American Journal of Botany 98(3): 397–403. 2011.

R EDUCE, REUSE, AND RECYCLE: D EVELOPMENTAL EVOLUTION OF TRAIT DIVERSIFICATION 1

Jill C. Preston2,4 , Lena C. Hileman 2 , and Pilar Cubas 3

2 Department of Ecology and Evolutionary Biology, University of Kansas, 1200 Sunnyside Avenue, Lawrence, Kansas 66045 USA; and 3 Departamento de Gen é tica Molecular de Plantas, Centro Nacional de Biotecnolog í a/CSIC, Campus Universidad Aut ó noma de Madrid 28049 Madrid, Spain

A major focus of evolutionary developmental (evo-devo) studies is to determine the genetic basis of variation in organismal form and function, both of which are fundamental to biological diversifi cation. Pioneering work on metazoan and fl owering plant systems has revealed conserved sets of genes that underlie the bauplan of organisms derived from a common ancestor. However, the extent to which variation in the developmental genetic toolkit mirrors variation at the phenotypic level is an active area of re- search. Here we explore evidence from the angiosperm evo-devo literature supporting the frugal use of genes and genetic pathways in the evolution of developmental patterning. In particular, these examples highlight the importance of genetic pleiotropy in dif- ferent developmental modules, thus reducing the number of genes required in growth and development, and the reuse of particular genes in the parallel evolution of ecologically important traits.

Key words: CRABS CLAW ; CYCLOIDEA ; evo-devo; FRUITFULL ; independent recruitment; KNOX1; parallelism; trait evolution.

Organisms show remarkable variation in phenotypic form in metazoan animals and nonfl owering plants (e.g., Rensing and function, both of which are fundamental to biological di- et al., 2008; Sakakibara et al., 2008; reviewed in Ca ñ estro versifi cation. A major focus of evolutionary developmental bi- et al., 2007 ). ology or “ evo-devo ” is to determine how and to what extent this phenotypic variation is refl ected at the level of underlying de- The developmental genetic toolkit— Ever since pioneering velopmental genetic pathways. Over recent years, tremendous work in the 1990s on angiosperm MADS-box genes, it has been advances in molecular phylogenetics have greatly facilitated clear that distantly related organisms share a common set of evo-devo studies by allowing more precise reconstruction of conserved genes — often referred to as the developmental genetic character evolution, thus fostering identifi cation of similar traits toolkit — which have been repeatedly modifi ed over evolution- that are either derived from a common ancestor (homologs) or ary time to affect trait diversifi cation (e.g., McGinnis et al., have evolved multiple times independently (analogs). For ex- 1984 ; Scott and Weiner, 1984 ; Utset et al., 1987 ; Duboule and ample, fl oral bilateral symmetry, an ecologically important trait Doll é , 1989 ; Coen and Meyerowitz, 1991 ; Purugganan et al., related to plant pollination syndromes, evolved once and was 1995 ; reviewed in Carroll et al., 2005 ; Degnan et al., 2009 ). lost multiple times within the plantain family (Plantaginaceae) Indeed, despite evidence for extensive gene/genome duplica- ( Reeves and Olmstead, 1998 ), whereas dehiscing fruits and tions in different lineages that can expand the genetic toolkit branched trichomes evolved several times independently within (e.g., Tang et al., 2008, 2010 ), recent comparative genomic the mustard family (Brassicaceae) ( Beilstein et al., 2006 ). These studies suggest that the generation of completely novel genes is different patterns of evolutionary lability likely refl ect differ- rare ( AGI, 2000 ; IRGSP, 2005 ; Paterson et al., 2009 ). Thus, the ences in selection and genetic constraint (reviewed in Langlade evolution of form predominantly occurs through the modifi ca- et al., 2005; Brakefi eld, 2006). In this review, we explore the tion or co-option of existing genetic pathways to different or basis of angiosperm (fl owering plant) biodiversity in terms of additional features, rather than to the de novo synthesis of genes the evolution of broadly conserved developmental genes, and and genetic pathways. attempt to discern whether phenotypic diversity is mirrored at Modifi cation of genetic pathways can occur through muta- the genetic level. Although we will focus on a few important tions within the protein-coding or cis -regulatory sequences angiosperm evo-devo studies, similar research is being done (e.g., promoters) of genes, resulting in biochemical or develop- mental function diversifi cation. For example, during Arabidop- sis [ Arabidopsis thaliana (L.) Heynh., Brassicaceae] and tomato 1 Manuscript received 28 July 2010; revision accepted 22 November ( Solanum lycopersicon L., Solanaceae) development, the par- 2010. alogous genes (i.e., derived from a duplication event), TERMI- The authors thank two anonymous reviewers for comments on a previous NAL FLOWER1/SELF PRUNING (TFL1/SP ) and FLOWERING version of the manuscript. In situ hybridizations in Schizanthus sp. were LOCUS T/SINGLE FLOWER TRUSS ( FT/SFT), are expressed carried out at the John Innes Centre in the laboratory of Enrico Coen. Their concurrently in the same tissues but have antagonistic func- work is supported by grants from the Ministerio de Ciencia e Innovaci ó n tions. Whereas TFL1 / SP stimulates growth and development of (GEN2006-7788-E, BIO2008-00581 and CDS2007-00057 to P.C.), Fundació n Ramó n Areces, and the National Science Foundation (IOS- apical meristems and leaf primordia, FT/SFT retards growth in 0616025 to L.C.H). these tissues, demonstrating that functional differences between 4 Author for correspondence (e-mail: [email protected] ) these paralogs are due to differences in their amino acid sequences (Shannon and Meeks-Wagner, 1991; Ruiz-Garc í a et al., 1997 ; doi:10.3732/ajb.1000279 Samach et al., 2000; Wigge et al., 2005; Shalit et al., 2009). By

