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Phylogeny of Isatis (Brassicaceae) and allied genera based on ITS sequences of
nuclear ribosomal DNA and morphological characters
Hamid Moazzeni
a,n
, Shahin Zarre
a
, Ihsan A. Al-Shehbaz
b
, Klaus Mummenhoff
c
a
Department of Plant Sciences, School of Biology, College of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran
b
Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri, 63166-0299, USA
c
Universit¨
at Osnabr¨
uck, Spezielle Botanik, Barbarastrasse 11, 49076 Osnabr¨
uck, Germany
article info
Article history:
Received 22 January 2009
Accepted 14 May 2009
Keywords:
Brassicaceae
Isatideae
Isatis
ITS
phylogeny
Iran
abstract
Systematics of the genus Isatis (Brassicaceae) is difficult and controversial, and previous studies were
based solely on morphological characters. Sequence variation of the internal transcribed spacer (ITS)
regions and the 5.8S gene of nuclear ribosomal DNA (nrDNA) were analyzed using parsimony and
Bayesian methods. Twenty-eight taxa of Isatis and related genera of the tribe Isatideae were sampled,
including 20 Isatis species representing almost all major morphological lineages, all three species of
Pachypterygium, two of nine species of Sameraria, and monospecific Boreava, Myagrum, and Tauscheria.
Two well-supported clades were resolved in the ITS tree, and they demonstrate the artificiality of the
present delimitation of the tribe. One clade includes I. emarginata, I. minima, I. trachycarpa,P. brevipes,
P. multicaule, P. stocksii, and T. lasiocarpa. The second clade includes I. buschiana, the polymorphic
I. cappadocica with five subspecies, I. gaubae, I. kotschyana, I. leuconeura,I. pachycarpa, I. takhtajanii,
I. tinctoria, and S. armena. Pachypterygium is polyphyletic and, together with Boreava,Sameraria, and
Tauscheria, all are nested within Isatis. This study is a continuation of our recent systematic survey
based on seed-coat microsculpturing (Moazzeni et al., 2007. Flora 202, 447–454) and reveals that fruit
characters mapped onto the molecular tree show considerable convergence. The reliance on fruit
characters alone in the delimitation of genera may well lead to erroneous phylogenetic results and thus
to incorrect taxonomic conclusions.
&2009 Elsevier GmbH. All rights reserved.
Introduction
Isatis L. (Brassicaceae or Cruciferae), a Eurasian genus of 79
species (Al-Shehbaz et al., 2006), is distributed primarily in
the Irano-Turanian region, where nearly 90% of its species
grow (Appel and Al-Shehbaz, 2003;Davis, 1965). Some species
(e.g., I. cappadocica) are highly polymorphic in fruit morphology,
the structures that provide the most diagnostic characters in the
genus (Davis, 1965;Hedge, 1968;Jafri, 1973). Intermediates have
been reported even among the most morphologically distinct
species (Davis, 1965;Hedge, 1968), and this suggests that
hybridization may have played an important role in the evolution
of the genus.
The genera Pachypterygium Bunge (3 spp.), Tauscheria Fisch. ex
DC. (1 sp.), Sameraria Desv. (9 spp.), and Chartoloma Bunge
(monospecific, not included in this study) were placed with Isatis
in the tribe Isatideae (Al-Shehbaz et al., 2006;Candolle, 1821), the
Arabideae subtribe Isatidinae (Hayek, 1911), and Lepidieae
subtribe Isatidinae (Schulz, 1936). These genera are delimited
solely on the basis of differences in single fruit characters. For
example, Sameraria differs from Isatis by its distinct (vs. obsolete)
style, and Pachypterygium is separated from Isatis by the presence
of thickened (vs. thin) fruit margin (Hedge 1968). Indeed, some
authors (e.g., Jafri, 1973;Rechinger, 1958;Sajedi et al., 2005)
reduced Pachypterygium to synonymy of Isatis and considered the
thickened fruit margin to be unreliable for the separation of these
genera.
