DISCORDANCE OF CHLOROPLAST AND NUCLEAR

Transcription

1 American Journal of Botany 89(6): DISCORDANCE OF CHLOROPLAST AND NUCLEAR RIBOSOMAL DNA DATA IN OSMORHIZA (APIACEAE) 1 KI-OUG YOO, 2 PORTER P. LOWRY II, 3 AND JUN WEN 2,4 2 Department of Botany, The Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, Illinois USA; 3 Missouri Botanical Garden, P. O. Box 299, St. Louis, Missouri USA; and Laboratoire de Phanérogamie, Muséum National d Histoire Naturelle, 16 rue Buffon, Paris, France Phylogenetic studies were conducted to evaluate interspecific relationships in Osmorhiza (Apiaceae: Apioideae) using sequences of the ITS regions of nuclear ribosomal DNA, the chloroplast ndhf gene, and two noncoding regions (trnl intron, and trnl [UAA] 3 exon-trnf [GAA] intergenic spacer). All data sets suggest the monophyly of the New World taxa and showed that Osmorhiza aristata from Asia is relatively divergent from other members of the genus, even though it is morphologically similar to the eastern North American O. claytonii and O. longistylis. The ITS and chloroplast DNA trees differ in the relationships among the New World taxa, especially the phylogenetic position of O. occidentalis, O. glabrata, and O. depauperata. The lack of congruence between the two data sets may be a result of hybridization or introgression. Although there is high discordance between nrits and two chloroplast DNA data sets, the latter two show similar topologies. Key words: Apiaceae; chloroplast DNA; ITS; ndhf, trnl-trnf noncoding region; Osmorhiza; phylogeny. Osmorhiza Raf., the sweet cicely genus, is a member of Apiaceae subfamily Apioideae consisting of ten species distributed in the temperate regions of North America, South America, and Asia (Lowry and Jones, 1984). Osmorhiza was first recognized as a distinct genus by Rafinesque in 1818, although species of the genus were referred to other genera by previous researchers (e.g., Thunberg, 1784; Michaux, 1803; Persoon, 1805; Sprengel, 1813). Constance and Shan (1948) conducted the first taxonomic study of the genus on a worldwide basis and proposed an infrageneric classification, which was adopted by Lowry and Jones (1984) with a few modifications. They recognized two subgenera (Glycosma and Osmorhiza), the latter subdivided into three sections (Osmorhiza, Nudae, and Mexicanae, each with three species), which are characterized by the morphology of involucres, bractlets, styles, and fruits (Lowry and Jones, 1984). Osmorhiza shows several interesting biogeographic disjunctions: (1) members of section Osmorhiza demonstrate the classical eastern Asian eastern North American disjunction; (2) two species (O. berteroi and O. depauperata) show an antitropical (often referred to as amphitropical) disjunction between temperate North and South America; and (3) O. berteroi and O. depauperata are also disjunct between eastern North America, the Great Lakes region, and western North America. The biogeographic diversification of Osmorhiza was recently evaluated using ITS sequences of the nuclear ribosomal DNA (Wen et al., 2002), revealing the relative antiquity of the eastern Asian eastern North American disjunction and the recent origin of both the antitropical as well as eastern and western North American disjunctions in Osmorhiza. The ITS phylog- 1 Manuscript received 31 May 2001; revision accepted 3 January The authors thank Fernando Chiang, Richard R. Halse, Lawrence Janeway, Jan Jorgensen, Sangtae Lee, Clodomiro Marticorena, Tod Stuessy, Jeff Walck, and Shiliang Zhou for their help in obtaining leaf material; Ron Hartman and Greg Plunkett for providing helpful comments; and the curators of the following herbaria for permitting the examination of their specimens: A, CS, F, MO, MSC, and RM. This study was supported in part by the Pritzker Laboratory of Molecular Systematics and Evolution of the Field Museum of Natural History and grants from the National Science Foundation (DEB and DEB ) to J. Wen. 4 Author for reprint requests ( wen@fieldmuseum.org). 