Catching a 'hopeful monster': shepherd's purse (Capsella bursa-pastoris) as a model system to study the evolution of flower development

Catching a ‘hopeful monster’: shepherd’s purse (Capsellabursa-pastoris) as a model system to study the evolution offlower development

Maren Hintz1, Conny Bartholmes1, Pia Nutt1, Janine Ziermann1, Steffen Hameister2,

Barbara Neuffer2 and Gunter Theissen1,*

1 Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Genetik, Philosophenweg 12, D-07743 Jena, Germany2 Universitat Osnabruck, Fachbereich Biologie/Chemie, Spezielle Botanik, Barbarastrasse 11, D-49076Osnabruck, Germany

Received 28 June 2006; Accepted 8 August 2006

Abstract

Capsella is a small genus within the mustard family

(Brassicaceae). Its three species, however, show many

evolutionary trends also observed in other Brassica-

ceae (including Arabidopsis) and far beyond, including

transitions from a diploid, self-incompatible, obligatory

outcrossing species with comparatively large and

attractive flowers but a restricted distribution to a poly-

ploid, self-compatible, predominantly selfing, invasive

species with floral reductions. All these evolutionary

transitions may have contributed to the fact that

Capsella bursa-pastoris (shepherd’s purse) has be-

come one of the most widely distributed flowering

plants on our planet. In addition, Capsella bursa-

pastoris shows a phenomenon that, although rare,

could be of great evolutionary importance, specifically

the occurrence of a homeotic variety found in relatively

stable populations in the wild. Several lines of evi-

dence suggest that homeotic changes played a consid-

erable role in floral evolution, but how floral homeotic

varieties are established in natural populations has

remained a highly controversial topic among evolu-

tionary biologists. Due to its close relationship with the

model plant Arabidopsis thaliana, numerous experi-

mental tools are available for studying the genus

Capsella, and further tools are currently being de-

veloped. Hence, Capsella provides great opportunities

to investigate the evolution of flower development from

molecular developmental genetics to field ecology

and biogeography, and from morphological refine-

ments to major structural transitions.

Key words: ABCmodel, flower development, homeosis, macro-

evolution, MADS-box gene, saltation, stamenoid petals.

Introduction: macroevolution, evo-devo,homeosis, and all that

As encompassed by the ‘Synthetic Theory’ (or ‘ModernSynthesis’) of evolutionary biology, the 20th century hasprovided a thorough understanding of the mechanisms ofmicroevolution (Dobzhansky, 1937; Mayr, 1942; Simpson,1944; Mayr and Provine, 1980). It is relatively well knownhow organisms adapt to their environment and, arguably,even how new species originate. However, whether thisknowledge suffices to explain macroevolution, narrowlydefined here to describe evolutionary processes that bringabout fundamental novelties or changes in body plans(Theissen, 2006), has remained highly controversial.

How fundamental innovations (or novelties) originate inevolution remains one of the most enigmatic questions ofbiology. According to the proponents of the SyntheticTheory, the gradual process of evolution by naturalselection that operates within populations and species alsocreates the unique traits recognizable at higher taxonomiclevels, meaning that macroevolution is just microevolutionextended over relatively long periods of time.

However, it has been repeatedly pointed out that in-novation is different from adaptation, and that the SyntheticTheory, which is largely based on population genetics,falls short of explaining innovations, novelties, and theevolution of body plans (Riedl, 1977; Gilbert et al., 1996;Bateman et al., 1998; Erwin, 2000; Wagner, 2000; Haag

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

Journal of Experimental Botany, Vol. 57, No. 13, pp. 3531–3542, 2006

Major Themes in Flowering Research Special Issue

doi:10.1093/jxb/erl158 Advance Access publication 3 October, 2006

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and True, 2001; Wagner and Muller, 2002; Wagner andLaubichler, 2004; Muller and Newman, 2005; Theissen,2006). These are not the only shortcomings of the SyntheticTheory. It considers evolution as the result of changes inallele frequency due to natural selection that engendersubtle modifications of phenotype. According to the Syn-thetic Theory evolution always occurs gradually, in a count-less number of almost infinitesimally small steps. Givensufficient time, these gradual changes accumulate and resultin the larger differences that typically seperate higher taxa.The fossil record, however, with its often abrupt transitions,provides limited evidence for the gradual evolution of newforms (Gould and Eldredge, 1993). In addition, the branch-ing patterns of higher taxa in both animals and plants, asrevealed by cladistics, do not support the view that themajor features of body plans and their constituent partsarose in a gradual way (Vergara-Silva, 2003).

These shortcomings of the Synthetic Theory in explain-ing evolutionary novelties led to the reintegration ofdevelopmental biology into evolutionary biology, givingrise to the discipline of ‘evolutionary developmental bio-logy’ (‘evo-devo’). The rationale of evo-devo takes intoaccount that multicellular organisms usually develop fromsingle cells (zygotes) in each generation anew. Thus, allevolutionary changes in the morphology of an organismoccur by changes in developmentalprocesses.Sincedevelop-ment is largely under genetic ‘control’, novel morpho-logical forms in evolution frequently result fromchanges in so-called ‘developmental control genes’, manyof which encode transcription factors. Consequently, evo-devo projects typically study the phylogeny of genesencoding certain classes of transcription factors (such ashomeodomain or MADS-domain proteins) and their rolein the evolution of morphological features (for furtherdetails and discussions of the evo-devo rationale see Gould,1977; Gilbert et al., 1996; Theissen et al., 2000; Carroll,2001; Arthur, 2002; Muller and Newman, 2005).

