3For correspondence (e-mail koch@ice.mpg.de; fax +49 (0) 3641 64 3668).

Introduction

In recent years Arabidopsis thaliana has become a model system for plant molecular biology. It is easy to cultivate, fast-growing, self-pollinating, and can be easily manipulated genetically. Mutants, mapping populations, DNA libraries, YACs, BACs, etc. are available for molecular analysis from the stock centers to address questions in genetics and development. Genome sequencing is ongoing in different parts of the world and data are increasing very rapidly. Data are available from numerous data bases, and researchers are using these data routinely. With the maturation of this system and the sequencing project arise important new challenges, including 1) elucidating the function of the approximately 21.000 genes in the Arabidopsis genome (Bevan et al., 1997), and 2) transferring information from model systems to related plant species.

There is increasing research focusing on crucifers, such as the agriculturally important Brassica species. A central question for this research is how to transfer knowledge from the Arabidopsis model system to other plant species. To understand evolutionary processes and interactions of plants with their environment, it is necessary to work with wild populations and to use other plant species. Relatives from the mustard family show remarkable variability in certain characters. Important variable traits such as insect and fungus resistance, heavy metal tolerance, salt tolerance, apomixis, annual versus perennial life cycles and morphological characters such as leaf morphology, flower archtitecture, fruit characters, development of woody tissues, and other traits are found in relatives of Arabidopsis and Arabis.

During the last five years knowledge about systematics and evolution of the genera Arabis, Arabidopsis and their relatives has increased greatly (O´Kane et al., 1996; O´Kane and Al-Shehbaz, 1997; Price, 1997; Price et al., 1994; Price and Palmer, 1996).

Genus Arabis has been assumed to be a well-defined genus (Al-Shehbaz 1988a) with more than 180 species distributed in the temperate areas of the Northern Hemispere and Arabis alpina and Arabis glabra which appear also at high mountains of tropical East Africa (Al-Shehbaz, 1988a). A compilation has been provided by Al-Shehbaz (1988a) calculating with about 75 taxa (with 60 endemic) from North America, 44 from Europe (30 endemic), 31 from Southwest Asia and the Caucasus (20 endemic), 19 from Central Asia (10 endemic), 28 from China and the Far East (22 endemic) and 15 in northwestern Africa (6 endemic). History of generic relationships of Arabidopsis is summarized in Price et al. (1994). And mostly on the basis of morphology Arabidopsis has been associated with European Cardaminopsis; Asian Cymatocarpus, Drabopsis, Microsisymbrium, Nasturtiopsis, and Neotorularia; American Halimolobus and Pennellia; circumboreal Braya; and the very widespread Arabis. A widely defined genus Arabidopsis is presented by Price et al. (1994) with 27 species, mostly distributed in the high mountain ranges from Southwest Asia to China. This distribution range is part of the overall distribution range of the 180 Arabis taxa and correlates with a conclusion drawn by Hedge (1968) that Arabis differs from Arabidopsis only in the position of the cotyledons relative to the radicle in the seed and that the Himalayan species Arabidopsis wallichii is essentially intermediate between the two.

However, there is no comprehensive concept of evolutionary relationships within the mustard family (Brassicaceae) or even the tribe Arabideae. Earlier tribal classification (Hayek, 1911; Janchen, 1942; Schulz, 1936), based mainly on morphological characters have been shown to be highly artificial (e.g. Koch et al., 1999; Mummenhoff et al., 1997; Price et al., 1994). A first molecular survey using chloroplast DNA sequences and restriction site analysis demonstrated that Arabidopsis is paraphyletic and that tribe Arabideae is not a natural group (Price et al., 1994). Taxa from tribes Sisymbrieae, Anchonieae, and Lepidieae (Price et al., 1994; Tsukaya et al., 1997) were combined into tribe Arabideae. Arabidopsis taxa such as A. himalaica (Tsukaya et al., 1997), A. gamosepala (Al-Shehbaz and O´Kane, 1997), A. parvula (Al-Shehbaz and O´Kane, 1995), A. erysimoides (Al-Shehbaz, 1994) and A. griffithiana (Price et al., 1994) were shown to be not related to Arabidopsis thaliana, and they were classified elsewhere.

