Estimation of the Age of Extant Ephedra Using Chloroplast rbcL Sequence Data

Jinling Huang1 and Robert A. Price

Department of Plant Biology, University of Georgia, Athens, Georgia The distinctive gymnosperm genus Ephedra is sometimes considered to have originated over 200 million years (Myr) ago on the basis of ephedroid fossil pollen. In this article we estimate the age of extant Ephedra using chloroplast rbcL gene sequences. Relative rate tests fail to reject the null hypothesis of equal rates of nucleotide substitution of the rbcL sequences among three landmark lineages (Gnetales, Pinaceae, and Ginkgo). The most divergent sequences we have found in Ephedra differ by only 7 bp for an 1,110 bp region of rbcL sequence, whereas the differences among genera range from 92 to 107 bp in Gnetales and from 35 to 92 bp in Pinaceae. Using three landmark events, the age of extant Ephedra is estimated to be approximately 832 Myr. Our result is consistent with the current distribution of many Ephedra species in geologically recent habitats and points out difculties in the identication of older ephedroid pollen fossils with the modern genus Ephedra.

Introduction Molecular sequence data have often been used to test the existence of molecular clocks among lineages and to estimate the approximate timing of evolutionary events. These molecular clocks assume the constancy of the rate of nucleotide substitution, and thus the sequence divergence between two lineages is approximately proportional to the time after they diverged from their most recent common ancestor. Methods used to test the existence of molecular clocks among biological lineages include the relative rate test and the likelihood ratio test (LRT). The relative rate test rst uses a reference taxon to derive a relative distance or rate of nucleotide substitution between a species (or lineage) and a reference taxon, and then compares the distances or rates of nucleotide substitution of two measured species (or lineages) (Sarich and Wilson 1973). A modied relative rate test method has also been proposed by Li and Bousquet (1992) to compare the rates of nucleotide substitution among several lineages simultaneously. The relative rate test method has been used in many studies, including dating the divergence of major lineages of extant seed plants (Savard et al. 1994). Tests of molecular clocks among lineages using the LRT method compare the likelihood of two hypotheses (molecular clock versus no molecular clock) for a given phylogenetic tree, and the likelihood ratio can be used to measure the support for one hypothesis versus the other (Huelsenbeck and Rannala 1997; Sanderson 1998). This method has been used to test the rate constancy of DNA sequence evolution in the studies of the diversication of Hawaiian silversword alliance from their mainland ancestors (Baldwin and Sanderson 1998) and the split of Adansonia (Bombacaceae) in major continents (Baum, Small, and Wendel 1998). Ephedra (Ephedraceae) is a gymnosperm genus of about 5060 species that are widely distributed in the temperate and subtropical areas of the world except
1 Present address: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia.

southern Africa and Australia (Kubitzki 1990; Price 1996). This genus has been of considerable scientic interest for its pharmaceutical value and evolutionary position. Many Eurasian species of Ephedra contain ephedrine and pseudoephedrine, alkaloids used to treat cough, asthma, and bronchitis (Caveney et al. 2001). Ephedra and the other gnetalean genera, Gnetum and Welwitschia, share a series of angiosperm-like features, such as presence of vessel members in the wood, double fertilization, and sometimes insect pollination; therefore they have often been considered to be the closest living relatives to the owering plants (see reviews in Doyle 1996; Price 1996), although more recent molecular phylogenetic studies suggest a closer relationship of Gnetales to conifers or other gymnosperms and parallel origins for these angiospermlike features (Goremykin et al. 1996; Winter et al. 1999; Bowe, Coat, and dePamphilis 2000; Chaw et al. 2000). The origin of Ephedra has sometimes been considered to be ancient, possibly as early as or prior to the breakup of Pangaea (ca. 230 Myr ago in the Middle Triassic) on the basis of the early occurrence of ephedroid pollen fossils (Hunziker 1995; Shen 1995), although no other conrmatory evidence has been found. However, because ephedroid pollen (elongate and marked by longitudinal ribs and furrows) was much more diverse during the Mesozoic and has been found in other plant groups both within and outside the Gnetales, these pollen fossils may not have any direct connection to the extant genus Ephedra (Hughes 1994). In addition, scarcity of megafossils of Ephedra, lack of occurrence of extant species or rigorously determined fossils of Ephedra in southern Africa and Australia, and little gross morphological differentiation within the genus seem to imply a more recent diversication of extant Ephedra. In studies on the molecular phylogenetics of Ephedra, we also found very little sequence divergence in the chloroplast matK and rbcL genes (Huang 2000). In this study, we use the relative rate test method to make a preliminary estimation of the age of extant Ephedra. Materials and Methods Taxa utilized in our rbcL sequence comparisons are listed in table 1. The chloroplast rbcL gene was chosen
435

