The JapaneseSocietyJapanese Society forforPlant Plant Systematics ISSN OOOI-6799 ActaPhytotax. Geobot. 49 (2): 89-97 (1998) Phylogenetic Relationships of Betulaceae Based on matKr Sequences with Particular Reference to the Position of 0str:yopsis HIDETOSHI KATOi, KAZUO OGINUMA2, ZHIJIAN GU3, BARRY HAMMEL` and HIROSHI TOBE5' iMakino Uerbarium, 7bkyo Metropolitan Uitiversity,Minami-Osawa, Hbchioji,7bkyo 192-039Z' 2Flaculty of Human L(fe and Environmentat Sciences, )<bchi Wbmen's U}ziversity, Kochi 780-8515; 3K"nming institute ofBotany, The Academy of Sciences of China, Kunming, Yitnnan 65a204, Peopte's `Missouri Republic of China; Botanical Garden, P, O. Box 299, St. Lo"is, MO 63166, U.S,A,; 'fucutty of integrated Human Studies, KYoto Uhiversity, Kyoto 606-8501 Abstract. Relationships among all six genera in Betulaceue, where the position of Osttlyopsts has been uncertain, were investigated on the basis of matK sequence clata. The study includes sequencing of 1260 bases in all six betulaceous genera, Casuarina (Casuarinaceae) and IVbthojbgus (Nothofagaceae), and of 250 bases in ficodendron (Ticodendraceae) lacking in the GenBank data. The maximum parsimony analyses using Nothojirgus as outgroup resulted in a single shortest tree, showing that Betulaceae are monophyletic with support of 100% bootstrap value and sister to Ticodendraceae. Betulaceae comprise two well-supported clades as already suggested by earlier cladistic studies, i,e,, the Alnus-Betula clade and the Carpinus-Ostrya-Corylus-Osttyopsis clade. Within the latter clacle, Ostryopsis forms a cornmon clade with Coryltts, rather than with the Carpinus-Ostrya cLade, contrary to results of any of the earlier cladistic analyses, Molecular evidence from matK sequenee data supports the reeognition of two subfamilies Betuloideae CAInus and Betula) and Coryloideae as in most of the current systems of classification, and furthermore the recpgriition of two tribes Carpnieae (Carpinus and Ostrya) and Coryleae (Corylus and Ostryopsis) in Coryloideae. Morpho]ogical congruency supporting close relatienships between Conylus ancl Ostryopsis is also discussed. Key words: Betulaceae, Coryloideae, matK, melecular phylogeny, Ostryqpsis, Tlcodendren Received September 11, l998; accepted December Z 1998 Betulaceae is a well-defined family distributed mainly in the Northern Hemisphere, and comprise about 110 species in six genera Alnus, Betula, Carpinus, Corzylus, Ostrya, and Ostryopsis (Mabberley, 1997). The family has been studied relatively well with respect to generic relationships. Crane (1989), on the basis of a cladistic analysis using 14 morphological and anatomical characters, showed that Betulaceae comprise two monophyletic tribes Betuleae (Alnus and Betuta) and Coryleae (Carpintts, Cocylus, 0strlya, and Ostryopsis), and that within the Coryleae Carpinus and Ostcya are sister taxa, Ostcyopsis sister to the Carpinus-Ostilya clade, and Corylus sister to the Caizpinus-Ostrzya-Ostr:yqpsis clade. Bousquet et * Author for correspondence NII-Electronic Library Service The JapaneseSocietyJapanese Society forforPlant Plant Systematics 90 Acta Phytotax. Geobot, Vol. 49 al. (1992), on the basis of a cladistic analysis of all six genera using 35 morphological, anatomical, and chemical characters, obtained a phylogenetic tree with the same topology as did Crane (1989). Bousquet et al. (1992) further attempted a molecular phylogenetic analysis using rbcL sequence data (not including data from Ostryopsis), and supported the generic relationships shown by the cladistic analysis based on non- molecular data set. Following results of the cladistic or the molecular phylogenetic studies, recent systems of classification mostly divide the Betulaceae into two subfamilies or tribes: Betuloideae or Betuleae (Alnus and Betula) characterized by having male flowers in groups of three, and Coryloideae or Coryleae (Carpinus, Co71ylus, Ostrlya, and Ostryopsis) characterized by having solitary male flowers (Thorne, 1992; Mabberley, 1997). Recently Takhatajan (1997) separated Coryloideae (or Coryleae) as a distinct family Corylaceae from Betulaceae sens. str. (Alntts and Betula), and he divided CoryIaceae into two subfamilies, Carpinoideae (Carpinus, Ostrlya, and Ostryopsis) and Coryloideae (Coryltas). The classification of Takhtajan greatly contrasts to that of Furlow (1990). Furlow (1990, p. 4), like Thorne (1992) and Mabberley (1997), assigned all the six genera to Betulaceae with two subfamilies, Betuloideae and Coryloideae, and he further divided the four genera of Coryloideae into two tribes Carpineae (Carpinus and Ostrya) and Coryleae (Coilylus and Ostryopsis), In order to supplement the molecular phylogenetic analysis of Betulaceae we analysed chloroplast DNA sequences of Ostryopsis, because