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Home , Acourtia, Acourtia microcephala, Ainsliaea, Arctotis stoechadifolia, Barnadesia, Blennosperma

Proc. NatI. Acad. Sci. USA Vol. 84, pp. 5818-5822, August 1987 Evolution A DNA inversion marks an ancient evolutionary split in the sunflower () (angiosperm evolution/molecular systematics//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 (receivedfor review February 10, 1987)

ABSTRACT We determined the distribution of a chloro- Cronquist's (1, 4, 7) subfamilial classification for the - plast DNA inversion among 80 representing 16 tribes of aceae have been proposed in the last 12 years (8-10). the Asteraceae and 10 putatively related families. Filter hy- We are investigating chloroplast DNA (cpDNA) variation bridizations using cloned chloroplast DNA restriction frag- in the Asteraceae to resolve phylogenetic relationships at ments oflettuce and petunia revealed that this 22-kilobase-pair higher taxonomic levels. Our previous study (11) showed that inversion is shared by 57 genera, representing all tribes of the the 151-kilobase (kb) cpDNAs of two species in the family Asteraceae, but is absent from the subtribe Barnadesiinae of (Lactuca sativa and caryophylla) are colinear the Mutisieae, as well as from all families allied to the throughout the genome, with the exception of a single 22-kb Asteraceae. The inversion thus defmes an ancient evolutionary inversion. The conservative organization of the chloroplast split within the family and suggests that the Barnadesiinae genome among land (12, 13) makes such rearrange- represents the most primitive lineage in the Asteraceae. These ments potentially valuable characters for phylogenetic stud- results also indicate that the tribe Mutisieae is not mono- ies. Here we report on the evolutionary direction of the phyletic, since any common ancestor to its four subtribes is also inversion in the Asteraceae by comparing the chloroplast shared by other tribes in the family. This is the most extensive genomes of Lactuca and Barnadesia with that of an out- survey of the systematic distribution of an organelle DNA group, Petunia hybrida (Solanaceae). We also examine the rearrangement and demonstrates the potential of such muta- distribution and phylogenetic significance of this rearrange- tions for resolving phylogenetic relationships at higher taxo- ment. nomic levels. MATERIALS AND METHODS The Asteraceae is one of the largest and economically most important families of flowering plants and consists of 12-17 cpDNAs were isolated by the sucrose gradient technique tribes, approximately 1100 genera, and 20,000 species (1). A (14). Where tissue amounts were limited, total DNA was combination of several specialized morphological character- isolated (15) and further purified by centrifugation in CsCl/ istics (e.g., capitula, highly reduced and modified , ethidium bromide gradients. Restriction endonuclease diges- inferior ovaries, syngenesious anthers) strongly supports the tions, electrophoresis, transfer of DNA fragments from naturalness of the family. Cronquist (1) emphasized the agarose gels to Zetabind filters (AMF Cuono), and - distinctness of the Asteraceae by placing it in a monotypic izations were performed as described (11, 14). Recombinant order at the most advanced position within the subclass plasmids containing cpDNA fragments from Lactuca and Asteridae. In addition to its large size, the family has a Petunia were described previously (11, 16). cosmopolitan distribution and is highly diversified in its preferences and life forms. This diversity includes RESULTS aquatics, herbs and shrubby in temperate, tropical, and Filter hybridizations using cloned restriction fragments (16) arid environments, and trees in tropical rain forests. Species from petunia (Petunia hybrida, Solanaceae) were performed ofAsteraceae are ofwide economic importance as vegetables to assess cpDNA genome arrangement in the Asteraceae. (lettuce, artichokes, endive), sources of oil (sunflower, saf- The petunia genome appears to have the ancestral cpDNA ) and insecticides (pyrethrum), and garden ornamen- arrangement for angiosperms, since it is colinear with the tals (, , marigold, and many others). genomes of a fern, a gymnosperm, and several diverse Although there is some controversy concerning its age (2, angiosperms (17-21). Barnadesia cpDNA is colinear with the 3), fossil evidence (4, 5) and biogeographical considerations petunia genome (Fig. 1) and therefore has the same gene (6) suggest that the Asteraceae originated in the middle to order as the ancestral angiosperm type. In contrast, lettuce upper Oligocene (30 million years ago) and subsequently (Lactuca sativa) cpDNA has a derived inversion in the large underwent rapid radiation. This rapid diversification has single copy region, as evidenced by the hybridization of posed special problems for understanding phylogenetic rela- nonadjacent petunia Pst I fragments of 9.0 and 15.3 kb to the tionships at higher taxonomic levels. Previous attempts (4, same two regions of the lettuce genome. For example, both 7-10) at constructing phylogenies have relied on comparative of these petunia probes hybridize to 7.5-kb Sac I-Sal I and anatomical, chromosomal, embryological, micromolecular, 6.7-kb Sac I lettuce restriction fragments (Fig. 1). Further- morphological, and palynological features. These studies more, the atpA through rpoB genes have an inverted order have been largely unsatisfactory because of the repeated and are transcribed in the opposite direction in lettuce parallel and convergent evolution of these characters. For relative to Barnadesia (Fig. 1; ref. 11). example, three major and highly divergent reformulations of Abbreviation: cpDNA, chloroplast DNA. The publication costs of this article were defrayed in part by page charge *Present address: Department of Ecology and Evolutionary Biology, payment. This article must therefore be hereby marked "advertisement" University of Connecticut, Storrs, CT 06268. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

