Phylogenetic relationships in the Corylopsis complex (Hamamelidaceae): Evidence from sequences of the internal transcribed spacers of nuclear ribosomal DNA and morphology

17 Pages·1997·6.7 MB·English

by  J H Li

^
-
RHODORA,
Vol. 99, No. 900, 302-318, 1997
pp.
PHYLOGENETIC
RELATIONSHIPS THE
CORYLOPSIS
IN
COMPLEX
(HAMAMELIDACEAE): FROM
EVIDENCE
SEQUENCES OF THE
INTERNAL TRANSCRIBED
SPACERS OF NUCLEAR RIBOSOMAL DNA AND
MORPHOLOGY
JlANHUA AND BOGLE
Li A. LlNN
1
Department New
of Plant Biology, University of Hampshin
NH
Durham,
03824
Herbaria
MA
22 Divinity Ave., Cambridge, 02138-2094
Anita
Klein
S.
Department
of Biochemistry and Molecular
Biology,
New NH
University of Hampshire, Durham, 03824
abstract.
Morphological and
nuclear ribosomal Transcribed
Internal
examine
genera
in the Corylopsis complex,
comprising
Corylopsis, Distylium, Eustig-
l™1 ^ « *-« m - «
The
and
similar
DNA
tionships within the complex. Both
morphological and ITS
data se-
quences
support
separation of Corylopsis from and
the other genera, a closer
relationship of Sinowilsonia
to Fortunearia than The monophy-
to Distylium.
of
ly Eustigma-Fortunearia-Sinowilsonia
was
supported by ITS sequence
the
data. This studv substantiates FnHre««'c ^f tUo w^tw*.
**<***** ir»t^«%».«*ot;^«
The
ports the hypothesis
that floral struct
plex appear to have been modified
evolution.
Key DNA
Words:
phylogeny,
Corylopsis complex, Hamamelidaceae, ITS
sequences
The
Hamamelidaceae
distributed
New
both the Old and Worlds Lu
(Endress Zhang and
1993; 1995).
Morphological
diversity
in this family, especially of reproductive
attributes, has prompted numerous
systematic (Bogle 1970,
studies
and Chang
Philbrick 1980;
Endress
Mione
and
— —
— — —— — — —
^_s 7 -m-
.
subfamilial
classification of
the family has been, and
302
—
1997] Li et Corylopsis Complex 303
al.
troversial, the subfamily Hamamelidoideae has been widely
ac-
cepted as a monophyletic clade (Bogle and Philbrick 1980; Endress
The
1989a, most
b). definitive characteristics of subfamily
this are
arpels, 2-carpellat
(Endress
persal
IS
Hamamelidoideae
One
problematic. unresolved problem of
is that the relationships
of the genera that have been considered more or less related to
Corylopsis Sieb. Zucc. The Corylopsis complex purpose
et for the
encompasses
Champ
Wils
The
Corylopsis complex shows a high diversity of floral struc
an epitome of family
the
am
and
petals are rather reduced in Eustigma, Fortunearia, Sinowil-
sonia; there are no sepals or petals in Distylium. Therefore, reso-
may
complex
lution of the intergeneric relationships within the
shed on of characters
light the evolutionary directionalities floral
in the Hamamelidaceae.
Harms made comprehensive taxonomic
(1930) the treat-
first
ment
of the Hamamelidaceae, placing 21 genera in five subfamilies
known
(two were were not placed).
additional genera
that
little
The
Hamamelidoideae was subfamily, containing 16
the largest
complex
genera in The genera of the Corylopsis fell
five tribes.
monogeneric Eustigmateae {Eustig-
into three of these the
tribes:
and Fortune and the Di-
ma)', the Corylopsideae (Corylopsis arid),
stylieae (Distylium, Sinowilsonia, and Sycopsis).
Harms's
Schulze-Menz adopted classification,
(1964) basically
tribe
Many
Harms
Chang
1973,
1979).
and
moved Fortunearia
both Si-
Endress however,
(1989a,
b),
Corylopsideae
nowilsonia Eustigmateae, thus treating the
into the
by Endress
admitted
as a monogeneric Nonetheless, as
tribe.
amon
well
resolved.
Internal
and almost
and
angiosperms specific
at
304 Rhodora
99
[Vol.
