The Ecology and Evolution of Cycads and Their Symbionts

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Citation Salzman, Shayla. 2019. The Ecology and Evolution of Cycads and Their Symbionts. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42013055

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA The ecology and evolution of cycads and their symbionts

ADISSERTATIONPRESENTED BY SHAYLA SALZMAN TO THE DEPARTMENT OF ORGANISMIC AND EVOLUTIONARY BIOLOGY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE SUBJECT OF BIOLOGY

The ecology and evolution of cycads and their symbionts

ABSTRACT

Interactions among species are responsible for generating much of the biodiversity that we see today, yet coevolved associations with high species speciﬁcity are rare in nature and have sometimes been considered to be evolutionary dead ends. The plant order

Cycadales is among the most ancient lineages of seed plants, and the tissues of all species are highly toxic. Cycads exhibit many specialized interactions, making them ideal for analyzing the causes and consequences of symbiotic relationships. In Chapter

1, I characterize the pollination mutualism between Zamia furfuracea cycads and their

Rhopalotria furfuracea weevil pollinators. I find that pollination in this New World species pair closely mirrors that of an Old World cycad and its thrips pollinators, and that this strict, pair-wise interaction is ancestral to the group. Molecular phylogenetics and fossil evidence suggest that it represents one of the earliest insect/plant pollination mechanisms, arising long before the evolution of visual signaling commonly employed by flowering plants. In Chapter 2, I identify systematic patterns in the gut bacteria of cycad herbivores. I survey the gut microbial communities of five insect species feeding on a diversity of tissues of cycads and collected world-wise, and discover a set of core bacteria that is shared amongst cycad herbivores and not found in their non-cycad feeding relatives. Some of these microbes have known anti-cancer and nitrogen-fixing capabilities, and may function to facilitate herbivory of these toxic plants. This is the

ﬁrst report of a core-microbiome amongst distantly related organisms that feed on a common plant family. Finally in Chapter 3, I synthesize all published literature on cycad feeding Lepidoptera and evaluate their diets and ecology in a phylogenetic framework.

Cycad feeding has independently evolved multiple times from angiosperm-feeding ancestors, representing a striking shift in host plant morphology and chemistry. As most cycad specialists are warningly colored and many are known to sequester cycad toxins, this presents an ideal system to further explore potential mechanisms of plant/insect coevolution.

iii “I consider cycads to be the Rosetta Stone for plant biology due the the wealth of information stored within them”

Knut Norstog “Cycads are the coolest plants in the world”

iv Contents

Abstract iii

Acknowledgements vii

Introduction 1

1 An ancient obligate pollination mutualism in cycads 7 1.1 Introduction ...... 7 1.2 Materials and methods ...... 10 1.2.1 Experimental design ...... 10 1.2.2 Study species ...... 10 1.2.3 Volatile collection ...... 11 1.2.4 Gas chromatography mass spectrometry ...... 12 1.2.5 Electrophysiological analysis: GC-EAD ...... 13 1.2.6 Weevil expulsion from pollen cones ...... 14 1.2.7 Behavioral analysis: Pit-tests of 1,3-octadiene concentrations 15 1.2.8 Behavioral analysis: Weevil movement in relation to 1,3- octadiene concentrations ...... 16 1.2.9 Phylogeny ...... 19 1.3 Results ...... 22 1.4 Discussion ...... 30

2 Cycad-feeding insects share a core gut microbiome 32

3 Ecology and Evolution of Cycad-Feeding Lepidoptera 44 3.1 Introduction ...... 44 3.2 Lepidopteran Cycad Herbivores ...... 47

v 3.3 Aposematism and Defensive Ecology ...... 59 3.4 Evolutionary Origins of Cycadivory ...... 64 3.5 Hostplant Use ...... 68 3.6 Management of Cycadivorous Species ...... 72 3.7 Discussion ...... 74 3.8 Conclusions ...... 80

Concluding Remarks 82

A Supplement Chapter 1 85

B Supplement Chapter 2 88

C Supplement Chapter 3 92 C.1 Evaluating hostplant records ...... 92 C.2 Cycadivorous Lepidoptera species synonyms ...... 101

Bibliography 102

vi Acknowledgements

As with a child, it takes a village to raise a PhD thesis and the student completing it. And as such, this work would have been impossible without the kindness, support, and guidance of many people. First and foremost I thank my advisors, Drs. Naomi Pierce and Robin Hopkins. Dr. Pierce has been a guiding force in my personal, professional, and educational life since before even the start of the degree program and in every sense of the word, is truly an excellent mentor. Dr. Hopkins embraced my interest and desire to join her lab group with open arms and has signiﬁcantly guided the scope and direction of this work as with all students she works with. Both Dr. Pierce and Dr. Hopkins have been endlessly supportive of me personally and my independent research program. Together, they have challenged me, encouraged me, and made me laugh. I cannot thank either of them enough for being such excellent examples of scientist, mentors, and human beings. Similarly I thank my thesis committee, Drs. David Haig, Brian Farrell, and Dennis Stevenson. Their perspectives and advice are clearly visible in the following work. I have been so lucky to have such a brilliant and supportive team to work with. Outside of campus, I have had the invaluable support and guidance from Drs. William Tang, Irene Terry, Chelsea Specht, Angélica Cibrián- Jaramillo, and Francisco Barona-Gómez. Drs. Thiago André and Amanda Mor- tati have been more than collaborators and ﬁeld guides, they are both brilliant scientists and mentors, excellent friends, and inspirational examples of human- ity.

vii My office mate and the administrative assistant in the Pierce lab, Maggie Starvish, has been my on campus family. She has kept me sane and laughing as well as kept my research supplies and reimbursements coming. She is always available to discuss all things Harvard, kitty, or moral. She is an integral part of both my time at Harvard and the ability to accomplish research. I am thankful for her presence on a daily basis. Many people assisted in the actual data collection or analysis that deserve extra acknowledgement. Dr. Melissa Whitaker has had her hand in almost every thing I have done and has established herself as not just a collaborator, but a mentor and friend as well. Her guidance has been priceless and life changing. Dr. Damon Crook at the USDA was an off campus collaborator for the entirety of my thesis and provided the opportunity for a complete line of work. Dr. James Crall provided a unique perspective and direction to this thesis. The staff at the Ernst Mayr library of the Museum of Comparative Zoology, especially Mary Sears, deserves thanks for their diligence and detail in hunting down obscure references. Dr. Steven Worthington at the Harvard Institute for Quantitative Social Science assisted with statistical analyses. Most of this work was done at the cycad conservation garden, Montgomery Botanical Center, where their dedicated and knowledgeable staff have helped in endless ways. At Montgomery Botanical Center, I thank Dr. Patrick Griffith, Dr. Michael Calonje, Claudia Calonje, Vicky Murphy, Tracy Magellan, Dr. Joanna Tucker Lima, and Xavier Gratacos for their dedication to both scientific research and to these incredible plants. I thank all the current and former members of the Naomi Pierce and Robin

viii Hopkins labs including Jon Sanders, Lori Shapiro, Leonora Bittleston, Jack Boyle, Chris Baker, Marjorie Lienard, João Tonini, Wei-Ping Chan, Richard Childers, Mark Cornwall, Kadeem Gilbert, Evan Hoki, Zhenyang Wang, Matt Farnitano, Federico Roda, Ben Goulet, Franchesco Molina-Henao, Austin Garner, Henry North, Heather Briggs, Sevan Suni, Callin Switzer, and Diana Bernal-Franco. I speciﬁcally thank Rachel Hawkins of the Pierce lab for her detailed help with museum collections and protocol as well as her endlessly cheerful attitude and persistent intellectual curiosity. Finally, I thank my family. My parents, Randy Salzman, Jeanne Liedtka, De- bra Brown, and Jim Brown have been endlessly supportive of this work both professionally and personally. As have my brothers Quinn Warner and Holland Brown and my extended family, Ginger and Dan Schaible. Most importantly, I thank my husband Rory Maher and daughter Laurel Maher. Rory has supported me and my dreams for almost two decades, providing much needed emotional support, child care, laughter, and stress relief. He is truly my rock and every- thing I do in life I do better because of and with him. Laurel has kept me present and always aware of the most important things in life. They are both a blessing in my life and my work and I am endlessly grateful for them.

ix Dedicated to Rory Maher without whom none of this would have been possible

x Introduction

Relationships are endlessly fascinating to us, and the causes and consequences of organismal interactions have long captivated naturalists. Darwin highlighted the importance of species interactions as agents of natural selection (Darwin, 1859), even writing an entire book on coevolution of orchids and their pollinators (Darwin, 1862). More than a century later, Ehrlich and Raven, 1964 described the coevolution of insects and plants as being responsible for generating the wealth of biodiversity as we know it. Despite their intrigue, strictly coevolved systems that involve a high degree of specialization by both partners are not common in nature and have sometimes been argued to lead to evolutionary dead ends (Vamosi, Armbruster, and Renner, 2014). A considerable body of work, however, suggests that specialized lineages may be able to thrive and diversify under natural selection (Day, Hua, and Bromham, 2016 and see Futuyma and Moreno, 1988). Controversy still exists regarding the potential evolutionary trajectory of these interactions as well as the degree to which they are mediated by third party associations such as other insects or microbes whose roles have been less tractable to study until now. The order Cycadales is among the most ancient lineages of seed plants, with a fossil record dating back over 265 my (Gao and Thomas, 1989). These dioecious gymnosperms dominated the terrestrial landscape (Taylor, Taylor, and

1 Introduction

Krings, 2009) and exhibited specialized insect pollination during the Mesozoic era, well before the rise of ﬂowering plants (Cai et al., 2018). While each of the ten living genera have persisted through geological time, the 350 modern species of cycad are reported to have arisen during synchronous global radiations in each genus ca. 10-15 million years ago (Nagalingum et al., 2011; Salas-Leiva et al., 2013; Condamine et al., 2015). Throughout this time, cycads have main- tained specialized relationships with insects (Cai et al., 2018; Terry et al., 2012a), making it a particularly suitable system in which to address the mechanisms underlying the origin and maintenance of specialized species interactions. The unique nature of toxicity (Vega and Bell, 1967; De Luca et al., 1980; Moretti, Sabato, and Gigliano, 1981; Moretti, Sabato, and Gigliano, 1983) exhibited by these plants may be responsible for both their evolutionary longevity and the specialized nature of their species relationships. All cycads produce two classes of carcinogenic and neurotoxic compounds, BMAA and MAM (Vega and Bell, 1967; De Luca et al., 1980; Moretti, Sabato, and Gigliano, 1981; Moretti, Sabato, and Gigliano, 1983). B-methylamino-L-alanine (BMAA) is a non-protein amino acid that can be misincorporated into proteins, altering their structure and function (Dunlap, Banack, and Rodgers, 2013). BMAA also disrupts glutamate receptor function with toxic effects reported in plants (Brenner et al., 2000), insects (Goto, Koenig, and Ikeda, 2012) and mammals (Spencer et al., 1987; Kisby, Moore, and Spencer, 2013). BMAA is the source of ongoing medi- cal research as it has been shown to produce symptoms similar to amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (Coz, Banack, and Murch, 2003).

2 Introduction

The other class of cycad toxins is methylazoxymethanol (MAM). The carcinogenic and neurotoxic (Morgan and Hoffman, 1983; Laquer and Spatz, 1968) effects of MAM result from its spontaneous degradation into formaldehyde and methyl-diazonium, a potent methylating agent (Morgan and Hoffman, 1983). MAM toxicity has been described in yeast, bacteria, insects, plants, and mammals (Morgan and Hoffman, 1983). The presence of these two potent toxins makes cycad tissue a distinctive food source and only a handful of specialized insects are able to feed on cycad tissues. Indeed, it appears that most species interactions involving cycads are highly specialized. For instance, the parasitic butterfly genus Eumaeus obligately feeds on Zamia cycad leaves and can do great damage to the plants. These butterflies in turn sequester Zamia plant toxins into their own tissues for defense against natural enemies (Rothschild, Nash, and Bell, 1986). Early work done by Norstog, Stevenson, and Niklas, 1986 and Tang, 1987b gave rise to an avalanche of cycad insect pollination studies until the paradigm shifted from a belief in strictly wind pollination for the group (Chamberlain, 1919) to an understanding that the system involved obligate insect pollination (Terry et al., 2012a). Norstog and Fawcett, 1989 first identified the specific and codependent nature of the pollinator relationship by showing that the Rhopalotria weevil pollinators live their entire lifecycle within the pollen cone tissue, staying faithful to their host plant species and even going dormant during the 10 months of the year when the plant does not have a pollen cone. In Chapter 1, I investigate the mechanism of pollination mutualism between

3 Introduction

Rhopalotria furfuracea and Zamia furfuracea first described by Norstog, Steven- son, and Niklas, 1986. Specialized coleopteran pollination has existed in the Cy- cadales since before the rise of flowering plants (Cai et al., 2018), yet the mechanism by which cycads entice pollination services from their coleopteran brood site mutualists remains unknown. I characterize a push-pull pollination mechanism between a New World cycad and their weevil pollinators that mirrors the mechanism between a distantly related Old World cycad and their thrips pollinators (Terry et al., 2007). The behavioral convergence between weevils and thrips, combined with molecular phylogenetic dating and a meta-analysis of thermogenesis and coordinated patterns of volatile attraction and repulsion suggest that a strict pollination mutualism strategy is ancestral in this ancient, dioecious plant group. As such, it represents one of the earliest insect/plant pollination mechanisms, arising long before the evolution of visual floral signaling commonly employed by flowering plants. Chapter 2 was published as an article in the Biological Journal of the Lin- nean Society (Salzman, Whitaker, and Pierce, 2018). In this chapter I identify a third potential level of specialization in the gut bacterial community of cycad feeding insects. I investigate the gut microbial communities of five insect species from across a diversity of cycad genera, feeding tissues, and geography, and uncover a set of core bacteria that is shared amongst cycad herbivores and not found in their non-cycad feeding relatives. The presence of a set of core bactieria, including a bacterium with known anti-cancer and nitrogen-fixing capabilities, suggests that they are important in helping cycad herbivores detoxify their poisonous host plants. This is the first reporting of a set of core bacteria

4 Introduction shared amongst herbivores of a group of phylogenetically and chemically related plants. Chapter 3 is a review of cycad feeding Lepidoptera. Cycads are chemically defended gymnosperms with highly specialized insect associations that include both pollinators and herbivorous parasites. Among parasitic species, the larvae of many moths and butterﬂies (order: Lepidoptera) feed exclusively on cycads, despite the presence of neurotoxic and carcinogenic compounds. The phylogenetic distribution of cycadivorous Lepidoptera suggests that cycad feeding has evolved independently in at least 7 lepidopteran families, with 36 substanti- ated reports of cycadivorous species across 12 genera. All cycad-feeding species appear to have evolved from angiosperm-feeding ancestors, such that shifts to cycads may represent particularly striking changes in phytochemistry relative to host switches within angiosperms, yet very little is known about the biology of these species. This review synthesizes published information about the ecology and evolution of cycadivory among Lepidoptera while highlighting issues relevant for species conservation and management and identifying outstanding questions and areas for future research. It is thanks to the pioneering work of Knut Norstog, Pracilla Fawcett, Den- nis Stevenson, Willie Tang, and Irene Terry that cycad mutualisms have gained attention and my work stands on the backs of these giants. Chapter 1 was done in collaboration with Damon Crook of the USDA who assisted in EAD-FID and GCMS and James Crall at Harvard who assisted with video analysis of weevil behavior. Chapters 2 and 3 were done in close collaboration with Melissa Whitaker while she was a postdoctoral fellow ﬁrst at Harvard and later at ETH

5 Introduction

Zurich. All of the unpublished chapters are written in the format for submission for peer-review publication. I conclude with a few remarks on future directions arising from this work.

6 Chapter 1

An ancient obligate pollination mutualism in cycads

Note: Supplemental materials included in Appendix A.

1.1 Introduction

Highly specialized coevolved systems are uncommon in nature and are sometimes considered evolutionary dead ends (Vamosi, Armbruster, and Renner, 2014). However, the plant order Cycadales is one of the oldest living lineages of seed plants (Chaw et al., 2000) and most species form obligate brood site pollination mutualisms with insects (Terry et al., 2012a; Tang, 2006). Here, we investigate whether unrelated pollination mutualists exhibit matching behavioral responses to conserved plant behaviors representing a shared pollination strategy.

The push-pull pollination mechanism of the Australian cycad, Macrozamia lucida is characterized by a daily peak in plant volatile production whereby thrips pollination mutualists are initially attracted to lower levels of a plant volatile

7 Chapter 1. An ancient obligate pollination mutualism in cycads and later repelled by high levels of the same compound (Terry et al., 2007). Across the Cycadales, reproductive cones undergo a daily cycle of thermogenesis (Tang, 1987a) and respiratory processes that similarly culminate in a peak of volatile production (Terry et al., 2016). By analyzing a distantly related New World cycad species that, like most cycads, is associated with a coleopteran pollination mutualist, we investigate whether this characteristic cycle of thermogenesis and coordinated volatile production is broadly indicative of an ancient and conserved pollination strategy. Specialized insect pollinators, mostly beetles, live and feed solely within the male pollen cone tissue of Cycadales (Terry et al., 2012a; Tang, 2006; Hall et al., 2004; Donaldson, 1997; Norstog and Fawcett, 1989). Successful cycad reproduction requires these brood site mutualists to leave the host pollen cone and trans- fer pollen to a female ovulate cone. Over the course of a day, reproductive cones produce a predictable peak in respiration, followed by a peak in thermogenesis, and finally a peak in volatile production, each separated by tens of minutes (Terry et al., 2016). Both pollen and ovulate cones of an individual species produce similar thermogenic and volatile profiles (Tang, 1987a; Pellmyr et al., 1991; Terry et al., 2004; Azuma and Kono, 2006; Proches and Johnson, 2009), yet across all species, pollen cones produce a higher temperature peak (Tang, 1987a) and more concentrated volatile compounds (Terry et al., 2012a; Pellmyr et al., 1991; Terry et al., 2004; Proches and Johnson, 2009). Pollinator exit from pollen cones has been observed to coincide with thermogenic and volatile peaks (Terry et al., 2007; Pellmyr et al., 1991; Terry, 2001; Azuma and Kono, 2006; Wallenius et al., 2012; Norstog, Stevenson, and Niklas, 1986). The specific behavioral mechanism

8 Chapter 1. An ancient obligate pollination mutualism in cycads by which these brood site mutualists are repelled from their host pollen cone and enticed into ovulate cones has been described in Macrozamia lucida (Terry et al., 2007) where Cycadothrips chadwickii thrips (Order: Thysanoptera) are attracted (“pulled”) by lower concentrations of the dominant plant volatile compound, -myrcene, and repelled (“pushed”) by higher concentrations of the same compound. The daily plant volatile cycle induces pollinator movement between pollen and ovulate cones enabling pollination and seed set (Terry et al., 2007). This push-pull pollination mechanism differs from most pollination syndromes because it has a repulsive component that expels pollinators from pollen cones at a certain point in the cycle in addition to the attractive mechanisms commonly seen in the visual and chemical cues of ﬂowers. Furthermore, the same chemical cue is used for attraction and repulsion.

The Mexican cycad Zamia furfuracea (Zamiacea), which lineage seperated over 150 million years ago from Macrozamia lucida, has an obligate pollination mutualism with Rhopalotria furfuracea (Coleoptera: Belidae) weevils (formally R. mollis) (Norstog, Stevenson, and Niklas, 1986). Rhopalotria furfuracea complete their entire development within the plants’ disposable pollen cone parenchyma tissue (Norstog and Fawcett, 1989) and the plants do not set seed without pollinators (Norstog, Stevenson, and Niklas, 1986). Specialized coleopteran pollination is widespread in extant Cycadales (Terry et al., 2012a; Tang, 2006) and has existed in the lineage since the mid-Mesozoic (Cai et al., 2018), well before the rise of ﬂowering plants (Niklas, Tiffney, and Knoll, 1983) or the ﬁrst evidence of possible thrips pollination (Peñalver et al., 2012). While push-pull pollination has also been hypothesized for Coleoptera-pollinated cycads (Valencia-Montoya

9 Chapter 1. An ancient obligate pollination mutualism in cycads et al., 2017; Suinyuy, Donaldson, and Johnson, 2013), the mechanism by which the plants manipulate their behavior to carry out pollination has not yet been explored.

