Phylogenetic analysis of Toxicodendron (Anacardiaceae) and its biogeographic implications on the evolution of north temperate and tropical intercontinental disjunctions

Abstract

Abstract Toxicodendron is a genus in the Rhus complex of Anacardiaceae with a disjunct distribution between eastern Asia and North America, extending to southeastern Asia and the neotropics. Nuclear (internal transcribed spacer, external transcribed spacer, and NIA-i3) and chloroplast (ndhF and trnL-F) sequences were used to construct phylogenetic relationships of Toxicodendron. Phylogenetic analysis of these data strongly support Toxicodendron as a monophyletic group distinct from other genera of the Rhus complex, and the phylogeny does not fully corroborate classification at the sectional level. Two temperate disjunct lineages were detected, one from section Toxicodendron and the other between the eastern North American Toxicodendron vernix and the eastern Asian Toxicodendron vernicifluum. Their divergence times were estimated to be 13.46 (7.95–19.42) and 7.53 (2.76–12.86) mya, respectively. The disjunction between section Griffithii (taxa from warm temperate to tropical Asia) and Toxicodendron striatum (from the neotropics) was supported and their divergence time was estimated to be 20.84 (11.16–30.52) mya in the early Miocene. Our biogeographic results and the paleontological data support the Bering land bridge as the most likely route to explain the temperate disjunctions, yet the tropical disjunction in Toxicodendron seems to be best explained by the North Atlantic land bridge hypothesis.

The cashew family (Anacardiaceae) consists of more than 80 genera and 600–750 species mostly distributed in tropical Africa, Asia, and the Americas, with a small number of species in subtropical and temperate areas (McNair, 1925; Cronquist, 1981; Pell, 2004; Wannan, 2006; Pell et al., 2008). Toxicodendron Mill. is one of a few genera with a primary distribution in temperate to subtropical regions (Gillis, 1971; Zomlefer, 1994). The genus comprises approximately 24 species showing a disjunct distribution in temperate North America and eastern Asia (Li, 1952; Gray, 1846; Graham, 1972; Wen, 1999, 2001). A few taxa of Toxicodendron also occur in the tropics ranging from Central America to northernmost South America and southeastern Asia.

Toxicodendron is well-known for possessing skin-irritating oil (urushiol) that can cause severe allergic reactions to humans. Species of the genus also have lacquer in the phloem, and the lacquer is important for making anticorrosives or decorative paint. Taxa of this genus were often included in Rhus L. s.l. (Barkley, 1937a, 1963; Young, 1979; Cronquist, 1981). Miller et al. (2001) showed that Rhus should be delimited more narrowly, and that Toxicodendron and several other genera including Actinocheita F. A. Barkley, Cotinus Mill., Malosma Nutt. ex Abrams, Melanococca Blume, Metopium P. Browne, and Searsia F. A. Barkley are best segregated from Rhus.

In comparison with other genera in the Rhus complex, Toxicodendron generally has axillary inflorescences, whitish to dune-colored fruits without glandular pubescence, and toxic catechol in its resin (Brizicky, 1963; Gillis, 1971). However, the delimitation of Toxicodendron remains controversial. For example, Brizicky (1963) suggested treating Toxicodendron as a subgenus of Rhus because of their overall similarities in the inflorescence and the structure of flowers and fruits. Gillis (1971), however, preferred to treat Toxicodendron as a separate genus because of its suite of distinctive characters (e.g., presence of poisonous resin, absence of red glandular hairs on pedicels and fruits, and significantly smaller pollen grains).