Herein we present a molecular analysis of systematic relationships focused on the close relatives of Arabidopsis. We sequenced the nuclear ribosomal internal transcribed spacer regions 1 and 2 and the 5.8S rDNA. The resulting phylogenetic hypothesis is discussed in light of convergent evolution of several important characters, and is compared to phylogenies derived from plastid DNA data.

Materials and Methods

We analyzed 35 accessions representing 30 taxa (Table 1). This collection comprises members of numerous Arabis and Arabidopsis lineages and some taxa from other tribes (Capsella rubella from tribe Lepidieae and Brassica oleracea from tribe Brassicinae) or the same tribe but another subtribe (Cardamine and Barbarea from tribe Arabideae subtribe Cardaminae) differing in morphological characters which have been used so far to identify Arabis and Arabidopsis.

Chromosome numbers were counted to identify polyploids. Determination of chromosome numbers followed the method as described by Koch et al. (1996).

Total DNA was isolated from leaf tissue by a modified CTAB procedure (Mummenhoff and Koch, 1994). Double stranded DNA of the complete ITS region including the 5.8 S rDNA gene was amplified directly by 35 cycles of symmetric PCR using the ITS primers 18For (5´-ccgtaggtgaacctcggaggg-3´) and 25Rev (5´-ggtgatcccgcctgacctgg-3´). PCR products were cloned into the pGEM-T cloning vector (PROMEGA, Mannheim, Germany). Two cloned ITS regions from two independent PCR reactions were sequenced with both amplification primers and two primers located in the flanking sites of the cloning vector (t7 forward: 5´-gtaacgatttaggtgacactatcg-3´ and M13-48 reverse: 5´-agcggataacaatttcacacagga-3´).

Boundaries of the ITS regions and rRNA sequences were determined by comparison to Sinapis alba (Rathgeber and Capesius, 1989) and other crucifers (Koch et al., 1999; Mummenhoff et al., 1997). DNA sequences were aligned by hand. Several types of phylogenetic methods were used to avoid problems inherent in each type of analysis. Parsimony analyses were performed with unordered Fitch-parsimony using PAUP (version 3.1; Swofford, 1993). The BRANCH-AND-BOUND algorithm was used to find maximally parsimonious trees. Gaps were treated as missing characters. Characters with an ambiguous alignment were removed from the data matrix (12 nucletoide sites within the ITS1 region, 24 sites within the ITS2 region). The alignment is not shown and is available on request. Bootstrap analysis (Felsenstein 1985) was performed using 1000 replicates and the HEURISTIC search algorithm with the MULPARS option. Phenetic analysis were performed using the PHYLIP software package (version 3.57c, Felsenstein 1995). Genetic distances were calculated using the Kimura-two-parameter option. Computation of genetic distances into a phenogram was done using the neighbor joining algorithm. Bootstrap analyses were performed using 1000 replicates. In addtion, a maximum-likelihood analysis was performed using the DNA-ML algorithm provided with the PHYLIP software package.

We initially rooted the tree using the sequence of Brassica napus (tribe Brassicinae; dbjD10840). All subsequent analyses positioned Arabis pauciflora outside the remaining ingroup taxa. In order to reduce the amount of homoplastic site changes, we removed Brassica napus from the data matrix and used Arabis pauciflora for the following analysis as outgroup.

The ITS alignment of 36 sequences is 643 bp in length. We excluded 36 nucleotide positions from further analysis (12 positions within the ITS1 region, characters 126-137; 24 positions within the ITS2 region, characters 457-463 and 629-645) because of ambiguous alignments at these positions.

