Phylogenetics of Acer (Aceroideae, Sapindaceae) Based on Nucleotide Sequences of Two Chloroplast Non-Coding Regions

Home , Acer caesium, Acer carpinifolium, Acer caudatum, Acer glabrum, Acer henryi, Acer monspessulanum

PHYLOGENETICS OF ACER (ACEROIDEAE, SAPINDACEAE) BASED ON NUCLEOTIDE SEQUENCES OF TWO CHLOROPLAST NON-CODING REGIONS

JIANHUA LI,1,3,4 JIPEI YUE,2 AND SUZANNE SHOUP 1

Abstract. Acer is one of the most diverse woody genera in the Northern Hemisphere. Recent phylogenetic studies support the placement of Acer and Dipteronia—sole members of the traditional Aceraceae—in the Sapindaceae. However, the monophyly of Acer and its sections remain to be tested. In this study, sequences of two chloroplast non-coding regions, psbM-trnD and trnD-trnT, are used to elucidate phylogenetic relationships of Acer and Dipteronia. Our results support the monophyly of Acer and sects. Arguta, Ginnala, Integrifolia, Lithocarpa, Macrantha, Palmata, Platanoidea, and Trifoliata. In contrast, sects. Acer, Goniocarpa, Parviflora, Saccharodendron, and Spicata are not monophyletic. Acer trautvetteri and A. opalus of sect. Acer are more closely related to A. monspessulanum of sect. Goniocarpa and A. saccharum of sect. Saccharina than to A. caesium and A. pseudoplatanus of sect. Acer. Acer distylium of sect. Parviflora is more closely related to sect. Platanoidea than to A. nipponicum of sect. Parviflora. Morphological species pairs between eastern Asia and North America are not sister species, including A. pycnanthum—A. rubrum and A. caudatum—A. spicatum. Acer ukurunduense is a distinct species from A. caudatum. Acer glabrum is most closely related to A. pseudoplatanus, whereas A. spicatum may be more closely related to A. carpinifolium than to A. caudatum. Section Hyptiocarpa is most closely related to sect. Rubra, and the two North American species of sect. Rubra (A. rubrum and A. saccharinum) are more closely related to each other than they are to the Japanese species (A. pycnanthum). Sections Integrifolia and Trifoliata are closely related, and so are Cissifolia and Arguta. Nevertheless, more data are needed to fully resolve intersectional relationships of Acer. Keywords: Acer, Dipteronia, chloroplast non-coding regions, psbM-trnD, trnD-trnT, Sapindaceae.

Maples (Acer L.) are one of the most impor- including more than 20 recently described taxa tant trees in the Northern Hemisphere, particu- (de Jong, 2002). Although species of Acer larly in the temperate regions of eastern Asia, occur mainly in temperate to subtropical areas, eastern North America, and Europe (van several species extend their distribution ranges Gelderen et al., 1994). During the fall season, to the tropics, such as A. laurinum Hassk. in the colorful foliage of maples paints the land- Thailand and Vietnam, and A. decandrum Merr. scape with red, yellow, and orange. Many in Hainan, China. species are also important sources of commercial Various authors have proposed classification products, for example, syrup from Acer sac- systems of Acer since the end of the 19th cen- charum Marshall and timber from A. saccha- tury (Table 1), including Pax (1885, 1886, rum, A. rubrum L., and A. pseudoplatanus L. 1902), Rehder (1905), Koidzumi (1911), Nakai Traditionally, Acer and Dipteronia Oliver (1915), Pojarkova (1933), Momotani (1962), have been placed in Aceraceae. Recent phylo- Fang (1966), Ogata (1967), Murray (1970), de genetic analyses, however, suggest that these Jong (1976, 1994, 2002), Delendick (1981), two genera be placed in Sapindaceae (Gadek et Mai (1984), and Xu (1996). al., 1996; Harrington et al., 2005). Dipteronia In recent years, four maple phylogenies have consists of two species, both of which are been published, greatly improving our under- endemic to China (Ying and Zhang, 1994). The standing of the evolutionary history of Acer most recent survey of Acer listed 156 species, (Huang et al., 2002). Hasebe et al.’s (1998) RFLP

