Copyright by Natalia Beatriz Biani 2009
The Dissertation Committee for Natalia Beatriz Biani Certifies that this is the approved version of the following dissertation:
Alliances and Struggles in the Miniature Ecosystem of a Socially Flexible Bee
Committee:
Ulrich G. Mueller, Co-Supervisor
Larry E. Gilbert, Co-Supervisor
Mike C. Singer
Lauren Ancel Meyers
John L. Neff
Alliances and Struggles in the Miniature Ecosystem of a Socially Flexible Bee
by
Natalia Beatriz Biani, M.S.
Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
The University of Texas at Austin August, 2009
Dedication
To my parents, for their gift of life.
To Evangelina, Marianela and Juan Francisco, the most marvelous siblings.
To Juan, my love.
To our son, Ignacio, who is loved even before existing.
Acknowledgements
Doing a PhD. is not a work of months, not even a year… it involves many years, many moments, many feelings, many struggles. And is not the work of a single person, it is a relatively collaborative process. This dissertation work would not have been accomplished without the guidance, support, and encouragement of Bill Wcislo and Ulrich Mueller. I am infinitely grateful to Bill, who in the midst of uncertainty suggested me to “watch those mites” in Megalopta bee nests. Bill is not only a brilliant and passionate scientist, but more importantly, he is a fantastic person. When I met him for the first time, we had a long talk, about graduate school and science, and it was the most peaceful and friendly talk I have ever had with a scientist. I am thankful to you for teaching me how to do science, for being always so supportive, understandable and kind person and for granting me the privilege to get you know you. I am extremely thankful to Ulrich, who timidly accepted me as one of his students, and rescue me from dropping this whole PhD. project. I am immensely grateful to you for your essentially kind and clever advice and your intellectual and moral support and friendship over these years. Thank you for allowing me to work in your lab and for honoring me with the privilege of being a “muellerita”. I thank Larry Gilbert, who opened the doors of UT and who introduced me to the beauty of tropical forests. The other members of my committee, Lauren Meyers, Jack Neff and Mike Singer were plentiful sources of support, advice and passion throughout this learning process.
v I am indebted to Juan Carlos di Trani and Fernando Soley for training me on finding and opening Megalopta’s nests. Field work was both possible and enjoyable thanks to the invaluable help, company and friendship of Hermogenes Fernandez-Marin, Adam Smith, Simon Tierney, Therany Gonzales, Marcos Rios, Cecilia Blundo, Gonzalo Rivas, Piotr Lukasik, and Juan Carlos Santos. Greg Gilbert was a wonderful fungi mentor and taught me the delicacies of these unique and ubiquitous organisms. Hermógenes Fernández-Marín and Julia Sarco provided essential logistic help and advice in the Gamboa mycology lab. Hans Klompen and Jon Shik were essential with the identification of the mite species. Andre Rodrígues suggested the best microbiology method to test the cleaning effect of mites. The Smithsonian community in Gamboa is the most stimulating, supporting and fun environment one can ever hope for during field work. I am thankful to the ones I had the privilege to overlap with. I am grateful to the Ryan Lab for accepting me as a “social parasite” during our summers in Gamboa. I am obliged to the Smithsonian helpful and friendly staff for their logistical support and for their help with the collection and exportation permits. I want to give special thanks to Paola Galgani who has not only allowed this research to be possible, but has been a wonderful friend. Oris Acevedo and
Belkys Jimenez are amazingly accommodating and caring persons. Oris, I will never forget the afternoon we returned from BCI in a little boat, avoiding transatlantic ships under a heavy tropical rain. The family who I stayed with in Gamboa, were constant source of laugh, support and gifted me with the thrilling experience of driving Emiliana all the way to Panama City while she was in labor.
vi The Mueller Lab is the most wonderful, supportive and fun atmosphere ever. I am thankful to all of you: Barrett Klein and Christian Rabeling, for being such enjoyable, funny and delightful friends and office mates, Rachelle Adams for her support during my first years and beyond, Alexander Mikheyev for his help and advice with the AFLP data and for his constant compliments about my baking skills, Ruchira Sen, Sze Huei, Heather Ishak the sweet and loving ladies of the lab, and Mike Cooper for his help during molecular work. Sandy Monahan could not be sweeter and more compassionate. I thank her for her friendship and for being always so helpful and kind. I will never forget the day you invited me to visit the tower! My friends in Austin and in Argentina are essential to me. I want to specially thank my friend Natalia Ruiz Juri, with whom I came to Austin for the first time, and who I lived with during the first mesmerizing and overwhelming years of grad school in a foreign country. My friend Lidia Marte, la “Arania”, with whom I share innumerable moments of joy, fear, nausea, hope, etc about grad school and life, who always gives me the anthropologist perspective and the beauty of her art, and with who I share the indescribable feelings of being a latinoamerican. My friends from the EEB cohort of the fall of 2002 and beyond are fundamental pillars enriching my life with theirs, and with their extravagant and delicious dishes from all over the world: Samraat, Catalina, Barrett, Simone, Christian, Juanita, Deepa, Evan, Sasha. I could not have made more marvelous friends. I thank Rachel Page, Ximena Bernal and Rachelle Adams, with whom I shared the adventures of grad school, field work, friendship and the amazing journey to motherhood. I am grateful to the music and the blessed musicians I love, for they make our life and our sometime tedious work in the lab, enjoyable and for they bring us awareness of vii unique memories. Borges, Galeano, Cortazar, Fontanarrosa, Maitena, Girondo, Gelman, Benedetti, Neruda, Garcia Marquez, Les Luthiers, are essential to rescue me from the hard questioning moments of grad school, and are sublime sources of inspiration. I feel eternal gratitude to my parents, Ines and Ricardo, who gifted me with the miracle of life, who taught me the love for the natural world and who instilled me the value of learning. I am immensely thankful to Evi, Marianela y Juan Francisco with whom I share the life, for their unique singularity, for their love and undoubtable fraternity. I thank their beloved ones, for making them truly happy. I am grateful to my grandmas for being so “grandmas”. I will never be able to thank all of them enough for their continual, essential and unconditional support and immense love; for giving me the most grandiose welcoming every time I went back to Rosario; and for the wonderful miracle of being present, connected and sharing the whole year throughout time and distance. Juan has being my friend, my guide, my extra advisor, my field assistant, my lab assistant, my personal statistical consultant, my fervent supporter, and above all, the love of my life. I could not have done this without you. Ignacio, our son, is the most wonderful being in my life and I thank him for being such a sweet and patient baby. Finally, I thank the beautiful bees and the amazing mites for allowing me to peep at their lives. This work was supported by the Smithsonian Tropical Research Institute (STRI) Short Term Fellowships and especially by the research grants from the Program of Ecology, Evolution and Behavior since without those little fellowships, none of this would have even started. I am grateful for the teaching assistantships that were granted to me during all these years. Being an independent international student, I would have been unable to support myself otherwise. viii Alliances and Struggles in the Miniature Ecosystem of a Socially Flexible Bee
Publication No.______
Natalia Beatriz Biani, PhD. The University of Texas at Austin, 2009
Supervisor: Ulrich G. Mueller Lawrence E. Gilbert
Cooperation is pervasive in nature but paradoxically also provides opportunity to cheaters. My dissertation involves the study of both cooperation and conflict in two species of Megalopta bees. Megalopta is a Neotropical genus of halictid bees whose biology is characterized by complex life cycles that can range from solitary to eusocial. These bees nest in dead wood and forage under dim light conditions. Megalopta ’s nests are inhabited by an extensive array of organisms and each nest therefore constitutes a miniature ecosystem providing opportunities for cooperation and conflict, both within and between species. I first delineate the social structure of M. genalis and M. ecuadoria nests in several Panamanian populations and integrate the factors that play a role in the behavioral decisions of females when joining a social group or not. Within a kin-selection framework, I discuss how genetic relatedness plays a role in the formation of social nests. Second, I investigate the conflict between host bees and a congener social parasite, and I elucidate reproductive structures that are relevant for understanding the evolution of ix parasitism. Finally, I describe a cleaning mutualism between Megalopta bees and their mite associates. Bee-mite associations encompass a broad spectrum of interspecific interactions. Some bee-mites are thought to perform cleaning services for their hosts in exchange for suitable environments for reproduction and dispersal. Field observations and experimental manipulation reveal a significant correlation between the presence of mites and the absence of fungi inside the brood cells, as well as between the absence of mites and increased bee mortality. This study therefore provides evidence of the sanitary effect of mites in nests of Megalopta bees. This bee-mite association constitutes one of the few examples of terrestrial cleaning mutualisms.
