Metabolites Between Wild Populations of Brass/Ca Oleracea and Its Implications for Plant—Herbivore Interactions
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Heredity 75 (1995) 472—484 Received 13 March 1995 Divergent selection for secondary metabolites between wild populations of Brass/ca oleracea and its implications for plant—herbivore interactions R. MITHEN*, A. F. RAYBOULD & A. GIAMOUSTARIS Brassica and Qilseeds Research Department, John Innes Centre, Colney Lane, Norwich NR4 7UH and tinstitute of Terrestrial Ecology, Furzebrook Research Station, Wareham, Dorset BH2O 5AS, U.K. Significantdifferences occur in the levels and types of aliphatic glucosinolates in leaves of plants of four Brassica oleracea populations in Dorset. Plants in grassland at St Aldhelm's Head and Winspit have high levels of 3-butenyl glucosinolate, whereas plants of an adjacent population growing on and along the top of cliffs at Kimmeridge have low levels of 2-hydroxy- 3-butenyl, 2-propenyl and methylsuiphinylalkyl glucosinolates. Plants growing in a variable habitat at Worbarrow Tout have intermediate levels. The differences in occurrence of indivi- dual glucosinolates result from allelic variation at four loci. The level of total aliphatic glucosi- nolates is under more complex genetic control, but is shown to be highly heritable. Allele frequencies at isozyme loci indicate that genetic variation for glucosinolate production is unlikely to have arisen or to be maintained by founder effects or genetic drift. It is suggested that there is selection for high levels of butenyl glucosinolates at St Aldhelm's Head and Winspit because of grazing by generalist herbivores, whereas there is selection for low levels of 2-hydroxy-3-butenyl and other non-butenyl aliphatic glucosinolates at Kimmeridge because of two factors. First, plants effectively escape from generalist herbivores because of physical aspects of the habitat and association with other plant species which provide physical and chemical defences. Thus there is selection for individuals which do not carry the hypothetical metabolic costs of glucosinolate biosynthesis. Secondly, herbivory by specialist cruciferous insects at Kimmeridge, which is enhanced because of the local abundance of B. nigra, selects for individuals which have low levels of 2-hydroxy-3-butenyl glucosinolates. Keywords:Brassica,coevolution, F-statistics, generalist herbivores, glucosinolates, specialist herbivores. Introduction nance of secondary metabolites and associated specialized structures. Secondly, many secondary Plantsecondary metabolites are widely regarded as metabolites attract and stimulate feeding behaviour being an important component of defence mechan- of insect herbivores which have become specialists isms against herbivoiy. Variation in secondary on taxa which contain the particular metabolite. metabolites in natural plant populations may there- There are, therefore, contrasting selection pressures fore indicate current and historical patterns of herbi- on secondary metabolism, and the metabolite com- vore activity, and provide insights into the selective position will be a result of the interaction of these forces acting within and between plant populations. different selective pressures and random genetic While secondary metabolites may provide effec- effects. Insufficient understanding of the genetic tive defence against generalist herbivores, invest- basis of secondary metabolite biosynthesis has ment in these metabolites incurs two major costs. limited studies on the ecological genetics of second- First, it is likely that there are significant metabolic ary metabolism. costs associated with the biosynthesis and mainte- Glucosinolates are secondary metabolites which occur in the Capparales and a few unrelated taxa. *Correspondence The molecule comprises a common glycone moiety 472 1995 TheGenetical Society of Great Britain. DIVERGENT SELECTION IN WILD BRASS/CA POPULATIONS 473 ,, S—/--D—Glucose CH =CH—CH —CH —C 2 2 2 CH2:CH_CH 2—CH 2—N=C=S (a) NOSO3 (b) CsL—oh /S—/'3--D—Glucose CH=CH—CH2 CH 2=CH—CH—CH2 -C 21 I OH 0 NH NOSO3 C (c) (d) ii S Methionine QTL CsL—elong Side chain elongation CsZ—pro Propyls Butyls Development of glycone moiety Fig. 1 Biochemical genetics of aliphatic glucosinolates. Aliphatic glucosinolates 3—Methyithiopropyl 4— Methylthobutyl such as 3-butenyl glucosinolate (a) are Csl-suip CsZ—sutp hydrolysed to the corresponding isothio- cyanate (b). Addition of a f-hydroxy 3—Methylsuiphinyipropyl 4—Methylsulphinylbutyt Side chain group (c) results in spontaneous cycliza- modification tion of the isothiocyanate to 5-vinylox- CsZ—atk Cst-atk azolidine-2-thione (d), which is nonvolatile. The side chain structure is 2—Propenyl 3—Butenyl controlled by alleles at two loci which Cst—oh regulate side chain length (pro and elong) and three loci which regulate