339.Why Are Orchid Flowers So Diverse Reduction (1)

7/27/2019 339.Why Are Orchid Flowers So Diverse Reduction (1)

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REVIEW

Why are orchid flowers so diverse? Reduction of evolutionary constraintsby paralogues of class B floral homeotic genes

Mariana Mondragon-Palomino* and Gu nter Theien

Department of Genetics, Friedrich-Schiller-University, D-07743 Jena, Germany

Received: 9 June 2008 Returned for revision: 29 July 2008 Accepted: 17 November 2008

Background The nearly 30 000 species of orchids produce flowers of unprecedented diversity. However,whether specific genetic mechanisms contributed to this diversity is a neglected topic and remains speculative.We recently published a theory, the orchid code, maintaining that the identity of the different perianth organs isspecified by the combinatorial interaction of four DEF-like MADS-box genes with other floral homeotic genes. Scope Here the developmental and evolutionary implications of our theory are explored. Specifically, it isshown that all frequent floral terata, including all peloric types, can be explained by monogenic gain- or-loss-of-function mutants, changing either expression of a DEF-like or CYC-like gene. Supposed dominance or reces-siveness of mutant alleles is correlated with the frequency of terata in both cultivation and nature. Our findingssuggest that changes in DEF- and CYC-like genes not only underlie terata but also the natural diversity of orchidspecies. We argue, however, that true changes in organ identity are rare events in the evolution of orchid flowers,even though we review some likely cases.Conclusions The four DEFparalogues shaped floral diversity in orchids in a dramatic way by modularizing thefloral perianth based on a complex series of sub- and neo-functionalization events. These genes may have elimi-nated constraints, so that different kinds of perianth organs could then evolve individually and thus often in dra-matically different ways in response to selection by pollinators or by genetic drift. We therefore argue that floraldiversity in orchids may be the result of an unprecedented developmental genetic predisposition that originatedearly in orchid evolution.

Key words: Orchidaceae, orchid evolution, evo-devo; perianth, class B genes, DEFICIENS, subfunctionalization,neofunctionalization, gene duplication, peloria, modularization.

O R C H I D F LO W ER S : EN D LES S F O R M S M O S TB EA U TI F U L B Y V A R I A TI O N O N A S C H EM E

The typical flower of a petaloid monocot consists of fivefundamentally 3-fold whorls or derivatives thereof. However,in contrast with other petaloid monocots, such as lilies ortulips, orchids have flowers of breathtaking morphologicaldiversity (Fig. 1A). This diversity is mainly brought aboutby variation on a relatively simple scheme (Fig. 1BD).Like flowers of lilies and tulips, those of orchids comprisetwo whorls of petaloid organs termed tepals surrounding thereproductive organs. In orchids, reproductive organs arespecial in that they constitute a gynostemium or column, acompound structure formed by adnation of male and female

organs (Dressler, 1993; Rudall and Bateman, 2002).Species-specific variation in the size and shape of thecolumn, together with the presence of appendages, the confor-mation, position and number of the anthers, as well as charac-teristics of the pollinia and other structures, make the column aremarkably complex organ. Evolution of the orchid columnis worthy of detailed study, and there is relatively detailedinformation on the molecular basis of its development (Yuand Goh, 2000; Johansen and Frederiksen, 2002; Tsai et al.,2004, 2005; Skipper et al., 2006; Song et al., 2006; Xu

et al., 2006; Kim et al., 2007); however, here the focus willbe on development and evolution of the perianth organs.

Although the perianth of most petaloid monocot familiesconsists of (almost) identical organs, three kinds of organidentity have been distinguished in the perianth of orchids:three outer tepals (T1T3; often also termed sepals) in thefirst floral whorl, and two lateral inner tepals (t1 and t2;petals) and a median inner tepal (t3) termed the labellumor lip in the second whorl (Rudall and Bateman, 2002;Mondragon-Palomino and Theien, 2008; Fig. 1BD).Although most descriptions of the orchid perianth considerthe outer tepals (T1T3) unlobed organs without adornments,they can be similar to the lateral inner tepals (Fig. 1A) or formnectar spurs (e.g. Plectophora, Oncidiinae). The lip is, withfew exceptions, always different from the other perianthorgans and elaborately adorned with calli, spurs, glands anda distinctive colour pattern (Fig. 1A). Although the lip isprobably homologous to the adaxial tepal of other monocotsand hence it should be the uppermost one, it is often thelowest one due to resupination (1808 developmental rotationin floral orientation; Arditti, 2002). The abaxial orientationof the resupinate lip and its location in direct oppositionto the fertile anther suggest that its strong degree of morpho-logical elaboration resulted from adaptations to specificpollinators.

* For correspondence: E-mail [email protected]

# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

For Permissions, please email: [email protected]

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doi:10.1093/aob/mcn258, available online at www.aob.oxfordjournals.org


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W H A T A R E TH E D R I V I N G F O R C ES O F O R C H I DF LO R A L D I V ER S I TY ?

Questions concerning causes of evolution are generally diffi-cult since they can address fundamentally different things.One can distinguish between ultimate causes such as selec-

tion or drift and proximate causes such as the moleculardevelopmental genetic mechanisms facilitating or constrainingevolutionary change. From Darwin onwards, orchid biologyhas focused on the ultimate causes of morphological diversityand species richness (Darwin, 1862; Cozzolino and Widmer,2005; Schluter and Schiestl, 2008). Specifically, the impressivefloral diversity of orchids has been attributed to adaptation tospecific pollinators (e.g. Johnson et al., 1998). Although inOrchidaceae there is a wide range of specificity in plantpollinator interactions, it has been estimated that about 60 %of orchid species have only one recorded pollinator species(Tremblay, 1992), and this specificity has been considered an

important ethological, prezygotic mechanism of reproductiveisolation (reviewed in Cozzolino and Widmer, 2005).Phenomena such as the attraction of pollinators by mimickingfood or mating partners without providing a reward (termedfood and sexual deception, respectively) have fascinated

many researchers and have been studied intensively(Jersakova et al., 2006).

