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Journal of Experimental Botany, Vol. 60, No. 5, pp. 1503–1513, 2009 Perspectives on Plant Development Special Issue doi:10.1093/jxb/ern304 Advance Access publication 3 December, 2008

REVIEW PAPER

Lepidium as a model system for studying the evolution of fruit development in Brassicaceae Klaus Mummenhoff1, Alexander Polster1,*, Andreas Mu¨hlhausen1 and Gu¨nter Theißen2,† 1 2

University of Osnabru¨ck, Department of Biology, Botany, Barbarastrasse 11, D-49076 Osnabru¨ck, Germany Friedrich Schiller University Jena, Department of Genetics, Philosophenweg 12, D-07743 Jena, Germany

Received 12 September 2008; Revised 31 October 2008; Accepted 10 November 2008

Abstract Fruits represent a key innovation of the flowering plants that facilitates seed dispersal. In many species of the plant family Brassicaceae dehiscent fruits develop in which seed dispersal occurs through a process termed ‘podshatter’. In the case of dehiscence, the fruit opens during fruit maturation. Phylogeny reconstructions using molecular markers indicate that the development of dehiscent fruits is the ancestral condition within the genus Lepidium s.l., but that indehiscent fruits evolved independently several times from dehiscent fruits. With Lepidium campestre and Cardaria pubescens (also known as Lepidium appelianum), very closely related taxa with dehiscent and indehiscent fruits, respectively, were identified which constitute a well-suited model system to determine the molecular genetic basis of evolutionary changes in fruit dehiscence. Following the rationale of evolutionary developmental biology (‘evo-devo’) phylomimicking mutants with indehiscent fruits of the close relative Arabidopsis have been used to define the candidate genes ALC, FUL, IND, RPL, and SHP1/2 which might be involved in the origin of indehiscent fruits in Cardaria. Comparative expression studies in L. campestre and C. pubescens are used to identify differentially expressed genes and thus to narrow down the number of candidate genes. Reciprocal heterologous transformation experiments may help us to distinguish direct from indirect developmental genetic causes of fruit indehiscence, and to assess the contribution of cis- and trans-regulatory changes. Key words: Arabidopsis, Brassicaceae, Cardaria, character evolution, genetic regulation, Lepidium, pod-shatter, phylomimicking mutant.

Introduction For more than 20 years the little weed Arabidopsis thaliana (L.) Heynh. (henceforth termed Arabidopsis) has been the major model system for investigating developmental and physiological phenomena in plant biology, and has led to the identification of genes and gene regulatory networks (GRNs) involved in numerous processes of plant life. Arabidopsis belongs to the Brassicaceae (mustard) family, comprising about 3700 species in 338 genera (Warwick et al., 2006), among them many economically important ornamental and crop plants such as cabbage, canola, and oil seed rape (Koch et al., 2003). In addition to their agronomic importance and given the large variation in physiology and in leaf and fruit shape within the Brassicaceae, there should

also be ample genetic material available to investigate the molecular bases of the changes in morphology over evolutionary time (Bowman, 2006). An interesting example is difference in leaf shape, which is now being intensively investigated using Cardamine hirsuta (which has a dissected leaf form) in comparison with Arabidopsis (which has a simple leaf form) (Hay and Tsiantis, 2006; Barkoulas et al., 2008). Indeed, recent molecular analyses have led to the knowledge that many morphological characters on which previous systematic relationships were based are, in fact, homoplasious. Fruit structures in particular have proved to be highly labile in evolutionary time; all molecular phylogenetic data

* Present address: Hannover Medical School, Centre for Physiology, Carl-Neuberg-Str.1, D-30625 Hannover, Germany. y To whom correspondence should be addressed: E-mail: [email protected] ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]

