 (OSC) ITS-1 AY146862 ITS-2 AY146928

Peucedanum japonicum Thunb ex A Murraymdashcult Botanical Conservatory Mulhouse France (no 97138) 8 Dec 1999 Reduronsn (Herb Reduron) ITS-1 AF495826 ITS-2 AF495827 P ostruthium (L) WDJ Koch (5Imperatoria ostruthium L)mdashDownie etal (1998) ITS-1 U78403 ITS-2 U78463

Podistera macounii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Alaska ridge W of Sheep Creek 30 Jun 1977 Reed etal 6047 (BRY) ITS-1 AY146863 ITS-2 AY146929 USA Alaska Mountains between Essie Creek and the headwaters of Seven-tymile Drainage 21 Jun 2002 Larsen et al sn (ALA) ITS-1 AY146864 ITS-2 AY146930 P nevadensis (A Gray) S WatsonmdashUSA California El Dorado Co Lake Tahoe Basin Management Unit Freel Peak 27 Jul 2002 Matson 634 (ILL) ITS-1 AY146865ITS-2 AY146931 P yukonensis Mathias amp ConstancemdashUSA Alaska Kathul Mountain along the Yukon River roughly halfwaybetween Eagle and Circle 11 Jun 2002 Larsen amp Batten 02-1263 (ALA) ITS-1 AY146866 ITS-2 AY146932

Polytaenia texana (JM Coult amp Rose) Mathias amp ConstancemdashUSA Texas Burnet Co E of Briggs 25 May 1985 Barrie 1403(RM) ITS-1 AY146867 ITS-2 AY146933

Pseudocymopterus longiradiatus Mathias Constance amp WL TheobmdashUSA New Mexico Otero Co Sacramento Mts ca 3mi SSW of Bent SW side of Domingo Peak 7 Aug 1990 Sivinski 1546 (UNM) ITS-1 AY146868 ITS-2 AY146934

Pteryxia davidsonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA New Mexico Grant Co Bear Mountain near SilverCity 17 Jun 1903 Metcalfe 165 (RM) ITS-1 AY146869 ITS-2 AY146935 P hendersonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Utah Duchesne Co 075 mi SE of Davis Lake Uinta Mtns 23 Aug 1996 Huber 3502 (BRY) ITS-1 AY146870 ITS-2AY146936 P petraea (ME Jones) JM Coult amp RosemdashUSA Nevada Nye Co Toiyabe National Forest Toquima Range IronSprings 5 Jun 1978 Goodrich 11292 (UTC) ITS-1 AY146871 ITS-2 AY146937 P terebinthina (Hook) JM Coult amp Rose varterebinthinamdashUSA Washington Benton Co Pit 30 between the 200 area 17 May 1993 McKinnon amp Sackschewsky 327 (BRY)ITS-1 AY146872 ITS-2 AY146938 P terebinthina var albiora (Nutt ex Torr amp A Gray) MathiasmdashUSA Wyoming SubletteCo Upper Green River Basin ca 95 air mi NNW of La Barge 20 May 1993 Hartman 37374 (RM) ITS-1 AY146873 ITS-2AY146939 USA Utah Daggett Co Flaming Gorge National Recreation area 5 Jun 1995 Refsdal amp Goodrich 3591 (RM) ITS-1AY146874 ITS-2 AY146940 P terebinthina var calcarea (ME Jones) MathiasmdashUSA Wyoming Uinta Co 275 mi N of Lone-tree 2 Jul 1999 Goodrich 26060 (BRY) ITS-1 AY146875 ITS-2 AY146941 USA Utah Daggett Co Ashley National Forest FlamingGorge National Recreation area E side of Flaming Gorge Reservoir 26 May 1992 Goodrich 24045 (BRY) ITS-1 AY146876 ITS-2 AY146942 USA Wyoming Big Horn Co Big Horn Mountains Cold Spring Road 26 May 1980 Hartman amp Dueholm 11224(RM) ITS-1 AY146877 ITS-2 AY146943 USA Wyoming Carbon Co ca 7 air mi NE of Savery 14 Jun 1979 Hartman amp Coffey8961 (RM) ITS-1 AY146878 ITS-2 AY146944 P terebinthina var californica (JM Coult amp Rose) MathiasmdashUSA CaliforniaMadera Co near Whiskey Creek 1 mi NW of Ellis Meadow Sierra Nevada 1 Jul 1938 Constance 2390 (RM) ITS-1 AY146879ITS-2 AY146945 P terebinthina var foeniculacea (Nutt ex Torr amp A Gray) MathiasmdashUSA Idaho Custer Co Morgan Creek10 mi N of Challis 26 Jun 1984 Atwood 10320 (RM) ITS-1 AY146880 ITS-2 AY146946

Selinum candollei DCmdashDownie and Katz-Downie (1996) ITS-1 U30564 ITS-2 U30565 S carvifolia (L) LmdashFrance Bas-Rhin between Herbsheim and Boofzheim 14 Aug 2001 Reduron sn (Herb Reduron) ITS-1 AY179028 ITS-2 AY179028

Seseli gracile Waldst amp KitmdashKatz-Downie et al (1999) ITS-1 AF008605 ITS-2 AF009084 S libanotis (L) WDJ KochmdashKatz-Downie et al (1999) ITS-1 AF008603 ITS-2 AF009082 S tortuosum LmdashPortugal Lisboa Sintra Praja das Macas cult BotanicalConservatory Mulhouse France (no 98042) 2 Aug 2001 Hildenbrand et al sn (Herb Reduron) ITS-1 AY179031 ITS-2 AY179031

Tauschia arguta (Torr amp A Gray) JF MacbrmdashUSA California San Diego Co Corte Madera Ranch near Pine Valley 22May 1983 Van der Werff amp Clark 4571 (MO) ITS-1 AY146881 ITS-2 AY146947 T glauca (JM Coult amp Rose) Mathias ampConstancemdashUSA California Trinity Co SE of Burnt Ranch 11 July 1990 Spellenberg 10254 (RM) ITS-1 AY146882 ITS-2AY146948 T kelloggii (A Gray) JF MacbrmdashUSA California Butte Co SW of intersection of Black Bart Road and ForbestownRoad about 35 air mi SW of Forgestown 16 Jun 1993 Ahart 6972 (UC) ITS-1 AY146883 ITS-2 AY146949

TABLE 2 Sequence characteristics of the two internal transcribed spacer (ITS) regions separately and combined for 150 terminals(159 accessions) of Apiaceae subfamily Apioideae

Sequence characteristic ITS-1 ITS-2Combined

(ITS-1 amp ITS-2)

Length variationNo of aligned positionsNo of aligned positions constantNo of aligned positions parsimony-informativeNo of aligned positions autapomorphicNo of unambiguous alignment gapsNo of unambiguous gaps parsimony-informativeMaximum pairwise sequence divergence ()

206ndash2182259189452111125

220ndash226233937961165

131

427ndash4414581841681063716121

424 [Volume 29SYSTEMATIC BOTANY

FIG 1 Strict consensus of 20000 minimal length 881-steptrees derived from equally-weighted maximum parsimonyanalysis of aligned ITS-1 and ITS-2 sequences from 150 ter-minals (159 accessions) of Apiaceae subfamily ApioideaeConsistency indices 5 04654 and 03762 with and without

not

uninformative characters respectively retention index506405 Numbers on branches represent bootstrap estimatesfor 100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-additionvalues 50 are not indicated All Cymopterus accessions areboldfaced Numbers in parentheses refer to numbers of ac-cessions bearing identical DNA sequences that were combinedto facilitate analysis

Thaspium and Zizia A large basal polytomy obscuresrelationships among these umbellifers yet four majorclades containing six or more taxa each are apparentThe rst and most distal of these clades comprises 37taxa representing 13 accessions of Cymopterus 16 ac-cessions of Lomatium 3 accessions each of Oreonanaand Pteryxia and 2 accessions of Orogenia A secondclade comprises all ve accessions of Cymopterus acau-lis plus C bulbosus C newberryi and Lomatium concin-num A third clade comprises some but not all infra-specic accessions of Pteryxia terebinthina plus Pteryxiapetraea Cymopterus corrugatus C coulteri and C ibapen-sis The fourth major clade contains Cymopterus con-stancei C macrorhizus C montanus C multinervatus Cpurpurascens and Aletes calcicola Several additionalclades of four to ve taxa each exist and are also ofheterogeneous generic composition

The results of the MP analysis revealed that Cymop-terus (boldfaced in all tree gures presented herein)and Lomatium the two largest genera of NA Apiaceaeare highly polyphyletic and that their species are in-extricably linked with those of the other endemic pe-rennial genera of the region In addition the generaAletes Musineon Oreonana Oreoxis Orogenia PodisteraPseudocymopterus Pteryxia and Tauschia are also notmonophyletic Constraining the 41 examined taxa ofCymopterus to monophyly and rerunning the MP anal-ysis resulted in trees 35 steps longer than those mostparsimonious (Table 3) Lomatium is monophyletic intrees 45 steps longer than those most parsimoniousSimilar analyses were performed by constraining eachof the other aforementioned genera with Aletes Glehn-ia Oreoxis Pteryxia and Tauschia each monophyletic intrees 10 or more steps greater than those most parsi-monious (Table 3) In contrast Polytaenia Thaspiumand Zizia are all monophyletic in unconstrainedsearches

The remaining 24 accessions examined formed a po-lytomy basal to this clade and included a monophy-letic Angelica with the inclusion of Sphenosciadium andPacic NA Glehnia littoralis subsp leiocarpa the cladeof Selinum candollei Seseli Peucedanum japonicum andPacic Asian Glehnia littoralis subsp littoralis the lattertwo taxa forming a strongly-supported sister group(97 bootstrap value) the Arracacia clade (ArracaciaPrionosciadium and Rhodosciadium) the clade of Ciclo-spermum 1 Spermolepis and Peucedanum ostruthium Se-

