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Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5163-5167, May 1994 Evolution Chloroplast and nuclear gene sequences indicate Late time for the last common ancestor of extant (noer/gym ne/molecular dock/rge subit of rlbulos,S php rboe/18S rRNA) LouISE SAVARD*t, PENG LI*, STEVEN H. STRAUSSt, MARK W. CHASE§, MARTIN MICHAUD*, AND JEAN BOUSQUET*¶ *Centre de Recherche en Biologie Forestitre, Facultd de Foresterie et de G6omatique, Universit6 Laval, Ste-Foy, Quebec, GIK 7P4; tDepartment of Science, Peavy Hall 154, Oregon State University, Corvallis, OR 97331-5705; and iDepartment of , University of North Carolina, Chapel Hill, NC 27599 Communicated by M. T. Clegg, February 17, 1994

ABSTRACT We have estimated the time for the last com- However, before applying molecular clocks to date evolu- moancor of extant seed plants by using molecular clocks tionary events, approximate constancy of evolutionary rate constructed from the ofthe chloroplastic gene coding over time needs to be established (13) by using procedures for for the large subunit of ribulose-1,5-blphosphate carboxyl- assessing rate homogeneity over taxa (14, 15) or over lineages ase/oxygenase (rbcL) and the nuclear gene coding for the snail (16). subunit of rRNA (Rrnl8). Phylogenetic analyses of nucleodde Here we estimate the time when extant seed plants shared sequences indicated that the earliest divergence of extant seed theirlast common ancestorby using molecularclocks derived plants is likely represented by a split between conufer- from nucleotide sequences ofthe chloroplast gene coding for and ansperm linea . Relative-rate tests were used to assess the large subunit of ribulose-1,5-bisphosphate carboxylase/ homogeneity of substution rates among lineages, and annual oxygenase (rbcL) and the nuclear gene coding for the small anglosperms were found to evolve at a faster rate than other subunit of rRNA (Rrnl8). 11 Using four different landmarks taxa for rbcL and, thus, these sequences were excluded from for the calibration of molecular clocks, we found this time to construction ofmolecular clocks. Five distinct molecular cocks be between 275 and 290 Myr, thus 85-100 Myr later than that were calibrated using substitution rates for the two genes and implied by Beck's hypothesis. four divergence times based on and published molecular cock estimates. The five mated times for the last common ancestor of extant seed plants were in agreement with one MATERIALS AND METHODS another, with an average of 285 million years and a range of For the chloroplast gene rbcL, the following nucleotide 275-290 million years. This implies a subsantily more recent sequences were used: monocots (Triticum aestivum, Oryza ancestor of all extant seed plants than suggsed by some sativa, and Sorghum bicolor) (17, 18), annual dicots (Pisum theories of evolution. sativum, Spinacia oleracea, and Nicotiana tabacum) (19- 21), perennial dicots (Magnolia macrophylla, Carpentaria Although are accepted as the ancestral californica, and Itea virginica) (22, 23), (Pinus group to all seed plants, the time when the five groups of edulis, , Pinus griffithii, Pinus pinea, Pinus extant seed plants (, conifers, , Gnetales, and radiata, , mensiezii, and angiosperms) shared their last common ancestor is unclear gracitior) (24-26), cycads ( inermis, Bow- because of uncertainty about ancestor-descendant relation- enia serrulata, Cycas circinalis, and Encephantos arenarius; ships between progymnosperms and seed plants (1, 2). Beck GenBank accession nos. L12683, L12671, L12674, and (2-5), although not discussing angiosperms, proposed that L12676), a (Angiopteris lygodiifolia) (27), a liverwort the two main lineages of , coniferopsids (coni- (Marchantia polymorpha) (28), and a green alga (Chla- fers, Ginkgo, and Gnetales) and cycadopsids (cycads), di- mydomonas reinhardtii) (29) as