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(American Journal of Botany. 1998;85:1787-1795.)
© 1998 Botanical Society of America, Inc.

Phylogenetic analysis of Artemisia section Tridentatae (Asteraceae) based on sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA1

Amy B. Kornkven3,a, Linda E. Watson2,b and James R. Estes6

a Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma, 73019; and b Department of Botany, Miami University, Oxford, Ohio 45056


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Artemisia sect. Tridentatae is composed of 11 species of xerophytic shrubs, which dominate much of western North America. Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were used to construct a phylogeny, examine circumscription of the section, resolve interspecific relationships, and test competing hypotheses on the origin of the section. The data support the monophyly of sect. Tridentatae, with the exclusion of A. bigelovii and A. palmeri—two historically, anomolous species. However, the ITS data provide insufficient variation to fully resolve interspecific relationships or to support major lineages within the Tridentata clade. Nuclear and chloroplast DNA phylogenies are discordant, which may be a result of interspecific gene flow and subsequent chloroplast capture, particularly related to the placement of A. filifolia and A. californica, in addition to A. bigelovii. Furthermore, the ITS data are in conflict with cpDNA data, providing equivocal evidence for competing hypotheses on the Old World vs. New World origin for the section and do not provide support for definitive subgeneric placement.

Key Words: Anthemideae • Artemisia • Asteraceae • ITS (internal transcribed spacer) nucleotide sequences • phylogeny


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Species of Artemisia sect. Tridentatae Rydb. (subg. Seriphidium) are some of the most ecologically and economically dominant shrubs in western North America. The section, broadly defined, encompasses 11 species of mostly xerophytic woody shrubs, which have an extensive distribution throughout western North America. However, despite extensive investigations since the early 1900s, sect. Tridentatae presents numerous taxonomic difficulties (Rydberg, 1916; Hall and Clements, 1923; Ward, 1953; Beetle, 1960; McArthur and Plummer, 1978; Shultz, 1983), with little agreement on circumscription, interspecific relationships, or subgeneric placement.

Previous studies, including morphology (Rydberg, 1916; Hall and Clements, 1923; Beetle, 1960), anatomy (Moss, 1940; Carlquist, 1966; Shultz, 1983), cytology (Ward, 1953; McArthur and Plummer, 1978; McArthur, Pope, and Freeman, 1981), and chemistry (Irwin, 1971; Hanks et al., 1973; Geissman and Irwin, 1974; Kelsey, 1974), support the monophyly and circumscription of sect. Tridentatae with two important exceptions. Both A. palmeri A. Gray and A. bigelovii A. Gray have been variously included within and excluded from sect. Tridentatae (subg. Seriphidium). The presence of homogamous flower heads has resulted in most authors including A. palmeri within sect. Tridentatae, an herbaceous species endemic to southern California (Hall and Clements, 1923; Ward, 1953; Bremer and Humphries, 1993; Ling, 1995a, b). However, A. palmeri is also morphologically similar to members of subg. Artemisia (Moss, 1940; Beetle, 1960; McArthur, Pope, and Freeman, 1981; Shultz, 1983). Artemisia bigelovii is included primarily in sect. Tridentatae, on the basis of overall morphological similarity to A. tridentata Nutt. (Beetle, 1960; Poljakov, 1961; McArthur and Plummer, 1978; McArthur, Pope, and Freeman, 1981; Bremer and Humphries, 1993). However, the presence of occasional heterogamous flower heads suggests an affinity to subg. Artemisia (Hall and Clements, 1923; Ward, 1953; Shultz, 1983; Ling, 1995a). Chloroplast (cp) DNA restriction site data support exclusion of A. palmeri and inclusion of A. bigelovii (Kornkven, 1997; Kornkven, Watson, and Estes, in press). Additionally, the monophyly of sect. Tridentatae was further called into question by the cpDNA phylogeny, which unexpectedly placed A. californica and A. filifolia within the Tridentata clade (Kornkven, 1997; Kornkven, Watson, and Estes, in press).

Two intrasectional lineages have been proposed for sect. Tridentatae on the basis of leaf morphology, habitat preference, and ability to root sprout (Ward, 1953; Beetle, 1960; Shultz, 1983). However, there is little agreement on the circumscription of the two lineages or on the relationships among species within each lineage, which may be a result of the little morphological divergence among species. Chloroplast DNA data failed to resolve relationships among the Tridentatae species (Kornkven, 1997; Kornkven, Watson, and Estes, in press), which may be a result of relatively recent diversification and rapid expansion of the Tridentata lineage throughout western North America during the Pleistocene.