1504 | Mummenhoff et al. consistently indicate that many species with similar fruits may be only distantly related, whereas species with dramatically different fruits can be very closely related (Koch et al., 2003; Mummenhoff et al., 2005; Bailey et al., 2006; Al-Shehbaz et al., 2006, and references therein). This implies that developmental processes controlling fruit shape and structure are extremely plastic. A good case in point are the structural prerequisites for mechanisms of seed dispersal in Brassicaceae. In many species of the Brassicaceae the fruit develops into a seed pod which opens because of mechanical forces which build up as the fruit dries out, in a process termed dehiscence (Spence et al., 1996). Fruit dehiscence effectively discloses the seeds upon maturation, which can eventually be dispersed, for example, by wind, rain or animals. Many other species of Brassicaceae, however, develop indehiscent fruits, in which the whole fruit is the dispersal unit, and the seeds are released upon decomposition of the fruit valves. The development of dehiscent versus indehiscent fruits was used for a long time as an important character in the systematics of Brassicaceae, before the repetitive origin of indehiscent fruits became obvious by phylogeny reconstructions employing molecular markers (Koch et al., 2003; Mummenhoff et al., 2005; Al-Shehbaz et al., 2006, and references therein). Fruit dehiscence in the Brassicaceae may thus represent a system for the study of parallel or convergent evolution of reproductive characters using the model plant Arabidopsis as a reference system. However, extending the ideas which have arisen from the knowledge of developmental genetic systems in Arabidopsis to other species of the Brassicaceae will require the application of genomic technologies developed in Arabidopsis, and the establishment of additional genetic systems and resources in other species. Thus, it is desirable to establish model systems in which genetics can be utilised in conjunction with the genomic resources available in Arabidopsis for the purpose of identifying the genetic changes that have contributed to morphological evolution. The current paper summarizes recent literature on fruit dehiscence/indehiscence in Arabidopsis and aims to present a convenient model system, the genus Lepidium sensu lato (s.l.), to shed further light on fruit evolution in wild Brassicaceae species. Lepidium s.l. comprises Lepidium, as traditionally delimited, and a number of nested genera, as identified by phylogeny reconstruction using molecular markers (Fig. 1). Lepidium s.l. is among the few well-supported monophyletic lineages found in all family-wide analyses (Bailey et al., 2006; Beilstein et al., 2006; Al-Shehbaz et al., 2006; Franzke et al., 2009) and is nested along with Arabidopsis in one well-supported clade (‘lineage one’ in Beilstein et al., 2006, 2008; Franzke et al., 2009). The genus Lepidium L. as traditionally delimited– Lepidium sensu strictu (s.str.)–is one of the largest genera of the Brassicaceae, consisting of approximately 175 species. It is distributed worldwide, primarily in temperate and subtropical regions; the genus is poorly represented in Arctic climates and, in tropical regions, grows only in mountain areas. Lepidium is well-known for its infrageneric systematic

difficulty, and traditional systematic concepts, mainly based on differences in fruit shape, have been criticised as being largely artificial (Mummenhoff et al., 2001, and references therein). Previous molecular analyses have concentrated on systematics (Mummenhoff et al., 2001), allopolyploidization events (Mummenhoff et al., 2004), and the evolution of flower structures (Bowman et al., 1999; Lee et al., 2002). Although these studies focused on different topics and were based on different taxon coverage, all molecular phylogenies supported three main lineages and indicated that few of the infrageneric taxa, as delimited in the traditional taxonomy, represent monophyletic groups. Coronopus Zinn, Stroganowia Kar. & Kir., Winklera Regel, Stubendorffia Schrenk ex. Fisch., and Cardaria Desv. are among the most closely related genera of Lepidium s.str. (Al-Shehbaz, 1986; Al-Shehbaz et al., 2006). These genera differ from Lepidium mainly in fruit characters, especially fruit dehiscence and inflation. Lepidium, Stroganowia, and Winklera are characterised by dehiscent fruits (but see Fig. 1 for Winklera), Cardaria, Stubendorffia, and a fraction of Coronopus species by indehiscent fruits, while the remaining Coronopus species have didymous fruits. In didymous fruits, the valve orifice that faces the replum is usually smaller than the seed and, therefore, the seeds are not readily released, and the fruit valves act as the dispersal unit (Al-Shehbaz et al., 2002). Thus, from a mechanistic point of view, didymous fruits are dehiscent but they are functionally indehiscent. Cardaria, Coronopus, and Stroganowia are hard to distinguish morphologically from Lepidium and the distinctions of all four genera are based solely on a few fruit characters (see above) of doubtful value. Along with preliminary molecular data it has been suggested that Cardaria, Coronopus, and Stroganowia should be united with Lepidium (Al-Shehbaz et al., 2002). However, in the provisional molecular analysis mentioned by Al-Shehbaz et al. (2002), only single representatives of the genera have been included and two other related genera, i.e. Winklera and Stubendorffia, were not considered at all. In the current study, nuclear (ITS) and cpDNA markers (trnT-trnL and trnL-trnF spacer, trnL intron) were used to construct robust phylogenies in order to learn more about fruit evolution in these taxa. Our taxon sampling of 124 species represents the full range of morphological variation in Lepidium and the five closest related genera. Our aim was to identify a suitable model system of closely related diploid (sister) species which differ in fruit morphology and dehiscence. As Lepidium and related genera are quite closely related to Arabidopsis, such a model system of diploid species should facilitate comparative genetic studies aimed at determining the molecular basis of evolutionary changes in fruit structure. The analysis presented here confirms our previous suggestions and clearly demonstrates that all related genera are well nested within Lepidium s.str.. Because not all of these genera have been assigned to Lepidium in previous analyses, the traditional names of all genera will be maintained here. Our phylogenetic analysis allowed a convenient model system for the study of fruit evolution to be identified. All