Key words: time of origin, Ephedra, rate of nucleotide substitution, rbcL, molecular clock. E-mail: tupistra@yahoo.com.
Mol. Biol. Evol. 20(3):435440. 2003 DOI: 10.1093/molbev/msg049 2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

436 Huang and Price

Table 1 GenBank Accession Numbers of the Species Used in the Study and the Nucleotide Substitutions per Site from Equisetum telmateia for a 1100 bp Region of the rbcL Gene
Accession Number AF489532 AF489533 U72819 AJ235814 AB019801 AF456381 AF145459 AB027311 AJ235804 AF313580 Observed Substitutions 165 167 188 173 184 182 173 180 183 0 Nucleotide Substitutions per Site 0.1652 0.1686 0.1928 0.1754 0.1858 0.1846 0.1743 0.1823 0.1858 0

Species Ephedra aspera (Ephedraceae) Ephedra fragilis (Ephedraceae) Gnetum gnemon (Gnetaceae) Welwitschia mirabilis (Welwitschiaceae) Pinus wallichiana (Pinaceae) Cedrus deodara (Pinaceae) Nothotsuga longibracteata (Pinaceae) Cathaya argyrophylla (Pinaceae) Ginkgo biloba (Ginkgoaceae) Equisetum telmateia (Equisetaceae)

for this study because it possesses a moderate rate of nucleotide substitution and sequences can be easily aligned among distant groups of seed plants. This is not the case for the more rapidly evolving chloroplast-encoded matK gene (Huang 2000). Sequences of the rbcL gene were obtained for approximately 20 species of Ephedra (Huang 2000) and the taxa with the maximum pairwise divergence, a representative Eurasian species (E. fragilis) and North American species (E. aspera), were used for our comparisons. Outgroup taxa from the Gnetaceae, Welwitschiaceae, Pinaceae, Ginkgoaceae, and Equisetaceae were obtained from previously published sequences in GenBank. Voucher specimens of E. aspera and E. fragilis are deposited in the herbarium of the University of Georgia (GA). The other groups were selected to use the following landmark events: (1) diversication of Pinaceae (ca. 140 Myr; Savard et al. 1994); (2) divergence of the Gnetales as approximated by the earliest occurrence of ephedroid pollen fossils in the Middle Permian (ca. 250 Myr ago; Hult and Crane 1988); and (3) divergence of the major lineages of extant seed plants (ca. 290 Myr ago; Savard et al. 1994). A sequence of 1,110 bp (bp 551164) of the rbcL gene (encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase) was used for the phylogenetic analysis. No length variation was found among the species sampled. The fern species Equisetum telmateia was used as a distinct outgroup in the phylogenetic analyses outside the Gnetales, Pinaceae, and Ginkgo. Both parsimony analysis using the exhaustive search algorithm and Neighbor-Joining analysis of the sequence data were conducted using PAUP* 4.0b10 (Swofford 2002). Bootstrap values (Felsenstein 1985) were obtained with 100 pseudoreplicates to assess relative support for the branches. To evaluate the constancy of the nucleotide substitution among lineages, the relative rate test using

