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J. Plant Res.

113: 259-269, 2000

Journal of Plant Research


63 bv The Botanical Society of JaDan 2000

A Comparison of ITS Nuclear rDNA Sequence Data and


AFLP Markers for Phylogenetic Studies in
Phy//mtachys (Barnbusoideae, Poaceae)
Trevor R. Hodkinson'", Stephen A. Renvoize', Grainne Ni Chonghaile',
Christopher M.A. Stapleton' and Mark W. Chase'
1

Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, United Kingdom

* Department of Botany, Trinity College, University of Dublin, Dublin 2. Ireland*


Two contrasting molecular techniques, namely DNA
sequences and amplified fragment length polymorphisms
(AFLP) were used to investigate phylogenetic relationships
of Phyllostachys, a large, economically important genus of
woody bamboos. DNA sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA
(nrDNA) were used in a parsimony analysis. PhyrrOStachys
was well supported as monophyletic with Chimonobambusa
as its closest allied genus. The 5s spacer region of nrDNA
was investigated but found unsuitable for this purpose. The
AFLP analysis showed much higher discriminating power
between species and was more useful for phylogenetic
reconstructionat this taxonomic level. The combined data
were used to review the previous infra-generic classifications. Section Heteroclada Wang & Ye is strongly supported and can be further divided into sub-groups. A group
within section Phyllosachys is strongly supported, but a
further group of taxa previously included in this section is
difficult to place. The ability of the methods to help separate species such as P.sulphurea and investigate genetic
diversity at the infra-specific level was also assessed. It is
argued that AFLPs could often be the method of choice for
phylogenetic studies of closely related taxa for which DNA
sequence data provide insufficient resolution.
Key words: AFLP -Bambusoideae-ITS-Molecular

-Phylogeny-PhyrrOSachys-Poaceae

Phy//ostachys Siebold & Zucc. is one of the largest genera


of woody bamboos, with over 75 species described. These
are a major source of raw material for domestic, commercial,
and industrial purposes, particularly in the temperate zone of
Asia. Two of the most important species, P. heterocycla
(=P.edulis) and P.rubromaginata, are commonly eaten as
bamboo shoots (Ohrnberger1996). With a genus of this size
and economic importance, the lack of a classification system is a serious obstacle to improving the exploitation of the

* Present address: Department of Botany, Trinity College, University of Dublin, Dublin 2, Ireland.

species. Most authors list the species alphabetically or in


the order in which they are keyed out (Chao 1989, Chen and
Chia 1988, McClure 1957, Ohrnberger 1987,1996, Okamura et
a/. 1991, Suzuki 1978, Wang and Shen 1987, Zhu et a/. 1994),
but in none of these works does the arrangement of the
species reflect natural groups. Phyllostachys was first described by Siebold and Zuccarini in 1843, a time when only
ten other genera of bamboos were known. A historical
outline of Phyllostachys classifications was given by Renvoize and Hodkinson (1997), who broadly retained the sectional
treatment of Wang et a/. (1980) but modified the species
allocated to each section on the basis of a new morphological comparison.
Phyllostachys is one of the easiest bamboo genera to
identify with its paired branches and sulcate, erect culms
arising from a leptomorph (running) rhizome and for these
reasons has been treated by taxonomists as a well-defined
monophyletic group. The availability of good taxonomic
characters for the further characterization of the genus is
limited, however. Culms offer variation in size, texture (scarified or smooth), and color (uniform, striped or blotched), but
their value is mostly at the infra-specific level. There is also
variation in nodal structure and prominence, although this is
often hard to quantify. Branch complements of two (or
occasionally three) at each culm node are uniform throughout the genus and appear to be of value primarily for generic
identity. Culm sheaths of the young shoots, on which most
keys have relied, are the most useful source of characters
(color, markings, auricles, ligules, and blade shape) but they
are only available for two to three weeks in the year. The
inflorescence is a useful source of characters but is only
available at intervals of many years, and in some species,
such as P. dulcis and P. viridiglaucescens, it is not known.
From a practical point of view, therefore, the most useful
characters lie in the culm sheaths. For a review of bamboo
morphology see Stapleton (1997).
Whereas circumscription of Phyllostachys is uncontroversial, how it relates to other genera is less clear. It has nearly
always been placed in a group of genera recognized as
Shibataeeae (Nakai 1933) or Shibataeinae (Soderstrom and
Ellis 1987), including variously some or all of the three-

260

T.R. Hodkinson et a/.