Based strictly on the overall morphology, Al-Shehbaz et al.
(2006) suggested that the Isatideae include the genera Pachypter-
ygium, Sameraria, Boreava Jaub. & Spach, Chartoloma,Glastaria
Boiss., Schimpera Hochst. & Steud. ex Endl., Spirorrhynchus Kar. &
Kir., and Tauscheria. Both Myagrum L. and Tauscheria were placed
by Candolle (1821) with Isatis (including Sameraria) in the
Isatideae. This morphologically well-defined and primarily central
and southwestern Asian tribe is characterized by having indehis-
cent, 1- or rarely 2-seeded angustiseptate fruits, yellow or rarely
whitish flowers, sessile and often auriculate cauline leaves, and
simple or no trichomes (Appel and Al-Shehbaz, 2003;Al-Shehbaz
et al., 2006).
ITS sequence data were shown to be useful in evaluating
relationships among several genera of the Brassicaceae, including
Arabidopsis (DC.) Heynh. (O’Kane and Al-Shehbaz, 2003), Cardamine L.
ARTICLE IN PRESS
Contents lists available at ScienceDirect
journal homepage: www.elsevier.de/flora
Flora
0367-2530/$ - see front matter &2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.flora.2009.12.028
n
Corresponding author. Tel.: + 98 2161112482; fax: +98 2166405141.
E-mail address: moazzeni@khayam.ut.ac.ir (H. Moazzeni).
Flora 205 (2010) 337–343
ARTICLE IN PRESS
(Franzke et al., 1998), Cochlearia L. (Koch et al., 1999b), Crambe L.
(Francisco-Ortega et al., 1999), Vella L. (Crespo et al., 2000), Yinshania
Ma&Y.Z.Zhao(Koch and Al-Shehbaz, 2000), Sisymbrium L.
(Warwick et al., 2002), Thlaspi (Koch and Mummenhoff, 2001), and
others (see Al-Shehbaz et al., 2006).
Convergence is quite widespread in almost every morpholo-
gical character in the Brassicaceae (Al-Shehbaz et al., 2006;
Dvora
´k, 1971;Hedge, 1976;Koch et al., 2003;Meyer 1973;
Mummenhoff et al., 1997b). Therefore, assessing relationships
based solely on morphology will very likely lead to erroneous
conclusions (Al-Shehbaz et al., 2006;Koch et al., 1999a;Meyer,
1991).
The principal goal of the present study is to investigate the
phylogenetic relationships within the complex and widespread
genus Isatis and to determine if the genera Boreava, Pachypter-
ygium,Sameraria, and Tauscheria are sufficiently distinct from it.
The study also focuses on the evolution of characters within the
tribe Isatideae and on their value in the delimitation of genera.
Material and methods
Plant material
DNAs were extracted from 28 Iranian taxa of Isatideae sensu
Al-Shehbaz et al. (2006), including Isatis (20 taxa representing the
major groups), Boreava (1 sp.), Pachypterygium (3 spp.), Sameraria
(2 spp.), and Tauscheria (1 sp.). Myagrum perfoliatum was sister to
Isatis in the molecular analyses of Beilstein et al. (2006) and Bailey
et al. (2006) and served herein as the outgroup. Because no
sequence differences were found among the subspecies of
I. cappadocica, they were merged in the analyses as I. cappadocica.
The same approach was used for the species pairs I. koeiei and
I. raphanifolia (the former was reduced to synonymy of the latter
recently, Moazzeni et al., 2008), I. koelzii and I. tinctoria,I. glauca
and I. kotschyana, and S. armena and S. elegans. The nomenclature
of taxa, collection data, and vouchers are given in Table 1.
