Intellectual Merit : Broader Impacts

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PROJECT SUMMARY Overview: Mimicry is a key topic in evolutionary biology. Despite much research, the diversity and macroevolution of mimicry and mimetic species remain poorly documented and understood. This is especially true for mimicry complexes outside the butterflies. Mimetic associations formed between the Hymenoptera (wasps, bees and ants) and numerous other insects are probably the most diverse, abundant and conspicuous mimicry system globally. This system presents an unparalleled yet underexplored opportunity for transforming the way in which we study and explain patterns and processes of mimicry evolution. The proposed project will take a phylogenetic approach to investigate the diversity and evolution of an intriguing mimicry complex formed by Neotropical braconid wasps and assassin bugs.

Intellectual Merit : The proposal project promises significant results as discussed below. (1) The investigators will study a large, novel, yet neglected Neotropical mimicry system. This system has many analogous mimicry complexes in other parts of the world. The investigators will target a total of >250 species in three study groups, and describe at least ~130 new species that may represent novel mimetic patterns. This activity will contribute greatly to the documentation of the diversity of mimetic species. (2) The investigators will examine >6,000 museum specimens. Previous collecting has generated ~2000 DNA-quality specimens of the target mimetic species. And new collecting trips will generate many more new samples to allow for extensively study of mimicry. (3) The investigators will thoroughly document and analyze the diversity of mimicry rings and mimetic types occurring in the target groups. This rich set of comparative information will be essential for understanding mimicry and inferring its evolutionary scenarios in the target groups. (4) The investigators will employ a phylogenetic approach towards an understanding of the deeper evolutionary patterns and history of mimicry. This approach has remained underutilized in mimicry research and has a great potential for advancing the understanding of mimicry and related biological phenomena. Phylogenies will be reconstructed for all three target groups and their ages will be estimated using molecular dating techniques. Comparative methods will be used to test evolutionary scenarios. The investigators will answer important and yet rarely explored questions such as (a) Did mimetic patterns evolve from a cryptic non-mimetic ancestor or from an existing mimetic condition? (b) Could species in a mimetic lineage revert to non-mimetic form? And (c) do age estimates of mimetic species conform to predictions of "advergent evolution" of mimicry? (5) The investigators will also devise or use innovative or cutting-edge methods including (a) phenotypic data visualization against a geographic background using Symbiota, (b) targeted amplicon sequencing of mimetic species, and (c) multispecies coalescence-based species delimitation using Bayes factors.

Broader Impacts : The proposed project offers many avenues for integrating research outcome with public education. (1) The investigators will use augmented reality technology to virtually connect the public with real specimens of mimetic species via mobile devices and/or desktops. (2) Two undergraduate research students will work together to implement an ’Evolve a Mimic’ educational game based on our results of character evolutionary analyses. (3) An undergraduate outreach assistant will demonstrate the mimicry visualization tool in Symbiota to visitors at the annual ’Night of the Open Door’ event at the insect collection at ASU, which attracts >1000 visitors. (4) Symbiota’s new visualization capability would represent enhancement of infrastructure. It would be useful for many research projects (e.g., for visualizing the massive amount of insect images generated by several current specimen digitization projects). The proposed project will also contribute to the training of future researchers and broad dissemination of scientific results. (5) At least 15-18 undergraduate students will be involved in laboratory or field-based research, as has been actively done by all PIs. (6) Two graduate students and one postdoctoral researcher will be trained in the systematics of two diverse insect orders and this would contribute to alleviating the taxonomic and biodiversity crisis. (7) All taxonomic, specimen, image and phylogenetic information will be made available through online publicly accessible databases (e.g. Symbiota, Morphbank, Encyclopedia of Life, GBIF, TreeBase).

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TABLE OF CONTENTS For font size and page formatting specifications, see GPG section II.B.2.

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Cover Sheet for Proposal to the National Science Foundation Project Summary

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Table of Contents

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Project Description (Including Results from Prior NSF Support) (not to exceed 15 pages) (Exceed only if allowed by a specific program announcement/solicitation or if approved in advance by the appropriate NSF Assistant Director or designee)

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References Cited

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Biographical Sketches

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Facilities, Equipment and Other Resources

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Appendix (List below. ) (Include only if allowed by a specific program announcement/ solicitation or if approved in advance by the appropriate NSF Assistant Director or designee)

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*Proposers may select any numbering mechanism for the proposal. The entire proposal however, must be paginated. Complete both columns only if the proposal is numbered consecutively.

Personnel PI and co-PIs Guanyang Zhang Institution: Arizona State University. Postdoctoral Research Associate Role: Lead PI Responsibilities: Oversee the project and coordinate between collaborators. Oversee and manage outreach/broader impacts activities. Perform taxonomic revision on mimetic assassin bugs. Analyze mimicry rings and mimetic traits using a quantitative method. Perform comparative phylogenetic analyses of character evolution and molecular dating on assassin bugs and braconid wasps (in collaboration with graduate students). Co-supervise two graduate students. Organize and participate in field trips. Michael Sharkey Institution: University of Kentucky. Professor. Role: co-PI Responsibilities: Conduct taxonomic revision of Alabagrus wasps. Supervise one PhD student on the taxonomy of Digonogastra wasps. Organize and participate in field trips. Christiane Weirauch Institution: University of California, Riverside. Associate Professor. Role: co-PI Responsibilities: Collaborate with PI Zhang on monographic revision of mimetic assassin bugs. Collaborate with Sharanowski and supervise one Master’s student on DNA sequencing. Organize and participate in collecting trips. Nico Franz Institution: Arizona State University. Associate Professor. Role: co-PI Responsibilities: Provide infrastructure support, including fully-equipped morphology and molecular labs to Zhang (as Postdoc in the lab). Oversee and participate in the implementation of mimicry visualization module in Symbiota.

Senior personnel Edward Gilbert Institution: Arizona State University. Biodiversity Informatician & Faculty Research Associate. Role: Senior personnel Responsibilities: Implement the mimicry visualization module in Symbiota. Barb Sharanowski Institution: University of Manitoba. Wallis-Roughley Museum of Entomology. Assistant Professor/Curator Role: Senior personnel Responsibilities: Provide consultation and training on ‘targeted amplicon’ sequencing method and related bioinformatics and analytical procedures.

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Specific Aims Some 150 years after Bates discovered mimicry1, biologists and the public continue to be fascinated by this spectacular phenomenon2,3,4. Mimicry is taxonomically widespread in insects5,6,7. However, recent research has concentrated on a few lineages, e.g., heliconiine8 and ithomiine butterflies9. The diversity of mimicry in insects remains poorly documented and understood10. Mimetic associations formed between the Hymenoptera (wasps, bees and ants) and numerous other insects are probably the most diverse, abundant and conspicuous mimicry system globally11. This system presents an unparalleled yet underexplored opportunity for transforming the way in which we study and explain patterns and processes of mimicry evolution. We will focus on one intriguing component within this system formed by two exceptionally speciose lineages: braconid wasps and assassin bugs (Hemiptera) (Fig. 1). We will unravel the splendid diversity of this amazing system and employ a phylogenetic approach to study mimicry evolution. This approach contrasts and complements the predominant focus on experimental studies of mimicry. We have the following specific aims. 1. Overcoming the taxonomic impediment in mimetic braconid wasp and reduviid species. We will provide taxonomic treatments for ~250 species of our target groups: two wasp genera from two subfamilies and a group of mimetic assassin bugs. We will describe as many as 130 new species that may represent novel mimicry patterns otherwise unknown to science. 2. Characterizing mimicry rings and mimetic types in this mimicry system. We will produce a rich set of comparative data to describe the diversity of mimicry rings, i.e., sympatric species sharing the same mimetic type, and additional phenomena such as sexual dimorphism and geographic polymorphism. These data are essential for testing evolutionary scenarios of mimicry in this large and diverse system. 3. Inferring phylogenetic frameworks for mimetic wasps and reduviids and testing hypotheses of character evolution of mimetic types and mimicry rings. Macroevolutionary patterns of mimicry have rarely been investigated. Our study will fill a major gap in the understanding of the evolution of mimicry. Background and Rationale The need for a phylogenetic approach to mimicry. Current mimicry research has mainly focused on ecological9, behavioral12, and genetic13,14,15 or genomic16,17 aspects, which has led to exciting discoveries. Nevertheless this research has seldom aimed at recovering patterns and processes of mimicry at deeper evolutionary scales. Consequently many interesting questions remain poorly studied, such as: (1) what is the character evolutionary history of mimetic types?; (2) what are the ages of mimetic associations?; and (3) is the community structure of mimetic species determined by mutualistic mimetic interactions, competition or phylogeny? A phylogenetic approach is required5,18,19,20,21, but to date this has not become common practice in mimicry research. Even in the best studied systems such as Heliconius and ithomiine butterflies, evolutionary reconstructions of mimetic types across an entire lineage are virtually nonexistent22,23,24,25,26. Age estimations in these complexes have been limited to populations or species pairs27. By contrast, research incorporating a strong phylogenetic component, albeit relatively scarce, has provided significant insights28,29,30,31,32. Furthermore, phylogenetic evidence has been invoked to support theoretical predictions of mimicry initiation and evolution21,33. With this background in mind we propose to study the (arguably) largest mimicry system in the Neotropics under a phylogeny-driven approach. Why braconid wasps and assassin bugs? One of the pioneers of mimicry research, E. B. Poulton described a Müllerian mimetic relationship34 between a braconid wasp and an assassin bug in the Neotropics in 193535. It has become clear now that several dozens of reduviid species participate in this mimicry system that may involve >1000 species of braconid wasps. The reduviids display aposematic color patterns strikingly similar to those seen in the braconid wasps (Fig. 1). The bug may even mimic the wasp ovipositor by sticking a hind tibia past the tip of abdomen (Fig. 1;36, C. Fischer, pers. comm.).

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Reduviids can inflict a painful bite and secrete noxious compounds when threatened. They may form Müllerian mimicry complexes among themselves and also with unpalatable braconid wasps, whereby these species mutually benefit one another by sharing the cost of 'educating' predators37. The wasp family Braconidae constitutes ~17,000 described species and is estimated to include up to 100,000 species. The strongly contrasting, bright yellow, black or red, and visually aposematic color patterns are found in a large percentage (50%) of the Neotropical members of several subfamilies; i.e., Agathidinae, Braconinae, Cenocoeliinae, and Orgilinae. To make this project tractable, we will investigate two clades of braconids - Alabagrus (Agathidinae) and Digonogastra (Braconinae). Members of Alabagrus are parasitoids of pyralid moths and those of Digonogastra attack wood-boring beetles. They are often found in the same habitats such as tree-falls of Neotropical rainforests where ground vegetation is lush. Species of Digonogastra have a potent but short-lived sting, probably functioning as Müllerian mimics, whereas Alabagrus do not sting and may be Batesian mimics taking advantage of the system. Research Objectives Our project will move towards a phylogenetically-informed paradigm for mimicry research. 1. Overcoming taxonomic impediment by conducting clade or area-based revisions. We face a taxonomic impediment in our study groups. The Digonogastra wasps and the mimetic reduviids have been largely neglected for more than 100 years38,39, and we estimate that they contain at least 60% undescribed species. The reduviid mimics appear to be a monophyletic clade (Fig. 2), and we will revise them as a whole, expecting to treat a total of ~50 species and describe at least 30 new species. Digonogastra currently contains >250 valid names. Treating the whole genus is beyond the scope of our project. Instead, we will perform area-based revisions, a proven effective method by co-PI Sharkey40. We will focus on four areas (Costa Rica, Northern Peru east of the Andes, the Guianas, and the Brazilian Atlantic Forest), which represent diverse habitats and allow us to sample mimicry patterns comprehensively. We expect to treat at least 150 species of Digonogastra. The other wasp genus Alabagrus was recently revised and comprises 110 species40,41. We will add at least 50 new species to this genus. We will follow the integrative taxonomy approach42 and use both morphology and DNA sequences. DNA-based methods will help us delimit cryptic species43, as has been noted in Alabagrus at even a single site (i.e., Guanacaste, Costa Rica; D. Janzen, pers. comm.). Preliminary results. Our DNA-quality reduviid specimens from four countries represent >25 species and at least 15 as new species. Co-PI Sharkey has amassed ~2000 alcohol-preserved specimens of Alabagrus and Digonogastra from the Neotropics. Sharkey is currently revising the species of Alabagrus of Costa Rica, building on his previous publications on this group40,41 and is treating slightly more than 60 species for that area alone. After sampled for four sites Alabagrus may reach at least 160 species. 2. Characterizing patterns of mimicry and visualizing mimicry on a virtual geographic background. We will address two important questions recognized in the literature29,44,45. First, how many mimicry rings can be found in an area and how many species and lineages may participate in the same mimicry ring? Local diversity of Müllerian mimicry rings is one of the biggest puzzles in mimicry research46. For heliconian and ithomiine butterflies a single site may contain three12 or eight47 mimicry rings, each consisting of several or more than a dozen species. Our three study groups seem to exhibit a rich local diversity of mimicry rings, participated by members from each group. Second, are there sexual dimorphism and geographically structured polymorphism, and if so, what are the patterns? Geographic color races have been observed in many species of Heliconius. Sexual dimorphism, especially femalelimited mimetic form is common in Batesian mimicry, but rare in Müllerian mimicry48. We are interested in exploring and documenting these patterns in our organisms, which contain both Müllerian and Batesian mimics. Besides generating comparative data, we will devise a novel tool to interactively visualize mimicry against a geographic background, allowing us to simultaneously visualize all mimicry rings and their participating species at our four study sites in a Google Earth environment. We can also interactively sort the mimicry data with various filters (e.g., taxonomy, locality and mimetic type). This may enable discoveries of geography or environment (habitat, climate)-associated patterns of mimicry as noted in braconids previously49. In contrast current methods typically involve coloring dots or areas on a map and displaying images on the side30,29, which is static and does not afford human-data interaction. Preliminary results. At one site in Costa Rica, Alabagrus species form at least seven mimicry rings40. In a small area in Southeast Peru (20 collections. Because of their relatively large size and bright coloration our focal taxa are well represented in collections of Neotropical insects. PIs Zhang and Weirauch have ~50 DNA-quality reduviid specimens representing >25 species. Co-PI Sharkey has ~2000 of DNA-quality target wasp specimens. For the reduviids we will cover the largest possible geographic range, but for the wasps we will primarily focus on four sites as aforementioned. Field work. We will conduct new field trips at two of the study sites (Atlantic forest, Espirito Santo, Brazil Reserva Biologica de Duas Bocas; and Nouragues Field Station, French Guiana). Based on experience and specimen records, these sites are rich in braconid and reduviid mimics. All PIs have extensive Neotropical field experience and have contacts to arrange logistics and permits for these sites. Integrative taxonomy. We will first form species hypotheses based on morphology. We will then use the “Bayes factors” coalescence-based species delimitation method as implemented in the program Beast55 . This method has the advantage of testing hypotheses of specimen assignment to alternative lineages56. We will use multi-locus data as described below. Taxonomic data management. To promote a data openness and to take advantage of the NSF-funded Hymenoptera Anatomy Ontology infrastructure, we will use MX (MatriX)57 as the taxonomic content management system (soon migrating to TaxonWorks).

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To widely disseminate data we will expose snapshots via Symbiota58 [http://taxonbytes.org/symbiota/], a specimen-based, and widely-used on-line collections portal platform. Mimicry characterization. We will follow an extensive color pattern study of bumble bees to quantitatively classify mimetic types44 (Fig. 3). In addition, the distinctiveness of mimicry rings will be statistically tested following methods used in Wilson et al.30. Mimicry visualization. Symbiota lead developer Gilbert (Senior Personnel) will implement a dynamic image visualization module in Symbiota. Specimen data will be exported from Symbiota into a KML (Keyhole Markup Language) file. Images of mimetic species or traits can be visualized on a Google Map background or overlaid with GIS layers in the interactive taxonomic environment in Symbiota. Molecular data: targeted amplicon sequencing. DNA extraction will follow protocols established by PIs Zhang and Weirauch. We will utilize a targeted amplicon sequencing approach59 using Life Sciences 454 GS FLX+ technology to obtain a ~30 gene dataset for ~140 taxa across all three focal groups at an estimated cost of ~$5,000. We will target standard genes used for species delimitation and species level phylogenetics (e.g. COI, COII, 28S and ITS1) but also target ~20 Exon-Primed Intron Crossing (EPIC) loci. EPIC loci have been shown to be effective for species and population-level insect phylogenetics60 and over 25 EPIC loci have already been developed and screened on multiple Hymenopteran taxa by Senior Personnel Sharanowski and her collaborators59,60,61,62. Phylogenetic reconstruction. We expect to sample 40 species of mimetic assassin bugs. We will include outgroup taxa from several closely related genera used in Zhang & Weirauch (2013)64. Alignment of the target sequences will be performed with MAFFT65. Phylogenetic reconstruction will be conducted using maximum likelihood methods and Bayesian approaches (with the programs RAXML66 and BEAST). For both Alabagrus and Digonogastra we will sample ~50 morphologically disparate species and exemplar species from related genera to obtain a skeletal phylogeny. Character evolution analyses. Two kinds of analyses will be performed, one on broadly defined mimetic types and another with individual color elements (Fig. 3). The former will allow us to infer whether similar mimetic types have repeatedly evolved. The latter will reconstruct the detailed pathways of trait evolution on a phylogeny. We will use stochastic character mapping methods implemented in BayesTraits67, which can infer character evolutionary pathways with probability estimates and has been used in several studies of coloration evolution48,68. Molecular dating. The ages of mimetic species will be estimated with the program BEAST, with fossil calibration points previously used in reduviids and braconids. Broader Impacts We will integrate our research with public education as outlined here. (1) We will use augmented reality technology [http://tblr.asu.edu/projects/insectarium] to virtually connect the public with real specimens of mimetic species via mobile devices and/or desktops, as has been done at co-PI Franz’s lab. (2) Two undergraduate research students will implement an ‘Evolve a Mimic’ educational game based on our results of character evolutionary analyses. In this game a player will have a simplified phylogeny, images of the tip species and a set of ancestral nodal mimetic patterns (as reconstructed by our analyses). The player will try to place the ancestral mimetic patterns at the nodes based on simple rules and information contained in the phylogeny and tip species. This game will introduce ‘tree-thinking’ to the general public and correct for the popular misrepresentation of evolution as linear69. (3) An undergraduate outreach assistant will demonstrate the mimicry visualization tool in Symbiota to visitors at the annual ‘Night of the Open Door’ event at the insect collection at ASU, which attracts >1000 visitors. Our research will also make significant contributions to the broader scientific community as discussed below. (4) SYMBIOTA’s visualization function would work for any kind of phenotypic data or images (e.g., for visualizing the massive amount of insect images generated by several current specimen digitization projects). (5) We will involve at least 15-18 undergraduate students (six per year for three years) in research, as has been actively done by all PIs. At least six of them would be from underrepresented background. (6) Two graduate students and one postdoctoral researcher will be trained in the systematics of two diverse insect orders and this would contribute to alleviating the biodiversity crisis. (7) All taxonomic, specimen, image and phylogenetic information will be made available through online publicly accessible databases (e.g. Symbiota, Morphbank, Encyclopedia of Life, GBIF, TreeBase).

