POPOVICH, encoding a C2H2 zinc-finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in Aquilegia Evangeline S. Ballerinia,1,2 , Ya Minb , Molly B. Edwardsb, Elena M. Kramerb , and Scott A. Hodgesa,1 aEcology, Evolution and Marine Biology Department, University of California, Santa Barbara, CA 93106; and bDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02318 Edited by Dominique C. Bergmann, Stanford University, Stanford, CA, and approved July 30, 2020 (received for review April 11, 2020) The evolution of novel features, such as eyes or wings, that allow traits, such as the powered flight of insects, birds, and bats, or organisms to exploit their environment in new ways can lead the pharyngeal jaws of cichlid fish, is recognized as contribut- to increased diversification rates. Therefore, understanding the ing to lineage diversification (13–15), discovering the genetic genetic and developmental mechanisms involved in the origin and developmental mechanisms that led to their evolution is of these key innovations has long been of interest to evolution- often difficult, in part, because many of these traits involve com- ary biologists. In flowering plants, floral nectar spurs are a prime plex developmental mechanisms that arose deep in evolutionary example of a key innovation, with the independent evolution of history. Given that the Aquilegia nectar spur evolved relatively spurs associated with increased diversification rates in multiple recently and is formed by modifications to a single floral organ, angiosperm lineages due to their ability to promote reproductive it provides a unique opportunity to begin to dissect the develop- isolation via pollinator specialization. As none of the traditional mental and genetic basis of a key innovation, which, in turn, will plant model taxa have nectar spurs, little is known about the provide insight into its origin. genetic and developmental basis of this trait. Nectar spurs are The development of the spurred petal in Aquilegia is relatively a defining feature of the columbine genus Aquilegia (Ranuncu- simple, facilitating the identification of key features. The Aqui- laceae), a lineage that has experienced a relatively recent and legia petal is composed of two components, the laminar blade at rapid radiation. We use a combination of genetic mapping, gene the distal end of the petal, and the spur, which forms adjacent expression analyses, and functional assays to identify a gene cru- to the attachment point as a tubular outgrowth with a nectary cial for nectar spur development, POPOVICH (POP), which encodes at the tip (SI Appendix, Fig. S1A). Previous studies of the Aqui- a C2H2 zinc-finger transcription factor. POP plays a central role legia nectar spur identified key cellular processes involved in in regulating cell proliferation in the Aquilegia petal during the its development, which can be broken down into two develop- early phase (phase I) of spur development and also appears to mental phases. During phase I, cell divisions that are initially be necessary for the subsequent development of nectaries. The dispersed throughout the petal become localized to the devel- identification of POP opens up numerous avenues for continued oping spur cup, where they continue until the spur is ∼7 mm scientific exploration, including further elucidating of the genetic to 10 mm in length (16, 17). As petal development proceeds, pathway of which it is a part, determining its role in the initial the spur enters phase II, in which mitotic activity ceases and evolution of the Aquilegia nectar spur, and examining its poten- the differentiating spur cells elongate anisotropically (16). Cell tial role in the subsequent evolution of diverse spur morphologies across the genus. Significance Aquilegia j petal development j nectar spur j key innovation j mitosis Throughout evolutionary history, organisms have evolved fea- tures that allow them to interact with their environment in he pace of species diversification varies across the tree of life, novel ways. When such features lead to increased rates of Twith some lineages exhibiting increased rates of speciation speciation in a lineage, we call them key innovations. Under- relative to others. The evolution of key innovations, that is, traits standing the genetic and developmental changes involved in thought to promote the process of diversification by increasing the origin of key innovations is of particular interest. Here ecological opportunities, have often been used to explain particu- we identify a gene, POPOVICH, that is crucial to the develop- larly species-rich clades (1–3). Floral nectar spurs are considered ment of a key innovation, floral nectar spurs, in the columbine to be a classic example of a key innovation (4). Nectar spurs are genus Aquilegia. While the function of POPOVICH orthologs in tubular structures, generally formed by floral tissue, that pro- other plant taxa suggests an ancestral function of the lineage duce a nectar reward