the sunflower family (Asteraceae)

Proc. NatI. Acad. Sci. USA Vol. 84, pp. 5818-5822, August 1987 Evolution

A chloroplast DNA inversion marks an ancient evolutionary split in the sunflower family (Asteraceae) (angiosperm evolution/molecular systematics/Mutisieae/Barnadesiinae)

ROBERT K. JANSEN*t

AND

JEFFREY D. PALMER

Department of Biology, University of Michigan, Ann Arbor, MI 48109

Communicated by Peter H. Raven, May 7, 1987 (received for review February 10, 1987)

ABSTRACT We determined the distribution of a chloroplast DNA inversion among 80 species representing 16 tribes of the Asteraceae and 10 putatively related families. Filter hybridizations using cloned chloroplast DNA restriction fragments of lettuce and petunia revealed that this 22-kilobase-pair inversion is shared by 57 genera, representing all tribes of the Asteraceae, but is absent from the subtribe Barnadesiinae of the tribe Mutisieae, as well as from all families allied to the Asteraceae. The inversion thus defmes an ancient evolutionary split within the family and suggests that the Barnadesiinae represents the most primitive lineage in the Asteraceae. These results also indicate that the tribe Mutisieae is not monophyletic, since any common ancestor to its four subtribes is also shared by other tribes in the family. This is the most extensive survey of the systematic distribution of an organelle DNA rearrangement and demonstrates the potential of such mutations for resolving phylogenetic relationships at higher taxonomic levels.

Cronquist's (1, 4, 7) subfamilial classification for the Asteraceae have been proposed in the last 12 years (8-10).

We are investigating chloroplast DNA (cpDNA) variation in the Asteraceae to resolve phylogenetic relationships at higher taxonomic levels. Our previous study (11) showed that the 151-kilobase (kb) cpDNAs of two species in the family (Lactuca sativa and Barnadesia caryophylla) are colinear throughout the genome, with the exception of a single 22-kb inversion. The conservative organization of the chloroplast genome among land plants (12, 13) makes such rearrangements potentially valuable characters for phylogenetic studies. Here we report on the evolutionary direction of the inversion in the Asteraceae by comparing the chloroplast genomes of Lactuca and Barnadesia with that of an outgroup, Petunia hybrida (Solanaceae). We also examine the distribution and phylogenetic significance of this rearrangement.

MATERIALS AND METHODS cpDNAs were isolated by the sucrose gradient technique (14). Where tissue amounts were limited, total DNA was isolated (15) and further purified by centrifugation in CsCl/ ethidium bromide gradients. Restriction endonuclease digestions, electrophoresis, transfer of DNA fragments from agarose gels to Zetabind filters (AMF Cuono), and hybridizations were performed as described (11, 14). Recombinant

The Asteraceae is one of the largest and economically most important families of flowering plants and consists of 12-17 tribes, approximately 1100 genera, and 20,000 species (1). A combination of several specialized morphological characteristics (e.g., capitula, highly reduced and modified flowers, inferior ovaries, syngenesious anthers) strongly supports the naturalness of the family. Cronquist (1) emphasized the distinctness of the Asteraceae by placing it in a monotypic order at the most advanced position within the subclass Asteridae. In addition to its large size, the family has a cosmopolitan distribution and is highly diversified in its habitat preferences and life forms. This diversity includes aquatics, herbs and shrubby trees in temperate, tropical, and arid environments, and trees in tropical rain forests. Species of Asteraceae are of wide economic importance as vegetables (lettuce, artichokes, endive), sources of oil (sunflower, safflower) and insecticides (pyrethrum), and garden ornamentals (chrysanthemum, dahlia, marigold, and many others). Although there is some controversy concerning its age (2, 3), fossil evidence (4, 5) and biogeographical considerations (6) suggest that the Asteraceae originated in the middle to upper Oligocene (30 million years ago) and subsequently underwent rapid radiation. This rapid diversification has posed special problems for understanding phylogenetic relationships at higher taxonomic levels. Previous attempts (4, 7-10) at constructing phylogenies have relied on comparative anatomical, chromosomal, embryological, micromolecular, morphological, and palynological features. These studies have been largely unsatisfactory because of the repeated parallel and convergent evolution of these characters. For example, three major and highly divergent reformulations of