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

422 [Volume 29SYSTEMATIC BOTANY

TABLE 1 Voucher information and GenBank accession numbers for those accessions of Apiaceae subfamily Apioideae examined fornuclear rDNA ITS sequence variation Equivalent information for those taxa considered in a previous phylogenetic study is presentedin Downie et al (2002)

Aletes calcicola Mathias amp ConstancemdashMexico Coahuila Sierra de San Marcos opposite Los Fresnos 4ndash5 Apr 1969 Pinkavaet al 6117 (UC) ITS-1 AY146818 ITS-2 AY146884 A lifolius Mathias Constance amp WL TheobmdashUSA Texas Culberson CoHunter Peak at Pine Top Guadalupe Mountains National Park 6 Sep 1987 Higgins 17453 (BRY) ITS-1 AY146819 ITS-2AY146885 A macdougalii JM Coult amp Rose WL subsp breviradiatus Theob amp CC TsengmdashUSA Utah San Juan Co Manti-LaSal National Forest Notch Canyon jct of Cottonwood Wash 5 Jun 1985 Atwood 11143 (BRY) ITS-1 AY146820 ITS-2 AY146886USA New Mexico San Juan Co Chaco Culture Nat Hist Park top of Chacra Mesa 2 May 1993 Heil 7374 (UNM) ITS-1AY146821 ITS-2 AY146887 USA Utah Gareld Co Dixie National Forest Death Hollow 28 May 1989 Franklin 6499 (BRY)ITS-1 AY146822 ITS-2 AY146888 USA New Mexico San Juan Co NM 575 015 mi NNW of US 64 NW of Blanco 10 Jul1999 Sun amp Hartman 649 (ILL) ITS-1 AY146823 ITS-2 AY146889 A macdougalii subsp macdougaliimdashUSA Arizona CoconinoCo Grand Canyon Kaibab Trail to Roaring Springs 23 Jun 1933 Eastwood amp Howell 986 (UC) ITS-1 AY146824 ITS-2 AY146890

Angelica grayi (JM Coult amp Rose) JM Coult amp RosemdashUSA Colorado Gareld Co Blair Mountain 17 mi NNW ofGlenwood Springs 24 Aug 1991 Vanderhorst amp Palaci 4490 (RM) ITS-1 AY146825 ITS-2 AY146891

Cymopterus aboriginum ME JonesmdashUSA California Mono Co ca 5 mi E of Benton along Hwy 6 21 Jun 1990 Atwood13838 (BRY) ITS-1 AY146826 ITS-2 AY146892 C acaulis (Pursh) Raf var greeleyorum JW Grimes amp PL PackardmdashUSAOregon Malheur Co McBride Creek Road 24 May 1989 Smithman et al LS-2045 (CIC) ITS-1 AY146827 ITS-2 AY146893 Cacaulis var higginsii (SL Welsh) S GoodrichmdashUSA Utah Kane Co E of Coyote Wash 8 Apr 1993 Chapman sn (BRY) ITS-1 AY146828 ITS-2 AY146894 C acaulis var parvus S GoodrichmdashUSA Utah Tooele Co Indian Rice Grass Duneeld NWof English village 7 Jun 1993 Johnson 351 (BRY) ITS-1 AY146829 ITS-2 AY146895 C beckii SL Welsh amp S GoodrichmdashUSAUtah San Juan Co SW of Abajo Mountains S of the Causeway 10 Jul 1992 Franklin 7564 (BRY) ITS-1 AY146830 ITS-2AY146896 C cinerarius A GraymdashUSA California Mono Co Sweetwater Canyon 15 Jul 1944 Alexander amp Kellogg 3893 (UTC)ITS-1 AY146831 ITS-2 AY146897 C constancei RL HartmmdashUSA Colorado Montrose Co San Miguel and lower DoloresRiver drainages Simbad Valley 30 May 1995 Hartman 50943 (RM) ITS-1 AY146832 ITS-2 AY146898 USA Wyoming SubletteCo Upper Green River Plain Cretaceous Mountain 19 May 1993 Hartman 37182 (RM) ITS-1 AY146833 ITS-2 AY146899 Ccorrugatus ME JonesmdashUSA Nevada Mineral Co Walker River Indian Reservation Rawhide Flats 15 May 1988 Tiehm 11640(BRY) ITS-1 AY146834 ITS-2 AY146900 C coulteri (ME Jones) MathiasmdashUSA Utah Sevier Co Triangle Mt Soldiers Canyon15 May 1990 Thorne amp Zupan 7377 (BRY) ITS-1 AY146835 ITS-2 AY146901 C davisii RL HartmmdashUSA Idaho Cassia CoN slope of Mt Harrison 30 Jul 1983 Cronquist 11840 (UTC) ITS-1 AY146836 ITS-2 AY146902 C deserticola BrandegeendashUSACalifornia San Bernardino Co 1 mi S of Kramer 21 Mar 1958 Raven 11891 (UTC) ITS-1 AY146837 ITS-2 AY146903 Cdouglassii RL Hartm amp ConstancemdashUSA Idaho Custer Co 2 mi N of Horseheaven Pass and just W of Spring Hill VABMPahsimeroi Mountains Challis National Forest 26 Jun 1984 Atwood 10302 (BRY) ITS-1 AY146838 ITS-2 AY146904 C gilmaniiC MortonmdashUSA Nevada Clark Co Desert Range SE base Desert National Wildlife Refuge 73 mi from Sheep Peak summit17 May 1987 Moreeld et al 4463 (BRY) ITS-1 AY146839 ITS-2 AY146905 C glaucus NuttmdashUSA Idaho Valley Co ridgebetween Fisher Creek Saddle and Black Tip along IdahoValley Co line 26 Jul 1989 Ertter et al 8742 (RM) ITS-1 AY146840ITS-2 AY146906 C goodrichii SL Welsh amp NeesemdashUSA Nevada Lander Co Toiyabe Range 2 mi N of Bunker Hill 16 Jul1981 Neese et al 10737 (BRY) ITS-1 AY146841 ITS-2 AY146907 C lapidosus (ME Jones) ME JonesmdashUSA Wyoming UintaCo Uinta Co Road 290 37 air mi W of Lonetree 7 Jul 1983 Hartman 15767 (UTC) ITS-1 AY146842 ITS-2 AY146908 Clongilobus (Rydb) WA WebermdashUSA Wyoming Sublette Co Wyoming Range 15 Aug 1993 Hartman 43986 (RM) ITS-1AF358482 ITS-2 AF358549 C macrorhizus BuckleymdashUSA Texas Tom Green Co Grandview campground on W side of FisherLake 19 Mar 1983 Ertter amp Strachan 4690 (RM) ITS-1 AY146843 ITS-2 AY146909 C minimus (Mathias) MathiasmdashUSA UtahIron Co Cedar Breaks National Monument just below N rim where rst reached by Rattlesnake Trail 25 Jul 1993 Stone 1656(BRY) ITS-1 AY146844 ITS-2 AY146910 C newberryi (S Watson) ME JonesmdashUSA Utah San Juan Co 45 mi downstreamfrom Bluff 20 May 1983 Barneby 17896 (BRY) ITS-1 AY146845 ITS-2 AY146911 C panamintensis JM Coult amp Rose varacutifolius (JM Coult amp Rose) MunzmdashUSA California San Bernardino Co south side of Ord Mtn north of Lucerne Valley8 Apr 1988 Boyd et al 1776 (OSC) ITS-1 AY146846 ITS-2 AY146912 C ripleyi BarnebymdashUSA Nevada Nye Co Stone CabinValley 24 May 1995 Holmgren amp Holmgren 12299 (BRY) ITS-1 AY146847 ITS-2 AY146913 C rosei (ME Jones ex JM Coult ampRose) ME JonesmdashUSA Utah Sanpete Co Hills W of Ninemile Reservoir 8 May 1987 Franklin 4604 (BRY) ITS-1 AY146848ITS-2 AY146914

Glehnia littoralis F Schmidt ex Miq subsp littoralismdashTaiwan Taitung Hsien Lanyu (Orchid Island) Liao et al 1235 (MO)ITS-1 AY146849 ITS-2 AY146915 G littoralis subsp leiocarpa (Mathias) HultenmdashUSA Oregon Tillamook Co by the beachon the south side of Cape Kiwanda 10 Aug 1975 Halse 1228 (OSC) ITS-1 AY146850 ITS-2 AY146916

Lomatium ambiguum (Nutt) JM Coult amp RosemdashUSA Montana Madison Co ca 15 mi from roadrsquos end on Bear TrapCanyon Trail 30 May 1979 Lowry 1867 (ILL) ITS-1 AY146851 ITS-2 AY146917 L bradshawii (Rose ex Mathias) Mathias ampConstancemdashUSA Oregon Benton Co NE Corvallis W of Canterbury Circle area around Frazier Creek 8 Jun 1980 Halse 2215(ILL) ITS-1 AY146852 ITS-2 AY146918 L brandegei (JM Coult amp Rose) JF MacbrmdashUSA Washington Chelan Co 5 mi SWof Leavenworth on Snow Creek Trail 29 May 1977 Lowry 621 (ILL) ITS-1 AY146853 ITS-2 AY146919 L grayi (JM Coult ampRose) JM Coult amp Rose var grayimdashUSA Wyoming Lincoln Co Tunp Range Preacher Hollow 16 mi NNE of Cokeville 22May 1993 Hartman 37444 (RM) ITS-1 AY146854 ITS-2 AY146920 L grayi var depauperatum (ME Jones) MathiasmdashUSA UtahJuab Co Gilson Mountains 7 mi from Lynndyl 7 May 1981 Goodrich 15335 (RM) ITS-1 AY146855 ITS-2 AY146921