Toxicodendron is commonly delineated by four sections (Venenata, Toxicodendron, Griffithii, and Simplicifolia). Section Toxicodendron (five species) comprises usually woody climbers with trifoliate leaves, short and subulate filaments, elongate anthers, and pendulous infructescences. The most well-known member of this section is Toxicodendron radicans, which is widely distributed throughout eastern North America and eastern Asia and is commonly known as poison ivy due to its superficial resemblance to English ivy (Hedera helix L. of Araliaceae) and Boston ivy [Parthenocissus tricuspidata (Siebold & Zuccarini) Planch. of Vitaceae]. Section Simplicifolia is represented by only one narrowly distributed species of northern Borneo in southeastern Asia. Its habit is very similar to that of section Toxicodendron as a deciduous woody climber, but its leaves are simple and subcoriaceous (Gillis, 1971). Section Venenata includes trees or woody shrubs with pinnately compound leaves, long filaments, and short globose anthers, mainly from eastern Asia. It is the largest section in the genus with approximately 18 species distributed throughout eastern Asia from Japan to Indonesia (Fig. 1). One species has a broad distribution within North America, ranging from southern Quebec to Florida, while another occurs in Central and South America from Veracruz, Mexico to the highlands of Colombia (Gillis, 1971; Min, 1980). Section Griffithii contains five species and has been segregated from section Venenata by Min (1980) based on their thick coriaceous leaves, erect infructescences, and mature fruits with irregular dehiscence of exocarps.

Figure 1. Distribution of sections within Toxicodendron showing biogeographic disjunctions between the Old and the New Worlds (modified from Min, 1980).

Two disjunct lineages between eastern Asia and North America have been reported in the genus, one from section Toxicodendron and the other from section Venenata. Toxicodendron radicans is perhaps one of the earliest examples cited by Gray (Gray, 1846, 1859) as showing a floristic connection between eastern Asia and eastern North America (also see Gillis, 1971, 1975; Wen, 1999). This is one of two examples that show such a disjunction at the species level, with the other being Phryma of Phrymaceae (Li, 1952; Wen, 1999; Nie et al., 2006). The distributional pattern in section Venenata is also intriguing with most species distributed throughout eastern Asia from Japan to Indonesia and only two species broadly distributed from North, to Central, to northern South America.

Most disjunct taxa between eastern Asia and North America are north temperate elements with only a few occurring in the subtropical and tropical regions (Li, 1952; Wen, 1999, 2001; Ying, 1983). Biogeographic studies so far have largely focused on temperate taxa in the Northern Hemisphere (Wen, 1999; Donoghue et al., 2001). However, it is important to include the close relatives from tropical and subtropical regions to further understand the evolution of the temperate disjunctions in the Northern Hemisphere (Lavin & Luckow, 1993; Wen, 1999, 2001; Ickert-Bond et al., 2007). A few examples of biogeographic disjunctions between temperate eastern Asia and North America with tropical extensions have been surveyed. One example can be found in Rhus s. str. Phylogenetic and biogeographic studies have suggested a migration route from North America into Asia by way of the Bering land bridge (BLB) with an ancient split in the late Eocene (Yi et al., 2004, 2007).

Although morphological data seem to support the separation of Toxicodendron from Rhus s.l., rigorous phylogenetic analyses of molecular sequence data and biogeographic analyses have never been used to test the status and evolution of Toxicodendron in the complex. The use of multiple genes from different genomes, especially low-copy nuclear genes, is needed to fully address these questions in a biogeographic context (Wang et al., 2000; Peng & Wang, 2008). In this study, we use nuclear [internal transcribed spacer (ITS), external transcribed spacer (ETS), and NIA-i3] and chloroplast data (trnL-F and ndhF) to: (i) test the monophyly of Toxicodendron in the Rhus complex; (ii) construct phylogenetic relationships within the genus; and (iii) reconstruct the biogeographic history of Toxicodendron, especially concerning the temperate and tropical intercontinental disjunctions between the Old and the New World.

1 Material and methods

1.1 Taxon sampling

Sequences of 68 accessions of Toxicodendron and some closely related taxa were used in the study (Table 1). Fifty samples representing 18 of the ca. 24 Toxicodendron species were sequenced, covering the species diversity of the three main sections (i.e., Venenata, Toxicodendron, and Griffithii) with an emphasis on the two main distributional centers of eastern Asia and North America. Our sampling of the three large sections represented the morphological and geographic diversity of the group. Rhus s. str., Actinocheita, Malosma, and Searsia of the Rhus complex have been considered to be the closest relatives of Toxicodendron (Miller et al., 2001; Yi et al., 2004). Representatives from the complex and the family were selected as outgroups (see Table 1).