American Journal of Botany 98(3): 397–403, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America 397 398 American Journal of Botany [Vol. 98 contrast, expression of APETALA3 -like ( AP3-like) and PISTIL- LATA -like ( PI -like) MADS-box genes in the second whorl of Arabidopsis fl owers compared to the fi rst and second whorls of tulip fl owers (Tulipa gesneriana L., Liliaceae), likely underlies interspecifi c differences in fi rst whorl morphology — sepals in Arabidopsis compared to petaloid tepals in tulip ( Jack et al., 1992 ; Kanno et al., 2003 ). Since these genes are likely to func- tion similarly to specify petal identity in the second whorl of both species, morphological differences in the fi rst whorl are due to changes in the regulation of these orthologous genes (i.e., derived from speciation events), rather than to differences in their protein-coding regions. The above examples highlight how fl ower developmental ge- netic pathways diversify through changes in gene regulation and protein function and the potential importance of gene dupli- cation for developmental evolution. These mechanisms appear to underlie much of organismal diversifi cation, but interestingly there does not seem to be an ever-expanding developmental ge- netic toolkit specifying novel and convergent phenotypes. Instead, growing evidence suggests extensive conservation through the reduction, reutilization , and recycling of developmental genetic programs. The following sections explore evidence for the re- utilization of existing genetic pathways in different develop- mental modules (e.g., leaves and fl owers) both within and between individuals, and the importance of independent coop- tion of the same genes or genetic pathways in the repeated evo- lution of similar traits. Pleiotropy in infl orescence and fl ower development— One way to reduce the number of genetic programs required for phe- notypic diversifi cation is to reuse specifi c gene products in differ- ent protein complexes, thus altering their developmental functions in time and space within an individual. One of the best exam- ples of this type of developmental pleiotropy is provided by the MADS-box transcription factors, including members of the Fig. 1. Expression patterns of Arabidopsis thaliana (Brassicaceae) well-studied APETALA1/FRUITFULL (AP1/FUL ) subfamily APETALA1/FRUITFULL (AP1/FUL ) genes illustrating developmental that are restricted to the angiosperms ( Litt and Irish, 2003 ). pleiotropy and functional diversifi cation following gene duplication. FUL Functional analyses across monocots and eudicots imply an (red) is expressed in the shoot apical meristem, where its protein product interacts with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ancestral function for AP1/FUL genes in meristem identity (SOC1) to induce infl orescence development, and later in carpel valves to specifi cation. However, the number and types of meristems in promote fruit elongation and differentiation. AP1 (green) is expressed which these genes function have been altered following both alongside its close paralog CAULIFLOWER ( CAL ) in fl oral meristems, duplication and speciation. Arabidopsis has three functionally where both genes specify fl oral meristem identity and later in sepals and characterized AP1/FUL genes —AP1, FUL and CAULI- petals to promote fl oral organ identity. FLOWER ( CAL ) — derived from an ancient duplication event at the base of core eudicots and a more recent duplication event within Brassicaceae ( Litt and Irish, 2003 ). In Arabidopsis , com- specifi cation, partially through the downregulation of infl ores- plexity of the developmental genetic toolkit is minimized cence meristem identity genes (e.g., AGAMOUS-LIKE 24 through the recycling of AP1 and FUL to specify different de- [ AGL24] and SHORT VEGETATIVE PHASE [ SVP]) and velopmental trajectories (Fig. 1). During fl oral induction FUL concomitant upregulation of the fl oral organ identity gene SE- is positively regulated in the shoot apical meristem (SAM), PALLATA3 (SEP3 ), the latter of which has been hypothesized where its protein interacts with SUPPRESSOR OF OVEREX- to heterodimerize with AP1 in sepals and petals (Gregis et al., PRESSION OF CONSTANS1 (SOC1) to initiate the transition 2008 , 2009 ; Liu et al., 2009 ; Kaufmann et al., 2010 ; Xu et al., to infl orescence development (Hempel et al., 1997; Ferr á ndiz et 2010 ). Finally, following fl ower maturation, FUL is re-utilized al., 2000 ; Melzer et al., 2008 ). After the production of an infl o- in fruit production during which it controls elongation and dif- rescence meristem, AP1 and CAL are upregulated in emerging ferentiation of the carpel valve ( Gu et al., 1998 ). lateral meristems to specify fl oral identity (Irish and Sussex, In addition to mediating fl owering time, evidence suggests 1990; Bowman et al., 1993; Kempin et al., 1995). This upregu- that FUL contributes to the annual herbaceous habit of Arabi- lation is achieved through the negative regulation of both FUL dopsis. Indeed, double soc1:ful mutants are very late fl owering and another MADS-box gene AGAMOUS ( AG ), which is in- under long-day conditions and have characteristics of perennial volved in fl oral organ production ( Mandel and Yanofsky, 1995 ; plants, including secondary growth, infl orescence meristems Sridhar et al., 2006 ). The next phase of fl ower development is that revert to vegetative meristems, and longer life cycles the production of fl oral organ primordia. At this stage AP1 ( Melzer et al., 2008 ). Because the perennial woody habit is switches function from fl oral meristem to sepal/petal identity likely ancestral to