Phylogenetic and biogeographic diversification of Rhus (Anacardiaceae) in the Northern Hemisphere

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 33 (2004) 861–879 www.elsevier.com/locate/ympev

Phylogenetic and biogeographic diversification of Rhus (Anacardiaceae) in the Northern Hemisphere Tingshuang Yia, Allison J. Millerb, Jun Wena,c,* a

Department of Botany, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, IL 60605-2496, USA Department of Biology, Washington University, Campus Box 1137, One Brookings Drive, St. Louis, MI 63130, USA Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Nanxinchun 20, Xiangshan, Beijing 100093, PR China b

c

Received 27 April 2004; revised 12 July 2004 Available online 11 September 2004

Abstract Sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA, the chloroplast ndhF gene, and chloroplast trnL-F regions (trnL intron, and trnL [UAA] 30 exon-trnF [GAA] intergenic spacer) were used for phylogenetic analyses of Rhus, a genus disjunctly distributed in Asia, Europe, Hawaii, North America, and Northern Central America. Both ITS and cpDNA data sets support the monophyly of Rhus. The monophyly of subgenus Rhus was suggested by the combined cpDNA and ITS data, and largely supported in the cpDNA data except that Rhus microphylla of subgenus Lobadium was nested within it. The monophyly of subgenus Lobadium was strongly supported in the ITS data, whereas the cpDNA data revealed two main clades within the subgenus, which formed a trichotomy with the clade of subgenus Rhus plus R. microphylla. The ITS and cpDNA trees differ in the positions of Rhus michauxii, R. microphylla, and Rhus rubifolia, and hybridization may have caused this discordance. Fossil evidence indicates that Rhus dates back to the early Eocene. The penalized likelihood method was used to estimate divergence times, with fossils of Rhus subgenus Lobadium, Pistacia and Toxicodendron used for age constraints. Rhus diverged from its closest relative at 49.1 ± 2.1 million years ago (Ma), the split of subgenus Lobadium and subgenus Rhus was at 38.1 ± 3.0 Ma. Rhus most likely migrated from North America into Asia via the Bering Land Bridge during the Late Eocene (33.8 ± 3.1 Ma). Rhus coriaria from southern Europe and western Asia diverged from its relatives in eastern Asia at 24.4 ± 3.2 Ma. The Hawaiian Rhus sandwicensis diverged from the Asian Rhus chinensis at 13.5 ± 3.0 Ma. Subgenus Lobadium was inferred to be of North American origin. Taxa of subgenus Lobadium then migrated southward to Central America. Furthermore, we herein make the following three nomenclatural combinations: (1) Searsia leptodictya (Diels) T. S. Yi, A. J. Miller and J. Wen, comb. nov., (2) Searsia pyroides (A. Rich.) T. S. Yi, A. J. Miller and J. Wen, comb. nov., and (3) Searsia undulata (Jacq.) T. S. Yi, A. J. Miller and J. Wen, because our analyses support the segregation of Searsia from Rhus.
2004 Elsevier Inc. All rights reserved. Keywords: Rhus; Anacardiaceae; Biogeography; Phylogeny; Northern hemisphere; Hawaii; Disjunction; Penalized likelihood

1. Introduction The sumac genus Rhus [Tourn.] L., emend Moench (Anacardiaceae), together with seven closely related genera: Actinocheita F. A. Barkley, Cotinus Mill., Malosma *

Corresponding author. Fax: +1312 665 7158. E-mail address: wen@fieldmuseum.org (J. Wen).

1055-7903/$ - see front matter
2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.07.006

Nutt. ex Abrams, Melanococca Blume, Metopium P. Browne, Searsia F. A. Barkley, and Toxicodendron Mill. form a heterogeneous aggregation commonly referred to as the Rhus complex (Barkley, 1937, 1963; Miller et al., 2001). The generic and infrageneric delimitation of the Rhus complex has been controversial for more than two centuries (Barkley, 1937, 1963; Brizicky, 1963; De Candolle, 1825; Engler, 1896; Gillis, 1971; Heimsch,

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1940; Linneaus, 1753; Miller et al., 2001; Tournefort, 1700; Young, 1975, 1978, 1979). The historical circumscription of Rhus (Rhus sensu lato) included the allied genera Actinocheita, Cotinus, Malosma, Melanococca, Metopium, Searsia, and Toxicodendron. Barkley (1937, 1963) removed the allied genera and defined Rhus (Rhus sensu stricto) based on the presence of red drupes with red glandular hairs on the fruit wall. Data from morphology, wood anatomy, and chemistry (Young, 1975, 1979), and nuclear ribosomal ITS sequences (Miller et al., 2001) support the monophyly of Rhus s. str. Rhus s. str. comprises approximately 35 species which fall into two subgenera, subgenus Rhus and subgenus Lobadium (Table 1). The two subgenera of Rhus were recognized first by Barkley (1937), and then by Young (1978). Rhus subgenus Rhus [= subgenus Sumac (DC.) Schneider (Barkley, 1937; Brizicky, 1963) is characterized by its deciduous, imparipinnately compound leaves. The flowers, which appear after the leaves, are arranged in terminal thyrses, and subtended by a linear-lanceolate deciduous bract (Barkley, 1937; Young, 1975). Subgenus Rhus includes about 10 species, with four in eastern Asia, four in North America, one in Europe, and one in Hawaii. Rhus subgenus Lobadium (Raf.) Torr. [= subgenus Schmaltzia Desv. (Barkley, 1937; Brizicky, 1963) has simple, trifoliolate, or pinnately compound leaves, with flowers appearing with or before leaves. Its flowers are mostly sessile, forming compound spikes, with a deltoid or ovate persistent bract and two bracteoles subtending each flower (Barkley, 1937; Young, 1975, 1978). Subgenus Lobadium consists of approximately 25 species primarily distributed in the southwestern United States, Mexico, and northern Central America. Although heart wood flavonoid data supported the treatment of the two subgenera (Young, 1979), recent ITS sequences data suggested the paraphyly of subgenus Rhus, with the monophyletic subgenus Lobadium nested within it (Miller et al., 2001). The relationships among species of subgenus Rhus have never been hypothesized, and the classification of this subgenus is not available at present. Based on distribution, habit, and morphology of leaves and inflorescences, Barkley (1937) divided subgenus Lobadium into five sections: Lobadium, Pseudoschmaltzia, Pseudosumac, Rhoeidium and Styphoniae. Building on BarkleyÕs work, Young (1978) added data derived from flavonoid chemistry and wood anatomical studies to test BarkleyÕs classification and updated the nomenclature. Young divided subgenus Lobadium into sect. Lobadium, sect. Styphoniae, and sect. Terebinthifolia (= sect. Pseudosumac). Section Styphoniae was then subdivided into subsections Compositae, Intermediae and Styphoniae (Table 1). YoungÕs sect. Terebinthifolia is identical to BarkleyÕs (1937) sect. Pseudosumac in circumscription, but the section was published as new because sect. Pseudosumac

was not validly published by Barkley. YoungÕs (1978) sect. Lobadium combined BarkleyÕs (1937) sects. Lobadium and Rhoeidium, and he also merged BarkleyÕs sects. Styphoniae and Pseudoschmaltzia. Many recent biogeographic studies have largely focused on the disjunct relationships between plants in eastern Asia and eastern North America (Donoghue et al., 2001; Wen, 1999). Further understanding of the biogeographic history of the intercontinental disjuncts between Asia and North America require the examination of the pattern in the broader context of the Northern Hemisphere. Important questions remain to be addressed concerning the migration routes, direction of migration, patterns of morphological differentiation (i.e., test of the morphological stasis hypothesis, see Wen, 1999, 2001). Rhus provides an ideal model for studying the evolution of intercontinental disjunctions in the Northern Hemisphere and the oceanic island flora of Hawaii. The 35 species of Rhus s. str. are disjunctly distributed in the Northern Hemisphere (Fig. 1), with one species in western Asia and southern Europe, one in Hawaii, four in eastern to southern Asia, and the remaining 29 species in North America and northern Central America (Miller et al., 2001). The fossil record indicates that Rhus was widely distributed in eastern Asia, Europe, and North America during the Eocene to the Miocene (Fig. 1). Furthermore, fossils of close relatives of Rhus including Pistacia and Toxicodendron are available to help calibrate the ages for Rhus diversification. Our goals in this study were to (1) construct the phylogeny of Rhus using both chloroplast (trnL-F and ndhF) and nuclear (ITS) sequences; and (2) elucidate the history of biogeographic diversification in Rhus in the North Temperate zone. Specifically, we employ phylogenetic data and the fossil record to address the evolution of subgenus Rhus in the Northern Hemisphere, the colonization of R. sandwicensis on the Hawaiian Islands, and the evolution of subgenus Lobadium in North America.