AA,
Table Vouchers for the species sampled. the Arnold Arboretum;
1.
GUNH, New
Wood-
the Greenhouse the University of Hampshire; WI,
at
landers Inc., SC. Each species name is followed by a 3-letter acronym used
and
in other tables figures.
GenBank
Species Voucher Source Accessions
AA
U65462
Corylopsis pauciflora (cpa) LiOl
AA
C. sinensis (csi) Li02 U65461
AA
C. spicata (csp) Li03 U65463
GUNH, WI
Distylium myricoides (dim) Bogle U65464
D. racemosum Bogle GUNH, WI U65465
(dir)
Eustigma Chung, Taiwan U65466
oblongifolia (eus) N.
J.
AA
Fortunearia sinensis Li04 U65467
(for)
AA
Sinowilsonia henryi Li05 U65468
(sin)
C
Mytilaria laosensis (myi) Luo, Xinning, China U65469
Y.
Wo
Campbell Suh
et 1995; 1993;
et
al. al.
we
In this studv. therefore, based our anal
>).
and
The
obiective of
this
among
the alternative hypotheses concerning the relationships
5
genera of the complex, which can be translated into the follow-
questions: 1) Should Corylopsis be isolated from the other gen-
and be treated as a monogeneric tribe? 2) reasonable to put
Is
it
tigma
together with Fortunearia and Sinowilsonia in the Eustig-
eae even though Eustigma somewhat from
the others
different
is
more
phologically and geographically? and Sinowilsonia
3) Is
iely related to Distylium than to Fortunearia?
MATERIALS AND METHODS
The
internal transcribed spacers ITS-1 and ITS-2 were se-
Cory-
My
and an
outgroup,
1).
>le
Three of the six genera sampled are monotypic, including Fortu
and
Mytilaria,
Corylop
and
Distylium
20), (10
Zhang Lu
(2-4; and
These
1995).
three
i g
been monographed
not
in recent years and
species debatable. Furthermore,
is materials
-
unavailable
for this study. Therefore,
the
r<
three genera unavoidablv een
is limited to
Complex
Li al.—Corylopsis 305
1997] et
which
valid species for samples could be obtained from cultivated
from
plants or accessible field locations.
The
genera of the Corylopsis complex represent three of the
four monophyletic Hamamelidoideae,
tribes in the but the
tribal
relationships are unresolved (Endress 1989a). Thus, uncertain
is
it
which genera should be used as outgroups from within the Ha-
mamelidoideae. Endress (1989a) used Exbucklandioideae
(incl.
Disanthus, and Exbucklandia) outgroup
Mytilaria, as the in his
phylogenetic evaluation of the Hamamelidoideae based on mor-
matK
phological data. Our preliminary results from sequences of
gene most
encoding plastid maturase suggested that the closely
related genera to the Hamamelidoideae were Mytilaria and Dis-
we
anthus chose Disanthus and Mytilaria as
(unpubl.). Therefore,
when
we same topology
outgroups. Although obtained the tree
using either or both of these two genera as outgroups, the confi-
when
dence of sequence alignment decreased both Mytilaria
the
we
and Disanthus were Thus, included only Mytilaria in the
used.
DNA
parsimony The sequences obtained in this study
analysis.
numbers
GenBank
have been submitted the and their accession
to
are Table (Disanthus not included).
listed in is
1
genomic
Molecular
techniques. Total
DNA
procedures
from young standard extraction
leaves using the
The
am
White were used
(1990) to
et —
— - • ——-» ^*mr ^** +* a^tlJi.m 1 -A. ^ ^ ^*r f w » — — — —- — — x ^
- >
DNA
Each
Reaction® (PCR).
Chain
Polymerase
region using the
WI)
Madison,
X
Taq
La CA),
4-5
units of Tag Extender (Stratagene, Jolla, 1
mM
DNA,
50-100 ng and 20 u.M
dNTPs,
extender
buffer, 2.5
94°C Hot
minute
were preceded by a three
Amplifications
ts.
Start
PCR
program
MA). The
(Watertown,
a MJ-Research thermocycler
45°C
30 seconds,
94°C denaturation for
consisted of 30 cycles with
seconds.
72°C extension for 115
and
annealing 115 seconds,
for
minute extension step at
The was by a ten
followed
cycle
final
72°C.