Historically, capitular and reproductive morphologies have been the diagnostic characters for the recognition of three subgenera in Artemisia s.l.: subg. Artemisia L. (disciform, heterogamous capitula, with pistillate ray florets, and perfect fertile disk florets); subg. Dracunculus (Besser) Rydb. (disciform, heterogamous capitula with pistillate ray florets and staminate disk florets); and subg. Seriphidium (Besser) Rouy (discoid, homogamous capitula with perfect, fertile disk florets). However, Artemisia has also been narrowly defined with several segregate genera recognized, including Seriphidium Polj., among others (Poljakov, 1961; Ling, 1991, 1995a, b; Bremer and Humphries, 1993; Bremer, 1994). Traditionally, sect. Tridentatae has been placed in Seriphidium, but has also been raised to subgeneric status within Artemisia s.l. (McArthur, Pope, and Freeman, 1981). However, the latter classification has not been widely adopted, with Tridentatae generally considered a section of Seriphidium (regardless of the subgeneric vs. segregate genus status for Seriphidium) (Ling, 1991, Bremer and Humphries, 1993; 1995b; Bremer, 1994).

Two conflicting hypotheses have been proposed to explain the origin of sect. Tridentatae within Artemisia s.l. Ling (1991, 1995b) suggests that the progenitors of sect. Tridentatae are members of Asian Seriphidium that migrated over the Bering Strait. In contrast, McArthur and Plummer (1978) suggest that sect. Tridentatae originated from herbaceous members of subg. Artemisia and differentiated in situ in North America during the Pleistocene, in response to the extreme climatic changes. Unfortunately, cpDNA restriction site data do not provide unequivocal evidence for support of either of these two hypotheses (Kornkven, 1997; Kornkven, Watson, and Estes, in press).

The internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA) have been widely used to resolve phylogenetic relationships for many plant taxa (reviewed in Baldwin, 1992; Baldwin et al., 1995). The objective of this study was to construct a phylogeny of sect. Tridentatae using nucleotide sequence data from ITS to (1) examine the monophyly and circumscription of the section, (2) resolve interspecific relationships, (3) examine congruence between ITS and cpDNA phylogenies in order to assess possible interspecific gene flow and/or chloroplast capture, and (4) evaluate alternative hypotheses on the origin and subgeneric placement of sect. Tridentatae within Artemisia s.l.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All 11 species of sect. Tridentatae s.l. were sampled, including two anomalous species, A. bigelovii and A. palmeri (Table 1). Multiple populations of each species were not sequenced because low levels of sequence divergence were observed during the early stages of the study. Thirteen outgroup species were sampled, including representatives from each subgenus of Artemisia and five genera from different subtribes of tribe Anthemideae (Table 1). All North American taxa were collected from natural field populations; the European and Asian taxa were obtained primarily from botanic gardens. Voucher specimens are deposited at the University of Oklahoma Robert Bebb Herbarium (OKL), unless otherwise noted (Table 1).


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  		Table 1. List of taxa used for the ITS analysis, with locality data, voucher specimens, and GenBank accession numbers. Collection and voucher information includes: AK = Amy Kornkven, vouchers located at the University of Oklahoma (OKL); LS = Leila Shultz, vouchers at Utah State University (UTC); and LW = Lind Watson, vouchers at Uppsala Herbarium (UPS; Botanic Garden Material) or KEW unvouchered DNAs (Accession numbers). The ITS spacers were submitted separately, with the first accession number referring to ITS1 and the second to ITS2. * indicates sequences from Francisco-Ortega et al., 1997.

 
Total DNA was extracted from ~1.5 g of frozen (-80°C) leaf tissue. The leaves were finely ground in liquid nitrogen with 4% w/v polyvinylpyrrolidone (PVP-40) with a mortar and pestle. Total DNA was extracted using 2x CTAB and further purified on cesium chloride/ethidium bromide gradients (Sambrook, Fritsch, and Maniatis, 1989; Palmer, 1986a, b; Doyle and Doyle, 1987). A single fragment containing both ITS regions and the 5.8S cistron was amplified via PCR (polymerase chain reaction), using primers ITS4 and ITS5 of White et al. (1990) and Baldwin (1992). The PCR reaction consisted of 1x buffer, 2.5 mmol/L MgCl2 solution, 10 mmol/L dNTPs in equimolar ratio, 10 µmol/L primer, 10–50 ng of template DNA, and 2.5 units of AmpliTaq DNA Polymerase (Perkin Elmer Corp., Branchburg, NJ). The samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 48°C for 2 min, 72°C for 2 min, with a final extension phase of 7 min at 72°C. The double-stranded PCR products were purified by electrophoresis on 2% low melting point agarose (NuSieve GTG, FMC Bioproducts, Rockland, MD). The DNA bands were then excised from the gel and melted at 65°–75°C for ~10 min. Either glassmilk (GeneClean, Bio101, Vista, CA) or Wizard columns (Promega, Madison, WI) were used to further purify and concentrate the amplified DNA.

