POPOVICH, encoding a C2H2 zinc-finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in

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 , 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 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 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 | petal development | nectar spur | key innovation | 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 , 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 | PNAS | September 8, 2020 | vol. 117 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.2006912117 Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 htaesrnl ifrnilyepesd(E ewe the between develop- petal (DE) of I in phase expressed ment during tissue blade differentially and spur strongly developing to genes An transition are identified development. experiment the that (RNAseq) of or sequencing II development, the RNA phase initial in of during mitosis expansion I little persistent cell phase anisotropic of However, during and control dif- (16). cup genetic length, the species spur the explains between about spur largely known length final is anisotropy spur to of in degree contributor ferences the major in a variation is anisotropy alrn tal. et Ballerini F the F in an segregated create phenotypes to petal selfed Various was tion. B), S1 Fig. Appendix, (SI spurs F the from individual in species, loss spur Candidate a controlling Identify Development. RNAseq Spur for and Gene Mapping Locus Trait Quantitative Results C2H2 I phase a the during encoding of mitosis development in regulating gene the available to a in crucial tools is identify of that to use TF era zinc-finger made and genomics (22) modern Prazmo genetic the similar of a how that made understanding to we in cross Therefore, step evolved. feature important novel development an this the innovation, critical in key a involved this provide network of genetic would devel- the spur element of early this in component role Identifying a crucial (20). be a may plays opment there that that factor indication genetic an single as interpreted been developmental has the petals, with spurless the combined between cross, similarities curva- genetic and This length spur (22). as ture such in traits morphological loss affecting spur loci that suggest findings ecalcarata These A. length. spur in variation the while that showed the in (22) samples Prazmo between petal W. 1960s by (21). between conducted tissue cross spur DE genetic lacking a consistently samples Interestingly, petal genes and tissue 35 spur only containing of list a the the of of morphology the and intact, ecalcarata remain development its the of and Although genetic (4). the development unravel ecalcarata spur to of helping in basis in key developmental as loss suggested spur been understanding has Nonetheless, (8). loss of that phenotype indicate evo- clearly spurless the analyses to the phylogenetic prior spurs, lineage nectar the of from lution diverged have to thought once gia China, development. spur revealed of have regulators far master so from potential explored functionally genes involved, candidates are DE the pathways of for of proliferation none cell set responsible that the be indicates While study to that 18). found (17, subsequently development were nectary that (TFs) spur, the tors of compartment the distal the and in proliferation cell restrain BRANCHED/CYCLOIDEA/PCF ewe ld n prtsu in DE tissue as spur identified and those blade with between spurless genes identified these was of Cross-referencing taxa (21). spurred petals divergent consistently phylogenetically developing three genes and the of set between A development DE expression. spur early gene in comparative involved using network genetic the of nents used n pce of species One ihptl htd o rdc pr rncais(9.While (19). nectaries or spurs produce not do that petals with .ecalcarata A. Aquilegia steol nw pce of species known only the is ecalcarata, Aquilegia AqSTYLISH qiei coerulea Aquilegia to sibirica, Aquilegia 5 ftesurdF spurred the of ∼75% ea sqiesmlrt h rmtvl prespetals spurless primitively the to similar quite is petal ea olne rdcsanca pr te aspects other spur, nectar a produces longer no petal 5 fteF the of ∼25% scue yasnl ou hti psai oother to epistatic is that locus single a by caused is .ecalcarata A. itrgenus, sister Aquilegia na fott ute arwi nkycompo- key on in narrow further to effort an in 1 ee,asalfml ftasrpinfac- transcription of family small a genes, eeain hs easdvlpdnectar developed petals whose generation, ecosdaspurred a crossed we ecalcarata, A. Aquilegia .ecalcarata A. nodrt dniytegntcregion genetic the identify to order In Oiai’icuigthe including ‘Origami,’ aiet otn ein fcentral of regions montane to native 2 n h pre species spurred the and niiul i o rdc spur, a produce not did individuals .ecalcarata A. 2 Semiaquilegia .coerulea A. niiul hwdcontinuous showed individuals gene etrspur. nectar hc csto acts which AqTCP4, ersnsasecondary a represents 2) epreviously We (20). .Asingle A 1A). (Fig. Oiai revealed ‘Origami’ and .ecalcarata A. .ecalcarata A. 2 .ecalcarata A. TEOSINTE generation, .vulgaris A. 2 genera- Aquile- A. A. etr,rsetvl,o h rxmlsd fteptl o 92 for petals or the apex of the side to proximal point the F attachment previ- on the the the respectively, from of evaluate nectary, spur length the to or measured pocket order we petal (22), In Prazmo tubular 1B). of developed observations clearly (Fig. ous with nectaries plants and as tubular well no spurs as but pocket nectary, small or a spur bore petals whose plants including Fig. Appendix, (SI loss spur causing locus single S1D; reces- a classes. a at spurless for allele expectations and with sive spurred consistent were into counts individuals Phenotype of binning guide Fig. Appendix, F (SI 334 individuals differ- S1C); spurless be and could spurred into phenotypes entiated that confirming histogram bimodal aafrom data 21). 1D; and (Fig. 17 development refs. of I taxa, phase during spurred levels, tissue ground the blade of petals the ecalcarata the and to formosa, in relative Aquilegia levels tissue higher spur at the in of Aqcoe3G231100, levels candidates, higher expression spur the for the in QTL of the occurs in one are 21) Only ref. –; loss. (spur tissue spur lacking 35 those between the DE of consistently any gia as whether identified determined first previously we more genes or a QTL, one the be of in sequence could coding proteins phenotype the affecting loss mutations spur of result the recombination Although low genes. a annotated has encompasses region we and This a rate phenotype, 3. to chromosome with interest of associated of middle region were genotype QTL that in our EE shift narrowed S2). and a Table in ES , Appendix resulted (SI between that spurs 71 events nectar whereas recombination spurs, lack Using produce individuals QTL EE the 79 at of individuals SE and SS all which 3, chromosome on as locus to the locus refer score, element the we on LOD genetic focus highest major we the the cross, with this on in in ). S2 development Fig. spur narrow controlling Appendix , sig- to (SI the order remains covariate, in 3 a Therefore, chromosome as on 2 when QTL chromosome however, nificant on locus; 2 genotype a chromosome the geno- as the treating at between QTL phenotype association 3 and the chromosome type eliminates the at completely genotype covariate the individ- treating one Indeed, only and an locus, had 2 for the chromosome homozygous for the ual homozygous at never (EE) were allele locus 3 mosome an with individuals example, For sibirica S1). Table Appendix, (SI loci deleterious a by caused between likely interaction is 2 genotype chro- chromosome between on association on phenotype the loci and that revealed 7. the 3 and at and 6, 2, 2 genotypes 5, mosomes chromosomes chromosome individual on of on effect examination small detected of Closer loci was several effect as 1 well moderate (Fig. as of 3 locus loss chromosome spur ond on with associated identified were the locus by major was (phenotypes predicted single loss As a individuals). counts, spur sequenced phenotype of and 10 for phenotyped mapping recorded were QTL not individuals conduct those to of used 276 and loss. spur genotyped, for (QTL) to locus chosen trait quantitative have the we on study, respon- first this locus focus In single presence/absence. this spur of for downstream sible contribute variation genes length multiple spur that indicate to data these time, same the 2 eoyea the at Genotype were individuals 286 sequencing, skim whole-genome Using niiul.Potn hs eghmaueet rdcda produced measurements length these Plotting individuals. .coerulea A. hs ea ape otiigsu ise(pr+ versus +) (spur tissue spur containing samples petal I phase n 334, = lee(ooyosS rhtrzgu S ttechro- the at ES) heterozygous or SS (homozygous allele .ecalcarata A. eas hr ti setal nyepesda back- at expressed only essentially is it where petals, 2 POPOVICH niiul eepeoye sn hsmti to metric this using phenotyped were individuals POP χ PNAS Oiai easadi ossetyexpressed consistently is and petals ‘Origami’ 2 .4 ereo reo 1, = freedom of degree 0.44, = POP T.Ti addt sepesda much at expressed is candidate This QTL. .ecalcarata A. | .sibirica A. lee(S ttecrmsm locus. 2 chromosome the at (ES) allele 0Mpo eunecontaining sequence of Mbp ∼20 etme ,2020 8, September hni spurless in than chrysantha , Aquilegia T shgl rdcieo phenotype— of predictive highly is QTL (POP S)a h hoooe3locus 3 chromosome the at (SS) ). and .sibirica A. | o.117 vol. .A paetsec- apparent An C). -Mrgo nthe in region <2-cM | lee tthese at alleles P o 36 no. .ecalcarata A. .1.At 0.51). = .sibirica, A. ∼1,100 Aquile- | 22553 A. A.

