Pl. Syst. Evol. 259: 143–174 (2006) DOI 10.1007/s00606-006-0417-x

Polyploidy, hybridization and reticulate : lessons from the Brassicaceae

K. Marhold1,2 and J. Lihova´1

1Institute of Botany, Slovak Academy of Sciences, Bratislava, Slovak Republic 2Department of Botany, Charles University, Praha, Czech Republic

Received November 26, 2005; accepted December 15, 2005 Published online: June 19, 2006 Springer-Verlag 2006

Abstract. The Brassicaceae is well known Introduction for its large variation in chromosome numbers, common occurrence of polyploids and many Polyploidization is undoubtedly a frequent reports of interspecific gene flow. The present mode of diversification and in review summarizes studies from the past decades plants. Otto and Whitton (2000) suggested on polyploidization and hybridization events, that polyploidization may be the most com- recognizing them as important evolutionary mon mechanism of in forces in the family. Attention is drawn to the plants. Moreover, recent data indicate that issue of the reconstruction of reticulated pattern most plants have undergone one or more of evolution resulting from allopolyploid and episodes of polyploidization during their evo- homoploid speciation. The research of lutionary history (Soltis et al. 2004b). For various authors on several Brassicaceae genera is many polyploids, multiple origins in space presented and discussed in the context of our and time have been proven, along with current understanding of polyploid and hybrid evolution. Model , Arabidopsis thaliana increased genetic diversity and complexity, and Brassica taxa, are referred to only margin- whereas in other cases a single origin is ally, major focus is on a comprehensive survey of proposed (Soltis and Soltis 1999, Soltis et al. studies on about a dozen best explored non- 2004b). Hybrid speciation is another impor- model genera (e.g. Cardamine, Draba, Rorippa, tant evolutionary phenomenon. It occurs in Thlaspi). The increasing amount of genetic and two possible ways, as allopolyploid or homop- genomic resources available for Brassicaceae loid hybrid speciation (Hegarty and Hiscock model species provides excellent opportunities 2005), thus hybridization and polyploidization for comparative genetic and genomic studies. events are usually tightly coupled. These Future research directions and challenges are processes often result in a reticulated pattern thus outlined, in to obtain more detailed of evolution. While in polyploids reticulation is insights into the evolution of polyploid and always suspected, homoploid hybrid speciation hybrid genomes. events may be more difficult to identify and Key words: Allopolyploidy, autopolyploidy, unravel. Linder and Rieseberg (2004), and Cruciferae, chromosomes, hybrids, phylogeny. Vriesendorp and Bakker (2005) have pointed 144 K. Marhold and J. Lihova´ : , hybridization and to the fact that the evolutionary history of database by Warwick and Al-Shehbaz 2006): many plant groups does not follow divergent Draba ) 25% diploid, 7% both diploid and evolutionary patterns, and hardly can be polyploid, 68% entirely polyploid taxa; Lepi- unravelled in a tree-building procedure. dium ) 34% diploid, 14% both diploid and Rather, it is like a network, which displays a polyploid, 52% entirely polyploid taxa; number of reticulate evolutionary events. The and Rorippa ) 48% diploid, 39% both diploid family Brassicaceae is not an exception, and and polyploid, and 13% entirely polyploid polyploidy has certainly played one of the key taxa. These estimates clearly point to the roles in its evolution. This is clearly illustrated evolutionary importance of polyploidy in by the recently discovered genome triplication, numerous Brassicaceae genera. Besides poly- recognized as a major evolutionary event that ploidization, hybridization and hybrid specia- gave rise to the whole Brassiceae. It has tion are common evolutionary phenomena in been assumed that the ancestral Brassiceae the Brassicaceae. Numerous examples of genome became triplicated via allohexaploidy hybrids are reported for this family (e.g. for that occurred 7.9–14.6 Mya (Lysak et al. the British Isles summarized by Stace 1975), 2005). and some genera, such as Rorippa (Bleeker and The large variation in base chromosome Hurka 2001), Cardamine (Lihova´ and Mar- numbers (x = 4–13 ()17); Warwick and Al- hold 2006) or Boechera (Koch et al. 2003b), Shehbaz 2006) in the family indicates complex are characterized by frequent hybridization evolution. Species with higher base and introgression. numbers are suspected to represent palaeopo- With the advent of new molecular tools, lyploids that have undergone substantial gen- especially in the past decade, our understand- ome diploidization (e.g. Brassica; Osborn ing of the above-mentioned evolutionary phe- 2004). Chromosome counts are currently avail- nomena has been significantly improved. able for 232 out of 338 accepted Brassicaceae Advances in Brassicaceae research in this genera (68.6%), and for 1558 out of the 3709 respect were recently summarized in Koch (42.0%) recognized species (Warwick and Al- et al. (2003a). The vast majority of studies on Shehbaz 2006). Approximately 37% of the polyploid origins and subsequent genetic and species are assumed to be polyploid (Warwick genomic evolution, have focused on model and Al-Shehbaz 2006). This estimate, however, plant species. The immensely increasing can be much higher, if we consider that several amount of genetic and genomic resources diploid species (e.g. from the genera Brassica, available for the model species Arabidopsis Physaria) are in fact palaeopolyploids. The thaliana provides an excellent opportunity to percentage of polyploids in the family can in transfer this knowledge to non-model relatives, such cases reach at least 50% (Koch et al. as well as to use the methodological progress in 2003a). Diploidization processes hamper the comparative studies with related genera. Com- identification of ancient polyploidization parative genetic and genomic analyses involv- events, which, however, can be suspected from ing this model species can reveal much about large-scale genome duplications (see Lysak the evolutionary patterns within this family et al. 2005). Polyploidy is widespread in many (Hall et al. 2002, Lysak et al. 2003). genera, and some of them (e.g. Crambe, The aim of this review is to focus on Moricandia) even seem to be exclusively poly- polyploidization and hybridization as two ploid (Koch et al. 2003a). In the Card- important evolutionary forces in the family amine we identified 32% diploid, 10% both Brassicaceae, as well as to explore the resulting diploid and polyploid, and 58% entirely poly- patterns of reticulate evolution. Case studies ploid taxa (Kucˇ era et al. 2005). For some are presented from the genus Cardamine, other Brassicaceae genera, the corresponding which has been extensively studied by our frequencies are as follows (based on the research group and other authors as well, and K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 145 from about a dozen other genera, which have reflects the occurrence of several cytogenetic received particular attention in the past years. phenomena, such as polyploidy, aneuploidy A better understanding of the evolutionary and dysploidy. Complex karyotype evolution patterns in Brassicaceae can be achieved only involving extensive chromosomal structural by a comprehensive survey of published stud- rearrangements can also be expected within ies, as conducted here. New challenges and the family, as already illustrated by compara- open questions are then identified, stimulating tive genetic mapping of Arabidopsis, Capsella further studies. and Brassica species (Lagercrantz 1998, Lan et al. 2000, Boivin et al. 2004, Kuittinen et al. 2004, Koch and Kiefer 2005). This approach, Chromosome number and genome size diversity together with cytogenetic techniques such as in A continuous of base chromosome situ hybridization or comparative chromosome numbers ranging from x = 4 to 13 (17) has painting, can elucidate significant patterns of been reported for the Brassicaceae, with a chromosome and karyotype evolution in the relatively high percentage of taxa (37%) based Brassicaceae (Schmidt et al. 2001, Hall et al. on x = 8 (Warwick and Al-Shehbaz 2006). 2002, Lysak et al. 2003, Lysak and Lexer The lowest chromosome number known for 2006). this family (n = 4) is found in two unrelated Along with chromosome number determi- genera, the Australian Stenopetalum and the nation and karyotype studies, techniques that western North American Physaria (Al-Sheh- measure DNA amount per nucleus (flow baz 1984). The highest chromosome numbers cytometry or microdensitometry) provide use- known in the family were reported in North ful tools to study cytogenetic diversity (Dolezˇ el American Cardamine diphylla and C. concate- and Bartosˇ 2005, Bennett and Leitch 2005b). nata (= Dentaria laciniata), both up to 2n = Genome size varies enormously among angio- 256 or n = 128, corresponding to 32-ploidy sperms, and several plant families, such as the level (Easterly 1963, Al-Shehbaz 1988). How- Fabaceae, Poaceae or Liliaceae, exhibit large ever, in the former species there is considerable ranges in DNA content (Bennett and Leitch intrapopulational and even intraindividual 2005a). Despite the extensive structural gen- variation in the chromosome numbers re- ome evolution observed in the Brassicaceae, ported, and thus, it is difficult to assess whether the species analyzed so far have shown small the high numbers represent regular stabilized nuclear DNA content levels, with a narrower chromosome sets. Arabidopsis thaliana has range in comparison to other families (up to x = 5 chromosomes, while many of its close 1C = 1.95 pg, Johnston et al. 2005). Super- relatives have x = 8. Several reductions in imposing genome size onto a robust phylogeny base chromosome number from x =8 to allows an assessment of ancestral DNA con- x = 5–7 have been recorded within the tribe tent, as well as the ability to trace genome size Arabideae, suggesting that numbers lower than evolution. Such studies have supported the x = 8 are phylogenetically derived. This also theory of bi-directional dynamic changes in holds true for the genome of A. thaliana, which genome size, i.e. that both genome size expan- is considered to be highly derived (Koch et al. sion and contraction occur commonly (see 1999). Chromosome number variation in the Wendel et al. 2002). A first attempt to trace family is indeed enormous (see Warwick and genome size evolution in Brassicaceae has been Al-Shehbaz 2006). Several genera exhibit very recently published by Johnston et al. (2005). large variation, as illustrated by Aethionema, Monoploid genome sizes were mapped onto a Draba, Erysimum, Cochlearia (Warwick single-most parsimonious tree based on the and Al-Shehbaz 2006 and references therein) internal transcribed spacer of nuclear ribo- and Cardamine (Kucˇ era et al. 2005, Lihova´ somal DNA (nrDNA ITS) sequence data from and Marhold 2006). This variation apparently 34 taxa, including both diploid and polyploid 146 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution taxa. Although the number of species analyzed of retroelements which might be activated by was not high, some general trends can be seen. allopolyploidization, while various mecha- The DNA content ranged over 8-fold (1C = nisms for the deletion of redundant genomic 0.16–1.31 pg) and 4.4 fold (1C = 0.16– sequences leading to genome downsizing have 0.71 pg) when allotetraploid Brassica species also been suggested (reviewed in Wendel et al. were excluded. The ancestral genome size was 2002, Kellogg and Bennetzen 2004, Leitch and estimated at ca. 1C = 0.2 pg, and the Bennett 2004). presented data unequivocally supported the Other aspects of genome size variation in concept of a dynamic nature of genome size the Brassicaceae than presented here, are evolution in this family, involving both discussed by Lysak and Lexer (2006). increases and decreases. The smallest genome was identified in Arabidopsis thaliana (1C = Spontaneous interspecific hybridizations 0.16 pg) that apparently underwent an evolu- and their evolutionary consequences: tionary decrease. A decrease in genome size molecular evidence was also revealed in extant allopolyploids of Brassica species and in Arabidopsis suecica The many examples of hybridization, intro- (that originated from A. thaliana and A. aren- gression and hybrid speciation reported for the osa, for details see below). In all these cases, Brassicaceae indicate that this is a significant the genome size of allopolyploids was reported evolutionary force in the family. The compre- to be less than the sum of their diploid hensive work on hybridization of the taxa ancestors (Johnston et al. 2005). occurring in the British Isles (Stace 1975) In polyploid series that are of recent origin, provided data on as many as 39 interspecific increase in the DNA content in polyploids is hybrids within this family. Yet, there are not so expected to be in direct proportion to the many recent studies, in which the hybrid origin ploidy level (Bennett et al. 2000). Indeed, such and the parentage of putative hybrid plants a pattern was found in several Draba species, have been proven using molecular or other which included diploids, triploids, tetraploids reliable evidence. Nevertheless, some genera and hexaploids, whose recent origin was sup- have been subjected to thorough studies ported by molecular markers (Grundt et al. exploring interspecific gene flow, and it is 2005). In some polyploids (as documented for apparent that hybridization has significantly Arabidopsis suecica, Johnston et al. 2005; and contributed to their evolution and species A. lyrata subsp. petraea, Dart et al. 2004), diversity. They certainly belong to the best- however, the additivity has not been retained, studied hybrid systems and provide interesting but genome size contraction has apparently examples of interspecific hybridization and occurred. Genome expansion following poly- hybrid speciation (e.g. hybrids in Rorippa, ploidization has been observed as well, Bleeker and Hurka 2001, Bleeker 2003; Boe- although it appears less common than genome chera, Schranz et al. 2005; Cardamine ·insueta contraction (Leitch and Bennett 2004). In our and C. schulzii, Urbanska et al. 1997). Inter- recent study focused on the allopolyploid specific hybridization has often been recog- Cardamine asarifolia (Lihova´ et al. 2006), we nized as a source of genetic variation and suggested that this hexaploid had experienced genetic novelties, and in some cases successful genome expansion subsequent to its origin. hybridization events have promoted rapid This particular case, however, has been com- radiation (Seehausen 2004). This seems to be plicated by the assumed extinction of one of the case for Australian/New Zealand Lepidium the parental species, and consequently, com- species originating in the Pliocene/Pleistocene, parisons were limited to other most closely for which hybridogenous genomic constitution related species. Increase in genome size has has been recently revealed (Mummenhoff et al. usually been associated with the accumulation 2004). Ancient trans-oceanic dispersals from K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 147