Different ITS types were found in all taxa except for Arabis lyrata ssp. lyrata, Arabidopsis thaliana, Arabis drummondii, Arabis lyalli, Arabis fendleri, Arabis microphylla, Arabis glabra (USA and Europe), Cardamine amara, Cardamine flexuosa, Aubrieta deltiodes, and Halimolobus perplexa. These different ITS types showed only low levels of intraspecific variation (1-8 site changes) and did not affect the topology of phylogenetic trees when all sequences were used for parsimony or phenetic analysis. All ITS types from one taxon always clustered together in one single clade when using all sequences. In order to reduce computional time for phylogenetic analyses, we used only one sequence per accession for the analysis shown. Gene accession numbers are given for all sequences (see Table 1).

Data analysis using Arabis pauciflora as an outgroup and removing Brassica oleracea from the data matrix resulted in 12 MPTs with a tree length of 559 and a consistency index of 0.67 autapomorphies included (0.53 autapomorphies excluded). Tree topology among the 12 MPTs differs only within terminal clades not supported by high bootstrap values (Fig. 1). The distribution of informative characters is as follows: 141 informative nucletide site changes within the ITS1 region (including 41 autapomorphies), 13 within the 5.8S rDNA (8 autapomorphies), and 85 within the ITS2 region (30 autapomorphies). Only one out of the 12 MPTs is shown in Fig. 1 to demonstrate relative branch lengths. Most clades are supported by high bootstrap values. Little phylogenetic information exists to estimate the positions of Capsella rubella, Arabidopsis griffithiana/korshinskyi, Arabidopsis himalaica/wallichii, and Arabis glabra. Along these branches bootstrap values are below 50 % and indicate a higher percentage of homoplastic character site changes.

Data analysis including Brassica napus as an outgroup resulted in 24 most parsimonious trees (MPTs) with a tree length of 600. The consistency index was 0.65 with autapomorphies included (0.50 autapomorphies excluded). Informative character site changes were distributed as follows: 149 informative nucleotide site changes in the ITS1 region (including 44 autapomorphies), 16 within the 5.8S rDNA (11 autapomorphies included), and 87 within the ITS2 region (39 autapomorphies included). The strict consensus tree is not shown. This analysis positioned Arabis pauciflora at a basal position with respect to all of the remaining ingroup taxa.

All distance analyses by neighbor-joining resulted in the same tree topology (Fig. 2). As in the parsimony analysis, the position of Arabis glabra and Arabidopsis himalaica/wallichii is only weakly supported by bootstrap values below 50 %. Similarly, the maximum likelihood analysis indicated that the corresponding branch lengths tend towards zero. However, this analysis showed higher resolution of the phylogenetic positions of Capsella rubella and Arabidopsis griffithiana/korshinskyi.

Phylogenetic analysis demonstrated that Arabis sensu lato is paraphyletic and consists of several different lineages: (1) European n = 8 Arabis including Arabis blepharophylla from North America, Aubrieta deltoides and Arabis turrita; (2) North American n = 7 Arabis; and (3) n = 6 Arabis glabra as well as; (4) n = 7 Arabis pauciflora as monotypic lineages.

Arabidopsis alsoconsists of several lineages, and the data support the concept that the genus should be restricted to taxa only of the Arabidopsis thaliana/Cardaminopsis clade as outlined by O´Kane & Al-Shehbaz (1997). Arabidopsis griffithiana and Arabidopsis korshinskyi are shown to be only distantly related to Arabidopsis thaliana. Arabidopsis wallichii and A. himalaica are more closely related to Arabis glabra and North American Halimolobus.