Considerable progress has recently been made in un-derstanding the genetic mechanisms that bring aboutdrastic, yet co-ordinated, changes in the adult phenotypeby modification of developmental processes. Changes inboth the timing (heterochrony) and the position (hetero-topy) of developmental events can occur, although, in thecase of plants, they are often difficult to distinguish(Kellogg, 2000). A considerable number of key innovationsand characteristics of major clades, most of which havebeen used as taxonomic characters for decades, can beexplained as the result of heterotopy or heterochrony,corroborating the importance of developmental shifts formacroevolution (Kellogg, 2000).

An important subset of heterotopic changes are hom-eotic transitions (Baum and Donoghue, 2002), whichdescribe a type of variation in which ‘something has beenchanged into the likeness of something else’ (Lewis,1994). Well-known examples include the Ultrabithorax

mutant of the fruit fly, Drosophila melanogaster, whereinhalteres (rudimentary wings) are replaced by a second pairof ‘true’ wings. Homeotic mutants also occur frequently inplants, affecting both vegetative and reproductive organs(Sattler, 1988; Meyerowitz et al., 1989).

Developmental genetics of floral homeoticmutants

Particularly well-known examples of homeotic mutants inplants are floral homeotic mutants: mutant plants withflowers that have more-or-less normal floral organs inplaces where organs of another type are typically found.Many flowers consist of four different types of organs,which are arranged in four (or more) whorls, with sepals inthe first, outermost whorl, followed by petals, then stamens,and finally carpels in the innermost whorl (an example isgiven in Fig. 1a). In the model plant Arabidopsis thaliana(thale cress), homeotic mutants can be categorized intothree classes, termed A, B, and C. Ideal Class A mutantshave carpels instead of sepals in the first whorl and stamensrather than petals in the second whorl. Class B mutants havesepals rather than petals in the second whorl and car-pels rather than stamens in the third whorl. Class C mutantshave flowers in which reproductive organs (stamens andcarpels) are replaced by perianth organs (petals and sep-als, respectively), and in which the determinacy of floralgrowth is lost, resulting in an increased number of floralorgans (Meyerowitz et al., 1989). Such ‘filled flowers’ arewell known from many wild and ornamental plants.

The defined classes of floral homeotic mutants areusually explained by simple combinatorial genetic modelssuch as the ABC model (Fig. 1a; reviewed by Theissen,2001a, b; Ferrario et al., 2004). This proposes threedifferent floral homeotic functions to explain how thedifferent floral organs adopt their unique identities duringdevelopment. Corresponding to the aforementioned mu-tant classes these functions are termed A, B, and C, with Aspecifying sepals in the first floral whorl, A+B petals in thesecond whorl, B+C stamens in the third whorl, and Ccarpels in the fourth whorl.

In Arabidopsis the Class A genes are represented byAPETALA1 (AP1) and APETALA2 (AP2), the Class Bgenes by APETALA3 (AP3) and PISTILLATA (PI), and the(single) Class C gene by AGAMOUS (AG), all encodingputative transcription factors (reviewed by Theissen,2001a; Krizek and Fletcher, 2005). Thus, the products ofthe ABC genes probably all control, and hence co-ordinate,the transcription of other genes (termed target genes),whose products are directly or indirectly involved in theformation or function of the different floral organs. Exceptfor AP2, all ABC genes are MADS-box genes encodingMIKC-type MADS-domain transcription factors (reviewedby Riechmann and Meyerowitz, 1997; Theissen et al.,2000; Becker and Theissen, 2003; De Bodt et al., 2003).

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Despite the fact that the ABC genes are required for thespecification of floral organ identity, it became clear thatthey alone are not sufficient to generate a typical flower.Moreover, the ABC model did not provide a molecularmechanism for the interaction of floral homeotic genesduring the development of floral organ identity (Theissen,2001b). These shortcomings of the ABC model havemeanwhile been overcome by the identification of ClassD genes involved in ovule development, and Class E genesrequired for development of all four kinds of floral organs.This led, step by step, to the extension of the ABC modelto the ABCDE model (Angenent and Colombo, 1996;Pelaz et al., 2000; Theissen, 2001a; Ditta et al., 2004). Trans-genic studies in Arabidopsis revealed that the ABCDEgenes are not only required, but are even sufficient, to atleast specify petal and stamen identity (Honma and Goto,2001). Moreover, the observation that floral homeoticproteins form multimeric complexes provided an explana-tion for the interaction of floral homeotic genes at themolecular level (Egea-Cortines et al., 1999; Honma andGoto, 2001), as described by the ‘floral quartet model’(Theissen, 2001a; Theissen and Saedler, 2001). However,all considerations that follow use the simpler ABC model.

Flowers on the evo-devo agenda: variations ona theme

Studies of homeotic genes reveal that major developmentalevents such as the specification of organ identity are often

controlled by a fairly small number of key developmentalcontrol genes. The analyses of both conventional mutantsand transgenic organisms, revealed that changes in such‘toolkit genes’ (Carroll, 2005) can cause profound, yet co-ordinated, morphological changes. Specifically, changes inthe expression domains of floral homeotic genes in mutantor transgenic plants cause homeotic transformations offloral organs. For example, ectopic expression of Class Cgenes in the perianth leads to a transformation of sepalsinto carpeloid organs and of petals into stamenoid organs(Bradley et al., 1993). Similarly, the expression of Class Bgenes in the first and fourth floral whorls of A. thalianaleads to a transformation of sepals into petaloid organs andof carpels into stamenoid organs (Krizek and Meyerowitz,1996).

