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Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa

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Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region for 189 accessions representing 184 species in 121 genera of Brassicaceae were used to determine monophyly of tribes and genera, tribal boundaries, and component genera. Parsimony analysis and Bayesian inference suggest that the tribes Camelineae and Arabideae are polyphyletic and should be subdivided into smaller tribes. The study also supports the recent recognition of the new tribes Aphragmeae, Biscutelleae, Buniadeae, Calepineae, Conringieae, Dontostemoneae, Erysimeae, Malcolmieae, Megacarpaeeae, and Turritideae. The data argue for the placement of Borodinia in the tribe Boechereae, Litwinowia and Pseudoclausia in the Chorisporeae, Atelanthera and Streptoloma in the Euclidieae, and Megacarpaea and Pugionium in the Megacarpaeeae, and exclusion of Asperuginoides, Didymophysa, and Ptilotrichum from the Alysseae, Macropodium, Pseudoturritis, and Stevenia from the Arabideae, and Crucihimalaya, Irenepharsus, Pachycladon, and Turritis from the Camelineae. Finally, the findings support the expansion of Stevenia to include both Berteroella and Ptilotrichum, Sterigmostemum to include Oreoloma and one species of Anchonium, Crucihimalaya to include Transberingia and several species of Arabis, and Parrya to include Pseudoclausia. The data also suggest that Calymmatium and Olimarabidopsis may be congeneric.
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ORIGINAL ARTICLE
Contribution to ITS phylogeny of the Brassicaceae, with special
reference to some Asian taxa
Dmitry A. German Æ Nikolai Friesen Æ
Barbara Neuffer Æ Ihsan A. Al-Shehbaz Æ
Herbert Hurka
Received: 9 February 2009 / Accepted: 20 August 2009
Ó Springer-Verlag 2009
Abstract Sequence data from the nuclear ribosomal inter-
nal transcribed spacer (ITS) region for 189 accessions repre-
senting 184 species in 121 generaofBrassicaceae were usedto
determine monophyly of tribes and genera, tribal boundaries,
and component genera. Parsimony analysis and Bayesian
inference suggest that the tribes Camelineae and Arabideae
are polyphyletic and should be subdivided into smaller tribes.
The study also supports the recent recognition of the new
tribes Aphragmeae, Biscutelleae, Buniadeae, Calepineae,
Conringieae, Dontostemoneae, Erysimeae, Malcolmieae,
Megacarpaeeae, and Turritideae. The data argue for the
placement of Borodinia in the tribe Boechereae, Litwinowia
and Pseudoclausia in the Chorisporeae, Atelanthera and
Streptoloma in the Euclidieae, and Megacarpaea and Pugio-
nium in the Megacarpaeeae, and exclusion of Asperuginoides,
Didymophysa,andPtilotrichum from the Alysseae, Macrop-
odium, Pseudoturritis,andStevenia from the Arabideae, and
Crucihimalaya, Irenepharsus, Pachycladon,andTurritis
from the Camelineae. Finally, the findings support the
expansion of Stevenia to include both Berteroella and Ptilo-
trichum, Sterigmostemum to include Oreoloma and one spe-
cies of Anchonium, Crucihimalaya to include Transberingia
and several species of Arabis,andParrya to include
Pseudoclausia. The data also suggest that Calymmatium and
Olimarabidopsis may be congeneric.
Keywords Brassicaceae ITS phylogeny Tribal limits
Asia
Introduction
The Brassicaceae (Cruciferae) are cosmopolitan but most
abundant in temperate regions of the northern hemisphere
and rather rare in the tropics except for the montane areas.
The family is very attractive for evolutionary studies (Koch
et al. 2003; Hurka et al. 2005; Mitchell-Olds et al. 2005)
because of its:
1. remarkable ecological amplitude;
2. economic importance as a source of edible and industrial
oils, condiments, vegetables, fodder, and ornamentals;
3. numerous species as colonizers or notorious weeds; and
4. most important model plant in molecular biology,
Arabidopsis thaliana (L.) Heynh.
However, the family is still in need of comprehensive
studies to resolve its major subdivisions and their rela-
tionships, to delimit its genera, and to understand its basal
Electronic supplementary material The online version of this
article (doi:10.1007/s00606-009-0213-5) contains supplementary
material, which is available to authorized users.
D. A. German (&)
South-Siberian Botanical Garden, Altai State University,
Lenin str. 61, 656049 Barnaul, Russia
e-mail: oreoloma@rambler.ru
N. Friesen H. Hurka
Botanical Garden, University of Osnabru
¨
ck, Albrechtstrasse 29,
49076 Osnabru
¨
ck, Germany
B. Neuffer
Department of Biology, Botany, University of Osnabru
¨
ck,
Barbarastrasse 11, 49076 Osnabru
¨
ck, Germany
I. A. Al-Shehbaz
Missouri Botanical Garden, 2345 Tower Grove,
St. Louis, MO 63110, USA
Present Address:
D. A. German
Department of Functional Genomics and Proteomics, Institute of
Experimental Biology, Masaryk University, Kamenice 5,
Building A2/214, 62500 Brno, Czech Republic
123
Plant Syst Evol
DOI 10.1007/s00606-009-0213-5
members. Substantial progress has been made in the last
few years, and a rather comprehensive framework of
phylogenetic relationships within the Brassicaceae is
emerging (Al-Shehbaz et al. 2006; Bailey et al. 2006;
Beilstein et al. 2006, 2008; Al-Shehbaz and Warwick 2007;
Koch et al. 2007; Warwick et al. 2007, 2008).
The most extensive molecular data set on the Brassica-
ceae is based on DNA sequences from the nuclear ribo-
somal ITS region. Although reliance on ITS as the sole
source of phylogenetic evidence may have its shortcomings,
because of homoplasy, orthology/paralogy, concerted evo-
lution, and alignment problems due to indel accumulation,
it remains the most commonly used marker for generating
family wide, species-level phylogenetic inferences (Koch
et al. 2003; Warwick and Sauder 2005; Bailey et al. 2006;
Warwick et al. 2007, 2008; Khosravi et al. 2009).
Although slightly more than 50% of the 338 genera of the
Brassicaceae have been surveyed for the ITS marker (Bailey
et al. 2006; Warwick et al. 2007, 2008; Khosravi et al. 2009),
most of the narrowly distributed or poorly known genera,
especially from Asia, have not been sampled; one of the
objectives of this study is to bridge that gap. The family has
recently been divided into 25 tribes (Al-Shehbaz et al. 2006),
and ten additional tribes have since been recognized
(Al-Shehbaz and Warwick 2007; German and Al-Shehbaz
2008a;German2009; Koch and Al-Shehbaz 2009). Obviously,
the vast majority of the species and genera of the family belong
to one of these tribes. However, the final number of tribes, the
phylogenetic relationships among them, and the number of their
component genera remain to be clarified. A step in the right
direction is taken herein by the inclusion of many genera not
previously studied, especially from central Asia, one of the most
important centers of species diversity of the Brassicaceae
(Hedge 1976; Appel and Al-Shehbaz 2003). Therefore the final
choice of taxa, aimed at maximum representation of the recently
recognized 28 tribes (Al-Shehbaz et al. 2006; Al-Shehbaz and
Warwick 2007; Warwick et al. 2007) and the seven tribes
(German and Al-Shehbaz 2008a;German2009; Koch and
Al-Shehbaz 2009) added on the basis of this study, provided the
backbone for assignment of the Asian taxa sampled herein.
Tribes either absent or poorly represented in central Asia
(e.g. Aethionemeae, Boechereae, Brassiceae, Halimolobeae,
Heliophileae, Malcolmieae, Physarieae, and Schizopetaleae)
were represented by the fewest species that ensure their
accurate topology in the overall phylogeny of the family.
Materials and methods
Material studied, sequence accession numbers
The sample comprised 184 species in 121 genera of
Brassicaceae, of which 80 species are represented by
original data, including 11 genera and 45 species studied
molecularly for the first time. Taxa, collection data,
vouchers, and EMBL accession numbers are listed in
Table 1. Species names follow the most recent world-wide
checklist of Warwick et al. (2006b) except for subsequent
changes in the generic placement of a few taxa. Sequence
data for other taxa were obtained from published EMBL/
GenBank/DDBJ accessions.
Sequencing, alignment
DNAs from herbarium specimens were isolated with the
NucleoSpin Plant Kit (Macherey–Nagel, Du
¨
ren, Germany)
in accordance with the instructions of the manufacturer and
were used directly in PCR amplifications. Double-stranded
DNA of the complete ITS region, including the 5.8S rDNA
gene, was amplified by 35 cycles of symmetric PCR using
ITS primers 18F and 26R. The PCR procedure involved a
hot start with 2 min at 94°C, and 35 cycles of amplification
(1 min 94°C, 45 s 55°C, 45 s 72°C), final elongation step
for 7 min at 72°C, and storage at 4°C. When amplification
of the complete ITS region was not possible, we used the
primer combination of 18F and ITS2 Primer (White et al.
1990) and 5.8F Primer (5
0
CTT CTG GCC GAG GGC ACG
TC 3
0
) with 26R. In some cases we applied a 5.8S primer
specific for Brassicaceae newly designed by us (Brass-
5.8R: 5
0
-TTGCGTTCAAAGACTCGATG-3
0
; Brass-5.8F:
5
0
-AGCGAAATGCGATACTTGGT-3
0
). The PCR prod-
ucts were separated on an agar gel and puried with the
NucleoSpin Gel Extraction Kit (Macherey–Nagel). After
checking DNA concentration on 1.8% agar gel, about 40 ng
PCR products were used in a 10-ll-cycle sequencing reaction
with the ABI BigDye Terminator Kit (ABI, Foster City, CA,
USA) in accordance with the instructions of the manufac-
turer. Products of the cycle sequencing reactions were run on
an ABI 377XL automatic sequencer. Forward and reverse
sequences from every individual were manually edited and
combined in single consensus sequences. The sequences of
all samples were aligned with CLUSTAL X (Thompson et al.
1997), and the alignment subsequently corrected manually.
Many published sequences from EMBL lacked the 5.8S
region, and to avoid the problems with incomplete sequences
and those that may represent heterogeneous sets of poly-
merase chain reaction products (Bailey et al. 2006), we cut
the 5.8S region from all used sequences. Alignment has been
submitted to the TreeBASE database.
Phylogenetic analyses
Phylogeny was estimated using parsimony and Bayesian
methods. Parsimony analyses were performed in Paup
4.0b10 (Swofford 2002). Heuristic searches were conducted
with simple and with 100 random addition sequences and
D. A. German et al.
123
Table 1 Taxa studied, geographical origin, source (either herbarium specimen or reference to published sequence), and GenBank accession no.
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Aethionema saxatile (L.) R. Br. Germany, Osnabru
¨
ck Bot.
Garden
Mummenhoff et al. (2005) AJ862697, AJ862698
Alliaria petiolata (Bieb.) Cavara &
Grande
Germany, Osnabru
¨
ck Mummenhoff et al. (2005) AJ862703, AJ862704
Alyssum cypricum Nya
´
r. Turkey, Burdur Mengoni et al. (2003) AY237948
Alyssum dasycarpum Steph. ex Willd. Kyrgyzstan Neuffer, Hurka, Friesen
(OSBU 15599a)
FM164502, FM164503
Alyssum klimesii Al-Shehbaz India, Ladakh Klimes
ˇ
2070 (ALTB) FM164504, FM164505
Alyssum lenense Adams Kazakhstan, Tarbagatai mts. Smirnov, German et al. B008
(ALTB)
FM164506, FM164507
Alyssum linifolium Steph. ex Willd. Kyrgyzstan Neuffer, Hurka, Friesen
(OSBU 15599b)
FM164508, FM164509
Alyssum montanum L. Italy, Tuscani Mengoni et al. (2003) AY237938
Alyssum obovatum (C.A. Mey.) Turcz. Russia, East Siberia, Chita
region
Golyakov and Mysik B033
(ALTB)
FM164510, FM164511
Andrzeiowskia cardamine Reichenb. Turkey, Vilayet Bailey et al. (2006) AJ628337, AJ628338
Anchonium elichrysifolium (DC.) Boiss. Turkey Warwick et al. (2007) DQ357514
Aphragmus eschscholtzianus Andrz. ex
DC.