We thank Kyle Port and Kathryn Richardson of the Arnold Arboretum of Harvard University, Howard Higson of the Quarryhill Botanical Garden, Sarah McNuall of Cornell Plantation, Peter Browns of the Royal Botanical Garden at Edinburgh, Xin Tian, Q. Fan of Zhongshan University, Aaron Liston of Oregon State University, and Tony Aiyello of the Morris Arboretum for providing material for this study. 1 Arnold Arboretum of Harvard University, Harvard University Herbaria, 22 Divinity Avenue, Cambridge, Massachusetts 02138, U.S.A. 2 Kunming Institute of Botany, Chinese Academy of Sciences, Heilongtan, Kunming, Yunnan, China. 3 Visiting Faculty of College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310029, China. 4 Address for correspondence. E-mail: [email protected]

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W t r i 9 r h r r r 5 3 u o a e u n o T s n 3 a s d n n e o u o y u i r l a a 0 u D m p m 0 s 6 i y t c A 6 e r i a a n s A N u u v d d i r d g a a h h 8 p u 8 a a r n a v 3 t h 8 r r o a d n r t n t O i i u r u s e c c m l t 3 e o e c r 3 & c o 3 s s u i & 0 t u u c a n n n f e i e n n b c c a r c a g t r C n a n . a i u u 3 . u k k y s l s e e e e S i t i p a a r o o u a y l r e r a o . d e k . r r u u w t t t s s s t p s p r s p s p p p p g r E o m u h r I 1 v n e e o m n n t l r r r r r r r r r r r r r r r r r r r r r x c i i t t C o e l o e M u n e s e e e e e e e e e e e e e e e e e e e e E a p p p E a i o i e c c c c c c c c c c c c c c c c c c c c c , L a P B H L O H K B X S D K D A A A A A A A A A A A A A A A A A A A A A S A A A M T 2006 LI ET AL., ACER PHYLOGENETICS 105 ) 2 0 0 a a a i l 2 a a a e t l ( p a h h a d n y r t t t i n e a a G h a a a n n o t a t a a l a c a i p t t t N c i a a a a t l a a a s n a a a a a r r r r r o O d r r r e a u o t n c c c c c r t m m J f b b b h e e e b i i i g n n l l i a t a a a c i c r c u u u i r l e u p a a p p E D M P A P P R R S P R L P A A G S S M A T W n n o o r r d d n n a a a e e a l a a a a e l p d d p r p h r h h a d y o o r t t t t i a a r r a h a a a n n n c o t l c a a i p c a a a a l a a a o a a a n o h h a r r r i r r r r r o r a r r o t n c c c c c t m n f b b b b b h c e e c c n n l i a t i a a a o i u u u c u u c i r a a e l a S R R R S P G P M R L G R M P M M T A A ...... t t t t t t t t t t t t t t t t t t t t c c c c c c c c c c c c c c c c c c c c e e e e e e e e e e e e e e e e e e e e a s s s s s s s s s s s s s s s s s s s s t r r r r r r r r r r r r r r r r r r r r u ) e e e e e e e e e e e e e e e e e e e e g 6 c c c c c c c c c c c c c c c c c c c r c 9 A A A A A A A A A A A A A A A A A A A A A 9 ...... 1 g g g g g g g g g g g g g g g g g g g g g ( b b b b b b b b b b b b b b b b b b b b b U u u u u u u u u u u u u u u u u u u u u u X S S S S S S S S S S S S S S S S S S S S S ) 6 7 9 a a a i 1 a a a a l e e ( a a a p h h h o a d d r r r r t t t t i i f a a G a o o o i a a n n n o o t t l l l l r i a N c f f f a a a a a l a a a n n a r g i i i r r r r r r o O r r r a a r o n b c c v c v v t t m m J f b b b h e e e e r n a l l r r i a t a a a a t c l i c c u u u i r l l a a a a a E n D M A P P I P R R R L P P G A G M A P T P M ) 7 a 6 a l a a a a a e 9 l n p h h h a d 1 y i r t t t t i ( r a a h a a n n n o t t a a a a a i p t t t c A a a a a a t l a a a n h a a a a r r r T r r r o r r a u o t c c c c c c c t m m A f b b b h e e i i i g n c l l i a a t a a G c r c u u u i r e l p a a a p p O M A P P — S P R R R P A L S — M A S S T M ) 2 6 9 1 ) ( ? a I ( a a a e N n p h a a d a i r t A t t i t r a a T a a a n o t t a a a a a a i i t t t c a O a a t l l a a a n b h a a a r r r r o r r a u o o o c M c c c c t m m f l f b b b h e e i i i g c l l i i i O a t a c r c u u u i r r r l p a a a p p M M A — P P P S T R R R A S L T — A S S T — a a a a v v v v a a a o o o o v v ) v k k k k o o r r r r 3 o k k a a a a 3 k r r j j j j r 9 a a o o o o a j j 1 . j P P P P ( o o T o a a a a a P P N A a P a a a a e p p p p O V a a n p r r h r r h h a d a t t i r t t t t i O t C a a a a r a a a a a n n n o t a c c c c K a . a i c a a a a t l a a a n b m o o m o o R h 1 r r r r r r o r r r r a u o o c A c c c t m l f b b b c c c c h m m E J g c l i i a i i a i i a t a e e r L u u u i r r O l a a B P M P P G R R R A S M M T L M M T M G M A T 106 HARVARD PAPERS IN BOTANY Vol. 11, No. 1