ITS amplification and sequencing
Leaves from herbarium specimens or dried in silica gel were
taken from individual plants. Total DNA was isolated following
Doyle and Doyle (1987) as modified in Mummenhoff and Koch
(1994). Double-stranded DNA of the ITS-1 and ITS-2 regions were
amplified using the polymerase chain reaction (PCR) protocol of
Mummenhoff et al. (1997a). PCR products were purified using the
purification kit (Roche Molecular Biochemicals). The four primers
used for sequencing both strands of the ITS-1 and ITS-2 regions
were 18 F, 5.8 F, 5.8 R and 25 R (for details, including modification
of the 18 F primer, see Mummenhoff et al., 1997a). Sequencing
reactions were run on an ABI 377XL automated sequencer.
Boundaries of the coding and spacer regions were determined
by comparison of our sequences to that of Sinapis alba L.
(Rathgeber and Capesius, 1989). DNA sequences were aligned
Table 1
Origin, collection data and GenBank accession numbers of taxa used in the current study
Taxon Collection data and collector Voucher number and
herbarium
Genbank accession
Borevea orientalis W. Azarbaijan; Chaldoran to Khoy, 57 km to Khoy, Moazzeni 35798-TUH GQ131309
Isatis buschiana E Azarbaijan; Mianeh to Tabriz, 20 km to Tabriz, Moazzeni 35800-TUH GQ131310
I. cappadocica subsp. besseri Ardebil; 5 km from Khalkhal to Rasht. Above Aznav spring,
Moazzeni
35756-TUH GQ131311
I. cappadocica subsp.
cappadocica
E Azarbaijan; Mianeh to Qareh Chaman, 26 km to Qareh Chaman,
Zarre & Moazzeni
35797-TUH GQ131312
I. cappadocica subsp.
macrocarpa
Kordistan; Sanandaj to Kamiaran, Noshor village, Awalan
mountain, Maroofi
2108-Hb. Kordistan GQ131333
I. cappadocica subsp.
stenophylla
Esfahan; Semirom, Vanak, Dalan kuh, Norouzi 14157-Esfahan
a
GQ131334
I. cappadocica subsp. subradiata Kordistan; SW Sanandaj, Dulab pass, Norouzi 6663-Hb. Kordistan GQ131335
I. emarginata Khorasan; SE Tabas, road of Nayband to Ali Abad, 47 km to Nistan,
Zangooie & Ayatollahi
24858-FUMH GQ131313
I. gaubae Gorgan; Azadshahr to Khosh Yelagh, Moazzeni 35781-TUH GQ131314
I. glauca Hamedan; Aq-Bolaq, Palat 15687-IRAN GQ375458
I. koeiei Kohgiluyeh; Yassuj, 5 km from Dehdasht to Behbahan 20309-TUH GQ131315
I. koelzii Khorasan; NE Bojnord, Tazeh kand to Gifan, Moazzeni 35803-TUH GQ131316
I. kotschyana Tehran; N Tehran, road of Firuzkuh, 20 km after Emmamzadeh
Hashem, Moazzeni
35777-TUH GQ131317
I. leuconeura Semnan; 32 km to Firuzkuh from Semnan, Moazzeni 35759-TUH GQ131318
I. lusitanica Markazi, Arak, Emarat, Ghahreman & Attar 12340-TUH GQ131319
I. minima Kerman, NE Bazman mt., Moazzeni 35765-TUH GQ131320
I. pachycarpa Kerman; Jebale-Barez, Moazzeni. 35785-TUH GQ131321
I. raphanifolia Tehran, Darake, Naqinezhad 29346-TUH GQ131322
I. takhtajanii Kordistan; Saqez to Baneh, Piramaran village, Nacaroz mountain,
Maroofi
1941-Hb. Kordistan GQ131332
I. tinctoria Khorasan; NE Bojnord, Tazeh kand to Gifan. Moazzeni 35779-TUH GQ131323
I. trachycarpa Khorasan; Torbat-e Jam to Afghanistan border, Cheshmeh-Zakani,
Ghahreman & Attar
28466-TUH GQ131324
Myagrum perfoliatum Kordistan; Sanandaj, near Qods hospital, Maroofi 6946-Hb. Kordistan GQ131325
Pachypterygium brevipes Kerman; Jupar mts, Mirtajedini 35788-TUH GQ131326
P. multicaule Kerman; Kuh Payeh, Mirtajedini. 35788-TUH GQ131327
P. stocksii Khorasan; Sarakhs to Sangar, 21 km to Sangar, Joharchi. 15060-FUMH GQ131328
Sameraria armena Khorasan; Torbat-e Jam to Afghanistan border, Cheshmeh-Zakani,
Ghahreman & Attar
28450-TUH GQ131329
S. elegans Khorasan, Birjand, Shokra mountain. Joharchi & Zangooie 14266-FUMH GQ131330
Tauscheria lasiocarpa Prov. Khorasan; NE Mashhad, Akhlamad, Joharchi & Zangooie 16774-FUMH GQ131331
a
Research Institute of Forest and Rangelands, Esfahan.
