Molecular Systematics of the Chinese Yinshania (Brassicaceae): Evidence from Plastid and Nuclear Its DNA Sequence Data

27 Pages·2000·21.9 MB·

by  Marcus Koch

MOLECULAR
SYSTEMATICS
Marcus Koch^ and Ihsan Al-Shehbar
A. J
OF THE CHINESE
YINSHANIA
EVIDENCE
(BRASSICACEAE):
FROM
AND
PLASTID
NUCLEAR DNA
ITS
SEQUENCE
DATA!
Abstract
Species of the Chinese endemic genera Yimhania, and were
Hilliella, Cochleariella originally i)laced in or closely
A
associated with Cochlearia. previous preliminary molecular study mainly on European Cochlearia and detailed
morphological studies by us showed that this complex was not affined to Cochlearia Depending on the authority
s. str.
number
consulted, the of taxa recognized in this complex ranged from 11 to 25 species in one to four genera. The
present phylogcnetic study is based on the analysis of the ITS (internal transcribed spacer regions of the nuclear
DNA)
ribosomal and the chloroplast frnL-intron sequences from 18 taxa. Resulting phylogenies were compared, and
the results demonstrate that there are two different lineages. One lineage combines exclusively the highly polyploid
taxa from Hilliella and Cochleariella. The second lineage includes the diploid taxa from Yinshania, However, incon-
when nrDNA-
gruencies and cpDNA-derived phylogenies were compared suggest hybridization between these two
We
lineages. followed a concept to combine all taxa of this complex into the genus Yirishania. Our results from
cpDNA
phylogcnetic analysis of nr and support the association of Yinshania with Cardamine and Rorippa, rather than
with Cochlearia, as was suggested by nearly previous authors.
all
Key
words: Brassicaceae, Cochleariella, Hilliella, molecular systematics, reticulate evolution, Yinshania,
Many
authors follow Schulz (1936) and Schultze- Rchb. (Koch et 1999a). Section Pseudosemper-
al.,
Motel
(1986) in dividing Cochlearia into the sec- vivum, which is centered in the Middle East and
Pseudosempervivum
tions Boiss., Glaucocochleria 0.
clearly unrelated to Cochlearia, most closely re-
is
E. Schulz, Cochlearia (= Eucochlearia and
Prantl), lated to Masmenia K. Mey. and Noccaea Moench
F.
Hilliella 0. E. Schulz. As shown by Koch
et al. (Koch et 1999a), both of which were segregated
al.,
(1999a), however, this sectional classification
is by Meyer
(1973, 1979, 1991) from Thlaspi L.
s.l.
highly Section Cochlearia widely
artificial. dis-
is
The
family of Brassicaceae divided into sev-
is
tributed in Europe and the circumpolar region,
^^^j ^^-^^^ ^^j Most them
subtribes. of are highly
whereas
section Glaucocochlearia, which was
artificial, such as tribe Arabideae (Koch et
al.,
raised the generic rank by Pobedimova
to
(1968),
1999b) or Lepidieae (Zunk 1996). Following
et al.,
is restricted to southwestern Europe. The latter sec-
classical concepts, Cochlearia Pseudo-
tribal sect.
me
and
L. C.
ifolia
sempervivum, sect. Cochlearia, and sect. Glauco-
perma
members
& ^''^ of tribe Lepidieae. Species
which was
Soulie, only recently included (Koch
^^f^/^^''^^
originally assigned by Schulz
(1923) to section Hil-
1996)
{Yinshania, and were
Hilliella, Cochleariella)
'^'^^^'^
related to the other two species (Koch
et
al.,
excluded from Cochlearia by Pobedimova
1999a). Section Cochleana consists of a species (1970),
complex ^^^ ^^^ ^^* assign them to any genus. However,
that demonstrates highly polymorphic
chromosome numbers, and diverse ecological ad- ^^^^^ species have recently been placed in three
&
Ma
Chinese endemic
aptation and geographic distributions. Morphologi- genera, Yinshania Y. C. Y.
&
cal differences between phylogenetically taxa Z. Zhao, Cochleariella Y. H. Zhang Vogt, and
sister
often weak and poorly defined (Koch et al., Hilliella (0, E. Schulz) Y. H. Zhang, each of which
1996). Both sections Cochlearia and Glaucococh- was assigned to a different subtribe. The genus Yin-
&
learia are closely related to the genus lonopsidium shania (Ma Zhao, 1979) was placed in subtribe
We
* are grateful to Zhang Yu-hua for providing some of the samples. The curators of A, B, BM, E, GH, HAST,
WU
IBSC, K, KUN, LE, MO, NAS, NY, P, PE, TAI, TI, TNS, US, W, and are thanked for the loan of specimens.