Fruit evolution in Brassicaceae | 1505

Fig. 1. Phylogenetic relationships among Lepidium species and related genera based on ITS sequence analysis. These genera are nested within and should be transferred to a broader defined Lepidium s.l.. For a better understanding of a broader readership, however, the traditional names of these genera are maintained in this study. Shown is the Bayesian 50% majority rule consensus tree with mean posterior probability values (clade credibilities) above branches, the distribution of species by continents and the assignment of fruit characters among the genera. Roman numerals I, II, and III refer to the main phylogenetic lineages. In Winklera, fruits may open but they stay indehiscent for a longer period and probably not all fruits finally open. In W. silaifolia, opening fruits are didymous. Species used for

1506 | Mummenhoff et al. Cardaria species with indehiscent fruits were found to be closely related to Lepidium species with dehiscent fruits. The diploid species Cardaria pubescens C.A. Meyer (Jarmolenko)¼Lepidium appelianum Al-Shehbaz with indehiscent fruits and Lepidium campestre (L.) R.Br. with dehiscent fruits were selected for anatomical analysis of the fruit. It has been possible to demonstrate that the anatomy of dehiscent Lepidium and indehiscent Cardaria fruits agrees completely with the pattern of Arabidopsis wild-type (dehiscent) and mutant (indehiscent) fruit types, respectively, suggesting hypotheses for the developmental genetic basis of the morphological difference that can be experimentally tested.

starting from different randomly chosen trees and with four independent chains with a heating temperature set to 0.2. Each chain was run for 2 500 000 update cycles and the chain states were sampled every 100th cycle. The first 6250 samples were discarded as the ‘burn-in’ of the Markov chain. The values sampled for different parameters were examined using TRACER 1.3 (Rambaut and Drummond, 2003). Maximum likelihood searches were conducted using a fast maximum likelihood ratchet approach (Morrison, 2007) as implemented in PRAP v.2.0 (Mu¨ller, 2004). PRAP-generated command files were handed over to PAUP (Swofford, 2002) as described by Wall et al. (2008).

Study group Ninety-seven Lepidium L. species from all continents representing all infrageneric taxonomic entities (Thellung, 1906) were analysed. Furthermore, species were included of the most closely related genera of Lepidium, i.e. Coronopus (eight species representing each of the four sections; AlShehbaz, 1986), Cardaria (two species, one with two subspecies), Winklera (both species; Schulz, 1936; Czerepanov, 1995), Stroganowia (ten species of each of the two sections; Botschantsev, 1984), and Stubendorffia (four species of the two sections; Bush, 1939). Thus, our taxon sampling of 124 species represents the full range of morphological variation in Lepidium and related genera, and the entire geographic distribution of these taxa on all continents. Following Mummenhoff et al. (2001), Hornungia petraea (L.) Reichenbach was used as an outgroup. Sources of plant material are given in Table S1 in the Supplementary data at JXB online. Methods for DNA extraction, PCR, and direct sequencing of ITS and non-coding cpDNA (trnT/trnL spacer, trnL intron, trnL/trnF spacer) have been presented elsewhere (Bowman et al., 1999; Mummenhoff et al., 2001). The DNA sequences were aligned by hand, and regions of ambiguous alignment eliminated. Insertions/deletions (indels) were treated as missing data from the data matrix.

Phylogenetic analyses The model of nucleotide substitution for the Bayesian analysis of phylogeny that best fitted the data was determined using MrModelTest 2.2 software (Posada and Crandall, 1998) and the Akaike information criterion (AIC). Bayesian inference of phylogeny was performed using MrBayes 3.1 (Huelsenbeck and Ronquist, 2003). Following MrModelTest, the symmetrical model of sequence evolution (SYM) was used in MrBayes, with an allowance for a gamma (G) distribution of rates and a proportion of invariant sites. Two separated runs were conducted, each

Anatomical analysis of fruits Infructescences from L. campestre with dehiscent fruits and C. pubescens (also known as L. appelianum) with indehiscent fruits were harvested from plants grown from seeds of known wild origin and stored in 70% (v/v) ethanol until further use. Fruits of both species were taken at different ontogenetic stages. The floral morphogenesis and fruit development of the model plant Arabidopsis has been described in detail (Spence et al., 1996). Smyth et al. (1990) divided the early flower development of Arabidopsis from initiation of the flower until anthesis into 13 stages, and this study has been used as a reference for the description of many mutants and the characterization of numerous Arabidopsis genes. Ferra´ndiz et al. (1999) divided post-fertilisation fruit development of Arabidopsis into seven further ontogenetic stages. In the current study, this approach has been followed and fruits of L. campestre and Cardaria pubescens have been harvested from stage 14 (beginning of fruit development subsequent to pollination) to stage 19 (fruit valves begin to separate from the replum). Transverse fruit sections approximately 10 lm in thickness were cut by hand using a single-sided razor blade. Each section was immersed for 1 min in phloroglucinol (mixed as described by Braune et al., 1994) and afterwards in diluted hydrochloric acid. Thickened and lignified cell walls were stained red. Transverse sections were analysed by a Leica DM/LS light microscope. A Sony F717 digital camera was used for photographic documentation.