966 eny also suggests a relatively rapid diversification of the western North American taxa, even though they show a high level of morphological diversity. The monophyly of Osmorhiza is well supported in a series of phylogenetic studies using ITS sequence data of Apiaceae tribe Scandiceae (Downie, Katz-Downie, and Spalik, 2000). Wen et al. (2002) clarified inter- and intraspecific relationships using a sample of 46 populations representing all ten species of Osmorhiza throughout its distribution based on ITS sequence data. This study suggested several relationships: (1) O. aristata from Asia occupies a basal position within the genus; (2) the nine New World species form a clade; (3) populations of O. berteroi from South America, western North America, eastern North America, and the Great Lakes region form a monophyletic group; (4) O. claytonii and O. longistylis from eastern North America form a clade; (5) O. brachypoda and O. purpurea are allied with a clade comprising O. depauperata and O. occidentalis; (6) several species (O. berteroi, O. brachypoda, O. depauperata, O. mexicana, O. mexicana subsp. bipatriata, O. occidentalis, and O. purpurea) form a largely western North American clade; and (7) O. glabrata from central Andes forms a trichotomy with the eastern North American clade and the western North American clade. The objectives of the present study are to (1) assess the phylogeny for Osmorhiza using the chloroplast ndhf gene and the trnl-trnf noncoding region (trnl intron, and trnl [UAA] 3 exon-trnf [GAA] intergenic spacer); (2) compare the cpdna phylogeny with the results of the previous ITS study; and (3) test the infrageneric classification of Lowry and Jones (1984) within a molecular phylogenetic framework. We selected the ndhf gene and the trnl-trnf noncoding regions of chloroplast DNA because they display relatively fast nucleotide substitution rates and have been employed successfully in many other studies at the interspecific level (e.g., Gielly and Taberlet, 1994, 1996; Kita, Ueda, and Kadata, 1995; Gielly et al., 1996; Bakker et al., 2000; Potter, Luby, and Harrison, 2000). Also, a comparison of phylogenetic inferences based on chloroplast and nuclear markers may provide important insights into relationships and patterns of evolution within Osmorhiza. For example, at lower taxonomic levels (genus and

2 June 2002] YOO ET AL. PHYLOGENETICS OF OSMORHIZA 967 TABLE 1. Taxa of Osmorhiza and their distributions following the classification scheme of Lowry and Jones (1984). Classification Taxon Distribution Subgenus Glycosma O. occidentalis (Nutt.) Torrey Western North America Subgenus Osmorhiza Section Mexicanae O. brachypoda Torrey California, Nevada and Arizona O. glabrata Philippi central Andes O. mexicana Griseb. subsp. bipatriata (Constance & Shan) Mexico and Texas Lowry & Jones subsp. mexicana Southwest Texas to northern Argentina Section Nudae O. berteroi DC. Western North America, Great Lakes area, northeast North America, and South America O. depauperata Philippi Western North America, Great Lakes area, northeast North America, and South America O. purpurea (Coult. & Rose) Suksd. Pacific Coast of northwest North America Section Osmorhiza O. aristata (Thunb.) Rydb. Eastern Asia O. claytonii (Michaux) C. B. Clarke Eastern North America O. longistylis (Torrey) DC. Eastern North America below) such comparisons have revealed high levels of concordance in several studies (e.g., Baldwin, 1992; Kim and Jansen, 1994; Bayer, Soltis, and Soltis, 1996; Bayer, Puttock, and Kelchner, 2000; Choi and Wen, 2000), but in a few cases, significant discordance has been found (e.g., Soltis and Kuzoff, 1995; Soltis, Johnson, and Looney, 1996). Such discordance may suggest hybridization or introgression (Soltis and Kuzoff, 1995). MATERIALS AND METHODS Twenty-three populations representing all ten species of Osmorhiza were sampled in this study. Taxa, voucher, source information, and accession numbers have been archived at the Botanical Society of America website [ The widespread O. berteroi was sampled from four different geographic areas and O. depauperata was represented by material from two disjunct populations. A possible hybrid (Halse 5560), which showed intermediate fruit morphology between the co-occurring O. berteroi (Halse 5559) and O. occidentalis (Halse 5561), was