American Journal of Botany 99(4): 778–794. 2012.

S YSTEMATICS AND EVOLUTION OF ARCTIC-ALPINE A RABIS ALPINA (BRASSICACEAE) AND ITS CLOSEST RELATIVES IN THE EASTERN MEDITERRANEAN 1

R OBERT K ARL 2 , C HRISTIANE K IEFER 3 , S TEPHAN W . A NSELL 4 , AND M ARCUS A . K OCH 2,5

2 Department of Biodiversity and Plant Systematics, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University 69120 Heidelberg, Germany; 3 Max-Planck-Institute for Plant Breeding 50829 Cologne, Germany; and 4 Natural History Museum London, Department of Botany, London, SW7 5BD, UK

• Premise of the study: The high mountains in southern Anatolia and the eastern Mediterranean are assumed to play a major role as a primary center of genetic diversity and species richness in Eurasia. We tested this hypothesis by focusing on the wide- spread perennial arctic-alpine Arabis alpina and its sympatrically distributed closest relatives in the eastern Mediterranean. • Methods: Plastid (trnL intron, trnL-F intergenic spacer) and nuclear (ITS) DNA sequence analysis was used for phylogenetic reconstruction. Broad-scale plastid haplotype analyses were conducted to infer ancestral biogeographic patterns. • Key results: Five Arabis species, identifi ed from the eastern Mediterranean (Turkey mainland and Cyprus), evolved directly and independently from A . alpina, leaving Arabis alpina as a paraphyletic taxon. These species are not affected by hybridiza- tion or introgression, and species divergence took place at the diploid level during the Pleistocene. • Conclusions: Pleistocene climate fl uctuations produced local altitudinal range-shifts among mountain glacial survival areas, resulting not only in the accumulation of intraspecifi c genotype diversity but also in the formation of fi ve local species. We also show that the closest sister group of Arabis alpina consists exclusively of annuals/winter annuals and diverged prior to Pleis- tocene climatic fl uctuations during the colonization of the lowland Mediterranean landscape. These fi ndings highlight that Anatolia is not only a center of species richness but also a center for life-history diversifi cation.

Key words: annual species; Arabis alpina ; Brassicaceae; eastern Mediterranean; evolutionary history; perennial species; Pleistocene.

In the last 15 years, phylogeographic studies have character- distributed throughout the alpine habitats of Europe and into the ized the evolutionary history of numerous Eurasian species arctic zones of Greenland and North America, the high moun- (Taberlet et al., 1998; Hewitt, 1999, 2000 ; Willis and Whittaker, tains of northern and eastern Africa, Anatolia, the Caucasus, 2000 ). Many of these studies have focused on time scales span- and the Near East ( Meusel et al., 1965 ; Koch et al., 2006 ). This ning the late Tertiary and Pleistocene (Comes and Kadereit, broad distribution makes this species an ideal system to study 1998 , 2003 ; Hewitt, 2004 ; Birks, 2008 ) and provided new in- the effect of Pleistocene climatic fl uctuations on speciation, ge- sights into the biogeography of various taxa during past climate netic differentiation, and adaptation over different geographic change. Organisms were forced either to escape, migrate, and scales, including those regions important for its long-term sur- eventually recolonize or to stay, adapt, and survive the glacia- vival and speciation. For example, studies on various organ- tions (Petit et al., 2003; Huck et al., 2009; Schmitt, 2009). isms have highlighted the importance of southern Anatolia Whereas most studies have used a single species approach or a (Taurus mountain systems) for Pleistocene survival and specia- comparative analysis of taxonomically unrelated organisms tion (e.g., Font et al., 2009 ; Parolly et al., 2010 ). Presently, Arabis (Taberlet et al., 1998, Hewitt 2004), comparative phylogeogra- alpina is mostly treated as a well-defi ned species without sub- phy focusing on closely related taxa should be informative for species or other segregates, although often the segregrate A . addressing evolutionary questions, especially concerning trait caucasica Willd. ( A. alpina subsp. caucasica) has been recog- and adaptive variation ( Hickerson et al., 2010 ). nized. This is despite few clear discriminating morphological In this paper, we focus on the arctic-alpine plant Arabis al- characters ( Plantholt, 1995 ) and a lack of genetic evidence to pina L. and its close annual and perennial relatives (see below). consistently separate it from A. alpina ( Plantholt, 1995 ; Koch Arabis alpina, the alpine rock cress (Brassicaceae), is widely et al., 2006 ; Ansell et al., 2011 ). Currently, the closest related sister taxa to A. alpina have not yet been identifi ed. Candidates for closest relatives to A. alpina 1 Manuscript received 14 September 2011; revision accepted 8 February 2012. based on morphological characters include the Central Asian In addition to the various collections mentioned, we especially endemic A . tianschanica Pavlov and the Cyprian endemics A . acknowledge receipt of plant material from D. German (South-Siberian cypria Holmboe and A . purpurea Sm. The latter species has Botanical Garden, Barnaul, Russia), M. Hoffmann (IPK Gatersleben, pink to purple petals, a character that is also shared by the Germany), B. Mutlu (In ö n ü University, Malatya, Turkey), and C. Kadis southern Anatolian endemic A . aubrietioides Boiss. Further (Nature Conservation Unit, Frederick University, Cyprus). Funding was candidates for close relatives include A . defl exa Boiss., A . fari- provided by the DFG (Deutsche Forschungsgemeinschaft) to M.A.K. nacea Rupr., and A . ionocalyx Boiss. Understanding the evolu- (KO2302/1-1, KO2302/11-1 and KO2302/12-1). 5 Author for correspondence (e-mail: [email protected]) tionary and geographic relationships of this species group will provide a framework for future studies focusing on different doi:10.3732/ajb.1100447 and fundamental aspects of plant evolution and adaptation.

American Journal of Botany 99(4): 778–794, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America 778 April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 779