However, increased understanding of the ultimate causes oforchid evolution tells us little about the reasons why orchids,but not, for example, Liliaceae or Hypoxidaceae, exhibitextreme floral diversification. One may hypothesize, forexample, that special genetic and developmental propertiescould have contributed to orchid diversity. In contrast to thegreat interest in the ultimate causes of orchid floral diversity,possible proximate causes are a severely neglected topic andremain speculative. Exceptionally, Bateman and Rudall(Bateman, 1985; Rudall and Bateman, 2002, 2003; Bateman

t2

T1

Column

Anther

Ovary

Pedicel

Pollinia

Callus

Lip

T2

T1

T2T3

t2t1

t3lip

A

B C D

F IG . 1. Structure and diversity of orchid flowers. (A) Sample of perianth diversity in Orchidaceae. Even though other families including Zingiberaceae,Corsiaceae and Cannaceae have independently evolved structures termed lips, the lip of orchids shows unprecedented morphological diversity. These examplesrepresent the wide degree of variation of the perianth in the five orchid subfamilies. From left to right, upper row: Apostasia wallichii (subfamily Apostasioideae);

Vanilla imperialis (subfamily Vanilloideae); Phragmipedium caudatum (subfamily Cypripedioideae); Ophrys apifera; lower row: Habenaria radiata (subfamilyOrchidoideae); Aerangis fastuosa, Telipogon intis, Cattleya tenebrosa, Psychopsis papilio (subfamily Epidendroideae). (B) Graphic representation of a transversesection through the flower of an orchid (Phalaenopsis hybrid) depicting the general arrangement of perianth organs, column and ovary. (C) Front view of anorchid flower (Phalaenopsis hybrid). The perianth is composed of six organs that are arranged in two whorls and represent at least three classes of organ identity.In the first (outer) floral whorl, there are three outer tepals (T1, T2 and T3; often also termed sepals), with T1 being a median and T2 and T3 being lateral outertepals; in the second floral whorl, there are two lateral inner tepals (t1 and t2; petals) and a median inner tepal (t3), called lip or labellum. (D) Schematic rep-resentation of organ identity in the orchid perianth. The three colours symbolize different organ identities [outer tepals green, lateral inner tepals yellow, lip(labellum) red] as possibly determined by a combinatorial code involving differential expression of four clades of DEF-like, MIKC-type, MADS-box genes.

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and Rudall, 2006) have integrated current knowledge aboutthe genetics of flower development in model species suchas Arabidopsis thaliana to predict the genetic basis oforganogenesis in wild-type and peloric orchid flowers. Thesecontributions, together with recent knowledge on floraldevelopmental control genes in orchids, enable an improved

understanding of orchid evolution.

A C O M B I N A TO R I A L C O D E F O R O R G A NI D E N T I T Y I N T H E O R C H I D P E R I A N T H

Recently, we proposed a theory on the developmental determi-nation and evolution of organ identity in the orchid perianth(Mondragon-Palomino and Theien, 2008). The underlyingdevelopmental-genetic code for organ identity in the orchidperianth (here termed the orchid code) assumes that identityof the different organs in orchid flowers is specified by thecombinatorial expression of orthologues of organ identitygenes known from eudicot model plants such as Arabidopsisthaliana and Antirrhinum majus. These include DEF-like (or

AP3-like) and GLO-like (or PI-like) genes specifying stamenand petal identity, collectively termed class B floral homeoticgenes. Although identity of petals in eudicot model plantsinvolves interaction and function of one DEF-like and oneGLO-like gene, our theory proposes that the identity of differ-ent petaloid tepals in the perianth of orchid flowers is specifiedby the combinatorial interaction of four paralogous DEF-likegenes with one GLO-like gene. Phylogenetic reconstructionshave indicated that these DEF-like genes fall into four distinctclades, termed PeMADS2-like (clade 1), OMADS3-like (clade2), PeMADS3-like (clade 3) and PeMADS4-like (clade 4)(Mondragon-Palomino and Theien, 2008). These genesshow highly conserved, clade-specific expression patterns.Based on these findings, our theory maintains that in the pri-

mordia of the first floral whorl, the combined expression ofclade 1 and clade 2 genes determines formation of outertepals (T1T3). In the second whorl, identity of lateral innertepals (t1 and t2) is determined by the combined action ofclade 1, clade 2 and clade 3 genes. Identity of the lip (t3) isspecified by the organ-specific expression of a clade 4 genein addition to expression of all other DEF-like genes(Fig. 2). Our theory implies that differential expression ofclade 3 genes distinguishes between inner and outer tepals,whereas differential expression of clade 4 genes distinguishesbetween identities of the lateral inner tepals and lip (Fig. 2).

Clade 1 and clade 2 DEF-like genes represent orchid-specific sister clades, and the same is true for clade 3 andclade 4 genes; with these and some other findings concerning

the phylogeny of orchid DEF-like genes, it was possible toestablish a relationship between molecular evolution of thesegenes and morphological differentiation of the orchid perianth(Mondragon-Palomino and Theien, 2008). We hypothesizedthat gene duplications played a fundamental role in perianthdifferentiation in the common ancestor of all Orchidaceae.The most recent common ancestor of orchids and the rest ofAsparagales, probably had an actinomorphic perianth com-posed of six almost identical tepals in which an ancestralDEF-like gene was uniformly expressed. According to ourtheory, the first duplication of the DEF-like gene gave riseto the ancestor of clade 1 and clade 2 genes and the ancestor

of clade 3 and clade 4 genes. Evolution of differentialexpression of the precursor of clade 3 and clade 4 genesmay have led to the establishment of different organ identitiesfor outer (gene expression off) and inner (gene expressionon) tepals. Similarly, another gene duplication gave rise toclade 3 and clade 4 genes, and differential expression of