plasmids containing cpDNA fragments from Lactuca and Petunia were described previously (11, 16). RESULTS Filter hybridizations using cloned restriction fragments (16) from petunia (Petunia hybrida, Solanaceae) were performed to assess cpDNA genome arrangement in the Asteraceae. The petunia genome appears to have the ancestral cpDNA arrangement for angiosperms, since it is colinear with the genomes of a fern, a gymnosperm, and several diverse angiosperms (17-21). Barnadesia cpDNA is colinear with the petunia genome (Fig. 1) and therefore has the same gene order as the ancestral angiosperm type. In contrast, lettuce (Lactuca sativa) cpDNA has a derived inversion in the large single copy region, as evidenced by the hybridization of nonadjacent petunia Pst I fragments of 9.0 and 15.3 kb to the same two regions of the lettuce genome. For example, both of these petunia probes hybridize to 7.5-kb Sac I-Sal I and 6.7-kb Sac I lettuce restriction fragments (Fig. 1). Furthermore, the atpA through rpoB genes have an inverted order and are transcribed in the opposite direction in lettuce relative to Barnadesia (Fig. 1; ref. 11). Abbreviation: cpDNA, chloroplast DNA. *Present address: Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06268. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5818

Proc. Nati. Acad. Sci. USA 84 (1987)

Evolution: Jansen and Palmer E E

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FIG. 1. Physical maps showing the arrangement of homologous sequences in the petunia and either lettuce or Barnadesia chloroplast Numbers indicate fragment sizes in kb. Each of 15 petunia fragments was hybridized to filter blots containing Nsi I and Sac I fragments of cpDNA from lettuce and Barnadesia. The lettuce or Barnadesia fragments to which the probes hybridize are indicated by lines leading from the petunia fragments to the lettuce or Barnadesia fragments. The heavy black lines on each map indicate the inverted repeat and the arrows at far right show the orientation (i.e., direction of transcription) of mapped genes. The enlargements of the 7.5-kb Sac I-Sal I and 6.7-kb Sac I restriction fragments show the four inversion endpoint fragments used as probes. Arrows pointing at the EcoRl sites indicate the approximate locations of the inversion endpoints. Lettuce and Barnadesia restriction site and gene mapping data are from ref. 11 and petunia data are from ref. 16. Restriction sites shown: m, Nsi I; A, Pst I; *, Sac I; *, Sal I; Bg, Bgl 1I; E, EcoRI; S, Sal 1.

genomes.

Many additional taxa were surveyed for the inversion by performing filter hybridizations using cloned lettuce cpDNA fragments that contain the inversion endpoints. The 7.5-kb Sac I-Sal I and 6.7-kb Sac I lettuce fragments were used as hybridization probes against filter blots containing 12 restriction enzyme digests of DNA from one species of each of 80 genera representing 10 putatively allied families and 16 tribes of Asteraceae (Table 1). The 7.5-kb Sac I-Sal I and 6.7-kb Sac I probes will hybridize to different restriction fragments

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FIG. 2. Physical maps showing the arrangement of homologous in the 22-kb inversion region of the lettuce and either Barnadesia or Vernonia chloroplast genomes. The Barnadesia and Vernonia Sac I fragments to which the 7.5-kb Sac I-Sal I and 6.7-kb Sac I probes hybridize are indicated by lines leading from the lettuce fragments to the Barnadesia and Vernonia fragments. Numbers indicate fragment sizes in kb. Restriction sites shown: e, Sac I; *, Sal I. sequences