x Table of Contents
List of Tables ...... xii
List of Figures ...... xiii
Chapter 1: Introduction ...... 1
Chapter 2: Nestmate relatedness in the socially flexible bees Megalopta genalis and Megalopta ecuadoria ...... 5 INTRODUCTION ...... 5 METHODS ...... 8 RESULTS ...... 12 DISCUSSION ...... 15 CONCLUSION ...... 17 ACKNOWLEDGEMENTS ...... 19
Chapter 3: Notes on the reproductive morphology of the parasitic bee Megalopta byroni (Hymenoptera: Halictidae), and a tentative new host record ...... 27
Chapter 4: Cleaner mites: sanitary mutualism in the miniature ecosystem of Neotropical bee nests ...... 32 ABSTRACT ...... 32 INTRODUCTION ...... 33 MATERIALS & METHODS ...... 36 RESULTS ...... 39 DISCUSSION ...... 41 ACKNOWLEDGMENTS ...... 45
References ...... 50
Vita 63
xi List of Tables
Table 2.1: AFLP scoring errors statistics...... 19
Table 2.2: Pairwise F st values between populations of M. genalis ...... 20 Table 2.3: Relatedness coefficient statistics for the M. genalis and M. ecuadoria
populations studied...... 20 Table 2.4: Bivariate Pearson correlation coefficients, Spearman’s correlation coefficients and significance level for several variables of M. ecuadoria
and M. genalis...... 20 Table 2.5: Cluster analysis of the reproductive variables of M. genalis and M.
ecuadoria...... 21 Table 2.6: Means and standard error of morphological variables measured in females
of M. ecuadoria and M. genalis ...... 21 Table 2.7: Mating frequency and number of patrilines estimated for each population
of M. ecuadoria and M. genalis ...... 22
xii List of Figures
Figure 2.1: Map of collection sites in Panama...... 23 Figure 2.2: Bivariate Pearson correlation between the head width and reproductive
variables of M. ecuadoria and M. genalis and M. ecuadoria ...... 24 Figure 2.3: Frequency distribution of the proportion of shared markers between reproductive individuals and their nest-mates (brood and non- reproductive adults), and among unrelated individuals in each population
of M. ecuadoria and M. genalis ...... 25
Figure 4.1: Megalopta nest structure and cleaning effects of the mites...... 46 Figure 4.2: Deutonymphs (dipersal stage of the mites) on thorax and metasoma of
Megalopta pupa...... 47
Figure 4.3: Artificial cell with a bee larva...... 47 Figure 4.5. Means ± 2 Standard Errors of Colony Forming Units in the four
experimental treatments...... 49
xiii
Chapter 1: Introduction
Despite the inherently selfish character of natural selection, cooperation is
pervasive in the natural world but, paradoxically, provides opportunity to cheaters
(Bronstein 2001; Sachs et al. 2004). Moreover, cooperation and conflict cross several level of organization from within a single cell to within and between species (Maynard
Smith & Szathmary 1997). Cooperation is quite conspicuous in insect societies, however, such alliances do not preclude the existence of intrasocietal conflicts (Ratnieks et al.
2006).
The most remarkable example of cooperation within species of eusocial organisms is reproductive altruism, where sterile workers forego their own reproduction in order to dedicate their lives to rearing siblings (Wilson 1971). Not coincidentally, one of the most widespread intraspecific conflicts in hymenopteran social groups is over the control of reproduction. There exists a vast literature regarding how relatedness asymmetry, the differential degree of relatedness between males and females, causes conflict in sex allocation, male parentage, and queen worker-conflict over reproduction
(reviewed in Bourke 2005; Ratnieks et al. 2006).
Mutualisms are reciprocally advantageous interactions among individuals of different species. Such interspecific cooperation is ubiquitous in nature and is one of the evolutionary driving forces in ecological communities (Maynard Smith 1989; Herre et al.
1999). More than 70% of all plant species rely on their relationships with mycorrhizal 1 fungi to obtain nutrients from the soil (Selosse et al. 2006). Similarly, most flowering
plants (90%) depend on animals for transporting pollen between flowers (Fægri & van
der Pijl 1979). The examples are countless.
Conflicts between species are best exemplified by predation, such as the deceptive
behavior of the bolas spider that not only spins their typical web, but also actively hunt
moths by sending specific pheromones that mimics those of their prey (Gemeno et al.
2000). Likewise, there are conflicts in animal communication when predator and parasite
eavesdroppers take advantage of the honest signals used by their preys to attract mates
(Bernal et al. 2007).
My dissertation work involves the study of both cooperation and conflict in two
species of Megalopta bees (Hymenoptera: Halictidae). Megalopta is a Neotropical genus of sweat bees (Moure & Hurd 1987; Engel 2000). Like many other species in the family,
Megalopta is characterized by complex life cycles that can range from solitary to eusocial. Unlike most bees, they forage under dim light conditions taking advantage of dawn and crepuscular blooms (Wcislo et al. 2004). Nests are constructed in dead branches or lianas intertwined in the vegetation, and consist of a single excavated tunnel with individual brood cells made of chewed wood (see Sakagami & Michener 1962;
Wcislo et al. 2004). Brood cells are provisioned with a mass formed from nectar and pollen. Following oviposition, the cell is sealed, and brood development from egg to adult eclosion takes about 35 days. Fungi are common in bee nests, including inside cells for developing brood (Batra 1965; Batra et al. 1973; Inglis et al. 1993; Hajek & Stleger
1994). Megalopta ’s nests are inhabited by an extensive array of organisms and each nest 2 thus constitutes a miniature ecosystem providing opportunities for cooperative and
antagonistic interactions, both within and between species.
In Chapter 2, I delineate the social structure of M. genalis and M. ecuadoria nests
in several Panamanian populations. Given the social flexibility of these species, every
new female that emerges in a nest has the opportunity to decide between staying in the
natal nest, helping their nest-mates and even reproducing, or start a new nest of her own.
There are benefits and drawbacks in whatever decision the bee makes. Multifemale nests
have higher survivorship because they are better defended against ant predation (Smith et al. 2003). Based on the framework of kin selection (Hamilton 1964), the cost and benefits of group living are two of the factors that need to be taken into account in order to understand the evolution of sociality. In Chapter 1, I outline the importance of the other parameter involved in the evolution of social groups, the degree of relatedness within nests. I calculated relatedness from information provided by AFLP markers, and discuss the possible social outcomes in these populations as well as several aspects of the reproductive biology of these bees.
Chapter 3 investigates the occurrence of brood parasitism in Megalopta , a common phenomenon not only in nest-making Hymenoptera (Wcislo 1987; Michener
2000; Stuart 2002) but in vertebrate groups as well (Jones 1997; Hauber et al. 2004). M.
byroni is one of the congeneric parasites of these bees. It is extremely rare and its biology
is almost unknown. Here, I provide notes on reproductive structures that are relevant for
understanding the evolution of parasitism, and provide a tentative new host record for M.
3 byroni in Panama . I also discuss how specific aspect of the biology of this parasite might contribute to its rarity and the appropriateness of classifying it as a social parasite, given the social flexibility of the host bees.
Finally, Chapter 4 describes an astonishing example of cooperation between
Megalopta bees and their mite associates. One of the most common arthropods inhabiting social insect nests are mites (Arachnida: Acari) (Eickwort 1990, 1993). Bee-mite associations encompass a broad spectrum of interspecific interactions. Some bee-mites are thought to perform cleaning services for their hosts in exchange for suitable environments for reproduction and dispersal (Ordway 1964; Eickwort 1979). Using field observation and experimental manipulation, I demonstrate that there is a significant correlation between the presence of mites and the absence of fungi inside the brood cells, as well as between the absence of mites and increased bee mortality. This study therefore provides clear evidence of the sanitary effect of mites in nests of Megalopta bees. This bee-mite association constitutes one of the few examples of terrestrial cleaning mutualisms.