1.2 Materials and methods

1.2.1 Experimental design

The purpose of this study was to determine the insect and plant behaviors responsible for the speciﬁc brood-site pollination mutualism seen in Zamia furfuracea cycads and their weevil pollinators, Rhopalotria furfuracea, and to contrast that with the behaviors seen in other cycad pollination systems, including Macrozamia lucida cycads and their thrips pollinators, Cycadothrips chadwickii (Terry et al., 2007). Plant volatile analysis was used to identify the volatile components produced by pollen and ovulate cones. Physiological analysis of R. furfuracea weevils was used to identify the electro-antennally active plant compounds, and behavioral responses of R. furfuracea to plant compounds. Field experiments matched plant volatiles corresponding to weevil behavior in order to conﬁrm laboratory experiments. Finally, thermogenic and volatile patterns across cycad genera were mapped onto a dated phylogeny to provide an evolutionary context.

1.2.2 Study species

Zamia furfuracea is an endangered dioecious gymnosperm native to Veracruz, Mexico (Calonje, Stevenson, and Osborne, 2019). Both pollen and ovulate cones

10 Chapter 1. An ancient obligate pollination mutualism in cycads are known to undergo a daily synchronous thermogenic peak at the time that they are reproductive, with a higher temperature spike in pollen cones (2.5 C above ambient, ovulate cones 0.8 C above ambient) (Tang, 1987a). Pollen cones produce two main volatile components, the hydrocarbon 1,3-octadiene and the alcohol linalool (Pellmyr et al., 1991).

Rhopalotria furfuracea is an obligate pollination mutualist with Z. furfuracea (Norstog and Fawcett, 1989; Norstog, Stevenson, and Niklas, 1986). It is required for Z. furfuracea reproduction (Norstog, Stevenson, and Niklas, 1986) and its entire lifecycle is tied to that of its host plant (Norstog and Fawcett, 1989). R. fufurfuracea adults feed, breed, and lay eggs in Z. furfuracea pollen cones and larvae feed and develop inside of microsporphylls, pupating inside of the microsporophyll stalk (Norstog and Fawcett, 1989). R. furfuracea visit ovulate cones of Z. furfuracea but do not feed or lay eggs on them. Late instar larvae go into diapause for 10 months out of the year when plants are not reproductive, remaining inside of the stalks of the dried, dead microsporphylls (Norstog and Fawcett, 1989).

1.2.3 Volatile collection

Zamia furfuracea plant volatiles collections were performed using headspace collection methods (Schiestl and Marion-Poll, 2002) on three pollen dehiscing male cones and two receptive female cones in situ. Accession information for all plants is found in Table 1.1. Cones remained attached to the plant and covered in oven bags (Reynolds Consumer Products, Lake Forest IL) that were tied shut at the bottom of the cone. Low-ﬂow vacuum air samplers (Gilian model

11 Chapter 1. An ancient obligate pollination mutualism in cycads

LFS-113DC) calibrated to 0.1 L/min pulled headspace air and volatiles for 45 min through filters made with 300 mg Porapak Q adsorbant mesh 80-100 75 cc (Sigma Aldrich, Saint Louis MO, part number 20331) held into glass pastuer pipettes size 5.75” (VWR International, Randor PA, part number 14672-200) with plugs of glass wool (Sigma Aldrich, Saint Louis MO, part number 20411). Prior to volatile collection, filters were soaked in HPLC grade dichlormethane (Sigma Aldrich, Saint Louis MO, part number 650463) for 48 hours and then prewashed ten times with full flow throughs of dichloromethane and dried with charcoal purified air pushed through at 0.4 L/min. After volatile collection, samples were eluted with 500 ul HPLC grade dicholomethane (Sigma Aldrich, Saint Louis MO, part number 650463) pushed through with charcoal purified air.

TABLE 1.1: Zamia furfuracea cones used for volatile analysis. Acces- sion is living plant identiﬁcation number at Montgomery Botanical Center in Miami Florida, USA.

Cone sex Accession Notes Pollen 981474*A Fully open, releasing pollen Pollen 20010214*M Fully open, releasing pollen Pollen 981743*A Fully open, releasing pollen Ovulate 951468*A Receptive, open at top Ovulate 20010214*D Receptive, open at top

1.2.4 Gas chromatography mass spectrometry

Initial chemical analyses were conducted using a combined Agilent Technolo- gies 6890 network gas chromatograph and 5973 mass-selective detector. The GC

12 Chapter 1. An ancient obligate pollination mutualism in cycads was equipped with a DB-5 column (J W Scientific Inc., Folsom CA); 30 m x 0.25 mm I.D.; film thickness, 0.25 m; splitless mode). Helium was the carrier gas at a constant flow rate of 0.7 ml/min. The injector temperature was 250 C. Oven temperature was held at 40 C for 1 min, programmed to 170 C at 10 C/min and held for 5 min. Volatile peaks were manually integrated. Volatiles were identified based on their mass spectra (NIST version 2.0,2002), Kovats indices (Piji, 1960; Kovats, 1965) and by comparison of the retention indices and mass spectra with those of available authentic synthetic compounds and a computer- ized data library (NIST version 2.0,2002). An authentic standard of 1,3-octadiene was obtained from ChemSampco Co. (Dallas, TX, Catalog number 7015.90). Au- thentic standards of (+/-) linalool were obtained from Sigma Aldrich Co. (Saint Louis, MO, Catalog number L2602-5G). Differences in volatile production between pollen and ovulate cones was determined using a weighted least squares model to account for the variation between groups. Similarly, differences in 1,3- octadiene increases over time in pollen cones was determined using a weighted least squares model to account for the variation between groups.

1.2.5 Electrophysiological analysis: GC-EAD

The physiological response of Rhoplaotria furfuracea weevils to host plant Za- mia furfuracea volatile compounds was determined using Gas Chromatography - Electroantenograph Detection (GC-EAD). The coupled GC-EAD system used was as previously described by Crook et al., 2008 with a few modiﬁcations. Sam- ples of aerations or standards were injected (two microliters), splitless, onto a

13 Chapter 1. An ancient obligate pollination mutualism in cycads

Hewlett Packard (Agilent Technologies, Santa Clara CA) 6890 gas chromatograph with a DB-5MS-DG column (J W Scientific Inc., Folsom CA, 0 m x 0.25 mm ID, 0.25 m film thickness) and a 1:1 effluent splitter that allowed simultane- ous FID and EAD detection of the separated volatile compounds. Helium was the carrier gas (2.5 ml/min). Oven temperature was held at 50 C for 1 min, programmed to 280 C at 10 C/min and held for 15 min. Injector temperature was

280 C. The GC outlets for the EAD and FID were 300 C. The column outlet for the EAD was held in a water-cooled humidified air stream (20 C) flowing at 20 ml/min over the antennal preparation of adult Rhopalotria furfuracea attached to an EAG probe (Syntech, Hilversum, the Netherlands). Whole heads of an adult beetle were removed so that both antennae could be used for recording. Tips of both antennae were cut off to make a clean opening for conducting gel (Spectra 360, Parker Laboratories, Fairfield, NJ) to form an uninterrupted con- nection to the EAG probe. The EAG probe was connected to an IDAC-232 serial data acquisition controller (Syntech). Signals were stored and analyzed on a PC equipped with the program EAD (version 2.6, Syntech). GC-EADs using whole

Zamia furfuracea plant volatile collections were performed on 6 males and 4 females and GC-EADs of the 1,3-octadiene standard were performed on 3 males and 3 females.

1.2.6 Weevil expulsion from pollen cones

Pollen dehiscing male cones were collected and videotaped throughout the course of the day in conjunction with hourly plant volatile collections to determine the correlation between plant volatile emission and weevil expulsion from male

14 Chapter 1. An ancient obligate pollination mutualism in cycads cones. Cones were placed into clear plastic containers 20.5 cm x 17 cm x 17 cm. One square side of the container was mesh to allow airﬂow and volatile dissipation and the cone was placed closest to this side. Cones used in video analysis were the same stage as those used for volatile collection, open and releasing pollen. These boxes were placed on top of a white sheet of paper set on a clear plastic sheet that was suspended over a 29.6 cm x 29.6 cm LED light panel (BK3301, US Solid State LLC, Shreveport, LA). This entire set up was covered with black cloth so that the only source of light was the even backlite LED panel. Cones were videotaped for 11 hours using a Sony Handycam HDR-CX260V. Weevil expulsion from the cone was manually counted at 30-minute intervals from the resulting videos.

1.2.7 Behavioral analysis: Pit-tests of 1,3-octadiene concentra-

tions

The behavioral response of Rhopalotria furfuracea weevils to the Zamia furfuracea plant volatile compound 1,3-octadiene was determined in pit-tests (Fig. A.1) using differing amounts of the compound along with a control of dicholomethane carrier solvent. Pits were constructed of 100x15 mm petri dishes with 1.5 cm circular holes cut into them and hot glued onto 5 cm deep plastic cups. These were set into larger glass cups so that all dishes were completely level. 10 ul of 1,3-octadiene standards (1 ng/ul, 10 ng/ul, 100 ng/ul, 1ug/ul, 10 ug/ul and 65.5 ug/ul) were applied to 1 cm by 4 cm pieces of ﬁlter paper (Whatman plc number 4, GE Healthcare Life Sciences, Chicago IL) handled with forceps and dropped into the pits. For each trial 1,3-octadiene concentrations and control

15 Chapter 1. An ancient obligate pollination mutualism in cycads were run simultaneously. R. furfuracea weevils were taken directly from Z. furfuracea pollen cones so as to be well fed. Five to ten weevils were placed at the edge of the petri dish which was then closed and placed in the dark at room temperature (22 C) for 30 minutes, at which time the number of weevils inside of the pits was counted and transformed into proportions (weevils in pits/total weevils). A likelihood ratio test was used to determine whether variances between the groups should be accounted for. No signiﬁcant difference was found between a weighted least squares model that allowed for different variances between groups and an ordinary least squares model (p=0.1044). Therefore, the ordinary least squares model with fewer parameters was used to determine sig- niﬁcant differences in proportions of weevils attracted to baits between the different amounts of 1,3-octadiene.

1.2.8 Behavioral analysis: Weevil movement in relation to 1,3-

octadiene concentrations

To determine whether the high concentration of 1,3-octadiene acts as a repel- lant, arenas were constructed in which 80-100 Rhopalotria furfuracea weevils were introduced to either 10,000 ng or 650,000 ng 1,3-octadiene and recorded for 30 minutes. Arenas were constructed of white acrylic sides and bottom with a clear acrylic top (Figure A.2). Dimensions were 14.5 cm x 9.5 cm and 3.5 cm tall. In order to allow airﬂow and the dissipation of volatile compounds, windows were cut into the sides and covered with white mesh fabric (13.5cm x 1 cm holes in the long sides and 8.2 cm x 1 cm holes in the short sides). Openings 2 cm x 5 cm were cut in the bottom of the arena for sample and control. These were covered with

16 Chapter 1. An ancient obligate pollination mutualism in cycads

fine white mesh fabric from the bottom. 10 ul of the 1,3-octadiene concentration was placed onto 1 cm x 4 cm filter paper (Whatman plc number 4, GE Healthcare Life Sciences, Chicago IL) and set under the opening in the bottom of the arena. A blank filter paper piece was placed under the other opening as a control. This entire setup was placed on a clear acrylic panel suspended over a 29.6 cm x 29.6 cm LED light panel (number BK3301, US Solid State LLC, Shreveport, LA) and encased in a black box covered with black fabric so as to be backlit. Weevils were allowed to feed freely on Z. furfuracea pollen cones prior to trials so as to be well fed. Videos were recorded using a Sony Handycam HDR-CX260V. Raw videos were converted from AVCHD to .mov, then processed in Matlab. The locations of the outer arena edges, and the left and right bait locations were manually located for each trial using custom scripts. Next, a background image was computed separately for each trial by calculating the median intensity at each pixel. This median-averaging method meant that weevils that were immo- bile are considered part of the background and excluded from further analysis. For each trial, we analyzed a subset of 4000 frames. For each analyzed frame, weevils were separated from the background by subtracting the frame’s pixel intensity values from the computed background image. This intensity-differential image was then turned into a binary map of weevils using an intensity thresh- old, which was subsequently digitally eroded and dilated to remove digital noise. After this segmentation step, image regions that were within a size thresh- old (i.e., between 150 and 800 pixels, manually calibrated) and located within the arena were considered separate weevils. For each identified weevil, distance was calculated to the nearest point on each bait. If weevils were located within

17 Chapter 1. An ancient obligate pollination mutualism in cycads the edges of the baits, the distance to that bait was set to zero. For each sampled frame, a mean distance to bait and to control was calculated across all the weevils tracked for that frame. Weevil movement in relation to 1,3-octadiene amount was determined by the change in asymmetry towards the bait (distance to bait box – distance to control control) from the start of the trial to the end. Rhopalotria furfuracea often play dead when startled and take upwards of 10 minutes to begin behaving again. Therefore, the ‘starting’ asymmetry towards bait was determined for each trial by subtracting the average distance to the control from the average distance to the bait over the ﬁrst 15 minutes of video (frame < 3000). Weevils were given 10 minutes to make a choice and the ‘ending’ asymmetry towards bait was determined beginning at 25 minutes (frame > 5000). Autocorrelation of the change in asymmetry was analyzed using the acf function in R separately for each trial. We then computed the average autocorrelation function, which showed a substantial diminishment in autocorrelation (correlation < 0.1) at a lag of 500 of the sampled frames. We then subsampled our raw tracking data over 500 sampled frames (or roughly one frame every 2.5 minutes) to minimize the impacts of autocorrelation. The ‘starting’ asymmetry towards bait value for the trial was then subtracted from the ‘ending’ asymmetry values of the subsampled frames. A likelihood ratio test was used to determine if variances between the groups should be accounted for, and no signiﬁcant difference was found between a weighted least squares model that allowed for different variances between groups and an ordinary least squares model (p=0.402). Therefore, the ordinary least

18 Chapter 1. An ancient obligate pollination mutualism in cycads squares model with fewer parameters was used to determine the signiﬁcance of change in asymmetry towards bait for the two amounts of 1,3-octadiene.

1.2.9 Phylogeny

Dated molecular phylogenetic analyses have been performed in the Cycadales with conﬂicting results (Condamine et al., 2015; Salas-Leiva et al., 2013; Na- galingum et al., 2011). The data set from Salas-Leiva et al., 2013 was used to construct a dated phylogeny using the methods and ‘traditional’ fossil calibrations suggested in Condamine et al., 2015. The Salas-Levia et al. data set was chosen over Condamine et al. due to its use of markers from more gene regions (5 verses 3) and the completeness of its matrix (100% gene coverage for all taxa versus 51% coverage). The two data sets were not combined because the represented taxa did not overlap completely and preference was given to a complete data matrix. Molecular dating was performed using BEAST v 1.10.0 (Suchard et al., 2018). Fossil calibrations are based on Hermsen et al., 2006 and were deﬁned as uniform priors as follows: stem node of Bowenia (lower=33.9, upper = 265.1 million years (MY)), stem node of Lepidozamia (lower= 33.9, upper =265.1 MY), stem node of Dioon (lower = 56, upper = 265.1 MY). Methods followed Con- damine et al., 2015 except for an unpartitioned analysis and the use of a random starting tree and a random local clock model for determining tree likelihood as suggested by Salas-Leiva et al., 2013. Literature used to determine the presence of pollinators and thermogenic and volatile patterning can be found in Table 1.2.

19 Chapter 1. An ancient obligate pollination mutualism in cycads

TABLE 1.2: Sources for pollinators and daily patterns of thermogenesis and volatile expression across the Cycadales. *Thermoge- nesis has been tested twice in Stangeria eriopus. Tang, 1987a found a very small thermogenic peak at 4:30 hrs. Proches and Johnson, 2009 did not take temperatures overnight and provide only one example of their temperature measurements in supplemental material that shows a small peak in late afternoon. **Suinyuy, Donald- son, and Johnson, 2013 ﬁnds a signiﬁcant pattern in volatile emissions in one population but not in another of Encephalartos villosus.

20 Chapter 1. An ancient obligate pollination mutualism in cycads This ** study 2013 2010 2012 2012 2018 2018 * 2018 2018 1987a 1987a 1987a 1987a 1987a 1987a 1987a 1987a 1987a 1987a * Suinyuy et al., Wallenius et al., Tang, Tang, Tang, Tang, Tang, Tang, Tang, Tang, Tang, Tang, Tang, 2009 Terry et al., 2007 Xaba and Donaldson, Suinyuy and Johnson, Suinyuy, Donaldson, and Johnson, Suinyuy, Donaldson, and Johnson, Terry et al., 2009 2015 1986 Proches and Johnson, 1999 2017 2005 2011a 1993 2007 2000 1997 2005 2009 1997 2018 2018 2018 2018 2004 1991 2002 2001 1987b 41 Tang et al., Terry et al., Terry et al., Farrera et al., Kono and Tobe, Skchez-Rotonda, Marler and Niklas, Norstog, Stevenson, and Niklas, BelidaeBelidae Tang, Tang et al., ErotylidaeErotylidaeErotylidae Hall et al., Chavez and Genaro, Erotylidae Tang et al., Erotylidae Suinyuy, Donaldson, and Johnson, ErotylidaeErotylidae Cosmopterigidae Valencia-Montoya et al., Tang et al., Vovides, Boganiidae Donaldson, Nitidulidae Nitidulidae Qian et al., Languriidae Thysanoptera CurculionidaeCurculionidae Terry, Suinyuy, Donaldson, and Johnson, Curculionidae Tang, Oberprieler, and Yang, Curcurlionidae Wilson, Cycas Dioon Zamia genus pollinators pollinators Pollinator Thermogenic Volatile Cycad Coleoptera Other Citations Bowenia Stangeria Microcycas Macrozamia Ceratozamia Lepidozamia Encephalartos

21 Chapter 1. An ancient obligate pollination mutualism in cycads

1.3 Results

We identify the chemical communication mechanism underlying the mutualism between R. furfuracea and Z. furfuracea. Using head-space collection methods, plant volatile compounds were collected from Z. furfuracea pollen and ovulate cones to determine the plant volatile proﬁle and the physiological response of

R. furfuracea to host plant volatiles. Zamia furfuracea pollen and ovulate cones produce two major compounds: the hydrocarbon 1,3-octadiene and the alcohol linalool (Figure 1.1). Electroantenograph detection demonstrated that Rhopalo- tria furfuracea male and female weevils are physiologically capable of perceiving only 1,3-octadiene (Figure 1.1, n=10), explaining why prior attempts to elicit behavioral responses in R. furfuracea using linalool were unsuccessful (Pellmyr et al., 1991).

22 Chapter 1. An ancient obligate pollination mutualism in cycads

FIGURE 1.1: Zamia furfuracea produce two main volatile components, 1,3-octadiene and linalool, yet Rhopalotria furfuracea only physiologically respond to 1,3-octadiene. Gas chromatography- ﬂame ionization detector (GC-FID) of Z. furfuracea plant pollen cone volatiles is shown at the top with R. furfuracea weevil electroantenograph detection (EAD) on the bottom. Scale bar is 1 mV for EAD channel. Red arrow denotes a positive physiological response to 1,3-octadiene that is not seen for linalool (n=10). Weevil response to 1,3-octadiene was conﬁrmed with a standard (n=6).