PCR
The am
The ITS bands
(pH ol
E buffer 8.0).
DNA
0X174
Haelll
comparison
to
30 minutes
.~a or»orocp_riiapstpd for
Then
the purified
306 Rhodora
99
[Vol.
stranded
terminator chemistry and
reactions,
an
ms, Foster City, CA). The procedures were carried
manufacturer's
instructions
Hampshir
White
(1990) were used
et as internal
al.
sequencing
primers.
DNA
sequence chromatograms were analyzed using the Seqed
program
(Applied Biosystems) and the sequences were then con-
DNA*
Seqman
tigged using the of software package (Madison,
The
WI).
boundaries of ITS-1 and ITS-2 were determined by com-
paring the limits of the 3' end of the 18S, 5.8S, and 5' end of the
rRNA
26S
sequences of ITS
the region Canella winterana
in
(GenBank
accession number: L03844).
Statistical analysis. Chi-square independence were
ap-
tests
plied to test whether the distribution of base compositions was
among
significantly different
the genera. Analysis of variance
(ANOVA)
was
utilized to test the null hypothesis that pairwise
among
divergences
the genera were between
significantly different
ITS-1 and ITS-2
(Zar
1996).
Morphological
data. Twenty-three morphological
the Corylopsis complex and
r the outgroup Mytilaria
based on own
:ted and our
literature observations B<
(
Chang
The and
characters
We
each genus
in the morphological analy
variation in the collected morphological
scores
tical for species within a senus.
rphological
data smaller than
set
is
number
of
species ITS
in the data
set.
DNA
Phylogenetic The
analyses.
ing
the clustal option of Megalign DNA*. The
the program
of
DNA
aligned sequences
from
are available
the authors. All se-
quence
and
characters
were
their states unweighted, and indels
were coded
as missing data. Phylogenies were using
reconstructed
PAUP
(Swofford
3.1.1. 1993) with
the exhaustive search option.
—
))
Complex
Li Corylopsis 307
1997] et
al.
Table Morphological and used parsimony
2. characters their states in the
analysis.
Code
Character and
State
Seed
1. dispersal Nonballistic (0), ballistic (1)
Venation Pinnate intermediate palmat<
2.
(0), (1),
(2)
Number
3. of ovules per carpel 3 ovules (0), 3 ovules (1), 1 ovule
(2)
Nodal anatomy Trilacunar multilacunar
4. (1)
(0),
andromonoecious
Sexuality Bisexual
5. (0),
( ),
1
monoecious
(2)
reduced absent
6. Petals Distinct (0), (1), (2)
7. Pollen apertures Tricolpate (0), tetracolpate (1)
wind
8. Pollination Insect (0), (1)
Smooth
9. Pollen surface (0), verrucate (1)
Ovary Semi-inferior superior
10. position (0), (1)
Filamentous leafy
11. Stipules (0), (1)
12. Trichome types Scale stellate (1)
(0),
Unexpanded expanded
Stigma strongly
13. (0),
(1)
Evergreen deciduous
14. Habit (0), (1)
Spike spadix
15. Inflorescence (0), (1)
absent
Staminodes Present
16. (0), ( 1
Absent present
17. Anther connective protrusion (0), (1)
Longer than or equal to anther
18. Filament (0),
than anther
shorter (1)
More than 30 30 or fewer (1)
19. Vessel bars (0),
Absent simple cluster
(2)
20. Crystal types (0), (1),
21. Flower Fixed variable (1)
parts (0),
C
Absent fusiform libriform
22. Foliar sclereids (0), (1),
Absent present
23. Lenticel on ovary (0), ( 1
and decay an
thousand bootstrap
replicate
formed
The
parsimony
and were unweighted to avoid biases in the
their states
The
program
MacClade
3.03
unambiguous
(Maddison and Maddison 1992) to an ilyze the
changes of morphological characters
the
<
RESULTS
Corylopsis
Sequence lengths in the
Sequence
characteristics-
from 224
and to
ITS-1
mnlex raneed from 236 to 275 bases in
308 Rhodora
99
[Vol.
sam
taxa
pled except Eustigma, Fortunearia, and whose two
Sinowilsonia,
GC
were more
spacers or equal
less in length. contents for ITS-1
57-64%
and ITS-2 were very and 61-67%,
similar, respectively
An
(Table Independence showed
of base
3). test that distribution
GC
compositions (A, G, C, and was
T, content) not significantly
>
different for the genera (P
all 0.8).