clade 4 genes led to distinction between lateral inner tepals(gene expression off) and the lip (gene expression on;Fig. 2). Based on these considerations, it is safe to assumethat different cis-regulatory elements evolved in the fourclades of DEF-like genes after their origin from a commonancestor gene, and these are now responsible for the differen-tial expression of these genes. Moreover, it also appears plaus-ible that these paralogous DEF-like genes must respond topositional cues in different ways to obtain their characteristicexpression patterns. We hypothesize that at least some oftheir cis-regulatory elements respond to positional cueswithin floral primordia, such as a basipetal acropetal gradientin the case of clade 3 genes and an adaxialabaxial (dorsiven-tral) gradient in the case of clade 4 genes. Two clines of gene

expression in the orchid flower were previously inferred byBateman and Rudall (2006).Dorsiventral pre-patterns existing throughout floral pri-

mordia due to dorsal expression of CYCLOIDEA-like tran-scription factors, which are members of the TCP family,appear to be good candidates for providing positional infor-mation for clade 4 gene expression (Mondragon-Palominoand Theien, 2008). Although there is no experimental evi-dence yet linking TCP-type transcription factors to dorsiventraldifferentiation of the orchid perianth, studies in model specieshave already suggested genetic mechanisms responsible forthis process. Specifically, early in flower development ofeudicot Antirrhinum majus, expression of CYCLOIDEA(CYC) and DICHOTOMA (DICH) in whorls 1, 2 and 3 deter-

mines differential growth and number of dorsal organ primor-dia in the floral meristem (Luo et al., 1996, 1999). Later, CYCexpression consolidates the zygomorphic configuration ofwhorls 2 and 3 by influencing differential growth and shapeof dorsal petals and arrest of the dorsal stamen (Luo et al.,1996). In A. majus, this last expression of CYC depends ontranscription of the class B floral organ identity gene DEF(Clark and Coen, 2002). These findings make it conceivablethat rather than being upstream of the DEF-like genes,TCP-type genes in orchids function as direct or indirecttarget genes of DEF-like genes or even both up- and down-stream of DEF-like genes. If TCP-type genes in orchids func-tion in a way similar to those in A. majus, they would beexpected to influence differential development of the lateral

outer tepals (T2 and T3) as compared with the median tepal(T1) and especially of the lip (t3) as compared with thelateral inner tepals (t1 and t2; as exemplified in Fig. 1C), aswell as being involved in developmental arrest and eventualsuppression of dorsal stamens in Orchidaceae. Research onCYC and its orthologue TCP1 from the distantly related andactinomorphic eudicot Arabidopsis thaliana showed that thesegenes are dorsally expressed in both floral meristems and axil-lary shoots (Cubas et al., 2001). Because axillary shoots donot have dorsiventral symmetry, this suggests that CYC andTCP1 respond to a pre-pattern in shoots and floral meristems,whereas effects are only obvious in floral development (Cubas

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et al., 2001). The actinomorphic symmetry of A. thaliana ispossibly the outcome of TCP1 being only transiently expressedin early stages of floral development.

So far, the genetic basis of dorsiventral pre-patterning in thefloral meristem of orchids remains unknown. However, exist-ence of a dorsiventral gradient of positional cues influencingspecification of distinct organ identities in the orchid perianthis corroborated by the developmental gradient observed in thefloral apex of monandrous orchids. Specifically, organ initiation

follows a dorsiventral sequence, with primordia of the outerlateral tepals (T2 and T3) emerging first, followed by primordiaof the lip (t3), lateral inner tepals (t1 and t2) and finally themedian outer tepal (T1) (Kurzweil, 1987a, b, 1988).

As mentioned in the previous section, the phenomenon ofresupination changes the orientation of many orchid flowersduring anthesis so the median inner tepal or lip, althoughinitially in an adaxial/dorsal position, becomes the lowermostperianth organ. This process also takes place in someother species with zygomorphic flowers such as Lobelia(Campanulaceae) and Orchidantha (Lowiaceae), but it isclearly more widespread in Orchidaceae. Assuming that thischaracter facilitates pollination by presenting the lip as alanding platform and nectar guide, it is reasonable to assume

that it evolved after the flower became zygomorphic. This iswell exemplified by Apostasioideae, the orchid subfamilysister to the other four, in which resupination occurs in thezygomorphic genus Neuwiedia but not in the actinomorphicApostasia (Kocyan and Endress, 2001). This scenario begsthe question of molecular mechanisms that associated zygo-morphy with the subsequent torsion of the pedicellate ovary.Experimental removal of column and pollinia indicate thatresupination occurs as a response of the flower to gravity(Nyman et al., 1985), which is mediated by auxin and otherhormones produced by orchid pollinia and associated withgravitropic phenomena in plants (Nyman et al., 1985; Nair

and Arditti, 1991). The link between pollinia and resupinationis further supported by observations in some species ofCatasetum, in which the female flowers are non-resupinate,whereas the male ones are resupinate even when they occurin the same inflorescence (Dressler, 1993). This suggests thatpollinia are somehow associated with the process ofresupination.

Although orientation of the lip in a way that suits pollinatorbehaviour provides a convincing ultimate explanation for the

evolution of resupination, we wonder why a similar morpho-logical change did not evolve by a much simpler mechanism,i.e. evolution of the abaxial/ventral median outer tepal (ratherthan the median inner tepal) into a lip, since this organ wouldalready have been in the correct position and orientation. Ourtheory about the evolution of organ identity in the orchidflower offers a proximate cause: the distinction between thelip and other tepals depends on the unique expression ofclade 4 DEF-like genes, which may have evolved under thecontrol of an adaxial/dorsal positional cue. Shortly afterorigin of the clade 4 gene by duplication from a clade 3 and4 precursor, all inner tepals probably had the same structure,but soon the lip may have become different from the twoother inner tepals to serve special functions. An important

new function was pollinator attraction, and resupination origi-nated as a secondary mechanism to make the lip also functionas a landing platform. Admittedly, this hypothesis is extremelyspeculative. However, to the best of our knowledge it providesthe first evolutionary developmental genetic explanation forresupination and could be experimentally tested once more isknown about the cis-regulatory elements of clade 4 DEF-likegenes and their trans-acting factors. A similar logic could beemployed to approach the evolution and development ofthe perianth of flowers in Orchidantha (Lowiaceae), whichsimilarly to orchid flowers are resupinate and have a medianinner tepal modified into a lip (Kirchoff and Kunze, 1995).

