MASARYK UNIVERSITY Faculty of Science and CEITEC

Chromosomal collinearity and karyotype

evolution in crucifers (Brassicaceae)

Ph.D. Thesis

Terezie Mandáková

Supervisor: Doc. Mgr. Martin A. Lysák, Ph.D. Brno 2013

2 Acknowledgements

I wish to thank Dr. Zuzana Münzbergová for introducing me into the mystery of plant science. I also have to thank Dr. John Bailey and other colleagues in the Heslop- Harrison’s lab for opening me the doors into the world of cytogenetics. It is difficult to overstate my gratitude to my supervisor Dr. Martin Lysák for giving me the opportunity to work in his group, for uncovering the discreet charm of cruciferous plants and their tiny chromosomes, for introducing me into the international scientific community, for constant guidance, inspiration and continuous support. I would like to give my thanks to colleagues of the Lysák lab for daily support, pleasant working environment and friendship. Particularly, I would like to thank all hardworking technicians accompanying me during last six years, Šarlota Štemberová Kateřina Toufarová and Petra Přikrylová, for their help with DNA isolation and labeling. I thank my colleague Petra Hloušková for her help with sequence data analyses. I am also grateful to Dr. Petr Mokroš and all members of the Fajkus lab for their support, willingness and help. I would like to thank all my international collaborators and co-authors. I wish to express my gratitude to my family and friends, they have been a great support for me.

This work was supported by the Grant Agency of the Czech Academy of Science (project no. KJB601630606, IAA601630902), the Czech Science Foundation (project no. P501/12/G090), the European Regional Development Fund (project no. CZ.1.05/1.1.00/02.0068), and the Systematics Research Fund. .

3 Bibliographic entry

Author: Terezie Mandáková Research Group Plant Cytogenomics Central European Institute of Technology (CEITEC) Masaryk University

Title of PhD thesis: Chromosomal collinearity and karyotype evolution in crucifers (Brassicaceae) Degree programme: Biology Field of Study: General and Molecular Genetics Supervisor: Doc. Mgr. Martin A. Lysák, Ph.D. Year of defence: 2013 Keywords: chromosomal collinearity, karyotype evolution, Brassicaceae, polyploidy, whole-genome duplication, chromosome rearrangements, comparative chromosome painting

4 Bibliografický záznam

Autor: Mgr. Terezie Mandáková Výzkumná skupina Cytogenomika rostlin Středoevropský technologický institut (CEITEC) Masarykova univerzita

Název disertacní práce: Evoluce karyotypu vybraných zástupců z čeledi Brassicaceae Studijní program: Biologie Studijní obor: Obecná a molekulární genetika Školitel: Doc. Mgr. Martin A. Lysák, Ph.D. Rok obhajoby: 2013 Klícová slova: chromosomová kolinearita, evoluce karyotypu, Brassicaceae, polyploidie, celogenomové duplikace, chromosomové přestavby, srovnávací malování chromosomů

5 Abstract

The Brassicaceae (Crucifereae) comprises 49 tribes, 321 genera and 3660 species, and belongs to the largest plant families. Whole-genome sequencing of the model plant Arabidopsis thaliana fuelled the interest of scientists in the mustard family as well as rapid development of comparative phylogenomics and cytogenomics, including the invent of chromosome painting in A. thaliana and comparative chromosome painting (CCP) in other Brassicaceae species. The Brassicaceae is the only plant family in which large-scale CCP is feasible. CCP provides unique insights into the karyotype and genome evolution in plants by comparing chromosome collinearity, identification of chromosome rearrangements, construction of comparative cytogenetic maps, and reconstruction of ancestral karyotype structures. This PhD thesis deals with the karyotype evolution in the Brassicaceae family uncovered by comparative chromosome painting. The introductory part is divided into four chapters. The first chapter introduces chromosomes, karyotypes, karyotypic variation, and the role of chromosome rearrangements and polyploidy in the karyotype and genome evolution. In the methodical second part, principles of chromosome painting are described. The core third chapter focuses on comparative cytogenomics in the Brassicaceae, and summerizes the current knowledge on karyotype and genome evolution in this family. The final chapter provides comparison of karyotype evolution in the Brassicaceae with evolutionary trends in other plant families. Eight publications document author’s contribution in the following research areas: 1) Optimization and application of the large-scale CCP in the Brassicaceae. 2) Identification of the mechanisms responsible for chromosome number reduction. 3) Evaluation of the role of polyploidy in karyotype and genome evolution. 4) Re-examination of the role of antient chromosome rearrangements in plant speciation.

6 Abstrakt

Čeleď brukvovitých (Brassicaceae, Crucifereae) patří k největším rostlinným čeledím; zahrnuje 49 tribů, 321 rodů a 3660 druhů. Zájem vědců o tuto čeleď vzrostl především díky ustanovení huseníčku rolního (Arabidopsis thaliana) modelovým druhem a sekvenování jeho genomu. To odstartovalo mimo jiné bouřlivý rozvoj srovnávací fylogenomiky a cytogenomiky, včetně úspěšného zavedení metody malování chromosomů (chromosome painting, CP) huseníčku a jejího rozšíření na další zástupce čeledi brukvovitých (srovnávací malování chromosomů, comparative chromosome painting, CCP). Metoda CCP umožňuje studium chromosomové kolinearity, rozpoznání přestaveb a rekonstrukci struktury karyotypů. Brukvovité jsou jedinou rostlinnou čeledí, u níž je v tomto rozsahu metoda CCP použitelná. Srovnávací cytogenetické mapy brukvovitých rostlin tak představují unikátní typ dat o evoluci rostlinných karyotypů a genomů. Předkládaná disertační práce se věnuje evoluci karyotypu v čeledi brukvovitých studované pomocí metody srovnávacího malování chromosomů. Úvod disertační práce je rozdělen na čtyři části. První kapitola představuje chromosomy, karyotypy, jejich variabilitu a evoluci ovlivněnou chromosomovými přestavbami a polyploidií. Druhá metodická část pojednává o principech malování chromosomů. Stěžejní třetí kapitola disertační práce shrnuje naši současnou znalost o evoluci karyotypu a genomu v čeledi Brassicaceae. Poslední kapitola poskytuje srovnání s ostatními rostlinnými čeleděmi. Osm přiložených publikací, které významně přispěly k poznání studované problematiky, dokumentuje vědecké výsledky autora disertační práce v těchto základních oblastech: 1) Optimalizace a použití metody CCP u různých brukvovitých druhů. 2) Určení mechanismu zodpovědného za snižování chromosomového počtu. 3) Studium role polyploidie v evoluci karyotypu a genomu. 4) Přezkoumání vlivu chromosomových přestaveb na rostlinnou speciaci.

7 Content

1 Chromosomes and karyotypes...... 9 1.1 The chromosome discovery ...... 9 1.2 Karyotype evolution ...... 9 1.3 Variation in the chromosome structure ...... 10 1.4 Chromosome rearrangements and speciation ...... 10 1.5 Variation in chromosome number ...... 11 1.6 The role of polyploidy in plant genome evolution ...... 12 2 Chromosome painting ...... 13 2.1 Chromosome painting in plants ...... 13 3 Comparative cytogenetics in the Brassicaceae ...... 15 3.1 Family Brassicaceae ...... 15 3.2 Chromosome painting in Arabidopsis thaliana ...... 17 3.3 Principles of comparative chromosome painting in the Brassicaceae ...... 17 3.4 Karyotype evolution in the Brassicaceae ...... 18 3.4.1 Concept of the Ancestral Crucifer Karyotype ...... 18 3.4.2 Karyotype evolution in species from the Brassicaceae lineage I and II ...... 19 3.4.3 Mesopolyploid evolution in the Brassicaceae ...... 23 3.4.4 Mechanisms of chromosome number reduction in the Brassicaceae ...... 28 3.4.5 Conclusions on the karyotype evolution in the Brassicaceae ...... 29 4 Trends in angiosperm karyotype evolution...... 30 4.1 Genome evolution in the Poaceae ...... 30 4.2 Genome evolution in the Solanaceae ...... 31 4.3 Genome evolution in the Fabaceae ...... 31 4.4 Genome evolution in the Rosaceae ...... 32 4.5 Contrasting karyotype evolution in angiosperms...... 32 5 References...... 34 6 Aims of the dissertation & author’s contribution...... 44 7 Selected publications of the author ...... 46 I. Lysak and Mandáková (2013) .……………...... ……………………………… 47 II. Lysak et al. (2010) ..………………………...... ……………..…………………61 III. Mandáková and Lysak (2008) ..…..……………....…………...……………….69 IV. Dierschke et al. (2009) ..……………………………………………...... ………83 V. Mandáková et al. (2010a) ..…………………...... ………………..….…………93 VI. Mandáková et al. (2012) ..……………………………………...... ………..….109 VII. Cheng et al., submitted ..……………………………………………...... …….123 VIII. Mandáková et al. (2010b) ..…………………………………………….…...... 153

8 1 Chromosomes and karyotypes

Motto: “I am so inconspicuous that no one can see me with the naked eye. For millenia no one knew of my existence until microscopes were developed allowing an object to be magnified as many as 1000 times. But it was not so easy. The organs and tissues, of plants and animals, had to be separated into that unit of life that is the cell. Subsequently, only by breaking the cell, could I be found, well encased in its bosom, in company with the other minute molecular structures. Lockes in this prison, with thick membranes and walls surrounding me everywhere, I looked pale, transparent and hardly distinguishable from my surroundings. Ladies, of the upper classes, had been colouring their cheeks and lips with a dye called carmine, extracted from the body of a scale-insect, which lived on the cactuses of tropical America. When a drop of carmine was placed over the disrupted cell I easily became coloured a bright red and could be well recognized.” (Lima-de-Faria 2008)

1.1 The chromosome discovery Heinrich von Waldeyer (1888) introduced the term chromosome (from the Greek chroma for colored and soma for body) to designate filaments in the cell nucleus involved in cell division (mitosis) previously described by Walther Flemming (1878). During the first decade of the twentieth century, the Mendel’s laws of heredity in which each individual presents two factors for each trait, with one factor coming from each parent, was rediscovered. In 1902, Walter Sutton (1902) observed chromosomes during meiosis to be organized in pairs, and gametes receiving only one of the two homologous chromosomes. This observation supported the idea that Mendel’s factors responsible for heredity are localized on chromosomes. Seven years later, Wilhelm Johannsen (1909) introduced the term gene (from the Greek genno for give birth) instead of Mendel’s factors to describe units of heredity. Thomas Morgan (1911) by his work on the Drosophila sex chromosome X proved Sutton’s theory (Sutton 1902) that chromosomes are carriers of genes and thus established the chromosomal theory of inheritance. In 1931, Harriet Creighton and Barbara McClintock (1931) demonstrated by an elegantly simple experiment in maize, that exchanges between genes are accompanied by exchange of cytologically visible chromosome parts, indicating that genes are physically aligned along the chromosome. Today it is a well-known fact that DNA is the hereditary material and that the vast majority of the DNA of an organism is housed in chromosomes.

1.2 Karyotype evolution The chromosomal constitution of each organism is reflected by its karyotype. It is characterized by a species-specific number of chromosomes of particular size and shape. Karyotypes may change via primary and secondary chromosome rearrangements (chapter 1.3 and 1.4), as well as ploidy mutations (chapter 1.5 and 1.6). Because karyotypes are dynamic structures, the reconstruction of ancestral karyotypes based on well described extant karyotypes is necessary for the understanding of trends in karyotype evolution (Schubert and Lysak 2011). The development of complementary techniques for studying chromosomes made possible to reconstruct and compare karyotypes between species, genera and even families to the extent not feasible before. The study of chromosome collinearity, and the identification of chromosome rearrangements became feasible with the invent of diverse chromosome banding techniques (reviewed by Bickmore 2001) and, more recently, fluorescence in situ hybridization methods like comparative chromosome

9 painting in mammals (reviewed by Ferguson-Smith and Trifonov 2007) and plants (e.g., Brassicaceae and grasses; Mandáková and Lysak 2008, Mandáková et al. 2010a, b, 2012, Idziak et al. 2011). Alternatively, karyotype and genome evolution can be studied by comparative genetic mapping (i.e., Devos and Gale 1997, Duran et al. 2009, Gale and Devos 1998, Paterson et al. 2000, Salse et al. 2008) and comparative analyses of whole-genome sequences (e.g., Cannon et al. 2009, Ming et al. 2008, Salse 2012, Schmutz et al. 2010).

1.3 Variation in the chromosome structure Structural chromosome alterations are the result of primarily or secondary rearrangements. Primary rearrangements are caused by illegitimate recombination during double-strand break repair, either via direct joining of ends between different double-strand breaks, or through recombination with ectopic homologous sequences. Primary rearrangements have breakpoints preferentially within heterochromatic regions enriched with repetitive sequences (reviewed by Bzymek and Lovett 2001, Lönnig and Saedler 2002, Schubert and Lysak 2011). Primary chromosome rearrangements are insertion, deletion, duplication, peri- and paracentric inversion, and intra- or interchromosomal reciprocal translocation. Deletion is the loss of genetic material. Two breaks can produce an interstitial deletion. In principle a single break can cause a terminal deletion but, because of the need for telomeres, it is likely that terminal deletions in fact include two breaks, one close to the telomere. Deletions are tolerated only in polyploids or when dispensable sequences are involved (Schubert and Lysak 2011). Duplication results in the origin of an additional copy that is free from selective pressure. The fate of the duplicated DNA, is either genetic deterioration of one of the gene copies, resulting in the formation of pseudogenes, or divergence in the function of the gene copies from each other (Schubert and Lysak 2011). Inversion occurs when a chromosomal segment is excised and reintergrated in opposite orientation into the same position, causing a reversed gene order. Paracentric inversions do not include the centromere and both breaks occur in one arm of the chromosome. Pericentric inversions include the centromere and there is a break point in each arm. Homozygotes for an inversion can have new linkage relations. When synapsis occurs in an inversion heterozygote, a loop is often formed. This looping tendency has a key evolutionary effect on crossover suppression within the inverted region (Kirkpatrick 2010, chapter 1.4). Reciprocal translocation is an aberration during which chromosomes mutually exchange segments. If it does not result in any loss or gain of chromosome material, the translocation is described as balanced (Schubert and Lysak 2011). Translocations have two genetic consequences. Translocation increases the linkage distance between two genes if a segment is translocated between the genes. Translocations can also define new linkage relationships among these genes (Rabkin and Janz 2008).

1.4 Chromosome rearrangements and speciation In plants, polyploidy and chromosome rearrangements are principal evolutionary forces generating the karyotypic variation. Whereas the role of polyploidy in reproductive isolation and plant speciation was unequivocally recognised (e.g., Comai 2005, Soltis et al. 2007), the evolutionary significance of chromosome rearrangements is less clear. A rare individual with a chromosome rearrangement generally shows reduced reproductive fitness (underdominance) and it will preferentially mate only with individuals not bearing the rearrangement. Hence, such plant is expected to be

10 eliminated from the population. Conversely, if the rearrangement is only weakly underdominant, it is not likely to cause a reproductive isolation leading to speciation. Regardless these caveats, chromosome repatterning has been often thought as key player in the intraspecific genetic diversification and plant speciation, having impact on the establishing of reproductive isolation barriers between populations (Faria and Navarro 2010, Levin 2002, Rieseberg 2001). The role of chromosome rearrangements in reproductive isolation has been described by the suppressed recombination model of speciation (Faria and Navarro 2010, Navarro and Barton 2003, Rieseberg 2001). This model explains the formation of reproductive isolation barriers through chromosome rearrangements impeding or inhibiting recombination. In the rearranged regions, alleles involved in reproductive isolation are accumulated which leads to increased levels of divergence (Faria and Navarro 2010). The recombinational speciation model is mainly based on studies in dipteran insect (e.g., Navarro and Barton 2003, Noor et al. 2001, Ranz et al. 2007) and sunflowers of the genus Helianthus (e.g., Gross and Rieseberg 2005, Lai et al. 2005, Rieseberg 2001). Lexer and Widmer (2008) reviewed the studies of the reproductive isolation genetics in monkeyflower, sunflower, iris, poplar and campion. They concluded that except sunflower, chromosome rearrangements are of limited importance in the origin of reproductive isolation barriers in plants. Recently, however, Lowry and Willis (2010) showed an inversion to be associated with the morphological and flowering time differences between populations of the yellow monkeyflower (Mimulus guttatus), and thus contributing to the adaptation and reproductive isolation between the annual and perennial ecotypes of the species. Besides sunflowers and monkeyflower, there are still limited data prooving the impact of chromosome rearrangements on reproductive isolation and speciation in plants which is probably caused by a limited number of comparative genetic maps in several congeneric species and technical impediments. Due to the feasibility of comparative chromosome painting in the mustard family, crucifers represent a superior plant group to address questions on the causal link between chromosome rearragements, intraspecific karyotypic variation, potential reproductive isolation barriers and speciation.

1.5 Variation in chromosome number An organism is considered as euploid when somatic cells contain two complete sets of homologous chromosomes (diploid, 2n) and gametes have half of this number (haploid, n). The ploidy level of cells can vary by an increase in complete chromosome sets (polyploidy) or by gain or loss of individual chromosomes (aneuploidy). Polyploids have been classified on the basis of genetic similarity. If a polyploid has the same genome in multiple copies, Stebbins (1947) has described it as an autopolyploid whereas if it has two distinctive genomes it has been described as an allopolyploid. Grant (1981) and others (Ramsey and Schemske 1998, 2002) proposed that autopolyploids arise within populations, whereas allopolyploids are the products of crossings between species. Polyploidy is more common in plants than in animals (Mable 2004). It can be restricted only to a specific tissue or group of cells, i.e., endopolyploidy (e.g., trichomes in plants or salivary glands of insect). Polyploidy can arise from i) a spontaneous somatic chromosome doubling during mitosis, ii) a non-disjunction/non-reduction of homologous chromosomes during meiosis resulting in unreduced gametes, or iii) in animals through the multiple fertilization of a single egg (reviewed by Ramsey and

11 Schemske 2002). Polyploidy can also be artificially induced by treatment with drugs inhibiting cell division (e.g., colchicine, an inhibitor of a microtubule polymerization).

1.6 The role of polyploidy in plant genome evolution Polyploidy (i.e., whole genome duplication, WGD) is broadly considered as one of the most important forces driving the genome evolution of plants. Athough polyploidy increases the cost of genome replication, it also generates evolutionary advantageous genetic diversity and may result in speciation events (Wood et al. 2009). Although one of the paralogues can be lost because deletions are generally tolerated in polyploid genomes, paralogous gene copies can acquire new functions. A plethora of research reports and reviews have reported on the importance of polyploidization events for generating physiological and morphological innovations, colonization of new ecological niches etc. (e.g., Soltis et al. 2009, Fawcett et al. 2009, Van de Peer et al. 2009). There is a compelling evidence that polyploidy may have caused the dramatic increase in species richness in several angiosperm lineages (Fawcett and Van de Peer 2010, Soltis et al. 2009, Wood et al. 2009). Polyploidy is probably also responsible for the evolutionary dominance of angiosperms over gymnosperms (Leitch and Leitch 2012). WGD events are followed by gene and genome-wide fractionation (diploidization process). Diploidization includes various processes, such as genome downsizing, gene diversification and loss, activation of transposable elements, epigenetic changes, chromosome rearrangements and/or chromosome number reduction (descending dysploidy). These processes can be specific for each polyploid population, species or clade, and result in reproductive isolation and speciation (Soltis et al. 2009). Perhaps all land plants have experienced one or several rounds of WGD events in their evolutionary history. The current view on the plant evolutionary history is based on phylogenomic analysis of sequenced plant genomes and several million expressed sequence tag (EST) sequences (Jiao et al. 2011, Zuccolo et al. 2011). Jiao et al. (2011) identified two ancient WGD events, one in the common ancestor of extant seed plants [c. 319 million years ago (mya)] and the other in the ancestor of extant angiosperms (c. 192 mya). The evolution of the eudicots is marked by the third polyploidization, the gamma (γ) whole-genome triplication (WGT) event. Recently, Jiao et al. (2012) and Vekemans et al. (2012) revisited the phylogenetic position of γ, and placed this event after the divergence of the Ranunculales and core eudicots, i.e., c.117 mya. Based on the sequence analysis of A. thaliana and papaya (Carica papaya) genomes, two additional WGD events, known as alpha (γ) and beta (β), have been uncovered as being specific for the Brassicales. Whereas all Brassicales families share the γ WGT, the younger β WGD occurred only in the core Brassicales clade, after its divergence from Caricaceae (papaya) (Barker et al. 2009). Interestingly, the occurrence of the β coincides with the mass K-T extinction caused by catastrophic events, such as an asteroid impact and/or volcanic activity, c. 65 mya (Van de Peer et al. 2009). It is hypothesized that duplicated Brassicales genomes had a greater adaptive potential to survive and diversify in the changed environment after the K-T extinction (Beilstein et al. 2010, Franzke et al. 2011). The alpha (α) WGD event, specific to the Brassicaceae, occurred 43 to 23 mya (Barker et al. 2009, Beilstein et al. 2010, Couvreur et al. 2010, Fawcett et al. 2009). Although the timing of the α event is debated, its position at the base of the family tree suggests its importance for promoting the radiation of the Brassicaceae genera and species (Franzke et al. 2011). An independent WGT event (c. 14 mya) has been identified in the Cleomaceae, a sister family to Brassicaceae (Barker et al. 2009, Schranz and Mitchell-Olds 2006).

12 2 Chromosome painting

Fluorescence in situ hybridization (FISH) is a method of microscopic detection of specific genome sequences on chromosomes using fluorescently labelled nucleic acid probes complementary to the target sequence. The term chromosome painting (CP) was introduced by Pinkel et al. (1988) to describe fluorescence in situ visualization of large chromosome segments or whole chromosome arms using sequence-specific probes. For CP in its original sense as applied to vertebrates and humans, probes are obtained from chromosomes or chromosome segments isolated either by flow sorting (i.e., separation of chromosomes in a suspension on the basis of their relative fluorescence using a flow cytometer/sorter) or microdissection (i.e., isolation of DNA from a particular chromosome type or its part using a fine microcapillary pipette). The isolated DNA is amplified by degenerate oligonucleotide primed-polymerase chain (DOP-PCR) reaction, fluorescently labeled, hybridized and visualized on chromosome preparations. The repetitive sequences often prevent the identification of complex DNA sequences. Because of their non-specificity, these sequences hybridize with their corresponding sequences present in the genome (reviewed by Sharma and Sharma 2001). To achieve chromosome specificity of hybridization signals, dispersed repeats contained in the painting probe and target chromosomes have to be prevented from hybridization by blocking with excess of unlabelled total genomic DNA or DNA enriched for repetitive fraction. Therefore, the term chromosomal in situ suppression was used as an equivalent of chromosome painting (Lichter et al. 1988). A broad spectrum of different CP-based techniques has been developed for applications in research and clinical diagnostics (Langer 2004). CP enables the identification of chromosomes and chromosome rearrangements (Blenow 2004), mutagenicity testing (Cremer et al. 1990), analysis of the chromosome organization in interphase (Cremer and Cremer 2001). Comparative chromosome painting (CCP) is based on cross-species hybridization of painting probes. It allows identification of entire chromosomes and large-scale chromosome regions shared among related species, study of chromosome collinearity, and reconstruction of ancestral karyotype structures (Ferguson-Smith and Trifonov 2007, Svartman et al. 2004).

2.1 Chromosome painting in plants Although CP is commonly used in animal and human cytogenetics, all attempts to establish CP in plants have failed. CP using flow-sorted or microdissected probes is not directly applicable in plants due to the high proportion of complex dispersed repeats evenly distributed throughout plant genomes (Schubert et al. 2001). Thus plants require painting strategies different from the protocols working in animals and humans. Alternative painting strategy is FISH with pools of large-insert DNA clones (Bacterial and Yeast Artificial Chromosomes, BACs and YACs). The BAC-FISH approach to identify specific chromosome region is successful in plant groups with small genome and relatively low content of repeats, e.g., Brachypodium (Febrer et al. 2010, Gu et al. 2009, Ma et al. 2010), Brassicaceae (Lysak et al. 2001, chapter 3.2), rice (Jiang et al. 1995), sorghum (Figueroa et al. 2011), potato and tomato (Szinay et al. 2008, Tang et al. 2009). The first large-scale painting of plant chromosomes was achieved by Lysak et al. (2001) in Arabidopsis thaliana. CP became feasible due to the A. thaliana genomic

13 resources and the specific organization of the repeats in its genome (chapter 3.2). In later studies, CP was also successfully applied to other Brassicaceae species (chapter 3.3). Comparative chromosome painting (CCP) allowed unprecedented analyses of the cruciferous genome evolution in this group at the chromosomal level (Lysak et al. 2006, 2010, Mandáková and Lysak 2008, Mandáková et al. 2010a, b, 2012, chapter 3.3). Brachypodium distachyon from the family Poaceae is a model system for temperate cereals and grasses. Like A. thaliana, B. distachyon possesses numerous model attributes, including small genome and low chromosome number (Draper et al. 2001, Garvin et al. 2008). The B. distachyon BAC DNA libraries and bioinformatic data from the completed genome sequencing project (International Brachypodium Initiative 2010) made it possible to paint entire B. distachyon chromosomes. CCP with BAC pools from B. distachyon was successfully used to investigate phylogenetic relationships between different Brachypodium species and the mechanisms which shaped their karyotypes (Wolny and Hasterok 2009). However, it has not been determined whether B. distachyon-specific BAC pools could be used for CCP of grasses outside the genus (Idziak et al. 2011).

14 3 Comparative cytogenetics in the Brassicaceae

3.1 Family Brassicaceae The mustard family (Brassicaceae, Crucifereae), comprising approximately 321 genera and 3660 species (Al-Shehbaz 2012), belongs to the largest plant families. The inconspicuously beautiful crucifer plants are recognised by their cruciform corolla, tetradynamous stamens and their characteristic siliques. Family-wide phylogenetic analyses by Beilstein et al. (2006) revealed three major, well supported phylogenetic clades named lineages I, II and III (Fig. 1). Al-Shehbaz et al. (2006) based on the phylogenetic study of Beilstein (2006) introduced phylogenetic tribal classification of the family in which 25 monophyletic tribes were recognized. Subsequent molecular phylogenetic studies further specified the limits of many genera and tribes (Al-Shehbaz 2012). Lineage I harboured eight tribes (Boechereae, Camelineae including A. thaliana, Cardamineae, Descurainieae, Halimolobeae, Lepidieae, Physarieae, and Smelowskieae), lineage II comprised five (Arabideae, Brassiceae, Isatideae, Schizopetaleae, and Sisymbrieae), and lineage III four tribes (Anchonieae, Chorisporeae, Euclidieae, and Hesperideae) (Al-Shehbaz 2006). The genus Aethionema was recognized as the tribe Aethionemeae sister to the rest of the family (e.g., Beilstein et al. 2006, Al-Shehbaz et al. 2006). With the doubling of the number of tribes in five years (German 2010, German and Al-Shehbaz 2010, Warwick et al. 2010, Franzke et al. 2011, Al-Shehbaz et al. 2011, Koch et al. 2012), lineage I includes 14 tribes (by the addition of Alyssopsideae, Crucihimalayeae, Erysimeae, Microlepidieae, Oreophytoneae, and Yinshanieae), and lineage II is expanded to include the recently recognized Stevenieae and all of the remaining 25 tribes of the family minus the Biscutelleae. Lineage III now includes seven tribes (by the addition of Anastaticeae, Buniadeae, and Dontostemoneae) which remain as part of the basal polytomy of the family (Fig. 1) (Franzke et al. 2011). Before A. thaliana, the interest in the mustard family was focused primarily on its economically important genera such as Brassica (oilseed rape, cabbage, cauliflower, broccoli), Raphanus (radish), Armoracia (horse-radish), Nasturtium (water-cress), Eutrema (wasabi), and Eruca (salad rocket). For a long time, Brassica was the prime genus of crucifer cytogenetics. However, due to the small genome size and rapid life cycle, A. thaliana has been selected as a model organism and its genome has been sequenced (Arabidopsis Genome Initiative 2000, Koorneef et al. 2003). This revolutionized plant experimental biology and stimulated the interest in the genome analysis of other crucifer species. The A. thaliana genome is currently one of the most intensively studied genomes and many breakthrough findings came from the A. thaliana research (reviewed by Koorneef et al. 2003). Although A. thaliana has been neglected by plant cytogeneticists for its tiny chromosomes, it has become a pre-eminent model in plant cytogenetics later on. It is due to the invent of FISH, substitution of the small mitotic chromosomes by extended meiotic chromosomes at the pachytene stage, availability of chromosome-specific BAC libraries, and intergration of cytogenetic data with genetic and whole-genome sequence data (Lysak et al. 2001).

Fig. 1 The Brassicaceae family tree with the main lineages and tribes (from Franzke et al. 2011). Grey numbers in the brackets represent number of genera/species in the individual tribes.

16 3.2 Chromosome painting in Arabidopsis thaliana The feasibility of CP in plants has dramatically changed when the A. thaliana-related genomic resources have become available. A. thaliana, like most Brassicaceae species, is favored for chromosome painting because of its small genome size, low amount of repetitive DNA (c. 15%) clustered mainly in the pericentromeric regions, low chromosome number (n = 5), and the public availability of chromosome-specific BAC libraries (Arabidopsis Genome Initiative 2000). CP in A. thaliana is based on the method previously applied to paint chromosomes of yeast (Scherthan et al. 1992). CP in A. thaliana involves labelling of chromosome- specific repeat-free individual BAC clones followed by FISH. Typically, overlapping and differently labeled BAC clones are pooled and simultaneously hybridized to chromosome preparations (Lysak et al. 2001, Schubert et al. 2001, Lysak and Mandáková 2013). As the exact position of BAC clones on chromosomes is known, these can be combined and differently labelled according to a required experimental scheme. Chromosomes and nuclei at different developmental stages can be painted in this way, however, the breakthrough was accomplished by the application of extended pachytene chromosomes, providing high-resolution painting signals (Lysak et al. 2001). A. thaliana chromosome 4 became the first entirely painted plant chromosome using different fluorochromes and chromosome-specific BAC contigs as painting probes (Lysak et al. 2001). Subsequently, all five A. thaliana chromosomes were painted, and A. thaliana became the first plant with an entirely painted karyotype (Pecinka et al. 2004, Lysak et al. 2006). Application of CP in A. thaliana includes chromosome structure analyses as well as studies of chromosome organization in interphase nuclei. CP represents a tool for study of intra- and interchromosomal rearrangements such as inversions and transloations (e.g., inversion after T-DNA insertion, Pecinka et al. 2005; a paracentric inversion identified in Shahdara × Columbia hybrid of A. thaliana, Lysak and Mandáková 2013). In interphase cytogenetics, CP enables the analysis of the spatial structure and association of individual chromosome territories (Berr et al. 2006, Berr and Schubert 2007, Pecinka et al. 2004, 2005).

3.3 Principles of comparative chromosome painting in the Brassicaceae CCP within the Brassicaceae takes advantage of the established multicolor chromosome painting in A. thaliana, available phylogenetic information, and comparative genetic maps developed for several crucifer species (e.g., A. thaliana, A. lyrata, Brassica rapa, Capsella rubella). CCP in the Brassicaceae is based on cross-hybridization of A. thaliana chromosome-specific BAC contigs to chromosomes of other crucifer species (Lysak et al. 2006, Mandáková and Lysak 2008, Mandáková et al 2010a, b, 2012). Although CCP using other than A. thaliana BACs was tested and successfully established in crucifer species other than A. thaliana (Lysak et al. 2010), A. thaliana BACs are still first-choice painting probes for CCP in Brassicaceae because they have many advantages compared to those of other species. A. thaliana BAC clones are assembled on chromosomes, entire BAC libraries are available to the public, and more importantly, the evolutionary young A. thaliana genome contains only limited amount of repetitive sequences. The absence of the ancestral repeats enables the CCP with hight number of BACs. CCP in the mustard family allows the identification of large-scale homeologous chromosome regions and entire chromosomes in different species, to reveal the extent of cross-species chromosomal homeology (chromosome collinearity), to identify chromosome rearrangements, to elucidate evolutionary mechanisms underlying the

17 extant karyotypic variation, and to acquire phylogenetically informative cytogenetic signatures (Lysak et al. 2006).

3.4 Karyotype evolution in the Brassicaceae 3.4.1 Concept of the Ancestral Crucifer Karyotype To determine the evolutionary progression of karyotype alterations, it is necessary to distinguish ancestral from derived karyotypes. The comparative genetic mapping between three species from the tribe Camelineae, A. thaliana (n = 5), A. lyrata (n = 8, Kuittinen et al. 2004), and Capsella rubella (n = 8, Boivin et al. 2004), revealed highly conserved genome structure shared between A. lyrata and C. rubella, and a relatively high level of chromosome collinearity between A. thaliana and the two n = 8 species (Koch and Kiefer 2005). Comparative genetic and cytogenetic analyses also showed that the compact A. thaliana genome is characterized by a highly reshuffled and derived karyotype (Boivin et al. 2004; Kuittinen et al. 2004; Koch and Kiefer 2005; Lysak et al. 2006), making this species inappropriate as a reference point for comparative studies across Brassicaceae (Schranz et al. 2006). The overall similarity between karyotypes of A. lyrata and C. rubella and the fact that n = 8 is the most common chromosome number found in Camelineae as well as across Brassicaceae (Warwick and Al-Shehbaz 2006), resulted in the concept of a hypothetical Ancestral Crucifer Karyotype (ACK) with eight chromosomes and genome resembling that of A. lyrata and C. rubella (Lysak et al. 2006, Schranz et al. 2006). The ACK concept was further expanded by defining apparently conserved genomic blocks (GBs) which make up individual ancestral chromosomes (Schranz et al. 2006). Conserved ancestral blocks were revealed through inter-specific genetic mapping between A. thaliana and Brassica napus. Parkin et al. (2005) identified a minimum of 21 conserved chromosomal segments within the A. thaliana genome, which can be duplicated and rearranged to build up the allopolyploid genome of B. napus. These chromosomal blocks were largely identical with the collinear chromosomal segments revealed by comparative genetic and cytogenetic mapping as shared between A. thaliana, A. lyrata, C. rubella, and other Camelineae and Descurainieae species (Boivin et al. 2004, Kuittinen et al. 2004, Lysak et al. 2006). These data have been integrated with Parkin et al.’s blocks into a set of 24 conserved genomic blocks (A to X) building up the eight AK chromosomes (Schranz et al. 2006; Fig. 2). The ACK karyotype with 24 ancestral genomic blocks has been successfully adopted as an ancestral reference genome in comparative genomics and cytogenomics studies in the Brassicaceae (e.g., Ma et al. 2012, Mandáková et al. 2010a, b, 2012, Mandáková and Lysak 2008, Nelson et al. 2011, Schranz et al. 2007, Wang et al. 2011).

18 ACK (n = 8) Arabidopsis BAC homeologous chromosome contigs used for regions identified by CCP CCP

Fig. 2 Ancestral Crucifer Karyotype (ACK) comprising eight chromosomes (AK1-8) and 24 conserved genomic blocks (A-X) has been proposed by Lysak et al. (2006) and Schranz et al. (2006). The level of chromosome collinearity shared between the ACK and karyotypes of extant crucifer species is explored by comparative chromosome painting (CCP). Fluorescently labeled A. thaliana BAC contigs arranged according to ancestral genomic blocks are hybridized to pachytene chromosomes of a crucifer species.

3.4.2 Karyotype evolution in species from the Brassicaceae lineage I and II The concept of the ACK has been for the first time applied in a multicolour CCP analysis of species from Brassicaceae lineage I with presumably reduced chromosome numbers from n = 8 towards n = 7, 6 and 5 (Lysak et al. 2006). This study elucidated the evolution of the A. thaliana karyotype marked by the reduction of chromosome number from n = 8 towards n = 5 (Fig. 3) through three reciprocal translocation-mediated chromosome fusions and at least three inversions. Although belonging to the same genus as the extremely reshuffled A. thaliana, the karyotype of A. lyrata (n = 8), has not undergone the process of descending dysploidy. This demonstrates that karyotype dynamics in some Brassicaceae species contrasts with the stability in the others. In species for which no genetic data were available, Neslia paniculata (n = 7, Camelineae), Turritis glabra (n = 6, Turritideae) and Hornungia alpina (n = 6, Descurainieae), CCP revealed largely preserved chromosomal colinearity between these species, A. thaliana and ACK. Despite some inversion events, six homeologous chromosomes in N. paniculata, and four in H. alpina and T. glabra resembled the structure of ancestral chromosomes (Fig. 3). Although some ancestral chromosomes participated in chromosome fusions more often than others, the chromosome fusion events were species-specific (Lysak et al. 2006). These data suggested that chromosome reductions from the ACK to evolutionary derived karyotypes with n = 7, 6 and 5 occurred independently and recurrently, and confirmed the ACK as a probable ancestral karyotype of lineage I (Fig. 3). Subsequently, karyotype evolution in the expanded lineage II and closely related tribes has been investigated by Mandáková and Lysak (2008). Tribes of expanded lineage II are characterized by karyotypes based on n = 7. Hence, question on the modes of karyotype evolution leading to the same chromosome numbers (2n = 14/28) in different species and tribes has been addressed by means of CCP. This study includes eight species from six different tribes; Calepina irregularis and Goldbachia laevigata (Calepineae), Conringia orientalis (Conringieae), Glastaria glastifolia (Isatideae), Noccaeae caerulescens (Coluteocarpeae), Ochthodium aegyptiacum (Sisymbrieae), and Thellungiella salsuginea (Eutremeae). All analyzed species shared a complex structure of two translocation chromosomes resulting from recombination involving three

19 ancestral chromosomes. This represents a characteristic cytogenetic signature proving the existence of a common n = 7 ancestor named Proto-Calepineae Karyotype (PCK; Fig. 3 and 4). The PCK remained preserved in tribes Calepineae and Conringieae, and has become modified by secondary inversions in Coluteocarpeae. An additional translocation involving one PCK-specific translocation chromosome and AK2-like chromosome, resulted in the origin of the evolutionary younger translocation PCK (tPCK) genome in Isatideae, Sisymbrieae and Eutremeae (Mandáková and Lysak 2008). As the PCK shares five chromosomes and two chromosome arms with the ACK, it has been suggested that either the PCK and ACK are descended from a common ancestor or, more likely, that the seven PCK chromosomes are derived from the eight chromosomes of the ACK (Mandáková and Lysak 2008). The tPCK was also tentatively suggested as an ancestral karyotype of the tribe Brassiceae (Mandáková and Lysak 2008). The released sequence of the triplicated Brassica rapa genome (chapter 3.4.3) allowed us to re-evaluate the genome evolution in the extant diploid Brassica species. We demonstrated that all three B. rapa paleogenomes resembled the n = 7 ancestral tPCK genome with seven chromosomes (Cheng at al., submitted). Recently, genomes of Schrenkiella (Thellungiella) parvula (Dassanayake et al. 2011) and T. salsuginea (Wu et al. 2012) have been sequenced. The comparative cytogenetic map of T. salsuginea reconstructed by Mandáková and Lysak (2008) facilitated the assembly of S. parvula sequence clusters into its seven chromosomes. The tPCK genome structure was confirmed by S. parvula whole-genome sequencing. This work demonstrated the accuracy of comparative cytogenetic maps, and the importance of cytogenetics and cytogenomics in the era of genome assembly projects, particularly in species for which no genetic maps are available. Similarly, comparative cytogenetic maps of Arabis alpina and Leavenworthia alabamica will represent a basis for whole-genome assemblies (Mandáková and Lysak, unpublished data). Considering 3,660 species in the mustard family, only negligible number of genomes has been reconstructed so far. No data on genome structure are currently available for lineage III species. However, some preliminary conclusions on karyotype evolution in lineage I and II could be drawn. The increasing body of evidence suggests the ACK with eight chromosomes and 24 genomic blocks is an ancestral genome of the lineage I and perhaps of the whole family. The PCK with seven chromosomes was identified as the ancestral genome of several tribes classified as or associated with expanded lineage II including the economically important tribe Brassiceae. The seven chromosomes of PCK were most likely derived from the eight chromosomes of the ACK (Fig. 3 and 4). Assuming the  WGD event at the base of the Brassicaceae family, the ACK genome itself most likely originated from a proto-genome with n = 4 via this genome duplication (Henry et al. 2006). Mechanisms of descending dysploidy from the ancestral n = 8 towards lower chromosome numbers are discussed in chapter 3.4.4.

20 Figure 3 The Cabagge Circle displaying collinear relationships between chromosomes of ACK (n = 8; Schranz et al. 2006), PCK (n = 7; Mandáková and Lysak 2008), and the modern karyotypes of Cardamine hirsuta (n = 8, Cardamineae), Boechera stricta (n = 7, Boechereae), Turritis glabra (n = 6, Turritideae), and A. thaliana (n = 5, Camelineae). Based on Lysak et al. (2006), Mandáková and Lysak (2008), and unpublished data of T.M.

Figure 4 (next page) A tentative scenario of karyotype evolution in the Brassicaceae assuming the ACK being ancestral for taxa of lineage I and II. Lineage II genomes descended from ACK via a common intermediate PCK. The 24 genomic blocks are indicated by A-X and colored according to their position on chromosomes AK1-AK8 of the ACK (Schranz et al. 2006). Downward-pointing arrows indicate the opposite orientation of genomic blocks compared with the position in the ACK. Based on Lysak et al. 2006, Mandáková and Lysak 2008, Parkin et al. 2005, and unpublished data of T. M.; adopted from Lysak and Koch (2011).

21 Arabidopsis thaliana (n=5)

At1 At2 At3 At4 At5 AK1/2 AK3/4/5 AK3/5 AK6/7 AK6/7/8 K G O A R F P H T

Q B I L U

M S J C N V

D W Hornungia alpina (n=6) Turritis glabra (n=6) E X AK1 AK2/5 AK3 AK4 AK6/8 AK7 AK1 AK2/8 AK3/5 AK4 AK6 AK7 Neslia paniculata (n=7) X O N I S I S Boechera stricta (n=7) A A AK1 AK2 AK3 AK4/5 AK6 AK7 AK8 R P F W F M T T AK1/2 AK1/2 AK3/8 AK4 AK3/5/8 AK6 AK7 J J Q V D Q(I) D O S ? C2 B G O(I) U B V V D G U A J P A1 I O V R F A2 S E T W P E H Q(II) H Q C1 F H (II) T O E I J Q C C X P B G B K L K K U D? R L K W L L G U H R M M M C W X E N N X N

Descurainieae (A) Camelineae (core) Camelineae (B) Camelineae Boechereae

Ancestral Crucifer Karyotype (ACK, n=8)

AK1 AK2 AK3 AK4 AK5AK6 AK7 AK8 K D I O V L S A P F W M T E J Q X N B G U R H C

/8 /6 /8 1 2 3 4 K5 6 7 AK AK AK AK A AK AK

D I O N S A P F E M T Proto-Calepineae Karyotype J W V B (PCK, n=7) G K U L R H Q C X

Brassiceae:Brassica rapa (n=10)

Br1 Br2 Br3 Br4 Br5Br6 Br7 Br8 Br9 Br10

Isatideae, Sisymbrieae, Eutremeae Calepineae, Conringieae, Noccaeeae

N H C O (n=7) (n=7) J A

U R R Q AK1AK2/5/6/8 AK3 AK4 AK2/5AK6/8 AK7 AK1AK2 AK3 AK4 AK5/6/8AK6/8 AK7

T T

A G

I O

S W S

W S

W B A A V

T U

B F M

I M

P T K B

T E

J W W

F E J R L

C V

I B

G B B Q

G U J K

R L

V R

I X

N H

K H

Q N

L O F C

P B Q L E I Glastaria glastifolia Calepina irregularis

X K V Myagrum perfoliatum Conringia orientalis

F A Ochthodium aegyptiacum Goldbachia laevigata Brassica oleracea(n=9) Thellungiella halophila Noccaea caerulescens T B. napus (n=19)

22 3.4.3 Mesopolyploid evolution in the Brassicaceae When polyploidization has occurred recently in evolutionary time, the corresponding chromosome number multiplication is evident (neopolyploidy, Fig. 5). Ancient polyploid WGD events (paleopolyploidy; e.g., α, β and preceding WGDs) are masked by an extensive genome diploidization and can be revealed only by a bioinformatic search for orthologous and paralogous sequences (Fig. 5). Ancient polyploidization followed by diploidization including chromosome number reduction but still detectable by comparative genomics and/or chromosome painting has been recently described as mesopolyploidy (Mandáková et al. 2010a, Fig. 5). Genetically diploid species with chromosome numbers as low as n = 4, 5, or 6 have never been suspected to be ancient polyploids on the road to genome diploidization. However, during a few recent years, several diploid-like crucifer species were shown to have experienced mesopolyploid whole-genome duplications or triplications postdating the three paleopolyploid WGDs (α, β and γ). Due to the feasibility of CCP, mesopolyploid WGD events were detected in several crucifer taxa containing diploid- like species, namely in Brassiceae (Lysak et al. 2005), Australian and New Zealand Microlepidieae (Mandáková et al. 2010a, b), Heliophileae (Mandáková et al. 2012), and other tribes (Mandáková and Lysak, unpublished data). The first characterized mesopolyploid event was the whole-genome triplication found at the base of the tribe Brassiceae through comparative genetic mapping (Parkin et al. 2005), cytogenetics (Lysak et al. 2005, 2007), and sequence genomics (Wang et al. 2011). Lysak et al. (2005, 2007) by analyzing several ancestral genomic blocks in eleven Brassiceae species spaning the entire range of chromosome numbers (from n = 7 to n = 34) gained compelling evidence for a tribe-specific triplication event. An independent mesotetraploid WGD has been identified in Orychophragmus violaceus, a species being frequently assigned to Brassiceae (Lysak et al. 2007). Later on, two mesotetraploid events were inferred to occur in two groups from the tribe Microlepidieae endemic to Australia and New Zealand, respectively (Mandáková et al. 2010a, b, Fig. 6). CCP analysis unexpectedly revealed that genomes of Australian species (Stenopetalum nutans, S. lineare, and Ballantinia antipoda) with low chromosome numbers (n = 4, 5, and 6, respectively) descended from the eight ancestral chromosomes of the ACK through an allopolyploid WGD event followed by the species divergence and species-specific genome diploidization (Mandáková at al. 2010a). Nuclear and maternal gene phylogenies corroborated the hybrid origin of the mesotetraploid ancestor and suggested that the WGD occurred c. 6 to 9 mya. Similarly, the ancestral genome of ten Pachycladon species (all with n = 10) has been reconstructed and suggested to originate through a mesopolyploid event involving two ACK-like genomes. The diversification of Pachycladon species was estimated to occur c. 1 to 2 mya (Mandáková et al. 2010b). The two, presumably independent, allopolyploid WGD events were followed by diploidization process towards diploid-like genomes, with the less extensive genome repatterning in the evolutionary younger genus Pachycladon. The duplicated ancestral chromosome number (n = 16) was reduced towards n = 10 and n = 6, 5, 4 in Pachycladon and Australian Brassicaceae species, respectively. Chromosome number was reduced via chromosome rearrangements typically including peri- and paracentric inversions and reciprocal whole-arm translocations mediating centromere losses. Alternatively, translocations between chromosome ends of two non-homologous chromosomes led to the origin of fusion chromosomes with two centromeres, one being inactivated and/or deleted (Mandáková et al. 2010a, b, see chapter 3.4.4). Such dramatic rearrangements reshuffled the parental genomes and genomic blocks building them. Consequently,

23 extant diploidized genomes are built up as complex mosaics of duplicated genomic blocks (Fig. 6). In some Stenopetalum species (n = 8, 10), additional (neopolyploid) genome duplications have been identified.

Fig. 5 A model of genome evolution through multiple WGD events (Mandáková et al. 2010a). Subsequent WGDs of different age shown as ploidy level increases (4x to 32x) are followed by genome diploidization towards diploid-like genomes. Chromosomes on the right represent duplicated and diploidized complements with three ancestral genomic blocks (GB) as revealed by comparative genetic and cytogenetic techniques. In paleopolyploids (e.g., A. thaliana, n = 5), paralogous regions are not detectable by (cyto)genetic analysis. In neopolyploids (e.g., A. suecica, n = 13), chromosome number is not reduced and most duplicated GBs are not reshuffled yet. In mesopolyploids (e.g., Stenopetalum nutans, n = 4), descending dysploidy results in diploid-like number of mosaic-like chromosomes and duplicated GBs are reshuffled by intra- and intergenomic rearrangements. The mesopolyploid genome experiences additional neopolyploidy (e.g., S. velutinum, n = 8).

Recently, seven Heliophila species from the endemic South African tribe Heliophileae possessing different diploid-like chromosome numbes (n = 8-11) and phylogenetic position within the tribe have been analyzed by CCP (Mandáková et al. 2012). In all species analyzed, 90% of painting probes unveiled three homeologous chromosome regions in Heliophila haploid chromosome complements. These results suggest that all the analyzed species, and probably the entire tribe Heliophileae, experienced a WGT event. As in Australian and New Zealand mesopolyploids, also WGT in Heliophila has been followed by species-specific chromosome rearrangements resulting in the extant diploid-like genomes with diverse chromosome numbers. More recent neopolyploid events in Heliophileae are reflected by increased chromosome

24 numbers (2n = 32-88). This study showed polyploidy as a potential major factor responsible for the diversification and species radiation in the species-rich tribe Heliophileae. The incidence of successive WGD events in several crucifer lineages allowed us to postulate the WGD/diploidization cyclic model (Fig. 6).