Phylogeny of Lepidium and related genera as revealed by molecular markers A comprehensive molecular systematic analysis has been performed in order to identify an appropriate model system for the analysis of the regulation of fruit dehiscence/ indehiscence in wild species. After eliminating regions with

anatomical fruit analysis, i.e. Cardaria pubescens and Lepidium campestre, are underlined and printed in bold type. Abbreviations: EA, Eurasia; AF, Africa; Aus/NZ, Australia/New Zealand; NA/ZA, North America/Central America; SA, South America; Pa, Pacific Islands (Hawaii); Did, didymous. Hornungia petraea was used as the outgroup.

Fruit evolution in Brassicaceae | 1507 ambiguous alignment, the aligned ITS data matrix consisted of 482 characters of which 285 were variable and 197 potentially informative. For the cpDNA marker, 1605 nucleotide positions were available for phylogenetic analysis, of which 563 characters were variable, and 235 potentially informative. The Bayesian tree generated from cpDNA or ITS data set was an almost exact match of the respective maximum– likelihood tree, with the exception of slight differences in branching among some terminal nodes. These differences, however, did not have any impact on the phylogenetic conclusions drawn in this study and, therefore, only the Bayesian tree for the cpDNA- and ITS data is shown. Both phylogenies consistently detect three main phylogenetic lineages, I–III. The grouping in lineage II generally follows the geographic distribution of the species by continents (Fig. 1). The minor incongruence of species relationships within lineage II based on two separate molecular markers, one of which is maternally inherited (cpDNA) and the other biparentally inherited, but with a potential for concerted evolution (ITS) (Wendel, 2000; Soltis et al., 2008), and the large number of polyploid species both suggest that the evolutionary history of Lepidium is reticulate (Mummenhoff et al., 2001, 2004; Lee et al., 2002). This was explicitly shown for the origin and evolution of the Australian/New Zealand endemic Lepidium species (Mummenhoff et al., 2004). The incongruence in the position of a group of three Stubendorffia and one Stroganowia species in the cpDNA(within lineage II) and ITS tree (within lineage I) also indicate a reticulate history, but will not be discussed further here. Both phylogenies clearly demonstrate that the genera suggested to be closely related to Lepidium, i.e. Cardaria, Coronopus, Stroganowia, Stubendorffia, and Winklera, are all well nested within the three main lineages of a broader defined Lepidium, and only one of these related genera (Winklera) is monophyletic. Instead of being separate from Lepidium, species of these genera group along with Lepidium species of the same continental distribution (Fig. 1). To accommodate a broader readership we have, however, maintained the traditional names of these genera here. In the traditional classification systems, these taxa have been recognised as separate genera mainly based on differences in fruit morphology. Therefore, fruit characters have been mapped onto the phylogenetic tree based on molecular markers (Fig. 1). The evolution of different fruit types was detected within one lineage (indehiscent and dehiscent fruits in Lepia) and parallel or convergent evolution of the same fruit type in different lineages (indehiscent fruits in Lepia and different clades of Lepidium s.str.; Fig. 1). Numerous studies (summarised in Koch and Mummenhoff, 2006) have amply demonstrated that morphological characters in the Brassicaceae, especially fruit characters, are highly homoplasious. So the questions arise: exactly how fluid is evolution of the fruit structure in Brassicaceae? Why is fruit form so variable in the Brassicaceae, and what is the underlying genetic basis for this variation? It has previously been hypothesised that dramatic differences in morphology

may result from mutations of so-called major morphogenetic genes, not accompanied by adequate change in the molecular marker system (Kadereit, 1994; Theißen et al., 2000; Bateman and DiMichele, 2002). The idea that major genes might be important in evolution is not new and has already been expressed by de Vries (1901) and Goldschmidt (1940). In the last few years developmental geneticists provided experimental support that the genetic basis of dramatic change in morphology might be relatively simple. A good example is the teosinte branched 1 (tb1) gene in maize and teosinte. Differences in the expression of tb1 result in very different morphotypes (Doebley, 2004). The distribution of dehiscent/indehiscent fruits was used as a key diagnostic character in traditional Brassicaceae classification to separate, for instance, Cardaria and Coronopus from Lepidium. In our phylogenetic tree, all Cardaria species with indehiscent fruits are sister to Lepidium species with dehiscent fruits (e.g. L. campestre). C. pubescens and L. campestre have been selected for further anatomical analysis of the fruit for the following reasons: both species are closely related but they differ in fruit morphology. The two species of this model system are diploid (2n¼2x¼16) and they are found in the same major phylogenetic lineage as Arabidopsis (Beilstein et al., 2006) facilitating comparative genetic studies.