angiosperms, independent modifi cations in March 2011] Preston et al. — Evo-devo of trait diversification 399 the expression or function of FUL-like genes may be associated suggests that CYC and DICH were recruited for specifying with repeated evolution of annual herbaceous taxa in different dorsal identity in snapdragon fl owers through temporal shifts in lineages ( Melzer et al., 2008 ). Although this hypothesis awaits their expression, rather than modifi cations to their biochemical rigorous testing, these data suggest that the re-utilization of function ( Cubas et al., 2001 ). FUL-like genes plays a role in the independent evolution of the In addition to snapdragon, recruitment of CYC -like genes to annual habit and that these genes are recycled during angio- establish dorsal identity in lineages with independently derived sperm ontogeny, both of which reduce the requirement of novel bilaterally symmetrical fl owers has been functionally demon- genes and genetic pathways. strated for candytuft ( Fig. 2D , Iberis amara L., Brassicaceae), and in the legumes pea and lotus (Fig. 2E, Pisum sativum L. and Repeated evolution of fl oral bilateral symmetry— Within Lotus japonicus L., Leguminoseae) ( Feng et al., 2006 ; Busch the angiosperms, bilaterally symmetrical (monosymmetric) and Zachgo, 2007; Wang et al., 2008). Importantly, phylogenetic fl owers have evolved multiple times from radially symmetrical analyses support the independent evolution of fl oral bilateral (polysymmetric) ancestors ( Fig. 2 ), and these shifts in symme- symmetry in the Plantaginaceae, Brassicaceae, and Legumino- try are strongly correlated with increased pollinator specializa- seae. As predicted, mutations in CYC-like genes of these spe- tion and higher speciation rates ( Donoghue et al., 1998 ; Ree cies result in radially symmetrical fl owers due to the loss of and Donoghue, 1999; Endress, 2001; Sargent, 2004; Knapp, dorsal identity. Interestingly, whereas CYC -like genes promote 2010). Recent studies in multiple fl owering plant lineages have increased petal size in snapdragon and legumes, orthologs in greatly advanced our understanding of how independent evolu- candytuft function to reduce petal size ( Luo et al., 1996 ; Busch tionary transitions to bilateral fl ower symmetry is established at and Zachgo, 2007; Wang et al., 2008). In addition to functional the developmental genetic level. Strikingly, these studies studies, a general role for CYC -like genes in establishing inde- strongly support a common genetic basis for bilaterally sym- pendently derived fl oral bilateral symmetry is supported by metrical fl owers, through parallel evolutionary shifts in class II asymmetric patterns of CYC -like gene expression in late stage TCP CYCLOIDEA ( CYC)-like gene expression (reviewed in bilaterally, but not radially, symmetrical fl owers within Mal- Preston and Hileman, 2009; Hileman and Cubas, 2009; Rosin pighiaceae ( Zhang et al., 2010 ). and Kramer, 2009 ). In other words, fl owering plants appear to reuse the same elements of the developmental genetic toolkit to Independently derived patterns of stamen abortion— Intra- establish independent transitions to bilateral fl ower symmetry. and interfl oral variation in stamen number and length is common The importance of CYC -like genes for specifying a dorsiven- within the angiosperms (Fig. 3), and is thought to increase indi- tral fl oral axis was fi rst identifi ed in the model organism snap- vidual fi tness by partitioning function between stamens (e.g., dragon ( Fig. 2C , Antirrhinum majus L., Plantaginaceae). In feeding stamens vs. pollinating stamens), maximizing pollen snapdragon, fl oral bilateral symmetry is established through the placement (e.g., dorsal staminodes vs. ventral stamens), and en- differentiation of the dorsal, lateral, and ventral petals and sta- suring reproduction through a mixed mating strategy (e.g., didy- mens and is partly mediated by the action of two recently dupli- namous stamens or dicliny) ( Endress, 1999 ; Escaravage et al., cated dorsal identity genes, CYC and DICHOTOMA (DICH ) 2001; Kalisz et al., 2006; Friedman and Barrett, 2009). In snap- (Luo et al., 1996, 1999 ). Both genes are expressed in the dorsal dragon, the dorsal stamen arrests early in development to leave a region of the fl ower during early to late stages of development, residual staminode, whereas the two lateral stamens develop to be with double cyc:dich mutant fl owers being fully radially sym- shorter than the two ventral stamens. Similar to petals, the resulting metrical due to the loss of dorsal identity (Luo et al., 1996, bilateral symmetry in the stamen whorl is controlled by the action 1999 ). By contrast, in radially symmetrical fl owers of the dis- of CYC, which inhibits cell division along the dorsilateral axis tantly related model species Arabidopsis , the CYC/DICH or- (Luo et al., 1996, 1999 ; reviewed in Kalisz et al., 2006). tholog TCP1 is expressed dorsally only in very early stages of Recent evo-devo studies are starting to reveal a role for CYC - fl ower development (Cubas et al., 2001). Thus, since class II like gene expression evolution in interspecifi c patterns of sta- TCP genes generally function as regulators of cell proliferation men number variation (reviewed in Hileman and Cubas, 2009). and expansion (Doebley et al., 1997; Crawford et al., 2004; First, in the close relative of snapdragon, desert ghostfl ower reviewed in Preston and Hileman, 2009; Mart í n-Trillo and [ Mohavea confertifl ora (Benth.) A. Heller, Plantaginaceae], Cubas, 2010 ), the early dorsal expression of TCP1 in Arabidopsis which has staminodes in both the dorsal and lateral positions,