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-

cycla (P. edu/is). Phyllostachys lithophila from Taiwan, was


distinct and could not be assigned to either of these. All the
species grouped by Chou et a/. (1984) are included in section
Phyllostachys as described by Wang et a/. (1980).
Friar and Kochert (1994) assessed variation at the DNA
sequence level using restriction fragment length polymorphisms (RFLPs) and found that the taxa clustered into two
broad groups when assessed with both genetic distance
methods and phylogenetic methods using maximum parsimony. Their results were in broad agreement with the
morphological classification of Wang et a/. (1980) except that
Phyllostachys nigra was placed in section Heteroclada and
not in section Phyllostachys. Internal support for their two
major clades was weak, with bootstrap values below 53%.
Gielis (1995) and Gielis et a/. (1997) assessed variation
within Phyllostachys using a multi-locus PCR fingerprinting
technique known as randomly amplified polymorphic DNA
(RAPD). Little variation was found within species, but considerable variation was found between species. RAPD
markers therefore showed great potential for the identification of taxa difficult to name on morphological grounds.
Sequencing of nrDNA has been used successfully for
phylogenetic reconstruction at the species and generic
levels in many plant groups (see Baldwin et a/. 1995 for a
review) and within the grass family (Buckler and Holtsford
1996, Hodkinson et a/. 1997, Hsiao et a/. 1995ab, Hsiao et a/.
1999, Sun et a/. 1994).
AFLP polymorphisms have also been used recently for
systematic study of plant groups (Hodkinson et a/. 1997, Lu
et a/. 1996, Paul et a/. 1997, Reeves et a/. 1998, Sharma et a/.
1996). AFLPs are a powerful marker system for closely
related plants and for infra-specific variation in particular
(Mueller and Wolfenbarger 1999, Vos et a/. 1995). The
complex multi-locus fingerprints produced by ,the AFLP
technique are highly reproducible and provide a targe number of informative markers derived from loci dispersed
throughout the nuclear genome (Ridout and Donini, 1999).
In barley, for example, AFLP markers were found to be
located on the long and short arms of all seven chromosomes, with a strong correlation between the number of
markers per chromosome and the length of the chromosome
(Waugh et a/. 1997). In rice, Mackill et a/. (1996) used AFLP
to map an F2 population from an indicaxjaponica cross
(each representing major breeding groups) and found that 50
AFLP markers were spread across all the chromosomes
except chromosome 12. However, clustering of AFLPs
around centromeric regions has also been reported (Qi et a/.
1998).
The fact that AFLP markers are generally distributed
across the genome gives the technique some advantages
over sequence analysis for closely related taxa. Sequence
analyses that relies on data from single DNA regions can
give misleading results because of hybridization (Riesberg
and Soltis 1991) and the presence of paralogous DNA regions
(Baldwin et a/. 1995). AFLPs are however limited to the
analysis of closely related species and infra-specific taxa
(Karp et a/. 1996). Above this level the multi-locus fingerprint becomes too variable, and markers are less likely to be

Phyllogenetic Studies in Phy//mtachys


homologous. AFLPs therefore seem a particularly appropriate and efficient method for systematic studies in plant
groups in which insufficient variation can be detected
through sequence analysis. The molecular markers generated in this study were used for the identification of individuals and taxa, to assess phylogenetic relationships within the
genus and to improve its taxonomy.