2. Materials and methods 2.1. Species examined All 10 species of Rhus subgenus Rhus recognized by Barkley (1937) and Young (1975, 1978, 1979) were included in this study (Table 1). Twelve (of 25) species of subgenus Lobadium were sampled, including three from sect. Lobadium, eight from sect. Styphoniae, and one from sect. Terebinthifolia (Table 1; sensu the classification of Young, 1978). This sampling scheme represents the taxonomic and biogeographic diversity of the genus. Specifically, we sampled both subgenera throughout their distributional range and included

Table 1 Accessions of Rhus and outgroup taxa Taxon

Voucher

Locality

Distribution

Wen 6389 (F) Wen 7310 (F) Wen 6526 (F) Wen 7134 (F) Wen 7165 (F) Wen 7150 (F) Wen 7171 (F) Wen 7277 (F) Hardin 13984 (F) Wen 7138 (F) Wen 7137 (F) Wen 7052 (F) Wen 7082 (F)

China, Yunnan Morton Arb., IL (cult.) China, Yunnan USA, Illinois USA, Alabama Oak Park, IL (cult.) USA, Alabama USA, Texas USA, North Carolina Morton Arb., IL (cult.) Morton Arb., IL (cult.) Hawaii: Hawaii USA, Wisconsin

E Asia to SE Asia

Rhus subgenus Lobadium Sect. Lobadium (Raf.) DC. R. aromatica Ait. #1 R. aromatica Ait. #2 R. microphylla Engelm. ex A. Gray #1 R. microphylla Engelm. ex A. Gray #2 R. trilobata Nutt. ex Torr. and Gray

Wen 7086 (F) Steinmann et al. 3697 (F) Wen 7288 (F) Steinmann et al. 3761 (F) Miller 21 (CS)

USA, Illinois Mexico, Quere´taro USA, Texas Mexico, Nuevo Leo´n USA, Colorado

E North America

Miller 27 (CS)

USA, Arizona

Miller 28 (CS) Ickert-Bond 1298 (F) Miller 6 (CS) Miller 22 (CS) Steinmann et al. 3724 (F) Steinmann et al. 3696 (F) Wen 7282 (F) Miller 6/22/97 (CS) Steinmann et al. 3719 (F)

Rancho Santa Ana Bot Gard, CA (cult.) USA, Arizona (cult.) USA, Arizona Phoenix Desert Bot Gard, (cult.) Mexico, Nuevo Leo´n Mexico, Quere´taro USA, Texas USA, Arizona Mexico, Tamaulipas

S Arizona and New Mexico to Sonora of Mexico S California to N Lower California S Arizona C Arizona to S California

Steinmann and Carranza 3146 (F)

Mexico, Michoaca´n

Sect. Styphonia Young R. choriophylla Woot. and Standl.

R. integrifolia (Nutt. ex Torr. and Gray) Benth. and Hook f. ex Rothr. R. kearneyi Barkl. R. ovata Wats. #1 R. ovata Wats. #2 R. pachyrrhachis Hemsl. R. schiedeana Schlecht. R. virens Lindh. ex Gray #1 R. virens Lindh. ex Gray #2 R. virens Lindh. ex Gray #3 Sect. Terebinthifolia Young R. rubifolia Turcz.

E Asia E North America W Asia to S Europe North America E North America E North America E Asia E Asia Hawaii E North America

SW America to N Mexico North America

NE Mexico S Mexico to Guatemala SW America to N Mexico

S Mexico

ITS

trnL-F

ndhF

AY641480 AY641481 AY641482 AY641483 AY641484 AY641485 AY641486 AY641487 AY641488 AY641489 AY641490 AY641491 AY641492

AY640435

AY643095

AY640436 AY640437 AY640438 AY640439 AY640440 AY640441 AY640442 AY640443 AY640444 AY640445 AY640446

AY633892 AY643097 AY643098 AY643099 AY643100 AY643101 AY643102 AY643103 AY643104 AY643105 AY643106

AY641493 AY641494 AY641495 AY641496 AY641497

AY640447

AY643107

AY640448

AY643108

AY640449

AY643109

AY641498

AY640450

AY643110

AY641499

AY640451

AY643111

AY641500 AY641501 AY641502 AY641503 AY641504 AY641505 AY641506 AY641507

AY640452 AY640453 AY640454 AY640455 AY640456 AY640457 AY640458

AY643112 AY643113 AY643114 AY643115 AY643116 AY643117 AY643118

AY641508

AY640459 AY643119 (continued on next page)

T. Yi et al. / Molecular Phylogenetics and Evolution 33 (2004) 861–879

Rhus subgenus Rhus R. chinensis Mill. #1 R. chinensis Mill. #2 R. chinensis Mill. var. roxburghii Steud. R. copallina L. #1 R. copallina L. #2 R. coriaria L. R. glabra L. R. lanceolata Gray ex Engler R. michauxii Sargent R. potaninii Maxim. R. punjabensis J. L. Stew. ex Brand R. sandwicensis A. Gray R. typhina Torner

GenBank Accession

863

864

Table 1 (continued) Taxon

P. texana Swingle P. vera L. Schinus molle L. Searsia ciliata (Licht. ex Schultes) A. J. Miller S. lancea (L. f.) F. A. Barkl. S. leptodictya (Diels) T. S. Yi, A. J. Miller and J. Wen S. pyroides (A. Rich) T. S. Yi, A. J. Miller and J. Wen S. quartiniana (A. Rich.) A. J. Miller #1 S. quartiniana (A. Rich.) A. J. Miller #2 S. undulata (A. Rich) T. S. Yi, A. J. Miller and J. Wen T. diversilobum (Torr. and A. Gray) Greene T. radicans (L.) Kuntze T. vernix (L.) Kuntze

Panero s.n. 44 (CS) Miller 34 (CS)

Locality

Distribution

GenBank Accession ITS

trnL-F

ndhF

S Mexico Rancho Santa Ana Bot Gard, USA (cult.) USA, Texas Israel, cultivated USA, Los Angeles, CA (cult.) USA, Arizona (cult.)

S Mexico S California and N Lower California S Texas and NE Mexico Mediterranean to C Asia California and Texas Africa

AY641509 AY641510

AY640460 AY640461

AY643120 AY643121

AY641511 AY677201 AY641512 AY641513

AY640462 AY677209 AY640463 AY640464

AY643122 AY677204 AY643123 AY643124

AZ (cult.) AZ

Africa Africa

AY641514 AY641515

AY640465 AY640466

AY643125 AY643126

AZ

Africa

AY641516

AY640467

AY643127

AZ

Africa

AY641517

AY640468

AY643128

AY541518

AY640469

AY643129

Africa

AY541519

AY640470

AY643130

Wen 6693 (F)

Phoenix Desert Bot Gard, Phoenix Desert Bot Gard, (acc. # 198007201) Phoenix Desert Bot Gard, (acc. # 197903786101) Phoenix Desert Bot Gard, (acc. # 1980007001) Phoenix Desert Bot Gard, (acc. # 1980007001) Phoenix Desert Bot Gard, (acc. # 19800071) USA, California

W North America

AY677202

AY677208

AY677205

Wen 6236 (F) Wen 7146 (F)

USA, Illinois Morton Arb., IL (cult.)