Anatomical basis of dehiscence in the Arabidopsis fruit Our first aim was to compare the anatomical pattern during fruit development of dehiscent and indehiscent fruit types in Arabidopsis (wild-type and mutant plants) and the Cardaria/ Lepidium model system. The development of the fruit in the model plant, Arabidopsis, provides an excellent system for analysing the mechanism that patterns a plant organ because of the distinctive morphological landmarks and the availability of reporter lines that mark specialised tissues (Dinneny et al., 2005). The region of the fruit that encloses the seeds is divided into three zones: the fruit valve, the replum, and the fruit valve margin (Fig. 2A). The valves are the seed pod walls and they encircle the developing seeds. The valves are connected with the replum, which forms the middle ridge of the fruit. A specialised strip of tissue, the valve margin, forms at the valve–replum boundary which is specialised for fruit opening, i.e. the detachment of the valves from the replum. The valve margin consists of two different cell types. On the replum side of the valve margin, the separation layer (sl) forms, which is responsible for the detachment of the valve from the replum through cell–cell separation. On the valve side of the margin, a layer of rigid lignified cells forms (lignified valve margin cells: vmc) which is connected with an adjacent layer of lignified tissue at the inner side of the fruit valves, termed the endocarp layer b (enb). The region of lignified valve margin cells, together with the separation layer is termed the dehiscence zone. A spring-like tension within the drying fruit eventually results in fruit opening and fruit valves detach from the replum by

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Fig. 2. Anatomy (cross-sections) of dehiscent and indehiscent fruits of Arabidopsis wild-type (A, B) and mutant plants (D, E), Lepidium campestre (C), and Cardaria pubescens (F). (A) Diagrammatic view of Arabidopsis wild-type gynoeceum after fertilization (left) and during fruit dehiscence together with factors contributing to fruit opening (right). (B) Section of dehiscence zones at the fruit valve margin of an Arabidopsis wild-type stage 17B fruit. Lignified cell walls have been traced in pink for clarity, and the fracture surface in the separation layer is noted by a blue line. (C) The valve margin region of L. campestre fruits at stage 19. Lignified tissue is selectively stained red by phloroglucinol treatment. As in the Arabidopsis wild-type fruit (B), the dehiscence zone is clearly visible, consisting of a strip of lignified valve margin cells (vmc), in addition to the lignified endocarp layer b (enb) and the vascular bundles of the replum. Cell–cell separation within the separation layer indicating the detachment of the fruit valves from the replum is marked by arrowheads. (D) Arabidopsis 35S::FUL mutant fruit (stage 17). Due to ectopic expression of FUL in the fruit valve margin, the dehiscence zone (sl, vmc) is not properly established resulting in indehiscence. (E) In Arabidopsis ind ful fruits (stage 17), the dehiscence zone is not formed. In addition, ectopic valve lignification does not occur. (F) In indehiscent fruit of C. pubescens (stage 17B) and Arabidopsis mutants (D, E), the dehiscence zone is not formed. Instead, a continuous strip of lignified cells (marked by an arrowhead) is stretching from one replar vascular bundle to the other one at the opposite side of the fruit. For genetic regulation of fruit valve margin differentiation see Fig. 3 and text. dz, dehiscence zone; ena, endocarp layer a; enb, endocarp layer b; r, replum; sl, separation layer; v, fruit valve; vb, vascular bundle; vmc, lignified valve margin cells. Scale bars: 100 lm. Reprinted and modified with permission from Macmillan Publishers Ltd, Liljegren et al. (2000) SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404, 766–769 (A), Oxford University Press, Ferra´ndiz (2002) Regulation of fruit dehiscence in Arabidopsis. Journal of Experimental Botany 53, 2031–2038 (B), American Association for the Advancement of Science, Ferra´ndiz et al. (2000) Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science 289, 436–438 (D), Elsevier, Liljegren et al. (2004) Control of fruit patterning in Arabidopsis by INDEHISCENT. Cell 116, 843–853 (E).

Fruit evolution in Brassicaceae | 1509 autolysis of middle-lamellae within the separation layer (Fig. 2A).

knocked out, or the ectopic expression of other genes in the valve margin suppresses the expression of valve margin determinating genes (for details, see below).