ITS sequences were obtained by either direct manual sequencing [A. tripartita Rydb., A. pygmaea Gray, A. longiloba (Oster.) Beetle, A. cana, A. rigida (Nutt.) Gray, A. arbuscula Nutt., and A. dracunculus L.], automated sequencing [A. tridentata, A. nova A. Nels., A. palmeri, A. abrotanum L., A. californica, A. ludoviciana Nutt., A. rupestris L., and A. sublessingiana (Kell.) Krasch. Ex Poljak.], or a combination of both [A. bigelovii, A. filifolia, A. rothrockii Gray, and A. vulgaris L.]. For manual sequencing, the purified double-stranded PCR products were sequenced directly using Sequenase 2.0 (U.S. Biochemical Corp., Cleveland, OH) and S dATP labeling. The standard dideoxy chain-termination method was followed using two forward (ITS1 and ITS3) and two reverse (ITS2 and ITS4) primers to sequence the entire ITS region (Sanger, Nicklen, and Coulsen, 1977; White et al., 1990; Baldwin, 1992; Francisco-Ortega et al., 1997). Primer concentrations of 2.5 µmol/L generated optimal sequencing product. The DNA/primer mixture was denatured by boiling the double-stranded DNA; the annealing mixture was then flash-frozen in liquid nitrogen and thawed at the beginning of the extension reaction (Gyllensten, 1989; Conti, Fischback, and Sytsma, 1993; Rodman et al., 1993; Baum, Sytsma, and Hoch, 1994). The labeled fragments were separated by gel electrophoresis on 6% polyacrylamide with 1x Tris Borate buffer (TBE). Both short and long gels were run to sequence both strands in their entirety. The gels were fixed for 30 min in 10% acetic acid/12% ethanol and transferred to 3 MM Whatman paper. The gels were dried under vacuum at 80°C and exposed to Kodak XAR film (Sigma Chemical Co., St. Louis, MO) for 24–72 h. For automated sequencing, the PCR products were purified on 2% low-melting-point agarose. The excised DNA bands were further purified and concentrated using Wizard Columns (Promega, Madison, WI). The purified double-stranded PCR products were sequenced using AmpliTaq DNA Polymerase FS (Perkin Elmer Corp., Branchburg, NJ), in a dye terminator mix, on an ABI automated sequencer (Model 373A). The two forward (ITS1 and ITS3) and two reverse (ITS2 and ITS4) primers (3.2 mol/L) were used to sequence both ITS regions and part of the 5.8S gene (White et al., 1990; Baldwin, 1992). Sequencher 2.1 (Gene Codes Corp. Inc., Ann Arbor, MI) was used to examine the resulting chromatograms and to align the sequences.

The boundaries of both ITS regions were determined by comparison with published and unpublished sequences from other Asteraceae taxa (Baldwin, 1992, 1993; Kim and Jansen, 1994; Bain and Jansen, 1995; Sang et al., 1994, 1995; Bayer, Hufford, and Soltis, 1996; Francisco-Ortega et al., 1997) and deposited in GenBank (Table 1). All ITS sequences for Artemisia species, including sect. Tridentatae and outgroup species, were aligned manually using sequential pairwise comparisons. The presence of several small insertions and deletions was not a significant factor in aligning the sequences. Each indel was excluded from the phylogenetic analysis and coded as missing data. The indels were then mapped onto the consensus tree to examine their phylogenetic distribution.

Phylogenetic analysis
The sequence data were analyzed with PAUP 3.1.1 (Swofford, 1993). Sequence divergence values were calculated for all pairwise comparisons over all characters using the DISTANCE MATRIX option in PAUP. A modified island search was conducted using the heuristic search option and TBR (Tree-Bisection-Reconnection) branch swapping, MULPARS on, ACCTRAN optimization, and 1000 replicate tree searches with random taxon addition and order (Maddison, 1991; Conti, Fischbach, and Sytsma, 1993). Bootstrap (Felsenstein, 1985) and decay values (Bremer, 1988) were calculated to obtain estimates of support for clades. The bootstrap option in PAUP was run with 100 replicates, simple taxon addition, and TBR branch-swapping. AutoDecay 2.3 (Eriksson and Wilkstrom, 1995), in conjunction with PAUP, to generate decay values for each clade. Strict and semistrict consensus trees were generated from each set of equally parsimonious trees.

