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Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, United Kingdom
-Phylogeny-PhyrrOSachys-Poaceae
* Present address: Department of Botany, Trinity College, University of Dublin, Dublin 2, Ireland.
260
stamened genera Shibataea, Chimonobambusa, Sinobambusa, Semiarundinaria, and Brachystachyum. These genera
share several morphological character states. Inflorescence branching is bracteate. Culms are sulcate with limited branching, erect and self-supporting. Rhizomes are
leptomorph and leaf blade venation is tessellate. In leaf
anatomy they apparently lack fusoid cells, adaxial ribs,
expanded leaf margins, and well-developed abaxial papillae
and prickles (Soderstrom and Ellis 1987). However, for most
of these characters it is not clear which state is ancestral,
and the group cannot presently be linked by any morphological synapomorphy. Indeed the character considered
paramount, the bracteate inflorescence, is most likely to be
ancestral within the grass family as a whole, and other
potential synapomorphies are common in the woody bamboos. Previous molecular investigations have failed to
support this grouping, and only one (Watanabe et a/. 1994)
has indicated a close relationship between Phyllostachys and
another genus of this group (Shibataea). To investigate the
relationship of Phyllostachys to these other potential sister
groups, the type species of Chimonobambusa, Shibataea,
and Sinobambusa were included in the ITS study. In addition, an Asian woody bamboo species that differed from
Phy//ostachys in all the macro-morphological character
states listed above, Neomicrocalamus andropognonifolius,
was included. This has ebracteate inflorescences, clambering, perfectly terete culms with multiple branches from
pachymorph rhizomes, non-tessellated leaf venation, and
six, rather than three, stamens.
The most important recent attempt to classify the species
of Phyllostachys was that of Wang et a/. (1980) who used
culm intranode, culm sheath, ligule, blade, inflorescence
shape, and spikelet dimensions. They formally identified
two major groupings within the genus at sectional rank,
Phyllostachys section Phyllostachys (type species: P. bambusoides) and Phyllostachys section Heteroclada Wang & Ye
(type species: P. heteroclada). Species of section Phy//ostachys have a lax inflorescence compared to a capitate form
in section Heteroc/ada (see illustrations in Renvoize and
Hodkinson 1997). They have larger spikelets (25-30 mm
compared to 15-20 mm), larger sheath ligules (>3 mm
compared to c. 2 mm), sparsely or densely spotted culm
leaves (compared to non-spotted), and a shorter culm
intranode (c. 3 mm compared to c. 5 mm). Recently, Ding et
al. (1993) have discovered that taxa of section Heteroclada
share rhizomes with air canals, a character that section
Phyllostachys lacks.
The scarcity of good morphological characters has
prompted a number of studies utilizing isozymes, secondary
metabolites, and DNA markers. Huang et a/. (1983) assessed variation of peroxidase isozymes and found three distinct
patterns each represented by Phy//ostachysviridis, P. vivax,
and P. heteroclada. However, they did not designate other
species to these groups and made no attempt at an infrageneric classification. Chou et a/. (1984) using peroxidase isozymes and flavanoids, recognized two groups. The
first group consisted of P. aurea, P, nuda and P. nigra and the
second group of P. makinoi, P, bambusoides, and P. hetero-
DNA extraction
DNA was extracted from 0.5-1.Og of fresh leaf material
using a modification of the 2XCTAB procedure of Doyle and
Doyle (1987) and precipitated using 100% ethanol for at least
48 hours at -20 C. The DNA was then pelleted, washed
with 70% ethanol and purified via cesium chloride/ethidium
Table 1. Accessions of Phyllostachys and related genera from the living collection at the Royal Botanic
Gardens, Kew, used for molecular analysis
ID
Voucher no.
1.
2.
3.
4.
5.
6.
7.
8.
9.
15.
16.
17.
18.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Renvoize 1986-2135
Renvoize 1991- 1526
Renvoize 1991- 1529
Renvoize 1991- 15f6
Renvoize 1986-2137
Renvoize 1973-20182
Renvoize 1973-14415
Renvoize 1984-2279
Renvoize 1983-3338
Renvoize 1973-14405
Renvoize 1991- 1517
Renvoize 1985-2084
Renvoize 1984-2763
Renvoize 1988-3113
Renvoize 1973-20209
Renvoize 1984- 1852
Renvoize 1985-2086
Renvoize 1988-3397
Renvoize 472-8907
Renvoize 1973-20180I
Renvoize 1994- 1217
Renvoize 731- 1243
Renvoize 1991-3178
26 1
Species name
P. rubromatginata McClure
P. glauca McClure
P. platyglossa Z.P. Wang & Z.H. Yu
P. rubicunda T.W. Wen
P. angusta McClure
P. mannii Gamble
P. dulcis McClure
P. viridiglaucescens (Carriere) A. & C. Riviere
P. bambusoides Siebold & Zuccarini
P. bissetii McClure
P. flexuosa (Carriere) A. & C. Riviere
P. arcana McClure
P. aurea Carriere ex A. & C. Riviere
P. humilis Muroi
P. nidularia Munro
P. aureosulcata McClure
P. sulphurea (Carriere) A. & C. Riviere
P. sulphurea (Carriere) A. & C. Riviere Robert Young
P. fulva Mitford
P. nuda McClure
Chimonobambusa marmorea (Miff.) Makino
Shibataea chinensis Nakai
Sinobambusa tootsik (Makino) Makino
Neomicmlamus andropcgonifolius (Griff.) Stapleton
All DNA samples have a corresponding herbarium voucher specimen stored in the Royal Botanic
Gardens, Kew, UK. Sample identification number (ID) refers to the DNA sample number.