Lepidium campestre versus Cardaria pubescens: anatomical analysis of dehiscent versus indehiscent fruits

Evolutionary developmental genetics of fruit dehiscence: general aspects

Following Fig. 2A, a sketch-map allowing orientation within the photographic illustrations of fruit transverse sections was created. Each photograph contains a complete, idealised, and exactly adjusted transverse section of the fruit under study, with cell types coloured as in Fig. 2A. Tissues shown in the respective photographic illustrations of fruit cross-sections are boxed in the corresponding sketch-map in the right corner of each photograph. Transverse sections of the replum-valve region of fruits of these two taxa demonstrate clear differences, although L. campestre and C. pubescens are closely related (Fig. 1). The anatomy of the replum-valve region of L. campestre dehiscent fruits (Fig. 2C) closely resembles the dehiscent wild-type fruit anatomy of Arabidopsis (Fig. 2B) (Liljegren et al., 2004). Welldeveloped dehiscence zones are apparent on both sides of the replar vascular bundles. Unlignified and, with increasing fruit ripening, successively lignified valve margin cells and endocarp layer b cells are discernible; at ontogenetic stage 19, lignification of endocarp layer b and the valve margin cells is completed and thus both tissues are connected to form a lignified strip of cells around both sides of the replum. Xylem cells of the replar vascular bundle lignify concomitantly with valve margin cells and endocarp layer b (Fig. 2B, C). Although the corresponding species are very closely related, transverse sections of indehiscent fruits of C. pubescens do not exhibit typical traits of the dehiscent L. campestre fruits. There are neither lignified valve margin cells nor the typical separation layer, although the replum is visible on the outside (Fig. 2F). One process associated with fruit evolution within the Brassicaceae is the loss of dehiscence through non-development of a separation tissue (Zohary, 1948). This is clearly seen in those indehiscent fruits in which the replum and the valves are still well marked. Furthermore, in Cardaria fruits, there is a continuous strip of lignified cells stretching from one replar vascular bundle to the other at the opposite side of the fruit, not interrupted by a dehiscence zone as in the dehiscent Lepidium fruits. It is interesting to note that the anatomy of indehiscent C. pubescens fruits (Fig. 2F) strongly resembles the anatomical pattern of indehiscent fruits of some Arabidopsis loss-offunction mutants (e.g. Fig. 2E). As in some Arabidopsis mutant (indehiscent) fruits, no dehiscence zone is formed. Instead, a continuous strip of lignified cells (enb) stretches from one replar vascular bundle to the other at the opposite side of the fruit (Fig. 2F). In the Arabidopsis mutant fruits, the dehiscence zone is not properly formed because the genes responsible for their proper establishment are

An important goal of contemporary evolutionary developmental biology (evo-devo, for short) is to identify the molecular genetic changes that led to evolution at the morphological level. One question of general importance from a heuristic point of view is whether mutations causing one taxon to mimic another (‘phylomimicking mutations’) define evolutionary relevant loci (Haag and True, 2001). Only if this is true in many cases, are candidate gene approaches effective strategies to clone genes of evolutionary importance. It is argued here that the genus Lepidium s.l. (with related genera included), for a number of reasons, is well-suited to address this question, using valve margin development and fruit dehiscence as exemplary characters. Firstly, Lepidium s.l. is closely related to Arabidopsis, which facilitates the transfer of knowledge and tools from one system to the other. Secondly, some Arabidopsis mutants develop indehiscent fruits that resemble the indehiscent fruits of the wildtype in some Lepidium s.l. species down to the cellular details (Fig. 2), making it conceivable that the genes which mutated during the evolution of fruit (in)dehiscence in Lepidium s.l. are orthologues of the phylomimicking Arabidopsis genes. Thirdly, dehiscent fruits are ancestral in Lepidium s.l and fruit indehiscence evolved several times independently within Lepidium s.l., enabling the study of the molecular mechanisms behind parallel or convergent evolution. Typical evo-devo approaches employ detailed knowledge about a developmental process of interest from a wellstudied model system, i.e. in this case Arabidopsis.

Regulation of valve margin development in Arabidopsis Based on mutant analysis, much has been learned in recent years about the molecular basis of fruit patterning in Arabidopsis (Fig. 3). Genes which control the differentiation of tissues during fruit development can be divided into two classes, those that promote valve-margin development and those that repress it (Dinneny and Yanofsky, 2004). The functionally redundant genes SHATTERPROOF1 and 2 (SHP1, 2), encoding MADS-domain proteins, are on top of a gene regulatory network composed of transcription factors that specify valve margin identity (Liljegren et al., 2000). Double loss-of-function mutants in which SHP activity is eliminated (shp1 shp2) develop indehiscent fruits that lack lignified valve margin cells and separation layer development. Both downstream and in parallel to SHP act INDEHISCENT (IND) and ALCATRAZ (ALC), two genes

