Background: The faunal diversity of has long been of interest to ecologists and conservationists, with a particular emphasis on the endemic of the island. The relatively recent arrival of humans on Madagascar had a profound effect on the lemurs, with the extinction of 17 species between 1,700 and 500 years ago1. The subfossil lemurs were larger in body size than current species of in Madagascar and could potentially disperse seeds of large plant species unable to be consumed by extant lemurs1. Plant species that now lack these megafaunal dispersers have been termed “orphaned” lineages, and their future on Madagascar remains uncertain2. Orphaned lineages have previously been identified in other parts of the world, such as the Neotropical plants of Central America eaten by the now extinct gomphothere3. In some cases orphaned lineages may be ‘recuperated,’ with the potential for selection for smaller seed size if the lower range of size variation overlaps the maximum gape of any smaller-bodied dispersers, as has been documented following the local extinctions of larger-bodied seed dispersing birds in the Brazilian Atlantic forest4. The specific ecological and potential evolutionary ramifications of the subfossil lemur extinctions (and thus, by proxy, the broader impact of the history of human- environment interactions on Madagascar), however, are not well understood. The Critically Endangered red (Varecia rubra) and the black-and-white ruffed lemur (Varecia variegata), two of the largest surviving lemur species, are of major ecological importance in Madagascar with respect to long-term forest survival (IUCN 2014)5,6. These lemurs are crucial seed dispersers for a variety of plant species – and the only remaining dispersers for many taxa – and their extinction could negatively affect the long-term health of Malagasy forests. Objectives and Overview: The purpose of this project is to determine whether V. rubra is preferentially consuming relatively smaller-sized fruits from otherwise larger-fruited tree species (i.e., those overlapping the predicted maximum size of Varecia consumption), which would represent a potential selection pressure towards smaller fruits and seeds for that species. If so, this could have far-reaching ecological implications, whereby the size-biased extinction processes (likely mediated at least in part by humans) that resulted in the survival of only smaller-bodied lemur taxa could be having under-appreciated effects on the co-evolutionary ecology of the Malagasy flora. This project would further highlight V. rubra as a conservation priority and illuminate the broader and longer-term ecological effects of its extinction. This work will be conducted via two approaches. First, working with a team of field assistants and Malagasy students for whom this project will provide field-based ecology training, I will combine behavioral observations of V. rubra groups in , Madagascar with an extensive ecological survey to investigate the sizes of their consumed fruits, including those of two species with fruit sizes that overlap the predicted maximum consumption size, Pandanus odoratissimus and Ampelosicyos humblotii. To determine consumed seed sizes of selected fruits I will regularly recover fecal samples from the observed individuals. Fruits of non-selected fruits (both from within the same tree, and from trees within the home ranges of the observed groups but that were not foraged) will also be collected, for measurement of seed size and comparison to the results from the selected and consumed fruits. Second, for the one tree species with the greatest differential between consumed and non-consumed seed size, I will perform a genome- wide association study (GWAS) to identify regions of the tree genome associated with seed size. For this analysis I will collect DNA samples and seed size data from a large number (n=500) of individual trees of the targeted species. Genome sequencing (low-coverage) and analysis on this scale is now feasible, even for non-model organisms. I will then use an evolutionary population genomics approach to test whether smaller seed-size alleles have been subject to recent positive selection for in these species. Methods: For this work, I will travel to Masoala National Park in Analanjirofo, Madagascar to study wild populations of V. rubra. Seed collections from various populations of lemurs will be conducted through fecal matter analyses and from behavioral observations for fruit selection in P. odoratissimus and A. humblotii. Fruit samples will be collected from bushels and trees that were explicitly avoided for consumption by V. rubra for seed size comparison to those found in fecal samples. A GWAS will be conducted in order to identify single nucleotide polymorphisms (SNPs) conducive to smaller seed size; results will be assessed for overlap with genes known to control seed size in the model plant organism Arabidopsis thaliana7. I will test the hypothesis of recent positive selection for alleles associated with smaller seed size using the Integrated Haplotype Score (iHS)8. This work will be conducted in the Anthropological Genomics Laboratory at Pennsylvania State University under the mentorship of Dr. George Perry, who has experience with combined GWAS-evolutionary genomic studies9 and has ongoing field and endemic species population genomics projects in Madagascar10,11. Intellectual Merit: The results of this work could provide a prime example of current evolutionary processes affecting phenotypic seed size in an active human-disturbance environment, and thus lay the foundation for the study of similar processes occurring in other geographic regions. This study could also shed light on a potential mechanism for the anachronistic fruits to regain seed dispersers. My project would further highlight the importance of conserving V. rubra, outside of strictly for the purpose of preserving biodiversity. If V. rubra is indeed exhibiting selective size pressure on species they currently consume, these results would have implications for the smaller orphaned lineages in Madagascar and their potential ability to recuperate from the extinction of their megafaunal dispersers through evolution. This phenomenon could have implications for the long-term health of Malagasy forests and thus for the island’s endemic taxa and Malagasy people who depend on these forests for resources. Broader Impacts: I currently work at the Center for Precollegiate Education and Training at the University of Florida as a program coordinator, where I have direct experience in conducting science education and outreach activities with high school students, as well as experience writing modules that incorporate high-level research into classroom friendly units. A similar center exists at Pennsylvania State University called Science University, a strong outreach center that focuses on science content for K-12 students and provides professional development resources for science teachers. I have established connections with the director of Science University at Penn State, Michael Zeman, and discussed opportunities for module implementation in their programs. As a graduate student, I will develop and deliver science modules to teach students about the value of insights from studying animal feces and other waste. For example, students will dissect owl pellets as an activity, and I will illustrate how I and my laboratory use lemur feces to ask broad-scale ecological questions. The students will learn of lemurs as seed dispersers and their role in the forests of Madagascar, and about seed dispersers in general. These modules will be incorporated in events such as the Expanding Your Horizons STEM Career Day for middle school girls, assimilated into the Science University summer camps, and used during teacher professional development workshops for potential implementation in local classrooms. References: 1. Federman, S. et al. PNAS (2016). 2. Crowley, B.E. et al. Am J Primatol (2011). 3. Janzen, D.H. et al. Science (1982). 4. Galetti, M. et al. Science (2013). 5. Razafindratsima, O.H. et al. Ecotropica (2012). 6. Perry, J.M. et al Am J Phys Anthropol (2010). 7. Luo, M. et al. PNAS (2005). 8. Voight, B.F. et al. PLoS Biol. (2006). 9. Perry, G.H. et al. PNAS (2014). 10. Perry, G.H. et al. PNAS (2013). 11. Thompson E.T. et al Int J Primatol (2016).