California and South Africa, respectively, Wendel 2003). Its high evolutionary rate followed by hybridization have been assumed permits discrimination of closely related puta- to occur. Low levels of sequence divergence tive parents and identification of additive observed among the species from Australia patterns in hypothesized hybrids. Although and New Zealand suggested a rapid and recent concerted evolution can erase nucleotide addi- radiation following the hybridization event, as tivity, bi-directional homogenization or inter- can be seen from the current genomic recombination resulting in a mosaic in this area (Mummenhoff et al. 2004). structure of ITS sequences would still retain Hybrid establishment and persistence im- traces of past hybridization. This marker has ply that the hybrid has become isolated from significantly contributed to the identification both of its parental taxa. This can be achieved of hybrids or hybridogenous origins in Card- either by chromosomal rearrangements and amine (Franzke and Mummenhoff 1999; subsequent postzygotic genetic isolation, Lihova´ et al. 2006), Boechera (Koch et al. which have been observed in newly formed 2003b), Lepidium (Mummenhoff et al. 2004), hybrids. Alternatively, ecological divergence, Thlaspi (Mummenhoff et al. 1997), and Draba e.g. spread into new ecological niches not (Widmer and Baltisberger 1999). Fingerprint- occupied by the parents results in spatial ing methods, such as RAPD, AFLP or ISSR, isolation and gene flow restriction. Other have been frequently used due to their high ecological barriers, such as temporal or polli- resolution. Intermediate genotypes and the nator divergence, can contribute to reproduc- additivity of individual fragments or alleles tive isolation as well (Arnold 1997, Gross and provided convincing evidence for hybrid ori- Rieseberg 2005, Hegarty and Hiscock 2005). gins (e.g. Rorippa austriaca · R. sylvestris, The role of environmental disturbance (often Bleeker and Matthies 2005; Cardamine ·insu- man-induced) for creating new habitats avail- eta, Urbanska et al. 1997; Diplotaxis muralis, able for hybrids is widely recognized, and Warwick and Anderson 1997a, Martı´ n and apparently was crucial also for the origin of Sa´ nchez-Ye´ lamo 2000; Erucastrum gallicum, several Brassicaceae hybrids, such as Card- Warwick and Anderson 1997b; Boechera ·di- amine ·insueta (Urbanska 1987) or Rorippa varicarpa, Dobesˇ et al. 2004a). Studies of austriaca · R. sylvestris hybrids (R. ·armora- cpDNA haplotype diversity can identify the cioides, Bleeker 2003). maternal parent, as well as demonstrate Molecular markers: detection of hybridiza- bi-directional and multiple hybrid origins. tion events in the Brassicaceae. A of Recently, we proved both bi-directional and molecular techniques has been used to detect recurrent polytopic hybridizations between the ancient or more recent hybridization events hexaploid Cardamine asarifolia and diploid and to identify parental taxa. Isozyme analyses C. amara. The hybrids (C. ·ferrarii) displayed and isoelectric focusing analysis of Rubisco multiple cpDNA haplotypes present in either (ribulose-1,5-bisphosphate carboxylase/oxy- of the parental species from the same or close genase) subunits have proven useful in many locality (Lihova´ et al. 2006). Similarly, bi- studies of the Brassicaceae genera, e.g. in directional hybridization has been assumed Rorippa (Bleeker and Hurka 2001), Nasturtium from the presence of both parental species- (Bleeker et al. 1999), Arabidopsis (Mummen- specific trnLortrnL-trnF length variants in hoff and Hurka 1995), Diplotaxis (Mummen- Rorippa amphibia · R. sylvestris (R. ·anceps, hoff et al. 1993, Eschmann-Grupe et al. 2003) Bleeker and Hurka 2001) and R. austriaca · and Cardamine (Urbanska et al. 1997), where R. sylvestris (R. ·armoracioides, Bleeker and species-specific alleles identified in the parents Matthies 2005). have been found in assumed hybrids. The As mentioned above, isozymes have the nrDNA ITS regions is among the most widely potential to confirm the parentage of putative used nuclear-encoded markers (A´ lvarez and hybrids, provided that enough variation and 148 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution especially species-specific alleles can be de- frequently involved in hybrid formation and tected. This is, however, not always the case. introgressive hybridization. An interesting Bleeker et al. (1999) aimed to confirm the model system to explore the dynamics of parentage of hexaploid Nasturtium ·sterile interspecific gene flow is represented by three (tetraploid N. officinale · octoploid N. micro- hybrid zones observed between invasive R. phyllum), first suggested by Manton (1935). austriaca and native R. sylvestris in Germany However, while N. microphyllum displayed (Bleeker 2003, Bleeker and Matthies 2005). In species-specific fixed alleles, no such alleles all three contact zones, hybridization was were found in N. officinale. Therefore, inferred from the additivity of diagnostic although N. ·sterile possessed the same band- parental AFLP markers of the local popula- ing patterns as N. microphyllum, providing tions, and from the overall genetic variation evidence that this species is one of the parents, patterns revealed by principal coordinate anal- it was not possible to confirm N. officinale as ysis (intermediate position of hybrids). In the second parent using available isozyme addition, three individuals, morphologically data. So far, no other marker systems have corresponding to R. sylvestris, possessed been applied that could provide more resolu- AFLP profiles attributable to hybrids, provid- tion. There is, however, circumstantial evi- ing evidence of introgression. Different cyto- dence originating from classical data that types of R. sylvestris (tetraploid and hexaploid) favours this parentage (Howard and Manton were found in the respective hybrid zones, 1946, Bleeker et al. 1999): (1) intermediate resulting also in different ploidy levels in the ploidy level of the hybrid, (2) morphological hybrid progeny (triploid and pentaploid). intermediacy, (3) experimental crosses result- While the origin of the triploid hybrid can be ing in progeny morphologically strongly easily explained by the fusion of reduced resembling N. ·sterile, and (4) the occurrence gametes, the prevalence of R. sylvestris mark- of N. ·sterile in areas where the putative ers and the lower level of additivity in the parents come into contact. pentaploids suggested that they may have In the following survey we focus on a few originated via first generation tetraploid plants Brassicaceae studies, where often multiple and further backcrosses with R. sylvestris molecular markers have been used to prove (Bleeker and Matthies 2005). Hybrid fitness the assumed hybridization events, and discuss differed between the triploids and pentaploids, resulting patterns. As demonstrated by these the latter showing better seed set and germi- studies, the use of additional data from other nation rate. Later generation hybrids are sources (e.g. chromosome numbers, morphol- expected to undergo , ogy, artificially-produced hybrids) often help which, together with backcrossing and selec- support the evidence of hybridization. tion, can result in a highly fertile and successful Hybridization and introgression in Rorippa: hybrid species (Song et al. 1995, Rieseberg hybrid fitness and intrapopulational variation. et al. 2000). The higher fitness found in the Hybridization among European species of pentaploid Rorippa hybrids can, thus, be Rorippa has been frequently reported by explained by these processes. The pentaploid several authors (e.g. Jonsell 1968, Tomsˇ ovic chromosome number may cause meiotic irreg- 1969). In recent years, more detailed studies ularities, however, viable gametes can still be employing molecular data have been published produced through polarized segregation of by Bleeker and co-authors (Bleeker 2003, 2004; chromosomes, as observed in Cardamine ·in- Bleeker and Hurka 2001; Bleeker and Matthies sueta (Urbanska et al. 1997). Another inter- 2005). Four closely related species belonging to esting issue that arose from these two Rorippa sect. Rorippa, R. amphibia (2n = 16, 32), R. studies (Bleeker 2003, Bleeker and palustris (2n = 32), R. sylvestris (2n = 32, 48) Matthies 2005) was the observed association and R. austriaca (2n = 16) have been of intrapopulational variation and invasive K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 149 success. R. austriaca, an introduction to described as C. ·insueta (Urbanska-Wor- Germany, showed different levels of intrapop- ytkiewicz and Landolt 1972). Autopolyploidi- ulational variation, with the highest one zation of these hybrids resulted in the forma- observed in areas where it behaves most tion of the highly fertile and viable hexaploid invasively. This observation seems to be in species C. schulzii (Urbanska-Worytkiewicz accordance with the assumption that accumu- 1977b). Since the discovery of this hybrid lation of genetic variation either through complex, extensive biosystematic and molecu- multiple introductions or hybridization with lar investigations have been performed to gain congeneric species may be a prerequisite for insights into its origin and ongoing microevo- the evolution of invasiveness (Lee 2002, Ell- lutionary processes. Earlier studies were fo- strand and Schierenbeck 2000). cused on cytogenetics and reproduction (chro- Another case, where interspecific gene flow mosome numbers, karyotype, meiosis and has shown a profound impact on intrapopula- chromosome segregation, pollen quality), as tional and intraspecific variation, is reported well as population biology (size and spatial for two self-incompatible species Rorippa structure of populations, population dynam- amphibia and R. sylvestris (Bleeker and Hurka ics, flowering intensity), and the role of human 2001). Both species are native in the area interference (Urbanska-Worytkiewicz and studied (Germany) and show different ecolog- Landolt 1972; Urbanska-Worytkiewicz ical preferences, but in periodically disturbed 1977a,b, 1980). It was concluded that the habitats the isolation barriers can temporarily triploid hybrid arose by fertilization of an breakdown. Hybridization and bi-directional unreduced C. pratensis gamete with a reduced introgression between these two species have C. amara gamete, and that it has only recently been confirmed at the river Elbe in areas established and expanded into suitable man- characterized by natural dynamic changes made habitats. More recent molecular investi- (Bleeker and Hurka 2001, Bleeker 2004). Both gations included several markers, RFLP of species exhibited increased intrapopulational cpDNA, isozymes, RAPD fingerprinting, and genetic variation in that hybrid zone, when ITS sequences (Neuffer and Jahncke 1997, compared with populations not influenced by Urbanska et al. 1997, Franzke and Mummen- hybridization. The same pattern was also hoff 1999). cpDNA restriction site mutations observed for seed set, being higher in the allowed the differentiation between the chlo- hybrid zone. Increased genetic variation dis- roplast genomes of the progenitor species C. played at the level of isozyme variability, thus, pratensis and C. amara, and showed that all might also affect variation at the S locus individuals of C. ·insueta and C. schulzii (through the exchange of alleles), which con- displayed patterns identical with C. pratensis, trols self-incompatibility. As a result, higher herewith providing the evidence for the mater- intrapopulational variation at the S locus may nal parent (Urbanska et al. 1997). Both RAPD positively affect successful fertilization and markers and isozymes revealed additivity in seed set (Bleeker 2004). the triploid and hexaploid, supporting their Hybridization and hybrid speciation in hybrid origin. The predominant vegetative Cardamine: the role of man-induced habitat reproduction observed in C. ·insueta, how- disturbances. A model of hybrid speciation ever, somewhat contradicted its high level of associated with man-induced habitat distur- genetic diversity, the latter being comparable bance is represented by triploid and hexaploid to that in the outcrossing parental species. Cardamine hybrids discovered in Switzerland. High genetic variation in the triploid can be Hybridization between two diploids, C. prat- most likely attributed to backcrossing and ensis (erroneously reported under the name recurrent hybridizations between the parental C. rivularis) and C. amara (subsp. amara) led species. In accordance with its assumed recent to successful establishment of triploid hybrids, origin (dated not earlier than 1900), only a few 150 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution non-parental RAPD markers were detected in and growing on somewhat man-influenced C. ·insueta, indicating that its genome has not sites. The hybrid status was confirmed with evolved far from the parental ones (Neuffer morphology, pollen sterility, and AFLP mark- and Jahncke 1997). ITS sequences revealed a ers. Similarly to C. ·insueta, the hybrid pop- rather pronounced sequence divergence be- ulations of C. ·enriquei exhibited considerable tween the diploids C. pratensis and C. amara morphological and genetic variation, suggest- and very rapid sequence homogenization in ing recurrent origins and/or backcrossing with both C. ·insueta and C. schulzii with a strong parents. Again, vegetative propagation has bias towards the maternal sequence type most likely made a major contribution to the (Franzke and Mummenhoff 1999). establishment of C. ·enriquei (Marhold et al. It seems that despite genetic differentiation 2002b). between congeneric species, incompatibility Recurrent and polytopic hybridizations: the barriers can occasionally break down, and case of Boechera ·divaricarpa. Hybridization plants with similar ecological requirements and and polyploidization, together with , sympatric occurrence hybridize. Similar to the have played an important role in the evolution case of Cardamine ·insueta, we can report on of the North American genus Boechera. This another example from this genus, where man- genus, originally classified within Arabis, induced environmental disturbances opened includes about 62 species (Al-Shehbaz 2003) the possibility for the establishment of a new which presumably radiated before Pleistocene hybrid. Inferring from our preliminary mor- glaciations. However, the frequent occurrence phological and molecular (AFLP markers) of hybrids and allopolyploids indicates that investigation, extensive hybridization, back- of these taxa is incom- crossing and introgression have occurred on plete (Schranz et al. 2005 and references there- several sites between high polyploids of Card- in). Most studies have focused on the wide- amine pratensis s.str. and C. raphanifolia s.str. spread and largely sympatric species, Boechera in the Cordillera Cantabrica Mts. (NW Spain; holboellii (= Arabis holboellii) and B. stricta Lihova´ et al., unpubl.). Parental species to- (= Arabis drummondii). Recent studies gether with the scarce occurrence of intermin- employing several molecular markers have gled hybrids or introgressed individuals have indicated a complex evolutionary history with- been observed to grow in relatively stable and in this group (Sharbel and Mitchell-Olds 2001; less disturbed habitats along brooks on pas- Koch et al. 2003b; Dobesˇ et al. 2004a, b; tures and wet meadows, while dense popula- Sharbel et al. 2005). As part of the thorough tions consisting exclusively of hybrids are studies, assumed hybridization between these spreading in ditches along the road. two species, resulting in conspicuous patterns, Natural hybridization at the diploid level has received much attention. The putative was documented between two eastern Pyre- hybrid (B. ·divaricarpa) showed substantial nean endemics, Cardamine amara subsp. pyre- part of the genetic variation present in its naea and C. crassifolia (Marhold et al. 2002b). parental species. Almost all chloroplast paren- Both species show very similar ecological tal haplotypes were found in the hybrid preferences and occupy almost the same dis- species. Their geographic distribution was tribution area. Recent molecular phylogenetic correlated with the distribution of the respec- analyses showed that they belong to two tive haplotypes found in the putative parents, different lineages, and thus are genetically and thus, suggested multiple hybridizations. A divergent (Lihova´ et al. 2004a). Nevertheless, very complex pattern was observed in ITS two hybrid populations (named as C. ·enri- sequences, with the following variants identi- quei) were found. In both cases they formed fied in different hybrid accessions: (1) a single small but rather dense populations that were sequence variant typically found in either of partially spatially separated from the parents, the parents, (2) multiple ITS sequence variants K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 151 of which at least one was found in one of the cultivated plants, resulting in a rather high parental species but not in the other, (3) chance of cross-fertilization. Recently, there multiple ITS sequence variants where both has been a major concern about transgenic parental ITS types were retained, and (4) a crop plants that could transfer engineered recombinant mosaic ITS sequence. Such a traits to weedy relatives and become beneficial pattern is consistent with different evolution- for them. The escape of the gene(s) from ary fates reported for divergent parental ITS transgenic plants into natural populations may sequences following their merger in the hybrids have unpredictable evolutionary and ecologi- (see A´ lvarez and Wendel 2003). Nevertheless, cal consequences (Warwick et al. 2003). This the occurrence of such a broad spectrum in a may be especially relevant for the oilseed rape single hybrid species is intriguing. Definitely, (Brassica napus), a major crop commonly hybridization must have been very common grown in Europe, North America, Argentina, and occurring multiple times and in multiple and China. This partially allogamous crop geographical locations (as seen from the plant is of hybridogenous allopolyploid origin cpDNA data). The most recent studies, how- (originated from wild B. rapa and B. oleracea) ever, have shown that the identification of the and has numerous compatible wild relatives parentage of B. ·divaricarpa may be even more which often occur sympatrically with the crop. complicated, and B. holboellii might not be one Several studies assessing the ability and fre- of its parents as currently believed. B. holboellii quency of interspecific gene flow and gene itself is a highly polymorphic and polyphyletic introgression from B. napus to various wild species that may be of hybrid origin (Dobesˇ relatives have been performed both in exper- et al. 2004a, b; Schranz et al. 2005). It has been imental field trials and in commercial fields recently suggested that B. stricta as the most (reviewed by Che` vre et al. 2004). While prob- widespread species of this genus hybridizes abilities of gene flow from transgenic B. napus with almost every sympatric diploid Boechera to several relatives (e.g. Raphanus raphani- species (Windham in Al-Shehbaz and Beilstein strum, Sinapis arvensis, Erucastrum gallicum) 2006). have been estimated to be very low, a high Gene flow between (transgenic) crop plants potential exists for hybridization with its and their wild relatives. An important aspect of progenitor B. rapa, as well as for the estab- interspecific hybridization that should also be lishment and spread of introgressed genes addressed here, is the possibility and the risk of within its natural populations (Warwick et al. gene flow from crop plants to their wild 2003, Che` vre et al. 2004). Weeds with novel relatives. The family Brassicaceae includes traits such as insect or disease resistance, or many economically important plants that are resistance to environmental stress, are likely to widely cultivated. Spontaneous hybridization display increased overall fitness and competi- between cultivated plants and their wild rela- tiveness, and their spread may have serious tives can occasionally occur, and has been agronomic and environmental implications indeed documented for several crops including (Warwick et al. 2003). Brassica species (Ellstrand et al. 1999, Che` vre Future tasks and challenges for hybridiza- et al. 2004). The probability of interspecific tion studies. Novel molecular and cytogenetic hybridization depends on various factors, such approaches provide the opportunity not only as phylogenetic relatedness, mating systems, to detect the origin and parentage of hybrids, and density and spatial distribution of wild but also to study hybrid speciation and the relatives (Warwick et al. 2003). Darmency impact of hybridization at the level of genome et al. (1998) pointed to the fact that a self- organization and gene expression (Hegarty incompatible wild species when growing and Hiscock 2005, Lysak and Lexer 2006). isolated within or near a crop field can receive Comparative genetic and physical mapping, a considerable amount of pollen from the chromosome painting and other methods 152 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution allow examination of genome evolution while structural genomic stasis has been following hybridization. There is increasing suggested in others (Adams and Wendel evidence that chromosomal rearrangements 2004, Ainouche et al. 2004). Numerous cases may take place rapidly in newly formed of changes in gene expression due to polyplo- hybrids. Other consequences reported are idization have been reported as well (e.g. transposon activation, gene silencing or Kashkush et al. 2002, Levy and Feldman sequence elimination (see Hegarty and Hiscock 2004, Soltis et al. 2004a). 2005). Within the Brassicaceae, extensive Allopolyploidy and long-distance dispersal, genetic and genomic analyses in this context respectively, are regarded as prominent factors have been undertaken primarily in Arabidopsis in plant evolution and biogeography. Mum- and Brassica species (see Schmidt et al. 2001, menhoff and Franzke (2006) review the rare Hall et al. 2002, Lysak and Lexer 2006). cases of prehistorical (not man-mediated) Expanding research activities to relatives of intercontinental long-distance dispersal of Arabidopsis thaliana is of great importance and plants combined with allopolyploidy. All opens future perspectives for studies within the examples given (Gossypium, Lepidium, Mi- Brassicaceae (Comai et al. 2003, Lysak et al. croseris, Nicotiana) indicate a Late Tertiary/ 2003, Lysak and Lexer 2006). Quaternary evolution of the polyploid lineages in the newly colonized continent. Late Ter- tiary, and especially Quaternary climatic fluc- Auto- and allopolyploidy – tracing the origin tuations affected all parts of the world and of polyploids these changes might have created novel habi- The high percentage of polyploids in the tats providing new niches for rapid radiation Brassicaceae clearly shows that polyploidiza- (Mummenhoff and Franzke 2006). tion processes must have been fundamental for Brassica, a model genus for the study of speciation in this family. The origin of polyp- polyploid evolution. Extensive genome rear- loids, their successful establishment, genetic rangements, suggested to occur in a number and genomic consequences, as well as evolu- of polyploid crops, including tobacco, maize, tionary implications of polyploidy are chal- soybeans and Brassica, are recognized as an lenging topics. Especially with the advances of important source of genetic novelty in the molecular biology techniques in recent dec- polyploids. Studies on synthetic experimental ades, such studies have received much atten- allopolyploids produced from interspecific tion (reviewed e.g. by Wendel 2000, Osborn crosses between Brassica rapa (AA genome, et al. 2003). Investigations of several model 2n = 20) and B. nigra (BB genome, 2n = 16) systems (e.g. Gossypium, Triticum, Aegilops, and between B. rapa and B. oleracea (CC Nicotiana, Brassica) have demonstrated the genome, 2n = 18) (and subsequent self- highly dynamic nature of polyploids, with pollinations up to the F5 generation) indicated increasing evidence that each polyploid system extensive genome changes in the early gener- may respond and evolve uniquely. Several ations after the polyploid formation (Song genetic and genomic attributes of polyploids et al. 1995). Southern hybridization studies have been suggested to account for their using several nuclear DNA probes have evolutionary success, such as increased hetero- revealed unexpected fragment profiles in the zygosity, increased genetic diversity through polyploids, involving loss and/or gain of multiple formation, genome rearrangements, parental restriction fragments and appearance and changes in gene expression (Soltis and of novel fragments. Song et al. (1995) dis- Soltis 2000). Major and rapid alterations to cussed several potential causes of these genome and chromosome organization have changes in RFLP patterns, including chromo- been observed in some allopolyploids (e.g. some rearrangements, point mutations, Levy and Feldman 2004, Lim et al. 2004), gene conversion or DNA methylation. They K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 153 concluded that polyploids can generate insights into the evolution of polyploid extensive genetic diversity within a short time genomes and to extend our current under- period, which could significantly contribute to standing of this evolutionary phenomenon. In their evolutionary success. Comparison of the following paragraphs, we will first present genetic linkage maps based on RFLP markers studies on several polyploid species illustrating between natural allopolyploid B. juncea their origins and evolutionary histories. Next (AABB, 2n = 36) and its parental species we will point to some common or contrasting (B. rapa and B. nigra) showed a high degree of patterns arising from those studies: the level of conservation of the A- and B-genomes in the genetic variation found in polyploids in com- allopolyploid and the diploids (Axelsson et al. parison with their diploid progenitors, differ- 2000). The collinearity observed in the genetic ences in mating systems in diploids and their maps indicated that the genome changes polyploid derivatives, the distinction between revealed by Song et al. (1995) in the synthe- auto- and allopolyploidizations, and patterns tized B. juncea are most probably not due to associated with single vs. polytopic and recur- extensive intergenomic translocations, but rent polyploid origins. originated from other genetic processes. Allopolyploid origins in Arabidopsis, Card- Studies on resynthesized Brassica polyp- amine and Diplotaxis. To unravel polyploid loids also provided evidence of novel pheno- origin is undoubtedly a challenging task. It is typic variation in several -history traits and not always straightforward, and in some cases changes in phenotypic plasticity, which have even multiple molecular approaches do not been associated with polyploidization and bring unequivocal results. Most paleopolyp- subsequent genetic changes (Schranz and Os- loids have undergone intergenomic recombi- born 2004). The research on Brassica species nation, genome rearrangement or diploidiza- reviewed by Osborn (2004) provides evidence tion, which often obscure their polyploid that several mechanisms can contribute to de origins and hamper identification of their novo variation observed in newly formed parentage. Genomes of both the original polyploids. The impact of polyploidy on dos- diploid progenitor and the polyploid have age-regulated genes was illustrated by studies become substantially differentiated since the on replicated FLC loci (flowering time regula- polyploid formation and much effort is needed tor) in Brassica polyploids. Variation in flow- to elucidate their origin and evolutionary ering time observed within palaeopolyploid history (Soltis et al. 2004b). Relatively recent Brassica rapa may be due to the increased allopolyploidization events, on the other hand, allelic variation at multiple FLC loci. New are usually much easier to reconstruct, as the phenotypic variation can be generated in affinities to diploids and overall additivity are polyploids also through intergenomic (homo- mostly still retained. The Brassicaceae family eologous) transpositions (suggested for B. offers examples of both well-explored and napus), or epigenetic changes and altered rather simple allopolyploid origins, and com- regulatory networks that affect gene expression plicated reticulate evolutionary histories of patterns (Osborn et al. 2003, Osborn 2004). high-polyploid complexes. Several other extensive genetic and molec- One of the best explored polyploids in the ular studies have been undertaken on Brassica family, Arabidopsis suecica (2n = 26), has species but it is not in the scope of this review attracted much interest, because one of its to deal with all of them in detail, instead we parents is the model species A. thaliana. With focus on non-model examples of Brassicaceae the immense knowledge gathered on A. thali- polyploids. They definitely represent new chal- ana, the tetraploid A. suecica offers unique lenges for future genetic and genomic studies opportunities for polyploid research, as comparable to those in Brassica and Arabid- presented by Comai et al. (2000, 2003), Ali opsis, and have the potential to provide new et al. (2004) and Madlung et al. (2005). 154 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution

The parentage of A. suecica was first suggested systems associated with polyploidization or by Hylander (1957), based solely on - hybridization have been found also in Capsella logical and phytogeographical evidence. A. sue- and Diplotaxis species (see below). Synthetic cica was suggested to be an allopolyploid that A. suecica-like allopolyploids were produced had arisen through hybridization between by Comai et al. (2000), which together with A. thaliana (2n =2x = 10 ) and Cardamin- natural A. suecica were subjected to cytoge- opsis arenosa (= A. arenosa,2n =2x, netic (GISH and FISH) studies (Comai et al. 4x = 16, 32). Isoelectric focusing analysis of 2003, Ali et al. 2004). Both natural and polypeptide composition of the large and small synthetic allopolyploids showed a pattern subunits of Rubisco provided molecular evi- consistent with a genomic constitution of 10 dence for A. thaliana as the maternal parent A. thaliana chromosomes and 16 A. arenosa and A. arenosa as the paternal one (Mummen- chromosomes. Homologous chromosome hoff and Hurka 1994). Further studies employ- pairing observed in the synthetic polyploid ing restriction site analysis of cpDNA, isozyme suggested that suppression of homoeologous analyses and ITS sequence data corroborated pairing occurs already in early generations this hypothesis (Mummenhoff and Hurka after polyploid formation, and was not 1995, O’Kane et al. 1996). As suggested for achieved by adaptive mutations or by genome several other Brassicaceae polyploids (e.g. in reshuffling as it might be expected (Comai Cardamine, Draba, see below), its origin and et al. 2003). spread to the current distribution area (south- Recent molecular studies showed that the ern Sweden and Finland) has most probably traditional taxonomical circumscription of been associated with Pleistocene glaciation, Arabidopsis and Arabis is largely artificial. more specifically, with glacier retreat providing Both were revealed to be polyphyletic, consist- large open areas. Interestingly, the parents of ing of several independent lineages (Koch A. suecica display different breeding systems et al. 1999, Koch et al. 2003a, Al-Shehbaz and extent of genetic variation. While A. tha- and Beilstein 2006). Al-Shehbaz et al. (1999) liana is a selfing species, characterized by high listed 59 binomials, previously assigned to individual homozygosity, low within- and high Arabidopsis, which are currently classified in between-population differentiation, A. arenosa 14 genera. On the other hand, taxa formerly is self-incompatible with high genetic diversity placed in Cardaminopsis are now included in (Lind-Hallde´ n et al. 2002, Sa¨ ll et al. 2004). In Arabidopsis, as well as several taxa, which were A. suecica, low RAPD variation was found originally described within Arabis. This holds (especially within populations, 80% of the true for the tetraploid taxa Arabis lyrata subsp. variation was distributed among populations) kamchatica and Arabis kawasakiana (Miyash- indicating selfing (Lind-Hallde´ n et al. 2002). ita et al. 1998; Koch et al. 2000, 2001; Savo- Even the occurrence of apomixis was lainen et al. 2000; Hoffmann 2005). There has discussed, which is extremely rare in the been much confusion about their taxonomic Brassicaceae, and otherwise observed only in position and circumscriptions, but the recent Boechera species (Schranz et al. 2005). Micro- study by Shimizu et al. (2005) resolved this satellite genetic segregation patterns studied in controversy and also shed some light on their A. suecica clearly showed that it reproduces polyploid origin. Recently, O’Kane and Al- sexually with a high level of selfing (Sa¨ ll et al. Shehbaz (1997) recognized three 2004). This observation implies that the self- under Arabidopsis lyrata, the North American incompatibility of A. arenosa has broken down subsp. lyrata (2n =2x=16), the European in the formation of the allopolyploid. The self- subsp. petraea (2n =2x,4x=16, 32), and incompatibility in Brassicaceae is apparently the Far East/North American subsp. kam- controlled by a multiallelic sporophytic sys- chatica (2n =4x=32). This taxonomic tem, and similar cases of shifts in mating concept has also been supported by recent K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 155