also included. Anthriscus cerefolium Hoffm. and Myrrhis odorata Scop., two close relatives of Osmorhiza (Downie, Katz-Downie, and Spalik, 2000), were selected as the outgroup. Total DNA was extracted with the cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1987) and purified over CsCl/ethidium bromide gradients. DNA amplifications were performed in 100- L reactions containing approximately 50 ng genomic DNA, 20 nmol/l Tris buffer (ph 8.3) with 50 mmol/l KCl, 1.5 mmol/l MgCl2, and 0.1% Tween 20 (buffer designed by C. Bult), 0.15 mmol/l of each dntp, 1 mol/l of each primer, 5 units of Taq polymerase (Promega, Madison, Wisconsin, USA), and 5% DMSO (dimethyl sulfoxide). The primers to amplify ndhf and the trnl-trnf noncoding regions were those used in Olmstead and Sweere (1994) and Taberlet et al. (1991), respectively. Double-stranded polymerase chain reaction (PCR) products were produced via 45 cycles of denaturation (94 C for 1 min), annealing (50 C for 2 min), and extension (72 C for 2 min). A 5-min final extension cycle at 72 C followed the 45th cycle to ensure the completion of novel strands. The PCR products were purified using Wizard PCR Preps DNA Purification System (Promega) prior to sequencing. The ndhf gene and the two noncoding regions were sequenced following the dideoxy chain termination method (Sanger, Nicklen, and Coulsen, 1977) using the Sequenase Version 2.0 DNA Sequencing Kit (cat. no. US70770, Amersham, Cleveland, Ohio, USA) and alpha 33 P-dATP as a radioactive tracer. Most mutations were base substitutions, thus allowing manual alignment. Phylogenetic analysis was performed with PAUP* (version 4.02b, Swofford, 1999) using maximum parsimony (Swofford et al., 1996) and maximum likelihood (Felsenstein, 1981) methods. Parsimony analyses were performed using a branch-and-bound search with MULPARS and furthest addition sequence options. The amount of support for monophyletic groups revealed in the maximally parsimonious tree(s) [MPT(s)] was examined with 500 bootstrap replicates (Felsenstein, 1985) with the random addition and the heuristic search options using parsimony. The proportions of site differences were estimated using the Kimura two-parameter distance (Kimura, 1980). A partition homogeneity test (Farris et al., 1995) was conducted with PAUP* (Swofford, 1999) to determine the congruence of the chloroplast and nuclear data sets. The test was performed with 100 replicates, using an heuristic search option with simple addition, tree bisection-reconnection (TBR) branch-swapping, and gaps as missing data. RESULTS ndhf data A total of 1979 base pairs (bp) of the ndhf gene were obtained for all taxa of Osmorhiza. No length mutations and only a low level of nucleotide substitution were observed. Of the 1979 aligned positions, 36 sites were variable, only 8 of which were phylogenetically informative. The parsimony analysis generated 7473 MPTs with a total length of 65 steps, a consistency index (CI) of 0.923, a retention index (RI) of 0.865, and a rescaled consistency index (RC) of Several relationships are suggested by the parsimony analyses (Fig. 1): (1) the two populations of O. aristata from Asia form a basally branching monophyletic group; (2) the nine New World species form a monophyletic group (bootstrap value 51%); (3) two subclades can be recognized among the New World taxa, O. claytonii O. purpurea (bootstrap value 58%) and O. depauperata O. mexicana subsp. bipatriata (bootstrap value 83%). Treating gaps as missing data, the Kimura two-parameter distance among species of Osmorhiza was estimated to be %. The highest divergence was between O. mexicana subsp. bipatriata from southwestern North America and O. claytonii from eastern North America; the lowest value was between O. purpurea from western North America and O. claytonii from eastern North America. Osmorhiza berteroi, sampled from five populations in South America and throughout North America, had a sequence divergence of only %. trnl (UAA)-trnF (GAA) intron and intergenic spacer data Lengths of the trnl-trnf noncoding region varied from 878 to 960 bases among species of Osmorhiza, with the trnl intron of bases, and the trnl [UAA] 3 exon-trnf

