PHYLOGENETIC POSITION Steve L. O’Kane, Jr.2 and Ihsan A. 3 AND GENERIC LIMITS OF Al-Shehbaz () BASED ON SEQUENCES OF NUCLEAR RIBOSOMAL DNA1

ABSTRACT

The primary goals of this study were to assess the generic limits and monophyly of Arabidopsis and to investigate its relationships to related taxa in the family Brassicaceae. Sequences of the internal transcribed spacer region (ITS-1 and ITS-2) of nuclear ribosomal DNA, including 5.8S rDNA, were used in maximum parsimony analyses to construct phylogenetic trees. An attempt was made to include all currently or recently included in Arabidopsis, as well as species suggested to be close relatives. Our findings show that Arabidopsis, as traditionally recognized, is polyphyletic. The , as recircumscribed based on our results, (1) now includes species previously placed in Cardaminopsis and Hylandra as well as three species of and (2) excludes species now placed in , Beringia, Olimar- abidopsis, Pseudoarabidopsis, and Ianhedgea. Key words: Arabidopsis, Arabis, Beringia, Brassicaceae, Crucihimalaya, ITS phylogeny, , Pseudoar- abidopsis.

Arabidopsis thaliana (L.) Heynh. was first rec- netic studies and has played a major role in un- ommended as a model for experimental ge- derstanding the various biological processes in netics over a half century ago (Laibach, 1943). In higher (see references in Somerville & Mey- recent years, many biologists worldwide have fo- erowitz, 2002). The intraspecific phylogeny of A. cused their research on this plant. As indicated by thaliana has been examined by Vander Zwan et al. Patrusky (1991), the widespread acceptance of A. (2000). Despite the acceptance of A. thaliana as a thaliana as a is attributed to the model organism and the sequencing and mapping discovery that it has one of the smallest genomes of its nuclear genome (The Arabidopsis Genome of any flowering plant, a low number Initiative, 2000; Cooke et al., 1996), little is known (n ϭ 5), and that its genome contains few repetitive about the other species of Arabidopsis sensu lato, sequences and little intergenic spacer DNA. A sur- and their closest relatives. prising recent finding by Blanc et al. (2000), how- A small number of molecular phylogenetic stud- ever, showed that although A. thaliana has a re- ies have included a few members of Arabidopsis markably small genome, much of the DNA is Heynh. sensu lato (Price et al., 1994; O’Kane et present in more than one copy. In addition to these al., 1996; Galloway et al., 1998; Koch et al., 1999, important attributes, A. thaliana has a short gen- 2000, 2001; Yang et al., 1999). However, none of eration time (four to six weeks), a small size (dozens these studies attempted to examine all of the taxa can be grown in a small pot), and can easily be either currently or previously included in the ge- grown on synthetic media (Meyerowitz, 1989; Mey- nus, and they included only a small number of oth- erowitz & Pruitt, 1985). The species has been used er, sometimes distantly related, genera. The last extensively in developmental, evolutionary, and ge- comprehensive taxonomic account (Schulz, 1924),

1 Research and fieldwork were supported by grants from the National Science Foundation (DEB-9208433), the National Geographic Society (NGS-5068-93), and the University of Northern Iowa. Special thanks to Barbara Schaal, in whose lab this work was initiated, and to the support of the Missouri Botanical Garden. We offer gratitude to the many hosts, field companions, and herbarium curators who aided in this study, especially Abdulla Abbas, Nogman Aralbaev, Isa O. Baitulin, Alexandra Berkutenko, Ram Chaudhary, Gheorghe Dihoru, Vladimir Dorofeyev, Yang Guang, Sun Hang, Josef Holub, H. Kato, the late Sigizmund Kharkevich, Franta Krahulec, Hanna Kuciel, Karol Marhold, Zbigniew Mirek, Klaus Mummenhoff, Noriaki Murakami, Nonna Pavlova, and Boris Syomkin. Reviews by Donovan Bailey and an anonymous reviewer improved this paper substantially. 2 Department of , University of Northern Iowa, Cedar Falls, Iowa 50614-0421, U.S.A. [email protected]. 3 Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A. [email protected].

ANN.MISSOURI BOT.GARD. 90: 603–612. 2003. 604 Annals of the Missouri Botanical Garden

which recognized 11 species, is unsatisfactory. As Molecular-based results (O’Kane et al., 1996; many as 50 species have been placed in the genus, Kamm et al., 1995; Mummenhoff & Hurka, 1994) and, although many of these are now placed in oth- agree with Lo¨ve’s (1961) and Hylander’s (1957) hy- er genera (Al-Shehbaz et al., 1999), their phylo- pothesis in showing A. thaliana to be most closely genetic relationships remain unresolved. Monophy- related to species placed in Cardaminopsis. In an- ly of the genus has not yet been critically ticipation of results published here and to make the determined, and even basic biological information, names available for floristic works in progress, we such as chromosome numbers, generation time, and previously published the needed nomenclatural in- breeding system of the members of the genus, is novations for the genus Arabidopsis (O’Kane & Al- lacking. Shehbaz, 1997) and have established several new Generic delimitation is perhaps one of the most genera to accommodate excluded species (Al-Sheh- difficult and frequently encountered problems