H. Moazzeni et al. / Flora 205 (2010) 337–343338
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visually by sequential pairwise comparison (Swofford and Olsen,
1990). The alignments required the introduction of seven indels of
one-bp length scattered among ITS-1, ITS-2, and 5.8S gene.
Empirical studies have shown that different approaches of gap
coding have only minimal, if any, effects on ITS tree topologies
(reviewed in Baldwin et al., 1995), and indels were coded as
missing data in parsimony analysis.
Phylogenetic analysis
Both maximum parsimony and Bayesian approaches to
phylogenetic estimation were used. The data matrix was analyzed
by assuming character states unordered and unweighted (i.e.,
Fitch parsimony) using the heuristic search strategy in PAUP
n
v.
4.0b10 (Swofford, 2002) with MULPARS, TBR (Tree Bisection-
Reconnection) branch swapping, and random taxon addition. Sets
of equally parsimonious trees were summarized by the strict
consensus approach. Bootstrap analyses (Felsenstein, 1985) with
1000 replicates were performed to obtain estimates of reliability
for each monophyletic group. Pairwise nucleotide differences
of unambiguously aligned positions were determined by the
DISTANCE MATRIX option in PAUP.
Bayesian inference of phylogeny using MrBayes 3.1 (Huelsen-
beck and Ronquist, 2003) was performed on the ITS alignment
using settings derived from MrModelTest 2.2 analysis (Nylander,
2004) and the Akaike information criterion (AIC). Following
MrModeltest, the symmetrical model of sequence evolution
(SYM) was employed in MrBayes, with an allowance for a gamma
(G) distribution of rates. The Markov chain Monte Carlo search
was run with 4 chains, one of which ‘‘cold,’’ for 1,000,000
generations, with trees being sampled every 100 generations.
After discarding the first 25% of trees as ‘‘burnin,’’ Bayesian search
results were summarized by 50% majority rule consensus and
posterior probability values (‘‘clade credibility’’) are indicated at
the branches (Fig. 1).
Morphological data
Patterns of morphological evolution were assessed for 28
characters emphasized in earlier taxonomic treatments of Isatideae
(e.g., Davis, 1965;Hedge, 1968;Sajedi et al., 2005). The characters
(Table 2) were compiled from original observations on field and
herbarium material and further discussed in Moazzeni (2006).The
polarity of character states was determined following Maddison
et al. (1984). The cladistic analysis of morphological characters is not
presented here, but four characters (Fig. 2,Table 2)previously
considered as taxonomically important in Isatis and allied genera
(see Davis, 1965;Hedge, 1968) were optimized onto the Bayesian
Fig. 1. Bayesian 50% majority rule consensus tree inferred from nuclear ITS sequences of selected Isatis species and related genera Boreava,Pachypterygium, Tauscheria and
Sameraria. Posterior probability values (clade credibility) are shown below branches. The sectional classification is that of Hedge (1968).Myagrum perfoliatum served as the
outgroup.