3 968 AMERICAN JOURNAL OF BOTANY [Vol. 89 Fig. 1. The strict consensus of 7473 most parsimonious trees from the ndhf data set obtained from Osmorhiza, with gaps treated as new character states (65 steps, consistency index 0.923, retention index 0.865, rescaled consistency index 0.798). Branch lengths are shown above the branches; the bootstrap values in 500 replicates are shown below the lines. [GAA] intergenic spacer of bases. A large 78-bp deletion was detected in seven species (O. berteroi, O. brachypoda, O. claytonii, O. glabrata, O. longistylis, O. mexicana, and O. purpurea) in the intergenic spacer. Of the 963 aligned positions, 41 sites were variable; of these 18 were phylogenetically informative. The parsimony analysis generated four MPTs with a total length of 49 steps, a CI of 0.857, an RI of 0.907, and an RC of Several relationships are suggested by parsimony analyses of the trnl-trnf data (Fig. 2): (1) O. aristata from Asia is basal within Osmorhiza; (2) the nine New World species form a monophyletic group (bootstrap 52%); (3) one accession of O. berteroi from Chile (Stuessy et al ) and O. glabrata (bootstrap 92%) form a subclade; (4) O. dapauperata and O. mexicana subsp. bipatriata form a subclade (bootstrap 64%); and (5) O. occidentalis is relatively divergent from the other New World taxa, and multiple accessions of this species form a well-supported monophyletic group (bootstrap 97%). The Kimura two-parameter distance among species of Osmorhiza by two noncoding regions was estimated to be %. The highest divergence was between O. mexicana subsp. bipatriata and O. brachypoda, both from western North Fig. 2. The strict consensus of four most parsimonious trees from data sets of the two noncoding regions (trnl intron and trnl [UAA] 3 exon-trnf [GAA] intergenic spacer) of chloroplast DNA in Osmorhiza with gaps treated as new character states (49 steps, consistency index 0.857, retention index 0.907, rescaled consistency index 0.777). Branch lengths are shown above the branches; the bootstrap values in 500 replicates are shown above the lines. America. Osmorhiza berteroi, collected from multiple populations, showed divergence values of %. Data incongruence Assessment of congruence among trees using the partition-homogeneity test of PAUP* showed that the ITS and the cpdna data sets were incongruent (P 0.01), whereas the two cpdna data sets were congruent (P 0.58), with no conflicting branches detected between them (cf. Figs. 1 and 2). The topology from trnl-trnf noncoding regions had a higher resolution than that of the ndhf tree. Of the 2942 aligned positions in the combined cpdna data set, 101 sites were variable and 30 sites were phylogenetically informative. Parsimony analysis of the combined chloroplast data set generated MPTs with a total length of 116 steps, a CI of 0.879, an RI of 0.875, and an RC of Phylogenies from the combined data (Fig. 3) and from separate analyses of the two noncoding regions (Fig. 2) had similar topologies, except that the combined analysis indicated the paraphyly of the two populations of O. longistylis. The maximum likelihood tree (MLT) has an identical topology to the MPT.

4 June 2002] YOO ET AL. PHYLOGENETICS OF OSMORHIZA 969 Fig. 3. The strict consensus of most parsimonious trees from the combined chloroplast DNA data sets (ndhf and two noncoding regions) in Osmorhiza, with gaps treated as new character states (116 steps, consistency index 0.879, retention index 0.875, rescaled consistency index 0.769). Branch lengths are shown above the branches; the bootstrap values in 500 replicates are shown above the lines. Fig. 4. The strict consensus of two most parsimonious trees from the internal transcribed spacer data set in Osmorhiza, with gaps being treated as new character states (110 steps, consistency index 0.918, retention index 0.905, rescaled consistency index 0.831). Branch lengths are shown above the branches; the bootstrap values in 500 replicates are shown below the lines. DISCUSSION Phylogenetic relationships and incongruence between nrits and cpdna data sets Both the cpdna and nrits data sets suggest that Osmorhiza aristata from Asia occupies a basally