Materials and Methods


Collection
Leaf material was extracted from 22 species of Phy//ostachys and four allied genera from the living collection at the
Royal Botanic Gardens, Kew (Table 1).

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

bromide (1.55 g/ml) gradient centrifugation with subsequent


dialysis. DNA was then stored in TE buffer (10 mM Tris-HCI;
1 mM EDTA; pH 8.0) at -20 C until use.
DNA sequencing
Two spacer regions were sequenced. The ITS region
was amplified using the primers described by Baldwin (1992)
and Sun et a/. (1994). ITS is approximately 600-700 base
pairs (bp) long and includes two spacer regions of less than
300 bp (ITS 1 and ITS 2) separated by a small coding region,
the 5.8s gene (163-164 bp). The 5s spacer was amplified
using the primers described by Cox et a/. (1992). The 5s
ribosomal spacer is approximately 200-500 bp long and
flanked on both sides by the 5s rRNA gene of approximately
120 bp (Cox et a/. 1992, Sastri et a/. 1992).
The thermal cycling parameters for both regions comprised 30 cycles, each with 1 min. denaturation at 97 C, 1 min.
annealing at 51 C and an extension of 3 mins. at 72 C. A
final extension of 7 mins. at 72 C was also included. Amplified, double-stranded, DNA fragments were purified using
Promega Wizard PCR mini-columns and sequenced using

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.

Renvoize 1991- 1521

2.

Renvoize 1991- 1524


Renvoize 1991- 1518
Renvoize 1986-2136

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. incarnata T.W. Wen


P. lithophylla Hayata
P. hetercclada Oliver

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.

T.R. Hodkinson et a/.

262

I I? viridiglaucescens

Taq Dye-Deoxy Terminator Cycle Sequencing Kits of PE


Applied Biosystems Inc. (ABI) on a ABI 373 or 377 automated
DNA sequencer.
AFLP analysis
AFLP fingerprinting is based on the selective amplification
of restriction fragments from a digest of total genomic DNA
(see Vos et a/. 1995, for a full description of the method).
AFLP reactions were performed using the AFLP Plant
Mapping Kit of ABI, and the DNA fragments were detected
on an ABI 377 automated DNA sequencer with ABI Genescan 2.0.2 and ABI Genotyper version 1.1 software. The DNA
was first digested with two restriction enzymes (fcoRI and
Msel). In the same reaction, oligonucleotide adaptors specific to these restriction sites were ligated to the fragments.
These fragments were then used for two successive PCR
reactions. The first PCR amplified a subset of the fragments, reducing them approximately 16-fold. The second
selective PCR amplified a subset of the fragments created in
the initial PCR, reducing them approximately 256-fold further,
as well as incorporating one primer with a fluorescent dye.
Two primer combinations were used for the selective PCR
step; B3 (fcoACA/MseCAA) and Y9 (fcoAAC/MseCAT).

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.

bootstrap value and its sister group was found to be


Chimonobambusa. However, an assessment of the character support for the separation of Phy/lostachys from these
postulated relatives (39-54 steps) does not suggest a particularly close relationship to any of the genera included in this
study.
There was a low degree of sequence divergence between
Phy//ostachys taxa with the majority falling into a large, poorly
resolved, clade (polytomy) in which many of the sequences
were almost identical. The remaining taxa, P. bissetii and P.
heteroclada, formed a monophyletic group sister to this
polytomy. The major clades correspond to the sections of
Wang and Ye. The highly divergent sequence of P. platyglossa relative to the other Phyllostachys species should be
noted because a similar result occurred in the AFLP analysis.
Sequencing of the 5s spacer was hindered by the highly
repetitive region of thymine residues near the 3-end of the
gene, a common feature in plants (Sastri et a/. 1992). The
5s DNA repeat units also showed sequence heterogeneity.
Only partial 5s sequences for Phyllostachys dulcis and P.
rubicunda were obtained. The sequences will be deposited
in GenBank for future use but are not discussed further here.
AFLP analysis
The AFLP analysis produced a large number of markers
for differentiatingthe species. The two selective primers 83
and Y9 produced 330 and 223 different DNA markers

