全 文 ：Journal of Systematics and Evolution 46 (3): 333–340 (2008) doi: 10.3724/SP.J.1002.2008.08026
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com
Sequences of low-copy nuclear gene support the monophyly of Ostrya
and paraphyly of Carpinus (Betulaceae)
1,2Jianhua LI*
1(The Arnold Arboretum of Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA)
2(Adjunct Faculty of College of Life Sciences, Zhejiang University, Hangzhou 310029, China)
Abstract Coryloideae consists of four genera: Corylus, Ostryopsis, Carpinus, and Ostrya. While both molecular
and non-molecular data support the close relationship of Carpinus and Ostrya, the monophyly of the two genera
has remained controversial. In this study, sequences of the nuclear nitrate reductase (Nia) were used to test the
naturalness of the two genera. Ostrya species form a robust clade, supporting the monophyly of the genus. The
clade, however, is located between Carpinus cordata and the remaining species of Carpinus, indicating that
Carpinus is paraphyletic, and Ostrya has evolved from within Carpinus. Within Carpinus, section Distegocarpus
is polyphyletic, whereas section Carpinus is a clade where subsections Polyneurae and Carpinus are more closely
related to each other than either is to subsection Monbeigianae.
Key words Carpinus, nitrate reductase (Nia), monophyly, Ostrya, paraphyly.
Betulaceae are a plant family of six genera and
about 130 species, and are small to large trees with a
predominant distribution in Northern Hemisphere
(Chen et al., 1999). Most authors recognize two
taxonomic groups in the family, either as tribes
Betuleae and Coryleae (Prantl, 1894; Winkler, 1904)
or subfamilies Betuloideae and Coryloideae
(Takhtajan, 1980; Thorne, 1983; Furlow, 1990).
However, others consider them as different families
(Hutchinson, 1967; Dahlgren, 1983). Phylogenetic
analyses based on morphology and sequences of
nuclear ribosomal (nr) DNA internal transcribed
spacers (ITS) and chloroplast gene rbcL support the
division of Betulaceae into two clades: 1) Betuloideae,
which includes Alnus Miller and Betula L. and 2)
Coryloideae, which contains Corylus L., Ostryopsis
Decne, Carpinus L., and Ostrya Scop. (Bousquet et
al., 1992; Chen et al., 1999). Within Coryloideae,
Corylus is basal, while Carpinus and Ostrya form a
clade with Ostryopsis (Chen et al., 1999; Yoo & Wen,
2002; Forest et al., 2005; Yoo & Wen, 2007). Se-
quences of chloroplast gene matK, however, suggest
that Corylus and Ostryopsis are more closely to each
other than either is to the other two genera of Cory-
loideae (Kato et al., 1998). Nevertheless, all morpho-
logical and molecular studies support the close affinity
of Carpinus and Ostrya. Morphologically, Carpinus
and Ostrya differ evidently in their infructescence
bracts, which are radially symmetrical and inflated
bladder-like in Ostrya and are open and flat in
Carpinus (Chen et al., 1999). Sequences of the
nrDNA ITS suggest that both Carpinus and Ostrya are
paraphyletic (Yoo & Wen, 2002). Sequences from
three chloroplast regions (matK, trnL-F, and
psbA-trnH) support the paraphyly of Ostrya and
monophyly of Carpinus (bootstrap support=63%),
while nrDNA ITS data recognize a weakly supported
clade (posterior probability of 53%) of Ostrya (Yoo &
Wen, 2007). Based on the nrDNA ITS and 5S spacer
data, however, Forest et al. (2005) found that both
Ostrya and Carpinus were monophyletic forming a
sister relationship. However, this relationship was
poorly supported.
Single and low copy nuclear genes have been in-
creasingly used in phylogenetic reconstruction be-
cause they contain large amount of genetic informa-
tion and are biparentally inherited (Small et al., 2004);
however, their application may encounter many
difficulties (e.g., paralogy and copy number). Few
nuclear genes have been used in molecular systemat-
ics of Betulaceae (Jarvinen et al., 2004). Nitrate
reductase (Nia) is a low copy nuclear gene catalyzing
the reduction of nitrate to nitrite in the nitrogen cycle
(Zhou & Kleinhofs, 1996). Introns of the gene are
apparently more variable than the nrDNA ITS
(Howarth & Baum, 2002), and phylogenetically
informative in Betula (Li et al., 2007).
The purpose of this study was to reconstruct the
phylogenetics of Coryloideae using sequence data of
nuclear gene nitrate reductase (Nia) with a focus on
testing the monophyly of Carpinus and Ostrya.
———————————
Received: 26 February 2008 Accepted: 6 May 2008
* E-mail: jli@oeb.harvard.edu; Tel: 617-496-6429; Fax: 617-495-9484.
Journal of Systematics and Evolution Vol. 46 No. 3 2008 334
1 Material and Methods
1.1 Plant material
Twenty-six samples (Table 1) were used in this
study representing 22 species belonging to four genera
of Coryloideae (Carpinus, Corylus, Ostrya, and
Ostryopsis) and two genera of Betuloideae (Alnus and
Betula). The latter two genera were used for rooting
purposes. Carpinus is a genus of about 25 species
with a broad distribution in North America (C. caro-
liniana Walter and C. tropicalis (Donn.Sm.) Lundell),
Europe (C. betulus L. and C. orientalis Miller), and
Asia (ca. 21 species). It has been divided into two
sections: Distegocarpus and Eucarpinus (=Carpinus)
(Winkler, 1904). Section Carpinus consists of three
subsections (Carpinus, Monbeigianae, and Polyneu-
rae) (Li & Cheng, 1979). In this study, seven species
of Carpinus were included to represent all sections,
subsections, and the clades recognized by recent
phylogenetic analyses (Yoo & Wen, 2007). Ostrya is
a disjunct genus of 5–7 species (Li & Cheng, 1979;
Furlow, 1990). Here three species representing the
paraphyletic lineages in the chloroplast phylogeny
(Yoo & Wen, 2007) were sampled from eastern Asia,
Europe, and North America to cover both geographic
and morphological diversity of the genus (Li, 1952).
Ostryopsis, with two Asian species, is sister to the
clade containing Ostrya and Carpinus based on
morphological and rbcL sequence data (Chen et al.,
1999). However, matK sequences support the sister
relationship of Ostryopsis and Corylus (Kato et al.,
1998), which has 15–20 species (Li & Cheng, 1979;
Furlow, 1990). Here Ostryopsis nobilis Balf. f. &
W.W.Sm. and six species of Corylus were sampled to
represent the two genera.
1.2 Molecular method
Genomic DNA was extracted from silica gel
dried leaves using a Qiagen DNeasy Plant Mini Kit
(Germantown, Maryland). The third intron of the Nia
was amplified using primers NiaF3 and NiaR3
(Howarth & Baum, 2002). The amplified products
were purified using a Qiagen Gel Purification Kit
(Santa Clarita, California). PCR products were
cloned using a pGEM-T vector system (Promega,
Madison), as in Li et al. (2004). Five clones were
obtained for each accession to detect whether there is
more than one type of the Nia sequences. Sequencing
reactions were conducted using the BigDye terminator
chemistry following manufacturer’s instructions (ABI,
Foster City, California). Sequences were analyzed
using an ABI 3100 or 3700 Genetic Analyzer, and
edited using Sequencher (version 4.1, GeneCode Inc.,
Ann Arbor, Michigan).