ITS sequence data, where the three subspecies from CHS sequencing. Three CHS homoeo- formed a cohesive and well-supported in logues were isolated from C. asarifolia, con- the including other Arabid- sistent with its hexaploid status and providing opsis species (Hoffmann 2005). In their treat- evidence for its allopolyploid origin. Phyloge- ment, O’Kane and Al-Shehbaz (1997) netic placements of the homoeologous se- considered Arabis kawasakiana as a synonym quences pointed to the potential parental of Arabidopsis lyrata subsp. kamchatica. How- species; C. amara s.l. was identified as the ever, as pointed by Shimizu et al. (2005), the maternal parent, while one CHS homoeo- former is a winter annual species occurring on logue close to C. hirsuta most likely origi- low-altitudinal sandy shores of western Japan, nated from an extinct parent (Lihova´ et al. while the latter is a mountain perennial more 2006). widely distributed (Eastern Asia, northern- The genus Diplotaxis comprising around 30 most North America). In addition, subsp. species distributed mainly in the European kamchatica appears to be distinct from A. Mediterranean has no economic importance, lyrata subsp. lyrata and subsp. petraea, and the but it represents one of the nearest wild best treatment reflecting its evolutionary his- relatives to Brassica. It may provide a valuable tory is as a separate species A. kamchatica. genetic resource for plant breeding programs Results from the nuclear single-copy gene of and crop improvement, and studies on this chalcone synthase (CHS; see Koch et al. 2000) genus may be therefore of special relevance showed the presence of two homoeologous loci (Go´ mez-Campo 1999, Martı´ n and Sa´ nchez- in the tetraploid A. kamchatica, which were Ye´ lamo 2000). Phylogenetic relationships highly homologous to sequences retrieved within the genus as well as the polyploid origin from diploids A. halleri subsp. gemmifera and of the tetraploid Diplotaxis muralis have been A. lyrata subsp. lyrata, respectively (Shimizu addressed in several studies employing differ- et al. 2005). This molecular data, together with ent markers. Based on morphological and the morphological and cytological study by cytological evidence, the allopolyploid origin Mulligan (1995) strongly suggest that A. kam- of D. muralis (2n = 42) from D. tenuifolia (2n chatica is an allotetraploid species originating = 22) and D. viminea (2n = 20) was first from the above mentioned diploids. There are suggested by Harberd and McArthur (1972). some preliminary indications that A. kawasa- Its genomic constitution has been subsequently kiana is a later derivative of A. kamchatica, studied by isoelectric focusing patterns of and their subspecific treatment as A. kamchat- Rubisco, isozymes, restriction site variation ica subsp. kawasakiana and subsp. kamchatica of the cpDNA, RAPDs, and microsatellites was suggested (Shimizu et al. 2005). (Warwick et al. 1992; Warwick and Anderson The use of low- or single-copy genes is 1997a; Mummenhoff et al. 1993; Martı´ n and strongly encouraged for studies on polyploids, Sa´ nchez-Ye´ lamo 2000; Eschmann-Grupe et al. as they usually better reflect biparental lin- 2003, 2004). All the data confirmed the allo- eages than commonly employed nrDNA and tetraploid origin of D. muralis with the par- cpDNA markers (Mort and Crawford 2004, entage as originally proposed by Harberd and Small et al. 2004). Despite the technical dif- McArthur (1972), and indicated that D. vim- ficulties associated with polyploidy, their inea was the maternal parent. Both the allote- application can provide valuable data on traploid D. muralis and diploid D. tenuifolia genome composition of polyploids. The sin- are successful colonizers; D. muralis indeed gle-copy gene CHS has proven useful in displays some common features of colonizing tracing the origin of the hexaploid Cardamine plants, such as polyploidy, annual to biennial asarifolia (Lihova´ et al. 2006). Phylogenetic life and selfing, while D. tenuifolia is incongruence observed between nrDNA and perennial and strictly allogamous (Eschmann- cpDNA markers were resolved by the results Grupe et al. 2004). Strong differences in 156 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution genetic variation between these two species 2004a, b). Although no substantial genetic were found. Tetraploid D. muralis in contrast differentiation has been found between these to D. tenuifolia displayed very low genetic two disjunct areas, Catalonian populations variation in RAPDs and isozymes. This displayed strong genetic depauperation in pattern may be explained by its young phylo- comparison with Italian ones. Alternative genetic origin, but also population history biogeographic hypotheses to explain the above (colonization history involving e.g. genetic pattern were discussed: (1) origin in Italy and drift, founder effects and local extinctions) later colonization of Catalonia either by long- might have caused the limited genetic variation distance dispersal or migration through the in this tetraploid (Eschmann-Grupe et al. regions connecting both peninsulas, associated 2003, 2004). with the reduction of genetic diversity through Biogeographic and evolutionary history founder effects; (2) fragmentation of original of polyploids in conjunction with Pleistocene larger distribution area, disappearance of con- climatic changes: Cardamine and Microthlaspi. necting populations and genetic impoverish- It has been widely recognized that the current ment in the western colonization route (Lihova´ distribution of genetic variation and geo- et al. 2004a, b). Despite the use of several graphic patterns in the Northern Hemisphere markers (AFLPs, cpDNA and ITS sequences), have been significantly shaped by Pleistocene the polyploid origin of C. amporitana could climatic changes. Plant migration during not be unequivocally proved, and both ancient glacial periods may have restricted or dis- autopolyploid and allopolyploid origin within rupted their original continuous range and led the C. amara group were considered as two to population diversification, but secondary equally plausible hypotheses (Lihova´ et al. contact has often occurred during recoloniza- 2004a, b). tion (Hewitt 2004, for Brassicaceae examples On the other hand, the resolution of the see Koch and Kiefer 2006). Geographic origin and parentage of polyploid Microthlaspi patterns and distribution of genetic diversity perfoliatum was more straightforward (Mum- in a given polyploid, particularly when menhoff et al. 1997, Koch et al. 1998b, Koch assessed from multiple markers, can help and Hurka 1999). Microthlaspi perfoliatum is elucidate both its evolutionary and biogeo- an annual species widely distributed in Europe graphic history, which are often tightly cou- and represented by diploid, tetraploid and pled. Two examples can be mentioned where hexaploid cytotypes. Together with three other hybridization and subsequent polyploidization diploids, it has usually been treated as the most likely predated Pleistocene glaciation M. perfoliatum aggregate. Although the differ- processes, and where climatic changes have ent cytotypes can be hardly distinguished significantly impacted on their geographic morphologically, based on molecular markers distribution and genetic variation patterns: they probably should be treated as two sepa- tetraploid Cardamine amporitana (Lihova´ rate species (diploid and polyploid). Several et al. 2004a, b) and diploid to hexaploid investigations using different markers (iso- Microthlaspi perfoliatum (Koch and Bernhardt zymes, cpDNA and nrDNA data) have docu- 2004). There have also been numerous studies mented the allopolyploid origin of M. dating the origin of polyploids to the time of perfoliatum populations from the diploids M. Pleistocene population migrations and second- perfoliatum and M. natolicum (Mummenhoff ary contacts, as also will be discussed for the et al. 1997, Koch et al. 1998b, Koch and hexaploid Cardamine silana (Perny´ et al. Hurka 1999). Compared to the diploids, the 2005a, Lihova´ et al. 2004a). polyploid populations displayed significantly Disjunct distribution has been observed in higher levels of genetic diversity as well as a Cardamine amporitana occurring in Catalonia wider distribution area. Apparently, glaciation (NE Spain) and central Italy (Lihova´ et al. must have had a greater influence on diploid K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 157 than on polyploid M. perfoliatum. Extinction found favourable conditions during colder of most of the genetic variability in the diploid periods (Taberlet et al. 1998, Fineschi et al. populations has been assumed, whereas polyp- 2002, Palme´ and Vendramin 2002). The pres- loids maintained genetically differentiated pop- ent data allow us only to speculate on where ulations (Koch and Hurka 1999). Different and when both presumable parental species biogeographic histories and glacial refugia met and whether the few remaining popula- were revealed for the diploid and polyploid tions are remnants of a previously more cytotypes. The polyploid populations showed widespread species, or were always restricted the classical European pattern with three main to the Sila Mts. Populations of C. silana do not diversity centers in Iberia, Italy and the Bal- appear genetically strongly depauperate (as kans, while the diploids experienced substan- shown by AFLP and cpDNA sequence data), tial fragmentation and extinction, and were but the number of individuals analyzed was forced into two refugia (SE France and Aus- too low to assess the level and distribution of tria/Croatia; Koch and Bernhardt 2004). genetic variation that could shed light on its Molecular marker and morphological stud- phylogeographic history (Perny´ et al. 2005a). ies have revealed the origin of the Calabrian Complex polyploid speciation: evolutionary (southern Italy) hexaploid endemic Cardamine scenaria in Draba, Cardamine and Cochlea- silana. The results provided strong support ria. When reviewing polyploidy as an impor- that two diploid species were involved in its tant speciation mode in the Brassicaceae, polyploid origin, the central Italian C. apenn- Draba, the largest genus in the family at ina and Balkan C. acris (Perny´ et al. 2005a, approx. 350 species, cannot be omitted. The Lihova´ et al. 2004a, see also Lihova´ and frequency of polyploid taxa is around 70% Marhold 2006). ITS sequences from C. silana (based on data from Warwick and Al-Shehbaz indicated that this multicopy DNA region, 2006), and both auto- and allopolyploidy have typically subjected to sequence homogeniza- apparently been common in its evolutionary tion, still retained (at least) two different history. The ploidy level ranges from diploids sequence variants. Intra-individual polymor- up to 18-ploids. The genus is well known for its phisms detected in C. silana displayed additive complexity in terms of morphological varia- patterns, strongly suggesting that one ITS tion, reticulate evolution and consequently sequence variant comes from C. apennina, taxonomy (Grundt et al. 2004). It has been while the other is shared by several related hypothesized that the complexity may be species. The AFLP-fingerprinting profile, rep- compounded by several factors, such as recur- resenting variation distributed throughout the rent formation of the polyploids, gene flow whole genome, on the other hand, revealed across ploidy levels, repeated migration and strong affinity to the Balkan C. acris. Geo- colonization resulting in secondary contacts graphic separation of both assumed parental between previously isolated populations species from C. silana might be at first sight (Brochmann et al. 1992a, d; Koch and Al- surprising. There is, however, clear evidence Shehbaz 2002; Grundt et al. 2004). There have for past migration and gene flow between the been numerous investigations, which aimed to Apennine and Balkan Peninsulas seen in phy- resolve this evolutionary and taxonomic com- logeographic studies (e.g. Fineschi et al. 2002), plexity in selected species groups (e.g. Broch- as well as Italian-Balkan distribution patterns mann et al. 1992a, b, c, 1993; Koch and Al- of numerous taxa (for examples see Perny´ Shehbaz 2002). While there is unequivocal et al. 2005a). In addition, Calabria is well evidence for the allopolyploid origin of the known as an important refugium during Swiss endemic D. ladina (Widmer and Baltis- Pleistocene climatic changes, where popula- berger 1999), much more complex pictures tions of several species distributed in higher have been found in arctic-alpine groups latitudes (including C. apennina) could have which involve high-polyploids. Additivity of 158 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution

ITS sequences and the cpDNA haplotype istics) mutually differentiated (Urbanska-Wor- found in the tetraploid D. ladina supported ytkiewicz and Landolt 1974). The molecular D. aizoides as the paternal and D. tomentosa as markers applied, however, did not yield the maternal parent. Despite the high to enough resolution to find genetic divergence moderate intraspecific genetic variation pres- among those diploid populations. ent in these widespread diploid parents, no Polyploidization has also been an impor- variation has been observed in D. ladina, tant speciation driving force in Cochlearia. The strongly supporting its single origin (Widmer Cochlearia is a highly polymorphic and Baltisberger 1999). As for the arctic-alpine with considerable cytogenetic group, fixed heterozygosity observed in the diversity (with two base chromosome numbers circumpolar tetraploid/hexaploid D. lactea and several polyploids), different ecological implied genetic allopolyploidy, and diploids and geographic patterns. It is D. fladnizensis, D. nivalis and/or D. subcapitata considered to be of recent Pleistocene origin, were suggested as the putative parents (Broch- showing only little morphological differentia- mann et al. 1992a). A recent study, however, tion and genetic divergence among the taxa based on several markers including the single- (Koch et al. 1996). Several studies devoted to copy nuclear gene RPD2 showed that D. lactea this species group (Koch et al. 1996, 1998a; and other related polyploids (D. turczaninovii, Koch 2002) indicated a complex evolutionary D. porsildii) may have originated from single history, including both auto- and allopolyploid diploid lineages. If allopolyploidization was origins. The scenario displayed in Fig. 1 has the case, this must have involved very similar been proposed based on molecular data from genomes (Grundt et al. 2004). It has been several markers, and is also supported by hypothesized that hexaploid D. lactea arose morphological, cytological and ecological evi- recurrently from the Beringian diploid D. dence. Apparently, the tetraploid C. officinalis, palanderiana via tetraploid D. lactea. distributed along the northern coasts of This apparent contradiction (genetic allopoly- Europe and assumed to originate via auto- or ploidy vs. taxonomic autopolyploidy) can be allopolyploidization from ancestors of the explained by the existence of cryptic species diploid C. aestuaria, have played a central role within diploid taxonomic species. Crossing in speciation processes in this section. This experiments have indicated that several diploid species, showing extensive morphological and inbreeding Draba species comprise numerous ecological variation, contributed to the origin ‘strains’, which are cross-incompatible. Occa- of several higher polyploids, both via auto- (C. sionally, the crossing barriers can break down anglica) and allopolyploidizations (C. polonica, and the cryptic species may produce a genetic C. bavarica, C. tatrae). An interesting biogeo- allopolyploid (Brochmann et al. 2004, Grundt graphic pattern has been observed within the et al. 2004). hexaploid C. bavarica restricted to two disjunct We may face a similar situation of cryptic regions in S Germany (Bavaria). Significant species also in diploid Cardamine pratensis genetic differentiation revealed between these s.str. This species includes several widely dis- two regions raised the question whether it tributed cytotypes from diploids up to heptap- reflects: (1) polytopic origin, (2) migration loids. Molecular studies suggest that the from the original range and subsequent genetic polyploids have evolved repeatedly from dip- differentiation, or (3) disruption of a former loids, and can be considered as taxonomic wider distribution area through population autopolyploids (Franzke and Hurka 2000, extinctions. The third scenario was favoured Lihova´ et al. 2003). However, diploid popula- based on recent isozyme data (Koch 2002). tions from the Alps and adjacent areas have Autopolyploid origins reported in Card- been reported to be ecologically and even amine, Biscutella and Capsella. Recent studies cytogenetically (based on karyotype character- from various angiosperm lineages indicate that K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 159