Discussion

There are a few notable published phylogenetic frameworks considering Arabis and Arabidopsis (Price et al., 1994; Tsukaya et al., 1997). These analyses used cpDNA, either by chloroplast DNA restriction site variation or sequence analysis of the rbcL gene. O´Kane et al. (1996) focused on Arabidopsis thaliana and its closest relatives from the genus Cardaminopsis using ITS sequence variation which are totally in congruence with our findings. Such studies (Mummenhoff and Hurka, 1994; Kamm et. al. 1995; O`Kane et al. 1996) show that Arabidopsis thaliana was one parent which gave rise to amphidiploid Cardaminopsis suecica (Fries) Hiitonen. The second parent was either the wide spread Arabidopsis [=Cardaminopsis] arenosa or A. neglecta from the Carpathian Mountains.

Data sets using cpDNA restriction site variation (Price et al., 1994) include also a broader range of taxa from tribe Arabideae. Our results agree with these studies, demonstrating strong correlation of cpDNA versus nuclear ITS based data. The same is true for the data presented by Tsukaya et al. (1997) showing that Capsella is closer to Arabidopsis than previously thought.

However, bootstrap re-sampling provides only weak statistical support for tree topologies using plastid DNA markers.

Price et al. (1994) listed 27 "Arabidopsis" species. Five of these taxa were recently merged into genera such as Neotorularia, Thellungiella, Erysimum, and Drabopsis. (Al-Shehbaz 1994; Al-Shehbaz and O´Kane 1995; Al-Shehbaz and O´Kane 1997; Price et al., 1994; Tsukaya et al., 1997). In addition, O´Kane and Al-Shehbaz (1997) proposed to merge the genus Cardaminopsis into Arabidopsis, thus increasing the number of Arabidopsis species.

Price et al. (1994) provided evidence for three separate lineages within Arabidopsis: Arabidopsis thaliana/Cardaminopsis, Arabidopsis griffithiana, and Arabidopsis wallichii. Our data clearly show that Arabidopsis griffithiana is more closely related to the Arabidopsis thaliana/Cardaminopsis lineage. Arabidopsis wallichii is closely related to North American Halimolobus and Arabis according to Price et al. (1994). Summarizing these results, it is obvious that current classification of genus Arabis and Arabidopsis is highly paraphyletic.

The molecular data clearly demonstrate that morphological variation does not provide phylogenetically informative characters among these taxa. All members of Arabis sensu lato are characterized by elongated, linear fruits. Taxa such as Aubrieta, Halimolobus perplexa, or Caspella have very different fruits (Fig. 3) and our molecular analysis demonstrates that they are closely related to different Arabis lineages. According to Rollins (1941) the essential characters differentiating Arabis sensu lato from other genera possessing linear siliques are its accumbent cotyledons and siliques flattened parallel to the septum (latiseptate). Even these characters changed several times during evolution within tribe Arabideae. Incumbent cotyledons are found in Halimolobus and rarely in Cardamine (Al-Shehbaz, 1988a), and siliques of Capsella are angustiseptate. Hedge (1968) suggested that Arabis differs from Arabidopsis only in the position of the cotyledons relative to the radicle in the seeds and that the Himalayan species Arabidopsis wallichii is essentially intermediate between the two. In our phylogenetic analysis Arabidopsis wallichii was placed between Arabidopsis thaliana and North American Arabis as well. In Arabis sensu lato a basal rosette is usually developed, and in all cases the basal leaves are differentiated from the cauline, but this is true for many cruciferae. Some authors tried to employ trichomes as taxonomic characters (Mulligan, 1995), but branched trichomes occur widely in the group including the expanded tribe Arabideae. Nectar-glands on the receptacle are thought to be an appropriate character when used in conjunction with other characters to aid in clarification of generic lines. In Arabis, the glandular tissue of the nectaries is well-developed except in one small group including Arabis glabra, Cardaminopsis, Arabis hirsuta, and Arabis blepharophylla (Rollins, 1941). But these taxa are representatives of very different lineages, and therefore this character is only useful to characterize North-American Arabis (Rollins 1993).

