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

SYSTEMATICS AND EVOLUTION OF ARCTIC-ALPINE ARABIS

ALPINA (BRASSICACEAE) AND ITS CLOSEST RELATIVES IN
THE EASTERN MEDITERRANEAN1

ROBERT KARL2, CHRISTIANE KIEFER3, STEPHAN W. ANSELL4, AND MARCUS A. KOCH2,5

2Department
of Biodiversity and Plant Systematics, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University
69120 Heidelberg, Germany; 3Max-Planck-Institute for Plant Breeding 50829 Cologne, Germany; and 4Natural 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, identified 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 fluctuations produced local altitudinal range-shifts among mountain glacial survival areas,
resulting not only in the accumulation of intraspecific genotype diversity but also in the formation of five 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 fluctuations during the colonization of the lowland Mediterranean landscape. These findings highlight that
Anatolia is not only a center of species richness but also a center for life-history diversification.

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

In the last 15 years, phylogeographic studies have character-
ized the evolutionary history of numerous Eurasian species
(Taberlet et al., 1998; Hewitt, 1999, 2000; Willis and Whittaker,
2000). Many of these studies have focused on time scales span-
ning the late Tertiary and Pleistocene (Comes and Kadereit,
1998, 2003; Hewitt, 2004; Birks, 2008) and provided new in-
sights into the biogeography of various taxa during past climate
change. Organisms were forced either to escape, migrate, and
eventually recolonize or to stay, adapt, and survive the glacia-
tions (Petit et al., 2003; Huck et al., 2009; Schmitt, 2009).
Whereas most studies have used a single species approach or a
comparative analysis of taxonomically unrelated organisms
(Taberlet et al., 1998, Hewitt 2004), comparative phylogeogra-
phy focusing on closely related taxa should be informative for
addressing evolutionary questions, especially concerning trait
and adaptive variation (Hickerson et al., 2010).
In this paper, we focus on the arctic-alpine plant Arabis al-
pina L. and its close annual and perennial relatives (see below).
Arabis alpina, the alpine rock cress (Brassicaceae), is widely
1 Manuscript received 14 September 2011; revision accepted 8 February 2012.
In addition to the various collections mentioned, we especially
acknowledge receipt of plant material from D. German (South-Siberian
Botanical Garden, Barnaul, Russia), M. Hoffmann (IPK Gatersleben,
Germany), B. Mutlu (Inönü University, Malatya, Turkey), and C. Kadis
(Nature Conservation Unit, Frederick University, Cyprus). Funding was
provided by the DFG (Deutsche Forschungsgemeinschaft) to M.A.K.
(KO2302/1-1, KO2302/11-1 and KO2302/12-1).
5 Author for correspondence (e-mail: marcus.koch@cos.uni-heidelberg.de)
doi:10.3732/ajb.1100447
distributed throughout the alpine habitats of Europe and into the
arctic zones of Greenland and North America, the high moun-
tains of northern and eastern Africa, Anatolia, the Caucasus,
and the Near East (Meusel et al., 1965; Koch et al., 2006). This
broad distribution makes this species an ideal system to study
the effect of Pleistocene climatic fluctuations on speciation, ge-
netic differentiation, and adaptation over different geographic
scales, including those regions important for its long-term sur-
vival and speciation. For example, studies on various organ-
isms have highlighted the importance of southern Anatolia
(Taurus mountain systems) for Pleistocene survival and specia-
tion (e.g., Font et al., 2009; Parolly et al., 2010). Presently, Arabis
alpina is mostly treated as a well-defined species without sub-
species or other segregates, although often the segregrate A.
caucasica Willd. (A. alpina subsp. caucasica) has been recog-
nized. This is despite few clear discriminating morphological
characters (Plantholt, 1995) and a lack of genetic evidence to
consistently separate it from A. alpina (Plantholt, 1995; Koch
et al., 2006; Ansell et al., 2011).
Currently, the closest related sister taxa to A. alpina have not
yet been identified. Candidates for closest relatives to A. alpina
based on morphological characters include the Central Asian
endemic A. tianschanica Pavlov and the Cyprian endemics A.
cypria Holmboe and A. purpurea Sm. The latter species has
pink to purple petals, a character that is also shared by the
southern Anatolian endemic A. aubrietioides Boiss. Further
candidates for close relatives include A. deflexa Boiss., A. fari-
nacea Rupr., and A. ionocalyx Boiss. Understanding the evolu-
tionary and geographic relationships of this species group will
provide a framework for future studies focusing on different
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
Various evolutionary studies have recently focused on the
evolution of Arabis alpina in more detail, addressing global
phylogeographic issues (Koch et al., 2006; Ansell et al., 2011)
or population differentiation on various spatial scales (Assefa
et al., 2007; Ehrich et al., 2007; Ansell et al., 2008). These stud-
ies revealed that this species originated in a defined area in cen-
tral Anatolia (Ansell et al., 2011), from where major waves of
emigration have shaped the current distribution range (Koch
et al., 2006; Ansell et al., 2011): (1) migration to central and
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
African high mountains. These waves of immigration span a
time scale of divergence of around 2 million years (Koch et al.,
2006; Assefa et al., 2007; Ansell et al., 2008), which is consid-
ered to be the minimum age of Arabis alpina.
Several studies have started to harness the potential of A. alpina
to investigate trait variation and adaptation in mountain habi-
tats. This started 50 years ago with the pioneering work of
Hedberg (1962), who indicated that there are reproductive bar-
riers between the European and East African material of A. al-
pina. More recently, Ansell et al. (2008) suggested putative
changes in the breeding system coincided with historical differ-
ences in glacial survival. This is supported by mating analysis,
which has identified a sporophytic self-incompatibility (SI) sys-
tem in some Italian populations and the loss of SI function in
several Alps populations (Tedder et al., 2011). Elsewhere AFLP
analysis of Alps populations has identified a remarkable num-
ber of polymorphic loci that are correlated to ecological varia-
tion (Manel et al., 2010; Poncet et al., 2010). There is also
increasing knowledge on the molecular basis of life-history
variation in Arabis alpina. One such trait is the shift from an-
nualism to perennialism and the regulation of perennial flower-
ing by PEP1 (PERPETUAL FLOWERING 1) in Arabis alpina
(Wang et al., 2009), which may be considered a “key character
trait” in the adaptation processes of an arctic-alpine plant.
Our study is part of a larger project to unravel the evolution-
ary history and systematics of the whole tribe Arabideae, which
consists of more than 500 species. A series of studies has al-
ready provided evidence for (1) the paraphyly of numerous taxa
within the tribe Arabideae (including Arabis) and that (2) the
tribe can be split into two subclades (Koch et al., 1999a, 2000,
2001, 2007, 2010; German et al., 2009; Al-Shehbaz et al.,
2011). The Arabideae consist mostly of taxa from the two
closely related genera Draba with more than 390 species and
Arabis with more than 65 species. The fully unexplored genus
Aubrieta represents another 10–20 species (Koch et al., ongo-
ing research). Preliminary phylogenetic analyses have focused
on establishing the tribal and generic relationships (Jordon-
Thaden et al., 2010; Koch et al., 2010). Consequently, in this
study we have excluded many taxa previously shown to be un-
related or only distantly related to Arabis alpina.