5818 Downloaded by guest on October 1, 2021 Evolution: Jansen and Palmer Proc. Nati. Acad. Sci. USA 84 (1987) 5819

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FIG. 1. Physical maps showing the arrangement of homologous sequences in the petunia and either lettuce or Barnadesia chloroplast genomes. 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.

Many additional taxa were surveyed for the inversion by in those genomes that contain the lettuce inversion. This performing filter hybridizations using cloned lettuce cpDNA situation is illustrated in Figs. 2 and 3, in which the two fragments that contain the inversion endpoints. The 7.5-kb inversion endpoint fragments from lettuce are hybridizing to Sac I-Sal I and 6.7-kb Sac I lettuce fragments were used as Sac I fragments of 14.7 and 17.0 kb in . Similar hybridization probes against filter blots containing 12 restric- hybridization results are evident for and tion digests of DNA from one species of each of 80 (Fig. 3), which are both members of the Asteraceae. In two genera representing 10 putatively allied families and 16 tribes contrast, in those genomes that are not rearranged, the Asteraceae 1). The 7.5-kb Sac I-Sal I and 6.7-kb lettuce probes will hybridize to two of the same restriction of (Table 6.7-kb Sac I probes will hybridize to different restriction fragments fragments. For example, the 7.5-kb Sac I-Sal I and Sac I lettuce probes both hybridize to Sac I fragments of 5.8 and 14.9 kb in Barnadesia (Figs. 2 and 3). The autoradi- ograms that of three related 5.8 _ t 14.9 (Fig. 3) reveal representatives tBarnadesia families, (Dipsacaceae), Pentas (Rubiaceae), and (), 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 9Lettuce of these taxa have been confirmed by constructing complete restriction maps (R.K.J., H. Michaels, and J.D.P., unpub- lished data). The inversion is absent from all putatively allied 14.7 Vernonia families and, within the Asteraceae, from the subtribe Barnadesiinae of the tribe Mutisieae. All other examined members of the Asteraceae, including the three other sub- tribes 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

7.5 6.7 JLettuce are confident that the selection of only one species from each is an adequate sampling because more extensive FIG. 2. Physical maps showing the arrangement of homologous studies of 60 species in Carthamus (R. Johnson and J.D.P., sequences in the 22-kb inversion region of the lettuce and either unpublished data), (D. Crawford and J.D.P., 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 unpublished data), (R.K.J. and J.D.P., unpub- Sac I probes hybridize are indicated by lines leading from the lettuce lished data), and Lactuca (E. Jandourek and J.D.P., unpub- fragments to the Barnadesia and Vernonia fragments. Numbers indi- lished data) have revealed no intrageneric variation in chlo- cate fragment sizes in kb. Restriction sites shown: e, Sac I; *, Sal I. roplast genome arrangement in the Asteraceae. Downloaded by guest on October 1, 2021 5820 Evolution: Jansen and Palmer Proc. Natl. Acad. Sci. USA 84 (1987)