North America E North America

AY677203 AY541520

AY677207 AY640471

AY677206 AY643131

Wen 7285 (F) Golan 1.539 (F) Wen 6686 (F) Miller 47 (CS) Miller 50 (CS) Miller s.n. (CS) Miller s.n. (CS) Miller 51 (CS) Miller s.n. (CS) Miller s.n. (CS)

AZ AZ

Barkley (1942, 1963) segregated Searsia from Rhus, and our analyses support the segregation. We herein make the following three nomenclatural combinations: (1) Searsia leptodictya (Diels) T. S. Yi, A. J. Miller and J. Wen, comb. nov.; basionym: Rhus leptodictya Diels, in Engler, Bot. Jahrb. Syst. 40: 86. 1907; (2) S. pyroides (A. Rich.) T. S. Yi, A. J. Miller and J. Wen, comb. nov.; basionym: R. pyroides A. Rich., Tent. Fl. Abyss. 1: 145. 1847; and (3) S. undulata (Jacq.) T. S. Yi, A. J. Miller and J. Wen, comb. nov.; basionym: R. undulata Jacq., Pl. Hort. Schoenbr. 3: 52. t. 346. 1798.

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Outgroups A. filicina (D. C.) Barkl. M. laurina (Nutt.) Nutt. ex Engl.

Voucher

T. Yi et al. / Molecular Phylogenetics and Evolution 33 (2004) 861–879

865

Fig. 1. Modern and fossil distribution of Rhus (Anacardiaceae). Light gray shading indicates the modern distribution; and the arrow shows the distribution of R. sandwicensis in Hawaii. I–IV represent four main distributing areas of Rhus, number species total in the area/number species endemic to the area in the parentheses are the numbers of extant species. 1–6 represent fossil records of different ages: 1, early Eocene; 2, middle Eocene; 3, late Eocene; 4, Oligocene; 5, Miocene; and 6, Pliocene.

representatives of all five sections of Barkley (1937) and all three sections of Young (1978). Because the sister group of Rhus has not been identified (Pell, 2004), outgroup sampling consisted six genera. Accessions of Actinocheita, Malosma, Searsia, and Toxicodendron were included as representatives of the Rhus complex. Additionally, Pistacia and Schinus were included as more distant outgroups. Although they were not considered part of BarkleyÕs Rhus complex (Barkley, 1937, 1963), these two genera are part of the Rhus tribe, Rhoeae, within the Anacardiaceae (Engler, 1896). Further, recent molecular analyses highlight the close relationship of Pistacia and Schinus with Rhus and allied genera (Miller et al., 2001; Pell, 2004). 2.2. DNA extraction, PCR amplification and sequencing Total DNA was extracted from silica-gel dried or fresh leaf material following the cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987). Amplification of selected DNA regions were performed in 20 lL reactions with approximately 10–50 ng of total DNA, 20 mM Tris buffer (pH 8.3, with 50 mM KCL, 1.5 mM MgCl2, and 0.1% Tween 20), 0.15 mM of each dNTP, 5 lM of each primer, 2 lL of Taq polymerase. The ITS region was amplified using primers ITS4 and ITS5 (White et al., 1990). The ndhF gene and trnL-F regions were amplified following Olmstead and Sweere (1994) and Taberlet et al. (1991), respectively. The PCR products were electrophoresed using 1.0% low-melting-point NuSieve GTG agarose gels (FMC BioProducts, Rockland, Maine, USA), in 1· Tris–ace-

tate buffer (pH 7.8), with one-tenth the EDTA concentration (Sambrook et al., 1989) and containing ethidium bromide. The amplicon was cut from the gel and digested using GELaseTM Agarose Gel-Digesting preparation, using the ‘‘Fast Protocol’’ method (Epicentre Technologies, Madison, WI, USA). The sequencing reaction was performed in a 10 lL final volume using the BigDye Terminator cycle sequencing kit (PE Applied Biosystems, Foster City, California, USA) following the manufacturerÕs instructions and viewed with an ABI 3100 automated DNA sequencer (Applied Biosystems). The resulting sequences were aligned and edited using Sequencher (version 3.1.1). Alignments were further adjusted by eye in PAUP*4.0b10 (Swofford, 2003). All sequences have been deposited at GenBank (see Table 1 for accession numbers). 2.3. Phylogenetic analysis Phylogenetic analyses of the ITS, the chloroplast (ndhF and trnL-F) and the combined ITS and chloroplast datasets were conducted with PAUP*4.0b10 (Swofford, 2003) using the maximum parsimony (Swofford et al., 1996) method. Parsimony analyses were performed with heuristic searches using TBR branch swapping, MULPARS option, and 100 random taxon addition. Internal branch support was estimated with 1000 bootstrap replicates (Felsenstein, 1985) with 100 random taxon addition and heuristic search options. A Bayesian analysis was conducted using MrBayes version 3.0 (Huelsenbeck and Ronquist, 2001) with the ITS, the chloroplast (ndhF and trnL-F) and the combined ITS

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and chloroplast. The analysis was conducted using a general time reversible (GTR + I + G) model [nst = 6; GTR + I + G experimentally determined to be the best-fit model using Modeltest version 3.06 (Posada and Crandall, 1998)] with gamma distributed rate variation across sites and initial estimate of equal base frequencies. The MCMC algorithm was run for 2,000,000 generations with 4 incrementally heated chains, starting from random trees and sampling one out of every 100 generations. A majority-rule consensus tree was calculated with PAUP* from the last 18,001 out of the 20,001 trees sampled. The first 2000 trees (burn-in) were excluded to avoid trees that might have been sampled prior to convergence of the Markov chains. The posterior probability of each topological bipartition was estimated by the frequency of these bipartitions across all 18,001 trees sampled. To evaluate the congruence of the chloroplast and nuclear data sets, we employed the partition homogeneity test or the incongruence length difference (ILD) test (Farris et al., 1995) and the Templeton test (Templeton, 1983). The ILD test has been criticized as an invalid method to test character incongruence (e.g., Barker and Lutzoni, 2002; Yoder et al., 2001). Hipp et al. (2004) argued that the ILD test can serve as a conservative initial test of data partition congruence, and can be used to determine which taxa are contributing most to the incongruence between data partitions. The partition homogeneity test was conducted with PAUP*4.0b10 (Swofford, 2003) with 100 replicates of heuristic search using TBR branch-swapping and gaps treated as missing data. Topological congruence between the gene trees was evaluated with the TempletonÕs test, as implemented in PAUP*. Maximum-likelihood (ML) analyses was performed in PAUP*4.0b10 (Swofford, 2003) for combined ITS and chloroplast data. The analyses were conducted using a general time reversible (GTR + I + G) model for that it is a model best fit our combined data set according to a hierarchical likelihood ratio test conducted in Modeltest version 3.06 (Posada and Crandall, 1998). 2.4. Biogeographic analyses The ML tree that resulted from the combined nuclear and chloroplast datasets was used for the dispersal-vicariance (DIVA) (Ronquist, 1997) and penalized likelihood (PL) (Sanderson, 2002) analyses. Because the tree has to be fully bifurcating in these analyses, we included only a single accession for Rhus chinensis, Rhus copallina, Rhus ovata and Rhus virens, and excluded Rhus pachyrrhachis in the analysis. Excluding the North American R. pachyrrhachis should not affect our biogeographic conclusions as it forms a clade with its North American relatives. We also excluded Rhus microphylla,