Table 1.  Voucher information and GenBank accession numbers for samples of Toxicodendron and outgroups used in this study

Taxon	Source	Locality	ITS	ETS	NIA-i3	trnL-F	ndhF
—, no data. ETS, external transcribed spacer; ITS, internal transcribed spacer.
T. delavayi (Franch.) F.A. Barkley	Nie & Meng 343 (KUN)	China: Yunnan	FJ945937	FJ945767	FJ945885	FJ945983	FJ945830
	Nie & Meng 409 (KUN)	China: Yunnan	FJ945933	FJ945763	FJ945881	FJ945979	FJ945826
T. diversilobum (Torr. & A. Gray) Greene	Wen 6693 (F)	USA: California	AY677202	—	DQ382328	AY677208	AY677205
T. fulvum (Craib) C.Y. Wu & T.L. Ming	Nie & Meng 332 (KUN)	China: Yunnan	FJ945921	FJ945751	FJ945869	FJ945967	FJ945814
T. grandiflorum C.Y. Wu & T.L. Ming	Nie & Meng 331 (KUN)	China: Yunnan	FJ945936	FJ945766	FJ945884	FJ945982	FJ945829
	Nie & Meng 350 (KUN)	China: Yunnan	FJ945926	FJ945756	FJ945874	FJ945972	FJ945819
	Nie & Meng 407 (KUN)	China: Yunnan	FJ945943	FJ945772	FJ945892	FJ945990	FJ945837
	Nie & Meng 447 (KUN)	China: Yunnan	FJ945945	FJ945774	FJ945894	FJ945992	FJ945839
	Nie & Meng 448 (KUN)	China: Yunnan	FJ945928	FJ945758	FJ945876	FJ945974	FJ945821
	Tibet 1738 (KUN, US)	China: Sichuan	FJ945956	FJ945791	FJ945909	FJ946009	FJ945856
T. griffithii (Hook. f.) Kuntze	Nie & Meng 347 (KUN)	China: Yunnan	FJ945925	FJ945755	FJ945873	FJ945971	FJ945818
T. hookeri (Sahni & Bahadur) C.Y. Wu & T.L. Ming var. microcarpum (C.C. Huang ex T.L. Ming) C.Y. Wu & T.L. Ming	Nie & Meng 396 (KUN)	China: Yunnan	FJ945935	FJ945765	FJ945883	FJ945981	FJ945828
	Nie & Meng 397 (KUN)	China: Yunnan	FJ945942	FJ945771	FJ945891	FJ945989	FJ945836
T. pubescens Mill.	Wen 9998 (US)	USA: South Carolina	—	FJ945788	FJ945906	FJ946006	FJ945853
T. radicans (L.) Kuntze subsp. hispidum (Engler) Gillis	Nie & Meng 213 (KUN)	China: Chongqing	FJ945924	FJ945754	FJ945872	FJ945970	FJ945817
	Wen 5463 (US)	China: Hubei	FJ945947	FJ945776	—	FJ945994	FJ945841
T. radicans subsp. radicans (L.) Kuntze	Nie & Meng 530 (KUN)	USA: Virginia	FJ945946	FJ945775	FJ945895	FJ945993	FJ945840
	Wen 6236 (F)	USA: Illinois	AY677203	FJ945809	DQ382329	AY677207	AY677206
	Wen 6271–1 (F)	USA: North Carolina	FJ945949	FJ945779	FJ945898	FJ945997	FJ945844
	Wen 8570 (US)	USA: Washington DC	FJ945919	FJ945749	FJ945867	FJ945965	FJ945812
	Wen 8747 (US)	Mexico: Chiapas	—	FJ945777	FJ945896	FJ945995	FJ945842
T. rydbergii (Small ex Rydb.) Greene	Wen 10495–4 (US)	Canada: Quebec	—	FJ945792	FJ945910	FJ946010	FJ945857