in baz et al., 1999). In brief, Arabidopsis includes only the systematics of the Brassicaceae (Al-Shehbaz, A. thaliana and species previously included, or 1973; Rollins, 1993), and Arabidopsis clearly dem- suggested to be, in Cardaminopsis (Jones & Ake- onstrates this problem. There has been a lack of royd, 1993a, 1993b). Species now excluded from agreement among taxonomists on the number of Arabidopsis are placed in O. E. species that belong to Arabidopsis and on the char- Schulz (Al-Shehbaz & O’Kane, 1995), Neotorularia acters that indicate its generic boundaries (e.g., Hedge & J. Le´onard (Al-Shehbaz & O’Kane, 1997), Ball, 1993; Lo¨ve, 1961; Rollins, 1993). The generic Ianhedgea Al-Shehbaz & O’Kane (Al-Shehbaz & limits of Arabidopsis have been highly unnatural, O’Kane, 1999), Crucihimalaya Al-Shehbaz et al., and there were no well-defined characters separat- Olimarabidopsis Al-Shehbaz et al., and Pseudoar- ing it from several closely associated genera (but abidopsis Al-Shehbaz et al. (Al-Shehbaz et al., see our taxonomic revision based on the results of 1999), and Beringia Price et al. (Price et al., 2001). this current work, O’Kane & Al-Shehbaz, 1997; Al- Our primary objectives are to determine the ge- Shehbaz & O’Kane, 2002a). Some individual Ara- neric limits of a morphologically coherent, mono- bidopsis species have been transferred among sev- phyletic Arabidopsis and to reconstruct a robust in- eral other genera. An example is A. thaliana, which terpretation of its phylogenetic neighborhood. A on the basis of Schulz’s (1924) synonymy was pre- well-corroborated phylogeny of the group will allow viously placed in at least nine other genera, in- better evolutionary interpretations to be made of the cluding Arabis L., Conringia Adans., Crucifera E. massive amounts of data now accumulating for A. H. L. Krause, Erysimum L., Hesperis L., Nasturtium thaliana. Workers will know which species to com- R. Br., Pilosella Kostel., Sisymbrium L., and Sten- pare to A. thaliana when making interpretations of ophragma Cˇ elak. evolutionary processes. Furthermore, these initial Arabidopsis has been closely associated with steps will provide a better understanding of mor- three different genera, Cardaminopsis (C. A. Mey.) phological character evolution in the Brassicaceae, Hayek, Arabis L., and Halimolobos Tausch. Schulz a family of great economic importance fraught with (1924, 1936) considered its nearest relative to be taxonomic problems related to an under-developed Halimolobos, and separated the latter as being understanding of character evolution and generic coarser herbs with the styles much narrower than delimitation. the , as opposed to Arabidopsis, which were seen as slender herbs with the styles slightly nar- MATERIALS AND METHODS rower than the fruit. These alleged differences are TAXON SAMPLING not mutually exclusive, and species recognized by him in one genus can easily be accommodated in We included representatives of all taxa (at least the other. Lo¨ve (1961) and Hylander (1957) indi- at the generic level) that are now or have been in- cated a relationship with Cardaminopsis based on cluded in Arabidopsis (e.g., Schulz, 1924; Hedge, natural interspecific hybridization. Hedge (1968) 1965; Jafri, 1973; Al-Shehbaz, 1988; Ball, 1993). suggested a closer relationship between Arabidopsis Taxa shown to lie near Arabidopsis in other molec- and Arabis and indicated that the two differ only in ular studies have also been included (Price et al., the cotyledonary position. He further suggested that 1994; O’Kane et al., 1996; Galloway et al., 1998), Arabidopsis wallichii (Hook. f. & Thoms.) Busch as have a sampling of taxa from elsewhere in the probably represents the link between the two gen- Brassicaceae. Phylogenetic trees were initially - era. An (1987) and Jafri’s (1973) transfer of several ed by Cleome lutea Hook. of the Cleomaceae, a species from Arabis to Arabidopsis was probably in- family basal to the Brassicaceae (Rodman et al., fluenced by Hedge’s view. 1993; Judd et al., 1994; Hall et al., 2002). Included Volume 90, Number 4 O'Kane & Al-Shehbaz 605 2003 Phylogenetic Position of Arabidopsis

taxa, as well as voucher information and some no- strap support was obtained from 500 replicates using menclatural comments, are given in Table 1. Where a single round of simple taxon addition. Decay values possible, plant materials were collected in the field (Bremer, 1988; Donoghue et al., 1992) were found and dried in powdered silica gel. In some cases using the AutoDecay program 4.01 of Eriksson tissue was obtained from plants grown from . (1998). Clade Significance (Lee, 2000) was imple- Where fresh or dried material was not available, we mented in PAUP by the AutoCladeS program (T. Er- used tissue from herbarium specimens; the se- iksson; available at Ͻhttp://www.bergianska.se/in- quence for All. was obtained from dex࿞forskning࿞soft.htmlϾ). We found that this new GenBank. measure of support indicates those clades that have the highest support based on the other two measures. DNA EXTRACTION, PCR AMPLIFICATION, AND The information content of the data was assessed by SEQUENCING the g1 statistic (Hillis & Huelsenbeck, 1992) based on 100,000 random trees and by the Permutation Tail Total DNA was extracted from dried tissue Probability (PTP) (Faith & Cranston, 1992) based on ground in a pinch of sterile sand by a modified 200 heuristic searches of randomized data (PAUP* CTAB procedure as previously described (O’Kane parameters as above except simple addition was used et al., 1996). Amplification of the internal tran- rather than random addition). scribed spacer region (including ITS-1, 5.8S, and ITS-2) was done using the conditions given in O’Kane et al. (1996) except that some ITS regions RESULTS were amplified as a single unit using primer ITS1– In nearly all samples there was no evidence of 18S (5Ј CGTAACAAGGTTTCCGTAGG 3Ј) and heterogeneity among individual ITS copies. Rarely, ITS-4 (White et al., 1990) rather than as two over- two different bases were present at a given position lapping pieces. PCR products were purified from as indicated by two bands on an autoradiograph or 0.8% agarose gels containing 1X TAE using Wizard as two clear peaks on a chromatogram. In these PCR Preps (Promega). Sequences were obtained ei- cases the base position was coded using the appro- ther manually using the fmol௢ DNA Sequencing priate ambiguity code. We interpret this rare ‘‘het- System (Promega) or from the automated sequencer erozygosity’’ as incomplete homogenization of the at the University of Iowa using the same primers ITS copies and not as evidence of hybridization; an as were used to amplify the product. GenBank ac- individual sequence would show much more vari- cession numbers are given in Table 1. Sequences ation if hybridization were involved. Although ini- of the allotetraploid Arabidopsis suecica (Fries) tial analyses used Cleome lutea as the outgroup, Norrl. were obtained from cloned PCR products as inclusion of this taxon added to the complexity of previously reported (O’Kane et al., 1996). sequence gaps and to areas with ambiguous align- ments. Of the taxa included in this study, Berter- SEQUENCE ALIGNMENT AND PHYLOGENETIC oella maximowiczii (Palib.) O. E. Schulz was found ANALYSES to be strongly supported as the basal-most taxon. Sequences were aligned with the computer program Further analyses, then, used B. maximowiczii as the MALIGN 2.7 (Wheeler & Gladstein; available at outgroup. The resulting multiple alignment of the Ͻftp://ftp.amnh.org/pub/people/wheeler/malign/Ͼ) us- internal transcribed spacer region (ITS) was 716 ing the following empirically determined parameters: base-pairs in length. Sequences are deposited in internal 7, extragap 5, leading 3, trailing 3, matrix 0 GenBank (accession numbers in Table 1), and the 3 2 3 3 0 3 2 2 3 0 3 3 2 3 0, aspr, spr, quick, full alignment is available from the first author. Two keepa2, keept3, and score 2. The matrix parameters regions of the alignment that were extremely sen- weight transversions as 3, transitions as 2, intitial gaps sitive to alignment parameters and could not be as 7, gap extensions as 5, and initial and ending gaps improved by eye (114–139 and 461–507) were not as 3 (not a factor in our sequences). Most-parsimo- used in the phylogenetic analyses. In all, 384 bases nious trees were found using PAUP* 4.0b4 (Swofford, were invariant, 79 were parsimony uninformative, 2000). In our analyses all characters were considered and 253 were parsimony informative. to be of equal weight and gaps were coded as missing Parsimony searches yielded 24 distinct most- data. Two hundred and fifty replicates of random ad- parsimonious trees of length 951, consistency index dition using Fitch parsimony were performed using (CI) 0.48, consistency index excluding uninforma- Tree Bisection Reconnection (TBR), Mulpars, multi- tive characters (CIU) 0.43, retention index (RI) state ϭ polymorphism, gaps coded as missing, and 0.73, and rescaled consistency index (RC) of 0.35. Collapse branches if maximum length is zero. Boot- The g1 statistic of the data was Ϫ0.5982, which 606 Annals of the Missouri Botanical Garden U52187 U43233 AF137542 AF137543 U96270 U96269 number, and herbarium of PolandRussia Poland JapanRussian Far East Russian U52188 Far East Russian Far EastJapanRussian Far AF137544 EastRussian Far EastRussian U96266 Far East Russian Far East Slovakia U96267 AF137539 U96268 U52186 4 4 2 3 3 4 2 (VLA) 1 (VLA) 1 (MO) Missouri, U.S.A. AF137570 (MO) (MO) (MO) 1 (MO) (VLA) (MO) Czech Republic U43224 (MO) Romania AF137540 (MO) Romania(MO) AF137559 Romania AF137574 (MO) (MO) 3682 (MO) Russian Far East AF137572 (MO) Japan AF137560 1 2 3 (MO) China AF137553 (GH) China AF137573 (LE) (H) Finland U52185 (1977) (BRA) (MO) Afghanistan U43225 (MO) (ISTC) Utah, U.S.A. AF137588 (MO) (MO) Montana, U.S.A. AF137561 (MO)(MO) Montana, U.S.A. Montana, U.S.A. AF137575 AF137562 (1977) (W) Yugoslavia AF137546 (CS) Colorado, U.S.A. AF137578 (MO) Poland AF137541 (LE) Russia AF137557 (1890) )BUCS) AF137545 kova s.n. ˇ ´c O’Kane & Dihoru 3611 O’Kane & Berkutenko 3679 O’Kane & Kuciel 3664 O’Kane & Probatova 3683 Probatova & Seledets 7-8-80 Coste s.n. Kummert s.n. O’Kane 3693 Hornı O’Kane 3773 Al-Shehbaz 9354 Tzvelev et al. 79 O’Kane & Berkutenko 3678 O’Kane 3684 O’Kane & Berkutenko 3681 Kharkevich et al. 22-7-83 Barkalov & Bezdeleva 6-8-89 Lampinen 2450 O’Kane 3673 Podlech 17544 O’Kane & Krahulec 3638 O’Kane & Dihoru 3618 O’Kane 3676 O’Kane & Kato 3689 O’Kane 3672 O’Kane & Berkutenko Neely 3174 O’Kane & Dihoru 3596 Al-Shehbaz & O’Kane 9401 O’Kane & Mirek 3650 Cheo & Yen 208 bursifolia Abolin 53 (Matsum.) O’Kane & Al-Shehbaz (Fisch. ex DC.) O’Kane & Al-Shehbaz (Wulfen) O’Kane & Al-Shehbaz (L.) O’Kane & Al-Shehbaz ovirensis gemmifera halleri Koteja 123 petraea kamchatica Taxon Voucher information Source locality Number ´e (Edgew.) Al-Shehbaz, O’Kane & R. A. Price (Palib.) O. E. Schulz (sample 2) (L.) Medik. Andrz. ex DC. (L.) Lawalre S. Watson (DC.) R. A. Price, Al-Shehbaz & O’Kane subsp. Miq. B. L. Rob. L. (Richardson) S. Watson (DC.) O’Kane & Al-Shehbaz L. All. From GenBank X98630 Hook. S. Watson (L.) Heynh. (sample 1) (Schult.) O’Kane & Al-Shehbaz (Schott) O’Kane & Al-Shehbaz (Fr.) Norrl. (L.) O’Kane & Al-Shehbaz subsp. (L.) O’Kane & Al-Shehbaz subsp. (L.) O’Kane & Al-Shehbaz subsp. (L.) O’Kane & Al-Shehbaz subsp. (L.) O’Kane & Al-Shehbaz subsp. Table 1. Voucher information and GenBank accession numbers for samples included in this study. Voucher information is given as collector, collector A. halleri A. lyrata deposit; identical superscripts indicate samples with identical rDNA sequences. Arabidopsis arenosa A. cebennensis A. croatica A. halleri A. halleri A. neglecta Crucihimalaya himalaica A. lyrata A. suecica Arabis nuttallii Camelina microcarpa Cleome lutea A. thaliana Arabis drummondii Arabis flagellosa Arabis lyallii Arabis pendula Arabis scabra Beringia bursifolia Berteroella maximowiczii Braya glabella bursa-pastoris Volume 90, Number 4 O'Kane & Al-Shehbaz 607 2003 Phylogenetic Position of Arabidopsis (LE) Tajikistan AF137594 (E) Afghanistan AF137549 (ISTC) Colorado, U.S.A. AF137584 (E) Afghanistan AF137552 (MO) Poland AF137576 (ISTC) Tennessee, U.S.A. AF137586 (BM) Turkey AF137579 (MO) Nuevo Leon, Mexico AF137590 (MO) Coahuila, Mexico AF137589 (MO) Nevada, U.S.A. AF137567 (LE) Kazakstan AF137563 (LE) Kyrgyzstan AF137548 (MO) Canada AF137564 (W)(MO) Pakistan Iran AF137554 AF137577 (W)(US) Iran Afghanistan AF137571 AF137547 (MO) China AF137556 (E) Afghanistan AF137565 (PE) China AF137566 (MO) Mexico AF137569 (ISTC) Tennessee, U.S.A. AF137585 (CS) Colorado, U.S.A. AF137580 (ISTC) Utah, U.S.A. AF137582 (ISTC)(MO) Montana, U.S.A. Montana, U.S.A. AF137583 AF137581 (1987) (MO) Kazakstan AF137558 (W) Afghanistan AF137568 (LE) Tajikistan AF137551 (SJC) New Mexico, U.S.A. AF137593 (S) Egypt (Sinai) AF137550 (K) Afghanistan AF137555 (SJC) New Mexico, U.S.A. AF137587 (SJC) Mexico AF137592 (SJC) Mexico AF137591 ˜a 5961 Kaiser 632 Al-Shehbaz 9333 Wendelbo & Ekberg 9642 Kamelin s.n. Rechinger 30655 Palmer 66 Porter 4467 Heil 6401 Rechinger 54514 O’Kane 2157 Chukavinah & Kenzikaeva 5473 O’Kane 3706 Magan Morefeld et al. 4549 Rasoul 3541 Heil 6399 Shao-Xing s.n. Davis & Dodds 18677 Ledingham 7937 Podlech 12379 Rechinger 40534 Sawyer 77 Grubov et al. s.n. Hedge & Wendelbo W8650 Rechinger 18359 Rebman & Dierig 2894 Rebman 2892 O’Kane & Anderson 3739 Skvortsov s.n. O’Kane 3675 Rollins & Roby 7489 Rollins & Tryon 5893 Pavlov et al. 1192 Rollins acutiloba ´onard didymocarpa O’Kane 3794 ´onard Taxon Voucher information Source locality Number (Munz) Rollins paniculata Bzowska & Rabczak 329 jaegeri (Rollins) Rollins (M. Bieb.) Al-Shehbaz, O’Kane & R. A. Price (Botsch.) Al-Shehbaz, O’Kane & R. A. Price (Popov) Ovcz. & Junussov (C. A. Mey.) Al-Shehbaz, O’Kane & R. A. Price (Hook. f. & Thoms.) Al-Shehbaz, O’Kane & R. A. Price (Hook. f. & Thoms.) Al-Shehbaz, O’Kane & R. A. Price (Botsch. & Vved.) Al-Shehbaz, O’Kane & R. A. Price (Hedge) Al-Shehbaz & O’Kane (Engelm.) Rollins (Bornm.) Al-Shehbaz, O’Kane & R. A. Price (Pall.) O. E. Schulz (Hook. f. & Thoms.) Al-Shehbaz, O’Kane & R. A. Price (Stephan) Al-Shehbaz, O’Kane & R. A. Price (C. A. Mey.) O. E. Schulz (S. Watson) Greene (Hook.) A. Gray var. (Hook. f. & Thoms.) Al-Shehbaz & O’Kane (Cambess.) Al-Shehbaz, O’Kane & R. A. Price (Schrenk) Al-Shehbaz & O’Kane (Desf.) Hedge & J. Le (C. A. Mey.) Hedge & J. Le (Stephan ex Willd.) C. A. Mey. (Hemsl.) O. E. Schulz var. Harv. A. Gray var. Rydb. (Rollins) O’Kane & Al-Shehbaz Rollins (Rollins) O’Kane & Al-Shehbaz A. Gray (Greene) O’Kane & Al-Shehbaz Hook. & Harv. (L.) Desv. subsp. (Belang. ex Boiss.) Stapf Table 1. Continued. Crucihimalaya mollissima Crucihimalaya kneuckeri Crucihimalaya lasiocarpa Crucihimalaya ovczinnikovii Crucihimalaya stricta Crucihimalaya wallichii Dichasianthus subtilissimus Dimorphocarpa wislizenii Dithyrea californica Drabopsis nuda Eutrema penlandii Halimolobos palmeri Olimarabidopsis pumila acutifolia Physaria didymocarpa Halimolobus diffusa Ianhedgea minutiflora Lyrocarpa coulteri Neotorularia gamosepala Neotorularia humilis Neotorularia torulosa Thellungiella parvula Thellungiella salsuginea Nerisyrenia linearifolia Neslia paniculata Olimarabidopsis cabulica Olimarabidopsis umbrosa densipila Paysonia stonensis Physaria pruinosa Pseudoarabidopsis toxophylla Smelowskia calycina Sphaerocardamum macropetalum Synthlipsis greggii Thellungiella halophila 608 Annals of the Missouri Botanical Garden