Because a physiological response does not equate with attraction, we analyzed the behavioral response of R. furfuracea to different amounts of 1,3-octadine. Using pit-tests (Figure A.1, Figure 1.2.A inset), we show that R. furfuracea are positively attracted to 1,3-octadiene and that weevil attraction to increasing amounts of 1,3-octadiene is nonlinear (Figure 1.2.A). Weevils are more attracted to the intermediate amount of 10,000 ng 1,3-octadiene than to all lower quantities (p<0.012

23 Chapter 1. An ancient obligate pollination mutualism in cycads for all lower amounts, Table 1.3) as well as to the highest quantity (p=0.0102 for 650,000 ng). The decrease in attraction suggests but does not show that weevils are repelled (i.e. “pushed”) by high concentrations.

TABLE 1.3: Sequential Bonferonni adjusted p-values for the proportion of weevils attracted to pits containing different amounts of 1,3-octadiene. 10,000 ng 1,3-octadiene is signiﬁcantly more attractive than all amounts except for 100,000 ng. The highest amount, 650,000 ng 1,3-octadiene, is not signiﬁcantly different from any amount except the most attract amount, 10,000 ng.

ng 1,3-octadiene 10 100 1,000 10,000 1e+5 6.5e+5 0 1.0 1.0 1.0 0.0044 0.4994 1.0 10 1.0 1.0 0.0087 0.6635 1.0 100 1.0 0.0051 0.4994 1.0 1,000 0.0110 0.7492 1.0 1e+5 1.0 6.5+e5 0.0102 0.6635

We therefore tested whether intermediate (10,000 ng) and high (650,000 ng) quantities of 1,3-octadiene elicited different movement responses in weevils. The push-pull hypothesis predicts that weevils should move towards 10,000 ng 1,3-octadiene and move away from 650,000 ng 1,3-octadiene. We set up enclosed arenas (Figure A.2, Figure 1.2.B inset) with 80 weevils, exposing them to either 10,000 ng of 1,3-octadiene plus a control, or 650,000 ng plus a control, and videotaped their movements over a period of 30 minutes. We measured the change in asymmetry towards the 1,3-octadiene bait (distance from bait box – distance from control box) from the beginning of the video to the end, and determined

24 Chapter 1. An ancient obligate pollination mutualism in cycads that the change in asymmetry is signiﬁcantly different between the two concentrations (p=0.0056). As predicted, Rhopalotria furfuracea move towards 10,000 ng 1,3-octadiene, but move away from 650,000 ng 1,3-octadiene (Figure 1.2.B). To- gether, these experiments demonstrate that weevil attraction preference is for an intermediate amount of 1,3-octadiene, and that their movement can be induced by varying the quantities of plant compounds, consistent with the push-pull pollination observed in Macrozamia lucida (Terry et al., 2007).

25 Chapter 1. An ancient obligate pollination mutualism in cycads

A. Weevil Attraction to 1,3−Octadiene

0.6

0.4 x e action Ind r

Att 0.2

0.0

0 10 100 1,000 10,000 100,000 650,000

B. Weevil Response to 1,3−Octadiene 150

100

0 action − Repulsion r −50 Att

−100

10,000 650,000 ng of 1,3−octadiene

FIGURE 1.2: Weevil behavior follows push-pull pollination where they are attracted to lower concentrations of 1,3-octadiene and repelled by higher amounts. (A) Weevils are preferentially attracted to mid-level amounts of 1,3-octadiene in pit-tests (shown in inset and Figure A.1) and less attracted to more concentrated amounts. Attraction index is the proportion of weevils found in pits after 30 minutes. (B) Weevils are attracted to and move towards 10,000 ng 1,3-octadiene and are repelled and move away from 650,000 ng in behavioral arenas (shown in inset and Figure A.2). Attraction and repulsion are determined by a change in asymmetry towards the volatile compound over the course of the trial. Values above the grey line show a change in orientation away from the 1,3-octadiene bait, and below the line a shift towards the bait. Amounts of 1,3- octadiene are shown on the x-axis.

26 Chapter 1. An ancient obligate pollination mutualism in cycads

Finally, we determined the pattern of Z. furfuracea volatile release and its correlation to R. furfuracea movement in the ﬁeld. We hypothesized that there would be an increase in volatile production and R. furfuracea activity around 19:00-20:00 after the thermogenic peak at 18:30 (6). We collected volatiles from reproductive pollen and ovulate cones (n=5, Table 1.1) at hourly intervals throughout the day and identiﬁed a typical diurnal pattern in volatile production in Z. furfuracea whereby cones change the ratio of the two major volatile compounds over the course of the day, approaching 100% production of 1,3-octadiene in the evening (Figure 1.3.A). Pollen cones consistently produce a greater quantity of volatile compounds (p=0.0007) than ovulate cones (Figure 1.3.B) and show a large increase in production of 1,3-octadiene in the evening (Figure 1.3.C, p=0.0137) that coincides with R. furfuracea expulsion from pollen cones around 20:00 (Figure 1.3.D).

27 Chapter 1. An ancient obligate pollination mutualism in cycads

A. Volatile Composition C. Pollen Cone 1,3−Octadiene Increase

1,3−octadiene linalool 40

30 100

Percent 10

octadiene Percent Increase 1,3 − octadiene Percent 0

1250 1500 1750 2000 2250 1250 1500 1750 2000

B. Total Volatile Amounts D. Weevil Expulsion from Pollen Cones

Ovulate Cone Pollen Cone 40

30 7.5e+07

20 5.0e+07

Number of Weevils 10 Total Peak Area Peak Total 2.5e+07

0.0e+00 0 1250 1500 1750 2000 2250 1250 1500 1750 2000 Time of Day Time of Day

FIGURE 1.3: An increase in production of 1,3-octadiene by Zamia furfuracea is positively correlated with an exodus of R. furfuracea weevils from the pollen cone. (A) The percent composition of the daily volatile proﬁle shifts towards 100% 1,3-octadiene after 20:00 hrs in both pollen and ovulate cones (n=5). (B) Pollen cones produce more volatile emissions throughout the day than ovulate cones (p=0.0007). Total peak area from GCMS analysis is shown on the y-axis. (C) The percent increase in 1,3-octadiene production in pollen cones rapidly increases after 20:00 hrs (p=0.0137). The smoothed conditional mean is shown (method=loess) with one standard error at 0.95 conﬁdence in A, B C. (D) Weevils housed in pollen cones are repelled from the cones at 20:00 hrs. Each colored line represents an individual pollen cone and numbers are raw counts of weevils seen in video recordings.

28 Chapter 1. An ancient obligate pollination mutualism in cycads

? ? Cycas (117)

? ? Dioon (16)

? ? Bowenia (2)

Macrozamia (41)

? ? Lepidozamia (2) Coleoptera pollinator

Lepidoptera pollinator ? Encephalartos Thysanoptera pollinator (65) Daily thermogenic pattern Daily volatile pattern ? ? Ceratozamia Push-pull pollination described (31)

? ? Stangeria (1) ? ? Microcycas (1)

Zamia (79) Specialized beetle pollination in cycads Rise of flowering plants This Study 350.0 300.0 250.0 200.0 150.0 100.0 50.0 0.0 Carboniferous Permian Triassic Jurassic Cretaceous Tertiary Paleozoic Mesozoic Cenozoic

FIGURE 1.4: Bayesian molecular dated phylogeny of Cycadales with all known information on pollinators and thermogenic and volatile patterns. The number of species in each genus is noted in parentheses. Grey bars show the 95% conﬁdence interval for dating at the nodes. Recently reported fossil evidence of specialized beetle pollination in cycads (Cai et al., 2018) as well as the esti- mated timing of the rise of ﬂowering plants are noted. Citations for metadata are provided in Table S3.

29 Chapter 1. An ancient obligate pollination mutualism in cycads

1.4 Discussion

Rhopalotria furfuracea weevils and Cycadothrips chadwickii thrips have converged on the same behavioral response to an ancient plant pollination mechanism. Both pollinators utilize the pollen cone as a larval development site, as do all known cycad pollinators (Terry et al., 2012a; Tang, 2006; Pellmyr et al., 1991; Terry et al., 2004; Azuma and Kono, 2006). Both insects show differential behavioral responses according to the quantities of one host plant volatile compound: attraction to intermediate amounts and repulsion to higher amounts. The increase in volatile production causes expulsion of pollinators from pollen cones, a pattern that has been observed in other cycad genera as well (Terry et al., 2007; Terry et al., 2004; Terry, 2001; Wallenius et al., 2012; Norstog, Stevenson, and Niklas, 1986). In both plants described here, as well as all cycad genera so far tested, the production of these compounds changes throughout the day (Figure 1.4) and is known to be caused by thermogenesis (Terry et al., 2016), an ancestral trait among the Cycadales (Tang, 1987a). The Cycadales are the basal gymnosperm lineage (Ran et al., 2018) and have an extensive fossil record stretching back 265 MY (Hermsen et al., 2006), placing them amongst the most ancient extant seed plants. Specialized beetle pollination has existed in this group well before the rise of ﬂowering plants (Niklas, Tiffney, and Knoll, 1983) from at least the mid-Mesozoic (Figure 1.4) (Cai et al., 2018) and perhaps as early as 245 MY (Klavins et al., 2005). Stereotypical sequential timing of thermogenesis in pollen and ovulate cones as well as specialized brood site pollination have been documented to occur across the Cycadales (Table 1.2). These observations, in combination with our discovery here of convergence in

30 Chapter 1. An ancient obligate pollination mutualism in cycads the behavioral responses of both thrips and weevils to volatiles released by the cones of Old World (Macrozamia) and New World (Zamia) lineages support the hypothesis that the push-pull system found in cycads represents the most ancient insect/plant pollination mechanism yet documented. These dioecious plants give us insight into the earliest forms of insect pollination prior to the prolific diversification of visual floral signaling and reward systems employed by angiosperms. Odor attraction has been thought to precede color attraction in angiosperms (Piji, 1960) and the coupling of thermogenesis with strong odors as seen in cycads has been suggested as an exaptation leading to early angiosperm pollination syndromes (Thien, Azuma, and Kawano, 2000). The hypothesis that plant volatiles attractive to pollinators are derived from herbivore deterrent secondary compounds (Pellmyr and Thien, 1986) is also supported in that attractive volatile compounds in cycad push-pull pollination maintain a repellent component for their specialized mutualists. Early insects found rewards such as pollination drops (Tang, 2006; Labandeira, Kvacek, and Mostovski, 2007), ovular exudates found in cycads and other gymnosperms that predate composition- ally similar angiosperm nectar, and high concentrations of starch in cone tissues (Tang, 2006). As they overcame the deterrent quality of host plant compounds and found brood sites in plant reproductive tissues, plant volatiles became the basis of chemical plant-insect communication, with signals for attraction and repulsion mediated in part by circadian cycles of thermogenesis.

31 Chapter 2

Cycad-feeding insects share a core gut microbiome

Published article: Cycad-feeding insects share a core gut microbiome

The following pages contain a reproduction of the paper. Supporting material is included as Appendix B.

32 applyparastyle "body/p[1]" parastyle "Text_First"

Chapter 2. Cycad-feeding insects share a core gut microbiome

Biological Journal of the Linnean Society, 2018, 123, 728–738. With 3 figures.

Cycad-feeding insects share a core gut microbiome

SHAYLA SALZMAN*, MELISSA WHITAKER and NAOMI E. PIERCE

Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02149, USA

Received 29 September 2017; revised 31 January 2018; accepted for publication 31 January 2018

Five insect species including three species of weevils (Coleoptera) and two species of lycaenid butterflies (Lepidoptera) that feed exclusively on the carcinogenic and neurotoxic tissues of cycads were found to share a core set of bacterial phylotypes, including the bacterium Raoultella ornithinolytica, which has known anti-cancer and nitrogen-fixing capabilities. The other shared bacteria belong to lineages that include insect-associated and extremophilic taxa. The presence of Raoultella ornithinolytica and an unknown Enterobacteriaceae was in contrast to a set of non-cycad- feeding relatives of these insects, none of which contained this same set of shared bacterial phylotypes. Given the considerable phylogenetic distance between the cycadivorous insect species as well as the fact that shared microbiota are not found in their non-cycad-feeding relatives, our data suggest that this core set of shared bacteria are important in helping cycad feeders detoxify their poisonous host plants.

ADDITIONAL KEYWORDS: BMAA – β-methylamino-L-alanine – coevolution – Cycadales – gut microbiome – herbivory – lycaenid – methylazoxymethanol.

INTRODUCTION not encounter these plant compounds in their diets, insects that have specialized on cycads must be cap- The plant order Cycadales comprises ten genera able of contending with both toxins concurrently, and with ~350 species found across the tropics (Calonje, each compound acts in very different ways. Stevenson & Stanberg, 2017). These dioecious gym- MAM has both carcinogenic and neurotoxic effects nosperms are often obligately insect-pollinated and in (Laqueur & Spatz, 1968; Morgan & Hoffmann, 1983). many cases provide a brood site for pollinators that This compound occurs in the plants in a non-toxic feed and develop on their tissue (Norstog, Stevenson form in which the toxic agent, MAM, is attached to & Niklas, 1989; Stevenson, Norstog & Fawcett, 1998; a glycoside. While MAM-glycosides are found in all Terry et al., 2012; Brookes et al., 2015; Valencia- cycad genera (De Luca et al., 1980; Moretti, Sabato & Montoya et al., 2017). Cycads are among the most Gigliano, 1981, 1983), the non-toxic storage form may ancient lineages of seed plants with a fossil record differ depending on the glycoside attached to MAM. extending back over 250 My (Mamay, 1969; Gao & The two most common MAM-glycosides are cyca- Thomas, 1989) and while they are currently the most sin, in which the glycoside is β-D-glucose (Nishida, endangered plant order in the world (IUCN, 2017), Kobayashi & Nagahama, 1955), and macrozamin, in they were once a dominant component of the Mesozoic which the glycoside is a disaccharide of glucose and flora (Friis, Chaloner & Crane, 1987; Thomas & Spicer, xylose (Lythgoe & Riggs, 1949). In both cases, toxicity 1987). Cycads produce many secondary metabolites results from cleavage of the glycoside from MAM by the (De Luca et al., 1982; Khabazian et al., 2002), includ- activity of endogenous glucosidase enzymes (Laqueur ing two highly toxic compounds found in species & Spatz, 1968; Schneider et al., 2002) that are pro- throughout the order: methylazoxymethanol (MAM) duced in the digestive tracts of mammals and insects (De Luca et al., 1980; Moretti, Sabato, Gigliano, 1983) (Conchie & MacDonald, 1959; Terra & Ferreira, 1994). and β-methylamino-L-alanine (BMAA) (Vega & Bell, Once cleaved, MAM spontaneously degrades into for- 1967). Whereas non-cycadivorous insect herbivores do maldehyde and methyl-diazonium, a potent methylating agent (Morgan & Hoffmann, 1983). MAM-induced *Corresponding author. E-mail: [email protected]. genetic alterations have been described in plants, edu mammals, yeast, bacteria and insects (Morgan &

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CYCAD-FEEDING INSECTS SHARE A CORE GUT MICROBIOME 729

Hoffmann, 1983). MAM-glycosides have been found Relatively little is known about potential toxin toler- in all cycad genera and in all plant tissues that have ance mechanisms in cycadivorous insects, which could been tested, including seeds, leaves, pollen and ovu- include methods to detoxify, sequester and/or avoid late cones, roots and stems (Cooper, 1941; Riggs, plant defensive chemicals. One study focused on BMAA 1954; De Luca et al., 1980; Moretti et al., 1981, 1983; avoidance in the pollinating weevil, Rhopalotria furfu- Blagrove, Lilley & Higgins, 1984; Rothschild, Nash racea (previously R. mollis), which feeds on pollen cone & Bell, 1986; Yagi & Tadera, 1987; Bowers & Larin, parenchyma tissue of Zamia furfuraceae (Norstog & 1989; Lindblad, Tadera & Yagi, 1990; Nash, Bell & Fawcett, 1989). In this case, staining experiments dem- Ackery, 1992; Vovides et al., 1993; Castillo-Guevara onstrated that plants sequester BMAA in specialized & Rico-Gray, 2003; Yagi, 2004; Prado, 2011; Nair & plant cells (idioblasts) that are able to pass through Staden, 2012; Prado et al., 2014, 2016). the insect gut intact. Interestingly, these idioblast cells The second class of cycad toxins, BMAA, has received were found intact in the pollen cones on which the wee- considerable attention by virtue of its implication as vils feed, but burst open in ovulate cones, which the the causative agent of amyotrophic lateral sclerosis- weevils visit but never eat. The authors suggested that parkinsonism-dementia, a human neurobiological this plant mechanism restricts pollinator herbivory to disease that is endemic to the island of Guam and is the expendable pollen cone tissue. No known or pro- also referred to as Guam’s dementia (Cox, Banack & posed mechanism exists for BMAA tolerance or avoid- Murch, 2003). BMAA was first isolated from cycads ance in leaf or ovulate cone feeders where the toxin is by Vega & Bell (1967) in response to an extremely not sequestered in plant idioblasts. detailed account of cycad consumption and toxic The only other investigation into cycad toxin tol- effects in humans and cattle (Whiting, 1963). BMAA’s erance mechanisms focused on MAM tolerance in a toxicity arises from its disruption of glutamate recep- leaf-feeding moth. A β-glucosidase enzyme was found tor function. Glutamate receptors have deep homology to be localized in the guts of the larvae of the ‘echo and are found across plants and animals (Lam et al., moth’, Sierarctia echo, feeding on leaves of Zamia inte- 1998; Chiu et al., 1999; Lacombe et al., 2001). BMAA grifolia, and this insect was shown to produce cycasin works as a neurotoxin in insects (Goto, Koenig & when fed the toxic MAM (Teas, 1967). Echo moths are Ikeda, 2012) and mammals, causing convulsions and aposematically coloured as both larvae and adults, and neural degeneration (Spencer et al., 1987) as well as are thought to sequester and utilize the host plant’s abnormalities in brain development (Kisby, Moore & cycasin for protection. Teas (1967) hypothesized that Spencer, 2013). In Arabidopsis, BMAA-induced glu- this endogenous β-glucosidase enzyme rehydrolyses tamate receptor blockage affects signal transduction the toxic MAM with a glycoside, reverting the com- causing hypocotyl elongation and inhibiting coty- pound into its non-toxic form, cycasin. Laqueur & ledon opening (Brenner et al., 2000). Moreover, as a Spatz (1968) suggested that this enzyme could be of non-protein amino acid, BMAA can be incorporated microbial origin, yet this remains untested, and it is into proteins, fundamentally altering their structure unknown whether the enzyme is present in the guts of and function (Dunlap et al., 2013). It is unknown how other cycadivorous insects. the cycad protects itself from this endogenous toxin. The suggestion that microbes may play a role in BMAA has been found in all cycad genera and in all toxin tolerance in Sierarctia echo moths is particularly tested tissues, including leaves, pollen and ovulate interesting given the growing evidence that many herb- cones, seeds, pollen and roots (Dossaji & Bell, 1973; ivorous insects rely on symbiotic gut bacteria to medi- Duncan, Kopin & Crowley, 1989; Norstog & Fawcett, ate the challenges associated with plant-based diets 1989; Vovides et al., 1993; Pan et al., 1997a, b; Banack (Douglas, 2013), including degrading plant secondary & Cox, 2003). metabolites (Boone et al., 2013; Ceja-Navarro et al., Given the highly toxic nature of these compounds 2015) and countering specialized plant defences (Chu and their presence throughout plant tissues, only et al., 2013). It is possible that cycadivorous insects specialized insects are able to utilize cycads as a rely on gut bacteria to ameliorate their highly toxic food source. Many of these are pollinating herbi- diets, and given the similar plant defensive chemistry vores, including species from several genera of bee- that all cycad herbivores are exposed to, even distantly tles that feed on pollen cone parenchyma tissue and related insects may have converged upon similar bac- coevolve with their host cycads (Donaldson, Nänni & terial associations. To investigate these possibilities, Bösenberg, 1995; Stevenson et al., 1998; Terry et al., we used 16S amplicon sequencing to characterize and 2012; Suinyuy, Donaldson & Johnson, 2015). In add- compare the gut bacterial communities of five spe- ition to pollinating herbivores, several folivorous cies of cycadivorous insects from two different orders, Lepidoptera are obligate cycad feeders for either their and to identify and investigate bacterial phylotypes entire larval development or for their first few instars that are shared across these phylogenetically distinct (e.g. Sihvonen, Staude & Mutanen, 2015). insect species.