Table 1. Distribution of a 22-kb cpDNA inversion Inversion absent Inversion present Rosidae Mutisieae (continued) Asteriodeae Apiaceae Gochnatiinae Angelica archangelica dissecta Achillea millefolium Hydrocotyle verticillata Gochnatia paucifolia Chrysanthemum maximum hyssopifolia Santolina chamaecyparissus chrysantha Asteridae Mutisiinae Aster cordifolius Brunoniaceae tomentosa Bellis perennis australis jamesondi Erigeron hybridus seemannii bergeriana Campanula ramosa acuminata sp. Jasione perennis Piloselloides hirsuta Lobelia ramosa Nassauviinae Calendula officinalis microcephala pluvialis Viburnum acerifolium multiflora muricatum Dipsacaceae Trixis californicum Cotuleae Cephalaria leucantha Arctotideae Cotula barbata Dipsacus sativus stoechadifolia atropurpurea Gazania splendens Coreopsis grandiflora Goodeniaceae scaposa Dahlia pinnata stricta Cardueae canescens hedera Centaurea montana Cirsium sp. emoryii Scaevola frutescens exaltatus Wedelia trilobata Rubiaceae Silybum marianum Inuleae Pentas lanceolata neodioica Psychotria bacteriophila Hieracium pratense Gnaphalium luteoalbum Lactuca sativa Inula helenium adnatum porrifolius Valerianaceae Eupatorieae nana Valeriana officinalis Chromolaena sp. pectinatus Asteraceae* Eupatorium atrorubens Senecio mikanioides Liatris spicata Mutisieae Dyssodia pentachaeta Barnadegiinae Cocosmia rugosa Tagetes erecta Barnadesia caryophylla glabrum Ursinieae Chuquiragua oppositifolia nana diacanthoides Lycnophora tomentosa 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 alternative explanations for the phylogenetic distribution of digest of DNA from the 80 species, using four restriction the inversion (Fig. 4). The most parsimonious interpretation fragments (whose sizes and locations are shown in the is that the three genera in the Barnadesiinae primitively lack enlargement at the top of Fig. 1) subcloned from the 6.7-kb the inversion and that this derived mutation groups all other Sac I and 7.5-kb Sac I-Sal I fragments. We previously Asteraceae together. Alternatively, the inversion occurred in showed (11) that the lettuce inversion endpoints are located the common ancestor of the entire family and subsequently very close to the EcoRI sites separating these two pairs of reverted in the Barnadesiinae. The former explanation seems adjacent fragments (Fig. 1, arrows). These smaller, more more likely, both on a parsimony basis (23) and, more precise probes hybridize to those genomes containing the compellingly, because cpDNA inversions are rare among inversion in exactly the same manner as to the parental land plants (12, 13). This particular inversion appears to have lettuce genome (data not shown). This gives us greater occurred only once in some 400 million years of land confidence that these taxa have the same inversion as lettuce, evolution. Furthermore, independent cladistic studies using rather than a similar but different inversion in the same region data from restriction site mapping (unpublished) and mor- of the chloroplast genome. phology (24) support the phylogeny shown in Fig. 4 and place the Barnadesiinae as an ancestral lineage within the Aster- aceae. DISCUSSION The distribution of the cpDNA inversion within the Aster- The 22-kb inversion must be derived within the Asteraceae aceae (Table 1, Fig. 4) defines the primary evolutionary split since all putative outgroup families lack this rearrangement. within this large and important family offlowering plants, and Furthermore, more inclusive outgroups, including 30 addi- thus it has significant phylogenetic implications. Indeed, one tional families of angiosperms, a gymnosperm, and a fern, of the most controversial systematic issues within the family also lack the inversion (12, 13, 17-20). There are two has been the identification of the most primitive lineage. A Downloaded by guest on October 1, 2021 Evolution: Jansen and Palmer Proc. Natl. Acad. Sci. USA 84 (1987) 5821 Cep Pen Sca Bar Tri Lac Ver Hel 6.7 7.5 6.7 7.5 6.7 7 5 6.7 7.5 6.7 7.5 6.7 7.5 6.7 7.5 6.7 7.5 25- qw _ O_ 15- - 4, __ 4, 9- da

