Proceedings of the X International Symposium on Biological Control of Weeds 281 4-14 July 1999, Montana State University, Bozeman, Montana, USA Neal R. Spencer [ed.]. pp. 281-288 (2000)

Know Your Enemy: The Use of Molecular Ecology in the Biological Control Project

P. C. O’HANLON 1, D. T. BRIESE 2, and R. PEAKALL 1

1 Division of Botany and Zoology, Australian National University, Canberra 0200, 2 CRC for Weed Management Systems and CSIRO Entomology, GPO Box 1700, Canberra 2601, Australia

Abstract Experience has repeatedly shown that accurate identification of the target weed(s) for a biological control project is critical to the success of a biological control project. This is particularly true where the weed may comprise different biotypes or be part of a species complex, where hybridisation is suspected or where the agent - host relationship is very tight. Molecular ecology provides a number of powerful tools for resolving the iden- tity of weeds, and their use in understanding a hybrid swarm of Onopordum spp. in Australia is described to illustrate some recently developed techniques. Some implica- tions of hybridisation between O. acanthium and O. illyricum for the impact of particular biological control agents are discussed. In addition to this applied aspect, molecular markers can be used to better understand the phylogeny of plant groups containing the tar- get weed(s). Onopordum is used as an example of how this can help our theoretical under- standing of the host-selection process by potential control agents.

Keywords: Biological control, molecular ecology, Onopordum spp., hybridisation, phylogeny

Introduction The host plant / control agent relationship is central to the science of weed biological control. From a practical viewpoint, it is critical to be certain of the identity of the target weed and of candidate agent. Correct identification can be used to pinpoint where in the native range an introduced weed comes from and can thus focus the search for potential control agents (Chaboudez et al. 1992, Scott et al. 1998). Failure to do so can lead to lack of agent establishment and /or incomplete control, particularly in cases involving biotypes of a single weed species (e.g. Chaboudez 1994), weed species complexes (e.g. leafy spurge (Mahlberg 1990), blackberries (Bruzzese and Hasan 1986)) or agent host-races (e.g. the capitulum , conicus (Goeden et al. 1985), skeleton weed rust, Puccinia chondrillina (Espiau et al. 1998)). From a more fundamental view- point, it is important to better understand how such host / agent relationships have evolved in oligophagous . Briese (1996) has shown how phylogenetic studies of plant and herbivore taxa give clues to the evolution of host-choice in agents. Such knowledge can help biological control practitioners to design better tests for evaluating host-range of can- didate agents and maximise the chances of safely introducing effective agents into a new habitat. 282 O’Hanlon et al.

Techniques used in biological control projects where the identity of and relationships between organisms have been studied vary from morphology (Hull and Groves 1973), chromosome counts (Morrison and Scott 1995), plant secondary chemicals (Mahlberg 1990) and protein electrophoresis (Goeden et al. 1985, Morrison and Scott 1996). However, recent developments in molecular techniques give the biological control practi- tioner a selection of powerful tools for resolving the identity and relationships of both tar- get weed and candidate agent (see reviews by Nissen et al. 1995, O’Hanlon and Peakall, 2000). The increased power of these methods is demonstrated by a recent study on black- berries by Evans et al. (1998), who, using DNA markers, identified 20 genotypes from 13 previously described morphological taxa (e.g. Evans et al. 1998). The importance of this knowledge is obvious in the light of differential susceptibility to a rust fungus control agent by different members of the blackberry species complex (Bruzzese and Hasan 1986). The present paper describes the use of molecular techniques to clarify doubts con- cerning the identity of Onopordum spp. in Australia and demonstrate the existence of widespread hybridisation between species. In doing so it discusses the implications of such hybridisation for the Onopordum biocontrol project. The use of molecular techniques to demonstrate phylogenetic relationships both within the Onopordum and between Onopordum and other genera of the Carduinae thistles is also described, and the impor- tance of knowing such relationships is discussed in terms of seeking to better understand host-range restrictions of candidate biological control agents.