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Fig. 3. Genetic pathway controlling fruit development in Arabidopsis. The differentiation of tissues is controlled by two sets of genes that either promote valve margin development, i.e. SHP1, 2 (MADS-box genes), IND and ALC (basic helix-loop-helix-type transcription factor genes) or restrict it from surrounding regions (FUL, RPL). Proper establishment of the dehiscence zone is controlled by SHP1, 2, IND, and ALC. IND, and ALC appear to act largely downstream of SHP1,2. SHP1, 2 and IND control the separation layer and lignified valve margin cell development, while ALC is controlling only the separation layer. The expression of these four genes is restricted to the valve margin through repression by FUL (MADS-box gene) in the valves, and by RPL (BELL-subfamily homeobox gene) in the replum. JAG, FIL, and YAB3 function redundantly to promote the expression of the valve margin identity genes and FUL. These activities are negatively regulated by RPL in the replum (Dinneny and Yanofsky, 2004; Dinneny et al., 2005). dz, dehiscence zone; enb, endocarp layer b; sl, separation layer; vmc, lignified valve margin cells. Reproduced and modified by permission of the Company of Biologists: Dinneny et al. (2005) Development 132, 4687-4696.

which encode basic helix-loop-helix-type (bHLH) transcription factors. IND and ALC also specify valve margin identity, with IND playing a crucial role in both lignified and separation layer development, while the function of ALC is restricted to controlling separation layer development (Rajani and Sundaresan, 2001; Liljegren et al., 2004). The expression of these ‘valve margin identity genes’ is restricted to the valve margin because of repression by FRUITFULL (FUL), another MADS-domain transcription factor, in the valves, and REPLUMLESS (RPL), a BELL-family homeodomain transcription factor, in the replum (Gu et al., 1998; Ferra´ndiz et al., 2000; Roeder et al., 2003). In ful loss-offunction mutants, the valve margin identity genes become ectopically expressed in the valves, which leads to a valve margin-like development, including the ectopic formation of lignified and separation layer-like cell types (Ferra´ndiz et al., 2000). Ectopic expression of FUL in the fruit valve margin in transgenic plants (35S::FUL) also results in indehiscent fruits (Fig. 2D) because FUL prevents the expression of SHP1, SHP2, IND, and ALC genes which are responsible for the proper establishment of the dehiscence zone. In rpl loss-of-function mutants, ectopic expression of SHP in the replum causes the valve margins

to coalesce into a single strip of tissues in place of the replum, leading to partially indehiscent fruits. Interestingly, the MADS-box genes SHP and FUL have ancestral functions in ovule and inflorescence meristem development, respectively, and have been recruited for derived functions during fruit development more recently (Theißen, 2000, 2001). Comparatively little is known about how the patterns of valve margin identity genes are initially established. However, it could already be demonstrated that SHP gene expression is activated by AGAMOUS, a MADS-box gene which specifies carpel identity (Savidge et al., 1995). Moreover, it was shown that FILAMETOUS FLOWER (FIL) and YABBY3 (YAB3), two YABBY genes which encode transcription factors, control the non-overlapping expression patterns of FUL and SHP (Dinneny et al., 2005). FIL and YAB3 activate the valve margin identity genes redundantly with JAGGED (JAG), a gene which encodes a C2H2 zinc-finger transcription factor that promotes leaf blade growth, suggesting that several genetic pathways converge to regulate these genes (Dinneny et al., 2005). The activities of FIL, YAB3, and JAG are negatively regulated by RPL, which divides FIL/JAG activity, thus generating two distinct strips of valve margin. YABBY genes control the polarity of tissues in lateral organs thus revealing a functional link between polarity establishment and the regulation of tissue identity in the developing Arabidopsis fruit (Dinneny et al., 2005).

Analysing the genetic basis of the origin of indehiscent fruits in Lepidium s.l. To further our understanding of the molecular mechanisms behind the transition from dehiscent to indehiscent fruits in the lineage that led to C. pubescens, the orthologues of six candidate genes, ALC, FUL, IND, RPL, SHP1, and SHP2, have been cloned from both L. campestre and C. pubescens. These genes have been chosen, because they are well-known to be involved in the differentiation of the dehiscence zone in the Arabidopsis fruit, and because the position of these genes within the gene regulatory network specifying valve margin identity is quite well established (Fig. 3). Moreover, anatomical analysis of the fruits of C. pubescens (Fig. 2F) revealed striking similarities to Arabidopsis mutants or transgenics in which the expression of these genes is changed, including shp1 shp2, ind, and ind ful (Fig. 2E) loss-of-function mutants as well as 35S::FUL transgenic plants (Fig. 2D) (Ferra´ndiz et al., 2000; Liljegren et al., 2000, 2004). Particularly because of the close relationship between Arabidopsis and Lepidium s.l. these findings strongly increase the likelihood that the candidate genes are involved in the origin of fruit indehiscence in Cardaria. To narrow down the number of promising candidate genes, their expression will be compared during development of the dehiscent fruits of L. campestre and the indehiscent fruits of C. pubescens using diverse techniques, including in situ hybridization analyses. The results may provide us with