its phylogenetic position suggested by non-molecular data has never been tested on the basis of molecular data. We also analysed chloroplast DNA sequences of 7'icodendron (Ticodendraceae). Manos and Steele (1997) demonstrated on the basis of matK sequences that, although its sequence data was incomplete, Ticodendron was sister to two examined genera (Betuta and Corytus) of Betulaceae. This paper provides more sequence data for Ticodendron and further confirms that it is never nested within Betulaceae when all the six genera are analysed together. We first sequenced rbcL gene of Ostryqpsis and 71codendron and, by putting their sequence data (accession number in GeneBank ABO15454 and ABO15455) together with the data of the five remaining genera registered in GenBank, analyzed relationships within the family. However, rbcL trees of Betulaceae including Ostryopsis (using 1290 bases and generated by the maximum parsimony method) did not resolve generic relationships within Coryloideae or Coryleae. In fact, a consensus tree of five shortest trees (with length=128, excluding autapomorphies, and CI=O.643) demonstrated that Coryloideae trifurcates into the Carpinus-Ostrya clade, Corylus, and Ostcyqpsis. In other words, in contrast to the cladistic analyses based on the non-molecular data set (Crane, 1989; Bousquet et al., 1992), rbcL sequence data did not clarify relationships withjn Coryloideae or Coryleae. We subsequently analysed matK sequences of all the six genera of Betulaceae and 71codendron, and found that matK sequence data yieids a robust resolution of relationships of CoTlylus and Ostryopsis as well as of NII-Electronic Library Service The JapaneseSocietyJapanese Society forforPlant Plant Systematics December 1998 KATO et al,: Phylogeny of Betulaceae 91 the relationships among the six genera of Betulaceae and Ticodendron. Concerning the position of Ostilyopsis, the matK tree was different from the tree based on the non-molecular data. This paper presents results of phylogenetic analysis of all the six genera'bf Betulaceae and 71codendron based on matK sequences and show how those and in Ostryopsis are related. On the basis genera particular'the of the results, we will discuss most appropriate infrafamilial classification of Betulaceae. Materials and Methods Plant materials One species each of all the six genera of Betulaceae, as well as of AJbthojlagus (N. solandri) selected as outgroup of the family, was studied with respect to chloroplast DNA sequences. Casuarina (Casuarinaceae) and 71codendron (Ticodendraceae) were included in the analysis to "rosid confirm a monophyly of Betulaceae. In rbcL trees of the I" generated by an extensive phylogenetic study of seed plants (Chase et al., 1993, p. 571), Casuarina is positioned nearest to Betula, and Nbthojizgus "higher" more distantly positioned. In the matK trees of Hamamelididae (Manos and Steele, 1997), 1'icodendron is sister to the Betula-Corylus ciade, and Casuarina sister to the Betula-Corylus-Ticodendron clade. All nucleotide sequence data of matK of the six genera of Betulaceae, Casuarina, and IVbthofagus were analyzed, while 250 bases from 526th to 1535th were analysed with respect to 7-icodendron because its matK data from GenBank (accession number U92855) represents only 1010 bases. The eollection data of the species examined and accession numbers in GenBank are presented in Table 1. In order to obtain nucleotide sequence data from fresh leaves, leaf pieces of Alnus, Betula, Carpunus, Corylus, and Ostto?a were collected from wild-growing or. cultivated plants and stored in a freezer controlled at -80 SC. In the case of 0stzyopsis, fresh leaves were preserved in NaCl- CTAB solution (Rogstad, 1992) at room temperature during a period when they were carried out from Kunming to Kyoto. Preserved leaves of Ostilyopsis were rinsed with distilled water in the laboratory and stored in -80OC. the freezer controlled at In the case of ficodendron, collected leaves were dried by silica gel powder in the field and transported to the laboratory. DNA extraction, amplijicats'on, and sequencing Total DNAs were extracted from the frozen and dried leaves following the modified method of Doyle and Doyle's (1990) using 2 X CTAB extraction buffer. Segments of double-stranded DNA containing most of sequences coding matK gene were amplified using the polymerase chain reaction (PCR). Table 2 and Fig. 1 show the primer designs used in this study. As PCR primers for matK gene, the combination of trnK- 3914F (Johoson and Soltis, 1994) and matK-8R (Ooi et al., 1995) were NII-Electronic Library Service The JapaneseSocietyJapanese Society forforPlantSystematics Plant Systematics 92 Acta Phytotax. Geobot, Vol. 49 TABLE 1. Taxa studied of Betulaceae and related genera, and theircoUection