H. Moazzeni et al. / Flora 205 (2010) 337–343 339
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tree using Mesquite version 1.11 (Maddison and Maddison, 2006).
Fruit shape is also outlined on the Bayesian tree (Fig. 1).
Results
The length of ITS-1 and ITS-2 regions and the 5.8S gene within
the Isatideae (sensu Al-Shehbaz et al., 2006) varied from 268 to
269 bp for ITS-1, 167 to 169 bp for ITS-2, and 173 to 174 bp for
5.8S gene. Proper alignments of ITS sequences resulted in a matrix
of 622 characters. Of these, 531 base positions were constant and
uninformative, 47 were variable but not parsimony informative,
and 44 were potentially parsimony informative. The sequence
alignments required the introduction of gaps in ITS-1 and ITS-2.
No alignment ambiguities were found.
The sequence divergences among the studied species varied
between 3.3% in the species pairs Isatis tinctoria-I. gaubae and
P. stocksii-P. multicaule to as high as 6.4% in the species pair
I. takhtajanii–I. emarginata.
Fitch parsimony analysis (heuristic search) resulted in three
maximally parsimonious topologies of 135 steps with a consis-
tency index (CI) of 0.793 and retention index (RI) of 0.818. The
Bayesian analysis of the ITS sequence data set is consistent with
that of maximum parsimony (MP) analysis. The tree topology of
the parsimony strict consensus tree (not shown) is very similar to
Bayesian tree (BT) in Fig. 1. The two differences observed are:
1-Tauscheria is not sister to the rest as appeared in the parsimony
tree (PT), and this node, with 57% bootstrap support, collapsed in
the BT (see clade C in Fig. 1); 2-Boreava is not sister to the
remaining species (clade I in Fig. 1), and the node, with bootstrap
below 50%, also collapsed in the BT. Both molecular analyses
suggest that analyzed taxa of the genus Isatis may be divided into
two main lineages (Fig. 1).
Clade I
This well-supported clade (98% posterior probability in BT and
87% bootstrap in MP) includes 12 species in the genera Isatis,
Boreava, and Sameraria (Fig. 1). It consists of two subclades (A and
B), and B. orientalis. Subclade A is well supported (100% posterior
probabilities in BT and 88% bootstrap in MP) and includes two
groups, of which one forms a polytomy of perennial species with
silicle fruits, and the other includes the sister species I. lusitanica
and I. raphanifolia that are annuals with silique fruits and unique
ocellate microsculpturing of seed surface (Moazzeni et al., 2007).
Subclade B, which is moderately supported (79% posterior
probabilities in BT and 57% bootstrap in MP), includes four
species of Isatis and S. armena and is characterized by the annual
or biennial habit and often siliquose fruits (S. armena has silicles).
Clade II
This clade of seven species is also well supported
(100% posterior probabilities in BT and 91% bootstrap in MP)
and includes Tauscheria lasiocarpa and three species each of Isatis
and Pachypterygium. Species of this clade are annual herbs with
either siliques (Isatis) or silicles (Pachypterygium and Tauscheria).
Patterns of morphological evolution
The evolution of morphological characters previously empha-
sized in taxonomic treatments of Isatideae genera (e.g., Davis,
1965;Hedge, 1968;Sajedi et al., 2005;Table 2) was investigated
by optimizing character-state changes onto the BT tree. The BT
tree (Fig. 1) shows a moderate congruency to the morphological
findings in the above studies. Among the 28 characters studied,
the optimization of four taxonomically important characters onto
the Bayesian tree is shown in Fig. 2AD and further discussed
below. These characters include habit (Fig. 2A), petal shape
(Fig. 2B), and fruit type (Fig. 2C and D).