'^ Institute of Botany, University for Agricultural Science, Gregor-Mendel-Str. 33, A-1180 Vienna, Austria.
USA
Missouri Botanical Garden. P.O. Box 299. Missmiri 6.'^166-n2QQ
•* St. Ixniis.
Ann. Missouri Bot. Card. 246-272.
87: 2000.
\
Volume Number &
2 Koch Al-Shehbaz 247
87,
2000
Chinese Yinshania
The
Descurainiinae, Sisymbrieae. genus
tribe tributions to a better understanding of the classifi-
Cochleariopsis (Zhang, 1985), renamed as Coch- cation, generic delimitation, and phylogenetic
re-
&
A
leariella (Zhang Cai, 1989), was placed in sub- lationships in the Brassicaceae. preliminary
nrDNA
tribe Cochleariinae, tribe Lepidieae, along with Hil- study (Koch et 1999a) utilizing ITS and
al.,
liella (Zhang, 1986). Although a few studies cp trnL intron sequence data of four species of the
s. str.
on taxonomy, evolution, and origin of these genera Yinshania complex (including and Coch-
Hilliella
&
Zhang
(Zhang, 1987; Xu, 1990; Zhang, 1993) leariella) clearly showed that the complex unre-
is
have been made, nothing was said about their sys- lated to Cochlearia. In this analysis has been
it
tematic position in relation to the remaining Asian shown that ITS and trnL intron sequence data pro-
and European
taxa of Cochlearia. Zhang's (1987) vide sufficient sequence variation to distinguish
division of Yinshania (excluding Hilliella) into two significantly between Yinshania and Cochleariellal
sections and two series and Zhao's (1992) classifi- Hilliella accessions with both data sets. In order to
cation of Yinshania (including Hilliella) into two gain a better insight of the phylogenetic relation-
sections and six series, were shown by Al-Shehbaz ships within this Chinese complex, we examined
et al. (1998) to be highly artificial. In fact, one of sequence variation of the internal spacer regions
the species assigned by Zhang (1987) to Hilliella (ITSl and ITS2) of nrDNA (Baldwin et 1995;
al.,
and by Zhao (1992) to Yinshania was placed by Al- Campbell et 1995) and of the cp trnh intron
al.,
&
Shehbaz and Yang synonymy
(1998) in the of Car- (Bohle 1994; Gielly Taberlet, 1994; van
et al.,
Ham
damine fragariifolia 0. E. Schulz. et al, 1994; Koch et al, 1999a), and com-
On
the basis of a comprehensive morphological pared the derived molecular phylogenies with
tra-
survey of Yinshania, Hilliella, and Cochleariella, ditional concepts based on morphological data.
Al-Shehbaz et al. (1998) reduced the latter two to This approach provided us with the opportunity to
synonymy of Yinshania, and concluded that there characterize species lineages and analyze incon-
to
is no need for infrageneric subdivisions that do not gruencies between different data sets in order to
reflect the phylogenetic relationships of this small test hypotheses of gene flow over lineages and chlo-
genus 13
of species. roplast capture.
They
also demonstrated that morphological char-
acters previously used in the delimitation of species MATERIALS AND Methods
on
density of papillae the valves,
(e.g., fruit fruit
PLANT MATERIAL
shape, and seed number per are highly
fruit) vari-
among and
able within different populations of the
DNA
Leaf material for extraction was obtained
same com-
species. Furthermore, differences in the
from herbarium specimens most which
(Table of
1),
pression of fruit (terete vs. latiseptate or angusti-
were provided and determined by Zhang Yu-hua
were emphasized by
septate) that heavily earlier
Academy
(Institute of Materia Medica, Zhejiang of
. .
authors (e.g., Schulz, 1936) in the delmeation of .* j. tj v d rr^u-
^' Medicm. e, Hangzhou, reopli e»s Kt>epubuih-c Lhma)\.
oi
^ n
1*1
r
-
eenera were not found1 1be taxonomically useful jj
to wr j
^^ • r 'yu
P "^*^ ^^^* examine vouchi,ers *to verity Zhang?s de-
in the Yinshani.a complex. As shown by Koch1
^ et al. .• rpi i u j
^ t. ermi•nations. Ihe samples represent* a broad spec-
placement heavy emphasis on
(1999a), the of
fruit ^^^
^f ^p^^j^^ ^^ Yinshania, and Coch-
Hilliella,
compression has led to the artificial integration of
Cardamine With, and Rorippa
i^^^^^u^^ fiexuosa
several taxa into Cochlearia sect. Pseudosempervi-
^^^^^^^ Besser served as the outgroups. The
(l.)
vum
and
instead of Thlaspi In fact, terete var-
s.l.