Equisetum telmateia as the reference group was employed. Nucleotide substitutions per site between Equisetum telmateia and other sampled species were calculated under both one-parameter (Jukes and Cantor 1969) and twoparameter (Kimura 1980) models using MEGA version 1.02 (Kumar, Tamura, and Nei 1993). Both synonymous and nonsynonymous substitutions were used for the calculation because of the overall sparsity of substitutions of the rbcL gene in Ephedra. The constancy of nucleotide substitutions per site between any two groups was evaluated using the standard two-sample z proportion test (Snedecor and Cochran 1989). The age of living Ephedra was estimated based on the time of a landmark event. If the molecular clock hypothesis is accepted between Ephedra and a landmark lineage, the landmark event would be used to estimate the rate of nucleotide substitution of the lineage (R) and the age of extant Ephedra. The rate of nucleotide substitution is dened as the number of nucleotide substitutions per site per year (Li and Graur 1991) and is calculated using the equation R 5 K/(2T), where K is the average number of nucleotide substitutions per site of the landmark lineage and can be obtained from all possible pairwise sequence comparisons among branching groups immediately above the landmark node (Savard et al. 1994), and T is the age of the landmark event. The age of extant Ephedra was estimated by dividing half of the maximum pairwise nucleotide substitutions per site (6 SE) of Ephedra by the rate of nucleotide substitution of the landmark lineage. The maximum pairwise nucleotide substitutions per site of Ephedra was obtained from E. fragilis and E. aspera as described at the beginning of this section. Results Maximum parsimony analysis yielded two minimum length trees of 498 steps with a consistency index excluding uninformative characters of 0.6395 and a retention index of 0.6460. The strict and the majority rule consensus trees provided the same topology and are shown in gure 1. The phenogram of the Neighbor-Joining analysis is shown in gure 2. The nucleotide substitutions per site between Equisetum telmateia and other sampled species using the oneparameter model and the test statistics on their constancy are given in tables 1 and 2, respectively. Those calculated under the two-parameter model are similar and not shown here. The results of the z tests using both one-parameter and two-parameter models fail to reject the constancy of nucleotide substitutions per site among the gnetalean genera Ephedra, Gnetum and Welwitschia, Pinaceae (Pinus, Cedrus, Nothotsuga, and Cathaya), and Ginkgo. Therefore all three landmark events were used to estimate the age of extant Ephedra. The average number of nucleotide substitutions per site was 0.1083 6 0.0104 in the Gnetales under the one-parameter model, which yielded a rate of nucleotide substitution of 2.166 6 0.21 3 1010 based on a landmark time of 250 Myr for the origin of the Gnetales. The number of nucleotide substitutions per site was 6.4 6 2.4 3 103 between E. aspera and E. fragilis, which provided an

Estimation of Age of Extant Ephedra 437

FIG. 1.Strict consensus of two equally parsimonious trees from rbcL gene sequence data. Tree length 5 498 steps. CI 5 0.6395. RI 5 0.6460. Bootstrap values out of 100 pseudoreplicates are given above the branches.

estimated age of extant Ephedra of 14.77 6 5.54 Myr. The two-parameter model gave a very similar result with an estimated age of extant Ephedra of 14.60 6 5.47 Myr. With a landmark time of 290 Myr for the divergence of seed plants, the average number of substitutions among major lineages of seed plants was 0.1365 6 0.0119 under the one-parameter model, and the rate of nucleotide substitution was 2.353 6 0.205 3 1010. This leads to an estimated age of 13.60 6 5.10 Myr for extant Ephedra. Under the two-parameter model, the age of extant Ephedra was estimated to be 13.34 6 5.00 Myr. To utilize the landmark event of the diversication of Pinaceae, four species representing the major groups of the Pinaceae, Pinus wallichiana, Cedrus deodara, Nothotsuga longibracteata, and Cathaya argyrophylla, were sampled. These four species showed a similar rate of nucleotide substitution for the rbcL sequence. The rbcL sequence of Cathaya is distantly related to those of other three species, and phylogenenetic analyses provided a topology that placed Cathaya sister to Ginkgo and other Pinaceae, which is contradictory to the popular hypothesis that Pinaceae are monophyletic. With Cathaya included in the analysis, the age of extant Ephedra was estimated at 10.36 6 3.88 Myr under the one-parameter model and 10.26 6 3.85 Myr under the two-parameter model based on a landmark time of 140 Myr for the diversication of Pinaceae. However, when Cathaya was excluded from analysis, the estimated ages of extant Ephedra were 23.58 6 8.84 Myr and 23.39 6 8.77 Myr under the two models. In contrast, if a hypothetical time of divergence between E. aspera and E. fragilis is posited at 50 Myr, the

