Interfamilial Relationships of the Asteraceae: Insights from rbcL Sequence Variation

Nucleotide sequences of the chloroplast gene rbcL were analyzed to examine relationships among the large, distinctive family Asteraceae and eight putatively closely related families. Phylogenetic analysis of a total of 24 sequences of rbcL identified a lineage consisting of two families, the Goodeniaceae and Calyceraceae, as the sister group to the Asteraceae. In addition, a strongly supported major monophyletic lade consisting of Asteraceae, Goodeniaceae, Calyceraceae, Corokia (Cornaceae sensu Cronquist), Menyanthaceae, Lobeliaceae, and Campanulaceae was found. These results clearly distance from the Asteraceae certain groups previously considered closely related; moreover, the results upport alternative hypotheses of affinity that were based upon floral and inflorescence morphology, biogeography, pollen morphology, chemistry, and pollen-presentation mechanisms. The angiosperm family Asteraceae has long been recognized as one of the large, "natural" families with well-established limits defined by several specialized floral characteristics and distinctive secondary chemistry. Many recent studies have illuminated numerous phylogenetic controversies within the family (Bremer, 1987; Bremer et al., 1992; Jansen et al., 1990, 1991a, b; Jansen & Palmer, 1987a, b, 1988; Karis et al., 1992; Keeley & Jansen, 1991; Kim et al., 1992; Watson et al., 1991). However, relationships among the Asteraceae and other families have remained obscure, due to considerable parallel and convergent evolution of conventional characters used to infer affinities, lack of recent studies employing modern methods of phylogenetic analysis, and substantial confusion as to relationships among the various families within the subclass Asteridae itself (but see Olmstead et al., 1992). At least 12 families have been proposed as closest relatives of the Asteraceae based on a variety of traditional taxonomic characters. Although Hutchinson (1969) noted the superficial similarity of the inflorescence of the Dipsacales (Caprifoliaceae, Valerianaceae, and Dipsacaceae) to that of the Asteraceae, he suggested convergence as the basis for this and identified the Campanulales (Campanulaceae and Lobeliaceae) as the closest relative. Aspects of the distinctive chemistry of the Asteraceae (e.g., alkaloids, polyacetylenes, terpenes, inulin for carbohydrate storage) have been noted in several members of the Campanulales (Campanulaceae, Lobeliaceae, Goodeniaceae, Stylidiaceae), while other chemical evidence has pointed to the Apiaceae and Araliaceae (Hegnauer, 1964, 1977). Cronquist (1955) advocated the Rubiales as closest relatives of the Asteraceae (grouped with the Gentianales in the system of Takhtajan, 1980), but also acknowledged strong similarities in floral and inflorescence morphology between the Asteraceae and the Calyceraceae (as did Takhtajan, 1980). An association with the Calyceraceae is also supported by biogeography and capitular structure (Turner, 1977) and pollen morphology (Skvarla et al., 1977). Others (reviewed in Skvarla et al., 1977) have noted a palynological resemblance of Valerianaceae, Goodeniaceae and Brunoniaceae to Asteraceae. In a morphological cladistic study of tribal relationships within the Asteraceae, stylar morphology, chemical characters, and pollen-presen1 We thank D. Morgan and D. Soltis for providing an unpublished rbcL sequence of Corokia. We are grateful to M. Chase, D. Soltis, and T. Lammers for comments on the manuscript. This research has been supported by Ohio Board of Regents Research Challenge and NSF grants BSR-8700195 to H. Michaels and BSR-8717600 to J. Palmer. 2 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403, U.S.A. 3Department of Biology, Indiana University, Bloomington, Indiana 47405, U.S.A. 4Department of E.P.O. Biology, University of Colorado, Boulder, Colorado 80309, U.S.A. 5 Department of Plant Pathology, University of California, Berkeley, California 94720, U.S.A. 6 Department of Botany, University of Texas at Austin, Austin, Texas 78713, U.S.A. ANN. MISSOURI BOT. GARD. 80: 742-751. 