indicates strong phylogenetic signal in the data (P included in Arabidopsis are now placed in Beringia, Ͻ 0.01). The Permutation Tail Probability (PTP) Crucihimalaya, Olimarabidopsis, Pseudoarabidop- also indicated strong signal (P ϭ 0.005). Figure 1 sis, Ianhedgea, Neotorularia, and Thellungiella. Ar- shows the strict consensus tree of the 24 most par- abidopsis sensu novo is distinguished from other simonious trees. genera in the Brassicaceae by having short petio- late but not auriculate or amplexicaul cauline DISCUSSION , the presence of simple trichomes, these of- ten mixed with few-forked ones but not stellate RELATIONSHIPS AND CIRCUMSCRIPTION OF hairs, well-defined basal rosettes at least in young ARABIDOPSIS plants, white to lavender (rarely almost purple) but The relationships among the species included in never yellow flowers, erect to slightly ascending this study are almost entirely consistent with results non-saccate or slightly saccate inner sepals, si- previously published for smaller taxon samples in liques at least slightly torulose, much longer than the Brassicaceae focusing on Arabidopsis (e.g., they are wide and glabrous, compressed (rarely Price et al., 1994; Galloway et al., 1998; Koch et subterete to terete), seeds uniseriate in the silique, al., 1999, 2000, 2001; Yang et al., 1999). Like and cotyledons accumbent or rarely incumbent those studies, our research indicates that Arabidop- (O’Kane & Al-Shehbaz, 1997). Habit ranges from sis as traditionally circumscribed is a highly artifi- annual to short- or long-lived perennials. Chromo- cial group. In fact, even the tribe Sisymbrieae, the some numbers vary from x ϭ n ϭ 5inA. thaliana traditional placement for Arabidopsis (Schulz, 1924, to x ϭ 8 in the remaining species except for A. 1936; Al-Shehbaz, 1984, 1988), is itself artificial. suecica, which is an allotetraploid (2n ϭ 26) de- The confused circumscription of Arabidopsis, as rived from A. thaliana (2n ϭ 10) and A. arenosa based on morphological grounds, was noted in pre- (L.) Lawalre´e (2n ϭ 16) (Mummenhoff & Hurka, vious taxonomic treatments. Hylander (1957: 602), 1994; O’Kane et al., 1996, and references therein). for example, recognized that if Cardaminopsis and Keys to the species and subspecies are given in Arabidopsis are combined, as seemed likely, the O’Kane and Al-Shehbaz (1997). As circumscribed limits of Arabidopsis ‘‘would thereby be consider- here, the genera Cardaminopsis and Hylandra A´ . ably widened—or, perhaps more correctly, drawn Lo¨ve are united with Arabidopsis. Arabidopsis is a in quite another way.’’ Jones (1964) also indicated monophyletic genus consisting of A. arenosa, A. ce- that two species of Arabis, A. pedemontana Boiss. bennensis (DC.) O’Kane & Al-Shehbaz, A. croatica and A. cebennensis DC., might best be included in (Schott) O’Kane & Al-Shehbaz, A. halleri (L.) Cardaminopsis. Thus, at least as early as 1964, tax- O’Kane & Al-Shehbaz, A. lyrata (L.) O’Kane & Al- onomic problems were anticipated in Arabidopsis, Shehbaz, A. neglecta (Schultes) O’Kane & Al-Sheh- Cardaminopsis, and Arabis. baz, A. suecica, and A. thaliana. Although we have Results from our study are sufficient to allow a no sequences of A. pedemontana (Boiss.) O’Kane & revision of the of the genus Arabidopsis. Al-Shehbaz, it, too, clearly belongs in Arabidopsis A strongly supported clade (bootstrap support 97%, based on its morphological relationship to A. ceben- decay index 6, clade support 0.014; see Fig. 1) nensis. containing A. thaliana, the type species of the ge- All other species previously included in Arabi- nus, defines the limits of a recircumscribed genus dopsis are more distantly related, especially those Arabidopsis. As stated above, we have anticipated Himalayan species now included in Crucihimalaya the publication of these results by redefining the and the Middle Eastern and central Asian Olimar- circumscription of Arabidopsis (O’Kane & Al-Sheh- abidopsis. Morphologically, Crucihimalaya differs baz, 1997), transferring species to previously rec- from Arabidopsis in that it has at least some stellate ognized genera (Al-Shehbaz & O’Kane, 1995, trichomes, whereas Arabidopsis has forked tri- 1997), and lastly by erecting several new genera chomes. Olimarabidopsis differs from Arabidopsis in for species previously included in Arabidopsis (Al- its yellow, rather than white or lavender petals, pu- Shehbaz et al., 1999; Al-Shehbaz & O’Kane, 1999, bescent, rather than glabrous , and auriculate 2002a; Price et al., 2001). Names in bold type in rather than attenuate or petiolate cauline leaves. Figure 1 represent new genera erected for species Al-Shehbaz et al. (1999) presented an analysis of previously included in Arabidopsis by other authors, all species formerly placed in Arabidopsis. Based and the bold letter ‘‘A’’ indicates species previously on the results presented here, Arabidopsis is a ge- included in Arabidopsis. In every case we have nus of circumboreal and circum-north-temperate made genera monophyletic (sensu Hennig, 1966; species. Most species, however, are confined to Eu- holophyletic of Ashlock, 1971). Species previously rope. Workers conducting comparative research us- Volume 90, Number 4 O'Kane & Al-Shehbaz 609 2003 Phylogenetic Position of Arabidopsis

Figure 1. Strict consensus tree of 24 most-parsimonious trees. Whole numbers indicate bootstrap support; values in parentheses are the Decay Index; decimal values are Clade Significance. Dashed branches indicate branches with Ͻ 50% bootstrap support. Large bold ‘‘A’’ indicates species variously placed in Arabidopsis in previous taxonomic treatments. Large asterisks indicate species traditionally placed in Arabis. Genera in bold type are segregates from Arabidopsis named elsewhere as a result of these analyses (see Discussion). 610 Annals of the Missouri Botanical Garden