PLANT BIOLOGY AB

CD

Fig. 1. Identifying a candidate locus for spur loss in A. ecalcarata. (A) Flowers from the cross parents, spurless A. ecalcarata (Top) and A. sibirica with curved spurs (Bottom). (B) Examples of variation in petal morphology segregating in the F2 generation of the cross between A. ecalcarata and A. sibirica, including plants that do not produce spurs at all (column 1) and plants exhibiting various spur morphologies (columns 2 to 4). (C) Genome-wide association of genotype with the presence/absence of spurs in the F2 generation. Logarithm of the odds (LOD) scores (y axis) for the presence or absence of spurs indicate a major QTL on chromosome 3 (x axis, genomic marker bin position by chromosome in centimorgans). The dashed line represents the 5% LOD false discovery rate. The association on chromosome 2 appears to be a result of a genomic incompatibility (SI Appendix, Table S1). (D) The expression of Aqcoe3G231100, plotted as mean normalized reads of multiple biological replicates (±SE), across several stages spanning phase I of petal development in various petal samples, including some containing spur tissue (spur +) and some lacking spur tissue (spur -). Aqcoe3G231100 is expressed at higher levels in spur + samples. Data for A. ecalcarata (spur–), A. sibirica (spur+), A. formosa (spur+), and A. chrysantha (spur+) are from ref. 21 where RNA was isolated from entire petals (n = 3 for each data point). Data for A. coerulea ‘Origami’ are from ref. 17 where RNA was isolated from petals dissected into blades (spur–) and spur cups (spur+; n = 4 for each data point). Developmental stage is represented numerically 1 to 5 as in ref. 21, and approximate spur lengths in millimeters corresponding to these stages are provided.

Aqcoe3G231100 Encodes a C2H2 Zinc-Finger Transcription Factor. PENTAFOLIATA1 (PALM1), functions in the regulation of Aqcoe3G231100 is annotated as a C2H2 zinc-finger TF, one of compound leaf development (25, 26). the largest TF families in plants (23). The subclade of C2H2 Analyzing the sequence of the A. ecalcarata and A. sibirica TFs containing Aqcoe3G231100, part of the C1-1i clade (23), Aqcoe3G231100 alleles segregating in the cross and from nine includes a number of genes with important roles in floral devel- additional phylogenetically distributed Aquilegia species shows opment (SI Appendix, Fig. S3). These genes contain LxLxL-type that, overall, the predicted protein is highly conserved across the EAR transcriptional repressor motifs, and related homologs genus, except for in a region of glutamine (Q) repeats near the from Arabidopsis have been found to function in regulating the N-terminus of the predicted protein (SI Appendix, Fig. S4). The transition between mitotic growth and cellular differentiation nucleotide and predicted protein sequence between the A. ecal- in various plant developmental processes by repressing either carata and A. sibirica alleles segregating in the F2 generation are genes responsible for cellular differentiation or genes involved nearly identical (both greater than 99% shared sequence iden- in the maintenance of mitosis (23). There is no functional tity) with the only amino acid variants occurring in this Q-repeat information for the Arabidopsis ortholog of Aqcoe3G231100, region (SI Appendix, Fig. S4). Comparing the A. ecalcarata allele AT4G17810, although it is expressed in leaves and cotyledons to the A. sibirica allele, there is a single nucleotide polymor- (24), and the ortholog in Medicago truncatula, PALMATE-LIKE phism causing an amino acid difference (E26Q) as well as three

22554 | www.pnas.org/cgi/doi/10.1073/pnas.2006912117 Ballerini et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 e ue–rmrts eut in results test 31.41, Tukey–Kramer = fied F (ANOVA, F genotype of the patterns ES expression in Normalized the Aqcoe3G231100 spurs. and had spurless, individuals SS were and individuals EE genotypes—the their QTL with consistent phenotypes presence/absence spur the had at (ES) heterozygous F 10 20 and of petals F the developing on homozygous in at RNAseq assayed genotypes was using allele- expression different explored gene we our with this, of test individuals patterns To QTL). expression a same specific that the likely in less encoded is be it possible, (while the tran- to sion in tissue, due by difference be petal part would functional spur– allele in the and that identified predicted spur+ first we was between gene differences this the scriptional in that effects and functional cause protein would that differences acid amino in Aqcoe3G231100 i.2. Fig. Cis S4 Fig. Appendix, (SI spurs in nectar found in that seen to identical loss sequence spur the the in as result ecalcarata, would variation acid amino this the in residues Q additional alrn tal. et (H Ballerini tip. spur the at cells of (floral layer primordia adaxial (car) the carplel to bars: is restricted (Scale and assessed signal. is (sd), plants Aqcoe3G231100 staminode three 10, (st), stage the in across (p), lengths (F petal flower. spur differentiating 9 of see stage fully early range 27; a throughout the ref. of on expressed proportions, length based spur various is stages the For Aqcoe3G231100 to length). meristem, relative spur length floral spur full 7 sample of (D–H the millimeters. proportion as in (i.e., presented provided, is plant also petals each of for staging petal Developmental plants. developed different three in stages developmental F the from heterozygous transcribed of 10 reads tissue the assignable Aqcoe3G231100 F of single of each a number of for The RNAseq (B) by genotype. assessed each as reads for normalized represents point Each QTL. F of 4) EFGH G F DE BC AB Rgltr hne otiuet ifrnilEpeso of Expression Differential to Contribute Changes -Regulatory 2 xrsino qo3210 nF in Aqcoe3G231100 of Expression niiul htaeete homozygous either are that individuals .coerulea A. .sibirica A. Late (G) flower. 10 stage early an in spur developing the of tip the to contracts expression Aqcoe3G231100 progresses, development As ) D, .ecalcarata A. .ecalcarata A. Oiai costetasto rmmttcgot paeI upe ocl ifrnito paeI,ga)a sae tavreyof variety a at assayed as gray) II, (phase differentiation cell to purple) I, (phase growth mitotic from transition the across ‘Origami’ and H, rgltr ifrne ngn expres- gene in differences cis-regulatory al S4 Table Appendix, SI S) homozygous 5 (SS), I, 100 2 infiatydfee yterQTL their by differed significantly s .ecalcarata A. nastage a In (D) hybridization. situ in using stages developmental various at petals in Aqcoe3G231100 of expression Spatial ) µm; . 2 in s leehsapeitdaioacid amino predicted a has allele ie htteeaen predicted no are there that Given POP E, ). P h ahdln ersnste11rtoo n ed.(C reads. S and E of ratio 1:1 the represents line dashed The A. hc has which japonica, Aquilegia and F, .As S3). Table Appendix, SI 2 .6-;Dnetmodi- Dunnett 1.96e-6; = T.AlF All QTL. yrd n in and hybrids lee ti nieythat unlikely is It allele. 200 G, .ecalcarata A. o tg oprsnwt Nsqsae.(E stage). RNAseq with comparison stage for rgltrwould trans-regulator .ecalcarata A. POP µm.) 2 lnsassayed plants .coerulea A. .ecalcarata A. T.Gene QTL. 2 (EE, yrd:5 hybrids: n (EE), ) homozygous 5), = POP ‘Origami.’ A. 2 2 xetd xrsino qo3210 a oetin lowest was carata Aqcoe3G231100 of expression expected, S5 does (R there sampled F Aqcoe3G231100 spurred spurred), were the between in are correlation length spur SS strong and level a and expression be ES to and appear spurless not are EE the cor- (i.e., at the from genotype Aside between 2A). relation (Fig. heterozygotes the in intermediate otiiganme fsnl uloieadidlvariants indel and nucleotide single of the number region in a a is variants there however, containing few site; start are transcription the there of upstream that that between cates sequence upstream ecalcarata, the A. Comparing 2B). F (Fig. the in grounds expression and in species parental difference ferent the the between both to seen contribute significantly alleles that suggesting the by further produced was reads of sibirica number F greater every a nearly assessed, for which, analy- gote in This pattern allele. strong parental a each showed of sis off transcribed been having as carata the between variants nucleotide sequence loci. multiple by niiul ihtebakbridctn enepeso falindividuals all of expression mean indicating bar black the with individual, ouigo h 0htrzgts h ml ubro coding of number small the heterozygotes, 10 the on Focusing ,cnitn ihteosrainta prlnt scontrolled is length spur that observation the with consistent ), xrsino qo3210 nptl dvlpetlstage (developmental petals in Aqcoe3G231100 of Expression (A) POP anfiaino h prtpin tip spur the of Magnification ) lee eeue oasg ed pnigteepositions these spanning reads assign to used were alleles .sibirica A. ooyoe,hgetin highest homozygotes, lee(ietoa icxnsge aktest, rank signed Wilcoxon (directional allele T eoye n tews aibegntcback- genetic variable otherwise and genotypes QTL .ecalcarata A. qo3210 sbodyepesdi h al ea fa of petal early the in expressed broadly is Aqcoe3G231100 ) n eea te pre pce indi- species spurred other several and sibirica, A. PNAS (SS, 2 0.21, = n | ) rhtrzgu (ES, heterozygous or 5), = eaieepeso fAceG310i spur in Aqcoe3G231100 of expression Relative ) etme ,2020 8, September rgltr ifrne ewe these between differences cis-regulatory lee( ed)v.the vs. reads) (E allele P 0.058, = .sibirica A. POP (I G. n h es rb hwdno showed probe sense The ) | T n prpresence spur and QTL I 14; = o.117 vol. .sibirica A. .sibirica A. n ooyoe,and homozygotes, 0 ttesu loss spur the at 10) = Fig. Appendix , SI . bdirectly kb ∼1.5 2 | niiul that individuals o 36 no. lee( reads) (S allele and P 2 2 ihdif- with s heterozy- 0.009), = .ecal- A. .ecal- A. | to ∼3 22555 A.