Musineon lineare (Rydb) MathiasmdashUSA Utah Cache Co Toney Grove Trailhead Trail to Naomi Peak 9 Jul 1989 Hartman24364 (RM) ITS-1 AY146856 ITS-2 AY146922

Oreonana clementis (ME Jones) JepsmdashUSA California Tulare Co Mt Guyot 22 Jul 1949 Howell 25604 (UTC) ITS-1AY146857 ITS-2 AY146923 O purpurascens Shevock amp ConstancemdashUSA California Tulare Co Sequoia National Forest SlateMountain along USFS trail 31E14 26 Apr 1977 Shevock 5428 (BRY) ITS-1 AY146858 ITS-2 AY146924 O vestita (S Watson)JepsmdashUSA California San Bernardino Co Angeles National Forest San Gabriel Mtns between Mt Harwood and Mt SanAntonio Peak 4 May 1977 Shevock 5433 (BRY) ITS-1 AY146859 ITS-2 AY146925

2004] 423SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 1 Continued

Oreoxis trotteri SL Welsh amp S GoodrichmdashUSA Utah Grand Co E of Courthouse Rock 20 May 1985 Welsh 23427 (BRY)ITS-1 AY146860 ITS-2 AY146926 USA Utah Grand Co 135 mi NNW of Moab 4 Jun 1985 Neese amp Welsh 16984 (BRY) ITS-1 AY146861 ITS-2 AY146927

Orogenia fusiformis S WatsonmdashUSA Oregon Josephine Co ats near S boundary with Siskiyou NF Oregon Caves NationalMonument 24 May 1996 Zika 12864 (OSC) ITS-1 AY146862 ITS-2 AY146928

Peucedanum japonicum Thunb ex A Murraymdashcult Botanical Conservatory Mulhouse France (no 97138) 8 Dec 1999 Reduronsn (Herb Reduron) ITS-1 AF495826 ITS-2 AF495827 P ostruthium (L) WDJ Koch (5Imperatoria ostruthium L)mdashDownie etal (1998) ITS-1 U78403 ITS-2 U78463

Podistera macounii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Alaska ridge W of Sheep Creek 30 Jun 1977 Reed etal 6047 (BRY) ITS-1 AY146863 ITS-2 AY146929 USA Alaska Mountains between Essie Creek and the headwaters of Seven-tymile Drainage 21 Jun 2002 Larsen et al sn (ALA) ITS-1 AY146864 ITS-2 AY146930 P nevadensis (A Gray) S WatsonmdashUSA California El Dorado Co Lake Tahoe Basin Management Unit Freel Peak 27 Jul 2002 Matson 634 (ILL) ITS-1 AY146865ITS-2 AY146931 P yukonensis Mathias amp ConstancemdashUSA Alaska Kathul Mountain along the Yukon River roughly halfwaybetween Eagle and Circle 11 Jun 2002 Larsen amp Batten 02-1263 (ALA) ITS-1 AY146866 ITS-2 AY146932

Polytaenia texana (JM Coult amp Rose) Mathias amp ConstancemdashUSA Texas Burnet Co E of Briggs 25 May 1985 Barrie 1403(RM) ITS-1 AY146867 ITS-2 AY146933

Pseudocymopterus longiradiatus Mathias Constance amp WL TheobmdashUSA New Mexico Otero Co Sacramento Mts ca 3mi SSW of Bent SW side of Domingo Peak 7 Aug 1990 Sivinski 1546 (UNM) ITS-1 AY146868 ITS-2 AY146934

Pteryxia davidsonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA New Mexico Grant Co Bear Mountain near SilverCity 17 Jun 1903 Metcalfe 165 (RM) ITS-1 AY146869 ITS-2 AY146935 P hendersonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Utah Duchesne Co 075 mi SE of Davis Lake Uinta Mtns 23 Aug 1996 Huber 3502 (BRY) ITS-1 AY146870 ITS-2AY146936 P petraea (ME Jones) JM Coult amp RosemdashUSA Nevada Nye Co Toiyabe National Forest Toquima Range IronSprings 5 Jun 1978 Goodrich 11292 (UTC) ITS-1 AY146871 ITS-2 AY146937 P terebinthina (Hook) JM Coult amp Rose varterebinthinamdashUSA Washington Benton Co Pit 30 between the 200 area 17 May 1993 McKinnon amp Sackschewsky 327 (BRY)ITS-1 AY146872 ITS-2 AY146938 P terebinthina var albiora (Nutt ex Torr amp A Gray) MathiasmdashUSA Wyoming SubletteCo Upper Green River Basin ca 95 air mi NNW of La Barge 20 May 1993 Hartman 37374 (RM) ITS-1 AY146873 ITS-2AY146939 USA Utah Daggett Co Flaming Gorge National Recreation area 5 Jun 1995 Refsdal amp Goodrich 3591 (RM) ITS-1AY146874 ITS-2 AY146940 P terebinthina var calcarea (ME Jones) MathiasmdashUSA Wyoming Uinta Co 275 mi N of Lone-tree 2 Jul 1999 Goodrich 26060 (BRY) ITS-1 AY146875 ITS-2 AY146941 USA Utah Daggett Co Ashley National Forest FlamingGorge National Recreation area E side of Flaming Gorge Reservoir 26 May 1992 Goodrich 24045 (BRY) ITS-1 AY146876 ITS-2 AY146942 USA Wyoming Big Horn Co Big Horn Mountains Cold Spring Road 26 May 1980 Hartman amp Dueholm 11224(RM) ITS-1 AY146877 ITS-2 AY146943 USA Wyoming Carbon Co ca 7 air mi NE of Savery 14 Jun 1979 Hartman amp Coffey8961 (RM) ITS-1 AY146878 ITS-2 AY146944 P terebinthina var californica (JM Coult amp Rose) MathiasmdashUSA CaliforniaMadera Co near Whiskey Creek 1 mi NW of Ellis Meadow Sierra Nevada 1 Jul 1938 Constance 2390 (RM) ITS-1 AY146879ITS-2 AY146945 P terebinthina var foeniculacea (Nutt ex Torr amp A Gray) MathiasmdashUSA Idaho Custer Co Morgan Creek10 mi N of Challis 26 Jun 1984 Atwood 10320 (RM) ITS-1 AY146880 ITS-2 AY146946

Selinum candollei DCmdashDownie and Katz-Downie (1996) ITS-1 U30564 ITS-2 U30565 S carvifolia (L) LmdashFrance Bas-Rhin between Herbsheim and Boofzheim 14 Aug 2001 Reduron sn (Herb Reduron) ITS-1 AY179028 ITS-2 AY179028

Seseli gracile Waldst amp KitmdashKatz-Downie et al (1999) ITS-1 AF008605 ITS-2 AF009084 S libanotis (L) WDJ KochmdashKatz-Downie et al (1999) ITS-1 AF008603 ITS-2 AF009082 S tortuosum LmdashPortugal Lisboa Sintra Praja das Macas cult BotanicalConservatory Mulhouse France (no 98042) 2 Aug 2001 Hildenbrand et al sn (Herb Reduron) ITS-1 AY179031 ITS-2 AY179031

Tauschia arguta (Torr amp A Gray) JF MacbrmdashUSA California San Diego Co Corte Madera Ranch near Pine Valley 22May 1983 Van der Werff amp Clark 4571 (MO) ITS-1 AY146881 ITS-2 AY146947 T glauca (JM Coult amp Rose) Mathias ampConstancemdashUSA California Trinity Co SE of Burnt Ranch 11 July 1990 Spellenberg 10254 (RM) ITS-1 AY146882 ITS-2AY146948 T kelloggii (A Gray) JF MacbrmdashUSA California Butte Co SW of intersection of Black Bart Road and ForbestownRoad about 35 air mi SW of Forgestown 16 Jun 1993 Ahart 6972 (UC) ITS-1 AY146883 ITS-2 AY146949

TABLE 2 Sequence characteristics of the two internal transcribed spacer (ITS) regions separately and combined for 150 terminals(159 accessions) of Apiaceae subfamily Apioideae

Sequence characteristic ITS-1 ITS-2Combined

(ITS-1 amp ITS-2)

Length variationNo of aligned positionsNo of aligned positions constantNo of aligned positions parsimony-informativeNo of aligned positions autapomorphicNo of unambiguous alignment gapsNo of unambiguous gaps parsimony-informativeMaximum pairwise sequence divergence ()

206ndash2182259189452111125

220ndash226233937961165

131

427ndash4414581841681063716121

424 [Volume 29SYSTEMATIC BOTANY

FIG 1 Strict consensus of 20000 minimal length 881-steptrees derived from equally-weighted maximum parsimonyanalysis of aligned ITS-1 and ITS-2 sequences from 150 ter-minals (159 accessions) of Apiaceae subfamily ApioideaeConsistency indices 5 04654 and 03762 with and without

not

uninformative characters respectively retention index506405 Numbers on branches represent bootstrap estimatesfor 100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-additionvalues 50 are not indicated All Cymopterus accessions areboldfaced Numbers in parentheses refer to numbers of ac-cessions bearing identical DNA sequences that were combinedto facilitate analysis

Thaspium and Zizia A large basal polytomy obscuresrelationships among these umbellifers yet four majorclades containing six or more taxa each are apparentThe rst and most distal of these clades comprises 37taxa representing 13 accessions of Cymopterus 16 ac-cessions of Lomatium 3 accessions each of Oreonanaand Pteryxia and 2 accessions of Orogenia A secondclade comprises all ve accessions of Cymopterus acau-lis plus C bulbosus C newberryi and Lomatium concin-num A third clade comprises some but not all infra-specic accessions of Pteryxia terebinthina plus Pteryxiapetraea Cymopterus corrugatus C coulteri and C ibapen-sis The fourth major clade contains Cymopterus con-stancei C macrorhizus C montanus C multinervatus Cpurpurascens and Aletes calcicola Several additionalclades of four to ve taxa each exist and are also ofheterogeneous generic composition

