Evolution. 44(2), 1990, pp. 390-402 EVOLUTIONARY SIGNIFICANCE OF THE LOSS OF THE CHLOROPLAST-DNA INVERTED REPEAT IN THE LEGUMINOSAE SUBFAMILY PAPILIONOIDEAE MAlT LAVIN,' JEFF J. DOYLE, L. H. Bailey Hortorium, Cornell University, Ithaca, NY 14853 AND JEFFREY D. PALMER2 Department ofBiology, University ofMichigan, Ann Arbor, MI48109 Abstract.-The distribution of a rare chloroplast-DNA structural mutation, the loss of a large inverted repeat, has been determined for 95 species representing 77 genera and 25 ofthe 31 tribes in the legume subfamily Papilionoideae. This mutation, which is regarded as a derived feature of singular origin within the subfamily, marks a group comprising six temperate tribes, the Galegeae, Hedysareae, Carmichaelieae, Vicieae, Cicereae, and Trifolieae, an assemblage traditionally con­ sidered to be monophyletic. This mutation also occurs in the chloroplast genome of Wisteria, a member ofthe tropical tribe Millettieae whose other members so far surveyed lack the mutation. These new DNA data, together with traditional evidence, support the hypothesis that Wisteria is an unspecialized member of a lineage that gave rise to the temperate tribes marked by the chlo­ roplast-DNA mutation; the probable paraphylesis of Millettieae is revealed. Two other tribes, Loteae and Coronilleae (traditionally regarded as a derived element of the aforesaid temperate tribes) do not possess this chloroplast-DNA structural mutation and, therefore, presumably rep­ resent a distinct temperate lineage. This hypothesis is supported by additional evidence from pollen, inflorescence, and root-nodule morphology that suggests that the Loteae and Coronilleae share a more recent ancestry with tropical tribes such as Phaseoleae and Millettieae than with other temperate tribes. Received January 13, 1989. Accepted November 17, 1989 The Leguminosae (or Fabaceae), with ap­ The higher-level systematics ofthe Papil­ proximately 18,000 species and 650 genera, ionoideae, particularly tribal circumscrip­ is the third largest plant family, after the tions and interrelationships, has been the Compositae and Orchidaceae. Its taxonom­ subject of much recent study (e.g., Polhill ic relationships with otherangiosperm plant and Raven, 1981; Stirton, 1987). Evidence families are obscure and, consequently, the presented in these investigations of chro­ family is the sole member of the order Fa­ mosome numbers, seed chemistry, anato­ bales (Cronquist, 1981). It is divided into my, pollen morphology, and phytogeogra­ three subfamilies, Caesalpinioideae, Mimo­ phy has provided a greater insight into the soideae, and Papilionoideae (Polhill and evolutionary patterns in Papilionoideae and Raven, 1981); of these, Papilionoideae is has resulted in a restructuring and rear­ the largest (with approximately 12,000 rangement ofmany tribes originally circum­ species and 440 genera) and economically scribed by Bentham (1865) in his taxonom­ the most important. Peanuts, beans, peas, ic system ofPapilionoideae. However, many sesban, guar, tragacanth, rosewood, indigo, characters used at the higher systematic and black locust are examples of econom­ levels, such as the degree ofstaminal fusion ically important papilionoids which pro­ or legume dehiscence, show a high degree vide food, fiber, oils, gums, hardwoods, dyes, ofhomoplasy, and evaluation ofsuch char­ and ornamentals. Species ofPapilionoideae acters affects not only tribal relationships, predominate in nearly every vegetation type but sometimes tribal circumscriptions as throughout the world, from tropical rain well. As stated by Polhill (198la), much still forests to deserts, and nearly all species root­ remains ill-defined in the subfamily. nodulate and fix atmospheric nitrogen. Recent work by Palmer and coworkers (summarized in Palmer et al. [1987] and Palmer et al. [1988b]) has shown that the , Present address: Department ofBiology, Montana State University, Bozeman, MT 59717. chloroplast genome ofdiverse Leguminosae 2 Present address: Department of Biology, Indiana possess several major structural mutations, University, Bloomington, IN 47405. including large inversions and a major dele- 390 LOSS OF cpDNA INVERTED REPEAT 391 168238 II 125 kb II .1 12 3 4 Carmichaelia sp. 12 3 214 II III II II • II II 150 kb 88e L8e region \rm 2 168238 region 238168 rm 2 1 r.Jl§ 19 iW:!A Coronilla varia FIG. 1. Linearized representation ofthe chloroplastgenome ofCoronilla and Carmichaelia showing locations, relative to the inverted repeats, ofgenes discussed in the text and the four probe fragments (numbers 1, 2, 3, and 4). The heavy lines connecting rpl2 with 23S indicate the region of the inverted repeated sequences. SSC = small single copy; LSC = large single copy. tion; such mutations have been shown in sess the taxonomic distribution of the loss other plant groups to be phylogenetically of the inverted repeat in the Leguminosae informative (Jansen and Palmer, 1987; and its evolutionary significance. Palmer et al., 1988a). Ofparticular interest here is the loss ofa large (ca. 25-kb) inverted