the outgroup. Complete rbcL verged independently from two lineages of the progymno- sequences for cycads were determined from both DNA sperms, with coniferopsids from Archaeopteridales and cy- strands following previously established procedures (30). To cads from Aneurophytales. Because Archaeopteridales and determine the node ofearliest divergence among extant seed Aneurophytales appeared in the Middle , Beck's plants sampled, phylogenetic analyses were conducted by the hypothesis implies that the two lineages di- neighbor-joining method (31) with numbers of nonsynony- verged about 375 million years (Myr) ago. Alternatively, mous nucleotide substitutions corrected for multiple hits (32) Rothwell (6, 7) hypothesized that the two gymnosperm and by parsimony analysis (33) of amino acid sequences in lineages both evolved from Aneurophytales via the seed fern order to consider the same level of sequence information. complex, suggesting a more recent diversification of extant The numbers of synonymous substitutions could not be used seed plants. for this study because they were saturated between seed Phylogenetic analysis of conserved gene sequences from extant plant taxa has contributed tangibly to our understand- Abbreviations: Myr, million years; rbcL, gene coding for the large ing of the early diversification of seed plants (8-11). Chlo- subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; roplast and nuclear gene sequences have also been used to Rrnl8, gene coding for the small subunit of cytoplasmic rRNA. construct molecular clocks and to estimate the times for key tPresent address: Institut de Recherche en Biologie Vdgdtale, Uni- events in the evolutionary history of flowering plants (12). versitd de Montrhal, 4101 Sherbrooke East, Montr6al, Qudbec, Canada H1X 2B2. 1To whom reprint requests should be addressed. The publication costs ofthis article were defrayed in part by page charge Il7he sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" GenBank data base (accession nos. L12683, L12671, L12674, in accordance with 18 U.S.C. §1734 solely to indicate this fact. L12676, L00970, L07059, and L12667). 5163 Downloaded by guest on September 25, 2021 5164 Evolution: Savard et A Proc. Nadl. Acad. Sci. USA 91 (1994) plants and nonseed plants (greater than one) and sometimes the construction of molecular clocks and in the estimation of were inestimable. A bootstrap procedure (1000 replicates) divergence times. based on the neighbor-joining analysis of numbers of amino acid substitutions was performed using the program NJBOOT2 (a gift from T. S. Whittam and M. Nei, Pennsylvania State RESULTS University). Deternination of the Node of Earlest Divergence Among For the nuclear gene Rrnl8, the following nucleotide Extant Seed Plants. For rbcL, both neighbor-joining analysis sequences were used: monocots (Oryza sativa andZea mays) of nonsynonymous rates of nucleotide substitution and par- (34), dicots (, Lycopersicon esculentum, Gly- simony analysis of amino acid sequences resulted in two cine max, and Arabidopsis thaliana) (34, 35), conifers clades of extant seed plants: (i) cycads and conifers and (ii) ( glyptostrobordes and ; Gen- angiosperms (Fig. 1). The bootstrap procedure based on the Bank accession nos. L00970 and L07059), a cycad (Zamia neighbor-joining analysis of numbers of amino acid substitu- pumila) (34), and Chlamydomonas reinhardtii (34) as the tions showed moderate to strong support for the seed plant outgroup. For Metasequoia glyptostroboldes and Picea mar- clade (bootstrap value of 80%o), the angiosperm clade (78%), iana, DNA was isolated from lyophilized needles following a and the -cycad clade (67%). Therefore, node A (Fig. CTAB method (36) and the gene Rrnl8 was amplified in two 1) likely represents the node of earliest divergence among overlapping fiagments by PCR using two sets ofprimers: NS1 extant seed plants