Fig. 2. Independent recruitment of CYC -like genes in the evolution of fl ower bilateral symmetry across core eudicots. (A) Example of a radially symmetri- cal rosid fl ower from sulphur cinquefoil (Potentilla recta L., Rosaceae) with multiple lines of symmetry (dashed lines). (B) Illustration of a typical core eudicot fl ower with strong bilateral symmetry along the dorsiventral axis due partly to the action of CYC protein function in the dorsal region (zigzag). (C – E) Func- tional studies have shown that CYC was recruited multiple times in the independent origin of (C) snapdragon ( Antirrhinum majus L.; Plantaginaceae; asterid I), (D) candytuft (Iberis amara L.; Brassicaceae; rosid II), and (E) pea (Pisum sativum L.; Leguminoseae; rosid I) fl ower bilateral symmetry. 400 American Journal of Botany [Vol. 98

Fig. 3. Patterns of stamen abortion and CYCLOIDEA (CYC ) expression in exemplary asterids. (A) Scanning electron micrograph (SEM) showing typi- cal Plantaginaceae fl ower morphology in Collinsia heterophylla Buist ex Graham, with two ventral and two lateral stamens (*), and a single dorsal stamin- ode (X). (B) SEM showing the divergent fl ower morphology of Gratiola offi cinalis L. (Plantaginaceae), with two lateral stamens and two ventral and one dorsal staminode. (C) Schizanthus Ruiz & Pav. fl ower showing strong dorsiventral bilateral symmetry. (D, E) Transverse section through a Schizanthus fl ower showing CYC expression in the (D) dorsal and (E) ventral staminodes. there has been an expansion of CYC -like gene expression into more, constitutive expression of KN1 causes ectopic shoot the lateral region of the stamen whorl ( Hileman et al., 2003 ). formation in simple leaves of tobacco ( Nicotiana tabacum L., Second, in Opithandra B. L. Burtt (Gesnericaceae) and Schi- Solanaceae) and Arabidopsis . These data from maize, to- zanthus Ruiz & Pav. (Solanaceae) ( Fig. 3C – E ), CYC -like gene bacco, and Arabidopsis suggest that expression of KN1 expression has expanded into the ventral region, correlating homologs during the development of leaves may contribute to with derived patterns of stamen abortion in both the dorsal and compound leaf development. This expression evolution may ventral regions (Song et al., 2009). Third, the maize ( Zea mays be due to changes in the cis -regulatory elements of KN1 genes L., Poaceae) CYC -like homolog TEOSINTE BRANCHED1 and/or to variation in their upstream regulators (e.g., CUC1 ( TB1) is strongly expressed in the stamen whorl of female, but and CUC2 ). not male, fl orets correlating with patterns of stamen abortion Comparative studies have revealed a role for differential (Hubbard et al., 2002). Although there are noteworthy excep- regulation of KN1 orthologs in the independent origins of sim- tions to this correlation— including the lack of CYC -like ex- ple and compound leaves across angiosperms, some Legumino- pression in ventral staminodes of Gratiola L. and Veronica L. seae being notable exceptions (Champagne et al., 2007; Chen (Plantaginaceae) (Preston et al., 2009) (Fig. 3B) — these results et al., 2010 ). In the simple leaf primordia of Amborella Baill. suggest