To examine congruence between the nuclear and organellar phylogenies, the cpDNA restriction site tree was rerooted with A. dracunculus as the outgroup. This was based on a greater sampling of outgroups for the ITS analysis, which placed A. dracunculus in a basal position. In addition, a combined analysis of the ITS sequence and cpDNA restriction site data sets was conducted using the heuristic search option of PAUP with TBR branch swapping, MULPARS on, and ACCTRAN optimization. The combined data set included 18 taxa and 541 characters (486 characters for ITS and 55 for cpDNA data), with A. dracunculus as the outgroup.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ITS sequences
The ITS spacers of Artemisia sect. Tridentatae are 253 and 222 base pairs (bp) in length for ITS1 and ITS2, respectively. Within Artemisia s.l., ITS1 ranges from 249 to 253 bp, while ITS2 ranges from 222 to 223 bp. The sizes of both ITS spacers in Artemisia s.l. fall within the range of sizes observed for other Asteraceae taxa (Baldwin, 1992; Kim and Jansen, 1994; Baldwin et al., 1995; Sang et al., 1995; Bayer, Hufford, and Soltis, 1996). The aligned sequences require four gaps of 1–2 bp each for ITS1, while the alignment of ITS2 requires only a single gap of 1 bp. Three indels are autapomorphic, while two are potentially phylogenetically informative. One indel, a 2-bp deletion, unites A. sublessingiana and A. canariensis. The second indel, a 1-bp deletion, was present in three Artemisia species (A. canariensis, A. rupestris, and A. dracunculus) and four of the five outgroup species (except Anthemis). Ambiguous nucleotide positions were infrequent in either ITS region, with 10 and 13 in ITS1 and ITS2, respectively. Two ITS length variants were observed in several populations of A. tridentata and A. nova. Polyploidy may have been a factor in these populations. Diploid (2x) and tetraploid (4x) populations have been documented within both species using both chromosomal and randomly amplified polymorphic DNA (RAPD) evidence (Ward, 1953; McArthur, Pope, and Freeman, 1981; McArthur et al., in press). Several additional populations of both species were then surveyed with no apparent length variation.

In ITS1, 78 (30.5%) nucleotide base positions are variable, 20 of which are potentially phylogenetically informative and 58 of which are autapomorphic. In ITS2, 59 (26.4%) positions are variable, 22 are potentially phylogenetically informative and 37 are autapomorphic. Within sect. Tridentatae s.s. (sensu stricto), and excluding the two anomalous species, A. bigelovii and A. palmeri, 14 positions (5.5%) are variable in ITS1 and 20 (9.0%) are variable in ITS2. The inclusion of A. bigelovii and A. palmeri increases the amount of interspecific variation to 35 (ITS1) and 32 (ITS2) positions, respectively, not including indels or ambiguous polymorphisms. Almost all of the variation observed among sequences was due to point mutations and was not a result of indels or other length variants. Pairwise divergence values within sect. Tridentatae s.s. range from 0.0 to 4.1%. Divergence values between A. bigelovii and A. palmeri to sect. Tridentatae s.s. range from 2.8 to 5.1% and from 5.8 to 8.0%, respectively. Divergence values within Artemisia s.l. range from 0.0 to 8.1% and 4.2 to 22% between Artemisia and the five outgroup genera, respectively.

Phylogenetic analysis
The ITS data produced 392 equally most parsimonious trees, 356 steps in length (CI [Consistency Index] = 0.71, RI [Retention Index] = 0.54, RC [Consistency Index Rescaled] = 0.38; Fig. 1). Monophyly of Artemisia s.l. is strongly supported, with subg. Dracunculus (represented by A. dracunculus and A. filifolia) and A. bigelovii basal within Artemisia s.l. Among the other 17 species of Artemisia, two clades are supported that include sect. Tridentatae s.s. and the North American A. vulgaris species complex. A close relationship among A. vulgaris, A. ludoviciana, and A. palmeri is weakly supported (bootstrap <50%). In addition, six species from subg. Artemisia and A. sublessingiana (subg. Seriphidium) form a weakly supported clade (2-bp substitutions; bootstrap <50%) on the semistrict consensus tree, which collapses on the strict consensus tree forming an unresolved polytomy with the Tridentata clade.



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  		Fig. 1. Strict consensus of 392 equally parsimonious trees from analysis of ITS sequence data for 20 Artemisia and five outgroup species (CI = 0.71, RI = 0.54, RC = 0.38, and tree length = 35). Numbers above the branches indicate the number of character state changes supporting each branch. Subgeneric delimitation indicated by shading, sect. Tridentatae s.l. indicated in bold, and * indicates outgroup species.

