Reproductive behaviour of male Xylocopa virginica and the influence of body size, nestmates, and siblings on territory defence Lyndon B. Duff, B.Sc. Department of Biological Sciences Submitted in partial fulfillment of the requirements for the degree of Master of Science Faculty of Mathematics and Science, Brock University, St. Catharines, Ontario © 2018 Abstract Males of Xylocopa virginica are territorial like many other bee species. Males interact aggressively to displace other males from territories. Body size is known to influence resource- holding potential in many other taxa, and studies of bees suggest that body size is important for territorial males. Familiarity and the avoidance of kin competition are also known to influence territorial behaviours in other taxa but has not been studied in male bees. Recent evidence suggests that nestmate recognition occurs in X. virginica and there is also evidence for the avoidance of kin competition. This thesis tests whether body size, familiarity, and kinship influence territorial interactions using social networking tools. Around half of all males attempted to establish or defend a territory. Males that established or defended territories are larger than males that did not. Male body size has a weak positive influence on hover rates, related to holding territories, the number of hovering neighbours each male had, and the number of males each male chased or fled in defence of territories. I found no evidence to support that familiarity influences aggressive behaviours, but there is a strong correlation with the number of neighbours a male had and the number of males it chased or fled. Brothers estimated from microsatellite genotypes are no more aggressive to each other than to non-siblings. However, the results indicate that several sets of brothers overwintered in different nests, which does not coincide with the behavioural patterns described in the literature. This study is the first step towards understanding the influence of familiarity and kin competition on male behaviour in a taxonomic group with a wide array of mate-locating strategies. Discussed herein are the importance of continued research on mating systems and mate-locating strategies of bees, as well as outlining future projects to address several gaps in knowledge that remain after this study. 2 Table of Contents Abstract ........................................................................................................................................... 2 List of Tables and Figures ........................................................................................................... 5 Acknowledgements ..................................................................................................................... 7 1 Introduction and literature review ............................................................................................ 8 1.1 Sexual selection ................................................................................................................. 8 1.2 Types of mating systems ................................................................................................... 8 1.3 The mate-locating strategies of bees ................................................................................. 9 1.4 Familiarity and kinship can influence territorial interactions .......................................... 13 1.5 Male reproductive behaviour in the genus Xylocopa ...................................................... 14 1.6 The behavioural biology of Xylocopa virginica .............................................................. 15 1.7 Objectives ........................................................................................................................ 17 2 Methods .................................................................................................................................. 18 2.1 Description of the field sites and timing of study ............................................................ 18 2.2 Bee capture, size measurement, and marking techniques ............................................... 22 2.3 Observations of male behaviour ...................................................................................... 25 2.4 Departures and arrivals of females to nests ..................................................................... 29 2.5 Microsatellite genotyping methods ................................................................................. 29 2.6 Statistical analyses ........................................................................................................... 33 3 Results .................................................................................................................................... 35 3.1 Male quality ..................................................................................................................... 35 3.2 The territory quality of female aggregations ................................................................... 43 3.3 Male behaviours and interactions occur in high-quality territories ................................. 49 3.4 Copulations and mating attempts .................................................................................... 51 3.5 The distribution of male nestmates and siblings for nests at each bridge ....................... 51 3 3.6 How does body size, familiarity, and male siblings influence male behaviours and interactions? ........................................................................................................................... 54 4 Discussion .............................................................................................................................. 80 4.1 The influence of body size on resource-holding potential and territorial interactions .... 80 4.2 Familiarity did not influence interactions ........................................................................ 83 4.3 Kinships did not influence interactions ........................................................................... 84 4.4 Conclusions and future directions ................................................................................... 86 Bibliography ................................................................................................................................. 88 Appendix ................................................................................................................................... 94 4 List of Tables and Figures Table 1: Ethogram of male Xylocopa virginica behaviours……………………………………. 26 Table 2: The percentage of success for genotyping males and females…………………………32 Table 3: Principal components analysis statistics of size variables in male Xylocopa virginica..40 Table 4: Bridge D is a higher-quality location for males to find females than bridge F………...46 Table 5: Overwintering patterns of brothers……………………………………………………..53 Table 6: ANOVA and regression coefficients of whether body size influences hovering rates...59 Table 7: ANOVA and regression coefficients of whether body size influences hovering neighbours……………………....………………………………………………………………..62 Table 8: ANOVA and regression coefficients of whether body size influences the number of males each male chased…………………………………………………………………………………69 Table 9: ANOVA and regression coefficients of whether body size influences the number of males each male fled……………………………………………………………………………………70 Table 10: ANOVA and regression coefficients of whether body size influences the combined number of males each male chased of fled…………………………………………....................71 Figure 1: Aerial view of the Glenridge Quarry Naturalization Site………………………………19 Figure 2: Side views of bridges containing Xylocopa virginica nests at the Glenridge Quarry Naturalization Site…………………………………………………………………………..........20 Figure 3: An aerial view of bridges containing Xylocopa virginica nests at the Glenridge Quarry Naturalization Site………………………………………………………………………………..21 Figure 4: Diagram of how size was measured for each bee………………………………………24 Figure 5: The cumulative number of emerging and disappearing males, total males present, and cumulative number of emerging females………………………………………………………...36 Figure 6: Male tenure at bridge D from 5 May – 9 June 2016…………………………………...37 Figure 7: Male tenure at bridge F from 9 May – 2 June 2016…………………………………....38 Figure 8: Histograms of male head width, inter-tegular width, and costal vein length…………..41 Figure 9: Principal components analysis plot of body size measurements of male X. virginica…42 Figure 10: Heatmaps to indicate high-quality locations for males to encounter females…………45 Figure 11: The number of females that departed or arrived and nests and the number departures and arrivals of females per hours of observation each day of study……………………………..47 Figure 12: The total number of departures and arrivals of females each hour……………………48 Figure 13: Heatmaps to show that males established and chase other males in high-quality locations to encounter females…………………………………………………………………... 50 5 Figure 14: Histogram of the frequency of hovering for all males………………………………...56 Figure 15: Males that hovered at least once are larger than males that never hovered…………..57 Figure 16: Body size positively influences the hovering rates of males ………….....……………58 Figure 17: The frequency and number of hovering neighbours males had……………………....60 Figure 18: Body size positively influences the number of hovering neighbours each male had...61 Figure 19: A social network