4 Chapter 2: Nestmate relatedness in the socially flexible bees Megalopta genalis and Megalopta ecuadoria
INTRODUCTION
Kin selection has recently been criticized as a sufficient explanation for the origin and maintenance of eusociality (Wilson 2005; Wilson & Hölldobler 2005; Wilson 2008a; Wilson 2008b), challenging a large body of evidence arguing for the role of genetic relatedness in the evolution of interactions in socially complex organisms or microbial communities (Jarvis 1981; Queller & Strassmann 1998; Faulkes & Bennett 2001; Griffin & West 2003; Bourke 2005; Gilbert et al. 2007) . Those defending the utility of kin selection argue that alternative explanations of eusociality (various forms of group selection) merely constitute a semantic rephrasing of selective processes that are best viewed as kin selection (Foster et al. 2006; West et al. 2007). A recent analysis of the importance of relatedness at the transition from solitary to eusocial life concluded that this transition occurred only in societies with singly-mated queens (high relatedness), whereas multiple mating (low relatedness) is invariably a derived condition arising within eusocial lineages. Hence, relatedness was essential in the evolution of eusociality (Hughes et al. 2008). The family of sweat bees (Hymenoptera, Halictidae) is one of most socially labile families within the order Hymenoptera, with obligate eusociality arisen (and lost) repeatedly within the family. Eusociality appear to have arisen five times among all the bees and three of these origins occurred within the family Halictidae (Danforth 2002). Moreover, sweat bees feature the greatest variability in life cycle and sociality of any bee,
5 sometimes even between and within populations of the same species (Wcislo 1997; Wcislo & Danforth 1997; Michener 2000; Danforth 2002). Megalopta is a neotropical genus of sweat bees (Hymenoptera: Halictidae) (Moure & Hurd 1987) that exhibit diverse social life cycles with alternative behavioral strategies between solitary and social life (Wcislo et al. 2004; Tierney et al. 2008), providing a window to view stages in the evolution of sociality. Unlike the typical ground-nesting species in the sweat bee family, Megalopta nests are constructed in dead branches or lianas intertwined in the profuse vegetation of primary and secondary forests. Megalopta nests are founded generally by a single female, however there is some evidence that co-founding by more than one female occurs in about 25-30% of the nests (Wcislo & Gonzalez 2006). At the beginning of the Panamanian dry season (mid December-mid January), nests are mostly solitary. As the season progresses, some nests become social (with more than one adult female), most likely because some of the emerging females decide to stay as helpers; other females might disperse and start new nests on their own. Males are more abundant at the beginning of the dry season, and most females are inseminated by the second half of the wet season (Wcislo et al. 2004). Solitary and social nests constitute two categorically distinct behaviors and are not developmental stages of a continuum. That is, nests that are solitary at the beginning of
the season tend to remain solitary at the end of the season. Similarly, social nests remain with several females at the end of the season (Smith et al. 2007). The founder female can
live an average of 97.1 ± 41.5 days, which is considerably longer than the mean egg to adult development time of about 35 days (Wcislo & Gonzalez 2006). Another estimate suggests that solitary females have one third chance of dying before their offspring emerge (Smith et al. 2007). Unrelated individuals joining nests or usurping nests have not
6 been reported (Wcislo et al. 2004), and unfortunately nothing is known about the mating behavior of these bees. In M. genalis , protection against ant predation and the risk taken by foraging adults seem to be strong selective forces favoring group living, consistent with the Assured Fitness Returns model (Queller 1989; Gadagkar 1990; Smith et al. 2003). Under this model, offspring depend on extended parental care in order to reach independence, and given a high risk of adult mortality, social groups composed of foundresses and helpers are favored over solitary nests (i.e., offspring survival is increased, or “assured” by the care provided by helpers). Solitary nesters are likely to lose all their investment because, if they die, they are not able to bring their offspring to independence. Smith et al.’s (2003) study on Megalopta is one of the few to assess the costs and benefits of social versus solitary life in a mass provisioning bee (Eickwort et al. 1996; Kukuk et al. 1998). The AFR model assumes some degree of relatedness between brood and workers (Queller 1989). We investigated the social status of several populations of M. genalis and M. ecuadoria in Panama to assess the importance of within-nest relatedness in the evolution of sociality of these bee species. Because the number of mates of a reproductive female might influence the degree of relatedness within her nest, we estimated the effective
mating frequency and determined if females mate singly or multiply in each of these two Megalopta species. We inferred reproductive dominance relationships in multifemale nests from morphological data on several reproductive physiology variables.
7 METHODS
Nest collections : Nests were collected in Soberanía National Park, Barro Colorado Island Natural Monument (BCI), and Bocas del Toro Province (Republic of Panamá), during June to August of 2005, December 2005 to January 2006, March to July 2006, and May to June of 2007. Nest collection sites were close to each other within each population (about 2.5 Km. apart), but distant between populations (Fig. 1, see Delineation of populations , below). Because Megalopta have crepuscular foraging habits (Wcislo et al. 2004), nests were collected during the day in order to assure capture of all resident bees inside the nests. Bees were individually preserved in 100% ethanol shortly after collection. Ecological information on these nests is reported in Biani et al. (2009). DNA extraction: A piece of muscle tissue was excised from the mesosoma to extract DNA. Use of thoracic muscle minimizes contamination with DNA from potential parasites (most frequently in the head or abdomen) or from food material in the crop or gut in the metasoma. The DNA extraction was performed with a Bioneer AccuPrep Genomic DNA Extraction Kit supplier, and the extracted DNA was diluted to 0.01µg/µl. AFLP marker development: We used a modified version of the Applied Biosystems (ABI) AFLP protocol for small plant genome
(http://www.appliedbiosystems.com , Protocol #: 4303146 Rev E). The DNA was
digested with EcoRI and Msel restriction enzymes. The EcoRI and Msel adaptor pairs were annealed using 10 µl of 10XT4 DNA ligase buffer, 10 µl of 0.5 M NaCl, 5 µl (1mg/ml) BSA, 100 Units Msel, 500 Units EcoRI, and 100 Units T4 DNA ligase. The restriction-ligation reaction was incubated at 37 °C for 3 hours instead of the recommended 2 hours to maximize production of template for amplification. Each restriction-ligation product was diluted in 60 µl of pure water instead of the recommended 189 µl of TE buffer. For the pre-selective and selective amplification, we 8 used 2 µl of 10X Taq buffer, 1.6 Units of Taq, 10mM dNTPs, all from New England Biolabs. The amplification of the pre-selective PCR was repeated for 25 cycles of 20s at 94 °C, 30s at 56 °C, and 2 min at 72 °C. The pre-selective PCR template was diluted by adding 40 µl of water to 10 µl of the pre-selective product. Primers pairs for selective amplification were EcoR1-TC x Msel-CAA, EcoR1- AA x Msel-CTC, and EcoR1-AT x Msel-CTG (Applied Biosystems). The samples were amplified by 11 cycles starting with 20s at 94 °C, 30s at 66 °C, and 2 min at 72 °C, but decreasing the annealing temperature by 1 °C each cycle until it reached 56 °C. The final selective PCR products were combined with 0.7 µl of Chimerex GeneFlo TM 625 (Rox label) as a size standard and 7.6 l of Hi-Di formamide, denatured at 95 °C for 5 minutes, and subsequently analyzed in an ABI Prism 3100 Genotyper. Scoring of AFLP markers: The AFLP banding pattern was scored with the program GeneMarker (AFLP/Genotyping ProgramVersion 1.51. Softgenetics LLC ®, State College, PA, 16803). A scoring panel for each primer combination was created where reliable alleles were first chosen from among all bands in an AFLP’s profile, then all samples were scored under that common pattern. We followed the recommendations of the software manual to create the panels and to run the program (SoftGenetics 2006). The two species were analyzed separately. A total of 236 markers was scored with 3
primers combinations for both species. We pursued the AFLP scoring strategy suggested by Bonin et al. (2007). We minimized the risk of homoplasy (non-homologous fragments migrating to the same position in the electrophoretic profile) by scoring only the fragments above 100 base pairs and,. With the exceptions of M. ecuadoria population from BCI (12 individuals) and M. genalis from Bocas del Drago (16 individuals), in all the other populations the number of individuals scored is greater than 30 for each species, which is the 9 recommended number (Bonin et al. 2007). The scoring was very conservative because we only used markers that could be scored unambiguously across all samples; markers that could not be scored reliably as present/absent were excluded from the analysis. Fifteen samples were processed blindly starting with DNA extraction. The samples were also blindly scored with respect to colony and population. The percentage of markers that differ between original and the blind rescoring correspond to the error rate in scoring the AFLP markers. We were able to unambiguously score 236 markers among the three primer combinations. We discarded 265 additional markers because they could not be scored unambiguously. For the 236 reliable markers, the average scoring error in the blind rescoring was 2.51 % (Table 1.1), which is comparable to error rates of microsatellite markers (Okello et al. 2005; Morin et al. 2007). Delineation of population boundaries : Before calculating within-nest relatedness, we defined the populations within which mating can be assumed to occur at random, using the program AFLPsurv (Vekemans et al. 2002). We used the option that assumes Hardy-Weinberg conditions within each population and non-uniform prior distribution of allele frequencies for the Bayesian analysis of the matrices (Zhivotovsky 1999). Relatedness calculation : The coefficient of relatedness was calculated following Reeve et al. (1992). Because this method obtains a single relatedness estimate without confidence intervals, we performed a bootstrap analysis to estimate the standard error of that single relatedness estimate. One hundred matrices were created in Mesquite by bootstrap re-sampling of markers from the original matrix (Maddison & Maddison 2009), calculating the average relatedness for each sub-sampled matrix, and then deriving the standard error across these 100 bootstrap averages. Morphological measurements : Several morphological variables were measured for each adult bee collected, including head width, intertegula distance, largest ovariole 10 width, Dufour gland width, and number of yellow bodies (degenerating nursing cells that indicate a recent egg laying history). These variables are known to correlate with the reproductive status of bees (Michener 1974; Arneson & Wcislo 2003), and therefore allow identification of the primary reproductive in each nest, as well as current and past reproductive activity. Bees were measured and dissected using an ocular micrometer mounted on a stereomicroscope. Bivariate Pearson correlation and non-parametric Spearman correlation were performed to assess the relationship between morphometric and reproductive variables. A cluster analysis determined which the reproductive individuals in each nest were. All tests were performed in SPSS (SPSS 2006). Estimation of mating frequency : Females that had the largest ovariole width, the largest Dufour gland, and the largest number of yellow bodies were assumed to be the primary reproductive in a given nest. The mating frequency was estimated in nests where reproduction by a single female (rather than several possible females) could be verified based on the above reproductive variables. AFLP markers present in the progeny but absent in the primary reproductive female must have been inherited paternally. If these sets of paternally-derived markers differ between brood, they might constitute an event of
polyandry. The frequency of the different sets of bands (p i) can then be used to estimate
2 the effective mating frequency in each nest [m e=1/ Σ(p i) (Starr 1984)]. We only considered paternal sets of markers consisting of more than one marker in order to avoid artificially created patterns that might be due to amplification or scoring errors of a single band. Alternatively, dissimilar banding patterns among brood can be due to events of queen supersedure (serial polygyny) when the original reproductive dies and another female takes over the reproduction in the nest (Mueller 1996; Paxton et al. 2003; Bargum et al. 2007). For the cases used in the mating-frequency analyses, we ruled out queen supersedure by also calculating the percentage of markers shared among all individuals 11 within each nest (see below). An event of polygyny would be detected by dissimilar banding pattern and low percentage of shared markers between the reproductive and some of the brood. Determination of potential intra-specific nest parasitism : We calculated the proportion of markers that were shared between the primary reproductive and her nestmates, as well as the proportion of shared markers among unrelated individuals in each population. By comparing the frequency distribution of these shared markers, it is potentially possible to detect cases of intra-specific nest parasitism. Identifying cases of intraspecific next parasitism is critical because the calculation of the mating frequency assumes correct identification of mother-daughter relationships within a given nest. We only considered the nests where the primary reproductive could be unambiguously identified and where workers did not seem to reproduce, based on the morphological and physiological criteria described above. Moreover, for these nests we ruled out the occurrence of nest usurpation, that is, when the primary reproductive is replaced by an unrelated individual that takes over reproduction of the nest. Under supersedure, older brood would have low proportion of band shared with respect to the identified primary reproductive, whereas younger brood would share a higher proportion of bands. Conversely, egg dumping (intra-specific nest parasitism) can be detected when a single individual share a low proportion of markers with the primary reproductive.