Fig. 6 Model of genome evolution in the Brassicaceae shaped by cyclic whole-genome duplication and diploidization events. A common paleopolyploid ancestor of the family, the ACK (n =8) experienced lineage-specific WGD event. The chromosome number of the tetraploid ACK-like karyotype (n = 16) was subsequently reduced during the process of diploidization towards genomes with diploid-like chromosome numbers (n = 6, 5, 4) and mosaic-like structure of the fusion chromosomes. Mesopolyploid karyotypes can undergo additional WGD and diploidization.

In Brassicaceae, several genera and tribes are polybasic (Fig. 7). Based on our published studies and preliminary data, we hypothesized that polybasic taxa have undergone mesopolyploid WGDs followed by extensive genome diploidization. From polybasic tribes listed in Fig. 7, WGDs have been already reported to occur in Australian and New Zealand Microlepidieae, Brassiceae and Heliophileae (Lysak et al. 2005, 2007, Mandáková et al. 2010a, b, 2012). Additional evidence of mesopolyploid WGD was revealed by CCP in the tribes Biscutelleae, Cardamineae, Cochlearieae, Erysimeae, Iberideae and Physarieae (Fig. 8, Mandáková and Lysak, unpublished data). These data support our hypothesis of a direct link between genome duplication events and the variation in basic chromosome numbers.

25 14

6 base chromosome number (x) 5

e e a a e eae eae eae eae dieae ri deae l i a i iceae ide er on epi e ber ss tatic ol rysimeae I iophi s Physarieae E el iscutelleae a nch icr H B Bra n Hesp A M Cochl ThelypodiA U A

Fig. 7 Selected polybasic cruciferous tribes based on Warwick and Al-Shehbaz (2006).

The available data on mesopolyploid events across Brassicaceae are sparse and yet insufficient to draw general conclusions about the frequency of lineage-specfic genome duplications and their long-term impact on crucifer genome evolution. However, recently uncovered mesopolyploid events i) indicate that ancient polyploidy events are more frequent than previously thought, ii) highlight the importance of multiple whole- genome duplication events in the angiosperm genome evolution, iii) elucidate diploidization mechanisms transforming polyploid into quasi-diploid genomes, and iv) demonstrate that chromosome number per se is not a reliable indicator of ploidy level.

Fig. 8 The Brassicaceae family tree, main lineages and tribes (from Franzke et al. 2011). Published or verified and purported tribe-specific mesopolyploid duplications are indicated as black and empty circles, respectively. Grey numbers in the brackets represent number of genera/species in the individual tribes (based on data from Lysak et al. 2005, 2007; Mandáková et al. 2010a, b, 2012; Mandáková and Lysak, unpublished data).

27 3.4.4 Mechanisms of chromosome number reduction in the Brassicaceae Uncovering the genetic mechanisms responsible for the descending dysploidy (i.e., reduction of the chromosome number) has been one of the most chalenging tasks of comparative cytogenetics in the Brassicaceae. The pericentric inversion - reciprocal translocation mechanism involves a pericentric inversion which moves the centromere of the (sub)metacentric chromosome towards the chromosome end, creating an acrocentric chromosome. A reciprocal translocation occurs between the centric end of the acrocentric and a subtelomeric region of another chromosome and results in the origin of a large fusion chromosome and small minichromosome. It is hypothesized that the minichromosome is free of essential genes, meiotically unstable, and hence eliminated (Fig. 9A). This Robertsonian-like translocation eliminating one ancestral centromere and decreasing chromosome number has been described for several crucifer species (Lysak et al. 2006, Mandáková and Lysak 2008, Mandáková et al. 2010a, b,) and represents a common mechanism of descending dysploidy in the Brassicaceae. Karyotypes are reshuffled independently in different clades, however, an apparent trend can be observerd in ancestral chromosomes AK5, AK8, and AK6 being more frequently involved in rearrangements than the other ancestral chromosomes (described for taxa of lineage I and II; Lysak et al. 2006, Mandáková and Lysak 2008, Mandáková et al. 2010a, b) Mandáková et al. (2010a, b) identified several ancestral chromosomes within the mosaic-like chromosomes in the mesopolyploid Australian and New Zealand Microlepidieae species. Ancestral associations of GBs in the karyotypes exhibit preserved collinearity corresponding to the structure of ancestral chromosomes. It cannot be ruled out that the ancestral associations of GBs were generated by the mechanism of a pericentric inversion-reciprocal translocation event but involving an additional inversion (Lysak et al. 2006, Schubert 2007). However, the occurrence of entire ancestral chromosomes within the composite chromosomes might more probably result from tandem end-to-end fusion of two chromosomes, i.e., end-to-end translocation with the breakpoints within the subtelomeric chromosome regions, producing a dicentric chromosome. Subsequent centromere inactivation and/or loss via recombination ensures regular meiotic segregation and stabilizes the composite chromosome (Fig. 9B). A similar telomere-to-telomere translocation has been assumed for an origin of a large metacentric chromosome of ants (Imai and Taylor 1989). Several cases of inactivated ancestral centromeres were reported in mammalian species (e.g., Ferreri et al. 2005, Ventura et al. 2007), and in the human chromosome 2 (Ijdo et al. 1991). Only a few examples of centromere inactivation are known in plants, including dicentric chromosomes of Trititiceae (Luo et al. 2009), maize B chromosomes (F. Han et al. 2006), and chromosomes of two cucurbit species (Y. Han et al. 2009). Besides Australian and New Zealand mesopolyploid Brassicaceae (Mandáková et al. 2010a, b), end-to-end chromosome fusions were not previously described in the family. Assuming an alternative scenario to explain the extant karyotype structures, centromere inactivation of AK4 can be suggested for the origin of chromosome At2 in A. thaliana (Lysak et al. 2006, Hu et al. 2011), centromere inactivation of AK5 for the origin of Bst5 in Boechera stricta (Schranz et al. 2007), and chromosome AK4/5 in Neslia paniculata (Lysak et al. 2006). Recently, new examples of putative paleocentromere inactivation were revealed in Arabis alpina (Arabideae; Mandáková and Lysak, unpublished data) and Cardamine rivularis (Cardamineae; Mandáková et al., submitted). Putting the evidence of paleocentromere inactivation in the Brassicaceae

28 together, centromere of the ancestral chromosomes AK5, AK3 and AK4 (in this order) seem to be inactivated preferentially. Except one heterochromatic knob in Stenopetalum nutans (Mandáková et al. 2010a), no heterochromatin was observed at sites of presumably inactivated centromeres in the Brassicaceae species (Mandáková et al. 2010a, b). Illegitimate recombination between (peri)centric repeats is supposed to gradually remove the repeats and heterochromatin from those regions (Ventura et al. 2004). Similarly, centromere inactivation accompanied by the loss of heterochromatin has been reportedted in cucumber (Y. Han et al. 2009).

Fig. 9 Putative mechanisms of chromosome number reduction in the Brassicaceae. (A) Pericentric inversion - reciprocal translocation mechanism. (B) End-to-end chromosome fusion accompanied by centromere inactivation (Schubert and Lysak 2011, modified).

3.4.5 Conclusions on the karyotype evolution in the Brassicaceae It can be concluded that descending dysploidy, one of the crucial features of the karyotype evolution in the Brassicaceae, is typically mediated by pericentric inversions, and reciprocal translocations resulting in centromere losses and origin of fusion chromosomes. Alternatively, translocations between chromosome ends lead to the formation of so-called fusion chromosomes with two centromeres, with one of the two being inactivated and/or deleted. Our data summarized above suggest, that rearrangement breakpoints were predominantly located in (peri)centromeric and subtelomeric regions. The rearrangements involving the whole chromosome arms played the prevalent role in the reduction of chromosome number. Clustering of breakpoints at (peri)centromeres and chromosome termini indicates a preferential involvement of repetitive sequences in the genome repatterning processes. Centromeres and telomeres can be considered as fragile sites and hotspots of structural rearrangements. The ACK karyotype is assumed to originated from a proto-genome (n = 4) by the Brassicaceae-specific  WGD (Henry et al. 2006). In A. thaliana (n = 5), the pre- ancestral chromosome number has been almost restored within 23-43 mya (Barker et al. 2009, Fawcett et al. 2009) via mechanisms described above. In the clade of mesopolyploid Australian Microlepidieae genera the evolutionary tempo was faster compared to A. thaliana. Despite an additional WGD in the ancestry of endemic Australian crucifers, the pre- chromosome number has been re-established in the same time frame as in A. thaliana.

29 4 Trends in angiosperm karyotype evolution

An intensive genetic mapping and genomics research has been carried out across other angiosperm families, particularly those with several crops, such as the Poaceae (Murat et al. 2010, Salse et al. 2008, 2012), Solanaceae (Mueller et al. 2005, Prince et al. 1993, Tanksley et al. 1992, Wu et al. 2009a, b), Fabaceae (Cronk et al. 2006, Zhu et al. 2005, Schoemaker et al. 2006), and Rosaceae (Considine et al. 2012, Dirlewanger 2004, Jung et al. 2012).

4.1 Genome evolution in the Poaceae Comparative genetic mapping in the grass family (Poaceae), which includes many important cereal and forage crops, resulted in the synthesis of the Crop Circle (Devos 2005, Devos and Gale 1997, 2000, Feuillet and Keller 2002, Moore et al. 1995). The Crop Circle demonstrates a large degree of chromosome collinearity among the grass genomes (Gaut 2002). The small-sized rice genome (n = 12) subdivided into c. 30 blocks was placed to the center of the Circle. Although rice has been shown to retain the genome structure of its n = 12 ancestor (Murat et al. 2010, Salse et al. 2008), extensive genome reshuffling marked the evolution of other grass genomes, such as maize, sorghum, Brachypodium, Triticeae species or sugar cane. The 30 ancestral blocks of rice represent an equivalent to the 24 genomic blocks of the Ancestral Crucifer Karyotype. In the grasses, the ancestral GBs have been shown as conserved in the extant karyotypes (Devos 2005). Similarly, cruciferous karyotypes are very stable in some clades (e.g., PCK-like karyotypes of lineage II, Arabidopsis lyrata, Capsella rubella), however, in the other clades rapidly evolving dynamic structures with GBs frequently disrupted via inversions and translocations, particularly in those with mesopolyploid WGD events. In grasses, genes and repeats are organized differently in small and large genomes. In small genomes, there is a clear partioning between gene-rich euchromatin and repeat-rich heterochromatin (Devos 2009). This kind of genome organization is similar to that frequently found in Brassicaceae. In grass species with larger genomes, repeats are interspersed between genes. As larger grass genomes have been considered as more dynamic compared to the small ones, dispersed repeats may be the source of chromosome rearrangements (Devos 2009). A common grass ancestor is reported to have n = 5 (or 7) chromosomes forming an allopolyploid genome of n = 10 (or 14) chromosomes (Devos 2009, Murat et al. 2010, Salse et al. 2008). The allopolyploidy in Poaceae was followed by diploidization process resulted in the origin of n = 12 karyotype of similar stucture as that of rice (Devos 2009, Murat et al. 2010, Salse et al. 2008). Multiple post-(n = 12) WGDs and subsequent chromosome number reduction responsible for the mosaic of parental genomic blocks have been well documented in grasses (Luo et al. 2009, Murat et al. 2010, Salse et al. 2008, 2012). Descending dysploidy in the Poaceae is caused mainly by so called nested chromosome fusions (Luo et al. 2009, Murat et al. 2010). Intra-chromosome crossover, produces ring chromosome, may form a chromosome with two telomere-free termini and a minichromosome containing two telomeres. The minichromosome is often lost. The major chromosome without telomeres attaches to another chromosome, often in a (peri)centromeric repet-rich region via illegitimate recombination between nonhomologous chromosomes. The attachment comprises an insertion, i.e., breakage of the recipient chromosome in its (peri)centromeric region and incorporation of the insertion chromosome. The nested chromosome fusion of one chromosome into

30 another’s (peri)centromeric region results in repeat-rich boundaries representing the traces of the split ancestral (peri)centromere of the recipient chromosome. In all observed nested fusions, centromeres of insertion but not recipient chromosome were preserved (Luo et al. 2009, Murat et al. 2010, Schubert and Lysak 2011). In the Brassicaceae, only two cases of chromosomal structure affected by nested fusion has been reported in Pachycladon (Mandáková et al. 2010b) and Hornungia alpina (Lysak et al. 2006). It suggests that although nested fusions have a major role in Poaceae, they represent an inconsiderable mechanism of the karyotype evolution in the Brassicaceae. The transposon-rich breakpoins are still detectable after the nested fusions in grasses (after ~70 mya) (Murat et al. 2010). This is in contrast to the evolutionary breakpoints in Brassicaceae in which are not reported to be enriched by repetitive sequences and thus detectable only based on the collapsed genome collinearity (Hu et al. 2011). The mechanism of end-to-end fusion accompanied by centromere inactivation is, however, reported to be quite rare in the Poaceae (Murat et al. 2010). By contrast, this mechanism is described to be the second most frequent type of chromosome rearrangement in the mustard family.

4.2 Genome evolution in the Solanaceae The Solanaceae is the third larger economically important plant family. Features and outcomes of chromosomal evolution in the family could be deduced from the extensive comparative mapping studies which have been performed for several major solanaceous crops relative to tomato (Prince et al. 1993, Tanksley et al. 1992, Wu et al. 2009a, b), from genome sequence of potato and tomato (Potato Genome Sequencing Consortium et al. 2012, Tomato Genome Consortium 2012), and from cross-species multicolor cytogenetic mapping using BAC clones from potato and tomato (Tang et al. 2008). The chromosome number in the Solanaceae is stable with actual karyotypes of eggplant, Nicotiana, pepper, potato and tomato sharing 12 chromosome pairs. Tomato and potato differ by 6 inversions, tomato and eggplant by 24 inversions and 5 translocations, tomato and pepper by 19 inversions and 6 translocations, tomato and Nicotiana by at least 10 inversions and 11 translocations (Prince et al. 1993, Tanksley et al. 1992, Wu et al. 2009a, b). This suggests that in the Solanaceae, inversions occur at a consistently higher rate than translocations. No complex chromosome changes as described in Brassicaceae or Poaceae were reported to occur in the Solanaceae. The major solanaceous crops diverged c. 30 mya and no recent WGD events played an important role in the genome evolution of the Solanaceae. Given the constant chromosome number across the Solanaceae, a relatively low rate of chromosome repatterning and absence of recent genome duplications, the family has experienced chromosomal changes at a moderate rate compared to Brassicaceae and Poaceae.

4.3 Genome evolution in the Fabaceae In legumes, genomes of soybean, Medicago truncata, pea and lotus have been sequenced, and detailed comparative genetic maps are available for bean and Mimosa. The legume crop species originated from a common ancestor which experienced a WGD c. 59 mya. The Fabaceae genomes underwent an additional, more recent genome doubling c. 13 mya (Schoemaker et al. 2006). Mosaic synteny blocks can be identified in the genomes of legumes. However, the genome synteny was shown to be limited compared to conserved large-scale genomic blocks in Brassicaceae, Poaceae and Solanaceae. This suggests substantial genome rearrangements to occur shortly after the ancestral legume-specific WGD event.

4.4 Genome evolution in the Rosaceae In the Rosaceae, genome sequencing of three crop species [apple (n = 17), peach (n = 8) and strawberry (n = 7)] showed different evolutionary patterns of genome evolution (Jung et al. 2012). The hypothetical ancestral genome of Rosaceae had seven chromosomes. The genome evolution in the family is characterized by shared conserved ancestral genomic blocks reshuffled by chromosome fusions and fissions. Chromosomal fusion-fission cycle was described as a reversible fusion of two telocentrics giving rise to the metacentric chromosome. The next step is a fission of this chromosome into two stable telocentrics eventually followed by a new fusion reconstituting the metacentric chromosome (Schubert et al. 1995). Jung et al. (2012) described strawberry lineage to experience at least five fission and seven fusion events, the peach lineage at least three fission and four fusion events. The apple genome to experienced a WGD after the divergence from peach and strawberry, and subsequently has undergone seven fission and nine fusion events (Jung et al. 2012). Thus, Rosaceae represents the only plant family in which chromosome fissions presumably were the crucial mechanism of karyotype evolution. Recently, however, the genome study in apple shed light on the mechanism responsible for the odd-numbered basic chromosome numbere (Considine et al. 2012). Considine et al. (2012) showed that diploid apple species produce gametes with different chromosome numbers. Fusions of these gametes result the formation of different aneuploid basic chromosome numbers, previously explained as the consequence of chromosome fissions (Jung et al. 2012).

4.5 Contrasting karyotype evolution in angiosperms These are great times for scientists as high-throughput genomic sequencing provides powerful new approaches to solve questions that puzzled scientists for centuries. Each crop species represents a model that offers unique opportunities to make a new progress in comparative plant biology. In the early 2000s, A. thaliana and rice genomes have been sequenced and since that time, 16 more plant genomes have become available (apple, barley, Brachypodium, cacao, cotton, grapevine, maize, papaya, pea, peach, poplar, sorghum, soybeen, strawberry, wheat) and high-resolution genetic and/or cytogenetic maps have been reconstructed for a number of species (Salse 2012). Such data provide an important insight into the plant genome organization and evolution. Although polyploidization has been repeatedly documented as the central process directing the genome evolution in land plants, there are obvious counteracting evolutionary forces towards lower chromosome numbers. Several rounds of WGD events have been followed by clade- and species-specific rearrangements leading to the pre-polyploid level. It is known that the rate with which genomes undergo and fix chromosome rearrangements varies between different lineages (Devos and Gale 2000, Ilic et al. 2003). The strong selection for diploid chromosome number restored via different mechanisms of chromosome number reduction after polyploidization is probably caused by the fact that massive structural and functional changes following WGD might provide an organism with a chance to adapt to changing environment. The available data on karyotype and genome evolution in plants support the widespread opinion that genomes evolve mainly by duplicating and/or reorganizing the existing genomic blocks rather than by inventing new ones (Salse 2012). Chromosome fissions probably do not occur at all because they produce potentially unstable chromosomes without telomere ends. In each analyzed angiosperm family, genomes were reshuffled by a different predominant type of chromosome

32 rearrangements. In the Brassicaceae, pericentric inversion-reciprocal translocation together with end-to-end chromosome fusions play the key role in chromosome number reductions, whereas nested chromosome fusions typical for Poaceae are very rare. Solanaceae karyotypes are stable, compared to other plant families, modified predominantly by inversions.

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43 6 Aims of the dissertation & author’s contribution

Author’s scientific contribution to the field of comparative cytogenetics in the Brassicaceae is documented by enclosed publications. The principal author’s contribution is summarized as follows:

6.1 Optimization and application of the large-scale comparative chromosome painting in different species from the family Brassicaceae... I, II

I. Lysak and Mandáková (2013) Step-by-step protocols of chromosome painting and comparative chromosome painting in plants.

II. Lysak et al. (2010) Fist large-scale comparative chromosome painting using non-Arabidopsis BAC contigs was established and tested in several crucifer species.

6.2 Identification of the mechanisms responsible for descending dysploidy in crucifer plants... III

III. Mandáková and Lysak (2008) This study represents the most comprehensive CCP study published in Brassicaceae. The whole-karyotype comparative analyses of species having identical chromosome number n = 7 from crucifer lineage II and affiliated tribes. The mechanism of chromosome number reduction from n = 8 to n = 7 is described and ancestral karyotype with n = 7 proposed.

6.3 Evaluation of the role of hybridization and polyploidy in karyotype and genome evolution... IV - VII

IV. Dierschke et al. (2009) Antient inter-species hybridization and polyploid evolution in the genus Lepidium revealed by genomic in situ hybridization.

V. Mandáková et al. (2010a) Mesopolyploid WGD in Australian crucifers revealed by CCP. It is demonstrated that the diploid-like genomes are originated from a common ancestral genome through a WGD followed by multiple rounds of chromosome rearrangements. Centromere inactivation was for the first time proposed as an important and common mechanism of karyotype and genome evolution in the Brassicaceae.

VI. Mandáková et al. (2012) Mesopolyploid WGT in the evolutionary history in the tribe Heliophileae, the most diversified Brassicaceae lineage, uncovered by CCP. This is the first study to reported polyploidy as a potential major mechanism for the radiation of a Cape plant group.

44 VII. Cheng et al., submitted. The reconstruction of the three diploid Brassica genomes with seven chromosomes involved in the origin of the hexaploid ancestror with 42 chromosomes.

6.4 The role of chromosome rearrangements in plant speciation... VIII

VIII. Mandáková et al. (2010b) An evidence that large-scale chromosomal rearrangements often thought as key players in plant speciation did not play a major role in species diversification in the genus Pachycladon endemic to New Zealand.

45 7 Selected publications of the author

I. Lysak M.A., Mandáková T. 2013. Analysis of plant meiotic chromosomes by chromosome painting. In: Pawlowski W.P, Grelon M. (Eds.). Plant Meiosis: Methods and protocols. Humana Press. NY.

II. Lysak M.A., Mandáková T., Lacombe E. 2010. Reciprocal and multi- species chromosome BAC painting in crucifers (Brassicaceae). Cytogenetic and Genome Research 129: 184-189.

III. Mandáková T., Lysak M.A. 2008. Chromosomal phylogeny and karyotype evolution in x = 7 crucifer species (Brassicaceae). Plant Cell 20: 2559-2570.

IV. Dierschke T., Mandáková T., Lysak M.A., Mummenhoff K. 2009. A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization. Annals of Botany 104: 681-688.

V. Mandáková T., Joly S., Krzywinski M., Mummenhoff K., Lysak M.A. 2010. Fast diploidization in close mesopolyploid relatives of Arabidopsis. Plant Cell 22: 2277-2290.

VI. Mandáková T., Mummenhoff K., Al-Shehbaz I.A., Mucina L., Mühlhausen A., Lysak M.A. 2012. Whole-genome triplication and species radiation in the southern African tribe Heliophileae (Brassicaceae). Taxon 64: 989-1000.

VII. Cheng F., Mandáková T., Wu J., Xie Q., Lysak M.A., Wang X. Deciphering the diploid ancestral genome of the mesohexaploid Brassica rapa. Submitted.

VIII. Mandáková T., Heenan P.B., Lysak M.A. 2010. Island species radiation and karyotypic stasis in Pachycladon allopolyploids. BMC Evolutionary Biology 10: 367.

46 I. Lysak M.A., Mandáková T. 2013. Analysis of plant meiotic chromosomes by chromosome painting. In: Pawlowski W.P, Grelon M. (Eds.). Plant Meiosis: Methods and protocols. Humana Press. NY.

47 48 Chapter 2 1

Analysis of Plant Meiotic Chromosomes 2 by Chromosome Painting 3

Martin A. Lysak and Terezie Mandáková 4

Abstract 5

Chromosome painting (CP) refers to visualization of large chromosome regions, entire chromosome arms, 6 or entire chromosomes via fluorescence in situ hybridization (FISH). For CP in plants, contigs of 7 chromosome-specific bacterial artificial chromosomes (BAC) from the target species or from a closely 8 related species (comparative chromosome painting, CCP) are typically applied as painting probes. Extended 9 pachytene chromosomes provide the highest resolution of CP in plants. CP enables identification and trac- 10 ing of particular chromosome regions and/or entire chromosomes throughout all meiotic stages as well as 11 corresponding chromosome territories in premeiotic interphase nuclei. Meiotic pairing and structural 12 chromosome rearrangements (typically inversions and translocations) can be identified by CP. Here, we 13 describe step-by-step protocols of CP and CCP in plant species including chromosome preparation, BAC 14 DNA labeling, and multicolor FISH (Fig. 1). 15

Keywords Chromosome painting, Fluorescence in situ hybridization, BAC FISH, Pachytene chro- 16 mosomes, DNA labeling 17

1 Introduction 18

The chromosome painting (CP) technique in human and animal 19 cytogenetics serves in situ identification of whole chromosomes 20 and specific chromosome regions using flow sorted, Degenerate 21 Oligonucleotide Primed PCR (DOP-PCR)-amplified, and 22 fluorescently labeled chromosomes or chromosome segments. Due 23 to abundant and diverse DNA repeats homogeneously distributed 24 across chromosome complements (1), flow sorted or micro 25 dissected chromosomes are not suitable for preparation of 26 chromosome-specific painting probes in plants. Instead, 27 chromosome-specific high-capacity DNA vectors have become 28 widely utilized in plant cytogenetics since the mid-1990. Specifically, 29 individual bacterial artificial chromosome (BAC) clones and BAC 30 contigs (continuous sets of BAC clones) are most frequently used 31

W.P. Pawlowski et al. (eds.), Plant Meiosis: Methods and Protocols, Methods in Molecular Biology 990, DOI 10.1007/978-1-62703-333-6_2, © Springer Science+Business Media New York 2013

49 M.A. Lysak and T. Mandáková

Fig. 1 Application of comparative multicolor chromosome painting for meiotic studies. (a) Chromosome painting in Shahdara × Columbia (Sha × Col) hybrid of Arabidopsis thaliana (n = 5, At1–At5). A 2.2 Mb paracentric inversion on the top arm of chromosome At3 is speciﬁc for Sha and absent in Col. Paired Sha and Col

50 Chromosome Painting

as chromosome-specific probes. Fluorescence in situ hybridization 32 (FISH) of single or several BAC clones is referred to as BAC FISH, 33 whereas BAC painting or chromosome painting applies to in situ 34 hybridization of BAC contigs covering larger chromosome regions 35 (e.g., chromosome arms) or whole chromosomes (Fig. 1). 36 Chromosome-specific BAC contigs are used as painting probes 37 either in the same species (2–5) or in species with sufficient chro- 38 mosome homeology (cross-species or comparative chromosome 39 painting, CCP) (3, 6–8). Recently, reciprocal BAC painting and 40 multi-species BAC painting (using painting probes of two or more 41 species to chromosomes of another species) on pachytene chromo- 42 somes has been established (9). 43 Although CP enables identification of chromosome regions 44 and whole chromosomes during all (pre)meiotic stages (2), 45 pachytene bivalents and multivalents usually offer the highest reso- 46 lution. Here, we provide a protocol to paint meiotic and mitotic 47 chromosomes of plants using chromosome-specific BAC clones 48 and BAC contigs. These procedures are essentially based on our 49 long-term experience with CP and CCP in crucifer species 50 (Brassicaceae) (2, 3, 6, 8, 9). 51

2 Materials 52

Prepare and store all reagents at room temperature (unless indi- 53 cated otherwise). 54

2.1 Collection of 1. Freshly prepared Carnoy’s I ﬁxative: 3 parts ethanol, 1 part 55 Floral Material glacial acetic acid; or Carnoy’s II ﬁxative: 6 parts ethanol, 3 56 parts chloroform, 1 part glacial acetic acid (see Note 1). 57

Fig. 1 (continued) homologues were identified by differentially labeled A. thaliana BAC contigs (inversion marked by arrowheads).(b–i) Comparative chromosome painting in Brassicaceae species using chromosome-specific A. thaliana BAC contigs. (b) Pachytene chromosome Sn4 of Stenopetalum nutans (n = 4, Sn1–Sn4) painted by 14 differentially labeled A. thaliana BAC contigs. Image of the same chromosome digitally straightened using the “straighten-curved- objects” plugin in the Image J software (12). (c–h) CCP of chromosome Ca1 in diploid Cardamine amara (n = 8, Ca1–Ca8) at pachytene (c), diplotene (d), diaki- nesis (e), metaphase I (f), telophase I (g), and anaphase/telophase II (h). (i) CCP of a pachytene tetravalent of Ca1 in autotetraploid C. amara (n = 16, Ca1–Ca16). Chromosome-specific Arabidopsis BAC contigs were labeled by biotin-dUTP (red) and digoxigenin-dUTP (green), and immuno-detected using antibodies coupled to Texas Red and Alexa Fluor 488, respectively. Yellow signals correspond to Cy3- dUTP-labeled contigs. In addition, BAC clones hybridized to chromosome Sn4 in (b) were also labeled by DEAC-dUTP (blue) and DNP-dUTP (Cy5, magenta). CENs refer to centromeres (not labeled by BAC clones). Size of BAC contigs in Mb according to http://www.arabidopsis.org. All meiotic chromosomes were isolated from anthers and counterstained with DAPI. Bars = 10 mm 51 M.A. Lysak and T. Mandáková

58 2. 70% ethanol.

59 3. Forceps.

60 4. Glass vials or microcentrifuge tubes.

61 2.2 Chromosome 1. 10× citrate buffer: 40 mL of 100 mM citric acid and 60 mL of 62 Preparation 100 mM trisodium citrate, adjust pH to 4.8; store at 4°C.

63 2. Pectolytic enzyme mixture in 1× citrate buffer: 0.3% pecto- 64 lyase, 0.3% cellulase, 0.3% cytohelicase (Sigma Aldrich, St. 65 Louis, MO, USA) prepared from frozen 1% stock solutions in 66 1× citrate buffer (see Note 2).

67 3. 60% glacial acetic acid in distilled water (see Note 3).

68 4. Freshly prepared ice-cold Carnoy’s I ﬁxative.

69 5. 4% freshly prepared formaldehyde in distilled water.

70 6. “Assistant” staining blocks with glass cover (Karl Hecht 71 Assistant, Sondheim/Rhön, Germany).

72 7. Humid box.

73 8. Incubator at 37°C.

74 9. Stereomicroscope, microscope with phase contrast.

75 10. SuperFrost microscope slides (Fisher Scientiﬁc, Suwanee, GA, 76 USA).

77 11. Dissection needles.

78 12. Fine forceps.

79 13. Glass Pasteur pipette.

80 14. Heating block.

81 15. Hair dryer.

82 2.3 Chromosome 1. 20× Saline Sodium Citrate (SSC): 3 M sodium chloride, 83 Preparation 300 mM trisodium citrate, pH 7.0. 84 Pretreatment 2. RNase: 100 mg/mL DNase-free ribonuclease A (AppliChem, 85 St. Louis, MO, USA). Make stock of 1 mg/mL in distilled 86 water. Store aliquots at −20°C.

87 3. Pepsin from porcine gastric mucosa (Sigma Aldrich, St. Louis, 88 MO, USA) 0.1 mg/mL in 10 mM HCl. Prepared from 89 100 mg/mL stock in 10 mM HCl. Store aliquots at −20°C.

90 4. 4% freshly prepared formaldehyde in 2× SSC.

91 5. 70%, 80%, and 96% ethanol.

92 6. 4¢, 6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. 93 Louis, MO, USA): 2 mg/mL in Vectashield antifade (Vector 94 Laboratories, Burlingame, CA, USA). Store at 4°C.

95 7. Coverslips (24 × 24 mm and 24 × 50 mm).

96 8. Coplin or Hellendahl jar (see Note 4).

52 Chromosome Painting

9. Humid box. 97

10. Incubator and water bath at 37°C. 98

11. Plastic tube rack. 99

100 2.4 Probe Labeling 1. 10× NT buffer: 500 mM Tris–HCl pH 7.5, 50 mM MgCl2, 0.05% bovine serum albumin. 101

2. Nucleotide mixture: 2 mM dATP, dCTP, dGTP, and 400 mM 102 dTTP (Roche Applied Science, Indianapolis, IN, USA). 103

3. 1 mM x-dUTP (x stands for biotin, digoxigenin, Cy3 or other 104 hapten/ﬂuorochrome; see Note 5). 105 4. 0.1 M b-mercaptoethanol. 106 5. DNase I (Roche Applied Science, Indianapolis, IN, USA). Use 107 a 1:250 dilution of a 1 mg/mL DNase stock in 0.15 M NaCl 108 in 50% glycerol. 109 6. DNA polymerase I (10 U/mL; Fermentas, Glen Burnie, MA, 110 USA). 111

7. 0.5 M EDTA, pH 8.0. 112

8. 100 bp DNA ladder. 113

9. 0.5 mL microcentrifuge tubes. 114

10. Thermocycler for 15°C and 60°C. 115

11. Electrophoresis system. 116

12. 1% agarose gel. 117

2.5 Probe 1. 3 M sodium acetate, pH 5.2. 118

Preparation and In 2. 70% ethanol, 96% ice-cold ethanol. 119 Situ Hybridization 3. Hybridization buffer: 50% formamide, 10% dextran sulfate in 120 2× SSC. 121

4. 2 mL microcentrifuge tubes. 122

5. Refrigerated centrifuge. 123

6. Vacuum desiccator or SpeedVac. 124

7. Thermomixer (a heating block with exact temperature control) 125 at 80°C. 126

8. 24 × 24 mm, 22 × 22 mm, or 24 × 32 mm coverslips. 127

9. Rubber cement. 128

10. Humid box. 129

11. Incubator at 37°C. 130

2.6 Fluorescence 1. 2× SSC. 131

Detection of 2. 50% or 20% deionized formamide in 2× SSC, pH 7.0 (see Note 6). 132 Hybridized Probes 3. 4T buffer: 4× SSC pH 7.0, 0.05% Tween-20. 133

53 M.A. Lysak and T. Mandáková

134 4. Blocking solution: 5% bovine serum albumin, 0.2% Tween-20 135 in 4× SSC.

136 5. TNT buffer: 100 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.05% 137 Tween-20.

138 6. TNB buffer: 100 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5% 139 blocking reagent (Roche Applied Science, Indianapolis, IN, 140 USA).

141 7. Antibodies: avidin–Texas Red (Vector Laboratories, 142 Burlingame, CA, USA), goat anti-avidin–biotin (Vector 143 Laboratories, Burlingame, CA, USA), mouse anti-digoxigenin 144 (Jackson ImmunoResearch Laboratories, West Grove, PA, 145 USA), goat anti-mouse–Alexa Fluor 488 (Invitrogen, Carlsbad, 146 CA, USA). 147 8. DAPI (2 mg/mL) in Vectashield antifade. 148 9. 70%, 80% and 96% ethanol.

149 10. Coplin or Hellendahl jars (see Note 2).

150 11. Water bath shaker at 42°C.

151 12. Coverslips (24 × 32 mm and 24 × 50 mm).

152 13. Humid box.

153 14. Incubator at 37°C.

154 15. Epifluorescence microscope equipped with optical filters for 155 DAPI and other fluorochromes, a digital charge-coupled 156 device (CCD) camera, and image acquisition software.

157 3 Methods

158 Carry out all procedures at room temperature unless otherwise 159 speciﬁed. To minimize ﬂuorochrome bleaching, avoid overexpo- 160 sure to light during procedures 3.4–3.6.

161 3.1 Collection 1. Fix entire inflorescences, individual flower buds, or anthers in 162 of Floral Material Carnoy’s fixative at room temperature or at 4°C overnight; 163 change the fixative several times.

164 2. Exchange the ﬁxative for 70% ethanol and store the ﬁxed mate- 165 rial at −20°C (see Note 7).

166 3.2 Chromosome 1. Rinse ﬂoral material with distilled water in a staining block or 167 Preparation small petri dish for 10 min. Under a stereomicroscope, remove 168 and discard unwanted parts (e.g., yellow anthers containing 169 pollen).

170 2. Replace water with 1× citrate buffer and wash two times, 5 min 171 each wash.

54 Chromosome Painting

3. Incubate the material in ~1 mL of pectolytic enzyme mixture 172 in a humid box at 37°C for 3 h (see Note 8). 173

4. Replace the enzymes with 1× citrate buffer and keep the mate- 174 rial on ice or at 4°C until use (see Note 9). 175

5. Put a single flower bud/anther on a microscope slide using a 176 Pasteur pipette, remove the excess fluid and add ~20 mL of 177 60% acetic acid. Disintegrate the bud by dissection needles 178 until a fine suspension is formed. 179

6. Place the slide on a heating block (50°C) and spread the 180 suspension by careful circular stirring with a needle for ~30 s 181 (see Note 10). 182 7. Fix the chromosomes by pipetting 100 mL of Carnoy’s I ﬁxative 183 around the suspension drop. Discard the ﬂuid by tilting the 184 slide and dry using a hair dryer. 185

8. Using a phase-contrast light microscope, examine the prepara- 186 tion for speciﬁc meiotic stages and the amount of cytoplasm. 187

9. Post-ﬁx the slides in a Coplin or Hellendahl jar with 4% form- 188 aldehyde in distilled water for 10 min and leave to air-dry. 189 Carry out this step in a fume hood. 190

10. Store dried slides in a dust-free box at 4°C (see Note 11). 191

3.3 Chromosome 1. Wash slides two times in 2× SSC in a Coplin jar, 5 min each 192 Preparation wash. 193 Pretreatment 2. Add 100 mL of RNase solution, cover with 24 × 50 mm cover- 194 slip and incubate the slides in a humid box at 37°C for 1 h (see 195 Note 12). 196

3. Tilt slides to let the coverslip fall off and wash slides as in step 1. 197

4. To remove cytoplasm, treat slides with pepsin at 37°C for 198 5 min in a Coplin jar placed in a water bath. 199

5. Wash slides as in step 1. 200

6. Dehydrate slides in an ethanol series (70%, 80%, and 96% etha- 201 nol, 3 min each) and leave them to air-dry. 202 7. Apply 15 mL of Vectashield with DAPI to a slide and cover it 203 with a 24 × 24 mm coverslip. Check the slide with a ﬂuorescence 204 microscope. Chromosomes should be undamaged and free of 205 cytoplasm. When cytoplasm is persistent, remove the coverslip 206 using running water and repeat steps 4–7. 207

8. Remove the coverslip using running water ﬂow and wash slides 208 as in step 1. 209

9. Post-ﬁx slides in a Coplin or Hellendahl jar with 4% formalde- 210 hyde in 2× SSC for 10 min. Carry out this and the following 211 step in a fume hood. 212

10. Wash slides as in step 1. 213

55 M.A. Lysak and T. Mandáková

214 11. Dehydrate slides in an ethanol series (70%, 80%, and 96% 215 ethanol, 3 min each) and leave them to air-dry.

216 3.4 Probe Labeling 1. Combine in an 0.5 mL microcentrifuge tube: 1 mg of BAC 217 by Nick Translation DNA in 32 or 29 mL distilled water, 5 mL of 10× NT buffer, 218 (See Note 13) 5 mL of nucleotide mixture, 1 mL of 1 mM comercial x-dUTP 219 or 4 mL of 1 mM custom-made x-dUTP, 5 mL of 0.1 M b-mer- 220 captoethanol, 1 mL of DNase I, and 1 mL of DNA polymerase 221 I. Vortex gently and spin brieﬂy (see Note 14).

222 2. Incubate at 15°C for 90 min. 223 3. Transfer the tube on ice and load 5 mL of the reaction volume 224 on a 1% agarose gel along with a 100-bp DNA ladder and run 225 the electrophoresis.

226 4. When the smear of labeled fragments is ~200–500 bp in size, 227 stop the nick translation by adding 1 mL of 0.5 M EDTA and 228 heating at 60°C for 10 min. When fragments are longer than 229 500 bp, extend the incubation at 15°C for further 30 min and 230 repeat steps 3 and 4.

231 5. Store the probe at −20°C until use.

232 3.5 Probe 1. Pool individually labeled BAC clones by pipetting 5 mL 233 Preparation and In (~100 ng of DNA) of each BAC into a 2 mL microcentrifuge 234 Situ Hybridization tube. 235 2. To reduce the probe volume and remove unincorporated 236 nucleotides, precipitate the DNA by adding 0.1 volume of 3 M 237 sodium acetate and 2.5 volume of ice-cold 96% ethanol. Vortex 238 and keep on ice or at −20°C for at least 30 min.

239 3. Centrifuge at 13,000 × g at 4°C for 30 min.

240 4. Carefully discard the supernatant. 241 5. Add 500 mL of 70% ethanol and centrifuge again for 5 min. 242 6. Repeat step 4.

243 7. Dry the pellet using a desiccator or SpeedVac. 244 8. Resuspend the pellet in 20 mL of hybridization buffer and 245 incubate at 37°C in a thermomixer (see Note 15). 246 9. Add 20 mL of probe to the chromosome preparation (see 247 Subheading 3.3), cover with a cover slip and seal with rubber 248 cement around the edges.

249 10. Denature the probe and the chromosomal DNA by placing the 250 slide on a heating block at 80°C for 2 min.

251 11. Incubate the slide in a humid box at 37°C overnight 252 (see Note 16).

56 Chromosome Painting

3.6 Fluorescence Signal detection and amplification is described here for hapten- 253 Detection of labeled probes (biotin- and digoxigenin-dUTP) visualized by indi- 254 Hybridized Probes rect immunofluorescence via Texas Red and Alexa Fluor 488, 255 respectively (dinitrophenyl (DNP)-dUTP can also be used as a 256 third hapten). This protocol can be also used with fluorochrome- 257 labeled probes (e.g., Cy3- or DEAC-dUTP) that require only 258 post-hybridization washing prior to microscopic evaluation (steps 259 1–4, and 14). All washing steps are carried out at 42°C in Coplin 260 or Hellendahl jars placed in a water bath shaker. All incubation 261 steps are carried out at 37°C on microscope slides covered with 262 24 × 50 mm coverslips and placed in a humid box. To minimize 263 unspecific background, do not let the slides dry during the entire 264 procedure. 265

1. Remove the rubber cement frame with forceps and let the 266 coverslip fall off. 267

2. Wash slides in 2× SSC for 2 min. 268

3. Wash slides three times in 50% or 20% formamide, 5 min each 269 wash (see Note 6). 270

4. Wash slides in 2× SSC for 2 min. If ﬂuorochrome-labeled 271 probes are used, proceed with step 14. 272

5. Wash slides in 4T buffer for 5 min. 273 6. Incubate slides in 100 mL of blocking solution for 30 min. 274 7. Wash slides in 4T buffer 2 × 5 min. 275 8. Incubate slides in 100 mL of avidin–Texas Red in TNB buffer 276 (1:1,000) for 30 min. 277

9. Wash slides in TNT buffer 2 × 5 min. 278 10. Incubate slides in 100 mL of mixed goat anti-avidin–biotin 279 antibody (1:200) and mouse anti-digoxigenin antibody (1:250) 280 in TNB buffer for 30 min (see Note 17). 281

11. Repeat step 9. 282 12. Incubate slides in 100 mL of mixed avidin–Texas Red (1:1,000) 283 and goat anti-mouse Alexa Fluor 488-coupled antibody 284 (1:200) in TNB buffer for 30 min. 285

13. Repeat step 9. 286

14. Dehydrate the slides in an ethanol series (70%, 80%, and 96% 287 ethanol, 3 min each) and leave them to air-dry. 288 15. Apply 15 mL of Vectashield with DAPI to each slide and cover 289 it with a 24 × 32 mm coverslip. 290

16. Observe and photograph slides under a ﬂuorescence micro- 291 scope equipped with appropriate optical ﬁlters, CCD camera, 292 and image acquisition software. 293

57 M.A. Lysak and T. Mandáková

294 4 Notes

295 1. Carnoy’s II is believed to be more suitable than the Carnoy’s 296 I fixative for fixation of floral material. However, we did not 297 observe a significant difference between the two fixatives and 298 use the 3:1 fixative routinely.

299 2. The enzyme mixture can be reused several times (store at −20°C). 300 Digestion time might need to be increased after each use.

301 3. Glacial acetic acid can be used at concentrations from 45% 302 to 60%.

303 4. A Coplin jar can hold up to nine slides, Hellendahl jar up to 15 304 slides.

305 5. Labeled nucleotides are available commercially or can be syn- 306 thesized by coupling allylamine-dUTP to succinimidyl-ester 307 derivatives of haptenes or ﬂuorochromes (10).

308 6. 50% formamide should be used for stringent post-hybridization 309 washing after same-species (homologous) in situ hybridization; 310 20% formamide should be used in case of cross-species (home- 311 ologous) in situ hybridization.

312 7. Fixed material can be used after several months or years of 313 storage. However, best quality preparations are usually obtained 314 from freshly ﬁxed material.

315 8. The amount of enzyme mixture should be proportional to the 316 amount of digested material (tissue should be submerged). 317 The incubation time of 3 h may not be appropriate to all spe- 318 cies/types of ﬂoral material. After the incubation, we make a 319 test preparation following protocol (3.2, steps 5–8). If the tissue 320 is hard to disintegrate and there is a large quantity of cytoplasm 321 and tissue fragments, we extend enzyme incubation for another 322 30 min or longer.

323 9. Digested ﬂoral tissue can be stored in 1× citrate buffer at 4°C 324 overnight or longer. Overnight storage further softens the 325 digested material. 326 10. The amount of acetic acid can be increased to 40–60 mL and 327 the duration of suspension spreading can be prolonged. The 328 duration of spreading can be shortened in case of very small 329 ﬂower buds or anthers. The needle should not touch the slide 330 surface.

331 11. Post-ﬁxed slides can be stored at 4°C for several weeks or 332 months. However, freshly prepared or few-days old slides 333 guarantee better results.

334 12. Omitting the RNase treatment may result in a longer pepsin 335 treatment used in Subheading 3.4, step 4 and/or increased 336 background.

58 Chromosome Painting

13. As Brassicaceae possess very small genomes and low amount of 337 repeats localized in pericentromeric heterochromatin, 338 chromosome-speciﬁc BAC clones from euchromatic regions 339 can be used without the need to block repetitive sequences 340 from cross-hybridization. For chromosome painting in other 341 plant taxa, BAC clones have to be screened for the presence of 342 dispersed repeats or genomic/Cot DNA needs to be applied 343 for blocking. 344

14. DNA of large-insert clones (usually BAC clones) can be iso- 345 lated by use of a standard alkaline lysis protocol (11) or using 346 a DNA isolation kit (e.g., the Qiagen Plasmid Midi Kit; Qiagen, 347 Hilden, Germany). A larger amount (4 mL) of custom-made 348 x-dUTPs must be used as custom dUTPs label DNA less 349 efﬁciently than commercial nucleotides (10). DNA of several 350 probes/BAC clones can be pulled and labeled together in a 351 single nick translation reaction. However, in our laboratory we 352 label each probe individually. 353

15. Time needed to resuspend the probe is dependent on the ini- 354 tial amount of precipitated DNA. DNA of several BAC clones 355 is well dissolved after several minutes or hours. Complex probes 356 comprising several dozen or hundred clones should be incu- 357 bated overnight. 358

16. Overnight incubation is usually sufﬁcient for in situ hybridiza- 359 tion of homologous (same species) sequences. Longer hybrid- 360 ization times (48–72 h) may be required to ensure hybridization 361 of homeologous (cross-species) sequences. 362

17. If the first detection step for hapten-labeled probes (see 363 Subheading 3.6, step 8) yields a sufficiently strong signal, 364 further signal amplification (e.g., by goat anti-avidin–biotin 365 antibody in this protocol) can be omitted. 366

Acknowledgments 367

We thank Dr A. Pecinka for providing seeds of the Sha × Col hybrid. 368 This work was supported by grants IAA601630902 and 369 P501/10/1014 from the Grant Agency of the Czech Academy of 370 Science and the Czech Science Foundation (GA CR), respectively. 371 German Science Foundation (DFG) and Alexander v. Humboldt 372 Foundation are acknowledged for supporting our research 373 (2003–2009). 374

59 M.A. Lysak and T. Mandáková

375 References

376 1. Schubert I, Fransz PF, Fuchs J, de Jong JH species. Proc Natl Acad Sci USA 103: 402 377 (2001) Chromosome painting in plants. 5224–5229 403 378 Methods Cell Sci 23:57–69 7. Ziolkowski PA, Kaczmarek M, Babula D, 404 379 2. Lysak MA, Fransz PF, Ali HBM, Schubert I Sadowski J (2006) Genome evolution in 405 380 (2001) Chromosome painting in Arabidopsis Arabidopsis/Brassica: conservation and diver- 406 381 thaliana. Plant J 28:689–697 gence of ancient rearranged segments and their 407 382 3. Lysak M, Fransz P, Schubert I (2006) breakpoints. Plant J 47:63–74 408 383 Cytogenetic analyses of Arabidopsis. In: Salinas 8. Mandáková T, Joly S, Krzywinski M, 409 384 J, Sanchez-Serrano JJ (eds) Methods in molec- Mummenhoff K, Lysak MA (2010) Fast dip- 410 385 ular biology, vol 323, Arabidopsis protocols. loidization in close mesopolyploid relatives of 411 386 Humana, Totowa, NJ, pp 173–186 Arabidopsis. Plant Cell 22:2277–2290 412 387 4. Szinay D, Chang S-B, Khrustaleva L, Peters S, 9. Lysak MA, Mandáková T, Lacombe E (2010) 413 388 Schijlen E, Bai Y et al (2008) High-resolution Reciprocal and multi-species chromosome 414 389 chromosome mapping of BACs using multi- BAC painting in crucifers (Brassicaceae). 415 390 colour FISH and pooled-BAC FISH as a back- Cytogenet Genome Res 129:184–189 416 391 bone for sequencing tomato chromosome 6. 10. Henegariu O, Bray-Ward P, Ward DC (2000) 417 392 Plant J 56:627–637 Custom fluorescent nucleotide synthesis as an 418 393 5. Febrer M, Goicoechea JL, Wright J, McKenzie alternative method for nucleic acid labeling. 419 394 N, Song X, Lin J et al (2010) An integrated Nat Biotechnol 18:345–348 420 395 physical, genetic and cytogenetic map of 11. Sambrook J, Russell DW (2001) Molecular clon- 421 396 Brachypodium distachyon, a model system for ing: a laboratory manual. Cold Spring Harbor 422 397 grass research. PLoS One 5:e13461 Laboratory Press, Cold Spring Harbor, NY 423 398 6. Lysak M, Berr A, Pecinka A, Schmidt R, 12. Kocsis E, Trus BL, Steer CJ, Bisher ME, Steven 424 399 McBreen K, Schubert I (2006) Mechanisms AC (1991) Image averaging of flexible fibrous 425 400 of chromosome number reduction in macromolecules: the clathrin triskelion has an 426 401 Arabidopsis thaliana and related Brassicaceae elastic proximal segment. J Struct Biol 107:6–14 427

60 II. Lysak M.A., Mandáková T., Lacombe E. 2010. Reciprocal and multi- species chromosome BAC painting in crucifers (Brassicaceae). Cytogenetic and Genome Research 129: 184-189.