Phyllogenetic Studies in Phyllosfachys


P. sulphurea
P. sulphurea
P. rubromarginata
P. mannii
P. lithophylla
Section
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

Fig. 2. Phylogenetic relationships of Pby//o.stacbys based on


AFLP data. One of two maximally parsimonious trees
(mid-point rooted). Numbers below branches are bootstrap percentages; numbers above branches are steps.
Length=1204, CI=O.35, RI=0.56. Three major clades are
indicated, sections Pby//ostacbysand Heteroclada and an
unnamed group (indicated by ?).

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

(Figs. 2 and 3). There is less than 50% bootstrap support to


indicate that this clade is sister to the remaining taxa of
section fhyllostachys Wang and Ye, but all optimal trees
show this pattern. The positions of these groups within the
genus or relative to section Heferoclada can not be determined from this analysis and neither can the position of P.
platyglossa which is sister to section Heferoclada in the AFLP
analysis but embedded within section Phyllostachys in the
ITS sequence analysis.
The results from the PCO analysis are illustrated in figure
4. The first two axes accounted for 28% and 20.3% of the
data variance, respectively. Cumulatively, this represents
48.3% and is sufficient to resolve the taxa into distinct
groups. The third and fourth axes represented 7.8and 5.7%
of the data variance, respectively, and do not significantly
improve the resolution of the taxa (plots not shown). The
groupings with the PCO analysis are congruent with the NJ
and parsimony analysis except that P, mannii and P. rubromarginata are not closely allied to section Heteroclada.
These two do not group with taxa from either section
Heferoclada or fhyllosfachys; they are positioned between
the two.
Minor groupings
A number of minor groupings are also evident. Within the
well supported section Heteroclada, two groups are resolved
(Figs. 2-4). One includes Phyllosfachys heteroclada, P. nigra,
and P. nidularia and the other P. bissetii, P. aureosulcata, and
P. humilis (the inclusion of P. humilis in this clade is supported by a 97% BP in the NJ analysis but does not obtain
greater than 50% support in the parsimony analysis).
Phyllosfachys nigra, which was included in section Phyllostachys by Wang and Ye, clearly belongs to section Heteroclada.
Phyllostachys arcana is sister to f. aurea, and P. flexuosa
sister to P. fulva (with AFLP and DNA sequence data), and
there is little genetic distance between these pairs; 0.025
(~2.5~0).
Phyllosfachys incarnata is sister to P. rubicunda.
We are currently investigating the morphology of these taxa
further to check their status as separate species.

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

T.R. Hodkinson et a/.

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).

supplied much better resolution and bootstrap support for


major groupings (Figs. 2-4).
ITS sequence data
Trees produced from the ITS sequences displayed low
levels of homoplasy and divergence. PhyIImfachys was
found to be a strongly supported monophyletic group with
Chimonobambusa as its sister group. Chimonobambusa is a
member of the same subtribe as PhyIIosfachys, Shibataeinae,
but was not the expected sister group. Shibataea was
expected to be the closest relative of PhyIIosfachys based on
morphology (Clayton and Renvoize 1986). Chimonobarnbusa has characteristics in common with Phy//ostachys, but
no morphological synapomorphies grouping the two genera
are evident. Branches of PhyIIosfachys arise in pairs from

the node, but a third branch is often present, albeit much


reduced in stature. In Chimonobambusa this third branch is
fully developed, and three to seven additional branches are
often produced. Leptomorphic rhizome structure is characteristic of the entire subtribe. Although Chimonobambusa
was seen here to be the closest relative from the subtribe in
which Phyllosfachys has been placed on morphological
grounds, this result should be treated with caution, as the
isolation of PhyIIosfachys from all the other genera included
in the study was substantial. Because the main character
used to combine these genera into a subtribe (bracteate
inflorescences) is likely to be ancestral, there has to be
cause for concern that the group as a whole may not be
monophyletic. The monophyly of PhyIIosfachys has been
demonstrated, but a much broader range of temperate Asian

Phyllogenetic Studies in Phyllostachys

P bambusoides
P viridiglaucescetu

02

-:

265

+ Section Phvllostachys group I

-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.