C. anglica 2n=6x 2n=8x,10x=48,60 C. polonica C. bavarica C. tatrae 2n=6x=36 2n=6x=42 C. groenlandica 2n=2x=14 2n=2x=12

2n=4x subsp. officinalis

C. officinalis subsp. norvegica 2n=4x=24 C. pyrenaica subsp. integrifolia C. macrorrhiza 2n=2x=12 2n=2x=12 C. excelsa 2n=2x=12 C. danica 2n=6x=42

C. aestuaria 2n=2x=12 diploid ancestral Cochlearia

Fig. 1. Evolutionary scenario in Cochlearia sect. Cochlearia according to Koch et al. (1998). Different colours indicate different ploidy levels in polyploids. Reproduced and modified with permission from the first author autopolyploidy is much more common than results showed very low genetic differentiation traditionally considered and apparently has between diploid C. amara subsp. amara and played an important role in plant evolution tetraploid subsp. austriaca, and only very few (Levin 2002, Soltis et al. 2004b). As in Coch- alleles or fragments unique to either of the learia (see above and Fig. 1), a few cases with subspecies (in contrast to other three subspe- evidence or indication for autopolyploidy have cies). This, together with only slight morpho- been documented in Cardamine and Biscutella. logical differentiation and clear geographical In Capsella, despite extensive studies, the patterns, strongly favoured autopolyploid polyploid origin of C. bursa-pastoris has not origin of the tetraploid subspecies. As the been unequivocally resolved, but ancient auto- distribution of subsp. austriaca coincides with polyploidization has been favoured by Hurka the area heavily affected by Pleistocene glaci- and Neuffer (1997). ation, we assume that the autopolyploidization Tetraploid populations of Cardamine amar- and establishment of this new tetraploid taxon a occupying the area of the Eastern Alps and took place in the last interglacial or postglacial adjacent regions (C. amara subsp. austriaca) period when new habitats became available have been studied using several molecular after glacier retreat. Whether the autotetra- markers, including isozymes, nuclear and chlo- ploid has a single origin and colonized the Alps roplast DNA sequences, as well as RAPD and spreading from a single refuge or whether AFLP fingerprinting (Marhold 1999; Lihova´ multiple events and different colonization et al. 2000, 2004b; Marhold et al. 2002a). The routes can be traced, remains an open question. 160 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution

Another suspected autopolyploid is Card- due to its ancient origin. New alleles identified amine majovskyi, a recently described tetraploid at isozyme loci not found in the diploids, species occurring in Central and southeastern presence of null alleles indicating gene silenc- Europe. C. majovskyi and its assumed progen- ing, and considerable isozyme and RAPD itor, diploid C. matthioli, were included in the variability favour an ancient polyploidization complex molecular systematic and biogeo- event, supported also from fossil records graphic study of the C. pratensis group (Franzke (Hurka and Neuffer 1997). Both allopolyploid and Hurka 2000, Lihova´ et al. 2003). All and autopolyploid origin from C. grandiflora analyses involving isozymes, nuclear and can be considered. Disomic allozyme inheri- cpDNA sequences, RAPD and AFLP markers tance and fixed heterozygosity would suggest showed a very close relationship between these the allotetraploid origin. Nevertheless, C. two species. Cardamine majovskyi is largely rubella seems to be a younger derivative of C. sympatric with C. matthioli. This fact, together grandiflora, and no other extant species from with slight morphological differences observed another genus appears to be closely related to among populations of C. majovskyi from dif- hybridize with Capsella. It can be hypothesized ferent parts of its distribution area (Lihova´ and that the second parent is extinct, but based on Marhold 2003; and K. Marhold, unpubl. data) available evidence, Hurka and Neuffer (1997) might indicate its recurrent and independent concluded that C. bursa-pastoris most likely origins. Thorough sampling and high-reso- arose via autopolyploidization from C. gran- lution molecular markers will be needed in diflora a long time ago. Recently published future studies to corroborate this assumption. reports on comparative chromosome painting In the highly variable species Biscutella of C. rubella and C. bursa-pastoris are prom- laevigata, genetic autopolyploidy has been ising and might in future studies bring more found in tetraploid populations (classified as resolution in this respect, provided that C. subsp. laevigata) based on isozyme data (Tre- grandifora will display a different painting metsberger et al. 2002). These populations pattern than C. rubella (Lysak et al. 2003). exhibited tetrasomic inheritance and thus Similar to Arabidopsis suecica, speciation pro- absence of fixed heterozygosity, and showed cesses in Capsella were accompanied by a only moderate divergence from the diploids, switch in mating systems. While C. grandiflora implying moreover their recent origin. As the is self-incompatible, both its assumed deriva- tetraploids showed genetic resemblance to tives, C. rubella and C. bursa-pastoris, are different diploid subgroups, their multiple predominantly selfing species (Hurka and origins have been hypothesized. Presumably, Neuffer 1997). the resulting high genetic variation and greater Contrasting patterns of genetic diversity in genomic plasticity have contributed to the polyploids and their possible determinants. The evolutionary success of tetraploids over dip- above survey of several recent studies focusing loids in alpine habitats (Tremetsberger et al. on the origin and evolution of polyploids in the 2002). Brassicaceae points to some interesting pat- An interesting case of polyploid speciation terns that can be further discussed. There have is also reported in Capsella. Only three species been numerous studies and reviews relating have been recognized in this genus, two geo- genetic diversity in polyploids to their evolu- graphically restricted diploids (C. rubella, C. tionary success (see e.g. Soltis and Soltis 2000, grandiflora) and a single tetraploid, the world- Soltis et al. 2004b). Increased genetic diversity wide successful colonizer C. bursa-pastoris. In in polyploids, when compared to putative spite of extensive studies (Hurka et al. 1989, diploid parents, has been observed in poly- Mummenhoff and Hurka 1990, Hurka and ploid Microthlaspi perfoliatum and it appar- Neuffer 1997), the origin of the tetraploid has ently correlates with increased morphological not been unequivocally resolved, most likely variation and larger geographic distribution. K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 161

Preglacial polyploid origin and different bi- a low frequency or single hybridization events. ogeographic history than in diploids have been In addition, theoretical models predict that proposed (Koch and Bernhardt 2004). In- polyploids relative to their diploid parent(s) creased variation and successful colonization should have reduced inbreeding depression, of predominantly alpine areas have also been allowing for increased selfing rates. Few revealed in tetraploid Biscutella laevigata empirical studies addressing this issue have subsp. laevigata that most likely reflect its been published (reviewed by Soltis and Soltis multiple origins (Tremetsberger et al. 2002). 2000, see also Rausch and Morgan 2005). In The best example is probably Capsella bursa- Draba, inbreeding is common, and as indicated pastoris, the tetraploid with high genetic var- by Brochmann (1993), diploid arctic Draba iation and world-wide distribution, which species are genetically depauperate due to contrasts with restricted areas of the related higher levels of selfing, while polyploids dis- diploids (Hurka and Neuffer 1997). Despite playing either predominantly autogamous or the limited sampling, several markers indicated mixed mating maintain high genetic variation. that the assumed autotetraploid Cardamine Although the hypothesis that polyploids ex- amara subsp. austriaca harbours genetic vari- hibit higher selfing rates than diploids has not ation comparable to that of its putative parent been supported for the studied Draba species, subsp. amara. This would favour multiple the allopolyploidy probably serves here as an autopolyploidization events, supported also escape from genetic depauperation caused by from the presence of several cpDNA haplo- drift and inbreeding at the diploid level. Fixed types in the tetraploid subspecies (Lihova´ et al. heterozygosity in allopolyploids ensures that 2000, 2004a, b; Marhold et al. 2002a). On the genetic diversity is maintained through gener- other hand, the allopolyploid Diplotaxis mu- ations despite inbreeding (Brochmann 1993, ralis distributed across most of Europe dis- Brochmann et al. 2004). In several Brassica- played surprisingly low genetic variation, ceae polyploid species, shifts in mating system attributable to its recent origin and/or loss of towards inbreeding have indeed been ob- genetic variation through extinctions, bottle- served, in contrast to outcrossing or even neck and genetic drift effects (Eschmann- self-incompatibility in their diploid parent(s). Grupe et al. 2004). Low genetic diversity due We can hypothesize that the high level of to single origins has also been found in Draba selfing found in the polyploids Arabidopsis ladina and Arabidopsis suecica, and both but suecica (Sa¨ ll et al. 2004), Capsella bursa-pasto- especially the former remained localized within ris (Hurka and Neuffer 1997) and Diplotaxis a small geographic area (Widmer and Baltis- muralis (Eschmann-Grupe et al. 2004) has berger 1999, Lind-Hallde´ n et al. 2002). In contributed to their evolutionary success. Arabidopsis suecica, the low level of genetic Distinction between auto- and allopolyploid variation is explained also by its autogamous origins. Auto- vs. allopolyploid origins can reproduction mode (Sa¨ ll et al. 2004). usually be distinguished from genetic or geno- Shifts in mating systems associated with mic patterns, nevertheless, in some cases polyploid speciation. An important determi- especially in ancient polyploidization events nant of the distribution of genetic variation this distinction may not be conclusive. Genetic and evolutionary success of a particular poly- allopolyploids are characterized by disomic ploid species is its reproduction mode. Mating inheritance at each locus and fixed (nonsegre- system can significantly influence polyploid gating) heterozygosity, while in autopolyploids establishment. It has been hypothesized that multisomic inheritance typically occurs (Soltis self-fertilization in polyploids may facilitate and Soltis 2000). In Biscutella laevigata subsp. their establishment because of limited back- laevigata the absence of fixed heterozygosity crossing to their parents resulting in sterile and tetrasomic inheritance observed in iso- progeny. Selfing also ensures the persistence of zyme data confirm its autopolyploid origin 162 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution

(Tremetsberger et al. 2002). Close genetic GISH can identify and separate parental resemblance and the absence of unique genomes in the studied polyploids, as was alleles/fragments in a polyploid when com- demonstrated for Arabidopsis suecica (Ali pared with a putative diploid progenitor can et al. 2004). also be considered a reliable indicator of Single vs. polytopic (multiple) origins of autopolyploidy, as suggested for Cardamine polyploid taxa. Molecular studies have contin- amara subsp. amara (Lihova´ et al. 2000, ued to reveal that multiple origins of either 2004b; Marhold et al. 2002a) and C. majovskyi auto- or allopolyploids from the same diploid (Franzke and Hurka 2000, Lihova´ et al. 2003). progenitors are common (Soltis and Soltis Very low genetic divergence between the 2000, Soltis et al. 2004b). It seems that poly- diploids, however, may hamper the distinction topic and recurrent polyploid origins are much between auto- and allopolyploidy, unless more widespread than traditionally consid- markers specific for either of the diploids are ered, and may represent a significant source of found. Although fixed heterozygosity has been genetic diversity in polyploids (Soltis et al. observed in Draba polyploids, results from the 2004b). To prove the single vs. multiple origins single-copy RPD2 gene implied that either unequivocally, however, it is crucial to sample autopolyploidy or allopolyploidy involving allelic (or haplotype) diversity in both the very similar genomes (from cryptic species) polyploid and its diploid progenitor(s) (Doyle had occurred (Grundt et al. 2004). Similarly, et al. 2004). Taxonomic and evolutionary the origins of tetraploids Capsella bursa-pasto- complexity in the highly polymorphic genus ris (Hurka and Neuffer 1997), Cardamine Draba has been explained by multiple origins amporitana (Lihova´ et al. 2004a) and Cochlea- of several widespread high-polyploid species, ria officinalis (Koch 2002) have not been as clearly proved in Draba lactea (Brochmann elucidated so far despite extensive studies. et al. 2004, Grundt et al. 2004). Multiple Future investigations employing other markers origins have also been proposed for Arabidop- or newly available techniques may provide sis suecica based on isozyme data (Mummen- more resolution. Allopolyploid origins can be hoff and Hurka 1995), although extremely low clearly proven from additive genetic patterns, RAPD variation may be an indication for a as observed at isozyme loci (Arabidopsis sue- single origin (Lind-Hallde´ n et al. 2002). The cica: Mummenhoff and Hurka 1995, Microthl- former scenario is favoured by the presence of aspi perfoliatum: Koch and Hurka 1999), in two fixed heterozygous genotypes in A. suecica multilocus fingerprinting marker systems of for the Pgm loci, corresponding to the allelic AFLPs or RAPDs (Cochlearia bavarica: Koch at the same locus in its progen- et al. 1996, Diplotaxis muralis: Eschmann- itors. Although this intraspecific variation in Grupe et al. 2004), and in nrDNA ITS A. suecica might be explained by evolution of a sequences (Cardamine silana: Lihova´ et al. new allele following a single polyploid origin, 2004a, Microthlaspi perfoliatum: Mummenhoff this hypothesis is unlikely (Mummenhoff and et al. 1997, Draba ladina: Widmer and Baltis- Hurka 1995). Multiple autopolyploid origins berger 1999). Nuclear encoded single-copy from genetically differentiated progenitor pop- genes have the potential to identify allopoly- ulations have been proposed for Biscutella ploid origins and involved parental species laevigata subsp. laevigata (Tremetsberger et al. more efficiently. The presence of two or more 2002). Single origin, on the other hand, has homoeologous loci in a polyploid, correspond- been strongly favoured for the local endemic ing to the sequences resolved from its diploid Draba ladina with less than 12 known popu- relatives has been documented for Cardamine lations. Lack of intraspecific variation in asarifolia (Lihova´ et al. 2006) and Ara- several individuals from two distinct locations, bidopsis kamchatica subsp. kamchatica (Shi- despite the significant variation present in both mizu et al. 2005). Genomic studies, such as parents, is a strong indication for the single K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 163 origin (Widmer and Baltisberger 1999). of incongruent patterns attributed to reticu- Although multiple cpDNA haplotypes with a lation can be found in recent Brassicaceae clear geographic structure were found in both studies, which are here shortly reviewed. the hexaploid Cardamine asarifolia and its A highly reticulate evolution has been maternal parent (C. amara), they shared only identified in Lepidium, a large worldwide one of them. The remaining haplotypes were distributed genus with conspicuous floral var- unique to C. asarifolia and, based on statistical iation and a predominantly autogamous mat- parsimony analysis, they were apparently ing system. The incongruence of species derived from the shared one. Thus, it has been relationships based on maternally inherited suggested that cpDNA diversification occurred cpDNA versus nrDNA ITS markers together after the polyploidization event, but further with the common occurrence of polyploids has studies are still needed (Lihova´ et al. 2006). suggested predominance of allopolyploid spe- The above studies illustrate that the Brass- ciation in this genus (Bowman et al. 1999, icaceae harbour polyploids with very different Mummenhoff et al. 2001, 2004). This indica- evolutionary and biogeographic histories, and tion has been further supported by the results provide an immense resource for future stud- from the single-copy nuclear gene PI (PISTIL- ies. As new molecular markers and techniques LATA), since multiple phylogenetically dis- for genomic studies become available, the tinct sequences have been found in several spectrum of powerful tools to study the investigated species from Americas and Aus- genomic composition and evolution of polyp- tralia (Fig. 2, designated as group 1; Lee et al. loids increases, and hopefully will soon find its 2002). On the basis of previous cpDNA and wide application beyond the model plants. ITS sequence data, and ploidy levels of those species, allopolyploidization (and thus homo- eologous origin of the multiple sequences) has Reticulate evolution been strongly favoured to explain the observed Polyploidization and hybridization are events, patterns. Four major groupings of allopolyp- which often result in a reticulated pattern of loids composed of two genomes, and one evolution. Reticulation complicates recon- grouping of taxa composed of even three struction of evolutionary relationships and genomes have been identified (Fig. 2). In some makes phylogeny inferences a challenging of them, multiple but very closely related undertaking. Reticulate evolution can be sequence types have been found even within detected via incongruence between gene trees, a clade, and these may have their origin either although other processes can sometimes pro- in allelic variation (proposed e.g. for L. monta- duce similar discordant patterns. Disentan- num) or in gene duplication (proposed for L. gling reticulate from divergent relationships densiflorum). The origin of several Australian/ requires use of multiple independent markers New Zealand polyploid species (Fig. 2, desig- with different modes of inheritance (Doyle nated as group 2) still remains unresolved. 1992, Linder and Rieseberg 2004, Vriesendorp Although only a single PI intron sequence has and Bakker 2005). Investigation of the addi- been resolved from each species, their positions tivity of molecular markers, as well as addi- in the ITS and cpDNA gene trees were tional data such as morphology, chromosome incongruent (Lee et al. 2002, Mummenhoff numbers, GISH or FISH patterns, C-values, et al. 2004). In addition, there are indications and geographic distribution, are often crucial from RFLP analyses that they possess two to unravel ancient hybridization events and to homoeologous loci, but one locus apparently distinguish them from population genetic has not been detected by PCR due to either processes that may confound the phylogenetic pseudogenization or interlocus recombination signal (Linder and Rieseberg 2004, Vriesen- (Lee et al. 2002). Inferring from these data, it dorp and Bakker 2005). Numerous examples is likely that they are allopolyploids as well. 164 K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution

L. phlebopetalum L. leptopetalum (3x) L. heterophyllum (2x) L. hirtum ssp. calycotrichum (2x) L. campastre (2x) L. perfoliatum (2x) L. ruderale (2x, 4x) L. cordatum L. latifolium (3x, 6x) group 1 group 2 L. pinnatifidum (2n=28) L. vesicarium (2x) A L. monoplocoides (4x) L. spinosum (2x, 3x) L. oxytrichum (3x) L. flexicaule L. pseudohyssopifolium (4x)

L. africanum (2x) L. densiflorum (4x) (4x) L. myriocarpum L. lasiocarpum L. armoracia L. virginicum (4x) L. allaudii (2x) L. montanum (4x) B L. flavum (4x) L. lyratum (2x) (4x, 8x) L. persicum (2x) L. bippinatifidum L. nitidum (4x) L. graminifolium (6x) L. sativum (3x, 4x) L. bonariense (4x, 8x) L. chichicara (4x, 8x) L. aschersonii L. apetalum (4x) L. desvauxii C L. pseudotasmanicum (2n=28) L. hyssopifolium (4x) L. oleraceum (4x) L. meyenii (8x) L. fremontii (4x) L. oblongum L. quitense D L. dictyotum

Fig. 2. Polyploid speciation in Lepidium basedonthePI intron gene tree. Group 1 is represented by allopolyploid species composed of genomes originating in two or three different (A-D). In taxa from the group 2, a single PI intron sequence has been resolved from each species, but at least for some taxa, their allopolyploid origin has been favoured from other evidence. Australian/New Zealand taxa are set in bold. Reproduced and modified with permission from Lee et al. (2002); 2002, National Academy of Sciences, U.S.A.