Identity of Australian Onopordum Pastures in south-eastern Australia are considered to have been invaded by two species of Onopordum of European origin, Scotch thistle (O. acanthium L.) and Illyrian thistle (O. illyricum L.) (Parsons and Cuthbertson 1992). These weedy thistles are currently the tar- get of a biological control project (Woodburn and Briese 1996). However, there has been some doubt concerning the identity of some populations (see Groves et al. 1990), as mor- phological analysis revealed that many infestations of Onopordum in Australia did not correspond with any of the known species. Instead, they were morphologically very diverse, but generally intermediate between O. acanthium and O. illyricum. Amplified

O. bracteatum

O. acanthium Australian Onopordum O. illyricum O. argolicum

O. corymbosum O. tauricum

Fig. 1. O. acaulon Non-metric multidimensional scaling scatterplot O. macracanthum of genetic distances between Australian Onopordum and putative European source taxa (shaded). Circles indicate individual Australian O. nervosum populations. Molecular ecology in Onopordum biocontrol 283 fragment length polymorphism (AFLP) was therefore used to determine the uniqueness of Australian Onopordum populations, whether they are descended from hybridisation between Scotch and Illyrian thistles, and how closely related they are to other species of Onopordum. Of more than 100 polymorphic DNA fragments generated by AFLP, those diagnostic for Scotch and Illyrian thistles were demonstrated to segregate in putative hybrids, con- firming their hybrid origin (Fig. 1) (O’Hanlon et al. 1999). Genetic exchange between these thistles may confer a fitness advantage to weedy Onopordum in the new Australian environment (see Vila and d’Antonio 1998), and also contribute to adaptive variation in the group. Indeed, preliminary transplant experiments suggest that hybrid genotypes have higher fitness in certain contexts and a demographic genetic study revealed heterozygote advantage within a hybrid population (O’Hanlon, unpublished data). The existence of widespread hybridisation amongst Onopordum in Australia also has implications for the management of these weedy thistles, as the wide variability exhibit- ed in certain plant characters may effect control methods, including the use of biological control agents, and their impact (Table 1). For example, both parent species of

Table 1. Differences between parental Onopordum taxa that could effect the impact of biological and other control methods on hybrid populations (hybrids have variable intermediate characteristics).

Character O. acanthium O. illyricum Possible effect

Life-history adapted to cooler adapted to warmer • selection of best-adapted hybrid continental Mediterranean populations in all infested localities climates climates • hybrid vigour

Life-history biennial facultative • rosette feeding by agents may lead to perennial varying degrees of perenniation with flow on effects for plant life-cycle

Capitula more, smaller, fewer, larger, • differential oviposition patterns and shorter time to longer time to survival of -feeders maturity maturity • differential impact on seed production

Architecture bushy, highly more open, fewer • differential survival of stem feeding branched branches agents • differential exposure of agents to predation

Leaf structure broader with indented with less • differential protection for eggs laid on dense woolly dense tomentosum tomentosum • differential palatability to grazing stock • differential uptake of herbicides 284 O’Hanlon et al.

Onopordum are monocarpic, but O. acanthium is a strict biennial while O. illyricum is a facultative perennial. Rees et al. (1999) found that in O. illyricum bolting is dependent on rosettes reaching a critical size, so that stresses such as rosette herbivory may reduce plant size and thus promote perenniation of rosettes. Three such rosette-feeding agents have been released, the weevil Trichosirocalus sp. nov., the moth Eublemma respersa and the fly, Botanophila spinosa, each with slightly different feeding niches and periods of attack. Perenniation induced by these agents could favour their population build-up by maintain- ing resource levels, but may also have complex flow on effects, through reduced seed pro- duction and input into soil seed reserves, which could affect capitulum attacking (the wee- vil, ) or stem-boring agents (the weevil, Lixus cardui). Another important difference between the two parent species is that O. illyricum has much larger capitula, which favours survival of L. latus (Briese 1996). Hybrid forms tend to show variability in capitulum size and number as well as varying degrees of perenniation (Pettit et al. 1996), and the presence of wide genetic variability in these and other characters will undoubted compound the plant / herbivore interactions alluded to above. Moreover, it could lead to selection for particular plant traits under continued herbivore pressure from introduced agents. Variability in other characters such as form and texture may also affect palata- bility and chemical uptake of hybrid thistle populations (see Pierce 1996), which needs to be considered in the integration of biological control with tactical grazing and herbicides, such as described by Huwer et al. (1999) for Onopordum thistles.