FIG. 2.Phenogram of Neighbor-Joining analysis of the rbcL gene sequence data. Numbers indicate bootstrap values out of 100 pseudoreplicates.

time of divergence of the Gnetales and that of seed plants would be pushed much earlier, ca. 846856 6 84 Myr and 10661087 6 97 Myr, respectively, well before the Cambrian period or the age of the earliest known vascular land plants, which is approximately 420 Myr ago (Stewart and Rothwell 1993).

Discussion Statistical analysis showed rate constancy of nucleotide substitution in the rbcL gene among the Gnetales, Pinaceae, and Ginkgo (table 2). Estimation using three landmark events suggested an age of the living Ephedra of approximately 832 Myr. The estimated age of extant Ephedra is much more recent than the split of Pangaea or the Triassic or Jurassic Periods, suggested by some authors as possible times of origin for the genus (Hunziker 1995; Shen 1995). The goal of this study is to estimate the age of extant Ephedra independent of the uncertainties of identication of possible ephedroid materials in the fossil record. It is not our intention to emphasize the exact time scale obtained from this analysis. Because only one chloroplast gene was used for the calibration, the result is subject to some uncertainty. However, we would expect the age of extant Ephedra to be less than 32 Myr, which

438 Huang and Price

Table 2 z Statistics for Tests of the Molecular Clock Among Genera

E. aspera E. fragilis G. gnemon W. mirabilis P. wallichiana C. deodara N. longibracteata Ca. argyrophylla G. biloba E. aspera E. fragilis G. gnemon W. mirabilis P. wallichiana Ce. deodara N. longibracteata Ca. argyrophylla G. biloba 0.2148 1.6972 1.4824 0.6393 0.4245 1.0579 1.2763 1.0615 0.4230 0.6370 1.2035 0.9887 0.4938 0.5642 0.0728 0.5711 0.3563 1.1262 0.0682 0.7052 0.6324 1.0635 0.8487 0.6338 0.4242 0.2128 0.1400 0.4924 1.2763 1.0615 0.4230 0.6370 0.0000 0.0728 0.7052 0.2128

NOTE.The null hypothesis assumes no signicant difference in nucleotide substitutions per site between two species, and the null hypothesis is rejected at the signicance level of a 5 0.05, i.e., z 5 1.96.