 
The combined analysis of cpDNA and ITS data sets produced 183 equally most parsimonious trees with two clades supported on the strict consensus tree (not shown; CI = 0.78, RI = 0.64, RC = 0.50, and tree length = 231 excluding invariant characters). Sect. Tridentatae forms a monophyletic clade, with A. rigida and A. californica sister to the remainder of the Tridentata clade, which includes both A. bigelovii and A. filifolia. The four species in subg. Artemisia also form a clade, with A. vulgaris and A. abrotanum sister to A. ludoviciana and A. palmeri. Artemisia dracunculus (subg. Dracunculus) forms a polytomy with A. sublessingiana (subg. Seriphidium), the Tridentata clade, and the subg. Artemisia clade. The combined molecular phylogeny has a topology most closely resembling the cpDNA based phylogeny.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Monophyly and circumscription of sect. Tridentatae
The ITS phylogeny supports the monophyly of sect. Tridentatae s.s., with the exclusion of both anomalous species, A. bigelovii and A. palmeri (Fig. 1). Historically, the phylogenetic relationship of these two species to sect. Tridentatae has been problematic, with both species possessing character states that have been variously interpreted as either plesiomorphic or secondary reversals (Hall and Clements, 1923; Ward, 1953; Beetle, 1960; McArthur and Plummer, 1978; McArthur et al., 1979; Shultz, 1983; Bremer and Humphries, 1993; Ling, 1991, 1995b). An understanding of the placement of these anomalous species may provide valuable information concerning the character state polarity of capitular morphology, a pivotal character to understanding and defining subgeneric and sectional limits within Artemisia s.l.

Artemisia palmeri has been previously included with the other North American Tridentatae (subg. Seriphidium) on the basis of several synapomorphies, including the presence of homogamous flower heads that possess perfect fertile disk florets, a 4–7 series of involucral bracts, and narrowly linear to lanceolate anther appendages (Hall and Clements, 1923; Ward, 1953; Ling, 1991, 1995b; Bremer and Humphries, 1993). However, an affinity of A. palmeri to subg. Artemisia is supported by the presence of receptacular bracts, highly polymorphic florets (in size and number, with ~12–25 disk florets per head), herbaceousness, lack of interxylary cork, and other anatomical characteristics associated with species found in mesophytic habitats (Moss, 1940; Beetle, 1960; McArthur and Plummer, 1978; Shultz, 1983). In the ITS tree, A. palmeri is sister to A. ludoviciana and A. vulgaris of subg. Artemisia, rather than to members of sect. Tridentatae or other members of subg. Seriphidium (Fig. 1). Chloroplast DNA restriction site data (Kornkven, 1997; Kornkven, Watson, and Estes, in press) also support their sister relationship and inclusion of A. palmeri in subg. Artemisia.

Artemisia bigelovii has been previously excluded from sect. Tridentatae, based on the presence of occasional heterogamous flower heads, typically possessing one pistillate ray floret and two perfect disk florets (Hall and Clements, 1923; Ward, 1953; Shultz, 1983; Ling, 1991, 1995a). However, chromosomal studies (karyotype analysis) and vegetative similarity to A. tridentata support its inclusion in sect. Tridentatae (Beetle, 1963; McArthur and Plummer, 1978; McArthur, Pope, and Freeman, 1981). Bremer and Humphries (1993) include A. bigelovii in the segregate genus Seriphidium on the basis of involucral and anther characters, and they suggest that heterogamy in A. bigelovii is either plesiomorphic or a secondary reversal. In contrast, the ITS data place A. bigelovii clearly outside of the Tridentata clade, with a sister-group relationship to A. dracunculus and A. filifolia (subg. Dracunculus) (Fig. 1). Although capitula in A. bigelovii are heterogamous, the disk florets are perfect and fertile, in contrast to the heterogamous capitula of subg. Dracunculus, which are characterized by sterile, functionally male disk florets. However, the cpDNA phylogeny differs from the ITS phylogeny in the placement of A. bigelovii, as well as for A. filifolia (Fig. 2). On the cpDNA tree, A. bigelovii is sister to A. filifolia and A. longiloba and is clearly embedded within the Tridentata clade, with a bootstrap value of 96%. The conflict between the ITS and cpDNA phylogenies may be a result of interspecific hybridization and transfer of a nrDNA (nuclear DNA) repeat type from a species in subg. Dracunculus to A. bigelovii, followed by fixation of the repeat in the nuclear genome, which would result in a gene tree that does not accurately reflect species relationships. If this occurred, it could explain the unexpected placement of A. bigelovii in the ITS tree. Concerted evolution, resulting from either unequal crossing-over and/or gene conversion, may result in the fixation of a gene within a population and ultimately within a species (Zimmer et al., 1980; Appels and Dvorak, 1982; Arnheim, 1983; Avise, 1989; Doyle, 1992; Wendel, Schnabel, and Seelanan, 1995). The presence of seven autapomorphies in the nrDNA repeat of A. bigelovii suggests past introgression or possible lineage sorting that may have occurred during the early diversification of Artemisia in North America. Alternatively, interspecific gene flow between A. bigelovii and an unsampled species is also possible, although the sister relationship of A. bigelovii and A. filifolia in both molecular trees supports gene flow between these two species. It is more likely that introgression and chloroplast capture of the Tridentatae chloroplast genome by A. filifolia occurred, which may explain the anomalous placement of A. filifolia within the Tridentata clade in the cpDNA tree since chloroplast capture has been documented in numerous taxa (Rieseberg and Soltis, 1991; Rieseberg and Brunsfeld, 1992; Soltis and Kuzoff, 1995; Bayer, Hufford, and Soltis, 1996; Soltis, Johnson, and Looney, 1996; Campbell et al., 1995). Therefore, bidirectional, interspecific gene flow between A. bigelovii and A. filifolia, involving exchange of both nuclear and chloroplast genomes can be invoked to explain the sister relationship and the anomalous placement of both species in the cpDNA and ITS trees. These two taxa occur sympatrically throughout much of their distribution, although no hybridization has been reported between the extant species. Both hybridization and introgression are common phenomena within Artemisia s.l. (Hall and Clements, 1923; Keck, 1946; Ward, 1953; Beetle, 1960; McArthur et al., 1979; McArthur, Pope, and Freeman, 1981; McArthur, Welch, and Sanderson, 1988; Freeman et al., 1991; Weber et al., 1994; Wang et al., 1997) and may be significant factors in many plant species (Anderson, 1949; Stebbins, 1950, 1969; Grant, 1981).