Ancestor of clades 1/2/3/4

Ancestor of clades1 and 2

Ancestor of clades3 and 4

Clade 4

Clade 3

Clade 2

Clade 1

Evolutionary time

Duplication of orchidDEF-like genes

T3t3lip

t1 t2

T2

T1

+++

TCP

Gene expression domainsInner tepals

Outertepals

(T1T3)

Lateraltepals(t1t2)

Lip

(t3) +

F IG . 2. Duplication and transcriptional divergence ofDEF-like genes and the origin of the orchid code. Duplications of orchid DEF-like genes resulting in fourclades of genes is schematically shown on the left. Different expression domains of extant genes and their impact on perianth organ identity (orchid code) isshown in the middle, next to the corresponding scheme of organ identity in the orchid perianth. Colours emphasize the correspondence between organ identityand patterns of gene expression, such that expression of clade 1 and 2 genes specifies outer tepals (T1, T2 and T3), expression of clade 1, 2 and 3 genes specifies

lateral inner tepals (t1, t2), and expression of clade 1, 2, 3 and 4 genes specifies the lip (t3). Differential expression of clade 3 and 4 genes is assumed to depend ona basipetalacropetal gradient (not shown), and an adaxialabaxial gradient, possibly composed of TCP-type proteins as indicated on the right. The orchid peri-

anth shown here is already resupinate (turned 1808), so the adaxial (dorsal-most) organ, the lip, adopts a final position nearest to the ground.



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H O M EO TI C V A R I A N TS I N C U LTU R EA N D N A TU R E

Previously, Bateman and Rudall categorized naturally occur-ring floral terata in Orchidaceae by systematically distinguish-ing six classes of peloria and pseudopeloria (Bateman, 1985;Rudall and Bateman, 2002, 2003; Bateman and Rudall,

2006). With the exception of type A pseudopeloria (assumedto originate by heterochrony), all terata involve homeotictransformation of one or more tepals. Although in peloricflowers the wild-type zygomorphic symmetry is completelylost due to development of an actinomorphic perianth, in pseu-dopeloric forms it is only reduced. The authors distinguishedthree categories each of peloric and pseudopeloric orchids byinvoking homeotic substitutions within the second whorl orbetween the first and second whorl (Bateman, 1985; Rudalland Bateman, 2002, 2003; Bateman and Rudall, 2006).

To generate hypotheses about developmental and geneticbases of teratological flowers in orchids, we inferred somesimple and plausible rules that may govern function ofDEF-like genes. One assumes that in any given organ, clade

3 gene function requires expression of clade 1 and clade 2genes, and clade 4 gene function requires expression ofclade 1, clade 2 and clade 3 genes. Another rule assumesthat loss of DEF-like gene function is recessive, whereasectopic expression of clade 3 or clade 4 genes within the peri-anth leads to a dominant gain-of-function. Here it is shownthat on these grounds, all categories of floral terata consideredby Rudall and Bateman (Rudall and Bateman, 2002; Batemanand Rudall, 2006) plus another one not considered by theseauthors can be explained by monogenic gain- or-loss-of-function mutants of clade 3 or clade 4 DEF-like genes.Intriguingly, supposed dominance or recessivity of mutantalleles is correlated with the frequency of the terata in bothcultivation and nature.

Type A peloria

In type A peloria the lateral inner tepals (petals; t1 and t2)are transformed into lip-like structures (t3; Fig. 1B). This is afrequent kind of peloria in cultivation (see, for example, Chenet al., 2005; and our own observations), where it can resultfrom somaclonal variation after tissue culture. Besides fulltransformants, semi-peloric variants in which the lateralinner tepals are partially transformed into lip-like structuresare frequent and commercially available (Wallbrunn, 1987;Chen and Chen, 2007). Type A peloria may also representthe most common category of natural terata affecting perianths

of orchids in nature; for example, it has been recorded in about25 % of British native orchid species, including several speciesof Dactylorhiza and Ophrys (Bateman and Rudall, 2006).

According to our theory on specification of organ identity inorchid flowers, type A peloria results from ectopic expressionof a clade 4 gene in the lateral inner tepals (Fig. 3B). Ourhypothesis is based on work by Tsai et al. (2004) inPhalaenopsis equestris, in which a variant of type A peloriashows this kind of ectopic gene expression. However, thisteratological orchid shows a mutant change in the putativepromoter region of its clade 2 gene that may have causedloss-of-function of that gene (Tsai et al. 2004). This may

indicate that clade 2 DEF-like genes regulate clade 4 genessuch that they prevent their ectopic expression in the lateralinner tepals, but this inference certainly requires furtherinvestigation.

Type B peloria

According to the combinatorial rules of the orchid code,type B peloria may result from loss-of-function of clade 4genes, leaving only expression of clade 1, 2 and 3 genes inthe median inner tepal that, therefore, acquires the same iden-tity as the lateral inner tepals (Fig. 3C). The phenotype of atype B peloria is here illustrated with Phragmipedium lindenii,a possible teratological form of Phragmipedium caudatum(Figs 3C and 1A, respectively) (Hurst, 1925; Bateman andRudall, 2006; Mondragon-Palomino and Theien, 2008).Another well-known case in point is Calochilus imberbis,which has been considered a hopeful monster (Burns-Balogh and Bernhardt, 1986) that like C. robertsonii mayproliferate autogamously (Tonelli, 1999) (Fig. 4). Overall,

examples of type B peloria are relatively frequent, but lesscommon than examples of type A peloria (Bateman andRudall, 2006).