Rhus michauxii, and Rhus rubifolia in our DIVA analysis because of their conflicting positions in ITS and cpDNA trees (we discussed this point carefully in Section 3.3). We are not certain at present on the relationships of Rhus to other taxa in the Rhus complex (Fig. 4; also see Pell, 2004). With our focus on the diversification of Rhus, we thus used the Rhus phylogeny alone based on the combined data set in the DIVA analysis. Our present sampling will not permit us to explore the biogeographic diversification of the Rhus complex, nor the origin of Rhus. To estimate the ages of diversification within Rhus, we sequenced several taxa of Pistacia (Pistacia texana and Pistacia vera) and Toxicodendron (Toxicodendron diversilobum, Toxicodendron radicans, and Toxicodendron vernix) of Rhoeae, which have good fossil record. Diverse representatives of both Pistacia and Toxicodendron were included so that the crown clade of each genus may be used as constraint points in the PL analysis due to problems associated with the uncertain phylogenetic positions of both genera (Miller et al., 2001; Pell, 2004). The DIVA analysis reconstructs ancestral distributions in a given phylogeny without any prior assumptions about area relationships, and considers vicariance, dispersal and extinction as viable biogeographic events (Ronquist, 1997). The maximum likelihood tree of the combined cpDNA and ITS data set was used as the framework for reconstructing the optimal ancestral distribution using DIVA 1.1 (Ronquist, 1996; http://www.ebc.uu.se/systzoo/research/diva/diva. html). Five areas of endemism were defined for Rhus based on the species distributions and our emphasis on the intercontinental diversification of the genus in the Northern Hemisphere: eastern Asia (A), Europe and western Asia (B), Hawaii (C), North America (D), and northern Central America (E). The fossils of Rhus were not distributed beyond these five areas of endemism. Because the relationships of Rhus fossils to extant taxa are not well understood, we did not include the fossil data in our DIVA analyses. Instead, we discuss the fossil distribution and their taxonomic affinities in the context of our DIVA results. Future analyses will test the biogeographic hypotheses developed in this study. To test whether these data conformed to the molecular-clock hypothesis, a likelihood ratio (LR) test was conducted by calculating the log likelihood score of the chosen model with the molecular clock enforced and comparing it with the log likelihood score without the enforced clock (Baldwin and Sanderson, 1998; Muse and Weir, 1992). The number of degrees of freedom is equivalent to the number of terminals minus two (Sorhannus and Van Bell, 1999). Divergence times were estimated using the penalized likelihood method (PL) (Sanderson, 2002) with the program r8s version 1.60 (Sanderson, 2003). We used the ML tree of combined ITS and cpDNA data in the esti-

T. Yi et al. / Molecular Phylogenetics and Evolution 33 (2004) 861–879

mates. A cross-validation analysis was performed to obtain the most likely smoothing parameter. We employed the TN algorithm, as recommended in r8s for PL, collapsing zero length branches. Based on fossils of Rhus and its close relatives, we were able to constrain the ages of three nodes in the phylogeny of the Rhus complex (Fig. 6). First, we fixed the age of the earliest divergence of the Rhoeae we sampled (the divergence of Malosma and the large clade of Rhus and close relatives including Searsia, Toxicodendron, Actinocheita, Schinus, Pistacia and Rhus) at 60 Ma in the Paleocene (point F in Fig. 6). Fossils of tribe Rhoeae date back to Paleocene (Cronquist, 1981). Younger fossils were reported by Chandler (1962), who described four taxa of fossil fruits as Rhus from the early Eocene of England, but she commented (p. 92) that these fossils belonged to the tribe Rhoeae, and there was uncertainty on the generic assignment. Our interpretation of geological ages followed the 1999 Geological Time Scale of the Geological Society of America (1999). Second, the oldest fossil for Rhus subgenus Lobadium was reported from the York Ranch flora (Montana, USA) during the Late Oligocene (Becker, 1973; leaf). We thus constrained the crown clade of Rhus subgenus Lobadium as 28 Ma. Third, Finally, the oldest fossil of Pistacia was from southern France during Oligocene (Zohary, 1952). We did not include all the genera of Rhoeae in our analysis, and the sister groups of Pistacia and Toxicodendron are unknown (Pell, 2004) and constraining the ages of the two genera with their sisters in Fig. 6 is thus problematic. We therefore constrained the minimum age of the crown group of Pistacia and Toxicodendron to be 30 and 50 Ma, respectively (Fig. 6). To estimate the standard errors associated with divergence times, we used a parametric bootstrapping strategy similar to that in Davis et al. (2002): (1) 100 data sets were simulated on the maximum likelihood tree with the computer program Seq-Gen version 1.2.7 (Rambaut and Grassly, 1997); (2) the divergence times were estimated on the original tree; and (3) the resulting simulated data sets were imported into PAUP*, and maximum likelihood trees were generated. The divergence times were estimated on each tree using r8s version 1.60, and the resulting ages of the notes from the simulated data sets were used to calculated the variance in divergence time estimates.

3. Results 3.1. ITS data The aligned matrix of the ITS1, 5.8S and ITS2 regions has a length of 742 characters, with 196 variable and 133 parsimony-informative sites. Within Rhus, the percentage of sequence divergence between taxa varied

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from 0 (between R. chinensis and R. chinensis var. roxburghii) to 6.08% (between R. coriaria and Rhus kearneyi). Between Rhus and the outgroup taxa, the percentage of sequence divergence varied from 4.96% (between Actinocheita filicina and R. copallina) to 12.46% (between R. kearneyi and Searsia pyroides). The alignment of the sequences of Rhus and outgroup taxa required 23 insertions and deletions (indels), most of which were 1–3 bp in size (12 1-bp, 4 2-bp, 3 3-bp, 2 4-bp, 1 5-bp, and 1 8-bp). Treating gaps as missing data or new characters did not change the tree topology. Maximum parsimony analysis produced 18 maximally parsimonious trees (MPTs) of 419 steps, with a consistency (CI) of 0.63, a retention index (RI) of 0.83, and a rescaled consistency index (RC) of 0.52. The 50% majority-rule consensus of 18,001 trees (20,001 trees minus 2000 burn-in trees) resulted from the Bayesian analysis was largely congruent with the trees of the parsimony analysis except that Rhus aromatica, R. microphylla, R. rubifolia, and Rhus trilobata formed a monophyletic group with the posterior probability (PP) value of 73%; and Searsia lancea, S. leptodictya, and Searsia undulata formed a clade (PP = 59%), which was sister to the clade composed by S. ciliata, S. pyroides, and S. quartiniana. In eight cases, nodes with P50% bootstrap support garnered PP values < 95% (Fig. 2). All clades except one within Searsia with P70% bootstrap support had PP values over 95% (Fig. 2). The ITS data strongly supported the monophyly of Rhus sensu Barkley (1937). Searsia from Africa was monophyletic and distinct from the Rhus clade (Fig. 2), as reported by Miller et al. (2001) and Pell (2004). Although species relationships within subgenus Rhus were not well resolved at the deep level, the expanded ITS sampling of this study is consistent with Miller et al.Õs, 2001) result that subgenus Rhus is paraphyletic with respect to subgenus Lobadium, (Fig. 2). The clade formed by R. copallina and Rhus lanceolata was weakly supported as sister to the remaining Rhus species (BS < 50%, PP = 84%). Within subgenus Rhus, the following clades of closely related species were suggested: (1) Rhus chinesis in eastern and southeastern Asia and R. sandwicensis in Hawaii; (2) Rhus punjabensis and R. potaninii from eastern Asia; (3) R. lanceolata and R. copallina from eastern North America; and (4) Rhus glabra, R. michauxii, and Rhus typhina from eastern North America. R. coriaria from southeastern Europe and western Asia was sister to this clade without support isolated from other clades (Fig. 2). Rhus subgenus Lobadium was monophyletic with a bootstrap support of 98%, and a Bayesian posterior probability value of 100% (Fig. 2). Within subgenus Lobadium, sect. Styphoniae was monophyletic, and sect. Lobadium was paraphyletic. YoungÕs circumscriptions of sect. Styphoniae subsects. Styphoniae and Compositae

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Fig. 2. The strict consensus tree of 18 MPTs of the ITS data of Rhus, with gaps treated as missing data (CI = 0.63, RI = 0.83, and RC = 0.52). The bootstrap values in 1000 replicates >50% are shown above the branches, and the Bayesian posterior probabilities >50% are indicated below the branches. * indicates the topological discordance of related clades between the MP and Bayesian trees.

were largely supported except that R. kearneyi of subsect. Styphoniae (Young, 1978) was nested within subsect. Compositae. R. rubifolia of sect. Terebinthifolia was supported as sister to R. microphylla (sect. Lobadium). 3.2. Chloroplast ndhF and trnL-F data The ndhF data set had 2126 aligned positions, with 172 variable sites, 98 of which were parsimony-informa-

tive. The aligned matrix of the trnL-F data had 998 positions with 109 variable and 52 parsimony-informative sites. Because there is no recombination in the chloroplast DNA, we combined the ndhF and trn L-F data in our analysis. The aligned matrix of combined cpDNA data had 3142 characters with 281 variable and 150 parsimony-informative sites. Within Rhus species, sequence divergence varied from 0 (between R. potaninii and R. punjabensis) to 1.85% (between R. chinensis and Rhus schiedeana). In comparison with the outgroups, se-