Some homeotic phenotypes in both animals and plantsresemble differences in character states between majorlineages of living organisms (e.g. consider the dragonfly-like appearance of the Ultrabithorax mutant of the Dipteranfruit fly). However, whether the genes underlying such‘phylo-mimicking mutants’ define loci that play an impor-tant role in character changes during macroevolution hasproven controversial (Haag and True, 2001). Nevertheless,much of the structural diversity of flowers can be explainedby modifications of the ABC system of floral organ identityspecification, especially by changes in the spatiotemporalexpression domains of the ABC genes. For example, in theflowers of tulips (Tulipa gesneriana) and other lily-likeplants, first-whorl organs are typically petaloid (Fig. 1c),

Fig. 1. Floral architectures in Capsella bursa-pastoris (a, b) and Tulipa gesneriana (c); upper parts show flowers, lower parts explanations of floralorgan identities by the ABC model. For simplicity, stamens of wild-type plants are considered as constituting only a single whorl. (a) Flower of C. bursa-pastoris wild-type, with four green, leaf-like sepals in the first, outermost whorl, four white and showy petals in the second whorl, six stamens in the thirdwhorl, and two fused carpels in the fourth, innermost whorl; the ABC model is the ‘classical’ one, with Class A genes specifying sepals, A+B petals, B+Cstamens, and C carpels. (b) Flower of the Spe variety, which has the same structure as the wild-type flower shown in (a), except that petals in the secondwhorl are transformed into stamens; the corresponding ABC model differs from the ‘classical’ one by expression of the Class C gene rather than Class Agenes in the organs of the second whorl (Nutt et al., 2006). (c) Flower of a tulip, with three white and showy petaloid organs termed ‘tepals’ in both thefirst and second whorls, six stamens in the third whorl, and three fused carpels in the fourth, innermost whorl; the corresponding ‘modified’ ABC model(Kanno et al., 2003) differs from the ‘classical’ one by expressing Class B genes not only in the organs of the second and third whorls, but also in thoseof the first whorl. Abbreviations: C, carpels; Pe, petals; Se, sepals; St, stamens; Te, tepals.

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quite similar to the petaloid sepals of eudicotyledonousflowering plants such as Arabidopsis or tobacco that arisewhen Class B genes are ectopically expressed in the firstfloral whorl (Davies et al., 1996; Krizek and Meyerowitz,1996). This suggests that a homeotic transition fromsepaloid to petaloid organ identity, or vice versa, occurredin the first floral whorl during the evolution of floweringplants. Petaloid organ identity requires the function of ClassB floral homeotic genes. Indeed, when Class B genes wereinvestigated in tulip, they were found to be expressed notonly in the petaloid tepals of the second floral whorl, butalso in the organs of similar identity in the first whorl(Kanno et al., 2003). This observation supports a ‘modifiedABC model’ for tulips and related plants, in which Class Bgene expression extends from the second and third into thefirst floral whorl (Fig. 1c; Kanno et al., 2003). Heterotopicexpression of Class B genes in the petaloid organs of thefirst floral whorl was also found in most, but not all, othermonocots with petaloid tepals investigated thus far (Parket al., 2003; Nakamura et al., 2005). Similarly, the petal-oidy of first-whorl organs in many flowers of the basaleudicot family Ranunculaceae appears to be due to a shiftof Class B gene expression towards the first floral whorl(Kramer et al., 2003). In a somewhat complementary way,reduction of Class B gene expression in petaloid organsof the floral perianth may have contributed to thesepaloid appearance of the perianth organs of some wind-pollinated flowers, such as those of sorrel (Rumex acetosa)(Ainsworth et al., 1995).

These inferences support the view that evolutionary‘tinkering’ with the boundaries of Class B floral homeoticgene expression prompted floral homeotic changes thatcontributed to the diversity of floral architecture. Similararguments can be made for changes in the (mutuallyexclusive) expression domains of Class A and Class Cgenes (Fig. 1b; see below for details). Theissen et al. (2000)and Kramer et al. (2003) developed models of howevolutionary variation of the ABC system of floral organidentity specification could explain floral diversificationduring evolution.

These considerations add to an increasing amount ofevidence provided by evolutionary analyses of morpholog-ical characters, indicating that homeosis played a signifi-cant role in plant evolution (Sattler, 1988; Iltis 2000;Kellogg, 2000; Baum and Donoghue, 2002; Rudall andBateman, 2002, 2003; Ronse De Craene, 2003; Rutishauserand Moline, 2005; Theissen, 2006). In principle, homeoticchanges could occur in a gradual mode of evolution,involving thousands or millions of generations of organ-isms (Sattler, 1988). However, given that complete tran-sitions in organ identity can occur in an individual carryinga mutation in a single homeotic gene, a saltational mode ofcharacter change appears more plausible from a develop-mental genetic viewpoint. Moreover, a saltational characterchange would help to overcome a notorious problem of

many gradualistic scenarios of evolutionary change, posedby the presumed low fitness of intermediate forms. Salta-tional changes, however, would be contradictory to theassumption of the Synthetic Theory that all kinds ofevolution are gradual and based on changes in allelefrequency at many loci. They would have a quite remark-able implication: homeotic mutants should represent criticalsteps during a macroevolutionary transition. Since hom-eotic mutants can be considered as profound variants oforganismic design, they might reasonably be called ‘hope-ful monsters’ (Bateman and DiMichele, 2002; Theissen,2006), reintroducing the provocative (and widely, if oftenthoughtlessly, rejected) term of Richard Goldschmidt(1940) (reviewed by Dietrich, 2003).

