COMPARATIVE MORPHOGENESIS OF CYNIPID LEAF GALLS INDUCED BY Diplolepis polita AND Diplolepis nebulosa (HYMENOPTERA: CYNIPIDAE) AND MODIFICATION BY INQUILINES OF THE GENUS Periclistus (HYMENOPTERA: CYNIPIDAE) by Brandy Fenwick Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biology The School of Graduate Studies Laurentian University Sudbury, Ontario, Canada © Brandy Fenwick, 2013 THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE Laurentian Université/Université Laurentienne School of Graduate Studies/École des études supérieures Title of Thesis Titre de la thèse COMPARATIVE MORPHOGENESIS OF CYNIPID LEAF GALLS INDUCED BY DIPLOLEPIS POLITA AND DIPLOLEPIS NEBULOSA (HYMENOPTERA: CYNIPIDAE) AND MODIFICATION BY INQUILINES OF THE GENUS PERICLISTUS (HYMENOPTERA: CYNIPIDAE) Name of Candidate Nom du candidat Fenwick, Brandy Degree Diplôme Master of Science Department/Program Date of Defence Département/Programme Biology Date de la soutenance May 27, 2013 APPROVED/APPROUVÉ Thesis Examiners/Examinateurs de thèse: Dr. Joseph D. Shorthouse (Supervisor/Directeur de thèse) Dr. Yves Alarie (Committee member/Membre du comité) Approved for the School of Graduate Studies Dr. Peter Ryser Approuvé pour l’École des études supérieures (Committee member/Membre du comité) Dr. David Lesbarrères M. David Lesbarrères Dr. Arthur E. Weis Director, School of Graduate Studies (External Examiner/Examinateur externe) Directeur, École des études supérieures ACCESSIBILITY CLAUSE AND PERMISSION TO USE I, Brandy Fenwick, hereby grant to Laurentian University and/or its agents the non-exclusive license to archive and make accessible my thesis, dissertation, or project report in whole or in part in all forms of media, now or for the duration of my copyright ownership. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also reserve the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that this copy is being made available in this form by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. ii ABSTRACT Cynipid galls are atypical plant growths induced by wasps in the family Cynipidae that provide larvae with shelter and nutrition. Larvae gain control of attacked plant organs and send them on a new developmental trajectory, with three developmental phases known as initiation, growth, and maturation. Each of the approximately 1400 species of cynipid gall wasps manipulates plant tissues in a slightly different manner such that galls of each species are structurally distinct. Although the means by which cynipids initiate galls has fascinated naturalists for hundreds of years, the basic events in gall induction are still poorly understood. In an attempt to understand the galling strategies and developmental processes responsible for species-specific galls, I chose to compare the intimate details of life history strategies of two taxonomically related species attacking the same plant organs. The strategies included host specificity, phenology, and oviposition strategies, along with gall development to highlight basic events in gall biology and reveal possible stages in past speciation events. It is argued that differences in phenology and gall development played a role in driving speciation and thus, the patterns observed today are a result of extensive ecological interactions in the past that have influenced the evolution of these complex insect-plant relationships. Two species of cynipid wasps of the genus Diplolepis that occur on the wild roses of central Ontario proved to be ideal candidates for the study. One species, Diplolepis polita, induces single-chambered, prickly galls found in clusters on the adaxial surface of leaflets of Rosa acicularis whereas, the other species, D. nebulosa, induces single- iii chambered, smooth-surfaced galls found in clusters on the abaxial surface of the leaflets of Rosa blanda. Galls at all stages of development, from freshly oviposited eggs to maturation, were found in large numbers for both species. Leaf tissues from the bud stage to maturity of both species of rose were fixed in FAA, embedded in paraffin, sectioned and stained for histological study. Likewise, leaf tissues with freshly oviposited eggs of both species and galls from immaturity to maturity were fixed, sectioned, and stained. Comparing the several thousand slides made for the study revealed that galls of D. polita and D. nebulosa differ in their developmental events as well as the anatomy of their mature galls. Like the galls of all species of cynipids, those of D. polita and D. nebulosa are composed of distinct layers of gall cells known as nutritive, parenchymatous nutritive, sclerenchyma, cortex, and epidermis. Galls of D. polita consist of nutritive cells, parenchymatous nutritive cells, and an epidermis throughout the initiation and growth phases. Larvae remain small in relation to chamber volume until the maturation phase, when a hard layer of sclerenchyma differentiates. In contrast, galls of D. nebulosa have a delayed initiation phase, where galls remain nearly undetectable on leaflets for several weeks after oviposition before they enter the growth phase. Freshly-hatched larvae are protected by two layers of bowl-shaped patches of sclerenchyma that differentiates soon after initiation, along with nutritive cells and parenchymatous nutritive cells that surround the larval chambers, but galls of this species develop without an external layer of epidermis. Galls become spherical as they mature and a second layer of sclerenchyma differentiates within the walls of the galls, as does a layer of spongy cortex that appears between the second layer of sclerenchyma and the gall exterior. Larvae of D. nebulosa occupy nearly the entire volume of their larval chambers throughout gall development. iv The size of each type of cell found within developing galls of both species, from gall initiation to gall maturity were measured and compared. Cells in the galls of both species continue to increase in size throughout development; however, the cells comprising the galls induced by D. polita are significantly larger than those induced by D. nebulosa. Comparing the biologies and galls of these two species, demonstrates how niche partitioning has occurred. It also shows that striking differences in structures occur within the galls of closely taxonomically related species as a result of differences in adult phenology, oviposition strategies, egg placement, and environmental conditions such as moisture levels. Although it has been proposed by other authors that diversity in cynipid galls results from differentiation of tissues found in the outer parts of galls, such as cortex and epidermis, the present study indicates the reasons are more complex. Furthermore, it is apparent that many aspects of gall development and anatomy have been overlooked by previous researchers and a variety of ecological factors contribute to differences in gall structure. To further complicate the already complex series of events that occur over the course of gall development, galls of D. polita and D. nebulosa are inhabited and structurally modified by inquilines of the genus Periclistus. These insects are also cynipid wasps, and have evolved a close relationship with Diplolepis galls whereby they kill the inducer larvae, feed on gall cells, and change the developmental trajectory of attacked galls. Galls of D. polita and D. nebulosa are attacked by two undescribed, but gall-specific species of Periclistus. Here, the inquiline associated with the galls of D. polita is referred to as Periclistus 1 and the inquiline associated with galls of D. nebulosa is referred to as Periclistus 2. The purpose of this study was to histologically examine all phases of v modification by the two species of Periclistus to establish the events that are developmentally unique to inquilines. Periclistus 1 and 2 are phenologically distinct as Periclistus 1 oviposits into immature galls of D. polita in late May soon after galls are induced, and Periclistus 2 oviposits into immature galls of D. nebulosa in July. Modified galls of D. polita differ from modified galls of D. nebulosa as they are significantly enlarged compared to normal galls. Periclistus 1 chambers are arranged around the periphery of the inner gall whereas modified galls of D. nebulosa are of a similar size to normal galls. Chambers of Periclistus 2 are evenly distributed throughout the inner gall. Periclistus 1 and 2-modified galls undergo four phases of development identified as the egg phase, gall enlargement, chamber formation, and maturation phases. Both Periclistus 1 and 2 oviposit into immature galls, killing the inducer larvae with their ovipositors, and then the presence of Periclistus eggs along the inner chamber surface cause changes in gall structure. Diplolepis-induced