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730 S. SALZMAN ET AL.

MATERIAL AND METHODS We collected three Curculionidae (Coleoptera) weevil species and two Lycaenidae (Lepidoptera) butterfly species that feed on a variety of cycad species and tissues (Table 1, Fig. 1). Insects were either immediately Singapore Miami, FL, USA FL, Miami, Miami, FL, USA FL, Miami, Naples, FL, USA FL, Naples, Miami, FL, USA FL, Miami, flash frozen whole in liquid nitrogen or dissected and Locality the gut preserved in ethanol, and all samples were stored at −80 °C. Whole insect samples were surface sterilized for 5 s in 10% bleach and rinsed in PBS prior to DNA extraction using the Powersoil DNA isolation kit and protocol (MoBio Laboratories, Carlsbad, CA, USA) with the addition of 60 µg proteinase K to the

lysis buffer. DNA concentration was assessed using a microsporophyll Leaf Leaf Subterranean stem Pollen cone peduncle Pollen Pollen cone Pollen Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) tissue Feeding and samples with low DNA yields were concentrated

using the MoBio protocol.

Extracted DNA was sent to Argonne National

Laboratories (Lemont, IL, USA) for library prepar-

ation and sequencing of the V4 region of the 16S rRNA gene. Library preparation used barcoded primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) and the meth- (Thunberg) (Aiton) (Urban) (Aiton) ods from Caporaso et al. (2012). Libraries were pooled, (Aiton) Cycas revoluta Zamia integrifolia Zamia aff. portoricensis Zamia aff. Zamia integrifolia Zamia furfuracea Host plant and 150-bp paired end reads were sequenced on an Illumina MiSeq sequencer. Raw sequences were preprocessed using previously published methods (Whitaker et al., 2016). Because the 16S universal primers we used are known to also amplify organellar DNA, we compared chloroplast

abundance across samples before removing non-target ethanol Gut dissected in Whole flash frozen Whole flash frozen Whole flash frozen Whole flash frozen sequences, which included chloroplasts and mitochon- Preservation dria as well as the common laboratory contaminants, Staphylococcaceae and Escherichia. We then applied a filtering method requiring bacterial operational taxo- 7 5 6 6 6 Number nomic units (OTUs) to be represented by at least ten sequences in the data set and at a minimum relative abundance of 0.05% per sample in order to be included in the analysis. Late instar Late instar Late instar Late instar Adult Core microbiomes were calculated using the filterfun Life stage function in the phyloseq package in R, requiring bacterial OTUs to be present in all replicates within a species (species cores) or all cycad herbivore replicates (cycad herbivore core, hereafter ‘shared OTUs’). The taxonomic assignments of all core OTUs were further checked using NCBI BLAST and Seqmatch from the Ribosomal Database project (Cole et al., 2014). Shared bacterial Lycaenidae Lycaenidae OTUs were further analysed using the Oligotyping pipe- Curculionidae Lepidoptera: Lepidoptera: Lepidoptera: Lepidoptera: Coleoptera: Coleoptera: Coleoptera: Erotylidae Coleoptera: Coleoptera: Belidae Coleoptera: line v2.1 (Eren et al., 2013) with a minimum substantive Order

abundance of 10 and the smallest number of entropy posi-

tions needed to properly decompose oligotypes. For com-

parison, we searched for these shared OTUs in published

(Casey)

surveys of the gut microbiomes of six species of non-cycad- sample numbers feeding tissues and localities; families and orders, two cycad genera and multiple insect species, Cycad herbivore sampling included

ivorous ‘outgroup’ insects: two Lycaenidae (Lepidoptera) and four Curculionidae (Coleoptera) (details in (Horsfield) (Poey) (Kirsh) floridana Supporting Information S1). For these comparisons, we furfuracea Tang) (O’Brian & Chilades pandava Eumaeus atala Eubulus sp. Pharaxanotha Rhopalotria Table in analyses consider only samples that passed the filtering requirements and were included Herbivore

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Figure 1. Cycad herbivores share five bacterial OTUs. Herbivore species are shown corresponding to the plant tissue on which they feed. Circles show core microbiome counts. Insets of adult insects are included in cases where we sampled the larval stage. Plant tissues are coloured to highlight the pollen cone (red) and stem (blue – underground for Zamia integrifolia). Herbivores of Zamia are (top to bottom): Eumaeus atala (note the aposematic colouring of both life stages), Rhopalotria furfuracea, Pharaxanotha floridana and Eubulus sp. The sole herbivore of Cycas is Chilades pandava.

processed and analysed raw 16S sequences of gut bac- Two additional samples were omitted following rar- teria in the same way that we did with the cycadivorous efaction for diversity analyses due to small library insects. sizes, one R. furfuracea and one Eum. atala. The For diversity analyses, libraries were first rarified to remaining samples clustered in a characteristic pat- 10 000 sequences, retaining any library with at least tern in non-metric multidimensional scaling (NMDS) 5000 sequences. The similarity of each sample’s bac- ordinations and hierarchical clustering (Fig. 2B, C). terial community composition was compared using The gut microbiota of all five cycad-feeding insects the Bray–Curtis dissimilarity metric and hierarch- were remarkably conserved, clustering mainly by spe- ical clustering based on weighted UniFrac distances cies except for an overlap between R. furfuracea and where OTU abundances were averaged across repli- C. pandava in NMDS ordinations of Bray–Curtis dis- cates within a species. All sequence data are deposited tances (stress 0.13; Fig. 2B). Rhopalotria furfuracea in the EMBL-EBI database. and C. pandava also grouped by similarity in hierarchical clustering (Fig. 2C). Taxonomy plots demonstrate a compositional similarity between these two species, driven by the dominance of Alicyclobacillaceae and RESULTS Comamonadaceae in the gut bacterial communities of Raw sequences clustered into 1789 unique OTUs both insects (Fig. 2A). across the 31 samples. As expected, chloroplast 16S The five most abundant bacterial families in our sequences were found in high abundance in caterpil- dataset were Alicyclobacillaceae, Enterobacteriaceae, lars of both leaf-feeding Lepidoptera, Eumaeus atala Moraxellaceae, Comamonadaceae and Enterococcaceae and Chilades pandava, but were unexpectedly preva- (Fig. 2A). Bacterial OTUs found in all samples of a lent in cone-feeding weevils, R. furfuracea, as well species are reported briefly in Table 2 and in detail (median 60% of the total sequences, 18% inter-quartile in Supporting Information S2. Most significantly, the range). After abundance filtering and removing non- microbiota of all five cycad-feeding species showed target OTUs, the filtered dataset lost one Eum. atala significant overlap for five OTUs that were present sample due to small library size but retained 177 taxo- in all replicates (Table 2). The greengenes taxonomic nomic OTUs across the remaining 30 samples. This assignments, however, did not match NCBI BLAST filtered dataset was used in all subsequent analysis. results for any of these five shared OTUs. Only one of

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Figure 2. (A) The relative abundance of the 20 most abundant bacterial families per sample shows that Enterobacteriaceae and Alicyclobacillaceae are dominant in the guts of several species. (B) In ordinations based on Bray–Curtis distances (stress 0.13), gut bacterial communities generally cluster according to species, except for a striking similarity in the gut communities of Rhopalotria furfuracea and Chilades pandava. (C) Hierarchical clustering of insect species by bacterial community composition also highlights the similarity between R. furfuracea and C. pandava.

the shared OTUs could be identified to species using Rickettsiaceae; Table 2). Unidentified bacteria are not NCBI BLAST (OTU 5: Raoutella ornithinolytica in the surprising when exploring novel environmental sam- Enterobacteriaceae). The four remaining OTUs could ples, and the four OTUs that could be identified only to only be identified to the family level using NCBI BLAST family belong to bacterial families that include insect- search (OTUs 4 and 249 in the Enterobacteriaceae, associated and extremophilic bacteria. Of these five OTU 3 in the Alicyclobacillaceae and OTU 85 in the shared OTUs, OTU 4 (Enterobacteriaceae) and OTU 5

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Table 2. Five bacterial OTUs are shared across five species of herbivores feeding on cycads

Chilades Eumaeus Rhopalotria Pharaxanotha Eubulus sp. pandava atala furfuracea floridana

Unknown Alicyclobacillaceae 3 3 3 3 3 Unknown Rickettsiaceae 85 85 85 85 85 Unknown Enterobacteriaceae 249 249 249 249 249 Enterobacteriaceae Raoultella 5 5 5 5 5 ornithinolytica Unknown Enterobacteriaceae 4 4 4 4 4 596 596 Comamonadaceae Curvibacter 13 13 13 Enterococcaceae Enterococcus 15 15 15 Comamonadaceae Lampropedia 36 36 Comamonadaceae Comamonas 432 432 Streptococcaceae Lactococcus 10 10 Alcaligenaceae Achromobacter 24 24 Unknown Blattabacteriaceae 240 240 Sphingobacteriaceae 17 17 Sphingobacterium Moraxellaceae Acinetobacter 763 187 2 2 91 37 21 47

OTUs that were found to be part of more than one species’ core microbiome are shown and highlighted in grey. Full species microbiota are listed in Supporting Information S2. OTUs were considered to be part of the species core microbiota when present in 100% of the samples for that species. They are designated here by their OTU number.

(Raoutella ornithinolytica) were found solely in cycad DISCUSSION herbivores. None was found in any of the outgroup Our results show that five cycad-feeding insect species weevils. However, OTUs 3, 85 and 249 were found in from two orders share a core set of bacterial OTUs in both outgroup butterflies. their gut microbiota, despite being generally distinct Oligotyping results for shared OTUs are pre- in overall bacterial community composition. While the sented in Figure 3 (data in Supporting Information functions of these OTUs remain unknown, our results S3). Twenty-one unique oligotypes were found for are consistent with the hypothesis that gut bacteria Raoutella ornithinolytica (OTU 5). Pharaxanotha may mediate herbivory of cycads, and they identify floridana had the most consistent composition of specific bacterial phylotypes as candidates for future Raoutella ornithinolytica oligotypes, with Eubulus functional study. sp. showing a similar although less constrained pat- In terms of the entire community of gut microbiota, tern (Fig. 3). In contrast, Eum. atala was dominated we found that each insect harbours a distinctive spe- (> 93%) by one Raoutella ornithinolytica oligotype cies-specific bacterial assemblage, with the exception across all replicates. Twenty unique oligotypes were of R. furfuracea weevils and C. pandava butterflies, found for OTU 4 (Unknown Enterobactereaceae). whose communities of microbiota were surprisingly Again, P. floridana and Eub. sp. showed consistent similar. For example, chloroplasts were found in high patterns of OTU 4 oligotype composition, whereas abundance in all Lepidoptera, as well as R. furfuracea, OTU 4 oligotype composition was highly variable but were not found in the other Coleoptera. In ordin- in the remaining species (Fig. 3). Only three oli- ation plots based on community dissimilarity met- gotypes were recovered for OTU 249 (Unknown rics, the gut bacterial communities of R. furfuracea Enterobactereaceae), one of which dominated all sam- weevils clustered more closely with C. pandava than ples. Finally, oligotyping analysis found no entropy with P. floridana, the other cone-feeding coleopteran peaks for either OTU 85 (Unidentified) or OTU 3 (Fig. 2B). Larvae and adults of R. furfuracea feed only (Unknown Alicyclobacillaceae).

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Figure 3. Oligotyping analysis of the two OTUs that were unique to cycad herbivores. Eumaeus atala was dominated by one oligotype for OTU 5 (Raoultella ornithinolytica). OTU 4 (Unknown Enterobacteriaceae) displayed highly constrained compositional patterns within Eubulus sp. and Pharaxanotha floridana. OTU 85 (Unidentified) and OTU 3 (Unknown Alicyclobacillaceae) were each represented by a single oligotype across all samples, and OTU 249 (Unknown Enterobacteriaceae) was dominated by a single oligotype across all species, so oligoptyping results for those three OTUs are not shown here.

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on the microsporophyll of the pollen cone (Fawcett & research to assess whether this bacterium provides Norstog, 1993), which is developmentally a modified critical benefit to cycadivorous insects, such as detoxi- leaf (Gifford & Foster, 1989), whereas P. floridana lar- fication of their poisonous cycad host plants. vae feed on cone peduncle tissue (Fawcett & Norstog, OTU 4 was identified with equal confidence to two 1993), which is developmentally a modified stem bacterial genera, Pantoea and Klebsiella, and in our (Gifford & Foster, 1989). It is possible that the bac- analysis it remains an unidentified member of the fam- terial compositional similarities we observe between ily Enterobacteriaceae. Oligotyping revealed conserved R. furfuraceae and C. pandava reflect developmen- strain-level compositional patterns in P. floridana and tally related chemical or nutritional similarities in the Eub. sp. for this OTU (Fig. 3). As these are the two plant tissues on which they feed. Further research on least mobile insects in our dataset, it is unclear if this the chemical composition of various cycad tissues will pattern arises from limited exposure to environmental be necessary to test this hypothesis. bacteria, or from selection on the part of the host or gut Ordination plots demonstrated that the microbiota of environment. While it is unknown whether this bac- P. floridana, and to some extent those of Eub. sp., were terium might contribute to host nutrition or fitness, more similar to each other than they were to those of we can make some inferences based on its similarity the remaining three species. In fact, whether looking at to Pantoea and Klebsiella. Pantoea is a known gut sym- the OTU (Fig. 2A) or sub-OTU level (oligotype; Fig. 3), biont in many insects, and it has been shown to confer P. floridana samples exhibited highly conserved bac- nutritional benefits including nitrogen fixation, toxin terial community compositions across replicates. It is degradation and hydrolysis of proteins (Sood & Nath, unclear if these conserved assemblages are a product 2002; MacCollom et al., 2009). Additionally, ingested of limited environmental exposure, vertical transmis- Pantoea and Klebsiella have both been shown to colon- sion or selection by the host. Pharaxanotha floridana ize the guts of insects and be subsequently vertically beetles feed on pollen for their first two instars, and transmitted (Lauzon et al., 2009). Future work should then spend the remainder of their larval development focus on isolating and identifying this OTU and on within the peduncle of the pollen cone, a fairly closed characterizing its metabolic capabilities and symbiotic environment (Fawcett & Norstog, 1993). Further sam- potential. pling of Pharaxanotha adults, which feed exclusively Of the remaining shared OTUs, two were initially on cycad pollen (Fawcett & Norstog, 1993), would help identified by greengenes taxonomic assignment as to elucidate whether these insects harbour consist- Buchnera (OTU 249) and Wolbachia (OTU 85), both ent bacterial communities during all life stages. This known insect symbionts. However, these assignments would enable us to assess the relative contributions of were not supported by the NCBI BLAST database, and host selection versus environmental exposure in deter- both OTUs displayed little oligotypic variation. OTU 3 mining gut bacterial community assemblages in these (Unidentified Alicyclobacillaceae) showed no oligotypic beetles. variation across samples, despite being represented by Four of the five shared OTUs found in the guts of a large number of reads (16 383). Bacteria in this fam- cycad-feeding insects were unidentifiable to genus. ily are found in extreme environments, often growing The one that could be identified, however, offers some at extreme temperatures or pH (Vos et al., 2009). With insight into potential activities of the gut bacterial no information about the function of these bacteria community. OTU 5 was identified as Raoultella orni- or their symbiotic potential as well as their presence thinolytica, a bacterium that has been shown to fix in the ‘outgroup’ butterfly samples, it is impossible to nitrogen in the guts of wild Ceratitis capitata fruit flies determine whether they have a functional relationship (Behar, Yuval & Jurkevitch, 2005) and to elicit cytotox- with their cycadivorous insects. icity and apoptotic and necrotic death of mammalian To our knowledge, this survey represents the first cancer cells due to the activity of a protein–polysac- report of gut bacterial associations that are conserved charide complex (Fiołka et al., 2013). Oligotyping of among a phylogenetically and geographically dispar- Raoultella ornithinolytica revealed consistent strain- ate set of herbivores that share a common toxic host level compositional patterns within Eum. atala, P. flor- plant. By comparing the composition of gut bacterial idana, and Eub. sp. (Fig. 3). The leaf- and cone-feeding communities of five species of cycad-feeding insects, Eumaeus larvae are more vagile than the cone- and we found that each insect harbours a distinctive spe- stem-feeding Pharaxanotha and Eubulus larvae, such cies-specific bacterial assemblage, with the exception that we might expect Eum. atala to be exposed to a of R. furfuraceaea weevil larvae and C. pandava cat- greater diversity of bacteria in the environment. It is erpillars that host similar communities of microbiota. therefore somewhat unexpected that the Eum. atala Most importantly, we found that all five of the insect caterpillars were dominated by only one oligotype. species share a core set of bacterial OTUs, which we Raoultella ornithinolytica warrants further functional believe warrant future functional study. We identified