The results of the MP analysis revealed that Cymop-terus (boldfaced in all tree gures presented herein)and Lomatium the two largest genera of NA Apiaceaeare highly polyphyletic and that their species are in-extricably linked with those of the other endemic pe-rennial genera of the region In addition the generaAletes Musineon Oreonana Oreoxis Orogenia PodisteraPseudocymopterus Pteryxia and Tauschia are also notmonophyletic Constraining the 41 examined taxa ofCymopterus to monophyly and rerunning the MP anal-ysis resulted in trees 35 steps longer than those mostparsimonious (Table 3) Lomatium is monophyletic intrees 45 steps longer than those most parsimoniousSimilar analyses were performed by constraining eachof the other aforementioned genera with Aletes Glehn-ia Oreoxis Pteryxia and Tauschia each monophyletic intrees 10 or more steps greater than those most parsi-monious (Table 3) In contrast Polytaenia Thaspiumand Zizia are all monophyletic in unconstrainedsearches

The remaining 24 accessions examined formed a po-lytomy basal to this clade and included a monophy-letic Angelica with the inclusion of Sphenosciadium andPacic NA Glehnia littoralis subsp leiocarpa the cladeof Selinum candollei Seseli Peucedanum japonicum andPacic Asian Glehnia littoralis subsp littoralis the lattertwo taxa forming a strongly-supported sister group(97 bootstrap value) the Arracacia clade (ArracaciaPrionosciadium and Rhodosciadium) the clade of Ciclo-spermum 1 Spermolepis and Peucedanum ostruthium Se-

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

2004] 423SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 1 Continued

Oreoxis trotteri SL Welsh amp S GoodrichmdashUSA Utah Grand Co E of Courthouse Rock 20 May 1985 Welsh 23427 (BRY)ITS-1 AY146860 ITS-2 AY146926 USA Utah Grand Co 135 mi NNW of Moab 4 Jun 1985 Neese amp Welsh 16984 (BRY) ITS-1 AY146861 ITS-2 AY146927

Orogenia fusiformis S WatsonmdashUSA Oregon Josephine Co ats near S boundary with Siskiyou NF Oregon Caves NationalMonument 24 May 1996 Zika 12864 (OSC) ITS-1 AY146862 ITS-2 AY146928

Peucedanum japonicum Thunb ex A Murraymdashcult Botanical Conservatory Mulhouse France (no 97138) 8 Dec 1999 Reduronsn (Herb Reduron) ITS-1 AF495826 ITS-2 AF495827 P ostruthium (L) WDJ Koch (5Imperatoria ostruthium L)mdashDownie etal (1998) ITS-1 U78403 ITS-2 U78463

Podistera macounii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Alaska ridge W of Sheep Creek 30 Jun 1977 Reed etal 6047 (BRY) ITS-1 AY146863 ITS-2 AY146929 USA Alaska Mountains between Essie Creek and the headwaters of Seven-tymile Drainage 21 Jun 2002 Larsen et al sn (ALA) ITS-1 AY146864 ITS-2 AY146930 P nevadensis (A Gray) S WatsonmdashUSA California El Dorado Co Lake Tahoe Basin Management Unit Freel Peak 27 Jul 2002 Matson 634 (ILL) ITS-1 AY146865ITS-2 AY146931 P yukonensis Mathias amp ConstancemdashUSA Alaska Kathul Mountain along the Yukon River roughly halfwaybetween Eagle and Circle 11 Jun 2002 Larsen amp Batten 02-1263 (ALA) ITS-1 AY146866 ITS-2 AY146932

Polytaenia texana (JM Coult amp Rose) Mathias amp ConstancemdashUSA Texas Burnet Co E of Briggs 25 May 1985 Barrie 1403(RM) ITS-1 AY146867 ITS-2 AY146933

Pseudocymopterus longiradiatus Mathias Constance amp WL TheobmdashUSA New Mexico Otero Co Sacramento Mts ca 3mi SSW of Bent SW side of Domingo Peak 7 Aug 1990 Sivinski 1546 (UNM) ITS-1 AY146868 ITS-2 AY146934

Pteryxia davidsonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA New Mexico Grant Co Bear Mountain near SilverCity 17 Jun 1903 Metcalfe 165 (RM) ITS-1 AY146869 ITS-2 AY146935 P hendersonii (JM Coult amp Rose) Mathias amp ConstancemdashUSA Utah Duchesne Co 075 mi SE of Davis Lake Uinta Mtns 23 Aug 1996 Huber 3502 (BRY) ITS-1 AY146870 ITS-2AY146936 P petraea (ME Jones) JM Coult amp RosemdashUSA Nevada Nye Co Toiyabe National Forest Toquima Range IronSprings 5 Jun 1978 Goodrich 11292 (UTC) ITS-1 AY146871 ITS-2 AY146937 P terebinthina (Hook) JM Coult amp Rose varterebinthinamdashUSA Washington Benton Co Pit 30 between the 200 area 17 May 1993 McKinnon amp Sackschewsky 327 (BRY)ITS-1 AY146872 ITS-2 AY146938 P terebinthina var albiora (Nutt ex Torr amp A Gray) MathiasmdashUSA Wyoming SubletteCo Upper Green River Basin ca 95 air mi NNW of La Barge 20 May 1993 Hartman 37374 (RM) ITS-1 AY146873 ITS-2AY146939 USA Utah Daggett Co Flaming Gorge National Recreation area 5 Jun 1995 Refsdal amp Goodrich 3591 (RM) ITS-1AY146874 ITS-2 AY146940 P terebinthina var calcarea (ME Jones) MathiasmdashUSA Wyoming Uinta Co 275 mi N of Lone-tree 2 Jul 1999 Goodrich 26060 (BRY) ITS-1 AY146875 ITS-2 AY146941 USA Utah Daggett Co Ashley National Forest FlamingGorge National Recreation area E side of Flaming Gorge Reservoir 26 May 1992 Goodrich 24045 (BRY) ITS-1 AY146876 ITS-2 AY146942 USA Wyoming Big Horn Co Big Horn Mountains Cold Spring Road 26 May 1980 Hartman amp Dueholm 11224(RM) ITS-1 AY146877 ITS-2 AY146943 USA Wyoming Carbon Co ca 7 air mi NE of Savery 14 Jun 1979 Hartman amp Coffey8961 (RM) ITS-1 AY146878 ITS-2 AY146944 P terebinthina var californica (JM Coult amp Rose) MathiasmdashUSA CaliforniaMadera Co near Whiskey Creek 1 mi NW of Ellis Meadow Sierra Nevada 1 Jul 1938 Constance 2390 (RM) ITS-1 AY146879ITS-2 AY146945 P terebinthina var foeniculacea (Nutt ex Torr amp A Gray) MathiasmdashUSA Idaho Custer Co Morgan Creek10 mi N of Challis 26 Jun 1984 Atwood 10320 (RM) ITS-1 AY146880 ITS-2 AY146946

Selinum candollei DCmdashDownie and Katz-Downie (1996) ITS-1 U30564 ITS-2 U30565 S carvifolia (L) LmdashFrance Bas-Rhin between Herbsheim and Boofzheim 14 Aug 2001 Reduron sn (Herb Reduron) ITS-1 AY179028 ITS-2 AY179028

Seseli gracile Waldst amp KitmdashKatz-Downie et al (1999) ITS-1 AF008605 ITS-2 AF009084 S libanotis (L) WDJ KochmdashKatz-Downie et al (1999) ITS-1 AF008603 ITS-2 AF009082 S tortuosum LmdashPortugal Lisboa Sintra Praja das Macas cult BotanicalConservatory Mulhouse France (no 98042) 2 Aug 2001 Hildenbrand et al sn (Herb Reduron) ITS-1 AY179031 ITS-2 AY179031

Tauschia arguta (Torr amp A Gray) JF MacbrmdashUSA California San Diego Co Corte Madera Ranch near Pine Valley 22May 1983 Van der Werff amp Clark 4571 (MO) ITS-1 AY146881 ITS-2 AY146947 T glauca (JM Coult amp Rose) Mathias ampConstancemdashUSA California Trinity Co SE of Burnt Ranch 11 July 1990 Spellenberg 10254 (RM) ITS-1 AY146882 ITS-2AY146948 T kelloggii (A Gray) JF MacbrmdashUSA California Butte Co SW of intersection of Black Bart Road and ForbestownRoad about 35 air mi SW of Forgestown 16 Jun 1993 Ahart 6972 (UC) ITS-1 AY146883 ITS-2 AY146949

TABLE 2 Sequence characteristics of the two internal transcribed spacer (ITS) regions separately and combined for 150 terminals(159 accessions) of Apiaceae subfamily Apioideae

Sequence characteristic ITS-1 ITS-2Combined

(ITS-1 amp ITS-2)

Length variationNo of aligned positionsNo of aligned positions constantNo of aligned positions parsimony-informativeNo of aligned positions autapomorphicNo of unambiguous alignment gapsNo of unambiguous gaps parsimony-informativeMaximum pairwise sequence divergence ()

206ndash2182259189452111125

220ndash226233937961165

131

427ndash4414581841681063716121

424 [Volume 29SYSTEMATIC BOTANY

FIG 1 Strict consensus of 20000 minimal length 881-steptrees derived from equally-weighted maximum parsimonyanalysis of aligned ITS-1 and ITS-2 sequences from 150 ter-minals (159 accessions) of Apiaceae subfamily ApioideaeConsistency indices 5 04654 and 03762 with and without

not

uninformative characters respectively retention index506405 Numbers on branches represent bootstrap estimatesfor 100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-additionvalues 50 are not indicated All Cymopterus accessions areboldfaced Numbers in parentheses refer to numbers of ac-cessions bearing identical DNA sequences that were combinedto facilitate analysis