(restriction fragment length polymorphism) used AFLP and chloroplast sequence data to study sampled 64 species of Acer, representing test the origin of endemic species of Acer from 17 sections of Ogata (1967), and generated 53 Ullung Island off South Korea. Tian et al. phylogenetically informative sites. Their data (2002) included 2 species of Dipteronia and 39 support the monophyly of sects. Arguta, species of Acer in their combined analyses of Cissifolia, Lithocarpa, and Spicata, and further sequences of nrDNA ITS and chloroplast non- suggest that sections Distyla and Parviflora coding trnL-trnF regions. Their results suggest may be closely related. Nonetheless, sectional that Dipteronia may be paraphyletic with D. relationships are generally unresolved or deyerana Henry embedded in Acer and that weakly supported. In that study, Hasebe et al. some sections sensu Xu (1996) need reevalua- (1998) did not sample outgroups from outside tion. Nevertheless, sectional relationships are Acer, and instead chose sect. Spicata as a func- hardly resolved. tional outgroup on the basis of the fossil record. The objectives of this study were (1) to fur- This may not be a warranted assumption, given ther test the monophyly of Dipteronia, Acer, that the basal position of sect. Spicata has not and sections of Acer, and (2) to provide insights been tested. Sequences of nuclear ribosomal into sectional relationships of Acer. We chose (nr) DNA ITS (internal transcribed spacer) to use sequences of two non-coding regions of regions support the monophyly of sects. the chloroplast genome including psbM-trnD Arguta, Cissifolia, Palmata, Integrfolia, and trnD-trnT, since these markers are among Trifoliata, Ginnala, Macrantha, Lithocarpa, the most variable regions in the chloroplast and Platanoidea (Ackerly and Donoghue, genome (Kelchner and Clark, 1997; Shaw 1998; Suh et al., 2000). Pfosser et al. (2002) et al., 2005).

MATERIALS AND METHODS Plant Material 1 µl of each primer (10 µM), and an appropri- In this study, we sampled 2 species of ate amount of sterilized distilled water. The Dipteronia and 52 species of Acer, representing PCR program included 3 m hotstart at 94˚C and all sections of previous classification systems 35 cycles of 1 min denaturing at 94˚C, 1.5 min (Pax, 1902; Rehder, 1905; Koidzumi, 1911; annealing at 46–50˚C, and 2 min extension at Nakai, 1915; Pojarkova, 1933; Momotani, 72˚C. The cycles were followed by an addi- 1962; Fang, 1966; Ogata, 1967; Murray, 1970; tional 7-min extension at 72˚C. For most taxa de Jong, 1976, 1994, 2002; Delendick, 1981; we used PCR products as templates for Mai, 1984; Xu, 1996). For rooting purposes we sequencing. However, for several taxa direct also included Aesculus glabra Willd., Sapindus sequencing PCR products did not work well, drummondii Hook. & Arn., Koelreulteria pan- either because of the sequence variation within iculata Laxm., and Xanthoceras sembifolia an individual or because of the long stretch of Bunge as outgroup taxa (Table 1), which are As or Ts that may have caused a polymerase representatives of the sister clade of Acer and slippage. In such circumstances, we cloned the Dipteronia (Harrington et al., 2005). PCR products using a standard pGEM-T Tail Molecular Techniques Vector System (Promega, Madison). Two or DNAs were extracted from silica-gel dried more clones were sequenced to detect sequence leaves using a DNeasy Plant Mini Kit (Qiagen, polymorphisms within each accession. Clones CA). The chloroplast DNA region between and PCR products were sequenced using the trnC and trnD was amplified using primers Dideoxy Terminator Chemistry with an ABI designed by Lee and Wen (2004), and that BigDye Cycle Sequencing Ready Kit. between trnD-trnT was obtained using primers Sequences were analyzed using an ABI 3100 or of Shaw et al. (2005). Polymerase chain reac- 3730 Genetic Analyzer, and were edited using tions (PCR) were carried out using either an Sequencher (Version 4.1, GeneCode Inc., Ann MJ-PT200 Thermocycler or an Eppendorff Arbor, Mich.). MasterCycler. Each 25-µl PCR reaction con- Phylogenetic Analyses tained 50–100 ng of genomic DNA, 4 µl of Sequence alignment of both non-coding DNTPs (2.5mM), 3 µl of MgCl2, 2.5 µl of Taq regions were readily done manually across all polymerase buffer (10), 0.3 µl of Taq (5 U/µl), taxa, including the outgroup taxa. Phylogenetic 2006 LI ET AL., ACER PHYLOGENETICS 107 analyses were conducted using neighbor-join- weighted and their states were unordered. For ing (NJ), maximum parsimony (MP), and max- ML analyses, Modeltest (Version 3.06; Posada imum likelihood (ML) methods as implemented and Crandall, 1998) was used to select the best in PAUP* (Version 4.0b10; Swofford, 2002). evolutionary model, and then the estimated For MP analyses, a heuristic tree search was parameters were used in the tree reconstruction. used with the following options: maxtrees = Bootstrap analyses of 100 replicates were car- 20,000, TBR (tree bisection reconnection) ried out to estimate support for individual branch swapping, random sequence addition clades (Felsenstein, 1985), and tree search with 5000 replicates and 1 tree held in each options for bootstrap analyses were the same as replicate, and steepest descent off. Gaps were in parsimony analyses except for simple treated as missing data. Characters were equally sequence addition.