262
I I? viridiglaucescens
Data analysis
Sequence editing and assembly of the complementary
strands used the Sequence Navigator and AutoAssembler
programs of ABI. DNA sequences were then aligned by eye
or CLUSTAL W (Thompson et a/. 1995) followed by further
manual alignment. Gaps were coded as missing values.
The resulting matrices were subjected to parsimony analysis
using PAUP 4.0 Beta 2 (Swofford 1998) using branch and
bound.
DNA fragments ranging from 50 to 500 bp in size from the
AFLP analysis were scored as presence/absence characters, and weak bands were removed from the matrix before
analysis with PAUP. Maximum parsimony and neighborjoining (NJ) analyses were applied (see Swofford et a/. 1996).
Internal support was assessed using the bootstrap procedure
(Felsenstein 1985). Since outgroups were not used in the
AFLP analysis, we used mid-point rooting (and the results
from the ITS analysis). Principal coordinates analysis (PCO)
was performed with Le Progiciel R v4.0d (Casgrain 1999)
using mean-character difference distances.
Results
ITS and 5s sequence data
ITS sequences were obtained for 12 species of Phyllostachys, three other members of the subtribe Shibataeinae, and
Neomicrocalamus androgonifolius, a member of subtribe
Racemobamosinae. A parsimony analysis with Neomicrocalamus andropogonifolius as an outgroup resulted in 101
equally parsimonious trees with a length of 192, consistency
index (CI) of 0.88 and retention index (RI) of 0.89 (Fig. 1).
Phyllostachys was well separated from the three other
genera placed in the same subtribe on morphological
grounds. Monophyly was strongly supported with a 100/~
I? platyglossa
I.! dulcis
+1
24
99
P heteroclada
21? bissetii
Seetion
Heteroclada
imonobambusa marmorea
Shibataea chinensis
Sinobambusa tootsik
Neomicrocalamus andropogonifolius
Fig. 1. Parsimony tree of ITS sequence data for Phyllostachys
species and related genera. One of 101 maximally parsimonious trees. Length=192, CI=O.88, RI=0.89. Values
above branches are steps. Numbers below branches are
bootstrap percentages. There are two major clades in
Phyllostachys (section Phyllostachys and section Heteroclada). Chimonohmbusa is sister to Phyllostachys.
P. angusta
O:;
EP.
incarnata
P. rubicunda
P. nuda
P. viridiglaucescens
P. dulcis
31
50
IL
2,
P. heteroclada
P. nigra
P. nidularia
P. humilis
P. bissetii
P. platyglossa
Section
Heteroclada
respectively, of which 357 were polymorphic and parsimonyinformative. The two different methods of phylogenetic
reconstruction (maximum parsimony and NJ) gave similar
results, the differences always occurring for groups that were
weakly supported. Three major monophyletic groups can
be identified (Figs. 2 and 3). In the parsimony analysis (Fig.
2) the tree length was 1204 with a CI of 0.35and a RI of 0.56.
One group, Phyllostachys heteroclada, P, nigm, P. nidularia, P.
bissefii, f .aureosulcata, and P. humulis, consists of taxa
predominantly from section Heferoclada of Wang and Ye,
with the exception of P. aureosulcata and P. platyglossa,
which were included by Wang and Ye in section fhyllosfachys. Evidence exists for subdivisions within this section.
In the NJ analysis strong internal support exists for two
groups within a revised section Heferoclada (Fig. 3).
The other two major groups consisted of taxa predominantly from section fhyllosfachys of Wang & Ye with the
exception of P. incarnata, P. mannii, and P. rubicunda (previously assigned to section Heferoclada). One group including P. viridiglaucescens, P. bambusoides, and P. dulcis, can
be identified in the parsimony analysis with 93 bootstrap
percentage (BP) support and the same group with the
BP)
addition of P. nuda is identified in the NJ analysis (9Io/o
263
Discussion
Both DNA sequencing of the ITS region of nrDNA and
AFLP analysis have provided new insights into the evolution
of the genus Phyllosfachys and allowed a review of its infrageneric classification. The most widely used infra-generic
classification is that of Wang & Ye, who divided the genus
into two groups assigned the rank of section. This system
was modified by Renvoize and Hodkinson (1997)using
morphology (Table 2).