61 62

Cytogenet Genome Res Published online: May 26, 2010 DOI: 10.1159/000312951

Reciprocal and Multi-Species Chromosome BAC Painting in Crucifers (Brassicaceae)

a a b M.A. Lysak T. Mandáková E. Lacombe a Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, b Masaryk University, Brno , Czech Republic; Laboratoire de Biochimie et Physiologie Moleculaire des Plantes, UMR Université Montpellier 2-CNRS-INRA-Montpellier SupAgro, Montpellier , France

Key Words Identification of plant chromosomes by chromosome- Arabidopsis halleri ؒ Arabidopsis thaliana ؒ BAC FISH ؒ specific painting probes is hampered by a wide spectrum Brassicaceae ؒ Collinearity ؒ Comparative chromosome of dispersed repetitive elements equally distributed over -painting ؒ Plant cytogenetics chromosomes of a given complement. Hence, all flow sorted or microdissected, and DOP-PCR amplified, DNA probes yielded unspecific cross-hybridization signals [re- Abstract viewed by Schubert et al., 2001]. An alternative strategy In crucifer cytogenomics, BAC contigs of Arabidopsis thaliana to paint chromosomes of plants has been found in the ap- have been used as probes for comparative chromosome plication of contigs and supercontigs of chromosome- painting among species. Here we successfully tested chro- specific BAC clones. This approach relies on the availabil- mosome-specific BAC contigs of A. thaliana (n = 5) and A. ity of chromosome-specific BAC libraries and a low halleri (n = 8) as probes for reciprocal BAC painting. Further- amount of repetitive elements in the donor and target ge- more, BAC contigs of both Arabidopsis species were applied nomes. as multi-species painting probes to a third crucifer species, Arabidopsis thaliana (n = 5) has become the first plant Noccaea caerulescens (n = 7), revealing their shared chromo- species with all chromosomes painted using chromosome homeology. Specifically, we found homeology across some-specific BAC supercontigs [Lysak et al., 2001; Pecin- portions of chromosomes At2, Ah4, and Nc4, which reflects ka et al., 2004]. Later the very same BAC contigs were ap- their shared common origin with chromosome AK4 of the plied as painting probes for comparative chromosome Ancestral Crucifer Karyotype (n = 8). We argue that multi- painting (CCP) across the mustard family (Brassicaceae) species and multi-directional painting will significantly ex- [e.g. Lysak et al., 2003, 2006; Mandáková and Lysak, pedite comparative cytogenomics in Brassicaceae and other 2008]. Recently, cross-species BAC FISH was successfully plant families. Copyright © 2010 S. Karger AG, Basel developed for sorghum and maize (Poaceae) [Amarillo and Bass, 2007], tomato and potato (Solanaceae) [Iovene et al., 2008; Tang et al., 2008] as well as for cucurbit species (Cucurbitaceae) [Han et al., 2009].

© 2010 S. Karger AG, Basel Martin A. Lysak 1424–8581/10/0000–0000$26.00/0 Department of Functional Genomics and Proteomics Fax +41 61 306 12 34 Institute of Experimental Biology, Masaryk University E-Mail [email protected] Accessible online at: Kamenice 5, Building A2, CZ–625 00 Brno (Czech Republic) www.karger.com www.karger.com/cgr Tel. +420 549 494 154, Fax +420 549 492 654, E-Mail lysak @ sci.muni.cz

63 In Brassicaceae, BAC clones of A. thaliana were exclu- P a i n t i n g P r o b e s sively used as painting probes for cross-species CCP as A. thaliana BAC clones were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH). The 3 BAC chromosome-specific BAC libraries for other crucifer contigs used [F3K23 (AC006841) – F8N16 (AC005727), F16P2 species were not developed. This is changing as genomes (AC004561) – F7F1 (AC004669), and F13P17 (AC004481) – T8I13 of an increasing number of cruciferous species are being (AC002337)] represent an almost complete BAC tiling path of A. sequenced and genomic resources become available. thaliana chromosome At2, corresponding to the collinear region Chromosome-specific BAC contigs were assembled for on the ancestral chromosome AK4 of the Ancestral Crucifer Karyotype [Schranz et al., 2006]. From this tiling path each third Brassica rapa [Mun et al., 2008] or A. halleri , a model spe- BAC was isolated and labeled. cies of heavy metal accumulation [Lacombe et al., 2008]. A. halleri BAC clones were selected from the genomic BAC In upcoming years we will witness a steadily increasing library [Lacombe et al., 2008] and 2 BAC contigs were built based number of whole-genome sequencing projects including on the chromosome collinearity existing between A. thaliana important crop and model crucifer species such as A. ly- and A. lyrata [Kuittinen et al., 2004]. The A contig (from AT2G31130 to AT2G32520) comprises BAC clones XIII-P-6, II- rata , Boechera spp., Capsella rubella , Eutrema ( Thellungi- F-23, XIV-I-23, II-K-14, V-A-2, I-E-5, I-K-8, and I-I-19. The B con- ella ) halophila (DOE Joint Genome Institute; http://www. tig (from AT2G32840 to AT2G33510) is composed of BAC clones jgi.doe.gov), and B. rapa (http://www.brassica.info). IV-K-16, III-M-8, and II-F-13. Furthermore, 2 repeat-rich BAC These sequencing efforts will generate new cytogenomic clones IV-E-9 and IX-B-21 [Lacombe et al., 2008] were tested resources and significantly facilitate family-wide com- ( fig. 1 A, D). DNA of individual BAC clones was isolated using a standard parative research. alkaline extraction omitting the phenol:chloroform purification In anticipation of newly available genomic resources step. BAC DNA was labeled by biotin-, digoxigenin-, and Cy3- for crucifer cytogenomics, we have tested chromosome- dUTP via nick translation as follows: 1 ␮ g of BAC DNA diluted specific BAC contigs of A. halleri (n = 8) and A. thaliana in distilled water to 29 ␮l, 5 ␮l of nucleotide mix (2 m M dATP, ␮ ␮ ! as probes for reciprocal CCP. Furthermore, the BAC con- dCTP, and dGTP, 400 M dTTP; all Roche), 5 l of 10 NT buffer (0.5 M Tris-HCl, pH 7.5; 50 m M MgCl2 , 0.05% BSA), 4 ␮ l of tigs of both Arabidopsis species were applied simultane- 1 m M x-dUTP (in which x was biotin, digoxigenin or Cy3), 5 ␮ l of ously as multi-species painting probes to reveal shared 0.1 M b-mercaptoethanol, 1 ␮ l of DNase I (Roche), and 1 ␮ l of chromosome homeology in a third species. DNA polymerase I (Fermentas). The nick translation mixture was

incubated at 15 ° C for 90 min (or longer) to obtain fragments of ϳ200 to 500 bp. The reaction was stopped by adding 1 ␮ l of 0.5 M

EDTA, pH 8.0, followed by incubation at 65 ° C for 10 min. Labeled Material and Methods DNA of individual BAC clones was stored at –20 ° C until use.

Plant Material Chromosome Painting: BAC FISH Inflorescences of A. halleri subsp. tatrica (Pawł.) Kolník (2n = The selected slides were treated by RNase (AppliChem; 100 16) were collected from a single wild population (Slovakia, Beli- ␮ g/ml in water) for 1 h at 37 ° C, and washed in 2 ! SSC for 2–5 anske Tatry Mts., Tatranská Javorina, the Zadné Meďodoly val- min. To remove cytoplasm, the slides were treated with pepsin ley). A. thaliana accession C24 (2n = 10) was used in the present (Sigma-Aldrich; 0.1 mg/ml) in 0.01 M HCl at 38 ° C for 10 min, fol- study. Flower buds of Noccaea caerulescens (2n = 14) were col- lowed by a wash in 2! SSC for 2–5 min. Subsequently, the slides lected from a population in the village of Kořenec, Czech Repub- were post-fixed in 4% formaldehyde in 2! SSC for 10 min, washed lic [Mandáková and Lysak, 2008]. in 2! SSC (2–5 min), and dehydrated in an ethanol series (70, 80, and 96%). Preparation of Pachytene Chromosomes Labeled BAC DNAs were pooled and precipitated to reduce the Entire inflorescences were fixed in freshly prepared etha- probe volume. For a single slide, the probe was dissolved in 20 ␮ l nol:acetic acid (3: 1) fixative overnight and stored in 70% ethanol of hybridization mix (50% formamide, 10% dextran sulfate in 2 ! at –20 ° C until use. Selected inflorescences were rinsed in distilled SSC) at 37 ° C overnight. Probe and chromosomes were denatured water and citrate buffer (10 m M sodium citrate, pH 4.8) and sub- together on a hot plate at 80 ° C for 2 min and incubated in a moist sequently incubated in the 0.3% enzyme mix (cellulase, cytoheli- chamber at 37 ° C for 48 h. Post-hybridization washing was per- case and pectolyase; all Sigma-Aldrich) in citrate buffer at 37 ° C formed in 50% or 20% (CCP in N. caerulescens ) formamide in 2! for 3–4 h. After digestion, individual flower buds were disinte- SSC for 3–5 min at 42 ° C. DNA labeled by biotin-dUTP was de- grated by a needle in a small drop of citrate buffer and the mate- ϳ ␮ tected using avidin Texas Red (Vector Laboratories) and ampli- rial spread in 20 l of 60% acetic acid on a hot plate (50 ° C). The ϳ ␮ fied by goat anti-avidin biotin (Vector Laboratories) and chromosomes were fixed using 100 l of ice-cold ethanol:ace- avidin ϳ Texas Red. Probes labeled by digoxigenin-dUTP were vi- tic acid fixative, then the slides were dried using a hair dryer. The sualized by mouse anti-digoxigenin (Jackson ImmunoResearch) preparations were staged using a phase contrast microscope and and goat anti-mouseϳ Alexa Fluor 488 (Molecular Probes). Chro- suitable slides were post-fixed in 4% formaldehyde in distilled wa- mosomes were counterstained with DAPI (2 ␮ g/ml) in Vecta- ter for 10 min and air-dried. shield (Vector Laboratories).

2 Cytogenet Genome Res Lysak /Mandáková /Lacombe

64 A

B C

Fig. 1. BAC painting in Arabidopsis halleri and A. thaliana . A Labelling scheme of A. halleri BAC contigs A and B, and BAC clone IV-E-9. B , C In situ hybridization of the labeled probes (A ) to pachytene chromosomes of A. halleri ( B ) and A. thaliana ( C ), respectively. D Labelling scheme of A. halleri BAC contigs A and B, and BAC clone IX-B-21. Black lines correspond to BAC clones labeled in (A ). E , F In situ hybridization of the labeled probes ( D) to pachytene chromosomes of A. halleri ( E ) and A. thaliana (F ), respectively. Bar (10 ␮ m) has the same magnification in ( B ) and (C ), and in (E ) and (F ), respectively.

Preparations were observed using an Olympus BX-61 epifluo- Results and Discussion rescence microscope, and images were acquired separately for all fluorochromes using appropriate excitation and emission filters (AHF Analysentechnik) using an AxioCam CCD camera (Zeiss). We have hybridized 2 A. halleri BAC contigs to pachy- The 4 monochromatic images were pseudocolored and merged tene chromosomes of A. halleri and A. thaliana . The 8 using the Adobe Photoshop CS2 software (Adobe Systems). and 3 BAC clones of the A (c. 0.55 Mb in A. thaliana ) and

Reciprocal and Multi-Species BAC Cytogenet Genome Res 3 Painting in Crucifers

65 A

Fig. 2. BAC chromosome painting in Arabidopsis halleri and multi-species chromosome painting in Noccaea caerulescens . A Labeling scheme of A. halleri BAC contigs A and B (green, yellow), and A. thaliana supercontigs corresponding to genomic blocks I and J (red) of the Ancestral Crucifer Karyotype [Schranz et al., 2006]. Si- multaneous in situ hybridization of A. halleri and A. thaliana BAC contigs to pachytene chromosome of A. halleri ( B ) and N. caerulescens (C ). Bar (10 ␮ m) has the same magnification in (B ) and (C ).

B (c. 0.27 Mb) contigs, respectively, have been differen- hybridization signal intensity was weaker than in A. hal- tially labeled (fig. 1 A, D), and tested in both Arabidopsis leri ( fig. 1 F). The hybridization of BAC IV-E-9 to centro- species in the absence of blocking DNA. In A. halleri , the meric heterochromatin is due to gypsy - and CACTA-like digoxigenin- (green) and Cy3-dUTP (yellow) labeled mobile elements, whereas the clone IX-B-21 contains pre- contigs A and B identified the expected chromosome re- sumably a copia -like retrotransposon(s) [Lacombe et al., gion on the bottom arm of chromosome Ah4 (fig. 1 B, E). 2008]. The same contigs cross-hybridized to A. thaliana pachy- Furthermore, we demonstrate how BAC contigs of tene chromosomes provided a comparably strong signal both Arabidopsis species can be combined and simulta- on the bottom arm of chromosome At2 (fig. 1 C, F), cor- neously used to paint specific chromosome regions in a responding to the collinear region of Ah4 [Kuittinen et third species ( fig. 2 A–C). The 2 A. halleri contigs along al., 2004; Schranz et al., 2006]. with 3 A. thaliana BAC supercontigs homeologous to the We have also tested 2 BAC clones positioned between genomic blocks I and J on ancestral chromosome AK4 the contigs A and B and presumably containing repetitive [Schranz et al., 2006], except the part covered by A. halleri elements [Lacombe et al., 2008]. In A. halleri , BAC clones BACs, have been hybridized to pachytene chromosomes IV-E-9 and IX-B-21 hybridized to all centromeres and of A. halleri and Noccaea (Thlaspi) caerulescens. The lat- pericentromeres, respectively (fig. 1 B, E). In A. thaliana , ter species is phylogenetically closely associated with the red signals of the IV-E-9 were irregularly scattered along crucifer Lineage II [Mandáková and Lysak, 2008], which all chromosomes with some preferential hybridization at is relatively distantly related to the genus Arabidopsis of pericentromeric regions (fig. 1 C), whereas BAC IX-B-21 the Lineage I. We assumed that the A. halleri karyotype (red) hybridized to pericentromeric regions, though the resembles that of A. lyrata (n = 8) [Kuittinen et al., 2004],

4 Cytogenet Genome Res Lysak /Mandáková /Lacombe

66 and hence the Ah4 chromosome will have a structure al., 2006], chromosome-specific BAC (super)contigs of similar to Al4 of A. lyrata. The overall conserved collin- closely related n = 8 species would be more suitable as earity between 2 other A. halleri chromosomes (Ah6 and CCP probes. The present data suggest that A. halleri BAC Ah7) and chromosomes Al6 and Al7 of A. lyrata has been contigs can be successfully employed as CCP probes, and confirmed previously [Lysak et al., 2003]. In N. caerule- even more extensive resources should become available scens (n = 7), chromosome Nc4 also resembles the ances- for A. lyrata and C. rubella (both n = 8) within the DOE tral structure of AK4 [Mandáková and Lysak, 2008]. In genome sequencing program (http://www.jgi.doe.gov). both analyzed species, a strong green/yellow signal of the Moreover, by hybridizing A. halleri and A. thaliana BAC A. halleri contigs was observed. Somewhat weaker red contigs to chromosomes of N. caerulescens, we showed fluorescence of the A. thaliana supercontigs labeled the for the first time that multi-species comparative chromo- remaining parts of chromosome Ah4 and Nc4, respec- some painting in a plant species is feasible. Current ex- tively (fig. 2 B, C). The weaker hybridization signal inten- periments prove that (i) painting BAC probes derived sity of A. thaliana contigs reflected the selective use of from a pair of species can be used reciprocally in the re- approximately each third clone of the BAC tiling path. spective species (A. halleri } A. thaliana ), and (ii) BAC Multi-species painting of chromosomes Ah4 and Nc4 contigs of one species can be substituted or supplemented along the entire length confirmed their conserved AK4- by painting probes of another closely related taxon (A. like structure. halleri + A. thaliana ] N. caerulescens ). We anticipate We have shown that BAC clones of A. halleri can be that the latter approach, namely multi-species CCP, will successfully used as reliable chromosome painting probes significantly facilitate and expedite comparative recon- in this species, congeneric A. thaliana as well as in the structions of karyotype evolution in Brassicaceae and more distantly related species N. caerulescens . Compara- other plant families. tive cytogenetics in the Brassicaceae has been until now As previously reported [Lacombe et al., 2008], physical limited to the use of chromosome-specific BAC super- mapping based on the conserved synteny among Arabi- contigs of A. thaliana. As the genome of A. halleri (0.24 dopsis species can be used to assemble BAC contigs. How- pg C–1 ) [Lysak et al., 2009] is 1.5-fold larger than the ever, the presence of repetitive elements such as transpo- A. thaliana genome (0.16 pg C–1 ) [Bennett et al., 2003], it sons or retrotransposons hampers the use of this strategy can be expected to find a higher percentage of repetitive for building supercontigs and constructing complete elements in the A. halleri genome. Indeed, the sequence physical maps. In the present study we showed that cyto- data analysis of Lacombe et al. [2008] corroborated in the genetic mapping can anchor individual BAC clones and present study identified 2 BAC clones containing trans- contigs on chromosomes, build supercontigs and identify poson and retrotransposon elements. These data suggest gaps between them. Therefore, BAC painting is an effi- that the selection of BAC clones suitable for chromosome cient alternative to chromosome walking in constructing painting in A. halleri and other crucifer species with a genome-wide physical maps. similar or larger genome must be carried out with even more caution than in A. thaliana [see Lysak et al., 2003]. In species with larger genomes, undesirable cross-hy- Acknowledgements bridization of repetitive sequences could be reduced through the application of a highly repetitive Cot-100 ge- The technical support of K. Matůšová and P. Mokroš is acknowledged. We thank Prof. I. Schubert for critical reading of the nomic fraction or sheared genomic DNA as shown for manuscript. This work was supported by reseach grants from the Solanaceae species [Iovene et al., 2008; Tang et al., 2008]. Grant Agency of the Czech Academy of Science (grant no. Repetitive elements present in 2 A. halleri BAC clones IAA601630902), the Czech Ministry of Education (grant no. (IV-E-9 and IX-B-21) are shared by the 2 Arabidopsis spe- MSM0021622415) and by a Humboldt Fellowship awarded to cies, though the weaker and more scattered hybridization M.A.L. signals in A. thaliana suggest that the repeats are less abundant and/or more divergent in A. thaliana as compared to A. halleri . As the karyotype structure of A. thaliana is relatively complex due to chromosome rearrangements accompanying the chromosome number reduction (from n = 8 to n = 5) in this species [Lysak et al., 2006; Schranz et

Reciprocal and Multi-Species BAC Cytogenet Genome Res 5 Painting in Crucifers

67 References

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69 70 The Plant Cell, Vol. 20: 2559–2570, October 2008, www.plantcell.org ã 2008 American Society of Plant Biologists

RESEARCH ARTICLES Chromosomal Phylogeny and Karyotype Evolution in x=7 Crucifer Species (Brassicaceae) W

Terezie Manda´ kova´ and Martin A. Lysak1 Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Masaryk University, Brno CZ-625 00, Czech Republic

Karyotype evolution in species with identical chromosome number but belonging to distinct phylogenetic clades is a long- standing question of plant biology, intractable by conventional cytogenetic techniques. Here, we apply comparative chromosome painting (CCP) to reconstruct karyotype evolution in eight species with x=7 (2n=14, 28) chromosomes from six Brassicaceae tribes. CCP data allowed us to reconstruct an ancestral Proto-Calepineae Karyotype (PCK; n=7) shared by all x=7 species analyzed. The PCK has been preserved in the tribes Calepineae, Conringieae, and Noccaeeae, whereas karyotypes of Eutremeae, Isatideae, and Sisymbrieae are characterized by an additional translocation. The inferred chromosomal phylogeny provided compelling evidence for a monophyletic origin of the x=7 tribes. Moreover, chromosomal data along with previously published gene phylogenies strongly suggest the PCK to represent an ancestral karyotype of the tribe Brassiceae prior to its tribe-speciﬁc whole-genome triplication. As the PCK shares ﬁve chromosomes and conserved associations of genomic blocks with the putative Ancestral Crucifer Karyotype (n=8) of crucifer Lineage I, we propose that both karyotypes descended from a common ancestor. A tentative origin of the PCK via chromosome number reduction from n=8 to n=7 is outlined. Comparative chromosome maps of two important model species, Noccaea caerulescens and Thellungiella halophila, and complete karyotypes of two purported autotetraploid Calepineae species (2n=4x=28) were reconstructed by CCP.

INTRODUCTION information on the direction of karyotype evolution. To ascertain the evolutionary progression of karyotypic alterations, it is nec- Comparative linkage mapping of shared genetic markers reveals essary to distinguish ancestral from derived karyotypes. Com- the extent of interspecies chromosome collinearity, as shown for parative genetic and cytogenetic analyses showed that the grasses (Devos, 2005; Wei et al., 2007) and crucifers (reviewed compact A. thaliana genome is characterized by a highly by Koch and Kiefer, 2005). Cytogenetic evidence for collinear reshuffled and derived karyotype (Boivin et al., 2004; Kuittinen chromosome segments among closely related plants is difficult et al., 2004; Koch and Kiefer, 2005; Lysak et al., 2006), making to obtain because ubiquitous and abundant dispersed repeats this species inappropriate as a reference point for comparative usually prevent sufficient chromosome specificity of DNA probes studies across Brassicaceae (Schranz et al., 2006). Therefore, an (Schubert et al., 2001). Crucifers (Brassicaceae) represent the Ancestral Crucifer Karyotype (ACK) with eight chromosomes only plant family to date in which homeologous chromosome (AK1 to AK8) and some 24 conserved genomic blocks (GBs; A to regions/chromosomes have successfully been analyzed with X) has been proposed (Figure 1A) (Lysak et al., 2006; Schranz chromosome-specific painting probes. Comparative chromo- et al., 2006). The ACK is based on the fact that x=8 is the most some painting (CCP) in Brassicaceae is feasible due to the common base number in the family (Warwick and Al-Shehbaz, availability of virtually repeat-free Arabidopsis thaliana BAC 2006) and that the eight chromosomes of Arabidopsis lyrata and clones and the preferential location of highly repetitive DNA Capsella rubella represent nearly identical linkage groups (Boivin sequences at pericentromeric heterochromatin in most crucifer et al., 2004; Kuittinen et al., 2004; Koch and Kiefer, 2005). Also species. Therefore, CCP with selected repeat-free Arabidopsis n=6 and n=7 karyotypes share ancestral chromosomes with the BAC contigs can reveal collinear chromosome regions in other ACK (Lysak et al., 2006). Moreover, the five studied extant crucifer species (Jiang and Gill, 2006; Lysak and Lexer, 2006). karyotypes (n=5 to n=8) (Lysak et al., 2006) as well as the Although CCP reveals the extent of chromosome collinearity allopolyploid genome of Brassica napus (Parkin et al., 2005) and between A. thaliana and other species, it provides only limited the ACK are composed of 24 evolutionarily conserved GBs (Schranz et al., 2006). 1 Address correspondence to [email protected]. The current intrafamiliar classification of the family Brassica- The author responsible for distribution of materials integral to the ceae (Al-Shehbaz et al., 2006), based on the ndhF phylogeny findings presented in this article in accordance with the policy described (Beilstein et al., 2006), includes at least 25 tribes clustered into in the Instructions for Authors (www.plantcell.org) is: Martin A. Lysak three major phylogenetic lineages (Lineages I to III, with A. ([email protected]). W Online version contains Web-only data. thaliana in Lineage I and Brassica in Lineage II) or having an www.plantcell.org/cgi/doi/10.1105/tpc.108.062166 unresolved position (Figure 1B). Genetic and cytogenetic

71 2560 The Plant Cell

Figure 1. ACK (n=8) and Phylogenetic Relationship of Selected Tribes and Species within the Family Brassicaceae.

(A) Scheme of the ACK of crucifer Lineage I comprising eight chromosomes (AK1 to AK8) and 24 GBs (A to X). Modiﬁed from Schranz et al. (2006). (B) Three major phylogenetic lineages (Lineages I to III) were recognized within Brassicaceae (Beilstein et al., 2006). The six analyzed x=7 tribes (in boldface) are embedded within an unresolved assemblage of tribes including Lineage II (in blue). A tentative phylogenetic position of the ACK, and species analyzed and/or discussed in this study, are given. Base chromosome number (x) is indicated for each tribe (mult., multiple base numbers). The tree is modiﬁed from Koch and Al-Shehbaz (2008).

analyses of cross-species chromosome homeology have mostly RESULTS focused on close relatives of A. thaliana within Lineage I (Lysak et al., 2003, 2006; Koch and Kiefer, 2005; Schranz et al., 2007) CCP Analysis Suggests a Common Ancestry of the x=7 and to a lesser extent on species of the tribe Brassiceae, Species and the ACK belonging to Lineage II (Lysak et al., 2005; Parkin et al., 2005). Here we investigate (1) to what extent tribes of Lineage II and The revealed CCP patterns are here described in relation to the potentially affiliated tribes (Figure 1B), mostly possessing x=7 ACK (n=8; Figure 1A) as a reference genome. All 24 (A to X) karyotypes, are congruous with the tentative ACK (n=8) of conserved GBs of the ACK were unambiguously identified in Lineage I; (2) whether the x=7 karyotypes descended from a chromosome complements of the analyzed species by CCP with common n=7 ancestral karyotype or originated repeatedly within A. thaliana BAC contigs as probes (see Methods and Supple- this group of tribes (involving different rearrangements each mental Table 1 online). In C. orientalis (2n=2x=14; Conringieae), time); and (3) to what degree collinear GBs are conserved among painting probes for ancestral chromosomes AK1 to AK4 and AK7 the chromosomes of x=7 species. Furthermore, we searched for labeled a single pachytene bivalent or mitotic chromosome pair clade- and tribe-specific chromosome rearrangements (cytoge- each (Figure 2A). C. irregularis and G. laevigata (2n=4x=28; netic signatures) that could help resolve the ambiguous tribal Calepineae) showed the karyotype structure similar to that polytomy associated with Lineage II and affiliated x=7 tribes described for Conringia, but all five AK-like chromosomes were (Figure 1B) (Bailey et al., 2006; Beilstein et al., 2006; Koch et al., present in two copies due to a putative genome duplica- 2007; Koch and Al-Shehbaz, 2008). tion (Figures 2B and 2C; see Supplemental Figure 1 online). To address the above questions, the pattern of karyotype Figure 2D shows the reconstructed karyotype of N. caerulescens evolution has been reconstructed for eight x=7 (2n=14, 28) (2n=2x=14; Noccaeeae). Similar to the pattern observed in species from six tribes by CCP with Arabidopsis BAC contigs Calepineae and Conringieae, five Noccaea homeologues resem- arranged according to the eight ancestral chromosomes (AK1 to ble AK1 to AK4 and AK7 chromosomes. However, all but one AK- AK8) and 24 ancestral GBs (A to X) of the ACK. We analyzed three like chromosomes exhibit specific inversions (Figure 2D; see species of Lineage II (Glastaria glastifolia, Myagrum perfoliatum [both of tribe Isatideae], and Ochthodium aegyptiacum [assigned Supplemental Figure 2 online). The repatterning of AK1- and to Sisymbrieae; K. Mummenhoff, personal communication]) and AK4-like homeologues was probably caused by two subsequent five species of associated tribes (Calepina irregularis, Gold- pericentric inversions, and the structure of the AK3-like chromo- bachia laevigata [Calepineae], Conringia orientalis [Conringieae], some can be explained by a paracentric and subsequent Noccaea [Thlaspi] caerulescens [Noccaeeae], and Thellungiella pericentric inversion (see Supplemental Figure 2 online). The halophila [=Eutrema salsugineum; Eutremeae]). Among these AK7-like homeologue probably underwent a single pericentric species, N. caerulescens is an important model of heavy metal inversion. The analyzed species from the three remaining tribes accumulation (Peer et al., 2006) and T. halophila is an important (all 2n=2x=14), M. perfoliatum and G. glastifolia (both Isatideae), model of salt and cold tolerance (Gong et al., 2005). O. aegyptiacum (Sisymbrieae), and T. halophila (Eutremeae),

72 Karyotype Evolution in x=7 Crucifers 2561

Figure 2. Reconstructed Karyotypes of Conringieae, Calepineae, and Noccaeeae Species. (A) Karyotype of C. orientalis (2n=14; Conringieae) and chromosomes of this species revealed by CCP. (B) Karyotype of C. irregularis (2n=28; Calepineae). (C) Karyotype of G. laevigata (2n=28; Calepineae). (D) Karyotype of N. caerulescens (2n=14; Noccaeeae) and Noccaea chromosomes after CCP. (E) Tentative scenario of the origin of translocation chromosomes AK6/8 and AK5/6/8 from ancestral chromosomes AK5, AK6, and AK8. The 24 GBs are indicated by uppercase letters (A to X) and colored according to their positions on chromosomes AK1 to AK8 of the ACK (Figure 1A). Downward-pointing arrows indicate the opposite orientation of GBs compared with the position in the ACK. Karyotypes in (A) to (D) are drawn to scale (bar = 5 Mb). In CCP images, arrowheads point to pericentromeric heterochromatin and the arrow indicates the heterochromatic knob. Bars = 10 mm.

73 2562 The Plant Cell

share four homeologues resembling the ancestral chromosomes corroborated by comparing the relative lengths of the 24 GBs AK1, AK3, AK4, and AK7 (Figures 3A and 3C to 3E). among all eight species (see Supplemental Table 2 online) and The identiﬁcation of at least four AK-like homeologues and GB with megabase size estimations for these segments in A. thaliana associations corresponding to those of the ACK across all (see Supplemental Table 1 online). There was no signiﬁcant analyzed tribes strongly suggests that the x=7 taxa and the difference between relative lengths of GBs among the analyzed ACK descended from a common ancestor. This was further taxa (analysis of variance; P = 0.008).

Figure 3. Reconstructed Karyotypes of Eutremeae, Isatideae, and Sisymbrieae Species. (A) Karyotype of M. perfoliatum (2n=14; Isatideae) and Myagrum chromosomes after CCP. (B) A tentative scenario of the origin of translocation chromosomes AK2/5 and AK2/5/6/8 from chromosomes AK2 and AK5/6/8. (C) Karyotype of G. glastifolia (2n=14; Isatideae). (D) Karyotype of O. aegyptiacum (2n=14; Sisymbrieae). (E) Karyotype of T. halophila (2n=14; Eutremeae). The 24 GBs are indicated by uppercase letters (A to X) and colored according to their positions on chromosomes AK1 to AK8 of the ACK (Figure 1A). Downward-pointing arrows indicate the opposite orientation of GBs compared with the position in the ACK. In O. aegyptiacum, it was not possible to distinguish whether the 5S rDNA loci are located within a pericentromeric region of short or long arms of the analyzed mitotic chromosomes; thus, the 5S rDNA positions are only approximate. Karyotypes in (A) and (C) to (E) are drawn to scale (bar = 5 Mb). In CCP images, arrowheads refer to pericentromeric heterochromatin and arrows indicate heterochromatic knobs. Bar = 10 mm.

74 Karyotype Evolution in x=7 Crucifers 2563

Structure and Origin of the Rearranged Chromosomes: the heterochromatin and repeat block landmarks of the analyzed Proto-Calepineae Karyotype species. Heterochromatic arrays of pachytene bivalents were identified by 49,6-diamidino-2-phenylindole staining and by fluo- Besides AK-like chromosomes, two chromosomes comprising rescence in situ hybridization localization of 5S and 45S rDNA. novel associations of GBs were identified in the analyzed x=7 Species-specific heterochromatin profiles were compared with species. The most parsimonious origin of the two chromosomes estimated genome size values. (AK6/8 and AK5/6/8) has been reconstructed assuming that the The number and position of rDNA loci found in the x=7 species two rearranged chromosomes arose as a result of chromosome are given in Table 1 and in Figures 2A to 2D, 3A, and 3C to 3E. In number reduction from n=8 to n=7 and that the ancestral eight all species, 45S rDNA loci (NORs) have been detected adjacent chromosomes resembled AK chromosomes of the ACK. to pericentromeric heterochromatic regions. In C. orientalis, C. In Conringieae (C. orientalis) and Calepineae (C. irregularis and irregularis, G. laevigata, and N. caerulescens, a 45S rDNA locus G. laevigata), chromosome AK6/8 consists of AK6-derived was consistently found adjacent to block D on the AK2-like blocks O, P, and R, with the block W assigned to AK8. The chromosome and frequently on the AK7-like chromosome. In G. AK6/8 chromosome probably originated from two subsequent glastifolia, M. perfoliatum, O. aegyptiacum, and T. halophila, AK2 reciprocal translocations between AK6 and AK8; the AK6/8 arms are separated by translocation and the 45S rDNA locus was centromere was derived from AK6 or AK8 (Figure 2E). The initial no longer found to be associated with block D on chromosome translocation event led to a whole-arm exchange followed by a AK2/5/6/8. Another locus is always associated with block S on reciprocal translocation involving blocks R and X. The translo- the AK7-like homeologue. Hence, the location of NORs on cation chromosome V/Q/X became acrocentric via a pericentric chromosomes AK2 and AK7 may represent an ancestral condi- inversion involving its short arm (block V). Then a reciprocal tion of the PCK (Figure 4). translocation transferred the long arm to the short arm end of The 5S rDNA loci are localized within pericentromeric hetero- AK5. The small translocation product (a minichromosome bear- chromatic regions of different homeologues in species of Con- ing the V/Q/X centromere) was apparently meiotically unstable ringieae, Calepineae, and Noccaeeae (Figures 2A to 2D). In tribes and thus became eliminated. The AK5/6/8 was further modified Eutremeae, Isatideae, and Sisymbrieae, the 5S rDNA is most by a paracentric inversion (Figure 2E). AK5/6/8 comprises the frequently found on the short arm of chromosome AK2/5/6/8 short arm and the centromere from AK5 (blocks M/N), whereas (adjacent to block D) and/or on the long arm of AK4-like its long arm is composed of GBs from AK5 (K/L), AK6 (Q), and homeologue (Figures 3A and 3C to 3E). Remarkably, 5S rDNA AK8 (V/X) (Figure 2). In Noccaeeae (N. caerulescens), AK6/8 loci are present within pericentromeric regions of all seven and AK5/6/8 chromosomes were altered by secondary intra- chromosome pairs in O. aegyptiacum (Figure 3D; see Supple- chromosomal rearrangements (Figure 2D). The structure of AK6/ mental Figure 3A online). 8 can be explained by a pericentric and a subsequent para- In tetraploid Calepineae species, rDNA loci were often located centric inversion, whereas the AK5/6/8 chromosome probably on only one of the two homeologues. In C. irregularis, both 5S experienced a single pericentric inversion (see Supplemental and 45S rDNA loci were located on only one of the two Figure 2 online). homeologues (Figure 2B; see Supplemental Figure 1 online), In Eutremeae, Isatideae, and Sisymbrieae (Figure 3), the AK6/8 whereas in G. laevigata, a 5S rDNA locus was situated within one chromosome is identical to that described in Conringieae, of the two homeologues, while 45S rDNA loci occupied pericen- Calepineae, and Noccaeeae. The translocation chromosome tromeric positions on both homeologues (Figure 2C; see Sup- AK2/5/6/8 bears blocks V/K/L/Q/X on its long arm, as in all other plemental Figure 1 online). taxa; however, the short arm comprises block D of AK2. Blocks Except for rDNA arrays, terminal and interstitial heterochro- M/N (from AK5) and E (from AK2) constitute the complementary matic knobs were discerned in three species. In C. orientalis,a product (AK2/5) of a reciprocal translocation between AK5/6/8 terminal knob is located on the short arm of AK2 homeologue and the ancestral chromosome AK2. The centromere identity in (Figure 2A). Three terminal knobs (on AK1-like, AK3-like, and the two translocation chromosomes cannot be inferred from the AK6/8 chromosomes) were revealed in M. perfoliatum (Figure present data (Figure 3B). In G. glastifolia, the AK2/5/6/8 chro- 3A). In T. halophila, three large interstitial knobs are located close mosome is modified by a paracentric inversion on its long arm to centromeres of AK1-, AK3-, and AK7-like chromosomes (Figure 3C). (Figure 3E). Based on the above data, a tentative Proto-Calepineae Kar- The monoploid genome size (Cx) varied from 0.20 pg (223 Mb) yotype (PCK; n=7) comprising five ancestral chromosomes (AK1 in C. orientalis to 0.66 pg (648 Mb) in T. halophila (Table 1). No to AK4 and AK7) and two translocation chromosomes (AK6/8 apparent relationship was found between genome size variation and AK5/6/8) has been deduced. Following the most parsimo- and heterochromatin amount, chromosome homeology pattern, nious interpretation, the PCK is likely the ancestor of all x=7 or the phylogenetic position of the x=7 species. Although the karyotypes analyzed (Figure 4). highest C value in T. halophila can be tentatively linked to the presence of three interstitial knobs and the large arrays of Comparative Analysis of Heterochromatic pericentromeric heterochromatin revealed in this species (see Chromosomal Landmarks Supplemental Figure 3B online), a comparable genome size (0.61 pg/600 Mb) was estimated in N. caerulescens possessing only Chromosome homeology and chromosome rearrangements one knob and less extensive pericentromeric heterochromatic revealed by CCP have been further compared with prominent regions (Figure 2D).

75 2564 The Plant Cell

Figure 4. Reconstruction of Karyotype Evolution in Six x=7 Tribes and Brassiceae from the PCK (n=7) and an Ancestral Karyotype (n=8) Based on CCP Data.

The 24 GBs are indicated by uppercase letters (A to X) and colored according to their position on chromosomes AK1 to AK8 of the ACK (Figure 1A). Downward-pointing arrows indicate the opposite orientation of GBs compared with the position in the ACK. A tentative number of translocations (transl.) and inversions (inv.) is given at the nodes of the chromosomal phylogeny. 45S rDNA loci are shown as cross-hatched boxes. For the tribe Brassiceae (exempliﬁed by the B. rapa karyotype, n=10; Parkin et al., 2005), only associations of GBs shared with the PCK are displayed; other chromosomes/chromosome regions are shown as black bars.

76 Karyotype Evolution in x=7 Crucifers 2565

Table 1. Chromosome Number, Monoploid Genome Size (Cx), and Number of rDNA Loci of the x=7 Species Analyzed

Cx Species 2n (pg/Mb)a No. of 45S rDNA Loci No. of 5S rDNA Loci

Species with two translocation chromosomes Conringia orientalis 14 0.228/223 4 2 Calepina irregularis 28 0.202/396 4 2 Goldbachia laevigata 28 0.218/427 8 2 Noccaea caerulescens (=Thlaspi caerulescens) 14 0.612/600 4 4 Species with three translocation chromosomes Myagrum perfoliatum 14 0.312/305 2 4 Glastaria glastifolia 14 0.425/416 4 2 Ochthodium aegyptiacum 14 0.336/329 4 14 Thellungiella halophila (=Eutrema salsugineum) 14 0.661/648 2 2 a Cx values based on the Cx value of A. thaliana (0.16 pg/157 Mb).

DISCUSSION (association of O/P/W/R blocks) and the collinearity of V/K/L/Q/X blocks found in all x=7 species serve as unique cytogenetic We have reconstructed the karyotype evolution of eight x=7 signatures unambiguously underlying the monophyletic origin of species from six Brassicaceae tribes, thereby reconstructing by all six tribes (Figure 4). Chromosome AK6/8 and the association chromosome analyses the extent of whole-genome collinearity of V/K/L/Q/X blocks must have been present already in the PCK. among a group of plant species for which no comparative Eutremeae, Isatideae, and Sisymbrieae share a younger recip- cytogenetic and genetic maps were previously available. CCP rocal whole-arm translocation between chromosomes AK5/8/6 using Arabidopsis BAC contigs arranged according to their and AK2 (Figure 4). position within the putative ACK of Lineage I (Lysak et al., 2006; Schranz et al., 2006) was applied to uncover homeologous chromosome regions of the x=7 species. Cytogenetic Signatures: Phylogenetic Implications

Cytogenetic signatures (e.g., novel associations of GBs) can be The ACK and the PCK used to delimit taxa, as chromosome rearrangements generally exhibit only a low level of homoplasy and thus have the power to Five chromosomes shared between the ACK (n=8) and the PCK disentangle unresolved or conflicting phylogenetic relationships. (n=7) unequivocally argue for a common origin of the two The tribe Eutremeae (;25 species) had an unresolved position karyotypes (Figure 4). As the phylogenetic relationship between within the group of x=7 tribes (Figure 1B) (Beilstein et al., 2006; Lineage I and tribes affiliated with Lineage II remains largely Koch et al., 2007; Koch and Al-Shehbaz, 2008). We have shown unresolved (Koch and Al-Shehbaz, 2008) (Figure 1B), two alter- that the karyotype of Eutremeae (T. halophila) is identical to that native scenarios reconstructing the origin of the translocation of Isatideae and Sisymbrieae but different from that of Calepi- chromosomes as well as the entire PCK complement have to be neae, Conringieae, and Noccaeeae. Hence, this supports the considered. The first assumes that a common ancestral karyo- inclusion of Eutremeae together with Brassiceae, Isatideae, type for both clades (i.e., Lineages I and II) possessed eight Schizopetaleae, and Sisymbrieae in Lineage II, forming a mono- chromosome pairs, and the second scenario implies a karyotype phyletic group (Figure 4). The data here corroborate the earlier of seven chromosome pairs (n=7). Based on our current under- exclusion of Calepineae and Conringieae from the Brassiceae standing of genome evolution in Brassicaceae, the former the- (Lysak et al., 2005). Although Calepineae and Conringieae pos- oretical model advocating an ancestral n=8 karyotype for both sess similar karyotypes, differences in morphological characters lineages is preferred. The most parsimonious scenario of chro- substantiate their recent circumscription as two closely related mosome number reduction from n=8 to n=7 requires only three tribes (German and Al-Shehbaz, 2008). CCP data are also translocations and two inversions (Figure 2E), compared with congruent with the close relationship between Conringieae and a higher number of less parsimonious steps necessary to re- the tribe Noccaeeae revealed by nuclear and chloroplast gene construct the ACK from the PCK. Moreover, the latter model phylogenies (Bailey et al., 2006; Beilstein et al., 2006; German (i.e., n=7 / n=8) would require an origin of new centromere by and Al-Shehbaz, 2008). The reconstructed chromosomal phy- an unknown mechanism. Although the emergence of de novo logeny elucidated relationships among the analyzed x=7 tribes centromeres (Nasuda et al., 2005) and centric fissions (Jones, and provided compelling evidence that tribes of Lineage II 1998) is documented in plants, these have not been observed in (including Eutremeae) and Calepineae, Conringieae, and Noc- crucifers as yet. caeeae have a monophyletic origin (Figure 4). Further CCP As ancestral chromosomes per se do not provide insight into analysis of other tribes currently placed into the phylogenetic the evolutionary relationship among the analyzed species, inter- proximity of Lineage II (Figure 1B) is needed to further clarify tribal relationships have been elucidated through evolutionarily phylogenetic relationships and genome evolution within this novel associations of crucifer GBs. The chromosome AK6/8 crucifer group.

77 2566 The Plant Cell

By analyzing species with an identical chromosome number rule out the possibility that the tribe-specific triplication was (2n=14 or 28) from six closely related but distinct tribes, we also preceded by another round of chromosome number reduction tested for the functional role of gross karyotypic alterations (n=7 / n=6), as x=6 was suggested as an ancestral base during speciation. Our data indicate that even morphologically chromosome number of Brassica species by several authors distinct groups such as Calepineae/Conringieae/Noccaeeae, (Catcheside, 1937; Ro¨ bbelen, 1960). However, the present data when compared with tribes of Lineage II, display almost com- do not warrant any conclusion regarding the chromosome num- plete genome collinearity and very similar karyotypes. It remains ber of the primary hexaploid (Lysak et al., 2007). elusive to what extent the Lineage II–specific translocation resulting in chromosomes AK2/5 and AK2/5/6/8 contributed to Mechanism of Chromosome Number Reduction from the divergence of Lineage II tribes. However, the resolution of n=8 to n=7 CCP analysis is constrained by the size of BAC painting probes, reducing the ability to detect minor rearrangements that might be Chromosome rearrangements revealed in the x=7 taxa can be of evolutionary significance. The above findings indicate that the reconstructed by considering only inversions and reciprocal speciation resulting in morphologically distinct but closely re- translocations. Chromosome number reduction from n=8 to lated taxa (Al-Shehbaz et al., 2006; German and Al-Shehbaz, n=7 implies the loss or inactivation of one centromere. Our 2008) is not necessarily accompanied or determined by major data indicate that the centromere loss occurred as a result of a karyotypic changes. reciprocal translocation between an acrocentric chromosome (formed via a preceding pericentric inversion) and a (sub)meta- Karyotype Evolution Preceding the Whole-Genome centric chromosome. This scenario yields a translocation chro- Triplication in Brassiceae mosome (AK5/6/8) and a minichromosome comprising mainly the centromere of the acrocentric chromosome (Figure 2E). The As Isatideae, Sisymbrieae, and Eutremeae along with tribes meiotically unstable minichromosome is eliminated. The same Brassiceae and Schizopetaleae form a monophyletic group mechanism has been argued as responsible for chromosome called Lineage II (Beilstein et al., 2006; Koch et al., 2007; this number reduction in tribes of Lineage I (Lysak et al., 2006) and is study), some conclusions on the early evolution of the whole assumed to be a prominent mechanism reducing chromosome lineage can be drawn from the data presented here. The chro- numbers in Brassicaceae (Schranz et al., 2006; Schubert, 2007). mosome homeology patterns of Isatideae, Sisymbrieae, and Other chromosome rearrangements identified in the x=7 species Eutremeae can be compared with the structure of Brassica included pericentric and paracentric inversions and whole-arm genomes (tribe Brassiceae) revealed by comparative genetic reciprocal translocations. No evidence for duplications or dele- analysis (Parkin et al., 2005). This comparison showed that the R/ tions of GBs has yet been detected. W block association (=long arm of AK6/8) as well as the V/K/L/Q/ We have scrutinized the number of rearrangement events and X association are present also in the Brassica rapa component corresponding break points necessary to explain the structure of (n=10) of the B. napus genome (n=19) (Figure 4) (Parkin et al., the extant x=7 karyotypes. The origin of the PCK (i.e., translo- 2005; Schranz et al., 2006). Three copies of the R/W block cation chromosomes AK6/8 and AK5/6/8) from an ancestral n=8 association are located on B. napus chromosomes N2, N3, and karyotype requires five events and nine break points (five in N10, whereas the V/K/L/Q/X association has been found in two centromeric, two in terminal, and two in interstitial regions at W/X copies on chromosomes N2 and N6 (only blocks Q and V of the and Q/R boundaries). The reciprocal translocation between third genomic copy were detected on chromosome N9 [Figure chromosomes AK2 and AK5/6/8 yielding the Lineage II–specific 4]). Due to the whole-genome triplication in the tribe Brassiceae chromosomes AK2/5 and AK2/5/6/8 involves another two peri- (Lysak et al., 2005; Parkin et al., 2005), three instead of two centromeric break points. In G. laevigata, six species-specific copies of the V/K/L/Q/X association are to be expected. Never- break points have been identified (one pericentromeric and five theless, this pattern corresponds well with the fragmentary interstitial, including one coinciding with the O/P boundary). In character of Brassica genomes, as the genomic redundancy Glastaria, 11 plus 2 species-specific break points at K/L and Q/X after the whole-genome triplication was followed by the diploid- boundaries were identified. The highest number of break points ization eroding the original triplicated pattern. Consequently, is in N. caerulescens, with 9 break points shared with the PCK some GBs were retained in three copies, other GBs as well as and 20 additional break points necessary to account for species- smaller segments were preserved as one or two copies, and specific pericentric and paracentric inversions (Figure 2D; see some were secondarily multiplied, probably through unequal Supplemental Figure 2 online). Of the 20 break points, 10 map to homeologous recombination (Lukens et al., 2004; Parkin et al., pericentromeric regions, whereas 10 are at interstitial positions 2005). The present structure of the B. napus genome does not (two at T/U and W/R boundaries). In total, of the 11 basic break allow us to arrive at a clear-cut conclusion whether the AK5/6/8– points necessary for the origin of the three translocation chro- AK2 translocation is shared by all tribes of Lineage II or whether it mosomes, 9 map to pericentromeric or telomeric regions and is specific only for Isatideae, Sisymbrieae, and Eutremeae. only 2 are interstitial. From a total of 28 secondary species- Regardless of this limitation, the aforementioned cytogenetic specific break points, 11 localize to centromeres. Thus, from all signatures provide convincing evidence that Brassiceae and the 39 break points, half (20 break points) involve telomeric/pericen- whole Lineage II descended from the PCK (n=7). In the clade tromeric regions and the other half (19 break points) occur at leading to Brassiceae, the PCK karyotype has undergone the interstitial positions. Although pericentromeric and telomeric whole-genome triplication. This parsimonious scenario does not regions characterized by clusters of tandem repeats are

78 Karyotype Evolution in x=7 Crucifers 2567

considered as chromosome regions prone to chromosome NORs were located interstitially, adjacent to pericentromeric rearrangements, most likely by etopic (nonallelic) homologous heterochromatic regions. This is in contrast with the prevalence recombination (Gaut et al., 2007; Schubert, 2007), the observed of terminal NORs found elsewhere in the family (Ali et al., 2005), equal frequency of interstitial break points suggests that large including Camelineae and Descurainieae species analyzed by clusters of repetitive sequence elements are not the exclusive CCP (Berr et al., 2006; Lysak et al., 2006). sites of chromosomal breakage. Segmental duplications were The number of 5S rDNA loci was two or four, except in O. proposed as regions facilitating chromosome rearrangements, aegyptiacum, with 14 loci (Table 1; see Supplemental Figure 3 such as inversions in primate genomes (Kehrer-Sawatzki and online). The remarkable localization of 5S rDNA on all chromo- Cooper, 2008). In Drosophila, duplications of nonrepetitive se- somes of a complement is not yet known in higher plants (Cloix quences at the break point regions of ;60% of the analyzed et al., 2003). This high number of heterochromatic 5S rDNA sites inversions were presumably caused by staggered breaks rather is most likely responsible for the severe stickiness of prophase I than by ectopic recombination between repetitive elements bivalents at pericentromeric heterochromatic regions in this (Ranz et al., 2007). Future genome sequencing of the x=7 species species. Similarly, better spread pachytene bivalents were (e.g., N. caerulescens and T. halophila) will unveil the detailed obtained in A. thaliana accessions with only two major 5S rDNA sequence structure of the break points. loci compared with the accessions having 5S rDNA on three chromosomes (Fransz et al., 1998). It remains unclear whether Diverse Routes of Chromosome Number Reduction from the chromosomal ubiquity of 5S rDNA in O. aegyptiacum was n=8 to n=7 caused by the mobility of transposable elements, similar to the So far, data on karyotype evolution in Brassicaceae suggest that at En/Spm transposon–mediated transfer of 5S rDNA observed in least two major phylogenetic branches (Lineage I and II) des- Aegilops (Raskina et al., 2004, 2008). cended from a common ancestral karyotype with eight chromo- The frequent mobility of 45S and 5S rDNA sites (Pontes et al., some pairs. We assume that in some crucifer taxa, the number 2004; Schubert, 2007; Raskina et al., 2008) precludes any of ancestral linkage groups was reduced toward karyotypes with plausible assumption on the ancestral distribution of rDNA sites. n=5 to n=7. One crucial question is whether chromosome num- While the loss of some NORs can occur during chromosome ber reduction within diverse phylogenetic lineages is based on the number reduction events (Lysak et al., 2006; Schranz et al., 2006; same or on different events. Among crucifer x=7 taxa, the genome Schubert, 2007) and other rDNA clusters can be amplified or structure of only two species from Lineage I, Neslia paniculata and transposed via ectopic recombination or due to the activity of Boechera stricta, was analyzed with a sufficient precision (Lysak transposable elements (Pontes et al., 2004; Raskina et al., 2004, et al., 2006; Schranz et al., 2007). In N. paniculata (Camelineae), the 2008), direct evidence for the involvement of rDNA repeats in reduction combined the ancestral chromosomes AK4 and AK5. crucifer chromosome rearrangements is lacking. Both chromosomes became acrocentric via pericentric and para- Three x=7 species possess heterochromatic knobs posi- centric inversions and fused by a translocation with break points tioned terminally or interstitially. In plants, knobs can originate close to the centromeres. The chromosome AK4/5 comprises through inversions, including pericentromeric heterochro- blocks I/J (from AK4) on the short arm and the K/L/M/N block matin (e.g., the hk4S knob in A. thaliana [Fransz et al., 2000]), association (from AK5) on its long arm (Lysak et al., 2006). In B. the insertion and subsequent silencing of repetitive elements stricta (Boechereae), chromosome number reduction followed a such as tandem repetitive transgenes (Probst et al., 2003), or more complex scenario than in Neslia (Schranz et al., 2007). The can be copied from other chromosomal regions due to the reduction involved translocations between five AK chromosomes physical proximity within interphase nuclei (Zhong et al., 1998). and resulted in translocation chromosomes AK1/2 (blocks A/B/C/ Apart from the hk4S knob in A. thaliana (Fransz et al., 2000) D), AK3/8 (blocks F/G/W/X), and AK3/5/8 (blocks K/L/M/N/V/H). In and small interstitial as well as distal knobs in Brassica species Neslia and Boechera (Lineage I) as well as in the x=7 tribes affiliated (Ro¨ bbelen, 1960; Lim et al., 2005; Lysak et al., 2007), large with Lineage II, the ancestral chromosome AK5 is always involved terminal heterochromatic knobs are not yet reported in Brassi- in chromosome number reduction; however, the changes leading caceae species. These terminal knobs resemble the knobs on to the reduction differ. In Neslia and Boechera,allAK5-derivedGBs chromosomes of maize (Zea mays; Lamb et al., 2007), rice (K/L/M/N) remain associated within new chromosomes, whereas (Oryza sativa; Ohmido et al., 2001), or tomato (Solanum lycoper- in the x=7 species analyzed here, the GBs are split up within AK5/6/ sicum; Zhong et al., 1998). As in these species, it is likely that 8 or between chromosomes AK2/5 and AK2/5/6/8 (Figure 4). The knobs in crucifer species will primarily comprise tandem repeats. comparison here clearly shows that chromosome number reduc- Satellite repeats composing distal knobs in the above species tion in Lineage I (Camelineae and Boechereae) is different from were classified as subtelomeric with a potential functional that of the x=7 tribes associated with Lineage II. The repeated role (Lamb et al., 2007). Three large interstitial knobs within the participation of certain ancestral chromosomes in diverse rear- T. halophila karyotype (Figure 3E) are not linked to a higher rangements was also observed in other groups such as grasses frequency of species-specific chromosome rearrangements, as (Srinivasachary et al., 2007). seen in N. caerulescens, a species with only a single knob (Figure 2D). Our data suggest that interstitial as well as termi- Heterochromatic Landmarks in the x=7 Species nal knobs, revealed only in C. orientalis (Figure 2A) and M. perfoliatum (Figure 3A), are apparently of more recent The analyzed x=7 species show a rather uniform pattern of rDNA origin and did not mediate the described chromosome repat- loci and heterochromatic knobs. In our analyzed x=7 species, all terning.