Presently, it is unclear as to whether Arabis alpina represents
a single well-defined species or a species complex. However,
there are various species that are obviously morphologically
very similar to Arabis alpina and are often distributed sympatri-
cally. On the basis of morphology and distribution, we have
selected candidate taxa that might be members of an Arabis
alpina species complex and asked how the morphologically
similar species relate to the evolutionary framework of the A.
alpina lineages, especially in connection to those found in the east-
ern Mediterranean, Anatolia and Central Asia. In answering the
question as to whether A. alpina represents a single well-defined
779
species or a species complex, we will explore the hypothesis
that the related species evolved in parallel in these regions from
different A. alpina populations. Furthermore, we will determine
whether any of the eight Mediterranean annual Arabis species
are sister to the perennial A. alpina group and test the hypothe-
sis that the divergence of annuals and perennial taxa predates
the Pleistocene and that they followed different evolutionary
pathways in the Mediterranean in response to the Pleistocene
temperature oscillations.
MATERIALS AND METHODS
Plant material—For the phylogenetic analysis, we included one outgroup
accession and 72 accessions of Arabis alpina and relatives of the tribe Ara-
bideae. These selected accessions comprised 29 species that have close mor-
phological similarity to Arabis alpina (e.g., Arabis tianschanica). The data set
also included all annuals assigned to Arabis [Arabis aucheri Boiss., Arabis au-
riculata Lam., Arabis erikii Mutlu, Arabis kennedyae Meikle, Arabis mont-
bretiana Boiss., Arabis nova Vill., Arabis parvula Dufour, and Arabis verna
(L.) R. Br.]. Six individuals of A. alpina were chosen as representatives of the
three major chloroplast DNA (cpDNA) haplotype lineages, which were identi-
fied 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.,
2010) and the “core-Arabis” clade (see Koch et al., 2010). We selected Pseudo-
turritis turrita (L.) Al-Shehbaz as the outgroup because this taxon was previ-
ously identified as belonging to the sister tribe of the Arabideae: the Stevenieae
(German et al., 2009, Al-Shehbaz et al., 2011). Plant material was obtained
from the Botanical Garden and Botanical Museum Berlin (B), the Natural His-
tory Museum London (BM), University of Copenhagen (C), Royal Botanical
Garden Edinburgh (E), Botanical Garden and Herbarium Heidelberg (HEID),
Botanische Staatssammlung Munich (M), Real Jardin Botanico Madrid (MA),
the Missouri Botanical Garden (MO), IPK Gatersleben, Inönü University
Malatya in Turkey, the Frederick University Cyprus and the South-Siberian
Botanical Garden Barnaul in Russia.
For the biogeographic analyses, we added a compilation of additional cpDNA
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-
sions from 329 locations.
Detailed accession information, including GenBank accession numbers, are
provided in the Appendix 1.
DNA extraction, PCR amplification and sequencing—DNA extractions
were carried out using the NucleoSpin Plant II Kit (Macherey-Nagel, Düren,
Germany) or according to an isopropyl alcohol variant of the CTAB method
(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-
filter and washing step. The internal transcribed spacer of nuclear ribosomal
RNA including the 5.8S rRNA (ITS), chloroplast trnL intron (trnL) and trnL-
trnF intergenic spacer (trnL-F) were selected as markers and were amplified by
PCR. Reactions were performed in a final volume of 25 µL, using 10 µM of
each primer, a total of 2.0 mmol/L MgCl2 and 0.5 U of MangoTaq polymerase
(Bioline). The primers used for ITS amplification were the 18F primer (5′-GGA
AGG AGA AGT CGT AAC AAG G-3′) as modified by Mummenhoff et al.
(1997a) and the 25R primer (5′-TCC TCC GCT TAT TGA TAT GC-3′) de-
signed by White et al. (1990). The trnL intron was amplified using the universal
primers c and d of Taberlet et al. (1991) (c: 5′-CGA AAT CGG TAG ACG CTA
CG-3′, d: 5′-GGG GAT AGA GGG ACT TGA AC-3′), while the trnL-F inter-
genic spacer was amplified with primer e of Taberlet et al. (1991) (5′-GGTTCA
AGT CCC TCT ATC ATC CC-3′) and a primer designed by Dobeš et al. (2004)
(5′-GAT TTT CAG TCC TCT GCT CTA C-3′). The amplifications were run on
a PTC 200 Peltier Thermal Cycler (MJ Research) under the following condi-
tions: 3 min initial denaturation at 95°C; followed by 30 cycles of 30 s at 95°C,
30 s at 48°C and 1 min at 72°C; and 10 min final elongation at 72°C. PCR
products were purified by centrifugation in a NucleoFast 96 PCR Plate (Mach-
erey-Nagel) and directly sequenced using the above primers by the commercial
sequencing service GATC (Konstanz, Germany). The ITS (Appendix S1, see
Supplemental Data with the online version of this article) and the combined
trnL intron and trnL-F intergenic spacer sequences (Appendix S2, see online
Supplemental Data) were checked and trimmed using the program SeqMan II
v.6.1 (DNAStar, Madison, Wisconsin, USA) (hereafter designated trnLF) and
780 AMERICAN JOURNAL OF BOTANY [Vol. 99

TABLE 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 + fixed

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
tmrca of Arabis alpina aggregate (Ma) 1.49 0.51 0.52
4.02 1.26 1.27
7.02 2.25 2.25
tmrca of the core-Arabis (Ma) 4.58 1.54 1.63
12.5 3.91 3.94
21.5 6.95 7.04
tmrca 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).
In addition, a combined data set was formed by merging the ITS and the trnLF
sequences (online Appendix S3). Identical sequences were excluded from the
final alignments prior to phylogenetic analysis.
Phylogenetic analyses—Bayesian Markov chain Monte Carlo (MCMC)
analyses (Yang and Rannala, 1997) were performed with the mpi (message
parsing interface) version of the program MrBayes v.3.1.2 (Ronquist and
Huelsenbeck, 2003). To substantiate the results of the Bayesian analyses, we under-
took additional analyses using maximum parsimony (MP) running the program
PAUP* 4.0b10 (Swofford, 2002) and maximum likelihood using the program
Garli v0.96 (Zwickl, 2006). The best-fitting nucleotide substitution models
were chosen using the program Modeltest v3.7 (Posada and Crandall, 1998),
which relied on the Akaike information criterion (AIC) and the Bayesian infor-
mation criterion (BIC). For the combined analyses, the data set was divided into
nrDNA and cpDNA partitions, so that each partition could be run under the
respective model. In the Bayesian analyses, four simultaneous runs with four
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
facilitated the most efficient chain-swapping. For each run, 1001 trees were
sampled, and the first 25% of the trees sampled were discarded (burn-in = 250)
when computing the consensus tree (50% majority rule) Completion of the
Bayesian runs was determined using the web-based program AWTY (Nylander
et al., 2007). Comparison of the posterior probabilities of all splits between the
respective runs and cumulative split frequency plots showed that convergence
between the Bayesian MCMC runs had been reached.
The parsimony heuristic search was performed under the following settings:
gaps were treated as missing data, the “mstaxa” option was set as “uncertain”,
tree construction was via stepwise addition, tree-bisection-reconnection (TBR)
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
during branch swapping). A strict consensus tree was calculated from most
parsimonious trees retained. For bootstrapping (Felsenstein, 1985), 100 repli-
cates with a maximum of 250 retained trees were run.