Various evolutionary studies have recently focused on the species or a species complex, we will explore the hypothesis evolution of Arabis alpina in more detail, addressing global that the related species evolved in parallel in these regions from phylogeographic issues (Koch et al., 2006; Ansell et al., 2011) different A. alpina populations. Furthermore, we will determine or population differentiation on various spatial scales (Assefa whether any of the eight Mediterranean annual Arabis species et al., 2007 ; Ehrich et al., 2007 ; Ansell et al., 2008 ). These stud- are sister to the perennial A . alpina group and test the hypothe- ies revealed that this species originated in a defi ned area in cen- sis that the divergence of annuals and perennial taxa predates tral Anatolia (Ansell et al., 2011), from where major waves of the Pleistocene and that they followed different evolutionary emigration have shaped the current distribution range (Koch pathways in the Mediterranean in response to the Pleistocene et al., 2006; Ansell et al., 2011): (1) migration to central and temperature oscillations. northwestern Europe and further into the arctic, (2) westward to Spain and north Africa, (3) migration into the Caucasus and the Near East, (4) two independent migration events to the east MATERIALS AND METHODS African high mountains. These waves of immigration span a time scale of divergence of around 2 million years (Koch et al., Plant material —For the phylogenetic analysis, we included one outgroup 2006; Assefa et al., 2007; Ansell et al., 2008), which is consid- accession and 72 accessions of Arabis alpina and relatives of the tribe Ara- ered to be the minimum age of Arabis alpina . bideae. These selected accessions comprised 29 species that have close mor- phological similarity to Arabis alpina (e.g., Arabis tianschanica). The data set Several studies have started to harness the potential of A. alpina also included all annuals assigned to Arabis [ Arabis aucheri Boiss., Arabis au- to investigate trait variation and adaptation in mountain habi- riculata Lam., Arabis erikii Mutlu, Arabis kennedyae Meikle, Arabis mont- tats. This started 50 years ago with the pioneering work of bretiana Boiss., Arabis nova Vill., Arabis parvula Dufour, and Arabis verna Hedberg (1962) , who indicated that there are reproductive bar- (L.) R. Br.]. Six individuals of A . alpina were chosen as representatives of the riers between the European and East African material of A. al- three major chloroplast DNA (cpDNA) haplotype lineages, which were identi- pina. More recently, Ansell et al. (2008) suggested putative fi ed earlier (Koch et al., 2006; Ansell et al., 2011). Three accessions were cho- sen from both the genus Draba (in its broadest sense, see Jordon-Thaden et al., changes in the breeding system coincided with historical differ- 2010) and the “ core- Arabis ” clade (see Koch et al., 2010). We selected Pseudo- ences in glacial survival. This is supported by mating analysis, turritis turrita (L.) Al-Shehbaz as the outgroup because this taxon was previ- which has identifi ed a sporophytic self-incompatibility (SI) sys- ously identifi ed as belonging to the sister tribe of the Arabideae: the Stevenieae tem in some Italian populations and the loss of SI function in (German et al., 2009, Al-Shehbaz et al., 2011). Plant material was obtained several Alps populations (Tedder et al., 2011). Elsewhere AFLP from the Botanical Garden and Botanical Museum Berlin (B), the Natural His- analysis of Alps populations has identifi ed a remarkable num- tory Museum London (BM), University of Copenhagen (C), Royal Botanical Garden Edinburgh (E), Botanical Garden and Herbarium Heidelberg (HEID), ber of polymorphic loci that are correlated to ecological varia- Botanische Staatssammlung Munich (M), Real Jardin Botanico Madrid (MA), tion (Manel et al., 2010; Poncet et al., 2010). There is also the Missouri Botanical Garden (MO), IPK Gatersleben, In ö n ü University increasing knowledge on the molecular basis of life-history Malatya in Turkey, the Frederick University Cyprus and the South-Siberian variation in Arabis alpina. One such trait is the shift from an- Botanical Garden Barnaul in Russia. nualism to perennialism and the regulation of perennial fl ower- For the biogeographic analyses, we added a compilation of additional cpDNA ing by PEP1 ( PERPETUAL FLOWERING 1 ) in Arabis alpina data from 483 accessions representing 296 locations (Koch et al., 2006; Assefa et al., 2007; Ansell et al., 2008, 2011 ), giving a combined total of 518 acces- (Wang et al., 2009), which may be considered a “ key character sions from 329 locations. trait ” in the adaptation processes of an arctic-alpine plant. Detailed accession information, including GenBank accession numbers, are Our study is part of a larger project to unravel the evolution- provided in the Appendix 1. ary history and systematics of the whole tribe Arabideae, which consists of more than 500 species. A series of studies has al- DNA extraction, PCR amplifi cation and sequencing — DNA extractions ready provided evidence for (1) the paraphyly of numerous taxa were carried out using the NucleoSpin Plant II Kit (Macherey-Nagel, D ü ren, within the tribe Arabideae (including Arabis ) and that (2) the Germany) or according to an isopropyl alcohol variant of the CTAB method tribe can be split into two subclades (Koch et al., 1999a, 2000 , ( Doyle and Doyle, 1987 ). The innuPREP Plant DNA Kit (Analytik Jena) was used for DNA extractions from seed material with the addition of an extra pre- 2001 , 2007 , 2010 ; German et al., 2009 ; Al-Shehbaz et al., fi lter and washing step. The internal transcribed spacer of nuclear ribosomal 2011 ). The Arabideae consist mostly of taxa from the two RNA including the 5.8S rRNA (ITS), chloroplast trnL intron (trnL ) and trnL - closely related genera Draba with more than 390 species and trnF intergenic spacer ( trnL - F) were selected as markers and were amplifi ed by Arabis with more than 65 species. The fully unexplored genus PCR. Reactions were performed in a fi nal volume of 25 µ L, using 10 µ M of each primer, a total of 2.0 mmol/L MgCl 2 and 0.5 U of MangoTaq polymerase Aubrieta represents another 10 – 20 species (Koch et al., ongo- ′ ing research). Preliminary phylogenetic analyses have focused (Bioline). The primers used for ITS amplifi cation were the 18F primer (5 -GGA AGG AGA AGT CGT AAC AAG G-3 ′ ) as modifi ed by Mummenhoff et al. on establishing the tribal and generic relationships (Jordon- (1997a) and the 25R primer (5 ′-TCC TCC GCT TAT TGA TAT GC-3 ′ ) de- Thaden et al., 2010; Koch et al., 2010). Consequently, in this signed by White et al. (1990). The trnL intron was amplifi ed using the universal study we have excluded many taxa previously shown to be un- primers c and d of Taberlet et al. (1991) (c: 5 ′ -CGA AAT CGG TAG ACG CTA related or only distantly related to Arabis alpina . CG-3 ′, d: 5 ′-GGG GAT AGA GGG ACT TGA AC-3 ′), while the trnL - F inter- genic spacer was amplifi ed with primer e of Taberlet et al. (1991) (5 ′-GGTT CA Presently, it is unclear as to whether Arabis alpina represents ′ a single well-defi ned species or a species complex. However, AGT CCC TCT ATC ATC CC-3 ) and a primer designed by Dobe š et al. (2004) (5 ′-GAT TTT CAG TCC TCT GCT CTA C-3 ′). The amplifi cations were run on there are various species that are obviously morphologically a PTC 200 Peltier Thermal Cycler (MJ Research) under the following condi- very similar to Arabis alpina and are often distributed sympatri- tions: 3 min initial denaturation at 95 °C; followed by 30 cycles of 30 s at 95 ° C, cally. On the basis of morphology and distribution, we have 30 s at 48 ° C and 1 min at 72 ° C; and 10 min fi nal elongation at 72 ° C. PCR selected candidate taxa that might be members of an Arabis products were purifi ed by centrifugation in a NucleoFast 96 PCR Plate (Mach- alpina species complex and asked how the morphologically erey-Nagel) and directly sequenced using the above primers by the commercial similar species relate to the evolutionary framework of the A . sequencing service GATC (Konstanz, Germany). The ITS (Appendix S1, see Supplemental Data with the online version of this article) and the combined alpina lineages, especially in connection to those found in the east- trnL intron and trnL - F intergenic spacer sequences (Appendix S2, see online ern Mediterranean, Anatolia and Central Asia. In answering the Supplemental Data) were checked and trimmed using the program SeqMan II question as to whether A. alpina represents a single well-defi ned v.6.1 (DNAStar, Madison, Wisconsin, USA) (hereafter designated trnLF ) and 780 AMERICAN JOURNAL OF BOTANY [Vol. 99

T ABLE 1. Parameters and results of the three divergence-time estimate approaches based on the nrDNA ITS marker. Mean values are indicated in boldface; the lower and upper bounds of the 95% highest posterior density (HPD) interval are given above and below that value, respectively. Enforced bounds are indicated in italics.

Using secondary calibration of Like previous + fi xed Fossil based calibration of node age of node age of tribe Arabideae (acc. mutational rates Parameter tribe Thlaspideae (acc. Beilstein et al., 2010) Couvreur et al., 2009) ( Kropf, 2002 ; Kay et al., 2006 )

Runs / Generations 4 independent runs with 4 independent runs with 4 independent runs with 30 million generations each 20 million generations each 20 million generations each Log likelihood − 3103.058 − 3103.198 − 3103.217 − 3090.494 − 3090.620 − 3090.576 − 3077.935 − 3078.138 − 3078.295 tmrca of Arabis alpina aggregate and annual 2.84 0.94 0.94 sister group: clade A ( Fig. 1 ) (Ma) 7.13 2.24 2.25 12.3 3.94 3.98 t mrca of Arabis alpina aggregate (Ma) 1.49 0.51 0.52 4.02 1.26 1.27 7.02 2.25 2.25 t mrca of the core-Arabis (Ma) 4.58 1.54 1.63 12.5 3.91 3.94 21.5 6.95 7.04 t mrca of Draba and its segregates (Ma) 8.41 2.98 3.03 19.3 6.09 6.09 31.7 10.2 10.2 tmrca of tribe Arabideae (Ma) 22.1 10.0 10.0 44.4 14.1 14.1 69.5 21.5 21.5 ucld.mean 0.85 × 10 − 9 2.66 × 10 − 9 2.69 × 10 − 9 1.60 × 10 − 9 5.10 × 10 − 9 5.08 × 10 − 9 2.47 × 10 − 9 7.37 × 10 − 9 7.41 × 10 − 9 meanRate 0.88 × 10 − 9 2.75 × 10 − 9 2.75 × 10 − 9 1.59 × 10 − 9 5.12 × 10 − 9 5.10 × 10 − 9 2.41 × 10 − 9 7.35 × 10 − 9 7.38 × 10 − 9 Bayes factors — / 1.09 / 1.38 0.92 / — / 1.26 0.73 / 0.79 / — then manually aligned using the program GeneDoc v2.6.002 ( Nicholas, 1997 ). The parsimony heuristic search was performed under the following settings: In addition, a combined data set was formed by merging the ITS and the trnLF gaps were treated as missing data, the “ mstaxa ” option was set as “ uncertain ” , sequences (online Appendix S3). Identical sequences were excluded from the tree construction was via stepwise addition, tree-bisection-reconnection (TBR) fi nal alignments prior to phylogenetic analysis. was used as the branch-swapping algorithm, the MaxTrees limit was set to 10 000, and the “ MulTrees ” option was in effect (saving all minimal trees found Phylogenetic analyses —Bayesian Markov chain Monte Carlo (MCMC) during branch swapping). A strict consensus tree was calculated from most analyses (Yang and Rannala, 1997) were performed with the mpi (message parsimonious trees retained. For bootstrapping (Felsenstein, 1985), 100 repli- parsing interface) version of the program MrBayes v.3.1.2 (Ronquist and cates with a maximum of 250 retained trees were run. Huelsenbeck, 2003 ). To substantiate the results of the Bayesian analyses, we under- For the maximum likelihood (ML) analyses, we ran two replicates per data took additional analyses using maximum parsimony (MP) running the program set, and each replicate was terminated under the condition that the ln likelihood PAUP* 4.0b10 ( Swofford, 2002 ) and maximum likelihood using the program value did not increase signifi cantly (0.01) for the previous 1 000 000 genera- Garli v0.96 (Zwickl, 2006). The best-fi tting nucleotide substitution models tions. Analyses were conducted using 1000 bootstrapping replicates with the were chosen using the program Modeltest v3.7 (Posada and Crandall, 1998), lowered termination condition of 10 000 generations without signifi cant ln L which relied on the Akaike information criterion (AIC) and the Bayesian infor- change. The corresponding alignments to the ITS, trnLF, and combined data mation criterion (BIC). For the combined analyses, the data set was divided into are provided with online Appendices S1, S2, and S3, respectively. Graphical nrDNA and cpDNA partitions, so that each partition could be run under the edition of all trees was executed in Adobe Illustrator CS4 (San Jose, California, respective model. In the Bayesian analyses, four simultaneous runs with four USA). chains each were run for 20 (ITS and trnLF data set) or 30 (combined data set) million generations. The temperature of the heated chain was set to 0.01, as this Congruency test— A Shimodaira-Hasegawa test ( Shimodaira and Hasegawa, facilitated the most effi cient chain-swapping. For each run, 1001 trees were 1999) was performed to identify congruencies between the tree topologies sampled, and the fi rst 25% of the trees sampled were discarded (burn-in = 250) of the ITS, trnLF, and combined marker data sets using PAUP* 4.0b10 when computing the consensus tree (50% majority rule) Completion of the (Swof ford, 2002 ). Before the tests, the number of accessions was reduced to the Bayesian runs was determined using the web-based program AWTY (Nylander 40 common to all three data sets. As a consequence, the number of tree to- et al., 2007 ). Comparison of the posterior probabilities of all splits between the pologies was very small (n = 2) and might have biased the levels of signifi - respective runs and cumulative split frequency plots showed that convergence cance. We therefore added 100 random topologies to the congruency tests to between the Bayesian MCMC runs had been reached. minimize this problem.