Discussion
The major classification systems of the Brassicaceae (e.g.,
Hayek, 1911;Janchen, 1942;Prantl, 1891;Schulz, 1936) variously
divided the family into 4–19 tribes based on a limited number of
morphological characters, and they did not pay enough attention
to convergence as a factor in the family evolution. However,
recent studies (e.g., Appel and Al-Shehbaz, 2003;Al-Shehbaz
et al., 2006;Koch et al., 2003;Price et al., 1994;Zunk et al., 1996)
have demonstrated the polyphyly and artificiality of almost all
tribes recognized in those earlier systems. Such artificiality was
also elucidated at the generic level, especially by molecular
studies on Cochlearia (Koch et al., 1999b), Thlaspi (Mummenhoff
et al., 1997a, b), Arabis (Koch et al., 1999a, 2001), and Arabidopsis
(O’Kane and Al-Shehbaz, 2003).
The Isatideae was suspected to be monophyletic based strictly
on morphology (Al-Shehbaz et al., 2006;Koch et al., 2003), but
prior to the present study no molecular phylogenetic analysis was
conducted on the tribe. The ITS data presented herein show that
Isatis sensu Schulz (1936) is not monophyletic because its species
form two major clades, within one of which (clade I, Fig. 1) the
genera Pachypterygium and Tauscheria are nested, and within the
other (clade II, Fig. 1) both Sameraria and Boreava are nested.
Table 2
Morphological characters used for the optimization onto the Bayesian tree.
Characters 1, 11, 17, and 25 are mapped onto the Bayesian 50% majority rule
consensus tree inferred from nuclear ITS sequence data (Fig. 2).
Habit
1. Annual or biennial (0), perennial (1)
2. Height: Z40 (0), o40 (1)
Basal leaves
3. Shape of blade: oblong (0), obovate to rounded (1)
4. Apex: obtuse to rounded (0), acute (1)
5. Blade length: 47 cm (0), r7 cm (1)
6. Petiole length: r7 mm (0), 47 mm (1)
Cauline leaves
7. Limb length: r40 mm (0), 440 mm (1)
8. Base: auriculate (0), not auriculate (1)
9. Apex of auricle: acute (0), obtuse to rounded (1)
10. Length of auricle: 45 mm (0), r5 mm (1)
Flower
11. Petal shape: oblong (0), obovate (1)
12. Size of petals: 43 mm (0), r3 mm (1)
13. Length of the longest filament: r3 mm (0), 43 mm (1)
Pedicel
14. Orientation: erect (0), patent to reflexed (1)
15. Apex: not thickened (1), thickened (0)
16. Size: r5 mm (0), 45 mm (1)
Fruit
17. Type: silicle (0), silique (1)
18. Length: r10 mm (0), 410 mm (1)
19. Width: r6 mm (0), 46 mm (1)
20. Locule: non-spongy (0), spongy (1)
21. Position of locule: apex or base (0), middle (1)
22. Base: cuneate (0), cordate or obtuse (1)
23. Apex: beaked (0), beakless (1)
24. Wing position: apical or basal (0), all around (1)
25. Rim of fruit: non thickened (0), thickened (1)
Seed
26. Length: 43 mm (0), r3 mm (1)
27. Seed shape: oblong (0), elliptic (1)
28. Reticulate to reticulate–areolate (0), lineate (1), ocellate (2)
H. Moazzeni et al. / Flora 205 (2010) 337–343340
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Previous molecular analyses (e.g., Beilstein et al., 2006, 2008;
Bailey et al., 2006) show that Isatis and Myagrum form a
monophyletic group (93% bootstrap support) sister to a clade
including the tribes Brassiceae, Schizopetaleae, and Sisymbrieae.
However, the phylogenetic relationships and delimitation of
genera within the Isatideae were not analyzed. According to the
present ITS sequence data (Fig. 1), Isatis should be broadly
delimited to include the genera Tauscheria,Boreava,Pachypter-
ygium, and Sameraria. The systematic position of various genera of
the Isatideae is discussed below in the light of morphological and
molecular data.
Boreava
Schulz (1936) placed Boreava and Tauscheria in the tribe
Euclidieae, whereas Koch et al. (2003) and Al-Shehbaz et al.
