American Journal of Botany 85 7 997 1006 1998 PARALLEL EVOLUTION OF GLUCOSINOLATE BIOSYNTHESIS INFERRED FROM CONGRUENT NUCLEAR AND PLASTID GENE PHYLOGENIES1 JAMES E RODMAN 2 6 PAMELA S SOLTIS 3 DOUGLAS E SOLTIS 3 KENNETH J SYTSMA 4 AND KENNETH G KAROL5 2 Division of Environmental Biology National Science Foundation Arlington Virginia 22230 3Department of Botany Washington State University Pullman Washington 99164 4238 4Department of Botany University of Wisconsin Madison Wisconsin 53706 1381 and 5Laboratory of Molecular Systematics Smithsonian Institution Washington DC 20560 The phytochemical system of mustard oil glucosides glucosinolates accompanied by the hydrolytic enzyme myrosinase b thioglucosidase the latter usually compartmented in special myrosin cells characterizes plants in 16 families of angiosperms Traditional classifications place these taxa in many separate orders and thus imply multiple convergences in the origin of this chemical defense system DNA sequencing of the chloroplast rbcL gene for representatives of all 16 families and several putative relatives with phylogenetic analyses by parsimony and maximum likelihood methods demonstrated instead a single major clade of mustard oil plants and one phylogenetic outlier In a further independent test DNA sequencing of the nuclear 18S ribosomal RNA gene for all these exemplars has yielded the same result a major mustard oil clade of 15 families Akaniaceae Bataceae Brassicaceae Bretschneideraceae Capparaceae Caricaceae Gyrostemonaceae Koeberliniaceae Limnanthaceae Moringaceae Pentadiplandraceae Resedaceae Salvadoraceae Tovariaceae and Tropaeolaceae and one outlier the genus Drypetes traditionally placed in Euphorbiaceae Concatenating the two gene sequences for a total of 3254 nucleotides in a data set for 33 taxa we obtain robust support for this finding of parallel origins of glucosinolate biosynthesis From likely cyanogenic ancestors the mustard oil bomb was invented twice Key words Capparales s l DNA sequencing glucosinolates phylogeny rDNA 18S Mustard oil glucosides also named glucosinolates Ettlinger and Kjaer 1968 are oxime derived sulfur containing compounds whose breakdown products include the familiar pungent principles of mustard radish and capers Fenwick Heaney and Mullin 1983 The compounds usually are accompanied in the plant by a hydrolytic enzyme myrosinase a b thioglucoside glucohydrolase E C 3 2 3 1 which may be compartmented in special myrosin cells Fig 1 Hypothesized to deter herbivores or pathogens this mustard oil bomb Lu thy and Matile 1984 is characteristic of all Brassicaceae including the genomic model Arabidopsis but occurs as well in 15 other angiosperm families Table 1 The phytochemical system of glucosinolates with accompanying myrosinase enzyme the latter compartmented in myrosin cells is believed to characterize all the species in these families except for Euphorbiaceae where Drypetes interpreted to include Guya and Putranjiva is the only established source of mustard oils Ettlinger and Kjaer 1968 Rodman 1981 Ettlinger 1987 Traditional classifications like Cronquist s 1981 1988 place these 16 families in several widely separate taxonomic orders and thus imply multiple origins for the glucosinolates withmyrosinase system In turn this multiple origin viewpoint has fragmented the study of host fidelity and evolution by herbivores and pathogens adapted to glucosinolate producing plants Chew 1988 In his early attempt to evaluate taxonomic relationships of angiosperms Dahlgren 1975 1977 challenged orthodox classifications of mustard oil plants by expanding the order Capparales to encompass nearly all families of glucosinolate taxa Later he retreated from this position Dahlgren 1980 1983 Dahlgren Rosendal Jensen and Nielsen 1981 and emphasized the considerable morphological and habital diversity among these taxa In a cladistic analysis of 90 morphological and phytochemical characters Rodman 1991b found weak support for Dahlgren s 1975 expanded Capparales To test Dahlgren s radical reclassification of glucosinolate taxa with an independent molecular data set representatives of all 16 families were sequenced for the chloroplast rbcL gene and analyzed with parsimony and maximum likelihood methods Gadek et al 1992 Rodman et al 1993 1994 1996a Comparisons were made with numerous putative relatives sequenced as part of a large collaborative analysis Chase et al 1993 Price and Palmer 1993 With one exception all mustard oil taxa united into a single major clade or lineage Fig 2 nested within a large rosid plexus Chase et al 1993 The sole exception is the genus Drypetes which is usually placed within the spurge family Euphorbiaceae and is affiliated with other 1 Manuscript received 28 July 1997 revision accepted 19 November 1997 The authors thank Ihsan Al Shehbaz Ray Cranfield Robert Hirano Hugh Iltis Ching I Peng Robert Price and Christopher Quinn for plant samples Kandis Elliot for graphics and Peter Raven and George Johnson for the idea behind our Fig 1 cartoon Support from the Andrew Mellon Foundation to P S Soltis and D E Soltis and the National Science Foundation DEB 9307000 to D E Soltis and DEB 9407270 to K J Sytsma advice from Elizabeth Zimmer and the facilities of the Smithsonian Institution s Laboratory of Molecular Systematics are appreciated The authors also thank reviewers Neil Harriman and Kathleen Kron for advice 6 Author for correspondence e mail jrodman nsf gov agency citation is solely for purposes of identification 997 998 AMERICAN JOURNAL OF BOTANY Vol 85 Fig 1 Biosynthesis of glucosinolates mustard oil glucosides and of cyanogenic glycosides from amino acids via aldoximes Du et al 1995 and hydrolysis of glucosinolates to isothiocyanates mustard oils not shown are glucose sulfate other potential breakdown products catalyzed by myrosinases a family of thioglucoside glucohydrolases Xue et al 1992 1995 associated with cell membranes Lu thy and Matile 1984 and concentrated in myrosin cells stained cells in insert Werker and Vaughan 1974 Ho glund Lenman and Rask 1992 Herbivore feeding is hypothesized to bring enzyme and substrate into contact thereby releasing toxic mustard oils Ehrlich and Raven 1965 analogous to cyanide release from cyanogenic plants Saupe 1981 rosids in lineages thus far only sparsely sampled for gene sequence diversity Chase et al 1993 Conti Litt and Sytsma 1996 Soltis et al 1997 A maximum likelihood analysis see Felsenstein 1985 1995 yielded a single fully resolved