side 2—Hydroxy—3—butenyt chain structure (suip, alk and oh). and a variable aglycone side chain, which is derived ambient conditions of temperature and pH. If the from amino acids (Fig. 1). Glucosinolates with an aliphatic side chain contains a JJ-hydroxy group, the aliphatic side chain are derived from methionine, isothiocyanate spontaneously cyclizes to produce the those with a indole side chain from tryptophan and corresponding oxazolidine-2-thione (Fig. 1). those with an aromatic side chain from phenyl- Glucosinolates have been shown to mediate herbi- alanine. Aliphatic glucosinolates are the most abun- vore interactions. They reduce palatability of leaf dant class of glucosinolates in leaves of Brassica and tissue to generalist herbivores, such as birds, mol- certain other cruciferous genera. Following tissue luscs and generalist insects (Chew, 1988; Glen et a!., damage, aliphatic glucosinolates undergo hydrolysis 1990; Louda & Mole, 1991; Mithen, 1992, Giamous- catalysed by the endogenous enzyme myrosinase and tans & Mithen, 1995),andattract and stimu- produce an array of products, of which isothiocya- late feeding and egg laying by insects which feed nates ('mustard oils') are the most prominent under specifically on crucifers, such as flea beetles The Genetical Society of Great Britain, Heredity, 75,472—484. 474 R.MITHENETAL. (Psylliodes spp. and Phyllotreta spp.), Pieris spp., herbivory and its consequences for the genetic struc- Delia spp, Brevycorne brassicae, Dasineura brassicae ture of wild Brassica populations, we have charac- and Ceutopyhncus assimilis (Hicks, 1974; Chew, 1988; terized the glucosinolate profiles and contents of Louda & Mole, 1991; Simmonds et al., 1994; Gia- four populations in an 11 km stretch of coastline in moustaris & Mithen, 1995). Both the total level of Dorset (Fig. 2) and compared this variation with aliphatic glucosinolates and the side chain structure that at isozyme loci. are important in mediating these interactions. Several recent studies have begun to elucidate the genetic basis of biosynthesis of aliphatic glucosino- Materials and methods lates (Magrath et al., 1993, 1994; Parkin et a!., 1994; Mithen et al., 1995) and have shown that variation in Plant habitats and sampling sites side chain structure is under simple genetic control Plants were sampled from four populations (Fig. 2). (Fig.1). Geographical and ecological variation in ali- At Kimmeridge, B. oleracea plants occur on and phatic glucosinolate composition has been described along the top of shale cliffs. The plant community in Brassica (Mithen et al., 1987), Cakile edentula on the cliff face has few component species. Daucus (Rodman, 1980), Arabidopsis thaliana (Bano, 1993; carota, Silene maritime and Crithmum maritimum are Mithen et al., 1995) and Cardamine cordifolia (Rod- frequent, but few other species are observed in signi- man & Chew, 1980). ficant numbers. Brassica nigra is common in places, Wild members of the Brassica oleracea n =9 particularly on the western cliff face of Kimmeridge species complex occur on maritime cliffs of southern Bay. On the top of the cliff between Kimmeridge and western Europe. Previous preliminary studies Bay and Rope Lake Head B. nigra, D. carota and have shown that there is considerable variation in Rubus are abundant and occur within and around the types of glucosinolates between taxa (Mithen et groups of B. oleracea plants. Rumex crispus, Plantago a!., 1987). In the United Kingdom, wild forms of B. maritima, Hordeum mureum and Daclylus glomerata oleracea, which are not thought to be introductions also occur in the plant community. Otherwise, the or recent escapes from domestication, occur on the vegetation is sparse. At St. Aldhelm's Head, B. oler- coasts of Kent, Dorset, Devon, Cornwall, Glamor- acea plants are found mainly in grassland. B. nigra gan and Gwynedd (Snogerup et a!., 1990). The occurs infrequently at St Aidhelm's sites A and B, demography and genetic structure of wild B. oler- and is absent from sites C and D. The plant commu- acea populations have received little attention. Indi- nity at Winspit is typical of grazed grassland, with vidual plants may be up to 20 years old. Each year, several dicotyledonous species including Be/usper- between one third and a half of the individuals in a ennis, Anthyllis vulneraria, Echium vulgare and Tnfo- population may flower during the summer months hum repens. Brassica oleracea on Worbarrow Tout and seedlings are found in autumn (Mitchell & grows in two distinct habitats. First, plants are found Richards, 1979). Casual observation suggests that on top of the small peninsular in relatively long seedling establishment is a rare event, although no grassland similar to St Aidheim's Head sites A and detailed data are available. As an initial study into B. A group of plants occurs in a patch of Rubus Fig.