^jsj^ sequences for the outgroups were obtained
m
occur numerous genera
lously flattened fruits of
f^^^ Franzke
et al. (1998).
many
and
the Brassicaceae, in cases this aspect of
morphology taxonomically
fruit is insignificant.
DNA EXTRACTION, PCR-AMPLIFICATION, AND
Morphological convergence and parallelism are
SEQUENCING
widespread in the Brassicaceae (Dvorak, 1971;
DNA
Meyer, 1973; Avetesian, 1983; Endress, 1992), and The total for the outgroups was isolated
the dependence on such characters construct from CTAB
to leaf tissues following the (cethyltriam-
phylogenies often leads erroneous conclusions moniumbromide) method Doyle and Doyle
to of
(Sytsma, 1990; Meyer, 1991). Recent molecular as modified by Mummenhoff and Koch
(1987),
DNA
Warwick
analyses (e.g., et al., 1992; Price et al., (1994). extraction from herbarium material
&
Mummenhoff Zunk
1994; Koch, 1994; performed
et
al.,
1996; Mummenhoff et 1997; Koch et 1998a, reaction tubes from 50-|xg dried tissue. Tissue was
al., al.,
b; Koch et 1999a, b) have made significant con- ground with sand and pre^ arme
al.,
;
248
Annals
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Volume Number 2 Koch & Al-Shehbaz 249
87,
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Chinese Yinshania
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250
Annals
the
of
Garden
Missouri Botanical
DNA
&
Organic extraction and isolation from herbar- to those of Sinapis alba L. (Rathgeber Capesius,
ium specimens followed Koch et al. (1996). 1989) and other Brassicaceae (Mummenhoff et al,
DNA
DNA
Double-stranded of the complete ITS re- 1997; Koch 1999a). sequences were
et al.,
rDNA
gion, including the 5.8S gene, was amplified aligned by hand. Parsimony analyses were
per-
PCR
by 30 cycles of symmetric using ITS primers formed with unordered Filch parsimony and
designed by White
et al. (1990) weighted parsimony with a transition transversion
:
ified by Mummenhoff etal. (1997). The 18F primer weighting of 1.0:1.08 using PAUP version 3.1
(5'.GGAAGGAGAAGTCGTAACAAGG-3')
BRANCH-AND-BOUND
is lo- (Swofford, 1993). The
al-
rDNA
cated the 3'-end of the 18S gene, and
at gorithm was used maximally parsimonious
to find
25R (5'-TCCTCCGCTTATTuAiAii,u-5
primer
trees. Bootstrap analysis (Felsenstein, 1985) was
J
25S rDNA.
is located at the 5'-end of the It has performed using 1000 replicates and the HEURIS-
PCR rDNA
been
reported that selection of para- MULPARS
TIC
search algorithm with the option,
logues has occurred (Buckler How- GAPMODE =
et al., 1997). We combined MISSING
the option
PCR
ever, selection might have only been impor-
^j^h the coding of the gaps as additional presence/
G+C
tant in high content sequences (Buckler et &
absence characters (Downie Katz-Downie,
1996).
Sequences from Yinshania and
1997).
al., Hilliella
number
xhis option decreases the of equally par-
G+C
(Koch 1999a) comparable
et al., are in con-
^i^onious because redundancy
trees of the result-
PCR
tent to sequences from Gossypium, which
in
-^^ f^^^ ^^^^^^ ^^^ ^^^^ ^^ ^^^^^j characters
for
selection was probably weak (Wendel 1995a)
et al.,
same
the indel events (Wojciechowski
et 1993).
al.,
The
resulting amplification product im-luded ITSl,
Evolutionary data are most often presented as a
PCR
rDNA,
5.8S and ITS2. Only those products
pni yliogenet^i. c ^tree, ,tihe undierli yi• ng assumpt.i• on ibei• ng
- ' ^T-./rr.