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quence divergence ranged from 0.77% (between Malosma laurina and Rhus integrifolia) to 2.22 % (between R. kearneyi and Searsia leptodictya). Maximum parsimony analysis produced 4049 MPTs of 348 steps, with a CI of 0.85, RI of 0.90, and a RC of 0.77, the strict consensus tree is presented in Fig. 3. The consensus of 18,001 trees (20,001 trees minus 2000 burn-in trees) resulting from the Bayesian inference was congruent with the MPTs. In four cases, nodes with P50% bootstrap support had PP values < 95% (Fig. 3). Both chloroplast data sets supported a monophyletic Rhus. Subgenus Rhus was paraphyletic with R. microphylla, a species historically included in subgenus Lobadium, nested within it (Fig. 3). Species of subgenus Rhus from Asia, Europe, and Hawaii formed a well-supported clade. The North American species of this subgenus formed two clades, one composed of R. copallina and

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R. lanceolata, another of R. glabra, R. michauxii and R. typhina. Two major subclades of subgenus Lobadium were detected (Fig. 3). The first subclade included R. aromatica, R. integrifolia, R. ovata, and R. trilobata; and the second group consisted of Rhus choriophylla, R. kearneyi, R. pachyrrhachis, R. schiedeana, and R. virens. These two subclades formed a trichotomy with subgenus Rhus (Fig. 3). 3.3. Data incongruence Both the partition-homogeneity test and TempletonÕs significantly less parsimonious test (SLPT) indicated that the cpDNA and ITS data sets were not congruent (P = 0.01). The conflicts between the trees of the two datasets (cf. Figs. 2 and 3) lied in the positions of R. michauxii, R. microphylla, and R. rubifolia. R. typhina

Fig. 3. The strict consensus tree of 4049 MPTs of the chloroplast ndhF and trnL-F data set of Rhus, with gaps treated as missing data (CI = 0.85, RI = 0.90, and RC = 0.77). The bootstrap values in 1000 replicates >50% are indicated above the branches, and the Bayesian posterior probabilities >50% are shown below the branches.

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and R. glabra were weakly supported as a monophyletic clade in the ITS data, R. michauxii was strongly suggested as sister to this clade. In contrast, the cpDNA data supported R. typhina as sister to the clade of R. glabra and R. michauxii. R. microphylla, and R. rubifolia formed a well-supported clade within subgenus Lobadium in the ITS data, yet R. microphylla was placed within subgenus Rhus in the cpDNA tree, and R. rubifolia was nested within subgenus Lobadium sect. Styphoniae sensu Barkley (1937). We then excluded R. microphylla, R. michauxii, and R. rubifolia, and re-analyzed the chloroplast and ITS data sets independently. Congruence of the resulting phylogenies was tested. The partition homogeneity test had a P value of 0.13 and about 50% of the pair-wise comparisons of the ITS and cpDNA trees of the TempletonÕs test had a P value over 0.01. We thus performed a combined analysis of the chloroplast and ITS sequences without R. microphylla, R. michauxii, and R. rubifolia. The aligned matrix of combined cpDNA data had 3966 characters with 461 variable and 273 parsimony-informative sites. Maximum parsimony analysis produced six MPTs of 742 steps, with a CI of 0.73, RI of 0.84, and a RC of 0.62. The strict consensus tree is presented in Fig. 4.

The consensus of 18 001 trees (20,001 trees minus 2000 burn-in trees) resulting from the Bayesian inference was congruent with the MPTs. In one case, nodes with P50% bootstrap support had PP values < 95% (Fig. 4). The topology from the combined analysis showed a similar relationship for subgenus Rhus to that in the cpDNA tree (cf. Figs. 3 and 4) except that R. coriaria was supported as sister to the clade of R. potaninii and R. punjabensis (Fig. 4). The combined tree also had a similar topology for subgenus Lobadium to that in the ITS tree (cf. Figs. 2 and 4). 3.4. DIVA analysis Optimization without constraints on the number of ancestral areas resulted in a single solution for the root of Rhus (ABCD), one for the root of subgenus Rhus (ABCD), one for the clade of subgenus Rhus in Asia, Europe and Hawaii (ABC), and one for subgenus Lobadium (D). When the maximum areas were constrained to four in the optimization, same results were obtained as that of optimization without constraints. When the number of areas was limited to three, four alternative solutions for the root of Rhus (D, AD, ABD, ACD),

Fig. 4. (A) The phylogram the maximum likelihood tree of Rhus of the combined ITS and cpDNA data showing the relative branch lengths. (B) The strict consensus tree of six maximally parsimonious trees of the combined ITS and chloroplast data (ndhF and trnL-F) of Rhus (excluding R. michauxii, R. microphylla, and R. rubifolia), with gaps treated as missing data (CI = 0.73, RI = 0.84, and RC = 0.62). The bootstrap values in 1000 replicates >50% are indicated above branches, and Bayesian posterior probabilities >50% are shown below the branches.

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four for the root of subgenus Rhus (AD, ABD, ACD, BCD), five for the clade of subgenus Rhus in Asia, Europe and Hawaii (A, AB, AC, BC, ABC), and one for subgenus Lobadium (D). When the number of areas was limited to two, the following results were obtained: the ancestral area for Rhus was North America, or both eastern Asia and North America; the ancestral area for subgenus Rhus was Asia and North America; and North America was the ancestral area of subgenus Lobadium (Fig. 5). The DIVA results also suggested that species of subgenus Rhus diverged early into two major lineages, which correspond to a major split between North America (D) and Asia (a vicariant event separating the eastern Asian and North American taxa, Fig. 5). The eastern Asian species further diversified into other areas: the ancestor of R. sandwicensis was dispersed into Hawaii; and the ancestor of R. coriaria was dispersed into Europe from Asia. The ancestor of R. schiedeana was dispersed from North America into northern Central America (Fig. 5).

from the cross-validation procedure, the PL analysis suggested Rhus diverged from its closest relative included in our analysis at 49.1 ± 2.1Ma, the split of the Eurasian and Hawaiian taxa (the R. chinensis–R. sandwicensis–R. potaninii–R. punjabensis–R. coriaria clade) from North American species (the R. copallina–R. lanceolata–R. glabra–R. typhina clade) occurred about 33.8 ± 3.1 Ma, and the split of the two clades (the R. copallina–R. lanceolata clade, and the R. glabra–R. typhina clade) of subgenus Rhus in North America about 33.0 ± 3.1 Ma (Fig. 6). The European and western Asian R. coriaria diverged from the eastern Asian R. potaninii– R. punjabensis clade at about 24.4 ± 4.2 Ma, and the Hawaiian R. sandwicensis diverged from the Asian R. chinensis at 13.5 ± 3.0 Ma.

3.5. Divergence times within Rhus

Rhus was characterized by its fruits with red hairs (Barkley, 1937). Data from morphology, anatomy, chemistry and ITS sequences (Gillis, 1971; Heimsch, 1940; Miller et al., 2001; Young, 1975, 1979) supported BarkleyÕs circumscription of the genus. Our ITS and cpDNA data again strongly supported the monophyly of Rhus. The chloroplast and ITS datasets support the segregation of historically allied genera Actinocheita, Malosma, Searsia, and Toxicodendron, from Rhus. In particular, these data provide strong evidence that Searsia, a group of more than 100 species distributed primarily in the southern Africa, is not closely related Rhus (e.g., De Candolle, 1825). Barkley (1942, 1963) was the first to treat the southern African Rhus as an independent genus. Searsia differs from Rhus by having white drupes, which are glabrous or sparingly pubescent; its mesocarp and endocarp are adhering (Barkley, 1963). Species of Searsia are still included within Rhus by some authors (e.g., Hutchinson et al., 1958; Kokwaro and Gillett, 1980; Moffett, 1993; Ozenda, 1977). Recent molecular analyses have found this genus to be highly distinct from Rhus (Miller et al., 2001; Pell, 2004). The present study included additional taxa of Searsia and provides futher support for the generic status of Searsia (Figs. 2–4). Some western North American species of Rhus subgenus Lobadium and Searsia share similar habit and leaf morphology. The similar arid climate in western North America and Africa perhaps caused the morphological convergence for species of Rhus and Searsia.