RESULTS Sequence Characteristics data sets of psbM-trnD and trnD-trnT did not PCR amplifications using primers psbM2 produce well-supported but conflicting clades and trnD produced a segment of 560–1200 base (tree not shown), the two data sets were com- pairs (bp), whereas primers trnD and trnT bined in phylogenetic analyses. The combined amplified a region of about 1400 bp. The data set had 3150 sites, 281 of which were par- lengths of psbM-trnD ranged from 560 to 616 simony informative. NJ analyses produced a bp in Acer and from 781 to 1167 bp in the out- cladogram (Fig. 1), and MP analyses generated group taxa, whereas those of trnD-trnT were 20,000 trees of 1018 steps; the strict consensus from 1255 to 1431 bp in Acer and outgroup is shown in Fig. 2 (CI = 0.83, RI = 0.79). taxa. The alignment across all taxa generated Modeltest indicated that the best fitting model 1424 and 1725 sites for the psbM-trnD and of evolution for the chloroplast regions was trnD-trnT, respectively. The alignment of TVM+G with estimated parameters as follows: psbM-trnD sequences across the outgroup taxa base frequencies (A = 0.3339, C = 0.1744, G = and Dipteronia and Acer required 18 indels 0.1601, T = 0.3316), rate matrix (A–C = ranging from 3 to 632 bp. Eight indels were 1.2302, A–G = 1.4401, A–T = 0.2821, C–G = parsimony informative. In the aligned data set 0.6580, G–T = 1.4401), and Gamma shape of trnD-trnT, there were 42 indels ranging from parameter = 0.6461. ML analyses with the 5 to 63 bp, and14 of these indels were parsi- selected model and estimated parameters pro- mony informative. A few indels were synapo- duced a single tree with likelihood of –ln = morphies and will be discussed in the context 10485.86 (Fig. 3). of tree structures. Both chloroplast regions Two species of Dipteronia formed a clade were AT rich, with an average of 65.6% and with moderate support (bs = 66%) in the NJ 64.8%, respectively. Sequence divergence of tree (Fig. 1), whereas in both MP and ML trees the psbM-trnD ranged from 5.9% to 10.2% these two species did not form a clade (Figs. between Acer and outgroup taxa, 1.4% to 3.8% 2–3). Species of Acer formed a clade in all trees between Dipteronia and Acer, and 0.2% to and this clade received little support in the NJ 3.1% within Acer. For the trnD-trnT region, tree, but in the MP tree it was moderately sup- sequences diverged from 4.6% to 10.6% ported (71% and 3 base substitutions). Within between Acer and outgroup species, 0.9% to Acer, there were 18 clades with moderate to 3.2% between Dipteronia and Acer, and 0.1% strong support (>70%) in the NJ and/or MP to 2.5% within Acer. trees , as well as in the ML tree (A–R). Acer Phylogenetic Analyses henryi of sect. Negundo formed a clade (Clade Clones of trnD-trnT in Acer palmatum A, bs = 80% in the NJ tree, bs = 75% and 3 base Thunberg ex Murray, A. henryi Pax, and A. dis- substitutions in the MP tree) with sect. Arguta tylium Sieb.& Zucc. had similar sequences; (B, 81%, 89% and 4). Acer distylium of sect. therefore, only one clone was used in the data Parviflora and A. pentapomicum Stewart & set for phylogenetic analyses. Because chloro- Brand. of sect. Pubescentia formed a clade (C, plast genes generally share a similar evolution- 92%, 90% and 4) with sect. Platanoidea repre- ary history and the trees based on individual sented by A. platanoides L. and A. campestre L. 108 HARVARD PAPERS IN BOTANY Vol. 11, No. 1