RESULTS
Scoring of AFLP markers: The average number of fragments per individual was 70.6 and 85.4 for M. genalis and M. ecuadoria , respectively, and the corresponding total
12 number of segregating (polymorphic) fragments was 184 (78%) and 163 (69.1%), respectively. Delineation of population boundaries: We measured population-genetic
differences with the F st statistic, which can be interpreted as the proportion of the total genetic diversity that occurs among populations (Excoffier et al. 1992). The AFLPsurv
program creates a distribution of F st through 1,000 random permutations of the matrix under a null hypothesis of no population differentiation. For M. genalis, the lower and upper 95% limits of the distribution of F st were -0.0082 and 0.0300 respectively. The observed F st was 0.0963, which fell outside these limits and therefore allowed the rejection of the null hypothesis of no population differentiation (p<0.001). The F st analyses defined three populations, Pipeline Road, BCI, and Bocas del Drago for M. genalis (Bocas del Toro Province; Table 1.2, Fig. 1.1). Likewise, for M. ecuadoria the observed F st of 0.0943 fell outside the lower (-0.0147) and upper (0.0291) 95% limit; hence we rejected the null hypothesis of no population differentiation (p<0.001). The two defined populations were Pipeline Road and BCI (Fig. 1). The close proximity of the collection sites within each population relative to the distance among populations supports the interpretation that within-population gene flow is very likely for both species. All subsequent analyses consider each population separately.
Within-nest relatedness: The genetic relatedness between individuals of the same nest, as estimated by the method of Reeve et al. (1992), varied from 0.25 (± 0.002 SE) to 0.37 (± 0.002 SE) for the three populations of M. genalis , and from 0.31 (± 0.003 SE) to 0.37 (± 0.002 SE) for the two populations of M. ecuadoria (Table 1.3).
Morphological measurements: The Dufour gland width was significantly correlated with head width in both species, while ovariole width was only significantly correlated with head width in M. genalis , but not in M. ecuadoria (Fig. 1.2, Table 1.4). 13 Non-parametric correlation assessed the relationship between the number of yellow bodies with the size of the bees. This correlation was significant only for M .genalis (Table 1.4). The cluster analysis divided the individuals into reproductives and non- reproductives based on values of the largest ovariole width, Dufour gland width, head width and intertegula distance (Table 1.5). Most of the nests seemed to have a primary reproductive (greatest ovariole development and largest Dufour gland development, highest number of yellow bodies; Table 1.6). However, 19% (N=21) of both M. ecuadoria and M. genalis nests may form more egalitarian societies, because all the adult females in these nests showed comparable reproductive measures (i.e., all could be potentially reproducing). In other nests, all the individuals were similar to each other but the morphological values were comparable to non-reproductive individuals, suggesting that none of them were reproducing yet (i.e., these nests did not have a primary reproductive at the time of collection). This could be due to the recent loss of a reproductive and the absence of a replacement individual. Estimation of mating frequency : The effective mating frequency varied between 1.80-2.67 for the two populations of M. ecuadoria and between 1.00-3.00 for the three populations of M. genalis (Table 1.7). Specifically, all M. ecuadoria brood in each nest came from at least 2 patrilines (N = 7, range 2-4 patrilines); in addition, the effective mating frequencies of all the females were above 2, except one that had an effective mating frequency of 1.8. In M. genalis , only one nest out of eight seemed to have brood from only a single patriline. Brood in all other M. genalis nests usually had 2 - 3 patrilines and most females had an effective mating frequency between 2 and 3. This indicates that most of the reproductive females of both species were using the sperm of several males. Because of our conservative approach in scoring the AFLP markers, it is possible that actual mating frequencies are higher than the estimated values. On the other 14 hand, if our analyses failed to identify cases of supersedure of primary reproductives, our results overestimate the actual mating frequencies. Determination of potential intra-specific nest parasitism: The proportion of markers shared among individuals of the same nest was above 87% for M. ecuadoria and above 84% for M. genalis (Fig. 1.3 A&B). Based on the comparison with the distribution of shared markers among unrelated individuals, there is no clear evidence for intra- specific nest parasitism (egg dumping) except for one M. genalis’ nest from Bocas del Drago where one individual shared only 70% of the markers compared to the rest of individuals in that nest and the population in general (marker sharing of above 90%). However, these estimates are based on only two nests from Bocas del Drago and hence might not be representative of the population as a whole. The analysis of the shared markers for all populations did not provide evidence of nest usurpation, because all brood appeared to share a high proportion of markers with the primary reproductive, as identified by the morphological and physiological data.
DISCUSSION
Our AFLP analyses indicate that cooperating nestmates of both M. ecuadoria and M. genalis have a considerable degree of genetic relatedness (0.31-0.37 and 0.25-0.37, respectively). Cooperating females are likely to be sisters or daughters of the primary reproductive. Moreover, the relatedness estimates are within the range of other primitively eusocial insects, including some Polistes wasps (Strassmann et al. 1989; Francescato et al. 2002; Landi et al. 2003) and bees (Richards & Packer 1998; Stow et al. 2007). The fact that the relatedness estimate is lower than the 0.75 value expected for full sisters in a single-mated single queen’s colony might be due to several factors, including
15 multiple mating, multiple reproductive females in a nest, and intraspecific nest parasitism. The estimated effective mating frequency and the existence of several patrilines in most of our surveyed nests suggest that females are multiply mated, consistent with studies of other halictid bees that also documented multiple mating (Page 1986; Kukuk 1989; Richards & Packer 1998). These studies were based on allozymes and behavioral observations of mating. A more recent study using microsatellite markers in Lasioglossum malachurum concluded that, even though most of the queens were singly- mated, up to four patrilines could be detected in a single nest. The effective mating frequency of L. malachurum varied between 1 and 3 with an average of 1.13 matings for 17 nests. L. malachurum therefore seems to be facultatively polyandrous or, the queens seem to use sperm from only one male even if they had mated with several males (Paxton et al. 2002). In another population of L. malachurum in Greece, eusocial colonies had mostly single-mated queens (97 %; Richards et al. 2005). A very closely related bee, Augochlorella striata , is the only halictid that seems to be clearly singly-mated, with consequently high within-colony relatedness (Mueller et al. 1994). Even though most nests had a female that dominated the reproductive output, some nests (19%) of each species might be more egalitarian societies in which more than one female reproduced in their nests. Based on the occurrence of aggressive interactions among individuals of M. genalis , it seems that egalitarian societies are rare (Arneson & Wcislo 2003); however, our morphological data suggest that they exist in a low frequency. As in egalitarian societies of other halictid species, several matrilines might be present in the nests (Wcislo 1997; Richards & Packer 1998; Richards 2000). Although egg dumping has not been directly observed in Megalopta , it is a common phenomenon among other bees (Kukuk et al. 1987; Wcislo 1987; Paxton et al. 16 2002), decreasing the relatedness between brood and adult females in affected nests. In our study, we were unable to detect intra-specific nest parasitism, except possibly in a single M. genalis nest in Bocas del Drago in which one of the individuals had a lower proportion of shared markers and therefore may have been unrelated. This single individual can also be the result of inter-specific parasitism by M. byroni (Biani & Wcislo 2007). Because we only found one such individual, it is impossible to reach any conclusion about the existence of social parasitism in M. genalis . Regarding the relationship between size and reproductive status of the bees, the only significant associations were between ovary width and Dufour gland width with head width in M. genalis , which is in agreement with a previous study on the same species (Smith et al. 2008). The relationship between ovariole width and head width was not significant in M. ecuadoria , perhaps because many large bees have small ovaries (the so-called “pre-reproductive”) or alternatively, because smaller individuals become dominant reproductives if they are the oldest (Arneson & Wcislo 2003; Smith et al. 2008; Tierney et al. 2008). Other factors such as parasitism and cephalic allometry present in these bees might affect the expression of body size and potentially the performance of certain social roles (Sakagami & Moure 1965; Smith et al. 2008).