79 2568 The Plant Cell

CytogeneticEvidenceofGenomeDiploidizationin acetic acid and spread by stirring with a needle on a hot plate at 508C for Autotetraploid Calepineae Species 0.5 to 2 min. Chromosomes were fixed by adding 100 mL of ethanol:acetic acid (3:1) fixative. The slide was tilted to remove the fixative and dried with In this study, the complete karyotype structure of two tetraploid a hair dryer. The dried preparation was staged with a phase contrast Calepineae species was analyzed. In C. irregularis, diploid microscope. Suitable slides were postfixed in 4% formaldehyde dis- (2n=2x=14) and tetraploid (2n=4x=28) cytotypes are known, solved in distilled water for 10 min and air-dried. whereas only tetraploids are recorded for G. laevigata (Warwick and Al-Shehbaz, 2006). As Calepina is a monospecific genus Painting Probes (German and Al-Shehbaz, 2008), we assume that the 4x genome was derived from the 2x cytotype via autopolyploidy relatively Arabidopsis thaliana BAC clones were obtained from the ABRC. The recently. Indeed, our CCP data corroborated the autopolyploid selection of clones suitable for chromosome painting and DNA isolation origin of the 4x cytotype, as all seven chromosomes showed the from individual BAC clones was performed as described by Lysak et al. (2003). For CCP, each third BAC was used and contigs were arranged and same structure in both genomes (Figure 2B). In G. laevigata, one differentially labeled according to the GBs of the ACK (Schranz et al., pair each of AK1, AK7, and AK6/8 differs from the other pair by 2006). For BAC contigs used for CCP, see Supplemental Table 1 online. pericentric inversions (Figure 2C). Although an allopolyploid origin To characterize species-specific inversions in N. caerulescens and G. of the tetraploid G. laevigata cannot be ruled out within the genus laevigata, the respective BAC contigs were split into smaller subcontigs comprising five other species (German and Al-Shehbaz, 2008), and used as CCP probes. The BAC clone T15P10 (AF167571) bearing autopolyploidization followed by three species-specific inversions 45S rRNA genes was used for in situ localization of NORs, and clone pCT is a more likely interpretation. No records of a diploid cytotype 4.2, corresponding to a 500-bp 5S rRNA repeat (M65137), was used for may indicate an earlier origin of the tetraploid G. laevigata localization of 5S rDNA loci. compared with the 4x cytotype of C. irregularis. This is underlined All DNA probes were labeled by nick translation with biotin-dUTP, m by the pericentric inversions that differentiate three of seven digoxigenin-dUTP, or Cy3-dUTP as follows: 1 g of DNA diluted in distilled water to 29 mL, 5 mL of nucleotide mix (2 mM dATP, dCTP, and homologous pairs of G. laevigata. Although chromosome rear- dGTP, 400 mM dTTP; all Roche), 5 mLof103 NT buffer (0.5 M Tris-HCl, pH rangements may reduce the risk of meiotic multivalent formation 7.5, 50 mM MgCl2, and 0.05% BSA), 4 mL of 1 mM X-dUTP (in which X was and thus contribute to the genome diploidization of autopoly- biotin, digoxigenin, or Cy3), 5 mL of 0.1 M b-mercaptoethanol, 1 mLof ploids (Ma and Gustafson, 2005), regular meiotic pairing and 14 DNase I (Roche), and 1 mL of DNA polymerase I (Fermentas). The nick bivalents have been observed in both species (see Supplemental translation mixture was incubated at 158C for 90 min (or longer) to obtain a Figure 1 online). This implies that the diploid-like meiosis in the fragment length of ;200 to 500 bp. The nick translation reaction was two autopolyploids is determined genetically rather than by stopped by adding 1 mL of 0.5 M EDTA, pH 8.0, and incubation at 658C for gross intrachromosomal rearrangements. A partial diploidization 10 min. Individual labeled probes were stored at 2208C until use. of meiosis was also observed in established autotetraploid lines of A. thaliana (Santos et al., 2003). CCP

To remove cytoplasm, the slides were treated with pepsin (0.1 mg/mL; METHODS Sigma-Aldrich) in 0.01 M HCl for 3 to 6 h, postfixed in 4% formaldehyde dissolved in 23 SSC (13 SSC is 0.15 M NaCl and 0.015 M sodium citrate) for 10 min, dehydrated in an ethanol series (70, 80, and 96%), and air-dried. Plant Material Selected BAC clones were pooled and precipitated by adding one- All but one species were grown from seeds obtained from the Millennium tenth volume of 3 M sodium acetate, pH 5.2, and 2.5 volumes of ice-cold Seed Bank, Royal Botanic Gardens, Kew (Glastaria glastifolia, Ochtho- 96% ethanol, kept at 2208C for 30 min, and centrifuged at 13,000g at 48C dium aegyptiacum), the Botanical Garden of Bordeaux (Calepina irregu- for 30 min. The pellet was resuspended in 20 mL of hybridization mix (50% laris, Conringia orientalis), the Hortus Botanicus Hauniensis, Copenhagen formamide and 10% dextran sulfate in 23 SSC) per slide. Cover slips (Goldbachia laevigata, Myagrum perfoliatum), and from D. Baum, Uni- were framed by rubber cement. The probe and chromosomes were versity of Wisconsin, Madison (Thellungiella halophila). Inflorescences of denatured together on a hot plate at 808C for 2 min and incubated in a Noccaea caerulescens were collected from a single wild population moist chamber at 378C for 39 to 63 h. (Korˇenec, Czech Republic). Plants grown from seeds were cultivated in a Posthybridization washing was performed in 50% formamide (A. greenhouse or growth chamber under long-day light conditions. Herbar- thaliana, which was used as a probe quality control) or 20% formamide ium vouchers of all analyzed species are deposited in the herbarium of (all other species) in 23 SSC at 428C. Fluorescent detection was as Masaryk University. follows: biotin-dUTP was detected by avidin–Texas Red (Vector Labora- tories) and amplified by goat anti-avidin–biotin (Vector Laboratories) and avidin–Texas Red; digoxigenin-dUTP was detected by mouse anti- Preparation of Pachytene Chromosomes digoxigenin (Jackson ImmunoResearch) and goat anti-mouse Alexa Entire inflorescences were fixed in ethanol:chloroform:acetic acid (6:3:1) Fluor 488 (Molecular Probes), and Cy3-dUTP labeled probes were ob- overnight and stored in 70% ethanol at 2208C until use. Selected flower served directly. Chromosomes were counterstained with 49,6-diamidino- buds were rinsed in distilled water (2 3 5 min) and in citrate buffer (10 mM 2-phenylindole (2 mg/mL) in Vectashield (Vector Laboratories). sodium citrate, pH 4.8; 2 3 5 min) and incubated in an enzyme mix (0.3% Fluorescence signals were analyzed with an Olympus BX-61 epifluo- cellulase, cytohelicase, and pectolyase; all Sigma-Aldrich) in citrate buffer rescence microscope and AxioCam CCD camera (Zeiss). Images were at 378C for 3 to 6 h, transferred into citrate buffer, and kept at 48C until use acquired separately for all four fluorochromes using appropriate excita- (overnight to 2 d). Individual anthers were put on a microscope slide of a tion and emission filters (AHF Analysentechnik). The four monochromatic stereomicroscope and disintegrated by a needle in a drop of citrate images were pseudocolored and merged using Adobe Photoshop CS2 buffer. Then the suspension was softened by adding 15 to 30 mL of 60% software (Adobe Systems).

80 Karyotype Evolution in x=7 Crucifers 2569

Chromosome Measurements and Genome Size Estimation Devos, K.M. (2005). Updating the ‘crop circle.’ Curr. Opin. Plant Biol. 8: 155–162. The length of painted GBs was measured using the ImageJ program Fransz, P., Armstrong, S., Alonso-Blanco, C., Fischer, T.C., Torres- (National Institutes of Health). The nuclear DNA content has been esti- Ruiz, R.A., and Jones, G.H. (1998). Cytogenetics for the model mated by flow cytometry at the Institute of Botany and Zoology, Masaryk system Arabidopsis thaliana. Plant J. 13: 867–876. University. For details, see Lysak et al. (2007). Fransz, P.F., Armstrong, S., de Jong, J.H., Parnell, L.D., van Drunen, G., Dean, C., Zabel, P., Bisseling, T., and Jones, G.H. (2000). Supplemental Data Integrated cytogenetic map of chromosome arm 4S of A. thaliana: Structural organization of heterochromatic knob and centromere The following materials are available in the online version of this article. region. Cell 100: 367–376. Supplemental Figure 1. Examples of CCP in Tetraploid Calepineae Gaut, B.S., Wright, S.I., Rizzon, C., Dvorak, J., and Anderson, L.K. Species (2n=4x=28). (2007). Recombination: An underappreciated factor in the evolution of Supplemental Figure 2. Species-Specific Chromosome Rearrange- plant genomes. Nat. Rev. Genet. 8: 77–84. ments (Inversion Events) in N. caerulescens. German, D.A., and Al-Shehbaz, I.A. (2008). Five additional tribes (Aphragmeae, Biscutelleae, Calepineae, Conringieae, and Erysimeae) Supplemental Figure 3. Heterochromatic Landmarks in O. aegyptia- in the Brassicaceae (Cruciferae). Harv. Pap. Bot. 13: 165–170. cum and T. halophila. Gong, Q., Li, P.H., Ma, S.S., Rupassara, S.I., and Bohnert, H.J. Supplemental Table 1. GBs of the ACK and Corresponding A. (2005). Salinity stress adaptation competence in the extremophile thaliana BAC Contigs Used as Painting Probes in This Study. Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J. 44: 826–839. Jiang, J., and Gill, B.S. (2006). Current status and the future of ACKNOWLEDGMENTS fluorescence in situ hybridization (FISH) in plant genome research. Genome 49: 1057–1068. We thank Petr Buresˇ for genome size estimates and Ingo Schubert, Jones, K. (1998). Robertsonian fusion and centric fission in karyotype Chris Pires, and M. Eric Schranz for their valuable comments on the evolution of higher plants. Bot. Rev. 64: 273–289. manuscript. Dmitry German, Marcus Koch, and Klaus Mummenhoff are Kehrer-Sawatzki, H., and Cooper, D.N. (2008). Molecular mechanisms acknowledged for providing unpublished phylogenetic data. This study of chromosomal rearrangement during primate evolution. Chromo- was supported by research grants from the Grant Agency of the Czech some Res. 16: 41–56. Academy of Science (Grant KJB601630606) and the Czech Ministry of Koch, M., and Al-Shehbaz, I.A. (2008). 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82 IV . Dierschke T., Mandáková T., Lysak M.A., Mummenhoff K. 2009. A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization. Annals of Botany 104: 681-688.

83 84 Annals of Botany Page 1 of 8 doi:10.1093/aob/mcp161, available online at www.aob.oxfordjournals.org

A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization

Tom Dierschke1,*, Terezie Manda´kova´2, Martin A. Lysak2 and Klaus Mummenhoff1 1Universita¨t Osnabru¨ck, Abteilung Botanik, Barbarastrasse 11, 49076 Osnabru¨ck, Germany and 2Masaryk University, Department of Functional Genomics & Proteomics, Institute of Experimental Biology, Kamenice 5, Building A2, CZ-62500 Brno, Czech Republic

Received: 23 March 2009 Returned for revision: 5 May 2009 Accepted: 1 June 2009

† Background and Aims Incongruence between chloroplast and nuclear DNA phylogenies, and single additive nucleotide positions in internal transcribed spacer (ITS) sequences of polyploid Australian/New Zealand (NZ) Lepidium species have been used to suggest a bicontinental hybrid origin. This pattern was explained by two trans-oceanic dispersals of Lepidium species from California and Africa and subsequent hybridization followed by homogenization of the ribosomal DNA sequence either to the Californian (C-clade) or to the African ITS-type (A-clade) in two different ITS-lineages of Australian/NZ Lepidium polyploids. † Methods Genomic in situ hybridization (GISH) was used to unravel the genomic origin of polyploid Australian/ NZ Lepidium species. Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) probes was applied to test the purported ITS evolution, and to facilitate chromosome counting in high-numbered polyploids. † Key Results In Australian/NZ A-clade Lepidium polyploids, GISH identiﬁed African and Australian/NZ C-clade species as putative ancestral genomes. Neither the African nor the Californian genome were detected in Australian/NZ C-clade species and the Californian genome was not detected in Australian/NZ A-clade species. Five of the eight polyploid species (from 7x to 11x) displayed a diploid-like set of rDNA loci. Even the undecaploid species Lepidium muelleriferdinandi (2n ¼ 11x ¼ 88) showed only one pair of each rDNA repeat. In A-clade allopolyploids, in situ rDNA localization combined with GISH corroborated the presence of the African ITS-type. † Conclusions The nuclear genomes of African and Australian/NZ C-clade species were detected by GISH in allopolyploid Australian/NZ Lepidium species of the A-clade, supporting their hybrid origin. The presumed hybrid origin of Australian/NZ C-clade taxa could not be conﬁrmed. Hence, it is assumed that Californian ancestral taxa experienced rapid radiation in Australia/NZ into extant C-clade polyploid taxa followed by hybridization with African species. As a result, A-clade allopolyploid Lepidium species share the Californian chloroplast type and the African ITS-type with the C-clade Australian/NZ polyploid and African diploid species, respectively.

Key words: Lepidium, Brassicaceae, FISH, GISH, hybridization, polyploidy, long-distance dispersal, ITS, rDNA, Australia, New Zealand.

INTRODUCTION levels of sequence divergence between Lepidium species from Lepidium is one of the largest genera in the Brassicaceae, different continents or islands suggest a rapid radiation of consisting of approximately 230 species distributed worldwide Lepidium by long-distance dispersal in the Pliocene/ (Al-Shehbaz et al., 2006). This genus is characterized by Pleistocene (Mummenhoff et al., 2001). The large number of reduced floral organs, an autogamous mating system and muci- polyploid species (Warwick and Al-Shehbaz, 2006) suggests laginous seeds (Al-Shehbaz, 1986). Recent molecular phylo- reticulate evolution in Lepidium and this was indicated also genetic studies using the nuclear ribosomal DNA (rDNA) in the analysis of PISTILLATA intron sequences, in which internal transcribed spacer (ITS), non-coding chloroplast many polyploid taxa harbour two or more phylogenetically DNA (cpDNA) and single-copy nuclear DNA sequences (an distinct sequences (Lee et al., 2002). Chromosome numbers intron of the PISTILLATA gene, PI), respectively, clarified have been reported for about 70 Lepidium species and subspe- only some relationships within the genus (Bowman et al., cies (Warwick and Al-Shehbaz, 2006), of which all except five 1999; Mummenhoff et al., 2001; Lee et al., 2002). All molecu- have x ¼ 8 chromosomes (Al-Shehbaz 1986). Vaarama (1951) lar phylogenies support three main infrageneric lineages, cor- speculated that species with 2n ¼ 24 (e.g. L. sativum) are hex- responding to (1) section Monoploca sensu stricto (s.s.) aploids based on x ¼ 4, but this interpretation was not sup- (Australia), (2) section Lepia with Cardaria included ported by any further research. More than half of Lepidium (Eurasia) and (3) Lepidium s.s. (referred to hereafter as species studied are polyploids, and chromosome numbers Lepidium) representing by far the bulk of Lepidium species vary widely (2n ¼ 16, 24, 28, 32, 40, 48, 64 and 80; on every continent. The fossil data, easily dispersible mucila- Warwick and Al-Shehbaz, 2006). ginous seeds, common autogamous breeding systems and low In Australia and New Zealand (NZ), Lepidium is represented by 19 and seven native species, respectively. Incongruence * For correspondence. E-mail [email protected] between the chloroplast and nuclear DNA phylogenetic # 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]

85 Page 2 of 8 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium

TABLE 1. Composition of the ITS sequences of Californian, Chromosome preparations Australian/New Zealand and African Lepidium species Plants were grown from seeds in plastic Petri dishes on ITS1 ITS2 sieved potting soil in a phytotron with long day illumination (16 h light at 20 8C, 8 h dark at 15 8C). Young inflorescences 1111111112233344 were fixed in ethanol/acetic acid (3 : 1, v/v) fixative for 24 h 67880013446775936933 at 4 8C. Fixative was replaced by 70 % ethanol and the material 83478965680241795402 stored at –20 8C until further use. Chromosome spreads were prepared as described by Lysak et al. (2006). Slides were Californian C-clade species L. nitidum, L. oxycarpum,L.dictyotum CATTAGCCCGTCATCGG–TA examined under phase contrast for the presence of suitable Australian/NZ C-clade polyploids mitotic metaphase spreads. Selected slides were post-fixed in L. aschersonii CATTATCCCGTCATCGG–TW 4 % formaldehyde in 2 sodium saline citrate (SSC, 5 min), L. muelleriferdinandi CATTRCCCCGTCATCGG–TA washed in 2 SSC (twice for 5 min), and dehydrated in an Australian/NZ A-clade polyploids ethanol series (70, 90 and 100 %, 2 min each). The prep- L. hyssopifolium (KM 1670) YWYKRSSYSSWACYTA-TCT 21 L. hyssopifolium (KM 1669) TTCGGCGTGCAACCTA–TCT arations were stained with 2 mgmL 4,6-diamino-2- L. pseudohyssopifolium TTCGGCGTGCAACCYA–TCT phenylindole (DAPI) in Vectashield antifade (Vector African A-clade species Laboratories, Burlington, Ontario, Canada) and screened for L. africanum, L. transvaalense TTCGGCGTGCAACCTA–TCT the quality of metaphase and the presence of cytoplasm under an epifluorescence microscope (BX-61, Olympus, This data matrix contains only those positions of a complete alignment (not shown) that distinguish Californian from African Lepidium taxa. Tokyo, Japan). If appropriate, the slides were treated with 21 Californian and African species are extant members of two different lineages pepsin (0.1g L )in0.01 M HCl at 39 8C for 15–45 min, suggested to have been involved in the hybridogenous origin of Australian/ and post-fixed and dehydrated as described above. NZ Lepidium species. R ¼ A and G; W ¼ A and T; Y ¼ C and T; K ¼ G and T; S ¼ G and C. Site numbers are those of the complete alignment. For details see Mummenhoff et al. (2004). DNA probes The Arabidopsis thaliana BAC clone T15P10 (AF167571) analyses indicated a hybridogenous genomic constitution of bearing 45S rRNA gene repeats was used for in situ localiz- Australian/NZ Lepidium species: all 18 studied species ation of 45S rDNA, and A. thaliana clone pCT 4.2 shared a Californian cpDNA-type, while 11 Australian/NZ (M65137), corresponding to a 500-bp 5S rRNA repeat, was species appeared to harbour a Californian ITS-type (C-clade) used for localization of 5S rDNA loci. For GISH, total and a group of the remaining seven species shared an genomic DNA (gDNA) was extracted from healthy young African ITS-type (A-clade) (Mummenhoff et al., 2004; leaves according to Dellaporta et al. (1983) followed by Table 1, Fig. 1). This pattern was explained by two trans- 2 RNase treatment (50 mgmL1). Extracted gDNA was oceanic dispersals of ancestral taxa from California and checked for protein, starch or RNA contamination using a Africa to Australia/NZ and subsequent hybridization followed Beckmann photospectrometer and run on a 1 % (w/v) by sequence homogenization of the rDNA either to the agarose gel in 1 Tris-acetate-EDTA (TAE) buffer. DNA Californian or to the African ITS-type in the two different probes were labelled either by biotin- or by digoxigenin-dUTP lineages, with single additive nucleotide positions remaining, using the Nick Translation Mix (Roche, Mannheim, Germany) representing the ancestral parental input (Table 1, Fig. 1). according to the manufacturer’s instructions. However, Mummenhoff et al. (2004) did not assess the ploidy level of Australian/NZ species and the conclusion of a bicontinental hybridogenous genomic constitution was In situ hybridization based only on two markers representing nuclear (ITS) and chloroplast genomes (three non-coding cpDNA regions). Labelled 5S and 45S rDNA probes were precipitated and the The present study aimed to (1) assess ploidy level and pellet was resuspended in 20 mL hybridization mixture con- chromosome number variation among Australian/NZ taining 50 % formamide and 10 % dextrane sulfate in 2 Lepidium species, (2) test the purported allopolyploid biconti- SSC. The probe was applied to the slide, covered with a nental origin of Australian/NZ species of the A- and C-clades cover slip and denaturated on a hot plate at 80 8C for 2 min. using genomic in situ hybridization (GISH) and (3) gain Slides were hybridized at 37 8C for approx. 12 h. To prevent insight into the physical organization of rDNA loci in the pre- drying, cover slips were framed by rubber cement. Labelled sumed parental species and their Australian/NZ hybrid gDNA probes were prepared and denatured as described derivatives. above. Hybridization time for GISH was between 12 and 48 h. Following hybridization, stringent washing was carried out in 50 % formamide in 2 SSC (v/v) at 42 8C three times for MATERIALS AND METHODS 5 min. Detection of hybridization signals was performed according to Lysak et al. (2006). The biotin-labelled probes Plant material were detected by avidin–Texas Red (Vector Laboratories), Collection data of selected species of Lepidium L. used in the and signals amplified by biotinylated goat anti-avidin current study are given in Table 2. The presumed parental and (Vector Laboratories) and avidin–Texas Red. Digoxigenin- hybrid species were used exemplarily, based on results of labelled probes were detected by mouse anti-digoxigenin previous studies (Mummenhoff et al., 2001, 2004, 2009). (Roche) and goat anti-mouse–Alexa Fluor 488 antibodies

86 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium Page 3 of 8

Californian genome cp CAL

African genome cp CAL

B Australia/NZ

(7) (11) Eurasia Africa (10) California (4) N-S America/ ITS-A ITS-C (1) ITS-A ITS-C Hawaii (11)

cp CAL A cp CAL Eurasia (2) C

N-S America (3) Eurasia (3)

Eurasia (3)

Eurasia Africa (10) Australia/NZ (7) Australia/NZ (11) California (4) N-S America/ (1) ITS-A ITS-A ITS-C ITS-C Hawaii (11)

cp CAL A cp CAL Eurasia (2) C

N-S America (3) Eurasia (3)

Eurasia (3)

F IG. 1. Organismal and geographical context of the genome evolution of polyploid Australian/NZ A- and C-clade Lepidium species. (A) Geographical context of the evolution of polyploid Australian/NZ Lepidium species further outlined in (B) and (C). (B) Evolution of polyploid Australian/NZ A- and C-clade Lepidium species as inferred from ITS- and cpDNA sequence data (Mummenhoff et al., 2004). All Australian/NZ species harbour a Californian chloroplast type (cp CAL), while bidirectional concerted evolution of ribosomal DNA (ITS) subsequent to hybridization between African and Californian parental species resulted in the presence of either of the parental ITS-types, i.e. African (ITS-A) and Californian (ITS-C), in the two lineages (Australian/NZ A- and C-clade species, respectively). Arrows indicate hybridization. Numbers in boxes represent number of analysed species of given geographical origin. Encircled letters A and C refer to A- and C-clade species, respectively. See text for more details. (C) Evolution of polyploid Australian/NZ A- and C-clade Lepidium species as inferred from ITS and cpDNA sequence data (Mummenhoff et al., 2004) and current GISH experiments. In this alternative scenario polyploid Australian/NZ C-clade species are the outcome of rapid radiation of an ancient Californian ancestor in Australia/New Zealand. Australian/NZ A-clade species originated from hybridization between African and Australian/NZ C-clade species, still harbouring the ancestral Californian chloroplast type. In this scenario concerted evolution operated unidirection- ally towards the African ITS-type in Australian/NZ A-clade hybrids. Arrows indicate hybridization. Numbers in boxes represent number of analysed species of given geographical origin. Encircled letters A and C refer to A- and C-clade species, respectively. See text for more details.

87 Page 4 of 8 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium

(Molecular Probes, New Haven, CT, USA). Chromosomes were counterstained with DAPI (2 mgmL21) in Vectashield. Fluorescence signals were analysed with an Olympus BX-61 No. of 5S rDNA loci epiﬂuorescence microscope and AxioCam CCD camera (Carl Zeiss, Jena, Germany). Individual images were merged and processed by using Photoshop CS software (Adobe Systems). rDNA loci No. of 45S RESULTS Chromosome counts 112 4 2 88 2 2 72 2 2 72 2 4 72 2 4 approx. 56 2 4 approx. 56 2 2 approx. 56 2 2 16 2 2 16 2 2 56 2 2 16 2 2 16 2 2 In the analysed Lepidium species, chromosome numbers ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ x x varied from 2n ¼ 16 to 2n ¼ 112 (see Table 2 for all chromo- x x x x x x x x x x x 11 14 9 9 9 7 7 7 2 2 7 2 2 some counts). The two African species (L. africanum, ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ n n n n n n n n n n n n n L. transvaalense) have a diploid chromosome number (2n ¼ 2 2x ¼ 16), whereas all Australian/NZ and Californian species

species studied are polyploid (Fig. 2). Four different ploidy levels (7x,9x, 11x and 14x) were discerned in Australian/NZ species. In the three Californian species, we could not obtain suitable chromosome spreads to determine exact chromosome Lepidium numbers. Observed chromosome numbers were tentatively interpreted as heptapolyploid (2n ¼ 7x ¼ 56). Mitotic chromosomes of Lepidium species are small (approx. 2–5 mm) with the bulk of heterochromatin concen- trated within pericentromeres, and often fuzzy euchromatic chromosome ends (Figs 2 and 3). In the two African species, chromosomes are larger and possess a less distinct eu-/heterochromatin proﬁle than chromosomes in the other two geographical groups. The presumably different chromosome structure of African species is particularly apparent upon GISH in the A-clade allopolyploids, whereby chromosomes of African origin show a more even labelling pattern and appear to be larger than the remaining chromosomes (Fig. 3).

Localization of rDNA loci Most Lepidium species exhibit one pair of 5S and 45S rDNA loci (Table 2 and Fig. 2). This pattern was also observed in high-numbered polyploid species such as L. nitidum, L. oxycarpum, L. pseudohyssopifolium, L. muelleriferdinandi and L. aschersonii. Two species (L. dictyotum and L. hyssopifolium) possess two pairs of 5S rDNA, and one species (L. ginninderrense) possesses two pairs of 45S rDNA loci (Table 2 and Fig. 2). In L. oxycarpum, 5S and

Creek/Scarlett, N. 45S loci were found to be partly co-localized (Fig. 2C). 5S Origin, chromosome number, ploidy level and number of rDNA loci for rDNA loci are positioned on chromosomes interstitially,

2. whereas 45S rDNA loci showed terminal localization (Fig. 2). no. Origin/Collector Chromosome no. ABLE KM 1670 Australia, Victoria, Belfast Lough, Port Fairy end of the Lough/Scarlett, N. 2 KM 1794 South Africa, Eastern Cape/Clark, V. R. 2 KM 1793 South Africa, Eastern Cape/Clark, V. R. 2 KM 1702 Australia, Melbourne, Royal Park, South of Park Street/Scarlett, N. 2 Accession T GISH in polyploid Australian/NZ Lepidium species With the aim to discern the origin of Australian/NZ Lepidium

* polyploids, labelled gDNA of putative African and Californian

Hewson KM 1666 Australia, Victoria, Melbourne, Fairﬁeld, Banks of the Yarra River/Parsons, R. F.parental 2 species was hybridized to mitotic chromosomes of Thell. KM 1710 Australia, Northern Territory, Alice Springs Desert Park, outside herbarium/Albrecht,Australian/NZ D. E. 2 polyploid A- and C-clade taxa. In two A-clade Scarlett KM 1673 Australia, Australian Capital Territory, Belconnen Naval Station, ﬂoodplain of Ginninderra is native to South Africa, and an introduced weed in Australia. Desv. KM 1669 Australia, New South Wales, Bathurst/Scarlett, N. 2 Marais KM 1792 South Africa, Eastern Cape/Clark, V. R.species (L. hyssopifolium, L. 2 pseudohyssopifolium), gDNA of Thell. KM 1668 Australia, Victoria, Lake Beeac/Scarlett, N. 2 Torr. & Gray KM 1713 USA, California, Central Valley, Los Banos/Bowman, J. 2

Gray KM 1714 USA, California, Carrizo plain/Bowman, J.presumed African parents (L. africanum, 2 L. transvaalense) (Burm.f.) DC.

Nutt. KM 1711 USA, California, Tucker Herbarium at UC Davis/Bowman, J.labelled 16 of the 72 2 chromosomes, whereas no chromosomes were identiﬁed using gDNA of presumed Californian parental L. africanum species (L. dictyotum, L. oxycarpum) (data not shown). No * L. muelleriferdinandi L. ginninderrense L. pseudohyssopifolium L. hyssopifolium L. dictyotum L. oxycarpum L. nitidum L. transvaalense L. aschersonii L.africanum Species chromosome-speciﬁc hybridization signals were observed in

88 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium Page 5 of 8

A D E

F IG. 2. DAPI-stained mitotic chromosome spreads from ﬂower bud tissue of Lepidium species. Chromosomes were hybridized with 5S (green) and 45S (red) rDNA probes and counterstained with DAPI. (A) L. africanum (2n ¼ 2x ¼ 16), (B) L. dictyotum (2n ¼ 7x ¼ 56), (C) L. oxycarpum (2n ¼ 7x ¼ 56), (D) L. aschersonii (2n ¼ 7x ¼ 56), (E) L. muelleriferdinandi (2n ¼ 11x ¼ 88), (F) L. ginninderrense (2n ¼ 14x ¼ 112). In (F) one pair of major 45S rDNA loci is decondensed around the nucleolus; two minor 45S rDNA loci are indicated by arrows. Scale bars ¼ 5 mm.

F IG. 3. GISH in two nonaploid (2n ¼ 9x ¼ 72) Australian/NZ Lepidium A-clade species. Chromosomes of L. hyssopifolium (A) and L. pseudohyssopifolium (B) hybridized with gDNA of L. africanum (16 red chromosomes) and gDNA of an Australian/NZ C-clade species, i.e. L. muelleriferdinandi (56 green chromosomes). Arrows mark 45S rDNA-bearing chromosomes (yellow/red). Scale bar ¼ 5 mm.

C-clade species (L. aschersonii, L. muelleriferdinandi) using the and L. pseudohyssopifolium has probably been contributed same probes as used in the A-clade species (data not shown). by the African parental genome(s). In follow-up GISH experiments, 56 of 72 chromosomes in the Australian/NZ A-clade species L. hyssopifolium and L. pseudohyssopifolium were detected using gDNA of two DISCUSSION Australian/NZ C-clade species, i.e. L. muelleriferdinandi rDNA patterns in polyploid Lepidium species (Fig. 3) and L. aschersonii (data not shown). In the two A-clade allopolyploids (2n ¼ 72), 16 chromosomes corre- The present study followed three principal goals as far as spond to L. africanum and 56 chromosomes were labelled by in situ localization of rDNA repeats is concerned. First, we gDNA of L. aschersonii/L. muelleriferdinandi (Fig. 3). This were interested to see if the three geographical species experiment also showed that 45S rDNA in L. hyssopifolium groups (Africa, Australia/NZ and California) are characterized

89 Page 6 of 8 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium

by specific rDNA patterns. Secondly, the number of rDNA loci chromosomes belonging to the 16 chromosomes of African in Australian/NZ polyploids was compared with the number of origin. The 56 remaining chromosomes displayed no 45S the same loci in the presumed progenitor species, and finally, rDNA loci (Fig. 3). This is in agreement with the ITS phylo- the origin of 45S rDNA loci in Australian/NZ polyploid genetic data showing that all Australian/NZ A-clade species species was inferred from GISH analysis and compared with harbour the African ITS-type (Mummenhoff et al., 2004; conclusions based on ITS sequence data (Mummenhoff Fig. 1C, Table 1). For these species, physical loss of C-clade et al., 2004). rDNA loci must be assumed subsequent to hybridization. From the limited data set (only ten of 230 Lepidium species Furthermore, the African ITS-type in polyploid Australian/ were analysed) no apparent geographical pattern of rDNA NZ A-clade species can be explained by unidirectional con- variation emerges. In all but one polyploid species analysed, certed evolution to the African ITS-type with single additive only a diploid-like number of 45S rDNA loci were found, nucleotides remaining (Table 1, L. hyssopifolium KM 1670). with two pairs of 45S identified in the tetradecaploid L. ginninderrense (2n ¼ 112). This finding is somewhat sur- Previous hypothesis on the origin of polyploid Australian/NZ prising given the high ploidy levels (from 7x to 14x) of the Lepidium species analysed taxa. Regardless of prevailing modes of polyploid evolution (auto- vs. allopolyploidy) in the Californian and The rDNA ITS regions are the most widely used nuclear- Australian/NZ groups, respectively, the present data indicate encoded phylogenetic markers (A´ lvarez and Wendel, 2003). that there is a strong tendency toward diploidization of Their high evolutionary rate permits discrimination of rDNA loci in both geographical species groups. Although closely related putative parental species and the identification less likely, preferential loss of rDNA-bearing chromosomes of additive patterns in hypothesized allopolyploids (Marhold in odd-numbered (7, 9 and 11x) Lepidium polyploids cannot and Lihova´, 2006). Although concerted evolution can erase be ruled out. nucleotide additivity, bidirectional, nearly complete Ali et al. (2005) analysed three different crucifer species homogenization or intergenomic recombination may result in with the same polyploid chromosome number (2n ¼ 48). a mosaic-like structure of ITS sequences, which has been In two species, the number of 45S was high (approx. 14 described in the literature (Marhold and Lihova´, 2006). in Camelina microcarpa, and 10 þ 6 minor loci in In the Brassicaceae, such analyses unravelled hybridization Olimarabidopsis cabulica), whereas only one pair has been events in Thlaspi (Mummenhoff et al., 1997), Cardamine recorded in Aethionema schistosum (Ali et al., 2005). It (Franzke and Mummenhoff, 1999; Lihova´ et al., 2006) and could be speculated that this limited comparison corroborates Boechera (Koch et al., 2003). Support for the bicontinental the generally accepted correlation between the number of hybrid origin of polyploid Australian/NZ Lepidium species 45S rDNA loci and the age of polyploid species as the was provided by bidirectional, nearly complete concerted evol- genus Aethionema is sister to and older than the rest of ution of the ITS to either of the two presumed parental Brassicaceae (Al-Shehbaz et al., 2006). However, the ITS-types (African and Californian) with single additive (par- Aethionema polyploid species may be of a similar age to ental) nucleotides remaining (Mummenhoff et al., 2004; two Camelineae species analysed. Hence, it appears to be dif- Table 1). It appears that all Australian/NZ polyploids share a ficult to establish a direct link between the age of a phyloge- Californian cpDNA-type (Mummenhoff et al., 2004), but netic split and the diploidization rate in crucifer taxa. this cpDNA evidence was detected only in 60 % of the maxi- With each polyploidization event the number of rDNA loci mally parsimonious trees (Mummenhoff et al., 2004). increases and some rDNA sequences become redundant. Furthermore, this scenario of a bicontinental hybridogenous These ITS sequences can be changed by bidirectional interlocus genomic constitution was based on only two markers repre- concerted evolution, resulting in complete homogenization to senting the nuclear (ITS) and chloroplast genome either of the parental types or to mosaic-like sequences with (Mummenhoff et al., 2004). Thus, GISH experiments were additive nucleotides from both parents (Wendel, 2000). In the performed to gain deeper insight into the origin and evolution long term, genome diploidization events as discovered for of polyploid Australian/NZ Lepidium species. ancient allopolyploid Nicotiana species of the section Repandae (4.5 Myr old) can occur, resulting in the loss of redun- Origin of Australian/NZ C-clade species: neither African nor dant rDNA loci and a diploid-like number of rDNA loci, which Californian progenitors detected by GISH can be even lower than the number of 45S loci found in the progenitor species (Clarkson et al., 2005). However, more recently In previous studies based on sequence analysis of nuclear formed Nicotiana allopolyploids (e.g. N. tabacum,0.2 Myr old) ITS and non-coding cpDNA (Mummenhoff et al., 2004), a display the sum of the rDNA loci of their progenitors (Clarkson bicontinental hybrid origin of polyploid Australian/NZ et al., 2005). As a maximum estimated age for the origin of Lepidium species has been suggested. In GISH experiments, polyploid Australian/NZ Lepidium species is approx. 1.3Ma gDNA probes of presumed African (L. africanum, (Mummenhoff et al., 2004), this or a shorter period of time L. transvaalense) and Californian parental species has been sufficient for (nearly) complete diploidization of (L. dictyotum, L. oxycarpum) did not label specific chromo- rDNA loci (Table 2). somes in the Australian C-clade species. This result is in con- Within the set of the 72 chromosomes of Australian/NZ trast to the ITS and cpDNA analysis (Mummenhoff et al., A-clade species (L. hyssopifolium, L. pseudohyssopifolium) 2004). The evidence of a bicontinental hybridogenous combined GISH and rDNA fluorescence in situ hybridization genomic constitution of C-clade species is based on weak (FISH) localization detected two 45S rDNA loci on two cpDNA evidence (see above), ITS sequence homology of

90 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium Page 7 of 8

Australian/NZ species with Californian taxa, and single addi- A-clades, respectively (Mummenhoff et al., 2004). These tive nucleotide positions representing both parental age differences might indicate that the radiation of ITS-types. If this additivity in single ITS nucleotide positions Californian ancestors into Australian/NZ C-clade species pre- is not random and indicates an African input, present-day dates the later arrival of African ancestors and the origin of African species used for GISH experiments do not represent A-clade allopolyploids. the original African progenitors. This means that the genome of the original African species contributing to the ITS additiv- Origin of Australian/NZ A-clade species: allopolyploid hybrids ity in the polyploid Australian/NZ C-clade species is too dis- between African and Australian/NZ C-clade species tinct from the genome of extant African taxa used in the present study (L. africanum, L. transvaalense), and thus GISH experiments clearly detected 16 chromosomes of gDNA probes of L. africanum/L. transvaalense could not be African origin (L. africanum, L. transvaalense) in the two used to detect the original African parental genome in polyploid Australian/NZ A-clade species analysed C-clade species. (L. hyssopifolium, L. pseudohyssopifolium). The remaining Ancestral Californian species (migrating to Australia) may 56 chromosomes in the A-clade allopolyploid genomes were have transmitted the Californian ITS-type to the allopolyploid not labelled by any of the Californian presumed parental Australian/NZ C-clade species via hybridization with an gDNAs used. Thus, the presumed direct Californian genomic unknown taxon, potentially of African origin. At the same input into the Australian/NZ A-clade Lepidium species could time the ancestral Californian taxa might have evolved into not be confirmed by GISH. the present-day polyploid Californian species, probably by One might thus speculate that Australian/NZ C-clade taxa several rounds of hybridization and polyploidization events, have contributed the Californian cpDNA-type to the accompanied by the dynamic evolution of genome-specific Australian/NZ A-clade taxa by hybridization with African dispersed repeats. As a consequence, the extant Californian species in Australia. To test this scenario, GISH experiments species differ significantly in genome constitution from their were conducted using gDNAs of Australian/NZ C-clade presumed polyploid Australian/NZ descendants, and thus species (L. aschersonii 2n ¼ 56, L. muelleriferdinandi their gDNAs do not reveal the ancestral Californian genome 2n ¼ 88) as probes onto the Australian/NZ A-clade polyploid in the polyploid Australian/NZ C-clade species. This can be species L. hyssopifolium and L. pseudohyssopifolium analogous to the decreased efficiency of GISH and its failure (2n ¼ 72). Of the 72 chromosomes, 56 were labelled by in approx. 1- and 5-Myr-old Nicotiana allopolyploids, respect- gDNA of Australian/NZ C-clade species and, as outlined ively (Lim et al., 2007). above, the remaining 16 chromosomes were labelled by gDNA of L. africanum/L. transvaalense. The remaining 32 chromosomes of the genome of present-day Australian/NZ C-clade species: simply descendants L. muelleriferdinandi (2n ¼ 88) not detected in Australian of Californian ancestors? A-clade polyploids (2n ¼ 72) could indicate some sort of Negative results using gDNA of Californian and African hybridization/polyploidization with unknown taxa in the evol- species in C-clade Australian/NZ polyploids suggest long- ution of L. muelleriferdinandi subsequent to the hybridization distance dispersal of ancestral Californian species to of an ancestor of L. muelleriferdinandi (presumably with 2n ¼ Australia followed by radiation into extant C-clade polyploid 56) with African species (2n ¼ 16). species without hybridization with African species being This general scenario is in agreement with the cpDNA involved. All Australian/NZ C-clade species appear to phylogeny in which the Australian/NZ A-clade hybrids harbour a Californian ITS- and cpDNA-type (Mummenhoff harbour a Californian chloroplast type (Mummenhoff et al., et al., 2004) and only one additive nucleotide position each 2004). There is some additional evidence for this alternative (representing African and Californian ancestral nucleotides, scenario: one accession of an Australian/NZ A-clade polyploid respectively) in the ITS sequences of two C-clade species (L. hyssopifolium, KM 1670) shows additivity in almost every studied (L. aschersonii, L. muelleriferdinandi). This single diagnostic nucleotide position that differs between the C-clade African nucleotide in these two C-clade species could also and A-clade species group (Table 1). Regardless, all GISH and be explained by random mutations or could represent an rDNA FISH experiments using two different accessions of input by hybridization with an unknown species. The remain- L. hyssopifolium (KM 1670, KM 1669) show the same ing 32 chromosomes of the genome of present-day results. As ITS homogenization can occur very rapidly, as L. muelleriferdinandi (2n ¼ 88) not detected in Australian also observed in experiments with recent and synthetic allopo- A-clade polyploids (2n ¼ 72) could indeed indicate some lyploids where 45S rDNA homogenization was apparent sort of hybridization/polyploidization with unknown taxa in within a few generations (Franzke and Mummenhoff, 1999; the evolution of L. muelleriferdinandi subsequent to the Skalicka´ et al., 2003; Kovarik et al., 2005; Shcherban et al., hybridization of an ancestor of L. muelleriferdinandi (presum- 2008), it is possible that additivity in almost all diagnostic ably with 2n ¼ 56) with African species (2n ¼ 16). GISH nucleotide position in L. hyssopifolium (KM 1670) indicates signals of an African genome input could not be detected, recent and/or recurrent hybridization between African and suggesting that Australian/NZ C-clade species are not the Australian/NZ C-clade species in Australia. outcome of a bicontinental allopolyploidization scenario as Thus, the main conclusions from GISH analysis are as originally described (Mummenhoff et al., 2004). Calibration follows. The nuclear genomes of African and Australian/NZ of molecular trees yielded ages of approx. 0.7–1.3 and C-clade species were detected in allopolyploid Australian/NZ 0.3–0.55 Ma for the Australian/NZ species of the C- and Lepidium A-clade species. The presumed hybrid origin of

91 Page 8 of 8 Dierschke et al. — Bicontinental origin of polyploid Australian/NZ Lepidium

Australian/NZ C-clade taxa with African and Californian Franzke A, Mummenhoff K. 1999. Recent hybrid speciation in Cardamine parents involved (Mummenhoff et al., 2004) could not be con- (Brassicaceae) conversion of nuclear ribosomal ITS sequences in statu nascendi. Theoretical and Applied Genetics 98: 831–834. firmed. This does not mean that hybridization processes did Kovarik A, Pires JC, Leitch AR, et al. 2005. Rapid concerted evolution of not play a role in the evolution of polyploid Australian nuclear ribosomal DNA in two Tragopogon allopolyploids of recent C-clade species, for example L. muelleriferdinandi with and recurrent origin. Genetics 169: 931–944. 2n ¼ 88 chromosomes. Hence, it is assumed that ancestral Koch M, Al-Shehbaz IA, Mummenhoff K. 2003. Molecular systematics, Californian taxa subsequent to their dispersal to Australia evolution, and population biology in the mustard family (Brassicaceae). Annals of the Missouri Botanical Garden 90: 151–171. experienced a rapid radiation in Australia and New Zealand Lee J-Y, Mummenhoff K, Bowman JL. 2002. Alloploidization and evolution into extant C-clade taxa that hybridized with African of species with reduced floral structures in Lepidium L. (Brassicaceae). species. As a result, A-clade allopolyploid Lepidium species Proceedings of the National Academy of Sciences 16: 835–840. in Australia/NZ share the Californian chloroplast type and Lihova´ J, Shimizu KK, Marhold K. 2006. Allopolyploid origin of Cardamine asarifolia (Brassicaceae): incongruence between plastid and the African ITS-type with polyploid Australian/NZ C-clade nuclear ribosomal DNA sequences solved by a single-copy nuclear and diploid African species, respectively. gene. Molecular Phylogenetics and Evolution 39: 759–786. Lim KY, Kovarik A, Matyasek R, et al. 2007. Sequence of events leading to near-complete genome turnover in allopolyploid Nicotiana within five ACKNOWLEDGEMENTS million years. New Phytologist 175: 756–763. Lysak MA, Berr A, Pecinka A, Schmidt R, McBreen K, Schubert I. 2006. We thank all collectors and institutions for providing plant Mechanisms of chromosome number reduction in Arabidopsis thaliana material, Ulrike Coja for technical assistance and Andreas and related Brassicaceae species. Proceedings of the National Academy of Sciences of the USA 103: 5224–5229. Franzke and two anonymous reviewers for valuable comments. Marhold K, Lihova´ J. 2006. Polyploidy, hybridization and reticulate evol- T.D. was supported by the DAAD (German Academic ution: lessons from the Brassicaceae. Plant Systematics and Evolution Exchange Service), and T.M. and M.A.L. were supported by 259: 143–174. research grants nos KJB601630606 and IAA601630902 from Mummenhoff K, Franzke A, Koch M. 1997. Molecular phylogenetics of the Grant Agency of the Czech Academy of Science, and a Thlaspi s.l. (Brassicaceae) based on chloroplast DNA restriction site variation and sequences of the internal transcribed spacers of nuclear riboso- grant from the Czech Ministry of Education (no. mal DNA. Canadian Journal of Botany 75: 469–482. MSM0021622415). Mummenhoff K, Bru¨ggemann H, Bowman JL. 2001. Chloroplast DNA phylogeny and biogeography of Lepidium (Brassicaceae). American Journal of Botany 88: 2051–2063. LITERATURE CITED Mummenhoff K, Linder P, Friesen N, Bowman JL, Lee J-Y, Franzke A. 2004. Molecular evidence for bicontinental hybridogenous genomic con- Al-Shehbaz IA. 1986. The genera of Lepidieae (Cruciferae; Brassicaceae) in stitution in Lepidium sensu stricto (Brassicaceae) species from Australia the southeastern United States. Journal of the Arnold Arboretum 67: and New Zealand. American Journal of Botany 91: 254–261. 265–311. Mummenhoff K, Polster A, Mu¨hlhausen A, Theißen G. 2009. Lepidium as a Al-Shehbaz IA, Beilstein MA, Kellogg EA. 2006. Systematics and phylogeny model system for studying the evolution of fruit development in of the Brassicaceae (Cruciferae): an overview. Plant Systematics and Brassicaceae. Journal of Experimental Botany 60: 1503–1513. Evolution 259: 89–120. Shcherban AB, Sergeeva EM, Badaeva ED, Salina EA. 2008. Analysis of Ali HBM, Lysak MA, Schubert I. 2005. Chromosomal localization of rDNA 5S rDNA changes in synthetic allopolyploids Triticum Aegilops. in the Brassicaceae. Genome 48: 341–346. Molecular Biology 42: 536–542. A´ lvarez I, Wendel JF. 2003. Ribosomal ITS sequences and plant phyloge- Skalicka´ K, Lim KY, Matyasek R, Koukalova´ B, Leitch A, Kovarik A. netic inference. Molecular Phylogenetics and Evolution 29: 417–434. 2003. Rapid evolution of parental rDNA in a synthetic tobacco allotetra- Bowman JL, Bru¨ggemann H, Lee J-Y, Mummenhoff K. 1999. Evolutionary ploid line. American Journal of Botany 90: 988–996. changes in floral structure within Lepidium L. (Brassicaceae). Vaarama A. 1952. Chromosome number and cryptic polyploidy in Lepidium International Journal of Plant Sciences 160: 917–929. sativum. Hereditas 37: 290–292. Clarkson JJ, Lim KY, Kovarik A, Chase MW, Knapp S, Leitch AR. 2005. Warwick SI, Al-Shehbaz IA. 2006. Brassicaceae: chromosome number index Long-term genome diploidization in allopolyploid Nicotiana section and database on CD-Rom. Plant Systematics and Evolution 259: Repandae (Solanaceae). New Phytologist 168: 241–252. 237–248. Dellaporta SL, Wood J, Hicks JB. 1983. A plant DNA minipreparation: Wendel JF. 2000. Genome evolution in polyploids. Plant Molecular Biology version II. Plant Molecular Biology Reporter 1: 19–21. 42: 225–249.