ing A. thaliana as a model organism can now con- Eutrema R. Br. also form a well-supported clade, fidently use the other species of a better- with Thellungiella being paraphyletic. circumscribed Arabidopsis, all of which are found A surprising result of our study was the discovery in the sister group to A. thaliana, as experimental of a clade of species all possessing with organisms. This sister group relationship implies more than the usual three colpi (see Polycolpate that all species of Arabidopsis are equally related clade, Fig. 1). Palynological studies (Rollins, 1979; to A. thaliana. Unfortunately, the sister group to the Rollins & Banerjee, 1979) showed that among some genus Arabidopsis cannot be given with confidence. genera thought not to be closely related in the Bras- A trichotomy appears below Arabidopsis (Fig. 1): sicaceae, colpi range from four to ten. These gen- (Arabidopsis clade)(‘‘Capsella–Pseudoarabidopsis’’ era, according to Schulz (1936), are as follows: clade)(‘‘Crucihimalaya–Olimarabidopsis’’ clade). Physaria (Nutt. ex Torr. & A. Gray) A. Gray (tribe Galloway et al. (1998), using sequences of arginine Lepidieae, subtribe Physariinae), Dithyrea Harv. decarboxylase and a much smaller taxon sample and its recent segregate Dimorphocarpa Rollins (28 species from throughout the family), confidently (Lepidieae, Iberidinae), Lyrocarpa Hook. & Harv. placed Capsella in a sister group relationship to (Lepidieae, Lyrocarpinae), Nerisyrenia Greene (as Arabidopsis. Unfortunately, their analysis did not Greggia A. Gray) and Synthlipsis A. Gray (Lepi- include any members of the ‘‘Crucihimalaya–Oli- deae, Capsellinae), and Lesquerella S. Watson (Dra- marabidopsis’’ clade. Koch et al. (1999) found, us- beae). The results presented here also suggest that ing ITS sequences (and with low bootstrap support), within this ‘‘polycolpate clade’’ taxonomic revisions Capsella to be sister to Arabidopsis and Olimara- are needed in Lesquerella and Physaria. We have bidopsis, but their analysis did not include any recently united these two genera (Al-Shehbaz & members of Crucihimalaya. Koch et al. (2001) ob- O’Kane, 2002b), except that the auriculate-leaved tained similar results using plastidic matK and nu- species formerly placed in Lesquerella are recog- clear Chs sequences. Additional work is needed to nized as a distinct genus, Paysonia O’Kane & Al- Shehbaz (O’Kane & Al-Shehbaz, 2002). resolve this issue, but assuming that the ‘‘Capsella– Future work in the family will certainly yield fur- Pseudoarabidopsis clade’’ is sister to Arabidopsis ther taxonomic alignments since there is rampant appears to be a valid working hypothesis. morphological convergence (Al-Shehbaz et al., 1999; Koch et al., 1999) and because previous tax- TAXONOMIC IMPLICATIONS ELSEWHERE IN THE onomy in the family has relied heavily on fruit mor- BRASSICACEAE phology (e.g., Rollins, 1993; Al-Shehbaz, 1984) to the exclusion of floral and vegetative features (Al- Although our intent was not to study the genus Shehbaz et al., 1999). Molecular techniques in con- Arabis in any detail, our results mirror those of cert with a reevaluation of morphological characters Koch et al. (1999, 2000, 2001) in showing Arabis, are rapidly reshaping our understanding of the fam- as traditionally recognized, to be polyphyletic even ily (e.g., Bailey & Doyle, 1999; Bailey et al., 2002; after A. lyrata L., A. pedemontana, and A. ceben- Bowman et al., 2000; Koch et al., 1999, 2000, nensis are transferred to Arabidopsis (Fig. 1). In our 2001; Mummenhoff & Koch, 1994; Mummenhoff et ´ analysis, the genus A.Lo¨ve & D. Lo¨ve al., 1997a, b, 2001a, b; Price & Palmer, 1996; ϭ seems to be the proper home for x 7 species like Rodman et al., 1996; Warwick & Black, 1993, A. lyallii A. Gray and A. drummondii S. Watson, 1997). Characterizing the membership of Arabidop- though not all of the necessary generic transfers sis and sketching its relationships to related genera, have been made. Arabis in its strictest interpreta- we believe, contributes to this growing body of tion will consist only of those species in the clade knowledge. with A. alpina L., the lectotype of Arabis. (L.) Bernh. belongs to Turritis L. and A. pau- Literature Cited ciflora (Grimm) Garcke belongs to Fourraea Greu- ter & Burdet (Koch et al., 1999). But to which ge- Al-Shehbaz, I. A. 1973. The biosystematics of the genus Thelypodium (Cruciferae). Contr. Gray Herb. 204: 3– nus does A. pendula L. or A. turrita L. belong (Koch 148. et al., 1999)? Including species once thought to be . 1984. The tribes of Cruciferae (Brassicaceae) in related to Arabidopsis in our study has also raised the southeastern United States. J. Arnold Arbor. 65: other taxonomic questions. Neotorularia, Braya 343–373. Sternb. & Hoppe, and Dichasianthus Ovcz. & Jun- . 1988. The genera of Arabideae (Cruciferae; Bras- sicaceae) in the southeastern United States. J. Arnold ussov form a well-supported clade (Fig. 1) with Arbor. 69: 85–106. Neotorularia being paraphyletic. Thellungiella and & S. L. O’Kane, Jr. 1995. Placement of Arabidop- Volume 90, Number 4 O'Kane & Al-Shehbaz 611 2003 Phylogenetic Position of Arabidopsis