Canada, Yukon Warwick et al. (2006a) DQ165334
Aphragmus involucratus (Bunge)
O.E. Schulz
Mongolia, Gobi Altai mts. Neuffer and Hurka (OSBU
12042)
FM164512, FM164513
Arabidopsis thaliana (L.) Heynh. Czech Republic O’Kane et al. (1996) U43224
Arabis alaschanica Maxim. China, Inner Mongolia, Henan
Shan mts.
Shurun (MO 04505376) FM164642
Arabis alpina L. Germany Koch et al. (1999) AJ232920
Arabis axillaris Kom. Korea, Unchkhen-gan (type) Komarov (LE) FM164643
Arabis blepharophylla Hook. & Arn. USA Koch et al. (1999) AJ232903
Arabis kokanica Regel & Schmalh. Tajikistan, Pamir mts. Ikonnikov 17756 (LE) FM164644
Arabis kamelinii Botsch. Tajikistan, Pamir mts. Ikonnikov 13877 (LE) FM164645
Arabis tibetica Hook. f. & Thomson Kyrgyzstan, Alai mts. Neuffer, Hurka, Friesen
(OSBU 15740)
FM164514, FM164515
Asperuginoides axillaris
(Boiss. &
Hohen.) Rauschert
Uzbekistan Vas
ˇ
a
´
k (WAG 0143231) FM164516, FM164517
Atelanthera perpusilla Hook.f. &
Thomson
Kyrgyzstan, Alai valley Shmakov et al. B068a (ALTB) FM164518, FM164519
Aubrieta deltoidea (L.) DC. Germany, Bot. Garden Jena Koch et al. (1999) AJ232909
Aurinia saxatilis (L.) Desv. [as Alyssum
saxatile L.]
Heenan et al. (2002) AF401115
Berteroa incana (L.) DC. Germany, Bot. Garden
Osnabru
¨
ck
Friesen (OSBU 16535) FM164646
Berteroella maximowiczii (Palib.)
O.E. Schulz
NE China Zimmermann 424 (P) FM164526, FM164527
Biscutella laevigata L. Europa Bailey et al. (2006) DQ452056
Boechera pinetorum (Tidestrom.)
Windham & Al-Shehbaz [incorrectly
reported as Arabis holboellii Hornem.]
USA, Nevada Roy (2001) AF183117
Boechera stricta (Graham) Al-Shehbaz
[as Arabis drummondii A. Gray]
USA, Wyoming Koch et al. (1999) AJ232887
Boreava orientalis Jaub. & Spach Turkey, Antalya Koch et al. (2007) DQ249859
Borodinia macrophylla (Turcz.) O.E.
Schulz
Russia, Far East Kharkevich and Vyshin 79
(LE)
FM164528, FM164529
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Table 1 continued
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Botschantzevia karatavica
(Lipsch. & Pavlov) Nabiev
Kazakhstan, Karatau Vvedensky and Kovalevskaya
73 (LE)
FM164530, FM164531
Brassica juncea (L.) Czern. Cultivated Yang et al. (1999) AF128093
Braya humilis (C.A. Mey.) Robins.
[as Neotorularia humilis (C.A. Mey.)
Hedge & J. Le
´
onard]
China O’Kane and Al-Shehbaz
(2003)
AF137566
Bunias cochlearioides Murr. (1) Kazakhstan, NW Sumnevich and Serkin (TK) FM958513
Bunias cochlearioides (2) Russia, SW Siberia Burdakova (TK) FM958514
Bunias orientalis L. (1) Germany, Osnabru
¨
ck Neuffer (OSBU 205) FM958515
Bunias orientalis (2) Russia, Altai Neuffer, Hurka, Friesen
(OSBU 13556)
FM958516
Bunias orientalis (3) Botanic Garden Dijon Koch et al. (2007) DQ249863
Calepina irregularis (Asso) Thell. Spain Francisco-Ortega et al. (1999) AF039995, AF040039
Calymmatium draboides (Korsh.)
O.E. Schulz (1)
Tajikistan, East Pamir Kuznetsov and Varivtseva s.n.
(MW)
FM958511
Calymmatium draboides (2) Tajikistan, Badakhshan Tzvelev (LE) FM958512
Camelina microcarpa Andrz. ex DC. Romania O’Kane and Al-Shehbaz
(2003)
AF137574
Capsella rubella Reut. Spain, Andaluz near Cadiz Neuffer 690 (Capsella-
collection Osnabru
¨
ck,
OSBU)
FM164647
Cardamine bellidifolia L. Russia, Altai mts. Neuffer, Hurka, Friesen
(OSBU 13254)
FM164648
Catolobus pendula (L.) Al-Shehbaz
[as Arabis pendula L.]
Russia, Far East O’Kane and Al-Shehbaz
(2003)
AF137572
Caulanthus inflatus S. Wats. USA, California Pepper and Norwood (2001) AF346653
Chorispora bungeana Fisch. & Mey. Kazakhstan Warwick et al. (2007) DQ357520
Chorispora macropoda Trautv. Afghanistan Warwick et al. (2007) DQ357522
Chrysochamela velutina Boiss. Botanic Garden, University of
Copenhagen
Koch et al. (
2007) DQ249856
Cithareloma lehmannii Bunge Iran Warwick et al. (2007) DQ357528
Clausia aprica (Steph). Korn.-Tr. Russia, East Europe Franzke et al. (2004) AY546110, AY546139
Clausia kazakhorum Pavlov Kazakhstan Warwick et al. (2007) DQ357530
Clausia trichosepala (Turcz.) Dvor
ˇ
a
´
k Mongolia, Eren-Daba mts. Kamelin, Gubanov et al. 754
(MW)
AJ628317, AJ628318
Clypeola aspera Turrill Iran Warwick et al. (2008) EF514642
Cochlearia danica L. Germany, Sylt Mummenhoff and Mu
¨
hlhausen
unpublished
AJ628283, AJ628284
Conringia perfoliata (C.A. Mey.) N.
Busch
Iran Warwick and Sauder (2005) AY722505
Crambe arborea Webb ex H. Christ Spain, Canary Islands, Tenerife Francisco-Ortega et al. (2002) AF450012
Crucihimalaya mollissima (C.A. Mey.)
Al-Shehbaz, O’Kane & R.A. Price
Afghanistan O’Kane and Al-Shehbaz
(2003)
AF137552
Crucihimalaya rupicola (Kryl.) A.L. Ebel
& D. German
Mongolia, Altai mts. German B056 (ALTB) FM164538, FM164539
Crucihimalaya wallichii (Hook.f. &
Thomson) Al-Shehbaz, O’Kane & R.A.
Price
Japan, Myagi University,
Sendai seed centre
Koch et al. (1999) AJ131396
Degenia velebitica (Degen) Hayek Hungary Warwick et al. (2008) EF514646
Descurainia pinnata (Walter) Britton USA, Utah Roy (2001) AF183122
D. A. German et al.
123
Table 1 continued
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Dichasianthus subtilissimus (M. Pop.)
Ovcz. & Junussov
Tajikistan O’Kane and Al-Shehbaz
(2003)
AF137594
Didymophysa fedtschenkoana Regel &
Schmalh.
Kyrgyzstan, Alai mts. Neuffer, Hurka, Friesen
(OSBU 15733)
FM164544, FM164545
Dilophia salsa Thomson Kyrgyzstan, Alai mts. Neuffer, Hurka, Friesen
(OSBU 16157)
FM164649
Diptychocarpus strictus (Fisch.) Trautv. Afghanistan Warwick et al. (2007) DQ357535
Dontostemon crassifolius Bunge Mongolia, O
¨
mno
¨
govi Batlai Oyuntsetseg (OSBU
12356)
AY558923, AY558951
Dontostemon dentatus (Bunge) Ledeb. Japan, Xoncio Anonymous (VLA) AY558915, AY558943
Dontostemon pinnatifidus (Willd.)
Al-Shehbaz & H. Ohba
Mongolia, Ho
¨
vsgol lake Gubanov et al. (MW) AY558935, AY558963
Dontostemon tibeticus (Maxim.)
Al-Shehbaz
China, Quinghai Ho et al. (MO) AY558942, AY558970
Draba doerfleri Wettst. Bot. Garden Osnabru
¨
ck (seeds
from Serbia)
Neuffer (OSBU 142) FM164650
Draba hystrix Hook.f. & Thomson Pakistan Koch and Al-Shehbaz
unpublished
AY134194
Draba oreades Schrenk Mongolia, Altai Hurka and Neuffer (OSBU
10433)
FM164651
Draba sibirica (Pall.) Thell. Russia, Altai Hurka, Neuffer, Friesen
(OSBU 12973)
FM164652
Draba nuda (Be
´
lang.) Al-Shehbaz &
M. Koch [as Drabopsis nuda (Be
´
lang.)
Stapf]
Iran O’Kane and Al-Shehbaz
(2003)
AF137577
Draba verna L. [as Erophila spathulata
La
´
ng]
Austria Koch and Al-Shehbaz (2002) AF377952
Eruca vesicaria (L.) Cav. subsp. sativa
(Mill.) Thell. [as E. sativa Mill.]
No locality given Francisco-Ortega et al. (1999) AF039996, AF040037
Erysimum cheiranthoides L. USA Kress et al. (
2005) DQ005989
Erysimum mongolicum D. German Mongolia, Altai mts. (type) German (OSBU 14802) FM164520, FM164521
Erysimum siliculosum (Bieb.) DC. Kazakhstan Smirnov and Antonyuk (OSBU
14155)
FM164522, FM164523
Euclidium syriacum (L.) W.T. Aiton Iran Warwick et al. (2007) DQ357544
Eutrema altaicum (C. A. Mey.)
Al-Shehbaz & Warwick
Russia, Altai Hurka, Neuffer, Friesen
(OSBU 13363)
FM164653
Eutrema botschantzevii (D. German)
Al-Shehbaz & Warwick
Russia, South-West Siberia
(type)
Shaulo et al. (NS) FM164654
Eutrema edwardsii R. Br. Mongolia, Altai, Tavan Bogd Hurka and Neuffer (OSBU
10405)
FM164655
Eutrema parvulum (Schrenk) Al-Shehbaz
& Warwick [as Thellungiella parvula
(Schrenk) Al-Shehbaz & O’Kane]
Turkey O’Kane and Al-Shehbaz
(2003)
AF137579
Fibigia clypeata (L.) Medik. Botanic Garden Bordeaux Koch et al. (2007) DQ249852
Fibigia spathulata (Kar. & Kir.)
B. Fedtsch.
Kazakhstan, Arkaly mts. Botschantzev and
Botschantzeva 1058 (LE)
FM164656
Fibigia suffruticosa (Vent.) Sweet Turkey Mummenhoff (OSBU 18314) FM164657
Fourraea alpina (L.) Greuter & Burdet
[as Arabis pauciflora (Grimm) Garcke]
Germany, Thu
¨
ringen, Jena Koch et al. (1999) AJ232890
Galitzkya potaninii (Maxim.) V. Bocz. Mongolia, Dzungarian Gobi German B161 (ALTB) FM164524, FM164525
Galitzkya spathulata (Steph. ex Willd.)
V. Bocz.
Russia, Altai foothills German et al. B163a (ALTB) FM164534, FM164535
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Table 1 continued
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Goldbachia pendula Botsch. Mongolia, Dzungarian Gobi Smirnov, German et al. A052
(OSBU 14762)
FM164536, FM164537
Halimolobos diffusus (A. Gray)
O.E. Schulz
USA, Nevada O’Kane and Al-Shehbaz
(2003)
AF137567
Heliophila arenaria Sond. South Africa Mummenhoff et al. (2005) AJ863600, AJ864811
Heliophila coronopifolia L. South Africa Mummenhoff et al. (2005) AJ863596, AJ864814
Hesperis laciniata All. Morocco Mummenhoff et al. (2005) AJ628315, AJ628615
Hesperis sibirica L. Russia, Altai Neuffer, Hurka, Friesen
(OSBU 13609)
FM164658
Hormathophylla purpurea (Lag. & Rodr.)