→ Fig. 1. Phylogenetic tree of the Bayesian analysis based on the nrDNA ITS data set. Posterior probability values (fi rst-mentioned) and bootstrap values of the maximum-likelihood analysis (middle-mentioned), and the maximum parsimony analysis (last-mentioned) of the nodes are given along the corre- sponding branches. Pseudoturritis turrita was used as the outgroup taxa. Accessions with a prefi xed asterisk had the same sequence as the top-listed acces- sion and were therefore excluded from the tree calculation. Purple- or pink-fl owered species are indicated with a black spot following the taxon label; species that occasionally produce pink petals are marked with a gray spot. Accessions without any spot correspond to primarily white-fl owered species. Annual/winter-annual taxa are labeled with the corresponding sign for annualism, also following the taxon label. April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 781 782 AMERICAN JOURNAL OF BOTANY [Vol. 99

Network analyses — Network analysis was performed with the program 2002 ; Mummenhoff et al., 2004 ; Kay et al., 2006 ). The most appropriate evolu- TCS v.1.21 ( Clement et al., 2000 ), which uses statistical parsimony ( Templeton tionary model was estimated using the program Modeltest v.3.7 (Posada and et al., 1992 ) as the underlying method of estimation. Taxa identifi ed to be dis- Crandall, 1998 ), based on the Bayesian information criterion (BIC). tantly related to Arabis alpina were excluded from the alignment previously Both ITS and trnLF divergence-time estimations were run under the lognor- used for phylogenetic analaysis. We utilized accessions of Arabis brachycarpa mal uncorrelated relaxed clock method and the birth – death speciation model Rupr., A . nordmanniana Rupr., A . graellsiiformis Hedge, and A . christiani (Gernhard, 2008), because the calculated Bayes factors (Nylander et al., 2004) N.Busch to root the network. For the ingroup, we included all cpDNA haplotypes favored their use over a strict clock and a pure birth model (Yule, 1924). Num- published earlier by Koch et al. (2006) (DQ060112– DQ060138, DQ060140– ber of runs and generations are given in Table 1 (ITS) and 2 (trnLF ). In each DQ060142, DQ060144 – DQ060145), Assefa et al. (2007) (EF449508 –EF449514), run, 10 001 generations were sampled. Each run was checked in the program Ansell et al. (2008) (EU403083 – EU403084), and Ansell et al. (2011) (JF705219 – Tracer v.1.4.1 (Rambaut and Drummond, 2007) for having reached stationary JF705252), giving a combined data set of 115 haplotypes from of 518 acces- phase and a suffi cient effective sample size (ESS over 200, as suggested by the sions, covering 329 locations and 10 taxa (A . cypria , A . purpurea , A . kennedyae , authors). All runs (log- and trees-fi les, respectively) were then combined with A . alpina , A . aubrietoides , A . tianschanica , A . defl exa , A . ionocalyx , A . mont- the LogCombiner program included in the Tracer package. bretiana , A . nova subsp. iberica Rivas Mart. ex Talavera). The alignment was adjusted further by excluding a microsatellite-like region Chromosome counts and ploidy level estimates— Most of the leaf material ′ at the 5 -end of the trnL-F intergenic spacer (alignment position 365 – 380, was obtained directly from wild sources (e.g., preserved herbarium vouchers) Appendix S2), and we also excluded the trnL gene from the analysis, so that all with supplementary material grown from seed. Root tips were incubated in sequences had comparable information content. This led to the exclusion of a 2 mmol/L 8-hydroxyquinoline for 3 h at 15 °C and subsequently transferred to series of nearly identical haplotypes shown in Ansell et al. (2011) (haplotypes freshly prepared ice-cold ethanol – acetic acid (3 : 1). After 2 h of incubation at 06, 22, 23, 24, 27, 32, 48, 52, 53, 55, 57, 64, 65, 66, 67, 71, B, and D). Redun- 4 °C, the root tips were transferred to 70% ethanol and stored at − 20 °C for sub- dant haplotypes from newly analyzed accessions of our study were also removed sequent analysis. Root tips were washed in water, equilibrated in 10 mmol/L (MG003, RK240, RK244, RK249, and RK252), as were haplotypes 16 and 31 citrate buffer, and then hydrolyzed with an enzyme solution (0.1% cellulase, from Koch et al. (2006). Furthermore, to be conservative when interpreting 0.1% pectolyase, 0.1% cytohelicase, and 1 mmol/L citrate buffer) at 37° C fol- nucleotide variation, we also excluded sequence types that differed by indels in lowed by squashing and fi xing in 45% acetic acid and staining with a 1 : 20 dilu- the beginning and end portions of the PCR fragments, where sequencing qual- tion of DAPI/Vectashield (Vector Laboratories, Burlingame, California, USA). ity may be insuffi cient. This excluded haplotypes E and F (Assefa et al., 2007) Additional information on chromosome numbers from various Arabis species and several newly analyzed accessions of our study (A53M, MA001, MA004, was obtained from published accounts (Warwick and Al-Shehbaz, 2006; Koch RK001, RK002, RK004, RK006, RK086, RK087, RK088, RK167, and et al., 2010 ). RK250). Finally, indels of more than one base pair in length were recoded with binary characters (online Appendix S4). This resulted in 73 unique haplotypes for minimum spanning network reconstruction. RESULTS Divergence-time estimates — Divergence-time estimates in the Brassicaceae have been discussed widely, because calculations led to highly diverging re- Nuclear ITS region — The ITS alignment comprised 45 se- sults, depending on the markers and calibration points used (refer to Franzke quences (representing 71 accessions) and 631 characters, of et al. [2011] for further discussion). Using the software package BEAST v.1.4.8 which 448 were constant. Of the 183 variable characters, 132 (Drummond and Rambaut, 2007), we performed three courses of divergence- time estimate calculations employing two different calibration points. (1) The were phylogenetically informative, and the remaining 51 were primary calibration was based on an Oligocene fruit fossil ( Thlaspi primae- autapomorphic positions (Appendix S1). The SYM+ Γ evolu- vum ), which was assigned to the tribe Thlaspideae ( Manchester and O ’ Leary, tionary model was applied to the Bayesian and ML analyses, 2010 ). This fossil, dated at 29.2 − 30.8 Ma ( Wing, 1987 ), has been recently used the results are shown in Fig. 1 . The resulting Bayesian trees had for age estimates of the Brassicales (Beilstein et al., 2010). (2) Secondary cali- a harmonic mean for the ln likelihood of − 2794.51 and an aver- bration used the estimated age of the most recent common ancestor of the tribe age standard deviation of split frequencies of 0.010. The corre- Arabideae (mean age 16.8 Myr) as shown by Couvreur et al. (2009) . (3) We additionally applied fi xed limits for the rates of variation on the secondary cali- sponding tree of the ML analysis had a ln likelihood of − 2678.34. bration point approach, following Koch et al. (2010). Two representatives of The maximum parsimony analysis resulted in 571 most parsi- the tribe Thlaspideae [Thlaspi arvense L. AF336152 (ITS1), AF336153 (ITS2), monious trees with a tree length of 326 (with “ maximum length = AY122461 (trnLF ); Alliaria petiolata (M.Bieb.) Cavara & Grande AF283492 0” rule applied), consistency index (CI) of 0.66 (autapomor- (ITS1), AF 283493 (ITS2), JN189740 ( trnL), JN189781 ( trnF)] were added to phies excluded) and retention index (RI) of 0.90. both the ITS and cpDNA alignments, used for the phylogenetic analyses. Both The ITS tree based on Bayesian analysis ( Fig. 1 ) is subdivided taxa were also used by Beilstein et al. (2010) to defi ne the tribe Thlaspideae. Both original alignments were modifi ed only by the introduction of additional into seven major clades, which are very well supported by both gaps. Taxon subsets were specifi ed for some clades as designated in the phylo- posterior probabilities and bootstrap values. Clade E comprises genetic analysis, including a denotation for the Thlaspideae (Thlaspi arvense the three species of the genus Aubrieta and the circum-Mediterra- and Alliaria petiolata ) and the Arabideae (excluding Pseudoturritis turrita and nean Arabis verna as sister to the genus. While this clade is both members of the Thlaspideae). For the fossil-based primary calibrations, basal to all other clades, it lacks strong statistical support. Clade the boundaries for the age of the most recent common ancestor (t mrca) of the D consists of Draba and its segregates and is sister to clade L, taxon set “ Thlaspideae ” were set between 29.2 and 30.8 Myr. For the secondary which contains four Arabis species from the Caucasus moun- calibration point, the boundaries for the t mrca of the taxon set “ Arabideae ” were set between 10.0 and 23.9 Myr. Additionally, for the calculations with fi xed tains (A. brachycarpa , A . christiani , A . graellsiiformis, and A . mutational rates these rates were fi xed within the range of 1.72 × 10 − 9 to 1.71 nordmanniana ). This clade (hereafter called A . nordmanniana × 10 − 8 (ITS) and 3.6 × 10 − 9 and 7.7 × 10 − 9 ( trnLF), respectively. These values group) shows strong congruence with the former section Allari- refl ect the published ranges for mutation rates in herbaceous plants ( Kropf, aopsis, as described by Busch (1906) and Schulz (1936). Clade