analyzed. We also included the rbcL and NS4; and NS5 and NS8 (37). Sequencing was conducted sequences from Ephedra tweediana (a Gnetales) (GenBank on single-stranded DNA products obtained from asymmet- accession no. L12667) and (sequence of 93% rical amplification (38), and complete nucleotide sequences ofthe gene; a gift from R. A. Price and J. D. Palmer, Indiana were determined from both DNA strands University) in phylogenetic analyses to further ascertain that using primers NS1 node A (Fig. 1) represents the earliest divergence among to NS8 (37). To determine the node of earliest divergence extant seed plants. Neighbor-joining and parsimony analyses among extant seed plants sampled, phylogenies were esti- placed Ginkgo in the conifer-cycad clade (bootstrap value of mated by the neighbor-joining method (31) with numbers of 68%). Ephedra emerged as a sister group to angiosperms in nucleotide substitutions corrected for multiple hits with the neighbor-joining analysis of nonsynonymous rates of substi- one- and two-parameter methods (39, 40) and by parsimony tutions (bootstrap value of 39%o), while it was placed within analysis (33) ofnucleotide sequences. A bootstrap procedure the conifer-Ginkgo-cycad dade in parsimony analysis of (1000 replicates) based on the neighbor-joining analysis of amino acid sequences. In no case was Ephedra found to numbers of nucleotide substitutions estimated with the one- branch out before node A. Because of uncertainties on the parameter method was performed with NJBOOT2. phylogenetic position of Ephedra whether in the gymno- To estimate the time at the node of earliest divergence sperm or the angiosperm clade, and because the available among extant seed plants, molecular clocks were constructed Ginkgo sequence was <95% ofthe open reading frame, rbcL by using numbers of nonsynonymous substitutions (32) for sequences from these two taxa were excluded from con- rbcL and by using one-parameter numbers of substitutions structing molecular clocks and estimating the time for the (39) for Rrnl8. For the gene rbcL, four distinct rates of earliest divergence among extant seed plants. nonsynonymous substitution per site per year (molecular For Rrnl8, both neighbor-joining analysis of one- and clocks) could be estimated from four landmark events: 1, the two-parameter numbers of nucleotide substitutions and par- diversification of the (140 Myr ago) (41, 42) (event simony analysis of nucleotide sequences resulted in two El, Fig. 1); 2, the split between monocots and dicots (200 Myr clades of extant seed plants: (i) conifer and cycads and (ii) ago) (event E2, Fig. 1)-this date was based on molecular angiosperms. The bootstrap procedure based on the neigh- clock estimates from chloroplast DNA (12), nuclear rRNAs bor-joining analysis of numbers of nucleotide substitutions (12), and mtDNA (P.L. and J.B., unpublished results), which estimated with the one-parameter method indicated a strong is about 75 Myr earlier than the oldest angiosperm (1), support for the conifer-cycad lade (bootstrap value of 95%) but nearly 150 Myr later than other more controversial and moderate support for the angiosperm clade (68%). There- molecular clock estimates (43-46); 3, the split between and seed plants (395 Myr ago) (1) (event E3, Fig. 1); and 4, E2 Angiosperms the split between liverwort and vascular plants (440 Myr ago) (47, 48) (event E4, Fig. 1). From the set of complete Rrnl8 gene sequences available, we could estimate only one rate of substitution per site per year from event E2 (Fig. 1). For a particular node-landmark used to derive a molecular clock, an average substitution rate per site was obtained from E3 Conifers all possible pairwise sequence comparisons between the two Cycads groups above the node-landmark. This rate was then divided by 2T, to obtain a rate per site per year, where T is the landmark time for the divergence