Results of the likelihood ratio test of the molecularclock hypothesis for the ITS and cpDNA data sets were as follows: ITS,
2 ln LR = 63.93, df =35, and P = 0.002 < 0.01; cpDNA, minus2 ln LR = 94.42, df = 35, and p < 0.001. The molecular clock of both ITS and cpDNA data was thus rejected at the level of P = 0.01. Using a smoothing value of 3162 as obtained

Fig. 5. Results of optimizations for ancestral distributions of Rhus using DIVA. The phylogeny used was the maximum likelihood tree of the combined cpDNA and ITS data set without R. michauxii, R. microphylla, and R. rubifolia (A, eastern Asia; B, Europe and western Asia; C, Hawaii; D, North America; and E, northern Central America).

4. Discussion 4.1. Monophyly of Rhus and its relationship with Searsia

4.2. Relationships within Rhus The classification of Rhus into two subgenera has been well supported by morphological and chemical

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Fig. 6. Chronogram of Rhus based on the maximum likelihood tree of the combined ITS and cpDNA data. Divergence times indicated by the chronogram were estimated using the penalized likelihood method. C1: constrained minimum age = 28 Ma; C1: constrained minimum age = 30 Ma; C3: constrained minimum age = 50 Ma; F: fixed root = 60 Ma.

data (Barkley, 1937; Young, 1979). The monophyly of subgenus Rhus was supported by combined cpDNA and ITS data, and largely supported in cpDNA data except that R. microphylla of subgenus Lobadium was nested within it. The ITS data did not resolve a monophyletic subgenus Rhus. Subgenus Lobadium was strongly supported as monophyletic in ITS analysis (Fig. 2) and in the combined analysis (Fig. 4). However, the cpDNA data revealed two main clades within the subgenus, which formed a trichotomy with the clade of subgenus Rhus plus R. microphylla (Fig. 3). 4.3. Relationships within subgenus Rhus Currently, there is no classification for subgenus Rhus. The results of this study allow us to examine the

morphological diversity of subgenus Rhus in a phylogenetic context, but do not provide the resolution required for a detailed subgeneric classification. Here we focus our discussion on the phylogenetic relationships suggested by the present study in the context of our morphological understanding. The close relationship between R. chinensis (eastern Asia to Indonesia) and R. sandwicensis (Hawaii) was well supported by both the ITS and cpDNA data. These two species have with ferruginous branches, imparipinnate leaves with 2–6 pairs of leaflets, and ovate-oblong and dark-green leaflets which are dark green above and tomentose underneath. R. chinensis differs from R. sandwicensis in the formerÕs bigger fruits (8 mm vs. 4–5 mm in diameter). R. sandwicensis was occasionally treated as variety of R. chinensis (Hillebrand, 1888).

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Rhus punjabensis and R. potaninii from eastern Asia were strongly supported as sister taxa. Both species have entire leaflets. R. punjabensis has pubescent twigs, winged uppermost rachis segments, and sessile or almost sessile leaflets. R. potaninii, on the other hand, has glabrous twigs, an unwinged rachis, and leaflets with short petiolules. Rhus lanceolata (native from southeastern North America to Mexico) and R. copallina (eastern North America) are well supported as sister taxa. Both species have a winged rachis and mostly entire leaflets. R. lanceolata has ovate-lanceolate leaflets with an obtuse apex. R. copallina has linear-lanceolate leaflets with an acuminate apex. The close relationship between R. copallina and R. lanceolata was also suggested by Hardin and Phillips (1985) based on foliar and fruit surface features. Hybrids between these two species were reported by Barkley (1937), and Hardin and Phillips (1985). Rhus coriaria is the only extant species in southern Europe and western Asia (Davis, 1967; Rechinger, 1969). Its position was unresolved in the ITS data (Fig. 2), but it formed a well-supported clade with the Asian species in the cpDNA tree (Fig. 3). The combined ITS and cpDNA data placed it as sister to the Asian R. potaninii–R. punjabensis clade. More detailed sampling and analysis are required to test the position of R. coriaria within subgenus Rhus. 4.4. Relationships within subgenus Lobadium The current classification of subgenus Lobadium includes three sections: sect. Lobadium, sect. Styphoniae, and sect. Terebinthifolia (Young, 1978). Young (1978) subdivided the morphologically diverse sect. Styphoniae into three subsections: Compositae, Intermediae, Styphoniae. Our previous ITS results (Miller et al., 2001) support YoungÕs sectional combination, but insufficient sampling precluded an assessment of his subsectional divisions. Subsections Styphoniae and Compositae were largely supported as monophyletic, except that R. kearneyi was nested within subsect. Compositae rather than subsect. Styphoniae in all analyses. We were not able to sample the monotypic subsect. Intermediae. The phylogenetic position of the subsection needs to be assessed in the future. YoungÕs (1978) sect. Styphoniae was not monophyletic in the cpDNA data. Both ITS and cpDNA data supported the simple-leaved R. kearneyi as a member of subsect. Compositae. Young (1978) combined BarkleyÕs (1937) sects. Lobadium and Rhoeidium based on morphological, anatomical and chemical data, to create sect. Lobadium (sensu Young). BarkleyÕs sect. Rhoeidium consists of only R. microphylla. Both ITS and cpDNA data suggested the monophyly of YoungÕs (1978) sect. Lobadium. R. micro-

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phylla (BarkleyÕs sect. Rhoeidium) differs from the other sect. Lobadium species by having very small, 3–9 foliolate leaves, winged rachis, bracts and bracteoles of similar sizes. In fact, the leaves of R. microphylla resemble miniaturized R. copallina leaves. R. microphylla was suggested to be a lineage of subgenus Lobadium in the ITS tree, but showed a close relationship to the species of subgenus Rhus in the cpDNA data. 4.5. Putative hybridization and the incongruence of nuclear ribosomal and chloroplast data The topologies from the ITS and cpDNA data sets were incongruent in some clades. The species relationships shown in Figs. 2 and 3 are based on gene trees, which represent the relationships of the alleles found in the different species at two different loci. Incongruence between these datasets may reflect the fact that that the two loci sequenced simply do not have congruent evolutionary histories. However, when the incongruence results from datasets derived from different genomes (e.g., nuclear and plastid), it is frequently attributed to introgression of the cytoplasmic genome from one species into the nuclear background of another (e.g., Ferris et al., 1983; Gyllensten and Wilson, 1987; Harrison et al., 1987; Rieseberg and Wendel, 1993; Soltis and Kuzoff, 1995; Soltis et al., 1991, 1996; Tegelstrom, 1987; Yoo et al., 2002). This hypothesis is strengthened when the putative hybrids are sympatric and interfertile. The phylogenetic positions of three Rhus taxa are different in the chloroplast and ITS trees. Here we examine the relationships of these taxa, and the potential role of hybridization in their evolutionary histories, in more detail. Natural hybridization has been reported for Rhus species. Barkley (1937) identified hybrids between R. copallina and R. lanceolata, Rhus lentii and R. integrifolia, R. ovata and R. integrifolia, R. vestita and R. pachyrrhachis, and R. aromatica and R. trilobata. Presumed natural hybrids of R. glabra · R. typhina were known as R. glabra var. borealis Britt. (R. borealis (Britt.) Greene, and R. pulvinata Greene) (Brizicky, 1963). Hybridization between R. michauxii and R. glabra was reported by Hardin and Phillips (1985). Hybrids of the two species were later confirmed by allozyme analysis (Burke and Hamrick, 2002). Rhus microphylla has been traditionally placed in subgenus Lobadium, as it shares several characteristics of the subgenus: flowers in terminal compound spikes and appearing before the leaves, with one bract and two bracteoles subtending each flower, and ovate and persistent bracts. However, it also shares morphological similarities with R. copallina and R. lanceolata of subgenus Rhus including a winged rachis, pinnately compound and deciduous leaves, and ovate to lanceolate sessile leaflets with entire margins. R. microphylla and