sis parvula in Thellungiella (Brassicaceae). Novon 5: uted by the author). Department of Botany, Stockholm 309–310. University, Stockholm. & . 1997. Arabidopsis gamosepala and A. Faith, D. P. & P. S. Cranston. 1992. Probability, parsi- tuemurnica belong to Neotorularia (Brassicaceae). No- mony, and Popper. Syst. Biol. 41: 252–257. von 7: 93–94. Galloway, G. L., R. L. Malmberg & R. A. Price. 1998. & . 1999. Ianhedgea (Brassicaceae), a Phylogenetic utility of the nuclear gene arginine decar- new generic name replacing the illegitimate Microsisym- boxylase: An example from Brassicaceae. Molec. Biol. brium. Edinburgh J. Bot. 56: 321–327. Evol. 15: 1312–1320. & . 2002a. Taxonomy and phylogeny of Hall, J. C., K. J. Sytsma & H. H. Iltis. 2002. Phylogeny Arabidopsis (Brassicaceae). (22 August, 2002), C. R. of Capparaceae and Brassicaceae based on Somerville & E. M. Meyerowitz (editors), The Arabidop- sequence data. Amer. J. Bot. 89: 1826–1842. sis Book. American Society of Plant Biologists, Rock- Hedge, I. C. 1968. Arabidopsis. Pp. 489–490 in K. H. ville, Maryland. doi/10.1199/tab. 0001, Ͻhttp:// Rechinger (editor), Flora Iranica, Vol. 57. Akademische www.aspb.org/publications/arabidopsis/Ͼ. Druck- u. Verlagsanstalt, Graz, Austria. & . 2002b. Lesquerella is united with Phy- Hennig, W. 1966 (1999 reissue). Phylogenetic Systemat- saria (Brassicaceae). Novon 12: 319–329. ics. Univ. Illinois Press, Chicago. , & R. A. Price. 1999. Generic placement Hillis, D. M. & J. P. Huelsenbeck. 1992. Signal, noise, of species excluded from Arabidopsis. Novon 9: 296– and reliability in molecular phylogenetic analysis. He- 307. redity 83: 189–195. An, Z. X. 1987. Arabidopsis. Pp. 280–288 in T.-Y. Cheo Hylander, N. 1957. Cardaminopsis suecica (Fr.) Hiit., A (editor), Flora Reipublicae Popularis Sinicae, Vol. 33. northern amphidiploid species. Bull. Jard. Bot. E´ tat 27: Science Press, Beijing. 591–604. The Arabidopsis Genome Initiative. 2000. Analysis of the Jafri, S. M. H. 1973. Brassicaceae. Pp. 1–308 in E. Nasir genome sequence of the flowering plant Arabidopsis & S. I. Ali (editors), Flora of West Pakistan, Vol. 55. thaliana. Nature 408: 796–815. Ferozsons, Karachi. Ashlock, P. D. 1971. Monophyly and associated terms. Jones, B. M. G. 1964. Arabis. Pp. 290–294 in T. G. Tutin, Syst. Zool. 20: 63–69. V. H. Heywood, N. A. Burges, D. H. Valentine, S. M. Bailey, C. D. & J. J. Doyle. 1999. Potential phylogenetic Walters & D. A. Webb (editors), Flora Europaea, Vol. utility of the low-copy nuclear gene pistillata in dicot- 1. Cambridge Univ. Press, Cambridge. yledonous plants: Comparison to nrDNA ITS and trnL & J. R. Akeroyd. 1993a. Cardaminopsis. Pp. 351– intron in Sphaerocardamum and other Brassicaceae. 352 in T. G. Tutin, N. A. Burges, A. O. Chater, J. R. Edmondson, V. H. Heywood, D. M. Moore, D. H. Val- Molec. Phylogenet. Evol. 13: 20–30. entine, S. M. Walters & D. A. Webb (editors), Flora , R. A. Price & J. J. Doyle. 2002. Systematics of Europaea, ed. 2, Vol. 1. Cambridge Univ. Press, Cam- the Halimolobine Brassicaceae: Evidence from three bridge. loci and morphology. Syst. Bot. 27: 318–332. & . 1993b. Arabis. Pp. 352–356 in T. G. Ball, P. W. 1993. Arabidopsis. Pp. 322–323 in T. G. Tutin, Tutin, N. A. Burges, A. O. Chater, J. R. Edmondson, V. N. A. Burges, A. O. Chater, J. R. Edmondson, V. H. H. Heywood, D. M. Moore, D. H. Valentine, S. M. Wal- Heywood, D. M. Moore, D. H. Valentine, S. M. Walters ters & D. A. Webb (editors), Flora Europaea, ed. 2, Vol. & D. A. Webb (editors), Flora Europaea, ed. 2, Vol. 1. 1. Cambridge Univ. Press, Cambridge. Cambridge Univ. Press, Cambridge. Judd, W. S., R. W. Sanders & M. J. Donoghue. 1994. Blanc, G., A. Barakat, R. Guyot, R. Cooke & M. Delseny. Angiosperm family pairs: Preliminary cladistic analy- 2000. Extensive duplication and reshuffling in the Ar- ses. Harvard Pap. Bot. 5: 1–51. abidopsis Genome. Pl. Cell 12: 1093–1101. Kamm, A., I. Galasso, T. Schmidt & J. S. Heslop-Harrison. Bowman, J. L., H. Bru¨ggemann, J.-Y. Lee & K. Mummen- 1995. Analysis of a repetitive DNA family from Arabi- hoff. 1999. Evolutionary changes in floral structure dopsis arenosa and relationships between Arabidopsis within Lepidium L. (Brassicaceae). Int. J. Pl. Sci. 160: species. Pl. Molec. Biol. 27: 853–862. 917–929. Koch, M., J. Bishop & T. Mitchell-Olds. 1999. Molecular Bremer, K. 1988. The limits of amino acid sequence data systematics and evolution of Arabidopsis and Arabis. Pl. in angiosperm phylogenetic reconstruction. Evolution Biol. 1: 529–537. 42: 795–803. , B. Haubold & T. Mitchell-Olds. 2000. Compar- Cooke, R., M. Raynal, M. Laudie´, F. Grellet, M. Delseny, ative evolutionary analysis of chalcone synthase and al- P.-C. Morris, D. Guerrier, J. Giraudat, F. Quigley, G. cohol dehydrogenase loci in Arabidopsis, Arabis and re- Clabault, Y.-F. Li, R. Mache, M. Krivitzky, I. J.-J. Gy, lated genera. Molec. Biol. Evol. 17: 1483–1498. M. Kreis, A. Lecharny, Y. Parmentier, J. Marbach, J. , & . 2001. Molecular systematics Fleck, B. Cle´ment, G. Philipps, C. Herve´, C. Bardet, D. of the Brassicaceae: Evidence from coding plastidic Tremousaygue, B. Lescure, C. Lacomme, D. Roby, M.- matK and nuclear Chs sequences. Amer. J. Bot. 88: F. Jourjon, P. Chabrier, J.-L. Charpenteau, T. Desprez, 534–544. J. Amselem, H. Chiapello & H. Ho¨fte. 1996. Further Laibach, F. 1943. (L.) Heynh. als progress towards a catalogue of all Arabidoposis genes: Objekt fu¨r genetische und entwicklungsphysiologische Analysis of a set of 5000 non-redundant ESTs. Pl. J. 9: Untersuchungen. Bot. Archiv 44: 439–455. 101–124. Lee, M. S. Y. 2000. Tree robustness and clade signifi- Donoghue, M. J., R. G. Olmstead, J. F. Smith & J. D. cance. Syst. Biol. 49: 829–836. Palmer. 1992. Phylogenetic relationships of Dipsacales Lo¨ve, A´ . 1961. Hylandra—A new genus of Cruciferae. based on rbcL sequences. Ann. Missouri Bot. Gard. 79: Svensk Bot. Tidskr. 55: 211–217. 333–345. Meyerowitz, E. M. 1989. Arabidopsis, a useful weed. Cell Eriksson, T. 1998. AutoDecay, ver. 4.01 (program distrib- 56: 263–269. 612 Annals of the Missouri Botanical Garden

