The Overcrowded Stage and the Evolutionary Play:

Resistance of Brassica rapa L. (Brassicaceae) to Multiple Enemies

A Dissertation Presented by

André Levy Coelho

To The Graduate School in Partial fulfillment of the Requirements for the Degree of

Doctor of Philosophy


Ecology and Evolution

Stony Brook University

August, 2004



Thesis Abstract

Chapter 1. Introduction                                                          


Chapter 2. Plant Interactions with Multiple Enemies: a review


Chapter 3. Correlated responses of rapid cycling Brassica rapa (Brassicaceae)

to artificial selection for resistance to Alternaria brassicicola (Deuteromycetes)


Chapter 4. Correlated responses to divergent artificial selection in vegetative anthocyanin expression                                        


Chapter 5. Conclusions                                                          



Rearing Trichoplusia ni (Cabbage looper)


Rearing Pieris rapae (Cabbage white)




Ecosystems contain complex networks of biotic interactions. In order to understand the ecology of a species and the evolution of many of its traits it is important to incorporate many of these interactions into our studies. Plants, for example, are attacked by a plethora of enemies, including vertebrate and invertebrate herbivores, and fungal, bacterial, and viral pathogens. Although we have accumulated knowledge about plant defense mechanisms to particular enemies, we are still far from understanding how plants cope with multiple enemies. Do plants evolve defenses in a specific manner to a given enemy, or do the effects of multiple enemies condition evolutionary responses? I contributed to this field of inquiry with artificial selection experiments using rapid cycling Brassica rapa L. (Brassicaceae). I selected populations of B. rapa for greater resistance to a fungal pathogen, the cabbage leaf spot, Alternaria brassicicola. Lines that evolved greater resistance to A. brassicicola did not exhibit correlated resistance to other enemies, particularly to larvae of three lepidopterans (Pieris rapae, Trichoplusia ni, and Spodoptera exigua), adults of a flea beetle (Phyllotreta cruciferae), or to the cabbage aphid (Brevicoryne brassicae). This suggests the independence of resistance to fungal pathogens and insect herbivores. In addition, I selected lines of B. rapa for divergent expression levels of anthocyanin pigments. These play important roles in response to abiotic factors, such as protection from UV light, but also in biotic interactions, for instance providing color to flowers that attract pollinators. I found that lines expressing higher levels of anthocyanins were more susceptible to P.rapae and P. cruciferae and less susceptible to T. ni and A.brassicicola. Feeding by S. exigua and colony size of B. brassicae did not differ among lines producing extreme anthocyanin contents. This presents a varied suite of resistance effects of anthocyanins, and illustrates the complexities of conflicting selection pressures that affect the evolution of plant defense.


















List of Figures


Fig. 2.1 Functional classification of identified Arabidopsis genes                                  50


Fig. 3.1 Disease severity scores between lines selected for resistance to

Alternaria brassicicola                                                                               121

Fig. 3.2   Distribution of disease severity scores caused by A. brassicicola          122

Fig. 3.3   Seed production between selected and control lines                        123

Fig. 3.4   Total glucosinolate content of each selection line                             124

Fig. 3.5   Glucosinolate profile expression                                                                 125

Fig. 3.6   Leaf area damaged by first instar larvae of Pieris rapae                             126

Fig. 3.7   Leaf area damaged by first instar larvae of Trichoplusia ni             127

Fig. 3.8   Leaf area damaged by adults of Phyllotreta cruciferae                             128

Fig. 3.9   Colony size of Brevicoryne brassicae                                                       129

Fig. 3.10 Leaf area damaged by T. ni under three fertilization treatments                   130


Fig. 4.1   Hypocotyl color score between base population and selected lines       168

Fig. 4.2   Frequency of color scores                                                                         169

Fig. 4.3   Seed production among lines                                                                     170

Fig. 4.4   Estimated leaf and stem anthocyanins concentrations                                  171

Fig. 4.5   Photosynthetic rates                                                                       172

Fig. 4.6   Total glucosinolate content of each selection line                                         173

Fig. 4.7   Glucosinolate profile of each selection line                                      174

Fig. 4.8   Leaf area damaged by first instar larvae of P. rapae                                   175

Fig. 4.9   Leaf area damaged by first instar larvae of T. ni                                         176

Fig. 4.10 Leaf area damaged by first instar larvae of S.  exigua                                 177

Fig. 4.11 Leaf area damaged by adults of P. cruciferae                                            178

Fig. 4.12 Colony size of B. brassicae                                                           179

Fig. 4.13 Disease severity score caused by A. brassicicola                                       180

Fig. 4.14 Distribution of disease severity scores caused by A. brassicicola          181

















List of Tables


Table 2.1 Survey of  correlations for resistance to natural enemies                               57

Table 2.2 Effects of sinigrin on plant natural enemies                                        61

Table 2.3 Effects of rutin on several plant natural enemies                                            61

Table 2.4 Effects of proteinase inhibitors on different plant enemies                  62

Table 2.5 Effects of lectins on plant natural enemies                                         65


Table 3.1 Analysis of disease severity score between lines, after 6 gen.                      117

Table 3.2 Analysis of disease severity score between lines, after 7 gen.                      117

Table 3.3 Average glucosinolate content                                                                   118

Table 3.4 Analysis of total foliar glucosinolate content                                    118