Table 1 Species of Coryloideae sampled in this study
Species Voucher and DNA No. Source GenBank accession #
Carpinus betulus L. AA 377-90B, 4282 Germany: Muhlhausen, Lengefeld EU692799–EU692801
Carpinus caroliniana Walter AA 970-79B, 4284 USA: North Carolina, Madison Co. EU692802–EU692805
Carpinus cordata Bl. AA 1468-77C, 4334; MA
84-183-A, 4345; MA 86-023-A,
4346
Japan: Hokkaido, Yamabe; Japan:
Hokkaido, Kameda-gun; South
Korea: Kyong Gi Do
EU692806–EU692812
Carpinus japonica Bl. AA 117-91A, 4296; MA
2001-292-A, 4347; MA
96-280-A, 4348
Japan: Hondo, Lake Chuzenji; Japan:
Honshu, Siga, Mt. Kira; Japan:
Kyoto, Ashiu Univ. Forest
EU692813–EU692819
Carpinus laxiflora Bl. AA 973-85A, 4292 South Korea: Kyong Gi Do EU692820–EU692823
Carpinus orientalis Miller AA 706-89A, 4285 Iran: Gorgan EU692824–EU692827
Carpinus pubescens Burkill Del Tredici & JLI 03 China: Guizhou EU692830
Carpinus tschonoskii Maxim. AA 72-68A, 4286 China EU692828–EU692829
Corylus americana Marsh. AA 1229A, 4301 USA: Virginia EU692831–EU692835
Corylus cornuta Marsh. AA 99-79A, 4303 Canada: Nova Scotia EU692836–EU692840
Corylus fargesii Schneid. AA 112-98A, 4288 China: Gansu, Mt. Xiaolong EU692841–EU692845
Corylus sieboldiana Blume AA 518-77B, 4304 South Korea: Mt. Sorak EU692846–EU692849
Corylus heterophylla Fisch. AA 15923C, 4295 China: Sichuan EU692850–EU692854
Corylus tibetica Batalin AA 113-98B, 4281 China: Shaanxi, Foping EU692855–EU692859
Ostrya carpinifolia Scop. AA 1295-83B, 4298 Czechoslovakia: Peninsula Lustica EU692860–EU692863
Ostrya rehderiana Chun AA 108-2002E, 4297 China: Zhejiang, Mt. Tianmu EU692864–EU692868
Ostrya virginiana (Miller) K. Koch AA 1538-83A, 4290 USA: Minnesota EU692869–EU692873
Ostryopsis nobilis Balf. f. & W.W.Sm. 4333, Zhiduan China: Yunnan EU692874–EU692878
AA, Arnold Arboretum; MA, Morris Arboretum. Vouchers are deposited at A and PE.
LI: Phylogenetics of Ostrya and Carpinus