for animal pollinators. Such spurs have in leaf development, in Aquilegia, POPOVICH also functions evolved independently many times in flowering plants, and, in to promote cell division in petals, a key cellular step in the nearly all cases, lineages with nectar spurs are more speciose than development of nectar spurs in the genus. their sister lineages that lack them (4–6). Spurs are hypothesized to increase speciation rates because changes in spur morphol- Author contributions: E.S.B. and S.A.H. designed research; E.S.B., Y.M., M.B.E., and S.A.H. ogy can lead to pollinator specialization—either through the performed research; E.S.B. analyzed data; and E.S.B. and E.M.K. wrote the paper.y differential placement of pollen on the body of a pollinator or The authors declare no competing interest.y visitation by a different animal pollinator altogether—resulting This article is a PNAS Direct Submission.y in increased reproductive isolation between plants with different Published under the PNAS license.y spur morphologies (5). A textbook example of a radiation fol- 1 To whom correspondence may be addressed. Email: [email protected] or hodges@ lowing the evolution of nectar spurs is the genus Aquilegia (7). lifesci.ucsb.edu.y Floral nectar spurs evolved as outgrowths of petals in the Aqui- 2 Present address: Department of Biological Sciences, California State University, legia ancestor ∼5 million to 7 million years ago (8), after which Sacramento, CA 95819.y modifications to spur morphology and other floral features, such This article contains supporting information online at https://www.pnas.org/lookup/suppl/ as color and orientation, allowed populations to adapt to differ- doi:10.1073/pnas.2006912117/-/DCSupplemental.y ent animal pollinators (9–12). Although the evolution of novel First published August 26, 2020. 22552–22560 j PNAS j September 8, 2020 j vol. 117 j no. 36 www.pnas.org/cgi/doi/10.1073/pnas.2006912117 Downloaded by guest on September 26, 2021 anisotropy is a major contributor to final spur length, and including plants whose petals bore a small pocket but no tubular variation in the degree of anisotropy largely explains the dif- spur or nectary, as well as plants with clearly developed tubular ferences in spur length between species (16). However, little spurs and nectaries (Fig. 1B). In order to evaluate the previ- is known about the genetic control of persistent mitosis in the ous observations of Prazmo (22), we measured the length of the spur cup during phase I of development, or the transition to petal pocket or spur from the attachment point to the apex or anisotropic cell expansion during phase II of development. An nectary, respectively, on the proximal side of the petals for 92 initial RNA sequencing (RNAseq) experiment identified genes F2 individuals. Plotting these length measurements produced a that are strongly differentially expressed (DE) between the bimodal histogram confirming that phenotypes could be differ- developing spur and blade tissue during phase I of petal develop- entiated into spurred and spurless individuals (SI Appendix, Fig. ment in Aquilegia coerulea ‘Origami,’ including the TEOSINTE S1C); 334 F2 individuals were phenotyped using this metric to BRANCHED/CYCLOIDEA/PCF gene AqTCP4, which acts to guide binning of individuals into spurred and spurless classes. restrain cell proliferation in the distal compartment of the spur, Phenotype counts were consistent with expectations for a reces- and the AqSTYLISH genes, a small family of transcription fac- sive allele at a single locus causing spur loss (SI Appendix, Fig. tors (TFs) that were subsequently found to be responsible for S1D; n = 334, χ2 = 0.44, degree of freedom = 1, P = 0.51). At nectary development (17, 18). While the set of DE genes from the same time, these data indicate that multiple genes contribute that study indicates that cell proliferation pathways are involved, to spur length variation downstream of this single locus respon- none of the candidates functionally explored so far have revealed sible for spur presence/absence. In this study, we have chosen to potential master regulators of spur development. focus first on the quantitative trait locus (QTL) for spur loss. One species of Aquilegia native to montane regions of central Using whole-genome skim sequencing, 286 individuals were China, Aquilegia ecalcarata, is the only known species of Aquile- genotyped, and 276 of those individuals were phenotyped and gia with petals that do not produce spurs or nectaries (19). While used to conduct QTL mapping of spur loss (phenotypes were once thought to have diverged from the lineage prior to the evo- not recorded for 10 sequenced individuals). As predicted by the lution of nectar spurs, phylogenetic analyses clearly indicate that phenotype counts, a single major locus associated with spur loss the spurless phenotype of A. ecalcarata represents a secondary was identified on chromosome 3 (Fig. 1C). An apparent sec- loss (8).