For the maximum likelihood (ML) analyses, we ran two replicates per data
set, and each replicate was terminated under the condition that the ln likelihood
value did not increase significantly (0.01) for the previous 1 000 000 genera-
tions. Analyses were conducted using 1000 bootstrapping replicates with the
lowered termination condition of 10 000 generations without significant ln L
change. The corresponding alignments to the ITS, trnLF, and combined data
are provided with online Appendices S1, S2, and S3, respectively. Graphical
edition of all trees was executed in Adobe Illustrator CS4 (San Jose, California,
USA).
Congruency test—A Shimodaira-Hasegawa test (Shimodaira and Hasegawa,
1999) was performed to identify congruencies between the tree topologies
of the ITS, trnLF, and combined marker data sets using PAUP* 4.0b10
(Swofford, 2002). Before the tests, the number of accessions was reduced to the
40 common to all three data sets. As a consequence, the number of tree to-
pologies was very small (n = 2) and might have biased the levels of signifi-
cance. We therefore added 100 random topologies to the congruency tests to
minimize this problem.

→
Fig. 1. Phylogenetic tree of the Bayesian analysis based on the nrDNA ITS data set. Posterior probability values (first-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 prefixed asterisk had the same sequence as the top-listed acces-
sion and were therefore excluded from the tree calculation. Purple- or pink-flowered 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-flowered 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
Network analyses—Network analysis was performed with the program
TCS v.1.21 (Clement et al., 2000), which uses statistical parsimony (Templeton
et al., 1992) as the underlying method of estimation. Taxa identified to be dis-
tantly related to Arabis alpina were excluded from the alignment previously
used for phylogenetic analaysis. We utilized accessions of Arabis brachycarpa
Rupr., A. nordmanniana Rupr., A. graellsiiformis Hedge, and A. christiani
N.Busch to root the network. For the ingroup, we included all cpDNA haplotypes
published earlier by Koch et al. (2006) (DQ060112–DQ060138, DQ060140–
DQ060142, DQ060144–DQ060145), Assefa et al. (2007) (EF449508–EF449514),
Ansell et al. (2008) (EU403083–EU403084), and Ansell et al. (2011) (JF705219–
JF705252), giving a combined data set of 115 haplotypes from of 518 acces-
sions, covering 329 locations and 10 taxa (A. cypria, A. purpurea, A. kennedyae,
A. alpina, A. aubrietoides, A. tianschanica, A. deflexa, A. ionocalyx, A. mont-
bretiana, A. nova subsp. iberica Rivas Mart. ex Talavera).
The alignment was adjusted further by excluding a microsatellite-like region
at the 5′-end of the trnL-F intergenic spacer (alignment position 365–380,
Appendix S2), and we also excluded the trnL gene from the analysis, so that all
sequences had comparable information content. This led to the exclusion of a
series of nearly identical haplotypes shown in Ansell et al. (2011) (haplotypes
06, 22, 23, 24, 27, 32, 48, 52, 53, 55, 57, 64, 65, 66, 67, 71, B, and D). Redun-
dant haplotypes from newly analyzed accessions of our study were also removed
(MG003, RK240, RK244, RK249, and RK252), as were haplotypes 16 and 31
from Koch et al. (2006). Furthermore, to be conservative when interpreting
nucleotide variation, we also excluded sequence types that differed by indels in
the beginning and end portions of the PCR fragments, where sequencing qual-
ity may be insufficient. This excluded haplotypes E and F (Assefa et al., 2007)
and several newly analyzed accessions of our study (A53M, MA001, MA004,
RK001, RK002, RK004, RK006, RK086, RK087, RK088, RK167, and
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.
Divergence-time estimates—Divergence-time estimates in the Brassicaceae
have been discussed widely, because calculations led to highly diverging re-
sults, depending on the markers and calibration points used (refer to Franzke
et al. [2011] for further discussion). Using the software package BEAST v.1.4.8
(Drummond and Rambaut, 2007), we performed three courses of divergence-
time estimate calculations employing two different calibration points. (1) The
primary calibration was based on an Oligocene fruit fossil (Thlaspi primae-
vum), which was assigned to the tribe Thlaspideae (Manchester and O’Leary,
2010). This fossil, dated at 29.2−30.8 Ma (Wing, 1987), has been recently used
for age estimates of the Brassicales (Beilstein et al., 2010). (2) Secondary cali-
bration used the estimated age of the most recent common ancestor of the tribe
Arabideae (mean age 16.8 Myr) as shown by Couvreur et al. (2009). (3) We
additionally applied fixed limits for the rates of variation on the secondary cali-
bration point approach, following Koch et al. (2010). Two representatives of
the tribe Thlaspideae [Thlaspi arvense L. AF336152 (ITS1), AF336153 (ITS2),
AY122461 (trnLF); Alliaria petiolata (M.Bieb.) Cavara & Grande AF283492
(ITS1), AF 283493 (ITS2), JN189740 (trnL), JN189781 (trnF)] were added to
both the ITS and cpDNA alignments, used for the phylogenetic analyses. Both
taxa were also used by Beilstein et al. (2010) to define the tribe Thlaspideae.
Both original alignments were modified only by the introduction of additional
gaps. Taxon subsets were specified for some clades as designated in the phylo-
genetic analysis, including a denotation for the Thlaspideae (Thlaspi arvense
and Alliaria petiolata) and the Arabideae (excluding Pseudoturritis turrita and
both members of the Thlaspideae). For the fossil-based primary calibrations,
the boundaries for the age of the most recent common ancestor (tmrca) of the
taxon set “Thlaspideae” were set between 29.2 and 30.8 Myr. For the secondary
calibration point, the boundaries for the tmrca of the taxon set “Arabideae” were
set between 10.0 and 23.9 Myr. Additionally, for the calculations with fixed
mutational rates these rates were fixed within the range of 1.72 × 10−9 to 1.71
× 10−8 (ITS) and 3.6 × 10−9 and 7.7 × 10−9 (trnLF), respectively. These values
reflect the published ranges for mutation rates in herbaceous plants (Kropf,
Fig. 2. Phylogenetic tree from the Bayesian analysis based on the cpDNA trnLF data set. Posterior probability values (first-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 prefixed asterisk had the same sequence than the top-listed
accession and were therefore excluded from the tree calculation. Purple- or pink-flowered 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-flowered species.
Annual/winter-annual taxa are labeled with the corresponding sign for annualism, also following the taxon label.
[Vol. 99
2002; Mummenhoff et al., 2004; Kay et al., 2006). The most appropriate evolu-
tionary model was estimated using the program Modeltest v.3.7 (Posada and
Crandall, 1998), based on the Bayesian information criterion (BIC).
Both ITS and trnLF divergence-time estimations were run under the lognor-
mal uncorrelated relaxed clock method and the birth–death speciation model
(Gernhard, 2008), because the calculated Bayes factors (Nylander et al., 2004)
favored their use over a strict clock and a pure birth model (Yule, 1924). Num-
ber of runs and generations are given in Table 1 (ITS) and 2 (trnLF). In each
run, 10 001 generations were sampled. Each run was checked in the program
Tracer v.1.4.1 (Rambaut and Drummond, 2007) for having reached stationary
phase and a sufficient effective sample size (ESS over 200, as suggested by the
authors). All runs (log- and trees-files, respectively) were then combined with
the LogCombiner program included in the Tracer package.