→ Fig. 2. Phylogenetic tree from the Bayesian analysis based on the cpDNA trnLF data set. Posterior probability values (fi rst-mentioned) and bootstrap values of the maximum-likelihood analysis (middle-mentioned) and the maximum-parsimony analysis (last-mentioned) of the nodes are given along the corresponding branches. Pseudoturritis turrita was used as the outgroup taxa. Accessions with a prefi xed asterisk had the same sequence than the top-listed accession and were therefore excluded from the tree calculation. Purple- or pink-fl owered species are indicated with a black spot following the taxon label; species that occasionally produce pink petals are marked with a gray spot. Accessions without any spot correspond to primarily white-fl owered species. Annual/winter-annual taxa are labeled with the corresponding sign for annualism, also following the taxon label. April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 783 784 AMERICAN JOURNAL OF BOTANY [Vol. 99

M is formed by the three representatives of the core Arabis , a and perennial as shown in the ITS analyses but rather formed a group that has been shown to contain the majority of Arabis single polytomy. The predominantly east African “ red-coded ” species (see Koch et al., 2010 ). Clade P comprises the Ibero- haplotype group (Fig. 3) is represented by accessions A22JO Moroccan Arabis taxa, A . parvula, and its east-Mediterranean and A40JO2 of A . alpina , to which one accession of the Ana- sister species A . aucheri . Clades D, L, M, and P form a weakly tolian endemic A . aubrietioides (A01M) and the Syrian ac- supported monophyletic group, as indicated by posterior prob- cession of A . ionocalyx (RK254) are basal. The mainly ability or bootstrap values. Arabis auriculata and A . nova subsp. western Asian “ green-coded ” haplotype group ( Fig. 3 ) is rep- nova are sister to each other in clade B. The accession of A . alpina resented by accessions A01JO2 and AW-30 of A. alpina and and all its morophologically similar species are confi ned to by three accessions of A . aubrietioides (MG003, RK244, clade A, which is further divided into subclades of annual and RK261) and one accession of each A . defl exa (RK249) and A . perennial taxa, respectively. The annual subclade consists of ionocalyx (RK253). Additionally, the Central Asian A . tians- one accession of the Spanish A . nova subsp. iberica (MA022), chanica (RK007) appears in this group. Several southern one accession of the endangered Cypriote endemic A . kenne- Anatolian accessions of A . defl exa (RK088, RK089, RK250, dyae (RK215), and four accessions of A . montbretiana from and RK251) and A . ionocalyx (RK117, RK118, RK252, and Western and Central Asia (A32MO, A59M, RK043, and RK121). RK255) plus the westernmost accessions of A . aubrietioides Two of these accessions (A32MO and A59M) were originally (RK240) are combined together. A group containing all wrongly determined as A . auriculata and A . nova , respectively. accession of A . cypria is amended by two accessions of The remaining accessions of A . auriculata (western Europe to A . aubrietioides (RK241, RK243), both being sampled on central Asia) and A . nova subsp. nova form a discrete clade the Turkish mainland opposite of the Kyrenia mountain (clade B) not related to other annuals within clade A. Finally, range. Finally, species-specifi c subgroups are formed by the the perennial subclade of clade A comprises the various A . alpina Cypriote endemic A . purpurea and by the annual A . mont- accessions and six other perennial species: Arabis aubrietioides bretiana . The accessions of A . nova subsp. iberica (MA022) (A01M, MG003, RK240, RK241, RK243, RK244, RK261), A . and A. kennedyae (RK215) are not directly connected to defl exa (RK088/89, RK248– 251), A. ionocalyx (RK117/18, A . montbretiana . RK252– 255), A. tianschanica (RK007), A. cypria (RK086/ 87, RK167, RK245/46), and A. purpurea (RK004, RK006, Combined data set — The alignment of the combined data set A53M, RK247, RK257 – 260). Arabis cypria and A . purpurea comprised 66 sequences (representing 70 accessions) and 1390 are characterized by discrete ITS sequences with the exception characters, of which 1066 were constant. Of the 324 variable of one accession of A . cypria (RK246), which shared its ITS- characters, 212 were phylogenetically informative and the re- type with A . purpurea . Most accessions of the remaining taxa maining 112 were autapomorphic positions (Appendix S3). The ( Arabis aubrietioides , A . defl exa , A . ionocalyx, and A . tians- SYM+ Γ evolutionary model was applied to the nrDNA parti- chanica) had an ITS sequence identical to the widespread ITS- tion, and the K81uf+ Γ evolutionary model was applied to the type A of A . alpina ( Koch et al., 2006 ). cpDNA partition in the respective ML and Bayesian analyses. The result of the Bayesian analysis is shown in Fig. 4. The re- Chloroplast trnLF region — The combined trnLF align- sulting trees had a harmonic mean for the ln likelihood of ment comprised 61 unique sequences (representing 72 acces- − 5422.36 and an average standard deviation of split frequencies sions) and 759 characters, 617 of which were constant. Of of 0.011. The corresponding tree of the ML analysis had a ln the 142 variable characters, 75 were phylogenetically infor- likelihood of − 5104.81. The maximum parsimony analysis re- mative, and the remaining 67 were autapomorphic positions sulted in 6134 most parsimonious trees with a tree length of 534 (Appendix S2). (with “ maximum length = 0” rule applied), CI of 0.68 (autapo- The K81uf+ Γ evolutionary model was applied to the ML and morphies excluded), and RI of 0.89. Bayesian analyses, the results of which are shown in Fig. 2. The The phylogenetic tree based on the combined data set ( Fig. 4 ) resulting Bayesian trees had a harmonic mean for the ln likeli- shows the same seven major clades as recovered by the ITS hood of − 2581.62 and an average standard deviation of split analysis (Fig. 1), refl ecting the high amount of parsimony infor- frequencies of 0.011. The corresponding tree of the ML analy- mative characters in the ITS data set. All of the clades were sis had a ln likelihood of − 2287.00. The maximum parsimony very strongly supported by their posterior probabilities (ppr = analysis resulted in 10 000 most parsimonious trees with a tree- 1.0) and bootstrap values (bootstrap > 98.5%). Clade δ ( Draba length of 195 (with “ maximum length = 0 ” rule applied) and an and its segregates) and clade ε ( Aubrieta and Arabis verna ) ensemble CI of 0.77 (autapomorphies excluded) and an ensem- remained unsupported. ble RI of 0.90. Similar to the ITS tree ( Fig. 1 ), the A . alpina containing The chloroplast trnLF Bayesian phylogenetic tree ( Fig. 2 ) clade (clade α ) is subdivided into statistically supported annual shows virtually the same seven major clades as the ITS tree and perennial subclades. The perennial subclade comprises (Fig. 1). Among these are the following: clade p ( A . aucheri and several subgroups recovered by both ITS and the trnLF analy- A . parvula ), clade m (core Arabis ), clade d (Draba and its seg- ses ( Figs. 1, 2 ). regates), and clade e (Aubrieta and Arabis verna ). The latter is sister to clade d and has high posterior probability support (ppr = Congruency test — The best tree topology was defi ned for 0.99), but only limited bootstrap support (maximum-likelihood each of the three data sets (ITS, trnLF , and combined) accord- bsML = 58.4, and maximum-parsimony, bsMP = 45.6). Arabis ing to the – lnL scores (ITS, trnLF , and 100 random topologies). auriculata (clade b1) and A . nova subsp. nova (clade b2) are not This was either the ITS topology (for the ITS and combined grouped together as in the ITS analyses, but form discrete data set) or the trnLF topology (for the trnLF data set). For all clades. Clade l (A . nordmanniana group) forms a well-supported other topologies including all random topologies, the associated clade sister to the clade comprising A . alpina and its closest p-value was used as a measure for the probability that this tree related species. This clade was not subdivided into the annual topology can also explain the data. All random tree topologies April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 785