& R. E. Pruitt. 1985. Arabidopsis thaliana and the rbcL gene indicate monophyly of mustard oil plants. plant molecular genetics. Science 22: 1214–1218. Ann. Missouri Bot. Gard. 80: 686–699. Mummenhoff, K. & H. Hurka. 1994. Subunit polypeptide , K. G. Karol, R. A. Price & K. J. Sytsma. 1996. composition of Rubisco and the origin of allopolyploid Molecules, morphology, and Dahlgren’s expanded order Arabidopsis suecica (Brassicaceae). Biochem. Syst. Ecol. Capparales. Syst. Bot. 21: 289–307. 22: 807–812. Rollins, R. C. 1979. Dithyrea and a related genus (Cru- & M. Koch. 1994. Chloroplast DNA restriction ciferae). Publ. Bussey Inst. Harvard Univ. 1979: 3–32. site variation and phylogenetic relationships in the ge- . 1993. The Cruciferae of Continental North Amer- nus Thlaspi sensu lato (Brassicaceae). Syst. Bot. 19: ica. Stanford Univ. Press, Stanford. 73–88. & U. C. Banerjee. 1979. of the Cruciferae. , A. Franzke & M. Koch. 1997a. Molecular data Publ. Bussey Inst. Harvard Univ. 1979: 33–64. reveal convergence in fruit characters used in the clas- Schulz, O. E. 1924. Cruciferae–Sismbrieae. In: A. Engler sification of Thlaspi s.l. (Brassicaceae). Bot. J. Linn. (editor), Pflanzenreich IV. 105(Heft 86): 1–388. Verlag Soc. 125: 183–199. von Wilhelm Engelmann, Leipzig. , & . 1997b. Molecular phyloge- . 1936. Cruciferae. In A. Engler & K. Prantl (ed- netics of Thlaspi s.l. (Brassicaceae) based on chloro- itors), Natu¨rl. Pflanzenfam., ed. 2, 17B: 227–658. Ver- plast DNA restriction site variation and sequences of lag von Wilhelm Engelmann, Leipzig. the internal transcribed spacers of nuclear ribosomal Somerville, C. R. & E. M. Meyerowitz (editors). 2002. The Ͻ DNA. Canad. J. Bot. 75: 469–482. Arabidopsis Book. http://www.aspb.org/publications/ Ͼ ,H.Bru¨ggemann & J. Bowman. 2001a. Chloro- arabidopsis/ . plast DNA phylogeny and biogeography of the genus Swofford, D. L. 2000. PAUP*. Phylogenetic Analysis Us- Lepidium (Brassicaceae). Amer. J. Bot. 88: 2051–2063. ing Parsimony (*and Other Methods). Version 4. Sin- ,U.Coja&H.Bru¨ggemann. 2001b. Pachyphrag- auer, Sunderland, Massachusetts. Vander Zwan, C., S. A. Brodie & J. J. Campanella. 2000. ma (Brassicaceae) revisited: Molecular data indicate The intraspecific phylogenetics of Arabidopsis thaliana close relationship to Thlaspi s.str. Folia Geobot. 36: in worldwide populations. Syst. Bot. 25: 47–59. 293–302. Warwick, S. I. & L. D. Black. 1993. Molecular relation- O’Kane, S. L., Jr. & I. A. Al-Shehbaz. 1997. A synopsis ships in subtribe Brassicinae (Cruciferae, tribe Brassi- of Arabidopsis (Brassicaceae). Novon 7: 323–327. ceae). Canad. J. Bot. 71: 906–918. & . 2002. Paysonia, a new genus segre- & . 1997. Phylogenetic implications of gated from Lesquerella (Brassicaceae). Novon 12: 379– chloroplast DNA restriction site variation in subtribes 381. Raphaninae and Cakilinae (Brassicaceae, tribe Brassi- , B. A. Schaal & I. A. Al-Shehbaz. 1996. The ceae). Canad. J. Bot. 75: 960–973. origins of Arabidopsis suecica (Brassicaceae) as indicat- White, T. J., T. Bruns, S. Lee & J. Taylor. 1990. Ampli- ed by nuclear rDNA sequences. Syst. Bot. 21: 559–566. fication and direct sequencing of fungal ribosomal RNA Patrusky, B. 1991. Drosophila botanica (the fruit fly of genes for phylogenetics. Pp. 315–322 in M. A. Innis, plant biology). Mosaic 22: 32–43. D. H. Gelfand, J. J. Sninsky & T. J. White (editors), Price, R. A. & J. D. Palmer. 1996. Chloroplast DNA based PCR Protocols. Academic Press, New York. phylogenies and fruit shape in the tribe Arabideae Yang, W.-W., K.-N. Lai, P.-Y. Tai, D.-P. Ma & W.-H. Li. (Brassicaceae). Amer. J. Bot. 83: S187–S188. 1999. Molecular phylogenetic studies of Brassica, Ro- , J. D. Palmer & I. A. Al-Shehbaz. 1994. System- rippa, Arabidopsis and allied genera based on the inter- atic relationships of Arabidopsis: A molecular and mor- nal transcribed spacer region of 18S–25S rDNA. Molec. phological perspective. Pp. 7–19 in E. M. Meyerowitz Phylogenet. Evol. 13: 455–462. & C. R. Somerville (editors), Arabidopsis. Cold Spring Harbor Laboratory Press, New York. Note added in proof. , I. A. Al-Shehbaz & S. L. O’Kane, Jr. 2001. Ber- ingia (Brassicaceae), a new genus of Arabidopsid affin- The genus Beringia was renamed in the following ities from Russia and . Novon 11: 332– article. 336. Al-Shehbaz, I. A. & S. L. O’Kane. 2003. Transberingia, a Rodman, J. E., R. A. Price, K. Karol, E. Conti, K. J. new generic name replacing the illegitimate Beringia Sytsma & J. D. Palmer. 1993. Nucleotide sequences of (Brassicaceae). Novon 13: 396.