(2006) suggested that they belong to the Isatideae. Our molecular
data support the latter conclusion. Boreava is readily separated
from the other genera of Isatideae by having a distinct style and
quadrangular, 4-winged fruits. Molecular data (Fig. 1) show that
Boreava clearly belongs to clade I.
Tauscheria
This genus clusters with Pachypterygium and three species of Isatis
(Fig. 1). The main differences between Boreava,Tauscheria and
Pachypterygium are fruit characters (e.g., presence vs. absence of fruit
wings or thickened margin). It was recently suggested that differences
in only a few genes can cause substantial alterations in fruit shape,
size, and dehiscence in the Brassicaceae (see Al-Shehbaz et al., 2006
and references therein). Therefore, the use of fruit characters alone for
the delimitation of genera must be critically evaluated.
Fig. 2. AD. Overlay of selected morphological characters on the Bayesian tree inferred from nuclear ITS sequences of selected Isatis taxa and related genera. A. Habit:
annual to biennial (open lines), perennial (black lines); B. petal shape: oblong (open lines), obovate (black lines); C. fruit type: silicle (open lines), silique (black lines) and D.
rim of fruit: not thickened (open lines), thickened (black lines).
H. Moazzeni et al. / Flora 205 (2010) 337–343 341
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Pachypterygium
The present molecular analysis demonstrates that Pachypter-
ygium is polyphyletic, and its three species group with Tauscheria
and three Isatis species in clade II (Fig. 1). The genus is
traditionally distinguished from other members of the Isatideae
by having thickened (vs. thin) fruit margins. As shown in Fig. 2D,
fruit thickening is clearly homoplasious in the tribe and therefore
is an unreliable phylogenetic character. Rechinger (1958) and Jafri
(1973) suggested that Pachypterygium should be united with
Isatis, but this position was not followed by other authors (e.g.,
Appel and Al-Shehbaz, 2003;Hedge, 1968). Based on our
molecular analysis, Pachypterygium cannot be maintained as
distinct from Isatis.
Sameraria
Sameraria is nested within Isatis (Fig. 1), and the two genera
differ solely by the presence in Sameraria of a distinct (vs.
obsolete) style (Davis, 1965;Hedge, 1968;Jafri, 1973). However,
field studies by one of us (H. M.) demonstrated that the style can
be highly reduced in some populations of S. armena. The
separation of these genera was highly questioned by Jafri (1973)
and Al-Shehbaz et al. (2006) who suggested uniting them under
Isatis, the earlier name. Our molecular data support this view, and
we recommend the reduction of Sameraria to synonymy of Isatis.
The recognition of mono- or oligospecific genera in the
Isatideae mirrors many other cases in the Brassicaceae where
the delimitation of such genera is based on only minor differences
in fruit characters that are overemphasized at the expense of
potentially more useful aspects of other structures. Examples
include Twisselmannia Al-Shehbaz vs. Tropidocarpum Hook.,
Lepidium vs. Coronopus Zinn., Heliophila L. versus the endemic-
related genera of the Cape region of South Africa Cycloptychis E.
Mey., Thlaspeocarpa C. A. Sm., Schlechteria Bolus, Silicularia
Compton, and Brachycarpaea DC. (Al-Shehbaz et al., 2006;Koch
et al., 2003;Mummenhoff et al., 2005).
Infrageneric classification of Isatis
Boissier (1867) divided Isatis into the four sections Eremoglas-
ton,Apterolobus,Sameraroides, and Glastum based on minor
differences in fruit size and nature of the seed locule (spongy
vs. membranous). He included Pachypterygium and Sameraria in
sections Sameraroides and Eremoglaston, respectively. Iran is the
only country in which all of Boissier’s sections are represented by
indigenous species, and taxa from all four sections were sampled
in the present study. As shown in Fig. 1, the molecular data
strongly suggest that all sections are polyphyletic and artificially
delimited. For example, I. minima (fruits narrowly winged or
wingless, locule spongy) and I. emarginata (fruits winged all
around, locule membranous), representing the polyphyletic
sections Apterolobus and Eremoglaston, respectively, are sister
taxa with 100% bootstrap support (Fig. 1). Finally, we suggest
considering a broadly defined genus Isatis which can be divided
into two monophyletic lineages (lineage I and II in Fig. 1).