Phylogeny of European Onopordum A previous taxonomic treatment of morphological variation in European Onopordum defined two subgenera, Onopordum comprising four sections, and Acaulon, comprising a single section (Amaral Franco 1975). However, phylogenetic analyses of AFLP data found this to be an unnatural classification, with section Echinata being non-mono- phyletic. In addition, the solitary species from section Acaulon, O. acaulon, grouped with other species in rather than forming a separate clade, as suggested from morphological studies. The AFLP phylogeny is supported by biogeographic evidence as the different clades correspond to regional distributions of Onopordum spp. in (Fig. 2).

Fig. 2. AFLP and morphological phylogenies of nine European species of Onopordum (Cynara spp. used as outgroups). AFLP Morphology Cynara (OUTGROUP )

O. acaulon AA O. nervosum O. macrocanthum O. corymbosum

O. tauricum B O. argolicum O. bracteatum O. illyricum

O. acanthium C

A = species restricted to Iberian peninsula B = species occurring Aegean region C = widespread species Molecular ecology in Onopordum biocontrol 285

Some Onopordum species showing strong morphological and habitat affinities are only distantly related. The discordance between molecular and morphological analyses may result from a pattern of colonisation and evolution (O’Hanlon et al. 1999). Given that the genus is considered to have originated in central , it could be hypothesised that Onopordum species invaded continental Europe from the east. During colonisation, dif- ferent species hybridised, resulting in genetically diverse founder populations. The Iberian Peninsula and Aegean regions became isolated, and speciation within these regions caused sorting of adaptive traits with ecological preferences, resulting in convergent mor- phological evolution. Some subsequent spread of species such as O. illyricum may have occurred along ecoclinal corridors such as the Mediterranean coastal strip. This proposed evolutionary pathway for European Onopordum spp. is concordant with the speciation of a group of capitulum-feeding of the genus Larinus into eastern and western European taxa specialising on the genus Onopordum (see Briese et al. 1996). Such “lineage sorting” is supported by the recurrent formation of hybrids in Europe (Gonzales Sierra et al. 1992) and the continual gene flow between species (O’Hanlon et al. 1999). This may help explain why the oligophagous fauna found on Onopordum spp. remains genus specific (Briese et al. 1994) rather than having developed more spe- cialised relationships with individual species.

Phylogenetic relationships between thistles Knowledge of the phylogenetic relationships of target weeds and related is important considering the need for very high degrees of specificity in biological control agents, particularly given current concern for non-target impacts (see Louda et al. 1997). Thistles appear to be a modern group with genera diverging relatively recently. Consequently, relationships between thistles have been difficult to resolve. Different data sets produced phylogenetic trees that, while differing in the levels of resolution obtained, were mostly congruent (Fig. 3). When combined, a “total evidence” tree produced a more

Fig. 3. A “total evidence,” chloroplast, and AFLP strict consensus tree. Bootstrap values given above branches. Grey branches indicate bootstrap values <50%. Total evidence cpDNA and morphology Echinops Jurinea Echinops Cousinia Arctium Jurinea Cynara Ptilostemon Cousinia Onopordum Notobasis Arctium Cynara Ptilostemon Galactites Onopordum AFLP Notobasis Echinops Arctium Cynara Picnomon Ptilostemon Onopordum Cirsium Cousinia Carduus Carduus Tyrimnus Galactites Tyrimnus Jurinea Cirsium Silybum Silybum Notobasis Galactites Picnomon 286 O’Hanlon et al. fully resolved than trees produced from individual data sources, with strong support for several clades. Arctium-Cousinia formed a monophyletic stem group to the remaining genera which were divided into two clades; (1) relatives of Carduus and (2) relatives of Cynara. Within the former clade, Picnomon-Notobasis, Carduus-Tyrimnus, and Silybum- Galactites were identified as sister genera. Within the latter clade, relationships between Cynara, Ptilostemon and Onopordum remained unresolved. Despite uncertainty within the clade, it is clear that Cynara and Ptilostemon are more closely related to Onopordum than are the other genera. Following Wapshere’s (1974) centrifugal phylogenetic testing procedure, this justifies the use of Cynara species as key test plants to determine whether it is worth pursuing a more generalised and expensive host-testing protocol. The restriction of a group of closely related seed weevils within the genus Larinus to the Cynara-Onopordum clade also supports this and provides clues to the evolution of host-choice (Briese et al. 1996).