PLANT BIOLOGY ∼2 kb upstream of the transcription start site (Chr03:27,452,100 ited to the adaxial surface of the petal spur cup (Fig. 2 G and to 27,452,400; SI Appendix, Fig. S6). This region may be a point H). This pattern of progressive spatial restriction to the spur tip of focus for future research into cis-regulatory elements related mirrors the contraction of mitotically active cells as indicated by to differences in allele expression. HISTONE4 (HIS4) expression (16, 17), with the notable excep- tion of the adaxial localization of Aqcoe3G231100 late in phase I, Temporal and Spatial Expression of Aqcoe3G231100 in A. coerulea which is not seen in cell division markers. In addition to expres- ‘Origami’ Is Broadly Consistent with Mitotic Activity during Spur sion in petals, Aqcoe3G231100 expression was also detected in Development. In order to further examine the temporal and spa- the placenta, ovules, and young leaves (SI Appendix, Fig. S7 A, tial expression patterns of Aqcoe3G231100, we used qRT-PCR D, F, and G), all of which are presumably mitotically active dur- and in situ hybridization to assess expression patterns in the hor- ing the phase in which expression was assayed (this has been ticultural variety A. coerulea ‘Origami.’ As prior RNAseq assays confirmed for young leaves in ref. 17). No signal was detected were conducted only on petals during phase I of development using a sense probe (Fig. 2I). Thus, both temporally and spa- (Fig. 1D and ref. 21), we used qRT-PCR to track the expres- tially, Aqcoe3G231100 expression is generally associated with sion profile of Aqcoe3G231100 in spurs as petal development mitotically active tissues. transitioned from mitotic growth (phase I) into cell differentia- tion and anisotropic cell elongation (phase II; Fig. 2C). In each Gene Knockdown Results in Dramatically Altered Spur Development plant assessed, the expression of Aqcoe3G231100 in nectar spurs Caused by a Reduction in Cell Number. We used Virus-Induced showed a similar pattern. Expression increased through phase Gene Silencing (VIGS) to transiently knock down the expression I but dramatically dropped as petals transitioned from mitotic of Aqcoe3G231100 in A. coerulea ’Origami.’ As the quantita- growth to the cell elongation phase (this phase transition was tive, spatial, and temporal degree of target gene knockdown previously identified in ref. 16 as occurring when A. coerulea (KD) is highly variable with VIGS, constructs were designed ‘Origami’ petals are ∼8 mm to 10 mm in length; Fig. 2C). to also target a reporter gene, ANTHOCYANIDIN SYNTHASE To assess spatial expression patterns of Aqcoe3G231100, we (AqANS), which is required to produce the red floral pigments used in situ hybridization across early developmental stages of in A. coerulea ‘Origami’ and petals and can be used as A. coerulea ‘Origami’ flowers. During floral development stages a general, although not exact, marker of tissue experiencing 3 to 7 (floral staging as in ref. 27; see SI Appendix, Table S4 for target KD versus wild-type (WT) tissue. A range of spur phe- developmental stage comparisons with RNAseq and qRT-PCR notypes were present in petals that showed evidence of VIGS data), the gene is broadly expressed in the floral meristem and in response when Aqcoe3G231100 was targeted. The strongest early differentiating petals, , staminodes, and carpels (SI spur phenotypes resulted in the near-complete loss of the nec- Appendix, Fig. S7 A and B and Fig. 2D). Although the expres- tar spur, including the nectary, while less severe phenotypes sion of Aqcoe3G231100 wanes through development in most included shorter spurs, some of which still produce nectaries floral organs, it is maintained in later stages of petal development (Fig. 3 A–F). As Aqcoe3G231100 is expressed only early in (Fig. 2 E–H and SI Appendix, Fig. S7 A–C). The locus is ini- petal development, largely before pigment is produced, petal tially expressed broadly in petals (Fig. 2E) but gradually becomes samples used to verify and assess the degree of gene KD were restricted to the spur cup (Fig. 2F and SI Appendix, Fig. S7 C identified based on morphology at earlier developmental stages and E). Toward the end of phase I, expression is eventually lim- (Fig. 3 E and F). In paired samples (WT vs. KD petals from

AC D EF

B G C’ D’

Fig. 3. Phenotypes of VIGS of Aqcoe3G231100 in A. coerulea ‘Origami.’ (A–D) Flowers at anthesis. (A) WT. (B) AqANS silenced VIGS control. (C) An Aqcoe3G231100-AqANS VIGS treated plant with two KD petals showing greatly reduced nectars spurs. (C0) Petals dissected from the flower in C with the two KD petals indicated (c1 and c2). (D) An Aqcoe3G231100-AqANS VIGS treated plant with four KD petals with variably reduced spurs. (D0) Petals dissected from the flower in D with the four KD petals indicated (d1 to d4). (E and F) Flowers at a developmental stage when Aqcoe3G231100 is normally expressed. (E) A WT flower with multiple sepals removed to see developing petal nectar spurs that have not begun producing anthocyanin pigments. (F) Example of an Aqcoe3G231100-AqANS VIGS treated plant used to verify gene KD, showing a strong reduction in spur development in two petals (indicated by arrows) relative to the other three. (G) Spur length plotted against cell number of WT (red) and Aqcoe3G231100-AqANS KD (gray) petals and the line of best fit, indicating a strong positive correlation between cell number and spur length (R2 = 0.95, P = 3.0e-9, n = 14). (Scale bars: A, B, C, C0, D, and D0, 1 cm; E and F, 1 mm.)