Thaspium and Zizia A large basal polytomy obscuresrelationships among these umbellifers yet four majorclades containing six or more taxa each are apparentThe rst and most distal of these clades comprises 37taxa representing 13 accessions of Cymopterus 16 ac-cessions of Lomatium 3 accessions each of Oreonanaand Pteryxia and 2 accessions of Orogenia A secondclade comprises all ve accessions of Cymopterus acau-lis plus C bulbosus C newberryi and Lomatium concin-num A third clade comprises some but not all infra-specic accessions of Pteryxia terebinthina plus Pteryxiapetraea Cymopterus corrugatus C coulteri and C ibapen-sis The fourth major clade contains Cymopterus con-stancei C macrorhizus C montanus C multinervatus Cpurpurascens and Aletes calcicola Several additionalclades of four to ve taxa each exist and are also ofheterogeneous generic composition

The results of the MP analysis revealed that Cymop-terus (boldfaced in all tree gures presented herein)and Lomatium the two largest genera of NA Apiaceaeare highly polyphyletic and that their species are in-extricably linked with those of the other endemic pe-rennial genera of the region In addition the generaAletes Musineon Oreonana Oreoxis Orogenia PodisteraPseudocymopterus Pteryxia and Tauschia are also notmonophyletic Constraining the 41 examined taxa ofCymopterus to monophyly and rerunning the MP anal-ysis resulted in trees 35 steps longer than those mostparsimonious (Table 3) Lomatium is monophyletic intrees 45 steps longer than those most parsimoniousSimilar analyses were performed by constraining eachof the other aforementioned genera with Aletes Glehn-ia Oreoxis Pteryxia and Tauschia each monophyletic intrees 10 or more steps greater than those most parsi-monious (Table 3) In contrast Polytaenia Thaspiumand Zizia are all monophyletic in unconstrainedsearches

The remaining 24 accessions examined formed a po-lytomy basal to this clade and included a monophy-letic Angelica with the inclusion of Sphenosciadium andPacic NA Glehnia littoralis subsp leiocarpa the cladeof Selinum candollei Seseli Peucedanum japonicum andPacic Asian Glehnia littoralis subsp littoralis the lattertwo taxa forming a strongly-supported sister group(97 bootstrap value) the Arracacia clade (ArracaciaPrionosciadium and Rhodosciadium) the clade of Ciclo-spermum 1 Spermolepis and Peucedanum ostruthium Se-

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

424 [Volume 29SYSTEMATIC BOTANY

FIG 1 Strict consensus of 20000 minimal length 881-steptrees derived from equally-weighted maximum parsimonyanalysis of aligned ITS-1 and ITS-2 sequences from 150 ter-minals (159 accessions) of Apiaceae subfamily ApioideaeConsistency indices 5 04654 and 03762 with and without

not

uninformative characters respectively retention index506405 Numbers on branches represent bootstrap estimatesfor 100000 replicate analyses using lsquolsquofastrsquorsquo stepwise-additionvalues 50 are not indicated All Cymopterus accessions areboldfaced Numbers in parentheses refer to numbers of ac-cessions bearing identical DNA sequences that were combinedto facilitate analysis

Thaspium and Zizia A large basal polytomy obscuresrelationships among these umbellifers yet four majorclades containing six or more taxa each are apparentThe rst and most distal of these clades comprises 37taxa representing 13 accessions of Cymopterus 16 ac-cessions of Lomatium 3 accessions each of Oreonanaand Pteryxia and 2 accessions of Orogenia A secondclade comprises all ve accessions of Cymopterus acau-lis plus C bulbosus C newberryi and Lomatium concin-num A third clade comprises some but not all infra-specic accessions of Pteryxia terebinthina plus Pteryxiapetraea Cymopterus corrugatus C coulteri and C ibapen-sis The fourth major clade contains Cymopterus con-stancei C macrorhizus C montanus C multinervatus Cpurpurascens and Aletes calcicola Several additionalclades of four to ve taxa each exist and are also ofheterogeneous generic composition

The results of the MP analysis revealed that Cymop-terus (boldfaced in all tree gures presented herein)and Lomatium the two largest genera of NA Apiaceaeare highly polyphyletic and that their species are in-extricably linked with those of the other endemic pe-rennial genera of the region In addition the generaAletes Musineon Oreonana Oreoxis Orogenia PodisteraPseudocymopterus Pteryxia and Tauschia are also notmonophyletic Constraining the 41 examined taxa ofCymopterus to monophyly and rerunning the MP anal-ysis resulted in trees 35 steps longer than those mostparsimonious (Table 3) Lomatium is monophyletic intrees 45 steps longer than those most parsimoniousSimilar analyses were performed by constraining eachof the other aforementioned genera with Aletes Glehn-ia Oreoxis Pteryxia and Tauschia each monophyletic intrees 10 or more steps greater than those most parsi-monious (Table 3) In contrast Polytaenia Thaspiumand Zizia are all monophyletic in unconstrainedsearches

The remaining 24 accessions examined formed a po-lytomy basal to this clade and included a monophy-letic Angelica with the inclusion of Sphenosciadium andPacic NA Glehnia littoralis subsp leiocarpa the cladeof Selinum candollei Seseli Peucedanum japonicum andPacic Asian Glehnia littoralis subsp littoralis the lattertwo taxa forming a strongly-supported sister group(97 bootstrap value) the Arracacia clade (ArracaciaPrionosciadium and Rhodosciadium) the clade of Ciclo-spermum 1 Spermolepis and Peucedanum ostruthium Se-

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

2004] 425SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

TABLE 3 The number of additional steps required to force eachgenus to monophyly in a maximum parsimony analysis with gapstates treated as missing data

Genus

No ofconstrainedaccessions

Additionalsteps

AletesCymopterusGlehniaLomatiumMusineonOreonanaOreoxisOrogeniaPodisteraPseudocymopterusPteryxiaTauschia

9412

30434252

105

1735114573

15174

1419

linum carvifolia occupied the rst branch above the rootof the tree

The single tree uncovered by ML analysis under abest-t model of DNA substitution had a 2Ln likeli-hood score of 58560673 (Fig 2A) The high-level re-lationships suggested by this tree are similar to thoseindicated by the MP analysis in the sense that all ofthe major clades identied previously also occur onthis tree The lack of resolution and generally low boot-strap support values preclude unambiguous hypothe-ses of relationship among these clades but do conrmthat many genera are not monophyletic Indeed spe-cies of Cymopterus Lomatium Pteryxia and Aletes areintertwined with many other genera Polytaenia Thas-pium and Zizia are all maintained as monophyleticand unite as a clade Outgroup relationships are alsosimilar to those inferred by the MP analysis with theexception that Angelica (with included Sphenosciadium)plus the closely allied Glehnia littoralis subsp leiocarpais sister group to the clade of NA endemics howeverthis relationship is only weakly supported (50bootstrap value)

Relationships inferred from the Bayesian tree (Fig2B) are similar to or consistent with those estimatedby MP (Fig 1) and ML (Fig 2A) methods The fourmajor clades of six or more taxa each are maintainedas is the clade comprising Polytaenia Thaspium and Zi-zia Bayesian posterior clade probabilities tend to behigher than corresponding nonparametric bootstrapsupport values (Huelsenbeck et al 2002) and this isreected in our results where many branches are sup-ported by high probability values For example mono-phyly of the perennial NA endemic taxa is supportedby a probability value of 78 whereas in the otheranalyses this clade is supported by bootstrap values50 Once more the large basal polytomy of in-group taxa is maintained and resolution of overall re-lationships is generally quite poor

DISCUSSION

Monophyly of the Perennial Endemic Genera of NAApioideae The phylogenetic results indicate that theendemic perennial apioid genera of NA (north of Mex-ico) constitute a monophyletic group albeit one that isweakly supported in all trees The similar life historyand overall general habit of many of its members theabsence of a prominent conical stylopodium in all gen-era except Podistera (where we presume it has beensecondarily derived Downie et al 2002) and uniqueamong umbellifers the shared presence of a protogyn-ous breeding system (Schlessman and Graceffa 2002)provide additional if not compelling evidence of theircommon ancestry In all trees except that of ML a po-lytomy arises sister to this clade and comprises thefollowing ve lineages (1) the perennial circumborealgenus Angelica (plus NA Sphenosciadium capitellatum)and the Pacic NA Glehnia littoralis subsp leiocarpa (2)the Old World Seseli Selinum and Peucedanum japoni-cum of which Seseli libanotis occurs in the eastern USand the Pacic Asian Glehnia littoralis subsp littoralis(3) the three examined members of the Mexican andCentral American Arracacia clade (4) the annual gen-era Spermolepis and Ciclospermum both distributedthroughout the southern US and other warm tem-perate areas and (5) Peucedanum ostruthium (5Impera-toria ostruthium L) an Old World perennial speciesdistributed sporadically in the northeastern US Sisterto this polytomy in all trees is Selinum carvifolia a pe-rennial herb common in Eurasia but restricted in NAto eastern Massachusetts In the ML tree (Fig 2A) theclade of Angelica (plus Sphenosciadium) and Glehnia lit-toralis subsp leiocarpa is sister to a clade comprising allperennial endemic NA genera lending some supportto Mathiasrsquo (1965) hypothesis that Cymopterus was de-rived from lsquolsquoAngelica-like stockrsquorsquo It has often been as-sumed that the indigenous Apiaceae of NA of whichmany were initially but erroneously referred to Euro-pean genera (eg Torrey and Gray 1840 Coulter andRose 1888) are indeed distinct morphologically fromOld World taxa (Coulter and Rose 1900 Mathias 19301971 Theobald et al 1963) The results presented here-in inferred on the basis of ITS data support this dis-tinction and suggest further that the perennial endem-ic apioid umbellifers of NA (north of Mexico) do in-deed comprise a (weakly supported) monophyleticgroup