	Wen 9698 (US)	USA: Texas	FJ945954	FJ945786	FJ945904	FJ946004	FJ945851
	Wen 9760 (US)	USA: Texas	FJ945955	FJ945787	FJ945905	FJ946005	FJ945852
T. striatum (Ruiz & Pavon) Kuntze	Wen 8712 (US)	Mexico: Chiapas	FJ945948	FJ945778	FJ945897	FJ945996	FJ945843
T. succedaneum (L.) Kuntze	Nie & Meng 334 (KUN)	China: Yunnan	FJ945923	FJ945753	FJ945871	FJ945969	FJ945816
	Nie & Meng 339 (KUN)	China: Yunnan	FJ945940	FJ945770	FJ945888	FJ945986	FJ945833
	Nie & Meng 377 (KUN)	China: Guizhou	—	—	FJ945890	FJ945988	FJ945835
	Nie & Meng 429 (KUN)	China: Yunnan	FJ945929	FJ945759	FJ945877	FJ945975	FJ945822
	Nie & Meng 431 (KUN)	China: Yunnan	FJ945931	FJ945761	FJ945879	FJ945977	FJ945824
	Nie & Meng 436 (KUN)	China: Yunnan	FJ945944	FJ945773	FJ945893	FJ945991	FJ945838
	Nie & Zhu 549 (KUN)	Nepal	—	FJ945789	FJ945907	FJ946007	FJ945854
	Nie & Zhu 573 (KUN)	Nepal	—	FJ945790	FJ945908	FJ946008	FJ945855
	Shui et al 82468 (KUN)	China: Yunnan	FJ945961	FJ945799	FJ945916	FJ946016	FJ945864
	Tibet 2196 (KUN, US)	China: Yunnan	—	FJ945793	FJ945911	FJ946011	FJ945858
	Wen 9273 (US)	China: Sichuan	—	FJ945781	FJ945900	FJ945999	FJ945846
T. sylvestre (Sieb. & Zucc.) Kuntze	Nie & Meng 383 (KUN)	China: Zhejiang	FJ945938	FJ945768	FJ945886	FJ945984	FJ945831
	Wen 9275 (US)	China: Hunan	—	FJ945780	FJ945899	FJ945998	FJ945845
T. trichocarpum (Miq.) Kuntze	Nie & Meng 388 (KUN)	China: Zhejiang	FJ945927	FJ945757	FJ945875	FJ945973	FJ945820
T. vernicifluum (Stokes) F.A. Barkley	Nie & Meng 229 (KUN)	China: Guizhou	FJ945939	FJ945769	FJ945887	FJ945985	FJ945832
	Nie & Meng 329 (KUN)	China: Yunnan	FJ945934	FJ945764	FJ945882	FJ945980	FJ945827
	Nie & Meng 376 (KUN)	China: Guizhou	FJ945941	—	FJ945889	FJ945987	FJ945834
	Tibet 1661 (KUN, US)	China: Yunnan	FJ945957	FJ945794	FJ945912	FJ946012	FJ945859
	Wen 9001 (US)	China: Shaanxi	FJ945951	FJ945783	FJ945902	FJ946001	FJ945848
T. vernix (L.) Kuntze	Wen 7146 (F)	USA: Illinois (cult.)	AY541520	FJ945810	DQ382330	AY640471	AY643131
T. wallichii (Hook. f.) Kuntze var. microcarpum T.L. Ming	Nie & Meng 430 (KUN)	China: Yunnan	FJ945930	FJ945760	FJ945878	FJ945976	FJ945823
	Nie & Meng 446 (KUN)	China: Yunnan	FJ945922	FJ945752	FJ945870	FJ945968	FJ945815
	Wen 10611 (US)	China: Yunnan	FJ945960	FJ945798	FJ945915	—	FJ945863
T. yunnanensis C.Y. Wu	Nie & Meng 426 (KUN)	China: Yunnan	FJ945932	FJ945762	FJ945880	FJ945978	FJ945825
	Wen 9148 (US)	China: Yunnan	FJ945950	FJ945782	FJ945901	FJ946000	FJ945847