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References 1. Bates, H. W. Contributions to an insect fauna of the Amazon valley. Lepidoptera: Heliconidae. Trans. Linn. Soc. Lond. 23, 495–566 (1862). 2. Mallet, J. & Dasmahapatra, K. Evolutionary biology: Catfish mimics. Nature 469, 41–42 (2011). 3. Pfennig, D. W. & Kikuchi, D. W. Evolutionary biology: Life imperfectly imitates life. Nature 483, 410– 411 (2012). 4. Forbes, P. Dazzled and deceived: mimicry and camouflage. (Yale University Press, 2009). 5. Ruxton, G. D. Avoiding attack: the evolutionary ecology of crypsis, warning signals, and mimicry. (Oxford University Press, 2004). 6. Komárek, S. Mimicry, aposematism and related phenomena in animals and plants. Bibliography 1800-1990. 296 pp. (1998). 7. Rettenmeyer, C. W. Insect Mimicry. Annu. Rev. Entomol. 15, 43–74 (1970). 8. Nadeau, N.J., Martin, S.H., Kozak, K.M., Salazar, C., Dasmahapatra, K.K., Davey, J.W., Baxter, S.W., Blaxter, M.L., Mallet, J., & Jiggins, C.D. (2013). Genome-wide patterns of divergence and gene flow across a butterfly radiation. Molecular Ecology 22, 814–826.Genome-wide patterns of divergence and gene flow across a butterfly radiation. Mol. Ecol. 22, 814–826 (2013). 9. Chazot, N., Willmott, K.R., Santacruz Endara, P.G., Toporov, A., Hill, R.I., Jiggins, C.D., & Elias, M. Mutualistic mimicry and filtering by altitude shape the structure of Andean butterfly communities. Am. Nat. 183, 26–39 (2014). 10. Polidori, C., Nieves-Aldrey, J. L., Gilbert, F. & Rotheray, G. E. Hidden in taxonomy: Batesian mimicry by a syrphid fly towards a Patagonian bumblebee. Insect Conserv. Divers. 7, 32–40 (2014). 11. Poulton, E. B. The Colours of Animals: Their Meaning and Use Especially Considered in the Case of Insects. (Keegan Paul, Trench, Trubner & Co., 1890). 12. Mallet, J. & Gilbert, L. E. Why are there so many mimicry rings? Correlations between habitat, behaviour and mimicry in Heliconius butterflies. Biol. J. Linn. Soc. 55, 159–180 (1995). 13. Joron, M., Papa, R., Beltrán, M., Chamberlain, N., Mavárez, J., Baxter, S., Abanto, M., Bermingham, E., Humphray, S.J., & Rogers, J. A conserved supergene locus controls colour pattern diversity in Heliconius butterflies. PLoS Biol 4, e303 (2006). 14. Hines, H.M., Counterman, B.A., Papa, R., Moura, P.A. de, Cardoso, M.Z., Linares, M., Mallet, J., Reed, R.D., Jiggins, C.D., Kronforst, M.R. Wing patterning gene redefines the mimetic history of Heliconius butterflies. Proc. Natl. Acad. Sci. 108, 19666–19671 (2011). 15. Reed, R.D., Papa, R., Martin, A., Hines, H.M., Counterman, B.A., Pardo-Diaz, C., Jiggins, C.D., Chamberlain, N.L., Kronforst, M.R., & Chen, R. optix Drives the repeated convergent evolution of butterfly wing pattern mimicry. Science 333, 1137–1141 (2011). 16. The Heliconius Genome Consortium, Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature, (2012). 17. Baxter, S.W., Nadeau, N.J., Maroja, L.S., Wilkinson, P., Counterman, B.A., Dawson, A., Beltran, M., Perez-Espona, S., Chamberlain, N., Ferguson, L. Genomic hotspots for adaptation: The population genetics of Müllerian mimicry in the Heliconius melpomene clade. PLoS Genet 6, e1000794 (2010). 18. Härlin, C. & Härlin, M. Towards a historization of aposematism. Evol. Ecol. 17, 197–212 (2003). 19. Oliver, J. C. & Prudic, K. L. Are mimics monophyletic? The necessity of phylogenetic hypothesis tests in character evolution. BMC Evol. Biol. 10, 239 (2010). 20. Monteiro, A. & Prudic, K. M. Multiple approaches to study color pattern evolution in butterflies. Trends Evol. Biol. 2, e2 (2010). 21. Balogh, A. C. V., Gamberale-Stille, G., Tullberg, B. S. & Leimar, O. Feature theory and the two-step hypothesis of Müllerian mimicry evolution. Evolution 64, 810–822 (2010). 22. Penz, C. M. Higher level phylogeny for the passion-vine butterflies (Nymphalidae, Heliconiinae) based on early stage and adult morphology. Zool. J. Linn. Soc. 127, 277–344 (1999). 23. Brower, A. V. Z. & Egan, M. G. Cladistic analysis of Heliconius butterflies and relatives (Nymphalidae: Heliconiiti): a revised phylogenetic position for Eueides based on sequences from mtDNA and a nuclear gene. Proc. R. Soc. B Biol. Sci. 264, 969–977 (1997). 24. Beltrán, M., Jiggins, C. D., Brower, A. V. Z., Bermingham, E. & Mallet, J. Do pollen feeding, pupalmating and larval gregariousness have a single origin in Heliconius butterflies? Inferences from multilocus DNA sequence data. Biol. J. Linn. Soc. 92, 221–239 (2007).

25. Brower, A.V.Z., Freitas, A.V.L., Lee, M.-M., Silva-Brandão, K.L., Whinnett, A., and Willmott, K.R. Phylogenetic relationships among the Ithomiini (Lepidoptera: Nymphalidae) inferred from one mitochondrial and two nuclear gene regions: Molecular systematics of ithomiine butterflies. Syst. Entomol. 31, 288–301 (2006). 26. de-Silva, D.L., Day, J.J., Elias, M., Willmott, K., Whinnett, A., and Mallet, J. Molecular phylogenetics of the neotropical butterfly subtribe Oleriina (Nymphalidae: Danainae: Ithomiini). Mol. Phylogenet. Evol. 55, 1032–1041 (2010). 27. Flanagan, N.S., Tobler, A., Davison, A., Pybus, O.G., Kapan, D.D., Planas, S., Linares, M., Heckel, D., and McMillan, W.O. Historical demography of Müllerian mimicry in the neotropical Heliconius butterflies. Proc. Natl. Acad. Sci. U. S. A. 101, 9704–9709 (2004). 28. Simmons, R. B. & Weller, S. J. What kind of signals do mimetic tiger moths send? A phylogenetic test of wasp mimicry systems (Lepidoptera: Arctiidae: Euchromiini). Proc. R. Soc. Lond. B Biol. Sci. 269, 983–990 (2002). 29. Marek, P. E. & Bond, J. E. A Müllerian mimicry ring in Appalachian millipedes. Proc. Natl. Acad. Sci. 106, 9755–9760 (2009). 30. Wilson, J. S., Williams, K. A., Forister, M. L., von Dohlen, C. D. & Pitts, J. P. Repeated evolution in overlapping mimicry rings among North American velvet ants. Nat. Commun. 3, 1272 (2012). 31. Symula, R., Schulte, R. & Summers, K. Molecular phylogenetic evidence for a mimetic radiation in Peruvian poison frogs supports a Müllerian mimicry hypothesis. Proc. R. Soc. Lond. B Biol. Sci. 268, 2415–2421 (2001). 32. Hines, H. M. & Williams, P. H. Mimetic colour pattern evolution in the highly polymorphic Bombus trifasciatus (Hymenoptera: Apidae) species complex and its comimics. Zool. J. Linn. Soc. 166, 805– 826 (2012). 33. Gamberale-Stille, G., Balogh, A. C. V., Tullberg, B. S. & Leimar, O. Feature saltation and the evolution of mimicry. Evolution 66, 807–817 (2012). 34. Müller, F. Ueber die Vortheile der Mimicry bei Schmetterlingen. Zool. Anz. 1, 54–55 (1878). 35. Poulton, E. B. A reduviid bug mimicking a braconid, and an asilid fly mimicking a bee, collected in British Guiana by Dr. J.G. Myers. Proc. R. Entomol. Soc. Ser. Gen. Entomol. 95–96 (1937). 36. Preston-Mafham, K. & Preston-Mafham, R. The natural world of bugs & insects. (Thunder Bay, 2001). 37. Sherratt, T. N. The evolution of Müllerian mimicry. Naturwissenschaften 95, 681–695 (2008). 38. Maldonado Capriles, J. Systematic catalogue of the Reduviidae of the world (Insecta: Heteroptera). (University of Puerto Rico, 1990). 39. Quicke, D. L. J. Reclassification of some new world species of braconinae hymenoptera braconidae. Entomol. Mon. Mag. 125, 119–121 (1988). 40. Leathers, J. W. & Sharkey, M. J. Taxonomy and life history of Costa Rican Alabagrus (Hymenoptera: Braconidae), with a key to world species. Contrib. Sci. 497, 1–78 (2003). 41. Sharkey, M. J. A taxonomic revision of Alabagrus (Hymenoptera: Braconidae). Bull. Br. Mus. Nat. Hist. Entomol. 57, 311–437 (1988). 42. Padial, J. M., Miralles, A., Riva, I. D. la & Vences, M. The integrative future of taxonomy. Front. Zool. 7, 16 (2010). 43. Bickford, D., Lohman, D.J., Sodhi, N.S., Ng, P.K.L., Meier, R., Winker, K., Ingram, K.K., and Das, I. Cryptic species as a window on diversity and conservation. Trends Ecol. Evol. 22, 148–155 (2007). 44. Williams, P. The distribution of bumblebee colour patterns worldwide: possible significance for thermoregulation, crypsis, and warning mimicry. Biol. J. Linn. Soc. 92, 97–118 (2007). 45. Brower, A. V. Rapid morphological radiation and convergence among races of the butterfly Heliconius erato inferred from patterns of mitochondrial DNA evolution. Proc. Natl. Acad. Sci. U. S. A. 91, 6491–6495 (1994). 46. Joron, M. & Mallet, J. L. B. Diversity in mimicry: paradox or paradigm? Trends Ecol. Evol. 13, 461– 466 (1998). 47. Elias, M., Gompert, Z., Jiggins, C. & Willmott, K. Mutualistic interactions drive ecological niche convergence in a diverse butterfly community. PLoS Biol 6, e300 (2008). 48. Kunte, K. The diversity and evolution of batesian mimicry in Papilio swallowtail butterflies. Evolution 63, 2707–2716 (2009).

49. Quicke, D. L. J. Preliminary notes on homeochromatic associations within and between the Afrotropical Braconinae (Hym., Braconidae) and Lamiinae (Col., Cerambycidae). Entomol. Mon. Mag. 122, 97–109 (1986). 50. Mallet, J. Shift happens! Shifting balance and the evolution of diversity in warning colour and mimicry. Ecol. Entomol. 35, 90–104 (2010). 51. Mallet, J. Causes and Consequences of a Lack of Coevolution in Müllerian mimicry. Evol. Ecol. 13, 777–806 (1999). 52. Brower, L. P. & Brower, J. V. Z. Parallelism, convergence, divergence, and the new concept of advergence in the evolution of mimicry. Trans Conn Acad Arts Sci 44, 59–67 53. Brower, A. V. Z. Parallel race formation and the evolution of mimicry in Heliconius Butterflies: A phylogenetic hypothesis from mitochondrial DNA sequences. Evolution 50, 195 (1996). 54. Quek, S.-P., Counterman, B.A., Moura, P.A. de, Cardoso, M.Z., Marshall, C.R., McMillan, W.O., and Kronforst, M.R. Dissecting comimetic radiations in Heliconius reveals divergent histories of convergent butterflies. Proc. Natl. Acad. Sci. 107, 7365–7370 (2010). 55. Drummond, A. J. & Rambaut, A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214 (2007). 56. Grummer, J. A., Bryson, R. W. & Reeder, T. W. Species delimitation using Bayes factors: simulations and application to the Sceloporus scalaris species group (Squamata: Phrynosomatidae). Syst. Biol. syt069 (2013). doi:10.1093/sysbio/syt069 57. Yoder, M. J., Mikó, I., Seltmann, K. C., Bertone, M. A. & Deans, A. R. A gross anatomy ontology for Hymenoptera. PLoS ONE 5, e15991 (2010). 58. Franz, N. & Gilbert, E. Symbiota – A virtual platform for creating voucher-based biodiversity information communities. Biodivers. Data J. at 59. Bybee, S.M., Bracken-Grissom, H., Haynes, B.D., Hermansen, R.A., Byers, R.L., Clement, M.J., Udall, J.A., Wilcox, E.R., and Crandall, K.A. Targeted amplicon sequencing (TAS): A scalable nextgen approach to multilocus, multitaxa phylogenetics. Genome Biol. Evol. 3, 1312–1323 (2011). 60. Lohse, K., Sharanowski, B. & Stone, G. N. Quantifying the pleistocene history of the oak gall parasitoid Cecidostiba fungosa using twenty intron loci. Evol. Int. J. Org. Evol. 64, 2664–2681 (2010). 61. Bowsher, J. H., Ang, Y., Ferderer, T. & Meier, R. Deciphering the evolutionary history and developmental mechanisms of a complex sexual ornament: the abdominal appendages of Sepsidae (Diptera). Evolution 67, 1069–1080 (2013). 62. Sharanowski, B. J. Four really cool things about introns: evolution, phylogenetic signal, species identification, and bio-monitoring. Entomological Society Annual Meeting (2012). 63. Ratnasingham, S. & Hebert, P. D. N. bold: The Barcode of Life Data System (http://www.barcodinglife.org). Mol. Ecol. Notes 7, 355–364 (2007). 64. Zhang, G. & Weirauch, C. Molecular phylogeny of Harpactorini (Insecta: Reduviidae): correlation of novel predation strategy with accelerated evolution of predatory leg morphology. Cladistics n/a–n/a (2013). doi:10.1111/cla.12049 65. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). 66. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690 (2006). 67. Pagel, M., Meade, A. & Barker, D. Bayesian estimation of ancestral character states on phylogenies. Syst. Biol. 53, 673–684 (2004). 68. Gluckman, T.-L. Pathways to elaboration of sexual dimorphism in bird plumage patterns. Biol. J. Linn. Soc. (2014). doi:10.1111/bij.12211 69. Baum, D. A. & Smith, S. D. Tree thinking: an introduction to phylogenetic biology. (Roberts Publisher, 2012).

PROJECT SUMMARY Overview: Mimicry is a key topic in evolutionary biology. Despite much research, the diversity and macroevolution of mimicry and mimetic species remain poorly documented and understood. This is especially true for mimicry complexes outside the butterflies. As a result, our understanding of mimicry remains rudimentary and is especially inadequate in addressing a large taxonomic diversity. Two exceptionally diverse lineages, braconid wasps (Hymenoptera: Braconidae) and assassin bugs (Hemiptera: Reduviidae) have evolved striking mimicry in the Neotropics, representing an ideal opportunity for investigating the evolution of mimicry interactions. The proposed project will take a comparative approach to study the diversity and evolution of this intriguing mimicry complex. The PIs are uniquely qualified to take on this project based on their combined taxonomic expertise in these two groups and their analytical skills.