Fig. 3. (A) Network of 73 unique cpDNA halpotypes, and B) regional distribution of haplotypes in the eastern Mediterranean. Shapes and colors cor- respond in both illustrations. The A . nordmanniana group (colorless circles) was used to root the network (see asterisk). Previously published A . alpina haplotypes are represented by fi lled circles; newly described haplotypes are represented either by triangles (for A . tianschanica , A . cypria , and A . purpurea ), empty circles (for A . aubrietioides , A . defl exa , and A . ionocalyx ) or gray diamonds (for the annual taxa A . montbretiana , A . nova subsp. Iberica , and A . kennedyae). (B) Some accessions are further specifi ed by an A (for A . aubrietioides), D (for A . defl exa) or I (for A . ionocalyx), if they belonged to one of these species. (C) shows the global distribution of Arabis alpina’ s three major cpDNA trnLF haplotype groups. The gray-shaded area in central Turkey contains members of all three haplotype groups. The locality sampled for A . tianschanica is indicated with a dark green triangle. 786 AMERICAN JOURNAL OF BOTANY [Vol. 99 had a p -value of 0.00, rejecting the hypothesis that any of to other haplotypes of A . alpina . One accession of A . defl exa them explain the data equally well. Conversely, the trnLF to- (RK248, haplotype 77) had a haplotype derived from central pology could explain the ITS (p = 0.22) and combined data blue haplotype 01, while two accessions of A . aubrietioides set (p = 0.50), and the ITS topology could explain the trnLF (RK241 and RK243) had haplotype 81, which is derived from data set (p = 0.30), as these values were above the threshold A . alpina haplotype 45 via two mutational steps. The annual of p = 0.05. species A . montbretiana (haplotypes 86, 87) and A . nova subsp. iberica (haplotypes 85 and 88) were also connected to haplo- Chloroplast DNA network — The modifi ed trnLF alignment type 01, whereas related A . nova subsp. iberica held an iso- for phylogeographic analysis resulted in 666 characters (660 lated position in the network ( Fig. 3 ). The three haplotypes regular bases, plus six positions used for gap-coding), from recovered from A . montbretiana were connected to haplotypes which 73 unique haplotypes were discriminated among the 518 20 and 03 of A . alpina, although an alternative mutational accessions of A. alpina and close relatives. The resulting mini- pathway linking to haplotype 41 was also proposed by the mum spanning network is shown in Fig. 3 . TCS program. Both of these pathways seem unrealistic based The network was almost identical to those previously pre- on the clear morphological and biogeographical differences sented based solely on accessions of A. alpina (e.g., Koch between the annual A . montbretiana and the perennial species et al., 2006; Assefa et al., 2007; Ansell et al., 2011), especially of the A . alpina group. Finally, the Cypriote A . kennedyae in recovering the three major cpDNA haplotype groups (blue, (haplotype 89) was connected with three mutational steps to red, green). The two haplotypes of the A . nordmanniana group A . montbretiana haplotype 86. (haplotypes 90, 91) were used to root the network; these types were connected through six mutational steps to central haplo- Divergence-time estimates — The results of the divergence- type 01 (blue). Four of the fi ve newly included A . alpina ac- time calculation are given in Table 1 (ITS) and Table 2 (trnLF ). cessions (MA001, MA004, RK001, and RK002) had the The results varied greatly among the three different ap- common blue haplotype (haplotype 01), and one new blue proaches, but two main conclusions could be drawn. (1) Esti- haplotype (haplotype 72) was identifi ed from the western Ana- mates calculated from the trnLF marker normally exceeded tolian accession RK001b. Two new haplotypes (83, 84) were those from the ITS marker by a factor of two. When the rates added to the base of the red haplotype group. Haplotype 84 of variation were fi xed, the estimates were congruent. Fixing from A . aubrietioides (accession A01M) formed a side branch these rates in a reasonable interval seems important, because to the red haplotypes 62 and 63 of A. alpina . The A . ionocalyx the calculations run without strict limits on the rates of varia- accession from Syria (RK254, haplotype 83) formed another tion showed nonconstant rates that dropped below all reported side branch within the red haplotype group and was closely values for this chloroplast marker (see Table 2). Therefore, the connected to the most basal haploytype 60 of A. alpina from corresponding divergence time values might be overestimated. northern Lebanon. Only one new haplotype was added to the (2) The calculated estimates using the Thlaspideae fossil as green haplotype group: the accession of A . tianschanica the primary calibration point according to Beilstein et al. (2010) (RK007, haplotype 82) from Kyrgyzstan, which occupied a tip were more than three times higher than those calculated with position in the green haplogroup (Fig. 3). In addition, several the secondary calibration point published by Couvreur et al. of the remaining relatives had green haplotypes that were (2009) . Although confi dence intervals are large, the overlap identical to those recovered from A. alpina : A . aubrietioides between fossil based calibration and secondary calibration is (MG003) had haplotype 04, A . defl exa (RK249) had haplotype small. Notably, the log likelihood values remain relatively 47, while accessions of A . aubrietioides (RK244, RK261) and constant for all dating approaches per marker, and therefore A . ionocalyx (RK253) held haplotype 51. All remaining acces- the calculated Bayes factors do not favor one approach over sions of A. alpina relatives were affi liated with the blue haplo- another. type group of Europe. The Cypriote species A . purpurea (haplotype 73) and A . cypria (haplotypes 75 and 76) along Chromosome numbers and ploidy levels — Known chromo- with one accession of A . purpurea (described as A . cypria some numbers and ploidy levels are summarized in Table 3 . [haplotype 74] formed a distinct “ purpurea haplotype group ” , Here we present counts for A . purpurea (accessions RK004, restricted to the Kyrenia mountains in Cyprus ( Fig. 3 ). Several RK006) and A . cypria (accession RK167) for the fi rst time; accessions of the A . alpina relatives from the Taurus Moun- both are 2n = 16. A hybrid plant of unknown origin (not in- tains shared haplotype 41, which is also common in accessions cluded in the analyses) between A . alpina and A . aubrietioides of A . alpina from this region (A . defl exa [RK088, RK250], A . was also counted as 2n = 16, indicating a hybridization event at ionocalyx [RK117, RK252, RK255] and A . aubrietioides the diploid level. According to the data available, there are no [RK240]). Other accessions of the relatives from the Taurus tetraploid or higher polyploid counts for any species closely re- region also have haplotypes that are closely related to haplo- lated to A . alpina . Although there are some species (A . defl exa , type 41 ( A . defl exa [RK089/haplotype 78 and RK251/haplotype A . tianschanica) whose chromosome numbers have not yet 80] and A . ionocalyx [RK118/haplotype 79]). Other acces- been determined. Hence it seems likely the “ A . alpina clade ” sions of the A . alpina relatives were connected genologically consists exclusively of diploids.

→ Fig. 4. Phylogenetic tree of the Bayesian analysis based on the combined data set (ITS plus trnLF). Posterior probability values (fi rst-mentioned) and Bootstrap values of the maximum-likelihood analysis (middle-mentioned) and the maximum-parsimony analysis (last-mentioned) of the nodes are given along the corresponding branches. Pseudoturritis turrita was used as the outgroup taxa. Accessions with a prefi xed asterisk had the same sequence than the top-listed accession and were therefore excluded from the tree calculation. Purple- or pink-fl owered species are indicated with a black spot following the taxon label; species that occasionally produce pink petals are marked with a gray spot. Accessions without any spot correspond to primarily white-fl owered species. Annual/winter-annual taxa are labeled with the corresponding sign for annualism, also following the taxon label. April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 787 788 AMERICAN JOURNAL OF BOTANY [Vol. 99

T ABLE 2. Parameters and results of the three divergence-time estimate approaches based on the cpDNA trnLF marker. Mean values are indicated in boldface; lower and upper bounds of the 95% highest posterior density (HPD) interval are given above and below that value, respectively. Enforced bounds are indicated in italics. Bayes-Factors with positive evidence against M 0 (i.e., both trees explain the data equally well) are marked with an asterisk.