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one of these shared bacteria as Raoultella ornithinolyt- Brenner ED, Martinez-Barboza N, Clark AP, Liang QS, ica, a species with documented anti-tumour and nitro- Stevenson DW, Coruzzi GM. 2000. Arabidopsis mutants gen fixation capabilities. Future comparative surveys resistant to S(+)-β-methyl-α-β-diaminopropionic acid, a of the microbiota of cycad herbivores should include cycad-derived glutamate receptor agonist. Plant Physiology a broader range of insect taxa, developmental stages 124: 1615–1624. and feeding ecologies, as well as an investigation into Brookes DR, Hereward JP, Terry LI, Walter GH. 2015. the functional profiles of core bacterial OTUs. Such Evolutionary dynamics of a cycad obligate pollination studies would be invaluable in elucidating the role of mutualism–Pattern and process in extant Macrozamia microbial symbionts in mediating cycadivorous diets. cycads and their specialist thrips pollinators. Molecular Phylogenetics and Evolution 93: 83–93. Calonje M, Stevenson DW, Stanberg L. 2017. The world list of cycads, online edition. Available at: http://www. ACKNOWLEDGEMENTS cycadlist.org. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, We thank Rory Maher for the use of his photographs Huntley J, Fierer N, Owens SM, Betley J, Fraser L, of Eumaeus atala, Pharaxanotha floridana and Bauer M, Gormley N. 2012. Ultra-high-throughput micro- Rhopalotria furfuracea and William Tang and Horace bial community analysis on the Illumina HiSeq and MiSeq Tan for the use of their photographs of Eubulus sp. platforms. ISME Journal 6: 1621. and Chilades pandava, respectively. We are grateful Castillo-Guevara C, Rico-Gray V. 2003. The role of mac- to the Montgomery Botanical Center for its helpful rozamin and cycasin in cycads (Cycadales) as antiherbi- and knowledgeable staff including Patrick Griffith, vore defenses. Journal of the Torrey Botanical Society 130: Michael Calonje, Claudia Calonje, Stella Cuestas 206–217. and Vickie Murphy and the use of their collections, Ceja-Navarro JA, Vega FE, Karaoz U, Hao Z, Jenkins S, as well as to Dennis Stevenson for his generosity and Lim HC, Kosina P, Infante F, Northen TR, Brodie EL. deep knowledge of cycad biology. The manuscript was 2015. Gut microbiota mediate caffeine detoxification in the greatly improved by the comments of three anonym- primary insect pest of coffee. Nature Communications 6: ous reviewers. The authors are aware of no conflicts 7618. of interest to report. SS was supported by a National Chiu J, DeSalle R, Lam HM, Meisel L, Coruzzi G. 1999. Science Foundation Graduate Research Fellowship and Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals MW was supported by an NSF Postdoctoral Research diverged. Molecular Biology and Evolution 16: 826–838. Fellowship in Biology (1309425) and grants from the Chu CC, Spencer JL, Curzi MJ, Zavala JA, Seufferheld Wildlife Reserves Singapore Conservation Fund and MJ. 2013. Gut bacteria facilitate adaptation to crop rotation the British Ecological Society. SS and NP conceived in the western corn rootworm. Proceedings of the National the project. SS and MW conducted the fieldwork, Academy of Sciences of the United States of America 110: laboratory work and analysed the data. All authors 11917–11922. contributed to the preparation of the manuscript. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM. 2014. Ribosomal Database Project: data and tools for high through- REFERENCES put rRNA analysis. Nucleic Acids Research 42: D633–D642. Conchie J, MacDonald DC. 1959. Glycosidases in the mam- Banack SA, Cox PA. 2003. Distribution of the neurotoxic non- malian alimentary tract. Nature 184: 1233–1235. protein amino acid BMAA in Cycas micronesica. 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Prado A. 2011. The cycad herbivores. Bulletin de la Société Terra WR, Ferreira C. 1994. Insect digestive enzymes: prop- d’entomologie du Québec 18: 3–6. erties, compartmentalization and function. Comparative Prado A, Sierra A, Windsor D, Bede JC. 2014. Leaf traits Biochemistry and Physiology Part B: Comparative and herbivory levels in a tropical gymnosperm, Zamia ste- Biochemistry 109: 1–62. vensonii (Zamiaceae). American Journal of Botany 101: Terry I, Tang W, Blake AST, Donaldson JS, Singh R, 437–447. Vovides AP, Jaramillo AC. 2012. An overview of cycad pol- Prado A, Rubio-Mendez G, Yañez-Espinosa L, Bede JC. lination studies. Memoirs of the New York Botanical Garden 2016. Ontogenetic changes in azoxyglycoside levels in the 106: 352–394. leaves of Dioon edule Lindl. Journal of Chemical Ecology 42: Thomas BA, Spicer RA. 1987. The evolution and palaeobiol- 1142–1150. ogy of land plants. London: Croom Helm. Riggs NV. 1954. The occurrence of macrozamin in the seeds of Valencia-Montoya WA, Tuberquia D, Guzman PA, cycads. Australian Journal of Chemistry 7: 123–124. Cardona-Duque J. 2017. Pollination of the cycad Zamia Rothschild M, Nash RJ, Bell EA. 1986. Cycasin in the incognita A. Lindstr. & Idárraga by Pharaxonotha beetles in endangered butterfly Eumaeus atala florida. Phytochemistry the Magdalena Medio Valley, Colombia: a mutualism depend- 25: 1853–1854. ent on a specific pollinator and its significance for conserva- Schneider D, Wink M, Sporer F, Lounibos P. 2002. Cycads: tion. Arthropod-Plant Interactions 2017: 1–17. their evolution, toxins, herbivores and insect pollinators. Vega A, Bell EA. 1967. α-Amino-β-methylaminopropionic Naturwissenschaften 89: 281–294. acid, a new amino acid from seeds of Cycas circinalis. Sihvonen P, Staude HS, Mutanen M. 2015. Systematic Phytochemistry 6: 759–762. position of the enigmatic African cycad moths: an integra- Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey tive approach to a nearly century old problem (Lepidoptera: FA, Schleifer KH, Whitman W, eds. 2009. Bergey’s man- Geometridae, Diptychini). Systematic Entomology 40: ual of systematic bacteriology Volume 3: The Firmicutes. New 606–627. York: Springer, 229–243. Sood P, Nath A. 2002. Bacteria associated with Bactrocera Vovides AP, Norstog KJ, Fawcett P, Duncan MW, Nash sp. (Diptera: Tephritidae)–isolation and identification. Pest RJ, Molsen DV. 1993. Histological changes during matur- Management and Economic Zoology 10: 1–9. ation in male and female cones of the cycad Zamia furfura- Spencer PS, Nunn PB, Hugon J, Ludolph AC, Ross SM, cea and their significance in relation to pollination biology. Roy DN, Robertson RC. 1987. Guam amyotrophic lateral Botanical Journal of the Linnean Society 111: 241–252. sclerosis-parkinsonism-dementia linked to a plant excitant Whitaker MR, Salzman S, Sanders J, Kaltenpoth M, neurotoxin. Science. 237: 517–522. Pierce NE. 2016. Microbial communities of lycaenid Stevenson DW, Norstog KJ, Fawcett PKS. 1998. Pollination butterflies do not correlate with larval diet. Frontiers in biology of cycads. In: Owens SJ, Rudall PJ, eds. Reproductive Microbiology 7: 1920. biology. Kew: Royal Botanic Gardens, 277–294. Whiting MG. 1963. Toxicity of cycads. Economic Botany 17: Suinyuy TN, Donaldson JS, Johnson SD. 2015. 270–302. Geographical matching of volatile signals and pollin- Yagi F, Tadera K. 1987. Azoxyglycoside contents in seeds of ator olfactory responses in a cycad brood-site mutualism. several cycad species and various parts of Japanese cycad. Proceedings of the Royal Society B 282: 2015–2053. Agricultural and Biological Chemistry 51: 1719–1721. Teas HJ. 1967. Cyasin synthesis in Seirarctia echo (lepidop- Yagi F. 2004. Azoxyglycoside content and β-glycosidase tera) larvae fed methylazoxymethanol. Biochemical and activities in leaves of various cycads. Phytochemistry 65: Biophysical Research Communications 26: 686–690. 3243–3247.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

S1. Non-cycadivorous lycaenid and curculionid samples that passed filtering requirements and were analysed for the presence of shared bacterial OTUs found in cycad herbivores. S2. Species core microbiome OTU and assigned taxonomy are listed with OTUs present in more than one species highlighted in grey. Species’ microbiome cores were determined by OTU presence in 100% of samples of that species after filtering and are represented by their OTU number. S3. Oligotyping results

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Ecology and Evolution of Cycad-Feeding Lepidoptera

Note: Supplemental materials included in Appendix C.

3.1 Introduction

Lepidoptera (butterflies and moths) have long been used to test theories about the evolutionary origins and consequences of ecological traits, and their larval associations with host plants have served as a scientific cornerstone of studies of coevolution. An extensive literature on the physiological, morphological, behavioral, genetic, and ecological mechanisms of plant-butterfly interactions has developed over the last half century, largely in response to Ehrlich’s & Raven’s seminal 1964 paper describing macroevolutionary patterns of host use among butterflies (Ehrlich and Raven, 1964). In observing that related butterfly groups tend to feed on related plant groups, Ehrlich & Raven proposed a step-wise process that is now commonly referred to as "escape and radiate"

44 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera coevolution (Thompson, 1989), in which plants diversify after evolving novel defenses that allow them to ’escape’ from herbivores, and herbivores diversify after evolving novel counter-adaptations to plants’ defenses. Thirty years later, advances in molecular phylogenetics provided the necessary tools to ex- amine trait variation from an evolutionary perspective, and phylogenetically explicit analyses of hostplant associations have since been conducted for numerous lepidopteran groups (see Mitter, Davis, and Cummings, 2017 and references therein), with cross-taxon analyses presented in several other papers (e.g., Janz and Nylin, 1998; Janz and Nylin, 2008; Fordyce, 2010; Menken, Boomsma, and Van Nieukerken, 2010). While these studies report different patterns at different taxonomic scales and for different lepidopteran lineages, most find general support for Ehrlich’s & Raven’s hypothesis that butterflies’ hostplant associations are significantly phylogenetically conserved, at least at higher taxonomic levels. Moreover, in cases where closely related butterflies use distantly related host plants, those plants often share phytochemical similarities rather than phylogenetic relatedness, underscoring the importance of plant defensive chemistry in determining the food preferences, dietary breadth, and host transitions of Lepidoptera. However, though phylogenetic and phytochemical conservatism appear to be widespread they are far from ubiquitous. Several explanations have been proposed for cases in which lepidopterans switch to plants that are phylogenetically and phytochemically dissimilar to their ancestral host, but a common challenge in identifying the factors underlying such host shifts is defining a "major" host shift in a biologically relevant way, including determining the appropriate taxonomic scale at which to assess

45 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera host shifts. This limitation is borne out of the fact that the overwhelming majority of previous studies have focused on Lepidoptera that feed on just a few angiosperm families, with comparatively little investigation into transitions to non-angiospermous diets (but see Braby, 2000; Pierce et al., 2002; Kaliszewska et al., 2015; Cong et al., 2016; Whitaker et al., 2016). This is a missed opportunity, as transitions to gymnosperms and other non-angiosperm plants likely represent some of the most drastic host switches in terms of phylogenetic, morphological and (potentially) phytochemical dissimilarity. We therefore suggest that study- ing the causes and consequences of transitions away from angiosperms—in this case, to a group of gymnosperms called cycads—could yield novel insight into the study of plant-insect interactions. Cycads (order: Cycadales) are a basal, pantropical group of dioecious gymnosperms with a fossil record extending back over 265 million years (Gao and Thomas, 1989). They possess an arsenal of distinctive chemical defenses that are themselves deserving of review, and yet a number of insects use cycads as larval and adult food plants. The great majority of cycad-feeding (cycadivorous) insects belong to a handful of lepidopteran families that exhibit varying degrees of dietary specialization and belong to multiple feeding guilds. However, the biology of cycadivorous Lepidoptera has never been reviewed, and the majority of relevant studies have concentrated on just a few focal species without exam- ining broader ecological or evolutionary patterns. The aims of this review are therefore to 1) assemble an authoritative list of cycadivorous Lepidoptera and distinguish veriﬁed from unveriﬁed records, 2) discuss key ecological and evolutionary implications of cycad feeding, 3) summarize conservation and pest

46 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera management concerns, and 4) highlight important data gaps and areas for future study.

3.2 Lepidopteran Cycad Herbivores

Cycadivory occurs in seven Lepidopteran families (Table 3.1), including the butterfly families Nymphalidae and Lycaenidae. Among nymphalid butterflies, larvae of two species in the Australasian genus Taenaris—T. onolaus and T. butleri—have been reported to feed on Cycas in Papua New Guinea (Parsons, 1984; Parsons, 1999). In addition to larval cycad feeding, some adult Taenaris butterflies imbibe cycad juices: T. onolaus and T. catops have been observed visiting fermenting cycad seeds, feeding on exudates from wounded cycad leaves, and even probing the fresh frass of cycadivorous beetle larvae with their probosces

(Parsons, 1984). This behavior is particularly remarkable in T. catops, the larvae of which feed on palms (Arecaceae) and are not known to be cycadivorous.

Three genera of lycaenid butterﬂies—Luthrodes, Eumaeus, and Theclinesthes— include species that are obligate cycad herbivores. The Luthrodes - Chilades clade is comprised of two sister genera that have historically been lumped together

(typically under name Chilades). Here we follow Talavera et al., 2013 and treat them as separate genera. Thus, we consider the cycadivorous lycaenid species that are typically referred to in the literature as Chilades to be properly placed in Luthrodes: L. pandava, L. peripatria, and L. cleotas. Luthrodes pandava is widespread across southern and southeast Asia and the larvae are often serious pests of

Cycas. Luthrodes cleotas also occurs in southeast Asia and feeds on Cycas (Parsons,

47 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

1999), but less is known about its life history. The third species, L. peripatria, is endemic to Taiwan and its taxonomic status is unclear: some authors treat it as a full species (Hsu, 1989; Talavera et al., 2013) while others consider it a subspecies of L. pandava (Wu et al., 2010; Ravuiwasa, Tan, and Hwang, 2011). The larvae of L. peripatria historically fed only on Cycas taitungensis, also endemic to Taiwan, though it now accepts the ornamental species Cycas revoluta (Ravuiwasa, Tan, and Hwang, 2011) that has been introduced to Taiwan in large numbers since the 1990s (Wu et al., 2010).

The neotropical lycaenid genus Eumaeus is comprised of six species distributed from Peru to the Caribbean (Lamas, 2004), with E. atala extending into southeastern Florida and some (perhaps dubious) records of rare strays of E. toxea into southern Texas (Kendall, 1984). All six Eumaeus species are obligate cycad herbivores, utilizing cycads in the neotropical genera Zamia, Dioon, and Ceratozamia (Koi and Daniels, 2015; Hammer, 1996; Contreras-Medina, Ruiz-Jiménez, and Luna Vega, 2003; Ramírez-Restrepo, Koi, and MacGregor-Fors, 2017; Comstock,

1948; Koi and Daniels, 2017). Larvae of several Eumaeus species have been observed feeding on plants’ fresh male and female reproductive cones in addition to stem and leaf tissue (Farrera et al., 2000; Cascante-Marín and Araya, 2012; González, 2004; Ruiz-García et al., 2015; Bosque and Rosales-Robles, 2015; Koi and Daniels, 2017), and we know of a single report of E. childrenae adults feeding on cycad exudates (Murillo, 1902). Finally, Theclinesthes is a mostly Australian genus of six species, of which one species, T. onycha, feeds on cycads in the genera Cycas and Macrozamia in eastern Australia (Braby, 2000). Among moths, 23 species from 8 genera have been recorded on cycads but

48 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera this is likely an underestimation, as many cycad-feeding moths remain poorly collected and understudied. An entire tribe of Geometrid moths, the Diptychini, consists of 17 cycadivorous species in 3 genera (Sihvonen, Staude, and Mutanen, 2015). Colloquially called "the cycad moths," these are the best studied of the cycadivorous moths and are the only cycadivorous Lepidoptera known from

Africa. The hostplants of all Diptychini larvae are Encephalartos and Stangeria cycads for the ﬁrst 3 instars, but larvae in later instars often switch to angiospermous host plants (Donaldson, 1991; Staude, 1994; Staude and Sihvonen, 2014). Hostplant species for Diptychini moths are therefore separated into primary (cycad) and secondary (non-cycad) hosts in Table 3.1.

49 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; ; 2015 2017 ; Parsons, ; Parsons, 1984 1984 1996 Continued on next page 1999 1999 Koi and Daniels, Koi and Daniels, Hammer, (species unknown)(species unknown) Parsons, Parsons, Cycas Cycas Zamia integrifolia, Z. ﬁs- cheri*, Z. loddigesii*,vasquezii*, Z. Z. furfuracea*, Cycas revoluta*,cairnsina*, Cycas Encephalartos villosus*, E. hildebrandtii*, Macrozamia lucida* the Supplementary Materials. *Introduced plant species. ( Kirsch, 1877) Hübner, [1819] 3.1: Larval hostplant records for cycadivorous Lepidoptera. Species synonyms are given in Hübner, [1819] (Oberthür, 1880) (Poey, 1832) ABLE T SpeciesNymphalidae Taenaris T. butleri T. onolaus Lycaenidae Cycad HostsEumaeus E. atala Other Hosts Sources

50 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; 1948 1992 ; Clark, 2015 2004 ; Bosque and 2012 2017 ; Ramírez-Restrepo, Continued on next page Comstock, Contreras-Medina, Ruiz- Jiménez, and Luna Vega, 2003 Koi, andFors, MacGregor- Rosales-Robles, Cascante-MarínAraya, and González, Clark, and Grayum, Dioon edule, D.Ceratozamia merolae, matudae, C. mexicana, C. norstogii, C. robusta, C. chimalapensis, Zamia ﬁscheri, Z. soconus- cencis, Cycas revoluta* Zamia acuminata,fairchildiana, Z. manicata, Z. Z. stevensonii Zamia encephalartoides, Z. skinneri (Gray, 1832) (Boisduval, 1870) (Hübner, [1809]) (Boisduval, 1870) Unknown Table 3.1 – continued from previous page SpeciesE. childrenae E. godartii Cycad HostsE. minyas Other HostsE. toxana Sources

51 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; ; ; ; 2003 2007 2015 1994 2015 ; Khew, 2010 1999 1993 1918 2012 Continued on next page Castillo-GuevaraRico-Gray, and Martínez-Lendech, Córdoba-Aguilar, and Serrano-Meneses, Ruiz-García et al., Burkill, Marler, Lindström, and Terry, Wu et al., Forster and Machin, Wilson, Cycas (species unknown) Parsons, Zamia furfuracea, Z. pauci- juga, Z. encephalartoides, Z. loddigesii Cycas Cycas taitungensis, Cycas revoluta* Cycas megacarpa, C. ophio- litica, C. media (Hewitson, 1865) (Röber, 1891) (Hsu, 1980) Druce, 1895 (Horsﬁeld, [1829]) >85 species of (Guérin-Méneville, [1831]) (Godart, [1824]) Table 3.1 – continued from previous page SpeciesE. toxea Luthrodes Cycad HostsL. cleaotas L. pandava Other HostsL. peripatria Sources Theclinesthes T. onycha onycha

52 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera 1994 Continued on next page Forster and Machin, Staude and2014 Sihvonen, 2014 Staude and2014 Sihvonen, 2014 in Secondary hosts:known un- inDiospyros the lycioides wild, captivity SecondaryApodytes dimidiata, Mimu- sops hosts: obovata En- Stangeria Macrozamia spiralis, M. communis, M.guilielmi pauli- cephalartos hildebrandtii Unknowneriopus, Encephalartos villosus Unknown Staude and Sihvonen, Sibatani & Grund, Staude & Sihvonen, (Prout, 1913) Primary hosts: (C. & R. Felder, 1874) Primary hosts: Felder, 1874 (Druce, 1896) Unknown Unknown Staude and Sihvonen, Table 3.1 – continued from previous page SpeciesT. onycha capricornia 1978 Geometridae Zerenopsis Z. costimaculata Cycad HostsZ. ﬂavimaculata Other2014 HostsZ. geometrina Sources Z. kedar

53 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; 2001 ; Staude, 1995 Continued on next page Donaldson and Basen- berg, Staude and2014 Sihvonen, 2014 Staude and2014 Sihvonen, Staude and2014 Sihvonen, in Carissa Secondary hosts: bispinosa, C. macrocarpa, C. bispinosa,lycioides, Diospyros D.Apodytes dimidiata, whyteana, Maesa alnifolia, M.Sclerocarya birrea lanceolata, Secondary hosts:known un- inDiospyros the lycioides wild, captivity En- (>20 Stangeria Cycas thouarsii, sp.* eriopus, Encephalartos species), C. circinalis*, C. revoluta*, Dioon cephalartos ferox Encephalartos hildebrandtii Adansonia digitata Warren, 1894 (Prout, 1928) Unknown Unknown Staude and Sihvonen, (Butler, 1878) (Walker, 1854) Primary hosts: Staude & Sihvonen, 2014 Primary hosts: Table 3.1 – continued from previous page SpeciesZ. lepida Z. meraca Cycad HostsZ. moi Other HostsZ. tenuis Sources Veniliodes

54 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; Staude, ; Staude, 2001 1994 2001 2001 2001 Continued on next page Staude, 2001 Staude, 2001 Staude, Staude, Staude, , sp. SecondaryApodytesDiospyros hosts: lycioides dimidiata, SecondaryApodytesDiospyros hosts: lycioides dimidiata, SecondaryApodytesDiospyros hosts: dimidiata, Carissa whyteana in in En- Stangeria Stangeria E. vollosus eriopus, Encephalartos villosus eriopus, Encephalartos villosus Stangeria eriopus cephalartos lebomboensis, E. altensteinii, E. villosus Stangeria eriopus &cephalartos En- tegulaneus the wild, captivity (C. & R. Felder, 1874) Primary hosts: Warren, 1894 Primary hosts: Prout, 1915 (C. & R. Felder, 1875) Staude, 2001 Primary hosts: C. & R. Felder, 1874 Table 3.1 – continued from previous page SpeciesV. inﬂammata V. pantheraria V. setinata Cycad HostsCallioratis C. abraxas Other HostsC. apicisecta Sources