Conclusions DNA markers have proved a powerful tool to better understand the system in which biological control of Onopordum spp. is being attempted. They provided, either alone or through combination with other character sets, a clearer picture of past and current evo- lution of this group of thistles, and the importance of hybridisation in this process. More precise knowledge of the phylogenetic relationships of Onopordum spp. have given a bet- ter idea of how the natural enemies of this group of thistles evolved with their hosts. The ability to unravel the existence of hybridisation and the extent of its occurrence in Australia means that researchers can better anticipate agent impact and modify control strategy if needed. Despite their power, the use of molecular techniques for better understanding the biol- ogy of weeds is often inhibited by the lack of expertise, and the time and cost involved in performing such studies. Furthermore, such costs are rarely included in biocontrol project budgets and often the benefits are not considered sufficiently important by outcome-ori- ented funding sources. As in this case study of Onopordum, this problem can be resolved through collaboration with researchers interested in plant population genetics and evolu- tion. Weed biological control, through targeting invasive plant species and through the deliberate transfer of organisms to new habitats, can provide excellent models for study in these important fields of considerable current research interest. In return, the biological control practitioner can obtain a much clearer picture of the system he hopes to manipu- late.

Acknowledgments Funding for this project was provided by the Australian National University and the Cooperative Research Centre for Weed Management Systems.

References Amaral Franco, J. 1975. Onopordum L., pp. 244-248. In T.G. Tutin et al. [eds.], Flora Europea vol. 4. Briese, D.T. 1996. Phylogeny: can it us to help understand host-choice by biological control agents?, pp 63-70. In V.C. Moran, and J.H. Hoffman [eds.], Proceedings, 9th International Symposium on Biological Control of Weeds, 19-26 Jan 1996, Stellenbosch, South . UCT, Cape Town. Molecular ecology in Onopordum biocontrol 287