Type C peloria

According to the orchid code, type C peloria may resultfrom lack of function of clade 3 DEF-like genes (Fig. 3D).Since clade 4 gene function is assumed to depend on clade 3genes, only clade 1 and clade 2 gene activity is left in alltepals that, therefore, adopt the identity of outer tepals(Fig. 3D). Loss of clade 3 DEF-like gene function leading totype C peloria (Fig. 3D) is possibly behind the independentemergence of rare actinomorphic genera within zygomorphic

groups of subfamily Orchidoideae. Examples of this are theAustralasian genus Thelymitra (tribe Diurideae; Fig. 3D) orthe monospecific genera from tribe Neottieae Diplandorchis,Tangtsinia, Sinorchis and Holopogon (Komarov, 1935;Chen, 1965, 1978, 1979). More specifically, Dressler (1993)and Rudall and Bateman (2002) argued that Diplandorchisand Holopogon are peloric forms of Neottia, whereasTangtsinia and Sinorchis may be actinomorphic variants ofCephalanthera. Possibly, autogamy and cleistogamy aidedthese putative hopeful monsters to form constant populations(Chen, 1965, 1979). Remarkably, Thelymitra diversified toform a new genus that evolved more complex pollinationsystems including floral mimicry (Burns-Balogh andBernhardt, 1986; Tonelli, 1999). A less parsimonious alterna-

tive to explain this phenotype would be to consider that allperianth organs have lateral inner tepal identity (yellow).This unclassified peloric form would require both ectopicexpression of the clade 3 DEF-like gene in all perianthorgans and complete disruption of the clade 4 DEF-likegene (not illustrated).

Type B pseudopeloria

Pseudopeloric orchid variants can also be explained by gainor loss of clade 3 gene function (Fig. 3E G). In type Bpseudopeloria, the lip adopts outer tepal identity by



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AInner tepals

Type B pseudopeloric

Type C pseudopeloric

Type D pseudopeloric

Type A peloric

Type B peloric

Type C peloric

Outer tepals(T1T3)

Lateral tepals(t1t2)

Lip(t3)

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loss-of-function of the clade 3 gene only in the lip, but not inthe lateral inner tepals, most likely by restriction of theexpression domain to the lateral inner tepals (Fig. 3E).Although the clade 4 DEF-like gene may still be expressedin the median inner tepal, lip identity does not developbecause this would require the combinatorial expression of

all DEF-like genes (Fig. 3E).

Type C pseudopeloria

In type C pseudopeloria, lateral inner tepals adopt outertepal identity by loss-of-function of the clade 3 gene only inthe lateral inner tepals, but not in the lip, most likely by therestriction of the expression domain to the lip (Fig. 3F).

Type D pseudopeloria

Although it is not clear whether type C pseudopeloria sensuBateman and Rudall (2006) is the result of homeotic conver-sion of inner lateral tepals into outer tepals or vice versa,

our theory on specification of organ identity in the orchidflower distinguishes between these two possibilities. This dis-tinction is a first step to testing these possibilities by compar-ing expression patterns ofDEF-like genes. Thus, in addition todefining type C pseudopeloria as described above we propose atype D pseudopeloria in which all outer tepals are transformedinto organs that adopt lateral inner tepal identity, possibly byectopic expression of clade 3 genes in the outer tepals(Fig. 3G).

We reason that type C and type D pseudopeloria, in whichall inner and outer tepals except the lip are highly similar toeach other (Fig. 3F, G), may be more common inOrchidaceae than previously recognized. For example, in thegroup of Brazilian Cattleya species (formerly Laelia), the

flowers of section Parviflorae have a perianth in which alltepals except the lip have similar shape, size and colour(Fig. 3G), giving the perianth a slight degree of actinomorphy.This morphology is in clear contrast to the related sectionsHadrolaelia and Cattleyodes (e.g. Cattleya tenebrosa inFig. 1A) in which all species (van der Berg et al., 2000)have distinct lateral inner and outer tepals. In this and otherexamples, the molecular phylogeny of the groups involvedand pattern of DEF-like gene expression would help toclarify whether apparent morphological differences betweenthese groups resulted from homeotic transitions (Fig. 3F, G)or from changes in downstream targets, as previously dis-cussed (Mondragon-Palomino and Theien, 2008).

Our hypothesis on the developmental genetic basis of organidentity in the orchid perianth explains, at least in part, the fre-quency of orchid terata. Assuming three classes of organ iden-tity (outer tepal-like, lateral inner tepal-like, and lip-like)on three kinds of positions (in the wild type occupied by threeouter tepals, two lateral inner tepals and a lip, respectively)

would allow for 27 (

3

3

) types of flowers; by definition,26 ( 33 1) are teratological configurations. Only a few of

these categories are frequent, and some others are extremelyrare. We assume that their relative frequency is largelyexplained by their mutant origin rather than their maintenanceby selection. This certainly applies in cultivation, but probablyalso in nature, where the vast majority of terata appears spor-adically and briefly and may presumably be eliminated due tolack of pollinators, if they are not autogamous.

It is thus probably not by chance that terata reported byBateman and Rudall (2006) are all predicted to be based onmonogenic changes (loss- or gain-of-function of a singleclade 3 or clade 4 DEF-like gene), as outlined above. Notethat terata requiring changes in more than one gene have not

been considered here. Moreover, terata supposed to be basedon dominant gain-of-function mutants, such as type Apeloria, are more frequent than terata supposed to be basedon recessive loss-of-function mutants, such as type Bpeloria, possibly because a mutant phenotype develops onlyin homozygous plants.