CONCLUSION
The evolution of social behavior is a very complex trait determined by intrinsic and extrinsic factors (Evans 1977). Some of the relevant extrinsic factors driving the evolution of sociality in Megalopta are predator pressure and risks of adult mortality (Smith et al. 2003, 2007). Here we attempted to evaluate the importance of intrinsic genetic factors in the formation of cooperative societies of M. genalis and M. ecuadoria
17 bees. Our results suggest that social cooperative groups are formed by related individuals in M. genalis and M. ecuadoria; individual females of these species therefore may gain inclusive fitness by staying in their natal nest rather than risking their fitness in an inadequately defended nest of her own (Smith et al. 2003). Each social outcome might be the product of a delicate combination of intrinsic and extrinsic factors mentioned above, plus other suites of characteristics determined by the organisms themselves: intra-specific interactions, the expression of dominance, their capability of nest mate recognition, and competition for limited resources (Wcislo 1997). Because cooperative females are related, this study provides additional support towards the possible importance of kin selection in the evolution of sociality. Even though environmental factors are known to play an important role in the shape of the social structure of halictid nests (Eickwort 1966; Packer 1990; Soucy 2002; Richards et al. 2005), most studies have been conducted on species from temperate regions or at high altitudes (but see Wcislo et al. 1993; Soucy & Danforth 2002; Tierney et al. 2008). In those species, the length of the reproductive season is determined mainly by temperature, which constrains the existence of social groups. In Panama, the active phase of nests is marked by the length of the dry season since there are fewer flower resources to provision the nests as the rainy season progresses (Wright & Calderon 1995).
Furthermore, the rains affect the resilience of the nesting substrates specifically in Megalopta , which in turn can also affect the expression of social behavior (Wcislo et al. 2004). The relatively long active season in central Panama generate opportunities for several overlapping generations, which is a prerequisite for the formation of social groups. This study constitutes another contribution to elucidate the social structure of tropical halictids.
18 ACKNOWLEDGEMENTS
This research was funded by the Smithsonian Tropical Research Institute (STRI) Short Term Fellowships and research grants from the Program of Ecology, Evolution and Behavior of the University of Texas at Austin to NBB. We are extremely grateful to Juan Carlos Santos for his statistical advice and help with the use of several software programs; to Adam Smith, Hermógenes Fernández-Marín, Therany Gonzales and Juan Carlos Santos for their invaluable help and company during nest collections; to Barrett Klein, Sze Huei Yek, Ruchira Sen, Heather Ishak and Alexander Mikheyev for their comments on earlier versions of the manuscript. We thank STRI staff for logistical support. The Autoridad Nacional del Ambiente de la República de Panamá provided collecting and export permits.
Table 2.1: AFLP scoring errors statistics.
Primer combination # of markers Average error Standard Deviation Blue (EcoR1-TC/Msel CAA) 117 2.05 % 0.026 Yellow (EcoR1-AT/Msel 41 3.09 % 0.023 CTG ) Green (EcoR1-AA/Msel 78 2.39 % 0.015 CTC) TOTAL 236 2.51 % 0.021
Error is estimated as the percentage of markers scored differently in blind repeat genotyping.
19 Table 2.2: Pairwise F st values between populations of M. genalis
BCI B. Drago Pipeline BCI 0 Bocas Drago 0.1051 0 Pipeline 0.0544 0.1259 0
Table 2.3: Relatedness coefficient statistics for the M. genalis and M. ecuadoria populations studied.
Species Population # nests Real Bootstrap S.E. average average M. ecuadoria Pipeline 13 0.369 0.340 0.002 M. ecuadoria BCI 3 0.309 0.314 0.003 M. genalis Pipeline 9 0.368 0.379 0.002 M. genalis BCI 10 0.348 0.339 0.002 M. genalis Bocas Drago 4 0.253 0.268 0.002
Bootstrap average and standard errors of the relatedness coefficients estimated from 100 bootstrap subsamples.
Table 2.4: Bivariate Pearson correlation coefficients, Spearman’s correlation coefficients and significance level for several variables of M. ecuadoria and M. genalis.
Species Bivariate Pearson Correlations Coefficients Significance Head width & Largest ovariole width 0.159 0.144 M. ecuadoria Head width & Dufour gland width 0.358 0.001 Head width & Largest ovariole width 0.423 0.001 M. genalis Head width & Dufour gland width 0.237 0.017
Non parametric Spearman correlations Coefficients Significance Head width & # yellow bodies 0.001 0.992 M. ecuadoria Largest ovariole width & # yellow bodies 0.480 0.001 Dufour gland width & # yellow bodies 0.478 0.001 Head width & # yellow bodies 0.204 0.041 M. genalis Largest ovariole width & # yellow bodies 0.458 0.001 Dufour gland width & # yellow bodies 0.318 0.015 20 Table 2.5: Cluster analysis of the reproductive variables of M. genalis and M. ecuadoria.
The two clusters below are statistically significant for both species.
M. genalis Intertegula Distance Head Width Ovariole Width Dufour Width Mean SD Mean SD Mean SD Mean SD Cluster 1 2.57 0.14 3.49 0.23 0.74 0.27 0.64 0.13 Cluster 2 2.22 0.22 2.82 0.28 0.36 0.19 0.45 0.17 Combined 2.36 0.26 3.09 0.42 0.51 0.29 0.52 0.18
Intertegula M. ecuadoria Distance Head Width Ovariole Width Dufour Width Mean SD Mean SD Mean SD Mean SD Cluster 1 2.08 0.15 2.84 0.25 0.64 0.14 0.53 0.15 Cluster 2 1.93 0.13 2.51 0.16 0.35 0.13 0.33 0.14 Combined 1.98 0.15 2.63 0.25 0.45 0.19 0.40 0.17
Table 2.6: Means and standard error of morphological variables measured in females of M. ecuadoria and M. genalis .
Intertegula Largest ovariole Dufour gland distance Head width width width mean SE mean SE mean SE mean SE M. ecuadoria (N=52) 1.98 0.02 2.63 0.04 0.45 0.03 0.40 0.02 M. genalis (N=58) 2.36 0.03 3.10 0.06 0.51 0.04 0.52 0.02
Measures are in mm.
21 Table 2.7: Mating frequency and number of patrilines estimated for each population of M. ecuadoria and M. genalis .
Variables Species Population # Nests Min. Max. Mean Std. Error Mating Freq. 6 1.80 2.67 2.12 0.13 Pipeline Rd. M. # Patrilines 2 4 2.50 0.84 ecuadoria Mating Freq. 1 N/A N/A 2.00* N/A BCI # Patrilines N/A N/A 2* N/A Mating Freq. 4 1.60 2.00 1.85 0.10 Pipeline Rd. # Patrilines 2 2 2.00 0.00 M. Bocas del Mating Freq. 2 1.00 3.00 2.00 1.00 genalis Drago # Patrilines 1 3 2.00 1.00 Mating Freq. 2 1.80 2.00 1.90 0.10 BCI # Patrilines 2 2 2.00 0.00
* estimate from a single M. ecuadoria nest in BCI.
22 Figure 2.1: Map of collection sites in Panama.
Bocas del Drago (Bocas del Toro Province), Pipeline Road, and Barro Colorado Natural Monument (BCI, Colon Province). Panama Canal Zone with details of collection locations on BCI and in Gamboa/Pipeline Road (modified from http://biogeodb.stri.si.edu/bioinformatics/maps ).
23 Figure 2.2: Bivariate Pearson correlation between the head width and reproductive
variables of M. ecuadoria and M. genalis and M. ecuadoria .
24 Figure 2.3: Frequency distribution of the proportion of shared markers between reproductive individuals and their nest-mates (brood and non-reproductive adults), and among unrelated individuals in each population of M. ecuadoria and M. genalis .
25 26
Chapter 3: Notes on the reproductive morphology of the parasitic bee Megalopta byroni (Hymenoptera: Halictidae), and a tentative new host record
Brood parasitism has evolved repeatedly in sweat bees (Halictidae) (Michener
2000), and in other nest-making Hymenoptera (bees, wasps, and ants) (Choudhary et al.
1994; Savolainen and Vepsalainen 2003; Wcislo 1987). However, many parasitic species are relatively rare and therefore little is known of their biology. Megalopta
(Augochlorini) is a Neotropical genus of sweat bees, most of which forage under extremely dim light conditions and nest in dead wood (reviewed in Wcislo et al. 2004). It also contains several parasitic species (see Michener 2000). Megalopta noctifurax Engel,
Brooks & Yanega and M. fununculosa Hinojosa-Díaz & Engel are putative obligate
parasites based on morphological features that are associated with parasitic behavior, but
there are no rearing records (Engel et al. 1997; Hinojosa-Díaz and Engel 2003). A third
parasitic species, M. byroni Engel, Brooks and Yanega, was reared from nests of M.
genalis Meade-Waldo, and is very rare: only three individuals have been found from
>300 nests between 1998 and 2001 (Wcislo et al. 2004). Here we provide notes on
reproductive structures that are relevant for understanding the evolution of parasitism,
and provide a tentative new host record for M. byroni in Panamá, which was collected
from a nest of M. ecuadoria Friese. Engel (2006) selected the synonymous name M.
centralis Friese as the name for this species, but a petition to conserve the commonly
27 used name M. ecuadoria will soon be before the International Commission on Zoological
Nomenclature for a decision; until a decision is rendered we use the name M. ecuadoria.