Fossil based calibration of node age of Using secondary calibration of node age of Like previous + fi xed mutational Parameter tribe Thlaspideae (acc. Beilstein et al., 2010 ) tribe Arabideae (acc. Couvreur et al., 2009 ) rates ( Mummenhoff et al., 2004 )

Runs / Generations 4 independent runs with 4 independent runs with 4 independent runs with 80 million generations each 20 million generations each 25 million generations each Log likelihood − 2604.064 − 2603.344 − 2602.146 − 2588.331 − 2588.106 − 2586.818 − 2574.108 − 2573.179 − 2471.985 t mrca of Arabis alpina agg. and annual 4.32 1.88 1.01 sister group: clade a ( Fig. 2 ) (Ma) 16.8 4.82 2.23 34.2 8.61 3.84 tmrca of core-Arabis (Ma) 3.99 1.65 0.53 19.3 5.50 2.42 40.0 10.2 4.75 tmrca of Draba and its segregates (Ma) 8.86 3.75 1.22 31.4 8.94 4.17 61.6 15.5 7.66 tmrca of tribe Arabideae (Ma) 16.8 10.0 mya 10.0 48.8 14.1 10.8 92.2 21.5 12.7 ucld.mean 0.15 × 10 −9 0.75 × 10 − 9 3.60 × 10 − 9 0.48 × 10 − 9 1.57 × 10 − 9 3.97 × 10 − 9 0.87 × 10 −9 2.44 × 10 − 9 4.77 × 10 − 9 meanRate 0.16 × 10 −9 0.84 × 10 − 9 2.41 × 10 − 9 0.49 × 10 − 9 1.63 × 10 − 9 3.15 × 10 − 9 0.86 × 10 −9 2.43 × 10 − 9 3.91 × 10 − 9 Bayes factors — / 0.93 / 0.24 1.08 / — / 0.26 4.10* / 3.81* / —

DISCUSSION it appears that the cpDNA results are more biologically meaning- ful, as all four species are confi ned to the Caucasus Mountains, Congruency between nuclear and cpDNA derived phyloge- which are themselves near to the center of origin and diversity nies and divergence-time estimates — In this study, which fo- for A . alpina ( Ansell et al., 2011 ). cuses on Arabis alpina and its closest relatives, the combined The phylogenetic position of the genus Aubrieta (including tree and the derived phylogenetic hypothesis (Fig. 4) help to Arabis verna ) formed a second incongruency between the two resolve phylogentic relationships within the tribe Arabideae markers. For the trnLF -based analyses, Aubrieta was placed and identify new divergent lineages ( Figs. 4, 5). We show that close to Draba ( Fig. 2 ). This relationship was not recognized by Arabis verna is more closely related to the genus Aubrieta than ITS based analyses ( Fig. 1 ). The low resolution of the ITS-based to genus Arabis (Figs. 2, 4) and that it forms a monophyletic analysis has previously been shown with two large-scale data clade (clade ε in Fig. 4), despite the different markers creating sets focusing on Draba and core-Arabis (Jordon-Thaden et al., uncertainty in the phylogenetically position of Aubrieta (e.g., 2010 ; Koch et al., 2010 ). Extensive hybridization and reticulate sister to Arabis alpina : Koch et al., 1999a , 2001 ; sister to core evolution within these groups has resulted in chloroplast cap- Arabis : Koch et al., 2000 ; sister to all Arabideae s.s.: German ture and incomplete lineage sorting of both nuclear and plastid et al., 2009 ). Although the phylogenetic relationship of A. verna markers. So far we have found no evidence for these complexi- and Aubrieta is based on limited species sampling for Aubrieta ties in the Aubrieta - Arabis verna and Arabis brachycarpa - A . ( n = 3), we assume that this pattern is signifi cant. Future work nordmanniana - A . christiani - A . graellsiiformis clades. Instead, will test whether Arabis verna is nested within the genus Aubrieta , it seems more likely that the mode of marker molecular evolu- which consists of ca. 15 species or whether it is a sister taxon to tion has contributed to the phylogenetic incongruencies; con- the genus Aubrieta . certed evolution for nuclear ITS regions (Koch et al., 2003) and The congruency test revealed no major incongruencies between the low mutation rates for plastidic genomes ( trnLF sequences). both DNA markers. However, several minor differences exist Consequently, experimental work adding single-copy nuclear between the nrDNA ITS (Fig. 1) and the cpDNA trnLF ( Fig. 2 ) genes is needed to further resolve these phylogenetic relation- sequence analyses, and these should be mentioned. On the basis ships at the tribal level (e.g., chs , adh ; M. A. Koch and R. Karl, of the cpDNA analysis, four Arabis species ( A. brachycarpa , A . unpublished manuscript). nordmanniana , A . graellsiiformis , and A . christiani ) are sister Our various estimates of divergence times varied greatly de- to A. alpina and its close relatives ( Fig. 2 ). However, this relation- pending on the approaches applied, making it diffi cult to deter- ship was not supported by the ITS analyses, which placed all mine the most reliable methodology. The primary fossil-based four species close to Draba . This difference probably refl ects calibrations following the approach of Beilstein et al. (2010) the variation in the mode of marker inheritance with the plastidic seem to have resulted in overly high divergence time values. trnLF being maternally inherited ( Harris and Ingram, 1991 ) and These estimates (ITS: 7.1 Ma with a 95% CI from 2.8– 12.3, the nuclear-encoded ITS being biparentally inherited. Overall, trnLF : 16.8 Ma with a 95% CI from 4.3 – 34.2) would scale most April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 789

T ABLE 3. Compilation of published chromosome numbers and ploidy The closest relatives of Arabis alpina: Parallel evolution of dis- levels of the taxa included in the study. tinct morphological characters — This study identifi ed Arabis alpina as a paraphyletic taxon, because six Arabis species (A. Taxon 2 n Ploidy level Reference aubrietoides, A . cypria, A. defl exa, A. ionocalyx, A. purpurea, and Arabis alpina 16 2 x # A. tianschanica , Figs. 2 – 5 ) are nested within the widely distributed Arabis aubrietioides 16 2 x this study A . alpina . All six species are perennials with small distribution Arabis aucheri 16 2 x # areas: Kyrgyzstan in the case of A . tianschanica and the eastern Arabis auriculata 16 2 x # Mediterranean for the remaining fi ve species. The eastern Mediter- Arabis blepharophylla 16 2 x # Arabis brachycarpa 16 2 x # ranean, more precisely Anatolia and the adjacent Levantine region, Arabis christiani 32 4 x # has been shown to be the center of origin and genetic diversity of Arabis cypria 16 (14*) 2 x Hand (2006) ; this study A. alpina (Ansell et al., 2011). As mentioned, we assume Pleisto- Arabis defl exa n.d. n.d. cene radiation events within A . alpina . The mountainous areas of Arabis erikii n.d. n.d. Anatolia in comparison to the European Alps and Fennoscandia, Arabis graellsiiformis n.d. n.d. are very diverse in terms of altitude range and geology and were Arabis hirsuta 32 4 x # Arabis ionocalyx 16 2 x # never covered by a continuous ice-shield during the Pleistocene Arabis kennedyae n.d. n.d. glaciation periods. Only the higher mountain peaks were glaciated Arabis montbretiana 16 2 x Hoffmann et al. (2010) (Erin ç , 1978; Atalay, 1996), and the vegetation belts shifted by up Arabis nordmanniana 16 2 x # to 500 m of elevation (Parolly et al., 2010). The existence of several Arabis nova subsp. iberica n.d. n.d. small-scale refugia in this area (M é dail and Diadema, 2009; Arabis nova subsp. nova 16 2 x # Parolly et al., 2010 ) allowed for the survival of various biota during Arabis parvula 32 4 x # Arabis purpurea 16 2 x #; this study the periods of temperature fl uctations. In the warmer periods, a dry Arabis sachokiana 16 2 x # steppe-like climate prevailed in the area (Webb and Bartlein, 1992), Arabis tianschanica n.d. n.d. and higher elevations would have provided suitable habitats for Arabis verna 16/32 2 x /4 x # temperate biota due to higher precipitations and more moderate Aubrieta canescens subsp. n.d. n.d. temperatures. For cold-tolerant biota, these microrefugia would be macrostyla further limited to the high alpine habitats of each mountain system, Aubrieta deltoidea 16 2 x # Arabis parvifl ora 16 2 x # thereby creating gene fl ow limitations and promoting local lin- Draba hederifolia 30 4 x # eages accumulation. Ansell et al. (2011) emphasized the roles of Draba hispanica 16 4 x # local survival centers in the coastal Taurus Mountains, which pro- Draba reptans 30/32 4 x # vided moister conditions compared to the drier interior climates of the Anatolian (Davis, 1971; Ekim and Gü ner, 1986). Accord- # Warwick and Al-Shehbaz (2006) ; * Yildiz and G ü cel (2006) ; n.d.: no data available ingly, the Turkish Arabis species with close relationships to A . alpina occur mainly within the distinct mountain ranges of the Taurus systems: the red-fl owering A . aubrietioides in the Central Taurus (Cilician sector) and Arabis defl exa and A. ionocalyx in the of the radiation of A . alpina to the late Miocene/Pliocene, a western parts (Lycian and Pisidian-Isaurian sectors) ( Davis, 1965 ). period with relatively stable, predominantly warm and moist A very similar spatial distribution of taxa from Heldreichia Boiss. climate ( Dowsett et al., 2005 ; Salzmann et al., 2009 ), which is has been recently reported along the Taurus systems ( Parolly et al., not in agreement with the biology of the various species. Fur- 2010 ). Although there are clear morphological characters that thermore, the uplift of mountain chains during the alpide orog- differentiate between the Taurus Arabis and A . alpina , so far it is eny was still in progress at that time ( Atalay, 2002 ). The Taurus not possible to discriminate between them using the molecular and the adjacent East Anatolian Plateau are considered to have markers employed by our investigation. Rather, A . aubrietioides , arisen in the late Miocene at the earliest or more likely origi- A. defl exa , and A . ionocalyx often bear local A . alpina haplotypes, nating in the late Pliocene (G ü ldali, 1979; Franzke et al., or closely related ones (see Fig. 3A, B ). Thus, for the mainland 2011 ). Consequently, the most likely divergence time esti- species of A . aubrietioides, A. defl exa, and A. ionocalyx, the mates might be close to the lower boundaries of the primary geographical footprint of A . alpina genotypes is stronger than fossil-based calibration method or the upper boundaries of the the morphological differentiation. secondary calibration method (Couvreur et al., 2009). Under Similar geographical separation of species fl ock members can these approaches, the split between Arabis alpina and its an- be seen on Cyprus, where endemites A . purpurea and A . cypria nual sister group would be dated between 2.84 – 3.98 (for ITS are restricted to the Troodos and Kyrenia mountain ranges, re- data) or between 3.84 – 4.32 ( – 8.6 Ma) (for trnLF data), respec- spectively. In contrast to the mainland A. alpina relatives, these tively. We recognize that the Bayes factors are insuffi ciently island relatives are genetically distinct from A. alpina in bear- high to support or reject either of the approaches. However, ing private chloroplast and ITS haplotypes. Together, these ob- the divergence times calculated under the secondary calibra- servations suggest that A . purpurea and A . cypria evolved tion approach correspond well to a previous cpDNA-based independently in disjunct mountain systems of Cyprus in the coalescence estimate of 0.80 – 6.43 Ma (mean of 2.69 Ma) for local absence of A. alpina populations. Hadjisterkotis and Bider the most distantly related Arabis alpina cpDNA haplotypes (1997) observed that many Cypriote species are closely related (Ansell et al., 2011). Furthermore, summary data from four to mainland taxa, despite Cyprus never being directly connected DNA markers indicates a split between the African and Eur- to the mainland during its geological history (Robertson, 1990; asian Arabis alpina of 0.28 – 1.39 Ma (mean of 0.70 Ma) ( Koch Hadjisterkotis, 2007 ). Hence, the modest distance to southern et al., 2006). These fi ndings place the subsequent radiation Anatolia (ca. 68 km) has probably allowed for rare long-distance events within the Arabis alpina clade into the Late Pliocene/ dispersal, but represents a suffi cient barrier to have promoted early Pleistocene. allopatric speciation. 790 AMERICAN JOURNAL OF BOTANY [Vol. 99