Despite the explanatory power of the orchid code, someorchids have variant flowers that do not easily fit into thescheme of organ identity discussed above (Bateman andRudall, 2006). Specifically, the lateral outer tepals (T2 andT3) are sometimes more similar to the lip (t3) than to themedian outer tepal (T1); likewise, the median outer tepal (T1)may resemble closely the lateral inner tepals (t1 and t2).Flowers of that type exist at the species level (e.g. Psychopsis

papilio; Fig. 1A) and at the level of mutants, such asHabenaria radiata Hishou (Fig. 5A), a horticultural variantof H. radiata Ginga (Fig. 1A). According to our orchidcode theory, in the case of H. radiata Hishou both lateralouter tepals and the lip should express DEF-like genes of allfour clades, whereas other tepals should express just the genesof clades 1, 2 and 3 (Fig. 5B). This would require both theectopic expression of a clade 3 gene in outer tepals and of aclade 4 gene in lateral outer tepals (Fig. 5B).

Kim et al. (2007) showed that clade 3 DEF-like genes areectopically expressed in the first floral whorl of Hishou,but more data are not presently available. In principle, parallelchanges in both clade 3 and 4 genes could underlie the floral

F IG . 3. Floral terata in orchids explained by changes in the expression of DEF-like genes. Colour-coding is as in Fig. 2; on the left, expression domains ofDEF-like genes are schematically shown; in the middle, organ identity is summarized; on the right, flowers of these types are shown. (A) The normal orchidperianth. The picture on the right shows a wild-type flower of Phalaenopsis equestris, the species in which expression patterns of the four DEF-like geneshas been characterized (Tsai et al., 2004). (B) Type A peloria, with petals (lateral inner tepals) transformed into lips. This phenomenon is assumed to becaused by the ectopic expression of a clade 4 gene in the lateral inner tepals. The picture on the right shows a peloric variant of Phalaenopsis equestris. (C)Type B peloria, with the lip transformed into a petal-like (lateral inner tepal-like) organ. This phenomenon is assumed to be caused by the loss-of-functionof a clade 4 gene. The picture on the right shows Phragmipedium lindenii a type B peloric variant of Phragmipedium caudatum (Fig. 1A) in which substitutionof the lip by a lateral inner tepal may be the result of losing the function of the DEF-like gene from clade 4. (D) Type C peloria, in which all perianth organs adoptouter tepal identity as the result of a loss-of-function mutation affecting the DEF-like gene from clade 3 with lateral inner tepals and lip transformed into outertepals. A possible candidate shown here is Thelymitra formosa. (E) Type B pseudopeloria with the lip adopting outer tepal identity by abolishment of clade 3

DEF-like gene expression only in the lip. This type is exemplified with a variant of Platanthera chlorantha. (F) Type C pseudopeloria. The lateral inner tepalsadopt outer tepal identity by loss-of-function of the clade 3 gene only in the lateral inner tepals, but not in the lip, due to the restriction of expression domain tothe lip, as exemplified by Epidendrum pseudoepidendrum. (G) Type D pseudopeloria. All outer tepals are transformed into organs that resemble lateral inner

tepals due to ectopic expression of clade 3 genes in the outer tepals. This putative example is the Brazilian Cattleya alvaroana.



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phenotype, but a single change in the common upstreamcontrol of both clade 3 and 4 genes appears more likely. Alikely candidate would be any gene that translates theprimary adaxialabaxial (dorsalventral) positional cue intoa gradient felt by clade 3 and 4 genes. As discussed in theprevious section, a feasible mechanism to form such a gradient

would be differential expression of CYC-like TCP-type genesin dorsal (strong expression) and ventral regions (lowexpression) of the floral meristem (Fig. 5C). We hypothesizethat deviant flowers such as those of Habenaria radiataHishou are dorsalized by overexpressing the materialbasis of the dorsalventral positional cue; i.e. they mayshow stronger TCP-type gene expression, especially in thedorsal part of the floral meristem (Fig. 5C).

Combining the dorsiventral patterning system with the organidentity system (the orchid code) may lead to a refined systemthat distinguishes four different types of organs. Dorsally, the lipis defined by clade 4 gene expression, whereas the lateral outertepals are defined by clade 1 and 2 expression and both types ofdorsal organs by high TCP-type gene concentration. Ventrally,

the lateral inner tepals are defined by clade 3 gene expressionand the median outer tepal is defined by clade 1 and 2 geneexpression, as well as by low TCP-type concentration affectingboth types of ventral genes (Fig. 5C).

Throughout this discussion, we have dealt with the geneticbasis of homeotic transitions affecting perianth organs.However, DEF-like genes, together with GLO-like genes, alsodetermine stamen identity in angiosperms. Thus, one mightexpect that changes in the expression of DEF-like genes associ-ated with development of peloric flowers may also affect malereproductive structures. Contrary to this general expectation,in the previously discussed examples of confirmed and likelyhomeotic transformations (Figs 3 and 5), male reproductivestructures do not seem to be equally affected. For instance, in

early developmental stages of type A peloria of Phalaenopsisequestris analysed with scanning microscopy by Tsai et al.(2004), lack of stamen and staminode development and fusionof adaxial carpels were observed. Dissection of adult flowersfrom other Phalaenopsis hybrids classified them as peloric orpseudopeloric, depending on the degree of identity betweenthe modified lateral inner tepals and lip. This analysis showedthat pseudopeloric flowers have normal stigmas and anthers,whereas fully peloric flowers lack stigmas and anther tissuesand are thus sterile (Wallbrunn, 1987), suggesting that theunderlying genetic causes may have different degrees ofphenotypic penetrance. Although it is clear that in all peloric

or pseudopeloric orchids the structure of the column remainszygomorphic, there are in each type of terata a few informativeexamples that suggest an association between developmentalchanges in the perianth and stamens. For instance,Phragmipedium lindenii (Fig. 3C), in which the lip is replacedby a lateral inner tepal (type B peloria), has a third fertile anther,

in contrast to all other members of Cypripedioideae that haveonly two (Hurst, 1925). Furthermore, the presumed type Cpeloric Diplandorchis sinica has two fertile median stamensfrom the first and second floral whorl growing on the endof the stigma, directly opposite the dorsal tepal and lip(Chen, 1979). In contrast, Tangtsinia nanchuanica, anotheractinomorphic species discussed above, has five staminodialprojections on the column that may represent three stamensof the inner whorl and two of the outer whorl (Chen, 1965).As the name of the potential type C pseudopeloric indicates,Prosthechea cochleata var. triandra has three anthers insteadof the one normally found in this species (Sauleda et al.,1985). Nevertheless, this general relationship between peloriain the perianth and changes in stamen structure might not

necessarily hold for Thelymitra, the most species-rich candidatecase of type C peloria. It is not yet clear whether the distinctiveand elaborated hood-like structure formed by the posterioranther lobe (the mitra) on the tip of the column representsmodified stamens or staminodes. Besides this, the reproductiveorgans in Thelymitra do not have any apparent modification innumber or configuration (Tonelli, 1999).