Chromosome counts and ploidy level estimates—Most of the leaf material
was obtained directly from wild sources (e.g., preserved herbarium vouchers)
with supplementary material grown from seed. Root tips were incubated in
2 mmol/L 8-hydroxyquinoline for 3 h at 15°C and subsequently transferred to
freshly prepared ice-cold ethanol–acetic acid (3 : 1). After 2 h of incubation at
4°C, the root tips were transferred to 70% ethanol and stored at −20°C for sub-
sequent analysis. Root tips were washed in water, equilibrated in 10 mmol/L
citrate buffer, and then hydrolyzed with an enzyme solution (0.1% cellulase,
0.1% pectolyase, 0.1% cytohelicase, and 1 mmol/L citrate buffer) at 37°C fol-
lowed by squashing and fixing in 45% acetic acid and staining with a 1 : 20 dilu-
tion of DAPI/Vectashield (Vector Laboratories, Burlingame, California, USA).
Additional information on chromosome numbers from various Arabis species
was obtained from published accounts (Warwick and Al-Shehbaz, 2006; Koch
et al., 2010).
RESULTS
Nuclear ITS region— The ITS alignment comprised 45 se-
quences (representing 71 accessions) and 631 characters, of
which 448 were constant. Of the 183 variable characters, 132
were phylogenetically informative, and the remaining 51 were
autapomorphic positions (Appendix S1). The SYM+Γ evolu-
tionary model was applied to the Bayesian and ML analyses,
the results are shown in Fig. 1. The resulting Bayesian trees had
a harmonic mean for the ln likelihood of −2794.51 and an aver-
age standard deviation of split frequencies of 0.010. The corre-
sponding tree of the ML analysis had a ln likelihood of −2678.34.
The maximum parsimony analysis resulted in 571 most parsi-
monious trees with a tree length of 326 (with “maximum length =
0” rule applied), consistency index (CI) of 0.66 (autapomor-
phies excluded) and retention index (RI) of 0.90.
The ITS tree based on Bayesian analysis (Fig. 1) is subdivided
into seven major clades, which are very well supported by both
posterior probabilities and bootstrap values. Clade E comprises
the three species of the genus Aubrieta and the circum-Mediterra-
nean Arabis verna as sister to the genus. While this clade is
basal to all other clades, it lacks strong statistical support. Clade
D consists of Draba and its segregates and is sister to clade L,
which contains four Arabis species from the Caucasus moun-
tains (A. brachycarpa, A. christiani, A. graellsiiformis, and A.
nordmanniana). This clade (hereafter called A. nordmanniana
group) shows strong congruence with the former section Allari-
aopsis, as described by Busch (1906) and Schulz (1936). Clade
→
April 2012] KARL ET AL.—EVOLUTIONARY HISTORY OF ARABIS ALPINA 783
784 AMERICAN JOURNAL OF BOTANY
M is formed by the three representatives of the core Arabis, a
group that has been shown to contain the majority of Arabis
species (see Koch et al., 2010). Clade P comprises the Ibero-
Moroccan Arabis taxa, A. parvula, and its east-Mediterranean
sister species A. aucheri. Clades D, L, M, and P form a weakly
supported monophyletic group, as indicated by posterior prob-
ability or bootstrap values. Arabis auriculata and A. nova subsp.
nova are sister to each other in clade B. The accession of A. alpina
and all its morophologically similar species are confined to
clade A, which is further divided into subclades of annual and
perennial taxa, respectively. The annual subclade consists of
one accession of the Spanish A. nova subsp. iberica (MA022),
one accession of the endangered Cypriote endemic A. kenne-
dyae (RK215), and four accessions of A. montbretiana from
Western and Central Asia (A32MO, A59M, RK043, and RK121).
Two of these accessions (A32MO and A59M) were originally
wrongly determined as A. auriculata and A. nova, respectively.
The remaining accessions of A. auriculata (western Europe to
central Asia) and A. nova subsp. nova form a discrete clade
(clade B) not related to other annuals within clade A. Finally,
the perennial subclade of clade A comprises the various A. alpina
accessions and six other perennial species: Arabis aubrietioides
(A01M, MG003, RK240, RK241, RK243, RK244, RK261), A.
deflexa (RK088/89, RK248–251), A. ionocalyx (RK117/18,
RK252–255), A. tianschanica (RK007), A. cypria (RK086/
87, RK167, RK245/46), and A. purpurea (RK004, RK006,
A53M, RK247, RK257–260). Arabis cypria and A. purpurea
are characterized by discrete ITS sequences with the exception
of one accession of A. cypria (RK246), which shared its ITS-
type with A. purpurea. Most accessions of the remaining taxa
(Arabis aubrietioides, A. deflexa, A. ionocalyx, and A. tians-
chanica) had an ITS sequence identical to the widespread ITS-
type A of A. alpina (Koch et al., 2006).
Chloroplast trnLF region— The combined trnLF align-
ment comprised 61 unique sequences (representing 72 acces-
sions) and 759 characters, 617 of which were constant. Of
the 142 variable characters, 75 were phylogenetically infor-
mative, and the remaining 67 were autapomorphic positions
(Appendix S2).
The K81uf+Γ evolutionary model was applied to the ML and
Bayesian analyses, the results of which are shown in Fig. 2. The
resulting Bayesian trees had a harmonic mean for the ln likeli-
hood of −2581.62 and an average standard deviation of split
frequencies of 0.011. The corresponding tree of the ML analy-
sis had a ln likelihood of −2287.00. The maximum parsimony
analysis resulted in 10 000 most parsimonious trees with a tree-
length of 195 (with “maximum length = 0” rule applied) and an
ensemble CI of 0.77 (autapomorphies excluded) and an ensem-
ble RI of 0.90.
The chloroplast trnLF Bayesian phylogenetic tree (Fig. 2)
shows virtually the same seven major clades as the ITS tree
(Fig. 1). Among these are the following: clade p (A. aucheri and
A. parvula), clade m (core Arabis), clade d (Draba and its seg-
regates), and clade e (Aubrieta and Arabis verna). The latter is
sister to clade d and has high posterior probability support (ppr =
0.99), but only limited bootstrap support (maximum-likelihood
bsML = 58.4, and maximum-parsimony, bsMP = 45.6). Arabis
auriculata (clade b1) and A. nova subsp. nova (clade b2) are not
grouped together as in the ITS analyses, but form discrete
clades. Clade l (A. nordmanniana group) forms a well-supported
clade sister to the clade comprising A. alpina and its closest
related species. This clade was not subdivided into the annual
[Vol. 99
and perennial as shown in the ITS analyses but rather formed a
single polytomy. The predominantly east African “red-coded”
haplotype group (Fig. 3) is represented by accessions A22JO
and A40JO2 of A. alpina, to which one accession of the Ana-
tolian endemic A. aubrietioides (A01M) and the Syrian ac-
cession of A. ionocalyx (RK254) are basal. The mainly
western Asian “green-coded” haplotype group (Fig. 3) is rep-
resented by accessions A01JO2 and AW-30 of A. alpina and
by three accessions of A. aubrietioides (MG003, RK244,
RK261) and one accession of each A. deflexa (RK249) and A.
ionocalyx (RK253). Additionally, the Central Asian A. tians-
chanica (RK007) appears in this group. Several southern
Anatolian accessions of A. deflexa (RK088, RK089, RK250,
and RK251) and A. ionocalyx (RK117, RK118, RK252, and
RK255) plus the westernmost accessions of A. aubrietioides
(RK240) are combined together. A group containing all
accession of A. cypria is amended by two accessions of
A. aubrietioides (RK241, RK243), both being sampled on
the Turkish mainland opposite of the Kyrenia mountain
range. Finally, species-specific subgroups are formed by the
Cypriote endemic A. purpurea and by the annual A. mont-
bretiana. The accessions of A. nova subsp. iberica (MA022)
and A. kennedyae (RK215) are not directly connected to
A. montbretiana.
Combined data set— The alignment of the combined data set
comprised 66 sequences (representing 70 accessions) and 1390
characters, of which 1066 were constant. Of the 324 variable
characters, 212 were phylogenetically informative and the re-
maining 112 were autapomorphic positions (Appendix S3). The
SYM+Γ evolutionary model was applied to the nrDNA parti-
tion, and the K81uf+Γ evolutionary model was applied to the
cpDNA partition in the respective ML and Bayesian analyses.