This overview should demonstrate that morphological variation in Arabis and Arabidopsis is extensive, and varies even within monophyletic groups. It will be interesting to discern whether parallel evolution of certain characters involved identical modifications in biochemical and regulatory pathways. Some additional characters are listed in Fig. 2. Evolutionary changes in life history traits such as self-incompatibility or annual life cycle occured several times, and characters such as leaf morphology (Hurka and Neuffer, 1997) showed a broad spectrum of variation.

Our results demonstrate that there is a need for drastic taxonomic revision among taxa usually combined the genera Arabis and Arabidopsis. The lectotype species for Arabis is Arabis alpina L. (Linne, 1753: 664) and the type species for Arabidopsis is Arabis thaliana L. [ = Arabidopsis thaliana (L.) Heynold in Holl et Heynh. Fl. Sachs. I. 1842: 538]. Some taxa of the genus Arabis in a broad sense had been already merged into different genera during the past (see Figure 1). Arabis pauciflora has been placed into a monotypic genus Fourraea (Greuter and Burdet, 1983). Arabidopsis himalaica and A. wallichii were merged from Sisymbrium to the genus Arabidopsis by Schulz (1924). Arabidopsis griffithiana is a member of the genus Microsisymbrium (Schulz, 1924). Both genera, Sisymbrium and Microsisymbrium, are part of tribe Sisymbrieae and not Arabideae. Arabis glabra has been described as Turritis glabra L. by Linné (1753). Members of North American Arabis have recently been almalgamated with genus Boechera (Price, 1997).

These examples raise the question whether tribal classification as proposed by several authors (Hayek 1911; Schulz 1936; Janchen 1942) reflects evolutionary history. In addition, weakly defined genus boundaries are well known among crucifers, such as among Thlaspi sensu lato or Cochlearia (Zunk et al. 1993; Zunk et al. 1996; Mummenhoff et al. 1997; Koch et al. 1999); in the past at least some tribes (e.g., Lepidieae) were assumed to reflect monophyletic lineages. Our results demonstrate that tribe Arabideae is interspersed with taxa from tribe Sisymbrieae and tribe Lepidieae (such as Capsella, and presumably Neslia and Camelina, Zunk et al. 1993). Price et al. (1994) showed that Malcolmia bicolor from tribe Anchonieae is close to Arabidopsis griffithiana based on chloroplast DNA restriction site variation. The limited value of morphological variation for phylogenetic inference makes it necessary to analyze as many taxa as possible with molecular markers to address evolutionary questions at a higher taxonomical scale.

Among the taxa under study we found several reductions of base chromosome number from

n = 8 to n = 5/6/7 (Figure 2). Base chromosome number reduction in Arabidopsis thaliana from n = 8 to n = 5, not seen in its closest relatives, clearly demonstrates the highly derived genome organization of Arabidopsis thaliana in respect to chromosome number. In our analysis we also investigated Brassica oleracea from tribe Brassiceae, which has generally been considered to be a natural group (Al-Shehbaz 1984) with the base chromosome number n = 8. This view has been confirmed by a series of papers summarized in Warwick and Black (1997). This tribe is characterized by multiplied genomes on the diploid level. Genome colinearity studies comparing the n = 5 genome of Arabidopsis thaliana with diploid Brassica genomes revealed up to three corresponding regions in the diploid Brassicas (Kowalski et al.,1994; Lagercrantz et al., 1996). Lagercrantz (1998) presented a hypothetical scenario of chromosome evolution of Arabidopsis thaliana compared to Brassica nigra based on extensive mapping results. This hypothesis also suggested a simple and "reconstructable" genome evolution on the chromosome level characterized by disruption and fusion of parts of the chromosomes.