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R. lanceolata are at least sympatric in the Davis Mountains area in Texas (J. Wen, pers. observ.). Our morphological and molecular results show that this unique species may be of a hybrid origin between species from the two subgenera. It is clearly placed within subgenus Rhus in the cpDNA tree (BS = 79%, PP = 100%). This suggests that the maternal parent of R. microphylla may be a species of subgenus Rhus, perhaps related to the common ancestor of the North American species. Because the ITS phylogeny reveals that R. rubifolia is most closely related to R. microphylla, we suggest that the paternal parent of R. microphylla is most likely a member of sect. Terebinthifolia. Our ITS and chloroplast DNA data support the close relationship among R. glabra, R. michauxii, and R. typhina. The ITS data placed R. michauxii sister to the R. glabra–R. typhina clade, whereas the cpDNA data strongly suggested R. glabra and R. michauxii as forming a clade (cf. Figs. 2 and 3). R. glabra, R. michauxii, and R. copallina can occur sympatrically in North Carolina (James Hardin, pers. comm.), and R. glabra and R. michauxii have been reported to co-occur in the same locality (Burke and Hamrick, 2002; Hardin and Phillips, 1985). Furthermore, natural hybrids were detected between R. glabra and R. typhina, as well as between R. glabra and R. michauxii (Hardin and Phillips, 1985). R. glabra and R. typhina are morphologically more similar to each other. These two species both have a unwinged rachis, lanceolate leaflets, and toothed leaflet margins. Barkley (1937) suggested that the closest relatives of R. michauxii were Rhus javanica L. (= R. chinensis) from eastern Asia and R. coriaria from Europe. However, Hardin and Phillips (1985) reported a close relationship among R. glabra, R. michauxii, and R. typhina of North America based on foliar glands and nonglandular trichomes of the fruit, and their red or orange pericarp. They further grouped R. glabra and R. michauxii together based on trichome morphology of the fruits. Rhus rubifolia also showed different species relationships between ITS and cpDNA trees. In the ITS tree, it was sister to R. microphylla of sect. Lobadium; whereas in the cpDNA tree, it formed a well supported clade with R. kearneyi, R. pachyrrhachis, and R. schiedeana of sect. Styphoniae (cf. Figs. 2 and 3). R. rubifolia and R. microphylla both have pinnately compound leaves, oval to ovate leaflets, entire leaflet margins, and sessile lateral leaflets. 4.6. Diversification of Rhus in the Northern Hemisphere The inference of the ancestral area of Rhus requires a broader analysis of the tribe Rhoeae, which is beyond the scope of our analysis. Our present discussion focuses on the diversification within Rhus in the North Temperate Zone. The optimization of ancestral distributions

using DIVA suggested North America and/or eastern Asia as the ancestral area of Rhus (Fig. 5). The fossil data are consistent with the hypotheses generated in DIVA analysis, but may favor North America as the ancestral area as it has the earliest known fossils. The oldest known Rhus fossils were found in western North America and date to the early Eocene (MacGintie, 1969; Wilf, 2000). North America possesses abundant fossil records of Rhus from the Lower Eocene to the Pliocene primarily from the west of the Rockies (Fig. 1) (e.g., Becker, 1969; MacGinitie, 1953; Manchester, 1994; Meyer and Manchester, 1997; Wolfe et al., 1966; see Fig. 1). European Rhus fossils resemble the North American species R. glabra and R. typhina, and the European species R. coriaria from the Middle to Late Miocene in Germany (Mai, 2001) and Hungary (Andrea´nszky, 1959). In Asia, the earliest Rhus fossils are dated to the Middle Eocene (Zhilin, 1989). Other records are known from Late Eocene and Early Oligocene (Zhilin, 1989), and Miocene (Huzioka, 1963; Ozaki, 1991; Tanai and Suzuki, 1963). Based on DIVA analyses and fossil evidence, we developed the following hypothesis on the biogeographic history of Rhus: the ancestral area of the Rhus clade is North America (D) or North America and eastern Asia (AD) based on the phylogeny and distribution of the extant species (Fig. 5). The occurrence of the earliest known Rhus fossils in western North America is consistent (in part) with this hypothesis. DIVA results of Xiang and Soltis (2001) indicated that the direction of intercontinental dispersal of temperate taxa was biased in one direction, mostly from the Old World to the New World. In contrast, available evidence for Rhus suggests that in this genus, the direction of migration was mostly likely from the New World to the Old World, or that there was an ancient vicariance between North America and Asia. Our phylogenetic and biogeographic analyses suggest that Rhus subgenus Rhus first diverged into two lineages: one in North America and one in eastern Asia, and that subgenus Lobadium had a North American origin (Fig. 5). Our penalized liklihood (PL) analysis and fossil evidence support North America as the ancestral area of Rhus. The PL analysis indicated Rhus diverged from its relative at 49.1 ± 2.1 Ma. Species of Rhus became widely disjunct in Asia and North America by the Middle Eocene. North American fossils of Rhus date back to the Early Eocene from the Little Mountain flora (Wyoming, USA; Wilf, 2000; leaf), and the Wind River flora (Wyoming, USA; MacGintie, 1969; leaf). A more reliable fruit fossil of Rhus was recorded from the Middle Eocene Nut Beds Flora in Oregon (Manchester, 1994). The eastern Asian species of subgenus Rhus diverged from the North American relatives at about 33.8 ± 3.1 Ma (the Late Eocene). The oldest fossil of Rhus from

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Asia is known from southern Turkmenistan and dates to the Middle Eocene (Zhilin, 1989). Rhus fossils from Alaska also date to the Middle Eocene. These fossil dates are slightly older than our PL results, indicating that the PL results may in fact underestimate the divergence time of eastern Asian and Northern American Rhus clades. Fossil evidence from the Eocene to the Miocene indicates that Rhus once had a wider distribution in northern and central Europe, high-latitude Asia and North America than it does today (Fig. 1). Subsequent climate changes during the Late Tertiary and the Quaternary (e.g., extreme cooling and drying, and uplift of the Rocky Mountains and the Himalayas) may have eliminated Rhus species from the high latitudes of Europe, Asia and North America, and survived in a few ‘‘refugia’’ of the Northern Hemisphere (Wen, 1999). Certain Rhus species have a contracted distributional range by comparison to fossil evidence. Fossils similar to the eastern North American R. glabra or R. typhina were reported from several floras in western North America throughout the Tertiary (Axelrod, 1944, 1956, 1964; Becker, 1969, 1972; Brown, 1934; Lakhanpal, 1958; MacGinitie, 1941, 1953, 1969; Wilf, 2000; Wolfe, 1966, 1977). Fossils attributable to R. copallina and R. lanceolata of eastern North America were described from the Florissant flora (Colorado, USA) of the Early Oligocene (MacGinitie, 1953), and from the Metzel Ranch flora (Montana, USA) of the Late Oligocene (Becker, 1972). The Bering Land Bridge (BLB) has been inferred to be open to terrestrial organisms from at least the Early Paleocene until its closure between 7.4 and 4.8 Ma (Tiffney and Manchester, 2001). Many temperate angiosperms and conifer taxa were suggested to have occupied the BLB in the Early Tertiary (Manchester, 1999, 2001; Wen, 1999, 2001; Xiang et al., 1998). Alaskan Rhus fossils were described from the Middle Eocene, the Late Eocene, and the Early Miocene (Wolfe, 1966, 1972, 1977; Wolfe et al., 1966). The Alaskan Rhus fossils, considered together with the occurence of eastern North American Rhus fossils (e.g., R. glabra or R. typhina, R. copallina, and R. lanceolata) in western North America implicate the BLB in the migration of Rhus species between Asia and North America. The North Atlantic land bridge (NALB), which opened at a lower latitude than the BLB, provided another important link between Eurasia and North America in the Early Tertiary (Davis et al., 2002; Tiffney, 1985, 2000; Tiffney and Manchester, 2001). Fossils similar to the North American R. glabra and R. typhina were reported from the late Miocene of Hungary (Andrea´nszky, 1959). Considering this fossil record, we cannot eliminate the NALB as a possible migration route for Rhus species moving between eastern North America and Europe. A close relationship between R. coriaria and the eastern Asian species of subgenus Rhus was suggested in the