an important role for recurrent evolutionary shifts in (Amborellaceae, basal dicot), grasses (Poaceae, monocot), and CYC-like gene expression for the independent patterning of sta- Arabidopsis (rosid), KN1-like gene expression is repressed men development across angiosperms. throughout leaf development, whereas in the complex leaf pri- mordia of Lepidium perfoliatum L. (Brassicaceae, rosid) and Parallel recruitment of KNOXI genes in leaf shape evolu- tomato (asterid) KN1 -like transcripts are abundantly expressed tion— Angiosperm leaves show a great diversity in shape, (Bharathan et al., 2002). The picture emerging from studies fo- largely resulting from differences in the number, arrangement, cusing on the role of KN1 -like genes in diverse angiosperms is and shape of blades or blade units (leafl ets) on the main leaf that multiple shifts in KN1 -like expression likely underlie par- axis. Simple leaves (e.g., Arabidopsis, snapdragon and maize, allel evolution of compound leaves. It is important to note that Fig. 4A, B) are borne in one piece, can be either entire or ser- simple leaves can develop from complex primordia and vice rated along the leaf margin, and are thought to be the ancestral versa and that KN1 -like gene expression strongly correlates with angiosperm leaf type. By contrast, compound (complex or dis- young, but not necessarily adult, leaf morphology ( Bharathan sected) leaves (e.g., tomato and giant starfruit [Averrhoa car- et al., 2002 ). This correlation between KN1 -like gene expres- ambola L., Oxalidaceae], Fig. 4C) are derived from several sion and young leaf morphology highlights the importance of leafl ets that dissect the leaf at the main axis. Across angio- careful morphological work in evo-devo studies. sperms, compound leaves have evolved independently multiple times ( Bharathan et al., 2002 ; reviewed in Blein et al., 2010 ). Regardless of form— simple or compound— leaf initials emerge similarly from groups of differentiated cells that subtend a zone of uncommitted (indeterminate) cells within the SAM. Indeter- minancy in the central zone of the SAM is maintained primarily by a group of related genes within the KNOX1 family of ho- meobox genes and their upstream regulators CUP-SHAPED COTYLEDON ( CUC) and CUC2 (Aida et al., 1999; Takada et al., 2001; Long et al., 1996; Williams, 1998; Hibara et al., 2003 ; reviewed in Langdale, 1994 ). One of the best-characterized KNOX1 genes, KNOTTED 1 (KN1 ), is expressed in both vegetative and fl oral meristematic Fig. 4. Variation in angiosperm leaf complexity can largely be ex- cells of maize, but KN1 transcripts are undetectable in regions plained by evolution of KN1 expression. (A) Simple snapdragon (Antir- of lateral organ formation ( Smith et al., 1992 ; Jackson et al., rhinum majus L., Plantaginaceae) leaf. (B) Simple dissected maple (Acer 1994). Ectopic expression of KN1 in simple maize leaves L., Sapindaceae) leaf. (C) Compound leaf of the giant starfruit ( Averrhoa causes cell proliferation around the lateral leaf veins. Further- carambola L.; Oxalidaceae). March 2011] Preston et al. — Evo-devo of trait diversification 401