in those genomes that contain the lettuce inversion. This situation is illustrated in Figs. 2 and 3, in which the two inversion endpoint fragments from lettuce are hybridizing to Sac I fragments of 14.7 and 17.0 kb in Vernonia. Similar hybridization results are evident for Helianthus and Trixis (Fig. 3), which are both members of the Asteraceae. In contrast, in those genomes that are not rearranged, the two lettuce probes will hybridize to two of the same restriction fragments. For example, the 7.5-kb Sac I-Sal I and 6.7-kb Sac I lettuce probes both hybridize to Sac I fragments of 5.8 and 14.9 kb in Barnadesia (Figs. 2 and 3). The autoradiograms (Fig. 3) reveal that representatives of three related families, Cephalaria (Dipsacaceae), Pentas (Rubiaceae), and Scaevola (Goodeniaceae), also lack the 22-kb inversion. The results of the inversion survey for all 80 examined taxa are summarized in Table 1. The genome arrangements for 69 of these taxa have been confirmed by constructing complete restriction maps (R.K.J., H. Michaels, and J.D.P., unpublished data). The inversion is absent from all putatively allied families and, within the Asteraceae, from the subtribe Barnadesiinae of the tribe Mutisieae. All other examined members of the Asteraceae, including the three other subtribes in the Mutisieae, were found to have the inversion. The 80 genera surveyed represent the major evolutionary lineages within the 16 tribes of Asteraceae and 10 related families. We are confident that the selection of only one species from each genus is an adequate sampling because more extensive studies of 60 species in Carthamus (R. Johnson and J.D.P., unpublished data), Coreopsis (D. Crawford and J.D.P., unpublished data), Hieracium (R.K.J. and J.D.P., unpublished data), and Lactuca (E. Jandourek and J.D.P., unpublished data) have revealed no intrageneric variation in chloroplast genome arrangement in the Asteraceae.

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Proc. Natl. Acad. Sci. USA 84

(1987)

Table 1. Distribution of a 22-kb cpDNA inversion Inversion present Inversion absent Asteriodeae Mutisieae (continued) Rosidae Anthemideae Gochnatiinae Apiaceae Achillea millefolium Ainsliaea dissecta Angelica archangelica Chrysanthemum maximum Gochnatia paucifolia Hydrocotyle verticillata Santolina chamaecyparissus Onoseris hyssopifolia Araliaceae Astereae Stifftia chrysantha Hedera helix Aster cordifolius Mutisiinae Asteridae Bellis perennis Chaptalia tomentosa Brunoniaceae Erigeron hybridus Gerbera jamesondi Brunonia australis Felicia bergeriana Leibnitzia seemannii Campanulaceae Solidago sp. Mutisia acuminata Campanula ramosa Calenduleae Piloselloides hirsuta Jasione perennis Calendula officinalis Nassauviinae Lobelia ramosa Dimorphotheca pluvialis Acourtia microcephala Caprifoliaceae Osteospermum muricatum Perezia multiflora Viburnum acerifolium Cotuleae Trixis californicum Dipsacaceae Cotula barbata Arctotideae Cephalaria leucantha Heliantheae Arctotis stoechadifolia Dipsacus sativus Coreopsis grandiflora Gazania splendens Scabiosa atropurpurea Dahlia pinnata Haplocarpha scaposa Goodeniaceae Geraea canescens Cardueae Dampiera stricta Helianthus annuus Centaurea montana Goodenia hedera Perityle emoryii Cirsium sp. Goodenia lanata Wedelia trilobata Echinops exaltatus Scaevola frutescens Inuleae Silybum marianum Rubiaceae Antennaria neodioica Cichorieae Pentas lanceolata Hieracium pratense Gnaphalium luteoalbum Psychotria bacteriophila Inula helenium Lactuca sativa Stylidiaceae Senecioneae Tragopogon porrifolius Stylidium adnatum Blennosperma nana Eupatorieae Valerianaceae Euryops pectinatus Chromolaena sp. Valeriana officinalis Senecio mikanioides Eupatorium atrorubens Asteraceae* Tageteae Liatris spicata Cichorioideae Dyssodia pentachaeta Liabeae Mutisieae Tagetes erecta Cocosmia rugosa Barnadegiinae Ursinieae Liabum glabrum Barnadesia caryophylla Ursinia nana Vernonieae Chuquiragua oppositifolia Lycnophora tomentosa Dasyphyllum diacanthoides Piptocarpha axillaris Stokesia laevis Vernonia mespilifolia Voucher specimens deposited at MICH (Ann Arbor, Ml). *The list of subfamilies and tribes follows Jeffrey (10). tThe list of subtribes follows Cabrera (22).