This support for monophyly of our native NA um-bellifers is in contrast to the results inferred from allother molecular systematic studies to date Prior high-er-level studies of the family using a variety of differ-ent molecular data sets but with often very limitedsampling of these indigenous NA taxa yielded resultsthat were equivocal in documenting the relationshipsamong the latter (Downie and Katz-Downie 1996

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

426 [Volume 29SYSTEMATIC BOTANY

FIG 2 A Single tree derived from maximum likelihood analysis of aligned ITS-1 and ITS-2 sequences from 150 terminals(159 accessions) of Apiaceae subfamily Apioideae under a TrN1G model of nucleotide substitution 2Ln Likelihood 5 58560673 Numbers on branches represent bootstrap estimates for 100 replicate neighbor-joining analyses using a maximum like-

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

2004] 427SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

not

lihood model of nucleotide substitution values 50 are not indicated B Bayesian inference tree Numbers on branchesrepresent Bayesian posterior probabilities All Cymopterus accessions are boldfaced Numbers in parentheses refer to numbersof accessions bearing identical DNA sequences that were combined to facilitate analysis

Plunkett et al 1996 Downie et al 1998 2000b Plunkettand Downie 1999 Downie et al 2002) However giventhe lack of resolution and (or) low levels of branch sup-port obtained in those clades comprising these nativeNA umbellifers none of the results actually precludedthis suggestion of monophyly Because thorough taxonsampling is essential to increasing the accuracy of aphylogenetic estimate (Rannala et al 1998 Zwickl andHillis 2002) our current study of 159 exemplars (rep-resenting extensive taxon sampling of Aletes Cymop-terus Oreonana Oreoxis Pseudocymopterus Pteryxiaand Musineon among other genera) which also in-cludes circumboreal genera having NA representation(ie Angelica Seseli Selinum and Peucedanum) offersthe best estimate yet of phylogenetic relationshipsamong NA umbellifers

Nevertheless additional study is required to evalu-ate the monophyly of these protogynous NA apioidumbellifers and to elucidate their sister group giventhe weak support for their monophyly and the lack ofresolution in the cladograms In particular greater rep-resentation of Old World species is necessary espe-cially those of the Angelica clade of the apioid super-clade The low ITS sequence divergence among thewestern NA meso-American and Eurasian taxa sam-pled herein and the morphological similarities of manysuggest that the Angelica clade may have diverged rap-idly Moreover biogeographic studies of the NorthernHemisphere have suggested frequent exchanges of o-ristic elements of western NA with other biogeograph-ic areas such as eastern NA eastern Asia and south-eastern Europe (Wen 1999) thus the assumption ofmonophyly of this large and widely distributed groupof NA umbellifers needs to be tested further by in-creased sampling of plants outside of western NA Inaddition data beyond those procured from the ITS re-gion will be required to increase the number of poten-tially parsimony-informative characters in order to in-crease resolution of the phylogenetic estimate and clar-ify sister group relationships

Polyphyly of Cymopterus The genus Cymopterusas currently treated (Kartesz 1994) is clearly notmonophyletic Expanding the limits of Cymopterus toinclude Oreoxis Pseudocymopterus and Pteryxia (Cron-quist 1997) or even placing Lomatium into this amal-gamation as intimated by Cronquist fares no better inyielding a group that is monophyletic upon consider-ation of ITS data In fact no historical treatment ofCymopterus and its allies reects our phylogenetic re-sults Jones (1908) recognized a number of sections and

subgroups within a highly variable and expanded Cy-mopterus (because of lsquolsquothe futility of trying to dividethe species into separate generarsquorsquo) but none of thesetaxa parallels the groupings obtained in the ITS-de-rived trees Given the estimates of phylogeny present-ed herein and the realization that trees of much great-er length than those most parsimonious are requiredto invoke monophyly the genus Cymopterus as pres-ently circumscribed is a highly articial assemblage ofspecies that must be abandoned in any revised systemof umbellifer classication

We are not aware of any morphological synapomor-phy supporting the monophyly of Cymopterus Thecharacters used traditionally to delimit the genus areprimarily those of the fruit and these display overlap-ping patterns of variation with several other umbelli-fers endemic to western NA (Downie et al 2002) Afunctional persistent 2-cleft carpophore once consid-ered a hallmark of Cymopterus (Mathias and Constance1944ndash1945) is lacking in nearly half of its speciesthrough partial or complete adnation of its halves tothe commissural surface of the mericarps and becom-ing deciduous with them (Hartman and Constance1985 Cronquist 1997 Hartman 2000 Downie et al2002) The presence of prominent wings on all ribs ofthe fruit has also been used as a distinguishing char-acter for the genus but in several species the dorsalwings are obsolete or reduced to three inconspicuousridges and thus show similarities to fruits of Loma-tium (Mathias and Constance 1944ndash1945 Hartman andConstance 1985 Goodrich 1986 Cronquist 1997Downie et al 2002) The lateral wings of Cymopterusare usually constricted at their base but in some spe-cies they may be inated at the base Traditionally ma-ture fruits of Cymopterus have been described as com-pressed dorsally (Mathias 1930 Mathias and Con-stance 1944ndash1945) yet fruit cross-sections reveal acomplex series from fruits that are subterete or some-what compressed laterally to those that are markedlydorsally compressed (Hartman 1985 Downie et al2002) The number of vittae (oil tubes) both on thecommissure and in the intervals between the ribs isanother diagnostic character but can show much var-iation even within a species (Downie et al 2002) Inlight of the extreme polyphyly of Cymopterus and itscomplex association with other perennial endemicgenera of NA further cladistic study of morphologicalcharacters is required However such an analysis mustconsider simultaneously all NA apioid umbellifersand not just focus on specic genera as monophyly of

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

428 [Volume 29SYSTEMATIC BOTANY

each of these endemic perennial genera should not betacitly assumed

Phylogenetic Relationships Within NA ApioideaeSeveral major clades of similar composition are appar-ent in all trees presented herein but each comprisesspecies of two or more genera and most are not verywell supported Therefore until these groups receiveconrmation through additional study we are cautiousnot to recognize new assemblages of taxa We high-light some of these clades below as well as discuss thephylogenetic placements of Glehnia and Oreonana Thephylogenetic relationships of the remaining NA en-demic genera as inferred through molecular studyhave been discussed previously (Downie et al 2002)

Six species of Cymopterus belong to a distinctive spe-cies group Phellopterus (Nutt ex Torr amp A Gray) JMCoult amp Rose or Cymopterus sect Phellopterus Nutt exTorr amp A Gray (Coulter and Rose 1900 Mathias 1930Hartman 2000) Its members include C bulbosus Cconstancei C macrorhizus C montanus C multinervatusand C purpurascens These species share large showybractlets that are often basally connate All species butC bulbosus are allied with one another in the molecularphylogenies with these ve species sister to Aletes cal-cicola The separation of C bulbosus from this clade issurprising given the similarity between this speciesand C constancei This may very well be the only pre-viously identied species group of Cymopterus that issupported in part by molecular evidence

The ve varieties of Cymopterus acaulis (vars acaulisfendleri greeleyorum higginsii and parvus) form astrongly supported clade with C newberryi and Lom-atium concinnum nested within This clade is sister toCymopterus bulbosus in all trees In vegetative condi-tion plants of L concinnum may easily be confusedwith those of C acaulis however in owering or fruit-ing condition the species differ in ower color andfruit morphology ITS data for L concinnum were ob-tained from GenBank (Hardig and Soltis 1999) and itsmorphological features could not be conrmed

No infrageneric classication exists for Lomatium al-though several informal species groups have been rec-ognized such as the Cynomarthrum group (Coulter andRose 1900) the lsquolsquotuberous lomatiumrsquorsquo group (Coulterand Rose 1888 Mathias 1938 Schlessman 1984) andthe Euryptera group (Mastrogiuseppe et al 1985 Hart-man and Constance 1988 Hardig and Soltis 1999) Inthe present study the Cynomarthrum group (repre-sented by L concinnum L junceum L nuttallii L sca-brum and L triternatum subsp platycarpum) the lsquolsquotu-berous lomatiumrsquorsquo group (represented by L ambiguumL bicolor and L cous) and the Euryptera group (rep-resented by L howellii L lucidum L parvifolium L re-postum L rigidum and L shevockii) are all polyphyleticin accordance with the ndings of others (eg Sch-lessman 1984 Soltis and Novak 1997) Several mem-

bers of the Euryptera group are closely allied with spe-cies of vegetatively similar Tauschia from coastal south-ern California

Plants of the maritime genus Glehnia initially butdubiously referred to Cymopterus because of their dor-sally attened fruits bearing prominent dorsal wavywings (Gray 1860 as cited by Mathias 1928) are dis-tributed on the sandy seashores of the northern PacicThey have been variously treated as two subspecies ortwo closely allied species (Mathias 1928 Hiroe 1962)Most authors however recognize a single species com-plex comprising two subspecies (subsp leiocarpa west-ern hemisphere subsp littoralis eastern hemisphere)Differences in fruit characters are apparent betweenthese taxa (Mathias 1928) but were thought not sig-nicant enough to warrant separate specic status(Hiroe 1962) However NA Glehnia can be distin-guished from Japanese specimens by their pattern ofaccumulation of secondary metabolites in the fruit(Hiraoka et al 2002) Our analyses of ITS data reveala separation of the subspecies with the NA G littoralissubsp leiocarpa allied with Angelica and the Asian Glittoralis subsp littoralis allied with Peucedanum japoni-cum Neither taxon allies with Cymopterus The positionof subsp leiocarpa in the ML and Bayesian trees (Figs2A and 2B) is consistent with its separation from An-gelica on the basis of its distinctive fruit morphologywhereas in the MP tree (Fig 1) it falls within Angelica(but with low bootstrap support) ITS sequences fromG littoralis subsp littoralis and P japonicum are almostidentical with only one nucleotide difference betweenthem The latter was chosen for inclusion in this studybecause of its similar distribution in Pacic Asia Italso shares a similar maritime habitat However giventhe differences in habit between G littoralis subsp lit-toralis and P japonicum additional study is required toconrm their sister group relationship