We collected 75 nests of M. genalis and M. ecuadoria Friese from June-August
2005, and 114 nests from March-July 2006, along Pipeline Road in Soberanía National
Park (Colón Province, Panamá, N 09.14778 W 079.72997), in nearby secondary forests near Gamboa, and in the Barro Colorado Nature Monument. Nests were collected in the field, and opened in the laboratory to collect resident adults and brood.
The metasoma of each parasite was preserved in formalin and later dissected under light microscopy. The number of ovarioles per ovary was counted, as well as the number of yellow bodies. The width of the largest ovariole and the width of the Dufour’s gland were measured using an ocular micrometer. Means are given with their standard deviations.
In 2005 only 2.7% of 75 nests contained an adult parasite, and in 2006 none of the
114 nests were parasitized (overall parasitism rate, ~1%). The first parasite was found in late June (beginning of the wet season) in a nest with two M. ecuadoria females and three open and empty brood cells. Thus, the new host record is tentative until it is confirmed with rearing records. The second parasite was found in a nest of M. genalis in early
August. The nest was inhabited by a single M. genalis female and there were no cells in the nest, so apparently it was in an early stage of construction. Almost nothing is known on the biology of M. byroni , and we do not know if it is a cleptoparasite or a social parasite. Given the social flexibility of the host bees (i.e., some bees are solitary and some social) (Wcislo et al. 2004), this classification might not fit at all, and host-parasite 28 relationships might depend also on the social status of particular nests. These two potential host species, M. genalis and M. ecuadoria, are sympatric in Panama, which is the only locality record for M. byroni .
Both parasites had three ovarioles per ovary, which is the plesiomorphic number for Halictidae, including parasitic forms (Rozen 2003), and the width of the largest ovariole was 0.45 mm for both parasites. By comparison, the resident M. ecuadoria had a mean ovariole width of 0.75 mm, slightly larger than the M. ecuadoria population mean of 0.37 mm ± 0.16 (N = 54) and the M. genalis population mean of 0.41mm ± 0.18
(N=59). Furthermore, the first parasite, collected in June, had 12 yellow bodies, while the second one, collected in August, had only one. In contrast, the nestmates had no yellow bodies, and the M. ecuadoria population mean was 0.32 ± 0.96 (N=54) yellow bodies, and 1.16 ± 2.65 (N=59) for M. genalis .
The Dufour’s gland, which produces secretions used in the internal lining of the cell walls (Cane 1983), was 0.15 and 0.20 mm, respectively for each parasite, which is much smaller than that of the resident host bees of M. ecuadoria (width, 0.37 mm ± 0.11,
N=2), implying that parasites do not use it in nest architecture or in antisocial communication. The M. ecuadoria population mean (N=54) for Dufour’s gland width was 0.41 mm ± 0.19, and 0.44 mm ± 0.19 for M. genalis (N=59).
In most bee taxa, ovariole number per ovary is constant among individuals and within taxa, except for highly social bees (e.g., Apis ) and some parasitic lineages that exhibit an increased number of ovarioles with respect to the plesiomorphic number in their free-living relatives (e.g., Alexander and Rozen 1987; Rozen and Ozbek 2003; 29 Serrão J. E. and Martins 2004). A larger number of ovarioles in parasitic species, or an increased number of oocytes per ovariole, might increase the probability of laying eggs both during a single nest usurpation event, as well as over the entire life of the parasite
(Ohl and D. 2003; Rozen 2003). However, other factors such as the number of mature oocytes, the size and thickness of the chorion, may also influence the time taken to produce eggs. Yellow bodies are remnants of re-absorbed oocytes (Bell and Bohm 1975).
The notable difference in the number of yellow bodies between the first and the second parasite (12 vs. 1 yellow body, respectively) might reflect the number of failures when trying to deposit an otherwise ready egg, given that the first parasite was found earlier in the season and in a well established nest.
The apparent rarity of this parasite might be explained by three aspects of the biology of hosts and parasite. First, the relative low degree of seasonality in tropical rain forests might diminish the synchronization of life cycles between hosts and parasites
(reviewed in Wcislo 1987), though central Panama is characterized by strong seasonal rhythms (Leigh 1999). Second, the lower probability of locating a suitable nest in a three dimensional space (nests are hung in the vegetation) than in a two dimensional space
(ground nests) may make host searching more difficult (Wcislo 1996). Finally, the fact that hosts fly at remarkably dim light levels and face severe challenges with respect to visual ecology (Kelber et al. 2006; Warrant et al. 2004), implies that the parasites also search for nests in the dark, which should be relatively more difficult. Alternatively, if parasites try to enter nests during the day, then hosts would always be present as functional guards. 30 ACKNOWLEDGMENTS
NBB is indebted for the training in finding and opening nests to Juan Carlos di Trani and
Fernando Soley, who participated in the research with WTW through STRI’s Behind-the-Scenes
Volunteer Program and STRI’s internship program, respectively. We thank STRI support staff and financial support from STRI general research funds to WTW. Funds from the Frank and Fern
Blair Fellowship, University of Texas at Austin to NBB are gratefully acknowledged. We are indebted to Ulrich Mueller, Michael Engel and an anonymous reviewer for their helpful comments. The Autoridad Nacional del Ambiente de la República de Panamá provided collecting and export permits.
31 Chapter 4: Cleaner mites: sanitary mutualism in the miniature ecosystem of Neotropical bee nests
ABSTRACT
Cleaning symbioses represent classic models of mutualism, and some bee-mites are thought to perform cleaning services for their hosts in exchange for suitable environments for reproduction and dispersal. These mutual benefits, however, have not been rigorously demonstrated. We tested the sanitary role of bee-mites by correlating mite loads with fungal contamination in natural nests of Megalopta genalis and
Megalopta ecuadoria, and by experimentally manipulating mite loads in artificial cells with developing brood. Field observations revealed significant correlations between the presence of mites and the absence of fungi inside the brood cells, as well as between the absence of mites and increased bee mortality. Likewise, experimental brood cells with mites have fewer fungal colonies than cells without mites. Field observations and experimental manipulations therefore provide clear evidence of the sanitary effect of mites in nests of Megalopta bees. This bee-mite association constitutes one of the few examples of terrestrial cleaning mutualisms.
32 INTRODUCTION
Cleaning symbioses represent classic models of mutualism (Becker & Grutter
2004; Östlund-Nilsson et al. 2005) in which individuals of one species obtain food by removing external parasites from those of another species. However, most examples are aquatic and restricted to fishes and shrimps (Becker & Grutter 2005). A commonly cited example of a terrestrial cleaning symbiosis involves birds that glean ectoparasites from large mammals, but empirical tests have shown that this relationship is more complicated and may not always be mutually beneficial (Weeks 2000). Mites associated with some stingless bees ( Trigona ) are thought to decrease larval mortality due to fungi, but this has not been rigorously demonstrated (Flechtmann & Camargo 1974). Parasitic mites associated with the wasp Allodynerus delphinalis act as ‘bodyguards’ and defend juvenile wasps from attacks by a hymenopteran parasitoid (Okabe & Makino 2008). Another example of mutualism includes mites associated with burying beetles. These mites reduce the number of fly larvae that compete with the beetles for food, and a lower number of flies on the carcasses increases beetle fitness (Wilson 1983; Wilson & Knollenberg
1987). This symbiosis, however, fluctuates between commensalism and mutualism over time. Here, we experimentally demonstrate that an association between bees and mites is a cleaning mutualism, adding to only a handful of documented examples of this kind of association.
Nests of Hymenoptera (ants, bees, wasps) are prone to attack by microorganisms
(Schmid-Hempel 1998). As a result, many species have evolved behavioral and chemical
33 traits to protect their brood and food. Examples include Philanthus wasps wrapping food
with postpharyngeal gland secretions that reduce the amount of mold growing on the
paralyzed prey (Herzner & Strohm 2007); wood ants furnishing nests with plant resins,
rich in secondary compounds (Chapuisat et al. 2007); and fungus-growing ants producing
metapleural gland secretions that decrease the amount of pathogenic fungi in the fungus
garden (Fernández-Marín et al. 2006). Some species have evolved symbiotic
relationships with other organisms that function to protect themselves from parasitic
attack, such as the antibiotic-producing bacteria associated with the beewolf Philanthus
triangulum (Kaltenpoth et al. 2005) or with fungus-growing ants (Currie et al. 1999; Mueller et al. 2005).
Both solitary and social bees construct brood cells within their nests (Michener
1974; Wcislo 2000), which creates distinctive microenvironments ( sensu Odling-Smee et al. 2003). Each cell is a miniature ecosystem, and the stored food sustains not only the development of the bee brood, but also an extensive array of commensal and parasitic organisms, such as other arthropods, fungi, bacteria and nematodes. One of the more common arthropod symbionts that live inside nests of bees, ants and wasps are mites
(Arachnida: Acari) (Eickwort 1990, 1993).
Bee-mite associations encompass a broad spectrum of interspecific interactions.