Intellectual Merit : The PIs will present unprecedented syntheses and discoveries in a large, diverse and complex mimicry system. The knowledge and data will be widely disseminated to facilitate further scientific advances. This project represents an effort to build a "paradigm shift" in mimicry research by addressing the long-neglected comparative perspective as outlined below. (1) The PIs will study a large, novel, yet neglected Neotropical mimicry system. In particular, the PIs will perform biodiversity syntheses and discoveries. A total of >250 species in three groups will be treated, and at least 130 species described as new to science. This activity will contribute greatly to the documentation of the diversity of mimetic species. (2) This project will produce primary biodiversity data based on >10,000 specimens to allow for extensively study of mimicry. (3) The project will resolve the taxonomic impediment to the study of mimicry by producing a series of large taxonomic monographs and publishing them in online open access journals. (4) This project will thoroughly document and analyze patterns of mimicry occurring in the target groups and investigate color pattern distribution, mimetic sexual dimorphism, local polymorphism, and taxonomic diversity of mimicry rings. (5) The PIs will also utilize the cutting edge RADseq method (Restriction site DNA sequencing) based on the Illumina next generation sequencing platform. (6) The PIs will employ a phylogenetic approach towards an understanding of the deeper evolutionary patterns of mimicry. This approach has remained underutilized in mimicry research and has great potential for advancing the understanding of mimicry and related biological phenomena. The investigators will answer important and yet rarely explored questions, such as (a) did mimetic patterns evolve more often from a cryptic non-mimetic ancestor or from an existing mimetic condition, (b) do color components evolve in a correlated manner, and (c) how do mimetic interactions shape phylogenetic community structuring?

Broader Impacts : The project will broaden public participation in science, and promote international collaboration through: (1) combining taxonomic expertise in two unrelated insect groups; (2) intensive training of PI Zhang as an emerging biodiversity research leader and one PhD student in the systematics and evolution of braconid wasps, thereby preserving taxonomic expertise in two large insect orders; (3) educating the public on natural selection and insect biodiversity using "Insect Mimicry and Camouflage" drawers; (4) introducing the idea of the "tree of life" to the general public using an innovative "Evolve a Mimic" educational game based on results of character evolutionary analyses produced from the proposed project; (5) training of 15-18 undergraduate students in research or outreach activities; (6) disseminating taxonomic, specimen, image and phylogenetic information and knowledge and promoting data reusability by using publicly accessible databases and sharing all project data and results (e.g. Symbiota, Morphbank, Encyclopedia of Life [EOL], TreeBase, Dryad, figshare); (7) international collaboration with scientists from four countries (Brazil, Colombia, Ecuador, and Peru); and (8) training of four South American graduate students in the taxonomy of Hemiptera and Hymenoptera and Neotropical biodiversity, thereby broadly disseminating biodiversity expertise more broadly.

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OVERVIEW AND SPECIFIC OBJECTIVES Some 150 years after Henry W. Bates discovered mimicry in the Amazon1, biologists and the public continue to be fascinated by this phenomenon2–4. For more than a century biologists have noted that mimicry is taxonomically widespread in insects6–8. However, researchers have so far focused on just a few lineages, e.g., heliconiine5 and ithomiine butterflies6. To date the diversity of mimicry in insects remains poorly documented and its deep evolutionary history little understood7. Mimetic associations formed between the Hymenoptera (wasps, bees and ants) and numerous other insects are probably the most diverse, abundant and conspicuous mimicry system globally8. These ubiquitous yet complex systems present an unparalleled and under-explored opportunity for transforming the way in which we study and explain the patterns and processes of mimicry. Within this system, two exceptionally diverse lineages, braconid wasps and assassin bugs (Hemiptera: Reduviidae) have evolved striking mimicry (Fig. 1) in the Neotropics, co-occurring with the famous Heliconius butterflies for millions of years. Our team combines expertise and resources to study these intriguing organisms. In the proposed project we will begin to unravel the vast taxonomic and phenotypic diversity of this mimetic complex and employ a phylogenetic approach to study mimicry diversification and evolution, as outlined below. 1. Advancing biodiversity synthesis of mimetic species with monographic and integrative taxonomy. We will provide taxonomic treatments of 250 species of two mimetic wasp genera from two subfamilies and a group of mimetic assassin bugs and expect to describe at least 130 species new to science. This will be a major advance in biodiversity discovery and synthesis of mimetic organisms. 2. Characterizing and analyzing patterns and diversity of mimicry. We will use quantitative methods to characterize and classify color patterns. We will explore, describe and analyze important properties of our mimicry system such as diversity and composition of mimicry rings, sexual dimorphism and polymorphism. These comparative data are essential for testing evolutionary scenarios of mimicry in this large and diverse system and will facilitate ecological and genetic research. 3. Reconstructing the “mimetic tree of life” and illuminating mimicry evolution. Macroevolutionary patterns of mimicry have rarely been investigated despite the recent rapid progress in sequencing techniques and phylogenetic methods. We will reconstruct phylogenies using next generation sequencing technologies and test evolutionary scenarios of patterns of mimicry.

Figure 1. Reduviid (top row) and braconid nd rd species (2 & 3 rows).

BACKGROUND AND RATIONALE Urgency of comparative and phylogenetic approach to mimicry research. Past and current research has continued to focus on the ecological6, behavioral9, genetic13-15, and genomic11,12 aspects of mimicry. These studies have not only advanced the understanding of this fascinating phenomenon, but also contributed to some of the most fundamental topics in biology. However, there are at least two major limitations in the predominant focus on a few model organisms. This research does not reveal how patterns of mimicry evolved over large evolutionary time-scales. Besides, some of the research requires live cultures and large taxonomic samples are not addressed. These limitations are evident in the literature. The majority of published Heliconius studies only investigated species pairs or populations. A literature survey using Google Scholar shows strong biases towards a few species within that genus: 75% of the 5075 articles published during the last 20 years (1994-2014) are associated with five species: H. erato, H. melpomene, H. cydno, H. sara and H. numata (Fig. 2). It is perhaps due to a lack of comparative research, that reconstructions of the evolution of mimetic color patterns across lineages are nearly non-existent13–18. 1

In order to achieve a more comprehensive, macro-evolutionary understanding of the patterns and processes of mimicry, both taxonomic and thematic scopes urgently need to be expanded and a comparative approach taken. This view has been shared by previous researchers19–23. British entomologist G. D. Hale Carpenter in an article published in Nature in 1946 remarked that “…conclusions drawn from the study of the color and pattern of a few dried butterflies do not cope with a vast number of cases among other creatures.”24 This statement still bears relevance to the contemporary mimicry research landscape. More work is needed to expand the taxonomic scope of research on mimetic organisms. A comparative approach will significantly advance our knowledge and understanding of mimicry in at least the following respects. (1) It will uncover the diversity of mimetic species and make this biodiversity knowledge available and accessible to all researchers regardless of their taxonomic focus. (2) It will provide fundamental data on the characteristics of mimicry, which will be essential for theoretical synthesis and comparative analyses. (3) Comparative phylogenetic analyses will enable us to answer questions at a deep temporal scale and gain insight into the macro-evolution of mimicry, otherwise not afforded by studying only a few model species. Significant patterns can be further investigated with ecological or genetic research. (4) By taking the recently developed theoretical framework of phylogenetic community ecology, we can test the role of mimetic interactions in shaping community structures, thereby providing a greater understanding of mimicry at the ecology-evolution interface. Why braconid wasps and assassin bugs? Edward B. Poulton, a pioneer of mimicry research, was the first to describe a mimetic relationship between a braconid wasp and an assassin bug in the Neotropics in 193525. It has now become clear that this system involves at least dozens of species of assassin bugs, perhaps >1000 species of braconid wasps, and an unknown number of other insects. Reduviid mimicry. Comprising ~6,800 species26,27, assassin bugs are a large group of primarily predaceous insects, but also contain blood-sucking, medically important species such as the kissing bugs (Triatominae). Based on a phylogenetic study by the lead PI (Zhang & Weirauch, 201428), resemblance to braconid wasps has evolved independently several times in the New World harpactorine assassin bugs, but a large radiation occurred in one lineage, which constitutes one focal group in the current proposal. These mimetic reduviids also belong to the “sticky bugs” which secrete a sticky substance on the legs for predation (Zhang & Weirauch, 201329). These reduviids display a variety of aposematic color patterns (Fig. 1). Some of the reduviids also exhibit structural modifications such as a narrowed anterior part of abdomen, elongated wings, and an enlarged head, characteristics that enhance their wasp-like appearances. Even more surprisingly, the assassin bugs sometimes raise a hind tibia past the tip of abdomen to resemble a wasp ovipositor (Fig. 130; and C. Fischer, pers. comm.). Interestingly, it is nearly certain that the assassin bugs function as Müllerian mimics31. Reduviids belong to the Heteroptera, one of the largest groups of hemi-metabolous insects also containing stink bugs, bed bugs, and water striders. Heteropteran insects are well known to be chemically defended32. When threatened, assassin bugs are capable of inflicting a painful bite and secrete noxious compounds from the Brindley’s glands33, which the lead PI had personally experienced several times. Braconid mimicry. The wasp family Braconidae comprises ~17,000 described species and may include up to 50,000 species34. These are solitary parasitic wasps that primarily parasitize the larvae of lepidopterans, beetles and flies, and are important biological control agents. The strongly contrasting, bright yellow, black or red, and visually aposematic color patterns are found in a large percentage (50%) of the Neotropical members of several subfamilies; i.e., Agathidinae, Braconinae, Cenocoeliinae and Orgilinae. To make this project tractable, we will investigate two speciose and distantly related groups of braconids – Alabagrus (Agathidinae) and Capitonius (Braconinae). Members of Alabagrus are parasitoids of pyralid moths and those of Capitonius attack wood-boring beetle larvae. They are often found in the same habitats such as tree-falls of Neotropical rainforests where ground vegetation is lush. Members of these two genera do not have a strong stinger and may be Batesian mimics taking advantage of better defended models. We argue below the advantages of our study system. System advantage: taxonomic diversity. Our focal groups exhibit species-level diversity much higher than that of Heliconius (~42 species), each with more than 50 or 200 species. Based on a preliminary sample, we are able to classify at least 24 major color forms and within each there exists much variation. 2

Also, unlike the butterflies where mimetic colors mainly reside on the wings, in the reduviids and braconids nearly all body segments or appendages, including thorax, abdomen, wings and legs, participate in color patterning. System advantage: mimetic complexity. A number of features may be difficult to understand with the current state of knowledge, which calls for more basic research. First, the assassin bugs and the two focal genera of braconids, other wasps and other insects may fall into a spectrum of unpalatability and may engage in Müllerian, Batesian or quasi-Batesian mimicry35 at the same time, thereby making it difficult to distinguish the model from the mimic. The mimetic reduviids appear to be more ‘courageous’ than the wasps, e.g., walking down a path they are very apparent and easily collected whereas braconids are relatively hidden, therefore the assassin bugs may be more often encountered by predators. Second, at least in the reduviids, sexual dimorphism exists (Fig.10), a paradoxical phenomenon for Müllerian mimics36. Third, some common mimetic color patterns are shared among all lineages, but some patterns are unique to certain lineages. Fourth, within a single wasp group, both cryptic color forms and aposematic forms exist, providing ideal comparative data for studying color evolution. System advantage: feasibility and research capacities. The wasps and assassin bugs occur synchronously in space and time and numerous specimens and species can be collected in large numbers using a combination of active and passive trapping methods. These are relatively large insects (10-30 mm) and can be observed in the field and studied in the lab easily. And we have years of experience working on the taxonomy of our study groups and closely related lineages. Our research team comprises world authorities on the systematics of Reduviidae and Braconidae and we are uniquely positioned to carry out the proposed project. PI Zhang is an up-and-coming young research leader in systematics and is well trained in the methods of taxonomy and phylogenetics. Co-PI Sharkey has worked on braconid wasps for over 34 years. This project is an unprecedented opportunity to combine our expertise and resources to study these fascinating organisms. RESEARCH OBJECTIVES & PRELIMINARY DATA We aim to take an integrative approach to reveal the taxonomic, phenotypic and phylogenetic diversity of mimicry and illuminate the evolutionary history of mimetic species. Our proposed research ranges from monographic revisions to comparative phylogenetics, our study approaches are both descriptive and analytical, and our questions are exploratory but also hypothesis-driven. In the following we outline and discuss three objectives, each with subordinate goals or research questions. And we describe the expected outcomes and also present pertinent preliminary results. Objective 1 – Advancing biodiversity synthesis of mimetic species with monographic revisions and integrative taxonomy. 1.1 Overarching goal - Resolving taxonomic impediment by conducting monographic revisions. We will lay a solid taxonomic foundation for phylogenetic, evolutionary or ecological analyses. The taxonomy of mimetic species can be specially challenging. Heliconius, arguably the best studied mimetic organisms, has 2000 published synonymic and intraspecific names associated with just ~40 valid species37 and cryptic species in this genus are still being uncovered38.We are similarly confronted with a taxonomic impediment to the documentation and analyses of mimicry in our study taxa. Two of our study lineages, Capitonius wasps and the mimetic reduviids, have suffered from taxonomic neglect for more than 100 years26,39 and monographic revisions are urgently needed for both groups. The reduviid mimics currently comprise 19 valid species classified into five genera, but the true diversity is likely to be two or three times higher. Capitonius may contain more than 200 species and has never been revised. Although 46 species are described, these are the product of numerous small papers 40–43, including previous works by co-PI Sharkey and his former students40,41. The other wasp genus, Alabagrus currently comprises ~110 species, mostly a result of work by co-PI Sharkey, his students or colleagues, providing much leverage for making further improvement. However, Sharkey’s ongoing work suggests at least 100200 new species await description in Alabagrus. In view of the urgency for biodiversity discovery and synthesis in our focal taxa, we will perform monographic taxonomic revisions. In the context of studying mimicry, we discuss the advantages of monographic revisions and specific outcomes in the following. 3

Product (1). Intensive biodiversity discovery and synthesis by describing 130 new species and treating 250 species in total. Our preliminary data indicate that the reduviid mimics appear to be a monophyletic clade and we will revise them as a whole. We expect to treat a total of ~50 species and describe at least 30 as new to science. In contrast, revising all wasp species in the two lineages is not feasible within a single project. These braconids are extraordinarily diverse, more than 20-30 species often existing in a small area, e.g., >35 species were documented in the La Selva biological station (~15 km2) in Costa Rica44. We will perform area-based taxonomic revisions for the wasps. We will sample extensively in four sites in the Amazon and lowland Andes, where the diversity is the highest. For each site, we expect to treat ~25 species of Alabagrus and ~25 species of Capitonius, hence a total of ~200 species. Of these we expect at least half or ~100 species to be new to science. These estimates are realistic and are based on previous sampling efforts in similar Neotropical habitats by co-PI Sharkey44. Product (2). A rich set of primary specimen-based biodiversity data. Using museum specimens and newly collected specimens, we expect to generate biodiversity data for at least 10,000 specimens. This data will provide the best knowledge of geographic information and habitat. Product (3). Electronic taxonomic descriptions and identification products. In order to increase the reusability and computability of taxonomic descriptions, we will use a recently developed ontology-based semantic representation approach for the taxonomic descriptions of the braconid wasps45,46. We will take advantage of the NSF-funded Hymenoptera Anatomy Ontology project47 and use the mx (Matrix; mx.phenomix.org/) online taxonomic content management platform. While a hemipteran ontology does not yet exist and construction of one is beyond the scope of the proposed project, we will closely follow anatomy ontology-like principles in preparing our descriptions (e.g., using EQ [entity-quality] coding), thereby maximizing downstream compatibility. We will create interactive taxonomic keys, enabling access to the knowledge of mimetic species and facilitating future species discovery. Product (4). Extensive morphological/phenotypical documentations. We will provide morphological documentation of taxonomically important characters and also mimetic traits. We will produce 2,400 photographs or illustrations. More than 1,000 images of Alabagrus wasps based on previous works by Sharkey have been made available at Morphbank and the Encyclopedia of Life (EOL). Product (5). Revision and construction of predictive phylogenetic classifications. Although the monophyly of Alabagrus is established, monophyly of the five reduviid genera and Capitonius has never been tested with formal phylogenetic analyses. Some characters such as color patterns used to delineate generic limits of the reduviids appear to be highly homoplastic resulting in possibly polyphyletic or paraphyletic genera. This makes the genus-names problematic for communicating about mimicry. Based on newly constructed robust phylogenies (see 3.1 below) we will test and redefine generic limits. Figure 3. Morphological diagnostic characters for species delimitation and identification in Alabagrus and Capitonius wasps. Arrows indicate differences. (A) Variation in postero-ventral apex of genae. (B) Differing degrees of sculpture on mesoscutum. (C) Differences in dimensions of metastomal median tergite and sculpture of propodeum. (D) Presence and absence of grooves on metacoaxe. (E) Diversity of sculpture and dimensions in the first metasomal median tergite. (F) Different sculptures on ventral surface of hind femora. Alabagrus – A, C, E & F. Capitonius – B & D.