between the two lineages. The standard error was calculated as the square root of the average variance ofall pairwise sequence comparisons used. Ferns To obtain an estimated time at the node ofearliest divergence among extant seed plants, the various rates per site per year were applied to half of the average (±SE) pairwise substitu- tion rate per site for that node, which was obtained similarly as above. Before constructing molecular clocks, lineage relative-rate Liverworts tests (16) were used to assess homogeneity of substitution FiG. 1. Consensus phylogenetic relationships among groups of rates. In these tests, the sequence from a green alga (Chia- land plants deduced from analyses of rbcL and Rrnl8 gene se- mydomonas reinhardtii), a close relative to land plants (1), quences. In the circles, A is the last common ancestor among extant was used as the reference . Ifa group of taxa was found seed plants, to be dated, and El to E4 represent the four landmark to evolve at a significantly different rate, it was excluded from events used for calibrating molecular clocks. Downloaded by guest on September 25, 2021 Evolution: Savard et aL Proc. Natl. Acad. Sci. USA 91 (1994) 5165

Table 1. Results from lineage relative-rate tests Lineages compared Rate difference per site* Probability rbcL Pseudotsuga/Pinaceae -0.00247 ± 0.00325 0.447 Pinaceae/Podocarpus -0.00011 ± 0.00435 0.976 Pinaceae/cycads 0.00164 ± 0.00358 0.646 Podocarpus/cycads 0.00175 ± 0.00413 0.674 Perennial angiosperms/gymnosperms 0.00243 ± 0.00396 0.542 Fern/perennial angiosperms + gymnosperms 0.00117 ± 0.00527 0.826 Liverwort/perennial angiosperms + gymnosperms -0.00197 ± 0.00612 0.749 Annual angiosperms/gymnosperms 0.01092 ± 0.00374 0.004 Annual angiosperms/fern 0.01161 ± 0.00552 0.036 Annual angiosperms/liverwort 0.01240 ± 0.00565 0.029 Rrnl8t Angiosperms/gymnosperms -0.00754 ± 0.00578 0.194 Monocots/dicots -0.00323 ± 0.00532 0.542 Chlamydomonas reinhardtii was used as reference taxon in all tests. *For rbcL, nonsynonymous numbers of substitutions per site (14); for Rrnl8, one-parameter numbers of substitutions per site (39). A negative value indicates that taxon or group on the left side of the pairwise comparison has a slower rate of substitution, while a positive value indicates a faster rate of substitution. tFor Rrnl8, because the estimated numbers ofnucleotide substitutions per site obtained from the one- and two-parameter methods (39, 40) were essentially the same, only results obtained with the one-parameter method are presented.

fore, node A (Fig. 1) was also confirmed by RrnJ8 gene estimated by dividing half of the average number of substi- sequences as the likely earliest divergence among extant seed tutions per site between angiosperms and conifers and be- plants analyzed. tween angiosperms and cycads (0.0803 ± 0.0075) by the Assessment of Rate Homogeneity. Before constructing mo- estimated substitution rate per year (Rrnl8, E2) (Table 2). lecular clocks, rate constancy over lineages was assessed for The estimated time obtained (287 Myr) was within the range nonsynonymous rates of substitution for rbcL and one- of time estimates derived from rbcL molecular clocks (Fig. parameter numbers of substitutions for Rrnl8. For rbcL, 2). rates were found homogeneous within gymnosperms as well Alternative Hypotheses. The time when extant seed plants as between gymnosperms, perennial angiosperms, fern, and shared their last common ancestor was also estimated under liverwort (Table 1). As previously reported for nonsynony- three alternative topologies, as suggested by cladistic anal- mous sites of rbcL (24), annual angiosperms were found to yses of morphological characters (49, 50) (Fig. 3). The evolve more rapidly than other taxa (Table 1) and, therefore, estimated times for topology I correspond to those in Fig. 2. they were excluded from the construction of all rbcL