MATERIALS AND METHODS repeated sequence containinga duplicate set Ninety-five species of Papilionoideae, ofribosomal-RNA genes (Fig. 1). This loss representing 77 genera and 25 ofthe 31 tribes is remarkable in that this repeat is an evo­ in this subfamily, have been surveyed for lutionarily conserved feature of land-plant the loss of the inverted repeat (Table 1; chloroplast DNA and is known to be absent voucher specimen information is available otherwise only from certain conifers (Strauss upon request from M. Lavin). In addition, et al., 1988). Structurally conserved features 12 species from the subfamilies Caesalpini­ of the chloroplast-DNA inverted repeat, oideae and Mimosoideae were tested. such as its size and position (dividing the Techniques. -A fraction enriched in genome into small and large single-copy re­ chloroplast-DNA sequences was obtained gions of approximately 20 kb and 80 kb, from most species by isolating a chloroplast respectively), location relative to such pellet in a high-salt buffer (Bookjans et al., flanking genes as psbA, and ribosomal-RNA­ 1984) at a ratio of approximately 30 g of gene transcriptional orientation (toward the leafmaterial to 500 ml ofbuffer. DNA was small single-copy region), suggest that the then isolated from the chloroplast pellet by inverted repeat was present in the common adding an equal volume of the CTAB iso­ ancestor of land plants (Palmer, 1985). lation buffer of Doyle and Doyle (1987) to Palmer et al. (1987), from a limited sam­ the pellet and incubating at 60°C for one­ pling oflegumes, hypothesized that the loss halfhour. Subsequent chloroform/isoamyl­ of the chloroplast-DNA inverted repeat in alcohol extraction, centrifugation, and al­ Leguminosae occurred but once during the cohol precipitation followed Doyle and evolution of the subfamily Papilionoideae. Doyle (1987). This method was especially The survey presented here attempts to as- useful for obtaining DNA from species with 392 M. LAVIN ET AL. resinous leaves or mucilaginous extracts should often detect the same two fragments. (e.g., those in the tribes Aeschynomeneae When the inverted repeat is absent, probes and Amorpheae). When leaftissue was lim­ 1 and 2 have only a single region of ho­ ited, total DNA was extracted by the CTAB mology on the chromosome and should de­ method of Doyle and Doyle (1987). tect only a single fragment. Only ifa restric­ DNAs were subjected to restriction-en­ tion site is located within its region of donuclease digestions and electrophoresis homology will such a probe detect more than in 0.7% agarose gels and transferred to MSI one fragment in a genome lacking the in­ (Fisher) or Zetabind (Cuno) nylon mem­ verted repeat, in which case the adjacent branes. Membrane-bound DNA fragments probe will detect only one of these frag­ were hybridized serially with each of the ments. clones described below that had been la­ Cloned restriction fragments in the sec­ beled with 32p by nick-translation. Electro­ ond set contain sequences flanking that seg­ phoresis, transfer, hybridization, and au­ ment of the inverted repeat that is deleted toradiography were performed as described in legumes (Palmer et al., 1987). These frag­ in Doyle and Beachy (1985), except that ments were used in this survey to test for nylon filters were used in place of nitrocel­ the absence of the inverted repeat (Fig. 1: lulose and Dextran sulfate was omitted from probes 3 and 4). Probe 3 (a 3.l-kb BamH the hybridization solution. I-Pst I fragment cloned in pUC 8 from Vig­ DNA of all species listed in Table 1 was na radiata; Palmer et al., 1987) is at the digested with at least two of the following margin ofthe small single-copy (SSC)region restriction enzymes: Hind III, Pst I, Pvu II, and probe 4 (a 1.2-kb Pst I-EcoR I fragment Sac I, Sal I, Xba I, and Xho 1.Two or more cloned into pUC 8 and containing the 3' single digests of a given DNA of a single end of psbA from Pisum sativum; Oishi et species were run side-by-side to maximize al., 1984) is at the margin ofthe LSC region reproducibility of the hybridization assays flanking the end ofthe inverted repeat seg­ that were used to diagnose the presence or ment. Each therefore has only a single re­ absence ofthe repeat. Additionally, a single gion of homology on a chloroplast chro­ digest ofthe DNA ofa species was run side­ mosome. These regions are normally by-side with other species in the same tribe separated by approximately 25 kb but are in order to assess more readily whether the known to be adjacent in species with chlo­ repeat was consistently present or absent in roplast DNA lacking an inverted repeat. Us­ a given tribe. ing a restriction enzyme with many recog­ Strategy. - Two sets of small cloned nition sites (e.g., Hind III or Xba I), these probes were used to diagnose the presence two probes should generally detect the same or absence ofthe inverted repeat. The first filter-bound fragment ofa DNA lacking the set included cloned restriction fragments inverted repeat but should detect two dif­ from Nicotiana tabacum (Fig.