Table 2. Sections of Phyllostachys (summarized by Renvoize and Hodkinson 1997)


Phyllostachys section Phyllostachys
P. acuta
P. angusta
P. arcana
P. aurea
P. bambusoides
P. circumpilis
P. dulcis
P. fimbriligula
P. flexuosa
P. glabrata

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

Phyllostachys section Heteroclada Wang and Ye


P. atrovaginata
P. aureosulcata

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).

bamboos still needs to be included before any definite


conclusions can be drawn about the generic affinities of
Phyllostachys.
Resolution of the ITS trees within Phyllostachys was limited
by the low levels of divergence. The majority of the taxa fell
into a large unresolved clade in which many of the

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

T.R. Hodkinson et a/.

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

placed in section Heteroclada but group here with the


species of section Phyllostachys. Some conflicts are also
evident with the groups recognized as sections in Geng and
Wang (1996). Four species, P. nigra, P. bissettii, P. aureosulcata, and P. platyglossa group with section Heteroclada in our
analysis but were included in section Phyllostachys by Geng
and Wang (1996). It is possible that these differences reflect
phylogenetic pattern or that they are the result of ITS capture
via hybridization. Conversely, P. rubicunda, placed in section Heteroclada by Geng and Wang (1996) is here placed in
section Phyllostachys.
Section Heteroclada is well supported in our analysis, but
would include Phyllostachys nigra and P. aureosulcata (previously designated to section Phyllostachys Wang and Ye).
Phyllostachys nigra has an intermediate inflorescence type
and has been shown to possess air canals in its rhizomes
(Ding et a/. 1993). This feature is a good diagnostic character (synapomorphy) for section Heteroclada and could be an
adaptation to wet soils. The common name for P. heteroclada is water bamboo (Gielis et a/.1997). The assertion that
P. nigra belongs to section Heteroclada is also congruent
with the results of a nuclear RFLP study by Friar and Kochert
(1994). Phyllostachys aureosulcata is intermediate in morphology between sections Heteroclada and Phyllostachys
and was placed by Renvoize and Hodkinson (1997) in a
revised section Heteroclada on the basis of its morphology.
Such a finding does not therefore conflict with morphological
data.
The inter-relationships of taxa from section Phyllostachys
have been partially resolved. Of particular interest is the
position of Phyllostachys 1(P. bambusoides, P, du/cis, P. nuda,
and P. viridg/aucescens) in relation to Phyllostachys 2 and
the Heteroclada group (figure 3). Phyllostachys 1 is strongly
supported but Phyllostachys 2 is less well-defined and
difficult to place. Adding more taxa to the analysis or
increasing the number of AFLP markers may help clarify
these ambiguities.
Phyllostachys viridig/aucescens, P. bambusoides, and P.
dulcis (Phyllostachys 1) share culm sheath auricles with the
taxa of section Heteroclada, but are more closely allied to
section Phyllostachys on the basis of other morphological
characters such as the spreading or deflexed arrangement of
the culm blades in the emerging shoot and the absence of
air canals in the rhizome. It is possible that Phyllostachys
groupl is of hybrid origin from Phyllostachys group2 and
section Heteroclada. This is hypothesis is supported by the
PCO analysis (figure4) in which Phyllostachys groupl is
positioned between the aforementioned groups on the first
axis.
Phyllostachys nuda, which groups with Phyllostachys 1 in
the NJ tree but with Phyllostachys 2 in the parsimony analysis, would on morphological grounds be better placed in
Phyllostachys 2. Phyllostachys nuda does not have auricles
on the culm sheaths and hence associates with Phyllostachys 2 in which all the taxa, with the exception of P. mannii
and P. lithophylla, lack auricles. In the PCO analysis P.
mannii and P, rubromarginata do not group closely with any of
the proposed subgroups. This is in contrast to the NJ

PhyIlogenetic Studies in Phyllostachys

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

T.R. Hodkinson et a/.