The result of the Bayesian analysis is shown in Fig. 4. The re-
sulting trees had a harmonic mean for the ln likelihood of
−5422.36 and an average standard deviation of split frequencies
of 0.011. The corresponding tree of the ML analysis had a ln
likelihood of −5104.81. The maximum parsimony analysis re-
sulted in 6134 most parsimonious trees with a tree length of 534
(with “maximum length = 0” rule applied), CI of 0.68 (autapo-
morphies excluded), and RI of 0.89.
The phylogenetic tree based on the combined data set (Fig. 4)
shows the same seven major clades as recovered by the ITS
analysis (Fig. 1), reflecting the high amount of parsimony infor-
mative characters in the ITS data set. All of the clades were
very strongly supported by their posterior probabilities (ppr =
1.0) and bootstrap values (bootstrap > 98.5%). Clade δ (Draba
and its segregates) and clade ε (Aubrieta and Arabis verna)
remained unsupported.
Similar to the ITS tree (Fig. 1), the A. alpina containing
clade (clade α) is subdivided into statistically supported annual
and perennial subclades. The perennial subclade comprises
several subgroups recovered by both ITS and the trnLF analy-
ses (Figs. 1, 2).
Congruency test— The best tree topology was defined for
each of the three data sets (ITS, trnLF, and combined) accord-
ing to the –lnL scores (ITS, trnLF, and 100 random topologies).
This was either the ITS topology (for the ITS and combined
data set) or the trnLF topology (for the trnLF data set). For all
other topologies including all random topologies, the associated
p-value was used as a measure for the probability that this tree
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 filled circles; newly described haplotypes are represented either by triangles (for A. tianschanica, A. cypria, and A. purpurea),
empty circles (for A. aubrietioides, A. deflexa, and A. ionocalyx) or gray diamonds (for the annual taxa A. montbretiana, A. nova subsp. Iberica, and A.
kennedyae). (B) Some accessions are further specified by an A (for A. aubrietioides), D (for A. deflexa) 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
them explain the data equally well. Conversely, the trnLF to-
pology could explain the ITS (p = 0.22) and combined data
set (p = 0.50), and the ITS topology could explain the trnLF
data set (p = 0.30), as these values were above the threshold
of p = 0.05.
Chloroplast DNA network— The modified trnLF alignment
for phylogeographic analysis resulted in 666 characters (660
regular bases, plus six positions used for gap-coding), from
which 73 unique haplotypes were discriminated among the 518
accessions of A. alpina and close relatives. The resulting mini-
mum spanning network is shown in Fig. 3.
The network was almost identical to those previously pre-
sented based solely on accessions of A. alpina (e.g., Koch
et al., 2006; Assefa et al., 2007; Ansell et al., 2011), especially
in recovering the three major cpDNA haplotype groups (blue,
red, green). The two haplotypes of the A. nordmanniana group
(haplotypes 90, 91) were used to root the network; these types
were connected through six mutational steps to central haplo-
type 01 (blue). Four of the five newly included A. alpina ac-
cessions (MA001, MA004, RK001, and RK002) had the
common blue haplotype (haplotype 01), and one new blue
haplotype (haplotype 72) was identified from the western Ana-
tolian accession RK001b. Two new haplotypes (83, 84) were
added to the base of the red haplotype group. Haplotype 84
from A. aubrietioides (accession A01M) formed a side branch
to the red haplotypes 62 and 63 of A. alpina. The A. ionocalyx
accession from Syria (RK254, haplotype 83) formed another
side branch within the red haplotype group and was closely
connected to the most basal haploytype 60 of A. alpina from
northern Lebanon. Only one new haplotype was added to the
green haplotype group: the accession of A. tianschanica
(RK007, haplotype 82) from Kyrgyzstan, which occupied a tip
position in the green haplogroup (Fig. 3). In addition, several
of the remaining relatives had green haplotypes that were
identical to those recovered from A. alpina: A. aubrietioides
(MG003) had haplotype 04, A. deflexa (RK249) had haplotype
47, while accessions of A. aubrietioides (RK244, RK261) and
A. ionocalyx (RK253) held haplotype 51. All remaining acces-
sions of A. alpina relatives were affiliated with the blue haplo-
type group of Europe. The Cypriote species A. purpurea
(haplotype 73) and A. cypria (haplotypes 75 and 76) along
with one accession of A. purpurea (described as A. cypria
[haplotype 74] formed a distinct “purpurea haplotype group”,
restricted to the Kyrenia mountains in Cyprus (Fig. 3). Several
accessions of the A. alpina relatives from the Taurus Moun-
tains shared haplotype 41, which is also common in accessions
of A. alpina from this region (A. deflexa [RK088, RK250], A.
ionocalyx [RK117, RK252, RK255] and A. aubrietioides
[RK240]). Other accessions of the relatives from the Taurus
region also have haplotypes that are closely related to haplo-
type 41 (A. deflexa [RK089/haplotype 78 and RK251/haplotype
80] and A. ionocalyx [RK118/haplotype 79]). Other acces-
sions of the A. alpina relatives were connected genologically
to other haplotypes of A. alpina. One accession of A. deflexa
(RK248, haplotype 77) had a haplotype derived from central
blue haplotype 01, while two accessions of A. aubrietioides
(RK241 and RK243) had haplotype 81, which is derived from
A. alpina haplotype 45 via two mutational steps. The annual
species A. montbretiana (haplotypes 86, 87) and A. nova subsp.
iberica (haplotypes 85 and 88) were also connected to haplo-
type 01, whereas related A. nova subsp. iberica held an iso-
lated position in the network (Fig. 3). The three haplotypes
recovered from A. montbretiana were connected to haplotypes
20 and 03 of A. alpina, although an alternative mutational
pathway linking to haplotype 41 was also proposed by the
TCS program. Both of these pathways seem unrealistic based
on the clear morphological and biogeographical differences
between the annual A. montbretiana and the perennial species
of the A. alpina group. Finally, the Cypriote A. kennedyae
(haplotype 89) was connected with three mutational steps to
A. montbretiana haplotype 86.
Divergence-time estimates— The results of the divergence-
time calculation are given in Table 1 (ITS) and Table 2 (trnLF).
The results varied greatly among the three different ap-
proaches, but two main conclusions could be drawn. (1) Esti-
mates calculated from the trnLF marker normally exceeded
those from the ITS marker by a factor of two. When the rates
of variation were fixed, the estimates were congruent. Fixing
these rates in a reasonable interval seems important, because
the calculations run without strict limits on the rates of varia-
tion showed nonconstant rates that dropped below all reported
values for this chloroplast marker (see Table 2). Therefore, the
corresponding divergence time values might be overestimated.