Oreonana comprises three species native to Califor-nia that are adapted to montane environments char-acterized by low-temperature and extensive wintersnow cover (Shevock and Constance 1979) Theseplants are cushion-forming and coarsely hairy or to-mentose Their globose to semihemispheric infructesc-ences with numerous persistent pedicels from the sta-minate and sterile owers aid in seed dispersal In allITS-derived trees O purpurascens and O clementis areclosely related in contrast O vestita is positioned awayfrom this group thus pending further study we pre-sume the genus is not monophyletic Oreonana hasbeen proposed to be closely related to Tauschia (Shev-ock and Constance 1979) but in all ITS trees its speciesally closely with Cymopterus and Lomatium

The lack of basal resolution among the taxa sampledcoupled with the presence of many distinct (yet weak-ly supported) lineages in all cladograms is consistentwith a pattern of rapid radiation in western NA

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

2004] 429SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

(Downie et al 2002) a mode of evolution suggestedpreviously for Lomatium (Soltis et al 1995 Hardig andSoltis 1999) and Osmorhiza Raf (Apiaceae Wen et al2002) Single trees uncovered in the MP analysis (notshown) were similar to that tree indicated by the MLanalysis (Fig 2A) in having short internal branchesand generally much longer terminal branches the lat-ter indicative of numerous autapomorphies This un-equal distribution of branch lengths suggests that thisgroup of umbellifers originated and diversied rela-tively quickly and that since their formation its majorlineages and species have persisted in isolation Manyspecies of Cymopterus and other western NA umbelli-fers are narrowly distributed and have strict edaphicrequirements (Mathias 1930 1965) suggesting thatspeciation in the group may be related to geologicalboundaries and edaphic shifts (Hardig and Soltis1999) While this group of largely aridity-adapted taxalacks a fossil record it may be assumed that their rapidradiation accompanied late Tertiary and Quaternarygeologic and climatological events that gave rise towidespread aridity and great habitat diversication inwestern NA that promoted speciation when new areasbecame available for colonization (Stebbins 1952 Ma-thias 1965 Delcourt and Delcourt 1993 Wen 2001)With approximately 200 species of Apioideae endemicto western NA the bulk of which comprises Cymop-terus Lomatium and their allies the region is recog-nized as a major center of differentiation for the sub-family (Mathias 1965) Rapid radiation is a commonpattern of long-term evolution (Futuyma 1997) amongplants it has been reported for several other taxa en-demic to continental or specically western NA (egRieseberg et al 1991 Schwarzbach and Kadereit 1995Wojciechowski et al 1999 Hershkovitz and Zimmer2000) Through additional gene sequencing and furtherevaluation of morphological characters (F-J Sun un-published data) we hope eventually to be able to re-solve the early branching patterns of these perennialendemic NA umbellifers although we realize that sucha study poses extraordinary challenges (Fishbein et al2001)

Generic delimitation in the Apiaceae is often vagueand arbitrary (Constance 1987 Cronquist 1997) andmany species-rich genera (and especially those of theapioid superclade) are polyphyletic (Downie et al2000b 2000c Spalik et al 2001) Unfortunately our re-sults do little to refute these statements Of all the pe-rennial endemic apioid genera of NA considered here-in only Polytaenia Thaspium and Zizia are each re-solved as monophyletic on the basis of phylogeneticanalyses of ITS data However the latter two generaare remarkably similar in appearance and their genericlimits too have been questioned (Ball 1979 Lindsey1982 Cooperrider 1985) Alas until we have evidenceto the contrary it appears that the vast majority of

perennial apioid genera endemic to NA are not mono-phyletic A complete reassessment of the generic limitsof all of our native umbellifers is clearly required asis a comprehensive simultaneous analysis of both Newand Old World Apiaceae

ACKNOWLEDGEMENTS The authors thank D S Katz-Downiefor assistance in the laboratory the curators of the herbaria citedin the text for access to specimens D Murray A Batten A LarsenC Roland and S Matson for supplying material of Podistera andJ Wen and one anonymous reviewer for comments on the manu-script This work was supported by NSF grants DEB 9407712 andDEB 0089452 to SRD and by Francis M and Harlie M Clark re-search support grants a Herbert Holdsworth Ross Memorial Fundaward a Program in Ecology and Evolutionary Biology SummerResearch Fellowship and travel grants from the Graduate Collegethe John R Laughnan Fund and the Program in Ecology and Evo-lutionary Biology to F-J Sun This paper represents a portion ofa PhD dissertation submitted by F-J Sun to the Graduate Collegeof the University of Illinois at Urbana-Champaign

LITERATURE CITED

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

BALL P W 1979 Thaspium trifoliatum (meadow-parsnip) in Can-ada The Canadian Field Naturalist 93 306ndash307

BARRIE F R and M A SCHLESSMAN 1987 The incidence of pro-togyny in the North American species of Apioideae (Api-aceae) American Journal of Botany 74 646 (Abstract)

CONSTANCE L 1987 Neogeozia (Apiaceae) a very distinct and el-egant genus of Mexican Umbelliferae Opera Botanica 92 59ndash71

COOPERRIDER T S 1985 Thaspium and Zizia (Umbelliferae) inOhio Castanea 50 116ndash119

COULTER J M and J N ROSE 1888 Revision of North AmericanUmbelliferae Crawfordsville Wabash College

mdashmdashmdash and mdashmdashmdash 1900 Monograph of the North American Um-belliferae Contributions from the U S National Herbarium 7 1ndash256

CRONQUIST A 1997 Apiaceae Pp 340ndash427 in Intermountain oravascular plants of the Intermountain West U S A Vol 3 partA eds A Cronquist N H Holmgren and P K HolmgrenBronx The New York Botanical Garden

DELCOURT P A and H R DELCOURT 1993 Paleoclimates pa-leovegetation and paleooras during the Late QuaternaryPp 71ndash94 in Flora of North America Vol 1 ed Flora of NorthAmerica Editorial Committee New York Oxford UniversityPress

DOWNIE S R and D S KATZ-DOWNIE 1996 A molecular phy-logeny of Apiaceae subfamily Apioideae evidence from nu-clear ribosomal DNA internal transcribed spacer sequencesAmerican Journal of Botany 83 234ndash251

mdashmdashmdash mdashmdashmdash and K SPALIK 2000a A phylogeny of Apiaceaetribe Scandiceae evidence from nuclear ribosomal DNA in-ternal transcribed spacer sequences American Journal of Bot-any 87 76ndash95

mdashmdashmdash mdashmdashmdash and M F WATSON 2000b A phylogeny of theowering plant family Apiaceae based on chloroplast DNArpl16 and rpoC1 intron sequences towards a supragenericclassication of subfamily Apioideae American Journal of Bot-any 87 273ndash292

mdashmdashmdash M F WATSON K SPALIK and D S KATZ-DOWNIE 2000cMolecular systematics of Old World Apioideae (Apiaceae)

430 [Volume 29SYSTEMATIC BOTANY

relationships among some members of tribe Peucedaneaesensu lato the placement of several island-endemic speciesand resolution within the apioid superclade Canadian Journalof Botany 78 506ndash528

mdashmdashmdash S RAMANATH D S KATZ-DOWNIE and E LLANAS 1998Molecular systematics of Apiaceae subfamily Apioideae phy-logenetic analyses of nuclear ribosomal DNA internal tran-scribed spacer and plastid rpoC1 intron sequences AmericanJournal of Botany 85 563ndash591

mdashmdashmdash G M PLUNKETT M F WATSON K SPALIK D S KATZ-DOWNIE C M VALIEJO-ROMAN E I TERENTIEVA A VTROITSKY B-Y LEE J LAHHAM and A EL-OQLAH 2001Tribes and clades within Apiaceae subfamily Apioideae thecontribution of molecular data Edinburgh Journal of Botany 58301ndash330

mdashmdashmdash R L HARTMAN F-J SUN and D S KATZ-DOWNIE 2002Polyphyly of the spring-parsleys (Cymopterus) molecular andmorphological evidence suggests complex relationshipsamong the perennial endemic genera of western NorthAmerican Apiaceae Canadian Journal of Botany 80 1295ndash1324

EVERT E F and L CONSTANCE 1982 Shoshonea pulvinata a newgenus and species of Umbelliferae from Wyoming SystematicBotany 7 471ndash475

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

FISHBEIN M C HIBSCH-JETTER D E SOLTIS and L HUFFORD2001 Phylogeny of Saxifragales (Angiosperms Eudicots)analysis of a rapid ancient radiation Systematic Biology 50817ndash847

FUTUYMA D J 1997 Evolutionary biology 3rd ed Sunderland Sin-auer

GILMARTIN A J and K S SIMMONS 1987 Relationships of Lom-atium among other genera of Apiaceae Plant Systematics andEvolution 157 95ndash103

GOODRICH S 1986 Utah ora Apiaceae (Umbelliferae) Great Ba-sin Naturalist 46 66ndash106

HARDIG T M and P S SOLTIS 1999 An ITS-based phylogeneticanalysis of the Euryptera species group in Lomatium (Api-aceae) Plant Systematics and Evolution 219 65ndash78