6- 4,

3-

1-

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 ofputatively primitive morphological and anatomical (8, 9, 26) that the Mutisieae contains the most primitive taxa characters have been used to hypothesize an ancestral in the family. position for the Heliantheae (2, 4, 7, 9, 25). However, four Our identification of the Barnadesiinae as the most prim- other tribes, the Cardueae, Mutisieae, Senecioneae, and itive lineage in the Asteraceae provides support for sugges- Vernonieae, have also been suggested as being most primi- tions that the Asteraceae originated in montane South Amer- tive (7-9, 26). The primary reasons for this lack of agreement ica (2, 6, 27, 28), as the eight genera of this subtribe are are the uncertainty about which family or families constitute centered in the northern Andes (22). Our results are consist- the best outgroup and the high incidence of parallel and ent with suggestions (8, 26, 29) that bilabiate (two-lipped) convergent evolution in the characters that have been used. flowers and woody habit, which are common features in the Identification of primitive character states has thus been Barnadesiinae, are primitive within the Asteraceae. This difficult, whereas the cpDNA inversion is unambiguously suggests affinities between the Asteraceae and the families with some bilabiate or woody members, including the rooted and appears free of parallelism and convergence. The Campanulaceae, Lobeliaceae, Goodeniaceae, and Stylidi- data presented here, together with the two recent morpho- aceae. logical and restriction site studies cited above, clearly indi- The distribution of the cpDNA inversion also provides cate that the Barnadesiinae is the primitive group within the insights into phylogenetic relationships within the Mutisieae. Asteraceae. This conclusion agrees with recent suggestions 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 any other tribes in Osmunda by the Asteraceae), since three of its four subtribes are more closely Ginkgo 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 (31-33) and floral 40 Families, 26 Orders, (30) morphology. and 9 Subclasses Inversion of Angiosperms Absent 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 Subtribe levels. Detailed studies of cpDNA inversions (12, 13) in other Barnadesiinae families, including the economically impor- of the Mutisieae tant grasses and legumes, should be equally valuable in making major phylogenetic groupings among and within these families. Asteraceae: 16 Tribes, Inversion - 57 Genera, 3 other Present We thank the following individuals for providing or live plant material: James M. Affolter, James A. Armstrong, Randall J. Bayer, Subtribes of the Mutisieae Roger C. Carolin, Nancy C. Coile, Daniel J. Crawford, Michael 0. Dillon, Charles Jeffrey, Samuel B. Jones, David J. Keil, Timothy K. FIG. 4. Evolutionary based on the 22-kb chloroplast DNA Lowrey, Guy L. Nesom, Tycho Norlindh, Robert Ornduff, James inversion. Data are summarized from Table 1 and refs. 12, 13, and 17. Price, Roger W. Sanders, John L. Strother. We also thank K. Bremer Downloaded by guest on October 1, 2021 5822 Evolution: Jansen and Palmer Proc. Natl. Acad. Sci. USA 84 (19.87)