92 V. Mandáková T., Joly S., Krzywinski M., Mummenhoff K., Lysak M.A. 2010. Fast diploidization in close mesopolyploid relatives of Arabidopsis. Plant Cell 22: 2277-2290.

93 94 The Plant Cell, Vol. 22: 2277–2290, July 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

Fast Diploidization in Close Mesopolyploid Relatives of Arabidopsis W OA

Terezie Manda´ kova´ ,a Simon Joly,b Martin Krzywinski,c Klaus Mummenhoff,d and Martin A. Lysaka,1 a Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic b Institut de Recherche en Biologie Ve´ ge´ tale, Universite´ de Montre´ al and Montreal Botanical Garden, 4101 Sherbrooke East, Montreal, Quebec, Canada H1X 2B2 c Canada’s Michael Smith Genome Sciences Center, Vancouver, British Columbia, Canada V5Z 4S6 d FB Biologie/Chemie, Botanik, Universita¨ tOsnabru¨ ck, D-49069 Osnabru¨ ck, Germany

Mesopolyploid whole-genome duplication (WGD) was revealed in the ancestry of Australian Brassicaceae species with diploid-like chromosome numbers (n = 4 to 6). Multicolor comparative chromosome painting was used to reconstruct complete cytogenetic maps of the cryptic ancient polyploids. Cytogenetic analysis showed that the karyotype of the Australian Camelineae species descended from the eight ancestral chromosomes (n = 8) through allopolyploid WGD followed by the extensive reduction of chromosome number. Nuclear and maternal gene phylogenies corroborated the hybrid origin of the mesotetraploid ancestor and suggest that the hybridization event occurred ;6 to 9 million years ago. The four, five, and six fusion chromosome pairs of the analyzed close relatives of Arabidopsis thaliana represent complex mosaics of duplicated ancestral genomic blocks reshuffled by numerous chromosome rearrangements. Unequal reciprocal translocations with or without preceeding pericentric inversions and purported end-to-end chromosome fusions accompanied by inactivation and/or loss of centromeres are hypothesized to be the main pathways for the observed chromosome number reduction. Our results underline the significance of multiple rounds of WGD in the angiosperm genome evolution and demonstrate that chromosome number per se is not a reliable indicator of ploidy level.

INTRODUCTION genome triplication in the sister family Cleomaceae (Schranz and Mitchell-Olds, 2006; Barker et al., 2009). The exact position of Hybridization and polyploidization (whole-genome duplication the b event within the order Brassicales has to be elucidated by [WGD]) are important evolutionary forces driving genetic diver- further research (Barker et al., 2009). sification and speciation in land plants. In angiosperms, nu- The a WGD has been dated to occur ;23 to 43 million years merous cases of speciation events following this pattern were ago (mya) (Barker et al., 2009; Fawcett et al., 2009), after the b documented (Grant, 1981; Soltis et al., 2007; Hegarty and duplication postdating the Brassicaceae-Caricaceae divergence Hiscock, 2008). Comparative research using whole-genome and 72 mya (Ming et al., 2008). The three rounds of paleopolyploid EST sequence data sets uncovered compelling evidence of WGDs (a, b, and g) were followed by diploidization (fractionation; multiple ancient WGD events in the ancestry of angiosperm Thomas et al., 2006) toward diploid-like genomes marked by lineages (De Bodt et al., 2005; Cui et al., 2006; Soltis et al., 2009; genome downsizing and chromosome rearrangements. For in- Jaillon et al., 2009). In crucifers (Brassicaceae), the analysis of the stance, the functionally diploid and extremely compact Arabi- Arabidopsis thaliana genome sequence (Arabidopsis Genome dopsis genome (n = 5, 1C = 157 Mb) is a result of the extensive Initiative, 2000) suggested the existence of three paleopolyploid karyotype reshuffling of already diploidized Ancestral Crucifer a b g WGDs ( , , and ; Bowers et al., 2003). Whereas the phyloge- Karyotype (n = 8) (Lysak et al., 2006). After a WGD, some g netic placement of the oldest event ( ) is still debated (Soltis duplicated genes are lost or subjected to sub- and neofunction- et al., 2009), the two more recent WGDs have been shown to alization (Se´ mon and Wolfe, 2007), dosage-sensitive genes postdate the split between Caricaceae and Brassicaceae (Tang such as transcription factors are preferentially retained in the a et al., 2008). The most recent ( ) duplication apparently occurred duplicated genomes, as described by the gene balance the- only within Brassicaceae, and it is equivalent to the whole- ory (Birchler and Veitia, 2007; Freeling, 2008; Veitia et al., 2008; Edger and Pires, 2009). 1 Address correspondence to [email protected]. Multiple and often lineage-specific WGD events uncovered in The author responsible for distribution of materials integral to the Asteraceae (Barker et al., 2008), Cleomaceae (Schranz and findings presented in this article in accordance with the policy described Mitchell-Olds, 2006; Barker et al., 2009), Solanaceae (Schlueter in the Instructions for Authors (www.plantcell.org) is: Martin A. Lysak et al., 2004), and other taxa (Soltis et al., 2009) imply a key role of ([email protected]). WGDs in the evolution of land plants. The steadily improving W Online version contains Web-only data. OA Open Access articles can be viewed online without a subscription. knowledge of crucifer genome evolution suggests that the www.plantcell.org/cgi/doi/10.1105/tpc.110.074526 duplication-diploidization process is ongoing and occurred

95 2278 The Plant Cell

several times across the family. In addition to the three afore- CCP experiments showed that most GBs, present as single metioned paleopolyploid events, several Brassicaceae groups copies in Arabidopsis and the ACK, were duplicated and hybrid- with diploid-like genomes have experienced additional, more ized to two chromosomes within pachytene complements of the recent WGD events, as exemplified by a whole-genome triplica- Australian crucifers (Figures 1A to 1C, 2A, and 3). In S. nutans, tion (;8 to 15 mya) most likely promoting the diversification blocks J, M, and R hybridized to three different chromosomes, within the tribe Brassiceae (Lysak et al., 2005, 2007; Parkin et al., and two copies of block U hybridized to a single chromosome 2005; Panjabi et al., 2008). Such duplications are younger than (SN4). Genomic blocks R and F labeled three and block J four paleopolyploid events but older than neopolyploid speciation chromosomes in S. lineare.InB. antipoda, block R hybridized events (e.g., the origin of Arabidopsis suecica, Brassica napus,or to three and blocks A and B to four different chromosomes, Cardamine schulzii) and can be detected by comparative genetic respectively. No obvious cross-hybridization of single Arabidop- and cytogenetic methods. sis BAC clones outside the homoeologous collinear regions was In this study, we used comparative chromosome painting to observed (Figure 2A). Whereas all 24 GBs were found to be analyze endemic Australian crucifers (Stenopetalum nutans, duplicated in S. nutans (Figures 1A, 2A, and 3A), only 22 GBs Stenopetalum lineare,andBallantinia antipoda) with low, diploid- were found duplicated in S. lineare (Figures 1B and 3B) and 20 like chromosome numbers (n = 4, 5, and 6, respectively). GBs in B. antipoda (Figures 1C and 3C) (as single copies were Comparison with the Ancestral Crucifer Karyotype (n =8) identified GBs P and Q in S. lineare, and D, E, P, and S in B. showed that the ancestor(s) of the analyzed species experienced antipoda). The position of each GB is compared for each pair of a mesopolyploid WGD followed by extensive karyotype reshuf- the Australian crucifers in Supplemental Figure 1 online. In all fling associated with genome diploidization. species, paired GB duplicates differed slightly but consistently in length and fluorescence intensity (see Figure 2A for examples). This difference was more pronounced in the case of longer GBs RESULTS (the longer and brighter copy always labeled as #1). The difference in intensity is likely due to differential origins of the two All or Most Genomic Blocks of the Ancestral Crucifer genome copies (but see Discussion). In B. antipoda, all distal Karyotype Are Duplicated in the Genomes of heterochromatic segments were labeled by painting probes Australian Species. comparably to euchromatin regions (Figure 1C). Ancestral ACK-like associations of many duplicated GBs were We analyzed chromosome structure and genome size of the retained in the extant complements (see Supplemental Table 2 three Australian crucifer species. Except distinct domains of online; Figures 1A to 1C). In S. nutans, we found seven AK-like pericentromeric heterochromatin, heterochromatic knobs and chromosomes [AK2(#2), AK3(#1, #2), AK4(#1), AK5(#2), AK7(#2), distal segments were detected in two species (Figures 1A to 1C). and AK8(#1)], 11 AK-like chromosome arms [upper arm of In S. nutans (n = 4; 477 Mb; accession 86929), chromosomes AK1(#1), AK2 (#1), AK4(#2), AK5(#1), AK6(#2), AK7(#1), and SN2 and SN4 possess terminal heterochromatic knobs on upper AK8(#2); bottom arm of AK1(AK1(#1, #2), AK6(#2), and AK8(#2)], arms, whereas chromosome SN3 has an interstitial heterochro- seven preserved GBs not forming any AK-like structure, and nine matic knob (hkSN3) on its bottom arm. No conspicuous hetero- split GBs. In S. lineare, five AK-like chromosomes [AK2(#2), chromatic knobs were observed in the S. lineare karyotype AK3(#2), AK5(#1, #2), and AK7(#1)], 15 AK-like chromosome (n = 5; 763 Mb). The B. antipoda karyotype (n = 6; 472 Mb) is arms [upper arm of AK1(#1), AK2(#1), AK4(#1, #2), AK6(#1), characterized by six large heterochromatic segments that ex- AK7(#2), and AK8(#1, #2); bottom arm of AK1(#1, #2), AK2(#1), tend from one-third up to the entire length of a chromosome arm AK3(#1), AK7(#2), and AK8(#1, #2)], six preserved GBs not (Figures 1C and 2D). In all three analyzed species, the Arabi- forming any AK-like structure, and six split GBs were identified. dopsis-type telomere repeat hybridized to chromosome termini. The B. antipoda complement comprises six AK-like homoeologs However, additional, remarkably strong hybridization signals [AK2(#2), AK3(#1, #2), AK4(#1), AK5(#2), and AK8(#1)], 12 AK-like were observed at centromeric regions of all B. antipoda chro- chromosome arms [upper arm of AK4(#2), AK5(#1), AK6(#1), mosomes (Figure 2E). AK7(#2), and AK8(#2); bottom arm of AK1(#1, #2), AK4(#2), As no genetic maps for the analyzed species were available, AK5(#1) AK6(#2), AK7(#1), and AK8(#2)], four preserved GBs not we used the Ancestral Crucifer Karyotype (ACK; Figure 1D) as a forming any AK-like structure, and six split GBs. Except AK1 and reference genome for the construction of molecular cytogenetic AK6, at least one copy of all other ancestral chromosomes maps by comparative chromosome painting (CCP). The ACK, is structurally preserved within the analyzed karyotypes, viz. inferred from (cyto)genetic comparisons between genomes of AK2(#2), AK3(#1, #2), AK4(#1), AK5(#1, #2), AK7(#1, #2), and Arabidopsis, B. napus, and other crucifer species, comprises AK8(#1). Homoeologs AK2(#2), AK3(#2), and AK5(#2) were eight ancestral chromosomes (AK1 to AK8) and 24 conserved shared by all three species. In all three genomes, homoeologs of genomic blocks (GBs) (Lysak et al., 2006; Schranz et al., 2006). AK6 and AK8 are rearranged, whereby the rearranged structure of Five hundred and forty seven chromosome-specific BAC clones AK8(#1) is shared by all three species (see Supplemental Figure 2 of Arabidopsis representing all genomic blocks of the ACK have and Supplemental Table 2 online). Although numerous AK chro- been differentially labeled and hybridized to pachytene chromo- mosomes and chromosome arms were found as conserved and somes. Conserved or rearranged ancestral GBs, their physical duplicated, they were rearranged (fused) to build extant chromo- size (bp), and correspondence to Arabidopsis BAC contigs are somes of the Australian species. For instance, the bottom arm shown in Supplemental Table 1 online for all three species. of S. nutans chromosome SN4 consists of three complete AK

96 Mesopolyploidy in Australian Crucifers 2279

Figure 1. Cytogenetic Maps of Stenopetalum and Ballantinia Species.

97 2280 The Plant Cell

homoeologs [AK4(#1), AK7(#2), and AK8(#1)], whereas the nine (19%) GBs (A1, B1, E1, J2, N1, M1, R1, W1, and X1) were upper arm comprises GBs of two other homoeologs [block U1 split. In S. lineare, out of 46 GBs, 40 (87%) were conserved and of AK7(#1) and K1, L1, and M1 of AK5(#1)] (Figures 1A and 2B). six (13%) blocks (F1, J1 and J2, R2, W1, and X1) were As duplicated and reshuffled karyotypes differ significantly partitioned. In B. antipoda, out of 44 GBs, 38 (86%) blocks even between congeneric taxa (S. nutans and S. lineare, Figure remained intact and six (14%) (A1 and A2, B1, R1, W1, and X1) 1), we investigated whether the cytogenetic maps based on a were split into sub-blocks. single accession represent species’ karyotype. Seven unique GB The AK8(#1) homoeolog exhibits a specific rearrangement of associations (A-B-D, C-K-L, U-K-L, V-S-T-U, R-S-O, P-O-V, and blocks W1 and X1 shared by all three taxa (Figures 1A to 1C; see R-V-C) from all but one chromosome arms within the S. nutans Supplemental Figure 2 and Supplemental Table 2 online). This #86929 cytogenetic map were tested in another S. nutans reshuffling of the AK8-like homoeolog has been analyzed also in population (#76272). All GB associations were conserved be- two other Australian crucifers, Arabidella eremigena (n = 5) and tween the two populations occurring >1000 km apart (see Blennodia canescens (n = 7). Although these two species have Supplemental Table 4 online). To get further insight into the pachytene complements not amenable to comprehensive cyto- origin of the revealed genome duplications, the seven unique GB genetic analysis, we could show that they also possess the associations of S. nutans were analyzed in two Stenopetalum rearrangement and four other GBs duplicated. These data sug- species with chromosome numbers doubled as compared with gest that the Australian crucifers share a common polyploid S. nutans and S. lineare.InStenopetalum velutinum (n =8)and ancestor, but with most genomic rearrangements being species Stenopetalum anfractum (n = 10), all tested GBs were found as specific. four genomic copies. Three and two GB associations in S. velu- Comparing the position and orientation (collinearity) of ances- tinum and S. anfractum, respectively, showed S. nutans–like pat- tral genomic blocks between the ACK and genomes of Australian terns; remaining duplicated GBs had the deviating arrangement. species, we attempted to reconstruct the fate of centromeres in Altogether, our data indicate that S. nutans, S. lineare, and Stenopetalum and Ballantinia species (Figure 1). Of the 15 active B. antipoda experienced a common WGD event, followed by re- centromeres in S. nutans, S. lineare, and B. antipoda, we could duction of chromosome numbers toward n = 6 to 4. We gained trace the putative origin of nine centromeres. Besides functional evidence that the common polyploid ancestor originated by a centromeres, we tentatively identified in total 12 positions of merger of two genomes resembling the ACK. The analysis of ancestral centromeres that were most likely lost via a series of S. velutinum and S. anfractum showed that n = 8 and n =10 pericentric inversions followed by unequal reciprocal translo- chromosome complements (containing four genomic copies of cations (white circles in Figures 1A to 1C). Furthermore, 18 an- GBs) are not ancestral but are derived through an additional, cestral GB associations containing an inactivated and/or lost more recent WGD. ancestral centromere have been discerned (black circles in Figures 1A to 1C). With the exception of the heterochromatic knob hkSN3 on the bottom arm of SN3, whose position corre- WGD Was Followed by Chromosome Fusions Accompanied sponds to the AK5 ancestral centromere (Figures 1A and 2C), by Genome Reshuffling and Loss or Inactivation GBs bordering the ancestral centromeres in the ACK were of Centromere adjacent to each other, without discernible heterochromatic domains resembling centromeres (Figures 1A to 1C, 2B, and 2C). Considering the low, quasidiploid chromosome numbers (n =4 to 6) of the analyzed Australian species, it is obvious that the assumed WGD event has been followed by species-specific Phylogenetic Analysis Corroborates the WGD in chromosome fusions. Chromosome number reduction was ac- Australian Crucifers companied by numerous intra- and intergenomic chromosome rearrangements reshuffling ancestral genomic blocks (Figures To elucidate their polyploid origin and phylogenetic relationship, 1 and 3; see Supplemental Table 2 online for details). In all three we sequenced S. nutans, S. velutinum, B. antipoda, and A. species, 60 to 64% of rearrangement breakpoints coincide with eremigena for three nuclear genes (chalcone synthase [CHS], centromeric and terminal chromosome regions, whereas 28 to malate synthase [MS], and cinnamyl alcohol dehydrogenase 5 33% of breakpoints occurred within genomic blocks splitting [CAD5]; GenBank accession numbers in Supplemental Table 5 them into two (a and b) or three sub-blocks (a, b, and c). In S. online) and four maternally inherited genes (chloroplast genes nutans, out of 48 GBs, 39 (81%) blocks remained intact, whereas rbcL, matK,andndhF and the mitochondrial nad4 intron 1;

Figure 1. (continued).

S. nutans (A), S. lineare (B), B. antipoda (C), and ACK (D) as a reference genome. The size of GBs A to X corresponds to the size of homoeologous blocks in Arabidopsis (http://www. Arabidopsis.org). Color coding of GBs A to X reﬂects their position on the eight ancestral chromosomes AK1 to AK8 of the ACK. Downward-pointing arrows denote the inverse orientation of GBs compared with their position in the ACK. Active centromeres are depicted as white double-triangle structures, and their presumed origins from AK chromosomes are given. White and black circles left of the chromosomes refer to ancestral centromeres presumably lost by a pericentric inversion followed by reciprocal translocation (white circles) or to centromere inactivation and/or loss (end-to-end fusion; black circles). rDNA loci shown as black ovals, heterochromatic knobs and segments shown as gray ovals and rectangles, respectively, and within the painted GBs in S. nutans.

98 Mesopolyploidy in Australian Crucifers 2281

GenBank accession numbers in Supplemental Table 6 online). These genes were analyzed along with representatives from the three major crucifer lineages, with a particular focus on Lineage I as some of the analyzed species were tentatively assigned to the tribe Camelineae (Al-Shehbaz et al., 2006; see Supplemental Text 1 online). The nuclear phylogenies (Figure 4; see Supplemental Figure 3 online) showed evidence of two different gene copies in the genome of the Australian species. The Camelineae gene copy (a1) is nested in the tribe Camelineae with affinities to the Boechereae and with close relationship with one genome copy of allopolyploid Pachycladon species endemic to New Zealand. The basal copy (a2) branches at the base of the Camelineae, close to Lepidieae, and with close affinities to the other genome copy of Pachycladon (see Supplemental Text 1 online for more details). The position of three paralogous copies in S. velutinum is congruent with the described pattern. Together, the presence of two or three gene copies in Australian species for most nuclear genes and their consistent phylogenetic position among trees suggest an allopolyploid origin of these species. The maternal phylogeny (see Supplemental Figure 4 online) agrees well with the nuclear gene trees and identifies the maternal parental genome as being closely related to New Zealand (Pachycladon) and Eurasian Camelineae taxa as well as to North American tribes Boechereae and Halimolobeae of Lineage I (the genome copy a1 in Figure 4). Based on CCP data, we expected to find two copies of each nuclear gene in S. nutans, B. antipoda, and A. eremigena and four copies in the neopolyploid S. velutinum. Concordant with these expectations, the nuclear phylogenies (Figure 4; see Supple- mental Figure 3 online) suggest that B. antipoda and A. eremigena possess two gene copies of CHS and CAD5 and one copy of MS, whereas S. nutans has two copies of CAD5 and one of CHS and MS. The presence of a single copy for some genes in the mesotetraploid species could imply a gene loss following the WGD, although it is not possible to rule out the possibility that these copies are present but that they were not sampled by the cloning procedure. In S. velutinum, three gene copies of CHS and CAD5 and one MS copy were identified. The presence of three gene copies for CHS and CAD5 is consistent with two WGD events. The presence of three gene copies instead of four could be explained by a loss of the fourth copy after the second WGD or

matic knob hkSN3, and inactive centromere on BA2 are shown. Genomic blocks of the four ancestral chromosomes are labeled, and the position of active/inactive ancestral centromere is indicated by arrowheads (see Figure 2. CCP in Stenopetalum and Ballantinia Species. Figures 1A and 1C for further details). (A) Examples of CCP in S. nutans. The eight ancestral chromosomes AK1 (D) Six heterochromatic segments in B. antipoda visible as condensed to AK8 represented by corresponding Arabidopsis BAC contigs revealed 4’,6-diamidino-2-phenylindole–stained structures at distal ends of homoeologous chromosome regions in Stenopetalum pachytene com- pachytene chromosomes. plements. (E) In situ localization of the Arabidopsis-like telomere repeat (green) on (B) Straightened pachytene S. nutans chromosome SN4 painted using meiotic (metaphase I, top) and mitotic chromosomes of B. antipoda. differentially labeled Arabidopsis BAC contigs corresponding to 14 Some heterochromatic segments indicated by arrowheads. The telo- complete or partial genomic blocks of the ACK (Figure 1A). mere repeat showed preferential hybridization to centromeres; minor (C) Ancestral centromeres in S. nutans and B. antipoda analyzed by CCP signals at chromosome termini are less prominent. Chromosomes and straightened. Active and inactive ancestral centromere on chromo- counterstained by 4’,6-diamidino-2-phenylindole. some SN2, inactive centromere on SN3 coinciding with the heterochro- Bars = 10 mm.

99 2282 The Plant Cell

one mesotetraploid with two copies and another with one copy could be in involved in the second WGD. Also, the fourth gene copy could be present but may not have been sampled. Overall, these observations corroborate the assumed mesotetraploid (S. nutans, B. antipoda, and A. eremigena) and neo-octoploid (S. velutinum) status of the analyzed species.

Hypothesis Testing and Divergence Time Estimates

We tested different evolutionary hypotheses regarding the possible number of the allopolyploidization event(s) at the origin of the Australian species using Bayes Factors. The analyses were performed with a particular emphasis on Pachycladon species (Camelineae; Al-Shehbaz et al., 2006), which were also shown to have an allopolyploid origin (Joly et al., 2009). The Bayes factor analyses could not reject the hypothesis that the Australian species and Pachycladon were formed from a single WDG event (see Supplemental Table 3 online). Although further data could eventually support more than one WDG event, we assumed a single origin when estimating the divergence times. The number of synonymous mutations in the nuclear genes suggests that the WGD at the origin of the Australian polyploids occurred ;5.9 mya (median range 3.7 to 8.7; see Supplemental Figure 5 online). This estimate is similar to that obtained by a relaxed clock phylogenetic analysis of the maternal data sets (8.5 mya, 95% credible interval: 4.7 to 12.3 mya; see Supple- mental Figure 4 online).

Karyotype Analysis of Purported Parental Species of Australian Mesopolyploids

Based on our phylogenetic hypotheses, comparative karyotypes of Crucihimalaya wallichii (n = 8) and Transberingia bursifolia (n = 8) were reconstructed (see Supplemental Figure 6 online). Both Camelineae genera were identiﬁed as harboring putative parental genomes, being closely associated with one paralogous gene copy of the Australian and Pachycladon species (Figure 4; see Supplemental Figures 3 and 4 online; Joly et al., 2009). CCP analysis showed that chromosome complements of both species are almost completely collinear with the ACK (Figure 1D). Both species differ from the ACK by a large pericentric inversion on the AK1 homoeolog and by another smaller pericentric inversion, speciﬁc for each species. Although neither Crucihi- malaya nor Transberingia species can be considered as direct

S. nutans (A), S. lineare (B),andB. antipoda (C). In each panel, ancestral and modern chromosomes are organizedcircularlyasideogramsanno- tated with their names and megabase scales. The relative scale for ACK and modern karyotype has been adjusted so that ACK ideograms occupy one-third of the figure. Ancestral chromosomes (AK1 to AK8) are further subdivided into 24 (A to X) GBs. Syntenic relationships between regions of ancestral and modern chromosomes are identified by connecting ribbons, whose colors correspond to the ancestral chromosomes. Labels inner to the modern chromosome ideograms correspond to the GBs identified in Figure 1. Outer labels identify Arabidopsis Figure 3. Collinear Relationships between ACK and Modern Karyotypes BAC clones that demarcate the boundaries of the GBs (see Supplemental of S. nutans, S. lineare,andB. antipoda, Based on the Cytogenetic Maps Table 1 online). Duplications within the modern karyotypes are shown by in Figure 1. thin black lines connecting the duplicated regions.

100 Mesopolyploidy in Australian Crucifers 2283

Figure 4. Phylogenies of the Nuclear Genes CAD5 (TrN + G) and CHS (GTR + G + I) Showing the Position of Sequences from the Australian Species (in Bold) in the Context of Other Brassicaceae Taxa. Clade posterior probabilities < 0.5 are not shown. “a1” and “a2” identify the two genome copies present in the genomes of Australian Brassicaceae species, whereas “b” indicates the most recent common ancestor for the two genome copies. Species within the crucifer Lineage I with karyotype resembling the ACK or karyotype derived from the ACK (in parenthesis with the extant chromosome number) are mapped onto the phylogenies (Lysak et al., 2006; Schranz et al., 2007; Roosens et al., 2008; see Supplemental Figure 6 online for karyotypes of T. bursifolia and of C. wallichii as a representative of the genus Crucihimalaya). Tribal classiﬁcation follows Al-Shehbaz et al. (2006).

ancestors of the Australian crucifers based on the CCP data, compelling evidence that the Australian species, despite being these results further corroborate the ACK as a presumable genetically diploid and having low chromosome numbers (n =4, ancestral genome of all Camelineae species. 5, and 6) experienced a WGD event postdating the three (a, b, and g) paleopolyploid WGDs in the ancestry of Brassicaceae (Bowers et al., 2003; Barker et al., 2009). We showed that DISCUSSION multiple paleopolyploid duplications can be followed by evolutionary younger WGD event(s) masked due to massive genome Mesopolyploid WGD repatterning and descending dysploidy. As shown for the Aus- tralian cruciferous species, even annual, self-compatible, bona CCP analysis with genomic blocks of the Ancestral Crucifer ﬁde diploid species with low chromosome number (n # 6) might Karyotype (n = 8) as probes revealed that all (S. nutans) or most have experienced several rounds of hidden polyploidy. Hence, (B. antipoda and S. lineare) blocks exist as two paralogous diploid-like chromosome number cannot be taken as a reliable copies in genomes of the three Australian crucifers. This is indicator of true genome diploidy if, for example, the polyploid

101 2284 The Plant Cell

incidence is appraised (e.g., Wood et al., 2009). Conversely, mesopolyploids are nested within the paraphyletic tribe Camel- polyploid-like increases of chromosome number can be caused ineae and the closely related Boechereae. It was shown that by serial centric fissions as reported for some orchids (Leitch karyotypes of all so far analyzed taxa from three tribes of Lineage et al., 2009) and cycads (Caputo et al., 1996). I (Camelineae, Boechereae, and Descurainieae) resemble or Considering the age of WGDs, polyploid species are tradition- were derived from the ACK (Figure 4; Lysak et al., 2006 and ally classified as paleopolyploid and neopolyploid. Recent poly- references therein; Schranz et al., 2007; Roosens et al., 2008). ploids with the increased genome size, chromosome number, Here, we showed that species of two other Camelineae genera gene copy number, and extant diploid ancestors are described as (Crucihimalaya and Transberingia), considered as potential pa- neopolyploids. As paleopolyploids were traditionally described, rental genomes of the Australian species, descended from the polyploid relics with high polyploid chromosome numbers and ACK. The identification of the ACK as an ancestral karyotype of extinct diploid ancestors (Guerra, 2008). This classification has the closest relatives of the Australian Camelineae species further been refined by Favarger (1961) who in addition to paleopolyploids corroborate our conclusion that these allopolyploids originated and neopolyploids recognized also mesopolyploid species of an from hybridization between taxa with ACK-derived comple- intermediate age and with diploid ancestors in the same or closely ments. Although a recurrent origin of allopolyploid species was related genus. Within the last decade, the definition of paleopoly- reported for Arabidopsis kamchatica (Shimizu-Inatsugi et al., ploidy has changed and the term is currently used for genetically 2009), Helianthus (Schwarzbach and Rieseberg, 2002), Trago- diploid species that experienced one or more rounds of ancient pogon (Lim et al., 2008), Persicaria (Kim et al., 2008), and other WGD and their polyploid past is becoming apparent only after a genera, we are unable to find clear evidence for recurrent careful sequence analysis (e.g., Jaillon et al., 2009; Soltis et al., polyploid formation from our data. On the contrary, two para- 2009). To differentiate between these differently aged WGD centric inversions on one of the AK8-like homoeologs shared by events, we resurrect and modify the polyploidy classification of five analyzed species (see Supplemental Figure 2 online) as well Favarger (1961). Figure 5 displays a model of multiple waves of as the estimated large evolutionary distance between the pa- WGD followed by genome diploidization and their impact on the rental genomes (;4 million years old at the time of the allo- extant genome structure detected through comparative genetic polyploidization; see Supplemental Figure 5 online) argue in favor mapping and cytogenetic techniques (CCP in particular). Meso- of a single origin. polyploid species exhibit diploid-like meiosis, disomic inheri- The fact that any two paralogous copies of GBs differed tance, and diploidized genomes up to quasidiploid complements consistently in size (and fluorescence intensity) could imply that with a very low number of chromosomes; however, the parental the WGD was an allopolyploidization event. Alternatively, the subgenomes are still discernible by comparative (cyto)genetic deviating size and fluorescence intensity of the two subgenomes and phylogenetic methods. In paleopolyploids, the long-term could be caused by a preferential fractionation (i.e., differentia- amalgamation of parental genomes is hampering their identi- tion) of paralogous sequences belonging to only one subge- fication by these methods, and ancient paleopolyploid WGD nome. Biased fractionation has been observed in Arabidopsis events can only be uncovered by comparsion of orthologous after the last (a) WGD event, targeting preferentially only one of sequences (Figure 5). the two paralogs and retaining mainly dose-sensitive regulatory The unexpectedly detected WGD shared by the diploid (connected) genes (Thomas et al., 2006). Such patterns support Australian crucifer species is analogous to the mesopolyploid the gene balance hypothesis (e.g., Birchler and Veitia, 2007; whole-genome triplication discerned in the tribe Brassiceae by Freeling, 2008; Veitia et al., 2008). As the paralogous gene copies comparative genetic mapping (Lagercrantz and Lydiate, 1996; cluster with two distinct species groups within the nuclear gene Parkin et al., 2005) and by comparative cytogenetic analysis phylogenies, we infer an allopolyploid origin of the Australian (Lysak et al., 2005, 2007; Ziolkowski et al., 2006). Several lineage- species. The fact that the other sequenced Brassicaceae spe- specific WGD events thus far documented across the angio- cies did not exhibit multiple gene copies makes the hypotheses sperms (Tang et al., 2008; Jaillon et al., 2009; Soltis et al., 2009) that this pattern is caused by gene duplication or lineage sorting represent only the tip of the iceberg, as potential other WGD untenable. Analysis of maternally inherited genes showed that events remain uncovered. We envisage that more mesopolyploid the Camelineae copy (a1) has been contributed by the maternal WGDs will be revealed in Brassicaceae and other families, such parent, whereas the basal copy (a2) is derived from the paternal as Asteraceae, where differently aged polyploidization events ancestor. (Barker et al., 2008) and descending dysploidy have a prominent role in the genome evolution. Evolutionary Scenario of the Mesopolyploid WGD Event

A Common Allopolyploid Ancestor? As the structure of all analyzed karyotypes is complex, only a tentative scenario of this polyploid event can be proposed. The Associations of genomic blocks corresponding to seven, ﬁve, simplest model assumes a merger of two ACK-like genomes ;6 and six AK-like chromosomes and to 11, 15, and 72 AK-like to 9 mya followed by a reduction from n = 16 toward n = 6 to 4. whole-arms preserved in the karyotypes of S. nutans, S. lineare, However, the two parental genomes while having the ACK-like and B. antipoda, respectively, strongly suggest the ACK (n =8)as structure could already have deviated by chromosome number. an ancestral genome of the analyzed species. Phylogenetic Chromosome number reductions from n =8ton = 7 to 4 occurred reconstructions showed that both ACK-like parental genomes repeatedly in Brassicaceae (Lysak et al., 2006; Schranz et al., (i.e., paralogous gene copies a1 and a2) of the Australian 2007; Manda´ kova´ and Lysak, 2008; this study).

102 Mesopolyploidy in Australian Crucifers 2285

Figure 5. A Model of Genome Evolution through Multiple WGD Events Followed by Diploidization.

Subsequent WGD events of different age shown as ploidy level increases (4x to 32x) are followed by genome diploidization associated with descending dysploidy toward quasidiploid chromosome complements. Diploidization expected to occur in neopolyploids is shown by dashed lines. Chromosomes on the right represent duplicated and diploidized complements with three ancestral GBs as revealed by comparative genetic and cytogenetic techniques. From the top to bottom: Paleopolypoidy is represented by two WGD events, paralogous regions not detectable by (cyto)genetic analysis (example: A. thaliana, n = 5). Neopolyploidy: two paleopolyploid WGDs followed by a recent neopolyploid event; chromosome number unreduced and most duplicated GBs not yet reshufﬂed (example: A. suecica, n = 13). Mesopolyploidy: a WGD event following two paleopolyploid WGDs. Descending dysploidy results in a quasidiploid number of fusion chromosomes; duplicated GBs reshufﬂed by intra- and intergenomic rearrangements (example: S. lineare, n = 5). Paleo-, meso-, and neopolyploid WGD events resulting in a duplicated mesopolyploid genome (example: S. anfractum, n =10).Possible rearrangements of GBs after the last (neopolyploid) WGD not shown.

As the WGD was detected only in the endemic Australian support the position of the maternal parent (the Brassica gene genera, we believe that it could have taken place in Australia after copy) close to the split of Lineage I and II (Joly et al., 2009). a long-distance dispersal of the parental genomes from Eurasia Although it is not possible to reject the null hypothesis of a single or North America. Alternatively, the mesopolyploid event could allopolyploid origin for Pachycladon and the Australian en- have occurred in Eurasia/North America and the allopolyploid demics, less diploidized (n = 10) Pachycladon species appear alone migrated to Australia. Several cases of intercontinental to originate and radiate on the South Island of New Zealand more long-distance dispersals were documented (Mummenhoff and recently (1.6 to 0.8 mya; Joly et al., 2009). Further studies of Franzke, 2007), including the crucifer genera Cardamine (Carlsen genome evolution within the genus Pachyladon are needed to et al., 2009) and Lepidium (Dierschke et al., 2009). A single or elucidate its origin and relationship to endemic Australian cruci- multiple polyploidization event(s) was followed by species radi- fer species. ation and reduction of chromosome number. More recently, reshuffled mesopolyploid genomes were involved in yet another Species-Specific Dysploidy Following the WGD round of auto- or allopolyploidy, as exemplified by four ACK- derived genomes identified in the neopolyploid species S. Multiple WGDs and subsequent chromosome number reduction anfractum (n = 10) and S. velutinum (n = 8). resulting in a mosaic of parental genomic blocks are well Present phylogenetic analyses uncovered the close relation- documented in grasses (Salse et al., 2008; Luo et al., 2009) ship between the Australian mesopolyploids and New Zealand and in the allopolyploid B. napus (Lysak et al., 2005; Parkin et al., Pachycladon allopolyploids (Figure 4). Our data strongly advo- 2005; Schranz et al., 2006). cate Pachycladon as a member of the paraphyletic tribe Camel- A full reconstruction of modes underlying chromosome num- ineae as proposed by Al-Shehbaz et al. (2006) and do not ber reduction in the three analyzed species is not yet feasible.

103 2286 The Plant Cell

The high numbers of evolutionary conserved chromosome arms 2009), and potentially also chromosomes of two cucurbit spe- (Figures 7A to 7C; see Supplemental Table 2 online) suggest that cies (Y. Han et al., 2009). Except for the heterochromatic knob rearrangement breakpoints were predominantly located in cen- on chromosome SN3 corresponding to the AK5 centromere, tromeric and terminal regions and that whole-arm rearrange- no heterochromatin was observed at sites of the other 17 pre- ments played a prevalent role in the reduction of chromosome sumably inactivated centromeres. Illegitimate recombination number. We attempted to get a deeper insight into karyotype between (peri)centromeric repeats is supposed to gradually reshuffling by scoring ancestral (AK-like) associations of ge- remove the repeats and heterochromatin from these regions nomic blocks and their orientation compared with the ACK (Ventura et al., 2004). Similarly, centromere inactivation accom- (ancestral versus inverted) and position of centromeres in the panied by the loss of heterochromatin has been recently envis- context of GB associations. AK-like association of GBs with one aged for cucumber (Cucumis sativus) chromosome 6 (Y. Han arm in inverted orientation compared with the ACK but without an et al., 2009). The knob hkSN3 might represent a recently inac- active centromere was interpreted as the result of chromosome tivated ancestral centromere with discernible heterochromatin fusion through a pericentric inversion followed by reciprocal trans- still present. location involving terminal breakpoints and the loss of one of the two resulting products (the minichromosome). This mechanism Diploidization: Retention versus Loss of Genomic Blocks has been described for several other crucifer species (Lysak et al., 2006; Schranz et al., 2006; Schubert, 2007; Manda´ kova´ As diploidization (fractionation; Thomas et al., 2006) are de- and Lysak, 2008). This type of rearrangement accounts for 72 scribed processes gradually transforming a polyploid into a chromosome fusion and centromere loss events in all three quasidiploid genome. In the analyzed mesopolyploid species, karyotypes. We speculate that the B. antipoda karyotype, con- diploidization could be documented at the chromosomal level taining two NOR-bearing telocentric chromosomes known only in and at the gene level. As all reconstructed chromosomes are Neslia paniculata (n = 7) (Lysak et al., 2006), might represent complex mosaics of ancestral genomic blocks, we assume that an evolutionary transient karyotype prone to further dysploidy the WGD was followed by lineage(species)-specific reductions through translocation-mediated fusions involving the telocentrics. of chromosome number accompanied by the extensive restruc- Seven, six, and five ancestral centromere positions were turing and, to a lesser extent, by the loss of duplicated GBs. Both identified in karyotypes of S. nutans, S. lineare, and B. antipoda, inter- and intragenomic translocations between nonhomoeolo- respectively, within entirely or partly preserved ancestral chro- gous chromosomes prevail in the three diploidized species. mosomes (Figures 7A to 7C). It cannot be ruled out that these Similar large-scale chromosome reshuffling was reported, for block associations were generated by the same type of rear- example, in natural and synthetic polyploid B. napus (Parkin rangement as described above but involving another inversion et al., 2005; Gaeta et al., 2007), Tragopogon allopolyploids (Lim (Lysak et al., 2006; Schubert, 2007). The frequent occurrence of et al., 2008), and in fertile Festulolium hybrids (Kopecky´ et al., this relatively complicated rearrangement in all three karyotypes 2008). is possible since the clustering of breakpoints around centro- Considering the different chromosome numbers (n =4,5,and meres and chromosome termini indicates a preferential involve- 6), the most severe chromosome reshuffling and loss of genomic ment of repetitive sequences in erroneous repair of double blocks are to be expected in S. nutans (n = 4), followed by S. strand breaks. Alternatively, the occurrence of entire ancestral lineare (n = 5) and B. antipoda (n = 6). However, all 24 duplicated chromosomes within the fusion chromosomes might result from ancestral GBs and seven conserved AK chromosomes were tandem end-to-end translocation, loss of (sub)telomeric repeat surprisingly found in the most reduced karyotype of S. nutans.In tracts at the breakpoints, and centromere inactivation/loss on S. lineare, five AK chromosomes are preserved and two GBs lost one of the chromosomes. A similar telomere-to-telomere trans- (1.3 and 2.6 Mb in the Arabidopsis genome), and in Ballantinia, six location has been assumed for an origin of the large metacentric AK chromosomes are conserved and four GBs were completely chromosome of the ant Myrmecia pilosula (n = 1; Imai and Taylor, (1.3, 2.3, 2.4, and 6.2 Mb) or partly (0.9 Mb) lost. In all three 1989) and of the human chromosome 2 (Ijdo et al., 1991b). In species, the high level of retained duplicated blocks (100 to 83%) Brassicaceae, end-to-end chromosome fusions were not de- contrasts with the reduction in chromosome number. Though scribed previously (Lysak et al., 2006; Schranz et al., 2006; we do not have a plausible explanation for these findings, the Schubert, 2007; Manda´ kova´ and Lysak, 2008). different degree of repatterning argues for independent ways Tandem chromosome fusions produce dicentric chromo- and different speed of descending dysploidy (diploidization), somes that have to be stabilized by inactivation of one centro- even within a well-defined genus. Future analysis of more en- mere. The centromere inactivation will ensure regular meiotic demic Australian species should reveal further patterns of chro- segregation of the fusion chromosome and can result in evolu- mosome number reduction. tionary fixed reduction of chromosome number. Several cases of Comparing the level of genome fractionation found in the three inactivated ancestral centromeres as well as the emergence species with another group of polyploids is problematic as the of neocentromeres (centromere repositioning) were reported age and character of genome duplication (duplication versus in mammalian species (e.g., Ferreri et al., 2005; Ventura et al., triplication), phylogenetic positions, and chromosome numbers 2007), but only a few examples of centromere inactivation (and have to be considered. The 6- to 9-million-year-old WGD char- reactivation; F. Han et al., 2009) in plants, including dicentric acterizing the Australian Camelineae species can be compared chromosomes of Trititiceae (Sears and Camara, 1952; Luo et al., with the 8- to 15-million-year-old whole-genome triplication in 2009), maize (Zea mays) B chromosomes (F. Han et al., 2006, the ancestor of the tribe Brassiceae (Lysak et al., 2005). Although

104 Mesopolyploidy in Australian Crucifers 2287

both mesopolyploid events are presumably of a comparable age, Prior to fluorescence in situ hybridization, ready-to-use slides were the level of genome redundancy should be different in the treated with pepsin (0.1 mg/mL; Sigma-Aldrich) in 0.01 M HCl for 3 to duplicated Camelineae versus triplicated Brassiceae genomes. 6 h, postfixed in 4% formaldehyde in 23 SSC (13 SSC: 0.15 M NaCl and Hence, assuming similar mechanisms and rate of chromosome 0.015 M sodium citrate) for 10 min, and dehydrated in an ethanol series (70, 80, and 96%). evolution in both groups, more extensive genome reshuffling is to be expected in Brassiceae. Triplication of 24 ancestral GBs Fluorescence in Situ Hybridization and CCP theoretically resulted in 72 GBs comprising quasidiploid Brassi- ceae genomes. Out of the 24 GBs, only 13 (54%) (Parkin et al., For localization of 5S and 45S rDNA loci, clone pCT 4.2 corresponding to 500-bp 5S rRNA repeat (M65137) and Arabidopsis thaliana BAC clone 2005; Schranz et al., 2006) and 14 (58%) (Panjabi et al., 2008) T15P10 (AF167571) were used, respectively. Telomeric probe was pre- blocks were found as three or more copies in the rapa (A) genome pared according to Ijdo et al. (1991a). A total of 547 Arabidopsis BAC of B. napus or B. juncea. In the nigra (B) and oleracea (C) clones were assembled to represent genomic blocks of the ACK (Schranz genomes, only seven (29%) (Panjabi et al., 2008) and eight (33%) et al., 2006). To further characterize the orientation and internal structure (Kaczmarek et al., 2009) GBs were reported as three or more of particular genomic blocks, the respective BAC contigs were arbitrarily homoeologous copies in B. juncea and B. oleracea, respectively. broken into subcontigs and differentially labeled. Supplemental Table Comparing these data with 83 to 100% of duplicated GBs 1 online shows the structure and position of ancestral genomic blocks in retained in the Australian species, triplicated Brassiceae ge- the karyotypes of Stenopetalum nutans, Stenopetalum lineare,and nomes show faster diploidization and/or originated earlier (13 to Ballantinia antipoda as well as corresponding Arabidopsis BAC contigs. 17 mya suggested by Yang et al., 2006). A loss of paralogous DNA probes were labeled with biotin-dUTP, Cy3-dUTP, digoxigenin- dUTP, and with diethylaminocoumarin (DEAC)-dUTP and Alexa Fluor gene copies was documented herein for all three single-copy 488-dUTP (S. nutans chromosome SN4; Figure 2B) by nick translation nuclear genes and all analyzed species. Extensive gene loss was and ethanol precipitated (Manda´ kova´ and Lysak, 2008). The hybridiza- also found in the mesopolyploid genomes of B. rapa (Yang et al., tion probes and chromosomes were denatured together on a hot plate 2006) and B. oleracea (Town et al., 2006). at 808C for 2 min and incubated in a moist chamber at 378C overnight Since the discovery of extensive segmental duplications in the or for 48 h. Arabidopsis genome (Arabidopsis Genome Initiative, 2000), mul- Posthybridization washing was performed in 50% formamide (rDNA and tiple paleo- and mesopolyploid WGD events have been revealed telomere probes) or 20% formamide (BAC contigs) in 23 SSC at 428C. across angiosperms (e.g., Soltis et al., 2009). Our data suggest Biotin-dUTP–labeled probes were detected by avidin;Texas Red (Vector ; that ancient paleopolyploid genomes have often undergone more Laboratories), goat anti-avidin biotin (Vector Laboratories), and avi- ; recent, mesopolyploid WGDs, which are discernible by genetic, din Texas Red; digoxigenin-dUTP was detected by mouse antidigoxigenin (Jackson Immunoresearch) and goat anti-mouse;Alexa Fluor 488 molecular phylogenetic, and cytogenetic methods. (Molecular Probes). Chromosome SN4 was painted using Arabidopsis BAC This CCP analysis showed that the diploid-like genomes (n =4 clones labeled with Cy3-dUTP, DEAC-dUTP, and Alexa Fluor 488-dUTP, to 6) of Australian Camelineae species experienced a meso- and biotin-dUTP detected by avidin;Texas Red and digoxigenin-dUTP polyploid WGD blurred by fast, species-specific, and extensive detected by mouse antidigoxigenin and goat anti-mouse conjugated with chromosome repatterning. Chromosome numbers become Alexa Fluor 647. Chromosomes were counterstained with 4’,6-diamidino- reduced and the polyploid character of the corresponding 2-phenylindole (2 mg/mL) in Vectashield (Vector Laboratories). genomes masked through inversions followed by reciprocal Fluorescence signals were analyzed with an Olympus BX-61 epifluo- translocations with breakpoints in pericentric and terminal repeat- rescence microscope and AxioCam CCD camera (Zeiss). The monochro- rich regions and by reciprocal translocations within terminal matic images were pseudocolored and merged using Adobe Photoshop repeat arrays with a subsequent centromere inactivation/loss. CS2 software (Adobe Systems). Pachytene chromosomes were straightened using the “straighten-curved-objects” plugin in the Image J soft- Present results lay a foundation for detailed whole-genome ware (Kocsis et al., 1991). Circular visualization of ancestral genomic sequence analyses of the ongoing diploidization processes in blocks in the ACK and genomes of the Australian crucifers (Figure 3; see this crucifer group. Supplemental Figure 1 online) was prepared using Circos (Krzywinski et al., 2009). Comparison of the position for each genomic block and each pair of the analyzed species (see Supplemental Figure 1 online) was METHODS created by a customized version of Circos, provided by M.K.