Table 3.5 Average resistance to different enemies                                                     119

Table 3.6 Analyses of resistance to different enemies                                     119

Table 3.7 Analysis of resistance to at 3 different fertilization regimes         120


Table 4.1 Analysis of seed set among lines                                                                163

Table 4.2 Analysis glucosinolate concentration among selection lines              163

Table 4.3 Average foliar glucosinolate content                                               164

Table 4.4 Average resistance to different enemies                                                     164

Table 4.5 Analysis of resistance to different enemies                                      165

Table 4.6 Analysis of disease scores                                                                         166

Table 4.7 Correlations among damage inflicted by different enemies and

anthocyanin content                                                                                      167


Table A1. Analysis of resistance to third instar Pieris larvae                          198

Table A2. Analysis of resistance to third instar Trichoplusia larvae                198

Table A3. Analysis of trichome density among full- and half-sib plants            198

Table A4. Analysis of T. ni damage among full- and half-sib plants                199

Table A5. Analysis of P. rapae damage among full- and half-sib plants                     199

Table A6. Analysis of P. rapae damage among control populations and lines

selected from greater resistance or susceptibility to Pieiris  damage         199

















A doctoral program is a very personal process and accomplishment that cannot be completed without the contributions and assistance of a great many people at different steps along the way, each in their unique fashion. I certainly could not have concluded my dissertation without the help and support of the following people.

My advisor, Doug Futuyma, was a true mentor. Part of my reasoning in coming to the US to get my degree had to do with escaping the European system, in which one simply follows in the footsteps of an advisor’s research. I sought Doug after he taught my Evolution class, because of his evident integrated and broad breadth of knowledge and his exceptional ability to communicate. From the start, he pedagogically facilitated the resolution of my struggle to find a specific topic of research, among my many interests. During difficult periods of my experiments, he helped me think through problems with a clear head. Indeed, Doug always approached advising in a deeply human manner, full of understanding for my personal barriers, but relentless when I was in need of a firm word. Doug has an uncanny eye for pointing out experimental caveats, editorial errors, and conceptual inconsistencies. I learned much from his intellectual example, from experiencing his mind at work and seeing him teach. And I gained much from his friendship.

My other committee members were also very helpful, each in his own way. Manuel Lerdau always gave me a fresh perspective on the direction of my research and on interpretation of results. He was generous to have served as my advisor, while Doug was at Michigan, and I am deeply thankful to him for having taken me under his wing when his hands were already full with several other students. James Thomson, an original committee member who subsequently left Stony Brook for Toronto, was pivotal in helping me in the early stages of thinking of a experimental approach and biological model system. Dan Dykhuizen was kind enough to join my committee midway, always had critical comments, and was an important force in keeping me on track. I am grateful for his insistence on the significance of the results of my ‘unfruitful’ experiments and on writing them as an appendix to my dissertation. Robert Fritz, my outside committee member, was very helpful in my final stages. His own work was an important influence and source of inspiration. His interest in my experiments, a source of encouragement. His timely reviews of my chapters and his incisive comments were an important contribution.

I must thank the Fulbright Commission that paved the way for my adventure to Stony Brook and the Portuguese Fundação de Ciência e Technologia that rewarded me with a doctoral scholarship. Michael Axelrod and John Klumpp, the greenhouse curators, were extremely helpful in my work and reliable sources of amusement. Dan Gilrein and Meg McGrath, of the Long Island Horticultural Research Laboratory (Riverhead, NY), and many people at the NYS Agricultural Experiment Station (Geneva, NY) assisted my collection of Brassica enemies and taught me much about their biology. Jonathan Gershenzon and Michael Reichelt, from the Max Planck Institute for Chemical Ecology (Jena, Germany) were extremely generous is performing the glucosinolate analysis.

In addition to my committee members, I must thank many other members from the Department of Ecology and Evolution. R. Geeta supported me for a couple of years as a Research Assistant, during which I learned molecular techniques and phlyogenetic methods. More importantly, she was a supportive and provocative friend, tempting me with new projects and tolerating my extra-curricular ventures. Martha Nolan and Marilyn Pakarklis were a family to me. Isabel Ashton and Thomas Merritt were exceptional friends, with a warm hearts and uninhibited mouths, reliable sources of companionship for having fun and helping me through very dark periods. My cohort members, particularly Luciano Matzkin, Amy Dunham, and Heather Throop and my labmates, particularly Lacey Knowles and Aaron Gassmann, were sources of ideas, goofiness and friendship. I should single out many others, but for the sake of brevity, I will thank everyone in E’n’E who has touched my life (you know who you are) and contributed to an indelible example of what a scientific community should be like.

I must also thank several institutions that helped keep my life in balance. The Plant-Insect Discussion Group and the people that contributed to the discussions, which helped me establish a broad foundation in a field largely new to me. The University Counseling Center for supplying free, quality psychotherapeutic support for the larger part of my stay, without which I might have managed to pull through, but certainly not without understanding as much about myself and graduating with the strength to face the forthcoming challenges. The Social Justice Alliance, the student political organization, which provided a much needed forum of political discussion on campus and an outlet for my political voice. Theatre Three, in Port Jefferson, for teaching me the thespian art, giving me the opportunity to exert it, and introducing me to wonderful and supportive friends.

Finally, I must thank my family for their support. My father and sister, for their love. And my mother, for everything.