Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5163-5167, May 1994 Evolution Chloroplast and nuclear gene sequences indicate Late Pennsylvanian time for the last common ancestor of extant seed plants (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, Canada GIK 7P4; tDepartment of Forest Science, Peavy Hall 154, Oregon State University, Corvallis, OR 97331-5705; and iDepartment of Biology, 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-cycad 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 fossil 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 plant evolution. sativum, Spinacia oleracea, and Nicotiana tabacum) (19- 21), perennial dicots (Magnolia macrophylla, Carpentaria Although progymnosperms are accepted as the ancestral californica, and Itea virginica) (22, 23), conifers (Pinus group to all seed plants, the time when the five groups of edulis, Pinus longaeva, Pinus griffithii, Pinus pinea, Pinus extant seed plants (cycads, conifers, Ginkgo, Gnetales, and radiata, Pinus thunbergii, Pseudotsuga mensiezii, and angiosperms) shared their last common ancestor is unclear Podocarpus gracitior) (24-26), cycads (Zamia 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 fern (Angiopteris lygodiifolia) (27), a liverwort the two main lineages of gymnosperms, 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 Devonian, Beck's plants sampled, phylogenetic analyses were conducted by the hypothesis implies that the two gymnosperm 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 (Alnus glutinosa, 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) (Metasequoia glyptostrobordes and Picea mariana; 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 conifer-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 Ginkgo biloba (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.