335
1.3 Phylogenetic analyses
Neighbor-joining (NJ), Maximum parsimony
(MP), and maximum likelihood (ML) analyses were
conducted using PAUP* 4.0 (Swofford, 2002). Both
MP and NJ analyses were performed for the data set
containing all sequences to test whether clones from
each sample form individual clades. Consensus se-
quence of clones from each sample was then created
using the standard ambiguity base coding (e.g., Y for
C and T, and R for A and G). MP and ML analyses
were done based on the reduced data set containing
consensus sequences. For MP analyses, characters
were equally weighted and their states were unor-
dered. To search for possible multiple islands
(Maddison, 1991), random sequence addition of 1000
replicates was used in the heuristic tree search with 10
trees held in each replicate. Other options were as
follows: TBR (tree bisection and reconnection) branch
swapping, steepest descent off, and MulTrees in
effect. ML analyses were carried out using the optimal
evolutionary model for the sequence data, as deter-
mined by the hierarchical likelihood ratio tests (hLRT)
implemented using the MODELTEST computer
program (Posada & Crandall, 1998). Tree search
options for ML analyses were as in MP analyses
except for simple sequence addition. Bootstrap analy-
ses were carried out to estimate the support for indi-
vidual clades (Felsenstein, 1985).
2 Results
2.1 Sequence characteristics
Eighty-eight sequences of the Nia gene were ob-
tained from the 26 samples, each with 2–5 clones.
Five clones were identical in Carpinus pubescens and
thus only one sequence was included in the data set.
The newly obtained sequences have been deposited in
the GenBank and their accession numbers are listed in
Table 1. There were 84 sequences in the complete data
set. Sequence lengths ranged from 648 base pairs (bp)
in Betula pendula to 901 bp in Ostryopsis nobilis. The
sequences were A+T rich and base frequencies did not
differ significantly across the taxa, as suggested by a
χ2 test (P=0.9). Clones from each accession varied
slightly (0.3%–1.5%). Sequence divergences ranged
from 1.5%–4.6%, 5.6%–6.9%, and 1.7%–7.5% within
Corylus, Ostrya, and Carpinus, respectively. They
were from 5.5%–8.9% between Ostrya and Carpinus,
7.3%–13.4% among Corylus, Carpinus+Ostrya, and
Ostryopsis, and 12%–14% between outgroup and
ingroup taxa. Sequences were readily aligned by sight
and the aligned data set had 1289 sites, 303 of which
were parsimony informative.
2.2 Phylogenetic relationships
MP and NJ analyses of the 86-sequence data set
generated congruent trees where clone sequences from
each sample formed individual clades. The NJ clado-
gram generated using the BioNJ method and the
Kimura 2-parameter distance is presented here (Fig. 1)
because bootstrap analyses using the MP method did
not run to completion due to small sequence variation
of clones and the large number of trees generated. For
further MP and ML analyses a consensus sequence
was used to represent each of the samples, resulting in
a reduced data set of 26 taxa and 1234 sites. Parsi-
mony analyses of the 26-taxon data set generated 180
trees of 506 steps, a consistency index (CI) of 0.85,
and a retention index (RI) of 0.87 (Fig. 2). Ostryopsis
nobilis was sister to the clade containing Corylus,
Ostrya, and Carpinus; however, the support was weak
(BS, bootstrap support=56%). Corylus species formed
a well-supported clade (BS=99%, labeled as A),
which was sister to the Ostrya + Carpinus clade (BS=
91%, clade B). Three accessions of Carpinus cordata
formed a well-supported clade (BS=100%, clade C),
which was sister to the group (BS=79%, clade D)
containing Ostrya and the remaining species of
Carpinus. Ostrya species formed a clade (BS=99%,
clade E) sister to the clade of the remaining species of
Carpinus (BS=67%, clade F). Within the Ostrya
clade, O. carpinifolia was sister to the clade of O.
rehderiana and O. virginiana. Three accessions of
Carpinus japonica were in a robust clade (BS=100%,
clade G) sister to clade H (BS=75%) consisting of C.
pubescens, C. betulus, C. orientalis, C. caroliniana,
and C. laxiflora, the latter four species formed a clade
(BS=100%, clade I). However, relationships within
clade H were not well resolved.
Modeltest suggested that the optimal evolution-
ary model for the sequence data was the HKY+G
model and the estimated parameters were as follows:
base frequencies (A=0.31, C=0.16, G=0.17, and T=
0.36), the ratio of transitional to transversional
changes=1.29, and gamma shape parameter=1.03. ML
analyses using the estimated parameters produced a
single tree with a likelihood of –lnL=4391.6955.
The tree topology was congruent to the MP tree with a
few exceptions. Within Coryloideae, Corylus as a
clade was sister to the clade consisting of Ostryopsis,
Ostrya, and Carpinus (Fig. 3). Ostryopsis was sister to
the latter two genera; however, the support was weak
(BS=53%).
Journal of Systematics and Evolution Vol. 46 No. 3 2008 336