HARTMAN R L 1985 A new species of Cymopterus (Umbelliferae)from southern Idaho Brittonia 37 102ndash105

mdashmdashmdash 2000 A new species of Cymopterus (Apiaceae) from theRocky Mountain region U S A Brittonia 52 136ndash141

mdashmdashmdash and L CONSTANCE 1985 Two new species of Cymopterus(Umbelliferae) from western North America Brittonia 37 88ndash95

mdashmdashmdash and mdashmdashmdash 1988 A new Lomatium (Apiaceae) from theSierran crest of California Madrono 35 121ndash125

HERSHKOVITZ M A and E A ZIMMER 2000 Ribosomal DNAevidence and disjunctions of western American Portulaca-ceae Molecular Phylogenetics and Evolution 15 419ndash439

HILLIS D M and J P HUELSENBECK 1992 Signal noise and re-liability in molecular phylogenetic analyses Journal of Hered-ity 83 189ndash195

HIRAOKA N J-I CHANG L R BOHM and B A BOHM 2002Furanocoumarin and polyacetylenic compound compositionof wild Glehnia littoralis in North America Biochemical System-atics and Ecology 30 321ndash325

HIROE M 1962 Supplementary notes on the genus Glehnia (Um-belliferae) Acta Phytotaxonomica et Geobotanica 19 39ndash44

HITCHCOCK C L and A CRONQUIST 1961 Vascular plants of thePacic Northwest Part 3 Saxifragaceae to Ericaceae Univer-sity of Washington Publications in Biology 17 506ndash587

HUELSENBECK J P and F RONQUIST 2001 MrBayes Bayesian in-ference of phylogeny Bioinformatics 17 754ndash755

mdashmdashmdash B LARGET R E MILLER and F RONQUIST 2002 Potentialapplications and pitfalls of Bayesian inference of phylogenySystematic Biology 51 673ndash688

JEANMOUGIN F J D THOMPSON M GOUY D G HIGGINS and TJ GIBSON 1998 Multiple sequence alignment with Clustal XTrends in Biochemical Sciences 23 403ndash405

JONES M E 1908 New species and notes Contributions to WesternBotany 12 1ndash81

KARTESZ J T 1994 A synonymized checklist of the vascular ora ofthe United States Canada and Greenland Second edition Biota ofthe North American Program of the North Carolina Botanical Gar-den Portland Timber Press

KATZ-DOWNIE D S C M VALIEJO-ROMAN E I TERENTIEVA AV TROITSKY M G PIMENOV B-Y LEE and S R DOWNIE1999 Towards a molecular phylogeny of Apiaceae subfamilyApioideae additional information from nuclear ribosomalDNA ITS sequences Plant Systematics and Evolution 216 167ndash195

LINDSEY A H 1982 Floral phenology patterns and breeding sys-tems in Thaspium and Zizia (Apiaceae) Systematic Botany 71ndash12

mdashmdashmdash and C R BELL 1980 Protogyny and associated reproduc-tive characters in Apiaceae P 269 in Second International Con-gress of Systematic and Evolutionary Biology The University ofBritish Columbia Abstract

MASTROGIUSEPPE J D S J GILL K S SIMMONS and G K BROWN1985 Morphologic and cytotaxonomic evaluation of Lomatiumtuberosum (Apiaceae) Brittonia 37 252ndash260

MATHIAS M E 1928 Studies in the Umbelliferae I Annals of theMissouri Botanical Garden 15 91ndash109

mdashmdashmdash 1930 Studies in the Umbelliferae III A monograph ofCymopterus including a critical study of related genera An-nals of the Missouri Botanical Garden 17 213ndash476

mdashmdashmdash 1938 A revision of the genus Lomatium Annals of the Mis-souri Botanical Garden 25 225ndash297

mdashmdashmdash 1965 Distribution patterns of certain Umbelliferae Annalsof the Missouri Botanical Garden 52 387ndash398

mdashmdashmdash 1971 Systematic survey of New World Umbelliferae Pp13ndash29 in The biology and chemistry of the Umbelliferae ed V HHeywood New York Academic Press

mdashmdashmdash and L CONSTANCE 1944ndash1945 Umbelliferae Pp 43ndash295 inNorth American Flora Vol 28B New York The New York Bo-tanical Garden

mdashmdashmdash and mdashmdashmdash 1981 Two new Umbelliferae of the Chihu-ahuan Desert Brittonia 33 342ndash346

MORT M E P S SOLTIS D E SOLTIS and M L MABRY 2000Comparison of three methods for estimating internal supporton phylogenetic trees Systematic Biology 49 160ndash171

PLUNKETT G M and S R DOWNIE 1999 Major lineages withinApiaceae subfamily Apioideae a comparison of chloroplastrestriction site and DNA sequence data American Journal ofBotany 86 1014ndash1026

mdashmdashmdash D E SOLTIS and P S SOLTIS 1996 Evolutionary patternsin Apiaceae inferences based on matK sequence data System-atic Botany 21 477ndash495

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

RANNALA B J P HUELSENBECK Z YANG and R NIELSEN 1998Taxon sampling and the accuracy of large phylogenies Sys-tematic Biology 47 702ndash710

RIESEBERG L H S M BECKSTROM-STERNBERG A LISTON and DM ARIAS 1991 Phylogenetic and systematic inferences fromchloroplast DNA and isozyme variation in Helianthus sect He-lianthus (Asteraceae) Systematic Botany 16 50ndash76

SCHLESSMAN M A 1984 Systematics of tuberous lomatiums (Um-belliferae) Systematic Botany Monographs Vol 4 55 pp

mdashmdashmdash and L M GRACEFFA 2002 Protogyny pollination and sexexpression of andromonoecious Pseudocymopterus montanus(Apiaceae Apioideae) International Journal of Plant Sciences163 409ndash417

mdashmdashmdash D G LLOYD and P P LOWRY II 1990 Evolution of sexual

2004] 431SUN ET AL RELATIONSHIPS AMONG ENDEMIC NA APIOIDEAE

systems in New Caledonian Apiaceae Memoirs of the New YorkBotanical Garden 55 105ndash117

SCHWARZBACH A E and J W KADEREIT 1995 Rapid radiation ofNorth American desert genera of the Papaveraceae evidencefrom restriction site mapping of PCR-amplied chloroplastDNA fragments Plant Systematics and Evolution 9 159ndash170

SHEVOCK J R and L CONSTANCE 1979 A new species of Oreon-ana a genus of snow-adapted Umbelliferae Madrono 26 128ndash134

SIMMONS K S 1985 Systematic studies in Lomatium PhD thesisWashington State University Pullman Washington

SOLTIS P S and R K KUZOFF 1993 ITS sequence variation withinand among populations of Lomatium grayi and L laevigatum(Umbelliferae) Molecular Phylogenetics and Evolution 2 166ndash170

mdashmdashmdash and S J NOVAK 1997 Phylogeny of the tuberous Loma-tiums (Apiaceae) cpDNA evidence for morphological con-vergence Systematic Botany 22 99ndash112

mdashmdashmdash mdashmdashmdash T M HARDIG and D E SOLTIS 1995 Phylogeneticrelationships and rapid radiation in Lomatium (Apiaceae)American Journal of Botany 82(6 supplement) 163

SPALIK K A WOJEWODZKA and S R DOWNIE 2001 Delimitationof genera in Apiaceae with examples from Scandiceae sub-tribe Scandicinae Edinburgh Journal of Botany 58 331ndash346

STEBBINS G L 1952 Aridity as a stimulus to plant evolutionAmerican Naturalist 86 33ndash44

SWOFFORD D L 1998 PAUP Phylogenetic analysis using parsi-mony (and other methods) Version 4 Sunderland SinauerAssociates

TAMURA K and M NEI 1993 Estimation of the number of nucle-otide substitutions in the control region of mitochondrialDNA in humans and chimpanzees Molecular Biology and Evo-lution 10 512ndash526

THEOBALD W L C C TSENG and M E MATHIAS 1963 A re-vision of Aletes and Neoparrya (Umbelliferae) Brittonia 16296ndash315

TORREY J and A GRAY 1840 A ora of North America Vol 1 NewYork Hafner Publishing Company

VARGAS P B G BALDWIN and L CONSTANCE 1999 A phylo-genetic study of Sanicula sect Sanicoria and S sect Sandwi-censes (Apiaceae) based on nuclear rDNA and morphologicaldata Systematic Botany 24 228ndash248

WEBER W A 1984 New names and combinations principally inthe Rocky Mountain orandashIV Phytologia 55 1ndash11

WEN J 1999 Evolution of eastern Asian and eastern North Amer-ican disjunct distributions in owering plants Annual Reviewof Ecology and Systematics 30 421ndash455

mdashmdashmdash 2001 Evolution of Eastern Asian-Eastern North Americanbiogeographic disjunctions a few additional issues Interna-tional Journal of Plant Sciences 162(6 supplement) S117ndashS122

mdashmdashmdash P P LOWRY II J L WALCK and K-O YOO 2002 Phylo-genetic and biogeographic diversication in Osmorhiza (Api-aceae) Annals of the Missouri Botanical Garden 89 414ndash428

WHITE T J T BRUNS S LEE and J TAYLOR 1990 Amplicationand direct sequencing of fungal ribosomal RNA genes forphylogenetics Pp 315ndash322 in PCR Protocols a guide to methodsand applications eds M A Innis D H Gelfand J J Sninskyand T J White San Diego Academic Press

WOJCIECHOWSKI M F M J SANDERSON and J-M HU 1999 Ev-idence of the monophyly of Astragalus (Fabaceae) and its ma-jor subgroups based on nuclear ribosomal DNA ITSand chlo-roplast DNA trnL intron data Systematic Botany 24 409ndash437

ZWICKL D J and D M HILLIS 2002 Increased taxon samplinggreatly reduces phylogenetic error Systematic Biology 51 588ndash598