The results of this study are discussed in relation to this
system of Phyllosfachys classification and to other major
publications (Friar and Kochert 1994,Geng and Wang 1996,
Ohrnberger 1996). DNA sequencing of the ITS region was
only partially successful in determining the inter-relationships of the taxa under study. The AFLP analysis supported
the groupings obtained by the ITS sequence data but
264
Fig. 3. Neighbor joining tree of AFLP data for Phyllostachys species. Numbers above branches are
bootstrap percentages. Branch length is proportional to genetic distance (mean-character difference).
Four major clades can be identified (fhy//ostachys 1 and 2; Heteroclada 1 and 2).
P bambusoides
P viridiglaucescetu
02
-:
265
-0.2
-0 3
-0. I
'+
+ P nigra
P nidularia
P. heteroclada
-0.1
P mannii
-__.
++ Psulphurea
-;
Psulphurea
+;
;
-02
-.
+ P rubromarginata
Axis2
Fig. 4. Principal coordinates analysis (PCO) of Phyllostachys AFLP data. The taxa can be divided into
subgroups using the first two axes of the PCO. These cumulatively account for 48.3% (28.0% and 20.
3% respectively) of the data variance, the third axis (not shown) representing 7.8%. Phyllostachys
mannii and P. rubromaginata do not group closely with any of the subgroups indicated by circles.
P. glauca
P. guizhouensis
P. iridescens
P. kwangsiensis
P. lithophylla
P. makinoi
P. meyeri
P. nigella
P. nuda
P. platyglossa
P. praecox
P. prominens
P. propinqua
P. rubromarginata
P. rutila
P. sulphurea
P. tianmuensis
P. viridiglaucescens
P. vivax
P. yunhoensis
P. robustiramea
P. rubicunda
P.
P.
P.
P.
P. stimulosa
aurita
bissetii
concava
heteroclada
P. heterocycla
P. humilis
P. incarnata
P. mannii
P. nidularia
P. parvifolia
P. nigra
P. rigida
P. rivalis
Phyllostachys species can be divided into the infra-generic sections Phyllostachys and Heteroclada,
first defined by Wang and Ye (Wang et a/. 1980).
sequences were almost identical. Two clades could, however, be recognized within Phyllostachys, and these correspond to the sections Phyllostachys and Heteroclada of
Wang et Ye (Fig. 1) with P. bissetii and P. heteroclada representing section Heteroclada. The distance between
these two sets of ITS sequences is much greater than that
266
in the AFLP trees despite the fact that little variation was
witnessed within these clades. There was some internal
support for the grouping of P. arcana and P. aurea within this
clade, a grouping also supported by AFLP analysis.
ITS has been successful for infra-generic classification in
other taxonomic groups, including grasses (Baldwin et a/.
1995, Hodkinson et a/. 1997, Hsiao et a/. 1999), but clearly
shows less discriminating power in this genus of woody
bamboos. One explanation is that the species are all of
recent origin and have undergone little molecular evolution.
It is also possible some of the taxa are not distinct species
and that some of the morphological characters differentiating the species are not suitable for that purpose. However,
there seems to be good evidence from both morphological
and molecular investigations to believe otherwise. A third
explanation is that the low substitution rate is correlated with
the life-form of the genus. There is evidence to indicate
that ITS sequences have evolved more slowly in some
ancient woody taxa within Viburnum, Nothofagus, and genera of Winteraceae, which diverged in the Cretaceous or the
early Tertiary, than between taxa of herbaceous plant lineages that diverged much more recently during the Pliocene
and Pleistocene (Baldwin et a/. 1995). Clark et a/. (1995)
suggested that such a process may explain the low level of
plastid ndhF sequence evolution found in woody bamboo
genera in comparison to herbaceous bamboos. Slower
molecular evolution may be due, in part, to longer generation
time.
AFLP
Faced with the problem of low genetic variability detected
within Phyllostachys by DNA sequencing of ITS nrDNA, AFLP
was chosen as a suitable technique to sample genetic
variation of the species. The AFLP analysis successfully
discriminated the species and reveals the inter-relationships
of the taxa. The AFLP analysis was more suitable than
DNA sequencing of the ITS region at this taxonomic level
because it produced far more informative markers. The
advantage of AFLPs compared to other multi-locus PCR
fingerprinting techniques such as RAPD and inter-microsatellite PCR is that far greater numbers of markers per
primer investigated are produced. Markers produced by
AFLP are also highly reproducible (Karp eta/. 1996, Vos eta/.