However, there cannot be much doubt that homeoticmutants often originate as rare individuals in populationsof wild-type organisms and hence, like all other mutants,are subject to the rules of population genetics. To qualify ashopeful monsters and to establish new evolutionarylineages, homeotic mutants must survive many years underconditions of natural selection until the mutant homeoticlocus achieves fixation and modifying mutations haveoptimized the new ‘body design’. How does this work?Unfortunately, evo-devo tells us little about the per-formance of homeotic mutants in natural environments(Theissen, 2000). In addition to theoretical discussionsof this issue, the question should be answered experimen-tally, since saltational modes of evolution involvinghomeotic mutants may not be generally accepted unlessa sufficient fitness of such organisms has been documentedin natural habitats. Therefore, the population dynamicsof homeotic mutants has to be studied through extensivefield work (Theissen, 2000, 2006; Bateman and DiMichele,2002; Vergara-Silva, 2003; Dietrich, 2003). However,probably only a small fraction of homeotic mutants willqualify as hopeful monsters. Most floral homeotic mutants,including the ‘classical’ mutants that gave rise to theABC model, have a strongly reduced fitness comparedwith the corresponding wild type; this almost certainlyhampers their long-term survival in nature (Nutt et al.,2006). So, how can a hopeful monster be distinguishedfrom a hopeless mutant?

Floral homeotic mutants in the wild, more thanhopeless cases?

One good way to identify floral homeotic mutants withevolutionary potential would be to seek populations of suchplants in the wild. If these populations prove more-or-lessstable for many years, the respective variants will havedemonstrated at least a minimum level of competitivenessunder natural growth conditions. However, the literatureon such populations is disappointingly thin. One caseis bicalyx, a recessive variety of Clarkia concinna

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(Onagraceae) in which the usually pink and showy trilobedpetals are transformed into sepaloid organs due to a muta-tion at a single genetic locus (Ford and Gottlieb, 1992). Thebicalyx variety occurs only in a small population at PointReyes (north of San Francisco, California, USA), whereit is accompanied by a majority (70–80%) of wild-typeplants. Ford and Gottlieb (1992) described the populationas being stable, probably due to the predominantly selfingmode of propagation. However, the population was ob-served over a period of just four years, so that the long-termstability of the bicalyx variant remained unknown (Fordand Gottlieb, 1992). Moreover, the bicalyx gene has notbeen molecularly characterized so far.

Another case of a naturally occurring floral homeoticmutant is a peloric variety of common toadflax (Linariavulgaris); it has actinomorphic rather than zygomorphicflowers and exists on a small island near Stockholm/Sweden (Cubas et al., 1999). Like the homeotic Clarkia,this Linaria is mutated at a single, recessive locus. It turnedout that the toadflax variety is affected in a CYCLOIDEA-like gene, but by methylation of DNA that leads to trans-criptionally silencing (epimutation) rather than a changein the DNA sequence (Cubas et al., 1999).

Both the Clarkia and Linaria varieties have a verylimited range of distribution, and their fitness in the wildis probably significantly lower than that of the wild-type.The Linaria epimutant, for example, may only propagatevegetatively (Theissen, 2000). Even though it has beenknown on the island for more than 200 years, it remainsunclear whether its long persistence is due to true longevityof an ancestral mutant or to frequent reappearance afterrepeated rapid extirpation (Theissen, 2000). Taken to-gether, the evolutionary potential of the wild floralhomeotic varieties reported thus far is doubtful (Theissen,2006). Better candidates for floral homeotic mutants thatmight qualify as hopeful monsters are thus welcome, butfinding them is not trivial. For a few promising cases inorchids see Bateman and Rudall (2006).

There could be two reasons for why descriptions of wildpopulations of floral homeotic mutants are rare. Suchpopulations could indeed be needles in haystacks (whichwould not, however, necessarily imply that they are oflow evolutionary importance; Theissen, 2006). They alsocould have been neglected by mainstream research, notleast because they were not considered crucial (or evenwere considered detrimental) to predominant scientifictheories such as the Modern Synthesis. As will be seenbelow, these ostensibly competing scenarios are notmutually exclusive.

Shepherd’s purse: from weed to model system

In biology, scientific progress often depends primarily onthe choice of a suitable model system. Imagine geneticswithout pea, maize, the fruit fly and E. coli! So, assuming

interest in the macroevolution of flowers, which speciesprovides us with a suitable hopeful monster?

Due to several well-known practical reasons (e.g.diploidy, short-life cycle, limited space requirements, andefficient transformability) a small member of the mustardfamily (Brassicaceae), the thale cress Arabidopsis thaliana,became the most favoured model plant. Its small genome ofjust about 130 million base pairs has (almost) been fullysequenced (Arabidopsis Genome Initiative, 2000), and arelatively large number of genetic and genomic tools, suchas T-DNA-, transposon insertion- and EMS-mutagenizedpopulations facilitating the investigation of gene function,have been developed. With A. thaliana it is possible toinvestigate traits from an ecological and evolutionary pointof view by studying them under different defined con-ditions in relation to their genetic background. UsingA. thaliana to understand the evolution of floral noveltiestherefore seems an obvious idea. However, even thoughA. thaliana has a global distribution and gave rise toa considerable number of so-called ‘ecotypes’, its floralarchitecture is remarkably standardized. Consequently, thenatural occurrence of floral homeotic varieties has not, tothe best of our knowledge, been reported so far. Arabi-dopsis thaliana appears to be a comparatively uncompet-itive plant and it is actually relatively rare in the wild. Howmany readers would find one in the wild within 10 minwalk from their home? In fact, one much more frequentlystumbles over a close relative, the shepherd’s purse,Capsella bursa-pastoris. Indeed, C. bursa-pastoris isone of the five most widespread flowering plants onour planet, the four others being Polygonum aviculare,Stellaria media, Poa annua, and Chenopodium album(Coquillat, 1951; Hurka et al., 2003). But C. bursa-pastorisis not only much more frequent than A. thaliana, italso appears to tinker more extensively with its flowerstructure. And thus it is C. bursa-pastoris where a floralhomeotic variety is found forming quite stable populationsin the wild.