T- 1
were cloned , pGLM-T-Easy .
into the clonine; vector
*v ^- tt
that^ evoli ution • a bi ranchi_i- ng process. However, em-
IS
/TiTyrwMj-r- \\
(PROMEGA)
i sihowedi • i ibandi on
that a single ethi-
and
pirical data rarely ideal often supports sev-
is
dium bromide stained agarose Two cloned ITS
gels.
eral trees instead of one unique Hence,
tree.
it
PCR
regions from two independent reactions were
makes
sense to consider tree reconstruction meth-
sequenced and
(forward reverse) with both ampli-
ods produce
that a tree the given data heavily
if
fication primers and two universal primers located
favor one tree over others. Otherwise, methods
all
pGEM-T-Easy
in the flanking sites of the vector
(t7-
that produce a more general graph that indicates
forward: 5'-gtaacgatttaggtgacactatcg-3, ml3-re-
different possible phylogenies are useful (Huson,
verse: 5'-agcggataacaatttcacacagga-3). This
One
1998). such method the Decomposition
is Split
that every single clone was sequenced four times
^"^roduced by Bandelt and Dress and
(1992)
its
to avoid sequence errors. The trnL (UAA) intron
^^^^^'^^^- '" ^'^^' ^o visualize conflicting phylo-
was amplified and sequenced by using
the univer-
^^ analyzed ingroup ITS
"^8"^^"' all se-
B49318 (5'-CGAAATCGGTAGACGCT- S^"^*^^
primer
sal
usmg
q^^^^^es (see Fig. the software program
ACG-3') fmL(UAA)5'- 6),
located the 3'-end of the
at
&
SplitsTree version (Huson Wetzel, 1995
1.0.3.
A49855 (5'-GGGGATAGAGGGACTTG-
exon and
shareware
ftp://ftp.uni-bielefeld.de/pub/math/spHts/).
AAC-3')
located the 5'-end of the frnL(UAA)3'-
at
DNA
imL
PCR intron data. sequences were aligned
exon (Taberlet 1991). The used
et al., profile
^^ ^^"^- Parsimony analysis was performed with
to amplify the trnL intron followed the following
^^^P
(version 3.1; for options, see ITS data). Gaps
profile: hot start with 5 min. at 94°C, and 35 cycles
^^^^ treated as additional unweighted binary char-
of amplification min. 94°C, 45 min. 50°C, 45
(1
These gaps were coded using
acters.
min. 72°C), final elongation step for 10 min. 72°C, strict criteria:
and 4^. DNAs ^"^^ ^^^^^ the same position and have the
storage were cycle-sequenced g^P^ ^^
at
same
using the Taq DyeDeoxy Terminator Cycle Se- aligned length to be treated as homologous,
and no one gap two more
quencing Kit (ABI Applied Biosystems, Prod- splitting of in or char-
Inc.).
was performed (Koch
ucts of the cycle sequencing reactions were run on acters et al., 1999a). Boot-
an ABI 377XL automated sequencer (ABI Applied strap analysis was performed as described above.
Biosystems, Material from accession numbers
Inc.).
22-26
(Table was only used for sequencing the
1) tribal relationships
trnL intron sequence, because amplification of the
ITS
regions failed totally. To estimate the tribal affinity of Yinshania, we
derived an ITS phylogeny with sequences from
DATA
ANALYSIS
Capsella rubella Reut. (Koch 1999b), Ara-
et
al.,
ITS data. Boundaries of the ITS regions and hidopsis thaliana Heynh. (GenBank U43224),
(L.)
coding sequences were determined by comparison Yinshania acutangula (0. E. Schulz) Y. H. Zhang,
Volume Number 2 Koch & Al-Shehbaz 251
87,
2000
Chinese
Yinshania
(EMBO
Barbarea vulgaris R. Br. X98632), Carda- eluding the additional 0/1 matrix for the gap posi-
mine flexuosa With. (Franzke et al., 1998), Thlaspi tion, resuhed in 24 most parsimonious trees (MPTs)
arvense L. (Koch et al., 1999a), Cochlearia aestu- with a length of 538 and a consistency index (CI)
1\A%
Heywood
aria (Lloyd) (Koch et 1999a), Brassica of (66.9% autapomorphies are excluded).
al., if
oleracea L. (GenBank AF039994/AF040038), and Of the 264 variable nucleotide positions, 166
in-
DNA
(EMBO
Sinapis alba X66325). sequences formative positions were in the ITSl region (in-
were aligned by hand, and the alignment is shown eluding 48 autapomorphies) and 98 in the ITS2
in Figure 4. Alignment positions 109—160 were re- region (including 25 autapomorphies). Four out of
moved
from subsequent analysis. Parsimony anal- 36 gap positions within the total sequence align-
yses were performed with unordered Fitch parsi- ment are unique to a particular sequence (Rorippa
PAUP A
mony
using version 3.1 (Swofford, 1993). palustris 2 gaps, ace. no. 3, and ace. no. 14).