Reticulation has been suggested to occur recently treated within three genera Yinshania, also in the evolution of the Eurasian/North Hilliella and Cochleariella. Molecular phylo- American group of Braya and Neotorularia genetic analyses of ITS and trnL sequence species, with the assumption of both recent data have identified two main lineages, one and more ancient hybridizations. Discordant corresponding to the diploid Yinshania, the phylogenetic placements of Braya rosea and second lineage including the remaining two Neotorularia brachycarpa in respect of the polyploid genera (Koch and Al-Shehbaz nrDNA ITS and trnL cpDNA gene trees, 2000). Comparisons of the two gene trees and an apparent ITS sequence homogenization revealed incongruences, which strongly sug- are in favour of an ancient reticulation. On the gested gene flow between these two main other hand, nucleotide additivity in ITS lineages, and all taxa should probably be sequences observed in several accessions of treated within a single widely conceived genus. various Braya species probably reflects recent Hybridizations within the polyploid Hilliella/ or even on-going hybridization (Warwick et al. Cochleariella clade and subsequent differential 2004). ITS sequence homogenization have been Much controversy has surrounded the proven as well. This has been illustrated also taxonomic treatment of the Chinese endemics by the split decomposition analysis, which has K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 165 the potential to display conflicting phyloge- and Bakker 2005). To decrease the risk of netic signal within a single marker. Accessions producing false phylogenies in such cases, one of hybrid origin with unidirectional ITS could suggest the removal of all known sequence homogenization as well as with the polyploids from the phylogenetic analyses, as presence of both paternal sequence types were strongly advocated by Bachmann (2000). This found. One accession of H. sinuata showed a approach was followed in the study on Card- mosaic recombinant sequence type (Koch and amine species traditionally treated within three Al-Shehbaz 2000). different polyploid complexes of mainly Euro- Complex evolutionary history including pean distribution, by including only diploids in hybridization, polyploidization and facultative the analyses (Marhold et al. 2004). The poly- apomixis in the North American genus Boec- ploid complexes of Cardamine amara, C. raph- hera has been recently addressed in several anifolia and C. pratensis have always been at studies. It has been assumed that extensive least implicitly considered to represent coher- reticulation has played an important role in the ent and monophyletic groups. To investigate evolution of the highly polymorphic taxon phylogenetic relationships among them, two B. holboellii, which includes diploids, triploids independent molecular data sets were used, and aneuploids. As many as 70 different ITS nrDNA ITS sequences and AFLP markers. sequence types have been found in this species, The phylogenetic trees obtained were largely which did not form a single well-supported congruent, and revealed two main lineages. clade, but were placed in several unresolved While the C. amara group was resolved as a branches sister to another species, B. stricta well-defined monophyletic group, neither of (Dobesˇ et al. 2004a). This, together with the the markers supported current taxonomic frequent occurrence of intra-individual ITS separation of remaining diploids into two polymorphism strongly suggests extensive groups (the C. pratensis and C. raphanifolia reticulation. ITS, cpDNA as well as microsat- groups). Instead, all these taxa formed a single ellite data provided clear evidence that trip- clade with poorly resolved relationships. It was loids have originated repeatedly from diploid hypothesized that either hybridization and lineages, and that hybridization between genet- introgression among the taxa obscured genetic ically distinct lineages most likely contributed differentiation between the two groups, or that to the high genetic diversity found in this traditional taxonomic treatment is incorrect species (Sharbel and Mitchell-Olds 2001, and the taxa form a single monophyletic group Dobesˇ et al. 2004a). In addition, occasional with a common ancestor. In addition, two introgression of B. holboellii towards B. stricta morphologically distinct Caucasian diploids has been detected from microsatellite variation (C. tenera and C. uliginosa) which are currently patterns (Dobesˇ et al. 2004a). The most recent allopatric and ecologically differentiated and study further suggests that B. holboellii is of thus do not come into contact, displayed traces polyphyletic hybrid origin, as its cpDNA of past hybridization events. They both pos- haplotypes have been found interspersed sessed intraindividual ITS polymorphisms, among the other investigated Boechera taxa and in both the ITS and AFLP trees they (Schranz et al. 2005). were found intermingled. It has been assumed Reconstruction of evolutionary history in that glacial-induced migrations may have genera strongly affected by reticulation and brought them together, and lack of reproduc- polyploidization is definitely not an easy task. tion barriers allowed them to hybridize (Mar- Forcing reticulation to be displayed in the hold et al. 2004). branching topology of a phylogenetic tree The lack of supported hierarchical struc- might lead to biased patterns, lack of support ture, together with the evidence for ancient for the resolved clades, and collapse of hierar- hybridization and introgression among chical structure (Bachmann 2000, Vriesendorp Caucasian diploids in that study, indicated 6 .MrodadJ Lihova J. and Marhold K. 166

AFLP nrDNA ITS tenera 94 seidlitziana uliginosa tenera 59 uliginosa uliginosa tenera uliginosa Caucasus uliginosa seidlitziana uliginosa uliginosa 2x uliginosa tenera Caucasus uliginosa 68 tenera tenera 2x uliginosa C. raphanifolia gr. tenera tenera acris 68 tenera tenera Balkan acris 61 uliginosa 2x acris 74 73 acris C. pratensis gr. 51 acris acris acris acris acris

acris

acris Balkan Europe matthioli 59 acris pratensis A acris 2x-7x 96 100 81 acris 2x pratensis 99 acris acris N Italy granulosa 100 73 acris granulosa 78 acris 2x acris acris castellana 100 62 acris castellana 79 acris Spain silana crassifolia 78 99 silana 2x silana S Italy crassifolia silana silana 6x C Italy apennina matthioli apennina matthioli 2x 69 majovskii apennina majovskii 85 majovskii S Italy silana 56 majovskii matthioli Europe 6x silana matthioli pratensis 2x-7x amara subsp. austriaca pratensis pratensis amara subsp. austriaca C. raphanifolia gr. pratensis pratensis amara subsp. opicii pratensis C Europe 62 pratensis amara subsp. opicii pratensis 2x 96 granulosa amara subsp. amara 100 granulosa N Italy 95 granulosa 2x amara subsp. amara C. pratensis gr. granulosa amara subsp. amara

crassifolia amara subsp. amara

crassifolia 64 A Balkan crassifolia amara subsp. balcanica 71 100 crassifolia crassifolia 2x amara subsp. balcanica

68 crassifolia amara subsp. pyrenaea ´

76 crassifolia Spain evolution reticulate and hybridization Polyploidy, : 58 castellana amara subsp. pyrenaea 80 castellana 2x 83 castellana amara subsp. pyrenaea 100 castellana Spain castellana amara subsp. pyrenaea 55 castellana 2x amara subsp. pyrenaea C. raphanifolia gr. amara subsp. pyrenaea apennina 55 apennina amporitana 99 apennina C Italy apennina amporitana 99 apennina 2x C. amara gr. 87 apennina amporitana apennina amporitana amporitana amara subsp. austriaca C Europe amara subsp. austriaca amporitana amara subsp. austriaca 69 B amara subsp. austriaca 2x amporitana amara subsp. balcanica 76 51 amara subsp. balcanica amporitana 54 78 amara subsp. balcanica Balkan amara subsp. balcanica amporitana amara subsp. amara 2x Italy&Spain amporitana amara subsp. opicii 57 amara subsp. opicii 4x amporitana amara subsp. opicii 52 amara subsp. opicii amporitana amara subsp. pyrenaea 96 55 amara subsp. pyrenaea amporitana amara subsp. pyrenaea Spain amara subsp. pyrenaea amporitana amara subsp. pyrenaea 2x 98 amara subsp. amara amporitana amara subsp. amara amporitana C. raphanifolia gr. 56 wiedemanniana 64 wiedemanniana Caucasus amporitana 100 wiedemanniana 66 wiedemanniana 2x amporitana wiedemanniana 72 amporitana amporitana 85 amporitana 97 amporitana amporitana C. amara gr. 82 amporitana Italy&Spain 98 87 amporitana barbaraeoides amporitana 4x Balkan barbaraeoides amporitana barbaraeoides 4x barbaraeoides 82 100 barbaraeoides barbaraeoides Balkan barbaraeoides B 68 barbaraeoides 71 barbaraeoides wiedemanniana 98 gallaecica 4x Caucasus 54 gallaecica wiedemanniana gallaecica 2x 99 gallaecica gallaecica gallaecica 92 gallaecica raphanifolia 65 Spain 64 raphanifolia Spain raphanifolia 60 raphanifolia raphanifolia 4x-8x 61 raphanifolia 4x-8x asarifolia 100 81 50 raphanifolia raphanifolia asarifolia raphanifolia raphanifolia corymbosa K. Marhold and J. Lihova´ : Polyploidy, hybridization and reticulate evolution 167 b Fig. 3. Relationships among taxa from three traditionally recognized polyploid complexes of mainly European distribution, the Cardamine amara, C. raphanifolia and C. pratensis groups. Neighbour-joining tree of AFLP data (left) and strict-consensus tree of parsimony analysis of nrDNA ITS sequence data (right) are presented. White, grey and black colours show assignments to the complexes, with the indication of ploidy levels and distribution areas. Bootstrap values are shown above the branches. The nrDNA ITS tree is based on data from Lihova´ et al. (2004a); the AFLP tree on re-analyses of separate data sets published in Lihova´ et al. (2003), Lihova´ et al. (2004b) and Perny´ et al. (2005b) extensive past reticulate evolution even at the reconstruct reticulate relationships resulting diploid level (Marhold et al. 2004). Neverthe- from either homoploid or allopolyploid hybrid less, we were interested to investigate genetic speciation more efficiently, network recon- affinities of polyploid representatives of the struction methods and approaches have often complex to the respective diploids. After proven useful. Numerous methods have been polyploids were included (see Fig. 3), the discussed by Vriesendorp and Bakker (2005) resolved phylogenetic pattern was largely that are powerful in detecting character con- retained. The distinction between the two main flict or in depicting alternative evolutionary lineages remained, but interestingly, polyploids scenaria. Statistical parsimony (applied e.g. in from the C. raphanifolia group were split the Boechera study, Dobesˇ et al. 2004b) and between the two clades. Iberian and Balkan split decomposition (see the Yinshania study by polyploids showed high affinity to the C. amara Koch and Al-Shehbaz 2000) have been most representatives, a pattern that would indicate often employed, but several other algorithms their progenitor(s) in the C. amara group, and software are available as well. Phyloge- whereas south Italian polyploid C. silana (as netic network reconstruction is, however, still discussed in more detail above) apparently at an early stage of development and we may originated within the C. pratensis + C. raph- expect that future studies will significantly anifolia clade (Fig. 3). The traditionally recog- contribute to this challenging issue (Linder nized polyploid complexes evidently do not and Rieseberg 2004). represent isolated lineages with their own This work was supported by the Scientific evolutionary history, as putative parents of at Grant Agency of the Ministry of Education of the least some polyploid taxa are likely to be found Slovak Republic and the Slovak Academy of in more than one of these groups. Although Sciences, VEGA, Bratislava (grant no. 3042 to K. two very different markers were used (ITS M.), by the Alexander von Humboldt foundation sequences known to undergo concerted evolu- (research fellowship to J. L.), by the German tion, and AFLP markers representing poly- Academic Exchange Service DAAD (project 323- morphisms across the major part of the PPP to K. M.), and by the Ministry of Education, genome), results of cladistic and distance- Youth and Sports of the Czech Republic (grant no. based analyses of respective data sets were 0021620828, to K. M.). The authors are grateful to mostly congruent and revealed a similar Suzanne Warwick and the editors of the present issue for useful comments on the manuscript. pattern. In the Brassicaceae, which comprises many genera with polyploid and hybridogenous References species, phylogenetic signatures of hybridiza- Adams K. L., Wendel J. F. (2004) Exploring the tion or hybrid speciation such as polytomies genomic mysteries of polyploidy in cotton. Biol. and character conflict are expected to be found J. Linn. Soc. 82: 573–581. rather frequently, but much caution is needed Ainouche M. L., Baumel A., Salmon A. (2004) to distinguish them from population genetic Spartina anglica C. E. Hubbard: a natural model processes (Linder and Rieseberg 2004). 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