22556 | www.pnas.org/cgi/doi/10.1073/pnas.2006912117 Ballerini et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 elnme (R (R number length cell cell in with both variation correlated that from not 0.60, show length is comparisons spur length These spur assessed petals. KD and counted and we cells that WT mitosis, spur suggest regulating would measured in inter- which and role with number, a seen cell plays due those in Aqcoe3G231100 are as or petals such KD variation, elongation, VIGS specific cell the in the in whether differences seen assess to to length order spur In differ- in (16). than differences number rather cell elongation, overall in cell ences anisotropic in attributed be differences largely can to length spur in the variation interspecific of that petals 36.1% WT average, with on comparison had, in (n petals Aqcoe3G231100 of KD expression flower), same the alrn tal. et Ballerini of feature common a is with Arabidopsis and saw of truncatula similarly leaves M. we developing as in expression (24), The leaves developing young in POP Arabidopsis in leaves truncatula compound of development the achieved during indeterminacy is This suppress- of (25). a by repression leaves direct development developing the in through leaf activity compound mitotic develop- ing of spur complexity controlling for the gene The candidate unlikely ment. an be to the in of division function cell promoting by gia development spur in Aqcoe3G231100 designating are we as evidence, of lines the multiple nectary— with the including consistent some spur, in traits the and, of loss spurs near-complete shortened the in cases, resulting produced, cells spur of variety spurred long the coerulea in A. gene the of expression in down expression knocking Aqcoe3G231100 het- of lack in the patterns that expression indicate vari- allele-specific Aqcoe3G231100 erozygotes genetic species, of development. the expression the spur differential between pinpointed of for not responsible phase have ants mitotic developing we the in although I, expression Moreover, phase expres- localized temporal during highly and spurs Spatial reveal spurs. patterns gene of sion absence The or (21). presence spurs petals the nectar the the under lost located in secondarily is expressed has is which gene carata, in spurs the spurred nectar shown, of of have development studies the prior to Aqcoe3G231100 that critical evidence of is lines multiple present we Here Discussion S9C). Fig. Appendix, (SI leaves such compound biternately compound exaggerated ternately to from were shifted presumably architecture leaf lobes overall in the leaflet that lanceolate between dissections and S9 cases, Fig. elongated Appendix, (SI to leaves rounded silenced leaves from altered control was these tissue shape In leaflet in floral S9A). of Fig. ratio in aspect Appendix, flo- the KD (SI leaves, in phenotype exhibiting leaf noted a plants were exhibited many also phenotypes however, obvious tissue; other other ral primarily situ in no in is Aqcoe3G231100 that organs, spurs fact of floral the KD expression Despite in cells. indicates fewer length hybridization of spur production the of to reduction due the that ing LEAFY eeomna tde fseveral of studies Developmental u eut ugs that suggest results Our POPOVICH 3 E14.4%; SE 13, = prcp ae oeyo htltl skonaotthe about known is little what on solely Based cup. spur n 14; = /FLORICAULA 2,2) ohn skonaottefnto fthe of function the about known is Nothing 28). (25, Oiai assadaai euto ntenumber the in reduction dramatic a causes ‘Origami’ .truncatula M. POP Aquilegia ,bti ihycreae with correlated highly is but S8B), Fig. Appendix, SI 2 (POP /PALM1 0.95, = rhlg nohrtx,ti ou ol seem would locus this taxa, other in orthologs i.S8A). Fig. Appendix, SI POP ). aabtnti h easof petals the in not but taxa POP rgltr ifrne otiueto contribute differences cis-regulatory P T,wihi togyascae with associated strongly is which QTL, rhlg lhuhi sas expressed also is it although ortholog, .ecalcarata A. rhlgof ortholog POP rhlgta ucin omaintain to functions that ortholog 3.0e-9, = rhlg nedct,ad although and, , in orthologs ugssta al efexpression leaf early that suggests ly e opoeei role morphogenetic key a plays IGELEAFLET1 SINGLE Aquilegia B n and POP, POP hntp.Gvnthese Given phenotype. ,indicat- 3G), Fig. 14; = Finally, ecalcarata. A. .I more-extreme In C). n t rhlg in orthologs its and pce aeshown have species regulates PALM1, 2 As Aquilegia. 0.02, = (SGL1), .ecal- A. Aquile- POP. P M. = POP of localization in program identity factor abaxial 3 the of ortholog an htohrlc otoln prmrhlg ayi hscross. whether this determine to in us allow vary will morphology loci such spur of identification controlling The loci F this segre- other the is of that that development, morphological individuals curvature spurred and continuous spur the length in to spur gating of as epistatic such phase traits be in variation earliest to the appears function in architecture regulatory role the pivotal into insight new of provide will POP, and AqTCP4, in observed parallel a is there adax- expression, the the its of of terms localization In must ial component. function non-cell-autonomous its a division, cell have of domain If broader organ. comparatively developing surface the later adaxial at the inside to and, restricted spur, becomes the petal it of stages, tip in developmental the toward particular, narrowly In more localized mark- 17). broad is (16, division show still spur cell that petal for stages developing observed the previously in have ers we the what in than differentiation rapid to species spurred spurless in division cell differentia- earlier (POP toward repressor shift that heterochronic in a hypothesized tion to study due That was (21). this stages genes more developmental 100% alent to 50 generally petal in were up-regulated of there study that previous found species The compared (23). that domain presence transcriptomics EAR the is repressive common in a have TFs of C2H2 these of of all KD What (32). as development, spur for AqJAG necessary is expansion spur laminar of not Although role petals. the including specific, organs, lateral 31). most ref. in ation (KRPs; inhibitors by cycle proliferation The cell cell several of repressing regulation the directly respec- C2H2, in development, broadly related more petal distantly functions and more leaf another in tively, roles the narrow of relatively compartment pro- distal Although cell (17). the spur termed down developing have shutting we for what responsible in is liferation gene the where spurs, factor of which differentiation cell petals, the in repressing to mitosis similar maintain more to of be role ultimate from to The shift appears (29). developmental differentiation the to of growth timing mitotic the controlling by shape EARS RABBIT organ in morphogenetic plant instance, cell For of regulating activity. genes aspects repressing crucial by control development homologs C2H2 related in spurs of evolution the with coincides Aquilegia. this whether and laceae petal for the evolved when develop- may determine domain leaf to in expression genera required be lineage both will gene study the in Further PALM1/POP ment. of phenotypes function normal ancestral leaf of an mutant reflect perturbation in the results that activity fact the in erably, phenotypes leaf the 3) osre eaierglto yteabaxial the by regulation negative Conserved (33). (ARF3) uuesuist isc h eei neatosamong interactions genetic the dissect to studies Future that notable is It closely several that note to interesting is it generally, More AqTCP4 Aquilegia Medicago Aquilegia rmtscl iiin oebodyi h organ. the in broadly more divisions cell promotes .ecalcarata A. lorslsi h oso pradncaydevelopment nectary and spur of loss the in results also .ecalcarata. A. aecnre aallfnto in function parallel a confirmed have prdvlpet Although development. spur ortholog swl sietfigadtoa agt of targets additional identifying as well as AqJAG, ortholog, PNAS u tl evsoe h usino how of question the open leaves still but POP, loiflecspetal influences also S4) Fig. Appendix , (SI (RBE) ol etebssfrti hf rmprolonged from shift this for basis the be could ) .ecalcarata A. eas oso xrsino transcriptional a of expression of Loss petals. POP AqJAG | Aquilegia etme ,2020 8, September hc sngtvl euae by regulated negatively is which PALM1, iial rmtscl prolifer- cell promotes similarly AqJAG, h lsl eae homolog related closely the Arabidopsis, Medicago xrsini ptal oerestricted more spatially is expression AqHIS4 npooigcl rlfrto and proliferation cell promoting in oprdt pre aaa equiv- at taxa spurred to compared easwudepanteadaxial the explain would petals POP PALM1 POP .ecalcarata A. POP UI EPNEFACTOR RESPONSE AUXIN and expression, rhlg nteRanuncu- the in orthologs nta ohlc function loci both that in satvl rmtn this promoting actively is RBE | Aquilegia and 2 o.117 vol. TCP4 eeain suggesting generation, POP civsb directly by achieves RBE JAGGED otrespurred three to 2,3) Studies 30). (29, POP | lal ly a plays clearly Aquilegia pert play to appear ifrconsid- differ o 36 no. expression | (JAG), POP, petal 22557 RBE