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cpDNA and the combined cpDNA–ITS data (Figs. 3,4). DIVA results indicated that this species was dispersed from eastern Asia to western Asia. The Turgai Strait was a biogeographic barrier between Asia and Europe in the Paleocene to the Middle Eocene. Its southward retreat in the Late Eocene allowed migration of plants between Asia and Europe (Tiffney and Manchester, 2001). The ancestor of R. coriaria perhaps migrated from Asia into Europe after the disappearance of the Turgai Strait, at 24.4 ± 3.2 Ma, as estimated in our study. For subgenus Lobadium, the oldest fossil is similar to R. ovata from the York Ranch flora (Montana, USA) during the Late Oligocene (Becker, 1973). Younger fossils similar to R. integrifolia and R. virens were described from the Tehuchapi flora (Washington, USA) during the Early Miocene (Axelrod, 1939). Fossils of R. ovata were described from California during the Pliocene (Dorf, 1930). Mexico is the center of modern diversity of subgenus Lobadium, but no fossils have been reported from this region. The age of subgenus Lobadium diverged from subgenus Rhus was inferred to be about 38.1 ± 3.0 Ma, which was lightly older than the fossil record of the subgenus. DIVA analysis suggested North America as the ancestral area of the subgenus and the Central American taxa were inferred to have dispersed from North America (Fig. 5). 4.7. Colonization of Rhus sandwicensis on the Hawaiian Islands The Hawaiian Islands, one of the most remote land masses in the world, are 3900 km from the closest continent (Kim et al., 1998). The Hawaiian flora is derived from progenitors that originated on several different continents (Wagner and Funk, 1995). For ca. 956 native species of flowering plants from 87 families and 216 genera (Wagner and Funk, 1995), 40.1% were found to have affinities with Indo-Pacific species, 16.5% with Austral species, 18.3% with American species, 12.5% pantropical, 2.6% boreal, and 10.3% unknown (Wagner, 1991). All Hawaiian taxa are hypothesized to be the result of long-distance disperal. Transoceanic long-distance dispersal from Africa, Americas, Arctic, Polynesia, Asia and Australia to the Hawaiian Islands have been suggested by numerous molecular phylogenetic studies (Baldwin et al., 1991; Ballard and Sytsma, 2000; Costello and Motley, 2001; Dejoode and Wendel, 1992; Ganders et al., 2000; Gemmill et al., 2001; Howarth et al., 1997; Howarth et al., 2003; Kim et al., 1998; Knox et al., 1993; Lindqvist and Albert, 2002; Lowrey, 1995; Pax et al., 1997; Seelanan et al., 1997; Wright et al., 2000). The single Hawaiian Rhus species, R. sandwicensis. occurs on five of the Hawaiian Islands (KauaÔi, OÔahu, MolokaÔi, Maui and HawaiÔi) (Wagner et al., 1990). Both cpDNA and ITS data sets strongly supported the

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close relationship between the Hawaiian R. sandwicensis with the Asian R. chinensis. Historical taxonomy of R. sandwicensis reflects this relationship. R. sandwicensis was treated as R. chinensis var. sandwicensis (Gray) Deg. and Greenwell, or R. semialata Murr. var. sandwicensis (Gray) Engler (R. semialata Murr. was merged with R. chinensis Mill.) (Hillebrand, 1888; Little and Skolmen, 1989). Our DIVA results favor a scenario in which the ancestor of R. sandwicensis dispersed from Asia at 13.5 ± 3.0 Ma (Fig. 5). This divergence time pre-dates the formation of the oldest extant island, Kauai (ca. 5 million years) (Carson and Clague, 1995). However, it is well documented that the Hawaiian chain of islands dates back to 70 Ma when the now submerged Emperor Seamounts are included (Kim et al., 1998; Stone and Pratt, 1994). It is unlikely that R. sandwicensis originated on one of the eight current high islands, because their geological age ranges only from 0.5 to 5.1 Ma (Kim et al., 1998). Perhaps the ancestor of R. sandwicensis split from R. chinensis on the mainland ca. 13.5 Ma, and then dispersed to Hawaii sometime following this divergence. Conceivably, the mainland ancestor of R. sandwicensis has since gone extinct. A more plausible and parsimonious explanation is that the ancestor of R. sandwicensis arrived on one of the older islands that has subsequently subsided within the archipelago, and then further dispersed onto younger islands. The similar pattern was suggested for the diversification of several other groups such as Geranium, the silverswords, and Platydesma (Funk and Wagner, 1995). Three primary modes of long-distance dispersal have been suggested for the colonization of flowering plants in Hawaii: transportation by birds, by air flotation, and by sea flotation (Baldwin et al., 1991; Carlquist, 1970, 1974; Howarth et al., 2003; Wright et al., 2000) The most plausible explanation for Rhus is long-distance dispersal via birds. All species of Rhus have red fleshy drupes. Fleshy fruits likely facilitate dispersal by migrating sea birds (Howarth et al., 2003). Although there is no record of birds feeding on fruits of R. chinensis nor R. sandwicensis, more than 50 bird species have been observed feeding on the fruits of species in the Rhus complex (Ridley, 1930), which are morphologically indistinguishable from the fruits of R. chinensis and R. sandwicensis. For example, fruits of R. copallina, R. glabra, and R. typhina are fed by Anas platyrrhyncha (American mallard), Corvus brachyrhncos (American crow), Dryobates villosus (woodpeckers), Pinicola enucleator (pine grosbeak), Sayornis phoebe, Sturnus vulgaris (starling), Turdus migratorius (American robin), and Vireo griseus. Rhus chinensis is reported to be dioecious (Min, 1979). Rhus sandwicensis was mentioned to be dioecious (Hillebrand, 1888); however, this species was found morphologically perfect (Wagner et al., 1990). Further analysis is needed on the reproductive biology of the species. If R. sand-

wicensis is dioecious, its establishment in Hawaii would have required at least two separate introductions for R. sandwicensis in Hawaii (one male and one female). One introduction would be required if the breeding system in Rhus is variable, including individuals with both female and hermaphroditic flowers (e.g., Lloyd, 1980) or if dioecy was derived independently in R. chinensis and R. sandwicensis.

5. Conclusions Rhus was well supported as a monophyletic group, and Searsia was shown to be a distinct genus. Both subgenus Lobadium and subgenus Rhus were monophyletic in combined chloroplast and nuclear data sets, although there are some conflicts with respect to the monophyly of subgenus Rhus in the individual ITS and cpDNA data sets. Additional field work, morphological studies, molecular data, and phylogenetic analyses are needed before a proper classification scheme for Rhus can be proposed. Our ITS data and DIVA analyses, interpreted together with the fossil record, suggest that Rhus maintained a wide, disjunct distribution in North America and Asia before and during the Middle Eocene. The earliest fossils of Rhus were from western North America. Rhus most likely migrated from North America into Asia via the Bering Land Bridge during the late Eocene (33.8 ± 3.1 Ma). R. coriaria, occupying western Asia to southern Europe, split from its eastern Asian relatives at the Early Oligocene (24.4 ± 3.2 Ma). Species similar to the North American R. glabra and R. typhina also occupied Europe by the late Miocene based on fossil evidence. The Hawaiian species diverged from its Asian relatives in the Middle Miocene (13.5 ± 3.0 Ma), and then colonized the Hawaiian Islands.

Acknowledgments We thank Jim Hardin, Alisha Holloway, John Mitchell, Jose Panero, Michael Vincent, Stefanie Ickert-Bond, the New York Botanical Garden, the Phoenix Desert Botanical Garden, the Rancho Santa Ana Botanic Garden, and the Santa Barbara Botanical Garden for providing leaf material. We are grateful to Jim Hardin for his advice on the morphology, ecology and distribution of Rhus michauxii and support of our study. This project was funded in part by the National Science Foundation (DEB-0108536), the Pritzker Laboratory for Molecular Systematics and Evolution of the Field Museum, and the Excellent Overseas Chinese Research Grant of the Chinese Academy of Sciences (to J. Wen).

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