P. Ku
¨
pfer
Spain, Granada Wieringa 2730 (WAG
0061830)
FM164546, FM164547
Hornungia petraea (L.) Reichenb. Morocco Staudinger (OSBU 13661) FM164548, FM164549
Ianhedgea minutiflora (Hook.f. &
Thomson) Al-Shehbaz & O’Kane
Afghanistan O’Kane and Al-Shehbaz
(2003)
AF137568
Iberis amara L. Botanical Garden University of
Mainz, Germany
Kropf et al. (2003) AJ440311
Ionopsidium prolongoi (Boiss.) Batt. Spain, Cadiz Bailey et al. (2006) AJ628303, AJ628304
Irenepharsus magicus Hewson Australia, NSW, South Coast Telford 9644 (CANB, CBG 84-
00-326)
FM164550, FM164551
Isatis tinctoria L. [as I. indigotica Fortune
ex Lindl.]
China, Jiangsu Bailey et al. (2006) AF384104
Iskandera alaica (Korsh.) Botsch. &
Vved.
Kyrgyzstan, Alai mts. Neuffer, Hurka, Friesen
(OSBU 15699)
FM164659
Kernera saxatilis (L.) Sweet subsp.
boissieri (Reut.) Nyman
Spain Kropf et al. (2003) AJ440313
Leiospora exscapa (C.A. Mey.) Dvor
ˇ
a
´
k Mongolia, Mongolian Altai
mts.
Hurka and Neuffer (OSBU
10410)
FM164552, FM164553
Leiospora pamirica (Botsch. & Vved.)
Botsch. & Pachom.
Central Asia Warwick et al. (
2007) AJ628329, AJ628330
Lepidium draba L. Romania Bot. Garden Jasi, Romania
(OSBU 95-0334-10)
FM164554, FM164555
Lepidium ruderale L. Germany, Oldenburg Mummenhoff et al. (2004) AJ582465, AJ582513
Lepidium paniculatum (Regel &
Schmalh.) Al-Shehbaz
Kyrgyzstan, Tin Shan Neuffer, Hurka, Friesen
(OSBU 15546)
FM164556, FM164557
Leptaleum filifolium (Willd.) DC. Turkmenistan Kutafiev and Shmakov (OSBU
14773)
FM164560, FM164561
Litwinowia tenuissima (Pall.)
Woron. ex Pavlov
Mongolia, Dzungarian Gobi Smirnov, German et al. A064
(OSBU 14774)
FM164562, FM164563
Lyrocarpa coulteri Hook. & Harv. Mexico O’Kane and Al-Shehbaz
(2003)
AF137591
Macropodium nivale (Pall.) W.T. Aiton Russia, Altai mts. Neuffer, Hurka, Friesen
(OSBU 12946)
FM164660
Malcolmia littorea (L.) W.T. Aiton Portugal Warwick et al. (2007) DQ357559
Mancoa bracteata (S. Wats.) Rollins Mexico Bailey et al. (2002) AF307633
Matthiola incana (L.) W.T. Aiton France, Bretagne Hurka (OSBU 96-0180-10) AJ628339, AJ628340
Matthiola lunata DC. Algeria Go
´
mez-Campo (OSBU 91-50-
0053-10)
AJ628341,AJ628342
Megacarpaea megalocarpa (Fisch. ex
DC.) Schischk. ex B. Fedtsch.
Mongolia, Dzungarian Gobi Smirnov, German et al. A066
(OSBU 14776)
FM164564, FM164565
Megadenia pygmaea Maxim. Russia, Buryatia Popov and Bardunov (NSK) FM164661
Microstigma brachycarpum Botsch. China Warwick et al. (2007) DQ357569
D. A. German et al.
123
Table 1 continued
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Microstigma deflexum (Bunge) Juz. Mongolia, Gobi Altai Batlai Oyuntsetseg 631 (OSBU
14828)
FM165293, FM165294
Microthlaspi perfoliatum (L.) F.K. Mey. Russia, Astrakhan Neuffer (OSBU 8811) FM164566, FM164567
Nasturtium microphyllum (Boenn. ex
Reichenb.) Reichenb.
Denmark, Ju
¨
tland, Helgenaes Franzke et al. (1998) AF078029, AF078030
Neotorularia torulosa (Desf.) Hedge &
J. Le
´
onard
Iran O’Kane and Al-Shehbaz
(2003)
AF137571
Neurotropis szowitsiana (Boiss.) F.K.
Mey. [as Thlaspi szowitsianum Boiss.]
Caucasus Koch and Mummenhoff (2001) AF336174, AF336175
Noccaea caerulescens (J. & C. Presl) F.K.
Mey.
Germany, Sauerland Koch (OSBU 95-0052-10) FM164568, FM164569
Olimarabidopsis cabulica (Hook.f. &
Thomson) Al-Shehbaz, O’Kane & R.A.
Price
Kyrgyzstan O’Kane and Al-Shehbaz
(2003)
AF137548
Olimarabidopsis pumila (Steph.)
Al-Shehbaz, O’Kane & R.A. Price
Arabidopsis Seed Stock Center,
Sendai, Japan
Johnston et al. (2005) AY662277
Oreoloma matthioloides (Franch.)
Botsch.
China Warwick et al. (2007) DQ357574
Oreoloma sulfureum Botsch. China, Dzungaria, Argalty mts. Rus/Sin Altai Exped. 2004-002
(ALTB)
FM164572, FM164573
Oreoloma violaceum Botsch. Mongolia, Dzungarian Gobi Smirnov, German et al. A089
(OSBU 14799a)
FM164574, FM164575
Orychophragmus violaceus (L.)
O.E. Schulz
E Asia Warwick and Sauder (2005) AY722506
Pachycladon wallii (Carse) Heenan &
A.D. Mitchell [as Cheesemania wallii
(Carse) Allan]
New Zealand Mitchell and Heenan (2000) AF100681
Pachyneurum grandiflorum (C.A. Mey.)
Bunge (1)
Mongolia, Mongolian Altai German A071 (OSBU 1481) FM164576, FM164577
Pachyneurum grandiflorum (2) Russia, Altai Neuffer, Hurka, Friesen
(OSBU 13117)
FM164578, FM164579
Parrya asperrima (B. Fedtsch.) M. Pop. Kyrgyzstan, Tian Shan Neuffer, Hurka, Friesen
(OSBU 16525)
FM164580, FM164581
Parrya lancifolia M. Pop. Kyrgyzstan, Terskei Alatau Lazkov (FRU) FM164582, FM164583
Parrya pulvinata M. Pop. Kazakhstan Warwick et al. (2007) DQ357579
Parrya stenocarpa Kar. & Kir. Kyrgyzstan, Terskei Alatau Lazkov s.n. (FRU) FM991736
Physaria didymocarpa (Hook.) A. Gray USA, Montana O’Kane and Al-Shehbaz
(2003)
AF137583
Pseudoarabidopsis toxophylla (Bieb.)
Al-Shehbaz, O’Kane & R.A. Price
Kazakhstan O’Kane and Al-Shehbaz
(2003)
AF137558
Pseudoclausia olgae (Regel & Schmalh.)
Botsch.
Uzbekistan, Nuratau Pimenov et al. (MW) FM991737
Pseudoclausia turkestanica (Lipsky)
A. Vassil.
Kyrgyzstan, Tian Shan Lazkov (FRU) FM164584, FM164585
Pseudosempervivum aucheri (Boiss.)
Pobed. [as Cochlearia aucheri Boiss.)]
Turkey, Tossia Koch and Mummenhoff (2001) AF336202, AF336203
Pseudoturritis turrita (L.) Al-Shehbaz
[as Arabis turrita L.)]
Bot. Gard. Berlin-Dahlem Koch et al. (1999) AJ232905
Ptilotrichum dahuricum Peschkova Mongolia, Pre-Hingan upland Gubanov et al. 1062 (MW) FM164588, FM164589
Ptilotrichum tenuifolium (Steph.)
C.A. Mey.
Russia, East Siberia, Baikal
lake
Bergmann (OSBU 14876) FM164590, FM164591
Pugionium pterocarpum Kom. Russia, Tuva Lomonosova and Danilov (NS) FM164592, FM164593
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Table 1 continued
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Rhammatophyllum fruticulosum (C.A.
Mey.) Al-Shehbaz
Kazakhstan, Tin Shan Smirnov, German et al. B061
(ALTB)
FM164594, FM164595
Rhammatophyllum kamelinii (Botsch.)
Al-Shehbaz & O. Appel
Mongolia, Dzungarian Gobi Smirnov, German et al. A075
(OSBU 14785)
FM164596, FM164597
Rorippa sylvestris (L.) Bess. Germany, Lower Saxony, Bad
Rothenfelde
Franzke et al. (1998) AF078023, AF078024
Sandbergia perplexa (L.F. Hend.)
Al-Shehbaz [as Halimolobos perplexus
(L.F. Hend.) Rollins]
USA, Idaho Koch et al. (1999) AJ232926
Sisymbriopsis yechengica (Z.X. An)
Al-Shehbaz, Z.X. An & G. Yang
China Warwick et al. (2004a) AY353161
Sisymbrium altissimum L. USA, California Francisco-Ortega et al. (1999) AF039997, AF040040
Sisymbrium brassiciforme C.A. Mey. India, Kashmir Koch et al. (2007) DQ518399
Sisymbrium linifolium (Nutt.) Nutt. [as
Schoenocrambe linifolia (Nutt.) Greene]
USA, Colorado Roy (2001) AF183088
Smelowskia flavissima (Kar. & Kir.) Kar.
& Kir.
Kazakhstan, Tarbagatai mts. Smirnov, German et al. A085
(OSBU 14795)
FM164598, FM164599
Smelowskia jacutica (Botsch. & Karav.)
Al-Shehbaz & Warwick [as Gorodkovia
jacutica Botsch. & Karav.]
Russia Warwick et al. (2004b) AY230646
Smelowskia tibetica (Thomson) Lipsky
[as Hedinia tibetica (Thomson) Ostenf.]
China Warwick et al. (2004b) AY230607
Solms-laubachia flabellata (Regel) J.P.
Yue, Al-Shehbaz & H. Sun [as
Desideria flabellata (Regel)
Al-Shehbaz]
Kyrgyzstan, Alai mts. Neuffer, Hurka, Friesen
(OSBU 15734)
FM164542, FM164543
Sphaerocardamum nesliiforme Shauer Mexico Bailey and Doyle (1999) AF055195
Spirorhynchus sabulosus Kar. & Kir. China, Dzungaria Rus/Sin Altai Exped. 2004-159
(ALTB)
FM164600, FM164601
Sterigmostemum caspicum (Lam.) Rupr. Kazakhstan, Arkaly mts. Smirnov, German et al. A087
(OSBU 14797)
FM164610, FM164611
Sterigmostemum fuhaiense H.L. Yang China, Altai mts. Rus/Sin Altai Exped. 2004-304
(ALTB)
FM164612, FM164613
Sterigmostemum ramosissimum (O.E.
Schulz) Rech.f.
Turkmenistan Shmakov et al. B238 (ALTB) FM164614, FM164615
Sterigmostemum schmakovii Kamelin &
D. German
Kazakhstan, Zaissan
depression
Smirnov, German et al. A088
(OSBU 14798)
FM164616, FM164617
Stevenia alyssoides Adams ex Fisch. Mongolia, Ho
¨
vsgol lake Gubanov 5226 (MW) FM164602, FM164603
Stevenia cheiranthoides DC. Mongolia, Pre-Hingan upland Kamelin et al. 1633 (MW) FM164604, FM164605
Stevenia incarnata (Pall. ex DC.)