PLANT BIOLOGY or not they function through POP to influence spur length. For of spur development provides us with a critical point of focus example, some of the variation in F2 spur length may be directly for future research. With the identification of POP, we can now related to cell number and POP expression levels, either con- work to elucidate the genetic and developmental pathway that trolled by the cis-regulatory differences at the POP locus itself or it regulates to better understand its role in spur development. through a combination of these and potential variants in trans- In addition, we can address questions regarding how nectar regulators. Even though the VIGS KD studies show that variable spurs evolved in Aquilegia by determining how and when POP levels of POP expression can dramatically alter spur length by acquired petal expression in the lineage leading to Aquilegia. The modulating cell number, the results of the limited analysis com- identification of POP will further allow us to explore questions paring POP expression with spur length in the 14 F2s suggest that concerning the genetic basis of speciation, as we seek to under- POP expression is unlikely to be a major determinant of spur stand the specific genetic basis for loss of POP expression in length variation in this cross. Alternatively, the spur length dif- A. ecalcarata. More broadly, it will also be of interest to deter- ferences among the F2s may be caused by changes in the degree mine whether variation in POP orthologs contributes to other of cell anisotropy, which has been shown to be the primary com- more subtle morphological variation in Aquilegia spur morphol- ponent of interspecific spur length differences in Aquilegia (16). ogy across the genus. Perhaps one of the most notable aspects The identification of such loci may also help explain the eight of this study is that it highlights the power of combining com- spurred F2s that are homozygous for A. ecalcarata alleles at POP. parative transcriptomics, genetic mapping, and analyses of gene These individuals all have at least one A. sibirica allele at both of function to discover the identity of a critical locus controlling the small-effect QTL on chromosomes 5 and 7. Although most of the development of a key innovation, which pure candidate gene the F2s with such an allelic combination lack spurs (30/38), under approaches would not have identified. certain circumstances, A. sibirica alleles at these or other loci affecting spur length variation may be able to generate a spur, Materials and Methods either by causing POP expression to pass above a threshold level Genetic Cross. Horticultural lines of A. sibirica and A. ecalcarata were or by affecting cell elongation such that a small spur appears due acquired from Suncrest Nurseries. An F1 generation was generated with A. to changes in cell length, rather than number. sibirica as the maternal parent and A. ecalcarata as the paternal parent. The Our studies also indicate that POP is necessary for nectary F2 generation was generated by selfing a single F1 plant. Approximately 1,000 seeds were sown in Sunshine #4 (Sun Gro Agriculture) soil in individual development, as both F2 plants homozygous for A. ecalcarata plug trays and stratified at 4 ◦C for 4 wk. After stratification, plug trays were alleles and stronger VIGS KD phenotypes lack nectaries. This moved to the University of California (UC), Santa Barbara greenhouse facil- places it, at least indirectly, up-stream of previously studied ities, and seeds were allowed to germinate under natural light conditions AqSTY homologs, which are essential to nectary, but not spur throughout the spring months. In total, approximately 500 seedlings germi- development (18). Interestingly, phenotypes of both the F2 and nated over a span of 3 mo. As vegetative plants matured, they were moved POP-silenced flowers suggest that there is a relationship between into successively larger pots, eventually ending up in gallon pots. After pro- spur length and proper nectary formation. There are several ducing at least 12 vegetative leaves, plants were vernalized at 4 ◦C for 8 wk possible explanations for this observation. First, it could be in batches of 100, to promote flowering. Each batch of 100 plants put into that a higher level of POP expression is required to activate vernalization was staggered by ∼1 mo. In total, ∼400 plants flowered. the nectary developmental program. Under this scenario, lower Phenotyping. For nearly every F2 plant that flowered, the first and second POP expression would promote only limited spur growth and flowers to mature were removed from the plant at anthesis. Petals were would be insufficient to promote nectary development. Alterna- removed, folded longitudinally, and placed onto the sticky side of clear tively, the interaction could be more indirect, such that there is packing tape that was then affixed to a piece of white paper. Sheets were some threshold of cell number or duration of expression that then scanned at 720 pixels per inch resolution on an Epson Perfection V370 is required before the nectary can be initiated. Previous stud- Photo scanner. As a first pass on phenotyping, 92 individuals were mea- ies have observed that mechanical removal of the nectaries at sured from the attachment point to the distal extent of the petal pocket early developmental stages does not stunt spur development or spur (ImageJ; SI Appendix, Fig. S1C). The distribution of these lengths (34), and KD of the essential nectary development loci AqSTY1 was clearly bimodal such that some F2s formed weakly defined petal pock- and AqSTY2 completely eliminates nectaries without impacting ets with a clear difference in length relative to spurred petals. These petals spur length. These results suggest that the nectary itself is not also differed in shape from spurred petals with short spurs (SI Appendix, Fig. S1 C and D) and always lacked nectaries. Based on this, petals with less required to promote spur growth. Further study of the function than 7-mm pockets were designated “spurless,” while those with greater of POP in spur development will help elucidate the relationship than 8-mm spurs were designated “spurred.” Spur presence or absence was between increasing spur length and nectary development. assessed from the scans and scored as a presence/absence binary trait (SI From an evolutionary perspective, a number of mutational sce- Appendix, Fig. S1D). narios leading to spur loss in A. ecalcarata can be considered. In one scenario, this cross indicates that the spur loss phenotype Genotyping. The F2s were genotyped using a whole-genome skim sequenc- in A. ecalcarata could have been largely achieved by mutations at ing method similar to that described in ref. 38, with variations outlined POP alone, rendering additional loci specific to spur morphology below. For the A. sibirica parent, DNA was extracted from desiccated leaf moot, at which point they could have secondarily accumulated tissue using Qiagen DNeasy reagents and MagAttract beads (Qiagen Inc.). There was no tissue available for the parent or the F . The mutations. On the other hand, spur loss could have happened in A. ecalcarata 1 sequencing library for the A. sibirica parent was constructed using a half- a stepwise fashion, with mutations occurring first in these other volume of the Illumina Nextera kit (Illumina Inc.) and sequenced as 100-bp loci affecting smaller aspects of spur morphology before muta- paired end reads to ∼50× coverage. For the F2s, DNA was extracted from tions eventually occurred at POP. Studies focusing on population young leaves that were flash frozen using the same protocol as for the genetics in A. ecalcarata and its closest relatives may help clarify A. sibirica parent. Sequencing libraries for the F2s were prepared using these scenarios (35). In either case, it is interesting to note that 2 ng of DNA as quantified using the Qubit 2.0 fluorometer (ThermoFisher the major component of spur loss in A. ecalcarata appears to be Scientific). Libraries were prepared using Illumina Nextera indices and caused by changes in POP expression, rather than function, per- reagents and a modified protocol allowing for library prep using 1/20th haps because POP has pleiotropic effects on other traits such as reaction volumes (Illumina Inc.) (36). Given that DNA input amounts were standardized during the library prep, it was initially assumed that library leaf development. coverage of the F2s would be relatively consistent, and equal volumes of libraries for each of 96 individuals were pooled and sequenced as 100-bp Conclusion paired end reads in a single lane. In processing the sequence data, it was While many questions regarding the evolution of nectar spurs apparent that there was high variation in coverage per individual. Three in Aquilegia still remain, the identification of this key regulator additional lanes of sequencing (100 bp paired end) were used to sequence

