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 bladder-like in Ostrya and are open and flat in about 130 species, and are small to large trees with a Carpinus (Chen et al., 1999). Sequences of the predominant distribution in Northern Hemisphere nrDNA ITS suggest that both Carpinus and Ostrya are (Chen et al., 1999). Most authors recognize two paraphyletic (Yoo & Wen, 2002). Sequences from taxonomic groups in the family, either as tribes three chloroplast regions (matK, trnL-F, and Betuleae and Coryleae (Prantl, 1894; Winkler, 1904) psbA-trnH) support the paraphyly of Ostrya and or subfamilies Betuloideae and Coryloideae monophyly of Carpinus (bootstrap support=63%), (Takhtajan, 1980; Thorne, 1983; Furlow, 1990). while nrDNA ITS data recognize a weakly supported However, others consider them as different families clade (posterior probability of 53%) of Ostrya (Yoo & (Hutchinson, 1967; Dahlgren, 1983). Phylogenetic Wen, 2007). Based on the nrDNA ITS and 5S spacer analyses based on morphology and sequences of data, however, Forest et al. (2005) found that both nuclear ribosomal (nr) DNA internal transcribed Ostrya and Carpinus were monophyletic forming a spacers (ITS) and chloroplast gene rbcL support the sister relationship. However, this relationship was division of Betulaceae into two clades: 1) Betuloideae, poorly supported. which includes Alnus Miller and Betula L. and 2) Single and low copy nuclear genes have been in- Coryloideae, which contains Corylus L., Ostryopsis creasingly used in phylogenetic reconstruction be- Decne, Carpinus L., and Ostrya Scop. (Bousquet et cause they contain large amount of genetic informa- al., 1992; Chen et al., 1999). Within Coryloideae, tion and are biparentally inherited (Small et al., 2004); Corylus is basal, while Carpinus and Ostrya form a however, their application may encounter many clade with Ostryopsis (Chen et al., 1999; Yoo & Wen, difficulties (e.g., paralogy and copy number). Few 2002; Forest et al., 2005; Yoo & Wen, 2007). Se- nuclear genes have been used in molecular systemat- quences of chloroplast gene matK, however, suggest ics of Betulaceae (Jarvinen et al., 2004). Nitrate that Corylus and Ostryopsis are more closely to each reductase (Nia) is a low copy nuclear gene catalyzing other than either is to the other two genera of Cory- the reduction of nitrate to nitrite in the nitrogen cycle loideae (Kato et al., 1998). Nevertheless, all morpho- (Zhou & Kleinhofs, 1996). Introns of the gene are logical and molecular studies support the close affinity apparently more variable than the nrDNA ITS of Carpinus and Ostrya. Morphologically, Carpinus (Howarth & Baum, 2002), and phylogenetically and Ostrya differ evidently in their infructescence informative in Betula (Li et al., 2007). bracts, which are radially symmetrical and inflated The purpose of this study was to reconstruct the ——————————— phylogenetics of Coryloideae using sequence data of Received: 26 February 2008 Accepted: 6 May 2008 nuclear gene nitrate reductase (Nia) with a focus on * E-mail: [email protected]; Tel: 617-496-6429; Fax: 617-495-9484. testing the monophyly of Carpinus and Ostrya. 334 Journal of Systematics and Evolution Vol. 46 No. 3 2008 1 Material and Methods clade containing Ostrya and Carpinus based on morphological and rbcL sequence data (Chen et al., 1.1 Plant material 1999). However, matK sequences support the sister Twenty-six samples (Table 1) were used in this relationship of Ostryopsis and Corylus (Kato et al., study representing 22 species belonging to four genera 1998), which has 15–20 species (Li & Cheng, 1979; of Coryloideae (Carpinus, Corylus, Ostrya, and Furlow, 1990). Here Ostryopsis nobilis Balf. f. & Ostryopsis) and two genera of Betuloideae (Alnus and W.W.Sm. and six species of Corylus were sampled to Betula). The latter two genera were used for rooting represent the two genera. purposes. Carpinus is a genus of about 25 species 1.2 Molecular method with a broad distribution in North America (C. caro- Genomic DNA was extracted from silica gel liniana Walter and C. tropicalis (Donn.Sm.) Lundell), dried leaves using a Qiagen DNeasy Plant Mini Kit Europe (C. betulus L. and C. orientalis Miller), and (Germantown, Maryland). The third intron of the Nia Asia (ca. 21 species). It has been divided into two was amplified using primers NiaF3 and NiaR3 sections: Distegocarpus and Eucarpinus (=Carpinus) (Howarth & Baum, 2002). The amplified products (Winkler, 1904). Section Carpinus consists of three were purified using a Qiagen Gel Purification Kit subsections (Carpinus, Monbeigianae, and Polyneu- rae) (Li & Cheng, 1979). In this study, seven species (Santa Clarita, California). PCR products were of Carpinus were included to represent all sections, cloned using a pGEM-T vector system (Promega, subsections, and the clades recognized by recent Madison), as in Li et al. (2004). Five clones were phylogenetic analyses (Yoo & Wen, 2007). Ostrya is obtained for each accession to detect whether there is a disjunct genus of 5–7 species (Li & Cheng, 1979; more than one type of the Nia sequences. Sequencing Furlow, 1990). Here three species representing the reactions were conducted using the BigDye terminator paraphyletic lineages in the chloroplast phylogeny chemistry following manufacturer’s instructions (ABI, (Yoo & Wen, 2007) were sampled from eastern Asia, Foster City, California). Sequences were analyzed Europe, and North America to cover both geographic using an ABI 3100 or 3700 Genetic Analyzer, and and morphological diversity of the genus (Li, 1952). edited using Sequencher (version 4.1, GeneCode Inc., Ostryopsis, with two Asian species, is sister to the 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 Japan: Hokkaido, Yamabe; Japan: EU692806–EU692812 84-183-A, 4345; MA 86-023-A, Hokkaido, Kameda-gun; South 4346 Korea: Kyong Gi Do Carpinus japonica Bl. AA 117-91A, 4296; MA Japan: Hondo, Lake Chuzenji; Japan: EU692813–EU692819 2001-292-A, 4347; MA Honshu, Siga, Mt. Kira; Japan: 96-280-A, 4348 Kyoto, Ashiu Univ. Forest 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 and the aligned data set had 1289 sites, 303 of which Neighbor-joining (NJ), Maximum parsimony were parsimony informative. (MP), and maximum likelihood (ML) analyses were 2.2 Phylogenetic relationships conducted using PAUP* 4.0 (Swofford, 2002). Both MP and NJ analyses of the 86-sequence data set MP and NJ analyses were performed for the data set generated congruent trees where clone sequences from containing all sequences to test whether clones from each sample formed individual clades. The NJ clado- each sample form individual clades. Consensus se- gram generated