55 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; Wagner, 1890 2001 2008 2001 2001 Continued on next page Staude, Staude, Staude, Staude, Packard, 2005 spp., Diospy- ﬂowers Lupinus in the wild, Quercus spp., spp.*, Secondary hosts: ros lycioides Tropaeolum majus in captivity ros Croton spp., many other woody plants, lettuce En- in cap- Stangeria in the wild, Stangeria eriopus,cephalartos En- guilielmi friderici- Encephalartos gratus Encephalartosguilielmi friderici- eriopus cephalartos villosus tivity Zamia integrifolia Sabal palmetto, Diospy- (Smith, 1797) Packard, 1864 Prout, 1922 Staude, 2001 Hampson, 1905 Primary hosts: Staude, 2001 Table 3.1 – continued from previous page SpeciesC. curlei C. grandis C. mayeri C. millari Cycad HostsErebidae OtherSeirarctia HostsSeirarctia echo Sources Cosmopterigidae

56 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera ; ; 2017 2006 2011b 2006 ; Marler ; Dawidowicz 2012b 1993 2018 ; Robinson and Hua, Salzman,Pierce, and and Rozwa ł ka, Bella and Mazzeo, terry2009cone and Muniappan, Marler and Niklas, Marler and Muniappan, 2006 Nielsen, 2006 Dozens of species, including both angiosperms and gymnosperms Unknown Dozens of plant species, mushrooms UnknownUnknown Marler and Muniappan, Terry et al., Zamia integrifolia, Cycas revoluta, C. circinalis Cycas micronesica Cycas micronesica Cycas micronesica Zamia pumila (Stainton, 1859) Meyrick, 1915 sp. (Hodges, 1962) sp Table 3.1 – continued from previous page SpeciesAnatrachyntis A. badia A. Cycad HostsTineidae Dasyses rugosella Other HostsErechthias Sources Blastobasidae Undetermined

57 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

In addition to these obligate cycad herbivores, a number of facultative cycad

Lepidoptera exist. Seirarctia echo (Erebidae) occurs in the southeastern United States where the larvae are highly polyphagous, feeding on leaves of the cycad Zamia integrifolia as well as plants in the families Arecaceae, Euphorbiaceae, Fabaceae, Fagaceae, and Ebenaceae. In captivity, they have even been reared on lettuce (Asteraceae) (Packard, 1890). One undetermined leaf-mining Erechthias moth (Tineidae) has been found feeding and pupating in the leaves of Cycas micronesica in Guam (Marler and Muniappan, 2006). Larvae of Dasyses rugosella (Tineidae) have been observed feeding on dead Cycas stems in India, Sri Lanka, Thailand, Indonesia, and Guam (Robinson, Tuck, and Shaffer, 1994; Marler and

Muniappan, 2006). Colloquially called "yam moths," D. rugosella are best known as pests of stored yams in West Africa (Iheagwam and Ezike, 1989; Ashamo, 2005), and are broad generalists on decaying vegetable matter (Robinson and

Nielsen, 1993). Larvae of Anatrachyntis badia, another highly polyphagous and cosmopolitan moth species, have been found in pollen cones of Zamia integrifolia in Florida, USA (Hua, Salzman, and Pierce, 2018) and feeding on leaves of Cy- cas revoluta and C. circinalis in Italy (Bella and Mazzeo, 2006). An undetermined Anatrachyntis species pollinates Cycas micronesica in Guam and feeds on pollen cones as larvae (Marler and Niklas, 2011b; Terry et al., 2009). Finally, larvae of an undetermined microlepidopteran in the family Blastobasidae have been found feeding in copious numbers on pollen cones of Zamia pumila in the Caribbean (Terry et al., 2012b). Many records exist for lepidopteran species feeding on cycads which are likely to be erroneous or require further conﬁrmation. We discuss these in the

58 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

Supplementary Materials. The species listed in Table 1 have been identiﬁed by experts, conﬁrmed by multiple sources, supported with photographic evidence, and in many cases their larvae have been reared in captivity on cycads. How- ever, while we feel that this paper serves as an authoritative list of cycadivory among Lepidoptera, it is likely not an exhaustive account of all cycadivorous species considering that new records of cycad-insect associations are still being reported (e.g., Hua, Salzman, and Pierce, 2018), particularly among cone- feeding microlepidoptera.

3.3 Aposematism and Defensive Ecology

Cycads produce several toxic compounds in their leaves and other tissues, including steryl glycosides, b-Methylamino-L-alanine (BMAA), and methylazoxymethanol acetate (MAM) (Morgan and Hoffman, 1983; Laquer and Spatz, 1968; Spencer et al., 1987; Kisby, Moore, and Spencer, 2013). These compounds are toxic to most animals and are therefore presumed to serve as anti-herbivore defenses, though MAM is the only compound for which experimental evidence exists for insect deterrence (Bowers and Larin, 1989; Castillo-Guevara and Rico-Gray, 2003; Prado et al., 2014). MAM is mutagenic and neurotoxic (Laquer and Spatz, 1968; Morgan and Hoffman, 1983) but occurs in plant tissues in a non-toxic form in which the toxic agent is attached to a glycoside. When ingested by insects or other animals, endogenous glucosidase enzymes cleave the glycoside from MAM (Laquer and Spatz, 1968; Rothschild, Nash, and Bell, 1986), which then spontaneously degrades into formaldehyde and methyldiazonium. Early

59 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera work by Teas (1966-7) showed that larvae of Seirarctia echo are able to chemically modify dietary MAM into its non-toxic, glycosylated form and accumu- late MAM-glycosides in their tissues after feeding on cycad leaves (Teas, Dyson, and Whisenant, 1966; Teas, 1967). It is presumed that other cycadivorus species are capable of similar chemical modiﬁcations though it has never been explic- itly tested in other species. Possible mechanisms of resistance to BMAA and steryl glycosides have not been investigated for any cycadivorous lepidopteran, though there is evidence that cycadivorous weevils are able to avoid BMAA by consuming only pollen cone parenchyma tissue where BMAA is sequestered in specialized cells that the weevils excrete in their frass (Vovides et al., 1993; Norstog, Stevenson, and Niklas, 1986). Even so, cycadivorous Lepidoptera appear to tolerate all cycad toxins and several species are brightly colored, diurnal, and gregarious—traits commonly associated with chemically defended Lepidoptera (Bowers, 2003). Indeed, several studies have shown that some cycadivorous species sequester MAM-glycosides into their larval and adult tissues. Rothschild, Nash, and Bell, 1986 found that

Eumaeus atala larvae, pupae, and adults contained MAM-glycosides in surprisingly high amounts relative to their hostplants, and Castillo-Guevara and Rico- Gray, 2003 detected MAM-glycosides in the eggs, larvae, pupae and adults of what is likely to be Eumaeus toxea in Mexico (identified by the authors as E. minyas, although the range of this specis is not thought to extend to Mex- ico). Nash, Bell, and Ackery, 1992 quantified MAM-glycosides in dried museum specimens of adult butterflies, including some specimens that were over

70 years old. The authors detected MAM-glycosides in Eumaeus minyas (male

60 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera and female), Luthrodes cleotas (male and female), Taenaris butleri (male and female), Taenaris catops (male), and Taenaris onolaus (female) but did not detect MAM-glycosides in Theclinesthes onycha (either gender), female Taenaris catops, or male Taenaris onolaus. They concluded that MAM-glycosides were not de- tectable from the latter two because of the advanced age of the museum specimens, but that Theclinesthes onycha probably do not sequester MAM-glycosides. Since several of the species that sequester MAM-glycosides are brightly colored, their coloration may be considered aposematic. Aposematism and chemical defense are exceedingly rare traits among lycaenid larvae (Rothschild, Nash, and Bell, 1986; DeVries, 1977; Fiedler, 1996), which typically rely on crypsis and ant association for protection against natural enemies (Pierce et al., 2002). Eu- maeus provide a striking exception in that they are gregarious and warningly colored in all lifestages, are known to sequester plant chemicals, and have larvae that do not associate with ants (Atsatt, 1981), whereas other cycadivorous lycaenid larvae commonly associate with ants (Wilson, 1993; Eastwood and Fraser, 1999; Tan and Sin Khoon, 2012) and most are not warningly colored. The adults of Theclinesthes onycha and Luthrodes pandava are typical of other Poly- ommatine butterﬂies with blue, brown, or purple uppersides and gray mottled undersides. The larvae of both species are polymorphic, with three main color forms: green, red, and mixed. This variation in coloration is common among Polyommatine larvae but it is not known if or how larval coloration affects survival and predation risk. Larvae of Luthrodes cleotas are cryptically colored but adults have much larger orange spots on their hindwings than do their con- geners, and it is possible that they are aposematic, particularly given that adults

61 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera have been shown to sequester MAM-glycosides (Nash, Bell, and Ackery, 1992). Larvae and adults of Dyptichini moths are brightly colored with gregarious larvae and diurnal adults, but it is unclear whether they sequester plant toxins at any life stage (Donaldson and Basenberg, 1995 suggest that Z. lepida sequester MAM-glycosides, but do not provide experimental evidence). Seirarctia echo larvae are warningly colored and covered with protective hairs. This species sequesters MAM-glycosides when feeding on cycads (Teas, 1967; Teas, Dyson, and Whisenant, 1966), but it remains unknown how feeding on non-cycad hostplants affects their palatability and predation risk. Finally, Anatrachyntis moths and the other microlepidoptera are not aposematic in any lifestage and many species spend their entire development concealed inside plants’ pollen cones, where they may avoid some cycad toxins (Vovides et al., 1993; Norstog and Fawcett, 1989) (see Hostplant Use section below). It is completely unknown whether leaf-mining Erechthias and detritivorous Dasyses encounter cycads’ defensive compounds while feeding. Unfortunately, records of predators and parasitoids are lacking for nearly all cycadivorous species. Natural enemies of Lepidoptera generally include birds, small reptiles, spiders, mantids, reduviid bugs, ants, and parasitic wasps and flies, though direct observations of attacks on larvae and adult butterflies are exceedingly rare in nature (Molleman, Whitaker, and Carey, 2010). The best- studied cycadivorous species with regard to defensive ecology is Eumaeus atala in southeastern Florida. Both native and non-native ants have been observed consuming E. atala eggs and pupae (Smith-Cavros, 2002), but are thought to avoid adult butterflies (Bowers and Larin, 1989). Some assassin and ambush

62 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera bugs (Reduviidae) will attack E. atala larvae (Koi and Hall, 2019) although published records are scarce. Unconfirmed reports exist of native and non-native reptiles attacking E. atala larvae and adults. Starlings, peacocks, and other non- native birds have been reported to attack caterpillars, though it’s possible that only naïve birds will attempt to eat E. atala, as adult butterflies were shown to be distasteful to grey jays (Bowers and Farley, 1990). Interestingly, all of the predators mentioned thus far are visual hunters, whereas many parasitoid wasps and flies use olfactory cues to locate their hosts. There are no reports of parasitoids using E. atala as hosts, a conspicuous absence given that parasitoids are typically significant natural enemies of lycaenid larvae. It may be that E. atala larvae utilize non-visual cues to advertise their chemical defensive status to potential parasitoids. Alternatively, parasitoids may simply not occur in the same places, or they occur but are sufficiently rare that they remain undetected. Given the history of this butterfly in Florida (see Conserva- tion & Management section below), it is also possible that a historical parasitoid occurred in Florida prior to the temporary extirpation of E. atala, but was not reintroduced along with its host. Proper evaluations of these possibilities would require a better understanding of parasitization of E. atala populations across its full geographic range. Among other cycadivorous species, Manners, 2015 reports that "high levels of parasitism" sometimes occur in Theclinesthes onycha larvae in Australia, and provides photographs of larvae parasitized by braconid wasps. Ruiz-García et

63 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera al., 2015 monitored survival and development of Eumaeus toxea larvae in Oax- aca, Mexico and observed Dasydactylus beetles preying on molting E. toxea larvae but did not report ﬁnding any parasitoids. The only published records of parasitization among cycadivorous moths come from Zerenopsis lepida: Staude and Sihvonen, 2014 reared a single parasitoid ﬂy (Tachinidae) from a late instar larva in South Africa, and Sommerer, 2014 reared 15 Z. lepida larvae and found more than 50 percent had been parasitized by Charops sp. (Ichneumonidae) or Drino sp. (Tachinidae). Aside from these scattered records we know relatively little about the natural enemies of cycadivorous Lepidoptera in the wild.

3.4 Evolutionary Origins of Cycadivory

To evaluate evolutionary origins of cycadivory and relationships among cycadivorous Lepidoptera, cycadivory was mapped on to a phylogenetic tree constructed by combining a Lepidoptera phylogeny (Regier et al., 2013) including butterflies and moths with a heavily sampled butterfly phylogeny (Espeland et al., 2018) (Figure 3.1). Both phylogenies were downloaded as .nex files from published sources and brought into R (version 3.5.1) (R Core Team, 2018) where the butterfly clade from Espeland et al., 2018 was substituted in place of the less sampled clade from Regier et al., 2013 using the R packages ape (Paradis and Schliep, 2018), GEIGER (Harmon et al., 2008), and ggtree (Yu et al., 2017; Yu et al., 2018). In cases where cycadivorous species were not represented as tips on the tree, the represented tip of the closest relative was identified using published

64 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera phylogenies of families or genera (Zahiri et al., 2012; Sihvonen et al., 2011; Ta- lavera et al., 2013; Wahlberg et al., 2009; Sihvonen, Staude, and Mutanen, 2015). A visual inspection of the Lepidoptera phylogeny suggests that cycadivory has evolved independently in multiple lepidopteran lineages, with several origins likely within single families and potentially even single genera. For example, a poorly resolved phylogenetic hypothesis based on morphological data for

Taenaris does not place the cycadivorous species within a monophyletic clade or closely related to each other (Parsons, 1999), suggesting multiple origins of cycadivory in the genus. Similarly, an unpublished molecular phylogeny that includes some species of Luthrodes does not place the two included cycadivorous species as sister taxa (Wu, 2009). Conversely, cycadivory appears to be an ancestral trait in Eumaeus butterflies (6 species) and Diptychini moths (17 species). Given that both of these clades are warningly colored and obligately cycadivorous, it seems likely that cycad feeding or defensive traits (or both) have led to limited radiations in these groups. Dated phylogenetic hypotheses for all genera would be required to understand the general evolutionary significance of cycadivory and why some lineages have diversified while others are represented by just one or two species nested within otherwise non-cycadivorous clades. It is possible that ancient lepidopteran herbivores fed on cycad species that are now extinct, as cycads were widespread and abundant from the late Trias- sic through the Cretaceous (Taylor and Taylor, 1993; Niklas, Tiffney, and Knoll, 1983), during which time the Lepidoptera originated and diversified (Espeland et al., 2018; Misof et al., 2014). However, cycads subsequently experienced high rates of extinction and low diversity until the late Miocene, when all extant

65 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera cycad species arose (Nagalingum et al., 2011; Condamine et al., 2015; Salas- Leiva et al., 2013). Given the evolutionary history of cycads and phylogenetic placement of cycadivorous Lepidoptera, it is likely that transitions to cycadivory among extant cycadivorous Lepidoptera occurred within the last 15 to 20 million years. Indeed, at least in lyceanid butterﬂies the evolutionary origins of cycadivory appear to be somewhat recent. Talavera et al., 2013 dates the split between Luthrodes and its sister genus Chilades at ~6 MY. Notably, the cycadivorous species of Luthrodes included in the analysis are derived, placing the evolution(s) of cycadivory in this lineage as even younger. Similarly, an unpublished molecular clock analysis in Theclinesthes puts the origin of the genus at 2-3 MY (Eastwood, 2006). Finally, Espeland et al., 2018 places the split between Eumaeus and Calycopis at ~18 MY, making the origin of Eumaeus even younger as this analysis did not include the sister genus Theorema.

66 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

(1/6) (6/6) (3/8) ?

Eumaeus TheclinesthesLuthrodes

Taenaris (2/25)

Zerenopsis (8/8) Callioratis (6/6) Veniliodes (3/3)

Sierarctia (1/1)

Undet. Blastobasidae (1/>250) Anatrachyntis (2/>50) Erechthias (1/>150) Dasyses (1/8)

Cone feeding Leaf feeding Dead tissue feeding Warning Coloration

FIGURE 3.1: Phylogenetic placement of cycadivorous Lepi- doptera. Genera containing cycadivorous species are shown by red tips, with the butterﬂy clade in black and moths in grey. Warning coloration is indicated symbolically, along with the feeding guild and whether the species is facultatively or obligately cycadivorous (black and green, respectively). The number of cycadivorous species and the total number of species in the genus are given in parentheses.

Improved phylogenetic estimates for extant cycadivorous Lepidoptera would

67 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera be useful for reconstructing and comparing historical diet evolution among cycadivorous lineages. Research in this area is hindered by incomplete or erroneous hostplant records for many species (see Appendix C). Some have spec- ulated that monocot-feeding may be an evolutionary precursor to cycadivory because non-cycadivorous Taenaris feed on monocots (Schneider et al., 2002), though there is little evidence from other groups to support this as a broad pattern. Among lycaenids, close relatives of cycadivorous species feed on dicots in the families Fabaceae, Amaranthaceae, Proteaceae, Sapindaceae, Myrtaceae, and Euphorbiaceae (Dunn and Dunn, 1991; Braby, 2000). Cycadivorous moths and their close relatives exhibit a broad range of hostplant preferences that includes both monocots and dicots. Improved knowledge of the evolutionary histories of cycadivorous lineages would provide a framework for testing hypotheses about evolutionary precursors to cycadivory and host breadth among extant species.

3.5 Hostplant Use

Based on the records reported here, cycadivorous Lepidoptera utilize 7 of the 10 recognized cycad genera (Calonje, Stevenson, and Osborne, 2019). Absent among accepted hostplant genera are Lepidozamia, Bowenia and Microcycas. These are all small genera (Lepidozamia: 2 species; Bowenia: 2 species; Microcycas:1 species) but are fairly well studied and co-occur with other cycad species that are known lepidopteran foodplants, making their omission all the more curi- ous.

