Karolina Heyduk and Jim Leebens-Mack Department of Biology, UGA C4 and CAM Plant Biology Symposium August 2013  ~640 , and 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 ofgenes 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