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  		Fig. 2. Comparison of strict consensus of analysis from both ITS sequence and cpDNA restriction site data, with the distribution of capitular morphology mapped onto each tree. (A) Strict consensus of 392 trees for the ITS sequence analysis (CI = 0.73, RI = 0.54, RC = 0.397, and tree length = 188). (B) Strict consensus of 144 trees for cpDNA analysis, rerooted with A. dracunculus (CI = 0.93, RI = 0.93, RC = 0.86, and tree length = 82). Capitular morphology indicated by shading, sect. Tridentatae indicated in bold, and * indicates problematic species.

 
Two divergent species, A. rigida and A. pygmaea, are united with the Tridentata clade in the ITS phylogeny. Both species are uniquely adapted to restricted habitats in western North America and exhibit specialized modifications to extreme conditions of aridity in both anatomy and morphology (Hall and Clements, 1923; Carlquist, 1966; Shultz, 1983). Artemisia rigida is restricted primarily to rocky outcrops along the Snake River plains in western Oregon and Washington. Artemisia pygmaea, a dwarf subshrub, is found in isolated populations on calcareous soils in the cold deserts of Nevada, Utah, and northern Arizona. Rydberg (1916) placed the species into two separate sections, sect. Rigidae Rydb. and sect. Pygmaea Rydb. in subg. Seriphidium, based on their specialized morphologies. However, the ITS data support a basal position of A. rigida and A. pygmaea to sect. Tridentatae (Fig. 1).

The cpDNA phylogeny places A. californica sister to A. rigida and basal within the Tridentata clade (Fig. 2). Artemisia californica, a low shrub that is endemic to the cismontane region of California, is traditionally included in subg. Artemisia on the basis of heterogamous flower heads that contain pistillate ray florets and perfect, fertile disk florets (Hall and Clements, 1923). Several possible explanations have been proposed for the placement of A. californica in the cpDNA tree, including a classification that does not accurately reflect relationships, lineage sorting, and introgression with subsequent chloroplast capture (Kornkven, 1997; Watson, Kornkven, and Estes, in press). In contrast to the cpDNA tree, the relationship of A. californica to sect. Tridentatae is unresolved on the ITS tree, with A. californica, the remaining six species of subg. Artemisia (including A. palmeri), sect. Tridentatae, and A. sublessingiana forming a polytomy. Artemisia californica and A. rigida do not share a sister relationship in the ITS tree.