Fig. 1. Neighbor-joining tree of Coryloideae based on sequences of nitrate reductase. Numbers at branches are bootstrap percentages. Numbers
after species names are accession number followed by clone numbers. Alnus and Betula are outgroups.
LI: Phylogenetics of Ostrya and Carpinus

337


Fig. 2. One of 180 parsimonious trees of 506 steps (CI=0.85 and RI=0.87) based on sequences of nitrate reductase. Numbers at branches are
percentages of bootstrap support. Asterisks denote clades absent in the strict consensus tree. Alnus and Betula are outgroups.

3 Discussion
3.1 Monophyly of Ostrya
Infructescence bracts of Ostrya are membrane-
ous, bladder-like completely enclosing nuts, while
those of Carpinus are leaf-like with or without side
lobes (Fig. 2). Therefore, the two genera have long
been recognized (Prantl, 1894; Winkler, 1904; Li &
Cheng, 1979; Mabberley, 1997). Nevertheless, se-
quences of the nrDNA ITS suggest that Ostrya is
paraphyletic since Asian species O. japonica Sarg.
and O. rehderiana form a robust clade with the Euro-
pean species O. carpinifolia, while North American
species O. virginiana and O. knowltonii Sarg. are in a
separate, well-supported clade (Yoo & Wen, 2002;
Table 2). However, support for the relationship of the
two clades with Carpinus species is weak
[BS=50%–65%, Fig. 1 in Yoo & Wen (2002)]. Recent
analyses of additional nrDNA ITS data suggest the
monophyly of Ostrya, but the support is also weak
(Yoo & Wen, 2007). Another phylogenetic analysis of
Betulaceae based on sequences of the nrDNA ITS and
5S spacer including two species of Ostrya (O. japon-
ica and O. virginiana) strongly supports the mono-
phyly of the genus [BS=93% in Fig. 1 of Forest et al.
(2005)]. In this study, I sampled three species of
Ostrya: O. carpinifolia from Europe, O. rehderiana
from eastern Asia, and O. virginiana from North
America. These species form a robust clade (BS=99%,
Figs. 2, 3), thus recognizing the genus Ostrya as
monophyletic (Table 2).
3.2 Paraphyly of Carpinus and implications for
character evolution and taxonomy
Sequences of the nrDNA ITS suggest that Ostrya
is derived from within the paraphyletic Carpinus
where species of section Distegocarpus are separated
into two clades (Yoo & Wen, 2002, 2007). However,
neither the first divergence of Carpinus japonica from
the rest of species (BS<50%) nor the sister relation-
ship (BS=65%) of Carpinus cordata and C. fangiana
with the clade containing Ostrya and the remaining
species of Carpinus is strongly supported (Yoo &
Wen, 2002; Table 2). The combined sequence data of
the nrDNA ITS and 5S spacer, nonetheless, support
the monophyly of Carpinus, and indicate that C.
cordata and C. fangiana form a clade that is sister to
the clade consisting of C. japonica, C. betulus, and C.
caroliniana (Forest et al., 2005). That study, however,
had a limited taxon sampling and focused mainly on
issues concerning molecular dating. In addition,
Journal of Systematics and Evolution Vol. 46 No. 3 2008 338