FIGURE 1. Neighbor-joining cladogram based on sequences of chloroplast non-coding regions of psbM-trnD and trnD-trnT. Numbers at branches are bootstrap percentages of 1000 replicates. Letters A–R indicate clades discussed in text. Section designations on the right follow de Jong (2002). 2006 LI ET AL., ACER PHYLOGENETICS 109

FIGURE 2. Strict consensus of 20,000 parsimonious trees of 1018 steps based on sequences of chloroplast non- coding regions of psbM-trnD and trnD-trnT (CI = 0.83 and RI = 0.79). Numbers above and below branches are bootstrap percentages and numbers of base substitutions. Letters A–R indicate clades discussed in text. Section designations on the right follow de Jong (2002). 110 HARVARD PAPERS IN BOTANY Vol. 11, No. 1

FIGURE 3. Maximum likelihood tree with a likelihood score of –ln = 10485.86 based on sequences of chloroplast non-coding regions of psbM-trnD and trnD-trnT. Letters A–R indicate clades discussed in text. Section designations on the right follow de Jong (2002). 2006 LI ET AL., ACER PHYLOGENETICS 111

(D, 100%, 100% and 4). Section Macrantha Diels formed a clade (L, Figs. 1–3) with weak formed a clade (E, 88%, 88% and 4), and so did (64%, Fig. 1) or moderate (79%, Fig. 2) sup- sect. Palmata (F, 94%, 100% and 7). Acer cau- port. The monotypic sect. Indivisa clustered datum Wall. clustered with sect. Palmata (G, with A. spicatum Lamarck (M, 67%, 80% and 54%, 98% and 3), and together they were 2). Section Lithocarpa was well supported as a weakly allied with A. ukurunduense (54%, 57% clade (N, 100%, 100% and 7). Section Rubra and 1). Section Ginnala was well supported as formed a clade (O, 100%, 100% and 12), which a clade (H, 100%, 100% and 15). Four sampled also contained A. garrettii Craib. of sect. species of sect. Pentaphylla formed a clade in Hyptiocarpa. Whereas A. saccharinum L. and the ML tree (Fig. 3), but in the NJ and MP trees A. rubrum of sect. Rubra formed a group (P, (Figs.1–2), A. paxii Franchet did not cluster 99%, 100% and 5), A. pycnanthum, another with the other three species of the clade (I, species of sect. Rubra, clustered with A. garret- 90%, 92% and 3). Section Trifoliata formed a tii in the NJ tree (Q, 95%). However, this clus- clade (J, 100%, 100% and 7) and so did sect. ter was absent in both MP and ML trees (Figs. Acer (K, 97%, 99% and 7), excluding A. 2–3). Acer pseudoplatanus of sect. Acer and A. pseudoplatanus and A. caesium Wall. ex Brand. glabrum Torr. of sect. Glabra formed a clade Clades I–K, A. paxii, and A. pentaphyllum (R, 100%, 100% and 14).