In summary, sect. Tridentatae is monophyletic, with the exclusion of A. palmeri and A. bigelovii. The inclusion of A. rigida and A. pygmaea in sect. Tridentatae is strongly supported by both molecular and nonmolecular data (Hall and Clements, 1923; Ward, 1950; Beetle, 1960; McArthur and Plummer, 1978; McArthur et al., 1981; Shultz, 1983; Ling, 1991, 1995b; Bremer and Humphries, 1993; Kornkven, 1997; Kornkven, Watson, and Estes, in press), and therefore both species should be retained in sect. Tridentatae. Interspecific gene flow is one possible explanation for the conflicting placement of A. bigelovii and A. filifolia on the ITS and cpDNA phylogenies. The placement of A. californica is unresolved by the molecular data.

Phylogenetic relationships within sect. Tridentatae
While the ITS sequence data support the monophyly of sect. Tridentatae, the results do not support recognition of either of the two previously proposed lineages within the section (Ward, 1953; Beetle, 1960; Shultz, 1983; Fig. 1). Although a close relationship between A. cana and A. tripartita has been previously proposed on the basis of nonmolecular data (= A. cana lineage), the two species only share a 1-bp substitution for ITS, and furthermore, no cpDNA restriction site mutations unite these two species. However, both molecular and nonmolecular data support A. rigida and A. pygmaea as basal within or sister to the section (Kornkven, 1997; Kornkven, Watson, and Estes, in press).

The difficulty in clearly defining species boundaries in sect. Tridentatae has resulted in both A. nova and A. longiloba being variously treated as subspecies of either A. arbuscula [i.e., A. arbuscula subsp. nova (Nels.) Ward and A. arbuscula subsp. longiloba (Osterh.) Shultz] or A. tridentata [i.e., A. tridentata subsp. nova (Nels.) Hall and Clements] (Hall and Clements, 1923; Ward, 1953; Shultz, 1983; Ling, 1991, 1995b). However, the molecular data support the distinctness of these two taxa in that they possess 11 and four autapomorphies, respectively, accounting for 37 and 13% of all autapomorphies in the Tridentatae. Therefore, based on the ITS data A. nova and A. longiloba appear to be highly divergent taxa within sect. Tridentatae.

In conclusion, neither ITS sequence nor cpDNA restriction site data have sufficient variation to resolve interspecific relationships within sect. Tridentatae, indicating that additional sampling within the section would not provide greater resolution. The level of phylogenetic resolution in the two molecular phylogenies is similar; however, some clades in the cpDNA tree are more strongly supported with higher bootstrap values (ranging from 55 to 98%). A relatively recent and rapid radiation of sect. Tridentatae species throughout the Intermountain region of western North America, may account for the low sequence divergence in both the nuclear and organellar genomes. Pollen records indicate that Artemisia is a relative newcomer to this region, with Artemisia pollen first evident in the Upper Miocene and only becoming widespread and significant during the Pleistocene (Gray, 1964; Mehringer, 1965; Tidwell, Rushforth, and Simper, 1972). Therefore, sect. Tridentatae s.s. appears to have diverged relatively recently in response to changing climatic conditions, which has resulted in a group of closely related shrub species that differ only slightly in morphology and habitat, but which are well defined morphologically from other Artemisia species.

Origin and relationship of sect. Tridentatae within Artemisia s.l.
Two contrasting hypotheses for the origin of sect. Tridentatae have been proposed. Based on floral morphology, Bremer and Humphries (1993) and Ling (1991, 1995b) support the inclusion of sect. Tridentatae within the segregate genus Seriphidium and propose that progenitors of the North American Tridentatae are Asian Seriphidium species that migrated over the Bering Strait and subsequently underwent rapid speciation in response to available habitats and climatic conditions throughout the West. In contrast, McArthur and Plummer (1978) propose that sect. Tridentatae evolved from North American progenitors in subg. Artemisia, and developed in situ in western North America during the Pleistocene in response to a rapidly changing environment. The ITS sequence and cpDNA restriction site data provide equivocal evidence for these two hypotheses on the origin and relationship of the North American sect. Tridentatae within Artemisia s.l. Unfortunately, despite numerous systematic investigations using both molecular and nonmolecular data, the origin and relationship of sect. Tridentatae within Artemisia remain unresolved (Rydberg, 1916; Hall and Clements, 1923; Ward, 1953; Carlquist, 1966; McArthur and Plummer, 1978; Shultz, 1983; Ling, 1991, 1995b; Bremer and Humphries, 1993; Kornkven, 1997; Kornkven, Watson, and Estes, in press).