However, formal taxonomic subdivision of Isatis should await
further studies including additional species.
Morphological character evolution
Among 28 morphological characters selected (Table 2) seven
characters including habit (character 1), shape of basal leaves (3),
blade length (5), petal shape (11), length of the paired filaments
(13), pedicel orientation (14), and fruit apex (23), do not show any
homoplasy and can be considered as synapomorphies of certain
monophyletic groups. Fifteen additional characters provided at
least some support for grouping of taxa. The remaining six
characters did not provide support for any grouping. These are:
apex of basal leaves (4), base of cauline leaves (8), length of
auricles (10), pedicel apex (15), pedicel length (16), and fruit base
(22). Only a few characters previously considered as taxonomi-
cally important in Isatis and allied genera (see Davis, 1965;Hedge,
1968) are discussed below in connection with the molecular data.
Habit
The distribution of this character is strikingly congruent with
the Bayesian tree (Fig. 2A). Most species of the Isatideae are
annuals, as in the outgroup, and the perennial habit is
apomorphic.
Petal shape
Both Myagrum perfoliatum and Boreava orientalis have oblong
petals, and the rest of the Isatideae have obovate petals (Fig. 2B).
It is not possible to determine the plesiomorphic state, but if the
oblong petals are plesiomorphic, then a reversal took place in
Boreava. On the other hand, if obovate petals are plesiomorphic,
then oblong petals evolved independently in the two species
above. The same can be said about pedicel orientation, which is
erect in these two species and reflexed in the rest of the tribe.
Fruit type
As shown in Fig. 2C, species with siliques apparently evolved
repeatedly from ancestors with silicles. The phylogenetic value of
such difference is highly questionable (see conclusions).
Fruit margin
Although a thin margin appears to be plesiomorphic (Fig. 2D),
it shows a reversal in the clade including I. minima, I. emarginata,
and I. trachycarpa.
Seed-coat microsculpturing
The reticulate or reticulate–areolate sculpturing of seed coat is
the plesiomorphic state in Isatideae. By contrast, the ocellate
seeds are uniquely restricted to the clade consisting of
I. raphanifolia and I.lusitanica, whereas lineate seeds evolved
independently in P. multicaule and P.brevipes.
Conclusions
The molecular data presented herein demonstrate that the
reliance on fruit characters alone in the delimitation of genera
may well lead to erroneous taxonomic results. Fruit characters
should be critically evaluated in light of molecular and other
morphological data (Al-Shehbaz et al., 2006;Koch et al., 2003;
Mummenhoff et al., 2005).
Molecular data show that the smaller genera Boreava,
Pachypterygium,Sameraria, and Tauscheria are nested within the
larger Isatis. The maintenance of these four genera as distinct
would make Isatis polyphyletic. Therefore, we suggest uniting
them with Isatis.
Acknowledgments
The authors are grateful to anonymous referees for critical
reviewing of the manuscript and for their useful suggestions and
improvement of the manuscript. We thank the curators of the
herbaria cited for providing plant material. We are grateful to
U. Coja for technical help and to Dr. A. R. Khosravi (University of
H. Moazzeni et al. / Flora 205 (2010) 337–343342
ARTICLE IN PRESS
Shiraz, Iran), Dr. Sh. Kazempour Osaloo (Tarbiat Modarres
University, Tehran) and M. Mehrnia (Potsdam University, Ger-
many) for advice. This work is partly supported by the Research
Council of the University of Tehran and is a portion of M.Sc. thesis
submitted by the first author to the University of Tehran.
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