Kamelin
Russia, East Siberia, Baikal
lake
Bergmann (OSBU 14841) FM164606, FM164607
Stevenia sergievskajae (Krasnob.)
Kamelin & Gubanov
Mongolia, Ho
¨
vsgol lake Gubanov et al. (MW) FM164608, FM164609
Streptanthus albidus Greene subsp.
peramoenus (Greene) Kruckeb
USA, California Pepper and Norwood (2001) AF346651
Streptoloma desertorum Bunge Turkmenistan, Ashkhabad
distr., Annau
Zhilenko (MO 3149466) FM164618, FM164619
Strigosella africana (L.) Botsch. [as
Malcolmia africana (L.) W.T. Aiton]
USA, Utah Warwick et al. (2007) AY237307
Strigosella brevipes (Bunge) Botsch. Mongolia, Dzungarian Gobi Warwick et al. (2007) AY558940, AY558968
Stubendorffia gracilis (Pavlov) Botsch.
& Vved.
Kazakhstan Bailey et al. (2006) DQ780944, DQ780945
D. A. German et al.
123
TBR branch swapping. Maximum tree limit was set at 50,000
most parsimonious trees. Statistical support of the branches
of the 50% majority-rule consensus tree was tested by
Bootstrap (1,000 replicates) and Jackknife (100 replicates)
analyses. Bayesian inference used the program MrBayes 3.1
(Ronquist and Huelsenbeck 2003). Sequence evolution
models were evaluated using the Akaike information crite-
rion with the aid of Modeltest 3.7 (Posada and Crandall
1998). The GTR?I?G model was chosen as the best fit.
There were two independent runs each of eight chains,
10 million generations, sampling every 100 trees, and 25%
of initial trees were discarded as burn-in. The remaining
28,000 trees were combined into a single data set, and a
majority-rule consensus tree was obtained. Bayesian pos-
terior probabilities were calculated for that tree in MrBayes
3.1.
Cleome lutea Hook. was used as outgroup, because
Cleomaceae was shown to be the sister family to the
Brassicaceae (Hall et al. 2002, 2004).
Results
Sequence data
The aligned ITS data matrix consisted of 473 characters
across 184 Brassicaceae taxa and Cleome lutea (outgroup)
(GenBank numbers are given in Table 1). Sixty-two char-
acters were constant, and of the 412 variable characters,
370 were potentially parsimony informative. Problems
were caused by Bunias orientalis L. (but not by B. coch-
learioides Murr.): in the two samples studied by us, PCR
amplification resulted in three fragments instead of one, as
for all other accessions studied, two weak bands of ca. 500
and 700 bp, respectively, and one stronger band of ca.
1,250 bp. Direct sequencing of the 700-bp fragment failed
except for ITS 2 when using the primer 26R (as for the
500-bp fragment, see below.) Sequencing the 1,250 bp
fragment revealed a sequence of 1,162 bp. The first 111 bp
were homologous with the beginning of the Brassicacean
ITS1 region, whereas the last 337 bp of the Bunias orien-
talis 1,162 bp sequence were homologous with the 5.8S
region and with the complete ITS2 region. The segment
from position 112 to position 825 (length 714 bp) is of
unknown homology. BLAST search gave no results. For
the alignment of the Bunias orientalis sequences, this
714 bp segment was discarded, and only the first 111 bp of
the ITS1 region and the ITS2 region were taken into
account. The Bunias orientalis sequence accession no.
DQ249863 taken from GenBank (Table 1) was submitted
by Koch et al. (2007). In their analysis, PCR amplification
resulted in a ca. 500-bp fragment, which corresponded to
our 500-bp fragment. The first 111 bp were homologous
with the beginning of ITS1, and the last 337 bp
Table 1 continued
Species Geographical origin Collector (herbarium)
or reference to published
sequences
GenBank
accession
no.
Synthlipsis greggii A. Gray Mexico, Nuevo Leon O’Kane and Al-Shehbaz
(2003)
AF137590
Tauscheria lasiocarpa Fisch. ex DC. Kazakhstan, Altai mts. Smirnov, German et al. B258
(ALTB)
FM164620, FM164621
Teesdalia nudicaulis (L.) R. Br. Germany, Bramsche Koch and Mummenhoff (2001) AF336214, AF336215
Thlaspi alliaceum L. Bot. Garden Du
¨
sseldorf,
Germany
Koch and Mummenhoff (2001) AF336156, AF336157
Transberingia bursifolia (DC.) Al-
Shehbaz & O’Kane [as Beringia
bursifolia (DC.) O’Kane, Al-Shehbaz &
R.A. Price]
Russia O’Kane and Al-Shehbaz
(2003)
AF137557
Turritis glabra L. [as Arabis glabra (L.)
Bernh.]
Germany Koch et al. (1999) AJ232925
Yinshania henryi (Oliv.) Y.H. Zhang China, Hubei, Shenlngjia Koch et al. (1999) AJ232930
Werdermannia anethifolia (Phil.) I.M.
Johnst.
Chile Warwick et al. (2002) AF531645
Zilla spinosa (L.) Prantl subsp.
macroptera (Coss.) Maire & Weiller
No locality given Crespo et al. (2000) AF263397
Zuvanda crenulata (DC.) Askerova Israel Warwick et al. (2007) DQ357606
Cleome lutea Hook. USA, Utah O’Kane and Al-Shehbaz
(2003)
AF137588
Herbaria acronyms according to Index Herbariorum
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
homologous with the 5.8S region and ITS2, comparable
with our results with the 1,250 bp fragment, although with
a much shorter insertion which was also of unknown
homology.
Apart from the problems with Bunias orientalis, we had
difficulties with the alignment of species of the tribe
Chorisporeae, except for species of Chorispora R.Br. ex
DC. An insertion of about 48 bp at the beginning of the
ITS2 region (position no. 322–369) prevented unequivocal
alignment. This region was cut off, and the alignment we
were working with is based on 473 bp. The alignment is
given in Supplement 2.
Phylogenetic analyses
The 50,000 most parsimonious trees had a length of 4,482
steps and a consistency index CI = 0.2048 (CI = 0.1951
excluding uninformative characters) and retention index
RI = 0.6319. These values are in the range of previously
studied family or ‘lineage’ wide ITS phylogenies (e.g.
Bailey et al. 2006; Warwick et al. 2007). The strict con-
sensus tree is available in Supplement 1 along with results
of Bootstrap and Jackknife analyses. Figure 1 presents the
Bayesian majority-rule consensus tree of the 28,000 post-
burn-in trees from that analysis. Bayesian posterior prob-
abilities and bootstrap supports from the parsimony anal-
yses are shown on the branches of the tree (see numbers
and asterisks above branches in Fig. 1; for Jackknife
support values, see Supplement 1).
Tree topology
Both Bayesian and parsimony analyses supported largely
congruent topologies (Fig. 1). In general, the tree topology
(Fig. 1) agrees fairly well with the molecular phylogenies
published to date (Koch 2003; Koch et al. 2003; Bailey
et al. 2006; Beilstein et al. 2006; Warwick et al. 2008;
Khosravi et al. 2009). Aethionema W.T. Aiton is sister to
an unresolved basal polytomy including the rest of the
family. Most of the tribes can be assigned to the three ndhF
‘lineages’ of Beilstein et al. (2006, 2008) which are also
supported by data from other markers (Koch et al. 2007;
Franzke et al. 2009; Lysak et al. 2009). The ‘core group’
of lineage I includes Camelineae sensu Al-Shehbaz et al.
(2006), Physarieae, Halimolobeae, and Boechereae, and is
mirrored in Fig. 1. The assignment of other tribes of this
lineage (Cardamineae, Lepidieae, Descurainieae, Sme-
lowskieae) was well supported by Bayesian values only
(Fig. 1). Lineage II comprises the tribes Brassiceae, Isati-
deae, Schizopetaleae, and Sisymbrieae, and this grouping is
fully supported by our analysis as is lineage III (Chori-
sporeae, Hesperideae, Euclidieae, Anchonieae; the last two
tribes sensu Al-Shehbaz et al. 2006) (Fig. 1).
The tree topology clearly support the view that the
Camelineae sensu Al-Shehbaz et al. (2006) is polyphyletic,
which agrees with Bailey et al. (2006), Clauss and Koch
(2006), Koch et al. (2007), Lysak et al. (2009), and some
earlier works (Heenan et al. 2002). The ITS data also show
that the Arabideae sensu Al-Shehbaz et al. (2006) is split into
two strongly supported monophyletic subclades (Fig. 1)
revealed in earlier studies (Heenan et al. 2002; Koch et al.
1999, 2000, 2001; Koch 2003). The study also supports the
recent recognition of the new tribes Aphragmeae, Biscu-
telleae, Buniadeae, Calepineae, Conringieae, Dontostemo-
neae, Erysimeae, Malcolmieae, Megacarpaeeae, and
Turritideae (Al-Shehbaz and Warwick 2007; German and
Al-Shehbaz 2008a; German 2009). Our data also argue for
tribal replacement of different genera and for the expansion
of some genera to include additional species or hitherto
independent genera. Monophyly of tribes and genera, tribal
boundaries, and component genera are discussed below,
especially when contradicting earlier work (Al-Shehbaz
et al. 2006; Bailey et al. 2006; Warwick et al. 2007, 2008;
Al-Shehbaz and Warwick 2007).
Discussion
It is well known that ITS-based phylogenies might be biased
because of homoplasy, orthology/paralogy, concerted evo-
lution, and alignment problems due to indel accumulation.
We also realize possible incongruencies between phyloge-
nies derived from different genomes (i.e., organelle versus
nuclear genomes), which, however, currently appear as only
minor in the Brassicaceae (Koch et al. 2007; Franzke et al.
2009). We are fully aware of all these possible shortcom-
ings, which, in general and often forgotten, more or less
apply to any molecular marker. Family-wide molecular
phylogenies in the Brassicaceae show major similarities in
the analyses of multiple molecular markers, and the overall
tree topology of our ITS data (Fig. 1) agrees fairly well with
the molecular phylogenies published to date: basal split
between Aethionemeae and the rest of the family; lack of
significant resolution of deeper nodes; assignment of the
majority of tribes to the three lineages of Beilstein et al.
(2006) and Al-Shehbaz et al. (2006) (section Tree topol-
ogy’). Some minor discrepancies compared with the phy-
logenies published to date are predominantly caused, as
tested by us at the preliminary stages of tree construction, by
differences in the taxon sampling, and, to much lesser
degree, by differences between alignments. This led us to
assume that our data, our alignments, and our phylogenetic
analyses are accurate and meaningful. The following dis-
cussion is not only based on molecular data but includes
evaluation of morphology and biogeography as important
and independent data sets.
