The Phylogenetic Utility of EPIC DNA Sequences in the Tribe Cecropieae

Mutualisms, interactions between species that are beneficial to both partners, are useful systems for investigating how interactions may affect the evolution and diversification of lineages. Interactions between ants and plant species of the genus Cecropia are a classic example of mutualism but the origin and evolution of the mutualism is poorly understood. We examined whether the inclusion of an exon-primed intron-crossing (EPIC) marker could improve our understanding of Cecropiaeae phylogeny. We performed a Bayesian phylogenetic analysis of 15 species from which EPIC sequences were obtained (Fig. 2). By comparing clade support from analyses with and without EPIC, we concluded that the addition of a third gene region strongly supports the hypothesis that the Afrotropical and antless genus Musanga was derived from a Cecropia ancestor and is most closely related to the antless neotropical species C. sciadophylla (Fig. 1). INTRODUCTION The study of mutualism involving neotropical trees of the genus Cecropia and their associated ants could provide insights into their coevolutionary relationship1, 2, 3. Distinctive features shared by Cecropia species—glycogen-rich Müllerian food bodies (Fig. 3 left panel) and internodal prostomata enabling ant queens to nest in the hollow stems of the plant—suggest that the mutualism evolved in the common ancestor of members of the genus3. We aimed to investigate phylogenetic relationships within the tribe Cecropieae of the plant family Urticaceae in order to identify the closest relatives of Cecropia. Previous work estimated the phylogeny based on 26S ribosomal DNA and ndhF chloroplast DNA sequences5 but results were not robust. In order to improve our understanding of the origin of the ant-Cecropia mutualism, we investigated the utility of EPIC DNA sequences6 had on for resolving and supporting phylogenetic relationships within the tribe. The five genera of Cecropieae are distributed in either the neotropics of Central and South America (Cecropia, Coussapoa, Porouma) or the afrotropics (Musanga, Myrianthus). Objective: The study examined the effect of including an exon-primed intron-crossing (EPIC) marker on resolution and support for Cecropieae phylogeny. RESULTS REFERENCES 1) Ayala, Francisco José, et al. "Molecular Phylogeny of Azteca Ants (Hymenoptera: Formicidae) and the Colonization of Cecropia Trees." Molecular Phylogenetics and Evolution 5.2 (1996): 423-428. 2) Bronstein, Judith L. "The Contribution of Ant‐Plant Protection Studies to Our Understanding of Mutualism1." Biotropica 30.2 (1998):150-161. 3) Davidson, D. W. and D. McKey. 1993. Ant-plant symbioses: Stalking the chuyachaqui. Trends in Ecology & Evolution 8:326-332. 4) Miller, M.A., Pfeiffer, W., and Schwartz, T. (2010) "Creating the CIPRES Science Gateway for inference of large phylogenetic trees" in Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA pp 1 8. 5) Strand, A. E., J. Leebens-Mack, and B. G. Milligan. 1997. Nuclear DNA-based markers for plant evolutionary biology. Molecular Ecology 6:113-118. 6) Yao, Xiaohong, Chenhong Li, and Christopher W. Dick. "Exon-Primed Intron-Crossing (EPIC) Markers for Evolutionary Studies of Ficus and Other Taxa in the Fig Family (Moraceae)." Applications in Plant Sciences 1.10 (2013). CONCLUSIONS With the inclusion of the EPIC markers, we observed augmented levels of support for the: •Musanga/Cecropia clade—suggesting that Musanga diverged from the Cecropia genus before adopting mutualistic associations with ants. •C. membranacea/C. putamayonis clade •C. peltata clade •C. multisecta/C. telealba clade Conversely, decreased resolution in clades was associated with the EPIC marker addition (for currently unknown reasons), including the underlying Pourouma, Myrianthus, Cecropia, and Musanga node. Figure 3: Tree-produced Food Bodies (left) and Cecropia Leaf (right). Figure 1: Geographic Range of the Tribe Cecropieae and Proposed Divergence Cecropia Coussapoa & Pourouma Musanga & Myrianthus Erin Treiber Fig. 2a: Bayesian phylogeny based on ndhF & 26S METHODS We sequenced 18 samples representing 15 species of Cecropieae and one outgroup taxon. Previously extracted DNA was amplified using a polymerase chain reaction mix and A167170b primers as outlined in Yao et al (2013). PCR products were run on a 0.8% agarose gel to confirm amplification and products were cleaned using ethanol precipitation. The cleaned PCR products were quantified to determine DNA concentration and were then used as template for Big Dye cycle sequencing reactions. Cleaned products were sequenced by the BioMedical Genomics Center core facility at the University of Minnesota. The data were analyzed and edited using Geneious with additional alignment done using Se-Al v2.0a1. Tree analysis was done on the CIPRES portal using Mr.Bayes v3.2.2. The analysis was run with four chains for 10,000,000 generations with a GTR model. Fig 2b. Phylogeny based on EPIC, ndhF & 26S Coussapoa villosa Pourouma myrmecophilia