BRANCH-AND-BOUND
The algorithm was used calculation of the transition/transversion ratio for
GAP- MPTs
to find maximally parsimonious trees with the the revealed a ratio of 1.00: 1.08. Therefore,
MODE = NEWSTATE
we
option. Bootstrap analysis used a weighted parsimony approach with a
(Felsenstein, 1985) was performed using 1000 rep- character state weighting of 1.00: 1.08 (transition:
MPT
HEURISTIC
licates and the search algorithm em- transversion), which resulted in one that
Is
MULPARS A
ployed with the option. decay anal- also represented among the 24 MPTs from the Fitch
ysis (Bremer, 1988) was performed in addition to parsimony with a consistency index of 67.7% (CI
We
57.9%
the bootstrap approach, in order to ss the con- autapomorphies were excluded). pre-
if
MPT
fidence that could be placed in the monophyly of sent the from the weighted parsimony ap-
clades. Decay indices (DI) were estimated accord- proach to demonstrate relative branch length (Fig.
Baum
ing to et al. (1994). 2). Bootstrap values are provided from 1000 repli-
cates using the weighted parsimony approach. For
Results we
most taxa identified only one ITS sequence type
within a single specimen. For Hilliella lichuanensis
DATA
ITS
Zhang
H.
Y. (ace, no. 13), Cochleariella zhejian-
&
The length of the alignment with accession gensis H. Zhang) Y. H. Zhang R. Vogt
total (Y. (ace.
numbers 1—21 and outgroups Cardamine Yinshania acutangula K
flexuosa no. 21), (ace. no. henryi
5),
and Rorippa palustris is 464 bp, with 283 and 181 (Oliv.) Y. H. Zhang (ace. no. and Y furcatopilosa
7),
nucleotides in ITSl and ITS2 spacer regions, re- (K. C. Kuan) Y. H. Zhang (ace. no. we found
8),
spectively. The alignment required 36 (7.8%) gap two very similar ITS sequences among the two
and shown num-
positions, including the outgroups, in clones sequenced. In the case of accession
is
Figure 1. Sequence data were submitted to Gen- bers 13, 5, 7, and 8, ITSl and ITS2 regions differed
Bank numbers AF100793-
with accession by
only a single site mutation. In the case of C.
AF100852 One
(Table 1). third of these gaps is zhejiangensis (ace. no. 21), the two ITS types from
located between positions 117 and 133 in the ITSl. the same individual differed by 15 mutations,
This region was completely excluded from subse- which might indicate that two different ITS loci
quent analysis. Additionally, we excluded nucleo- were cloned and sequenced. Both sequences clus-
473^84
465—467
tide position and from subse- tered within the same clade. In Hilliella fumarioi-
&
quent data analysis because of an ambiguous des (Dunn) Y. H. Zhang H. W. Li (ace. no.
17),
Gaps we
alignment (Fig. from nucleotide positions detected two ITS sequences from a single in-
1).
11-14 293-294
and were treated as one single gap, dividual that clustered in different clades (Figs.
2,
respectively. The number of introduced gaps Both sequences differed by 76 mutations. The
is 6).
comparable to a phylogenetic analysis of Thlaspi ITS data clearly support a separation of two clades
s.L with 4.8% of the sites (Mummenhoff consisting of Yinshania Zhang (1987) and
et al.,
Kim
1997). In Krigia and outgroups, and Hilliellal Cochleariella, Different accessions from
Ji
3.9%
(1994) had to introduce gaps in of the sites. one taxon (sensu Al-Shehbaz et al., 1998, refer to
Total lengths of ITSl and ITS2 are nearly identical grouped at different positions in the ease of
fig. 2)
among and between 447 bp
the taxa surveyed, vary C. zhejiangensis (ace. no. 20, not related to ace. nos.
&
Zhang
(Hand.-Mazz.) H. H.
{Hilliella alatipes Y. 18, 19, 21).
W. Li var. micrantha Y. H. Zhang [= Yinshania Using the taxonomic concept of Al-Shehbaz et
rivulorum (Dunn) Al-Shehbaz accession and merging changhuaensis
et al.], no. al. (1998) Hilliella Y.
10) and 457 bp {Cardamine flexuosa and Rorippa H. Zhang, H, guangdongensis Y. H. Zhang, and H,
palustris). lichuanensis (ace. nos. 11, 12, 13, respectively) into
Phylogenetic analysis using Fitch parsimony, in- H. lichuanensis, only accession numbers 11 and 13
Annals
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