Further systematic description of different degrees ofperianth modification and associated variation in reproductivestructures is needed for more types of orchid peloria andpseudopeloria. Certainly, the fact that the cases previouslydiscussed (Fig. 3) include cases of autogamy, cleistogamyand animal pollination suggests that peloric changes of repro-ductive organs do not necessarily result in sterility.

Considering the incredible diversity of orchid flowers, it isclear that nature provided us with a lot of material to test theorchid code theory by determining organ-specificity ofexpression of different DEF-like and CYC-like genes via, forexample, northern hybridization, in situ hybridization orquantitative RT-PCR.

TH E O R C H I D F LO W ER : A R EP R O D U C TI V ES TR U C TU R E D EC O N S TR A I N ED

As outlined above, homeotic mutations probably contributedto diversification of orchid flowers during evolution. It is

A B C

F IG . 4. Calochilus robertsonii wild type and two peloric forms found in nature. The normal form (A), peloric type A, in which inner lateral tepals are trans-formed into lip-like structures, (B), and peloric type B, in which the lip is transformed in a lateral inner tepal-like structure, (C), share the same range in

Australasia.



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assumed that morphological changes are based on changes inexpression of DEF-like genes or genes controlling theirexpression. These increases or decreases in domains or inten-sities of expression of developmental control genes led tochanges in organ identity, such as transformation of lateral

inner tepals into lip-like organs or vice versa. However, eventhough homeotic mutants occur sporadically in many (if notall) populations of orchids, they tend to be ephemeral,presumably because they are often not pollinated (Batemanand Rudall, 2006). For example, even though occurring most

FIRST WHORL SECOND WHORL

VENTRAL

DORSAL

Median tepal Inner lateral tepal

Outer lateral tepal Lip

B

C

Inner tepalsOuter tepals

Lateral(T2T3)

Median(T1)

Lateral(t1t2)

Lip(t3)

A

F IG . 5. A dorsalized mutant orchid flower. (A) Flower of mutant Habenaria radiata Hishou in which lip and outer lateral tepals (dorsal organs) adopt a lip-likemorphology, whereas the lateral inner tepals and the median outer tepal (ventral organs) adopt lateral inner tepal identity (modified from Kim et al., 2007). Notethat, in addition to these transformations, the peloric flower shown here is non-resupinate. (B) Modified scheme of DEF-like gene expression in the dorsalizedflower, indicating ectopic expression of clade 3 and clade 4 genes. (C) The dorsal (adaxial, bottom)ventral (abaxial, top) gradient controlling DEF-like geneexpression, shown for the wild type (left) and a dorsalized mutant (right). This gradient is possibly composed of TCP-type proteins, as indicated by triangles

superimposed on the perianth schemes, with higher TCP concentrations in dorsal regions and mutant flowers.



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frequently, type A peloria does not result in new species. Eventhe famous naturally occurring Cattleya intermedia var.aquinii is considered only a variety. Type B peloria is alsofrequent, but it too only rarely causes speciation, a remarkableexception being Phragmipedium lindenii (Hurst, 1925;Bateman and Rudall, 2006; Mondragon-Palomino and

Theien, 2008). Thus, generation of homeotic variants is notthe most efficient mechanism for diversification of orchidflowers, and true changes in organ identity are probably rareevents in orchid evolution. This is not to say, however, thatthey have not been crucial for the origin of some other plantgroups (Ronse de Craene, 2003; Theien, 2006).

Considering the floral diversity of orchids (incompletelycaptured in Fig. 1A), it is likely that differential elaborationof specific perianth organs without change of organ identityhas been a much more important mechanism of floral diversi-fication. For example, although still recognizable as a lip, themedian inner tepal can develop in different ways. At maturity,it may be similar to the lateral inner tepals or outer tepals, butit can also appear dramatically different (Fig. 1A). The lip can

be much larger or much smaller than other tepals, have adifferent colour or the same, have different patterns of color-ation and ornamentation or may or may not bear nectarspurs. It may develop into an almost flat organ like the othertepals, but it may also form elaborate structures such as atube or a ladys-slipper. In essence, it is obviously the poten-tial of different kinds of petals, especially the lip, to developindependently from other types of organs that has made amajor contribution to evolutionary diversification of orchidflowers. Assuming that our theory on development of organidentity in the orchid perianth (the orchid code) is correct,it appears not too far-fetched that it was the origin of fourparalogous classes of DEF-like genes that enabled the orchidflower to address the different types of tepals individually

and hence allowed them to evolve independently. Althoughall tepals of flowers such as lilies and tulips (like the petalsof eudicots) are probably under developmental control ofone and the same set of floral homeotic genes, includingDEF-like and GLO-like genes (Kanno et al., 2003), the outertepals, lateral inner tepals and lip of orchids are controlledby different sets of genes with nested expression domains(Fig. 2). Thus, mutational changes in the orchid perianth caneasily be restricted to the inner tepals by means of clade 3gene mutation, or targets thereof, or to the lip by means ofclade 4 gene mutation, or targets thereof. This may provide aproximate explanation for why the lip is the most diverseorgan. A similar scenario is unlikely in petaloid monocotswith identical tepals, such as lilies and lily-like species.