1.2 Addressing specific challenges – Delimiting species with integrative taxonomy. We will follow the well-established integrative taxonomy paradigm48,49 and use both morphology and DNA data to formulate and test species hypotheses. Utility and limitations of morphology in mimetic species. Longterm taxonomic research conducted by the PIs and other past and contemporary workers demonstrates that morphology holds great utility in delimiting species boundaries in the target lineages or close relatives44,50,51. Diagnostic morphological characters of braconids and reduviids can often effectively delimit species and assign specimens to species membership even across large geographic ranges 4

(e.g., >3000km for Zelus renardii [Reduviidae]). In the wasps, readily observable external structures such as topology of metascutum, configuration of metasomal median tergite, and sculpturing of propodeum (Fig. 3) are excellent diagnostic characters. In the reduviids, external coloration and structure can be useful, but male or female genitalic structures often offer much greater resolution and accuracy. We will fully exploit the utility of morphology, but we are aware of and also have evidence showing the limitations of morphology in our target taxa. And we propose to use DNA data to corroborate or revise morphology-based species hypotheses. The integrative approach. Species hypotheses will be initially formed based on morphology using a unique combination of diagnostic characters. DNA data will be used to validate or revise these morphospecies. This two-step discover-validation strategy has been well established in the species delimitation literature52. In our project DNA data will be most useful for associating sexes, polymorphic forms and delimiting cryptic species. Based on DNA data, we have found sexual dimorphism in two putative reduviid species (Fig. 10). Sharkey and colleagues’ previous research indicates presence of cryptic species in braconids, and DNA data has proved effective for resolving such difficult cases53–55. We will target 3-5 specimens per morpho-species to sample for DNA data. We will use the DNA barcoding gene COI. We will follow Carsten et al (2013)52’s argument and use multiple algorithmic methods. We will place our trust in delimitations that are congruent with both morphology and DNA. Two general scenarios of incongruences are expected. When variations are subtle, initial morphological observations may result in ‘lumping’ of species. Under the light of DNA data we will reexamine the morphological evidence and revise species diagnosis. An example is provided here comparing two species with Figure 4. Two similar/cryptic Alabagrus minimal morphological variations but differing in COI by species. Morphological variations were initially 6.3% (Fig. 4). Contrastingly, morphology might ‘split’ considered to be intraspecific, but redefined as species, usually due to large discrete variation, interspecific under the light of DNA data. polymorphism, especially between allopatric populations, or sexual dimorphism. We emphasize that COI will not be a stand-alone data source for forming species boundaries, as some authors have argued that single locus is often not sufficient56. Rather, COI will mainly be used as a tool for testing and revising morphology-based species hypotheses and associating dimorphic sexes or polymorphic forms. Preliminary results. As many as ~2,000 specimens of Alabagrus and Capitonius have been amassed by Sharkey from the Neotropics (including but not limited to Mexico, Costa Rica, Panama, Colombia, Peru, Brazil, French Guiana and Guyana) and are maintained in a -80 °C freezer, ready for species boundary testing and formal description. Sharkey is currently finishing a revision of the species of Alabagrus of Costa Rica based on a multi-year sample, building on his previous publications on this group50,44 and is treating slightly more than 60 species from one area alone, i.e., Guanacaste State, Costa Rica. ~200 specimens of mimetic assassin bugs have been loaned from two collections (Texas A&M Insect Collections) and INBio (National Biodiversity Institute, Costa Rica). These specimens, together with previously collected ethanol-preserved samples, have been sorted and assigned to 31 species, a value already much higher than the current known diversity (19 species). We analyzed a data set of ~450 Alabagrus COI sequences representing 53 morpho-species, using a maximum likelihood-based Poisson Tree Process (PTP) approach57. We Figure 5. DNA-based species delimitation of Alabagrus using COI, with ~450 sequences, representing 53 morpho-species. (1) DNA and morphology are congruent for most species (green branches). (2) DNA detects cryptic species, which morphology failed to recognize (branches in other colors; e.g., blue branches were considered as one species by morphology, but represent two species according to DNA. Poisson Tree Process method (Zhang et al. 201257)

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show here COI-based species has produced results largely congruent with morphology based delimitation, but is mainly effective at identifying morphological invariable species (Fig. 5). DNA data delimited 15 species out of five of the morpho-species. Only two morpho-species were not considered as unique species by DNA. Objective 2. Connecting taxonomy, phenotype and phylogeny – characterizing and analyzing patterns and diversity of mimicry. This research objective will build upon the extensive taxonomic work and specimen data accumulated in the previous object, and will also provide essential comparative data for testing hypotheses of mimicry evolution using phylogenetic methods. The documentations of diversity and patterns of mimicry have far-reaching implications. For example, observations of local polymorphism have led to the hypothesis of a supergene controlling color patterning58,59, which has recently been confirmed with massive genomic mapping efforts and the supergene loci have been found in various butterfly species60,61. Other interesting aspects of mimicry include: mimetic sexual dimorphism, sex-limited mimicry, divergent patterns between sister Figure 6. Lateral surface can be into 17 regions and species and geographical races. We will gather a large number of specimens divided scored for 4 color classes/hues. and a diverse taxonomic sample, and we will explore and Table 1. Color space coding and descriptions. document important aspects of mimetic patterns. The data Color dimension No. Description (x, y, z) and findings will not only be useful for our comparative (X) Forewing 0 uniformly dark 1 dark, single apical or medial clear band phylogenetic analyses, but also provide fresh information pattern 2 dark, single subcostal yellow spot for ecological and genetic aspects of mimicry research. 3 alternating yellow and dark double bands 4 yellow, single apical or medial dark band 2.1. Quantifying and classifying mimetic color 5 clear, colorless patterns. What are the most common color patterns? Do (Y) Dark areas 0 absent color patterns reoccur among different species more often on body 1 present only on thorax 2 present only on abdomen than would be expected by chance alone? To answer these surface 3 present on thorax and abdomen questions we will need to quantify and classify colors. We 4 over entire surface follow and modify a method used to study bumble bees (Z) Dominant 0 yellow or orange of light 1 reddish colors62 to quantitatively classify mimetic color patterns at hue colors on body 2 absent (body entirely dark) both a fine scale and a coarse level. First, to achieve a refined classification, the body surface will be divided into color regions and each will be scored for a color class/dominant hue (Fig. 6). This will generate numerous mimetic color patterns. Subsequently these patterns will be categorized into major color-pattern groups according to a scheme focusing on the major body parts (Table 1) and visualized on a three-dimensional ‘color space’ (Fig. 7). 2.2. Documenting the diversity of Figure 7. Three-dimensional color pattern space. See Table 1 for color coding and descriptions. (1) A total of 52 combinations of color patterns mimicry rings and mimetic sexual are theoretically possible. (2) 24 patterns are observed and 28 theoretical dimorphism and polymorphism. combinations are not actually observed (denoted by dots). (3) DarkSeveral important questions, mainly winged (X=0) and banded winged (X=3) forms are most diverse. concerning the community structure and phenotypic diversity of mimicry patterns, will be investigated. (1) How many mimicry rings can co-exist in a local community and how many species and lineages participate in each mimicry ring? The local diversity of Müllerian mimicry rings is one of the greatest puzzles in mimicry research63. This empirical observation contradicts the theoretical prediction that comimics should converge on the same pattern in order to maximize predator learning. Contrasting to 6

Müllerian mimics, Batesian mimics are under negative frequency-dependent selection on mimetic phenotypes, which may favor the evolution of multiple color forms that mimic different models so that predators cannot easily associate palatability with any single form64. Our three target groups contain both Müllerian and Batesian mimics and all seem to exhibit a rich local diversity of mimicry rings. We will sample extensively from four distinct localities to assess the diversity and constituency of mimicry rings. (2) Do sexual dimorphism and intraspecific local polymorphism exist? Sexual dimorphism, especially female-limited mimicry is common in Batesian mimicry, but rare in Müllerian mimicry65 2.3. Assessing the exclusiveness of mimetic associations between reduviids and braconids - are there other secret players? Our observations indicate that the mimicry complexes possessed by reduviids and the two target braconid genera include other lineages of reduviid bugs and braconid wasps, and also insects form completely different families or orders, e.g., ichneumonid wasps, beetles, plant bugs, and cockroaches. However, this speculation has never been tested with synchronous sampling from local communities and we aim to address this knowledge gap. We will collect a large number of specimens representing diverse insect lineages from four long-term target collecting sites. We will sort, identify and analyze the diversity of mimicry rings, their taxonomic compositions and their relative abundance in term of species numbers as well as specimen quantities. We will classify the color patterns to major groups according to the scheme proposed in 2.1 (Table 1, Fig. 7). Preliminary results. Color pattern classification. Based a sample of 60 Capitonius species, 50 Alabagrus and 30 species of reduviids, we were able to classify 24 major color patterns (Fig. 7). This is about half of the 52 theoretical combinations of color patterns. In other words, 28 theoretical forms were not observed. The color forms with double-banded or black fore wing appear to be most diverse, occupying >65% of the observed major color patterns (16 out 24). Diversity and composition of mimicry rings. At one site in Costa Rica, Alabagrus species form at least seven mimicry rings44. In a small area in Southeast Peru (i.e., Los Amigos Biological Station, $83 ($25,000/300) or 330 times more expensive. 3.2 Hypothesis testing – Inferences of ancestral states and evolutionary pathways. We will address a series of fundamental questions concerning the evolution of mimicry. (1) Do shared mimetic forms result from common ancestry or convergent evolution? (2) How many times did mimetic coloration evolve, what is the most ancient form, and have species reverted to a non-mimetic or cryptic color condition? (3) Did novel mimetic forms arise more often from a cryptic or an existing mimetic ancestor? (4) Can we trace the sequence(s) of evolutionary transitions between the various mimetic forms, leading to the current diversity of mimicry, and are there biases in the evolutionary transitions to and from certain mimetic color forms? The answers to these questions bear special significance to understanding the patterns and processes of mimicry evolution as we discuss below. Repeated or convergent evolution is strong evidence for adaptive evolution offered by phylogenetic comparative analyses75. The evolution of aposematic or mimetic coloration from cryptic ancestors within a linage appears to be rare, at least in the butterflies, counting for only 12 origins in 18,000 species. By contrast, nearly 400 color variants may have evolved repeatedly from existing mimetic forms Figure 8. Ancestral state reconstructions of 10 major color in just ~40 Heliconius species76. Last, loss of mimicry patterns in Capitonius using Mesquite based on maximum has been occasionally documented in species likelihood phylogeny. Colored branches indicate parsimony reconstruction and pie charts indicate relative likelihood of occurring in areas without the model77,21. ancestral states with maximum likelihood reconstruction and Predictions or expectations to be tested. Taken all are placed on nodes leading to pattern changes. together, we hypothesize that (1) shared mimetic forms are mainly a result of convergent evolution, but may also result from shared phylogenetic history; (2) the evolution of mimetic types from a cryptic ancestral condition happened infrequently and loss of mimetic condition is possible but rare; (3) novel mimetic color types evolved from existing mimetic conditions more often than from cryptic ancestors; and (4) transitions to and from color forms are not randomly distributed on the phylogeny. 3.3 Additional phylogenetic hypothesis testing of correlated evolution between mimetic color components. Evolutionary correlations between phenotypic characters can arise by natural selection or linkage between the genes regulating the characters78. Major color patterns may represent Figure 9. Transitions between color forms in separate peaks in the mimicry/warning color adaptive Capitonius. Arrow thickness is proportional to 76 landscape , i.e., alterations of the individual components are relative transition frequency. Transitions among disadvantageous. Recent research on the genetic mechanisms forms are unevenly distributed.

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for controlling mimetic color patterns13,15,17, 77–81 have revealed that much of the color pattern diversity and variation in Heliconius can be attributed to just a few loci, which are often tightly linked and control stereotypical expressions of correlated color components83. We will test correlation between color components in reduviids and braconids. We will target distinct color patterns that may represent adaptive peaks and examine whether their formation is created by linked color components. According to theories of coloration and empirical observations84, yellowblack bands are great warning colors because they create high contrast against the background and can be detected under varied environmental conditions. And the black and red combination has both high chromatic and luminance contrast. We formulate two specific hypotheses: yellow-black banding on the wings is correlated with yellow-black banding on the body (abdomen, thorax or both) and red colors on body surface are correlated with black wings. 3.4 Expanding the mimicry paradigm – Phylogenetic community ecology of mimicry – how do mimetic interactions affect community structure? The role of mimetic interactions in shaping community structure has seldom been investigated using the approach of phylogenetic community ecology. Under this paradigm, patterns of phylogenetic relatedness of species from a local community can be characterized as phylogenetically clustered (co-existence of close relatives) or over-dispersed (coexistence of distant relatives)85,86. While the former suggests environmental filtering as the dominant force in shaping community structures, the latter invokes competition. Interactions between Müllerian mimics are supposed to be positive or mutualistic and thereby help ecologically similar species co-exist87. . In a local community, Müllerian co-mimics are expected to be phylogenetically more closely related than randomly distributed on the phylogeny, resulting in a pattern of “phylogenetic clustering”. Contrastingly, Batesian mimics functions much like competition because negative frequency-dependent selection keeps the mimics in low numbers. Batesian co-mimics are expected to be phylogenetically dispersed. To date, only two empirical studies focusing on Müllerian mimicry systems have been conducted. However, they showed exactly opposite results. Alexandrou et al (2012) showed that in Neotropical catfish “resource partitioning [competition] and phylogeny determine community structure and outweigh the positive effects of Müllerian mimicry”. Contrastingly, Elias et al (2008) provided Figure 10. (1) Reduviid co-mimics from the same community may evidence from ithomiine butterflies that appear to be phylogenetically dispersed or clustered. (2) Sexual “[mimetic] mutualistic interactions can drive dimorphism is observed at least twice, both with the female having black-wings. Maximum clade credibility tree generated with MrBayes. convergence along multiple ecological axes, outweighing both phylogeny and competition in shaping community structure”. We have not yet seen tests in Batesian mimicry systems. The lack of consensus and studies calls for more empirical testing. Our sampling strategies will provide 12 empirical tests (four sites multiplied by three groups). We will utilize the global phylogenies newly reconstructed in this project. We will use the well-established net relatedness index (NRI) and nearest taxon index (NTI)85,88 to determine the phylogenetic relatedness among co-mimics in local communities. Positive values of these indices indicate phylogenetic clustering, and negative values indicate phylogenetic over-dispersion. Preliminary analyses and results. Phylogenies. We obtained a five-gene data set of 60 species of Capitonius wasps and a four-gene data set of 20 mimetic reduviid species based on previous or ongoing research89. We reconstructed the phylogenies using maximum likelihood (with RAxML) and Bayesian inference (with MrBayes) methods, which produced highly congruent or identical topologies. We performed comparative analyses to investigate some of the questions posed here. Ancestral state 9

reconstructions and evolutionary transitions. Based on maximum likelihood and parsimony reconstructions using the program Mesquite90 (A Modular System for Evolutionary Analysis, http://mesquiteproject.org/) of major color patterns in Capitonius, a cryptic color form (clear wings with dark body) is the ancestral state, and conspicuous or mimetic conditions have evolved at least nine times independently in that group (Fig. 8). The reconstructions also support the idea that new color patterns have evolved more readily from a mimetic ancestor than from non-mimetic/cryptic ancestors. In lineages that have generally retained the cryptic ancestral condition, only four mimetic forms have evolved in 12 species (out of 37 in total). By contrast, color patterns seem to have radiated in one clade which has the “single-banded forewing & orange body” mimetic form as the ancestral condition. Eight different color forms exist in this clade containing just 25 species, some forms have evolved repeatedly, and twelve shifts in color forms have occurred (Fig. 8). Also, a single reversal to a cryptic form also occurred in this clade. By summarizing color pattern changes in Mesquite over 3,000 trees from posterior sampling generated by MrBayes, we show that transitions among color forms are not randomly or equally distributed among forms (Fig. 9). Interestingly, there is no transition between two apparently similar forms (Dark-winged with red colors on body, 2 & 3 in Fig. 9). For Capitonius we tested for correlation and character dependence between wing banding and body banding, and between red abdomen and black wings using Pagel’s correlation test75. Interestingly, the results show that presence of red abdomen is dependent on black wings, but there is no correlation between wing and body banding, warranting further investigations. The reduviid co-mimics from two local communities may show either phylogenetic overdispersion or clustering (Fig. 10), and stronger sampling of the proposed project should provide a clearer picture. RESEARCH APPROACH & EXPECTED OUTCOMES Specimen sampling strategies. We will use existing or newly collected ethanol-preserved DNAquality specimens for molecular work as well as for morphological examinations. We have observed that the colors of both braconid and reduviid specimens can be well-preserved in ethanol. Field work. We will perform extensive new field trips in four South American countries (Brazil, Colombia, Ecuador and Peru). We will adopt two complementing collecting strategies: long-term local collecting and active broad-range collecting to obtain both revisionary and phylogenetic samples. For the former, we will run five Malaise traps at each of the four targeted collecting sites for at least a continuous 6-month duration in four countries. We will hire local collecting assistants to collect the trap samples every week to ensure the qualities of specimens. Co-PI Sharkey has used this collecting method in three massive multi-year insect biodiversity inventory projects, including two in Colombia and the specimens have generated great DNA data in several publications27,28. Our targeted collecting sites are: (1) Colombia: Amacayacu National Park, 3°29’0”S, 70°12’0”W ; (2) Brazil: Reserva Florestal Adolpho Ducke (Amazonas State), 2°57'42"S 59°55'40"W; (3) Ecuador: Tiputini Biodiversity Station, 0°38'18.0"S 76°09'00.0"W; and (4) Peru: Los Amigos Biological Station, 12°34'09.0"S 70°06'00.4"W. For active collecting, we will sample more broadly in the four target countries to cover large geographic ranges in order to attain the largest phylogenetic diversity possible. This combination of samples will not only allow us to reconstruct global phylogenies and perform clade and area-based taxonomic revisions, but also to investigate mimicry in local communities. We have secured collaboration agreements from local contacts in all four target countries (Dr. Dimitri Forero, Colombia; Dr. Frank Azorsa, Peru; Dr. Tânia Mara Guerra, Brazil; and Dr. Kelly Swing, Ecuador; collaborator letters included), and they will help us with obtaining collecting and export permits, arranging local logistics, and recruiting local collecting assistants and graduate students for training in the field and the PIs’ home institutions. Museum specimens. Additional sources of specimens, useful at least for taxonomic revisions, will also come from museum loans. We will examine ~1,000 museum specimens from >20 museums or for each focal taxonomic group, as we have routinely done in other similar taxonomic projects, therefore a total >5,000 specimens. Because of their relatively large size and bright coloration, our focal taxa are well represented in collections of Neotropical insects. Outcome: ~10,000 specimens as a basis for primary biodiversity information. We will amass the largest samples of the focal taxa ever possibly known and create an invaluable set of biodiversity data, which will be a significant advance to documenting mimetic species diversity. Monographic revisions and integrative taxonomy. Zhang will perform revision of all reduviid 10

