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 of and paraphyly of Carpinus () 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, , 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 , supporting the monophyly of the . 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 family of six genera and bladder-like in Ostrya and are open and flat in about 130 species, and are small to large 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 : 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). , which are radially symmetrical and inflated The purpose of this study was to reconstruct the ——————————— 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 # 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 using the BioNJ method and the quence of clones from each sample was then created Kimura 2-parameter distance is presented here (Fig. 1) using the standard ambiguity base coding (e.g., Y for because bootstrap analyses using the MP method did C and T, and R for A and G). MP and ML analyses not run to completion due to small sequence variation were done based on the reduced data set containing of clones and the large number of trees generated. For consensus sequences. For MP analyses, characters further MP and ML analyses a consensus sequence were equally weighted and their states were unor- was used to represent each of the samples, resulting in dered. To search for possible multiple islands a reduced data set of 26 taxa and 1234 sites. Parsi- (Maddison, 1991), random sequence addition of 1000 mony analyses of the 26-taxon data set generated 180 replicates was used in the heuristic search with 10 trees of 506 steps, a consistency index (CI) of 0.85, trees held in each replicate. Other options were as and a retention index (RI) of 0.87 (Fig. 2). Ostryopsis follows: TBR (tree bisection and reconnection) branch nobilis was sister to the clade containing Corylus, swapping, steepest descent off, and MulTrees in Ostrya, and Carpinus; however, the support was weak effect. ML analyses were carried out using the optimal (BS, bootstrap support=56%). Corylus species formed evolutionary model for the sequence data, as deter- a well-supported clade (BS=99%, labeled as A), mined by the hierarchical likelihood ratio tests (hLRT) which was sister to the Ostrya + Carpinus clade (BS= implemented using the MODELTEST computer 91%, clade B). Three accessions of Carpinus cordata program (Posada & Crandall, 1998). Tree search formed a well-supported clade (BS=100%, clade C), options for ML analyses were as in MP analyses which was sister to the group (BS=79%, clade D) except for simple sequence addition. Bootstrap analy- containing Ostrya and the remaining species of ses were carried out to estimate the support for indi- Carpinus. Ostrya species formed a clade (BS=99%, vidual clades (Felsenstein, 1985). 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. 2 Results rehderiana and O. virginiana. Three accessions of 2.1 Sequence characteristics Carpinus japonica were in a robust clade (BS=100%, Eighty-eight sequences of the Nia gene were ob- clade G) sister to clade H (BS=75%) consisting of C. tained from the 26 samples, each with 2–5 clones. pubescens, C. betulus, C. orientalis, C. caroliniana, Five clones were identical in Carpinus pubescens and and C. laxiflora, the latter four species formed a clade thus only one sequence was included in the data set. (BS=100%, clade I). However, relationships within The newly obtained sequences have been deposited in clade H were not well resolved. the GenBank and their accession numbers are listed in Modeltest suggested that the optimal evolution- Table 1. There were 84 sequences in the complete data ary model for the sequence data was the HKY+G set. Sequence lengths ranged from 648 base pairs (bp) model and the estimated parameters were as follows: in Betula pendula to 901 bp in Ostryopsis nobilis. The base frequencies (A=0.31, C=0.16, G=0.17, and T= sequences were A+T rich and base frequencies did not 0.36), the ratio of transitional to transversional differ significantly across the taxa, as suggested by a changes=1.29, and gamma shape parameter=1.03. ML χ2 test (P=0.9). Clones from each accession varied analyses using the estimated parameters produced a slightly (0.3%–1.5%). Sequence divergences ranged single tree with a likelihood of –lnL=4391.6955. from 1.5%–4.6%, 5.6%–6.9%, and 1.7%–7.5% within The tree topology was congruent to the MP tree with a Corylus, Ostrya, and Carpinus, respectively. They few exceptions. Within Coryloideae, Corylus as a were from 5.5%–8.9% between Ostrya and Carpinus, clade was sister to the clade consisting of Ostryopsis, 7.3%–13.4% among Corylus, Carpinus+Ostrya, and Ostrya, and Carpinus (Fig. 3). Ostryopsis was sister to Ostryopsis, and 12%–14% between outgroup and the latter two genera; however, the support was weak ingroup taxa. Sequences were readily aligned by sight (BS=53%). 336 Journal of Systematics and Evolution Vol. 46 No. 3 2008