Although the monophyly of Artemisia s.l. is supported by the ITS sequence data (bootstrap < 50%; decay = 1), further studies that include additional representative species of subtribe Artemisiinae are clearly needed to more adequately assess subgeneric relationships within this large and diverse genus of over 400 species. However, several preliminary comments can be made concerning relationships within Artemisia s.l. Hall and Clements (1923), in the first phylogenetic treatment of the genus, consider subg. Artemisia (as sect. Abrotanum) as ancestral and the reduction of the capitulum in both subg. Dracunculus and subg. Seriphidium as derived. In a more recent evolutionary treatment of Artemisia, Ling (1991, 1995b) concurs and proposes a complicated scenario concerning the evolution of the group, treating members of both subg. Seriphidium and subg. Dracunculus as derived. Specifically, Ling suggests that members of sect. Abrotanum were the progenitors of subg. Seriphidium, and that there was a reduction in both ray florets and number of disk florets within subg. Seriphidium. In sharp contrast to Ling's hypothesis, subg. Dracunculus is basal in the ITS phylogeny, raising questions concerning the polarity and evolution of capitular morphology within Artemisia s.l. (Figs. 1, 2).

The segregation of subg. Seriphidium from Artemisia s.l. is not supported by either cpDNA restriction site or ITS sequence phylogenies (Figs. 1, 2). This is in contrast to previous classifications of tribe Anthemideae (Poljakov, 1960; Ling, 1991, 1995a, b; Bremer and Humphries, 1993), which narrowly define Artemisia and segregate numerous genera, including Seriphidium. The relationship of the North American sect. Tridentatae to the Eurasian subg. Seriphidium remains unresolved, with sect. Tridentatae s.s., A. sublessingiana (subg. Seriphidium), and representatives from subg. Artemisia forming a polytomy in both molecular phylogenies. Further studies are clearly needed to examine not only subgeneric relationships within Artemisia s.l. and generic circumscription, but also to examine generic relationships and monophyly of subtribe Artemisiinae.

Character evolution
The ITS and cpDNA phylogenies provide an independent framework in which to examine the evolution of capitular morphology within Artemisia s.l. Capitular morphology encompasses at least four different, variable characters, including (1) the presence or absence of ray florets in the capitula, (2) disk florets that are either perfect and fertile or sterile and functionally staminate, with reduced ovaries, (3) a glabrous or hairy receptacle, and (4) a reduction in the total number of florets per head. Historically, sectional classification in Artemisia s.l. has been based on the presence of four types of capitula, with more recent studies that either combine the sections into three subgenera or narrowly define Artemisia s.l. and segregate several genera (Rydberg, 1916; Hall and Clements, 1923; Poljakov, 1961; Ling, 1991, 1995a, b; Bremer and Humphries, 1993). The complete reduction of marginal ray florets (to discoid, homogamous capitula) and an overall reduction in the number of disk florets (i.e., 2–15 florets per capitulum) is considered derived within Artemisia s.l. (Hall and Clements, 1923; Ling, 1991, 1995a, b), a conclusion that is further supported by the molecular data (Fig. 2). Bremer and Humphries (1993) support a single origin of homogamy in the segregate genus Seriphidium, include all 11 Tridentatae species (including A. palmeri), and consider heterogamy in A. bigelovii as either plesiomorphic or a secondary reversal. In contrast, molecular data support at least two independent origins of homogamy, with the placement of A. palmeri in subg. Artemisia. The presence of sterile disk florets in subg. Dracunculus can be interpreted as either plesiomorphic within the genus or as an independent loss of fertility from perfect, fertile disk florets to sterile florets. In the Bremer and Humphries (1993) cladistic analysis, the presence of disciform, heterogamous capitula with pistillate ray florets and perfect, fertile disk florets is plesiomorphic within subtribe Artemisiinae, supporting multiple origins of heterogamous capitula that possess sterile disk florets. Examination of capitula morphology across the entire genus, using an independent assessment of phylogeny (e.g., molecular markers), is essential to understanding this pivotal character in the classification and evolution of Artemisia s.l.


   FOOTNOTES
 
1 The authors thank Timothy Evans for laboratory assistance, Leila Schultz for discussion on taxonomy and species identifications, Sara Hoot for access to laboratory and automated DNA sequencing facilities, Angie Fox for producing the illustrations, Robert Jansen and Javier Francisco-Ortega for primer and ITS sequences of some species, and Leila Schultz, E. Durant MacArthur, and an anomymous reviewer for carefully reading the manuscript and providing useful comments.

This paper is a portion of a dissertation by the senior author, submitted to the University of Oklahoma in partial fulfillment of a Ph.D. This research was supported by National Science Foundation grants DEB9311086 to LEW and ABK; BSR9110375, DEB9408019, and DEB9596274 to LEW. Back

2 Author for correspondence (email:watsonle{at}muohio.edu ). Back

3 Current address: Department of Biology, University of Wisconsin–Milwaukee, Milwaukee, Wisconsin 53201. Back

6 Current address: University of Nebraska State Museum and School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588. Back


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 MATERIALS AND METHODS
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