Capsella in brief: it is a small genus of Brassicaceae thatmay only contain three species; these, however, showremarkable differences in ploidy level, breeding systems,and habitat range (Hurka and Neuffer, 1997; Zunk et al.,1999; Hurka et al., 2005). Two of the species, C. grandifloraand C. rubella, are diploid, whereas the third, C. bursa-pastoris, is tetraploid. Capsella grandiflora is obligatelyoutbreeding due to a sporophytic self-incompatibility (SI)system; it grows only in a limited habitat in Albania, westernGreece, and northern Italy. Compared with the other twospecies, C. grandiflora—nomen est omen—has relativelylarge, fragrant flowers with showy petals to attract polli-nators. By contrast, C. rubella is a completely selfing plantwith relatively small flowers that originally grew around theMediterranean Sea, but it colonized nearly all Mediterraneanclimatic regions worldwide during historical times (Hurkaet al., 1989). The predominantly selfing C. bursa-pastoris


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generally prefers disturbed, ‘man-made’ habitats, like themargins of agricultural fields (Fig. 2a), and grows all overthe world except in the hot and humid tropics (Hurka andNeuffer, 1997).

How did C. bursa-pastoris originate, and what allowed itto spread over most of the planet? Even though therelationships between the three species in the genusCapsella have been investigated for some time, they remainsurprisingly unclear. Based on the results of isozymeelectrophoresis and isoelectric focusing experiments ofRubisco, it has been suggested that C. bursa-pastorisis an allopolyploid of C. rubella and C. grandiflora(Mummenhoff and Hurka, 1990). With additional datafrom restriction enzyme site variation in the chloroplastgenome, it was then assumed that C. bursa-pastoris is anautopolyploid of C. grandiflora (Hurka and Neuffer, 1997).More recently, a phylogenetic analysis based on dataobtained with chloroplast and nuclear DNA markerssuggested a sister relationship between C. bursa-pastorison the one side and C. rubella plus C. grandiflora on theother. From their data, Slotte et al. (2006) concluded that C.bursa-pastoris is not an autopolyploid of either C. rubellaor C. grandiflora, nor did either of these species playthe maternal role in an allopolyploidization event (Slotteet al., 2006). Instead, these authors suggested two alter-native scenarios. One is that C. bursa-pastoris originatedby allopolyploidization, with C. rubella as the paternalancestor and with an unknown or extinct diploid ancestoras the mother plant. In the alternative scenario, C. bursa-pastoris originated wholly from extinct diploid ancestors,followed by repeated hybridization and backcrossing with

C. rubella, leading to introgression of C. rubella allelesinto the C. bursa-pastoris genome.

Whatever the origin of C. bursa-pastoris, comparisonwith outgroup species strongly suggests that C. grandiflorarepresents the most ancestral character states concerningreproductive biology and ploidy level within Capsella andthat C. bursa-pastoris is the most derived species (Hurkaet al., 2005). That implies that an obligate outbreedingdiploid Capsella has evolved into the self-compatibletetraploid C. bursa-pastoris. But what makes this charactercombination a ‘formula for success’ at least in terms ofexpanding its geographical range? A crucial event for thesuccessful distribution of C. bursa-pastoris was probablythe breakdown of the sporophytic self-incompatibility (SI)system that is active in C. grandiflora (Paetsch et al., 2006).According to the ‘reproductive assurance hypothesis’, thecapability to self-fertilize enables the plant to reproduceeven under poor outcrossing conditions, for example, inthe absence of either conspecific neighbours or suitablepollinators, and thereby allows colonization of unoccupiedniches (Shimizu et al., 2004; Shimizu and Purugganan,2005; and references cited therein). In addition, floweringand reproduction become less season-dependent, becausethe plant no longer depends on the timing of other plantsor pollinator activity. Several ecotypes of C. bursa-pastorisare facultative winter annuals that are able to survive asover-wintering leaf rosettes (Neuffer and Hurka, 1986,1999; Neuffer and Bartelheim, 1989; Neuffer, 1990;Neuffer and Hurka; Linde et al., 2001). This rapid re-productive cycle enables C. bursa-pastoris plants thatgerminate in early spring to produce up to three generations

Fig. 2. Spe plants of Capsella bursa-pastoris growing outside the laboratory. (a) A mixture of Spe and wild-type plants in a ‘natural’ (actually,man-made) habitat, a vineyard close to Gau-Odernheim (Germany). (b) Investigations on floral visitation in the Botanical Garden of Jena; field plots(535 plants) of the wild-type (in the middle, 2) compared with the Spe variety from Gau-Odernheim (in the front, 1) and Warburg (in the back, 3).