1993. Volume 80, Number 3 Michaels et al. 743 1993 Interfamilial Relationships of Asteraceae tation mechanism were identified by Bremer (1987) as potential synapomorphies linking the Lobeliaceae and Asteraceae. Finally, strong similarities to the highly specialized secondary pollen-presentation mechanism of the Asteraceae have been documented in Goodeniaceae, Brunoniaceae, Campanulaceae, and Lobeliaceae (Leins & Erbar, 1990). As noted in many of the above attempts to resolve this controversy, most of the morphological or chemical characters that support a particular hypothesis of ancestry are often also found in several related groups, and some must certainly be the product of parallel evolution. Several of the recent molecular advances in elucidating phylogenetic relationships within the family have employed restriction site analysis of chloroplast DNA. However, this approach is generally unsuitable at the interfamilial level because the homology of site changes becomes doubtful due to increased levels of both nucleotide sequence divergence (causing multiple "hits" within restriction sites) and length variation causing problems in alignment of sites (Downie & Palmer, 1992b; Palmer et al., 1988). At higher taxonomic levels, restriction site analysis of only the more conserved inverted repeat region of chloroplast DNA may be used to circumvent hese problems (e.g., Downie & Palmer, 1992a), but fewer characters are generated than in whole genome surveys. DNA sequence analysis of the slowly evolving chloroplast gene rbcL and nuclear rRNA genes has proven highly effective in resolving higher-level relationships in plants (Chase et al., 1993; Hamby & Zimmer, 1992; Palmer et al., 1988; Ritland & Clegg, 1987; Zurawski & Clegg, 1987). In particular, recent studies by a number of researchers employing comparative sequencing of the chloroplast gene encoding the large subunit of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase (rbcL) indicate an appropriate size and rate of evolution for providing a sufficient number of characters for phylogenetic studies at the familial and ordinal levels (Donoghue et al., 1992; Kim et al., 1992; Olmstead et al., 1992; Soltis et al., 1990). In this paper we analyze nucleotide sequences for rbcL from representatives of the Asteraceae and eight putatively related families to determine evolutionary relationships among the families and identify the sister group of the Asteraceae. MATERIALS AND METHODS New rbcL sequences were determined for six taxa in the Asteraceae and eight representatives of putatively closely related families in the subclass Asteridae. For these sequences, fresh leaf material obtained either from the field or seedlings was used to isolate DNA as purified chloroplast DNA by the sucrose gradient method (Palmer, 1986) or as total cellular DNA by a modified CTAB procedure (Doyle & Doyle, 1987) followed by CsCl gradient purification. The rbcL gene was isolated for cloning by one of two methods. (1) For most taxa, fragments containing the entire rbcL gene were gel-isolated from either Sac I or Sac I/BamHI digests and ligated into the plasmid vector Bluescript (Stratagene, Inc., LaJolla, California). Recombinant, rbcLcontaining colonies were confirmed by Southern hybridization to cloned rbcL fragments from peas. The coding region was sequenced from a singlestranded template by the dideoxy chain termination method (Sanger et al., 1977) using a series of primers based on rbcL sequences from maize and spinach (obtained from G. Zurawski, DNAX). (2) Sequences from Pentas and Boopis were obtained following amplification and cloning of a doublestranded fragment using the polymerase chain reaction following the methods of Olmstead et al. (1992). Preliminary analyses included 14 new sequences (see Table 1 and Appendix to this issue) and one previously published sequence from Flaveria in the Asteraceae (Hudson et al., 1990) and, to serve as outgroups, sequences from Spinacia in the Caryophyllidae (Zurawski et al., 1981) and Nicotiana in the Asteridae (Shinozaki et al., 1988). These analyses (Michaels & Palmer, 1990) on only a subset of the data reported here identified a closest sister group identical to the present expanded analysis. The results of concurrent studies of rbcL sequences in the Saxifragaceae (Soltis et al., 1990), Asteridae (Olmstead et al., 1992), and 499 angiosperms (see Chase et al., 1993) have motivated the inclusion of data from other outgroups and from taxa not previously suspected to be associated with the Asteraceae. Data for Heuchera (Soltis et al., 1990), Magnolia (Golenberg et al., 1990), Villarsia, Menyanthes, Hedera, and Coriandrum (Olmstead et al., 1992) were obtained from published reports, whereas an unpublished sequence for Corokia was made available by D. Morgan and D. Soltis. Although the coding regions for these taxa ranged from 1428 to 1458 bp, only a 1428 bp region was analyzed because no major insertions or deletions were found in this region, allowing alignment by eye, and because homology of positions beyond 1428 is uncertain. Analyses over longer sequences did, however, produce virtually the same results. 744 Annals of the Missouri Botanical Garden TABLE 1. Species of Asteraceae, related families, and outgroups compared by rbcL sequence. * = New sequence obtained for this study.