means that the diversication of extant species of Ephedra would have begun in a period from the Early-Middle Oligocene to the Miocene. Otherwise, the age of the Gnetales would be far beyond that of the earliest known vascular land plants, which is impossible. Comparisons of nuclear ribosomal ITS sequences for a broad sampling of Ephedra species suggests an early split between Old World and New World groups in the genus (Huang 2000), and our nding that representative rbcL sequences from these groups differ by only 7 bp for the 1,110 bp region of our comparisons in contrast to a range of 92 to 107 bp among the three gnetalean genera and 35 to 92 bp among groups of Pinaceae strongly suggests a relatively recent origin for extant Ephedra. The recent origin of living Ephedra may imply a possibly much younger age for the genus or an adaptive radiation of the living species after recent extinctions or longtime stasis. Although we cannot exclude the possibility, little concrete evidence has been found to support the ancient age of Ephedra. One way of estimating the age of Ephedra is through the use of rigorously determined fossils, which could yield earlier estimates of the age of the genus from possible detection of extinct groups of Ephedra or later estimates from the incomplete fossil record. A major difculty with the fossil record is the uncertainty in identication of fossil ephedroid pollen or fragmentary vegetative macrofossils with any specic genus. As already mentioned, ephedroid pollen fossils appeared as early as in the Middle Permian (250 Myr ago), but may not bear any direct connection to Ephedra or the Gnetales. Macrofossils of Ephedra would be extremely helpful in evaluating the age obtained from this study, but available materials with possible afnity to Ephedra are scarce and incomplete. The fragments of Ephedrites stotzkianus from the Eocene, which is supercially similar to E. fragilis, and Ephedrites antiquus from the Jurassic, are incomplete and unable to be rigorously identied (Arber and Parkin 1908; Seward 1963). Those of Ephedrites johnianus and Ephedra mengeana from the Oligocene (24.638 Myr ago) have since been found to actually represent the angiosperm genus Patzea (Loranthaceae) (Seward 1963). According to Crane (1996) and James Doyle (personal communication), several gnetalean macrofossils are known from the Lower Cretaceous, but none of them seems to belong to Ephedra. The suggestion of recent origin of extant Ephedra is consistent with the apparent recency of many habitats

occupied by living species of Ephedra as determined by geological evidence. For example, the North American species E. funerea is native to Death Valley, California, and E. californica is endemic to the Mojave Desert of California. Much of that region was covered by lakes during Pleistocene glaciations (Ellwood 1996, page 258), and the climate was very humid. Recession of these lakes and subsequent desertication occurred only about 2 Myr ago. Therefore, these locally endemic species may have originated after the formation of the recent deserts. In Asia, E. saxatilis, E. likiangensis, E. minuta, and E. gerardiana are native to the Himalayan and the Hengduan mountains that began to rise in the Oligocene. The western Asian species E. transitoria and E. sarcocarpa are restricted to an area that was part of the ancient Tethys Sea in the Miocene (24.65.1 Myr ago). Because these species have very little or no rbcL sequence divergence from other morphologically similar species in the genus (Huang 2000), it is likely that a number of current species of Ephedra are of very recent origin. Living species of Ephedra are widely distributed on the Eurasian continent, northern Africa, and North and South America. Based upon the widespread occurrence of eshy and often red-colored cones in the genus, it is possible that bird dispersal has played an important role in the distribution of Ephedra. This hypothesis is supported by the occurrence of Ephedra on a number of offshore islands, e.g., on many of the islands of the Aegean (Freitag and Maier-Stolte 1989). Most species of Ephedra grow in open and arid habitats, such as deserts and rocky slopes. Although similar suitable habitats are widespread in southern Africa and Australia, no extant species occur in these areas and no well-authenticated fossils are known. Considering the intercontinental distribution pattern of Ephedra and the possibility of long-distance dispersal events by birds in the genus, a lack of sufcient time for colonization may be a plausible explanation for the absence of living species of Ephedra in Australia and southern Africa. The Gnetales is very diverse in many important features. In fact, the extant gnetalean genera, Ephedra, Gnetum, and Welwitschia, are so different in habitat, vegetative morphology, pollen shape, karyotype, anatomy, and embryogenesis that there was a long controversy over whether they are closely related (see review in Doyle 1996). Equally prominent is the great uniformity in the macroscopic morphological characters among species of