for providing an unpublished cladistic analysis of the Asteraceae, M. Allard, R. W. (1984) Proc. Natl. Acad. Sci. USA 81, 8014- Hommel and the Matthaei Botanical Garden for expert care and 8018. growth of plants, E. Clark and M. Hanson for petunia Sal I clones, 16. Palmer, J. D., Shields, C. R., Cohen, D. B. & Orton,, T. J. and W. Brown, T. Bruns, M. Chase, D. Crawford, J. Manhart, H. (1983) Theor. Appl. Genet. 65, 181-189. Michaels, B. Milligan, C. Moritz, W. Wagner, Jr., and M. Zolan for 17. Palmer, J. D. & Stein, D. (1986) Curr. Genet. 10, 823-833. critical reading ofthe manuscript. This study was supported by Grant 18. Palmer, J. D. & Thompson, W. F. (1982) Cell 29, 537-550. BSR-8415934 from the National Science Foundation. 19. Fluhr, R. & Edelman, M. (1981) Nucleic Acids Res. 9, 6841-6853. 1. Cronquist, A. (1981) An Integrated System ofClassification of 20. De Heij, H. T., Lustig, H., Moeskops, D. J. M., Bovenberg, Flowering Plants (Columbia Univ. Press, New York), pp. W. A., Bisanz, C. & Groot, G. S. (1983) Curr. Genet. 7, 1-6. 1020-1028. 21. Sytsma, K. J. & Gottlieb, L. D. (1986) Evolution 40, 1248- 2. Turner, B. L. (1977) in The Biology and Chemistry of the 1201. Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, 22. Cabrera, A. L. (1977) in The Biology and Chemistry of the B. L. (Academic, London), Vol. 1, pp. 21-39. Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, 3. Boulter, D., Gleaves, J. T., Haslett, B. G., Peacock, D. & B. L. (Academic, London), Vol. 2, pp. 1039-1066. Jensen, U. (1978) Phytochemistry 17, 1585-1589. 23. Crisci, J. V. (1982) J. Theor. Biol. 97, 35-41. 4. Cronquist, A. (1977) Brittonia 29, 137-153. 24. Bremer, K. (1987) Cladistics, in press. 5. Muller, J. (1981) Bot. Rev. 47, 1-142. 25. Koch, M. F. (1930) Am. J. Bot. 17, 938-952. 6. Raven, P. H. & Axelrod, D. 1. (1974) Ann. Mo. Bot. Gard. 61, 26. Jeffrey, C. (1977) in The Biology and Chemistry of the 539-673. Compositae, eds. Heywood, V. H., Harborne, J. B. & Turner, 7. Cronquist, A. (1955) Am. Midi. Nat. 53, 478-511. B. L. (Academic, London), Vol. 1, pp. 111-118. 8. Carlquist, S. (1976) Aliso 8, 465-492. 27. Bentham, G. (1873) J. Linn. Soc. Lond. Bot. 13, 335-577. 9. Wagenitz, G. (1976) Plant Syst. Evol. 125, 29-46. 28. Small, J. (1919) New Phytol. 18, 1-29. 10. Jeffrey, C. (1978) in Flowering Plants of the World, ed. 29. Carlquist, S. (1966) Aliso 6, 25-44. Heywood, V. H. (Mayflower, New York), pp. 263-268. 30. Small, J. (1918) New Phytol. 17, 13-40. 11. Jansen, R. K. & Palmer, J. D. (1987) Curr. Genet. 11, 553-564. 31. Wodehouse, R. P. (1928) Bull. Torrey Bot. Club 55, 449-462. 12. Palmer, J. D. (1985) Annu. Rev. Genet. 19, 325-354. 32. Wodehouse, R. P. (1929) Am. J. Bot. 16, 297-313. 13. Palmer, J. D. (1985) in Molecular Evolutionary Genetics, ed. 33. Skvarla, J. J., Turner, B. L., Patel, V. C. & Tomb, A. S. MacIntyre, R. J. (Plenum, New York), pp. 131-240. (1977) in The Biology and Chemistry of the Compositae, eds. 14. Palmer, J. D. (1986) Methods Enzymol. 118, 167-186. Heywood, V. H., Harborne, J. B. & Turner, B. L. (Aca- 15. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A. & demic, London), Vol. 1, pp. 141-248. Downloaded by guest on October 1, 2021