Fig. 3. Single ML tree based on sequences of nitrate reductase. Numbers at branches are bootstrap indices. Alnus and Betula are outgroups.

LI: Phylogenetics of Ostrya and Carpinus

339
Table 2 Summary of phylogenetic studies of the monophyly and paraphyly of Ostrya and Carpinus
Literature and markers used Ostrya Carpinus
Yoo & Wen, 2002: nrDNA ITS Paraphyly Paraphyly

Monophyly, bs=53%

Paraphyly
Yoo & Wen, 2007:
1. ITS
2. matK, trnL-F, psbA-trnH Paraphyly Monophyly, bs=63%
Forest et al., 2005: nrDNA ITS and 5S spacer Monophyly, bs=93% Monophyly, bs=65%
Li, 2008, this study: nuclear Nia Monophyly, bs=99% Paraphyly
bs, Bootstrap percentages.

bootstrap support for the monophyly of Carpinus was
65% and that for the early divergence of C. cordata
and C. fangiana was 52%. In the present study,
Carpinus cordata splits first from the rest of species,
while C. japonica is sister to a clade containing C.
betulus, C. caroliniana, C. orientalis, C. pubescens,
and C. tschonoskii Maxim. (Figs. 2, 3). Therefore,
sequences of the Nia gene support the paraphyly of
Carpinus, as shown in Yoo and Wen (2002), and the
early divergence of C. cordata from the rest species of
Carpinus, as indicated in Forest et al. (2005).
Carpinus has generally been divided into two
sections based on infructescence bracts (Winkler,
1904; Nakai, 1915; Hu, 1964; Li & Cheng, 1979):
sections Distegocarpus and Carpinus (with crowded
vs. open bracts, respectively). In the Nia trees (Figs.
1–3), section Distegocarpus is not monophyletic; C.
cordata is basal in the clade of Carpinus+Ostrya and
C. japonica forms a clade with the monophyletic
section Carpinus. The Nia tree, therefore, suggests
that the crowded arrangement of bracts may have
evolved independently in C. cordata and C. japonica.
It is worth noting that the asymmetry of infructescence
bracts supports the close relationship of C. japonica
and section Carpinus. In addition, the bracts are
symmetrical in Carpinus cordata and Ostrya. The Nia
sequence data, thus, suggest that bract asymmetry may
be a derived feature in the Ostrya-Carpinus clade
(Fig. 2).
Within section Carpinus, C. tschonoskii of sub-
section Polyneurae is embedded within subsection
Carpinus, supporting the merger of the two subsec-
tions (Yoo & Wen, 2002). C. pubescens of subsection
Monbeigianae is sister to subsections Carpinus and
Polyneurae. However, the support is weak. More
comprehensive taxon sampling is needed to further
test subsectional relationships of Carpinus.
If the derivation of Ostrya from within the para-
phyletic Carpinus is correct, it becomes necessary to
modify the definition of Carpinus to include Ostrya.
Interestingly, Ostrya had been treated as belonging to
Carpinus (Linnaeus, 1753; Miller, 1768) before
Scopoli (1772) made it a separate genus. Similarities
of Ostrya and Carpinus in inflorescences, infructes-
cences, and vegetative features support such the
treatment (Furlow, 1990). An alternative is to recog-
nize Carpinus and Ostrya as rankless clades with the
latter embedded within the former clade. This avoids
nomenclatural changes at the species level. Neverthe-
less, it is necessary to sample more species from both
genera and gather additional data from both nuclear
and chloroplast genomes to further test their relation-
ships before a formal taxonomic treatment can be
proposed.
Acknowledgements I thank Suzanne Shoup for lab
assistance, Dr. Zhiduan Chen for providing material of
Ostryopsis nobilis, and Tony Aiello of Morris Arbore-
tum for Carpinus cordata and C. japonica.
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