eudicots, this function appears to have been recycled multiple times in developmentally distinct tissues. For example, petunia [ Petunia hybrida (Hook.) Vilm., Solanaceae] nectaries are as- sociated with the base of the ovary wall, and are therefore only serially homologous to nectaries of Arabidopsis . Regardless, the petunia CRC ortholog is strongly expressed in growing nectary tissue, and gene silencing causes loss of nectary devel- opment (Lee et al., 2005b). By contrast, CRC -like genes have not been demonstrated to function in nectary development in monocots. Instead, evidence from lily (Lilium longifl orum Thunb., Liliaceae) and rice (Oryza sativa L., Poaceae) suggests neo-functionalization of CRC in monocot leaf midrib develop- ment and carpel identity ( Ishikawa et al., 2009 ; Wang et al., 2009) (Fig. 5). Thus, data suggest that CRC -like genes have been reutilized and neo-functionalized multiple times, indepen- dent of gene duplication, within the angiosperms.

Concluding remarks— The examples outlined above illus- trate the frugal use of genes and genetic pathways in different developmental modules during the ontogeny of individual plants and during the evolutionary diversifi cation of form and function— both of which are critical components of biodiver- sity. In the case of angiosperm AP1/FUL genes, recycling of Fig. 5. Major hypotheses of CRABS CLAW (CRC ) functional evolu- gene function during ontogeny has occurred partly through tion across the angiosperms based on gene expression, interspecifi c genetic gene duplication, allowing expansion of the genetic toolkit complementation tests, and mutant analyses (for references, see section without the de novo synthesis of genes (Rosin and Kramer, Reuse of CRABS CLAW (CRC) during angiosperm diversifi cation ). Dotted 2009 ). Therefore, to reuse developmental genetic programs line at the basal node represents uncertainty due to lack of functional anal- within ontogeny and over evolutionary diversifi cation is not al- yses in Amborella Baill. and other basal dicots. ways to reduce total gene number. By contrast, developmental pleiotropy in AP1/FUL and CRC genes has fostered reduction in the genetic toolkit through the reuse of specifi c genes within Reuse of CRABS CLAW (CRC) during angiosperm diversi- different modules of an individual. Similarly, repeated diversi- fi cation— The independent recruitment of CRC -like YABBY fi cation of CYC , CRC , KN1, and AP1/FUL genes has resulted in family transcription factors in carpel, leaf, and nectary develop- the independent evolution of fl ower bilateral symmetry, necta- ment exemplifi es the frugal use of genes in plant evolution and ries, compound leaves, and possibly the annual habit, across the development (Fig. 5). Indeed, not only is CRC used at different angiosperms, respectively. These examples highlight the ability stages during ontogeny (i.e., within an individual), but it has of evolutionary forces to reduce the number of tools within the also been recruited multiple times independently to perform genetic toolkit while simultaneously increasing biodiversity, similar functions in different lineages of angiosperms (Lee and together resolve the apparent mismatch between diversity et al., 2005b; Orashakova et al., 2009). CRC was fi rst identifi ed at the phenotypic and genetic levels. through mutant screens of Arabidopsis plants that showed defects in carpel development ( Alvarez and Smyth, 1999 ; Bowman and Smyth, 1999). Mutants occasionally develop su- LITERATURE CITED pernumerary carpels that are shorter and wider than normal and that are unfused at the apex. These mutant phenotypes suggest Aida , M. , T. Ishida , and M. Takaka . 1999 . 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