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of progenitors in just one year (Neuffer and Hurka, 1988;Neuffer and Bartelheim, 1989; Hurka and Neuffer, 1991;Aksoy et al., 1998).

Most annual selfers show rapid floral maturation facil-itated by reproducing at a small overall size and bydeveloping smaller flowers and seeds compared with theiroutcrossing annual relatives (Snell and Aarssen, 2005).This generalization applies to both Capsella (see above)and members of the genus Arabidopsis. The combination ofself-compatibility (SC) and flower-size reduction seems tobe a general trend; the latter promotes selfing by reducingthe distance between anthers and stigma in smaller flowers.Since self-fertilizing plants do not need to attract pollinatorsan energy cost that is required for developing large flowerscould be saved (Snell and Aarssen, 2005).

However, many flowering plants make ambitious effortsto prevent selfing in bisexual flowers. Such species evolvedself-incompatibility systems (like SI in C. grandiflora),time-shifted maturation of pollen and ovules (dichogamy)or structural barriers (herkogamy) to promote outbreeding.Outbreeding leads to allelic variation in the population,which is an important prerequisite for reacting to changesin the environment and to avoid inbreeding depression.That being self-compatible was particularly advantage-ous for C. bursa-pastoris may be due to the fact that out-crossing persists; rates in the field are up to 20% (Hurka andNeuffer, 1997).

But selfing cannot be the only reason why C. bursa-pastoris is so widely distributed, because it also applies toC. rubella. Perhaps the tetraploidy of C. bursa-pastorisprovided another important advantage. Polyploid plantsoften show broader ecological tolerances and the ability tocope with a wider range of conditions compared with theirdiploid progenitors. Since, in polyploids, deleterious muta-tions will be masked by the extra genome, polyploidy ishypothesized to result in reduced inbreeding depressioncompared with diploid parents (reviewed by Soltis andSoltis, 2000). Therefore, becoming self-fertile is particu-larly advantageous for polyploid plants such as C. bursa-pastoris, as it results in higher progeny without the negativeeffects of inbreeding depression.

Thus, within the species-poor genus Capsella, thetransition from a diploid, self-incompatible, obligatoryoutcrossing species with comparatively large and attractiveflowers but a restricted distribution (C. grandiflora) toa tetraploid, self-compatible, predominantly selfing specieswith relatively small flowers but exceptional colonizationability (C. bursa-pastoris) can be inferred. Similar evolu-tionary scenarios are also inferred in other plant groups,including both close and distant relatives of Capsella(Barrett, 2002; Mable et al., 2005). Capsella might thusserve well in comparative studies aiming to understandparallel and convergent evolution of floral features.

Nevertheless, one might remain reluctant to chooseC. bursa-pastoris as a new model plant for studying

developmental and evolutionary processes because of thelimited number of molecular genetic tools available a priori.However, there is more to this issue than meets the eye.As a result of its close relationship with the model plantA. thaliana, numerous experimental tools are available tostudy the genus Capsella and more are currently beingdeveloped. The order, orientation, and sequence of genes isvery similar in the genomes of Arabidopsis and Capsellaand their exons show over 90% sequence identity (Acarkanet al., 2000; Koch and Kieffer, 2005). This allows theidentification of genes within Capsella with the help of thethoroughly mapped Arabidopsis genome. Experimentationalong these lines will be further boosted by the ongoingsequencing of the Arabidopsis lyrata and Capsella rubellagenomes (Joint Genome Institute, United States Depart-ment of Energy).

Like A. thaliana, C. bursa-pastoris is self-compatible,and easy to cultivate and propagate. Although longer thanthat of A. thaliana, the life cycle of C. bursa-pastoris is stillsufficiently short to allow the production of three to fourgenerations per year (depending on the variety). Andalthough C. bursa-pastoris is a tetraploid, it has alreadyevolved disomic inheritance (Hurka et al., 1989; Hurka andDuring, 1994), which makes crossing experiments easierto interpret. Moreover, C. bursa-pastoris is amenable topowerful techniques such as genetic transformation by the‘floral dip’ method and gene knockdown via RNA inter-ference (RNAi) (Bartholmes, 2006; C Bartholmes andG Theissen, unpublished data), which greatly facilitatesthe analysis of gene function.

The authors are thus confident that, due to its interestingbiology, close relationship with Arabidopsis, and an in-creasing number of molecular tools available, the success-ful globetrotter shepherd’s purse will start a second careeras a satellite model system of Arabidopsis.

A hopeful monster in a shepherd’s purse

In addition to representing a derived state in a typicalevolutionary process affecting floral biology, C. bursa-pastoris shows a rare phenomenon which, nevertheless,could be of great evolutionary importance, specifically theoccurrence of a homeotic variety in stable populations inthe wild (Nutt et al., 2006; Theissen, 2006). This wasbrought to our attention when we searched for naturalhomeotic mutants that show competitiveness under naturalgrowth conditions.

The remarkable variety that became our study object hasbeen described for almost two centuries at different placesin Europe (Opiz, 1821; Trattinnick, 1821; Schlechtendahl,1823; Wiegmann, 1823; Murbeck, 1918) and is still foundin substantial stable populations. The four petals of itsflowers are transformed into stamens, whereas all otherfloral organs are unaffected, leading to apetalous flowerswith ten stamens (Fig. 1b). Therefore, the mutant has been


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termed ‘dekandrisch’ (‘decandric’) and the phenomenon‘Staminale Pseudapetalie’ (‘Stamenoid pseudo-apetaly’);the mutant variety has even been considered being a newspecies, ‘C. apetala’ (Opiz, 1821; Murbeck, 1918). Re-cently, we termed the locus/loci affected in the mutantvarieties ‘Stamenoid petals’ (Spe) (Nutt et al., 2006).