The
among
pairwise divergences
tl
from
and
outgroup
the Mytilaria varied
Corylop
and where more
Distylium,
than
4%
vergences
within each genus were
generally lower than (Table
(ANOVA)
Analysis of variance
demonstrated
3). pairwise
that di-
vergences between ITS- and ITS-2 were
not significantly different
1
=
(P nor were among
0.8), the pairwise divergences genera
the in
=
combined
the data of ITS-1 and ITS-2
(P
0.1).
Alignment
of
the sequences required 14 of which
indels, six
were two more
or bases in length. Table 4 the largest six
lists
indels in these ITS sequences. Noticeably, deletion 4 occurred only
in Corylopsis, whereas was
deletion 36
consisting of bases,
2,
found
in Eustigma, Fortunearia, and These
Sinowilsonia. three
common
B
with
Corylopsis.
sessed deletion and shared
5,
paucifl,
was
found
in Mytilaria
When
Phylogenetic
trees. only ITS-1 sequences were
uti-
lized in the parsimony analysis, an exhaustive search found one
shortest of
tree 131
steps (Figure la) in which a clade of Cory-
and
lopsis a clade of
Distylium-Eustigma-Fortunearia-Sino-
wilsonia were
well
resolved, with bootstrap values and decay
indices of 100%,
11 steps, and 98%,
six steps, respectively.
However,
Eustigma
and
Fortunearia were grouped within a
clade weakly 60%
supported by
a bootstrap value and one step
of decay.
The
consensus
strict of two
tree the shortest of 14 steps
trees
1
based on ITS-2
data
(Figure lb) did not
resolve the relationships
of
the three groups,
Corylopsis, Distylium, and Eustigma-For-
tunearia-Sinowilsonia. However,
the internal relationships of the
were itc ^u^^rr^nv
three clades hasirallv cam*, ac ;«
th*» *u<*
i
—
<
1997] Li Corylopsis Complex 309
et
al.
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310 Rhodora
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DNA
Table 4. Indels of two or more bases in length of ITS sequences
-
+
sampled **
in the species presence; absence; * deletion number; base
(
Taxon
range; abbreviations Table
as in
1).
1*
2 4
3 6
5
63-67**
Species 110-145 302-323 334-337 338-339 490-49
—
cpa + ^~ + + +
— —
^~ + +
csi ^—
— — —
+
csp ^— +
—
+
dim
^^^^ -1-
dir + +
+ +
eus +
+
+
for +
myt
+
+
sin +
with
the exception of Fortunearia and Sinowilsonia forming a
61%
clade supported by
a bootstrap value of and decay index
a
of one
step.
When
ITS-1 and ITS-2 were combined
in the analysis, a sin-
gle shortest tree of 246 steps was generated (Figure showing
lc),
relationships
similar to those of the ITS-1 but with For-
tree,
tunearia and
Sinowilsonia forming
a clade sister to Eustigma.
The
combination two
of
the spacers resulted in stronger support
group
for the of Eustigma,
Fortunearia, and Sinowilsonia
rela-
tive to either of the separate data
sets.
The
single
shortest phylogenetic produced,
tree (34 steps)
based on
morphological
data (Figure contained two clades
2),
complex:
in the Corylopsis and
Eustigma-Distylium-Fortunear-
ia-Sinowilsonia. Within the second clade, Eustigma was the ba-
genus
sal followed by Distylium, and was
Distylium the
sister to
clade of Fortunearia and The and
Sinowilsonia. bootstrap value
decay 75%
index were
and two
Eustigma-
steps for the clade of
Distylium-Fortunearia-Sinowilsonia,
and these supporting val-
ues were
small
all for the internal
relationships.
A
parsimony
analysis using combined mor-
the data of
set
phology and ITS
sequences produced same cladogram
the (Fig-
ure combined
as the ITS
3) phylogeny
(Figure
lc).
As shown
in Figure 3, mapping unambiguous changes of the
morph1oloi gical«
combined
the
morphological
clade of Distylium and was
Eustigma-Fortunearia-Sinowilsonia
—
Complex
1997] Li Corylopsis
et 311
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