species (~50 spp.), but also collaborate with Dr. Christiane Weirauch (University of California, Riverside, letter included). Sharkey and Zhang will revise Alabagrus (~100 spp.). Zhang will receive training from Sharkey on braconid taxonomy. One PhD student will be trained and will revise Capitonius (~100 spp.) with supervision and assistance from Sharkey. Online specimen and taxonomic content database or management systems. We will create and manage all of our specimen-based biodiversity and taxonomic data using structured online databases and content management systems to allow for maximum data availability and reusability. We choose to use the NSF-supported, widely used Symbiota database91 to manage specimen-based primary biodiversity data, including geo-referenced locality data, images, and taxon profiles. Both institutions (ASU & UK) have established a collection portal at Symbiota, which is ready to incorporate more specimen data. To manage taxonomic content, especially character descriptions and documentations, we will use mx47 (matrix; http://mx.phenomix.org; soon migrating to TaxonWorks, Yoder, pers. comm.). This system supports ontology-driven or matrix-based character descriptions, dichotomous and interactive keys, image linking, and multi-user online collaboration, and suites the needs of the project. Databasing and georeferencing. We target to database ~10,000 specimens (5,000 museum specimens, 2,000 existing ethanol specimens, and 3,000 newly collected specimens). All specimens will receive a unique identifier and databased using the Symbiota portal. Historical specimens will be carefully geo-referenced following protocols established at the Franz Lab (ASU). All specimen data will be harvested by the national iDigBio (integrative Digital Biocollections) portal. Character documentation. We will use a combination of high-resolution digital photography and illustrations to document morphological characters, techniques proven effective and used in previous studies by the PIs44,92,93. We will produce altogether >2,400 morphological documentations (10 images/wasp species; 8 images/redvuiid species). All images will be uploaded to Symbiota, which will link to primary specimen records, and also morphbank.net (e.g., Sharkey Group: http://www.morphbank.net/?id=7), which automatically feeds to the Encyclopedia of Life. Sharkey has already uploaded ~1,000 images representing 110 species of Alabagrus based on previous work in EOL [http://eol.org/collections/233/images?sort_by=1&view_as=3]. Taxonomic descriptions. For wasps, we will utilize the Hymenoptera Anatomy Ontology framework as implemented in MX to create structured descriptions that enable standardized character and character state definitions. There is currently no hemipteran or reduviid anatomy ontology, but we will follow the ontology-like entity-quality description principles45 to code and describe characters, which will facilitate downstream processing. Publication. To disseminate knowledge widely, we will publish all biodiversity data and taxonomic works with open access publications (e.g., Zookeys, Biodiversity Data Journal, PLoS series or PeerJ; the last is free for ASU authors). We will also generate species pages or taxon profiles using Symbiota or specie-id.net. Integrative taxonomy: species delimitation and hypothesis testing. We will first form species hypotheses based on morphology, which are termed morpho-species. We will then use COI DNA barcodes to corroborate or revise the morpho-species delimitations. To obtain this molecular data set, we will use established primers and protocols of DNA extraction, PCR and sequencing, previously tested and used in wasps55 and hemipterans94. We will use Sanger sequencing, which still remains an effective method for obtaining single-locus data. For each species, we will target 35 specimens for DNA barcodes. We will use at least three algorithmic delimitation methods: the “Bayes factors” coalescence-based species delimitation method as implemented in the program BEAST95, which has the advantage of testing hypotheses of specimen assignment to alternative lineages96, the Generalized Mixed Yule Coalescent (GMYC) approach97, and the Poisson Tree Process (PTP) method57, which does not require an ultrametric input tree, thereby alleviating time calibration errors. Outcome: unprecedented taxonomic synthesis of focal taxa and rigorous tested species hypotheses. We will produce at least nine monographs and make all information widely available. Mimicry classification and characterization. We will photograph the dorsal and lateral surfaces of the insects using high-resolution digital SLR cameras (available at both labs) on 18% grey card background, which does not affect the colors/hues of the insect. We will take two steps to first maximally recognize the diversity of color patterns, and then classify these patterns into major color groups. In the first step, the body surface including wings will be divided into color regions, and the dominant hue (yellow, orange, red or black [lack of hues]) of each region will be scored. These individual color patterns 11

will then be classified into major groups as previously described (Table1, Fig. 7). Using the long-term trap samples collected from four sites, we will collect and analyze the following information. (1) The diversity/number of mimicry rings (major color patterns) at each site. (2) The taxonomic composition of each mimicry ring, including braconid wasps, reduviids, and other insects. All insects other than braconids and reduviids involved in the mimicry ring will be photographed and identified to at least the family or genus level. (3) The quantities of co-mimics in the same mimicry ring. And (4) the relative abundance of each mimicry ring in a local community. Outcome: advanced understanding of mimicry in nature. Our data and analyses will provide fundamental information on one of the largest mimicry complexes in the Neotropics and these data or knowledge will inspire other research on these fascinating organisms. Next generation sequencing using ddRADseq. We will perform wasp and reduviid molecular work respectively in the two participating laboratories. All PIs have fully equipped molecular labs. We will sample 96 taxa of each wasp genus (192 in total,) and 48 taxa of the reduviids (both including outgroups). These samples will represent a large taxonomic and geographic diversity. We will select closely related outgroup taxa according to higher-level phylogenies28,98,99 previously published by the PIs. We will collaborate with Dr. Barbara Sharanowski (University of Manitoba), who has extensive experience with next generation sequencing methods100–102. One PhD student at UK will perform molecular work on the Capitonius wasps (96 samples) and an existing full-time technician (Eric Gordon [UK]; supported by other sources) will perform the remaining wasp molecular work. Zhang will perform molecular work on the reduviids. We will extract total genomic DNA using the Qiagen DNeasy Blood and Tissue DNA extraction kit using a non-destructive extraction method (whole-body soak) to preserve the intactness of voucher specimens. DNA quality and quantity will be tested using a NanoDrop and gel electrophoresis. We will follow the ddRADSeq protocol outlined in Peterson et al (2012)72 and use the bioinformatic analysis pipeline employed in a recent empirical phylogenetic study70. Briefly, DNA will be digested with two restriction enzymes and then individual multiplex identifiers and sequence adapters will be ligated to genomic DNA (barcodes and sequence adapters designed by Peterson et al, 2012). Samples will then be pooled, cleaned, size selected for fragments between 200-400bp, and enriched with PCR amplification. The enriched RADseq libraries will then be sequenced 48 taxa per lane using paired end sequencing on the Illumina MiSeq (available at both institutions, ASU and UK). Prior to creating our RADSeq libraries for all taxa, we will test several digest combinations with four test taxa to ensure an adequate number of loci that will achieve appropriate coverage. Sequences will be de-multiplexed using Python scripts designed by Peterson et al (2012) that recognize the specific adapters and oligos that were ligated to the genomic DNA for multiplexing. Sequencing reads for individuals will then be filtered for quality and assembled de novo into individual clusters (aka loci) using the program pyRAD103. Compared to the program ‘Stack’, commonly used for calling SNPs for population-level studies, the pyRAD program allows for lower similarity clustering thresholds and the inclusion of indel variation, and therefore proves to perform better at identifying and assembling orthologs for phylogenetic analyses103,104. The clustered data sets will be individually aligned using MUSCLE105, a fast program ideal for dealing with large numbers of loci, and concatenated into a single supermatrix (three matrices in total, each for the two wasp genera and the reduviids). Phylogenetic reconstructions. We will use RAxML106, a maximum likelihood program to analyze the RADseq data and reconstruct the phylogenies because its computational power has proved to be able handle the large number of taxa and sequence data produced from RADseq70,73. The general time reversible model, with Gamma-distributed rate heterogeneity and invariant sites (GTRGAMMAI) will be used for DNA sequence evolution. Branch support will be evaluated with at least 200 nonparametric bootstrap replicates (larger values may become computationally prohibitive for the large target data sets). We will also use a Bayesian inference method of multilocus species trees107 that applies coalescent theory to multiple unlinked loci, and infers the species tree while accounting for incomplete lineage sorting of individual genes. This will also generate a set of trees drawn from the posterior distribution, which can be used in comparative analyses that accommodate phylogenetic uncertainty (e.g., BayesTraits). We will use the program BEAST2108 [http://beast2.org/] to implement the multilocus Bayesian inference and estimate 12

the species trees. Because of computational limitations, reduced matrices derived from ddRADseq (~10% of full matrix) will be used in the Bayesian inferences following a procedure described in Rubin et al (2012)73. In BEAST, the lognormal relaxed-clock model will be used to allow rate variation among branches without a prior assumption. The Yule speciation process will be used as the tree prior. The analyses will run four times each for at least 50 million generations in BEAST, sampling every 5,000 generations, but higher numbers may be used to achieve chain convergence. We will assess convergence of model parameter values and node-height estimates by plotting the marginal posterior probabilities versus the generation state in the computer program Tracer [http://beast.bio.ed.ac.uk/Tracer]. The posterior probability density of the combined tree and log files will be summarized as a maximum clade credibility tree by using TreeAnnotator [http://beast.bio.ed.ac.uk/TreeAnnotator]. We will use the program FigTree [http://beast.bio.ed.ac.uk/FigTree] to visualize the phylogeny with posterior probabilities. Outcome: robust phylogenies and novel phylogenetic pipelines in insects. This project will be among the first to use the novel RADseq method in insects on empirical phylogenetic reconstructions, thereby providing much baseline data to future researchers. Comparative phylogenetic analyses. Ancestral state reconstruction. We will code and analyze two character systems: major color pattern groups and individual color elements or regions. The former will allow us to infer whether major mimetic patterns have repeatedly evolved. The latter will be useful for reconstructing the details of trait evolution on a phylogeny and testing correlations between color components. We will use parsimony and likelihood ancestral state reconstruction methods implemented in Mesquite, using the likelihood phylogeny produced from RAxML. To account for phylogenetic uncertainty, we will also use BayesTraits109 and the sample of trees from posterior distribution produced by BEAST. Both likelihood and Bayesian reconstruction methods can infer character evolution with probability estimates. Testing convergent evolution of color patterns. Based on the character mapping results of ancestral state reconstruction, we will be able to infer whether shared color forms are due to common ancestry or represent convergent evolution. This is an easy and fast method. However, this does not provide statistical significance support. We will also use two other methods: Slatkin-Maddison test110 (implemented in the program Mesquite [a modular program for evolutionary analysis] http://mesquiteproject.org/) and Bayesian Tip Signifcance testing (BaTS, 111 http://evolve.zoo.ox.ac.uk/Evolve/BaTS.html) . The former utilizes a single phylogeny, uses an ‘association index’ within clades recursively, and compares observed value with a null (expected) value obtained by bootstrapping. The BaTS method tests for significant phylogeny-trait associations and accounts for phylogenetic uncertainty by integrating over the posterior sampling of trees. In BaTS 1,000 randomization tests will be generated to create the null distribution to test the significance of the observed color patterns distributed on the phylogeny. Character transition and pathway analyses. To test the hypothesis that transitions between major color forms are not randomly distributed on the phylogeny and to quantify the rates of color changes, we will use the program BayesTraits. The relative MCMC (Markov Chain Monte Carlo) transition rate coefficients will be summarized over the collection of posterior samples of trees. Testing correlation between color components. We will use Pagel’s correlation method75 as implemented in Mesquite to test for correlations between color components. For each correlation test, we will use eight parameter models, with an intensity of likelihood search equal to ten and 1000 simulations conducted. Phylogenetic community ecology analyses. We will use the open source software package Phylocom112 to calculate the NRI and NTI indices. We will perform analyses with four randomization models and determine significance with 999 randomization runs. Positive NRI and NTI values indicate phylogenetic clustering and negative values indicate overdispersion. Outcome: paradigm shift towards hypothesis-driven phylogenetic studies of mimicry. Our methods and questions may be applied to numerous other mimetic insects or organisms, and thus generating a community interest in the comparative aspects of mimicry. BROADER IMPACTS We will integrate our research with training, public education, knowledge dissemination and international as outlined here. (1) The project will offer intensive training to PI Zhang (postdoc) as an emerging biodiversity research leader, strengthening his existing taxonomic expertise, broadening his 13

taxonomic scope to also include parasitic hymenopterans, training him on cutting-edge next generation sequencing technologies, deepening his analytical skills, and fostering or promoting his project leadership, scientific communication, and student mentoring skills. (2) We will train one PhD student in the systematics and evolution of braconid wasps, in phylogenetic techniques, and in Neotropical biodiversity, thereby preserving taxonomic expertise in this large and important insect group. (3) We will generate a substantial amount of comparative data and biodiversity information on mimicry, laying down a foundation for future ecological and genetic research. (4) This project will allow us to combine and synthesize taxonomic expertise in two unrelated insect groups, thereby facilitating taxonomic knowledge integration. (5) We will design and assemble Insect Mimicry and Camouflage drawers and display them to the general public at the ASU Biodiversity and Bioinformatics Center (also housing the Hasbrouck Insect Collection) and at the University of Kentucky. We will utilize existing specimen resources available at both institutions, but also incorporate material newly collected in this project. Three undergraduate students (two at ASU and one at UK) will assist the PIs with making the drawers and also showcasing to the public. One special event to display the drawers to a large number of the public is the annual ASU Open Door Night. This event draws at least 1,500 visitors to the insect collection based on past statistics. Zhang had participated in this event in 2014 and educated the public about the diversity of hemipteran insects. (6) We will design and implement an ‘Evolve a Mimic’ educational game based on our results of character evolutionary analyses. Models of mimetic species or ancestral nodal patterns will be made from painted wooden or styrofoam boards and simplified phylogenetic trees made form PVC pipes. The player will try to place the ancestral mimetic patterns at the nodes based on simple rules and information contained in the phylogeny and tip species. The players will learn basic concepts of the phylogeny/tree of life such as “common ancestor”, “sister species/groups” and “cladogenesis”. This game will introduce ‘tree-thinking’ to the general public and correct for the popular misrepresentation of evolution as linear113 (e.g., the popular illustration of monkey-ape-human evolution). This activity will involve at least three undergraduate students. We will collaborate with Dr. Melody Basham on outreach activities; she is an outreach specialist at the Franz Lab and has a strong background in technology-driven outreach (letter included). (7) We will train at least 15-18 undergraduate students in research and outreach, as has been actively done by all PIs (e.g., Zhang had trained seven undergraduate research students since Aug 2013 at ASU, each for at least one semester; and one minority student won a NESCent travel award to attend the Evolution2014 meeting). At least six of them will be from underrepresented background. (8) We will disseminate taxonomic, specimen, image, comparative and phylogenetic information and knowledge widely and promoting data reusability by using publicly accessible databases and sharing all project data and results (e.g. Symbiota, Morphbank, Encyclopedia of Life, TreeBase, Dryad and figshare). (9) The project will promote international collaboration with scientists from four countries (Brazil, Colombia, Ecuador, and Peru). (10) We will train South American students in the taxonomy and evolution of Hemiptera and Hymenoptera, thereby disseminating biodiversity expertise more broadly to developing tropical countries in much need of knowledge and expertise. PRIOR SUPPORTS 1. PI Zhang. N/A (Beginning investigator). 2. Co-PI Sharkey. NSF- DEB 0542864. 2006-2010, $649,999. TIGER: Thailand Inventory Group for Entomological Research. PI: M.J. Sharkey and B.V. Brown. Training and Development: One undergraduate (minority), two graduate students, and a technician worked with the PI in Thailand. One trainee has started a PhD at the University of Arkansas, and the other is an assistant professor at the University of Manitoba. About forty Thai National Reserve employees were trained to collect and prepare insect samples. Collections: Many collaborators have incorporated specimens into their collections where they will be available to future revisers. For example, Paul Freytag (U. Kentucky) prepared more than 20,000 Cicadellidae (leafhoppers), James Pitts has curated a similar number of Pompilidae (spider wasps), and at the U.K. Hymenoptera Institute, more than 15,000 Braconidae have been mounted and hundreds have been sequenced for 28S and COI. Electronic Products: We developed a large database on the web with tools for our collaborators and results of our sampling efforts, e.g., species distributions with Berkeley Mapper. Also, we have published interactive keys to the Oriental genera and species of the 14