This variety became obscure, receiving only occasionalattention in the literature (for details on the history of Spesee Nutt et al., 2006). Fortunately, a new population of Speplants was discovered in 1991 by Reichert (1998) on fieldpaths in vineyards in Gau-Odernheim (Rheinhessen,Germany). Reichert’s report, which covered several yearsof observations, showed that the Spe plants are stableconcerning number and local distribution, even thoughthey are mixed with wild-type plants (Fig. 2a). AlreadyReichert (1998) considered this wild growing homeoticvariety as a very remarkable case due to its ability toestablish whole populations rather than merely singleplants. The long-lasting existence of the Spe variety inthe wild at least indicates that it has a fitness similar to thatof the wild-type. Sadly, Reichert’s observations attractedlittle interest, perhaps because floral homeotic mutants inthe wild are rare, and rarity is commonly confused withunimportance (Theissen, 2006). Most developmental ge-neticists might lack interest, because they fail to recognizethe difference between Spe plants and their more familiar‘laboratory artefacts’. Most evolutionary biologists willprobably question the scientific relevance of Spe. For us,however, the decandric C. bursa-pastoris variety appearsto be an optimal model for studying non-gradualisticchanges in floral architecture (Nutt et al., 2006). Hence,it was recently made a research focus in the authors’laboratories. A brief progress report is presented below.

Studying a hopeful monster in the laboratory andin the field

Additional decandric C. bursa-pastoris populations havebeen identified throughout Europe (P Nutt, B Neuffer, andG Theissen, unpublished data), revealing that Spe plantsare more frequent than was initially assumed. Lack of atten-tion evidently contributed to the rarity of descriptionsin the literature. Current studies are concentrating on theabove-mentioned population in Gau-Odernheim and onone population found on the Desenberg, near Warburg(Westphalia, Germany). We have begun carefully toanalyse the phenotype and the mode of inheritance of Spe.The decandric C. bursa-pastoris plants from bothGau-Odernheim and Warburg show almost complete trans-formation of petals into stamens (Nutt et al., 2006). Thatapplies also to the functional level, because second-whorlstamens of Spe plants produce viable pollen that cansuccessfully pollinate gynoecia of C. bursa-pastoris andthus generate viable seeds (P Nutt, unpublished data).

By crossing true-breeding mutant and wild-type plants,an intermediate phenotype was obtained in the F1 genera-tion; the second-whorl organs were smaller than petals,slightly curled and some showed yellow-coloured edges,thus revealing an organ identity intermediate betweenpetals and stamens (P Nutt, unpublished data). Segregationof the mutant phenotype in the F2 generation varied depen-ding on the parents: stamenoid versus petaloid second-whorl organs were observed in a ratio of about 3:1, orstamenoid versus intermediate versus wild-type second-whorl organs in a ratio of about 1:2:1 (P Nutt, unpub-lished data). A similar segregation pattern was reportedby Dahlgren (1919) for the decandric C. bursa-pastorisvariety that he analysed. This suggests that the mutantphenotype of a Spe plant is caused by a co-dominant mutantallele conferring stamen identity at a single locus inone of the two disomically inherited genomes of C.bursa-pastoris. Tests for allelism of the mutant loci in thetwo investigated populations are under way. Molecularmarkers, including isozymes and AFLPs, are being used todetermine the origin from wild-type and the geographicdistribution of the different Spe varieties in detail.

We are interested in both the molecular identity of theSpe locus and the mechanisms by which it generates themutant phenotype. According to the ABC model, forstamens to develop, expression of both Class B and ClassC floral homeotic genes is required (Fig. 1). Therefore, it ishypothesized that in the Spe mutants ectopic expression ofa Class C gene, or a closely related gene, is extended fromthe third and fourth whorl towards the second whorl,thereby suppressing Class A genes in this whorl (Fig. 1b).The most obvious candidate genes for this ectopic expres-sion are an orthologue of the Arabidopsis Class C geneAGAMOUS (AG) or one of its closely related paraloguesSHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK(STK), which share high sequence similarity with AG(Becker and Theissen, 2003). For all of these genes, exceptSTK, ectopic expression in A. thaliana leads to theformation of stamenoid organs in the second whorl (Favaroet al., 2003). To test this hypothesis, investigations weremade into the mRNA expression patterns of the ortho-logues of Class A and Class C floral homeotic genes, andclosely related paralogues, in wild-type and Spe flowersof C. bursa-pastoris. Moreover, experiments to knock-down AG-like genes in Spe plants employing RNAi tech-nology are underway to determine whether stamenoidy ofsecond-whorl organs depends on the activity of Class Cor related genes.

Assuming that the Spe phenotype is brought about bythe ectopic expression of a Class C gene (Fig. 1b) does notimply that the Spe locus is an AG-like gene. Ectopicexpression of a Class C gene could of course be caused bya change in a cis-regulatory element of an AG-like geneitself, but also by a trans-acting factor functioning upstreamof the AG-like gene (and not necessarily directly binding

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to the AG-like gene itself). Such an upstream-actingfactor would probably be a protein, but could also be aregulatory RNA.