Briese, D.T., A.W. Sheppard, H. Zwölfer, and P.E. Boldt. 1994. The phytophagous insect fauna of Onopordum thistles in the northern Mediterranean basin. Biol. J. Linn. Soc. 53: 231-253. Briese, D.T., C. Espiau, and A. Pouchot-Lermans. 1996. Micro-evolution in the weevil genus Larinus: host-race formation and speciation. Mol. Ecol. 5: 531-545. Bruzzese, E., and S. Hasan. 1986. Collection and selection in Europe of isolates of Phragmidium violaceum (Uredinales) pathogenic to species of European blackberry naturalised in Australia. Ann. Appl. Biol. 108: 527-533. Chaboudez, P. 1994. Patterns of clonal variation in skeleton weed (Chondrilla juncea), an apomictic species. Aust. J. Bot. 42: 283-295. Chaboudez, P., S. Hasan, and C. Espiau. 1992. Exploiting the clonal variability of Chondrilla juncea to collect virulent strains of Puccinia chondrillina for use in Australia, pp. 118-121. In Proceedings, 1st International Weed Congress, Melbourne, Australia, Vol. 2. Espiau, C., D. Riviere, J.J. Burdon, S. Gartner, B. Daclinat, S. Hasan, and P. Chaboudez. 1998. Host-pathogen diversity in a wild system - Chondrilla juncea and Puccinia chondrillina. Oecologia 113: 133-139. Evans, K.J., D.E. Symon, and R.T. Roush. 1998. and genotypes of the Rubus fruticosus L. aggregate in Australia. Plant Prot. Quart. 13: 152-156. Goeden, R.D., D.W. Ricker, and B.A. Hawkins. 1985. Ethological and genetic differences among three biotypes of (Coleoptera: Coleoptera) introduced into for the biological control of asteraceous thistles, pp. 181-189. In E.S. Delfosse [ed.], Proceedings, 6th International Symposium on Biological Control of Weeds, 19-25 August 1984, Vancouver, Canada, Agriculture Canada, Ottawa. Gonzalez Sierra, G., C. Perez Morales, A. Peñas Merino, and S. Rivas-Martinez. 1992. Revisión taxonomica de las especies ibericas del género Onopordum L. Candollea 47: 181-213. Groves, R.H., J.J. Burdon, and P.E. Kaye. 1990. Demography and genetics of Onopordum in southern New South Wales. J. Appl. Ecol. 18: 649-658. Hull, V.J., and R.H. Groves. 1973. Variation in Chondrilla juncea L. in south-eastern Australia. Aust. J. Bot. 21: 113-135. Huwer, R.K., D.T. Briese, A.W. Sheppard, T.L.Woodburn, W.M. Lonsdale, D. Kemp, P. Dowling, D. Michalk, and B. Jacobs. 2000. IWM: The key to control of broadleaf pasture weeds, (this volume). In Neal R. Spencer [ed.], Proceedings, Xth International Symposium on Biological Control of Weeds, 4-14 July 1999, Bozeman, Montana USA, pg. 966. Louda, S., D. Kendall, J. Connor, and D. Simberloff. 1997. Ecological effects of an insect introduced for the biological control of weeds. Science 277: 1088-1090. Mahlberg, P.G. 1990. Chemotaxonomic affinities of Eurasian leafy spurges (Euphorbia spp.) in relation to a biological control program, pp. 145-154. In E.S. Delfosse [ed.], Proceedings, 7th International Symposium on Biological Control of Weeds, 6-11 March 1988, Rome, Italy, MAF, Rome. Morrison, S.M., and J.K. Scott. 1995. Chromosome numbers of Cape tulips (Homeria species) in Australia. Plant Prot. Quart. 10: 96-98. Morrison, S.M., and J.K. Scott. 1996. Variation in Australian and world-wide populations of Tribulus terrestris (Zygophyllaceae). 3. Isozyme analysis. Aust. J. Bot. 44: 201-212. Nissen, S.J., R.A. Masters, D.J. Lee, and M.L. Rowe. 1995. DNA-based marker systems to determine genetic diversity of weedy species and their application to biological control. Weed Sci. 43: 504-513. O’Hanlon, P.C., and R. Peakall. 2000. New PCR-based genetic markers and their utility to weed research. Weed Res. 40: (in press). O’Hanlon, P.C., D.T. Briese, and R. Peakall. 1999. Historical and contemporary colonization of novel environments by hybrid Onopordum thistles. In Proceedings, 7th Congress of\ 288 O’Hanlon et al.

the European Society for Evolutionary Biology, Barcelona, Spain, in press. O’Hanlon, P.C., R. Peakall, and D.T. Briese. 1999. AFLP reveals introgression in weedy Onopordum thistles: hybridisation and invasion. Mol. Ecol. 8: 1239-1246. Parsons, W.T., and E.G. Cuthbertson. 1992. Noxious Weeds of Australia. Inkata Press, Melbourne. Pettit, W.J., D.T. Briese, and A. Walker. 1996. Aspects of thistle population dynamics with reference to Onopordum. Plant Prot. Quart. 11: 232-235. Pierce, J.R. 1996. The relevance of variation in thistles to herbicidal control. Plant Prot. Quart. 11: 277-279. Rees, M., A.W. Sheppard, D.T. Briese, and M. Mangel. 1999. Evolution of size-dependent flowering in . Amer. Nat. 154: 628-651. Scott,, L.J., B.C. Congdon, and J. Playford. 1998. Molecular evidence that fireweed (Senecio madagascariensis, ) is of South African origin. Plant Syst. Evol. 213: 251-257. Vila, M., and C.M. D’Antonio. 1998. Hybrid vigor for clonal growth in Carpobrotus (Aizoaceae) in coastal California. Ecol. Applic. 8: 1196-1205. Wapshere, A.J. 1974. A strategy for evaluating the safety of organisms for biological weed control. Ann. Appl. Biol. 77: 201-211. Woodburn, T.L., and D.T. Briese. 1996. The contribution of biological control to the management of thistles. Plant Prot. Quart. 11: 250-253.