braconid subfamily Agathidinae. Outreach activities: We have produced 10 newsletters that kept collaborators informed and involved, and presented lectures to reserve staff on insect diversity and conservation on every visit. Publications: Since the inception of the project, more than 70 publications have been generated from specimens collected through the TIGER project. Sharkey has co-authored seven papers on braconids as a direct product of the collections (van Achterberg et al. 2014, Sharkey and Stoelb, 2013, 2012a,b, Sharkey and Clutts 2011, Stary et al. 2010, 2008; see References114–119). 3. Co-PI Franz. All as lead PI unless otherwise noted. (1). NSF-DEB 0641231. RIG: Towards a Systematic and Evolutionary Synthesis of the Neotropical Exophthalmus Genus Complex (Coleoptera: Curculionidae: Entiminae). 2007–2010. $174,755. IM (Intellectual Merit): Twelve peer-reviewed articles published; BM (Broader Impacts): two graduate/eight undergraduate students trained. (2). NSF DBI0749434. BRC: a New Infrastructure for Invertebrate Biodiversity Research in Puerto Rico. 2008–2012; $318,292. IM: Moved > 175,000 specimens into new storage system; created first Caribbean GBIF Arthropod node; BM: 58 undergraduate students trained. (3). NSF DEB-1155984. CAREER: Systematics of Eustyline and Geonemine Weevils: Connecting and Contrasting Caribbean and Neotropical Mainland Radiations. 2011–2016; $639,747. IM: Eight papers published, two in review; BM: one postdoc, two graduate students; and six undergraduate students in training. (4). NSF EF-1207107. Digitization TCN: Collaborative Research: Southwest Collections of Arthropods Network (SCAN): a Model for Collections Digitization to Promote Taxonomic and Ecological Research. 2012–2015; $284,613. IM: Reached all digitization benchmarks after 2/3 project years; links to Filtered Push, created Weevils of North America (WoNA) checklist; BM: > 15 undergraduate students mentored. (5). NSF DBI-1342595. Collaborative Research: ABI: Innovation: The Global Names Architecture, an infrastructure for unifying taxonomic databases and services for managers of biological information. Original PI D. Patterson (MBL/ASU, 2011–2013), completed by PI Franz (ASU, 2014); 2011–2014; $1,044,563. See http://globalnames.org/; IM: Franz with two articles published, two in review. (6). NSF DEB-1258154. ARTS: Systematics of the Darkling Beetle Genus Eleodes: Integrating Morphology, DNA, and Biodiversity Informatics to Resolve a Taxonomically Impeded Genus. PI A. Smith, co-PIs N. Franz, Q. Wheeler; 2013–2015; $458,104. IM: Three articles published; BM: one Ph.D. student in training and one undergraduate trained. TIMELINE & DELIVERABLES We will produce at least 17 publications (nine taxonomic monographs; three systematic studies focusing on phylogenetic reconstructions, each for one focal group; three comparative evolutionary analyses of mimicry; one publication on the characterization of color patterns; and a synthesis of the diversity of mimicry rings in the four collecting sites). The timeline of the project is shown in Table 2. Table 2. Work package and deliverables time table (diamonds indicate deliverables: manuscripts/outreach products). Work package

Personnel

Year

05/2015 - 04/2016 05/2016 - 04/2017 05/2017 - 04/2018

Month 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 36

Task 1-Field work 1.1 Field trips in four countries

All

1.2 Return to sites to retrieve samples All Task 2-Monographic revision of assassin bugs Zhang Task 3-Monographic revision of braconids 3.1 Revision of Capitonius from four sites PhD stu, Sharkey 3.2 Revision of Alabagrus from Ecuador and Peru Sharkey 3.3 Revision of Alabagrus from Colombiaand Brazil Zhang Task 4-DNA sequencing 4.1 DNA extraction of existing and new samples PhD stu, Zhang, technichian 4.2 Sequencing PhD stu, Zhang, technichian Task 5-Comparative analyses and phylogenetics All Training, collaboration & presentations Taxonomy & phylogenetics workshops @ UK & ASUAll Field techniques training in Peru All South American student visits to ASU & UK Zhang, Sharkey RAD sequencing training & colllaboration Zhang, student, Sharanowski Undergraduate student research All Presentations at national or international conferencesAll Outreach Insect Mimicry & Camouflage drawers All (ASU&UK) ASU Night of Open Door, school visits Zhang, undergraduates Evolve a Mimic' Tree-of-Life game All (ASU&UK)

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15

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73. Rubin, B. E. R., Ree, R. H. & Moreau, C. S. Inferring Phylogenies from RAD Sequence Data. PLoS ONE 7, e33394 (2012). 74. Bacon, C., McKenna, M., Simmons, M. & Wagner, W. Evaluating multiple criteria for species delimitation: an empirical example using Hawaiian palms (Arecaceae: Pritchardia). BMC Evol. Biol. 12, 23 (2012). 75. Pagel, M. Detecting Correlated Evolution on Phylogenies: A General Method for the Comparative Analysis of Discrete Characters. Proc. R. Soc. Lond. B Biol. Sci. 255, 37–45 (1994). 76. Mallet, J. Shift happens! Shifting balance and the evolution of diversity in warning colour and mimicry. Ecol. Entomol. 35, 90–104 (2010). 77. Rettenmeyer, C. W. Insect Mimicry. Annu. Rev. Entomol. 15, 43–74 (1970). 78. Revell, L. J. & Collar, D. C. PHYLOGENETIC ANALYSIS OF THE EVOLUTIONARY CORRELATION USING LIKELIHOOD. Evolution 63, 1090–1100 (2009). 79. Jones, R. T., Salazar, P. A., ffrench-Constant, R. H., Jiggins, C. D. & Joron, M. Evolution of a mimicry supergene from a multilocus architecture. Proc. R. Soc. B Biol. Sci. 279, 316–325 (2012). 80. Martin, A. et al. Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand. Proc. Natl. Acad. Sci. 109, 12632–12637 (2012). 81. Reed, R. D. et al. optix Drives the Repeated Convergent Evolution of Butterfly Wing Pattern Mimicry. Science 333, 1137–1141 (2011). 82. Papa, R. et al. Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies. BMC Genomics 9, 345 (2008). 83. Papa, R., Martin, A. & Reed, R. D. Genomic hotspots of adaptation in butterfly wing pattern evolution. Curr. Opin. Genet. Dev. 18, 559–564 (2008). 84. Stevens, M. & Ruxton, G. D. Linking the evolution and form of warning coloration in nature. Proc. R. Soc. B Biol. Sci. rspb20111932 (2011). doi:10.1098/rspb.2011.1932 85. Campbell O. Webb, Ackerly, D. D., McPeek, M. A. & Donoghue, M. J. Phylogenies and community ecology. Annu. Rev. Ecol. Syst. 33, 475–505 (2002). 86. Emerson, B. C. & Gillespie, R. G. Phylogenetic analysis of community assembly and structure over space and time. Trends Ecol. Evol. 23, 619–630 (2008). 87. Elias, M., Gompert, Z., Willmott, K. & Jiggins, C. Phylogenetic community ecology needs to take positive interactions into account. Commun. Integr. Biol. 2, 113–116 (2009). 88. Webb, C. O. Exploring the Phylogenetic Structure of Ecological Communities: An Example for Rain Forest Trees. Am. Nat. 156, 145–155 (2000). 89. Pitz, K. M. Systematic and taxonomic revision of the subfamily Cenocoeliinae (Hymenoptera: Braconidae). (2006). 90. Maddison, W. & Maddison, D. Mesquite: a modular system for evolutionary analysis. (2011). at 91. Franz, N. & Gilbert, E. Symbiota – A virtual platform for creating voucher-based biodiversity information communities. Biodivers. Data J. at 92. Sharkey, M. Two new genera of Agathidinae (Hymenoptera: Braconidae) with a key to the genera of the New World. Magnolia Press Zootaxa 1185, 37–51 (2006). 93. Zhang, G. & Weirauch, C. Matching dimorphic sexes and immature stages with adults: resolving the systematics of the Bekilya group of Malagasy assassin bugs (Hemiptera: Reduviidae: Peiratinae). Syst. Entomol. 36, 115–138 (2011). 94. Park, D.-S., Foottit, R., Maw, E. & Hebert, P. D. N. Barcoding Bugs: DNA-Based Identification of the True Bugs (Insecta: Hemiptera: Heteroptera). PLoS ONE 6, e18749 (2011). 95. Drummond, A. J. & Rambaut, A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214 (2007). 96. Grummer, J. A., Bryson, R. W. & Reeder, T. W. Species Delimitation Using Bayes Factors: Simulations and Application to the Sceloporus scalaris Species Group (Squamata: Phrynosomatidae). Syst. Biol. syt069 (2013). doi:10.1093/sysbio/syt069

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97. Fujisawa, T. & Barraclough, T. G. Delimiting Species Using Single-Locus Data and the Generalized Mixed Yule Coalescent Approach: A Revised Method and Evaluation on Simulated Data Sets. Syst. Biol. 62, 707–724 (2013). 98. Sharanowski, B. J., Dowling, A. P. G. & Sharkey, M. J. Molecular phylogenetics of Braconidae (Hymenoptera: Ichneumonoidea), based on multiple nuclear genes, and implications for classification. Syst. Entomol. 36, 549–572 (2011). 99. Sharkey, M. J., Laurenne, N. M., Sharanowski, B., Quicke, D. L. J. & Murray, D. Revision of the Agathidinae (Hymenoptera: Braconidae) with comparisons of static and dynamic alignments. Cladistics 22, 546–567 (2006). 100. Lohse, K., Sharanowski, B., Blaxter, M., Nicholls, J. A. & Stone, G. N. Developing EPIC markers for chalcidoid Hymenoptera from EST and genomic data. Mol. Ecol. Resour. 11, 521–529 (2011). 101. Sharanowski, B. J. et al. Expressed sequence tags reveal Proctotrupomorpha (minus Chalcidoidea) as sister to Aculeata (Hymenoptera: Insecta). Mol. Phylogenet. Evol. 57, 101–112 (2010). 102. Lohse, K., Sharanowski, B. & Stone, G. N. Quantifying the pleistocene history of the oak gall parasitoid Cecidostiba fungosa using twenty intron loci. Evol. Int. J. Org. Evol. 64, 2664–2681 (2010). 103. Eaton, D. A. R. PyRAD: assembly of de novo RADseq loci for phylogenetic analyses. Bioinformatics 30, 1844–1849 (2014). 104. Torres-Miranda, A., Luna-Vega, I. & Oyama, K. New Approaches to the Biogeography and Areas of Endemism of Red Oaks (Quercus L., Section Lobatae). Syst. Biol. 62, 555–573 (2013). 105. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). 106. Stamatakis, A. RAxML Version 8: A tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies. Bioinformatics btu033 (2014). doi:10.1093/bioinformatics/btu033 107. Heled, J. & Drummond, A. J. Bayesian Inference of Species Trees from Multilocus Data. Mol. Biol. Evol. 27, 570–580 (2010). 108. Bouckaert, R. et al. BEAST 2: A Software Platform for Bayesian Evolutionary Analysis. PLoS Comput. Biol. 10, e1003537 (2014). 109. Pagel, M., Meade, A. & Barker, D. Bayesian estimation of ancestral character states on phylogenies. Syst. Biol. 53, 673–684 (2004). 110. Slatkin, M. & Maddison, W. P. A cladistic measure of gene flow inferred from the phylogenies of alleles. Genetics 123, 603–613 (1989). 111. Parker, J., Rambaut, A. & Pybus, O. G. Correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 8, 239–246 (2008). 112. Webb, C. O., Ackerly, D. D. & Kembel, S. W. Phylocom: software for the analysis of phylogenetic community structure and trait evolution. Bioinformatics 24, 2098–2100 (2008). 113. Baum, D. A. & Smith, S. D. Tree thinking: an introduction to phylogenetic biology. (Roberts, 2012). 114. Van Achterberg, K., Sharkey, M. & Chapman, E. Revision of the genus Euagathis Szépligeti (Hymenoptera, Braconidae, Agathidinae) from Thailand, with description of three new species. J. Hymenopt. Res. 36, 1–25 (2014). 115. Sharkey, M. & Stoelb, S. Revision of Zelodia (Hymenoptera, Braconidae, Agathidinae) from Thailand. J. Hymenopt. Res. 26, 31–71 (2012). 116. Stoelb, S. & Sharkey, M. Revision of Therophilus s.s. (Hymenoptera, Braconidae, Agathidinae) from Thailand. J. Hymenopt. Res. 27, 1 (2012). 117. Clutts, S. & Sharkey, M. A revision of Thai Agathidinae (Hymenoptera, Braconidae), with descriptions of six new species. J. Hymenopt. Res. 22, 69 (2011). 118. Starý, P. et al. Review and Key to the World Parasitoids (Hymenoptera: Braconidae: Aphidiinae) of Greenideinae Aphids (Hemiptera: Aphididae), Including Notes on Invasive Pest Species. Ann. Entomol. Soc. Am. 103, 307–321 (2010).

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119. Starý, P., Sharkey, M. J. & Hutacharern, C. Aphid parasitoids sampled by Malaise traps in the national parks of Thailand (Hymenoptera, Braconidae, Aphidiinae). Thai J. Agric. Sci. 41, 37–43 (2008).

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Biographic Sketch – Guanyang Zhang School of Life Sciences Arizona State University 85282, Tempe AZ Tel: 480-965-2850 Email: [email protected] A. PROFESSIONAL PREPARATION Institution

Major

Degree & Year

National University of Singapore

Life Sciences

University of California, Riverside

Entomology

BSc (Honors) 2007 PhD, 2012

B. PROFESSIONAL APPOINTMENTS 05/2013-present

Postdoctoral Associate, Arizona State University, Major: Systematics.

01-04/2013

Postdoctoral Associate, University of California, Riverside

C. PUBLICATIONS 1. Zhang, G. & Weirauch, C. 2014. Molecular phylogeny of Harpactorini (Insecta: Reduviidae): correlation of novel predation strategy with accelerated evolution of predatory leg morphology. Cladistics 30, 339–351. 2. Zhang, G., Weirauch, C. 2013. Sticky predators: a comparative study of sticky glands in harpactorine assassin bugs (Insecta: Hemiptera: Reduviidae). Acta Zoologica 94 (1): 1–10. 3. Zhang, G. and C. Weirauch, 2011. Matching dimorphic sexes and immature stages with adults: resolving the systematics of the Bekilya group of Malagasy assassin bugs (Hemiptera: Reduviidae: Peiratinae). Systematic Entomology 36: 115–138. 4. Zhang, G. 2009. Specimens versus sequences. Science 323(5922): 1672. 5. Meier, R., Zhang, G., Ali, F. 2008. The use of mean instead of smallest interspecific distances exaggerates the size of the “barcoding gap” and leads to misidentification. Systematic Biology 57: 809–813. OTHER PUBLICATIONS Weirauch, C., Alvarez, C., Zhang, G. 2012. Investigating dispersalist potential: observations on the natural enemy assassin bugs Zelus renardii and Zelus tetracanthus. Florida Entomologist 95(3): 641–649. Meier, R., Zhang, G. DNA barcoding and DNA taxonomy: An assessment based on 4261 COI sequences for 1001 species, in Diptera Diversity: Status, Challenges and Tools edited by Pape, T., Bickel, D., Meier, R. Brill Academic Publishers, 2009. Hwang, W. S., G. Zhang, D. Maslov and C. Weirauch 2009. Infection rates of Trypanosoma cruzi in Triatominae (Hemiptera: Reduviidae) in Southern California. American Journal of Tropical Medicine and Hygiene 83: 1020–1022.

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D. SYNERGISTIC ACTIVITIES 1. Journal editing 09/2013- Subject Editor, Biodiversity Data Journal 2. Conference organizing and society duties 11/2013 Student Presentation Competition Judge, Session Moderator. Entomological Society of America, Austin, TX, USA 06/2012 Organizing Committee. XXXI Willi Hennig Society Annual Meeting, Riverside, CA, USA. 07/2011 Second International Workshop of Reduviidae, Riverside, CA, USA. 02/2010 Assistance with organizing the Heteroptera Synthesis Meeting (sponsored by the Biodiversity Synthesis Center, Field Museum, Chicago & ‘Encyclopedia of Life’), Riverside, CA, USA. 2012/06 Organizing Committee. XXXI Willi Hennig Society Annual Meeting, Riverside, CA, USA. 3. Student mentoring 08/2014 – Mentoring seven undergraduate research students. Under my supervision, one minority undergraduate student, Usmaan Basharat, won a national competitive travel award from the National Evolutionary Synthesis Center (NESCent) to present at the Evolution 2014 meeting in North Carolina. 08/2014 – Co-supervising graduate students at the Franz Lab. 4. Outreach “Bad Bugs” Mesa Youth Museum, AZ, 10/2013; UC Riverside Annual Plant Sale, 2010-11; Discover Day, College of Agricultural and Natural Sciences, UC Riverside, 2011; Caryn Elementary School, Rancho Cucamonga, CA, 2011; McKinley Elementary School, Corona, CA, 2010; College of Desert student visit to UC Riverside, 2010-11; Pomona Insect Fair, CA, 2008. 5. Science communication 04/2014 –

Entomological Collections Network, Twitter Account Coordinator.