(2) The calculated estimates using the Thlaspideae fossil as
the primary calibration point according to Beilstein et al. (2010)
were more than three times higher than those calculated with
the secondary calibration point published by Couvreur et al.
(2009). Although confidence intervals are large, the overlap
between fossil based calibration and secondary calibration is
small. Notably, the log likelihood values remain relatively
constant for all dating approaches per marker, and therefore
the calculated Bayes factors do not favor one approach over
another.
Chromosome numbers and ploidy levels— Known chromo-
some numbers and ploidy levels are summarized in Table 3.
Here we present counts for A. purpurea (accessions RK004,
RK006) and A. cypria (accession RK167) for the first time;
both are 2n = 16. A hybrid plant of unknown origin (not in-
cluded in the analyses) between A. alpina and A. aubrietioides
was also counted as 2n = 16, indicating a hybridization event at
the diploid level. According to the data available, there are no
tetraploid or higher polyploid counts for any species closely re-
lated to A. alpina. Although there are some species (A. deflexa,
A. tianschanica) whose chromosome numbers have not yet
been determined. Hence it seems likely the “A. alpina clade”
consists exclusively of diploids.

→
Fig. 4. Phylogenetic tree of the Bayesian analysis based on the combined data set (ITS plus trnLF). Posterior probability values (first-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 prefixed asterisk had the same sequence than the
top-listed accession and were therefore excluded from the tree calculation. Purple- or pink-flowered 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-flowered
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

TABLE 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 M0 (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 + fixed 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
tmrca 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
Congruency between nuclear and cpDNA derived phyloge-
nies and divergence-time estimates— In this study, which fo-
cuses on Arabis alpina and its closest relatives, the combined
tree and the derived phylogenetic hypothesis (Fig. 4) help to
resolve phylogentic relationships within the tribe Arabideae
and identify new divergent lineages (Figs. 4, 5). We show that
Arabis verna is more closely related to the genus Aubrieta than
to genus Arabis (Figs. 2, 4) and that it forms a monophyletic
clade (clade ε in Fig. 4), despite the different markers creating
uncertainty in the phylogenetically position of Aubrieta (e.g.,
sister to Arabis alpina: Koch et al., 1999a, 2001; sister to core
Arabis: Koch et al., 2000; sister to all Arabideae s.s.: German
et al., 2009). Although the phylogenetic relationship of A. verna
and Aubrieta is based on limited species sampling for Aubrieta
(n = 3), we assume that this pattern is significant. Future work
will test whether Arabis verna is nested within the genus Aubrieta,
which consists of ca. 15 species or whether it is a sister taxon to
the genus Aubrieta.
The congruency test revealed no major incongruencies between
both DNA markers. However, several minor differences exist
between the nrDNA ITS (Fig. 1) and the cpDNA trnLF (Fig. 2)
sequence analyses, and these should be mentioned. On the basis
of the cpDNA analysis, four Arabis species (A. brachycarpa, A.
nordmanniana, A. graellsiiformis, and A. christiani) are sister
to A. alpina and its close relatives (Fig. 2). However, this relation-
ship was not supported by the ITS analyses, which placed all
four species close to Draba. This difference probably reflects
the variation in the mode of marker inheritance with the plastidic
trnLF being maternally inherited (Harris and Ingram, 1991) and
the nuclear-encoded ITS being biparentally inherited. Overall,
it appears that the cpDNA results are more biologically meaning-
ful, as all four species are confined to the Caucasus Mountains,
which are themselves near to the center of origin and diversity
for A. alpina (Ansell et al., 2011).
The phylogenetic position of the genus Aubrieta (including
Arabis verna) formed a second incongruency between the two
markers. For the trnLF -based analyses, Aubrieta was placed
close to Draba (Fig. 2). This relationship was not recognized by
ITS based analyses (Fig. 1). The low resolution of the ITS-based
analysis has previously been shown with two large-scale data
sets focusing on Draba and core-Arabis (Jordon-Thaden et al.,
2010; Koch et al., 2010). Extensive hybridization and reticulate
evolution within these groups has resulted in chloroplast cap-
ture and incomplete lineage sorting of both nuclear and plastid
markers. So far we have found no evidence for these complexi-
ties in the Aubrieta-Arabis verna and Arabis brachycarpa-A.
nordmanniana-A. christiani-A. graellsiiformis clades. Instead,
it seems more likely that the mode of marker molecular evolu-
tion has contributed to the phylogenetic incongruencies; con-
certed evolution for nuclear ITS regions (Koch et al., 2003) and
the low mutation rates for plastidic genomes (trnLF sequences).
Consequently, experimental work adding single-copy nuclear
genes is needed to further resolve these phylogenetic relation-
ships at the tribal level (e.g., chs, adh; M. A. Koch and R. Karl,
unpublished manuscript).
Our various estimates of divergence times varied greatly de-
pending on the approaches applied, making it difficult to deter-
mine the most reliable methodology. The primary fossil-based
calibrations following the approach of Beilstein et al. (2010)
seem to have resulted in overly high divergence time values.
These estimates (ITS: 7.1 Ma with a 95% CI from 2.8–12.3,
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

TABLE 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 identified Arabis
alpina as a paraphyletic taxon, because six Arabis species (A.
Taxon 2n Ploidy level Reference
aubrietoides, A. cypria, A. deflexa, A. ionocalyx, A. purpurea, and
Arabis alpina 16 2x # A. tianschanica, Figs. 2–5) are nested within the widely distributed
Arabis aubrietioides 16 2x this study A. alpina. All six species are perennials with small distribution
Arabis aucheri 16 2x # areas: Kyrgyzstan in the case of A. tianschanica and the eastern
Arabis auriculata 16 2x # Mediterranean for the remaining five species. The eastern Mediter-
Arabis blepharophylla 16 2x #
Arabis brachycarpa 16 2x # ranean, more precisely Anatolia and the adjacent Levantine region,
Arabis christiani 32 4x # has been shown to be the center of origin and genetic diversity of
Arabis cypria 16 (14*) 2x Hand (2006); this study A. alpina (Ansell et al., 2011). As mentioned, we assume Pleisto-
Arabis deflexa 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 4x #
Arabis ionocalyx 16 2x # 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 2x Hoffmann et al. (2010) (Erinç, 1978; Atalay, 1996), and the vegetation belts shifted by up
Arabis nordmanniana 16 2x # 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 (Médail and Diadema, 2009;
Arabis nova subsp. nova 16 2x # Parolly et al., 2010) allowed for the survival of various biota during
Arabis parvula 32 4x #
Arabis purpurea 16 2x #; this study the periods of temperature fluctations. In the warmer periods, a dry
Arabis sachokiana 16 2x # 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 2x/4x # 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 2x #
Arabis parviflora 16 2x #
thereby creating gene flow limitations and promoting local lin-
Draba hederifolia 30 4x # eages accumulation. Ansell et al. (2011) emphasized the roles of
Draba hispanica 16 4x # local survival centers in the coastal Taurus Mountains, which pro-
Draba reptans 30/32 4x # vided moister conditions compared to the drier interior climates
# Warwick and Al-Shehbaz (2006); * Yildiz and Gücel (2006); n.d.: no of the Anatolian (Davis, 1971; Ekim and Güner, 1986). Accord-
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-flowering A. aubrietioides in the Central
Taurus (Cilician sector) and Arabis deflexa 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. deflexa, and A. ionocalyx often bear local A. alpina haplotypes,
nating in the late Pliocene (Güldali, 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. deflexa, 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 flock 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 insufficiently 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 findings 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 sufficient barrier to have promoted
early Pleistocene. allopatric speciation.