D. A. German et al.
123
0.85
0.86
**
**
**
Cardamine bellidifolia
Nasturtium microphyllum
Rorippa sylvestris
Andrzeiowskia cardamine
Cardamineae
Lepidium paniculatum
Lepidium ruderale
Lepidium draba
St b d ffi ili
Lepidieae
(a)
1.00
**
**
**
**
*
*
Stubendorffia gracilis
Ianhedgea minutiflora
Descurainia pinnata
Hornungia petraea
Descurainieae
Smelowskia jacutica
Smelowskia flavissima
Smelowskia tibetica
Yinshania henryi
Smelowskieae
Unresolved
Crucihimalaya wallichii
Arabis tibetica
0.85
**
**
**
**
**
Lyrocarpa coulteri
Synthlipsis greggii
Physaria didymocarpa
Physarieae
Irenepharsus magicus
Pachycladon wallii
Ex-Camelineae II
Arabis tibetica
Crucihimalaya mollissima
Transberingia bursifolia
Crucihimalaya rupicola
Alyssum klimesii
Ex-Camelineae I
Calymmatium draboides 1
**
*
*
**
**
*
***
Pachycladon wallii
Halimolobos diffusus
Turritis glabra
Mancoa bracteata
Halimolobeae
Sphaerocardamum nesliiforme
Boechera pinetorum
Boechera stricta
Borodinia macrophylla
Sandbergia perplexa
Boechereae
Turritideae (Ex-Camelineae III)
0.97
Calymmatium draboides 1
Olimarabidopsis pumila
*
*
*
*
**
**
**
**
Olimarabidopsis cabulica
Arabidopsis thaliana
Camelineae
Calymmatium draboides 2
Chrysochamela velutina
Catolobus pendulus
Camelina microcarpa
Pseudoarabidopsis toxophylla
Capsella rubella
**
Erysimum mongolicum
Erysimum siliculosum
Erysimum cheiranthoides
Erysimeae (Ex-Camelineae IV)
Aethionema saxatile
Cleome lutea
Outgroup
Aethionemeae
Capsella rubella
Fig. 1 ac Phylogenetic tree resulting from a Bayesian analysis of
the ITS sequences of the 184 Brassicaceae species from 121 genera
and Cleome lutea as outgroup taxon. For taxa origins, etc., see
Table 1. The taxa represent all the 35 currently recognized tribes, and
their tribal assignment is given on the right. Asterisks above branches
indicate statistical support (**Bayesian posterior probabilities (BI)
C0.90, bootstrap values (BS) C90%; *BI between 0.80 and 0.90, BS
between 75 and 90%). Some branches are only supported by BI
(shown as figures on branches if C0.80) but not by BS C75%
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Tribe Camelineae DC.
The Camelineae s.l. were split into five clades intermixed
with the tribes Boechereae, Halimolobeae, and Physarieae
(Fig. 1) to form a well-resolved major clade corresponding
in part to lineage I of Beilstein et al. (2006, 2008). Erysi-
mum L., which was also assigned to the Camelineae by Al-
Shehbaz et al. (2006), formed an independent, well-
resolved clade within the family polytomy (Fig. 1); our
preliminary analyses were consistent with results summa-
rized by Koch and Al-Shehbaz (2009) regarding its closer
relationship to the tribes Descurainieae, Smelowskieae,
Lepidieae, and Cardamineae. The molecular and morpho-
logical data (German and Al-Shehbaz 2008a) clearly
*
**
Isatideae
Boreava orientalis
Isatis tinctoria
*
*
**
**
Tauscheria lasiocarpa
Sisymbrieae
Sisymbrium brassiciforme
Sisymbrium altissimum
Sisymbrium linifolium
Schizopetaleae
Werdermannia anethifolia
Streptanthus albidus subsp. peramoenus
Caulanthus inflatus
Fourraea alpina
Brassiceae
Crambe arborea
Brassica juncea
Eruca vesicaria subsp sativa
(b)
0,99
*
**
**
**
**
Eutremeae
Eutrema edwardsii
Eutrema altaicum
Eutrema botschantzevii
Eruca vesicaria subsp. sativa
Zilla spinosa subsp. macroptera
Orychophragmus violaceus
Eutrema parvulum
Brassiceae?
Thlaspideae
Alliaria petiolata
Thlaspi alliaceum
Didymophysa fedtschenkoana
Pseudosem
pervivum aucheri
Eutremeae?
0,93
Bunias cochlearioides 1
Bunias cochlearioides 2
*
*
*
*
**
**
**
**
Megadenia pygmaea
Bi t ll
Calepineae
Noccaeeae
Conringieae
Spirorhynchus sabulosus
Goldbachia pendula
Calepina irregularis
p
Noccaea caerulescens
Microthlaspi perfoliatum
Zuvanda crenulata
Conringia perfoliata
Neurotropis szowitsiana
*
**
**
**
**
**
Biscutella laevigata
Megadenia pygmaea
Biscutelleae
Megacarpaeeae
Pugionium pterocarpum
Megacarpaea megalocarpa
Heliophileae
Heliophila arenaria
H li hil if li
Cochlearieae
Cochlearia danica
Ionopsidium prolongoi
Aphragmus eschscholtzianus
Aphragmus involucratus
Cithareloma lehmannii
Malcolmia littorea
Malcolmieae
Aphragmeae
Berteroa incana
*
**
**
*
Heliophileae
Heliophila coronopifolia
Iberideae
Teesdalia nudicaulis
Iberis amara
Asperuginoides axillaris
Kernera saxatilis subsp. boissieri
Galitzkya spathulata
Galitzkya potaninii
Aurinia saxatilis
Alyssum obovatum
Alyssum cypricum
Unresolved
Unresolved
Clyopela aspera
Degenia velebitica
*
**
**
**
**
**
Fibigia clypeata
Fibigia spathulata
Hormathophylla purpurea
yyp
Alyssum linifolium
Alyssum lenense
Alyssum dasycarpum
Alyssum montanum
Fibigia suffruticosa
Alysseae
Fig. 1 continued
D. A. German et al.
123
support the recognition of the large genus Erysimum (223
spp. sensu Warwick et al. (2006b), but certainly smaller)
into the unigeneric Erysimeae Dumort.
Erysimum siliculosum (Bieb.) DC. and E. mongolicum
D. German formed with E. cheiranthoides L., the generic
type, a monophyletic clade with 100% BS support (Fig. 1).
The first species is a type of a previously accepted genus,
Syrenia Andrz. ex Bess., but our data clearly support its
union with Erysimum, a position taken earlier by Appel and
Al-Shehbaz (2003).
Crucihimalaya Al-Shehbaz, O’Kane & R. A. Price &
Transberingia Al-Shehbaz & O’Kane were placed by
Pseudoclausia olgae
Parrya stenocarpa
Parrya pulvinata
**
Parrya lancifolia
Parrya asperrima
Pseudoclausia turkestanica
Lit ino ia ten issima
Chorisporeae
(c)
Chorispora macropoda
Diptychocarpus strictus
**
*
**
**
**
**
Litwinowia tenuissima
Dontostemon tibeticus
Chorispora bungeana
Clausia kasakhorum
Clausia aprica
Clausia trichosepala
Dontostemon pinnatifidus
Dontostemon crassifolius
Dontostemon dentatus
Dontostemoneae
Neotorularia torulosa
Streptoloma desertorum
1.00
0.87
0.98
0.86
**
*
*
*
*
Streptoloma desertorum
Leiospora pamirica
Leiospora exscapa
Dichasianthus subtilissimus
Euclidium syriacum
Atelanthera perpusilla
Dilophia salsa
Rhammatophyllum kamelinii
Sisymbriopsis yechengica
Solms-laubachia flabellata
Braya humilis
Strigosella africana
Strigosella brevipes
Euclidieae
0.80
0.84
**
**
**
*
*
*
*
**
**
**
Dichasianthus subtilissimus
Leptaleum filifolium
Sterigmostemum fuhaiense
Sterigmostemum schmakovii
Oreoloma matthioloides
Sterigmostemum caspicum
Oreoloma violaceum
Oreoloma sulfureum
Sterigmostemum ramosissimum
Matthiola incana
Matthiola lunata
Microstigma brachycarpum
Anchonieae
Anchonium elichrysifolium
Bunias orientalis 2
Bunias orientalis 3
Bunias orientalis 1
***
**
**
**
**
Iskandera alaica
Microstigma brachycarpum
Microstigma deflexum
Hesperis laciniata
Hesperis sibirica
Hesperideae
Draba oreades
Draba doerfleri
Draba sibirica
Draba nuda
Draba verna
Buniadeae
092
*
**
**
*
*
**
Aubrieta deltoidea
Stevenia incarnata
Arabideae s. str.
Arabis alaschanica
Arabis kokanica
Arabis kamelinii
Draba hystrix
Botschantzevia karatavica
Pachyneurum grandiflorum 1
Pachyneurum grandiflorum 2
Arabis blepharophylla
Rhammatophyllum fruticulosum
Arabis alpina
0.92
**
**
**
**
Macropodium nivale
Pseudoturritis turrita
Stevenia sergievskajae
Stevenia alyssoides
Arabis axillaris
Stevenia cheiranthoides
Ptilotrichum dahuricum
Ptilotrichum tenuifolium
Berteroella maximowiczii
Arabideae II
Arabideae II?
0.91
Fig. 1 continued
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Al-Shehbaz et al. (2006) in the tribe Camelineae, but our
data do not support that assignment. Instead, these two
genera plus Arabis tibetica Hook.f. & Thomson, A. rupi-
cola Kryl., and Alyssum klimesii Al-Shehbaz formed a
well-defined clade (100% support in all analyses; Fig. 1;
Supplement 1) that merits recognition as an independent
tribe. Arabis rupicola and Transberingia bursifolia (DC.)
Al-Shehbaz & O’Kane have already been transferred to
Crucihimalaya (German 2005; German and Ebel 2005),
but A. tibetica has not. The position of Alyssum klimesii
was quite perplexing, although it has recently been estab-
lished with Crucihimalaya by Koch et al. (2007). Alyssum
klimesii differs from the rest of this clade by being a dwarf
(1–3 cm high) pulvinate plant with entire fleshy leaves,
few-flowered racemes, and few-seeded silicles (Al-She-
hbaz 2002). By contrast, Crucihimalaya consists of taller
(up to 1 m), annuals or non-pulvinate perennials with non-
fleshy, dentate to pinnately divided leaves, many-flowered
racemes, and many-seeded linear siliques (Al-Shehbaz
et al. 1999; Appel and Al-Shehbaz 2003). Our data suggest
that either all taxa above be united into one highly heter-
ogeneous genus, or at least two or three genera be recog-
nized. The systematic position of A. klimesii will be dealt
with in a forthcoming paper.
Finally, the position of Turritis L. within lineage I was
established earlier (Koch et al. 1999, 2000), and is closely
related to the other tribal-rank clades within this lineage.
Therefore, we suggest its placement in the monogeneric
tribe Turritideae (German 2009).
Another clade of the Camelineae sensu Al-Shehbaz
et al. (2006) is formed by Irenepharsus Hewson and
Pachycladon Hook.f. Although the phylogeny of Austra-
lian taxa is beyond the scope of this study, the position of
both genera emphasizes the need for further phylogenetic
studies of the tribe.
Eight genera (Calymmatium O.E. Schulz, Olimarabid-
opsis Al-Shehbaz, O’Kane & R. A. Price, Chrysochamela
Boiss., Arabidopsis (DC.) Heynh., Catolobus (C.A. Mey.)
Al-Shehbaz, Camelina Crantz, Pseudoarabidopsis
Al-Shehbaz, O’Kane & R. A. Price, and Capsella Medik.)
formed a monophyletic clade recognized herein as Came-
lineae s.str. Although previous ITS (Bailey et al. 2006) and
combined (Koch et al. 2007) studies fully agree with ours
on the polyphyly of Camelineae s.l., further studies are
needed to properly assign additional genera and establish
the monophyly of Camelineae s.str.
The closer phylogenetic relationship of Calymmatium to
Olimarabidopsis and their morphological similarities (e.g.
both annuals with short-stalked to subsessile 2–5-rayed
trichomes, usually auriculate-sagittate stem leaves, usually
yellow flowers, and incumbent cotyledons) suggest they
may be congeneric. As recently revealed by Khosravi et al.
(2009), morphologically similar SW Asian Alyssopsis
Boiss. also belongs to this group of affinity. However,
further studies are needed to finally clarify relationships
among these genera.
Tribe Boechereae Al-Shehbaz, Beilstein & E. A.
Kellogg
On the basis of morphological and cytological (2n = 14)
data, German (2004) suggested close affinity of the
monospecific Borodinia N. Busch (E Siberia, Russian Far
East) and the primarily North American Boechera A
´
.Lo
¨
ve
&D.Lo
¨
ve. This relationship was also suspected by Bailey
et al. (2006) and recently demonstrated in a significant way
by Kiefer et al. (2009). Our ITS data support the placement
of Borodinia in the tribe Boechereae. Borodinia macro-
phylla (Turcz.) O.E. Schulz and Boechera falcata (Turcz.)
Al-Shehbaz (E Siberia, Russian Far East) are the only
representatives of the tribe Boechereae in the Old World.