DISCUSSION Monophyly of Acer with strong support (B, bs = 81%–84%, Figs. Dipteronia differs distinctively from Acer in 1–2). This is consistent with previous phyloge- its unique combination of pinnately compound netic studies (Hasebe et al., 1998; Tian et al., leaves and circular fruit wings. In the nrDNA 2002). tree, D. dyeriana is sister to the monophyletic Pax (1902) put Acer henryi in sect. Trifoliata Acer. However, in the trnL-trnF phylogeny, on the basis of leaf morphology. Later, this species is embedded within Acer, indicat- Koidzumi (1911) separated this species from ing that Acer is paraphyletic (Tian et al., 2002). Trifoliata and recognized it as a new sect. Dipteronia species do not form a clade in either Cissifolia. This taxonomic treatment has gener- the MP or ML tree (Figs. 2–3), whereas in the ally been accepted (Pojarkova, 1933; NJ tree the two species cluster together with Momotani, 1962; Ogata, 1967). Cissifolia may weak support (bs = 66%). In all trees (Figs. be closely related to A. negundo because they 1–3), Acer species form a clade, and in the MP share similar morphology in buds and leaves tree this clade has moderate support (bs = 71% (de Jong, 1976, 2002; Xu, 1996). However, in and 3 base substitutions). Thus, our results sup- Figs. 1–3, A. henryi forms a moderately support the monophyly of Acer, which is consis- ported clade (A, bs = 75%–80%) with species tent with the unique fruit morphology of Acer, of sect. Arguta (B) and is remotely related to A. that is, samaras, each with 2 one-seeded meri- negundo L. Morphologically, A. negundo dif- carps. Nevertheless, more data are needed to fers from sect. Arguta in having apetalous strengthen the support for the monophyly of flowers (vs. petalous flowers in sect. Arguta). Dipteronia and Acer. Nevertheless, the systematic position of A. Sections of Acer negundo remains unresolved. The genus Acer has been divided into sec- Section Platanoidea, with 13 species, is tions and series on the basis of morphological, unique in having a milky sap in the leaf petiole. anatomical, and chemical characters by various Pax (1902) placed Acer campestre and A. mon- authors (Pax, 1902; Rehder, 1905; Koidzumi, spessulanum L. in sect. Campestria and sepa- 1911; Nakai, 1915; Pojarkova, 1933; rated them from sect. Platanoidea. However, Momotani, 1962; Fang, 1966; Ogata, 1967; this treatment has not gained wide support. Murray, 1970; de Jong, 1976, 1994, 2002; Instead, A. campestre has been put in sect. Delendick, 1981; Xu, 1996). Platanoidea (Pojarkova, 1933; Momotani, There are 4 species in sect. Arguta (de Jong, 1962; Ogata, 1967; de Jong, 1976, 2002; Xu, 2002), which is defined by dioecy and raceme 1996). The placement is supported by recent inflorescence with 4-merous flowers. Three molecular evidence (Hasebe et al., 1998; Tian, sampled species of sect. Arguta form a clade 2002). Our results provide further evidence for 112 HARVARD PAPERS IN BOTANY Vol. 11, No. 1 the close relationship of A. campestre and A. Palmata share three indels, two (9 and 17 bp) platanoides (Fig. 1). Acer pentapomicum also in the psbM-trnD region and one (6 bp repeat) has the milky sap in the leaf petiole and has in the trnD-trnT. been recognized as a series in sect. Platanoidea Pojarkova (1933) placed Acer spicatum in (de Jong, 1976), as a member of sect. her sect. Microcarpa with A. erianthum Goniocarpa (Xu, 1996), or as constituting a Schwerin and A. sinense Pax. In Figs. 1–2, A. separate section (de Jong, 2002). Here, it forms erianthum and A. sinense are closely allied a clade with sect. Platanoidea and A. distylium within sect. Palmata, whereas A. spicatum (D, Figs. 1–3), supporting the inclusion of A. forms a moderately supported clade with A. pentapomicum in Platanoidea (de Jong, 1976). carpinifolium Sieb.& Zucc. (M). Therefore, our Acer distylium is unique in linden-like leaves data do not support sect. Microcarpa. Acer cau- and has been placed in sects. Indivisa (Pax, datum has been treated as a species of sect. 1902; Koidzumi, 1911) or Spicata (Momotani, Spicata (Pax, 1902; de Jong, 2002), 1962). It has also been treated as its own sect. Microcarpa (Pojarkova, 1933), and Parviflora Distyla (Ogata, 1967; Xu, 1996). De Jong (de Jong, 1976). Acer caudatum and A. spica- (1976), however, recognized the close relation- tum have been considered as a species pair ship of A. distylium and A. nipponicum Hara between eastern Asia and North America (de and considered both species as belonging in Jong, 1976, 2002), and A. ukurunduense as a sect. Parviflora. This treatment is weakly sup- subspecies or variety of A. caudatum (de Jong, ported by two RFLP markers (Hasebe et al., 1994). However, in