Here, mutant changes in one tepal are likely accompanied bythe same (pleiotropic) changes in all other tepals because ofcommon developmental genetic control. Thus, evolution ofthe four classes of paralogous DEF-like genes modularizedthe orchid perianth in such a way that the inner tepals couldevolve semi-independently of the outer ones and the lip semi-independently of the lateral inner tepals. In this way, evolutionof the paralogous DEF-like genes may have deconstrained alily-like floral perianth that was limited in its evolutionarypotential by the pleiotropic interdependence of tepals. Oncethese constraints were reduced by modularization, the differentclasses of tepals thus generated were capable of evolving in a

semi-independent way, and an almost explosive morphologicaldiversification occurred, largely driven by adaptation oforchid flowers to specific classes of pollinators. Independentevolution was probably still restricted by the fact, however,that some developmental control genes (such as clade 1 andclade 2 DEF-like genes and GLO-like genes) are required for

all petals to develop.Origin and evolution of the four classes of paralogousDEF-like genes were probably not simple processes(Mondragon-Palomino and Theien, 2008). We assume thatthe four DEF paralogues were subject to a complex seriesof sub-functionalization events that mainly affected cis-regulatory elements and led to changes in gene expressiondomains, as well as neo-functionalization events that couldalso involve the coding region. During these events, someupstream regulators and the target genes of DEF-like genesmay have changed.

For instance, imagine an orchid with white tepals (e.g. somePhalaenopsis) or greenish tepals (e.g. Vanilla imperialis inFig. 1A) in which only the lip is coloured purple by anthocya-

nins. Work on model plants such as Antirrhinum majus andZea mays (Grotewold, 2006), and also on orchids (Chiou andYeh, 2008), revealed that anthocyanin production depends onthe expression of key enzymes including chalcone synthase,chalcone isomerase (CHI), flavanone 3-hydroxylase, dihydro-flavonol 4-reductase (DFR) and others. Expression of the cor-responding genes is under control of some transcription factorsof the bHLH and MYB families; the latter includes OgMYB1of the orchid hybrid Oncidium Gower Ramsey, which activatesCHI and DFR (Chiou and Yeh, 2008). Anthocyanins can beproduced in different floral organs and other parts of theplant, depending on expression of the appropriate enzymes,so in cases in which only the lip produces anthocyanins,clade 4 DEF-like genes are probably required and sufficient

to activate the whole anthocyanin pathway, possibly by activat-ing the regulatory bHLH and MYB genes as shown for otherpetal-specific genes in Antirrhinum majus (Perez-Rodriguezet al., 2005). In cases where all inner tepals, or even alltepals, produce anthocyanins (a frequent situation inorchids), clade 3 genes or even clade 1 or 2 genes might besufficient to activate the anthocyanin pathway. This simpleexample would explain how evolutionary changes in the linkbetween paralogous DEF-like organ identity genes and theirtarget genes may have contributed to diversification oforchid flowers.

C O N C LU S I O N S A N D P ER S P EC TI V ES

We have outlined the hypothesis that four paralogues ofDEF-like class B floral organ identity genes have modularizedthe perianth of orchid flowers by a complex series of sub- andneo-functionalization events. These genes may have elimi-nated constraints, so that the different kinds of tepals couldevolve individually and often in dramatically different ways.We thus argue that a developmental genetic predispositionunique to orchids may have played an important role infloral diversification in this family.

It is an important insight provided by evolutionary develop-mental biology (evo-devo) that the internal organization oforganisms, especially their developmental genetic systems,



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can influence the tempo and direction of evolutionary change(e.g. Brakefield, 2003). Generally, gene duplications increasethe mutational robustness of organisms and thereby facilitateevolutionary innovations (Wagner, 2008). However, assumingthat our hypothesis about the orchid code is correct, we arenot aware of many other systems that show such a close associ-

ation between gene duplications and origin of evolutionarynovelties directly involved in speciation. We are therefore con-vinced that the orchid flower is an extremely well-suited systemfor future evo-devo studies aimed at a better understanding ofthe relationship between developmental gene evolution andchanges in morphology leading to speciation, especially oncemethods for validation of orchid gene function, such as trans-formation and virus-induced gene silencing, are optimized (Luet al., 2007). Developing a diploid orchid with a rapid lifecycle and a small genome size into a tractable model systemis another urgent goal for the near future, which is alreadyunderway (Mark Chase, Royal Botanic Gardens, Kew, UKand B. Gravendeel, National Herbarium of the Netherlands,pers. comm. November 2007). The great number of orchids

with peloric or pseudopeloric flowers, however, can alreadybe used to test the orchid code hypothesis and the impact ofthis system on evolution of orchid flowers.

A C K N O W LED G EM EN TS

Many thanks to Mark Chase and Michael Fay (Royal BotanicGardens, Kew) and the Linnean Society for inviting M.M.-P.to participate in the Symposium Orchid evolutionarybiology and conservation: From Linnaeus to the 21stcentury, at which this paper was presented. We thank MarkChase, Paula Rudall, Richard Bateman and Rainer Melzerfor helpful comments on a previous version of this manuscript,

Pia Nutt for producing an orchid line drawing and ThomasWolf for assistance with photography. We thank the followingcolleagues for providing permission to use their valuablephotographic material: A. Kocyan (Apostasia wallichii),A. Kanno and S. Y. Kim (Habenaria radiata in Figs 1 and 4),T. Kusibab (Phragmipedium caudatum and Phragmipediumlindenii in Figs 1 and 3), H. Schildauer and W. Schraut(Vanilla imperialis, Ophrys apifera, Aerangis fastuosa,Telipogon intis, Cattleya tenebrosa, Epidendrum pseudoepiden-drum and Cattleya alvaroana in Figs 1 and 3), Hans Wapstra forthe picture of type B peloric Calochilus robertsonii (from thecollection of the late Les Rubach), James Wood (wild-typeand type A peloric Calochilus robertsonii), Michael Pratt(Thelymitra formosa) and Richard Bateman and Robin Bush

(peloric Platanthera chlorantha). This work was supported bythe VolkswagenStiftung (I/81 901 to M.M.P. and G.T.).

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