Berkutenko (2003, 2005) reduced Borodinia to synon-
ymy with Arabis L. on the basis of having branched tric-
homes, white petals, latiseptate siliques, and accumbent
cotyledons. As shown by Al-Shehbaz (2003, 2005), Koch
et al. (2003), and Al-Shehbaz et al. (2006), that combina-
tion of characteristics is highly homoplasious and evolved
many times across the whole family. Berkutenko (2003,
2005) placed A. alaschanica Maxim. (N China) in synon-
ymy with B. macrophylla, but the molecular data (Fig. 1)
and morphological differences (fruit type and width, seed
shape, indumentum) strongly support their placement in the
remotely related tribes Arabideae and Boechereae. Further
studies are needed to clarify the phylogenetic position of
Borodinia within the Boechereae, especially with regard to
the larger (110 spp.) and taxonomically difficult Boechera
(Windham and Al-Shehbaz
2006, 2007a, b).
Tribe Arabideae DC.
The core Arabideae (Arabideae s.str.) consisted of Arabis,
Draba L., Aubrieta Adans., monospecific Pachyneurum
Bunge, Botschantzevia Nabiev, and Dendroarabis (C.A.
Mey.) D. German & Al-Shehbaz. The core of the second
subclade, hereafter Arabideae-II, is a strongly supported
group of predominantly E/C Asian-Siberian taxa which
consists of Stevenia Fisch., Ptilotrichum C.A. Mey.,
monospecific Berteroella O.E. Schulz, and bispecific
Macropodium W.T. Aiton. Monospecific and predomi-
nantly Mediterranean Pseudoturritis Al-Shehbaz had weak
but constant affinity to this subclade.
Arabideae s.str
Aubrieta was invariably sister to the rest of this core
group. Draba (including Drabopsis C. Koch, Erophila
D. A. German et al.
123
DC., and Schivereckia Andrz. ex DC. and excluding
D. hystrix Hook.f. & Thomson) showed 100% BS support
(Fig. 1), and these findings agree with earlier studies (e.g.
Al-Shehbaz et al. 2006; Bailey et al. 2006;Koch2003;
Koch and Al-Shehbaz 2002;Kochetal.2003, 2007).
Draba hystrix is anomalous in the genus, because of its
needle-like leaves, large fruits and seeds, and long-setose
trichomes (Jafri 1973), and we suggest its exclusion from
Draba.
Despite extensive morphological (Al-Shehbaz 2003,
2005) and molecular (Koch et al. 1999, 2000, 2001;
O’Kane and Al-Shehbaz 2003) studies, the limits of Arabis
remain problematic (Al-Shehbaz et al. 2006; Koch et al.
2003; Zhou et al. 2001). The addition herein of taxa not
previously studied molecularly associate the generic type,
A. alpina L., with Pachyneurum and Botschantzevia,
although their positions relative to each other varied
according to the algorithm used.
The monospecific Pachyneurum, together with the
central Asian Dendroarabis and western North American
Arabis blepharophylla Hook. & Arn., formed a well-
defined subclade with 100% BS support (Fig. 1). The
subclade included A. hirsuta (L.) Scop. (not shown here)
and other species of Arabis (Clauss and Koch 2006; Koch
2003; Koch et al. 1999, 2000).
The position and status of Pachyneurum have been
treated in different ways by various authors (Table 2) but
our ITS (Fig. 1) and cpDNA trnL intron data (not shown)
strongly support the recognition of Pachyneurum as a
distinct genus in the Arabideae, as suggested by Vassilyeva
(1969) and Botschantzev (
1972) and later accepted by
Al-Shehbaz et al. (2006). A closer relationship between
Pachyneurum and Noccaea Moench (tribe Noccaeeae)
revealed by Koch et al. (2007) is a consequence of misi-
dentification of the cited voucher specimen (M. Staudinger
5493, deposited in W with duplicates in WU and HEID)
which, in fact, belongs to Noccaea cochleariformis (DC.)
A
´
.Lo
¨
ve & D. Lo
¨
ve (see GenBank annotation by M. Koch).
Overall morphology (e.g. the presence in Pachyneurum of
latiseptate siliques vs. angustiseptate silicles and malpig-
hiaceous trichomes vs. simple ones or none, etc.) support
the placement of Pachyneurum in the Arabideae and not in
Noccaeeae.
Species of the Central Asian Rhammatophyllum O.E.
Schulz were assigned to various genera in the tribes An-
chonieae, Arabideae, Alysseae, and Hesperideae (Al-She-
hbaz and Appel 2002). The placement herein of R.
kamelinii (Botsch.) Al-Shehbaz & O. Appel, a very close
relative of the generic type R. pachyrrhizum (Kar. & Kir.)
O.E. Schulz, into the Euclidieae agrees with Al-Shehbaz
et al. (2006). However, the placement herein of R. fruti-
culosum in the Arabideae demonstrates that the genus
sensu Al-Shehbaz (2005) is polyphyletic. Our molecular
data prompted German and Al-Shehbaz (2008b) to place
the species in the monospecific Dendroarabis.
Three species of Arabis (A. alaschanica, A. kamelinii
Botsch., and A. kokanica Regel & Schmalh.), Draba hys-
trix, and Botschantzevia karatavica (Lipsch.) Nabiev form
a well supported (Fig. 1), predominantly central Asian
subclade. These woody perennials (B. karatavica is a
subshrub) grow in rock crevices and have well-developed
rosettes and few, non-auriculate cauline leaves. Although
the generic placement of the last species fluctuated between
Arabis, Erysimum, and Parrya, the molecular data (Fig. 1)
and trichome morphology (Dvor
ˇ
a
´
k 1968; Nabiev 1972)
strongly support its recognition in a monospecific
Bots-
chantzevia assigned to the Arabideae, rather than to the
Erysimeae or Chorisporeae. These three species of Arabis
strongly resemble A. karategina Lipsky, A. popovii Botsch.
& Vved., A. saxicola Edgew., A. setosifolia Al-Shehbaz,
especially in their morphology, ecology, and distribution,
and it is likely that these seven species form an independent
genus.
As delimited herein, the Arabideae s.str. remain in need
of further phylogenetic studies and considerable taxonomic
adjustments. Depending on the algorithm used, the sister
position of Arabis alpina L. was unstable in relation to the
subclades including Pachyneurum, Botschantzevia, or even
the rest of Arabideae s.str. minus Aubrieta (Clauss and
Koch 2006; Heenan et al. 2002; Koch et al. 1999, 2000).
We believe that a monophyletic Arabis would be a rather
small genus consisting of its type, A. alpina, and its nearest
relatives (e.g. A. caucasica Willd., A. tianschanica Pavlov).
The vast majority of Arabis species must be placed in other
genera, and systematics can be only solved by including
the genus Draba and its various segregates (Koch et al.,
work in progress), and several taxa will have to be cir-
cumscribed in a different generic context, as was partially
done by Al-Shehbaz (2003, 2005).
Arabideae-II
This clade is strongly supported (Fig. 1), and its component
genera are also highly resolved. The southern Siberian
Macropodium nivale (Pall.) W.T. Aiton is sister to a
polytomy including four species of Stevenia, two of
Ptilotrichum, Arabis axillaris Kom., and monospecific
Berteroella (hereafter the Stevenia alliance). Macropodium
pterospermum F. Schmidt (eastern Asia) has not yet been
studied molecularly.
Macropodium was assigned to the Thelypodieae, a tribe
considered by many to include most extant primitive
Brassicaceae (Hayek 1911; Busch 1939; Takhtajan 1986;
Dvor
ˇ
a
´
k 1972
; Al-Shehbaz 1973; Hedge 1976; Avetisyan
1990) because of the superficial floral similarities of its
genera and Cleome. However, others (Prantl 1891; Busch
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Table 2 Revised tribal assignment of the studied Brassicaceae genera and their previous taxonomic treatments
Genus De
Candolle
(1821)
Bentham
and Hooker
(1862)
Prantl (1891) Hayek (1911) Schulz (1936) Janchen
(1942)
Dvor
ˇ
a
´
k
(1972)
Kamelin
(2002)
Al-Shehbaz
et al. (2006)
Subsequent
treatments
a
This study
Aphragmus Andrz. ex DC. Camelineae
(Braya p.
p.)
Sinapeae/
Alliariinae
Arabideae/
Arabidinae
Sisymbrieae/
Pachycladinae
Sisymbrieae/
Brayinae
Aphragmeae
Asperuginoides Rauschert
(Buchingera Boiss. et
Hohen.)
Alysseae Hesperideae/
Alyssinae
Alysseae/
Alyssinae
Alysseae Alysseae/
Alyssinae
Clypeoleae ? Alysseae Unresolved Unresolved
Atelanthera Hook. f. et
Thomson
Arabideae Hesperideae/
Malcolmiinae
Alysseae/
Hesperedinae
Hesperideae Sisymbrieae/
Brayinae
Sisymbrieae/
Brayinae
? Euclidieae Euclidieae
Berteroella O. E. Schulz Sisymbrieae/
Brayinae
Sisymbrieae/
Brayinae
? Arabideae ? Arabideae Arabideae II
Biscutella L. Thlaspideae Thlaspideae Sinapeae/
Lepidiinae
Lepidieae/
Lepidiinae
Lepidieae/
Iberidinae
Lepidieae/
Iberidinae
Megacarpaeae Unresolved Unresolved Biscutelleae
Boreava Jaub. et Spach Isatideae Sinapeae/
Sisymbriinae
Arabideae/
Buniadinae
Euclidieae Sisymbrieae/
Buniadinae
Parabrassiceae/
Buniadinae
Isatideae Isatideae Isatideae
Borodinia N. Busch Arabideae Arabideae/
Drabinae
? Boechereae Boechereae
Botschantzevia Nabiev Arabideae/
Arabidinae
Arabideae Arabideae
Bunias L. Buniadeae Isatideae Hesperideae/
Hesperedinae
Arabideae/
Buniadinae
Euclidieae Sisymbrieae/
Buniadinae
Parabrassiceae/
Buniadinae
?Anchonieae Buniadeae Buniadeae
Calepina Adans. Zilleae Isatideae Sinapeae/
Sisymbriinae
Brassiceae/
Raphaniinae
Brassiceae/
Raphaninae
Brassiceae/
Raphaninae
Parabrassiceae/
Buniadinae
Unresolved Unresolved Calepineae
Calymmatium O. E.
Schulz
Sisymbrieae/
Brayinae
Lepidieae/
Cochleariinae
Camelineae
Chorispora R. Br. ex DC. Cakileae Raphaneae Hesperideae/
Hesperedinae
Alysseae/
Brayinae
Matthioleae Hesperideae/
Matthiolinae
Hesperideae/
Anchoniinae
Parabrassiceae/
Chorisporinae
Chorisporeae Chorisporeae Chorisporeae
Clausia Korn.-Tr. Sisymbrieae
(Hesperis
p. p.)
Hesperideae/
Hesperedinae
Alysseae/
Hesperedinae
Hesperideae
(Hesperis
p. p.)
