Karolina Heyduk and Jim Leebens-Mack Department of Plant Biology, UGA C4 and CAM Plant Biology Symposium August 2013 ~640 species, Agave and Yucca comprise half Taxa have worldwide distribution APGIII Historically, assumed to be an all-CAM clade . Park Nobel Evidence for CAM: P. Nobel 1988 Mesic Xeric Agavoideae Intermediate Michael McKain 2012, Dissertation, UGA Understand the number of gains or losses of CAM How has CAM contributed to species diversity? Can we use the Agavoideae as a tool to understand the transition from C3 to CAM? Focus has been on Agave and Yucca Imperfect resolution within genera despite their importance in this subfamily Duplications/ploidy complicate inference McKain et al. 2012 Utilizes a large number of loci Takes into account gene tree incongruence . Hybridization . Gene duplication/loss . Deep coalescence A B C A B C A B C RNA baits designed complementary to 910 single copy loci (in 10 plant genomes) Adjacent intron sequence also captured RNA baits Exon Intron Intron Sequenced on a Distribution of number of taxa per gene 35 HiSeq, PE 100 Ignored for Missing data due 30 Combined de-novo phylogenetics to duplicate 25 copies of genes and reference 20 based assembly 15 10 98 genes, 23 Number of genes 5 species 0 1 3 5 7 9 11 13 15 17 19 21 23 25 Number of taxa Filtered Contigs with exon Genes Exon cov. Intron cov. reads sequence Max. cov: 862,742 1733 557 92.76 29.09 Min. cov: 1,593,568 745 449 18.97 5.66 Average 5,761,427 986 424 44.56 14.76 112,073 bp aligned 5,690 informative sites All 98 genes combined ML tree estimated Generated from 98 genes Each gene had ML tree estimate Use of carbon isotopes as a proxy for C3 vs. CAM Currently sparse sampling across the family Future directions for isotopes: . More dense sampling within Agave and Yucca Yucca currently indicates phylogenetic signal for CAM and C3 species C13 isotope ratio in Yucca subgenera Chaenocarpa Sarcocarpa -30.00 -25.00 -20.00 -15.00 -10.00 Unique hybrid system between C3 (Y. filamentosa) and CAM (Y. aloifolia) parents Offspring considered intermediate or facultative (Y. gloriosa) C13 ratios between Y. filamentosa, Y. aloifolia, and Y. gloriosa Series1Y. gloriosa Y. aloifolia Y. filamentosa -30.00 -28.00 -26.00 -24.00 -22.00 -20.00 -18.00 -16.00 Investigate genetic basis for evolution of CAM Use clonal offshoots – 1 under drought, 1 well- watered >6 pairs for all three species from region of sympatry Y. aloifolia Y. gloriosa Y. filamentosa Drought treated for ~2-3 weeks Assays for day/night transpiration and titratable leaf acidity Multiple samples during the drought period taken for RNAseq Potentially 3 origins of CAM within the subfamily Expand phylogeny to include better sampling within genera Likewise, expansion of isotope sampling is underway Analyze key photosynthetic genes RNAseq experiment will examine what occurs during transition to CAM Jim Leebens-Mack Lisa Donovan Michael McKain Jeremy Rentsch Olle Pellmyr Desert Botanical Garden Herbarium . Wendy Hodgson Funding from NSF and UGA Department of Plant Biology Palfrey Award .