22558 | www.pnas.org/cgi/doi/10.1073/pnas.2006912117 Ballerini et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 faioai elcmn sipeetdi h IRSwbportal web ica from CIPRES sequences the of SRR7965809, published in species previously different implemented nine used as model We Jones–Taylor–Thornton replacement the list, (http://www.phylo.org/). acid with sequence amino 44) the ref. of trimming max- the (RAxML, accelerated After random likelihood to to outgroup. using imum related an related estimated were as those closely relationships used as phylogenetic clades was well several which as JAG, in Aqcoe3G231100 sequences containing was list one include sequence in relationships to implemented the Initial (43) and trimmed regions. https://www.geneious.com method ), alignable Neighbor-Joining (v9.1.16, to the Geneious using trimmed estimated and were https://www.geneious.com), eye, (v9.1.16, by Geneious adjusted in (42) the using algorithm aligned ClustalW were proteins Predicted (https://phytozome.jgi.doe.gov). vinifera Vitis of proteomes the for Tree. cutoff Gene LOD for Ten 5% scores the algorithm. determine (LOD) (EM) to odds QTL. used expectation-maximization the significant were command of the permutations R/qtl logarithm using thousand the calculate and phenotype marker to (presence/absence), spur each used trait The The binary was 41). (41). a phe- scanone ref. as v1.35-3 presence/absence v1.35-3; scored spur R/qtl R/qtl was gen.w.phen.csv, the package S1; with R (Dataset combined F the and then using notypes was map map genetic genetic a estimate Mapping. the QTL in appropriately cross map to previous bins a marker these map. of allow genetic to map created genetic were sizes the the and between map genome. between inconsistent physical the as of v3.1 identified regions ‘Goldsmith’ were low-recombination crosses, Mb previous in regions 1 from used Several or estimates windows Mb rate larger 0.5 recombination with either on were depending size Windows nonoverlapping in heterozygosity. across indicating 0.6 parent and each either for from gosity frequencies reads read with of windows, genomic frequency the mine F informa- each at of for reads indicative of sites number tive the (available scripts https://github.com/anjiballerini/POP), custom and at 0.1.19 SAMtools Using 39). (38, 0.1.19 SAMtools the and alrn tal. et Ballerini the to aligned were reads single Raw 50-bp Center. as Genome coerulea Inc.) Davis (Illumina A. UC HiSeq4000 the quanti- the at were on reads, lane Libraries end single and 21. samples, a across ref. in representation equal in sequenced for aiming as pooled were qPCR, construction using quality Qiagen library fied and the quantity to and RNA using prior sample, 21 extracted each assessed ref. was For in (Qiagen). RNA kit Total 3 Micro nitrogen. stage RNeasy liquid to in equivalent frozen stage flash developmental the at collected ecalcarata RNAseq. the across Aqcoe3G231100 phylogeny. in variation sequence compare to cross carata the to addition in SRR413921, ances- determining for sites Informative F 38). the ref. in in try (details Burrows–Wheeler (37) the aligner using (https://phytozome.jgi.doe.gov) genome ence F mock F a reconstitute to F the Berkeley). (UC Laboratory Sequencing Genomics Coates usable J. coverage Vincent 0.1x the than greater (n having F genotyping those for 287 with 1x, total, of In pooling depth individuals. to coverage 96 prior qPCR of using groups quantified into were libraries these however erage, F additional 1 n h F the and , nodrt dniyifraiestst eemn netyi h F the in ancestry determine to sites informative identify to order In SRR413499, - 2 a eunerasfo igeln 9 niiul)wr merged were individuals) (96 lane single a from reads sequence raw ln SR915 rmtesm rwra h aetue nthe in used parent the as grower same the from (SRR892115) plant ea isefo 0F 20 from tissue Petal .sibirica A. .formosa A. 2 ooyoe,ad1 eeoyoe ttesu osQLwas QTL loss spur the at heterozygotes 10 and homozygotes, hs edcut eete rcse nR(0 odeter- to (40) R in processed then were counts read These . .pubescens A. 2 2 h qo3210 rdce rti a ure against queried was protein predicted Aqcoe3G231100 The Glsih 31rfrnetasrpoe(https://phytozome. transcriptome reference v3.1 ‘Goldsmith’ .chrysantha A. irre,icuigtoefo h rtln hthdlwcov- low had that lane first the from those including libraries, eeainwr hs hr h okF mock the where those were generation 2 h F The 2 GVV)uigteBATagrtmi htzm 12 Phytozome in algorithm BLAST the using (GSVIVG) eeaindt the to aligned were s eoye o 7 niiul ocnutQLmapping QTL conduct to individuals 276 for genotypes .longissima A. .sibirica A. .sibirica A. 8) l eunigwscnutdo ie30 at HiSeq3000 a on conducted was sequencing All 286). = rbdpi thaliana Arabidopsis 2 aeti ooyos hs eeietfidusing identified were These homozygous. is parent and eoye fdfeetmre iswr sdto used were bins marker different of genotypes .sibirica A. R7494 and SRR7943924, - Aquilegia .pubescens A. 1 SRR408559, - ed o the for Reads . aet(R1581)ada individual an and (SRR11508011) parent or SRR7965810, - .ecalcarata A. 2 lns 5 plants, or .aurea (A. .ecalcarata A. .coerulea A. 3) nteergos utmbin custom regions, these In (38). .formosa A. (AT), . or >0.9 .vulgaris A. .sibirica A. 2 .sibirica A. n rqece ewe 0.4 between frequencies and eesqecdamn for aiming sequenced were s .oxysepala A. .truncatula M. SRR405095, - . niaighomozy- indicating <0.1 netywr counted were ancestry Glsih 31refer- v3.1 ‘Goldsmith’ SRR408554, - R444;rf 38), ref. SRR404349; - ooyoe,5 homozygotes, aet h mock the parent, 1 sheterozygous is var. .barnebyi A. Mdr,and (Medtr), .coerulea A. .thaliana A. oxysepala Aquilegia .japon- A. .ecal- A. 2 A. s, - - i n xdoengti A 37 omleye %gailaei acid, acetic glacial 5% formaldehyde, 4 (3.7% at FAA ethanol) in 50% overnight fixed and sis also WT (n as a classified petals. across WT petals effect true some KD to that in relative out variation KD ruled some is experienced be there cannot and what it be know flower, not would single do length we As spur production. petals final pigment Aqcoe3G231100, the of lack on of the primarily based than KD development rather in targeted shape early antho- identified test were producing phenotype to KD begin order with petals in before pigments, KD largely cyanin in development, gene petal protocol the in same early of the using expression qRT-PCR using in comparing flower as same by the samples from petals petal WT and of those of subset (89.1% a phenotype in spur a an 21.25% exhibited showing plants, 17.22% spurs and treated petals, Of in rounds. notype multiple in treated an exhibited were 4 at plants vernalized 331 then and approxi- of leaves had true they seven to until five grown mately were plants Syngenta) con- TRV2 (Goldsmith, the a ‘Origami’ similarity into from cloned sequence have then containing 3 was not fragment struct does The the homologs. region C2H2 This other with of respectively. sites, sequence enzyme restriction 223-bp a TCTTGATGATCC-3 exon, from CGGAATTCCATCCTGCCCAACCCTCAAT-3 single directly a amplified of Aqcoe3G231100 As consists 50). (49, protocols published previously following ducted Assessment. Phenotypic and VIGS the using cal- University. taken Harvard 1% were of Arboretum in Images Arnold the stained visualization. at before were microscope AxioImager min Slides Zeiss 5 48. steps for ref. hybridization white by situ coflour described In protocol Fisher). the (Thermo primers followed vector using pCR4-TOPO amplified into PCR cloned was region), nonconserved 5 a is (which frame 5 the of Hybridization. Situ In the using correction. expression efficiency calculated relative was The (27). Aqcoe3G231100 expression of Aqcoe3G231100 normalize was to used ISOMERASE2) PHYROPHOSPHATE PYROPHOSPHATE:DIMETHYLALLYL SYBR of 5 Universal fragment primers iTaq A and 3 using Fisher). (Bio-Rad) quantified (Thermo Supermix and primers Green amplified dT was oligo Verso Mini and Aqcoe3G231100 the Plus using Kit RNeasy synthesized Synthesis the was cDNA (cDNA) using DNA done Complementary was (Qiagen). removal Kit –80 point, DNA at attachment genomic kept the and and at isolation frozen petal flash each was developmental from tissue for final isolated and used for was was length tissue measure spur measured, final Spur a were to staging. spurs length get plant, collected, spur to to of flower ratio each used plant the For and was from plant. each anthesis length for at spur length spur flower in developed variability fully some a was there three As of period. each from collected test to qRT-PCR. Developmental used was test rank Genomics signed Integrative Wilcoxon of the one-tailed overrepresentation for a using and counted (47), were so Viewer individual, the Aqcoe3G231100 ES between of sites one alleles variant for was spanning anthesis test reads at this als, scans for petal size sample have spur and the not on expression did Aqcoe3G231100 were nectary between We measurements correlation the length. these a and for to ImageJ, test using point to spur used attachment the of SS R the edge proximal in and from the package ES measured DTK the was the for individuals using length Tukey–Kramer implemented modified Spur sizes) Dunnett (https://CRAN.R-project.org/package=DTK). sample the R, unequal using in done (for implemented was test as testing ANOVA hoc using post expres- assessed and normalized were mean in Aqcoe3G231100 Differences as of 46). sion (45, transcript command) edgeR calcNormFactors package the per R using (via the estimated method using counts M-values were of transcript read mean per trimmed counts the generate Normalized 38. to ref. in described processed and jgi.doe.gov/) 0 0 -GTATTCGGAGCGAGGTTCACT-3 )adK (n KD and 4) = elcutadlnt esrmnswr aeo ul eeoe WT developed fully on made were measurements length and count Cell n 5 and eas qo3210 sol expressed only is Aqcoe3G231100 Because qRT-PCR. Developmental 0 0 -CGGGGTGGTCTTGATGATCC-3 nrnltdrgo n h einn fteoe reading open the of beginning the and region untranslated AqANS 0 PNAS 0 eas TadK easwr avse tanthe- at harvested were petals KD and WT petals. 10) = hc nld eune obidi cR n XbaI and EcoR1 in build to sequences include which , AqANS ◦ .Tetsu a eyrtdt 5 tao n then and ethanol 95% to dehydrated was tissue The C. hntp nspl,1.4 xiie an exhibited 19.34% sepals, in phenotype 5-pfamn fAceG310 nldn part including Aqcoe3G231100, of fragment 250-bp A | orflwr tvrosdvlpetlsae were stages developmental various at flowers Four .sibirica A. .coerulea A. hntp) Do qo3210 a verified was Aqcoe3G231100 of KD phenotype). etme ,2020 8, September .coerulea A. n 0 IStreigAceG310wscon- was Aqcoe3G231100 targeting VIGS n 5 and 4 o h 0htrzgu individu- heterozygous 10 the For 14. = eie reads. derived qiei ANS Aquilegia 0 0 Oiai N sn rmr 5 primers using DNA ‘Origami’ xrsinof Expression . 0 -AACCCTACCAGGCAAAAC-3 n 5 and 0 -GACCAATAACCTGATGGCATCCT- ◦ Oiai lnsdrn 1-h a during plants ‘Origami’ ro oRAioain RNA isolation. RNA to prior C | ∆ ∆ .sibirica A. 0 -CGGCTCTAGACGGGGTGG- o.117 vol. tmto ihprimer with method Ct ee(49). gene AqIPP2 ◦ | o k total A wk. 4 for C and o 36 no. .ecalcarata A. AqANS 0 (ISOPENTYL .coerulea A. n was end | 0 22559 and , phe- 0 -