68 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

All cycadivorous butterflies appear to be obligate cycad specialists while cycadivorous moths exhibit a broader range of dietary preferences. Seirarctia echo is the only confirmed facultative cycad folivore, accepting leaves from a wide variety of hostplants from several plant families. The ecological causes and consequences of feeding on cycad versus non-cycad plants are completely unex- plored in this species. Diptychini moths are facultatively polyphagous in their 4th-6th instars but all species are obligate cycad specialists for the first 3 instars. Donaldson and Basenberg, 1995 found no significant differences in survival rate, developmental duration or pupal mass between 4th instar Z. lepida larvae reared on angiosperm versus cycad hosts. Staude and Sihvonen, 2014 have suggested that some Diptychini moths may not require cycads even in their early stages, as they collected a single final-instar Z. tenuis larva feeding on the leaves of a baobab tree (Adansonia digitata, Malvaceae) on Misali Island, Tanzania, where no cycads were found. The remaining cycadivorous moth species are either highly polyphagous (e.g. Dasyses rugosella) or their host breadth is unknown (e.g. Erechthias sp.). Whereas not all cycadivorous Lepidoptera are specialists of cycads, their larvae are specialized on particular plant tissues and can therefore be categorized into discrete feeding guilds. These guilds include leaf chewers, leaf miners, ovulate cone feeders, pollen cone feeders, and detritivores, and the larvae in each of these guilds likely experience qualitative and quantitative differences in exposure to cycad toxins. For example, pollen cone feeders may experience reduced exposure to toxins since pollen cones generally have lower toxin concentrations than leaves or ovulate cones, and at least one cycad toxin, BMAA, appears to

69 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera be sequestered in specialized cells in the pollen cones that can pass through the guts of other insects intact (Vovides et al., 1993; Norstog and Fawcett, 1989). Detritivorous species feed on decaying cycad pollen cones and stems that may also harbour lower concentrations of toxins. In contrast, Eumaeus butterﬂy larvae feed on both ovulate and pollen cones as well as leaves (González, 2004;

Cascante-Marín and Araya, 2012), and some evidence suggests that Z. lepida moths also feed on ovulate cones in addition to leaves (Donaldson, 1991). Among cycad specialists, it appears that larvae can accept diverse cycad species and hostplant breadth is expanding for several species, particularly as exotic cycads are planted as ornamentals in gardens worldwide. The Caribbean species Eumaeus atala, for example, historically fed only on Caribbean cycads in the genus Zamia, but have been obsereved laying eggs and feeding on cultivated Central American cycad species that are outside of the native range, as well as some species of African, Australian, and Asian cycads. The ability to feed on non-native cycads has been observed in other Eumaeus species as well (Bosque and Rosales-Robles, 2015), and increased hostplant breadth has been reported for Luthrodes pandava, a widespread species that feeds on numerous native and exotic cycads across Asia and the Middle East (Wu et al., 2010; Tiple et al., 2009; Fric et al., 2014; Feulner et al., 2014).

Contemporary host use may challenge the species status of Luthrodes peripatria, which some authors consider to be a subspecies of Luthrodes pandava. The natural range of L. pandava is widespread across southern Asia (excluding Tai- wan), whereas L. peripatria is endemic to Taiwan and has historically fed on a single cycad species restricted to southeastern Taiwan, Cycas taitungensis (Shen,

70 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

Hill, and Chen, 1994). In the past 30 years, L. pandava has been introduced to Taiwan along with several exotic Cycas species. As both Luthrodes species accept native and non-native Cycas species as hostplants, expanded hostplant use and range overlap likely provide opportunities for interbreeding. Further assessment of the population structure, introgression, and species status of L. pandava and L. peripatria would be fruitful (but see Wu et al., 2010). Hostplant specialization may promote divergence in the Australian species

Theclinesthes onycha, for which two subspecies are recognized, T. onycha onycha and T. onycha capricornia. T. o. onycha feeds only on Cycas species distributed from Queensland to southern New South Wales, whereas T. o. capricornia feeds only on Macrozamia cycads found in central Queensland. They overlap in their distributions in a narrow region in central Queensland, though microhabitat preferences may maintain allopatry even within this contact zone. Patterns of hostplant use and mate choice are not well described within the contact zone, though Eastwood, 2006 found considerable genetic differentiation in the mitochondrial genes of each subspecies, suggesting that there is little to no gene ﬂow between them. Careful analysis of hostplant use, species relationships, and reproductive barriers would also be useful for the two pairs of sympatric species of Eumaeus butterﬂies in Central and South America. Eumaeus childrenae and E. toxea co- occur in some parts of their ranges in Mexico, where they are easily distinguished based on wing pattern. These species are likely quite diverged and they utilize different cycad genera as hostplants throughout much of their range, though detailed studies of host use in areas of sympatry and allopatry have not

71 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera been carried out. Eumaeus toxana and E. minyas both occur in South America and according to published records their ranges overlap in Peru. However, it is difﬁcult to glean even basic natural history information for these two species due to widespread mistakes in species identiﬁcations in the published literature.

Eumaeus minyas is commonly confused with several other Eumaeus species, especially E. toxana and the isthmus species E. godartii, but also E. toxea and even E. atala. Credible accounts of the distributions and range limits for these two species are needed, with E. toxana being particularly under-collected and poorly studied.

Among Caribbean species, the ranges of Eumaeus atala and Seirarctia echo overlap in southern Florida but there are very few records of both species occurring in the same place, suggesting that there is some displacement at a relatively

fine spatial scale. Since S. echo is broadly polyphagous, hostplant competition is unlikely to be a sufficient explanation for this displacement. Furthermore, the range of E. atala does not occupy the entire range of its hostplants in Florida, and a better understanding of the factors that determine the range boundaries of these species would be very valuable for the management of local butterfly and cycad populations.

3.6 Management of Cycadivorous Species

With 75% of the 355 extant cycad species threatened with extinction, cycads are the most imperiled plant order in the world (Calonje, Stevenson, and Osborne, 2019; Gilbert, 2010; Baillie, Hilton-Taylor, and Stuart, 2004). The lepidopteran

72 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera herbivores of cycads therefore present something of a conservation conundrum: obligate cycad specialists almost certainly warrant formal protection, yet they are natural enemies of endangered plants. Conservation efforts that prioritize plants over insects (or vice versa) could have unintended negative consequences (e.g. Wu et al., 2010); instead, conservationists should prioritize the co-existence of these interacting species (Marler, Lindström, and Terry, 2012). However, co- management of multiple interacting species typically requires extensive knowledge of the natural history and population dynamics of each target species.

The lycaenid butterﬂy Eumaeus atala has served as a ﬂagship species for local conservation efforts in Florida, USA. Once common across the southeastern parts of the state, overharvesting of Florida’s cycad populations in the early

1900’s led to the extirpation of E. atala from Florida. In 1959 a small breeding population was discovered near Miami (Rawson, 1961), and subsequent grass- roots conservation efforts have led to the dramatic recovery of Florida’s E. atala populations. However, overzealous reintroductions of E. atala have the potential to be catastrophic for local cycads, including both native species and ornamental exotics. Like many cycadivorous Lepidoptera, E. atala larvae are prodigious feeders and a single clutch can decimate a large cycad, which are exceedingly slow to grow and reproduce. The African cycad moths are also of serious conservation concern due to habitat loss and large-scale poaching of cycads from the wild (Staude and Sihvonen, 2014), and one species, Callioratis millari, is considered critically endangered in South Africa (Mecenero et al., 2013). Conservation of cycads and their herbivores is made more challenging by the emerging pest status of some cycadivorous species. Luthrodes pandava is already

73 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera considered a terrible pest of Cycas plants in India, Taiwan (Wu et al., 2010), Sin- gapore (Marler, Lindström, and Terry, 2012), Papua New Guinea (Tennent, 2014) and Guam (Moore et al., 2005), and has recently been introduced in Egypt (Fric et al., 2014) and possibly the United Arab Emirates (Feulner et al., 2014). While there are no reports of L. pandava in Australia or the neotropics, its introduction would likely present a signiﬁcant threat to cultivated and perhaps native cycads in these regions. The invasiveness of this species is exacerbated by its broad hostplant use: L. pandava larvae have been recorded feeding on 85 species of Cycas in a tropical botanical garden in Thailand (Marler, Lindström, and Terry, 2012) and can feed on other cycad genera as well. Theclinesthes onycha are also prone to localized population outbreaks in Australia (Rathie, 2004), and even

Eumaeus atala have been known to reach destructive population sizes at highly localized scales in southeastern Florida. While occasional outbreaks may represent natural population cycles, the increasing availability of non-native cycads that are sold as garden ornamentals could lead to novel population dynamics even within species’ native ranges, with unknown consequences for local cycad populations.

3.7 Discussion

Cycadivorous Lepidoptera comprise a ’component community’ of distinct lineages with varying degrees of specialization and diverse feeding ecologies, and therefore present numerous opportunities for comparative studies of ecoevo- lutionary dynamics (e.g., Farrell, 2001). With improved molecular phylogenies

74 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera and natural history information researchers can begin to identify the factors that have led to adaptation and the consequences of speciﬁc adaptations on diversi- ﬁcation in cycadivorous lineages. Until then, we speculate here on some of the salient questions regarding cycad-Lepidoptera interactions.

Is cycad-feeding adaptive? Herbivory is considered an adaptive trait among insects when evolutionary shifts to herbivory are associated with increased diversification in phytophagous lineages relative to their aphytophagous sister groups (Mitter, Farrell, and Wiegmann, 1988; Wiens, Lapoint, and Whiteman, 2015). Evolutionary transitions to feeding on plants that contain defensive secondary compounds that deter other herbivores are claimed to promote diversification of Lepidoptera through escape and radiation (e.g., Braby and Trueman, 2006). If cycadivory has similarly promoted diversification in lepidopteran lineages, then it might be considered an adaptive trait. Based on the phylogenetic pattern shown in Figure 3.1, Eumaeus butterflies and Diptychini moths exhibit modest radiation following their transition to cycad-feeding, whereas other cy- cadivores remain as only one or two species at the tips of otherwise angiosper- mivorous clades. Why have some cycadivorous lineages diversified while others have not? That the largest clades of cycadivorous Lepidoptera are also aposematic suggests that defensive ecology may play a role in diversification: perhaps it is not cycadivory per se that leads to diversification in some lineages, but rather the subsequent evolution of aposematism. This explanation is consistent with the cryptic coloration of cycadivorous species that have not radiated, though

75 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera a few exceptions must be considered. Luthrodes cleotas and Seirarctia echo are both known to sequester cycad toxins and could be considered warningly colored; why have these species not diversiﬁed? Cycadivorous Taenaris species are also warningly colored but do not appear to have radiated (though even non- cycadivorous Taenaris are considered aposematic so this situation may be more complicated). It may be that evolutionary trade-offs or constraints have limited diversiﬁcation in these groups, that other cycadivorous relatives once existed but have gone extinct, or that cycadivory has evolved too recently for diversi-

fication to have yet taken place. Indeed, cycadivorous species of Luthrodes and Theclinesthes appear to be very young, and it would be interesting to compare their ages to those of Eumaeus butterflies and Diptychini. Among generalists, cycadivory is not expected to significantly influence speciation rates (at least for detritivorous moths), though Seirarctia echo and Erechthias sp. may be exceptions given that they possess adaptations for feeding on cycads’ fresh leaf tissue of cycads.

Is there evidence of coevolution between cycads and their lepidopteran herbivores? All cycadivorous Lepidoptera must possess adaptations to circumvent or tolerate cycad-speciﬁc defenses, and the selective value of cycad defensive traits against herbivores seems clear. Do cycadivorous Lepdioptera exert selective pressure on their host cycads to become more toxic? While there is little de- bate about the importance of plant defensive traits for herbivore ﬁtness (Janz

76 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera and Nylin, 1998; Futuyma and Agrawal, 2009), the importance of insect herbivores as selective agents is less clear as most plants seem able to tolerate intermediate levels of herbivory without a significant reduction in fitness (Cornell and Hawkins, 2003). Evidence of reciprocal adaptation between pairs of plants and herbivores has been relatively scarce (Farrell, 1993) , and the step-wise selection scenario initially envisaged by Erlich & Raven appears to be extremely asymmetrical: shifts to chemically novel hosts lead to bursts in diversification in many herbivore groups, but escape from herbivores through chemical nov- elty seems to have had little impact on diversification rates in most plant groups (Farrell, 1998; Wheat et al., 2007) (but see Berenbaum and Zangerl, 1998 and Edger et al., 2015). Yet damage inflicted by folivorous Lepidoptera can be so extreme that just a few generations can kill a large cycad, which are exceedingly slow to grow and reproduce. Selective pressures exerted by specialist herbivores may therefore be especially severe for cycads relative to other plants groups, raising the possibility that some lepidopteran herbivores could select for escalated chemical defenses and perhaps influence the diversification of their cycad hosts. Previous work has identified diverse secondary compounds in cycads (Pan et al., 1997; Snyder and Marler, 2011; De Luca et al., 1982) that appear to be evolving (De Luca et al., 1982), but phylogenetically explicit comparisons of cycad defensive chemistries (toxins, antinutritive compounds, and volatile organic compounds) would be required to look for evidence of phytochemical escalation and coevolution sensu Ehrlich & Raven.

77 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

Of course, the phylogenetic distribution of cycadivory in Lepidoptera suggests repeated, independent colonizations of cycads from distantly related angiosperm hosts, and co-speciation with cycads is reasonably plausible only among

Eumaeus butterflies and Diptychini moths. Attention should therefore be focused on assessing coevolution between Eumaeus with the new world cycad genera Zamia, Dioon, and Ceratozamia, and between the African Diptychini moths with cycad genera Encephalartos and Stangeria. All cycad genera likely diversified within the last 10-20 MY (Nagalingum et al., 2011; Condamine et al., 2015; Salas-Leiva et al., 2013; Calonje et al., 2019). While we do not have age estimates for Eumaeus we know that it is less than 18MY old (Espeland et al., 2018) and likely much younger, though dated phylogenetic analyses are needed to properly evaluate the possibility of co-diversification in any lineage pair.

How does cycadivory evolve? Identifying evolutionary and ecological precursors to cycadivory could help explain the repeated transitions to cycads among Lepidoptera. For example, did the host plants of ancestral species somehow facilitate shifts to cycad feeding, either through chemical similarity or other fea- tures? From the data gathered and presented here, there is no evidence that cycadivory has evolved from a single, shared host lineage. The ancestors of cycadivorous taxa likely fed on diverse angiosperms including both monocots and dicots, though improved phylogeographic and life history information will be required to infer the most likely ancestral food plants of cycadivorous lineages. Hypotheses regarding what the ancestors of cycadivorous species ate prior to their transitions to cycads may suggest as yet unknown chemical similarities

78 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera between cycads and some angiosperm groups. Or, if no chemical similarities are found, it raises questions about the origins of potentially novel adaptations for overcoming cycads’ defenses. Lepidoptera are known to employ numerous adaptations for feeding on chemically defended host plants. These include behavioral adaptations (Dussourd and Denno, 1991), physiological mechanisms (Hartmann et al., 2005), and perhaps even associating with symbiotic gut bacteria (e.g. Salzman, Whitaker, and Pierce, 2018, Chapter 2), though this is considered rare in Lepidoptera (Whitaker et al., 2016; Hammer et al., 2017). Increased host breadth and feeding on select plant tissues can also minimize an insect’s exposure toplant defensive compounds. Even Diptychini moths – which we consider to be obligate cycad specialists – often switch to feeding on angiospermous plants and thereby potentially reduce their exposure to cycad defenses. It is presently unknown which specific adaptations might be required for cycadivory, or how widely specific adaptations are shared across and within feeding guilds, e.g., among specialized folivores. Sierarctia echo are capable of modifying dietary MAM into its non-toxic form (Teas, 1967), but it is unknown whether other herbivores actively detoxify MAM using a similar mechanism. Moreover, no adaptations have been identified to date that would enable herbivores to cope with BMAA, steryl glycosides, or other defensive compounds, let alone complex phytochemical mixtures. Fi- nally, herbivores need to locate and discriminate between potential host plants, and while previous work has described chemical cues used by the insect pollinators of cycads (Terry et al., 2005), no work has addressed chemical communication between cycads and lepidopteran herbivores.

79 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera

Different lepidopteran lineages may also experience different evolutionary constraints in their ability to feed on cycads. Among butterflies, the Nymphal- idae appear to be particularly constrained in their ability to colonize new hostplant families (Hamm and Fordyce, 2015), whereas the Lycaenidae exhibit enor- mous trophic diversity that includes both phytophagous and aphytophagous diets (Pierce et al., 2002). Indeed, Ehrlich & Raven were able to identify few phylogenetic patterns in lycaenids’ host use and were puzzled by their ’bewildering array’ of host plant affiliations. The only published lycaenid genome demon- strates significant expansion in detoxification and digestion enzymes (Cong et al., 2016), which, if shared broadly across the family might explain why lycaenid butterflies seem predisposed to trophic innovation, including repeated coloniza- tion of cycads over the last 20 MY. Even so, feeding on chemically defended host plants and sequestration of host plant defensive chemicals is rare among lycaenids (Fiedler, 1996).

3.8 Conclusions

Cycadivorous Lepidoptera are remarkably diverse in their defensive strategies, life histories, and hostplant relationships, providing numerous opportunities for research. Despite several decades of research on a handful of focal species, many cycadivorous Lepidoptera remain understudied, undersampled, and un- described. Additional systematic surveys of herbivore diversity and host breadth, studies of predator and parasitoid pressures in natural habitats, and investiga- tions into insects’ adaptations to cycad toxins would be valuable, along with

80 Chapter 3. Ecology and Evolution of Cycad-Feeding Lepidoptera genus- and tribe-level phylogenies of cycadivorous groups and their sister taxa. Future studies should consider other insect groups as well, as cycadivory has been reported among larvae and adults of non-pollinating beetles (Coleoptera) (Marler and Muniappan, 2006); bees (Hymenoptera) (DeVries, 1983; Ornduff, 1991; Terry, 2001; Valencia-Montoya et al., 2017); leaf-mining larvae of an uniden- tiﬁed ﬂy (Diptera) (DeVries, 1983); termites (Blattodea) (Marler, Yudin, and Moore, 2011); and phloem-feeding scale insects, aphids, and mealybugs (Hemiptera). In summarizing what is known about the phylogenetic placement of cycadivorous Lepidoptera, along with their hostplant relationships, defensive ecology, and management, we hope to encourage more research on the insect herbivores of cycads and other gymnosperms.

81 Concluding Remarks

In this thesis I have used the plant order Cycadales as a backdrop to study symbiosis. I choose cycads because of their long evolutionary history and many understudied and highly speciﬁc species interactions, but also because I fell in love with these plants the ﬁrst time that I heard about them in Chelsea Specht’s undergraduate plant morphology course at UC Berkeley. Dr. Specht is the academic granddaughter of Knut Norstog, whose wonderful quote opens this thesis and guides my work. Dr. Norstog believed that we should view the living cycads as the Rosetta Stone for plant biology, and I would add, symbiosis biology and evolutionary biology.

The Neotropical genus Zamia maintains multiple specialized organismal relationships. Rhopalotria weevils have an obligate brood site pollination mutualism with Zamia in which the entire genus of weevil feeds solely on Zamia male cone parenchyma tissue, and each Zamia species is pollinated by a single Rhopalotria species (Tang, 1987b; Norstog and Fawcett, 1989). A phylogenetic analysis of these beetles and their hosts will enable us to assess whether the cospeciation that we expect to find between them also reflects the kind of coevolution we might expect from such an exquisetley co-evoloved system. Bee- tles in the genus Pharaxanotha also feed on and pollinate Zamia, although less is known about their specificity (Fawcett and Norstog, 1993; Tang et al., 2018),

82 Concluding Remarks and they would also be fruitful candidates for further phylogenetic analysis, in part so that their histories might be compared with those of Rhopalotria and their hosts. In contrast to these specialized pollination mutualisms, Zamia cycads also possess a number of specialized insect herbivores such as those found in the lycaenid butterﬂy genus Eumaeus. This thesis found yet a third layer of specialization by identifying key gut bacterial species shared by unrelated and geographically distant cycad herbivores (Chapter 2, Salzman, Whitaker, and Pierce,

2018). Zamia and its multiple specialized organismal relationships represent a natural simplified interaction network. The simplicity of the network and specialization of the interactions allows for evolutionary analysis of an entire network of specialized interacting partners that could ultimately provide insight into the the causes and consequences of specialized species interactions. Assessing the population structure of Rhopalo- tria weevils that are shifting host plants could elucidate the potential for specialized species interactions to lead to lineage diversification. Understanding the mechanisms of host plant perception and acceptance in these populations will also illuminate how insect host preference might contribute to partner fidelity as well as lineage diversification. This work has already spurred multiple lines of research. The work presented in Chapter 1 prompts the obvious question: Is there evidence of coevolution between pollinators and plants? In work done during my thesis and continued in my postdoc, I am undertaking systematic surveys of plant volatiles and insect response to look for reciprocal evolution of those phenotypes. The results found in Chapter 2 instigated a project with Drs. Angélica Cibrián-Jaramillo,

83 Concluding Remarks and Francisco Barona-Gómez, investigating the potential for the gut bacterial communities to aid in detoxiﬁcation of cycad toxins and identifying emergent bacterial gene clusters unique to the core bacteria found in cycad herbivores. Chapter 3 introduces the concept of cycadivorous lineages as excellent candi- date systems for comparative life history strategies and has lead to phylogenetic analyses of lyceanid cycad herbivores with Dr. Melissa Whitaker. As a graduate student, I have had the great good fortune to study a system that I love, investigating questions that intrigue me, and working with collaborators who inspire me. I look forward to continuing my research on this beauti- ful system with these incredible researchers during my postdoctoral studies.