Figs. 1–2, A. caudatum is 1998) and nrDNA ITS sequence data (Suh et sister to sect. Palmata, and together they are al., 2000; Tian et al., 2002). Sequences of trnL- weakly allied with A. ukurunduense (bs = trnF, however, did not support this relationship 51%–54%). Acer spicatum, however, clusters (Tian et al., 2002). Our chloroplast sequence with A. carpinifolium. Therefore, Acer cauda- data place A. distylium in a clade with sect. tum and A. spicatum may not be as closely Platanoidea with strong support (D, bs = 90%, related as was previously thought (de Jong, Fig. 1), whereas the relationship of A. nippon- 2002), and A. ukurunduense is probably a dis- icum is not resolved. Here, we do not attempt to tinct species from A. caudatum. explain the contrasting hypotheses of relation- Acer carpinifolium is a unique species with ships of A. distylium and A. nipponicum, since simple and serrate leaves. Pax (1902) placed it more accessions are needed to account for together with other simple-leaved species (e.g., potential sequence polymorphisms within A. crataegifolium Sieb.& Zucc., A. davidii species, and we need more data from additional Franchet, A. distylium, and A. stachyophyllum chloroplast and nuclear markers to generate a Hiern). However, it has generally been robust phylogeny. accepted as the sole species of sect. Indivisa Section Macrantha consists of 21 species (Koidzumi, 1911; Ogata, 1967; Pojarkova, distributed in eastern Asia and North America. 1933; van Gelderen et al., 1994), or subgenus Six sampled species form a clade (E, bs = 88%, Carpinifolia (Momotani, 1962; Xu, 1996). Our Figs. 1–2). This clade is characterized by results indicate that A. carpinifolium may be raceme inflorescences, horizontally spreading closely related to A. spicatum. This relationship fruit wings, and buds with stalks (de Jong, has not been proposed before and needs rigor- 1976). Previous phylogenetic analyses also rec- ous test from additional data. ognized this section (Hasebe et al., 1998; Pax (1902) placed all species with undivided Ackerly and Donoghue, 1998; Suh et al., 2000; simple leaves in sect. Indivisa. Pojarkova Tian et al., 2002; Pfosser et al., 2002). (1933) revised sect. Indivisa by transferring A. Section Palmata is the largest section, with crataegifolium to sect. Macrantha and A. 41 species, and is characterized by a few poten- stachyophyllum to sect. Arguta. Nevertheless, tial synapomorphies including 4-pair bud she considered the rest of the species as belong- scales and abortive terminal buds. Our data ing to sect. Integrifolia (e.g., A. fabri Hance, A. support the monophyly of the section (F, bs > garrettii, A. cinnamomifolium Hayata, and A. 98%) as did previous molecular studies lucidum Metcalf). De Jong (1976) moved A. (Hasebe et al., 1998; Suh et al., 2000; Tian et fabri from sect. Integrifolia to sect. Palmata, al., 2002; Pfosser et al., 2002; Ackerly and whereas Fang (1966) established sect. Donoghue, 1998). In addition, species of sect. Hyptiocarpa for A. garrettii. Molecular data 2006 LI ET AL., ACER PHYLOGENETICS 113 support the separation of A. fabri and A. garret- Jong, 2002). Our results, therefore, reject sects. tii from Integrifolia (Hasebe et al., 1998; Suh et Saccharodendron of Xu (1996), Goniocarpa al., 2000; this study). However, results from (Pojarkova, 1933), and Acer (de Jong, 2002). Tian et al. (2002) suggest that A. fabri belongs Sections Integrifolia, Trifoliata, and Acer in sect. Integrifolia, which might have resulted (excluding A. caesium and A. pseudoplatanus) from species misidentification. Acer lucidum form a moderately supported clade (L, bs = has been placed in sect. Palmata (de Jong, 64%–79%, Figs. 1–2). These sections also 2002). But, our data recognize it as a species of share an indel (14 bp). sect. Integrifolia (Figs. 1-3). All molecular evi- The phylogenetic position of Acer caesium is dence supports the close relationship of sect. unclear, whereas A. pseudoplatanus forms a Trifoliata and Integrifolia (Hasebe et al., 1998; clade with A. glabrum (R, bs = 100%). Acer Suh et al., 2000; Tian et al., 2002; this study). glabrum is unique in having variable leaves One potential synapomorphy for this clade is from 3- to 5-lobed to partly trifoliate. It has the pointed bud with multiple pairs of imbricate been treated as its own sect. Glabra (Pax, 1902; scales. Pojarkova, 1933; Momotani, 1962), or as a sec- Acer pentaphyllum is characterized by its tion also containing ser. Arguta. Sequences of compound leaf with five leaflets and has been nrDNA ITS suggest a close but weakly sup- placed in sect. Trifoliata (Momotani, 1962) or ported affinity of A. glabrum with A. ginnala