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

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 Yoo & Wen, 2007: 1. ITS Monophyly, bs=53% Paraphyly 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 Scopoli (1772) made it a separate genus. Similarities 65% and that for the early divergence of C. cordata of Ostrya and Carpinus in , infructes- and C. fangiana was 52%. In the present study, cences, and vegetative features support such the Carpinus cordata splits first from the rest of species, treatment (Furlow, 1990). An alternative is to recog- while C. japonica is sister to a clade containing C. nize Carpinus and Ostrya as rankless clades with the betulus, C. caroliniana, C. orientalis, C. pubescens, latter embedded within the former clade. This avoids and C. tschonoskii Maxim. (Figs. 2, 3). Therefore, nomenclatural changes at the species level. Neverthe- sequences of the Nia gene support the paraphyly of less, it is necessary to sample more species from both Carpinus, as shown in Yoo and Wen (2002), and the genera and gather additional data from both nuclear early divergence of C. cordata from the rest species of and chloroplast genomes to further test their relation- Carpinus, as indicated in Forest et al. (2005). ships before a formal taxonomic treatment can be Carpinus has generally been divided into two proposed. sections based on infructescence bracts (Winkler, 1904; Nakai, 1915; Hu, 1964; Li & Cheng, 1979): Acknowledgements I thank Suzanne Shoup for lab sections Distegocarpus and Carpinus (with crowded assistance, Dr. Zhiduan Chen for providing material of vs. open bracts, respectively). In the Nia trees (Figs. Ostryopsis nobilis, and Tony Aiello of Morris Arbore- 1–3), section Distegocarpus is not monophyletic; C. tum for Carpinus cordata and C. japonica. cordata is basal in the clade of Carpinus+Ostrya and C. japonica forms a clade with the monophyletic References section Carpinus. The Nia tree, therefore, suggests Bousquet J, Strauss SH, Li P. 1992. Complete congruence that the crowded arrangement of bracts may have between morphological and rbcL-based molecular evolved independently in C. cordata and C. japonica. phylogenies in birches and related species (Betulaceae). It is worth noting that the asymmetry of infructescence Molecular Biology and Evolution 9: 1076–1088. bracts supports the close relationship of C. japonica Chen Z-D, Manchester SR, Sun H-Y. 1999. Phylogeny and and section Carpinus. In addition, the bracts are evolution of the Betulaceae as inferred from DNA symmetrical in Carpinus cordata and Ostrya. The Nia sequences, morphology, and paleobotany. American Journal of Botany 86: 1168–1181. sequence data, thus, suggest that asymmetry may Dahlgren R. 1983. General aspects of angiosperm evolution be a derived feature in the Ostrya-Carpinus clade and macrosystematics. Nordic Journal of Botany 3: (Fig. 2). 119–149. Within section Carpinus, C. tschonoskii of sub- Felsenstein J. 1985. Confidence limits on phylogenies: An section Polyneurae is embedded within subsection approach using the bootstrap. Evolution 39: 783–791. Carpinus, supporting the merger of the two subsec- Forest F, Savolainen V, Chase MW, Lupia R, Bruneau A, Crane PR. 2005. Teasing apart molecular-versus fossil-based tions (Yoo & Wen, 2002). C. pubescens of subsection error estimates when dating phylogenetic trees: A case Monbeigianae is sister to subsections Carpinus and study in the birch family (Betulaceae). Systematic Botany Polyneurae. However, the support is weak. More 30: 118–133. comprehensive taxon sampling is needed to further Furlow JJ. 1990. The genera of Betulaceae in the southeastern test subsectional relationships of Carpinus. United States. Journal of the Arnold Arboretum 71: 1–67. If the derivation of Ostrya from within the para- Howarth DG, Baum DA. 2002. Phylogenetic utility of a nuclear intron from nitrate reductase for the study of closely phyletic Carpinus is correct, it becomes necessary to related plant species. Molecular Phylogenetics and modify the definition of Carpinus to include Ostrya. Evolution 23: 525–528. Interestingly, Ostrya had been treated as belonging to Hu H-H. 1964. The materials on the monography of gen. Carpinus (Linnaeus, 1753; Miller, 1768) before Carpinus Linn. of China. Acta Phytotaxonomica Sinica 9: 340 Journal of Systematics and Evolution Vol. 46 No. 3 2008

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