Plant Material and Chromosome Preparation Phylogeny Reconstruction

For the origin of the analyzed species accessions, see Supplemental We sequenced Arabidella eremigena, B. antipoda, S. nutans,and Table 4 online. Herbarium vouchers were deposited in the herbarium of Stenopetalum velutinum for three nuclear and four maternal genes, the Masaryk University. Inflorescences of the analyzed accessions were respectively. Because of the limited supply of material, we were unable fixed in ethanol:acetic acid (3:1) overnight and stored in 70% ethanol at to include three species (partly) analyzed by CCP (Blennodia canescens, 2208C. Selected inflorescences were rinsed in distilled water and in Stenopetalum anfractum, and S. lineare). DNA extractions, PCR amp- citrate buffer (10 mM sodium citrate, pH 4.8; 2 3 5 min) and incubated in lifications, cloning, and sequencing followed standard procedures an enzyme mix (0.3% cellulase, cytohelicase, and pectolyase; all Sigma- (Franzke et al., 2009; Joly et al., 2009). Clone sequences that had Aldrich) in citrate buffer at 378C for 3 to 6 h. Individual flower buds were >99% similarity with each others were assumed to come from a single disintegrated on a microscope slide in a drop of citrate buffer and 15 to 30 gene copy, and only one representative clone was retained for the mL of 60% acetic acid. The suspension was spread on a hot plate at 508C analyses (the sequence showing the fewer autapomorphies). By doing so, for 0.5 to 2 min. Chromosomes were fixed by adding 100 mL of ethanol: we hoped to minimize the impact of allelic variation and PCR-induced acetic acid (3:1) fixative. The slide was dried with a hair dryer, postfixed in mutations on the results. Nuclear introns were removed because they 4% formaldehyde dissolved in distilled water for 10 min, and air-dried. were too divergent across the Brassicaceae. Data sets were aligned with

105 2288 The Plant Cell

MUSCLE (Edgar, 2004), and substitution models were selected with the Supplemental Figure 4. Maternal Phylogeny (Chronogram) of Aus- AIC in Modeltest 3.7 (Posada and Crandall, 1998) on a GTR+G+I phyml tralian Genera Stenopetalum, Ballantinia,andArabidella in the Con- tree (see Supplemental Data Sets 1, 2, and 3 online). Phylogenies were text of Other Brassicaceae Taxa Resulting from the Partitioned estimated in BEAST 1.4.8 (Drummond and Rambaut, 2007) using the best Analysis of the Genes rbcL (K81uf + G + I), nad4 (TVMef + G + I), ﬁt substitution model, a lognormal relaxed molecular clock, and a birth matK (TVM + G + I), and ndhF (TVM + G + I) in BEAST. and death model. The root of the phylogeny was estimated using a Supplemental Figure 5. Comparison of the Estimated Time of relaxed molecular clock model as part of the phylogenetic analysis. For Original Divergence Between the Parents That Were Involved in the the maternal data set, a partitioned analysis was performed where each Allopolyploid Event (A) and the Estimated Time of the Event (B) Based gene had its own substitution model. Two independent MCMC chains on the Analysis of Three Nuclear Genes. were run for108 generations (2 3 108 for CHS), sampling trees every 5000 generations and discarding 10% of the samples as burnin for the Bayes Supplemental Figure 6. Reconstructed Karyotypes of Transberingia factor analyses and for building the maximum sum of clade credibility tree bursifolia (n = 8) and Crucihimalaya wallichii (n =8). (the chains always reached their stationary phase well before this point). Supplemental Table 1. Ancestral Genomic Blocks (GB) of the For the CHS and the maternal data set, a weight of 50 was given to the Ancestral Crucifer Karyotype (ACK) Identiﬁed on Chromosomes of operators internalNodeHeights, subtreeSlide, and narrowExchange and Stenopetalum nutans (chromosomes SN1-SN4), S. lineare (SL1-SL5), 40 for wideExchange and wilsonBalding. Convergence was always and Ballantinia antipoda (BA1-BA6); Details on Corresponding BAC reached for all parameters among independent runs and estimated Contigs of Arabidopsis thaliana Are Given. sample sizes were always >>100. Evolutionary hypotheses were tested Supplemental Table 2. Structure and Position of the Eight Ancestral using Bayes Factors (following 8) by performing BEAST analyses with Chromosomes (AK1 to AK8) and 24 Genomic Blocks (GBs) of the appropriate monophyly constraints. Uncertainty around the log likelihood Ancestral Crucifer Karyotype (n = 8) within Duplicated Genomes of harmonic means was assessed with 1000 bootstrap replicates. Stenopetalum and Ballantinia Species.

Divergence Time Estimates Supplemental Table 3. Bayes Factor (BF) Scores for Different Evolutionary Hypotheses Regarding the Number of Independent For the nuclear genes, minimal age estimates for the WGD was approx- Polyploid Events at the Origin of the Australian Species Relative to imated by estimating the age of the most recent common ancestor the Null Hypothesis of Four Independent Origins. (MRCA) of all Australian species and Pachycladon for each gene paralog. Supplemental Table 4. Collection Data of Australian Crucifer Species This was done by comparing the divergence between sequences that Used in the Present Study. branched back to these MRCA. We assumed a molecular clock with a mutation rate of 1.5 3 1028 synonymous (syn.) substitutions per syn. site Supplemental Table 5. GenBank Accession Numbers for Sequences

(dS) per year (Koch et al., 2000). dS was estimated following Goldman and Used in the Phylogenetic Analyses of the Nuclear Genes CHS, CAD5, Yang (1994) in paml (Yang, 1997). Divergence times (T) were obtained and MS. given that syn. substitution rate = dS/2T. Supplemental Table 6. GenBank Accession Numbers Lineage and Because syn. substitution rates for chloroplast genes are not well Tribal Assignments for Taxa Included in the Maternal Phylogenetic documented in Brassicaceae, divergence times were estimated using soft Analysis. calibration points within the BEAST phylogenetic analysis (see Supplemen- tal Figure 4 online). Three calibration points were used: (1) a normal prior of Supplemental Data Set 1. Text File of the CAD5 Alignment (Figure 4). mean 89.5 and SD of 1 was given to the crown Brassicales group to reflect a Supplemental Data Set 2. Text File of the CHS Alignment (Figure 4). Turonian Dressiantha fossil (»89.5 mya; Gandolfo et al., 1998); (2) a normal Supplemental Data Set 3. Text File of the MS Alignment (Supple- prior with a mean 35 and SD 6 was enforced to the crown Brassicaceae mental Figure 3). node to reflect that the oldest reliable Brassicaceae fossil occurs in Oli- gocene deposits (22 to 34 mya; Cronquist, 1981); (3) a normal prior of mean Supplemental Data Set 4. Text File of Alignments of Chloroplast 6andSD 2 was given to the MRCA of Rorippa and its closest relative Genes rbcL, matK,andndhF and the Mitochondrial nad4 Intron to reflect Rorippa fossils in Pliocene deposits (2 to 5 mya; Mai, 1995). 1 (Supplemental Figure 4). Supplemental Text 1. Phylogenetic Relationships and Comparison Accession Numbers with Previous Studies. Sequence data from this article can be found in the GenBank data libraries under accession numbers GQ926501 to GQ926568 (see Sup- plemental Tables 5 and 6 online). ACKNOWLEDGMENTS

Supplemental Data We acknowledge Millenium Seed Bank Project (Royal Botanic Gardens, Kew) and Janet Terry for providing seeds of Australian species. We The following materials are available in the online version of this article. thank Neville Scarlett for providing remaining seeds, Petr Buresˇ for Supplemental Figure 1. A Three-Way Comparison of the Relative genome size estimates, Ulrike Coja for technical assistance, and Ingo Position of Corresponding Synteny Blocks of Stenopetalum nutans Schubert for valuable comments on the manuscript. This work was (SN), S. lineare (SL), and Ballantinia antipoda (BA) Relative to the supported by research grants from the Grant Agency of the Czech Reference Ancestral Crucifer Karyotype (ACK). Academy of Science (IAA601630902) and the Czech Ministry of Educa- tion (MSM0021622415) and by a Humboldt Fellowship awarded to Supplemental Figure 2. The Unique Rearrangement of the AK8(#1)- M.A.L. Like Homoeolog Shared by All Analyzed Species. Supplemental Figure 3. Phylogeny of the Malate Synthase (MS) (TrN + G + I) Showing the Position of Sequences from the Australian Received February 6, 2010; revised June 9, 2010; accepted June 22, Species (in Bold) in the Context of Other Brassicaceae Taxa. 2010; published July 16, 2010.

106 Mesopolyploidy in Australian Crucifers 2289

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108 VI. Mandáková T., Mummenhoff K., Al-Shehbaz I.A., Mucina L., Mühlhausen A., Lysak M.A. 2012. Whole-genome triplication and species radiation in the southern African tribe Heliophileae (Brassicaceae). Taxon 64: 989-1000.

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Whole-genome triplication and species radiation in the southern African tribe Heliophileae (Brassicaceae)

Terezie Mandáková,1 Klaus Mummenhoff,2,5 Ihsan A. Al-Shehbaz,3 Ladislav Mucina,4 Andreas Mühlhausen2 & Martin A. Lysak1,5

1 RG Plant Cytogenomics, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic 2 Osnabrück University, Biology Department, Botany, Barbarastrasse 11, 49076 Osnabrück, Germany 3 Missouri Botanical Garden, St. Louis, Missouri 63166-0299, U.S.A. 4 Curtin Institute for Biodiversity & Climate, Department of Environment & Agriculture, Curtin University, GPO Box U1987, Perth, 6845, Australia 5 contributed equally to this work Authors for correspondence: Martin A. Lysak, [email protected] Klaus Mummenhoff, [email protected]

Abstract The unigeneric tribe Heliophileae includes ca. 90 Heliophila species, all endemic to southern Africa. The tribe is morphologically the most diverse Brassicaceae lineage in every aspect of habit, foliage, flower and fruit morphology. Despite this diversity, virtually nothing is known about its origin and genome evolution. Here we present the first in-depth information on chromosome numbers, rDNA in situ localization, genome structure, and phylogenetic relationship within Heliophileae. Chromosome numbers determined in 27 Heliophila species range from 2n = 16 to 2n = ca. 88, but 2n = 20 and 22 prevail in 77% of the examined species. Chromosome-number variation largely follows three major lineages (A, B, and C) resolved in the ITS phylogeny. Clade A species mostly have a chromosome number of 2n = 20, whereas 2n = 22 is the dominant number in clade C (2n = 16 and 22 were counted in two diploid species of clade B). The number and position of 5S and 45S rDNA loci vary between species and cannot be employed as phylogenetically informative characters. Seven species with different chromosome number and from the three ITS clades were analyzed by comparative chromosome painting. In all species analyzed, 90% of painting probes unveiled three homeologous chromosome regions in Heliophila haploid chromosome complements. These results suggest that all Heliophila species, and probably the entire tribe Heliophileae, experienced a whole-genome triplication (WGT) event. We hypothesize that the mesohexaploid ancestor arose through hybridization between genomes resembling the Ancestral Crucifer Karyotype with n = 8. The WGT has been followed by species-specific chromosome rearrangements (diploidization) resulting in descending dysploidy towards extant quasi-diploid genomes. More recent neopolyploidization events are reflected by higher chromosome numbers (2n = 32–88). The WGT might have contributed to diversification and species radiation in Heliophileae. To our knowledge, this is the first study to document polyploidy as a potential major mechanism for the radiation of a Cape plant lineage.

Keywords Cape flora; chromosome painting; comparative phylogenomics; Heliophila; ITS; karyotype evolution; phylogenetics; polyploidy; rDNA; whole-genome duplication

Supplementary Material Figure S1 and Table S1 (both in the Electronic Supplement) and the alignment are available in the Supplementary Data section of the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

INTRODUCTION foliage (entire to variously dissected), floral size (1.2–25 mm) and color, petal and stamen appendages, inflorescence position There are multiple reasons why the unigeneric tribe (terminal vs. intercalary; Marais 1970), flower number (few to Heliophileae DC. (hereafter alternatively referred to under numerous), ovule number (2–80), fruit size (2–120 mm long), its generic type Heliophila L.) is a truly remarkable group in shape and type of fruit (silique, silicle, samara, schizocarp), the context of the entire Brassicaceae (Cruciferae). Heliophila fruit flattening (terete, latiseptate, angustiseptate), and dehis- (> 90 species), the 9th-largest genus in Brassicaceae, is also one cence (Mummenhoff & al., 2005). of the most complex genera from a taxonomic point of view, as Heliophila is mainly native to South Africa; the distribu- suggested by more than 400 scientific names listed in the Inter- tion ranges of three species extend into Lesotho, seven into national Plant List Index (http://ipni.org), as well as 15 generic Namibia, and one into Swaziland. The center of the greatest names currently placed in its synonymy (Al-Shehbaz, 2012). diversity is the Cape Floristic Region (CFR) which has 87 spe- Heliophila is morphologically by far the most diverse genus in cies (77 endemic), and only three species (H. formosa Hilliard the family, especially in habit (tiny herbs to shrubs or lianas), & Burtt, H. obibensis Marais, H. scandens Harv.) are endemic

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to KwaZulu-Natal Province (compiled from Marais, 1970). One Systems), and counted. Multiple chromosome figures were species (H. pusilla L. f.) is naturalized in Australia. analyzed per species. These chromosome spreads were probed Despite two molecular phylogenetic studies (Mummen- with 5S and 45S rDNA probes to identify rRNA gene loci. hoff & al., 2005; Verboom & al., 2009) and recent taxonomic FISH: rDNA localization and comparative chromosome accounts (Marais, 1970; Al-Shehbaz & Mummenhoff, 2005), painting (CCP). — The Arabidopsis thaliana (L.) Heynh. BAC Heliophila remains under-explored in many respects, includ- clone T15P10 (AF167571) containing 45S rRNA genes was ing genome evolution, pollination and reproductive biology, used for fluorescence in situ localization of nucleolar organiz- dispersal ecology, character development and evolution, rapid ing regions (45S rDNA). Arabidopsis thaliana clone pCT4.2 radiation and speciation, phylogeography, etc. Chromosome (M65137), corresponding to a 500-bp 5S rRNA repeat, was counts have been reported for only four unvouchered of the used to identify 5S rDNA loci. For CCP, we used A. thaliana 90 species (Heliophila africana (L.) Marais, H. amplexicau- chromosome-specific BAC contigs corresponding to ances- lis L. f., H. crithmifolia Willd., and H. linearis DC.; Jaretzky, tral genomic blocks on chromosomes AK1 and AK8 of the 1932; Manton, 1932). Assessing chromosome and genome evo- Ancestral Crucifer Karyotype (n = 8, Schranz & al., 2006). In lution in the tribe thus remained unfeasible for the last eighty the A. thaliana genome (http://www.arabidopsis.org/), AK1 years. The closest relatives of Heliophileae remain unknown, corresponds to 17.1 Mb (block A: 6.7 Mb, B: 5.7 Mb, C: 4.7 Mb) as the tribe groups with many others in a basal hard polytomy and AK8 to 9.2 Mb (block V: 2.4 Mb, W: 4.3 Mb, X: 2.5 Mb). (expanded lineage II) in a multi-gene Brassicaceae phylogeny BAC contigs were differentially labeled with biotin-dUTP, (Couvreur & al., 2010; Franzke & al., 2011). These unexplored digoxigenin-dUTP, and Cy3-dUTP by nick translation, etha- features, substantial taxonomic complexity and the very high nol precipitated, and hybridized to pachytene chromosomes of endemism (97%) of the genus in a relatively small CFR (only Heliophila species (for details see Mandáková & al., 2010a). ~90,000 km²; Goldblatt & Manning, 2000), all make Heliophila Pachytene chromosome spreads were prepared as described by a challenging study system. Mandáková & al. (2010a). DAPI-stained and painted chromo- Here we present the first in-depth information on chromosome complements were photographed, pseudo-colored and some number variation, localization of ribosomal RNA genes analyzed in Adobe Photoshop. Some chromosome images were (rDNA), and genome structure in the light of an updated tax- straightened (Fig. 2) using the plugin ‘Straighten Curved Ob- onomy and a comprehensive phylogeny. We characterized the jects’ (Kocsis & al., 1991) in ImageJ v.1.38 program (Abramoff extent of chromosome number variation across the tribe by & al. 2004). analyzing approximately one third of its species. Unexpectedly, Phylogenetic analyses. — The ITS analysis presented here a comparative chromosome painting (CCP) analysis revealed is based on the taxon sampling of Mummenhoff & al. (2005; 47 that diploid-like genomes of Heliophila species have undergone accessions) and 43 newly acquired accessions. Thus the current a whole-genome triplication (WGT) event, which along with phylogeny comprises 90 accessions, representing 57 species. other factors might have promoted the unique radiation of this This represents 63% of the 90 known species. Brassicaceae lineage. Methods for DNA extraction, PCR, and ITS sequencing follow Mummenhoff & al. (2001, 2004). The DNA sequences were aligned automatically using the MAFFT v.6.0 multiple Materials and methods alignment programme (http://mafft.cbrc.jp/alignment/server/) and edited by hand. The model of nucleotide substitution for Plant material. — Plant material for the phylogenetic the Bayesian analysis was determined using MrModeltest and chromosomal analyses (including CCP) was obtained v.2.2 software (Nylander, 2004; Posada, 2008) and the Akaike from herbarium specimens and from two field expeditions in information criterion (AIC). Bayesian inference of phylogeny 2008 and 2009, respectively. Ninety accessions, representing was performed using MrBayes v.3.1 (Ronquist & Huelsenbeck, 57 Heliophila species sensu Marais (1970) and Al-Shehbaz 2003). Following MrModeltest, the General Time Reversible & Mummenhoff (2005), were analysed. Chamira circaeoides model with a proportion of invariable sites and a gamma- (L. f.) A. Zahlbr. the only species of this genus without tribal shaped distribution of rates across sites (GTR + R + I) was used assignment (Al-Shehbaz, 2012), was chosen as outgroup for the in MrBayes. Two separate analyses were conducted, each start- Bayesian analysis as done in previous molecular phylogenetic ing from a different randomly chosen tree, on four parallel studies (Mummenhoff & al., 2005). Origin and collection data chains with a temperature for the heated chain set to 0.2. Each of plant material are given in Table S1 (Electronic Supplement). chain was run for 2,500,000 update cycles and every 100th Chromosome counts. — Mitotic chromosomes were cycle sampled. The initial 6250 samples (25%) were discarded counted from enzymatically digested young flower buds col- as burn-in. Bayesian search results were summarized by a 85% lected in the field. They were fixed in 3 : 1 (ethanol : acetic majority-rule consensus tree. Tracer v.1.5 (Rambaut & Drum- acid) fixative and stored in 70% ethanol at −20°C until use. mond, 2009) was used to check for convergence of the model For details of the protocol, see Mandáková & al. (2010a). DAPI- likelihood and parameters after each run until reaching sta- stained (4ʹ,6-diamidino-2-phenylindole in Vectashield, 2 μg/ml) tionary status. chromosome figures were photographed with an Olympus BX- Molecular dating. — Node ages were estimated as fol - 61 epifluorescence microscope and CoolCube CCD camera lows: We incorporated the Cleomaceae/Brassicaceae split as (Metasystem), processed in Adobe Photoshop CS2 (Adobe a secondary calibration point using values of 19 Ma (Franzke

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& al., 2009) and 32 Ma (Couvreur & al., 2010) for this split. In the auto-optimization option, the final analysis was done with addition, we simultaneously applied a minimum age constraint the MCMC chain-length set to thirty million. These settings of 2.58 and 5.33 Ma for the clade containingRorippa Scop. and resulted in ESS (effective sampling size) values, determined in Cardamine L., as this corresponds to the lower and upper age Tracer, for all estimated parameters and node ages above 100, estimate of Pliocene dated Rorippa fruit fossils (Mai, 1995). indicating a sufficient posterior distribution quality. A relaxed-clock approach was chosen to infer the ages of the Heliophileae radiation. Tarenaya spinosa (Jacq.) Raf. (= Cleome spinosa Jacq.), Aethionema grandiflorum Boiss. RESULTS & Hohen., Alliaria petiolata (M. Bieb.) Cavara & Grande, Rorippa amphibia (L.) Besser and Cardamine matthioli Moretti ITS phylogeny and dating. — Bayesian analysis of ITS were used as outgroup taxa. The analyses were performed in data resulted in a robust phylogenetic tree of 57 Heliophila BEAST v.1.7.1 (Drummond & Rambaut, 2012) following an species, with a basal trichotomy comprising sublineages A approach by Drummond & al. (2006) under a relaxed-clock (19 spp.), B (17 spp.) and C (19 spp.) (Fig. 1). Using different ap- model. The BEAST input file was created using the BEAST proaches to estimate and to calibrate the radiation of Heliophila user interface BEAUti v.1.7.1. In this approach, a relaxed-clock in southern Africa, multiple previous analyses arrived at age model is incorporated into a Bayesian phylogenetic inference. estimations of 1.0–4.4 Ma for the crown group of Heliophileae The analyses were conducted with a relaxed molecular clock (Table 1) (Mummenhoff & al., 2005; Verboom & al., 2009). model drawing uncorrelated rates from a log-normal distribu- The analysis of our new and expanded dataset using different tion. We created a normal prior with an age of 32/19 Ma for the calibration settings and BEAST confirms previous estimates, Cleomaceae/Brassicaceae split and a normal prior with an age all of which date the crown group age of Heliophila in the of 2.58/5.33 Ma for the Rorippa/Cardamine split, respectively. Pliocene and Pleistocene (1.22–5.64 Ma). Two runs were calibrated with two calibration points (32 Ma Chromosome-number variation (Figs. 1 & 2; Table 2). — and 5.33 Ma; 19 Ma and 2.58 Ma) in the same analysis. All Chromosome numbers were determined in 72 populations analyses were started with a random tree. We used the General representing 27 Heliophila species, representing all three in - Time Reversible model of nucleotide substitution with a gamma fratribal clades. Chromosome numbers range from 2n = 16 (G) distribution with four gamma categories of rates and a pro- in H. juncea (P.J. Bergius) Druce to 2n = ca. 88 inH. pubescens portion of invariant sites (I). After tuning the operators using Burch. (Fig. 2C, D). Twenty-one species (78%) have 2n = 20

Table 1. Age estimates for the Heliophileae (Heliophila) clade and its three main lineages A, B, and C using different methods and calibration options. Numbers indicate million years ago (Ma). Node Calibration Heliophila A B C Mummenhoff & al. (2005) Wikström & al.a NPRSb 4.2/4.6 3.4/3.7 2.9/3.3 3.9/4.3 NPRS jackknifec 3.7–5.4 2.8–4.5 2.2–3.9 n.a. Forced clockd 1.9 1.3 1.0 1.7 rDNA ITS ratese Non-ultrametric distances 2.7–5.8 1.1–2.3 1.5–3.2 2.2–3.8 Forced clockd 2.3–4.9 1.6–3.3 1.3–2.7 2.1–4.4 Current Study (expanded dataset) Franzke & al. (2009)f 3.77 1.70 1.22 2.03 Couvreur & al. (2010)g 7.84 5.60 3.99 5.64 a The Brassica/Cleome clade was dated to 22 Ma (from Wikström & al., 2001). b Tree made ultrametric using NPRS (Non-Parametric Rate Smoothing); additional calibration by constraining the Rorippa/Cardamine clade to be minimally 2.5/5.0 Ma. c Estimated on jackknife re-sampled branch lengths under NPRS. d Tree made ultrametric assuming a global clock. e (3.9–8.3) 3 × 10–9 substitutions/site/year (from Sang & al., 1994; Zhang & al., 2001). f The Brassicaceae/Cleomaceae split was dated to 19 Ma; additional calibration by constraining the Rorippa/Cardamine clade to be minimally 2.58 Ma. This age of the Rorippa/Cardamine split corresponds to the lower limit of the minimal age estimate of a Rorippa macrofossil (Mai, 1995). g The Brassicaceae/Cleomaceae split was dated to 32 Ma; additional calibration by constraining the Rorippa/Cardamine clade to be minimally 5.33 Ma. This age of the Rorippa/Cardamine split corresponds to the upper limit of the minimal age estimate of a Rorippa macrofossil (Mai, 1995)

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Fig. 1. Phylogeny of Heliophileae (Helio C. circaeoides KM1164 2n phila). 85% majority-rule consensus tree H. glauca KM1091 H. scoparia KM1065 from a Bayesian analysis of ITS sequence H. cedarbergensis KM997 LM7 data. This is a conservative estimate of H. esterhuyseniae KM1104 H. scoparia KM1480 the phylogeny, where nodes with posterior H. tulbaghensis KM1047 probability (PP) values < 0.85 were col- H. dregeana LM7 H. dregeana KM982 22 lapsed. Nodes with PP 0.95–1.0 are shown B H. hurkana KM1014 as thick lines, and normal lines represent PP H. maraisiana KM1015 H. juncea KM2144 LM23 0.94–0.85. Capital letters A, B, and C refer H. juncea LM5 to the main phylogenetic lineages. Chamira H. juncea KM2170 16, 32, 64 circaeoides was used as outgroup. Chromo- H. juncea KM1009 H. macra KM1088 some numbers (2n) and images of flowers H. elongata KM1097 are given for selected species. H. elongata KM1484 H. elongata KM1488 H. macrosperma KM1087 H. ephemera KM1090 H. monosperma KM1010 LM12 H. polygaloides KM1156 H. nubigena KM1060 H. tricuspidata KM1071 H. cf. descurva OSBU9253 20 H. acuminata KM1000 H. macowaniana KM2169 H. macowaniana LM6 H. macowaniana LM12 20, 80 LM21 H. macowaniana KM1086 H. elata KM2162 H. cf. elata KM1487 20 H. arenaria LM21 H. cf. arenaria KM1006 20 H. arenosa KM2160 20 H. cf. digitata KM1483 LM13 H. linoides KM2163 20 H. bulbostyla LM13 H. bulbostyla LM24 20 H. coronopifolia KM984 H. coronopifolia OSBU17187 H. coronopifolia LM2 20 H. coronopifolia OSBU817 H. africana KM1002 LM1 H. africana LM1 H. africana LM11 20 H. africana LM35 H. linearis LM3 H. linearis KM1083 36, 44 A H. cornuta KM985 20, 40, 60 H. namaquana KM1061 LM17 H. lactea LM17 18 H. refracta KM2105 H. pusilla KM1049 H. pusilla KM1497 H. subulata KM1078 H. subulata KM1505 H. pinnata LM4 H. pinnata KM2143 18, 36 H. pinnata LM8 H. gariepina KM1094 22 H. cornellsbergia KM998 LM33 H. amplexicaulis LM33 H. amplexicaulis KM2174 22 H. amplexicaulis LM25 H. eximia KM1092 H. rigidiuscula KM1050 H. carnosa KM994 H. carnosa KM1478 LM18 H. seselifolia LM18 22 H. nigellifolia KM1499 H. trifurca OSBU9280 22 C H. crithmifolia KM1069 H. crithmifolia KM989 H. crithmifolia KM2181 22 H. crithmifolia LM9 H. suborbicularis KM2146 LM9 H. latisiliqua KM2127 22 H. suborbicularis KM1012 22 H. minima KM1057 H. suavissima KM1073 H. variabilis KM1046 H. variabilis LM15 22 H. variabilis LM31 LM15 H. deserticola KM977 26 H. pectinata KM1062 H. pubescens KM1053 ~88 H. collina LM20 H. collina KM 2154 H. collina KM2154 22, 44 H. collina KM999

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(11 spp.) or 2n = 22 (10 spp.) chromosomes; two species have 2n (Fig. 1). The lowest chromosome count (2n = 16) was found = 18 and one has 2n = 26. Five species were found to have two only in H. juncea (clade B). In clade A, 2n = 20 is the dominat- or three intraspecific cytotypes. For example, the morphologi- ing number (78% of species analyzed), and 2n = 18 and 20 were cally well-defined H. juncea has diploid (2n = 16), tetraploid found exclusively in this clade. Species of clade C mostly have (2n = 32) and octoploid (2n = 64) cytotypes, and in H. cornuta 2n = 22 (91% of species analyzed). Sond. diploid (2n = 20), tetraploid (2 n = 40) and hexaploid rDNA (Fig. 2; Table 2). — Within diploid chromosome (2n = 60) populations were found. In H. linearis, two chromo- complements, the number of 45S rDNA loci varies from one some counts (2n = 36 and 44) apparently reflect the unsettled to four pairs. Six loci were observed in tetraploid (H. collina taxonomy of this species. The size of mitotic chromosomes O.E. Schulz, 2n = 44) and hexaploid (H. cornuta, 2n = 60) cyto- (ca. 2–13 μm in H. latisiliqua E. Mey. ex Sond.) is largely de- types, and eigth loci in tetraploid (H. pinnata L. f., 2n = 36) and pendent on the level of chromatin compaction, whereby less octoploid (H. macowaniana Schltr., 2n = 80) cytotypes. Several condensed prophase and pre-metaphase chromosomes are clade A species have two 45S rDNA loci, whereas four loci were longer than metaphase chromosomes (Fig. 2). In most (if not detected in most clade C species. However, this pattern is incon- all) species, mitotic chromosome complements comprise large sistent between the two clades. The same is true for the number of and small chromosomes (e.g., Fig. 2A). Heterochromatin is 5S rDNA loci, which varies from 1 to 14 pairs (in H. pinnata, 2n = localized in (peri)centromeric regions and, in some species, 36), although two pairs are found most frequently. The tetraploid in distinct interstitial or terminal heterochromatic knobs (Figs. cytotype of H. pinnata stands out from all other species as it has 2B & 3; Fig. S1 in the Electronic Supplement). Some trends the highest number of both 5S and 45S rDNA loci. Interestingly, in chromosome-number variation in Heliophila are detected all 45S and 5S rDNA loci are localized interstitially on chromo- when superimposing chromosome numbers on the ITS tree somes in all Heliophila species analyzed (Fig. 2C–H). The size

Fig. 2. Chromosome and rDNA variation in Heliophila. A, Differences in chromosome size in H. latisiliqua LM36 (2n = 22); arrowheads point to the smallest (4 μm) and largest (13 μm) chromosomes; B, numerous interstitial and terminal heterochromatic knobs on pachytene chromosomes of H. linearis LM3B (2n = 44); C–H, chromosomal localization of 5S rDNA (green) and 45S rDNA (red): C, one chromosome pair with interstitial 45S rDNA loci and six chromosomes with 5S rDNA in H. juncea KM2144 (2n = 16); D, twelve 5S rDNA loci in H. pubescens KM2137 (2n = ~88), the species with the highest chromosome number (the number of 45S rDNA loci has not been determined); E, H. africana LM11 (2n = 20); 5S and 45S rDNA loci are partly colocalized; F, H. bulbostyla LM24 (2n = 20); one chromosome pair with interstitial loci of 5S and 45S rDNA; one chromosome with a 45S locus flanked by 5S rDNA loci on both sides; G, unusually large 45S rDNA loci in H. amplexicaulis LM25 (2n = 22); H, eleven loci of 5S and three interstitial loci of 45S rDNA in H. crithmifolia KM2136 (2n = 22). — Chromosomes were counterstained with DAPI and B/W images inverted in Adobe Photoshop. Scale bars = 10 μm.

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of the 45S rDNA locus can differ significantly between species Chromosome painting in diploids (Fig. 3; Fig. S1). — Two (compare H. amplexicaulis and H. crithmifolia, Fig. 2G & H), chromosomes of the Ancestral Crucifer Karyotype (ACK), AK1 but rarely differs within a chromosome complement (H. juncea, and AK8, were chosen to get a glimpse of genome structure Fig. 2C). In two species, 5S and 45S rDNA loci are co-localized and karyotype evolution in seven Heliophila species. These (H. africana, Fig. 2E) or reside in close proximity (H. bulbostyla species were chosen such that they would represent the three P.E. Barnes, Fig. 2F). In H. bulbostyla, one 45S locus is flanked ITS clades and have both different ploidy levels and chromo- on both sides by 5S rDNA, probably due to an inversion within some numbers (Fig. 1). Chromosome-specific A. thaliana co-localized 5S and 45S rDNA loci. BAC contigs, representing the two AK chromosomes and six

Table 2. Chromosome numbers and numbers of rDNA loci (vouchers at MO and OSBU). rDNA loci Chromo- rDNA loci Chromo- somes with somes with Accession Species 2n 45S 5S 45S and 5S Accession Species 2n 45S 5S 45S and 5S KM2166 H. africana 20 2 4 2 KM2120 H. lactea 18 2 4 2 LM1 H. africana 20 2 4 2 LM17 H. lactea 18 2 2 2 LM11 H. africana 20 2 4 2 KM2127 H. latisiliqua 22 4 4 2 LM35 H. africana 20 2 4 2 LM34 H. latisiliqua 22 4 2 0 KM2161 H. africana 20 2 4 2 LM3B H. linearis agg. 44 6 4 – LM25 H. amplexicaulis 22 2 4 0 LM3A H. linearis agg. >30 4 10 4 LM33 H. amplexicaulis 22 2 4 0 KM2163 H. linoides 20 2 2 2 LM16 H. arenaria 20 2 4 2 KM2140 H. macowaniana 20 2 4 2 LM21 H. arenaria 20 2 4 2 KM2169 H. macowaniana 80 8 >8 0 KM2156 H. arenaria var. acocksii 20 2 4 2 LM12 H. macowaniana 20 2 4 2 KM2160 H. arenosa 20 2 2 0 LM6 H. macowaniana 20 2 4 2 LM13 H. bulbostyla 20 2 4 2 KM2143 H. pinnata 18 2 8 2 LM24 H. bulbostyla 20 2 3 2 KM2150 H. pinnata 18 4 > 6 4 LM20 H. collina 22 2 4 0 LM10 H. pinnata 36 – – – KM2101 H. collina 22 4 – – LM4 H. pinnata 36 8 14 8 KM2154 H. collina 44 6 4 0 LM8 H. pinnata 18 4 4 2 KM2094 H. cornuta 20 2 – – KM2137 H. pubescens ~88 >22 18 – KM2117 H. cornuta 60 6 – – KM2130 H. seselifolia 22 4 – – KM2134 H. cornuta 40 4 – – KM2131 H. seselifolia 22 – – – LM2 H. coronopifolia 20 2 4 2 KM2133 H. seselifolia 22 4 4 2 KM2116 H. crithmifolia 22 4 2 0 KM2135 H. seselifolia 22 4 – – KM2119 H. crithmifolia 22 4 4 2 KM2148 H. seselifolia 22 4 4 2 KM2136 H. crithmifolia 22 3 8 0 LM18 H. seselifolia 22 4 4 2 KM2147 H. crithmifolia 22 4 8 1 KM2138 H. schulzii 20 2 – – KM2247 H. crithmifolia 22 4 4 0 KM2146 H. suborbicularis 22 4 4 2 LM9 H. crithmifolia 22 4 10 0 KM2107 H. trifurca 22 2 4 2 LM14 H. descurva 20 2 4 2 KM2100 H. variabilis 22 4 4 – KM2106 H. deserticola 26 4 4 2 KM2112 H. variabilis 22 4 – – KM2108 H. deserticola 26 4 – – KM2113 H. variabilis 22 4 – – LM7 H. dregeana 22 – – – KM2139 H. variabilis 22 4 4 or 6 0 KM2162 H. elata 20 2 4 2 LM15 H. variabilis 22 4 4 0 KM2115 H. gariepina 22 2 – – LM22 H. variabilis 22 – – – KM2121 H. gariepina 22 2 4 2 LM30 H. variabilis 22 4 6 0 KM2129 H. juncea 32 4 4 0 LM31 H. variabilis 22 4 6 0 KM2144 H. juncea 16 2 6 0 LM32 H. variabilis 22 4 4 0 LM23 H. juncea 32 4 9 0

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A B cen C AK1

H. dregeana LM7 Fig. 3. Examples of comparative chro- (2n = 22) A1 B1 mosome painting in Heliophila species/ cytotypes with different chromo- C1 C2 some numbers (2n = 16–64) and from B3 B2 the three infratribal clades (A–C). A3 Arabidopsis thaliana BAC contigs cor- A2b C3 A2a responding to AK1-specific genomic

H. juncea KM2144 blocks A, B, and C were differentially B2 labeled and polarized, and used as (2n = 16) C2 A2 painting probes on pachytene bivalents. A A1 Triplicated GBs were tentatively as- B1 A signed, for example, as A1, A2, and A3. C1 B In H. juncea with 2n = 64, the complex B C3 hybridization pattern has not been analyzed. Small capitals refer to rear- H. juncea LM23 ranged/split blocks. Blue arrows mark (2n = 32) C1 heterochromatic knobs. The AK1 ho- A1 B1 meologue in the diploid H. juncea and in H. amplexicaulis was straightened and C2 shown as enlarged images on the right. B2 Chromosomes were counterstained with A2 DAPI. Scale bars = 10 μm.

H. juncea LM5 (2n = 64)

H. macowaniana LM6

(2n = 20) C3 C2

A3 B1 B2 A2

C1 A1

H. pinnata LM8 (2n = 18)

A1 C1 C B1 A2 C A3

H. variabilis LM31 (2n = 22) C1 C2 B1 A1 B2 B A3 A2

H. amplexicaulis LM25 (2n = 22) C3 A2 B3 A1 A3a A3b B2 B1 C1

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ancestral genomic blocks (GB), were used as painting probes infrageneric classification, morphological character evolution, on Heliophila pachytene chromosomes. Painting probes were and eco-geographical evolution of Heliophila have already been polarized and differentially labeled to facilitate identification of discussed in detail by Mummenhoff & al. (2005). GBs building up the ancestral chromosomes (AK1: blocks A, B, Chromosome-number variation. — While chromosome and C, Fig. 3; AK8: V, W, and X, Fig. S1). The fluorescent paint- numbers of only four Heliophila species have been published in ing signals were strong and unambiguously identified respec- the last eighty years (Jaretzky, 1932; Manton, 1932), the present tive homeologous chromosome regions. Most GBs were found study reports chromosome counts for 27 species. This still only triplicated in the analysed Heliophila accessions with 2n = 16, represents less than one-third of all known Heliophileae species. 18, 20, and 22 (100% of A and W, 97% of C, V and X, and 93% Similar to many other Brassicaceae groups with small chro- of B were triplicated). In most species, one homeologous copy mosomes, heterochromatic knobs and nucleolar organizing re- was on average longer than the other two genomic copies (see gions (often detached from chromosomes) are often erroneously the diploid H. juncea cytotype in Fig. 3). In 37% of AK1 copies, counted as chromosomes or interphase chromocenters. Jaretzky A and B form the ancestral association corresponding to the up- (1932) already reported on difficulties with chromosome count- per arm of AK1. Less frequently (13% of AK1 copies) the whole ing “in cells filled with an achromatic mass” (= chromocenters). AK1-like chromosome structure remained preserved (see the Although chromosome numbers are known for only one-third of straightened chromosomes of H. juncea and H. amplexicaulis in Heliophileae species, counts of 2n = 20 and 22 dominate (77% of Fig. 3). AK8 painting probe revealed one conserved VW and one species). These chromosome numbers, though not exceptional in rearranged WX association, respectively, as well as partly re- Brassicaceae, are restricted only to a few tribes and genera such arranged AK8-like chromosome in H. macowaniana. In both as Anastaticeae, Brassiceae, Iberis L., Leavenworthia Torr., the AK1- and AK8-like chromosomes, the position of the func- Menonvillea DC. and Pachycladon Hook. f. (Warwick & Al- tional centromere does not match the position of the ancestral Shehbaz, 2006). Interestingly, as in Heliophileae, 2n = 20 and centromere in AK1 and AK8. The ancestral structure of AK1 22 are found in taxa of mesopolyploid origin, i.e., in Brassiceae and AK8 in Heliophila was frequently altered by intrachromo- (Lysak & al. 2005), Pachycladon (Mandáková & al. 2010a) and somal rearrangements and translocations with other (unpainted) Leavenworthia (T. Mandáková and M.A. Lysak, unpub. data). chromosomes, whereby AK8 homeologous copies are signifi- Therefore, other species and genera with n = 11 might also have cantly more often rearranged than segments homeologous to experienced mesopolyploid evolution. AK1. Interestingly, in H. dregeana Sond., H. pinnata, H. varia- Higher chromosome counts (2n = 32, 36, 40, 44, 60, 64, bilis Burch. ex DC. and H. amplexicaulis, in addition to three 80 and 88) represent tetra-, hexa- and octopolyploids derived homeologous copies of blocks X and W, one or two very short from mesopolyploid diploidized cytotypes, as was shown extra copies of these blocks were also found (Fig. S1). for H. juncea and H. pinnata by our CCP results. We consider Chromosome painting in polyploids (Fig. 3). — Patterns these accessions as very recent neopolyploids, representing of inter-species chromosome homeology to AK1 were analyzed intraspecific karyological variation. The geographic distribu- in two polyploid accessions of H. juncea (2n = 4x, 8x = 32, 64) tion of the neopolyploid cytotypes remains to be studied, and and in the tetraploid accession of H. pinnata (2n = 4x = 36). In it needs to be established whether there is gene flow between the tetraploid H. juncea genome, one AK1-like knob-bearing different ploidy levels. chromosome and one AB association observed in the diploid rDNA. — We analyzed the number and position of rDNA cytotype were found as two identical genomic copies. In the loci in 72 accessions of 27 Heliophila species with the aim octoploid cytotype of H. juncea, at least twelve homeologous of obtaining phylogenetically informative markers. Although copies of blocks A, B and C were found. At least six copies of some differences in the number of 45S rDNA loci between these blocks were observed in the tetraploid accession of H. pin- clades and individual species were observed, often identical nata. However, the complexity of painting results prevented us numbers of rDNA loci preclude these markers as species- from clear conclusions on the exact number and structure of specific characters. The interstitial position of 45S rDNA, homeologous segments in these polyploid accessions. otherwise rare in Brassicaceae (Ali & al., 2005), apparently is common in Heliophileae. Whole-genome triplication. — Triplicated homeologous DISCUSSION chromosome regions corresponding to ancestral chromosomes AK1 and AK8 found in Heliophila quasi-diploid species (2n = Infratribal phylogeny. — The present ITS study corrobo- 16–22) argue for a whole-genome triplication (WGT) shared by rates the results published by Mummenhoff & al. (2005) and all three clades. This was an unexpected finding, as all known demonstrates the monophyly of the unigeneric tribe Helio- chromosome numbers (2n = 20 and 22) as well as 2n = 16 and 18 phileae, with Chamira circaeiodea as sister species. Mono- were implicitly considered diploid. Also, the genome size (ca. phyly of Heliophileae was recently also confirmed by Couvreur 400 Mb) of two Heliophila species with 2n = 20 and 22 is com- & al. (2010) based on a multi-gene dataset representing the nu- parable to many truly diploid crucifer taxa (Lysak & al., 2009). clear, chloroplast and mitochondrial genomes. The phylogeny As the WGT was detected by CCP in seven Heliophila species presented here recognizes three main lineages (A, B and C), from all three clades, it is apparently shared by the whole tribe which correspond to the main groups detected by Mummenhoff and predates the diversification of Heliophileae (Fig. 4). As the & al. (2005) based on a smaller taxon sampling. Aspects of homeologous chromosome regions identified correspond to

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ancestral GBs and GB associations (i.e., AB of AK1 and VW of have been accompanied by extensive chromosome rearrange- AK8) in several species, we assume that the hexaploid ancestor ments, centromere loss, and by changes at the epigenetic and of Heliophileae was derived from hybridization events between sequence levels. Similar to the Australian and New Zealand two or three species with genomes resembling the Ancestral crucifers (Mandáková & al., 2010a, b), the overall collinearity Crucifer Karyotype (n = 8; Schranz & al., 2006). The fact that of AK1 and AK8 in Heliophila species was retained, whereas the two homeologous copies are on average shorter than the the ancestral centromeres were deleted or lost their function. In third one, and that the chromosomes are of different size in the some species analyzed, the positions of inactive paleocentro- triplicated genomes (Jaretzky, 1932; this study), argues for an meres coincide with interstitial heterochromatic knobs (Fig. 3; allopolyploid origin of the mesohexaploid ancestor. Similarly, Fig. S1), and knobs on several other chromosomes may also the different size of homeologous chromosome segments was be remnants of inactive centromeres (e.g., Fig. 2B). The exact also reported in other mesopolyploid taxa, such as Brassiceae mechanism of centromere elimination in Brassicaceae taxa (Lysak & al., 2005) and Australian crucifers (Mandáková & al., remains to be studied. 2010b). Although biased (sub)genome fractionation that usually Has the whole-genome triplication been a driver of follows ancient polyploidization events (e.g., Schnable & al., the species radiation in Heliophileae? — Factors that might 2011) may explain the subgenome difference in Heliophila, we promote speciation and/or determine speciation rates of Cape argue that a more likely scenario is that proposed for the three clades are intrinsic and extrinsic (Barraclough, 2006). These subgenomes in Brassica rapa L. (Tang & al., 2012). Analogous factors include climatic changes supposed to generate an array to B. rapa, the two more fractionated (“shorter”) genomic cop- of responses including phenology shifts and migration facili - ies might represent a more diploidized tetraploid ancestral ge- tating both fragmentation as well as re-union of populations, nome prior the WGT, whereas the least fractionated (“longer”) potential geographical (topographic in the first place) isolating subgenome was brought into a hexaploid Heliophila by subse- barriers (e.g., mountain ranges, deep river valleys), steepness of quent hybridization (Fig. 4). One or two very short extra copies ecological and altitudinal gradients, regional edaphic (e.g., soil of blocks X and W found in four diploid Heliophila species are types, geology) and disturbance heterogeneity (specially fire re- more intriguing. We hypothesize that these segments may be gimes differing in spatial distribution, frequency and severity) relics of a whole-genome duplication (WGD) event predating and last but not least a plethora of biotic interactions including the triplication or large segmental duplications. Clearly, more mycorrhiza and related phenomena and animal–plant interac- detailed analyses are needed to disentangle the genome structions especially pollinators, pollination and dispersal (Linder, ture of ancient diploids and the hexaploid ancestor. 2005; Barraclough, 2006; Verboom & al., 2009; Warren & al., The Heliophileae-specific WGT is yet another example of 2011; Schnitzler & al., 2011; Mucina & Majer, 2012), but gener- a lineage-specific mesopolyploid event recently identified in ally also intrinsic factors such as WGD events (Franzke & al., Brassicaceae. Mesopolyploid triplication has been described 2011). Consequently, the speciation rate of a particular lineage in the ancestry of Brassiceae (Lysak & al., 2005, 2007; Tang will depend on the interaction between a wide range of intrinsic & al., 2012), whereas mesotetraploid events mark the evolution and extrinsic factors. of Orychophragmus Bunge (Lysak & al., 2007), Australian Heliophila, as many other lineages in the CFR, has prob- endemic crucifer genera (Mandáková & al., 2010b) and Pachy- ably undergone an adaptive radiation and has diversified at cladon Hook. f. (Mandáková & al., 2010a). the highest rates calculated for a dated Cape radiation (Warren WGT and karyotypic diploidization. — The WGT has & Hawkins, 2006). Using different methodologies and calibra- been followed by descending dysploidy (chromosome-number tion concepts, the radiation was dated to 1.0–5.64 Ma (Mum - reduction) towards the diploid-like chromosome complements menhoff & al., 2005; Verboom & al., 2009; this study, see found in extant Heliophila species. The diploidization process Table 1), and all datings indicate a recent diversification against during which the presumably more than 40 chromosomes of a background of increased aridity in the Late Pliocene and the mesohexaploid ancestor were reduced to only 16 to 22 must of alternation of cold and warm (= dry and wet, respectively)

WGD 2x (4x) 4x (8x) meso- neo-mesopolyploid genomes WGD WGD clade 6x (10x) 12x (20x) 24x (40x) B 2n = 16 2n = 32 2n = 64 6x (10x) species A 2n = 18 2n = 36 2x radiation A 2n = 20 2n = 40 2n = 80 chromosome number B+C 2n = 22 2n = 44 2n = 88 reduction C 2n = 26

Fig. 4. Model of genome evolution in Heliophileae (see Discussion for details). A haploid genome is shown for each putative ancestral species. Ad- ditional genome copies detected in some diploid species by CCP (Fig. S1) are depicted as grey bars and reflected by ploidy levels in parentheses.