sect. Integrifolia (de Jong, 1976). Ogata (Suh et al., 2000). The alliance of A. glabrum (1967), however, recognized it as its own sect. with A. pseudoplatanus (Figs. 1–2) is new and Pentaphylla. Our results support the close rela- thus requires further tests with more data. tionship of A. pentaphyllum with sects. Nevertheless, our data do not support the close Trifoliata and Integrifolia (Suh et al., 2000; relationship of A. glabrum with sect. Arguta or Tian et al., 2002). Nevertheless, the support for Macrantha, as suggested by de Jong (1976). the relationship is weak. Section Hyptiocarpa consists of two species, Pojarkova (1933) established sect. A. garrettii and A. laurinum. However, some Goniocarpa for Acer monspessulanum and A. authors have treated them as a single species opalus P. Miller, and sect. Gemmata for A. cae- (van Gelderen et al., 1994). On the basis of sium, A. pseudoplatanus, and A. trautvetteri chemical and morphological characters, Medvedev. However, Momotani (1962) placed Delendick (1981) and de Jong (1976) consid- A. monspessulanum in sect. Platanoidea. This ered Hyptiocarpa as closely related to sect. treatment has not received wide support Rubra, which has three species (two in eastern (Ogata, 1967; de Jong, 1976; Xu, 1996). North America and one in Japan). The sugges- Instead, A. caesium, A. monspessulanum, A. tion gets support from sequence data of nrDNA pseudoplatanus, and A. trautvetteri have been and trnL-trnF (Tian et al., 2002). Acer rubrum placed in sect. Acer (de Jong, 1976). In Fig. 1, of eastern North America and A. pycnanthum A. monspessulanum, A. opalus, A. trautvetteri, K. Koch of Japan have been considered as a and A. saccharum form a well-supported clade sister species pair (de Jong, 1976; Barnes et al., (K, bs = 97%), excluding A. caesium and A. 2004). This implies that these two species are pseudoplatanus. Section Acer, therefore, is not more closely related to each other than either is monophyletic. Acer saccharum is a species to A. saccharinum, the other eastern North complex with five subspecies in North America American species of sect. Rubra. However, our (de Jong, 2002). Pax (1902) erected sect. data indicate that the two North American Saccharina for A. saccharum and its varieties. species, A. saccharinum and A. rubrum, are This treatment has been followed (Pojarkova, phylogenetically closer to each other than 1933; Momotani, 1962; Ogata, 1967). either is to the Japanese species (Figs. 1–3). However, de Jong (1976) put A. saccharum in Section Ginnala has one species with four sect. Acer, whereas Xu (1996) recognized sect. subspecies (de Jong, 2002). Previous molecular Saccharodendendron consisting of A. saccha- studies have suggested that it may be closely rum and A. saccharinum. In Fig. 1, A. saccha- related to Rubra (Hasebe et al., 1998) or rum is embedded in a well-supported clade Glabra (Suh et al., 2000). Nevertheless, the containing sect. Goniocarpa, and A. monspes- support for the relationships is weak. Our data sulanum and A. trautvetteri of sect. Acer (de do not resolve the relationship of sect. Ginnala 114 HARVARD PAPERS IN BOTANY Vol. 11, No. 1 either (Figs. 1–2). Therefore, more data are monophyletic. Section Cissifolia is more needed. closely related to sect. Arguta than to A. Section Lithocarpa consists of eight Asian negundo. Acer caudatum is sister to sect. species (de Jong, 2002) and has been consid- Palmata, and A. saccharum clusters with sect. ered to be closely related to sect. Macrophylla, Acer (excluding A. caesium and A. pseudopla- which has a single species from western North tanus). Acer carpinifolium is closely related to America. Two sampled species of sect. A. spicatum, whereas A. pseudoplatanus is Lithocarpa form a clade (N, bs = 100%), sup- allied with A. glabrum. Section Hyptiocarpa is porting the monophyly of the section. closely related to sect. Rubra, and A. sacchar- However, the relationships of sect. inum and A. rubrum are more closely related to Macrophylla remain unresolved (Figs. 1–2). each other than either is to A. pycnanthum. In summary, our data from two non-coding Nevertheless, intersectional relationships are chloroplast regions support the monophyly of generally weakly supported. More data are the genus Acer and sects. Arguta, Integrfolia, needed to obtain a fully resolved phylogeny of Trifoliata, Platanoidea, Macrantha, Acer, which will then provide a backdrop for Lithocarpa, Palmata, Rubra, and Ginnala. In better understanding of evolutionary and bio- contrast, sects. Goniocarpa, Spicata, Acer, geographic history of the genus. Parviflora, and Saccharodendron are not

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