branching position within the genus, and the New World species form a monophyletic clade. The Asian O. aristata is morphologically similar to the eastern North American O. claytonii and O. longistylis, and these three species were placed together in Osmorhiza section Osmorhiza by Lowry and Jones (1984). Although no morphometric analyses have been performed, these taxa clearly form a morphologically coherent group and have in the past been treated as a single species by some authors (e.g., Clarke, 1879; Kuntze, 1891; Boivin, 1968). Wen et al. (2002) suggested, however, that the morphological similarities exhibited by these species may involve shared plesiomorphic characters. In contrast to the well-supported basal position of Osmorhiza aristata, the relationships among the New World species showed high discordance between cpdna and nrits phylogenies (cf. Figs. 3 and 4). For example, O. occidentalis is highly distinct from the other North American taxa in the cpdna data sets and represents a major subclade. The ITS data (Fig. 4), on the other hand, suggest that O. occidentalis forms a clade with western North American O. depauperata. Osmorhiza occidentalis was first described in the monotypic genus Glycosma (Torrey and Gray, 1840) and is distinguished from other Osmorhiza species by having unappendaged, glabrous fruit. Constance and Shan (1948) treated Glycosma as a section within Osmorhiza, whereas Lowry and Jones (1984) recognized it as a monotypic subgenus. Osmorhiza glabrata, restricted to the central Andes, forms a subclade with the South American populations of O. berteroi (Fig. 3). The ITS phylogeny shows that all four populations of O. berteroi form a monophyletic clade; O. brachypoda is sister to O. occidentalis and O. glabrata forms a trichotomy with the eastern North American clade (Fig. 4). Osmorhiza depauperata is disjunctly distributed in the Great Lakes region, northeastern North America and southern South America, whereas O. mexicana subsp. bipatriata is known from only a few localities in Coahuila and Nuevo León, Mexico, and in Texas. These two taxa form a clade in the chloroplast phylogeny, but the ITS data show that O. depauperata is sister to O. occidentalis. The eastern North American taxa Osmorhiza claytonii and O. longistylis are placed in section Osmorhiza, along with the

5 970 AMERICAN JOURNAL OF BOTANY [Vol. 89 Asiatic O. aristata (Lowry and Jones, 1984). In the ITS phylogeny, however, the section is paraphyletic. By contrast, the chloroplast DNA data sets suggest that O. claytonii and O. longistylis form a monophyletic group with O. purpurea, although this relationship is not strongly supported (bootstrap 60%) (Fig. 3). Osmorhiza mexicana subsp. mexicana, a member of section Mexicanae, is distributed in scattered populations from northern Mexico to North Argentina, thus occupying a much larger range than O. mexicana subsp. bipatriata. None of the analyses, however, suggest that these taxa form a monophyletic group. In the ITS phylogeny, the position of the two subspecies is unresolved, although both appear to be closely related to other western North American species. The chloroplast data sets also suggest that the subspecies of O. mexicana are not monophyletic, with the position of the typical subspecies resolved (Fig. 3) and O. mexicana subsp. bipatriata closely allied to O. depauperata (Figs. 1 3). Comparisons of phylogenies based on ITS and chloroplast DNA data have been made in several previous studies (e.g., Franzke et al., 1998; Choi and Wen, 2000; McDade et al., 2000; Potter, Luby, and Harrison, 2000; Schwarzbach and Ricklefs, 2000; Smith, 2000). In the present analysis, phylogenies from the two data sets are largely incongruent. If separate data sets are incongruent as a result of evolutionary independence, then analysis of combined data sets may result in reduced or erroneous resolution with respect to the true organismal phylogeny (Hardig, Soltis, and Soltis, 2000). We thus have not attempted a combined analysis of the data obtained on Osmorhiza. Incongruence between nuclear and organellar phylogenetic trees is typically attributed to introgression of a cytoplasmic genome from one