molec- Time for the earliest divergence among extant seed plants ular clocks. ForRrnl8, substitution rates were homogeneous under topologies II and III (node A) was estimated in a similar between monocots and dicots as well as between an- fashion as for topology I. For topology IV with the chloro- giosperms and gymnosperms (Table 1). plast gene rbcL, the time for the earliest divergence among Determination ofthe Time at the Node ofEarliest Divergence extant seed plants was obtained by dividing half of the Among Extant Seed Plants. The four nonsynonymous rates of average number of nonsynonymous substitutions per site substitution per site per year obtained for the chloroplast between perennial angiosperms and conifers, between pe- gene rbcL differed by <6% (Table 2). For this gene, the time rennial angiosperms and cycads, and between conifers and when extant seed plants shared their last common ancestor cycads (0.0245 ± 0.0048) by the four estimated substitution (node A, Fig. 1) was estimated by dividing halfofthe average rates per year (rbcL, El to rbcL, E4, Table 2). For topology number of nonsynonymous substitutions per site between IV with the nuclear gene Rrnl8, the time for the earliest perennial angiosperms and conifers and between perennial divergence among extant seed plants (node A) was obtained angiosperms and cycads (0.0264 ± 0.051) by the four esti- by dividing half of the average number of substitutions per mated substitution rates per year (rbcL, El to rbcL, E4) site (one-parameter method) between angiosperms and co- (Table 2). The four time estimates ranged from 275 Myr to 290 nifers, between angiosperms and cycads, and between coni- Myr (Fig. 2). fers and cycads (0.0756 ± 0.0074) by the estimated substitu- For the nuclear gene Rrnl8, the time when extant seed tion rate per year (Rrnl8, E2, Table 2). For the topology plants shared their last common ancestor (node A, Fig. 1) was supported by our phylogenetic analyses of rbcL and RrnJ8 Table 2. Molecular clocks for rbcL and Rrnl8 Divergence No. of nucleotide Substitution rate per Gene Landmark event time, Myr substitutions per site* site per year rbcL El, Pinus-Pseudotsuga split 140 0.0130 ± 0.0034 4.64 ± 1.21 x 10-11 rbcL E2, Monocot-dicot split 200 0.0096 ± 0.0018t 4.80 ± 0.90 x 1O-1't rbcL E3, Fern-seed plant split 395 0.0361 ± 0.0058 4.57 ± 0.73 x 10-11 rbcL E4, Liverwort- split 440 0.0399 ± 0.0061 4.54 ± 0.69 x 10-11 Rrnl8 E2, Monocot-dicot split 200 0.0558 ± 0.0059 1.40 ± 0.15 x 10-10 *For rbcL, nonsynonymous numbers of substitutions; for Rrnl8, one-parameter numbers of substitutions. tFor molecular clock rbcL, E2, fast-evolving annual angiosperms were excluded (see text), and the rate per year was estimated by dividing the average path length (0.0096) from the node at the origin of the dicots to extant perennial species (Magnolia, Carpenteria, and Itea), as estimated by the neighbor-joining method (results not shown; method described in ref. 24), by the divergence time between monocots and dicots. Downloaded by guest on September 25, 2021 5166 Evolution: Savard et al. Proc. Natl. Acad. Sci. USA 91 (1994)

Time Geological (n Estimated time for event A: Ephedra rbcL sequence was not included in divergence (myr) epoch last common ancestor of time estimates because of uncertainties on its position, Present - E extant seed plants c whether within the gymnosperm or as a group to (U sister the Tertiary -J angiosperm lade. Although phylogenetic analysis of 18S Molecular clocks rRNA and 26S rRNA sequences and cladistic analysis of 100 - morphological characters placed Ephedra and other Gnetales wNL as a sister group to the angiosperms (8, 49, 50), results from w* w . IA rbcL sequences are more variable, with the Gnetales as either 200 - a sister group to the angiosperms (10) or nested within the 1=- gymnosperms (9). An important assumption for the calculation of our esti- I