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climatic periods of the Pleistocene. As suggested by Linder Dating the radiation of Heliophileae. — Molecular dating (2003), the radiation of many lineages in the Cape region re- within Brassicaceae has been limited and controversial due sembles evolutionary processes of island-species radiation. The to a lack of fossils and calibration options. Different age esti - Cape seed plant endemism of almost 70% is comparable to that mates for the origin of Brassicaceae and its major lineages are found on islands. Furthermore, a high contribution by a very given in Beilstein & al. (2010) and Franzke & al. (2011). Using small number of clades to overall species richness is also typi- different approaches to calibrate phylogenetic trees, or differ- cal of island floras (Linder, 2003). This indicates a great degree ent algorithms, Mummenhoff & al. (2005) arrived at age esti- of isolation of the CFR. While islands are isolated by water, mates of 1.0–4.4 Ma for the crown group age of Heliophileae the CFR differs in climate, topography and soil types from the (Table 1). Our new and expanded ITS dataset was dated with rest of southern Africa and is surrounded by oceans on three an uncorrelated relaxed molecular clock approach (BEAST) sides (Marloth, 1929; Linder, 2003). To our knowledge, no other using different calibration settings, e.g., direct or primary fossil example in Brassicaceae exhibits such a species-rich radiation calibration (Rorippa/Cardamine split) and secondary calibra- confined to a relatively small region as does tribe Heliophileae. tion (age of the Cleomaceae/Brassicaceae split). The age of the Similar to the WGT event specific for tribe Brassiceae and radiation of Heliophila was inferred to be ca. 3.4 (1.22–5.64) its apparent impact on the infratribal diversification (238 spp. Ma, which agrees with previous studies (Table 1), all dating the in 47 genera), we link cladogenesis, species radiation and the diversification of Heliophila to the Pliocene and Pleistocene. WGT event in Heliophileae. Ancient polyploidization and sub- This corresponds with findings by Warren & Hawkins (2006) sequent diploidization have been repeatedly shown as driving who suggested that Heliophila diversified at the highest rates forces of genetic diversification and radiation in plant lineages calculated for a dated Cape radiation. (e.g., Barker & al., 2008; Soltis & al., 2009; Jiao & al., 2011). Phylogenetic relationships of Heliophileae. — Nothing For example, gene duplication, neofunctionalization or loss is known about the closest relatives of Heliophileae. How- may facilitate reproductive isolation and speciation. The paleo- ever, based on the average age of the origin of Heliophila polyploid event (a) that occurred very early in the history of (1.9–7.8 Ma; Table 1), one might speculate that ancestors of Brassicaceae provided the genetic raw material for radiation, Heliophila—like those of South African endemic Lepidium and diversification rates that are among the highest reported species—entered the African continent from southwest Asia, for flowering plants (Franzke & al., 2011). We suggest that the presumed cradle of Brassicaceae (Franzke & al., 2011). the Miocene onset of global aridity (Zachos & al., 2001; Guo From here they may have reached southern Africa by several & al., 2002; Liu & al., 2009) heralded the advent of a pro- putative routes, including the East African route (Hedge, 1976) longed climatically unstable period punctuated by periods of and the (more probable) Arid Corridor (Verdcourt, 1969). The enhanced aridity and later (towards Pliocene) associated with latter may have functioned as an intermittent link between the climatic oscillations. These dynamics, driven by changes in semi-deserts and deserts of North Africa and Arabia and the periodicity of Milankovich oscillations (Raymo & Huybers, emerging arid regions of southern Africa since the Late Mio- 2008), and boasts of increased aridity during the Pleistocene cene (about 7 Ma) when the Sahara Desert started to emerge as (deMenocal, 2004), set the scene for the remarkable radiation a major arid biome (Schuster & al., 2006; Mummenhoff & al., of Heliophila. The progressing aridity presumably resulted in 2001). Clearly, the closest relatives of Heliophileae are in the opening of the formerly forested landscapes (Axelrod & Raven, expanded lineage II sensu Franzke & al. (2011), and of the other 1978) and the creation of more sun-exposed (‘heliophilous’) 24 tribes included in that lineage, Brassiceae and Sisymbrieae, habitats, which underwent further changes (including altera- which also show some diversification in South Africa, are more tions such as expansion, contraction, obliteration, formation) likely candidates than other tribes. in reaction to the dry-wet cycles of the Pleistocene. Repeated Polyploidy in the evolution of Cape flora. — Based on isolation of populations (fragmentation) and putative reunion of available surveys of chromosome numbers and ploidy levels, migrating populations could then have acted as an ‘evolutionary polyploidy was considered to have only minor importance for pump’ (see also Plana, 2004 and Compton, 2011 for applica- radiations of genera centered in the CFR (Goldblatt & al., 1993; tions of this concept). Environmental heterogeneity, including Goldblatt & Takei, 1997; Goldblatt & Manning, 2011; Prebble geological and topographical complexity of the region, steep & al., 2011; P. Goldblatt, pers. comm.). The Heliophileae is ecological gradients (see Linder, 2003; Linder & al., 2010), the first group of the Cape flora where the prominent role of various large-scale disturbance factors (Rebelo & al., 2006) ancient and more recent polyploidization events has been dem- and small-scale animal–plant interactions (Van der Niet & al., onstrated. Multiple base numbers in Bruniaceae (Goldblatt 2006) are presumed to have been of vital importance in pro- 1981) suggest that ancient WGD events played an important pelling this evolutionary pump. Phenological responses, i.e., role in the evolution of this family, an endemic to the CFR. In shifts in mean flowering time (from summer towards spring) Oxalis L., remarkable intraspecific ploidy variation was found and flowering time duration, consistent with wet/dry climatic in nearly 30% of Cape species investigated (Suda & al., 2012). cycles, may also have contributed to the species diversity in However, it is premature to draw major conclusions on the Heliophila and other Cape lineages (Warren & al., 2011). We role of WGD events in the evolution of Cape genera as karyo- argue that the purported whole-genome triplication occurred logical data are limited and no in-depth genomic studies are prior to the origin of the three Heliophila lineages and facili- available. Prior to this study only four chromosome numbers tated radiation in changing environment. were known for Heliophila (> 90 spp.), nine species have been

998 120 Version of Record (identical to print version). TAXON 61 (5) • October 2012: 989–1000 Mandáková & al. • Whole-genome triplication in Heliophileae

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1000122 Version of Record (identical to print version). VII. Cheng F., Mandáková T., Wu J., Xie Q., Lysak M.A., Wang X. Deciphering the diploid ancestral genome of the mesohexaploid Brassica rapa. Submitted.

123 124 Deciphering the diploid ancestral genome of the mesohexaploid

Brassica rapa

Feng Cheng,a Terezie Mandáková,b Jian Wu,a Qi Xie,c Martin A. Lysak,b,1 and Xiaowu Wanga,1

a Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; b RG Plant Cytogenomics, CEITEC and Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic; c State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Synopsis The ancestral genome of Brassica oil and vegetable crops has undergone an ancient whole-genome triplication. This work reports on the reconstruction of the three diploid Brassica genomes with seven chromosomes involved in the origin of the hexaploid ancestor with 42 chromosomes. The hexaploid genome evolved through extensive genome diploidization toward extant diploid-like Brassica genomes.

Abstract The genus Brassica includes several important agricultural and horticultural crops. Their current genome structures were shaped by whole-genome triplication followed by extensive diploidization. The availability of several crucifer genome sequences, especially that of Chinese cabbage (Brassica rapa), enabled novel insights regarding the evolution of the mesohexaploid Brassica genomes from their “diploid” progenitors. We reconstructed three ancestral subgenomes of B. rapa (n = 10) by comparing its whole-genome sequence to ancestral and extant Brassicaceae genomes. All three B. rapa paleogenomes apparently consisted of seven chromosomes, similar to the ancestral translocation Proto-Calepineae Karyotype (tPCK, n = 7), which is the evolutionarily younger variant of the Proto-Calepineae Karyotype (n = 7). Based on comparative analysis of genome sequences or linkage maps of B. oleracea, B. nigra, Raphanus sativus, and other closely related species, we propose a two-step merger of three tPCK-like genomes to form the hexaploid ancestor of the tribe Brassiceae with 42 chromosomes. Subsequent diversification of the Brassiceae was marked by extensive genome reshuffling and chromosome number reduction mediated by translocation events and followed by loss and/or inactivation of centromeres. Furthermore, via interspecies genome comparison, we refined intervals for seven of the genomic blocks of the Ancestral Crucifer Karyotype (n = 8), thus revising the key reference genome for evolutionary genomics of crucifers.

125 Introduction

The genus Brassica currently comprises 38 species and numerous varieties, many of which are important crops or weeds. The six species that make up the so-called “Triangle of U” are of particular economic importance because of their cultivation as vegetables, condiments, and sources of oilseed (Nagaharu, 1935). These species are the “diploids” Brassica rapa (AA genome), B. nigra (BB), and B. oleracea (CC), and the allotetraploid species B. juncea (AABB), B. napus (AACC), and B. carinata (BBCC). Brassica is one of 47 genera in the monophyletic tribe Brassiceae. This tribe, which contains about 238 species primarily distributed in the Mediterranean region, southwestern Asia, and northern Africa (Prakash and Hinata, 1980; Al-Shehbaz, 2012), belongs to lineage II, one of three well-supported lineages recognized within the Brassicaceae family (Figure 1A). Within lineage II, the Brassiceae have been grouped together with tribes Sisymbrieae, Isatideae, and Thelypodieae (Schizopetaleae) (Al-Shehbaz et al., 2006; Beilstein et al., 2006; Beilstein et al., 2008; Franzke et al., 2011). However, the phylogenetic relationships within lineage II, in particular the position of the closely related tribes Eutremeae and Thlaspideae, remain unclear, and more than 20 loosely interrelated tribes have been described within an “expanded” lineage II (Figure 1B) (Franzke et al., 2011).

Figure 1. Currently hypothesized phylogenetic relationships among the Brassicaceae taxa discussed in this study and purported evolutionary relationships among extant and ancestral crucifer genomes (adopted and modified from Franzke et al. [2011] and Mandakova and Lysak [2008]). (A) Schematic phylogenetic relationships among the three major Brassicaceae lineages (I, II, and III). (B) Tentative genome evolution in Lineage II and five closely related tribes. The star refers to the Brassiceae-specific whole-genome triplication. Question marks indicate unknown or assumed ancestral genomes. ACK: Ancestral Crucifer Karyotype (n = 8); tPCK/PCK: (translocation) Proto-Calepineae Karyotype (n = 7).

126 Early chromosomal studies indicated that the diploid Brassica species might actually represent “balanced secondary polyploids” (e.g., Catcheside, 1934; Röbbelen, 1960). Later comparative RFLP mapping among these species and between Brassica species and Arabidopsis thaliana (Lagercrantz and Lydiate, 1996; Lagercrantz, 1998) suggested that “diploid” Brassica genomes descended from a hexaploid ancestor. All Brassiceae species analyzed to date contain either three or six copies of orthologous genomic regions of A. thaliana (Lysak et al., 2005; Parkin et al., 2005; Lysak et al., 2007). These findings provide compelling evidence for an ancestral whole-genome triplication in the ancestry of the monophyletic tribe Brassiceae. More recently, analysis of the draft genome sequence of B. rapa (n = 10) revealed the three subgenomes of the hexaploid ancestor (Wang et al., 2011). A comparison of collinearity between the 10 B. rapa chromosomes and the 5 A. thaliana chromosomes using 24 ancestral genomic blocks (GBs) of the Ancestral Crucifer Karyotype (ACK, n = 8; Schranz et al., 2006) revealed three syntenic copies of each A. thaliana GB, with only one exception, in the B. rapa genome (Wang et al., 2011). Because the three GB copies differ regarding their rate of gene loss (fractionation), blocks were classified according to their gene density as belonging to the least fractionated (LF), the medium fractionated (MF1), and the most fractionated (MF2) subgenome. Wang et al. (2011) suggested a two-step origin of B. rapa involving a tetraploidization followed by substantial genome fractionation (subgenomes MF1 and MF2), and subsequent hybridization with a third, less-fractionated genome. This two-step model gained support by comparing the frequency of deletions in exon sequences between the three B. rapa subgenomes (Tang et al., 2012). Karyotype and genome evolution in the expanded lineage II were analyzed by Mandakova and Lysak (2008). Comparative chromosome painting using A. thaliana BAC contigs demonstrated that the six analyzed tribes (i.e., Calepineae, Coluteocarpeae, Conringieae, Eutremeae, Isatideae, and Sisymbrieae) descended from a common ancestor with the Proto-Calepineae Karyotype (PCK) (n = 7). While Calepineae, Coluteocarpeae (syn. Noccaeeae), and Conringieae taxa retain the PCK genome structure, the tribes Eutremeae, Isatideae, and Sisymbrieae display an additional whole-arm translocation (Figures 1B and 2A). This younger ancestral genome is henceforth referred to as the translocation PCK (tPCK). The tPCK genome structure was recently confirmed by whole-genome sequencing of Schrenkiella parvula (syn. Thellungiella parvula, formerly placed in the Eutremeae) (Dassanayake et al., 2011) and Thellungiella salsuginea (Eutremeae) (Wu et al., 2012). The PCK shares five chromosomes with the ACK and structurally closely resembles the extant karyotypes of Arabidopsis lyrata and Capsella rubella (Lysak et al., 2006; Schranz et al., 2006; Hu et al., 2011). It has therefore been suggested that either the PCK and ACK are descended from a common ancestor or, more likely, that the seven PCK chromosomes are derived from the eight chromosomes of the ACK (Mandakova and Lysak, 2008) (Figure 1B). For more than half a century, the chromosome number and genomic structure of the hexaploid Brassica ancestor and its three parental subgenomes have been a matter of debate. Several chromosome numbers, ranging from x = 3 to 7, have been proposed for ancestral Brassica subgenomes (e.g., Röbbelen, 1960; Truco et al., 1996; reviewed by Prakash and Hinata, 1980). Based on a comparison of B. rapa, B. oleracea, and B. nigra

127 linkage maps, Truco et al. (1996) suggested that all three subgenomes were derived from an ancestor with five, six, or seven chromosomes. Although those authors favored six ancestral chromosomes (W1–W6), a seventh chromosome (W7) could not be ruled out. Comparison of the PCK with the structure of the B. rapa subgenome in B. napus (Parkin et al., 2005) revealed two shared rearrangements, thus suggesting that two or all three subgenomes of the hexaploid ancestor of the Brassiceae corresponded to either PCK or tPCK (Mandakova and Lysak, 2008). The aim of this study was to elucidate the structure of the three ancestral subgenomes of the hexaploid ancestor of the Brassiceae. Toward that aim, structural genome analyses of B. rapa in comparison with ancestral and extant crucifer genomes were performed. Specifically, we tested whether the PCK and/or tPCK represent the karyotype of the diploid ancestors of the mesohexaploid B. rapa, the genus Brassica, and the tribe Brassiceae.

128 Results Deciphering the Ancestral Diploid Karyotypes of B. rapa

Figure 2. Ancestral Brassicaceae genomes and the distribution of ancestral genomic blocks (GBs) along 10 chromosomes of B. rapa. (A) The ancestral genomes ACK, PCK, and tPCK, each comprising 24 ancestral GBs. In the PCK and tPCK, chromosome labels reflect the presumed origin of the two karyotypes from the more ancestral ACK genome. (B) Ten B. rapa chromosomes (A01–A10) consisting of 24 triplicated GBs (71 blocks in total, as one copy of block G was lost) assigned to subgenomes least fractionated (LF, red), medium fractionated (MF1, green), and most fractionated (MF2, blue). Two or more segments of a single block are labeled by lowercase letters (a, b, etc.). Downward pointing arrows are adjacent to GBs that were inverted relative to other block(s) which originated from a single ACK chromosome. Detected and undetected ancestral centromeres in the B. rapa genome are shown as black and white small rectangles, respectively.

Based on the previously determined syntenic relationship of the three B. rapa subgenomes (LF, MF1, and MF2) to the A. thaliana genome (Wang et al., 2011; Cheng

129 et al., 2012b), 71 of 72 expected GBs (3 × 24) were detected in the B. rapa genome (Figure 2B, Suppl. Table S1). Of the 24 blocks, 23 were present in three copies and one (block G) in only two copies. Among the 71 GBs, 48 (67.6%) exhibited continuous sequence collinearity between B. rapa and A. thaliana, 21 (29.6%) were disrupted into at least two segments, and two (2.8%; one copy each of blocks E and T) contained sequence deletions (Suppl. Tables S1 and S2). Analysis of GB integrity did not reveal significant differences in the degree of rearrangements among the three B. rapa subgenomes (Suppl. Tables S3 and S4). We compared the structure of the ACK, PCK, and tPCK (Figure 2A) with that of the three B. rapa subgenomes (Figure 2B). Using gene densities characteristic for each of the three B. rapa subgenomes and methods previously described (Wang et al., 2011; Cheng et al., 2012b), we then reconstructed three tentative B. rapa ancestral subgenomes by projecting the 71 GBs onto the ACK genome and the seven chromosomes of the PCK and tPCK. When a modern genome is compared with ancestral ones, the more recent the ancestor, the greater the expected degree of genomic continuity with the present-day genome. To examine genome continuity, we defined two adjacent genomic blocks as a GB association and compared all GB associations between B. rapa and the ancestral genomes ACK, PCK, and tPCK. There are 16 GB associations in the ACK genome and 17 associations in the PCK and tPCK genomes (Table 1, Figure 2A). In B. rapa, 10, 15, and 16 GB associations were identified between ACK, PCK, and tPCK, respectively. Of the 10 ACK-specific block associations, 4 were present in three copies in B. rapa, while 6 were present in two copies. Of the 15 PCK-specific GB associations, 8 were present in three copies, 6 in two, and 1 as a single copy. Of the 16 tPCK-specific GB associations, 8 were present in three copies, 6 in two copies, and 2 as single copies (Table 1, Suppl. Table S5). Block associations V/K/L/Q/X and O/P/W/R, specific for both PCK and tPCK, were identified in all three B. rapa subgenomes (Figure 2B, Table 1). Three genomic copies of the V/K/L/Q/X association were found on chromosomes A02 (MF1), A06 (LF), and A09 (MF2). Two copies of the O/P/W/R association were rearranged on chromosomes A02 (MF2) and A03 (MF1). The third O/P/W/R copy was split between chromosomes A09 (blocks O/P) and A10 (W/R). tPCK contained two specific GB associations—D/V and M/E—resulting from a whole-arm reciprocal translocation between chromosomes AK2 and AK5/6/8 within the PCK genome (Figure 2A; Mandakova and Lysak, 2008). In the MF2 subgenome, the D/V association was identified on chromosome A09. In the LF subgenome, blocks D and V were split between chromosomes A03 (MF1) and A06 (LF) due to an intersubgenomic translocation (Suppl. Figure S1A). The M/E association is not present in B. rapa. We propose, however, that the present-day block associations M(LF)/M(MF2) (A06) and E(LF)/E(MF2) (A07) are the products of intersubgenomic translocation between two ancestral M/E associations (Suppl. Figure S1B). Furthermore, evidence that the MF1 subgenome also originated from the tPCK arises from a comparison of B. rapa and B. nigra genome structures with respect to the M/E association (Panjabi et al., 2008). Based on chromosome synteny, the block association R/W/M/O/P/E on B2 in B. nigra is syntenic to R/W/E( MF1)/O/P on chromosome A02 in B. rapa (Suppl. Figure S2).

130 Although the M/E association was interrupted by blocks O and P on chromosome B2, the colocalization of M/E with blocks O, P, R, and W on the same chromosome supports the idea that it originated from the ancestral associations O/P/W/R and M/E. The larger number of GB associations shared between B. rapa and PCK/tPCK compared with those between B. rapa and ACK, as well as the tPCK-specific GB associations and chromosome breakpoints found in B. rapa, indicate that the three B. rapa subgenomes bear a close structural resemblance to the seven chromosomes of the translocation Proto-Calepineae Karyotype. To facilitate the retrieval of gene sets of the three tPCK-like subgenomes in B. rapa (Suppl. Figure S3), with mutual homoeology and synteny to A. thaliana orthologs, we developed a webtool—“Search Syntenic Genes Br-tPCK”— embedded in the BRAD Brassica Database (BRAD; http://brassicadb.org/brad/searchSyntenytPCK.php). There was a total of 12,914, 8,905 and 7,719 genes in LF, MF1 and MF2 subgenome, respectively, syntenic to A. thaliana (Suppl. Table S6).

Paleocentromeres in the B. rapa Genome Paleocentromere sequence analysis indicated that the 10 B. rapa centromeres were all inherited from the triplicated tPCK-like genomes. Using the B. rapa genome sequence, we analyzed the fate of paleocentromeres corresponding to 21 ancestral chromosomes of the three tPCK-like genomes. After aligning (peri)centromere-specific repeats, including centromeric satellite repeats CentBr (Lim et al., 2005; Koo et al., 2011), PCRBr, and TR238 (Lim et al., 2007) to the B. rapa genome sequence, signals were detected for 18 paleocentromere regions. The paleocentromeres of three ancestral chromosomes (AK3(MF1), AK3(MF2), and AK6/8(MF1)) could not be located (Figures 2B and 3, Suppl. Table S7). Among the 18 detected paleocentromeres, 10 were involved in the origin of the 10 B. rapa centromeres (Table 2, Figures 2B and 3). The centromeres on chromosomes A04, A05, A07, and A09 correspond to ancestral centromeres of AK7, AK1, AK3, and AK2/5/6/8, respectively, with two ancestrally positioned GBs facing the centromeres on either sides. The centromeres of A01, A03, and A10 were derived via a translocation or pericentric inversion event from paleocentromeres of AK2/5/6/8, AK2/5/6/8, and AK6/8, respectively, leaving only one of the two GBs associated with the ancestral centromere. The origin of centromeres on A02, A06, and A08 is directly linked to a reduction in chromosome number. For these centromeres we inferred translocation events with breakpoints within the (peri)centromeric regions of two different ancestral chromosomes resulting in the loss of one of the two paleocentromeres. The available data do not allow to decide which three of the six participating paleocentromeres remained active in B. rapa (Table 2, Figure 3).

131 Figure 3. The fate of 21 paleocentromeres in the triplicated ancestral genome of B. rapa. The central column shows triplicated tPCK-like chromosomes with paleocentromeres and two adjacent genomic blocks highlighted. The left and right columns display the extant or purported location of the ancestral centromeres on B. rapa chromosomes. All blocks that are not ancestrally adjacent to paleocentromeres are plotted in semitransparent colors. Three instances of extant centromeres having an equally possible origin from any of two different paleocentromeres are marked by connecting dashed lines.

Of the eight remaining ancestral centromeres, six were disrupted by translocation events, with sequence traces detectable at four paleocentromere regions (Figure 3). Chromosomes A03 and A04 contained two entirely conserved ancestral chromosomes with inactivated/deleted centromeres. AK3 centromere left no sequence remnants between blocks GMF2 and HMF2 within A03, whereas centromere sequence traces (PCRBr and TR238 in the boundary of J/IMF1) corresponding to AK4 (Suppl. Table S7) were detected on A04 (Figures 2B and 3, Figure 3).

132 Comparison of B. rapa Ancestral Genomes with Other Brassicaceae Genomes To obtain further insights into the origin and phylogenetic context of the B. rapa ancestral genomes, we investigated whether the tPCK-specific GB associations D/V/K/L/Q/X, O/P/W/R, M/E, and D/V are present in genomes of other Brassica and Brassiceae species, as well as other lineage II species (i.e., B. oleracea, B. nigra, Raphanus sativus, Sinapis alba, Caulanthus amplexicaulis, S. parvula, and T. salsuginea; Figure 1B). Three copies of the first two tPCK-specific block associations were identified in B. oleracea assembled scaffolds (Suppl. Table S8). In the B. nigra genome analyzed within the allotetraploid B. juncea, two copies of V/K/L/Q/X were located on chromosomes B1 and B6, whereas the third copy was split between B4 and B8. Similarly, two copies of O/P/W/R were located on B2 and B3, while the third copy was rearranged on chromosome B8 (Panjabi et al., 2008) (Suppl. Figure S2). Furthermore, the D/V association was found on one scaffold of B. oleracea (Suppl. Table S8) as well as on chromosomes B1 and B6 in B. nigra (Panjabi et al., 2008) (Suppl. Figure S2). Similar to B. rapa, the M/E association was not found in B. oleracea; however, one copy of M/E was rearranged with O/P/W/R on B2 in B. nigra (Panjabi et al., 2008) (Suppl. Figure S2). These analyses suggest that the three Brassica genomes (A, B, and C) descended from a common hexaploid ancestor that originated from the merger of three tPCK-like ancestral genomes. For radish (R. sativus), we used an EST linkage map (Shirasawa et al., 2011) to align EST sequences to A. thaliana genes. These results were then used to determine the position of 24 GBs on the nine R. sativus chromosomes (Suppl. Table S9). Most of the 24 GBs (75%) were present in three copies in the radish genome. The two tPCK-specific block associations (V/K/L/Q/X and O/P/W/R) were detected in triplicate on chromosomes LG2, LG4, and LG6, and LG5, LG6, and LG8, respectively. In addition, two copies of V/K/L/Q/X blocks were associated with D on chromosomes LG2 and LG6, and one copy of O/P/W/R was rearranged with M/E on LG5 (Suppl. Table S9). This suggests that at least two tPCK-like genomes were involved in the origin of the radish genome. In the diploid genome of S. parvula, tPCK-specific GB associations D/V/K/L/Q/X, O/P/W/R, and M/E were located on chromosomes SpChr2, SpChr6, and SpChr5, respectively (Dassanayake et al., 2011). tPCK-specific block associations were found to be conserved also in the T. salsuginea genome (Wu et al., 2012). Comparative genome mapping in white mustard (S. alba; Nelson et al., 2011) and C. amplexicaulis (Burrell et al., 2011) also revealed tPCK-specific arrangements of genomic blocks. Because of extensive genome rearrangements and the limited resolution of the linkage maps, however, no clear conclusions could be drawn for the two species. In S. alba, V/K/L/V/Q on chromosomes S10 and S11 and O/P/W/R on S09 were rearranged and not readily identifiable. The M/E association was identified on S02 (Nelson et al., 2011), but the D/V association was not found. In Caulanthus, the block association D/V/K/L/Q/X on LG3 was fragmented to such extent that it could not be clearly identified (i.e., blocks V, L, and Q were disrupted, while K remained undetected). The O/P/W/R association was observed on LG2 (with blocks P and R fragmented), while the M/E association was not present (Burrell et al., 2011).

133 Analysis of Synonymous Substitution Rates Using the syntenic orthologs identified for B. rapa, A. thaliana, A. lyrata, S. parvula, and T. salsuginea, we calculated Ks values (the number of substitutions per synonymous site) for B. rapa relative to the other crucifer species. Ks for B. rapa relative to S. parvula was smaller (~0.29–0.31, ~10 million years of divergence; Koch et al., 2000) than that relative to T. salsuginea (~0.34–0.36, ~11.7 million years), A. lyrata (~0.41–0.45, ~14.3 million years), and A. thaliana (~0.42–0.45, ~14.5 million years) (Figure 4A). Based on these calculations, B. rapa is thus phylogenetically closer to S. parvula than to T. salsuginea or to the genus Arabidopsis. Ks values for each B. rapa subgenome relative to S. parvula ranged from 0.29 to 0.31 (Figure 4B), while those for the subgenomes relative to one another ranged from 0.29 to 0.33 (i.e., ~10.3 million years) (Figure 4C). To investigate the evolutionary relationship of A. thaliana, A. lyrata, S. parvula, T. salsuginea, and the three B. rapa subgenomes, we carried out a phylogenetic analysis using shared synonymous SNPs for these five genomes and the two outgroup species Carica papaya and Vitis vinifera. From the 591 syntenic orthologs shared by the three B. rapa subgenomes and the six other species, 63,239 Ks loci (i.e., synonymous SNPs; see Methods) were identified and concatenated into a dataset that was subjected to phylogenetic analysis. Based on the resulting tree, Brassiceae and Arabidopsis ancestors diverged prior to the Brassiceae-Thellungiella split. The Brassiceae-specific whole-genome triplication occurred near the time of the split between the progenitors of the Brassiceae and Schrenkiella (Figure 4D).

134

Figure 4. Pairwise comparison of Ks values and a phylogenetic tree based on Ks loci. (A) The distribution of Ks values between B. rapa and crucifer species T. salsuginea, S. parvula, A. thaliana, and A. lyrata. (B)

The distribution of Ks values between each of the three B. rapa subgenomes and S. parvula. (C) The distribution of Ks values between pairs of the three B. rapa subgenomes. (D) Phylogenetic tree for the four crucifer species and the three B. rapa subgenomes, with non-Brassicaceae species Carica papaya and Vitis vinifera used as outgroups. The tree was constructed based on Ks loci for all syntenic orthologs shared among the seven species.

135 Reevaluation of Block Association V/K/L/Wa/Q/X and Interval Refinement of Seven Genomic Blocks

Figure 5. Redefinition of intervals for genomic blocks (GBs) H, I, Q, R, V, W, and X, and the interspecies collinearity of the diagnostic block association V/K/L/Wa/Q/X. Block intervals were redefined based on interspecies comparison among genomes of A. lyrata (Scaffold_8 on chromosome AK8), A. thaliana (At5), S. parvula (Sp2), and B. rapa (A02, A06, A09). (A) The diagnostic block association V/K/L/Wa/Q/X. The split of block W into Wa and Wb preceded the formation of the block association V/K/L/Wa/Q/X in B. rapa, S. parvula, and probably all species of the expanded lineage II. Block V is inverted in A. thaliana, S. parvula, and B. rapa compared with the ancestral ACK-like orientation in A. lyrata. (B) Refined intervals of seven ancestral GBs shown as corresponding A. thaliana genes located on chromosomes At2 and At5. GB intervals as defined by Schranz et al. (2006) appear in black on the left, whereas newly refined intervals are listed in red on the right. The boundary between Wa and Wb is located between AT5G49630 and AT5G49640.

The results of the multispecies comparison prompted a reevaluation of the V/K/L/Q/X block association considered specific for the x = 7 tribes of expanded lineage II (Mandakova and Lysak, 2008). This association was identified on chromosome Sp2 in S. parvula, albeit with a fragment of block V between L and Q (V/K/L/V'/Q/X) (Dassanayake et al., 2011). The same rearrangement was found on B. rapa chromosomes

136 A02, A06, and A09 (Figure 5A), B. oleracea (Suppl. Table S8) and R. sativus (Li et al., 2011; Shirasawa et al., 2011). We analyzed this chromosome region by performing an interspecies comparison between B. rapa, B. oleracea, S. parvula, A. thaliana and A. lyrata, with the A. lyrata genome (Hu et al., 2011) serving as a proxy for the ACK genome (n = 8). The presumed V' region turned out to be part of block W, located on the bottom arm of the AK8 (Al8) chromosome and adjacent to the centromere (Figure 5A). The origin of the V/K/L/Wa/Q/X rearrangement, which is specific to the ancestral PCK genome, is consistent with the previously proposed scenario (Mandakova and Lysak, 2008), except that one of the breakpoints is actually positioned within block W (Figure 5B and Suppl. Figure S4A). An alternative scenario involves a paracentric inversion of blocks V/K/L followed by inactivation/removal of the AK8 paleocentromere (Suppl. Figure S4B). These results, together with multispecies comparisons of syntenic chromosomes, allowed us to redefine the boundaries of blocks V and W (Table 3 and Figure 5A). By comparing the boundaries of blocks H, I, Q, R, V, W, and X reported by Schranz et al. (2006) with corresponding regions and/or breakpoints within genomes of B. rapa, A. lyrata, S. parvula, and A. thaliana (Suppl. Table S10), we revised intervals of the seven blocks of ACK (Table 3 and Figure 5B).

137 Discussion

We compared the whole-genome sequence of B. rapa (Wang et al., 2011) with genome sequences or genetic maps of other crucifer species, including A. thaliana, A. lyrata, B. oleracea, B. nigra, C. amplexicaulis, R. sativus, S. alba, S. parvula, and T. salsuginea, as well as with previously-proposed Brassicaceae ancestral genomes (Schranz et al., 2006; Mandakova and Lysak, 2008; Panjabi et al., 2008; Burrell et al., 2011; Dassanayake et al., 2011; Hu et al., 2011; Li et al., 2011; Nelson et al., 2011; Shirasawa et al., 2011; Wu et al., 2012). We have provided conclusive evidence that the B. rapa genome arose via a whole-genome triplication event involving three n = 7 genomes structurally resembling the ancestral translocation Proto-Calepineae Karyotype (tPCK). tPCK-like Ancestral Genomes and the Origin of the Mesohexaploid Brassica Genome Since the hypothesis was proposed that diploid Brassica species represent “balanced secondary polyploids” (e.g., Catcheside, 1934; Röbbelen, 1960), numerous lines of evidence have supported the occurrence of an ancestral whole-genome triplication prior to species radiation of the tribe Brassiceae (e.g., Lagercrantz, 1998; Lysak et al., 2005; Parkin et al., 2005; Wang et al., 2011). In this study, we inferred the tPCK-like genome with seven chromosomes to be the basic diploid genome of the hexaploid ancestor of Brassiceae. The hexaploid genome likely evolved through hybridization between a tetraploid and diploid progenitor genome. Evidence for this is based on genetic mapping (Lagercrantz, 1998; Babula et al., 2003), comparative cytogenetic analyses (Lysak et al., 2005; Ziolkowski et al., 2006), and, most convincingly, whole-genome sequence analysis. Wang et al. (2011) found that the three subgenomes in B. rapa have experienced different levels of gene loss. The more extensive fractionation of two subgenomes (MF1 and MF2) compared with the third subgenome (LF) suggest a two-step origin for the hexaploid genome. Several recent investigations (Cheng et al., 2012b; Tang and Lyons, 2012; Tang et al., 2012) convincingly support the origin of the Brassiceae ancestral genome according to the formula 4x × 2x  3x  6x. Here, we provide evidence that all three participating ancestral genomes had seven chromosomes and that the hexaploid ancestor probably possessed 42 chromosomes (2n = 6x = 42). tPCK in a Phylogenetic Context Because species of the genus Brassica and closely related genera, such as Raphanus and Sinapis, can be intercrossed, and generic circumscriptions based on morphological characters are unreliable, congeneric species have frequently been assigned to two different sister phylogenetic clades within Brassiceae: Rapa/Oleracea and Nigra (e.g., see Warwick and Black, 1991). To better understand the structure of Brassica progenitor genomes, we compared tPCK-specific block associations with chromosome structures of Rapa/Oleracea (B. oleracea, B. rapa, and R. sativus) and Nigra (B. nigra) species. We found that all three Rapa/Oleracea species (B. oleracea, B. rapa, and R. sativus) retained three copies of the V/K/L/Wa/Q/X and O/P/W/R associations. D/V or M/E associations

138 were also found in the three species. In addition, chromosome synteny among B. nigra, B. oleracea, and B. rapa, and the existence of the tPCK-specific associations D/V and M/E in B. nigra and D/V in B. oleracea and R. sativus indicate that B. nigra, B. oleracea, and R. sativus had the same origin as B. rapa. Furthermore, some tPCK-specific associations observed within the comparative linkage map of S. alba (Nelson et al., 2011), a species from the Nigra clade, suggest that the genome of this species is also derived from a triplicated tPCK-like ancestral genome. The tribes Brassiceae, Isatideae, Thelypodieae, and Sisymbrieae were designated as lineage II of the Brassicaceae (Al-Shehbaz et al., 2006; Beilstein et al., 2006; Beilstein et al., 2008). The relationship, however, of these tribes to the two other Brassicaceae lineages and to the remaining 45 tribes remains uncertain. Franzke et al. (2011) grouped lineage II with 18 other tribes to form an expanded lineage II. Using comparative chromosome painting, Mandakova and Lysak (2008) provided valuable insight into the phylogenetics of expanded lineage II. They demonstrated that tribes Calepineae, Coluteocarpeae, Conringieae, Eutremeae, Isatideae, the genus Ochthodium (formerly Sisymbrieae), and probably the Brassiceae descended from an ancestral PCK genome. In Eutremeae, Isatideae and Ochthodium, PCK was altered by a whole-arm translocation giving rise to tPCK. The tPCK genome structure was recently confirmed by draft genome assemblies in T. salsuginea (from Eutremeae) (Wu et al., 2012) and in S. parvula (Dassanayake et al., 2011). Although the tribal placement of Schrenkiella is still uncertain (Al-Shehbaz, 2012), our results with respect to Ks values place Brassiceae closer to Schrenkiella than to Eutremeae (Thellungiella) and indicate that the Brassiceae triplication event occurred near the time of the divergence between Brassiceae and Schrenkiella (~10 million years ago). Collectively, our data suggest that the ancestor of Brassiceae, Isatideae, Schrenkiella, Ochthodium, and Eutremeae, and probably also Thelypodieae (Caulanthus), descended from the same diploid tPCK-like ancestral genome. While the triplicated tPCK genome has undergone extensive repatterning in Brassiceae, the ancestral structure of the tPCK genome has been conserved in diploid x = 7 taxa, such as Schrenkiella or Thellungiella, where no diploidization of a polyploid genome could occur.

Descending Dysploidy in the Hexaploid Ancestor of Brassiceae The chromosome number of 2n = 42 of the hexaploid ancestor has been reduced by a factor of two during the evolution of Brassiceae (Warwick and Al-Shehbaz, 2006). Such reductions are associated with the origin of composite chromosomes and loss of chromosome segments such as centromeres, telomeres, and nucleolar organizing regions. In B. rapa, descending dysploidy led to the origin of 10 “fusion” chromosomes and the elimination of 11 paleocentromeres. Some paleocentromeres were eliminated through symmetric translocations, thus resulting in products of unequal size: a large “fusion” chromosome, and minichromosome which contains mainly a centromere and telomeres and usually gets lost during meiosis (Lysak et al., 2006; Schranz et al., 2006; Schubert and Lysak, 2011). Other ancestral chromosomes were combined by asymmetric end-to-end translocations (e.g., between AK6/8 and AK3; in A03; Figure 2B). End-to-end translocation events must be accompanied by centromere loss or

139 inactivation on one of the participating chromosomes to prevent unstable dicentric chromosomes. Centromeres were apparently lost from the collinear GBs F/G/H (AK3) of A03 and I/J (AK4) of A03 and A04 (Figure 2B). Nested chromosome fusions (i.e., insertion of one chromosome into the centromere of a recipient chromosome), which occurred frequently in the evolution of grass species (Luo et al., 2009; Salse et al., 2009; Murat et al., 2010), have not been detected in the karyotype of B. rapa. In Brassicaceae, single such events apparently occurred in Pachycladon species (Mandakova et al., 2010a) and in Hornungia alpina (Lysak et al., 2006). We also analyzed whether recombination between homoeologous chromosome segments of the three B. rapa subgenomes exceeded recombination between non-homoeologous segments. If recombination between homoeologous segments was prevalent, we would expect to find the same GBs located adjacent to one another (e.g., association A(MF1)/A(MF2)). In B. rapa, only four such associations were detected (S(LF)/S(MF2) on A04, M(LF)/M(MF2) on A06, E(MF2)/E(LF) on A07, and D(MF1)/D(MF2) on A09). These data suggest that interchromosomal rearrangements were not mediated preferentially by recombination between homoeologous GBs. Nevertheless, the true picture may have been blurred by numerous rearrangements (typically inversions) that may have occurred later and reshuffled originally adjacent homoeologous genomic blocks.

Whole-Genome Sequence Assembly Improved the Resolution of Comparative Paleogenomics in Brassicaceae The B. rapa genome sequence (Wang et al., 2011) provided much more detailed information on genome structure than previously constructed linkage maps. Although most genomic blocks and parts thereof (68%) were detected by genetic mapping (Parkin et al., 2005; Panjabi et al., 2008; Kim et al., 2009; Trick et al., 2009), some blocks and block associations were missed or reported as being present in less than three copies. A comparison of the B. rapa genome sequence with previously reported linkage maps shows that block D on chromosome A01; blocks W, Q, X, M, and D on A03; S on A05; M, H, and U on A6; F, X, and S on A07; S on A08; K, L, and M on A09; and C on A10 were not detected by genetic mapping (Parkin et al., 2005; Panjabi et al., 2008; Kim et al., 2009; Trick et al., 2009). Although the assembly of B. rapa chromosome A03 by Mun et al. (2010) identified 14 GBs or block segments, three additional blocks on the same chromosome were detected within the B. rapa reference genome (Wang et al., 2011). Similarly, the 2.4-Mb fragment of block W within the V/K/L/Wa/Q/X association was not detected by genetic mapping. Moreover, two adjacent copies of the same GB belonging to different subgenomes have often been regarded as a single block in previous genetic maps, including blocks S(LF)/S(MF2), E(LF)/E(MF2), and D(MF1)/D(MF2) on chromosomes A04, A07, and A09, respectively (see Panjabi et al., 2008 and Figure 2B). Furthermore, we found that earlier reports on GBs represented by more than three copies were mostly due to misinterpretation of block segments as entire blocks. For instance, Panjabi et. al (2008) had observed four copies of block A located on chromosomes A06, A08, A09, and A10. Analysis of B. rapa genome sequences revealed, however, that the two copies of block A on chromosomes A06 and A10 were in fact two

140 segments of this block (Figure 2B). These examples demonstrate that whole-genome sequence assemblies largely eliminate the risk of misinterpreting low-resolution genetic data.

Refining Intervals of Ancestral Genomic Blocks The original concept of 24 GBs (Schranz et al., 2006) had resulted from the synthesis of comparative genetic and cytogenetic data for A. thaliana, A. lyrata, C. rubella, the A and C genomes of B. napus, and other lineage I species (Boivin et al., 2004; Kuittinen et al., 2004; Parkin et al., 2005; Lysak et al., 2006). Because A. thaliana had been the only crucifer species with a sequenced genome at that time, chromosome collinearity between all Brassicaceae species had been inferred by mapping homoeologous genetic markers from A. thaliana and by chromosomal location of A. thaliana BAC clones and contigs. This approach was largely influenced by the density of genetic markers and the selection of chromosome-specific BAC contigs. Without sufficiently accurate information on cross-species collinearity, only approximate or minimal intervals could be defined for some GBs (Schranz et al., 2006) and GB associations. This is exemplified by the PCK/tPCK-specific chromosome rearrangement initially described as V/K/L/Q/X (Mandakova and Lysak, 2008) and V/K/L/V'/Q/X (Dassanayake et al., 2011), and eventually refined as V/K/L/Wa/Q/X in this study. The extensive repatterning of ancestral GBs within the B. rapa genome, along with newly available sequence information for Brassicaceae genomes, allowed us to refine the intervals of seven genomic blocks. This was particularly feasible when individual GBs were found to be relocated and forming a non-ancestral GB association with corresponding breakpoints at syntenic positions in two or three B. rapa subgenomes. The interval refinement of seven GBs will improve the resolution of interspecies comparisons of genome collinearity across Brassicaceae.

141 Methods

Syntenic Ortholog Determination Syntenic orthologs between B. rapa, A. thaliana, A. lyrata, S. parvula, T. salsuginea, C. papaya, and V. vinifera were identified using the tool SynOrths (Cheng et al., 2012a), with B. rapa as the query genome and each of the other species as the subject genome. SynOrths was run with default parameters (m = 20, n = 100, r = 0.2). Under these settings, SynOrths first identifies homoeologous gene pairs between the two genomes using BLASTP, defining gene pairs as those with an E-value more significant than 1x10-20 or representing the best hits through the entire genome. The 20 closest genes (m = 20) flanking either side of the gene in the query B. rapa genome are then compared with the 100 closest genes (n = 100) flanking either side of the gene in the subject genome. If at least 20% (r = 0.2) of the best hits for the 40 genes (20 × 2 for both sides) in the query B. rapa genome are found within the 200 genes (100 × 2 for both sides) in the subject genome, the original pair of homoeologous genes are designated as a syntenic ortholog candidate. The increase in the number of flanking genes searched in the subject genomes was necessary to compensate for the fractionation that has reduced the gene content of each B. rapa subgenome.

Comparison of Arrangements of GB Associations We searched the distribution of predefined GBs in the three genomes of the ACK, PCK, and tPCK for adjacent pairs of GBs (GB associations). These GB associations were compared with the extant arrangement of GBs within B. rapa. GB associations in ACK, PCK, and tPCK that were also immediately adjacent to each other in the B. rapa genome were counted and recorded.

Reconstruction of Subgenomes Based on the tPCK As previously reported (Wang et al., 2011; Cheng et al., 2012b), the B. rapa genome is composed of three subgenomes each of the 24 GBs. We annotated these blocks along the seven chromosomes of the tPCK and reconstructed three tPCK-like subgenomes. At first we identified all continuous chromosome segments corresponding to genomic blocks and belonging to the same chromosome of the tPCK. Then, chromosome segments were concatenated according to the following rules: (i) reconstructed chromosome could not contain redundant copies of the same syntenic segment, and (ii) the concatenated chromosome segments had to have a comparable gene density corresponding to the three B. rapa subgenomes (LF, MF1, and MF2).

Ancestral Centromere Detection Centromere-specific sequences, including CentBr, PCRBr, and TR238, were obtained from previous studies (Koo et al., 2004; Lim et al., 2005; Lim et al., 2007; Koo et al., 2011). These sequences were aligned to the B. rapa genome using Nucmer (Kurtz et al., 2004) with parameters “-maxmatch -g 500 -c 16 -l 16 ” (i.e., find all matches with minimal exact match size of 16 bp regardless of uniqueness, merge exact matches

142 distributed less than 500 bp apart into clusters, and report all clusters >16 bp). The nearest genes to the best Nucmer hits were determined and used as labels of genomic coordinates for the detected centromere traces (Suppl. Table S7).

Identification of tPCK-specific Block Associations in B. oleracea Because a pseudomolecule-level assembly for the B. oleracea genome is not yet available, we used a different method to determine local syntenic relationships between B. rapa and B. oleracea. Thirty B. rapa genes located on either side of the boundaries between GBs within the GB associations O/P/W/R and D/V/K/L/Q/X were selected. These genes were compared using BLAST against B. oleracea genes via the web interface provided by BRAD (http://brassicadb.org/brad/blastPage.php) (Cheng et al., 2011). Two blocks were classified as being syntenic in B. oleracea when more than 60% of the B. rapa block boundary genes from each GB had BLAST hits (identity and coverage) on the same B. oleracea scaffold. For example, if 1) 60% of 30 B. rapa genes at the boundary of block O (near P) were homoeologous to genes on Scaffold000203 of B. oleracea, 2) 30 B. rapa genes at the block P boundary (near O) were also homoeologous to B. oleracea genes in Scaffold000203, and 3) these genes were adjacent to each other on Scaffold000203, then the O/P association was considered to be present also in the B. oleracea genome.

Ks Analysis Protein sequences from B. rapa were aligned with those of A. thaliana, A. lyrata, S. parvula, and T. salsuginea using MUSCLE (Edgar, 2004). Protein alignments were translated into coding sequence alignments using an in-house Perl script. Ks values were calculated based on the coding sequence alignments using the method of Nei and

Gojobori as implemented in KaKs_calculator (Zhang et al., 2006). Ks values of all syntenic orthologs between B. rapa and each of the four species were then plotted as histograms.

Phylogenetic Analysis

For phylogenetic analysis, the same MUSCLE alignments as for Ks analysis were used. The genotypes of synonymous loci in 591 syntenic orthologs among B. rapa, S. parvula, T. salsuginea, A. thalaliana, A. lyrata, C. papaya, and V. vinifera were extracted from multiple alignments and concatenated into a single sequence for each species. Phylogenies were constructed, based on the character states of these 63,239 synonymous loci using the neighbor-joining method, and plotted under a linearized tree model as implemented in MEGA (Tamura et al., 2011).

Accession Numbers Gene and protein sequences for B. rapa were obtained from BRAD (V1.2; http://brassicadb.org) (Cheng et al., 2011). Gene and genome datasets for A. thaliana were downloaded from TAIR (TAIR9; http://www.arabidopsis.org/index.jsp). Datasets for A. lyrata were downloaded from the JGI database (Gene models 6; http://genome.jgi-psf.org/Araly1/Araly1.home.html) (Hu et al., 2011). S. parvula

143 datasets (TpV7) were obtained from Dassanayake et al. (2011). T. salsuginea data were downloaded from NCBI (Accession no. AHIU00000000) (Wu et al., 2012).