268

Provosts Academic Development Fund, Trinity College


Dublin and The Royal Botanic Gardens Kew, UK. We would
also like to thank Mary Thorpe and Ray Townsend of the
Living Collections, Mike Fay for assistance with the AFLP
analysis, and the technical staff in the Jodrell Laboratory at
Kew (Jeff Joseph and Anette de Bruijn) for assistance with
the DNA sequencing.

References
Baldwin, B.G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an
example from the Compositae. Molec. Phylogenetics
EvoI. 1, 1: 3-16.
Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski,
M.F., Campbell, C.S. and Donoghue, M.J. 1995. The
ITS region of nuclear ribosomal DNA: a valuable source
of evidence on angiosperm phylogeny. Ann. Missouri
Bot. Gard. 82: 247-277.
Baum, B.R. and Appels, R. 1992. Evolutionary change at
the 5s DNA loci of species in the Triticeae. Plant Syst.
EvoI. 183 195-208.
Buckler, E.S. and Holtsford, T.P. 1996. Zea systematics:
ribosomal ITS evidence. Molec. Biol. Evol. 13, 4 612622.
Casgrain, P. 1999. Le Progiciel Rv4.Odl. Development
Release.
Chao, C.S. 1989. A Guide to Bamboos Grown in Britain.
Royal Botanic Gardens, Kew, UK.
Chen, S.L and Chia, L.C. 1988. Chinese Bamboos. Dioscorides Press, Portland.
Chou, C.H., Young, C.M. and Sheen, S.S. 1984. A biochemical aspect of phylogenetic study of Bambusaceae in
Taiwan. Part 1 The genus Phyllostachys. Proceedings of the National Science Council ROC(B).$: 89-98.
Clark, L.G., Weiping, 2. and Wendel, J.F. 1995. A
phylogeny of the grass family (Poaceae) based on ndhF
sequence data. Syst. Bot. 20, 4: 436-460.
Clayton, W.D. and
Renvoize, S.A. 1986. Genera
Graminum, grasses of the world. Kew Bulletin Additional Series XIII, Royal Botanic Gardens Kew, UK.
Cox, A.V., Bennett, M.D. and Dyer, T.A. 1992. Use of the
polymerase chain reaction to detect spacer size heterogeneity in plant 5s-rRNA gene clusters and to locate
such clusters in wheat (Triticum aestivum L.). Theor.
Appl. Genet. 83: 684-690.
Ding, Y., Grosser, D., Liese, W. and Hsiung, W. 1993.
Anatomical studies on the rhizome of some monopodial
bamboos. Chinese J. Bot. 5, 2 122-129.
Doyle, J.J. and Doyle, J.L. 1987. A rapid DNA isolation
procedure for small quantities of fresh leaf tissue.
Phytochem. Bull. Bot. SOC.Amer. 19 11-15.
Felsenstein, J. 1985. Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39 783-791.
Friar, E. and Kochert, G. 1994. A study of genetic variation
and evolution of Phyllostachys (Bambusoideae:
Poaceae) using nuclear restriction fragment length
polymorphisms. Theor. Appl. Genet. 8 9 265-270.
Geng, B. and Wang, Z.P. 1996. Gramineae Bambusoideae.
Flora Reipublicae Popularis Sinicae 9,1 Beijing Science
Press.