mated dates was the observed monophyly of conifers and 300 - Pennsylvanian 1 cycads in the phylogenetic analyses, which was also sup- Mississippian I ported by previous analyses of nuclear 18S rRNA and 26S Devonian U) rRNA sequences (8) as well as sequences from the chloro- c ) 400 - U) U) plastic genes rbcL (9) and chiB (R. Boivin, Laval University, 40 4-e personal communication). However, cladistic analyses of ~ 4", c 0 morphological traits did not support the monophyly of coni- )m 's~~~~~~~U G V) U) fers and cycads and revealed several alternative topologies 500 -t - with regard to conifers, cycads, and angiosperms (49, 50), 9, I. E E which differed by only one or few steps. We have considered these alternative topologies in our study. When these alter- native topologies were considered with regard to the para- FIG. 2. Estimated time when extant seed plants shared their last common ancestor. phyly or monophyly of conifers and cycads, the estimated divergence times were all more recent, with some estimates sequences (topology I, Fig. 3), the estimated times were postdating the first reliable appearances ofthe five groups of greater than those under the three alternative topologies (Fig. extant seed plants in the fossil record (cycads from the Lower 3). Permian, conifers from the Lower Triassic, Ginkgo from the Permian, Gnetales as early as Middle Permian, and an- giosperms from the Lower Cretaceous) (1, 41, 49-51). Thus, DISCUSSION estimates under topology I most likely represent the time for Phylogenetic analyses of gene sequences from rbcL and the earliest divergence among extant seed plants. Rrnl8 resulted in two clades ofextant seed plants: (i) cycads By using the various molecular clocks, the estimated times and conifers and (ii) angiosperms (Fig. 1). This confirmed when extant seed plants shared their last common ancestor previous results based on nuclear 18S rRNA and 26S rRNA were in good agreement. For the chloroplast gene rbcL, the sequences (8) as well as rbcL sequences (9). Given that four estimates come from four different landmark events and Ginkgo and Ephedra (a Gnetales) did not branch out before are highly congruent, while for the nuclear gene Rrnl8, the the angiosperm-conifer-cycad lade (Fig. 1), a result also estimate for the initial divergence of extant seed plants was reported from morphological and molecular studies (8, 9, 49, within the range of rbcL estimates. Because they are based 50), the earliest divergence among extant seed plants is likely on different genomes and four calibration events, the odds of to be represented by the split between the conifer-cycad obtaining such high congruence among the five estimates by lineage and the angiosperm lineage (node A in Fig. 1). chance alone are small. I II III IV

Angiosperms ,Angiosperms Angiospemns Angiosperms .SIF22*~. Cycads

Conifers Cycads Conifers Molecular clocks rbcL-El 284±55 262±52 245+51 264±52 rbcL-E2 275±53 253+50 236±49 255±50 Rrn18-E2 287±27 265±27 259±25 270±26 rbcL-E3 289±55 266±53 248+51 268±53 rbcL-E4 290±56 268+53 250±51 269±53

Estimated time for node A FIG. 3. Estimated time when extant seed plants shared their last common ancestor under four topologies of conifers, cycads, and angiosperms. The estimated times for topology I correspond to those in Fig. 2. Downloaded by guest on September 25, 2021 Evolution: Savard et al. Proc. NatL Acad. Sci. USA 91 (1994) 5167 We consider the more recent estimate derived from one 16. Li, P. & Bousquet, J. (1992) Mol. Biol. Evol. 9, 1185-1189. molecular clock (rbcL, E2) less reliable than the estimated 17. Terachi, T., Ogihara, Y. & Tsunewaki, K. (1987) Jpn. J. Genet. times from the other four molecular clocks (rbcL, El; rbcL, 62, 375-387. E3; rbcL, E4; and Rrnl8, E2). This is so because of slight 18. Doebley, J., Durbin, M., Golenberg, E. M., Clegg, M. T. & acceleration of nonsynonymous rate of substitution of rbcL Ma, D.