Some mites are parasites, some are predators on other arthropods, including other mites, and others are hypothesized to be mutualists. One kind of mutualism may involve cleaning services of mites by suppressing or curbing fungal growth within nests; hosts, in exchange, provide a suitable environment for mite reproduction, as well as an effective 34 dispersal service (mites ride on the dorsal part of the bee’s metasoma or mesosoma)
(Ordway 1964; Eickwort 1979). The associated sanitary benefits, however, have not been
rigorously demonstrated until now (Flechtmann & Camargo 1974; see Discussion). This
mutualism hypothesis makes two critical predictions. First, if cells contain mites, the
incidence of contaminant fungal growth should be lower compared to cells without mites.
Second, bee brood should be healthier when mites are present in a cell, and brood
survivorship therefore should be higher if raised in the presence of mites, compared to
mite-free brood. We tested these two predictions by correlating natural mite loads with
fungal contamination of cells in natural nests of Megalopta genalis and M. ecuadoria , and by experimentally manipulating mite load in artificial cells with developing brood.
Overview of bee-mite natural history
Megalopta Smith 1853, is a Neotropical genus of halictid bees (Moure & Hurd
1987), which exhibit a continuous spectrum from solitary to social life cycles, and forage under dim light (Wcislo et al. 2004). Nests are usually constructed in dead wood and consist of a single excavated tunnel with individual cells made of chewed wood (Fig. 4.1
A&B) (see Sakagami & Michener 1962; Wcislo et al. 2004). Cells are provisioned with a mass formed from nectar and pollen. Following oviposition, the cell is sealed, and brood development from egg to adult eclosion takes about 35 days. Fungi are common in bee nests given the nutritious substrate available (Batra 1965; Batra et al. 1973; Inglis et al. 1993; Hajek & Stleger 1994).
35 In Megalopta , a tight synchrony between bee and mite ontogeny exists: each developmental stage (stadium) of the host bee is often associated with a specific mite stage (Biani and Wcislo, unpublished data). When callow bees emerge, 91% of the mites are deutonymphs, the dispersal stage of certain mites (NBB pers. obs.; see Fig. 4.2). A preliminary identification of the tritonymphs (i.e., the penultimate mite stage before adulthood) collected from Megalopta nests places them in the genus Laelaspoides
(Mesostigmata; H. Komplen, pers. com.), which are known to be obligate bee-mites
associated with other closely related halictid genera, Augochlorella and Caenaugochlora
(Eickwort 1966, 1979).
MATERIALS & METHODS
Nests collection and field surveys
Nests were collected in Soberanía National Park, Barro Colorado Natural
Monument, San Lorenzo Protected Area, and Bocas del Toro Province (Republic of
Panamá), during June through August of 2005, December 2005 to January 2006, March
through July 2006 and May to June of 2007. Throughout these field seasons, a total of
294 nests were collected, opened in the lab and scanned under a light microscope for the
presence of mites and fungi (39 nests were not included in this study because they were
either abandoned or without brood, leaving 255 nests for analysis).
36 Experimental brood cell procedures
Nests were opened in the lab and the contents of natural cells were transferred into artificial brood cells made of paraffin, cover slips, and modeling clay (Fig. 4.3). In order to empirically test the hypothesis that the presence of mites reduces fungal infection, we experimentally manipulated the presence/absence of mites in artificial brood cells. Mites were removed or added with a fine paintbrush and all cells were opened and exposed for the same amount of time, even in control cells (sham treatment).
The paintbrush was disinfected before each manipulation. Between seven and ten tritonymphs were transferred to the artificial cells. No cells were experimentally inoculated with fungi, and we relied on natural rates of brood cell contamination.
For the experimental manipulations, we collected 27 nests that had mites and 23 nests that did not have mites. We then divided the group of nests with mites into two experimental groups: one was used as control (N = 10, sham-opened but otherwise not manipulated), and the other one had mites removed (N = 17). Similarly, the group of nests that did not have mites was divided in two experimental groups: one was used as control (N = 12, sham-opened but otherwise not manipulated), and one had mites added
(N = 11). One independent variable was the presence/absence of mites at the time of collecting. The second, critical, independent variable was the experimentally controlled presence/absence of mites at the end of the experiment.
37 Assessing levels of fungi contamination with colony-forming units procedure
Bees were reared in artificial brood cells and after 10 days (approximately a third of the bee life cycle), fungal growth was quantified by plating diluted cell content and counting the number of colony-forming units (CFU) (Smith 1980; Christe et al. 2003).
This method consists of diluting and homogenizing a sample (in this case 0.01g of pollen mass or bee feces) in 10 ml of a solution with 0.85% sodium chloride, 0.2% peptone and
0.05% Tween 80 (a surfactant that facilitates the suspension of fungi spores in water,
Sigma, St. Louis, MO). Successive dilutions were made and then an aliquot of 100 l was plated on a PDA (Potato Dextrose Agar) medium with 0.2g/L of chloramphenicol as antibiotic. The optimal dilution is the one that allows the growth of 30 to 300 colonies per plate; we used the dilution 1x10 -4 for all the samples. All the cultures were done under a sterile laminar-flow hood. Fungi were allowed to grow in a humidity and temperature- controlled room and then the number of CFU was counted blindly with respect to treatment. Two replicates were made for each dilution per sample and the CFU counts were averaged. The CFU of cells that received the same treatment and that belonged to the same nest were averaged in order to control for within-nest correlations.
Statistical analysis
All data analyses were performed using SPSS 15.0 for Windows (SPSS 2006).
Means are given with their standard errors. For the field survey, a Chi-square test ( χ2) was used. The effect of mites in the experimental brood cells was tested with a 2-way 38 ANOVA. The data met all the respective assumptions for these two tests. Voucher
specimens of the mites and bees are deposited in the Museo Fairchild, Universidad de
Panamá (MFUP), and the Dry Reference Collection of the Smithsonian Tropical
Research Institute (STRI).
RESULTS
Field Observations
Nests of M. genalis and M. ecuadoria were often colonized by mites carried by
bees ( M. genalis 63%, N=88; M. ecuadoria 17%, N=79). The number of mites per cell
depended on the instar of both bee brood and mites, but it ranged from 0 to 25, with an
average of 8.3 ± 5.8, N=35 tritonymphs per cell. Mites were more common in nests of M.
genalis than in nests of M. ecuadoria (Fig. 4.4 A). During four field surveys in 2005,
2006, and 2007, spanning both wet and dry seasons, we observed a significant correlation
between the presence of mites and the absence of fungi growing inside the brood cells of
both M. genalis and M. ecuadoria ’s nests (Fig. 4.4 A, B &C). This provides support for
the first prediction that the incidence of contaminant fungal growth is lower in cells with
mites than in those without them.
There was no significant association between presence of mites and sex of brood
(χ2=0.838, p=0.6577, N=25; χ2=1.311, p=0.2522, N=13; χ2=0.009, p=0.9229, N=97;
χ2=1.543, p=0.2142, N=54, respectively, for the seasons mentioned in Fig. 4.4 A). Both male and female brood therefore become equally infested by mites.
39 Although most brood are successfully reared in the absence of mites, there was a significant association between healthy bees (i.e. bees that reach adulthood and are not infected by fungi) and the presence of mites ( χ2=5.665, p=0.0173, N=283). A total of 13 bees died out of 178 bees growing in the absence of mites, while only one pupa died out of 105 bees in association with mites (the nest with the dead pupa had been abandoned by the resident adults and the brood cell was broken, so it is likely that other factors were responsible for the death of this pupa). This finding implies that the mites serve some sanitary function, supporting the second prediction of healthier bees when mites are present (Fig. 4.1 C&D).
Experimental brood cell test
The effect of mites on the incidence of fungal growth in cells was tested by removing or adding mites to cells that naturally did or did not have mites, respectively.
The control cells that naturally had mites throughout the experiment yielded an average of 130.6 ± 26.5, N=10 fungal colonies (CFU). Cells that naturally did not have mites but had them added yielded an average of 190.6 ± 26.4 CFU, N=11. In contrast, cells that never had mites had an average of 224.4 ± 25.6 CFU, N=12, and cells that naturally had mites but had them removed had an average of 230.1 ± 16.3 CFU, N=17 (Fig. 4.5).
A two-way ANOVA assessed the effect of the presence of mites before and after experimental manipulation on the number of fungal colonies. Cells that always had mites or had mites added later, have a significantly lower number of CFU than cells that never
40 had mites or had mites removed (F 1,46 =8.227, p=0.006). There was no significant effect on the CFU counts due to the handling of the cells (F 1,46 =1.366, p=0.249), and no interaction between the presence/absence of mites at the beginning of the experiment and at the end (F 1,46 =1.994, p=0.165).
The effect size ( η2p) is 0.152, which means that most of the variation can be explained by the treatments, and that the size of the difference between means is moderate to large. The power is about 80% and so it is reasonable to conclude that there are biologically real differences between the means.