To clone the Spe locus, a combined candidate geneand map-based cloning approach is currently being ap-plied, which is facilitated by the close relationship be-tween Capsella and Arabidopsis. Candidate genes for Spe,currently being tested for co-segregation with the mutantphenotype in F2 populations, include all of the AG-typegenes mentioned above (orthologues of AG, SHP1, SHP2,STK), but also orthologues of some trans-acting regula-tors of AG that show stamenoid petals upon mutation(for details see Nutt et al., 2006). These putatively trans-acting candidates are considered a lower priority, how-ever, because their mutant alleles are usually recessiveand mutant plants often display considerable pleiotropiceffects beyond stamenoidy of petals, sometimes even out-side the flower.

There are many floral homeotic mutants available thatcan be studied from a developmental genetics point of view.What makes Spe so special, however, is its continuous pre-sence in the wild, and hence the fact that both proximateand ultimate causes of evolution can be studied using thesame model system. In addition to trying to understandthe molecular mechanism behind Spe we are, therefore, alsodeeply interested in investigating the performance of Speplants outside the greenhouse. Hence, the performance ofSpe and wild-type plants is being compared, both in the‘wild’ in Gau-Odernheim and Warburg (where the plantsgrow, albeit in typical man-made or disturbed habitats)and in ordered field plots in Botanical Gardens (Fig. 2).

As a rough proxy of reproductive fitness, the number offruits and seeds produced by wild-type and Spe plants isbeing determined. So far, significant differences have notbeen observed in our garden experiments (J Ziermann,unpublished data). Although C. bursa-pastoris is predom-inantly self-pollinating, even very low rates of outcrossingcould help to avoid inbreeding depression and hence mightbe of considerable evolutionary importance. Therefore,outcrossing rates between Spe and wild-type plants arebeing determined under controlled conditions in theBotanical Garden of Osnabruck using molecular markers.In addition, floral visitation by insects is being investigatedas a possible mechanism of outcrossing facilitated bypollinators. Wild-type inflorescences display white petalsand hence might be quite appealing to some visuallyoriented visitors like bees (Fig. 3a); by contrast, inflor-escences of Spe plants lack petals, but offer more pollenthan wild-type plants (Fig. 3b). Compared with the wild-type, Spe inflorescences might be slightly less attractive tobees but slightly more appealing to beetles. In the case ofthe similarly predominantly selfing A. thaliana, flowervisits by potential pollinators such as solitary bees,dipterans, and thrips have been observed in the field(Mitchell-Olds, 2001; Hoffmann et al., 2003). In field plotsin the Botanical Garden of Jena (Fig. 2b) very similarobservations were made for C. bursa-pastoris (J Ziermann,unpublished data). A slightly higher preference of bees forwild-type inflorescences and of beetles for Spe inflorescen-ces may have been observed in one year but needs moredata for confirmation (J Ziermann, unpublished data). Sofar a dramatic change has not been observed in the species

Fig. 3. Inflorescences of Capsella bursa-pastoris. (a) Wild-type inflorescence that displays petals and hence might be quite appealing to visuallyoriented visitors such as bees. (b) Inflorescence of a Spe plant lacking petals but offering more pollen; compared to the wild-type, it might be somewhatless attractive to bees but somewhat more appealing to beetles.


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spectrum or the number of floral visitors when comparingSpe with wild-type plants.

Overall, evidence that the Spe variety is significantlyhandicapped in its reproductive fitness has not been found,a conclusion supported by its persistence in wild habitatsfor many years. To corroborate that view, it is intended tomonitor the development of Spe populations in their naturalhabitats for as long as possible. Taken together, these en-deavours will help us to learn more about the evolutionarypotential of the Spe variety and, hopefully, of floralhomeotic mutants in general.

Concluding remarks

Due to the large time-scales and diverse mechanismsinvolved, studying macroevolution is hardly a trivial task.Reconciling macroevolution with population geneticsappears to be a considerable challenge for the future(Nutt et al., 2006; Theissen, 2006). The Spe variety of C.bursa-pastoris shows a rare, yet potentially evolutionarilyimportant, phenomenon, specifically the occurrence of ahomeotic variety apparently forming stable populations inthe wild. A detailed study of the Spe variety may notonly tell us more about the developmental genetic me-chanisms that generate novel structures in the first place,but also indicate whether, and if so how, drastic morpho-logical variants are established in natural populations.

However, in discussions with colleagues we are occa-sionally told that even clarifying the molecular mechanismthat brings about the Spe phenotype, and demonstrating thatSpe plants currently have a fitness in the wild that is at leastas high as that of wild-type plants of C. bursa-pastoris,would not suffice to make a convincing case for Spe beinga hopeful monster. Rather, one would have to demonstratethat Spe is still flourishing in, say, one million years time.However, we do not agree, because it is a characteris-tic feature of homeosis or other saltational changes inevolution that they readily produce morphological novelt-ies within a short period of time. While these noveltiesmay strongly influence the probability of short-term survivalof the affected population, its long-term survival willmuch more depend on all kinds of contingencies ratherthan on the newly established morphological feature;hence long-term survival is irrelevant for the plausibilityof saltational evolution.

Acknowledgements

We thank Hannelore Simon for providing Fig. 1c. We are gratefulto Richard M Bateman and an anonymous reviewer for helpfulcomments on a previous version of the manuscript. One of us(GT) is grateful to Paula J Rudall and Nick H Battey for theirinvitation to present our project in Session P5 (Flowering) at theSEB 2006 Meeting in Canterbury (UK) and hence to contribute tothis volume. Work on Spe in the authors’ laboratories is supported

by grants TH 417/4-1 and NE 314/7-1 from the DeutscheForschungsgemeinschaft (DFG).

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