Hesperideae/
Matthiolinae
Hesperideae/
Matthiolinae
Arabideae/
Hesperidinae
Anchonieae Dontostemoneae Dontostemoneae
Conringia Heist. ex Fabr. Sisymbrieae Hesperideae/
Moricandiinae
Brassiceae/
Moricandiinae
Brassiceae/
Moricandiinae
Brassiceae/
Moricandiinae
Brassiceae/
Moricandiinae
Unresolved Unresolved Conringieae
Cryptospora Kar. et Kir. Raphaneae Hesperideae/
Malcolmiinae
Alysseae/
Hesperedinae
Hesperideae Sisymbrieae/
Brayinae
Arabideae/
Hesperidinae
Euclidieae Euclidieae Euclidieae
Dichasianthus Ovcz. et
Junussov
Sisymbrieae/
Brayinae
Euclidieae Euclidieae Euclidieae
Didymophysa Boiss. Thlaspideae Sinapeae/
Cochleariinae
Lepidieae/
Thlaspidinae
Lepidieae/
Physariinae
Lepidieae/
Physariinae
Lepideae/
Lepidiinae
? Alysseae Unresolved Thlaspideae
Dilophia Thomson Lepidieae Sinapeae/
Cochleariinae
Lepidieae/
Thlaspidinae
Lepidieae/
Cochleariinae
Lepideae/
Lepidiinae
Euclidieae Euclidieae Euclidieae
Dontostemon Andrz. ex
C. A. Mey.
Sisymbrieae Hesperideae/
Hesperedinae
Alysseae/
Brayinae
Arabideae Hesperideae/
Anchoniinae
Arabideae/
Hesperidinae
Anchonieae Dontostemoneae Dontostemoneae
D. A. German et al.
123
Table 2 continued
Genus De
Candolle
(1821)
Bentham
and Hooker
(1862)
Prantl (1891) Hayek (1911) Schulz (1936) Janchen
(1942)
Dvor
ˇ
a
´
k
(1972)
Kamelin
(2002)
Al-Shehbaz
et al. (2006)
Subsequent
treatments
a
This study
Erysimum L. Sisymbrieae Sisymbrieae Hesperideae/
Erysiminae
Arabideae/
Erysiminae
Hesperideae Hesperideae/
Hesperidinae
Erysimeae Arabideae/
Erysiminae
Camelineae Camelineae Erysimeae
Euclidium W. T. Aiton Euclidieae Isatideae Hesperideae/
Malcolmiinae
Alysseae/
Euclidiinae
Euclidieae Hesperideae/
Euclidiinae
Parabrassiceae/
Buniadinae
Euclidieae Euclidieae Euclidieae
Goldbachia DC. Anchonieae Raphaneae Sinapeae/
Sisymbriinae
Arabideae/
Buniadinae
Hesperideae Sisymbrieae/
Buniadinae
Parabrassiceae/
Chorisporinae
? Isatideae Unresolved Calepineae
Iskandera N. Busch Hesperideae/
Matthiolinae
Arabideae/
Hesperidinae
Anchonieae Anchonieae Anchonieae
Leiospora (C. A. Mey.)
Dvor
ˇ
a
´
k
Hesperideae/
Matthiolinae
Arabideae/
Hesperidinae
Euclidieae Euclidieae Euclidieae
Leptaleum DC. Sisymbrieae Camelineae Hesperideae/
Malcolmiinae
Alysseae/
Brayinae
Hesperideae Hesperideae/
Hesperidinae
Sisymbrieae/
Brayinae
Arabideae/
Hesperidinae
Euclidieae Euclidieae Euclidieae
Litwinowia Woron. Euclidieae
(Euclidium p.
p.)
Hesperideae/
Euclidiinae
Parabrassiceae/
Buniadinae
Chorisporeae
Macropodium W. T. Aiton Arabideae Arabideae Hesperideae/
Turritinae
Thelypodieae Stanleyeae Macropodieae Arabideae Arabideae Arabideae-II
Megacarpaea DC. Thlaspideae Thlaspideae Sinapeae/
Lepidiinae
Lepidieae/
Lepidiinae
Lepidieae/
Iberidinae
Lepidieae/
Iberidinae
Megacarpaeeae Unresolved Megacarpaeeae
Megadenia Maxim. Sinapeae/
Cochleariinae
Lepidieae/
Thlaspidinae
Lepidieae/
Iberidinae
Megacarpaeae Biscutelleae
Microstigma Trautv. Matthioleae
(Matthiola p.
p.)
Matthioleae Anchonieae Anchonieae Anchonieae
Oreoloma Botsch. Arabideae/
Hesperidinae
Anchonieae Anchonieae Anchonieae
Orychophragmus Bunge Brassiceae Hesperideae/
Moricandiinae
Brassiceae/
Moricandiinae
Brassiceae/
Moricandiinae
Brassiceae/
Moricandiinae
Brassiceae/
Vellinae
Unresolved
Pachyneurum Bunge Arabideae
(Parrya p.
p.)
Hesperideae/
Hesperedinae
(Parrya p. p.)
Alysseae/
Hesperedinae
(Parrya p. p.)
Arabideae Arabideae/
Arabidinae
? Arabideae ? Noccaeeae Arabideae
Parrya R. Br. (Neuroloma
Andrz., Achoriphragma
Soja
´
k)
Arabideae Hesperideae/
Hesperedinae
Alysseae/
Hesperedinae
Matthioleae Hesperideae/
Matthiolinae
Hesperideae/
Matthiolinae
Arabideae/
Hesperidinae
Chorisporeae Chorisporeae Chorisporeae
Pseudoclausia M. Pop. Arabideae/
Hesperidinae
Anchonieae Chorisporeae
Ptilotrichum C. A. Mey. Alysseae
(Alyssum
p. p.)
Hesperideae/
Alyssinae
Alysseae/
Alyssinae
Alysseae Alysseae/
Alyssinae
Arabideae/
Alyssinae
? Arabideae Arabideae Arabideae-II
Pugionium Gaertn. Euclidieae Isatideae Hesperideae/
Malcolmiinae
Lepidieae/
Thlaspidinae
Lepidieae/
Isatidinae
Lepidieae/
Pugioniinae
Parabrassiceae/
Chorisporinae
Megacarpaeeae
Rhammatophyllum O. E.
Schulz
Hesperideae Sisymbrieae/
Brayinae
Arabideae/
Arabidinae
Euclidieae Euclidieae Euclidieae
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
Table 2 continued
Genus De
Candolle
(1821)
Bentham
and Hooker
(1862)
Prantl (1891) Hayek (1911) Schulz (1936) Janchen
(1942)
Dvor
ˇ
a
´
k
(1972)
Kamelin
(2002)
Al-Shehbaz
et al. (2006)
Subsequent
treatments
a
This study
Sisymbriopsis Botsch. et
Tzvel.
Sisymbrieae/
Brayinae
Euclidieae Euclidieae Euclidieae
Solms-laubachia Muschl. Matthioleae Arabideae/
Hesperidinae
Euclidieae Euclidieae Euclidieae
Spirorhynchus Kar. et Kir. Isatideae Sinapeae/
Sisymbriinae
Arabideae/
Buniadinae
Euclidieae Sisymbrieae/
Buniadinae
Parabrassiceae/
Chorisporinae
? Isatideae Calepineae
Sterigmostemum Bieb.
(Sterigma DC.)
Anchonieae Raphaneae Hesperideae/
Hesperedinae
Alysseae/
Brayinae
Hesperideae Hesperideae/
Hesperidinae
Hesperideae/
Anchoniinae
Arabideae/
Hesperidinae
Anchonieae Anchonieae Anchonieae
Stevenia Fisch. Arabideae Arabideae
(Arabis p. p.)
Hesperideae/
Turritinae
(Arabis p. p.)
Arabideae/
Arabidinae
(Arabis p. p.)
Arabideae Arabideae/
Arabidinae
? Arabideae Arabideae-II
Streptoloma Bunge Sisymbrieae Hesperideae/
Malcolmiinae
Alysseae/
Brayinae
Sisymbrieae/
Brayinae
Sisymbrieae/
Brayinae
Sisymbrieae/
Brayinae
Euclidieae
Strigosella Boiss.
(Fedtschenkoa Regel et
Schmalh.)
Sisymbrieae
(Malcolmia
p. p.)
Hesperideae/
Malcolmiinae
Alysseae/
Brayinae
Hesperideae
(Malcolmia
p. p.)
Hesperideae/
Hesperidinae
(Malcolmia p. p.)
Sisymbrieae/
Brayinae
Arabideae/
Hesperidinae
Euclidieae Euclidieae Euclidieae
Tauscheria DC. Isatideae Isatideae Sinapeae/
Sisymbriinae
Arabideae/
Isatidinae
Euclidieae Sisymbrieae/
Isatidinae
Parabrassiceae/
Isatidinae
? Isatideae Isatideae Isatideae
Zuvanda (Dvor
ˇ
a
´
k)
Askerova
Unresolved Conringieae
a
Al-Shehbaz and Warwick (2007), Koch et al. (2007), Warwick et al. (2007, 2008)
D. A. German et al.
123
1926; Al-Shehbaz et al. 2006) placed it in the Arabideae
s.l., a position supported by our molecular data.
The Stevenia alliance is characterized by a uniform in-
dumentum (sessile or subsessile 2–4-branched with pinnate
rays), absence of simple hairs, entire and sessile or sub-
sessile cauline leaves, dilated filaments, lack of median
nectaries, often latiseptate fruits, usually long and slender
styles, minute stigmas, and non-mucilaginous, usually
flattened seeds. This characteristic combination, with the
molecular data herein, support the treatment of this alliance
as one genus, the oldest generic name in which is Stevenia.
The Mediterranean Pseudoturritis turrita (L.) Al-She-
hbaz showed a weak relationship to Berteroella (Koch
2003), which is somewhat confirmed by our analysis
(Fig. 1). Despite the support only in Bayesian analysis
(0.91; Fig. 1), the association of Pseudoturritis with Ara-
bideae is stable, and we believe that its morphological
similarity to the tribe was the product of parallel evolution.
If the Arabideae s.l. is maintained, Pseudoturritis would
most likely be one of its members. However, our data
support the division of this tribe into at least two, of which
the Arabideae-II needs a new tribal name.
Tribe Alysseae DC.
As delimited by Dudley and Cullen (1965) and Al-Shehbaz
et al. (2006), the Alysseae are polyphyletic. The tribe has
recently been studied phylogenetically by Warwick et al.
(2008), and our data (Fig. 1) fully agree with their findings
in excluding Didymophysa Boiss., Asperuginoides, Ptilo-
trichum, and Alyssum klimesii from the tribe. With the
exclusion of these four taxa and placement of Lobularia
Desv. and Farsetia Turra in the Malcolmieae (Warwick
et al. 2008), the tribe becomes monophyletic.
The position of Alysseae relative to the other tribes varied
substantially depending on the marker used. For example, in
the ndhF phylogeny (Beilstein et al. 2006), the tribe was
related to the Brassiceae in lineage II, whereas in the ITS
phylogeny (Bailey et al. 2006) it appeared either related to
Erysimum (their Camelineae; our Erysimeae) or unrelated to
any tribe, and in the trnLintrontrnL-F intergenic spacer
analysis (Koch et al. 2007) it was related to the Noccaeeae,
and in ITS-based tree of Khosravi et al. (2009)itgrouped
together with the tribes Chorisporeae, Hesperideae, and
Malcolmieae. Our ITS data did not shed additional light.
Although Alyssum has been estimated to include ca. 195
species (Warwick et al. 2006b), the genus sensu Appel and
Al-Shehbaz (2003
) is polyphyletic (Bailey et al. 2006;
Koch et al. 2007; Warwick et al. 2008; Khosravi et al.
2009), and our tiny sample of six species supports that. Our
preliminary data agree with Warwick et al. (2008) that
species of sect. Alyssum (A. montanum L., the generic type,
and A. lenense Adams) grouped together with a member of
sect. Psilonema (C.A. Mey.) Hook.f. (A. dasycarpum
Steph. ex Willd.) in a well-resolved clade (Fig. 1), whereas
those of sect. Odontarrhena (C.A. Mey.) Hook.f. (A.
cypricum Nya
´
r., A. obovatum (C.A. Mey.) Turcz.) grouped
with A. linifolium Steph. ex Willd. (sect. Meniocus (DC.)
Hook.f.) in a similarly strongly supported clade (Fig. 1).
Extensive sampling, however, is needed before any
meaningful generic adjustments are made.
Both Fibigia suffruticosa (Vent.) Sweet and F. clypeata
(L.) Medik. fell in a polytomy (Warwick et al. 2008;hereinin
Fig. 1) confirming the polyphyly of this genus of 13 species.
Better sampling is needed to resolve its polyphyly and the
position of F. spathulata (Kar. & Kir.) B. Fedtsch. (Kazakh-
stan), which was transferred by Boczantzeva (1976, 1977)to
the independent genus Pterygostemon V. Bocz.