Supplemental Data

The following materials are available in the online version of this article. Supplemental Figure S1. Purported translocation events reshuffling tPCK-specific associations D/V and M/E in the B. rapa genome. (A) Translocation between block UMF1 and block association D/VLF resulted in block associations Ua/D and V/Ub on B. rapa chromosomes A03 and A06, respectively. (B) Translocation between two M/E associations from subgenomes LF and MF2 resulted in associations MLF/MMF2 and ELF/EMF2 on chromosomes A06 and A07, respectively. Supplemental Figure S2. Chromosome collinearity comparison among A, B, and C genomes, revised from Panjabi et al. (2008). PCK-specific GB associations O/P/W/R and V/K/L/Q/X are colored to distinguish subgenomes: red = LF, green = MF1, blue = MF2. Each copy of block D associated with block V is indicated in yellow. The entire chromosomes A1 and C1, and A2 and C2 have a similar block orders between A and C genomes; the structures of A4 and B4, A5 and B5, and A6 and B6 are shared between genomes A and B; A3, B3, and C3 share similar chromosome structures between A, B, and C genomes. Supplemental Figure S3. Reconstruction of three tPCK-like ancestral subgenomes in B. rapa. tPCK-like genomes each comprising 24 genomic blocks (A–X) were reconstructed for the three B. rapa subgenomes (least fractionated [LF], medium fractionated [MF1], and most fractionated [MF2]). Ancestral centromeres still active in B. rapa, traces of ancestral centromeres, and theoretically purported but undetected ancestral centromeres are represented by ovals of different colors. Red arrows highlight breakpoints shared by two or three subgenomes. The downward pointing arrow refers to the genomic block inverted relative to the position within the ACK. Supplemental Figure S4. Two alternative origins of translocation chromosomes AK5/6/8 (PCK) and AK2/5/6/8 (tPCK). Genomic blocks are indicated by capital letters and colored according to their positions on ancestral chromosomes (see Figure 2A). Downward pointing arrows indicate the opposite orientation of GBs compared to their ancestral position within the ACK. Scenario (A) involves a double inversion of genomic block V, whereas alternatively (B) is based on a paracentric inversion of V/K/L and accompanying inactivation of the AK8 paleocentromere. The position(s) of the inactivated centromere depend(s) on the position of the inversion breakpoint within the AK8 (peri)centromere. Supplemental Table S1. Summary of genomic blocks identified in B. rapa. Supplemental Table S2. Genomic blocks E, G, and T showing sequence deletions in B. rapa (Br) as compared with A. thaliana (At). Supplemental Table S3. Number of genomic blocks with different fragmental status in B. rapa subgenomes. Supplemental Table S4. Syntenic fragments comparison between B. rapa and the Ancestral Crucifer Karyotype (ACK). Supplemental Table S5. Frequency of tPCK-specific block associations in each of the three B. rapa subgenomes. Supplemental Table S6. Number of syntenic genes shared between each B. rapa

144 subgenome and the A. thaliana genome (At). Supplemental Table S7. Centromere-specific sequences detected in the B. rapa genome. Supplemental Table S8. The frequency of tPCK-specific block associations in B. rapa and B. oleracea. Supplemental Table S9. List of genomic blocks comprising the nine linkage groups of Raphanus sativus (Shirasawa et al., 2011). Supplemental Table S10. Redefined intervals of seven ancestral genomic blocks.

Acknowledgements

We thank Drs. Michael Freeling and James Schnable for their helpful suggestions concerning this work. This study was funded by the National Program on Key Basic Research Projects (The 973 Program: 2012CB113900, 2013CB127000), and the National High Technology R&D Program of China (2012AA100201). Research was carried out in the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, P. R. China. T. M. and M. A. L. were supported by a research grant from the Czech Science Foundation (Excellence Cluster P501/12/G090), and by the European Regional Development Fund (CZ.1.05/1.1.00/02.0068).

145 Tables

Table 1 Comparison of genomic block (GB) associations shared between B. rapa and three ancestral crucifer genomes (Ancestral Crucifer Karyotype [ACK], Proto-Calepineae Karyotype [PCK], and translocation Proto-Calepineae Karyotype [tPCK]).

ACK (n = 8) PCK (n = 7) tPCK (n = 7) Chra GB assoc.b #c B. rapa Chr Chr GB assoc.# B. rapa Chr Chr GB assoc.# B. rapa Chr A/B 2 A06, A08 A/B 2 A06, A08 A/B 2 A06, A08 AK1 AK1 AK1 B/C 2 A05, A08 B/C 2 A05, A08 B/C 2 A05, A08 AK2 D/E 0 - AK2 D/E 0 - N/M 3 A01, A03, A09 AK2/5 F/G 2 A03, A07 F/G 2 A03, A07 M/E 0 - AK3 AK3 G/H 2 A03, A07 G/H 2 A03, A07 F/G 2 A03, A07 AK3 AK4 I/J 2 A03, A04 AK4 I/J 2 A03, A04 G/H 2 A03, A07 K/L 3 A02, A06, A09 S/T 2 A04, A08 AK4 I/J 2 A03, A04 AK7 AK5 L/M 0 - T/U 3 A01, A03, A08 S/T 2 A04, A08 AK7 M/N 3 A01, A03, A09 O/P 3 A02, A03, A09 T/U 3 A01, A03, A08 O/P 3 A02, A03, A09AK6/8 P/W 1 A03 O/P 3 A02, A03, A09 AK6 P/Q 0 - W/R 3 A02, A03, A10 AK6/8 P/W 1 A03 Q/R 0 - N/M 3 A01, A03, A09 W/R 3 A02, A03, A10 S/T 2 A04, A08 M/V 0 - D/V 1 A09 AK7 T/U 3 A01, A03, A08 V/K 3 A02, A06, A09 V/K 3 A02, A06, A09 AK5/6/8 V/W 0 - K/L 3 A02, A06, A09 AK2/5/6/8 K/L 3 A02, A06, A09 AK8 W/X 0 - L/Q 3 A02, A06, A09 L/Q 3 A02, A06, A09 Q/X 3 A02, A03, A09 Q/X 3 A02, A03, A09 aancestral chromosome. bgenomic block association. ccopy number of GB association in the B. rapa genome.

146 Table 2 The inheritance of 10 B. rapa centromeres from the triplicated ancestral tPCK-like genome (n = 21).

B. rapa Ancestral B. rapa chromosome chromosome subgenome A01 AK2/5/6/8 MF1 A02 AK6/8 or AK2/5 MF2 or MF1 A03 AK2/5/6/8 LF A04 AK7 LF A05 AK1 MF2 A06 AK2/5 or AK2/5 LF or MF2 A07 AK3 LF A08 AK1 or AK7 MF1 or MF2 A09 AK2/5/6/8 MF2 A10 AK6/8 LF

147 Table 3 Interval redefinition of seven genomic blocks (GBs) in the Ancestral Crucifer Karyotype (ACK). Newly defined GB intervals based on multi-genome comparison are underlined and shown in bold. Intervals of the 17 remaining blocks follow Schranz et al. (2006). Each GB interval is defined by two A. thaliana genes.

AK GB Interval chromosome A 1 AT1G01560 AT1G19330 B 1 AT1G19850 AT1G36240 C 1 AT1G43600 AT1G56120 D 2 AT1G63770 AT1G56530 E 2 AT1G65040 AT1G80420 F 3 AT3G01040 AT3G25520 G 3 AT2G05170 AT2G07690 H 3 AT2G15670 AT2G20900 I 4 AT2G20920 AT2G28910 J 4 AT2G31040 AT2G47730 K 5 AT2G01250 AT2G03750 L 5 AT3G25855 AT3G29770 M 5 AT3G43740 AT3G49970 N 5 AT3G50950 AT3G62790 O 6 AT4G00030 AT4G04955 P 6 AT4G12070 AT4G08690 Q 6 AT5G28885 AT5G23010 R 6 AT5G23000 AT5G01010 S 7 AT5G41900 AT5G33210 T 7 AT4G12750 AT4G16143 U 7 AT4G16250 AT4G38770 V 8 AT5G42130 AT5G47810 W 8 AT5G47820 AT5G60800 X 8 AT5G60805 AT5G67385

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152 VIII. Mandáková T., Heenan P.B., Lysak M.A. 2010. Island species radiation and karyotypic stasis in Pachycladon allopolyploids. BMC Evolutionary Biology 10: 367.

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RESEARCH ARTICLE Open Access Island species radiation and karyotypic stasis in Pachycladon allopolyploids Terezie Mandáková1, Peter B Heenan2, Martin A Lysak1*

Abstract Background: Pachycladon (Brassicaceae, tribe Camelineae) is a monophyletic genus of ten morphologically and ecogeographically differentiated, and presumably allopolyploid species occurring in the South Island of New Zealand and in Tasmania. All Pachycladon species possess ten chromosome pairs (2n = 20). The feasibility of comparative chromosome painting (CCP) in crucifer species allows the origin and genome evolution in this genus to be elucidated. We focus on the origin and genome evolution of Pachycladon as well as on its genomic relationship to other crucifer species, particularly to the allopolyploid Australian Camelineae taxa. As species radiation on islands is usually characterized by chromosomal stasis, i.e. uniformity of chromosome numbers/ploidy levels, the role of major karyotypic reshuffling during the island adaptive and species radiation in Pachycladon is investigated through whole-genome CCP analysis. Results: The four analyzed Pachycladon species possess an identical karyotype structure. The consensual ancestral karyotype is most likely common to all Pachycladon species and corroborates the monophyletic origin of the genus evidenced by previous phylogenetic analyses. The ancestral Pachycladon karyotype (n = 10) originated through an allopolyploidization event between two genomes structurally resembling the Ancestral Crucifer Karyotype (ACK, n = 8). The primary allopolyploid (apparently with n = 16) has undergone genome reshuffling by descending dysploidy toward n = 10. Chromosome “fusions” were mediated by inversions, translocations and centromere inactivation/loss. Pachycladon chromosome 3 (PC3) resulted from insertional fusion, described in grasses. The allopolyploid ancestor originated in Australia, from the same or closely related ACK-like parental species as the Australian Camelineae allopolyploids. However, the two whole-genome duplication (WGD) events were independent, with the Pachycladon WGD being significantly younger. The long-distance dispersal of the diploidized Pachycladon ancestor to New Zealand was followed by the Pleistocene species radiation in alpine habitats and characterized by karyotypic stasis. Conclusions: Karyotypic stasis in Pachycladon suggests that the insular species radiation in this genus proceeded through homoploid divergence rather than through species-specific gross chromosomal repatterning. The ancestral Pachycladon genome originated in Australia through an allopolyploidization event involving two closely related parental genomes, and spread to New Zealand by a long-distance dispersal. We argue that the chromosome number decrease mediated by inter-genomic reshuffling (diploidization) could provide the Pachycladon allopolyploid founder with an adaptive advantage to colonize montane/alpine habitats. The ancestral Pachycladon karyotype remained stable during the Pleistocene adaptive radiation into ten different species.

Background eroded by genomic fractionation toward diploid-like Multiple rounds of WGD events (both allopolyploidy genomes. Lineage-specific WGD events followed by gen- and autopolyploidy) cyclically increase the genetic diver- ome repatterning and descending dysploidy toward sity in vascular plants, and subsequently this is steadily diploid-like genomes have been revealed in several angiosperm groups and are probably best characterized * Correspondence: [email protected] in grasses [1-5] and Brassicales [6-11]. Despite still scant 1Department of Functional Genomics and Proteomics, Masaryk University, knowledge of the number and genealogical context and CEITEC, Masaryk University, Brno, Czech Republic of ancient WGD episodes [12], c. 15% of speciation Full list of author information is available at the end of the article

© 2010 Mandáková et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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events among extant angiosperms are associated with that one of the purported parents comes from the poly- polyploidy [13]. phyletic Camelineae or the genus Boechera (i.e. from Polyploidy has also played a significant role in coloni- crucifer lineage I [29]), whereas the Brassica copy zation and species radiation on islands. Multiple exam- from the crucifer lineage II. Our recent comparative ples of long-distance dispersals of diploid progenitors or phylogenomic study of some allopolyploid Australian polyploid founders followed by adaptive radiation are Camelineae species (Ballantinia antipoda,2n=12; documented on well-studied archipelagos (Canary Stenopetalum nutans,2n=8andS. lineare, 2n = 10) Islands, New Zealand, Hawaiian Islands) [14-17]. revealed their close phylogenetic affinity to Pachycladon Remarkably, species radiation on islands is usually char- and other Camelineae taxa [10]. The ~6 to 9 million old acterized by chromosomal stasis, i.e. uniformity of chro- allopolyploid event in the ancestry of Australian genera mosome numbers/ploidy levels [15-17]. This means that was found to be obscured by extensive chromosome adaptive or species radiations proceed through homo- repaterrning leading to the extant diploid-like karyo- ploid divergence, rather than by changing the number of types (n = 4-6). Such concealed WGD episodes still linkage groups by dysploidy and/or polyploidy. The rea- detectable by comparative genetic and cytogenetic analy- sons for insular chromosomal stasis are most likely sis were classified as mesopolyploid [10]. Although both complex and lineage-specific, albeit the young age of recent studies [10,23] argued for an allopolyploid origin radiating polyploid lines and the adaptive advantage of of the New Zealand and Australian Camelineae genera, successful polyploid founders and their descendants are the unknown genome structure of Pachycladon species suspected as crucial factors. Although chromosomal sta- did not yet allow to elucidate the relationship between sis does not necessarily imply karyotypic stasis [17], only the two polyploid Camelineae groups. a handful of reports deal with the evolution of whole In the present paper comparative chromosome paint- chromosome complements in island endemics. With the ing (CCP) has been applied to four Pachycladon species exception of the Hawaiian silverswords (Asteraceae), (Pachycladon cheesemanii, P. enysii, P. exile,and analyzed through inter-species crossing experiments and P. novae-zelandiae) that represent the morphological, meiotic chromosome pairing configurations [18], none ecological and phylogenetic diversity of the genus of the homoploid species complexes on islands has been (Figure 1 and [21]), and for which genetic maps are not analyzed for whole-genome collinearity. available. Pachycladon enysii is a monocarpic, lanceolate The genus Pachycladon (Brassicaceae) comprises nine and serrate-leaved, stout terminal inflorescence species morphologically and ecologically diverse species in of high altitude (975-2492 m) alpine greywacke rock; mainly alpine habitats of the South Island of New Zeal- P. novae-zelandiae is a polycarpic, lobed-leaved, lateral and, and a single species occurs in alpine habitats in inflorescence species of mid-altitude (1080-2031 m) Tasmania [19,20]. The morphology of Pachycladon is so diverse that prior to the genus being recircumscribed by [21], species were also placed in Cheesemania and Isch- P. fasciarium nocarpus. Pachycladon is monophyletic [21-23], characterized by little genetic variation amongst species at a P. fastigiatum variety of genetic loci [24], and the species are interfer- P. stellatum tile [25,26]. Furthermore, six Pachycladon species analyzed karyologically all have the same chromosome P. enysii number of 2n = 20 [27,28] and comparable genome sizes (430 to 550 Mb [26,28]). Pachycladon is related to P. latisiliquum Arabidopsis [21,23], with both these genera belonging to the polyphyletic tribe Camelineae [29,30]. The close P. radiatum relationship between these genera is underlined by the P. wallii generation of a sexually derived intergeneric hybrid between A. thaliana and P. cheesemanii [31]. P. novae-zelandiae Based on chromosome counts and preliminary cytogenetic data, Pachycladon species were thought to have a P. cheesemanii polyploid origin (M. Lysak and P. Heenan, unpublished P. exile results). Indeed, an allopolyploid origin of the genus during the Pleistocene between ~0.8 and 1.6 mya (mil- Figure 1 Phylogenetic relationships in Pachycladon.Strict lion years ago) has been confirmed through identifica- consensus tree of the six most parsimonious trees based on the tion of two paralogous copies of five single copy nuclear internal transcribed spacer (ITS) region of 18S-25S ribosomal DNA. Species analyzed herein are in bold. Adapted from [[21], Figure 2]. genes [23]. Phylogenetic data of Joly et al. [23] suggested

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alpine schist rock; and P. chessemanii and P. exile are Evolution of the ten Pachycladon chromosomes polycarpic, heterophyllous, slender terminal inflorescence We have reconstructed the origin of the nine “fusion” and generalist species of high fertility rock such as lime- chromosomes of the ancestral Pachycladon karyotype stone, schist, and volcanics and occur from near sea-level using the minimal number of rearrangements and to the alpine zone (10-1600 m altitude) [19]. We used assuming that the ten PC chromosomes originated from CCP to study the extent of chromosome collinearity the duplicated ACK (i.e. from 16 AK chromosomes). between the ten chromosomes of Pachycladon species PC1 and PC2 chromosomes and the eight chromosomes of the theoretical Ancestral (Figure 4A). PC1 originated via a reciprocal transloca- Crucifer Karyotype (ACK [32,33]). Combining compara- tion between chromosomes AK1 and AK2 with break- tive cytogenetic data with already published accounts on points in the (peri)centromeric region of AK1 (close to phylogenetics, biogeography, and ecology of the genus we block B) and in the block D of AK2. The second trans- addressed (i) genome structure and evolution of Pachy- location product harbouring the AK1 centromere has cladon species, (ii) the genome relationship to other cru- been involved in a subsequent reciprocal end-to-end cifer species, particularly to the endemic Australian translocation with AK5, resulting in chromosome PC2. Camelineae taxa, and (iii) the role of major karyotypic As the four GBs (K-N) of AK5 have the ancestral posi- reshuffling in the species radiation in the island setting. tion within PC2 chromosome, we infer an inactivation and/or loss of the AK5 centromere. Results PC3 chromosome Comparative structure of Pachycladon karyotypes (Figure 4B). The origin of PC3 can be reconstructed as a The karyotype structure of four Pachycladon species (P. paracentric inversion of the block D on AK2 followed by cheesemanii, P. enysii, P. exile,andP. novae-zelandiae) nested “fusion” of this chromosome into the (peri)cen- has been reconstructed by comparative chromosome tromere of AK3. The nested “fusion” required three or painting (CCP) (Figure 2). Considering the close phylo- four breakpoints: two at the chromosome termini of genetic relationship between Pachycladon and Arabidop- AK2 and one or two at the centromere of AK3. One sis [21,23], we assumed that both genera descended breakpoint would presumably disrupt the AK3 centro- from the Ancestral Crucifer Karyotype (ACK) with eight mere, whereas two breaks at pericentromeric regions of ancestral chromosomes AK1 to AK8 [32,33]. Hence A. the opposite arms would yield a dispensable minichro- thaliana BAC clones and contigs corresponding to the mosome as a second translocation product. 24 conserved genomic blocks (GBs) of the ACK were PC4 and PC5 chromosomes used as painting probes to identify collinear chromo- (Figure 4C). PC4 and PC5 were generated through the some regions in Pachycladon species. The four reshuffling of ancestral chromosomes AK4 and AK6, reconstructed karyotypes showed overall similarity, com- and AK4, AK5 and AK6, respectively. A pericentric prising seven (sub)metacentric (PC1, PC3, PC4, PC6 - inversion (GBs O and P) transforming AK6 into a telo- PC8, and PC10) and three acrocentric (PC2, PC5, and centric chromosome was followed by a reciprocal trans- PC9) chromosomes with the identical arrangement of location between this chromosome and AK4. This ancestral GBs (Figure 3). The structural uniformity of all translocation joined the long arm of AK4 (block J) with reconstructed karyotypes suggeststhatthisstructureis the AK6 telocentric (= PC4). The AK4-derived telo- the ancestral Pachycladon karyotype. centric chromosome comprising only the centromere All 24 GBs were found to be duplicated within the and block I has undergone a reciprocal end-to-end analyzed pachytene complements displaying regular translocation with AK5. As the GB collinearity around meiotic pairing (Figure 2 and 3). The Pachycladon kar- the AK5 centromere between blocks L and M remained yotype comprises one AK chromosome (PC7), seven conserved, we inferred an inactivation and/or loss of AK-like chromosomes discernible within the composite this centromere on PC5. A small reciprocal transloca- Pachycladon chromosomes (four chromosomes modified tion between the bottom arm of PC4 (block R) and the by inversions), and 14 AK-like chromosome arms upper arm of PC5 occurred after the major reshuffling (Figure 3 and Table 1). Thus, in total forty-three ances- steps. A paracentric inversion between GBs K and L on tral GBs (90%) remained intact and only five blocks PC5 could have occured before the origin of both PC were split within one chromosome arm (block L on chromosomes or it is a later event. PC5), between two arms of the same chromosome (W PC6 chromosome on PC6), or between two different chromosomes (D to (Figure 4D). This chromosome most likely originated via PC1 and PC2, J to PC9 and PC10, R to PC4 and PC5). a reciprocal end-to-end translocation between AK6 and Except chromosome PC7 resembling chromosome AK7, AK8 and was probably followed by a concurrent or sub- all Pachycladon chromosomes originated through sequent inactivation and/or loss of the AK8 centromere, “fusion” of two or three AK chromosomes (Figure 3). reflected by the ancestral position of blocks V and Wa

157 Mandáková et al. BMC Evolutionary Biology 2010, 10:367 Page 4 of 14 http://www.biomedcentral.com/1471-2148/10/367 R NO E Db B A Da CKLM N

PC9 PC1 PC2 PC2 PC3 PC1 PC3 PC10 PC5

H DEGF J P OQ Ra

PC10

PC1 PC4 PC3 PC2 PC6 PC4 PC9

PC3

PC10

NOR Rb I La K Lb M N X WbO P V Wa Q R

PC5 PC5

PC6 PC5 PC9 PC6 PC6 PC2 PC4 PC8

S T U UVWXTS

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PC7 PC7 PC8

PC7 PC8

PC6

I Ja B A Jb CHGF

PC5

PC10

PC1 PC4

PC9 PC2 PC10 PC4

PC3 PC9

PC9

Figure 2 Comparative chromosome painting (CCP) in Pachycladon cheesemanii. Labelling scheme, in situ localization within a pachytene complement and straightened pachytene bivalent for each of the ten chromosomes (PC1-PC10) are shown. Chromosomes were identified by CCP with Arabidopsis BAC clones and contigs labelled by biotin-dUTP (red), digoxigenin-dUTP (green), and Cy3-dUTP (yellow). Due to the duplicated nature of Pachycladon genomes, each painting probe labels two homeologous chromosome regions on different chromosomes (white and yellow acronyms). Chromosomes counterstained by DAPI. NOR: nucleolar organizing region. Scale, 10 μm.

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PC3 PC4 PC1 (AK2/3) (AK4/6) (AK1/2) PC2 AK4 H AK2 (AK1/2/5) PC5

Da J (AK4/5/6) AK2 E E D J Rb AK3 D

H D I C I G E AK6 Db AK1 E La AK5 K K K C AK5 F P L R Lb L K B L O M M G Q B M M N Q P N O A N N A F Ra 1Mb

PC8 PC10 PC6 (AK7/8) (AK1/3/4) (AK6/8)

X AK4 PC7 PC9 I Jb (AK1/4) AK8 Wb (AK7) U AK7 X I J AK7 W S O U S AK6 Ja C V P P T T T AK1 V O T S C AK3 Q U B H Wa S H

R B Q G G U AK8 V V F A A W R W F X

Figure 3 Comparative cytogenetic map of the ancestral Pachycladon karyotype based on CCP data. Collinearity relationship of the ten Pachycladon chromosomes (PC1 - PC10) to the duplicated Ancestral Crucifer Karyotype (ACK) comprising two sets of eight ancestral chromosomes (AK1- AK8). Dashed lines connect collinear regions shared by the two genomes. Duplicated 24 conserved genomic blocks (A-X) of the ACK are colored according to their position on chromosomes AK1 to AK8 [33]. Blocks split into two parts are labeled as „a” and „b”. Centromeres of Pachycladon chromosomes are depicted as sandglass-like symbols colored according to their presumed origin from AK chromosomes.

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Table 1 Comparison of ancestral genomic features Chromosome landmarks (heterochromatin, telomeres and between Pachycladon and Australian Camelineae species rDNA) PK BA SL SN We have analyzed mitotic and pachytene chromosome n=10 n=6 n=5 n=4 complements of the four Pachycladon species for the entirely conserved AK chromosomes 3 443distribution of heterochromatin domains, localization of AK chromosomes modified by 4 214ribosomal RNA genes (rDNA) and the Arabidopsis-type inversions telomere repeat (Figure 5). Except for prominent hetero- AK chromosome arms 14 12 15 11 chromatin of pericentromeres and terminal nucleolus GBs not forming any AK-like structure 2 467organizing regions (NORs) (Figure 2), a single hetero- split GBs 5 669chromatic knob occurs in P. enysii and two knobs were lost GBs 0 420found in P. exile. Whereas two of the three knobs reside non-ancestral associations of GBs 18 29 30 38 within genomic blocks (B on PC1 in P. enysii,UonPC7 This table shows the extent of conservation of the eight ancestral in P. exile), the knob on the bottom arm of PC10 in P. chromosomes (AK1-8) and 24 genomic blocks (GBs) of the duplicated exile is localized between blocks G and H, i.e. at the site Ancestral Crucifer Karyotype (ACK) in Pachycladon and Australian Camelineae species [10]. PK- Pachycladon karyotype (n = 10), BA - Ballantinia antipoda (n of presumably inactivated centromere of AK3 (Figure = 6), SL - Stenopetalum lineare (n = 5), and SN - S. nutans (n = 4). 4F). No heterochromatic domains were observed at the sites of other presumably inactivated and/or lost paleo- on PC6. This event was followed by a pericentric inver- centromeres. The telomere (TTTAGGG)n repeat hybri- sion with breakpoints in the (peri)centromeric region dized only to chromosome ends and no interstitial (close to block Q) and within block W. telomeric signals were observed (data not shown). PC8 chromosome Whereas the four species have a single 5S rDNA locus (Figure 4E). PC8 originated via a reciprocal translocation at the same position, the number of terminal 45S rDNA between AK7 and AK8, yielding the fusion PC8 chro- loci varies. P. novae-zelandiae has one, P. cheesemanii mosome and a meiotically unstable minichromosome and P. exile possess two, and P. enysii has three 45S containing the centromere of AK8. The translocation rDNA loci, with 45S locus on the upper arm of PC2 was preceded by a paracentric inversion on AK7 (block being common to all species (Figure 5). Thus, the cross- S) and pericentric inversion on AK8 (block V). species karyotypic stasis does not apply to the number PC9 and PC10 chromosomes of terminal 45S rDNA loci. (Figure 4F). Chromosome PC9 originated through a reciprocal translocation between AK1 and AK4 with Discussion breakpoints in the AK1 pericentromere (close to block We have used comparative chromosome painting to B) and the proximal part of the bottom arm of AK4 reconstruct karyotype structure and evolution in the (block J). The second translocation product (GBs C and genus Pachycladon. Interestingly, our analysis showed Jb, and the AK1 centromere) participated in a reciprocal that the four analyzed species representing the phyloge- end-to-end translocation with AK3 which resulted in netic, ecological and morphological diversity of the the origin of PC10 and small acentric fragment. Ances- genus possess an identical karyotype, which is also most tral arrangement of AK3-derived GBs suggests that the likely to be the ancestral karyotype of the genus AK3 centromere has been lost or inactivated. Pachycladon. The reconstructed chromosome origins are congruent with the reduction of 16 ancestral chromosomes (cen- Chromosomal and karyotypic stasis in Pachycladon tromeres) to only 10 in Pachycladon. Centromeres of The present study of four Pachycladon species is the both homeologues of AK1, AK2, AK4, AK6 and AK7 first whole-genome analysis of an island species radia- remained functional, whereas six centromeres were lost tion. Pachycladon species have uniformly ten chromo- (Figure 3). The centromere of one AK3 homeologue was somes [27,28] and this infrageneric chromosomal stasis eliminated due to the nested chromosome fusion, one has been now extended for karyotypic stasis. Overall AK8 centromere was eliminated via symmetric translo- similar genome structures supported the monophyletic cation, and both AK5 centromeres and centromeres of origin of the genus [21-23] and allowed inference of the second homeologues of AK3 and AK8 were inactivated/ ancestral Pachycladon karyotype whose structure lost (Figure 4). remained conserved in the extant species. Karyotypic Out of the 33 breakpoints inferred for the origin of stasis revealed in Pachycladon clearly indicates that the ten Pachycladon chromosomes, 12 (36%) map to peri- Pleistocene species radiation on the South Island of centromeric regions, 16 (49%) to telomeric regions, New Zealand [19] was not associated with major chro- whereas only five (15%) occurred within GBs (Figure 4A mosome rearrangements. The four karyotypes differ to 4F). only by the number of heterochromatic knobs and

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A B PC3 PC1 (AK2/3) (AK1/2) AK1 PC2 (AK1/2/5) H Da AK3 E AK5 D A AK2 K AK2 L C D Da end-to-end D D Db translocation F & centromere K paracentric inactivation nested fusion E translocation M inversion B C L B E E E G G N M

H C N A F

PC4 C (AK4/6) PC5 J AK6 (AK4/5/6) AK5 AK4 O P J Rb La I K K O I P Lb translocation L La I & end-to-end P K Q translocation Lb La Q para- and pericentric & centromere K M O P inversion inactivation translocation Lb M M J O Q M N R N R N Q N R Ra

PC6 PC8 (AK6/8) (AK7/8) D X E X AK6 W Wb AK7 U O AK8 S P AK8 V S P V end-to-end O V V translocation O T & centromere pericentric T Q translocation inactivation inversion V T para- and pericentric P inversion & centromere loss W W W Wa S Q U R X Q U X X V

R R W

PC10 F (AK1/3/4) PC9 AK1 (AK1/4) AK3 Jb I AK4 A I Ja Jb F end-to-end translocation C & centromere translocation B inactivation B J H G

C G A H C

Figure 4 Tentative scenarios of the origin of nine Pachycladon chromosomes (PC1- PC6, PC8-PC10) from the duplicated Ancestral Crucifer Karyotype (ACK). Duplicated 24 conserved genomic blocks (A-X) of the ACK are colored according to their position on chromosomes AK1 to AK8 [33]. Blocks split into two parts are labeled as „a” and „b”. Centromeres of Pachycladon chromosomes are depicted as sandglass-like structures colored according to their presumed origin from AK chromosomes. Inactivated and/or lost ancestral centromeres are shown outside the modern Pachycladon chromosomes. Downward-pointing arrows indicate the opposite orientation of genomic blocks as compared to their position within the ACK [33]. Jagged lines mark purported breakpoints of inferred chromosome rearrangements.

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PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 Hawaiian mints [37]) or diploid ancestors hybridize in P. enysii 45S rDNA 5S rDNA situ after long-distance dispersal (e.g. the New Zealand pericentromeric heterochromatin heterochromatic knob P. enysii P. exile and Australian Lepidium species [38]). The allopoly- P. exile ploidy-driven speciation on islands is frequently asso- P. cheesm. P. cheesm. P. enysii P. enysii P. exile ciated with chromosomal stasis as shown for the P. exile P. novae-z. Hawaiian flora with the high incidence of polyploidy (> Figure 5 Idiogram of ten Pachycladon pachytene chromosomes 80% [15]). (PC1 to PC10) showing the positions of rDNA loci and Polyploidy is also a pronounced feature of the New heterochromatic arrays. 5S and 45S rDNA loci on chromosome Zealand flora, with 72% of the species being polyploid in PC2 occur in all species analyzed; positions of other 45S rDNA loci and heterochromatic knobs are species-specific. families with 25 or more species [39]. Chromosomal features of New Zealand plants indicative of polyploidy are the high number of species with even haploid numbers NORs, without an apparent link to infrageneric phyloge- and/or haploid numbers n = > 10-14 [40]. Many of the netic relationships (Figure 1). Hence, the speciation pro- polyploid genera that are like Pachycladon exhibiting ceeded through homoploid divergence from the chromosomal stasis are species-rich and generally con- ancestral allopolyploid genome. sidered to be recent species radiations, often into mainly Perhaps with the exception of meiotic studies in the alpine-montane habitats. They include, for example, Aci- Hawaiian silversword alliance [18] there is virtually no phylla (42 species, 2n = 22), Brachyglottis (30 species, data on karyotype evolution during island angiosperm 2n = 60), Chionochloa (22 species, 2n = 42), Gentianella speciation. Hence, only the variation in chromosome (40 species, 2n = 36), Epilobium (38 species, 2n = 36), number/ploidy level can be discussed more extensively. and Ourisia (20 species, 2n = 48) (data from [27]). Several surveys of angiosperm chromosome numbers Chromosomal stasis is also observed in the few cruci- showed the trend of chromosomal stasis during species fer genera that have species radiations on islands. All radiation on islands (see reviews by [15-17]). This ten- seven Parolinia species endemic to the Canary Islands dency might appear paradoxical considering geographi- probably have 2n = 22 (4 species counted [41]), and cal isolation and a wealth of diverse insular seven shrubby species of Descurainia endemic to the environments potentially promoting the origin of novel Canary Islands share 2n = 14 [42]. Similarly, of nine chromosomal races and karyotypes. However, genomes Diplotaxis species in the Cape Verde Islands, five have diverging on islands are under multiple constraints 2n = 26 [43]. Unfortunately, insufficient chromosomal determining chromosomal stasis or chromosomal varia- data are available for c. 40 Cardamine species endemic tion. As self-evident factors influencing the insular spe- to New Zealand (P. Heenan, unpublished data) as well cies radiation and genomic stability are the age of as for most crucifer genera endemic to Australia [41,44]. islands and their distance from the mainland, the number of colonization events, the incidence of polyploidy Pachycladon karyotype is derived from the duplicated and phylogenetic constraints. Colonizations followed by Ancestral Crucifer Karyotype adaptive radiation on (volcanic) islands represent often Our data suggest that the ancestral Pachycladon karyo- relatively young evolutionary events and therefore many type (n = 10) was derived from the duplicated Ancestral island endemics represent monophyletic lineages com- Crucifer Karyotype (n = 8) through allopolyploidy. The prising closely related species with uniform chromosome ACK was expected to be inferred as an ancestral gen- numbers. Furthermore, it was concluded that chromoso- ome of Pachycladon, as all of the Camelineae genomes mal stasis vs. lability is under a strong phylogenetic con- analyzed thus far have descended from the ACK (for straint as some lineages (e.g. Asteraceae, Sideritis)seem instance, Arabidopsis, Capsella, Turritis,andNeslia to be more prone to genome reshuffling than others [32,45,46], including the analyzed Australian Camelineae [15,16,34]. species [10]). Furthermore, the karyotypes of Crucihima- Generally the low incidence of polyploidy has been laya and Transberingia, two genera often found as being claimed for island floras [16]. These estimates collated the closest relatives of Pachycladon [21,23], resemble the prior to the era of indepth whole-genome analyses ACK structure [10]. Similarly, the ACK was proposed as revealing multiple whole-genome duplications of a dif- an ancestral karyotype for tribes Boechereae and Carda- ferent age (e.g., [7,12,10]) had to be, by definition, too mineae [[47], Mandáková and Lysak, unpublished data]. conservative. Recent studies suggest that colonization of It is likely, therefore, that the ancestral Pachycladon islands has been frequently associated with hybridization genome has been derived from the hybridization and allopolyploidy (see [35,36] for examples). Allopoly- between two ACK-like genomes. The primary allopolyploid ancestors originated either on continents and ploid had either the structure of duplicated ACK with n spread to islands (e.g. the allopolyploid ancestor of the = 16 or the participating genome(s) were reduced (n = 8

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® n = 7-5?) prior to the hybridization event and the (except inverted block D) translocation events with allopolyploid had less than 16 chromosome pairs. The breakpoints at chromosome termini of AK2 and centro- fact that paralogous genomic blocks do not lay on mere of AK3 seems to be the parsimonious scenario. In the same chromosome suggests that the modern Pachy- grasses (Poaceae), insertional chromosome “fusion” has cladon chromosomes were reshuffled prior to the hybri- been proposed as a general mechanism of descending dization event, rather than due to homeologous dysploidy [4,50], whereas in crucifers it can be suggested recombination between two ACK-like genomes within only for the origin of chromosome AK2/5 in Hornungia the allopolyploid ancestor. alpina [32]. Thus, Pachycladon chromosome PC3 is The ten composite Pachycladon chromosomes origi- most likely the first instance of reconstructed insertional nated through inversions, reciprocal translocations and dysploidy in Brassicaceae. An alternative mechanism of centromere inactivation/loss events within the dupli- the PC3 origin via end-to-end reciprocal translocation cated ACK complement (Figure 4). Chromosome coupled with the elimination of the AK3 centromere “fusions” were mediated by reciprocal translocations requires two more breakpoints. with or without preceding para- and pericentric inversions. These translocations yielded a “fusion” chro- Common origin of Pachycladon and Australian mosome and (a)centric fragment as the second translo- Camelineae species? cation product. Small acentric fragments and the Based on the phylogenetic analysis of Australian Cameli- minichromosome harbouring one AK8 centromere were neae taxa and Pachycladon species, [10] concluded that meiotically unstable and eliminated. Whereas Robertso- both groups might originate from a very similar allopoly- nian-like translocations eliminating one AK centromere ploid ancestor. Although the authors could not reject a together with the minichromosome is a common single origin of both lineages, they considered two succes- mechanism of the karyotype evolution in Brassicaceae sive allopolyploidization events as more likely, i.e. mesopo- [32,48,49], asymmetric translocation events yielding lyploid Australian Camelineae species originated and miniature acentric fragments and dicentric chromo- radiated in continental arid habitats before the mesopoly- somes with one AK centromere apparently inactivated ploid ancestor of Pachycladon. The present data corrobo- or removed by recombination were proposed for the rate this conclusion. Specifically, the two species groups origin of composite chromosomes in the Australian do not share any cytogenetic signature, i.e. a taxon/line- Camelineae species [10]. Centromere inactivation and/or age-specific chromosome rearrangement, such as the rear- loss has been inferred on bottom (long) arms of four ranged AK8 homeologue shared by five Australian species Pachycladon chromosomes (PC2, PC5, PC6, and PC10) analyzed [10]. In the Australian species, any two paralo- based on the absence of ancestral centromeres and con- gous GBs differ by the length and fluorescence intensity as served organization of adjacent genomic blocks (Figure revealed by CCP [10]. This difference was either present 3 and 4). The incidence of centromere inactivation in already in the hybridizing progenitors or was caused by Australian and New Zealand Camelineae species might preferential fractionation of paralogous regions belonging be tentatively related to the common ancestry of both to only one subgenome [51]. In Pachycladon, two paralo- lineages and/or to the duplicated character of the allo- gous copies of all GBs cannot be distinguished upon CCP polyploid ancestral genomes. Centromere inactivation of analysis. Furthermore, higher chromosome number AK4 can also be suggested for the origin of chromo- in Pachycladon species(n=10)thanintheAustralian some At2 in A. thaliana [32], and centromere inactiva- species (n = 4-7) implies a more recent origin and less tionofAK5fortheoriginofBst5inBoechera stricta extensive diploidization in Pachycladon. Indeed, the signif- [47] and chromosome AK4/5 in Neslia paniculata [32]. icantly lower number of non-ancestral junctions of geno- Nevertheless, an alternative mechanism of centromere mic blocks in Pachycladon compared to Ballantinia removal through subsequent paracentric and pericentric antipoda and the two Stenopetalum species (Table 1 and inversions followed by a symmetric translocation (Figure [10]) underlines the less extensive genome reshuffling in 2C in [32]) is also plausible, though more breakpoints Pachycladon. Also the number of split GBs in Pachycladon have to be considered. A dicentric chromosome could (10%) is lower than in the Australian species (13% to 19%; also be stabilized by intrachromosomal translocation, [10]). Interestingly, both groups do not differ substantially with breakpoints in pericentromeric region of one of the by the number of preserved AK chromosomes, chromo- ancestral centromeres, followed by a loss of the resulting some arms and GBs (Table 1). This comparison suggests centric fragment. that the most recent steps of chromosome number reduc- Chromosome PC3 originated probably through a tion in the Australian Camelineae species have been nested “fusion” of chromosome AK2 between chromo- mediated by tandem end-to-end translocations followed some arms of AK3. As both AK chromosomes within by centromere inactivation/loss, not disrupting the struc- PC3 possess the ancestral structure of genomic blocks ture of AK-like chromosomes and chromosome arms.

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Altogether, the differences in genome structure and Ranunculus). For these shared genera, species diver- between the mesopolyploid Australian and New Zealand sity is often highest in New Zealand and the Australian lineage indicate two successive WGD events involving species are considered to be the result of westward dis- the same pool of parental species. The existence of the persal from New Zealand and subsequent speciation progenitor species in Australia for a long period of time (e.g. [64,65]). Indeed, P. radicatum occurs in the Tasma- is a credible assumption considering the remarkable sta- nian mountains and is considered to have dispersed sis of the ACK and AK chromosomes across crucifer there and diverged contemporaneously with the radia- lineages I and II [32,33,49]. Further research is needed tion of Pachycladon in New Zealand [21,22]. Other taxa to elucidate if the ancient ACK-like karyotype could be are also shared between the two countries, but these are found in some not yet analyzed Australian crucifer spe- considered to have dispersed eastward from Australia to cies. Recurrent formation of allopolyploids from the New Zealand and include, for example, Craspedia [66], same or closely related parents has been documented, e. Montigena [67], Poranthera [68], Scleranthus [69], and g. in the North American allopolyploid species of Trago- Stylidiaceae [70]. This pattern of eastward dispersal pogon [52], in Persicaria [53] or Arabidopsis kamchatica means it is plausible that Pachycladon could have origi- [54], and also proven by the generating synthetic allopo- nated in Australia and then subsequently dispersed to lyploids as Arabidopsis suecica [55], tobacco [56] or New Zealand. Tragopogon mirus and T. miscellus [57]. An alternative scenario of the origin of the Pachycla- Although less likely, we cannot rule out that karyoty- don allopolyploid ancestor in (eastern) Eurasia followed pic change in the Australian Camelineae species and in by a later dispersal to New Zealand is unlikely and Pachycladon had significantly different dynamics. The incongruent with the close phylogenetic ties of Pachy- Australian Brassicaceae species exhibit a predominantly cladon to Australian Camelineae. Also, the origin of annual growth habit [44] in comparison to the perennial Pachycladon and Australian crucifer species in New Pachycladon [21], and a more rapid rate of genome evo- Zealand is very unlikely, considering the diversity of lution could therefore be brought about with faster endemic Australian Brassicaceae taxa (15 genera and 65 nucleotide substitution rates that occur in many annuals species [44]). [58,59]. Perennials are thought to have greater chromo- Many of the Australian Camelineae are distributed in somal stasis than annuals [60,61]. Certainly the annual- the arid Eremaean Zone and/or the southeastern tempe- ity could have accelerated genome reshuffling in the rate biome [44], whereas in New Zealand Pachycladon Australian lineage. However, for Brassicaceae we have mainly occupies montane-alpine habitats. These three insufficient data on large-scale genome evolution in rela- environments have expanded in both Australia and New tion to the life forms, reproduction systems and ecologi- Zealand during the Pliocene and Pleistocene and are cal factors, and as noted by [15] and [62] chromosomal generally considered important drivers of species radia- evolution is often stochastic and does not obey the tions (e.g. [71,72]). For the Australian Camelineae their models. origin and diversification ~6 to 9 mya [10] is consistent with other dated molecular phylogenies of a diverse Phylogeographic scenario of the origin of Pachycladon range of arid-adapted taxa [73]. These dated phylogenies Pachycladon istheonlyNewZealandgenusfromthe show the deepest divergences of taxa are consistent with polyphyletic tribe Camelineae (other endemic crucifer the beginning of the formation of the arid zone in the species belong to Cardamineae, Lepidieae, and Nototh- mid-Miocene and that most arid-zone species lineages laspideae), and therefore an in situ origin seems unli- date to the Pliocene or earlier. The molecular clock date kely. The closest Camelineae relatives of Pachycladon of 0.8 to 1.6 mya for the origin of Pachycladon [23] is occur in Australia (e.g., Arabidella, Ballantinia, and Ste- also consistent with its alpine distribution and habitats nopetalum) and Eurasia/Beringia (e.g., Arabidopsis, Cru- in the Southern Alps in the South Island of New Zeal- cihimalaya, Transberingia)[10,23].Itseemsmore and [19]. Uplift of the Southern Alps occurred over the plausible that the hybridization event giving rise to last 8 million years, but only reached a suitable height Pachycladon has taken place on the Australian to permanently support alpine plants during the continent. Pleistocene. There are strong taxonomic and biogeographic links between Australia and New Zealand and dispersal across Reconstructed genome evolution corroborates the close the Tasman Sea can occur in both directions. Tasmania relationship of Pachycladon to Arabidopsis and other and New Zealand have about 200 species in common Camelineae species [63], and there are many genera in continental Australia The phylogenetic position of Pachycladon has been and New Zealand that have species that are closely investigated repeatedly using various nuclear, chloro- related (e.g., Aciphylla, Celmisia, Gentianella, Melicytus, plast, and mitochondrial genes [10,21,23]. All studies are

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congruent in placing the genus into the crucifer lineage allopolyploid Australian Camelineae taxa. CCP data I, within the polyphyletic tribe Camelineae [21,29,30,74]. demonstrate that both mesopolyploid groups most likely Although the phylogenetic relationships within Cameli- originated from two different WGD events that involved neae are unclear, these studies have shown Transberin- identical or very similar diploid parents. We argue that gia and Crucihimalaya (Camelineae), Sphaerocardamum the Pachycladon ancestor has its origin in Australia and (Halimolobeae), Physaria (Physarieae), and Boechera later dispersed to the South Island of New Zealand. The (Boechereae) to be among the closest relatives of Pachy- endemic Australian and New Zealand Camelineae pro- cladon. Based on the analysis of five single-copy nuclear vide an excellent framework to examine the nature and genes, [23] showed that Pachycladon has an allopoly- consequences of differently-aged WGD events within a ploid origin and that the two genomes were associated complex of closely related species. with two divergent Brassicaceae lineages (lineage I and II [29,75]). One putative parental genome was associated Methods with Camelineae sensu lato (and Boechereae) and the Plant material second genome being related to Brassiceae, Sisymbrium, The four species of Pachycladon included in this study Eutremeae, Thlaspideae, and remarkably also to Carda- represent the morphological and ecological diversity of mineae on the chalcone synthase gene tree. This pattern the genus [19]. Plants were cultivated in a glasshouse at has been interpreted as the evidence of an inter-tribal Landcare Research, Lincoln, New Zealand. All species allopolyploidization event at the origin of Pachycladon. have known wild origins: P. cheesemanii (Bobs Cove, A recent study using nuclear, mitochondrial and Queenstown, Otago; 168°37’E, 47°08’S), P. enysii (Mount chloroplast genes, as well as significantly increasing the Potts, Canterbury; 170°55’E, 43°30’S), P. exile (Awaho- sampling of Australian Camelineae (in comparison to komo, Otago; 170°23E, 44°42’S), and P. novae-zelandiae that of [23]), has confirmed the allopolyploid origin of (Old Man Range, Otago; 169°12’E, 45°20’S). Pachycladon and provides confidence that the two gene paralogues that constitute Pachycladon are derived from Chromosome preparation within lineage I [10]. Most importantly, this study has Entire inflorescences were fixed in ethanol:acetic acid disclosed the close relationship of Pachycladon to the (3:1) fixative overnight and stored in 70% ethanol at -20° Australian genera Arabidella, Ballantinia,andStenope- C until use. Selected flower buds were rinsed in distilled talum, and the maternal gene paralogues of Pachycladon water and citrate buffer (10 mM sodium citrate, pH 4.8) and these three genera clustered with Eurasian Cameli- and incubated in an enzyme mix (0.3% cellulase, cytohe- neae (Arabidopsis, Capsella, Crucihimalaya, Olimarabi- licase, and pectolyase; all Sigma) in citrate buffer at 37°C dopsis, Transberingia) and North American Boechereae. for 3 h. Individual flower buds were disintegrated on a The position of the paternal gene copy was less evident, microscopic slide by a needle in a drop of citrate buffer but it was always embedded within lineage I, and there- and the suspension softened by adding 20 μL of 60% fore different from the study by [23]. Mandakova et al. acetic acid. The suspension was spread on a hot plate at [10] and the present study convincingly show that the 50°C for ~0.5 min. Chromosomes were fixed by adding Pachycladon ancestor orginated from hybridization of ethanol:acetic acid (3:1, 100 μL) and dried with a hair between a Camelineae species and either another species dryer. Suitable slides were postfixed in 4% formaldehyde of that tribe or a very closely related tribe of lineage I. in distilled water for 10 min and air-dried. Future phylogenomic analyses of other Australian crucifer genera are likely to further resolve the parentage and DNA probes for fluorescence in situ hybridization (FISH) phylogenetic relationships of Pachycladon. For CCP in P. exile, on average each third Arabidopsis thaliana BAC clone was used to establish contigs corre- Conclusion sponding to the 24 genomic blocks of the ACK [33]. For We have shown that the remarkable infrageneric mor- the detail composition of the BAC contigs see [49]. phological and ecological differentiation in Pachycladon After initial CCP experiments in P. exile,someBAC is characterized by the genome stability manifested as contigs were split into smaller subcontigs to pinpoint chromosomal and karyotypic stasis. The monophyletic rearrangement of ancestral blocks. (Sub)conting charac- Pachycladon species descended from a common allopo- terizing chromosome rearrangements in P. exile were lyploid ancestor (n = 10) through a whole-genome used as CCP probes to reconstruct karyotypes of duplication of the Ancestral Crucifer Karyotype (n = 8) P. cheesemanii, P. enysii and P. novae-zelandiae.The and subsequent diploidization by descending dysploidy. A. thaliana BAC clone T15P10 (AF167571) containing Furthermore, the present study and the phylogenetic 45 S rRNA genes was used for in situ localization of data of [10] clearly demonstrate the close relation- NORs, and A. thaliana clone pCT4.2 (M65137), corre- ship between the allopolyploid Pachycladon and the sponding to a 500-bp 5 S rRNA repeat, was used for

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doi:10.1186/1471-2148-10-367 Cite this article as: Mandáková et al.: Island species radiation and karyotypic stasis in Pachycladon allopolyploids. BMC Evolutionary Biology 2010 10:367.

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