species into the nuclear background of another (e.g., Ferris et al., 1983; Gyllensten and Wilson, 1987; Harrison, Rand, and Wheeler, 1987; Tegelstrom, 1987; Soltis et al., 1991; Rieseberg and Wendel, 1993; Soltis and Kuzoff, 1995; Soltis, Johnson, and Looney, 1996). No interspecific hybridization, however, has been reported for Osmorhiza. Rare morphological intermediates have been observed among some species of Osmorhiza (Constance and Shan, 1948; Lowry and Jones, 1984), including between O. berteroi and O. occidentalis, O. glabrata and either O. berteroi or O. depauperata, and O. glabrata and O. mexicana subsp. mexicana. The high level of incongruence between nrdna and cpdna suggests that these morphological intermediates may be the result of hybridization and/or introgression. The morphologically variable taxa need to be examined along with their close relatives in areas where they co-occur. In this study, we examined a sample (Halse 5560) from a plant that cooccurs with O. berteroi and O. occidentalis and that shows some morphological intermediacy (especially the presence of some bristles on its fruit similar to those found in O. berteroi), but which is otherwise very similar to O. occidentalis. We examined the ITS and cpdna profiles of three samples representing the presumed hybrid and typical O. berteroi (Halse 5559) and O. occidentalis (Halse 5561). Both cpdna and nrits data showed that the presumed hybrid had an ITS and cpdna profile identical to that of O. occidentalis. There is thus no molecular evidence that hybridization has occurred in these populations. However, the data do not exclude the possibility that recent hybridization has occurred in which O. occidentalis is the maternal parent. An independent study will be undertaken with at least one additional nuclear marker, such as waxy (Mason-Gamer and Kellogg, 1996) or adh (Gaut and Clegg, 1993), to test the phylogenetic hypotheses and to examine the possible role of hybridization in the evolution of Osmorhiza. Biogeography The basally branching position of the Asiatic Osmorhiza aristata and the monophyly of the diverse New World clade (Figs. 1 4) support the hypothesis of a relatively ancient origin of the eastern Asian-eastern North American disjunction. The cladogenesis of Osmorhiza clearly appears to have been more rapid in the New World than in Asia. The relatively low DNA sequence divergence seen among the New World taxa, especially those in western North America, suggest rapid evolutionary radiation. Regarding the likely place of origin of the genus, the apparent close relationship of Osmorhiza with the Old World genera Myrrhis and Geocaryum (Downie, Katz-Downie, and Spalik, 2000), coupled with the basal position of O. aristata, suggest that it took place in the Old World (Wen et al., 2002). This interpretation differs from the one offered by Lowry and Jones (1984), who hypothesized a New World origin of Osmorhiza. More recent diversification among western North American Osmorhiza may have been facilitated by the availability of a broad array of habitats associated with the uplifting of the Rocky Mountains and the western cordillera in general, during the Tertiary (Barbour and Christensen, 1993; Graham, 1993). Osmorhiza berteroi and O. depauperata show a similar pattern of antitropical disjunction between the temperate western North America and South America. Little sequence divergence was found among the populations of these species from the two hemispheres, a pattern similar to that observed in the nrits sequences. Constance (1963) suggested that these species might have migrated to South America in a step-wise manner along the western American cordillera during the Tertiary, with subsequent elimination of populations from the intervening tropical areas. However, the sequence divergence of cpdna ( %) and nrits ( %) between the western North American and South American populations of both species suggests a more recent origin. As indicated by Raven (1963), this pattern of disjunction corresponds closely to the migration routes of many bird species, which likely accounts for the distributions of O. berteroi and O. depauperata (Lowry and Jones, 