790 AMERICAN JOURNAL OF BOTANY
Within the red-flowering species, there is some interesting
vicariance of A. purpurea on Cyprus and A. aubrietoides on the
Turkish mainland. We speculate that the red flowers may be
indicative of outbreeding. Because the phylogenetic trees do
not support a shared origin of the Cypriote and Turkish main-
land populations within the predominantly white-flowering
Arabis alpina, this phenological change may have occurred in
parallel. This observation is supported also by the diploid chro-
mosome numbers of these taxa (Table 3), indicating an absence
of hybridization or polyploidy, in contrast to the other major
clades in the Arabideae for which various ploidy level series
indicate extensive hybridization and reticulation (Jordon-Thaden
and Koch, 2008; Koch et al., 2010). Further ecological and
molecular research may unravel new and interesting aspects of
parallel evolution among sister-species pairs that have been
derived from white-flowering Arabis alpina. More widely
red-flowering taxa within the genus Arabis are typically not
sympatrically distributed with other red-flowering species: e.g.,
in Europe the red-flowering endemic of Arabis stenocarpa
Boiss. & Reut. from Spain, Arabis cretica Boiss. & Heldr. from
Crete (Koch et al., 2010), and Arabis rosea DC. from southern
Italy. Furthermore, all European red-flowering Arabis species
with the exception of Arabis alpina have failed to developed
red-flowering sister species pairs (M. A. Koch and R. Karl,
unpublished data). These multiple changes in flower color
might be indicative of genetic/geographic isolation caused by
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 significance.
[Vol. 99
the habitat fragmentation during Pleistocene glaciations, and
possibly reinforced by breeding system shift.
In contrast to most members of the Arabis alpina species
flock, A. tianschanica is located in Uzbekistan and western
Kyrgyzstan, several hundred kilometers east of the nearest lo-
calities for A. alpina in northeast Iran. German et al. (2009)
have already suggested a close relationship between this en-
demic and A. alpina based on morphological characters. Geneti-
cally, A. tianschanica has a private haplotype that is derived
from the common green haplotype 04, which suggests this taxa
might have evolved from a remnant population of a former
more easternly distributed A. alpina.
Southern refuges as centers of origin and evolutionary
starting points for different life-history strategies— The occur-
rence of species-poor annual clades being sister to species-rich
perennial clades is a common feature within the tribe Arabideae.
This trend is also found in many other tribes and genera of the
Brassicaceae (e.g.: Arabidopsis/tribe Sisymbrieae [Koch and
Matschinger 2007]; Noccaea, Microthlaspi/tribe Coluteocarpeae
[Mummenhoff et al., 1997a, b; Koch et al., 1998]; Cochlearia,
Ionopsidium/tribe Cochlearieae, [Koch et al., 1999b]).
Notably, this is pattern is also found in Draba (Arabideae),
where we found that the ca. 20 annual species are restricted to a
single clade that is sister to the clade comprising the remaining
300 perennial taxa, and which underwent a dramatic Pleistocene
April 2012] KARL ET AL.—EVOLUTIONARY HISTORY OF ARABIS ALPINA
radiation (Jordon-Thaden and Koch, 2008; Jordon-Thaden et al.,
2010). This pattern is also true for the Arabis alpina species
group, although less pronounced, whereby the seven perennial
species are sistered by three annual taxa: Arabis montbretiana,
A. nova subsp. Iberica, and A. kennedyae (Fig. 4). The five re-
maining annual species included in this study (Arabis aucheri,
Arabis auriculata, Arabis erikii, Arabis nova subsp. nova, Ara-
bis parvula) show no close relationships to any other perennial
Arabis species but form two distinct clades (Fig. 4). Future phy-
logenetic research may help clarify their systematic and life his-
tory relationships.
The evolutionary success of the genus Arabis as a whole may
be attributed to a combination of adaptive characters: (1) High-
elevation plants that are outcrossing, long-lived perennials and
produce large flowers to attract insects and winged seeds suit-
able for long-distance dispersal. (2) Plants, mostly from mon-
tane regions, produce medium-sized flowers and narrowly
winged seeds and tend to shift from perennial to biennial growth
forms. (3) Low-elevation plants are primarily selfing annuals
with small flowers and small seeds (Koch et al., 2010). Conse-
quently, we find annuals primarily in lowland regions and with
more southern distribution areas than the present-day distribu-
tion areas of the perennials. The generally higher number of
species in the perennial groups reflect their alpine and montane
habitats, creating opportunities for lineage diversification during
the Pleistocene environmental fluctuations. These features have
recently been shown to have accelerated speciation and poly-
ploidization rates in Draba and core Arabis (Jordon-Thaden
and Koch, 2008; Koch et al., 2010).
Conclusion and taxonomic remarks— The phylogenetic
results presented revealed that Arabis alpina in its current
limits is a paraphyletic species and the origin of several evo-
lutionary lineages located in Anatolian mountain regions and
the eastern Mediterranean. Whereas the four mainland spe-
cies (A. aubrietioides, A. deflexa, A. ionocalyx, and A. tians-
chanica) are completely nesting within A. alpina, the two
Cypriote endemics (A. cypria and A. purpurea) are both geo-
graphically and geneologically, much more distinct. Pres-
ently, there is no evidence of a common evolutionary history
for these two Cypriote taxa, suggesting that the shift from
white to red flowers has evolved independently more than
once. Our results imply the need for a number of taxonomic
changes within Arabis alpina and its closest relatives. The
recognition of the perennial relatives as subspecies of A. alpina
would fit the observed pattern of genetic distribution and re-
store monophyly for A. alpina. Furthermore, as A. alpina is
the type species of the genus Arabis, major adjustments within
the whole tribe Arabideae seem to be necessary as well. How-
ever, to make any profound suggestions in this respect requires
additional data gathering and experimental work focusing on
the tribal phylogeny.
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794 AMERICAN JOURNAL OF BOTANY
APPENDIX 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 defined 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;
Spain], [MA004; HQ200931; GU181906; GU181974; MA; 224236;
Canary Islands], [RK001; GU182066; GU181934; GU181999; HEID;
809695; Slovakia], [RK001b; GU182067; GU181935; GU182000; HEID;
502914; Turkey], [RK002; GU182068; GU181936; GU182001; NSK;
A. Telpukhovskaya, 1964, s.n.; Siberia/Russia], [A01JO2; DQ060109;
DQ060115 (trnLF); BM; T.R.I. Woods, 09.05.1975, no. Y/75/176;
Yemen], [A19JO2; DQ060102; DQ060113 (trnLF); BM; J.R. Press & M.J.
Short, 29.03.1984, no. 398; Madeira], [A22JO; DQ060101; DQ060116
(trnLF); BM; A. Hemp, s.n.; Tanzania], [A36JO2; DQ060100; DQ060112
(trnLF); BM; N.K.B. Robson, 07.04.1973, no. 3004; Turkey], [A40JO2;
DQ060105; DQ060128 (trnLF); BM; A. Hemp, no. 3676; Ethiopia],
[AW-30; DQ060101; DQ060117 (trnLF); WU; K.H. Rechinger, 28.07. -
01.08.1957, no. 1960-3968; Iraq]; Arabis aubrietioides Boiss., [A01M;
FJ187845; FJ188142; FJ187991; M; ex. cult., 1976; Turkey], [MG003;
HQ646647; HQ646768; HQ646707; C; Th. Kotschy, 25.06.1862, no.