We also performed filter hybridizations to a single enzyme digest of DNA from the 80 species, using four restriction fragments (whose sizes and locations are shown in the enlargement at the top of Fig. 1) subcloned from the 6.7-kb Sac I and 7.5-kb Sac I-Sal I fragments. We previously showed (11) that the lettuce inversion endpoints are located very close to the EcoRI sites separating these two pairs of adjacent fragments (Fig. 1, arrows). These smaller, more precise probes hybridize to those genomes containing the inversion in exactly the same manner as to the parental lettuce genome (data not shown). This gives us greater confidence that these taxa have the same inversion as lettuce, rather than a similar but different inversion in the same region of the chloroplast genome.

alternative explanations for the phylogenetic distribution of the inversion (Fig. 4). The most parsimonious interpretation is that the three genera in the Barnadesiinae primitively lack the inversion and that this derived mutation groups all other Asteraceae together. Alternatively, the inversion occurred in the common ancestor of the entire family and subsequently reverted in the Barnadesiinae. The former explanation seems more likely, both on a parsimony basis (23) and, more compellingly, because cpDNA inversions are rare among land plants (12, 13). This particular inversion appears to have occurred only once in some 400 million years of land plant evolution. Furthermore, independent cladistic studies using data from restriction site mapping (unpublished) and morphology (24) support the phylogeny shown in Fig. 4 and place the Barnadesiinae as an ancestral lineage within the Aster-

DISCUSSION

aceae. The distribution of the cpDNA inversion within the Asteraceae (Table 1, Fig. 4) defines the primary evolutionary split within this large and important family of flowering plants, and thus it has significant phylogenetic implications. Indeed, one of the most controversial systematic issues within the family has been the identification of the most primitive lineage. A

The 22-kb inversion must be derived within the Asteraceae since all putative outgroup families lack this rearrangement. Furthermore, more inclusive outgroups, including 30 additional families of angiosperms, a gymnosperm, and a fern, also lack the inversion (12, 13, 17-20). There are two


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Proc. Natl. Acad. Sci. USA 84 (1987)

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FIG. 3. Hybridization of cloned (11) lettuce restriction fragments to Sac I digests of DNA from eight representative species from the Asteraceae and related families. Bar, Barnadesia; Cep, Cephalaria; Hel, Helianthus; Lac, Lactuca; Pen, Pentas; Sca, Scuevola; Tri, Trixis; and Ver, Vernonia. Numbers above the filters refer to hybridization probes. Numbers alongside the filters indicate fragment sizes in kb.

number of putatively primitive morphological and anatomical characters have been used to hypothesize an ancestral position for the Heliantheae (2, 4, 7, 9, 25). However, four other tribes, the Cardueae, Mutisieae, Senecioneae, and Vernonieae, have also been suggested as being most primitive (7-9, 26). The primary reasons for this lack of agreement are the uncertainty about which family or families constitute the best outgroup and the high incidence of parallel and convergent evolution in the characters that have been used. Identification of primitive character states has thus been difficult, whereas the cpDNA inversion is unambiguously rooted and appears free of parallelism and convergence. The data presented here, together with the two recent morphological and restriction site studies cited above, clearly indicate that the Barnadesiinae is the primitive group within the Asteraceae. This conclusion agrees with recent suggestions Osmunda Ginkgo

40 Families, 26 Orders, and 9 Subclasses

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of Angiosperms

Absent

Subtribe Barnadesiinae of the Mutisieae

Asteraceae: 16 Tribes, - 57 Genera, 3 other Subtribes of the Mutisieae

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Present

FIG. 4. Evolutionary tree based on the 22-kb chloroplast DNA inversion. Data are summarized from Table 1 and refs. 12, 13, and 17.

(8, 9, 26) that the Mutisieae contains the most primitive taxa in the family. Our identification of the Barnadesiinae as the most primitive lineage in the Asteraceae provides support for suggestions that the Asteraceae originated in montane South America (2, 6, 27, 28), as the eight genera of this subtribe are centered in the northern Andes (22). Our results are consistent with suggestions (8, 26, 29) that bilabiate (two-lipped) flowers and woody habit, which are common features in the Barnadesiinae, are primitive within the Asteraceae. This suggests affinities between the Asteraceae and the families with some bilabiate or woody members, including the Campanulaceae, Lobeliaceae, Goodeniaceae, and Stylidiaceae.