1984). Thus, the sequence divergence, the monophyly of both species, and their fruit morphology support the recent origin of the antitropical disjunction, perhaps by long-distance dispersal via birds from western North America to South America. LITERATURE CITED BAKKER, F. T., A. CULHAM, C. E. PANKHURST, AND M. GIBBY Mitochondrial and chloroplast DNA-based on phylogeny of Pelargonium (Geraniaceae). American Journal of Botany 87: BALDWIN, B. G Phylogenetic utility of the transcribed spacers of nuclear ribosomal DNA in plants: an example from the Compositae. Molecular Phylogenetics and Evolution 1: BARBOUR, M. G., AND N. L. CHRISTENSEN Vegetation. In Flora of North America Editorial Committee [eds.], Flora of North America, vol. 1, Oxford University Press, New York, New York, USA. BAYER, R. J., C. F. PUTTOCK, AND S. A. KELCHNER Phylogeny of South African Gnaphalieae (Asteraceae) based on two noncoding chloroplast sequences. American Journal of Botany 87: BAYER, R. J., P. S. SOLTIS, AND D. E. SOLTIS Phylogenetic inferences in Antennaria (Asteraceae: Gnaphalieae: Cassininae) based on sequences from nuclear ribosomal DNA internal transcribed spacer (ITS). American Journal of Botany 83:

6 June 2002] YOO ET AL. PHYLOGENETICS OF OSMORHIZA 971 BOIVIN, B Flora of the prairie provinces. Phytologia 17: CHOI, H.-K., AND J. WEN A phylogenetic analysis of Panax (Araliaceae): integrating cpdna restriction site and nuclear rdna ITS sequence data. Plant Systematics and Evolution 224: CLARKE, C. B Umbelliferae. In J. D. Hooker [ed.], Flora of British India, vol. 2, Reeve, Ashford, Kent, England. CONSTANCE, L Amphitropical relationships in the herbaceous flora of the Pacific Coast of North and South America: a symposium. Introduction and historical review. Quarterly Review of Biology 38: CONSTANCE, L., AND R. H. SHAN The genus Osmorhiza (Umbelliferae), a study in geographic affinities. University of California Publications in Botany 23: DOWNIE, S. R., D. S. KATZ-DOWNIE, AND K. SPALIK A phylogeny of Apiaceae tribe Scandiceae: evidence based from ribosomal DNA internal transcribed spacer sequences. American Journal of Botany 87: DOYLE, J. J., AND J. L. DOYLE A rapid DNA isolation procedure for small quantities of fresh leaf material. Phytochemical Bulletin 19: FARRIS, J. S., M. KALLERSJO, A. G. KLUGE, AND C. BULT Testing significance of incongruence. Cladistics 10: FELSENSTEIN, J Evolutionary trees from DNA sequences: maximum likelihood approach. Journal of Molecular Evolution 17: FELSENSTEIN, J Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: FERRIS, S. D., R. D. SAGE, C. M. HUANG, J. T. NIELSON, U. RITTE, AND A. C. WILSON Flow of mitochondrial DNA across a species boundary. Proceedings of the National Academy of Sciences, USA 80: FRANZKE, A., K. POLLMANN, W. BLEEKER, R. KOHRT, AND H. HURKA Molecular systematics of Cardamine and allied genera (Brassicaceae): ITS and non-coding chloroplast DNA. Folia Geobotanica 33: GAUT, B. S., AND M. T. CLEGG Molecular evolution of the ADH1 locus in the genus Zea. Proceedings of the National Academy of Sciences, USA 90: GIELLY, L., AND P. TABERLET The use of chloroplast DNA to resolve plant phylogenies: noncoding versus rbcl sequences. Molecular Biology and Evolution 11: GIELLY, L., AND P. TABERLET A phylogeny of the European gentians inferred from chloroplast trnl (UAA) intron sequences. Botanical Journal of the Linnean Society 120: GIELLY, L., Y.-M. YUAN, P. KUPFER, AND P. TABERLET Phylogenetic use of noncoding regions in the genus Gentiana L.; chloroplast trnl (UAA) intron versus nuclear ribosomal internal transcribed spacer sequences. Molecular Phylogenetics and Evolution 5: GRAHAM, A History of the vegetation: Cretaceous (Maastrichtian)- Tertiary. In Flora of North America Editorial Committee [eds.], Flora of North America north of Mexico, Oxford University Press, New York, New York, USA. GYLLENSTEN, U., AND A. C. WILSON Interspecific mitothondrial DNA transfer and the colonization of Scandinavia by mice. Genetical Research 49: HARDIG, T. M., P. S. SOLTIS, AND D. E. SOLTIS Diversification of the North American shrub genus Ceanothus (Rhamnaceae): conflicting phylogenies from nuclear ribosomal DNA and chloroplast DNA. American Journal of Botany 87: HARRISON, R. G., D. M. RAND, AND W. C. WHEELER Mitochondrial DNA variation in field crickets across a narrow hybrid zone. Molecular Biology and Evolution 4: KIM, K.