Gielis, J. 1995. Bamboo and Biotechnology. Eur. Bamboo


SOC. J. 1995: 27-39.
Gielis, J., Everaert, 1. and De Loose, M. 1997. Genetic
variability and relationships in Phyllostachys using random amplified polymorphic DNA. In G.P. Chapman,
ed., The Bamboos: Linn. SOC.Symp. Ser. 19: 107-124.
Hodkinson, T.R., Renvoize, S.A. and Chase, M.W. 1997.
Systematics of Miscanthus. Aspects. Appl. Biol. 49:
189-198.
Hsiao, C., Chatterton, N.J., Asay, K.H. and Jensen, K.B.
1995a. Molecular phylogeny of the Pooideae
(Poaceae) based on nuclear rDNA (ITS) sequences.
Theor. Appl. Genet. 90: 389-398.
Hsiao, C., Chatterton, N.J., Asay, K.H. and Jensen, K.B.
1995b. Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae),
inferred from nuclear rDNA (internal transcribed spacer)
sequences. Genome 38: 2l1-223.
Hsiao, C., Jacobs, S.W.L, Chatterton, N.J. and Asay, K.H.
1999. A molecular phylogeny of the grass family
(Poaceae) based on the sequences of nuclear ribosomal
DNA (ITS). Austral. Syst. Bot. 11: 667-668.
Huang, M.R., Chen, D.M. and Xu, N. 1983. An application
of isozymes in identificationof species of Phyllostachys.
Bamboo Research 2,l: 132-136.
Karp, A., Seberg, 0. and Buiatti, M. 1996. Molecular techniques in the assessment of botanical diversity. Ann.
BOt. 78: 143-149.
Li, W-H. 1997. Molecular Evolution. Sinauer Associates,
Sunderland MA.
Lu, J.M.R., Ambrose, M.J., Brown, J.K.M. and Ellis, T.H.N.
1996. Comparative analysis of the genetic diversity in
pea assessed by RFLP- and PCR-based methods.
Theor. Appl. Genet. 9 3 1103-1111.
Mackill, D.J., Zhang, 2, Redona, E.D. and Colowit, P.M. 1996.
Level of polymorphism and genetic mapping of AFLP
markers in rice. Genome 39, 5: 969-977.
McClure, F.A. 1957. Bamboos of the genus Phyllostachys
under cultivation in the United States. United States
Department of Agriculture Handbook l14.
Mueller, U.G. and Wolfenbarger, L. 1999. AFLP genotyping
and fingerprinting. Trends Ecol. Evol. 14,lO 389-394.
Nakai, T. 1933. Bambusaceae in Japan proper II. J. Jap.
BOt. 9 77-95.
Ohrnberger, D. 1987. Bamboos of the World: Phyllostachys. Verlag, Langweid am Lech.
Ohrnberger, D. 1996. The Bamboos of the World: Genus
Phyllostachys. Verlag Langweid am Lech.
Okamura, H., Tanaka, Y., Konishi, M. and Kashiwagi, H.
1991. Illustrated Horticultural Bamboo Species in
Japan. Haato Limited, Shinzaiki Wakaiama.
Paul, S., Wachira, F.N., Powell, W. and Waugh, R. 1997.
Diversity and genetic differentiation among populations
of Indian and Kenyan tea (Camellia sinensis (L.) 0.
Kuntze) revealed by AFLP markers. Theor. Appl.
Genet. 94: 255-263.
Qi, X., Stam, P. and Lindhout, P. 1998. Use of locus specific markers to construct a high density map of barley.
Theor. Appl. Genet. 9 6 376-384.
Reeves, G., Francis, D., Davies, M.S., Rogers, H.J. and
Hodkinson, T.R. 1998. Genome size is negatively