12/2013 –

Contributing author. BioDiverse Perspectives. Biodiversity science and research blog contributed by students and researchers supported by NSF grants. http://www.biodiverseperspectives.com/

06/2012 –

Online research communication and blogging. www.taxonbytes.org www.somanyinsects.org

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Biographical Sketch - Nico M. Franz Nico M. Franz, Ph.D. Associate Professor & Curator of Insects School of Life Sciences PO Box 874501 Arizona State University Tempe, AZ 85287-4501

Office: (480) 965-2036 Collection: (480) 965-2850 Fax: (480) 965-6899 E-mail: [email protected]

A. PROFESSIONAL PREPARATION Institution University of Hamburg University of Ulm University of Costa Rica Cornell University

Major Biology Plant Ecology Biology Entomology

Degree & Year Prediploma 1996 1 semester 1996 M.Sc. 1999 Ph.D. 2005

B. APPOINTMENTS Period 2011 – 2011 – 2008 – 2011 2006 – 2008 2006 – 2011 2004 – 2006

1999

Appointment Curator, Frank F. Hasbrouck Insect Collection, School of Life Sciences, ASU, AZ Associate Professor, School of Life Sciences, Arizona State University, AZ Director, Invertebrate Collection, Department of Biology, UPR Mayagüez, PR Curator of Entomology, Department of Biology, UPR Mayagüez, PR Assistant Professor, Dept. of Biology, University of Puerto Rico at Mayagüez, PR Andrew W. Mellon Foundation Postdoctoral Research Fellow, Science Environment for Ecological Knowledge Project (SEEK, DBI-0225676), National Center for Ecological Analysis & Synthesis, University of California at Santa Barbara, CA Graduate Research Fellow, Smithsonian Tropical Research Institute (STRI), Panama

C. PRODUCTS (total = 38 peer-reviewed publications + 3 textbook chapters + 5 published reviews) Five Products Most Closely Related 1.

2.

3. 4.

Chen, M., S. Yu, N. Franz, S. Bowers & B. Ludäscher. 2013. Euler/X: a toolkit for logicbased taxonomy integration. WFLP 2013 – 22nd International Workshop on Functional and (Constraint) Logic Programming. http://taxonbytes.org/pdf/ChenEtAl2013EulerToolkit.pdf Franz, N.M. & J. Cardona-Duque. 2013. Description of two new species and phylogenetic reassessment of Perelleschus Wibmer & O'Brien, 1986 (Coleoptera: Curculionidae), with a complete taxonomic concept history of Perelleschus sec. Franz & Cardona-Duque, 2013. Systematics and Biodiversity 11: 209–236. http://www.tandfonline.com/doi/full/10.1080/14772000.2013.806371#.UdxmD21nf15 Franz, N.M. & D. Thau. 2010. Biological taxonomy and ontology development: scope and limitations. Biodiversity Informatics 7: 45–66. https://journals.ku.edu/index.php/jbi/article/view/3927/3852 Franz, N.M. & R.K. Peet. 2009. Towards a language for mapping relationships among taxonomic concepts. Systematics and Biodiversity 7: 5–20. http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=3760372

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5.

Franz, N.M., R.K. Peet & A.S. Weakley. 2008. On the use of taxonomic concepts in support of biodiversity research and taxonomy; pp. 63–86. In Wheeler, Q.D. (Ed.): The New Taxonomy, Systematics Association Special Volume Series 74. Taylor & Francis, Boca Raton, FL. http://www.crcnetbase.com/doi/book/10.1201/9781420008562

Five Further Products 1. 2.

3. 4. 5.

Franz, N.M. 2013. Anatomy of a cladistic analysis. Cladistics 29. (Early View) http://onlinelibrary.wiley.com/doi/10.1111/cla.12042/abstract Cardona-Duque, J. & N.M. Franz. 2012. Description and phylogeny of Azotoctla, a new Neotropical genus of Acalyptini (Coleoptera: Curculionidae: Curculioninae) associated with the staminodes of Cyclanthaceae. Zoological Journal of the Linnean Society 166: 559–623. Girón, J.C. & N.M. Franz. 2012. Phylogenetic assessment of the Caribbean weevil genus Lachnopus Schoenherr, 1840 (Coleoptera: Curculionidae: Entiminae). Invertebrate Systematics 26: 67–82. Franz, N.M. 2012. Phylogenetic reassessment of the Exophthalmus genus complex (Curculionidae: Entiminae: Eustylini, Geonemini). Zoological Journal of the Linnean Society 164: 510–557. Franz, N.M. 2005. On the lack of good scientific reasons for the growing phylogeny/ classification gap. Cladistics 21: 495–500.

D. SYNERGISTIC ACTIVITIES 1. 2. 3. 4. 5.

Primary workshop organizer and instructor, The Weevil Course, Southwestern Research Station, Portal, AZ (2012, 2014); http://research.amnh.org/swrs/weevil-course Project co-leader, InsectAR Project, Teaching Insect Diversity with Augmented Reality, Arizona State University & SkySong (2012); https://sites.google.com/site/asuinsectarium/home Member and lead collaborator for Filtered Push integration and outreach, Southwest Collections of Arthropods Network (SCAN), Northern Arizona University (2012); http://www.scanbugs.org/ Workshop instructor, USDA Southern Plant Diagnostic Network Weevil Workshop, University of Georgia, Athens, GA (2010); http://www.entnemdept.ufl.edu/Hodges/Weevils.htm Principal coordinator for zoology, HHMI ROLE-MODEL, integrating biodiversity research modules in UPRM undergraduate laboratory curriculum (2008); http://rolemodel.uprm.edu/

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Biographical Sketch – Michael J Sharkey Contact Info: Professor Department of Entomology Phone: (859) 257-9364 University of Kentucky Lexington Fax (859) 323-1120 E-mail Kentucky 40546-0091 USA E-mail [email protected] Home Page: www.sharkeylab.org A. PROFESSIONAL PREPARATION Institution Major University of Guelph, Guelph Entomology McGill University, Montreal Entomology McGill University, Montreal Entomology

Degree & Year B. Sc. 1977 M.Sc. 1981 Ph.D. 1984

B. APPOINTMENTS 2004-present Professor, Department of Entomology, University of Kentucky. 1998-2004 Associate Professor, Department of Entomology, University of Kentucky. 1996-98 Assistant Professor, Department of Entomology, University of Kentucky. 1983-1996 Curator and Research Scientist, Canadian National Collection of Insects. Agriculture Canada. 1987-1996. Adjunct Professor. McGill University, Ste. Anne de Bellevue, P.Q. 1981-1983 Biologist, Agriculture Canada. C. PUBLICATIONS (i) 5 publications most closely related to proposed research 1. Butcher BA, Smith AM, Sharkey MJ, Quicke DL. 2012. A turbo-taxonomic study of Thai Aleiodes (Aleiodes) and Aleiodes (Arcaleiodes) (Hymenoptera: Braconidae: Rogadinae) based largely on COI barcoded specimens, with rapid descriptions of 179 new species. Zootaxa 3457: 1–232. ISBN 978-1-77557-000-4 2. Sharkey MJ, Carpenter JM, Vilhelmsen L, Heraty J, Liljeblad J, Dowling APG, Schulmeister S, Murray D, Deans AR, Ronquist F, Krogmann L, Wheeler WC. 2011. Phylogenetic relationships among superfamilies of Hymenoptera. Cladistics 27: 1-33. DOI: 10.1111/j.1096-0031.2011.00366.x 3. Sharkey MJ, Clutts Stoelb SA, Tucker EM, Janzen D, Hallwachs W, Dapkey T, Smith MA. 2011. Lytopylus Forster (Hymenoptera, Braconidae, Agathidinae) species from Costa Rica, with an emphasis on specimens reared from caterpillars in Area de Conservacion Guanacaste. Zookeys 130:379-419. DOI: 10.3897/zookeys.130.1569 [Open Access] 4. Heraty J, Ronquist F, Carpenter JM, Hawks D, Schulmeister S, Dowling AP, Murray D, Munro J, Wheeler WC, Schiff N, Sharkey MJ. 2011. Evolution of the hymenopteran megaradiation. Molecular Phylogenetics and Evolution 60(1): 73-88. DOI: 10.1016/j.ympev.2011.04.003

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5. Sharanowski BJ, Dowling AP, Sharkey MJ. 2011. Molecular phylogenetics of Braconidae (Hymenoptera: Ichneumonoidea), based on multiple nuclear genes, and implications for classification. Systematic Entomology 36:549-572. DOI: 10.1111/j.1365-3113.2011.00580.x (ii) 5 additional publications 1. Sharkey, M.J. and Stoelb, S.A.C. 2013. Revision of Agathacrista New Genus (Hymenoptera, Braconidae, Agathidinae, Agathidini). Journal of Hymenoptera Research 33:99-112 . Doi:10.3897/JHR.33.4373 [Open Access] 2. Wei Shu-jun, Min Shi, Jun-hua He, M. Sharkey and Xue-xin Chen. 2009. The complete mitochondrial genome of Diadegma semiclausum (Hymenoptera: Ichneumonidae) indicated extensive independent evolutionary events. Genome 52:308-319. DOI: 10.1139/G09-008 3. Quicke, D.L.J., M.J. Sharkey, N.M. Laurenne and A. Dowling. 2008. A preliminary molecular phylogeny of the Sigalphinae (Hymenoptera: Braconidae), including Pselaphanus Szepligeti, based on 28S rDNA, with descriptions of new Afrotropical and Madagascan Minanga and Malasigalphus species. Journal of Natural History 42(43-44):2703-2719. DOI:10.1080/00222930802364042 4. Sharkey, M.J. 2007. Phylogeny and Classification of Hymenoptera. Zootaxa 1668: 521-548. http://www.mapress.com/zootaxa/2007f/zt01668p548.pdf [Open Access] 5. Fernandez, F. & M.J. Sharkey (eds). 2006. Introduccion a los Hymenoptera de la Region Neotropical, Serie Entomologia Colombiana, Sociedad Colombiana de Entomologia, Bogota D.C., Colombia. 800++ pages. (In Spanish). D. SYNERGISTIC ACTIVITIES 1. Editorial Responsibilities: Subject editor for Zookeys, frequent reviewer for many journals. 2. Professional Society Responsibilities: Active in Entomology Collection Network, e.g., chaired and organized annual meetings. Active in the Entomological Society of America, organizing symposia, evaluating student posters, oral presentations, committee work etc. 3. Taxonomic Consulting: Active in the ATBI effort in the Great Smoky Mountains National Park where I was the coordinator of all of the Taxonomic Working Groups and a member of the board for many years. 4. Professional Leadership: Current president of the International Society of Hymenopterists. 5. Online knowledge and data dissemination: a. Interactive illustrated keys and other resources www.sharkeylab.org. b. ~900 images uploaded to Morphbank.org (NSF-supported) http://www.morphbank.net/?id=7

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SUMMARY PROPOSAL BUDGET

YEAR

1

FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

Arizona State University PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Guanyang Zhang A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Guanyang Zhang - PI 12.00 0.00 2. 3. 4. 5. 6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 12.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 0 ) GRADUATE STUDENTS 4. ( 2 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 0 1. STIPENDS $ 4,800 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 2) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL PARTICIPANT COSTS

SUMR

Funds Requested By proposer

0.00

45,000

0.00 0.00

0 45,000

0.00 0.00

0 0 0 2,000 0 0 47,000 10,521 57,521

Funds granted by NSF (if different)

0 1,200 9,500

4,800 3,101 0 7,400 0 102,196 2,000 114,697 187,718

MTDC (Rate: 54.5000, Base: 105722) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Guanyang Zhang ORG. REP. NAME*

57,618 245,336 0 245,336 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno 1 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

SUMMARY PROPOSAL BUDGET

YEAR

2

FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

Arizona State University PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Guanyang Zhang A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Guanyang Zhang - PI 12.00 0.00 2. 3. 4. 5. 6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 12.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 0 ) GRADUATE STUDENTS 4. ( 2 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 0 1. STIPENDS $ 2,400 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 1) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL PARTICIPANT COSTS

SUMR

Funds Requested By proposer

0.00

46,350

0.00 0.00

0 46,350

0.00 0.00

0 0 0 2,060 0 0 48,410 11,163 59,573

Funds granted by NSF (if different)

0 1,500 1,900

2,400 8,300 3,000 1,800 0 91,200 0 104,300 169,673

MTDC (Rate: 54.5000, Base: 76073) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Guanyang Zhang ORG. REP. NAME*

41,460 211,133 0 211,133 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno 2 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

SUMMARY PROPOSAL BUDGET

YEAR

3

FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

Arizona State University PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Guanyang Zhang A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Guanyang Zhang - PI 12.00 0.00 2. 3. 4. 5. 6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 12.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 0 ) GRADUATE STUDENTS 4. ( 1 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 0 1. STIPENDS $ 0 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 1) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

SUMR

Funds Requested By proposer

Funds granted by NSF (if different)

0.00

47,741

0.00 0.00

0 47,741

0.00 0.00

0 0 0 2,122 0 0 49,863 11,843 61,706

0 3,000 0

0

TOTAL PARTICIPANT COSTS

0 4,500 0 0 81,542 0 86,042 150,748

MTDC (Rate: 54.5000, Base: 69206) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Guanyang Zhang ORG. REP. NAME*

37,717 188,465 0 188,465 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno 3 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

SUMMARY PROPOSAL BUDGET

Cumulative FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

Arizona State University PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Guanyang Zhang A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Guanyang Zhang - PI 36.00 0.00 2. 3. 4. 5. 6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 36.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 0 ) GRADUATE STUDENTS 4. ( 5 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 0 1. STIPENDS $ 7,200 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 4) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Guanyang Zhang ORG. REP. NAME*

TOTAL PARTICIPANT COSTS

SUMR

Funds Requested By proposer

0.00

139,091

0.00 0.00

0 139,091

0.00 0.00

0 0 0 6,182 0 0 145,273 33,527 178,800

Funds granted by NSF (if different)

0 5,700 11,400

7,200 11,401 7,500 9,200 0 274,938 2,000 305,039 508,139

136,795 644,934 0 644,934 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno C *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

Budget justification, Guanyang Zhang, Arizona State University A. Senior Personnel: $139,091 Guanyang Zhang, Principal Investigator. Funds are requested to fund the salary of the PI for 12 months for each year of the project. The PI meets all the criteria to be a PI on this project; however, he currently does not hold a faculty position. Lead PI Zhang is currently a postdoctoral researcher at the lab of co-PI, Nico Franz and funds are required to fund his salary full-time. ASU is aware of the 2-month salary limitation for PIs who hold a faculty position, but given the unique position of PI Zhang, an exception is requested. Zhang will act as the lead PI on this proposed project and will work full-time as a postdoctoral researcher. Zhang will oversee the project, conduct monographic revisions of assassin bugs and wasp species (total ~100 species), perform molecular work (DNA extraction and sequencing) on assassin bug samples, organize and participate in field trips, design and conduct comparative phylogenetic analyses, enforce data quality standards, co-supervise a PhD student with co-PI Sharkey and one foreign student participant form Ecuador, and publish results. B. Other Personnel: $6182 Undergraduate research/outreach students: $6,182. Five undergraduate assistants will be involved in research and outreach activities: two in year 1, two in year 2 and one in year 3. Two students will participate in research-related activities such as databasing and geo-referencing specimens and photographing specimens (Year one). Two students will design and assemble ‘Mimicry & Camouflage’ drawers and perform outreach activities to the general public including K-12 students (Year two). One students will be assisting PI Zhang with implementing the ‘Evolve-aMimic’ educational game and educating and engaging the public at the ASU Biodiversity and Bioinformatics Center (Year three). A total 600 hours are expected at $10/hour.

The standard increase of 3% per year for inflation has been added to salaries in subsequent years. C. Fringe benefits: 33,527 The standard fringe benefits rates for ASU have been applied for budget planning, and increase at a rate of 3% per year. The rates are as follows: Post-Doc – 23.28% in FY16, 23.98% in FY17, 24.70% in FY18. Hourly Students – 2.27% in FY16, 2.33% in FY17, 2.40% in FY18. E. Travel: $17,100 (1) Domestic: $5,700 (i) $4,500. PI Zhang will attend and present research findings at the Entomological Society of American annual meetings, the International Congress of Entomology, and the Evolution 2018 meeting. $1,500 per meeting per person is requested to cover registration ($300), airfare ($500), hotel ($500), and per diem ($200). Details of the meetings are below. -Year two, Sept 2016, Orlando, Florida. Entomological Society of American annual meeting in conjunction with the International Congress of Entomology. $1,500. -Year three, Nov 2017, Denver, Colorado. Entomological Society of American annual meeting. $1,500. - Year three, 2018, venue TBD, Evolution 2018 (joint meeting of the Evolution Society, the Society of Systematic Biologists and the American Naturalists Society). $1,500. (ii) $1,200 (Year one). PI Zhang will organize and attend a week-long taxonomy and data management workshop at the University of Kentucky in Sept 2015 designed to enforce work and data standards of the project. Cost items: airfare ($400), hotel ($500), and per diem ($300). (2) International: $11,400 $11,400 for five trips to Peru, Colombia and Brazil. Zhang will carry out field trips extensively in South America in order to collect fresh specimens for molecular work and for revisionary taxonomy. Both

F-5

active and passive collecting methods will be used. Long-term passive collecting traps will be set up at a target site during the first trip and another trip is needed to return to retrieve the samples. We prefer to go back to the sites ourselves to retrieve the samples over having the samples shipped from the collecting sites to the US. This is because shipping may risk damage to or loss of the samples. Our strategy would incur a slightly higher cost, but will ensure the quality and the safety of the samples. Table 1. Field trip budgeting. Airfares based on Orbitz.com quotes for traveling in May 2015. Country Peru Colombia Colombia Brazil Brazil

Month, Year June 2015, year one July 2015, year one Dec 2015, year one Jan 2016, year one June 2016, year two

Activities

Cost (one participant)

Perform extensive active collecting. Joint field training. ~20 days. Set up long-term traps and perform extensive active collecting. ~20 days.