Within the red-fl owering species, there is some interesting the habitat fragmentation during Pleistocene glaciations, and vicariance of A . purpurea on Cyprus and A . aubrietoides on the possibly reinforced by breeding system shift. Turkish mainland. We speculate that the red fl owers may be In contrast to most members of the Arabis alpina species indicative of outbreeding. Because the phylogenetic trees do fl ock, A. tianschanica is located in Uzbekistan and western not support a shared origin of the Cypriote and Turkish main- Kyrgyzstan, several hundred kilometers east of the nearest lo- land populations within the predominantly white-fl owering calities for A. alpina in northeast Iran. German et al. (2009) Arabis alpina , this phenological change may have occurred in have already suggested a close relationship between this en- parallel. This observation is supported also by the diploid chro- demic and A. alpina based on morphological characters. Geneti- mosome numbers of these taxa ( Table 3 ), indicating an absence cally, A. tianschanica has a private haplotype that is derived of hybridization or polyploidy, in contrast to the other major from the common green haplotype 04, which suggests this taxa clades in the Arabideae for which various ploidy level series might have evolved from a remnant population of a former indicate extensive hybridization and reticulation (Jordon- Thaden more easternly distributed A. alpina . and Koch, 2008; Koch et al., 2010). Further ecological and molecular research may unravel new and interesting aspects of Southern refuges as centers of origin and evolutionary parallel evolution among sister-species pairs that have been starting points for different life-history strategies — The occur- derived from white-flowering Arabis alpina . More widely rence of species-poor annual clades being sister to species-rich red-fl owering taxa within the genus Arabis are typically not perennial clades is a common feature within the tribe Arabideae. sympatrically distributed with other red-fl owering species: e.g., This trend is also found in many other tribes and genera of the in Europe the red-fl owering endemic of Arabis stenocarpa Brassicaceae (e.g.: Arabidopsis /tribe Sisymbrieae [ Koch and Boiss. & Reut. from Spain, Arabis cretica Boiss. & Heldr. from Matschinger 2007] ; Noccaea , Microthlaspi/tribe Coluteocarpeae Crete ( Koch et al., 2010 ), and Arabis rosea DC. from southern [ Mummenhoff et al., 1997a , b ; Koch et al., 1998 ]; Cochlearia , Italy. Furthermore, all European red-fl owering Arabis species Ionopsidium /tribe Cochlearieae, [ Koch et al., 1999b ]). with the exception of Arabis alpina have failed to developed Notably, this is pattern is also found in Draba (Arabideae), red-fl owering sister species pairs (M. A. Koch and R. Karl, where we found that the ca. 20 annual species are restricted to a unpublished data). These multiple changes in fl ower color single clade that is sister to the clade comprising the remaining might be indicative of genetic/geographic isolation caused by 300 perennial taxa, and which underwent a dramatic Pleistocene

Fig. 5. Diagram of systematic relationships within the tribe Arabideae based on data summarized from various sources ( German et al., 2009 ; German and Al-Shehbaz, 2008 ; Koch et al., 1999a , 2000 , 2001 , 2007 , 2010 ; Jordon-Thaden et al., 2010 ; Warwick et al., 2010 ; Al-Shehbaz et al., 2011 ) and comple- mented with the data presented in this study. l.s. = low signifi cance. April 2012] KARL ET AL. — EVOLUTIONARY HISTORY OF ARABIS ALPINA 791 radiation ( Jordon-Thaden and Koch, 2008; Jordon-Thaden et al., mountains as the cradle of global diversity in Arabis alpina, a key 2010 ). This pattern is also true for the Arabis alpina species arctic-alpine species. Annals of Botany 108 : 241 – 252 . group, although less pronounced, whereby the seven perennial A SSEFA , A . , D . E HRICH , P . T ABERLET , S . N EMOMISSA , AND C . B ROCHMANN . species are sistered by three annual taxa: Arabis montbretiana , 2007 . Pleistocene colonization of afro-alpine ‘ sky islands ’ by the A . nova subsp. Iberica , and A . kennedyae ( Fig. 4 ). The fi ve re- arctic-alpine Arabis alpina. Heredity 99 : 133 – 142 . maining annual species included in this study (Arabis aucheri , A TALAY , I. 1996 . Palaeosoils as indicators of the climatic changes during Quaternary period in S. Anatolia. Journal of Arid Environments 32 : Arabis auriculata , Arabis erikii , Arabis nova subsp. nova , Ara- 23 – 35 . bis parvula ) show no close relationships to any other perennial A TALAY , I. 2002 . Mountain ecosystems of Turkey. In M. F. Buchroithner [ed.], Arabis species but form two distinct clades (Fig. 4). Future phy- Kartographische Bausteine, vol. 28, 29 – 38. Institute for Cartography, logenetic research may help clarify their systematic and life his- Dresden University of Technology, Dresden, Germany. tory relationships. B EILSTEIN , M. A. , N. S. NAGALINGUM , M. D. CLEMENTS , S. R. MANCHESTER , The evolutionary success of the genus Arabis as a whole may AND S . M ATHEWS . 2010 . Dated molecular phylogenies indicate a be attributed to a combination of adaptive characters: (1) High- Miocene origin for Arabidopsis thaliana. Proceedings of the National elevation plants that are outcrossing, long-lived perennials and Academy of Sciences, USA 107 : 18724 – 18728 . produce large fl owers to attract insects and winged seeds suit- B IRKS , H. H. 2008 . The late-quaternary history of arctic and alpine plants. able for long-distance dispersal. (2) Plants, mostly from mon- Plant Ecology & Diversity 1 : 135 – 146 . tane regions, produce medium-sized fl owers and narrowly B USCH , N. A. 1906 . Sistematika i botaniceskaja geografi ja kavkazskich winged seeds and tend to shift from perennial to biennial growth vidov roda Arabis L. osobjenno sekzii Alliariopsis m. Moniteur du Jardin Botanique de Tifl is 6 : 3 – 23 . forms. (3) Low-elevation plants are primarily selfi ng annuals C LEMENT , M . , D . P OSADA , AND K. A. C RANDALL. 2000 . tcs: A computer program with small fl owers and small seeds ( Koch et al., 2010 ). Conse- to estimate gene genealogies. Molecular Ecology 9 : 1657 – 1659 . quently, we fi nd annuals primarily in lowland regions and with C OMES , H. P. , AND J. W. KADEREIT . 1998 . The effect of Quaternary cli- more southern distribution areas than the present-day distribu- matic changes on plant distribution and evolution. Trends in Plant tion areas of the perennials. The generally higher number of Science 3 : 432 – 438 . species in the perennial groups refl ect their alpine and montane C OMES , H . P . , AND J . W . K ADEREIT . 2003 . Spatial and temporal patterns in habitats, creating opportunities for lineage diversifi cation during the evolution of the fl ora of the European alpine system. Taxon 52 : the Pleistocene environmental fl uctuations. These features have 451 – 462 . recently been shown to have accelerated speciation and poly- C OUVREUR , T. L. P. , A. FRANZKE , A . I . 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A PPENDIX 1. Species sampled in this study, GenBank accession numbers for ITS, trnL , trnL - F sequences, and voucher information. Herbarium abbreviations follow Holmgren et al. (1990) and are defi ned in the chapter “ Plant material ” in the “ Materials and methods ” section.