-P. (1990) Evolution 44, 1097-1108. 19. Zurawski, G., Whitfeld, P. R. & Bottomley, W. (1986) Nucleic in perennial angiosperms (24), although not shown significant Acids Res. 14, 3975. in our relative rate tests. Furthermore, it is unlikely that 20. Zurawski, G., Perrot, B., Bottomey, W. & Whitfeld, P. R. saturation effects contributed to the observed differences (1981) Nucleic Acids Res. 9, 3251-3269. among rbcL molecular clocks, because all pairwise numbers 21. Shinazoki, K. & Sugiura, M. (1982) Gene 20, 91-102. of nonsynonymous substitutions per site used to construct 22. Golenberg, E. M., Giannasi, D. E., Clegg, M. T., Smiley, these clocks were below 0.05. C. J., Durbin, M., Henderson, D. & Zurawski, G. (1990) Our results indicate that the last common ancestor to all Nature (London) 344, 656-658. extant seed plants occurred most likely during the Upper 23. Soltis, D. E., Soltis, P. S., Clegg, M. T. & Durbin, M. (1990) Pennsylvanian. This postdates by about 85-100 Myr that Proc. Natl. Acad. Sci. USA 87, 4640 -4644. implied under Beck's hypothesis (2-5) and is slightly earlier 24. Bousquet, J., Strauss, S. H., Doerksen, A. H. & Price, R. A. than the first appearances of the five groups of extant seed (1992) Proc. Natl. Acad. Sci. USA 89, 7844-7848. plants in the fossil record. Our estimated time when extant 25. Mukai, Y., Yamamoto, N., Odani, K. & Shinohara, K. (1991) seed plants shared their last common ancestor supports the Plant Physiol. 32, 273-282. 26. Hipkins, V. D., Tsai, C.-H. & Strauss, S. H. (1990) Plant Mol. conclusion from cladistic and stratigraphic analyses of ex- Biol. 15, 505-507. tinct and extant taxa (49, 50) that all major groups of extant 27. Yoshinaga, K., Kubota, Y., Isshii, T. & Wada, K. (1992) Plant seed plants, with the possible exception ofangiosperms, were Mol. Biol. 18, 79-82. products of diversification that occurred between the Lower 28. Ohyama, K., Fukazawa, H., Kohchi, T., Shirai, H., Sano, T., Pennsylvanian and the Upper Triassic (51). The recentness of Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., this event also implies that all extant seed plants, including Aota, S.-i., Inokuchi, H. & Ozeki, H. (1986) Nature (London) conifers and cycads, are likely to have evolved from seed 322, 572-574. ferns, which derived from one lineage, most 29. Dron, M., Rahire, M. & Rochaix, J.-D. (1982) J. Mol. Biol. 162, probably the Aneurophytales (6, 7). The identification of the 775-793. 30. Albert, V. A., Williams, S. E. & Chase, M. W. (1992) Science seed fern group from which extant seed plants derived awaits 257, 1491-1495. further studies on the morphology and relationships of Palae- 31. Saitou, N. & Nei, M. (1987) Mol. Biol. Evol. 4, 406-425. ozoic and seed ferns (51). 32. Li, W.-H., Wu, C.-I. & Luo, C.-C. (1985) Mol. Biol. Evol. 2, 150-174. We thank R. A. Price and J. D. Palmer (Department of Biology, 33. Swofford, D. L. (1993) PAUP-Phylogenetic Analysis Using Indiana University) for use of their unpublished Ginkgo rbcL se- Parsimony (Illinois Natural History Survey, Champai, IL), quence, M. Nei and T. S. Whittam (Institute of Molecular Evolu- Version 3.1. tionary Genetics, Pennsylvania State University) for providing com- 34. Neefs, J.-M., Van der Peer, Y., De Rjk, P., Goris, A. & De puter programs, and anonymous reviewers for their suggestions to Wachter, R. (1991) Nucleic Acids Res. 19, 1987-2015. improve the manuscript. This work was supported by Qu6bec 35. Savard, L. & Lalonde, M. (1991) Plant Mol. Biol. 16, 725-728. Research Fund (FCAR) and Canadian Natural Sciences and Engi- 36. Bousquet, J., Simon, L. & Lalonde, M. (1990) Can. J. For. Res. neering Research Council grants to J.B., U.S. National Science 20, 254-257. Foundation grants to S.H.S. and M.W.C., and a Canadian Natural 37. White, T. J., Bruns, T., Lee, S. & Taylor, J. 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