PLANT BIOLOGY stained overnight in 1% acid fuchsin. Petals were mounted on microscope ACKNOWLEDGMENTS. We thank J. Wechter, D. Birdseye, and J. Pretorius slides using Cytoseal 60 (Electron Microscopy Sciences no. 18006) with the for contributing to planting, extracting DNA, and conducting qPCR. Mem- proximal side of the spur facing up, and left to dry overnight underneath a bers of the S.A.H. and Finkelstein laboratories and M. Shahandeh provided coverslip secured by binder clips. Each spur was imaged continuously from constructive feedback throughout the project. D. Taber and C. Hannah- Bick were invaluable in maintaining the F population and VIGS plants. × 2 the attachment point to nectary at 20 magnification under brightfield We thank G. Popovich for being an inspirational leader. The NSF (Grant illumination using a Zeiss AxioCam 512 mounted on a Zeiss LSM700 con- OIA-0963547) provided funds to construct the greenhouses that facilitated focal microscope. Cells were counted and their lengths measured using the plant growth. E.S.B. was supported by the NIH under the Ruth L. Kirschstein straight line selection tool in FIJI (51, 52). National Research Service Award (F32GM103154). Research was funded by the UC Santa Barbara Harvey Karp Discovery award to E.S.B., NSF Grant 1456317 to S.A.H., and NSF Grant 1456217 to E.M.K. The sequencing was Data Availability. Raw sequence data used for genetic mapping and the F2 RNAseq is available in the Sequence Read Archive (SRA) at the National carried out by the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center, supported by NIH Shared Instrumentation Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/sra) Grant 1S10OD010786-01, and the Vincent J. Coates Genomics Sequencing under the following BioProject: PRJNA623619. Scripts for processing map- Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumen- ping data can be found at https://github.com/anjiballerini/POP and the tation Grant. These funding bodies had no role in the design of the genotype-phenotype file used for mapping is in the SI Appendix (Dataset study, collection, analysis, and interpretation of data, or in writing the S1). Otherwise, study data are included in the article and SI Appendix. manuscript.