-J., AND R. K. JANSEN Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia, Asteraceae): additional information from internal transcribed spacer sequences of nuclear ribosomal DNA. Plant Systematics and Evolution 190: KIMURA, M A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: KITA, Y., K. UEDA, AND Y. KADATA Molecular phylogeny and evaluation of the Asian Aconitum subgenus Aconitum (Ranunculaceae). Journal of Plant Research 108: KUNTZE, O Revisio generum plantarum. Felix, Leipziger, Germany. LOWRY II, P. P., AND A. G. JONES Systematics of Osmorhiza Raf. (Apiaceae: Apioideae). Annals of the Missouri Botanical Garden 71: MASON-GAMER, R. J., AND E. A. KELLOGG Potential utility of the nuclear gene waxy for plant phylogenetic analysis. American Journal of Botany (supplement) 83: 178. MCDADE, L. A., S. E. MASTA, M.L.MOODY, AND E. WATERS Phylogenetic relationships among Acanthaceae: evidence from two genomes. Systematic Botany 25: MICHAUX, A Flora boreali-americana, vol. 1. Levrault, Paris, France. OLMSTEAD, R. G., AND J. A. SWEERE Combining data in phylogenetic systematics: an empirical approach using three molecular data sets in the Solanaceae. Systematic Biology 43: PERSOON, C. H Synopsis plantarum seu enchiridium botanicum, vol. 1. J. G. Cotta, Tübingen, Germany. POTTER, D., J. J. LUBY, AND R. E. HARRISON Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from noncoding nuclear and chloroplast DNA sequences. Systematic Botany 25: RAFINESQUE, C. S A review of Pursh s Flora of North America. American Monthly Magazine and Critical Review 2: RAVEN, P. H Amphitropical relationships in the floras of North and South America. Quarterly Review of Biology 38: RIESEBERG, L. H., AND J. F. WENDEL Introgression and its consequences in plants. In R. G. Harrison [ed.], Hybrid zones and the evolutionary process. Oxford University Press, New York, New York, USA. SANGER, F., S. NICKLEN, AND A. R. COULSEN DNA sequencing with chain-terminating inhibitors. Proceeding of the National Academy of Sciences, USA 74: SCHWARZBACH, A. E., AND R. E. RICKLEFS Systematic affinities of Rhizophoraceae and Anisophylleaceae, and intergeneric relationships within Rhizophoraceae, based on chloroplast DNA, nuclear ribosomal DNA, and morphology. American Journal of Botany 87: SMITH, J. F Phylogenetic resolution within the tribe Episcieae (Gesneriaceae): congruence of ITS and ndhf sequences from parsimony and maximum-likelihood analyses. American Journal of Botany 87: SOLTIS, D. E., L. A. JOHNSON, AND C. LOONEY Discordance between ITS and chloroplast topologies in the Boykinia group (Saxifragaceae). Systematic Botany 21: SOLTIS, D. E., AND R. K. KUZOFF Discordance between molecular and chloroplast phylogenies in the Heuchera group (Saxifragaceae). Evolution 49: SOLTIS, D. E., P. S. SOLTIS, T.G.COLLIER, AND M. L. EDGERTON Variation within and among genera of the Heuchera group: evidence of chloroplast capture and paraphyly. American Journal of Botany 78: SPRENGEL, C Plantarum Umbelliferarum. Hendel, Halle, Germany. SWOFFORD, D. L PAUP*: phylogenetic analysis using parsimony, version 4. Sinauer, Sunderland, Massachusetts, USA. SWOFFORD, D. L., G. J. OLSEN, P.J.WADDELL, AND D. M. HILLIS Phylogenetic inference. In D. M. Hillis, C. Moritz, and B. K. Mable [eds.], Molecular systematics, 2nd ed., Sinauer, Sunderland, Massachusetts, USA. TABERLET, P., L. GIELLY, G. PAUTON, AND J. BOUVET Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: TEGELSTROM, H Transfer of mitochondrial DNA from the northern red-backed vole (Clethrionomys rutilus) to the bank vole (C. glareolus). Journal of Molecular Evolution 24: THUNBERG, C. P Flora Japonica. Müller, Leipziger, Germany. TORREY, J., AND A. GRAY Flora of the North America, vol. 1, part 4. Wiley and Putnam, New York, New York, USA. WEN, J., P. P. LOWRY II, J. L. WALCK, AND K.-O. YOO Phylogenetic and biogeographic diversifications in Osmorhiza (Apiaceae). Annals of the Missouri Botanical Garden, in press.