84 Appendix A

Supplement Chapter 1

85 Appendix A. Supplement Chapter 1

FIGURE A.1: Schematic of pit test

86 Appendix A. Supplement Chapter 1

FIGURE A.2: Schematic of behavioral arena

87 Appendix B

Supplement Chapter 2

Published article: Cycad-feeding insects share a core gut microbiome supporting material

The following pages contain a reproduction of the paper supporting material.

88 Appendix B. Supplement Chapter 2

Appendices

Supplementary Material 1: Non-cycadivorous lycaenid and curculionid samples that passed filtering requirements and were analyzed for the presence of shared bacterial OTUs found in cycad herbivores.

Outgroup herbivore Order # Host plant Locality Citation Jalmenus daemeli Lepidoptera: 3 Acacia Queensland, Whitaker et al. 2016 (Lucas) Lycaenidae (von Martius) Australia Anthene usamba Lepidoptera: 4 Vachellia Laikipia, Kenya Whitaker et al. 2016 (Talbot) Lycaenidae (Wight & Arn.) Hypothenemus Coleoptera: 4 Coffea arabica Chiapas, Mexico Ceja-Navarro et al. 2015 hampei (Ferrari) Curculionidae (Linnaeus) Hypothenemus Coleoptera: 4 Cecropia Chiapas, Mexico Ceja-Navarro et al. 2015 eruditus (Westwood) Curculionidae (Loefl.) Hypothenemus Coleoptera: 4 Cecropia Chiapas, Mexico Ceja-Navarro et al. 2015 crudiae (Panzer) Curculionidae (Loefl.) Scolytodes maurus Coleoptera: 5 Cecropia Chiapas, Mexico Ceja-Navarro et al. 2015 (Blandford) Curculionidae (Loefl.)

Supplementary Material 2: Species core microbiome OTU and assigned taxonomy are listed with OTUs present in more than one species highlighted in grey. Species’ microbiome cores were determined by OTU presence in 100 percent of that species samples after filtering and are represented by their OTU#.

Chilades Eumaeus Rhopalotria Pharaxanotha Eubulus pandava atala furfuracea floridana sp. Unknown 3 3 3 3 3 Alicyclobacillaceae Unknown 85 85 85 85 85 Rickettsiaceae Unknown 249 249 249 249 249 Enterobacteriaceae Enterobacteriaceae Raoultella 5 5 5 5 5 ornithinolytica Unknown 4 4 4 4 4 Enterobacteriaceae 596 596 Comamonadaceae 13 13 13 Curvibacter Comamonadaceae 36 36 Lampropedia Comamonadaceae 432 432 Comamonas Enterococcaceae 15 15 15 Enterococcus Streptococcaceae 10 10 Lactococcus Alcaligenaceae 24 24 Achromobacter Unknown 240 240 Blattabacteriaceae

89 Appendix B. Supplement Chapter 2

Sphingobacteriaceae 17 17 Sphingobacterium 187 37 Moraxellaceae 763 91

Acinetobacter 2 2 21 47 9 Pseudomonadaceae 443 123 Pseudomonas 1045 Rhodobacteraceae 212 65 Rhodobacter Enterobacteriaceae 487 Pantoea Enterobacteriaceae 1526

Serratia 654 Sphingobacteriaceae 75 Novosphingobium Weeksellaceae 7 Weeksella Weeksellaceae 88 Chryseobacterium Flavobacteriaceae 251 Flavobacterium Xanthomonadaceae 1555

Stenotrophomonas 6 Microccaceae 279 Arthrobacter Brucellaceae 14 Ochrobactrum Enterobacteriaceae 12 Erwinia Unknown 642 Enterobacteriaceae Alcaligenaceae 112 Alcaligenes Porphyromonadaceae 78

Dsygonomonas 77 Porphyromonadaceae 159 Parabacteroides Bacteroidaceae 94 Bacteroides Weeksellaceae 167 Wautersiella Hyphomicrobiaceae 132 Devosia Cytophagaceae 18 Leadbetterella Erysipelotrichaceae 23 Erysipelothrix Rhizobiaceae 30 Agrobacterium

90 Appendix B. Supplement Chapter 2

Supplementary Material 3: Oligotyping results

Seven entropy peaks were necessary to decompose oligotypes for OTU5 (Raoutella ornithinolytica). Twenty one unique oligotypes were found and represented 16,154 of the 16,383 sequence reads (98.60%). Eubulus sp. and Eumaeus atala are most strongly represented in this OTU (34.73% and 33.03%) with Pharaxanotha floridana close behind (22.11%). Rhopalotria furfuraceae is represented by 9.50% and Chilades pandava by only 0.64%. P. floridana has the most consistent composition of oligotypes across all species with Eub. sp. showing a similar although less constrained pattern (Figure 3). Eum. atala is completely dominated (> 93%) by one oligotype across all samples.

Six entropy peaks were necessary to decompose oligotypes for the 16,383 sequence reads for OTU 4 (Unknown Enterobactereaceae). The resulting 20 unique oligotypes represent 16,223 reads (99.02%). P. floridana and Eub. sp. represent the majority of sequences in this OTU (60.65% and 28.57% respectively) with relatively low abundances in R. furfuraceae (7.71%) and Eum. atala (2.77%) and very few in C. pandava (0.30%). P. floridana and Eub. sp. show consistent patterns of oligotype composition, with P. floridana again more constrained (Figure 3). OTU 4 oligotypes are varied across the remaining species.

Three entropy peaks decomposed oligotypes for the 522 reads for OTU 249 (Unknown Enterobactereaceae). The resulting three oligotypes represented 464 reads (88.89%). All species are fairly evenly represented in the oligotyping (C.s pandava, Eum. atala, and R. furfuraceae each 15.52%, Eub. sp. 28.66% and P. floridana 24.79%). One oligotype dominated across all samples with no species showing a consistent pattern regarding the remaining two oligotypes.

Oligotyping analysis found no entropy peaks for either OTU 85 (Unknown Rickettsiaceae) (1,092 reads) or OTU 3 (Unknown Alicyclobacillaceae) (16,383 reads) suggesting that these two OTUs are represented by one oligotype each across all herbivore samples or that rare oligotypes are not present in enough abundance in the dataset to be distinguished from sequencing noise.

91 Appendix C

Supplement Chapter 3

C.1 Evaluating hostplant records

In preparing this review, we found it challenging to confirm the accuracy of published hostplant records, even for relatively well-studied organisms such as butterflies. In the words of Robinson et al., 2010, "erroneous hostplant records are cumulative." Through repeated citation these errors propagate throughout the literature, such that the original errors become increasingly difficult to find and correct. In an effort to confirm as many records of cycadivory as possible, we have traced back every citation (when possible) until original errors were found. We successfully located original sources in all cases unless otherwise noted. Table C.1 shows unconfirmed hostplant association(s) with the original source and citing authors. An explanation for each record follows. In compiling this list we noticed a few common errors that were made by multiple recorders. For example, many authors list Cycas circinalis as a larval hostplant even in areas where C. circinalis does not occur (e.g., Brazil). We gather that Cycas circinalis

92 Appendix C. Supplement Chapter 3 was something of a catchall name for cycads prior to improved knowledge of cycad taxonomy and distributions. Additionally, many authors attempt to identify Lepidoptera based on larval morphology alone. Doing so is especially error- prone for moths and lycaenid species, as these larvae are exceedingly difﬁcult to identify even by trained experts. Such records therefore require additional scrutiny. Importantly, not all records listed here are taken to be erroneous. Several records simply lack necessary data and are therefore provisionally listed here pending further conﬁrmation.

93 Appendix C. Supplement Chapter 3

TABLE C.1: Unveriﬁed host plant records

Species Host(s) Sources

Nymphalidae 1. Opsiphanes invirae Cycas circinalis Schneider et al., 2002; Robinson et al., 2010; Peña and Espeland, 2015

2. Elymnias agondas Cycas circinalis, C. revoluta Robinson et al., 2010; Merrett, 1993; Peña and Espeland, 2015

3. Faunis eumeus Cycas revoluta Robinson et al., 2010; Easton and Pun, 1997

Lycaenidae 4. Eumaeus atala Manihot Comstock, 1948; Ehrlich and Raven, 1964

5. Eumaeus childrenae Amaryllis Ehrlich and Raven, 1964; Schnei- der et al., 2002; Contreras- Medina, Ruiz-Jiménez, and Luna Vega, 2003

6. Euchrysops cnejus Cycas Robinson et al., 2010; Cayabyab, 1993; Io, 1994

7. Acytolepis puspa Cycas rumphii Robinson et al., 2010; Cey- lon Department of Agriculture, 1924; Vane-Wright and Jong, 2003

8. Everes lacturnus Cycas Antony, Prasad, and Kalesh, 2016

9. Chilades kimurae Cycas revoluta Wakabashi and Yoshizaki, 1967 10. Luthrodes pandava Non-cycad plants in the fam- Tiple et al., 2009; Schneider et al., ilies Fabaceae and Sapan- 2002; Ehrlich and Raven, 1964; daceae Nitin et al., 2018 94

Pyralidae 11. Unknown Zamia amblyphyllidia, Z. por- Franz and Skelley, 2008 tophylla 12. Cryptoblabes plagi- Cycas rumphii Hinckley, 1964 oleuca

Erebidae 13.Orvasca subnotata Cycas circinalis Robinson et al., 2010; Tiple et al., 2009; Sevastopulo, 1941; Moore et al., 2005

Tortricidae 14.Cryptoptila immersana Bowenia serrulata Common, 1990; Wilson, 1993

Psychidae 15.Oiketicus kirbyi Cycas revoluta Robinson et al., 2010 Appendix C. Supplement Chapter 3

Explanations

1. The neotropical nymphalid species Opsiphanes invirae is often claimed to feed on Cycas circinalis. Authors typically cite Ackery, 1988 or Penz, Aiello, and Srygley, 1999, although the latter source also highlights this hostplant record as dubious. The original source is a catalog of Brazilian insects and

their host plants (Silva et al., 1968) that states that O. invirae larvae feed on "palmeira de jardim," which translates simply to "garden palm." How this

was interpreted to mean Cycas circinalis is unknown, though hostplants for O. invirae do include several palm species. In due diligence, we sought all original sources for the Silva et al., 1968 publication, many of them obscure Brazilian agricultural articles from the turn of the century and were successfully able to access 16 of the 21. None of these mentioned cycads as a host plant.

2. Merrett, 1993 reports ﬁnding a single pupa of another nymphalid butter-

ﬂy, Elymnias agondas, on Cycas circinalis in a ﬁeld study in New Guinea. He then attempted to rear Elymnias agondas larvae on cycads but observed 100% mortality among larvae. We suspect that the pupa fed on palms and merely wandered onto the cycad to pupate. Interestingly, this species co- occurs with and is thought to be involved in mimetic relationships with

Taenaris catops, which feeds on cycad exudates as an adult.

3. Easton and Pun, 1997 reared larvae of the nymphalid Faunis eumeus on Cy- cas revoluta and several palm species in Macau, China, noting that these

95 Appendix C. Supplement Chapter 3

are not the normal hostplants for this species. The authors report noth- ing about the performance of larvae fed on cycad versus non-cycads, so we assume that larvae were able to develop. Even so, considering that this species does not normally feed on cycads we do not consider it to be naturally cycadivorous, though further study would be helpful.

4. The larvae of Eumaeus atala have been said to feed on introduced Manihot species (cassava) by Comstock, 1948 and Ehrlich and Raven, 1964 (citing "Comstock unpubl."). Comstock does not provide any citations or details about the observation so the basis of this hostplant association is unclear, but given that cassava is widely cultivated throughout the Caribbean yet

no other records exist for E. atala feeding on them, we classify this as an erroneous record.

5. Ehrlich and Raven, 1964 note that the larvae of Eumaeus childrenae feed on Amaryllis (Amaryllidaceae), citing only "Comstock unpubl." Schneider et

al., 2002 also mention that E. childrenae feed on Amaryllis , citing Draudt, 1921. However, Comstock, 1948 highlights this as a likely dubious report:

"Dr. M. Draudt, in Seitz, says that "the carmine, black-belted

[E. childrenae] larva lives gregariously on an Amaryllis standing in water." His reference does not give the speciﬁc name of the plant and the designation of "black-belted" does not tally with

the [E. childrenae] larva reared by us, unless the sparse black hairs crossing the center of the segments gives an impression of black bars."

96 Appendix C. Supplement Chapter 3

Based on the physical description of the larva and the fact that no further observations have been recorded since 1921, it is likely that Draudt

misidentiﬁed the larva found feeding on Amaryllis as Eumaeus childrenae.

6. The lycaenid butterﬂy Euchrysops cnejus presents a somewhat tricky case. Vane-Wright and Jong, 2003 state that the larvae of this species accept Cy- cas (species not given) in Sulawesi, but the authors provide only a single citation (Watson et al. 1995). However, Watson, Ooi, and Girling, 1995 do

not mention Euchrysops cnejus feeding on Cycas, so perhaps this record is based on the authors’ own ﬁeld observations. Cayabyab, 1993 also claim

to have found E. cnejus on Cycas revoluta but provide no details, vouchers, or photographs. Euchrysops cnejus is quite polyphagous among lycaenids, so it is certainly possible that the larvae accept cycads as larval foodplants.

On the other hand, E. cnejus larvae are nearly identical to Luthrodes pandava larvae, so it is also possible that the authors confused these two species.

Finally, Chou, 1994 Monograph of Chinese Butterﬂies also lists Cycas as a foodplant of E. cnejus but we are unable to further evaluate the this record.

7. The lycaenid species Acytolepis puspa was reported to feed on Cycas (species not given) by Vane-Wright and Jong, 2003, citing Fukuda et al., (1992) and Fiedler, unpubl. We were unable to ﬁnd a publication by Fukuda et al. (1992). However, (Fukuda et al., 1984 does not list cycads as a food source

for Acytolepis puspa, nor do more recent accounts of this species’ larval foodplants by the same author (Igarashi and Fukuda, 2000). We are therefore unable to trace the original source for this hostplant record. A second publication, a list of agricultural pests in India published in 1924, states

97 Appendix C. Supplement Chapter 3

that Cyaniris puspa (a former synonym for Acytolepis puspa) feeds on Cycas circinalis and Cycas rumphii (Ceylon Department of Agriculture, 1924), but does not provide citations or details. We conclude that further documen- tation is needed regarding hostplant use in this species.

8. A single record exists for Everes lacturnus (Lycaenidae) feeding on Cycas (species not determined) from a survey of butterflies found on the Kerala University Campus in India (Antony, Prasad, and Kalesh, 2016). Given the difficulty of identifying Polyommatine larvae and that this is an isolated record, we suspect the larva was misidentified.

9. Wakabashi and Yoshizaki, 1967 reported feeding by "Chilades kiamurae" on Cycas revoluta in Japan, providing extensive photographic evidence of larval feeding and female oviposition on the plants. However, Chilades kiamurae is not a recognized species (Talavera et al., 2013), and though it is sometimes considered a subspecies of Luthrodes mindora, L. mindora does not occur in Japan. Based on the photographic evidence provided by Wak- abayashi & Yoshizaki, we suspect that the larvae they observed feeding on

cycads were most likely to be Luthrodes pandava, a conﬁrmed cycadivorous species.

10. The lycaenid species Luthrodes pandava has been recorded feeding on numerous native and introduced cycads across its large range, and several

authors claim that L. pandava larvae have been found feeding on non-cycad hostplants as well. Tiple, Khurad, and Dennis, 2011 note that they were

98 Appendix C. Supplement Chapter 3

able to rear L. pandava larvae on non-cycad hosts in captivity. We tenta- tively list these non-cycad host records here due to the extreme difﬁculty

of identifying juvenile stages of Luthrodes larvae, and the high likelihood of authors confusing L. pandava larvae with other co-occurring lycaenid species. However, it may indeed be true that L. pandava feed on both cycad and non-cycad hosts, in which case they would provide the only example of polyphagy among cycadivorous butterflies. We encourage efforts to confirm the host breadth of this species through continued captive rearing efforts combined with expert identification and molecular sequencing.

11. Larvae of an unidentified moth were found feeding on Zamia amblyphyllidia and Z. portophylla male cones in Puerto Rico (Franz and Skelley, 2008). The authors placed the moth in the family Pyralidae but did not provide details about the method of identification, and we wonder if they may have mistaken the larvae of Blastobasidae moths, which have been confirmed on the these same plant populations by Terry et al., 2012a, who provide photograph evidence and means of identification. Further study is needed to confirm the identity of this species.

12. A single record exists of Cryptoblabes plagioleuca (Pyralidae) larvae feeding on Cycas rumphii in Fiji (Hinckley, 1964). Further reports of this hostplant association have not been published.

13. Several authors claim that the moth Orvasca subnotata (Erebidae) feeds on Cycas circinalis in Borneo. However, Holloway, 1999 points out that this moth is often misidentiﬁed, cautioning that:

99 Appendix C. Supplement Chapter 3

"The possibility that this species has been confused with others means that lists of host records need to be treated with some cau-

tion and may particularly overlap with those for Somena similis. No larval description has been located apart from those of Moore

(1883), who may have confused Somena species and subnotata".

14. Cryptoptila immersana (Tortricidae) is a highly polyphagous leaf roller that Common, 1990 records from Bowenia in Australia, though he does not provide any supporting evidence. Further conﬁrmation of this record would be particularly valuable, as this would present the ﬁrst and only record of

a lepidopteran feeding on Bowenia cycads.

15. The Natural History Museum’s HOST database lists Cycas revoluta as a hostplant for Oiketicus kirbyi (Psychidae) (Robinson et al., 2010) but we can ﬁnd no original reports of this hostplant association.

100 Appendix C. Supplement Chapter 3

C.2 Cycadivorous Lepidoptera species synonyms

TABLE C.2: Species synonyms

Accepted Species Name Synonyms / Previous Names

Lycaenidae E. atala (Poey, 1832) E. ﬂoridana, Eumenia atala E. childrenae (Gray, 1832) E. deborah, Eumaea debora, Eumenia childrenae E. toxea (Godart, [1824]) Eumenia toxea, erroneously as E. minyas E. godartii (Boisduval, 1870) E. costaricensis, erroneously as E. minyas E. minyas (Hübner, [1809]) E. minijas E. toxana (Boisduval, 1870) E. minyas obsoleta, E. giganteus, E. sara, Eumenia toxana, erroneously as Eu- maeus minyas L. cleaotas (Guérin-Méneville, [1831]) Chilades cleotas, Polyommatus cleotas L. pandava (Horsﬁeld, [1829]) Chilades pandava, Lycaena pandava, Edales pandava, Catochrysops pandava, Catochrysops nicola, Lampides pandava, Catochrysops bengalia, Euchrysops pandava, Euchrysops insularis Geometridae Z. lepida (Walker, 1854) Z. leopardina, Deiopeia lepida Z. tenuis (Butler, 1878) Z. fulva C. abraxas Staude, 2001 C. boisduvalii V. setinata (Felder & Rogenhofer, 1875) Durbana setinata

101 Bibliography

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