DISCUSSION
Our study provides the first rigorous demonstration of the repeatedly hypothesized cleaning mutualism between mites and bees. A single natural history account suggested that stingless bees benefit from the presence of mites inhabiting their nests because the latter clean the nest of fungal contaminants, but this account presented neither methodological details nor statistical analyses (Flechtmann & Camargo 1974). In contrast, our tests provide clear evidence of the cleaning effect of mites in nests of
Megalopta bees. Laboratory results demonstrate that there are fewer fungal colonies
(CFU) in cells with mites than in cells without mites. This outcome mirrors the observational data from natural nests of both M. genalis and M. ecuadoria . Experimental manipulation of mite load further confirms that the absence or presence of mites is a causal factor in the growth of fungi (or not) in brood cells. It is unclear why M. genalis has generally higher mite loads compared to M. ecuadoria . This difference might be due 41 to the incipience of the mutualism or perhaps M. genalis spreads mites more effectively between nests. Future studies should address the ecological and species-specific reasons for the species differences in mite load between M. genalis and M. ecuadoria .
Symbiotic relationships range through a wide continuum from parasitic to mutualistic, as well as from diffuse to highly coevolved associations between pairs of species (Herre et al. 1999). Of these relationships, mutualisms are widespread, and some
are thought to be one of the driving forces of diversification and stability in complex
ecological systems (Boucher et al. 1982; Leigh 1991; Bronstein et al. 2006). The fact that
not all the nests were colonized by mites, and that brood can be reared successfully even
in the absence of mites, may indicate that this bee-mite relationship is a facultative
mutualism, at least from the bee perspective. However, as far as it is known, these mites
are obligate bee-associates (Eickwort 1966), and it remains unclear if they can survive in
favorable habitats outside of bee nests, such as flowers.
Host sex preference has evolved in some mites associated with hymenopterans
(Cowan 1984; Hunter & Rosario 1988; Dawicke et al. 1992). For instance, some parasitic
mites are found preferentially on female hosts, presumably to disperse to new nests,
while others are found in males, and are transferred to females during copulation
(OConnor 1982; Cowan 1984). Other bee mites do not exhibit such sex-based
associations (Cross & Bohart 1969; OConnor 1982; Houston 1987; Cross & Bohart 1992;
Okabe & Makino 2003). Our results show that cells with male brood are as equally likely
to have mites as are those cells that produce females. This prompts the question of the
mode of transmission of the mites associated with Megalopta . Based on the ontogeny of 42 the mites relative to that of the bees (Biani et al., unpublished data), we assume that bees
are infested by mites in their natal nests during development. Nothing is known about
how mites are transferred from males to females. Since only females potentially found
new nests, mites developing in males brood cells would constitute an evolutionary dead
end unless there is a mechanism that allows their transference to females. The mating
behavior of Megalopta is unknown, but venereal transmission during mating is possible, as occurs in mites associated with an eumenine wasp (Cooper 1955; Cowan 1984).
Alternatively, both male and female Megalopta remain in the natal nest for up to 10 days after they eclose (Wcislo & Gonzalez 2006), a period that could constitute an opportunity for mites to transfer from males to females. Furthermore, M. genalis and M. ecuadoria are socially flexible species, meaning that some newly emerged females might disperse to found new nests, while others will remain in their natal nest to become part of a social unit. Hence, it is possible that mites attached to dispersing females will be developing a new generation in a brand new nest, whereas mites attached to daughter bees remaining in their natal nest might colonize new brood cells in the same nest.
Mites are present in the nests of bees from diverse families, including Halictidae,
Megachilidae, and Apidae; many taxa possess pouch-like structures (acarinaria) that localize mites to certain body regions, and museum specimens often house numerous mites (Batra 1965; Eickwort 1966; Cross & Bohart 1969; Eickwort 1979; Mc Ginley
1986; Houston 1987; Eickwort 1990; Cross & Bohart 1992; Eickwort 1993; Okabe &
Makino 2002, 2005). Yet ecological and behavioral studies of bee-mite symbioses are scarce (Woodring 1973; Rack & Eickwort 1979; Houston 1987; Cross & Bohart 1992; 43 Walter et al. 2002; Okabe & Makino 2008). Acarinaria were interpreted as structures that evolved to transport beneficial mites. However, due to the dearth of examples of such beneficial interactions, this notion has recently been challenged by an alternative hypothesis that the acarinarium is a defense mechanism against harmful mites because it decreases the likelihood that mites will leave any given host and thus infest other cells in the same nests (Klimov et al. 2007). The evolution of these morphological structures needs to be examined in more depth, for example by exploring the percentage of species where acarinaria are exclusively a female attribute (Okabe & Makino 2002). If bees are carrying harmful mites in the acarinaria, there would be strong sexual selection against males with these structures.
Coevolutionary interactions between wild bees and mites have been largely neglected, except for a few notable examples (Rack & Eickwort 1979; Houston 1987;
Cross & Bohart 1992). Future studies need to investigate experimentally the nature of the relationships based on fitness advantages for each of the players; identify the mode of transmission and behavioral repertoires leading towards the association; and examine phylogenetic convergences or ecological constraints that may be affecting the system.
The mechanisms that curb fungal growth in brood cells need to be further investigated, as our study did not address whether the mites are eating fungal spores or hyphae, or if they might be secreting biochemical substances with fungistatic activity. This study is the first experimental demonstration of the mutualistic nature of a bee-mite association, and one of the few examples of terrestrial cleaning mutualisms.
44 ACKNOWLEDGMENTS
This work was supported by the Smithsonian Tropical Research Institute (STRI)
Short Term Fellowships as well as research grants from the Program of Ecology,
Evolution and Behavior of the University of Texas at Austin to NBB. Additional support came from general research funds from STRI to WTW; support for an intern came from
STRI’s Office of Education. We are extremely grateful to Juan Carlos Santos for his statistical advice and help with figures; Andre Rodrígues for his suggestions on microbiology methods; Adam Smith, Simon Tierney and Therany Gonzales for their invaluable help and company during nest collections; Hermógenes Fernández-Marín for logistic help in the lab; Hans Klompen and Jon Shik for their help in the identification of the mite species; Barrett Klein, Christian Rabeling, Rachelle Adams, Alexander
Mikheyev and Heather Ishak for their comments on earlier versions of the manuscript.
Dr. Gard Otis and an anonymous reviewer offered very helpful suggestions. We thank
STRI staff for logistical support. The Autoridad Nacional del Ambiente de la República de Panamá provided collecting and export permits. And of course, we thank the bees and the mites.
45 Figure 4.1: Megalopta nest structure and cleaning effects of the mites.
A. Section through nest in dead branch. Brood cells are constructed out of chewed wood. B.
Nest entrance. C. Brood cell completely overgrown with fungi. D. A dead pre-pupa with fungi growing on the bee feces.
46 Figure 4.2: Deutonymphs (dipersal stage of the mites) on thorax and metasoma of Megalopta pupa.
Figure 4.3: Artificial cell with a bee larva.
Artificial cells are made of paraffin, a cover slip, and modeling clay. Natural cells can be transferred into these artificial cells to allow for observation and rearing of bee brood.
47 Figure 4.4 A. Frequency distribution of brood cells with or without mites (M) and/or
fungi (F) in M. genalis and M. ecuadoria for all four field seasons.
(X2* p<0.05, ** p<0.001). In M. genalis mites are more abundant and fungi are scarce, while in M. ecuadoria , mites are less abundant and fungi are more pervasive. The negative relationship between these organisms holds for within species associations. B. & C. Brood cells with and without mites, respectively. Both cells are of approximately the same age. The pellets in the cells are feces deposited by the bee larvae. Cells with mites tend to be fungus-free (B), whereas cells without mites show significant fungal growth (C) that may harm the developing bee brood. Cell diameters are on average 8 mm.
48 Figure 4.5. Means ± 2 Standard Errors of Colony Forming Units in the four experimental treatments.
“Control with Mites” are cells that naturally had mites. Cells that did not have mites received them experimentally. “Control without Mites” are cells that naturally did not have mites throughout the experiment, and other cells had mites experimentally removed. (Two-way ANOVA, F 1,46 =8.227, p=0.006).
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62 Vita
Natalia Beatriz Biani was born in Rosario, Argentina, on September 28 th 1972. She is one of the daughters of Ines Cavo and Ricardo Biani. She has three wonderful siblings. During her childhood she lived in several countryside places of Brazil and Northern Argentina, which together with her parents’ influence, inspired her love for nature. She is married to Juan Carlos Santos and the fruit of their love gave origin to Ignacio, their adorable son. She graduated from the National University of Rosario where she earned an Agricultural Engineering degree in 1997. She worked as a teaching assistant in the Botany department at that university and as a technical promoter in small family farms in Rosario. Later she earned a Master in Conservation Biology in the International University of Andalucia in Spain. While doing her master’s field work in Argentina, she was marveled by the amazing interactions among ants and plants, and decided to pursue a PhD. In the fall of 2002 she entered the Program of Ecology, Evolution and Behavior at the University of Texas at Austin under the co-supervision of Dr. Ulrich Mueller and Dr. Larry Gilbert. She was a teaching assistant for many courses such as Principles of Animal Behavior, Biostatistics, Genetics and Genetics Lab, and Introductory Biology courses. She also participated in the program of Supplemental Instruction which is a nationally recognized, assistance program aimed at improving student performance and increasing retention. She conducted her doctorate field work in Panama, but Ecuadorian and Costa Rican tropical forests were stirring sources of wonder.
Permanent address: San Juan 2906, Rosario, Santa Fe, Argentina.
This dissertation was typed by the author.
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