Taxon , [internal code; GenBank accessions: ITS; trnL ; trnL - F ; Herbarium; Voucher number if provided or collector details; origin; ploidy level if determination was possible].

Arabis alpina L., [MA001; GU182038; GU181905; GU181973; MA; 321468; [JR048; FJ187938; FJ188235; FJ188084; HEID; 400762; Croatia]; Spain], [MA004; HQ200931; GU181906; GU181974; MA; 224236; Arabis ionocalyx Boiss., [RK117; HQ200936; HQ200950; HQ200943; Canary Islands], [RK001; GU182066; GU181934; GU181999; HEID; B; 10 0201502; Turkey], [RK118; HQ200937; HQ200951; HQ200944; 809695; Slovakia], [RK001b; GU182067; GU181935; GU182000; HEID; B; 10 0201504; Turkey], [RK252; JN189752; JN189731; JN189772; E; 502914; Turkey], [RK002; GU182068; GU181936; GU182001; NSK; 00381016; Turkey], [RK253; -; JN189732; JN189773; E; 00381020; A. Telpukhovskaya, 1964, s.n.; Siberia/Russia], [A01JO2; DQ060109; Turkey], [RK254; JN189753; JN189733; JN189774; E; 00381021; DQ060115 (trn LF); BM; T.R.I. Woods, 09.05.1975, no. Y/75/176; Syria], [RK255; JN189754; JN189734; JN189775; E; 00381025; Turkey]; Yemen], [A19JO2; DQ060102; DQ060113 (trn LF); BM; J.R. Press & M.J. Arabis kennedyae Meikle , [RK215; HQ646643; HQ646764; HQ646703; Short, 29.03.1984, no. 398; Madeira], [A22JO; DQ060101; DQ060116 HEID; 809841; M. Andreou & C. Constantinou, 28.04.2010, AR-23-4; (trn LF); BM; A. Hemp, s.n.; Tanzania], [A36JO2; DQ060100; DQ060112 Cyprus]; Arabis montbretiana Boiss. , [A32MO; GU182046; GU181912; (trn LF); BM; N.K.B. Robson, 07.04.1973, no. 3004; Turkey], [A40JO2; GU181980; MO; 1262438; Pakistan], [A59M; FJ187865; FJ188162; DQ060105; DQ060128 (trn LF); BM; A. Hemp, no. 3676; Ethiopia], FJ188011; M; Sh. Zarre, 06.04.1999, no. 258; Iran], [RK043; HM046193; [AW-30; DQ060101; DQ060117 (trn LF); WU; K.H. Rechinger, 28.07. - HM046245; HM046218; HUB; B. Mutlu, 30.05.2002, no. BM-7968; 01.08.1957, no. 1960-3968; Iraq]; Arabis aubrietioides Boiss. , [A01M; Turkey], [RK121; HQ200938; HQ200952; HQ200945; B; 10 0201515; FJ187845; FJ188142; FJ187991; M; ex. cult., 1976; Turkey], [MG003; Iran]; Arabis nordmanniana Rupr., [A54MO; FJ187924; FJ188221; HQ646647; HQ646768; HQ646707; C; Th. Kotschy, 25.06.1862, no. FJ188070; MO; 05060970; Georgia]; Arabis nova subsp. iberica Rivas 88; Turkey], [RK240; -; JN189720; JN189761; E; 00381050; Turkey], Mart. ex Talavera , [MA022; GU182039; GU181907; GU181975; MA; [RK241; JN189742; JN189721; JN189762; E; 00381053; Turkey], 732814; Spain]; Arabis nova subsp. nova Vill. , [A34M; GU182063; [RK243; JN189743; JN189722; JN189763; E; 00381056; Turkey], GU181929; GU181995; M; Doppelbaur, 06.08.1963, no. 11160; Italy], [RK244; JN189744; JN189723; JN189764; E; 00381057; Turkey], [MA024; GU182041; GU181909; GU181977; MA; 48509; France]; [RK261; JN189759; JN189739; JN189780; MA; 765874; Turkey]; Arabis parvula Dufour , [A38M; FJ407224; GU181931; FJ407244; M; Arabis aucheri Boiss. , [A06MO2; FJ187881; FJ188178; FJ188027; D. Podlech, 11.04.1993, no. 51465; Morocco]; Arabis purpurea Sm. , MO; 2363411; Syria]; Arabis auriculata Lam. , [A56M; FJ187863; [A53M; FJ187861; FJ188158; FJ188007; M; W. Lang, 04.04.1999, s.n.; FJ188160; FJ188009; M; F.A. Tscherning, 09.06.1895, s.n.; Austria], Cyprus], [RK004; GU182070; GU181938; GU182003; HEID; 809699; [A58M; FJ187864; FJ188161; FJ188010; M; D. Podlech, 28.04.1987, no. Cyprus; diploid], [RK006; GU182072; GU181940; GU182005; HEID; 43319; Morocco], [RK027; GU182091; GU181959; GU182024; HEID; 809703; Cyprus; diploid], [RK247; JN189747; JN189726; JN189767; E; 809774; Uzbekistan]; Arabis blepharophylla Hook. & Arn. , [Arab1795; FJ187986; FJ188287; FJ188136; GH; R.C. Rollins, 30.04.1942, no. 00381038; Cyprus; diploid], [RK257; JN189755; JN189735; JN189776; 3006; California/USA]; Arabis brachycarpa Rupr. , [A35MO; FJ187913; E; 00124777; Cyprus], [RK258; JN189756; JN189736; JN189777; E; FJ188210; FJ188059; MO; 04958380; Armenia]; Arabis christani 00381041; Cyprus], [RK259; JN189757; JN189737; JN189778; HEID; N. Busch , [JR099; GU182044; FJ188271; FJ188120; HEID; 502919; 00381043; Cyprus], [RK260; JN189758; JN189738; JN189779; HEID; Armenia]; Arabis cypria Holmboe, [RK086; HQ200932; HQ200946; 00381048; Cyprus]; Arabis sachokiana N. Busch , [A69MO; FJ187929; HQ200939; B; 10 0206185; Cyprus], [RK087; HQ200933; HQ200947; FJ188226; FJ188075; MO; 05060963; Georgia]; Arabis tianschanica HQ200940; B; 10 0206362; Cyprus], [RK167; JN189741; JN189719; Pavl. , [RK007; GU182073; GU181941; GU182006; OSBU; 16518; JN189760; HEID; 809784; Cyprus; diploid], [RK245; JN189745; Kyrgyzstan]; Arabis verna (L.) R. Br., [A20MO2; FJ187890; FJ188187; JN189724; JN189765; E; 00381031; Cyprus], [RK246; JN189746; FJ188036; MO; 05040147; Greece]; Aubrieta canescens subsp. JN189725; JN189766; E; 00381036; Cyprus]; Arabis defl exa Boiss. , macrostyla Cullen & Hub. , [RK214; HQ646642; HQ646763; HQ646702; [RK088; HQ200934; HQ200948; HQ200941; B; 10 0212852; Turkey], HEID; ex cult., 2010; Turkey]; Aubrieta deltoidea (L.) DC. , [AJ232909; [RK089; HQ200935; HQ200949; HQ200942; B; 10 0212853; Turkey], DQ180257; DQ180303; sequences from GenBank]; Aubrieta parvifl ora [RK248; JN189748; JN189727; JN189768; E; 00381005; Turkey], Boiss. , [DQ357518; — ; — ; sequence from GenBank]; Draba hederifolia [RK249; JN189749; JN189728; JN189769; E; 00381008; Turkey], (Coss.) Hyam & Jury, [L241; DQ467438; DQ467072 (trn LF); BM; [RK250; JN189750; JN189729; JN189770; E; 00381009; Turkey], Ait-Lafkikh et al., 01.10.1991, no. 4920; Morocco]; Draba hispanica [RK251; JN189751; JN189730; JN189771; E; 00381014; Turkey]; Boiss. , [B76; DQ467289; DQ466988 (trn LF); B; 10 0127430; Spain]; Arabis erikii Mutlu, [RK042; HM046192; HM046244; HM046217; Draba reptans (Lam.) Fernald , [72; AF146495; AF146942; AF146979; HUB; B. Mutlu, 10.06.1999, BM-4900; Turkey]; Arabis graellsiiformis Muehlenbach, 20.04.1971, no. 3498; Missouri/USA]; Pseudoturritis Hedge , [MG056; HQ646648; HQ646769; HQ646708; C; P.H. Davis & turrita (L.) Al-Shehbaz , [A52JO; GU592179; GU592180; GU592181; O.V. Polunin, 08.07.1954, no. 22574; Turkey]; Arabis hirsuta (L.) Scop. , BM; J. Vogel, s.n.; Italy].