Tribe Chorisporeae C.A. Mey.
The Chorisporeae sensu Al-Shehbaz et al. (2006) included
Chorispora and Diptychocarpus Trautv. Warwick et al.
(2007) added Parrya R.Br., and herein we add Pseudo-
clausia M. Pop. (10 spp.), monospecific Litwinowia
Woron., and Dontostemon tibeticus (Maxim.) Al-Shehbaz.
Dontostemon Andrz. ex C.A. Mey. becomes polyphy-
letic (Fig. 1)ifD. tibeticus (Al-Shehbaz 2000) is main-
tained in it. On the basis of recent molecular studies
(Warwick et al. 2007), the genus is now recognized in the
tribe Dontostemoneae (Al-Shehbaz and Warwick 2007).
Our molecular data and morphology (strongly two-lobed
stigmas with subdecurrent lobes and deeply pinnatifid
leaves with numerous decurrent lobes) support the place-
ment of the species into a new genus in the Chorisporeae.
Parrya was reduced to a monospecific genus (Bots-
chantzev 1972), and its remaining species were recognized
in Achoriphragma Soja
´
k(Neuroloma Andrz., nom. illeg.).
Parrya is heterogeneous and much in need of thorough
phylogenetic and systematic studies to test its monophyly,
to determine its component species, and to check the merit
of the genera segregated from it. Our preliminary cpDNA
data (not shown here) indicate that P. arctica R.Br., the
generic type, and some Parrya species belong to the
Chorisporeae, but more species should be examined to
address these concerns. The current data indicate paraphyly
of Parrya with Pseudoclausia nested within it. The latter
genus was considered to be related to Clausia (e.g. War-
wick et al. 2007), but our data do not support that. Further
studies should help in testing the relationships between
Parrya and Pseudoclausia.
Tribe Euclidieae DC.
The Euclidieae sensu Al-Shehbaz et al. (2006) was shown
by Warwick et al. (2007) to be polyphyletic and split into
Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa
123
two clades recognized by Al-Shehbaz and Warwick (2007)
as the tribes Euclidieae s.str. and Malcolmieae. Both tribes
were identifed in our studies, and the Euclidieae s.str. was
monophyletic with moderate support (Fig. 1) The mono-
specific Atelanthera Hook.f. & Thomson is studied herein
for the first time, and it belong to the Euclidieae. A similar
position is revealed for Streptoloma Bunge (two spp.)
first studied independently by Khosravi et al. (2009).
Both genera have features typical of the tribe as redefined
by Al-Shehbaz and Warwick (2007).
As for the tribe Malcolmieae, represented in our analysis
by Cithareloma lehmannii Bunge and Malcolmia littorea
(L.) W.T. Aiton, its position within a polytomy of other
tribes was unstable depending on the sampled taxa and the
algorithm used.
Tribe Anchonieae DC.
The current data agree well with earlier studies (Warwick
et al. 2007; Al-Shehbaz and Warwick 2007) in recognizing
two tribes, Anchonieae and Dontostemoneae, instead of
one (sensu Al-Shehbaz et al. 2006). However, the cir-
cumscription of Oreoloma Botsch. and its relationship to
Stergimostemum Bieb. varied in the two studies. Oreoloma
was monophyletic in Warwick et al. (2007), but in our
analysis (Fig. 1) it formed a monophyletic lineage and
together with Anchonium elichrysifolium (DC.) Boiss. was
nested in Sterigmostemum. Botschantzev (1980) separated
Oreoloma from Sterigmostemum by having long-clawed
(vs. short-clawed) petals abruptly (vs. gradually) expanded
to limb and basally saccate (vs. non-saccate) lateral sepals.
As indicated by Kamelin and German (2001), these dif-
ferences are trivial because some species of Sterigmoste-
mum (e.g. S. schmakovii Kamelin & D. German) have
petals well differentiated into a claw and limb and have
non-saccate sepals. Based on morphology and our molec-
ular data and those of Warwick et al. (2007), we recom-
mend the expansion of Sterigmostemum to include both A.
elichrysifolium, as initially placed in Sterigmostemum (de
Candolle 1821), and Oreoloma. As a result, Anchonium
becomes monospecific, with its type A. billardieri DC.
sister to the expanded Sterigmostemum (Warwick et al.
2007).
Tribe Buniadeae DC.
Our data show that the recently recognized monogeneric
tribe Buniadeae (Al-Shehbaz and Warwick 2007)is
monophyletic if Bunias cochlearioides Murr. is excluded
from it. The latter species differs from the other two
congeners, B. erucago L. (generic type) and B. orientalis
L., in the lack of multicellular glands and branched
trichomes. With members of the recently restored
tribe Calepineae Horan. (German and Al-Shehbaz 2008a)
Bunias cochlearioides formed a highly resolved clade
(Fig. 1).
It is interesting to mention that the diploid (2n = 14)
B. orientalis and B. erucago share the biggest genome sizes
(GS) among all Brassicaceae species studied to date (Lysak
et al. 2009).
Tribe Brassiceae DC.
This tribe was subjected to extensive molecular studies
summarized by Warwick and Sauder (2005) who retained
within it the genera Calepina Adans., Conringia Heist. ex
Fabr., and Orychophragmus Bunge. Subsequent studies
(Bailey et al. 2006; Beilstein et al. 2006, 2008; Koch et al.
2007; Lysak et al. 2005, 2009; Manda
´
kova
´
and Lysak
2008) strongly supported the exclusion of Calepina and
Conringia from the tribe.
Calepina and Conringia formed a well-supported
monophyletic group in Warwick and Sauder (2005) but not
in Lysak et al. (2005), who suggested that this discrepancy
in the ITS phylogeny probably resulted from concerted
evolution. Our ITS-based phylogeny (Fig. 1) agrees with
that of Bailey et al. (2006), and with the ndhF phylogeny of
Beilstein et al. (2006), the trnL-F phylogeny of Koch et al.
(2007), and chromosome painting of Lysak et al. (2005)in
the placement of Calepina (together with
Goldbachia DC.,
Spirorhynchus Kar. & Kir., and Bunias cochlearioides) and
Conringia (with Zuvanda (Dvor
ˇ
a
´
k) Askerova) in unrelated
clades recognized by German and Al-Shehbaz (2008a)as
the tribes Calepineae and Conringieae, respectively.
Warwick and Sauder (2005) found Orychophragmus
nested within the Brassiceae, but our data show O. vio-
laceus (L.) O.E. Schulz to be outside the tribe, though its
position remains unresolved. Other lines of evidence
regarding its phylogenetic position are controversial. For
example, successful experimental hybridization between
the species and different members of Brassica (Warwick
and Sauder 2005) support its retention in the tribe, whereas
its East Asian distribution (vs. predominantly Mediterra-
nean/Saharo-Sindian distribution for the Brassiceae) agree
with our molecular data. Further studies are needed to
unravel the phylogenetic position of this genus. Its group-
ing with Conringia planisiliqua Fisch et Mey., recently
found by Khosravi et al. (2009) and recognized as Ory-
chophragmus clade’’, seems to be a good step in this
direction.
Tribe Eutremeae Al-Shehbaz, Beilstein & E.A. Kellogg
This tribe includes the monospecific Chalcanthus Boiss.
and recently expanded Eutrema R.Br. (Al-Shehbaz and
Warwick 2005; Al-Shehbaz et al. 2006; Warwick et al.
D. A. German et al.
123
2006a). Our study suggests the tribe is polyphyletic if E.
parvulum (Schrenk) Al-Shehbaz & Warwick is maintained
within the genus. In O’Kane and Al-Shehbaz (2003) and
Koch (2003) the species (as Thellungiella parvula (Sch-
renk) Al-Shehbaz & O’Kane) formed a well-supported
clade with species later assigned to Eutrema. Discrepancies
among these data and ours may represent a case of gene-
tree/species-tree incongruence similar to that proposed for
Draba funiculosa Hook.f. by Bailey et al. (2006). Alter-
natively, E. parvulum may represent a new monospecific
genus, and some morphological data (e.g. mucilaginous
seeds, reduced petals) and geographical distribution (Irano-
Turanian region) support that.
Other clades
Our current data (Fig. 1) on the genera Biscutella with
Megadenia; Goldbachia together with Spirorhynchus,
Calepina, and Bunias cochlearioides; Aphragmus Andrz.
ex DC.; and Conringia with Zuvanda have resulted in the
recognition (respectively) of the tribes Biscutelleae, Cal-
epineae, Aphragmeae, and Conringeae (German and Al-
Shehbaz 2008a). These genera and tribes were discussed at
some length in that publication, and the interested reader
should consult it for prior tribal assignments, generic and
tribal boundaries, number of species, distinguishing char-
acteristics, and distribution.
The remaining discussion focuses on Pugionium Gaertn.
and Megacarpaea DC., both of which were not subjected
to prior molecular studies. They were assigned to different
tribes or subtribes (Table 2), and in a few accounts only
(Grubov 1982; Kuan 1987; Zhou et al. 2001) were placed
next to each other. Because of its angustiseptate silicles,
Megacarpaea was traditionally placed in the Thlaspideae
or Lepidieae (Table 2), but Kamelin (2002) suggested that
it, with genera having didymous silicles (e.g. Biscutella,
Megadenia), should be assigned to the new tribe Mega-
carpaeeae (nom. nud.). Our molecular data do not support
the association of the last two genera with Megacarpaea,
and didymous fruits evolved independently several times in
the Brassicaceae (Al-Shehbaz et al. 2006).
The systematic position of Pugionium varied substan-
tially in prior classification systems, and the genus was not
mentioned by Al-Shehbaz et al. (2006). Its winged and/or
spiny fruits are bizarre in the Brassicaceae, which led
Hedge (1976
, p 22) to consider it ‘a genus without any
obvious allies.’ By contrast, Janchen (1942) placed
Pugionium in a monogeneric subtribe Pugioniinae (nom.
nud.). Molecular data (Fig. 1) place Pugionium and
Megacarpaea in a well-resolved clade recognized as the
tribe Megacarpaeeae Kamelin ex D. German (German
2009). However, further studies on the remaining two
species of Pugionium and eight of Megacarpaea are
needed to firmly establish their monophyly and position in
relation to other tribes.
Supratribal classification
Although 35 tribes are currently recognized (Al-Shehbaz
et al. 2006; Al-Shehbaz and Warwick 2007;Germanand
Al-Shehbaz 2008a;German2009; Koch and Al-Shehbaz
2009; Warwick et al. 2008), it is obvious that several
additional ones will be established upon completion of a
molecular generic survey of the entire family. Supertribes
were proposed by Avetisyan (1990) mostly on the basis of
morphological characteristics, and a similar approach was
taken by Dorofeyev (2004) who ignored the wealth of
molecular data available. These systems are equally
artificial to all prior ones. A few major groups above the
tribal rank are gradually emerging, especially with regard
to the sister relationship of the Aethionemeae to the rest
of the family and the three major lineages recognized by
Beilstein et al. (2006, 2008) and Koch and Al-Shehbaz
(2009). However, it is premature to go beyond the tribal
rank because of the need for a complete molecular
phylogeny based on a combination of several nuclear,
plastidic, and mitochondrial markers for most if not all
genera.
Acknowledgments The authors are grateful to Ulrike Coja for
extensive help in the laboratory and to Andreas Franzke for fruitful
discussion. We appreciate the comments of two anonymous reviewers
and Marcus Koch which helped us to improve the paper. The first
author thanks the Deutscher Akademischer Austauschdienst (DAAD,
Germany) and the Russian Federal Agency for Science and Innova-
tions for supporting his visits and laboratory research at Osnabru
¨
ck
University. Financial support for B. Neuffer, H. Hurka, and N. Friesen
by the Deutsche Forschungsgemeinschaft (DFG) and the Ministry of
Science and Culture (MWK) of Lower Saxony, Germany, for field
work and herbaria visits in Mongolia, Kyrgyzstan, and Russia, is
gratefully acknowledged. We gratefully thank all the herbaria men-
tioned for supplying plant material.
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