Fruit evolution in Brassicaceae | 1511 clues as to whether changes in the temporal or spatial expression patterns of some genes contributed to the evolution of indehiscence. Only genes that are differentially expressed in L. campestre and C. pubescens will be selected for the next series of experiments. To distinguish direct from indirect developmental genetics causes, and to assess the contribution of cis- and transregulatory changes, reciprocal heterologous transformation experiments will be carried out. If transformation protocols for L. campestre and C. pubescens can be established, preferably by the ‘floral dip’ method, that works for a number of Brassicaceae already (Clough and Bent, 1998; Bartholmes et al., 2008), transformation experiments will include these species. General strategies to identify the genetic causes of morphological evolution have been reviewed by Baum (2002) and were successfully applied to the analysis of rosette flowering in diverse Brassicaceae taxa by Yoon and Baum (2004). Specifically, once promising candidate genes have been identified which are differentially expressed in the ancestral (L. campestre) and derived species (C. pubescens) in a way that makes their involvement in the evolution of fruit dehiscence conceivable, the genomic loci, including putative regulatory regions upstream and downstream of the coding regions (such as promoters), will be cloned from the derived species and transformed into the ancestral species and into Arabidopsis. To understand the rationale behind these experiments better, let us consider one example. Let us assume that gene expression studies suggest that heterotopic expression of the FUL orthologue of C. pubescens (CpFUL) in the valve margin is involved in the origin of indehiscent fruits. In that case, it appears likely (even though not certain) that CpFUL exerts a dominant and gain-of-function effect when brought into Arabidopsis or L. campestre. So the CpFUL locus (including regulatory sequences) would be transformed into the ful mutant of Arabidopsis. As controls, the FUL locus of L. campestre (LcFUL) and of Arabidopsis (FUL) would also be transformed into the ful mutant. If Arabidopsis ful FUL and LcFUL transgenics and their descendants show wildtype phenotypes, this would indicate that Arabidopsis and Lepidium s.l. are not too distantly related for these kinds of complementation experiments. If, then, Arabidopsis ful mutants carrying the CpFUL transgene develop indehiscent fruits as do C. pubescens, this would indicate that changes in cis-regulatory sites of the CpFUL gene caused the transition from dehiscent to indehiscent fruits. If Arabidopsis ful CpFUL plants develop dehiscent wild-type fruits, however, this would suggest that changes in trans-acting factors caused the changes in the expression (and possibly function) of the CpFUL gene. If possible, CpFUL should also be transformed into L. campestre (or a lcful mutant generated, for example, by RNAi), which may result in indehiscent fruits if changes in cis-regulatory sites of the CpFUL gene caused the transition from dehiscent to indehiscent fruits. In this case, transformation of the ancestral LcFUL into the wild-type C. pubescens should not bring about a mutant

phenotype, but C. pubescens cpful LcFUL plants should develop dehiscent fruits like L. campestre. If, however, changes in trans-acting factors caused the transition from dehiscent to indehiscent fruits, C. pubescens cpful LcFUL plants should develop indehiscent fruits like C. pubescens wild-type plants do.

Fruit indehiscence in Lepidium s.l. to study parallel and convergent evolution Indehiscent fruits evolved independently several times within Brassicaceae (Appel and Al-Shehbaz, 2003; Fig. 1). In general, the repeated evolution of specific morphologies could be due to parallel or convergent evolution. According to the definition reviewed by Yoon and Baum (2004), parallelism refers to the independent evolution of the same derived trait via the same developmental changes, whereas convergence refers to superficially similar traits that have a distinct developmental basis. Thus, if the molecular genetic basis of fruit indehiscence is analysed for additional pairs of closely related species with indehiscent and dehiscent fruits, it will also be possible to determine whether the independent evolution of indehiscent fruits represent cases of parallel or convergent evolution.

Supplementary data Supplementary data are available at JXB online. Supplementary Table S1. Lepidum (Brassicaceae) as a model system for studying the evolution of fruit development in wild species.

Acknowledgements We thank Ulrike Coja for technical assistance, numerous herbaria and collectors for providing plant material, the staff of the Botanical Garden Osnabru¨ck for plant cultivation, Andreas Franzke for critical discussion, and Lucille Schmieding for correcting style and grammar. Many thanks also to Teresa Lenser, Pia Nutt, and Diana Lobbes for their contributions to the fruit dehiscence project. Work in the authors’ laboratories on the evo-devo of fruit dehiscence in Brassicaceae is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to KM (MU 1137/8-1) and GT (TH 417/6-1).

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