Some taxa considered in this study are ideal study species to examine a variety of traits and characters, and an international collaboration is underway to establish a comparative genetical map of a cross originated from Arabis lyrata X Cardaminopsis petraea (n = 8) using markers from Arabidopsis thaliana (n = 5). Studies of ecology and evolution of natural plant populations will be facilitated by availability of a model species with modern molecular tools. Because Arabidopsis thaliana is the best-known experimental plant system, its close relatives Arabidopsis lyrata (formerly Arabis lyrata from North America) and Arabidopsis petraea (formerly Cardaminopsis petraea from Eurasia) have been chosen for an international collaborative project to develop infrastructure for molecular ecology and evolution. These two taxa are assumed to be conspecific (O´Kane and Al-Shehbaz, 1997). Arabidopsis lyrata, petraea, and halleri constitute the closest known outcrossing relatives of Arabidopsis thaliana. Populations of Arabidopsis lyrata and Arabidopsis petraea are interfertile and are fairly similar across continents. As sequence information increases in the Arabidopsis Genome Initiative, there is greater focus on determining the function of newly discovered genes. Besides mutant analysis, genetic segregation in wide crosses can identify functional differences between gene homologues from two different species. Therefore, recombinant inbred lines from crosses among Arabidopsis relatives are being developed for the stock centers (for detailed information see web page: http://www.arabis.net/).

Crossing experiments have been performed with intercontinental populations of Arabis alpina (Hedberg 1962), and within European Arabis hirsuta (Titz; 1970; Titz, 1978). Comparative mapping efforts in Arabidopsis and Brassica (e.g. Kowalski et al., 1994; Lagercrantz et al., 1996) are in progress. But because of the great phylogenetic distance between Arabidopsis thaliana and Brassica relatives, the resulting comparative genetic maps are complicated by polyploidization, widespread genomic rearrangements, and difficulties finding the most homologous and/or orthologous regions.

Phylogenetic information is clearly important for comparative analysis, as done for alcohol dehydrogenase (Miyashita et al., 1996). From a comparison of Adh alleles in Arabidopsis thaliana and Arabis gemmifera (= Arabidopsis halleri ssp. gemmifera, O´Kane and Al-Shehbaz, 1997) the authors concluded that these species can be regarded as sibling species. The authors chose Arabis gemmifera because of the presumed close relationships of Arabis to Arabidopsis. The results would have been very different had they choosen an Arabis taxon such as Arabis pauciflora for their analysis. Our phylogenetic analysis demonstrates that indeed Cardaminopsis halleri/Arabis gemmifera is one of the most closest relatives of Arabidopsis thaliana.

Evolution of microsatellites (van Teuren et al., 1997) or repetitive DNA (Kamm et al., 1995) in close relatives of Arabidopsis was studied based on knowledge from Arabidopsis thaliana. These analyses provided new insights into genome evolution and may demonstrate a useful route to study function and evolution of different types of repetitive DNA sequences.

The use of biogeographic data in combination with a phylogenetic framework provides researchers with additional tools to calibrate molecular clock models or to test hypotheses on modes of gene evolution. One such example is the evolution of the Adh gene family. Although locus duplications have been found in Leavenworthia (Brassicaceae), only limited conclusions could be drawn about evolution of these genes with respect to speciation in related taxa (Charlesworth et al., 1998), because closely related genera such as Cardamine or Barbarea were not considered.

Acknowledgments

We thank all colleques and institutes that provided plant and seed material (Table 1). This work was supported by the Max-Planck-Gesellschaft and by grants DEB-9527725 (to TMO) and BIR-9626079 (to JGB) from the US National Sience Foundation. Thanks to D. Schnabelrauch for technical assistance and S. Dix for secretarial help.

International Stock Centers provide seeds for most of the species studied here. The Ohio Stock Center (ABRC, USA) provide some seed material from Cardaminopsis and Arabidopsis griffithiana. The SENDAI Arabidopsis Seed Stock Center (SASSC, Japan) provides seeds from Arabidopsis himalaica, A. griffithiana, A. wallichii, Cardaminopsis and several others. Arabis seed material is also obtainable from numerous botanic gardens.

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