1. G. Simpson, The Major Features of Evolution (Columbia University Press, New York, 27. E. S. Ballerini, E. M. Kramer, Environmental and molecular analysis of the floral NY, 1953). transition in the lower eudicot Aquilegia. EvoDevo 2, 4 (2011). 2. J. Cracraft, “The origin of evolutionary novel pattern and process at different hier- 28. H. Wang et al., Control of compound leaf development by FLORICAULA/LEAFY archical levels” in Evolutionary Innovations, M. H. Niteki, Ed. (University of Chicago ortholog SINGLE LEAFLET1 in Medicago truncatula. Plant Physiol. 146, 1759–1772 Press, Chicago, IL, 1990), pp. 21–46. (2008). 3. S. B. Heard, D. L. Hauser, Key evolutionary innovations and their ecological 29. J. Li et al., RABBIT EARS regulates the transcription of TCP4 during petal development mechanisms. Hist. Biol. 10, 151–173 (1995). in Arabidopsis. J. Exp. Bot. 67, 6473–6480 (2016). 4. S. A. Hodges, Floral nectar spurs and diversification. Int. J. Plant Sci. 158, S81–S88 30. A. Nag, S. King, T. Jack, miR319a targeting of TCP4 is critical for petal growth and (1997). development in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 106, 22534–22539 (2009). 5. S. A. Hodges, M. L. Arnold, Spurring plant diversification: Are floral nectar spurs a key 31. K. Schiessl, J. M. Muino,˜ R. Sablowski, Arabidopsis JAGGED links floral organ pattern- innovation?Proc. Biol. Soc. B Biol. Sci. 262, 343–348 (1995). ing to tissue growth by repressing Kip-related cell cycle inhibitors. Proc. Natl. Acad. 6. K. M. Kay et al., “Floral characters and species diversification” in Ecology and Evo- Sci. U.S.A. 111, 2830–2835 (2014). lution of Flowers, L. Harder, S. C. H. Barrett, Eds. (Oxford University Press, Oxford, 32. Y. Min, E. M. Kramer, The Aquilegia JAGGED homolog promotes proliferation of United Kingdom, 2006), pp. 311–325. adaxial cell types in both leaves and stems. New Phytol. 216, 536–548 (2017). 7. D. Schluter, The Ecology of Adaptive Radiation (Oxford University Press, Oxford, 33. J. Peng, A. Berbel, F. Madueno,˜ R. Chen, AUXIN RESPONSE FACTOR3 regulates United Kingdom, 2000). compound leaf patterning by directly repressing PALMATE-LIKE PENTAFOLIATA1 8. S. Fior et al., Spatiotemporal reconstruction of the Aquilegia rapid radiation through expression in Medicago truncatula. Front. Plant Sci. 8, 927–932 (2017). next-generation sequencing of rapidly evolving cpDNA regions. New Phytol. 198, 34. J. R. Puzey, “Plant microRNA evolution and mechanisms of shape change in plants,” 579–592 (2013). PhD thesis, Harvard University, Cambridge, MA (2012). 9. V. Grant, Isolation and hybridization between Aquilegia formosa and A. pubescens. 35. L. Huang et al., Genetic diversity and evolutionary history of four closely related Aqui- Aliso A J. Syst. Evol. Bot. 2, 341–360 (1952). legia species revealed by 10 nuclear gene fragments. J. Systemat. Evol. 56, 129–138 10. V. Grant, Origin of floral isolation between ornithophilous and sphingophilous plant (2018). species. Proc. Natl. Acad. Sci. U.S.A. 90, 7729–7733 (1993). 36. M. Baym et al., Inexpensive multiplexed library preparation for megabase-sized 11. M. Fulton, S. A. Hodges, Floral isolation between Aquilegia formosa and Aquilegia genomes. PloS One 10, e0128036 (2015). pubescens. Proc. Biol. Soc. B Biol. Sci. 266, 2247–2252 (1999). 37. R. Durbin, H. Li, Fast and accurate short read alignment with Burrows-Wheeler 12. J. B. Whittall, S. A. Hodges, Pollinator shifts drive increasingly long nectar spurs in transform. Bioinformatics 25, 1754–1760 (2009). columbine flowers. Nature 447, 706–709 (2007). 38. D. L. Filiault et al., The Aquilegia genome provides insight into adaptive radiation 13. D. B. Nicholson, A. J. Ross, P. J. Mayhew, Fossil evidence for key innovations in the and reveals an extraordinarily polymorphic chromosome with a unique history. eLife evolution of insect diversity. Proc. Biol. Soc. B Biol. Sci. 281, 20141823 (2014). 7, e36426 (2018). 14. F. P. Peixoto, P. H. P. Braga, P. Mendes, A synthesis of ecological and evolutionary 39. H. Li et al., The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, determinants of bat diversity across spatial scales. BMC Ecol. 18, 18 (2018). 2078–2079 (2009). 15. F. Galis, E. G. Drucker, Pharyngeal biting mechanics in centrarchid and cichlid fishes: 40. R Core Team, R: A Language and Environment for Statistical Computing, v.3.6.3 (R Insights into a key evolutionary innovation. J. Evol. Biol. 9, 641–670 (1996). Foundation for Statistical Computing, Vienna, Austria, 2017). 16. J. R. Puzey, S. J. Gerbode, S. A. Hodges, E. M. Kramer, L. Mahadevan, Evolution 41. K. W. Broman, H. Wu, S.´ Sen, G. A. Churchill, R/qtl: QTL mapping in experimental of spur-length diversity in Aquilegia petals is achieved solely through cell-shape crosses. Bioinformatics 19, 889–890 (2003). anisotropy. Proc. Biol. Sci. 279, 1640–1645 (2012). 42. J. D. Thompson, D. G. Higgins, T. J. Gibson, CLUSTAL W: Improving the sensitivity 17. L. Yant, S. Collani, J. Puzey, C. Levy, E. M. Kramer, Molecular basis for three- of progressive multiple sequence alignment through sequence weighting, position- dimensional elaboration of the Aquilegia petal spur. Proc. Biol. Sci. 282, 20142778 specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (2015). (1994). 18. Y. Min, J. I. Bunn, E. M. Kramer, Homologs of the STYLISH gene family control nectary 43. N. Saitou, M. Nei, The neighbor-joining method: A new method for reconstructing development in Aquilegia. New Phytol. 221, 1090–1100 (2019). phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987). 19. L. L. Tang, Q. Yu, J. F. Sun, S. Q. Huang, Floral traits and isolation of three sympatric 44. A. Stamatakis, P. Hoover, J. Rougemont, A rapid bootstrap algorithm for the RAxML Aquilegia species in the Qinling Mountains, China. Plant Systemat. Evol. 267, 121–128 web servers. Sys. Bio. 57, 758–771 (2008). (2007). 45. M. D. Robinson, D. J. McCarthy, G. K. Smyth, edgeR: A bioconductor package for 20. S. C. Tucker, S. A. Hodges, Floral ontogeny of Aquilegia, Semiaquilegia, and Enemion differential expression analysis of digital gene expression data. Bioinformatics 26, (). Int. J. Plant Sci. 166, 557–574 (2005). 139–140 (2010). 21. E. S. Ballerini, E. M. Kramer, S. A. Hodges, Comparative transcriptomics of early petal 46. M. D. Robinson, A. Oshlack, A scaling normalization method for differential development across four diverse species of Aquilegia reveal few genes consistently expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010). associated with nectar spur development. BMC Genom. 20, 668 (2019). 47. J. T. Robinson, H. Thorvaldsdottir,´ A. M. Wenger, A. Zehir, J. P. Mesirov, Variant review 22. W. Prazmo, Genetic studies on the genus Aquilegia L. I. Crosses between Aquilegia with the Integrative Genomics Viewer. Canc. Res. 77, e31–e34 (2017). vulgaris L. and Aquilegia ecalcarata Maxim. Acta Soc. Bot. Pol. 29, 423–442 (1960). 48. E. M. Kramer, “Methods for studying the evolution of plant reproductive structures: 23. C. C. Englbrecht, H. Schoof, S. Bohm,¨ Conservation, diversification and expansion of Comparative gene expression techniques” in Molecular Evolution: Producing the Bio- C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genom. 5, 39 chemical Data, E. A. Zimmer, E. H. Roalson, Eds. (Elsevier Academic, San Diego, CA, (2004). 2005), pp. 617–635. 24. A. V. Klepikova, A. S. Kasianov, E. S. Gerasimov, M. D. Logacheva, A. A. Penin, A high 49. B. Gould, E. M. Kramer, Virus-induced gene silencing as a tool for functional analyses resolution map of the Arabidopsis thaliana developmental transcriptome based on in the emerging model plant Aquilegia (columbine, Ranunculaceae). Plant Methods RNA-seq profiling. Plant J. 88, 1058–1070 (2016). 3, 6 (2007). 25. J. Chen et al., Control of dissected leaf morphology by a Cys(2)His(2) zinc finger tran- 50. B. Sharma, E. M. Kramer, Virus-induced gene silencing in the rapid cycling columbine scription factor in the model legume Medicago truncatula. Proc. Natl. Acad. Sci. U.S.A. Aquilegia coerulea ‘Origami,’Methods Mol. Biol. 975, 71–81 (2013). 107, 10754–10759 (2010). 51. J. Schindelin et al., Fiji: An open-source platform for biological-image analysis. Nat. 26. L. Ge, J. Chen, R. Chen, Palmate-like pentafoliata1 encodes a novel Cys(2)His(2) zinc Methods 9, 676–682 (2012). finger transcription factor essential for compound leaf morphogenesis in Medicago 52. C. A. Schneider, W. S. Rasband, K. W. Eliceiri, NIH Image to ImageJ: 25 years of image truncatula. Plant Signal. Behav. 5, 1134–1137 (2010). analysis. Nat. Methods 9, 671–675 (2012).

22560 | www.pnas.org/cgi/doi/10.1073/pnas.2006912117 Ballerini et al. Downloaded by guest on September 26, 2021