1995). AFLP requires no prior knowledge of the target
genome and can be applied universally to plant groups. In
our opinion AFLP is the fingerprinting method of choice for
establishing genetic distances and phylogenetic relationships between closely related taxa in which DNA sequencing
is not informative or efficient. Of course, higher numbers of
DNA basepairs can be sequenced, but this is often neither
practical nor cost-effective.
The major. monophyletic groups produced by the AFLP
analysis are broadly consistent with previous infra-generic
classifications based on morphology by Wang & Ye and
Renvoize and Hodkinson (1997) and with the trees produced
by the ITS sequence data, with the exception of Phyllostachys platyglossa (now included in section Heteroclada), and
P. rubicunda, P. mannii, and P. incarnata, which were formerly
267
Table 3. Revised treatment of Phyllostachysspecies based on new sequence and AFLP data in conjunction with
morphology
Section Hetemlada
Blades of sheaths at the apex of the young shoot
forming a tight, erect bunch; inflorescence congested or glomerate. Air canals present in the
rhizomes.
Heteroclada group 1
Hetemlada group 2
Section Phyllostachys
Blades of sheaths at the apex of the young shoot
forming a l m e bunch; inflorescence lax. Air
canals lacking in the rhizomes.
Phyllostachys group 1
Phyllostachys group 2
P. angusta
P. arcana
P. aurea
P. heteroclada
P. nidularia
P. aureosulcata
P. bissetii
P. dulcis
P. viridiglaucescens
P. nigra
P. humilis
P. bambusoides
P. nuda
P. flexuosa
P. fulva
P. glauca
P. incamaia
P. lithophylla
P. mannii
P. rubicunda
P. rubmmarginata
P. sulphurea
The groups outlined above are based primarily on major well supported (BP) clades from the molecular analysis
with some reference to morphology and previous classifications where support is missing.
analysis where they cluster with taxa from section Phyllostachys but are highly divergent from these. It is possible that
these taxa are of hybrid origin and thus have characters in
common with both section Phyllostachys and Heteroclada.
We are currently investigating the morphology of these
taxa further to check their species status and better understand the results of this molecular analyses. Phyllostachys
fulva had previously been synonymized with P. sulphurea
(Renvoize and Hodkinson, 1997), but AFLP data show that it
is more closely related to P. flexuosa and clearly distinct from
P, sulphurea.
It appears therefore that four clades can be identified, two
corresponding to section Heteroclada and two to section
Phyllostachys of Wang and Ye. Determining the positioning
of these clades relative to one another and the positioning of
some of the other taxa such as P, platyglossa will require
further research. Phyllostachys platyglossa is problematic
because it is sister to section Heteroclada in the AFLP
analysis, but was embedded within section Phyllostachys in
the ITS sequence analysis and the morphological treatment
of Renvoize and Hodkinson (1997). It has a longer branch
length in the ITS sequence analysis than the other terminal
taxa of section Phyllostachys. Whether this has influenced
its positioning within the genus is doubtful however because
the grouping has high bootstrap support. It is an issue that
may be resolved by increased sampling of taxa. It is also
possible that the taxon is a hybrid and shares an ITS copy
type with one of the parental species and not the other.
However, in the PCO analysis, P. platyglossa groups closely
with taxa from section Heteroclada. Thus the AFLPs show
no evidence of hybridization.
Conclusions
Our molecular analysis indicates that the taxonomic treatment of Wang et a/. (1980) needs revision. Despite the fact
that four major clades were detected, there are sufficient
levels of bootstrap support to retain the two sections,
Heteroclada and Phyllostachys. A subset of taxa within
section Phyllostachys has strong support (Phyllostachys 1) but
the remaining taxa are less easy to place and could possibly
group closer to section Heteroc/ada than to Phyllostachys. It
is also clear that section Phyllostachys group1 could have
resulted from hybridization between taxa of sections Phyllostachys group 2 and Heteroclada. Chromosome number
information is not available for these taxa. Future
cytogenetic research should target these taxa to determine
ploidy and investigate hybridization phenomena. At this
stage it would be prudent to retain the sections but modify
them in light of the new evidence and create new subgroups
reflecting such groups (Table 3).
It remains to be seen where the taxa within the section
Phyllostachys 1 are placed. Increased sampling of Phyllostachys species combined with more AFLP data may resolve
this problem. The position of Heteroclada within the genus
also requires further analysis. The phylogenetic relationships of Phyllostachys and closely related genera in the subtribe Shibataeinae, such as Chimonobambusaand Shibataea,
are currently being assessed in more detail. The AFLP
analysis has proven to be well-suited to phylogenetic analysis in this genus. In general, it can be a useful technique to
establish the inter-relationships of closely related taxa,
particularly if DNA sequence data have failed to provide
sufficient resolution.
We would like to acknowledge the financial support of the
268
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