88; Turkey], [RK240; -; JN189720; JN189761; E; 00381050; Turkey],
[RK241; JN189742; JN189721; JN189762; E; 00381053; Turkey],
[RK243; JN189743; JN189722; JN189763; E; 00381056; Turkey],
[RK244; JN189744; JN189723; JN189764; E; 00381057; Turkey],
[RK261; JN189759; JN189739; JN189780; MA; 765874; Turkey];
Arabis aucheri Boiss., [A06MO2; FJ187881; FJ188178; FJ188027;
MO; 2363411; Syria]; Arabis auriculata Lam., [A56M; FJ187863;
FJ188160; FJ188009; M; F.A. Tscherning, 09.06.1895, s.n.; Austria],
[A58M; FJ187864; FJ188161; FJ188010; M; D. Podlech, 28.04.1987, no.
43319; Morocco], [RK027; GU182091; GU181959; GU182024; HEID;
809774; Uzbekistan]; Arabis blepharophylla Hook. & Arn., [Arab1795;
FJ187986; FJ188287; FJ188136; GH; R.C. Rollins, 30.04.1942, no.
3006; California/USA]; Arabis brachycarpa Rupr., [A35MO; FJ187913;
FJ188210; FJ188059; MO; 04958380; Armenia]; Arabis christani
N. Busch, [JR099; GU182044; FJ188271; FJ188120; HEID; 502919;
Armenia]; Arabis cypria Holmboe, [RK086; HQ200932; HQ200946;
HQ200939; B; 10 0206185; Cyprus], [RK087; HQ200933; HQ200947;
HQ200940; B; 10 0206362; Cyprus], [RK167; JN189741; JN189719;
JN189760; HEID; 809784; Cyprus; diploid], [RK245; JN189745;
JN189724; JN189765; E; 00381031; Cyprus], [RK246; JN189746;
JN189725; JN189766; E; 00381036; Cyprus]; Arabis deflexa Boiss.,
[RK088; HQ200934; HQ200948; HQ200941; B; 10 0212852; Turkey],
[RK089; HQ200935; HQ200949; HQ200942; B; 10 0212853; Turkey],
[RK248; JN189748; JN189727; JN189768; E; 00381005; Turkey],
[RK249; JN189749; JN189728; JN189769; E; 00381008; Turkey],
[RK250; JN189750; JN189729; JN189770; E; 00381009; Turkey],
[RK251; JN189751; JN189730; JN189771; E; 00381014; Turkey];
Arabis erikii Mutlu, [RK042; HM046192; HM046244; HM046217;
HUB; B. Mutlu, 10.06.1999, BM-4900; Turkey]; Arabis graellsiiformis
Hedge, [MG056; HQ646648; HQ646769; HQ646708; C; P.H. Davis &
O.V. Polunin, 08.07.1954, no. 22574; Turkey]; Arabis hirsuta (L.) Scop.,
[JR048; FJ187938; FJ188235; FJ188084; HEID; 400762; Croatia];
Arabis ionocalyx Boiss., [RK117; HQ200936; HQ200950; HQ200943;
B; 10 0201502; Turkey], [RK118; HQ200937; HQ200951; HQ200944;
B; 10 0201504; Turkey], [RK252; JN189752; JN189731; JN189772; E;
00381016; Turkey], [RK253; -; JN189732; JN189773; E; 00381020;
Turkey], [RK254; JN189753; JN189733; JN189774; E; 00381021;
Syria], [RK255; JN189754; JN189734; JN189775; E; 00381025; Turkey];
Arabis kennedyae Meikle, [RK215; HQ646643; HQ646764; HQ646703;
HEID; 809841; M. Andreou & C. Constantinou, 28.04.2010, AR-23-4;
Cyprus]; Arabis montbretiana Boiss., [A32MO; GU182046; GU181912;
GU181980; MO; 1262438; Pakistan], [A59M; FJ187865; FJ188162;
FJ188011; M; Sh. Zarre, 06.04.1999, no. 258; Iran], [RK043; HM046193;
HM046245; HM046218; HUB; B. Mutlu, 30.05.2002, no. BM-7968;
Turkey], [RK121; HQ200938; HQ200952; HQ200945; B; 10 0201515;
Iran]; Arabis nordmanniana Rupr., [A54MO; FJ187924; FJ188221;
FJ188070; MO; 05060970; Georgia]; Arabis nova subsp. iberica Rivas
Mart. ex Talavera, [MA022; GU182039; GU181907; GU181975; MA;
732814; Spain]; Arabis nova subsp. nova Vill., [A34M; GU182063;
GU181929; GU181995; M; Doppelbaur, 06.08.1963, no. 11160; Italy],
[MA024; GU182041; GU181909; GU181977; MA; 48509; France];
Arabis parvula Dufour, [A38M; FJ407224; GU181931; FJ407244; M;
D. Podlech, 11.04.1993, no. 51465; Morocco]; Arabis purpurea Sm.,
[A53M; FJ187861; FJ188158; FJ188007; M; W. Lang, 04.04.1999, s.n.;
Cyprus], [RK004; GU182070; GU181938; GU182003; HEID; 809699;
Cyprus; diploid], [RK006; GU182072; GU181940; GU182005; HEID;
809703; Cyprus; diploid], [RK247; JN189747; JN189726; JN189767; E;
00381038; Cyprus; diploid], [RK257; JN189755; JN189735; JN189776;
E; 00124777; Cyprus], [RK258; JN189756; JN189736; JN189777; E;
00381041; Cyprus], [RK259; JN189757; JN189737; JN189778; HEID;
00381043; Cyprus], [RK260; JN189758; JN189738; JN189779; HEID;
00381048; Cyprus]; Arabis sachokiana N. Busch, [A69MO; FJ187929;
FJ188226; FJ188075; MO; 05060963; Georgia]; Arabis tianschanica
Pavl., [RK007; GU182073; GU181941; GU182006; OSBU; 16518;
Kyrgyzstan]; Arabis verna (L.) R. Br., [A20MO2; FJ187890; FJ188187;
FJ188036; MO; 05040147; Greece]; Aubrieta canescens subsp.
macrostyla Cullen & Hub., [RK214; HQ646642; HQ646763; HQ646702;
HEID; ex cult., 2010; Turkey]; Aubrieta deltoidea (L.) DC., [AJ232909;
DQ180257; DQ180303; sequences from GenBank]; Aubrieta parviflora
Boiss., [DQ357518; —; —; sequence from GenBank]; Draba hederifolia
(Coss.) Hyam & Jury, [L241; DQ467438; DQ467072 (trnLF); BM;
Ait-Lafkikh et al., 01.10.1991, no. 4920; Morocco]; Draba hispanica
Boiss., [B76; DQ467289; DQ466988 (trnLF); B; 10 0127430; Spain];
Draba reptans (Lam.) Fernald, [72; AF146495; AF146942; AF146979;
Muehlenbach, 20.04.1971, no. 3498; Missouri/USA]; Pseudoturritis
turrita (L.) Al-Shehbaz, [A52JO; GU592179; GU592180; GU592181;
BM; J. Vogel, s.n.; Italy].