$2,300 (1200 airfare, 400 lodging, 300 local transportation and 400 per diem) $2,400 (600 airfare, 600 lodging, $600 local transportation and 600 per diem)

Retrieve samples. ~7 days.

$1,400 (800 airfare, 200 lodging, $200 local transportation and 200 per diem) $3,400 (1200 airfare, 800 lodging, 800 local transportation and 600 per diem) $1,900 (1200 airfare, 300 lodging, 200 local transportation and 200 per diem)

Set up long-term traps and perform extensive active collecting. ~20 days. Retrieve samples. ~7 days.

Subtotal

$11,400

F. Participant Support Costs: $7,200 1. Three graduate students from South American collaborating institutions will travel to ASU to receive training on taxonomy and systematics. The training will facilitate knowledge dissemination to tropical countries with rich biodiversity, but with limited taxonomic expertise, thereby help with alleviating the taxonomic impediment and biodiversity crisis. Costs per person per trip include lodging: $1200, airfare: $800, per diem $400. G. Other Direct Costs 1. Material and supplies: $11,401 (i) Year one – Field collecting related supplies: $3,101. (a) Townes Style Malaise traps: $2,500. Ten units of Malaise traps are needed for long-term collecting at two local sites (five traps each site). Each costs ~$250 from the supplier SanteTraps (http://www.santetraps.com/). (b) Vials: $228. 5 ml conical vial, four bags (1000 vials/bag) for $57/bag from USA Scientific. (c) 200-proof ethanol: $300. 15 gallons at $20/gallon. (d) Whirl-Pak bags: $73. For storing Malaise trap samples. Available from http://www.enasco.com/whirlpak/ (ii) Year two – DNA sequencing and molecular supplies: $8,300. (a) DNA extraction and sequencing: $6723. See Table 2 below for details.

Table 2. Molecular work budget. RAD Sequencing & DNA barcoding budget cost/sample Oligos (including adapters and PCR primers and biotinylated oligos and barcodes). Ailquotes from co-PI Sharkey Illumina sequencing ($ ~2000 per run - 48 taxa per lane) 42 Total all reactions not including sequencing (items listed below) 12.4 -Extraction 2.2 -Enzymes 1.2 -T4 DNA ligase 2 -Ampure XP beads (cleanup) 2 -Size selection 3 -PCR 1 -Consumables 1 DNA barcodes for 150 samples 10 Subtotal

No. samples total 48 48

2016 595.2 2611.2

150

1500 6722.4

(b) Molecular supplies: $1,577.

F-6

-

$595. TipOne filter tips, 10 boxes, unit price $59.5. http://www.usascientific.com/10ultipone-filtertip.aspx - $482. ThermoScientific. Matrix 2D barcoded storage tubes for storing DNA extracts. http://www.matrixtechcorp.com/storage-systems/solutions.aspx?id=14 - $500. Other molecular consumables (agarose, dye and DNA ladder) 2. Publication Costs: $7,500 We plan to publish primarily using Open Access journals to promote accessibility of our research findings to the scientific community and the public. A budget of $7,500 is requested to publish five manuscripts, each for $1,500 (1-assassin bug revision, 2-Colombia Alabagrus wasp revision, 3- Brazil Alabagrus wasp revision, 4-mimiry characterization analyses, and 5-phylogenetics). 3. Consultant Services: 9,200 Consulting travel: $9,200 (i) $4,800 (Year one). Dr. Christiane Weirauch (University of California, Riverside) will provide consulting services on field collecting based on her extensive expertise in collecting insects in the Neotropics. More specifically she will collect assassin bug specimens used for this project. A budget is requested for her to participate in two field trips to Brazil (Year one). Cost estimates: $4,800 (2400 airfare, 1000 lodging, 400 local transportation and 1000 per diem). (ii) $2,600 (Year one) Dr. Barbara Sharanowski will travel to Peru to participate in a field training session aimed to reciprocally train participants on collecting braconid and reduviid specimens (1200 airfare, 500 lodging, 400 local transportation and 500 per diem). (iii) $1,800 (Year two). Sharanowski will provide training and consulting services at a phylogenetics workshop. A budget is requested for her to travel from University of Manitoba (Canada) to ASU (600 air ticket, 800 lodging, and 400 per diem) 4. Computer Services: N/A 5. Subcontracts: $274,938 A subcontract is budgeted for co-PI Michael Sharkey at the University of Kentucky for a total amount of $274,938 (total direct and indirect costs). A separate budget justification is provided. 6. Other: 2,000 (i) Year one. $2,000 for hiring two local field assistants to collect from long-term Malaise traps for at least a continuous 6-month duration at two sites (Colombia and Brazil). The trap samples will be collected every week and it requires less than a day to do the colleting each time. H. Total Direct Costs: $508,138 I. Indirect Costs The Arizona State University indirect rate agreement approved by DHHS on May 29, 2013 is 54.5% based on Modified Total Direct Cost (MTDC). Equipment, capital expenditures, tuition remission, rental costs, participant support, scholarships and fellowships, and the portion of subgrants and subcontracts in excess of $25,000 are excluded from MTDC. J. Total Direct And Indirect Costs: $644,934

F-7

SUMMARY PROPOSAL BUDGET

YEAR

1

FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

University of Kentucky PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Michael Sharkey A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Michael J Sharkey - Co-PI 1.00 0.00 2. 3. 4. 5. 6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 1.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 1 ) GRADUATE STUDENTS 4. ( 1 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 0 1. STIPENDS $ 0 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 0) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

SUMR

Funds Requested By proposer

Funds granted by NSF (if different)

1.00

8,787

0.00 1.00

0 8,787

0.00 0.00

0 0 18,000 2,000 0 0 28,787 6,594 35,381

0 4,000 11,300

0

TOTAL PARTICIPANT COSTS

10,602 0 0 0 0 15,000 25,602 76,283

MTDC (Rate: 40.0000, Base: 64783) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Michael Sharkey ORG. REP. NAME*

25,913 102,196 0 102,196 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno 1 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

SUMMARY PROPOSAL BUDGET

YEAR

2

FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

University of Kentucky PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Michael Sharkey A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Michael J Sharkey - Co-PI 1.00 0.00 2. 3. 4. 5. 6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 1.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 1 ) GRADUATE STUDENTS 4. ( 1 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 2,400 1. STIPENDS $ 0 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 2) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL PARTICIPANT COSTS

SUMR

Funds Requested By proposer

1.00

9,050

0.00 1.00

0 9,050

0.00 0.00

0 0 18,540 0 0 0 27,590 6,752 34,342

Funds granted by NSF (if different)

0 3,000 3,700

2,400 7,815 6,000 0 0 0 12,000 25,815 69,257

MTDC (Rate: 40.0000, Base: 54858) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Michael Sharkey ORG. REP. NAME*

21,943 91,200 0 91,200 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno 2 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

SUMMARY PROPOSAL BUDGET

YEAR

3

FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

University of Kentucky PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Michael Sharkey A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Michael J Sharkey - Co-PI 1.00 0.00 2. 3. 4. 5. 6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 1.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 1 ) GRADUATE STUDENTS 4. ( 1 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

SUMR

Funds Requested By proposer

1.00

9,322

0.00 1.00

0 9,322

0.00 0.00

0 0 19,096 0 0 0 28,418 7,083 35,501

0 0 0

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 0 1. STIPENDS $ 0 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 2) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

Funds granted by NSF (if different)

0

TOTAL PARTICIPANT COSTS

7,815 6,000 0 0 0 12,500 26,315 61,816

MTDC (Rate: 40.0000, Base: 49315) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Michael Sharkey ORG. REP. NAME*

19,726 81,542 0 81,542 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno 3 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

SUMMARY PROPOSAL BUDGET

Cumulative FOR NSF USE ONLY PROPOSAL NO. DURATION (months) Proposed Granted AWARD NO.

ORGANIZATION

University of Kentucky PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR

Michael Sharkey A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets)

NSF Funded Person-months

CAL

ACAD

1. Michael J Sharkey - Co-PI 3.00 0.00 2. 3. 4. 5. 6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 3.00 0.00 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( 0 ) POST DOCTORAL SCHOLARS 0.00 0.00 2. ( 0 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 0.00 0.00 3. ( 3 ) GRADUATE STUDENTS 4. ( 3 ) UNDERGRADUATE STUDENTS 5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( 0 ) OTHER TOTAL SALARIES AND WAGES (A + B) C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. FOREIGN

F. PARTICIPANT SUPPORT COSTS 2,400 1. STIPENDS $ 0 2. TRAVEL 0 3. SUBSISTENCE 0 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 4) G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER TOTAL OTHER DIRECT COSTS H. TOTAL DIRECT COSTS (A THROUGH G) I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE) TOTAL INDIRECT COSTS (F&A) J. TOTAL DIRECT AND INDIRECT COSTS (H + I) K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) M. COST SHARING PROPOSED LEVEL $ PI/PD NAME

Michael Sharkey ORG. REP. NAME*

TOTAL PARTICIPANT COSTS

SUMR

Funds Requested By proposer

3.00

27,159

0.00 3.00

0 27,159

0.00 0.00

0 0 55,636 2,000 0 0 84,795 20,429 105,224

Funds granted by NSF (if different)

0 7,000 15,000

2,400 26,232 12,000 0 0 0 39,500 77,732 207,356

67,582 274,938 0 274,938 0

AGREED LEVEL IF DIFFERENT $ FOR NSF USE ONLY INDIRECT COST RATE VERIFICATION Date Checked

Date Of Rate Sheet

fm1030rs-07

Initials - ORG

Eric Moreno C *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

Budget Justification University of Kentucky Subcontract – Michael Sharkey, co-PI A. Senior Personnel: $27,159 Dr. Michael Sharkey will commit 1 month effort to this project. He will conduct taxonomic revision of Alabagrus wasps. Supervise one PhD student on the taxonomy and phylogenetics of Capitonius wasps. Organize and participate in field trips. Participate in and supervise outreach activities at UK. B. Other personnel: $57,636 1. Graduate research assistant (PhD student): $55,636. Stipend support is requested for one PhD graduate research assistant to work on the taxonomy of Capitonius, obtain molecular data and perform comparative analyses. An annual salary of $18,000 is requested for three years, with 3% escalation/year. 2. Undergraduate research/outreach students: $2,000. Undergraduate assistants will be involved in databasing, photographing and outreach activities. A total 200 hours are expected at $10/hour. C. Fringe benefits: $20,429 Fringe benefits for faculty include 7.65% FICA; 10% retirement; 3.4% other fringe plus a prorated portion of the $10,260 life and health insurance (increased 3% per year for years 2 & 3). The student fringe rates include 7.65% FICA and 0.8% other fringe. The graduate student will also receive student health insurance each year ($2,200 year 1; $2,400 year 2; $2,600 year 3). D. Equipment: None Requested E. Travel: $22,000 (1) Domestic: $7,000 (i) $6,000. Co-PI Sharkey and the PhD student will attend and present research findings at the Entomological Society of American annual meetings and the International Congress of Entomology Conference in 2016 and 2017. $1,500 per meeting per person is requested to cover registration ($300), airfare ($500), hotel ($500), and per diem ($200). Details of the meetings are below. x x

Sept 2016, Orlando, Florida. Entomological Society of American annual meeting in conjunction with the International Congress of Entomology. $3,000. Nov 2017, Denver, Colorado. Entomological Society of American annual meeting. $3,000.

(ii) $1,000. The student will travel to Arizona State University in Oct 2016, to attend a workshop on comparative analyses and phylogenetics (airfare $400, hotel $400 and per diem $200). (2) International: $15,000 Sharkey and PhD student will carry out field trips extensively in South America in order to collect fresh specimens for molecular work and for revisionary taxonomy. (i) $15,000 for five trips to Peru, Ecuador and Colombia. Sharkey and student will perform both active collecting and long term passive trapping at the target sites. Active collecting will involve travels across the target countries. Long-term passive collecting traps (Malaise traps) will be set up at a collecting site during the first trip and another trip is needed to return to retrieve the samples. We prefer to go back to the sites to retrieve the samples ourselves than having the samples shipped from the collecting sites to the US. This is because shipping may risk damage to or loss of the samples, which are critical to the success of the proposed project. This strategy would incur a slightly higher cost, but will ensure the quality and the safety of the samples. See trip budget details in Table 1 below.

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Table 1. Field trip budget. Airfares based on Orbitz.com quotes for traveling in May 2015. Country Peru Colombia Ecuador Peru Ecuador

Month, Year June 2015 July 2015 Dec 2015 Jan 2016 June 2016

Activities

No. participants & Cost

Field training with Sharkey and student. Set up traps & extensive active collecting. ~20 days. Set up traps & extensive active collecting. ~20 days. Set up traps & extensive active collecting. ~20 days. Retrieve samples. ~7 days.

Two persons. $4,600 (2400 airfare, 800 lodging, 600 local transportation and 800 per diem)

Retrieve samples. ~7 days.

Subtotal

One person. $1,900 (900 airfare, 600 lodging, 400 per diem) Two persons. $4,800 (2200 airfare, 800 lodging, 1000 local transportation and 800 per diem) One person. $1,900 (1200 airfare, 200 lodging, 200 local transportation and 200 per diem) One person. $1,800 (1100 airfare, 200 lodging, 300 local transportation and 200 per diem) $15,000

F. Participant Support Costs: $2,400 1. A student from Ecuador will travel to UK to receive training on the taxonomy and systematics of braconid wasps. The student will assist with local field work in Ecuador. Also, the training will facilitate knowledge dissemination to a tropical country with rich biodiversity, but with limited taxonomic expertise, thereby help with alleviating the taxonomic impediment and biodiversity crisis. Costs include lodging: $1200, airfare: $800, per diem $400. G. Other direct costs: 1. Material and supplies: $26,232 (i) Molecular work: $23,445. See Table 2 below for details. Table 2. Molecular work budget. RAD Sequencing & DNA barcoding budget cost/sample One time Cost for Oligos (including adapters and PCR primers and biotinylated oligos and barcodes) Illumina sequencing ($ ~2000 per run - 48 taxa per lane) 42 Total all reactions not including sequencing (listed below) 12.4 -Extraction 2.2 -Enzymes 1.2 -T4 DNA ligase 2 -Ampure XP beads (cleanup) 2 -Size selection 3 -PCR 1 -Consumables 1 DNA barcodes for 600 samples 10 Subtotal

No. samples -

total 7000

192 192

8064 2380.8

600

6000 23444.8

(ii) Malaise trap: $2,500. Ten units of Malaise traps are needed for long-term collecting at two local sites (five traps each site). Each costs ~$250 from the supplier: http://santetraps.com/. (iii) Vials: 5 ml conical vial, two bags (1000 vials/bag) for $114 from USA Scientific. (iv) 200-proof ethanol for preserving samples: $300. 15 gallons at $20/gallon. (v) Whirl-Pak bags: $73. For storing Malaise trap samples. Available from http://www.enasco.com/whirlpak/

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2. Publication Costs: $12,000 We plan to publish primarily using Open Access journals to promote accessibility of our research findings to the scientific community and the public. $12,000 are requested to publish eight manuscripts each for $1,500 (Six wasp taxonomic revision manuscripts and two phylogenetic or comparative analyses manuscripts). 3. Consultant Services: None 4. Computer Services: None 5. Subcontracts: None 6. Other: $39,500 a. Tuition support for one graduate student research assistant is requested for 3 years at $11,500 year 1; $12,000 year 2; and $12,500 year 3. b. Field Assistants: $2,000. The funds will be used to hire two local field assistants at two international sites to collect from Malaise traps for a continuous duration of at least 6 months at each site. The trap samples will be collected every week and it requires less than a day to do the collecting each time. c. Maintenance & repair of scientific equipment $1,500: Funds are requested to repair any equipment needed for this project. Repairs are routinely less expensive than purchasing new equipment. H. Total Direct Costs: $207,356 I. Indirect Costs: $67,582 The University of Kentucky’s federally negotiated indirect cost rate for research in the College of Agriculture is 40% of modified total direct costs ($168,956). J. Total Direct And Indirect Costs: $274,938 K. Residual Funds: N/A L. Amount of This Request: $274,938

F-15