The distribution of the cpDNA inversion also provides insights into phylogenetic relationships within the Mutisieae. Our data confirm previous suggestions (22, 30, 31) that the Mutisieae is not a monophyletic group (i.e., one derived from a common ancestor not shared by any other tribes in the Asteraceae), since three of its four subtribes are more closely related cladistically to 15 other tribes than they are to the Barnadesiinae (Table 1, Fig. 4). The uniqueness of the subtribe Barnadesiinae is evident in its lack of the 22-kb cpDNA inversion and its distinctive pollen (31-33) and floral (30) morphology. This is the most extensive survey of the systematic distribution of a structural mutation in an organelle genome and clearly demonstrates the potential of rearrangements for resolving phylogenetic relationships at higher taxonomic levels. Detailed studies of cpDNA inversions (12, 13) in other flowering plant families, including the economically important grasses and legumes, should be equally valuable in making major phylogenetic groupings among and within these families. We thank the following individuals for providing seeds or live plant material: James M. Affolter, James A. Armstrong, Randall J. Bayer, Roger C. Carolin, Nancy C. Coile, Daniel J. Crawford, Michael 0. Dillon, Charles Jeffrey, Samuel B. Jones, David J. Keil, Timothy K. Lowrey, Guy L. Nesom, Tycho Norlindh, Robert Ornduff, James Price, Roger W. Sanders, John L. Strother. We also thank K. Bremer

5822

Proc. Natl. Acad. Sci. USA 84


for providing an unpublished cladistic analysis of the Asteraceae, M. Hommel and the Matthaei Botanical Garden for expert care and growth of plants, E. Clark and M. Hanson for petunia Sal I clones, and W. Brown, T. Bruns, M. Chase, D. Crawford, J. Manhart, H. Michaels, B. Milligan, C. Moritz, W. Wagner, Jr., and M. Zolan for critical reading of the manuscript. This study was supported by Grant BSR-8415934 from the National Science Foundation. 1. Cronquist, A. (1981) An Integrated System of Classification of Flowering Plants (Columbia Univ. Press, New York), pp. 1020-1028. 2. Turner, B. L. (1977) in The Biology and Chemistry of the Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, B. L. (Academic, London), Vol. 1, pp. 21-39. 3. Boulter, D., Gleaves, J. T., Haslett, B. G., Peacock, D. & Jensen, U. (1978) Phytochemistry 17, 1585-1589. 4. Cronquist, A. (1977) Brittonia 29, 137-153. 5. Muller, J. (1981) Bot. Rev. 47, 1-142. 6. Raven, P. H. & Axelrod, D. 1. (1974) Ann. Mo. Bot. Gard. 61,

539-673. 7. Cronquist, A. (1955) Am. Midi. Nat. 53, 478-511. 8. Carlquist, S. (1976) Aliso 8, 465-492. 9. Wagenitz, G. (1976) Plant Syst. Evol. 125, 29-46. 10. Jeffrey, C. (1978) in Flowering Plants of the World, ed. Heywood, V. H. (Mayflower, New York), pp. 263-268. 11. Jansen, R. K. & Palmer, J. D. (1987) Curr. Genet. 11, 553-564. 12. Palmer, J. D. (1985) Annu. Rev. Genet. 19, 325-354. 13. Palmer, J. D. (1985) in Molecular Evolutionary Genetics, ed. MacIntyre, R. J. (Plenum, New York), pp. 131-240. 14. Palmer, J. D. (1986) Methods Enzymol. 118, 167-186. 15. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A. &

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22. Cabrera, A. L. (1977) in The Biology and Chemistry of the Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, B. L. (Academic, London), Vol. 2, pp. 1039-1066. 23. Crisci, J. V. (1982) J. Theor. Biol. 97, 35-41. 24. Bremer, K. (1987) Cladistics, in press. 25. Koch, M. F. (1930) Am. J. Bot. 17, 938-952. 26. Jeffrey, C. (1977) in The Biology and Chemistry of the Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, B. L. (Academic, London), Vol. 1, pp. 111-118. 27. Bentham, G. (1873) J. Linn. Soc. Lond. Bot. 13, 335-577. 28. Small, J. (1919) New Phytol. 18, 1-29. 29. Carlquist, S. (1966) Aliso 6, 25-44. 30. Small, J. (1918) New Phytol. 17, 13-40. 31. Wodehouse, R. P. (1928) Bull. Torrey Bot. Club 55, 449-462. 32. Wodehouse, R. P. (1929) Am. J. Bot. 16, 297-313. 33. Skvarla, J. J., Turner, B. L., Patel, V. C. & Tomb, A. S. (1977) in The Biology and Chemistry of the Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, B. L. (Academic, London), Vol. 1, pp. 141-248.