Phyllogenetic Studies in Phy//ostachys


correlated with altitude in natural populations of Dactylis
glomerata. Ann. Bot. 82 (supplement A): 99-105.
Renvoize, S.A. and Hodkinson, T.R. 1997. Classification of
Phyllostachys. In G.P. Chapman, ed., The Bamboos:
Linn. SOC.Symp. Ser. 19 95-106.
Ridout, C.J. and Donini, P. 1999. Use of AFLP in cereals
research. Trends Plant. Sci. 4, 2: 76-79.
Riesberg, L.H. and Soltis, D.E. 1991. Phylogenetic consequences of cytoplasmic gene flow in plants. Evol.
Trends Plant 5, 1: 65-84.
Sastri, D.C., Hilu, K., Appels, R., Lagudah, S., Playford, J. and
Baum, B.R. 1992. An overview of evolution in plant 5s
DNA. Plant Syst. Evol. 183 169-181.
Sharrna, S.K., Knox, M.R. and Ellis, T.H.N. 1996. AFLP
analysis of the diversity and phylogeny of Lens and its
comparison with RAPD analysis. Theor. Appl. Genet.
93:751-758.
Siebold, P.F. and Zuccarini, J.G. 1843. Plantarium quas in
Japonia Collegit Dr. PF Siebold genera nova. Abhandl.
Bayer. Akad. Wiss. 3: 745-749.
Soderstrom, T.R. and Ellis, R.P. 1987. The position of the
bamboo genera and allies in a system of grass classification. In T.R. Soderstrom, K.W. Hilu, C.S. Campbell
and M.E. Barkworth, eds., Grass Systematics and Evolution. Smithsonian InstitutionPress, Washington DC pp.
225-338.
Stapleton, C.M.A. 1997. The morphology of woody bamboos. In G.P. Chapman, ed., The Bamboos: Linn. SOC.
Symp. Ser. 19 251-267.
Sun, Y., Skinner, D.Z., Liang, G.H. and Hulbert, S.H. 1994.
Phylogenetic analysis of Sorghum and related taxa
using internal transcribed spacers of nuclear ribosomal
DNA. Theor. Appl. Genet. 89: 26-32.
Suzuki, S. 1978. Index to Japanese Bambusaceae. Gakken Co. Limited, Tokyo.

269

Swofford, D.L., Olsen, G.J., Waddell, P.J. and Hillis, D.M.


1996. Phylogenetic inference. In D.M. Hillis, C. Moritz
and B.K. Mable, eds., Molecular Systematics, Sinauer
Associates, pp. 507-514.
Swofford, D.L. 1998. Phylogenetic analysis using Parsimony (PAUP) version 4.0. Sinauer Associates, Sunderland MA.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1995.
CLUSTAL W: improving the sensitivity of progressive
sequence alignment through sequence weighting, position, specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22: 4673-4680.
Vos, P.R., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T.,
Hornes, M., Frijters, A., Pot, J., Kuiper, M. and Zabeau,
M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407-4414.
Wang, C.P., Yu, Z.H., Ye, G.H., Chu, C.D., Chao, C.S., Chen,
S.Y., Yao, C.Y. and Zhao, H.R. 1980. A taxonomical
study of Phyllostachys, China. Acta Phytotaxon. Sin.
18, 1: 15-19; 168-193.
Wang, D.J. and Shen, S.J. 1987. Bamboos of China.
Christopher Helm, London.
Watanabe, M., Ito, M. and Kurita, S. 1994. Chloroplast DNA
phylogeny of the Asian bamboos (Bambusoideae,
Poaceae) and its systematic implication. J. Plant Res.
107: 253-261
Waugh, R., Bonar, N., Baird, E., Thomas, B., Graner, A.,
Hayes, P. and Powell, W. 1997. Homology of AFLP
products in three mapping populations of barley.
Molec. Gen. Genet. 2 5 5 311-321.
Zhu, S.L., Ma, N.X. and Fu, M.Y. 1994. A Compendium of
Chinese Bamboo. China Forestry Publishing House
References, Beijing.
(Received April 18, 2000; accepted June 10, 2000)