Estimation of Age of Extant Ephedra 439

Ephedra (Huang 2000). What can be inferred from these striking intergeneric dissimilarities and infrageneric similarities? On the one hand, while we cannot assume that the evolution of morphology, pollen shape, karyotype, anatomy, and embryogenesis is directly proportional to time, the diverse intergeneric features are in keeping with a great age for the Gnetales as suggested by the sequence comparisons of Savard et al. (1994). On the other hand, the great uniformity of the infrageneric characters of Ephedra, combined with the lack of occurrence in Australia and southern Africa, is more consistent with the recent diversication of living species of the genus. An ancient age for the Gnetales does not necessarily imply a great age for Ephedra. Although ephedroid pollen fossils were much more widespread in the Mesozoic, their abundance decreased dramatically in strata after the Cretaceous (Crane 1996). The pre-Cenozoic ephedroid pollen fossils were also very diverse, ranging from standard Ephedripites grains to forms in which the thickened exine ribs are expanded into hornlike projections (e.g., Galeacornea) or become unraveled from the body of the grain to form elater-like structures analogous to those of Equisetum (Crane 1996). According to Shen (1995), about 50 ephedroid pollen types have been identied in the Cretaceous strata in China. Considering the morphological similarity of the pollen grains of the living species of Ephedra, apparently some major extinctions have occurred during the course of evolution of the Gnetales. Ephedra has also recently been characterized as a crown group of the Gnetales (Doyle 1998), implying that the living species of Ephedra might have originated after the extinction of the stem lineages of the Gnetales. Diversication of a biological group is often related to the changes in physical environments, which can cause extinctions of some groups but create suitable habitats for others, for further radiation. If our estimates of the age of extant Ephedra are valid, one can place the diversication of the living Ephedra in the context of geological and climatic changes during the past 32 Myr. The Oligocene and the Miocene witnessed major climatic and geological changes (Graham 1993; Novacek 1999). The terrestrial temperature evidently decreased dramatically in the boundary of the Eocene and the Oligocene and then increased again from the Early Oligocene and uctuated thereafter. Meanwhile, the weather became drier and the seasonality increased. Cooling temperature and increased seasonality and aridity during that period caused a great reduction in species diversity and a major shift of the vegetation from tropical or subtropical forests to temperate deciduous forests in the higher latitudes of the Northern Hemisphere (Graham 1993). During the Miocene, the C4 grasses originated and underwent a rapid expansion, and the C4 grass-dominated ecosystems began to appear in the late Miocene (Jacobs, Kingston, and Jacobs 1999). Geologically, continuous drift and collisions of land blocks caused some major mountain building events during that period, including the rising of the Himalayas and the Andes and their associated ranges. The buildup of ice cover in Antarctica lowered sea level and caused the disappearance of the Turgai Strait in interior Asia, allowing Europe and Asia to become one continent

(Briggs 1995). The Mediterranean also became landlocked in the Miocene (Mintz 1981). These regions are the major distribution areas of the living Ephedra. Thus, it is quite possible that the diversication of the extant species of Ephedra was associated with the major climatic and geological changes that would have opened up new arid or mountainous habitats for the genus in the Oligocene and the Miocene. Our results do not support the ancient origin of Ephedra, but instead point to a relatively recent diversication of extant Ephedra or a possibly recent origin of the genus Ephedra, and therefore, tend to modify the interpretation of character evolution in the genus. Because Ephedra has often been assumed to be ancient, the uniformity of some characters in the genus is sometimes considered to be the result of very long-term stasis. For example, the karyotype of Ephedra has occasionally been called an example of karyotype conservation (Hunziker 1995). Nevertheless, the morphological similarity of Ephedra is often attributed to reduction in leaves and cones (Arber and Parkin 1908). Our results suggest that recent origin of the extant taxa be considered an important factor in the relative uniformity of the genus, along with the presumed stabilizing selective effects of the harsh arid habitats typical of the genus and a preponderant wind pollination syndrome. In addition, if the origin of Ephedra is more recent than assumed previously, characters of Ephedra used to interpret the origin and evolution of owers should be carefully evaluated. Acknowledgments We are grateful to Helmut Freitag for providing plant materials of several Old World Ephedra species and for his valuable comments and discussion, and to W. E. Friedman for useful discussions and encouragement of the study. We thank David E. Giannasi and Wendy Zomlefer for their critical reading of the manuscript and their comments, and Lilin Xiang and R. C. Jackson for their help during eld collections in the western United States. This study was partially supported by the Palfrey Small Grant program of the Department of Plant Biology, University of Georgia. Literature Cited
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Julian Adams, Associate Editor Accepted November 18, 2002