Actinocheita filicina (D.C.) F.A. Barkley	Panero s. n. (CS)	Mexico	AY641509	FJ945802	DQ382321	AY640460	AY643120
Lithrea ternifolia (Gillies) F.A. Barkley	Nee & Wen 53849	Bolivia	—	FJ945796	FJ945913	FJ946014	FJ945861
Malosma laurina (Nutt.) Nutt. ex Engl.	Miller 34 (CS)	USA: California (cult.)	AY641510	FJ945803	DQ382322	AY640461	AY643121
Mauria heterophylla Kunth	Nee & Wen 53811	Bolivia	FJ945958	FJ945795	—	FJ946013	FJ945860
Pistacia chinensis Bunge	Wen 7090 (F)	USA: California (cult.)	DQ390466	—	DQ382323	DQ390470	DQ390462
Pistacia mexicana Kunth	Parfitt 27 (F)	USA: California (cult.)	DQ390468	—	DQ382325	AY640462	DQ390464
Pistacia mexicana Kunth	Wen 8675 (US)	Mexico: Oaxaca	FJ945959	FJ945797	FJ945914	FJ946015	FJ945862
Pistacia weinmannifolia Poisson	Ji 0174 (KUN)	China: Yunnan	DQ390469	—	DQ382327	DQ390473	DQ390465
Poupartia minor (Bojer) L. Marchand	Wen 9481 (US)	Madagascar: Ihosy	FJ945964	FJ945811	—	FJ946019	—
Rhus choriophylla Wooton & Standl.	Miller 27 (CS)	USA: Arizona	AY641498	FJ945804	DQ382287	AY640450	AY643110
Rhus integrifolia Benth. & Hook f. ex S. Watson	Miller 28 (CS)	USA: California (cult.)	AY641499	FJ945805	DQ382294	AY640451	AY643111
Rhus sandwicensis A. Gray	Wen 7052 (F)	USA: Hawaii	AY641491	FJ945806	DQ382316	AY640445	AY643105
Rhus trilobata Nutt. ex Torr. & Gray	Miller 21 (CS)	USA: Colorado	AY641497	FJ945807	DQ382317	AY640449	AY643109
Rhus typhina L.	Wen 8557 (US)	USA: Washington DC	FJ945920	FJ945750	FJ945868	FJ945966	FJ945813
Schinus molle L.	Wen 6686 (F)	USA: California (cult.)	AY641512	FJ945808	DQ382333	AY640463	AY643123
Searsia dentata (Thunb.) F.A. Barkley	Wen 10090 (US)	South Africa: Kwazulu Natal	FJ945963	FJ945801	FJ945918	FJ946018	FJ945866
Searsia glauca (Thunb.) Moffett	Wen 10051 (US)	South Africa: Western Cape	FJ945962	FJ945800	FJ945917	FJ946017	FJ945865
Searsia natalensis (Bernh. ex Krause) F.A. Barkley	Luke 11694 (US)	Kenya: Nairobi	FJ945953	FJ945785	—	FJ946003	FJ945850
Searsia pyroides (Burch.) Moffett	Luke 11693 (US)	Kenya: Nairobi	FJ945952	FJ945784	FJ945903	FJ946002	FJ945849

1.2 DNA extraction, amplification, and sequencing

Genomic DNA was extracted from silica-gel dried leaf material using the DNeasy extraction kits following the manufacturer's protocol (Qiagen, Mississauga, Canada). The nuclear internal transcribed spacer (ITS) region was amplified using primers ITS4 and ITS5 (White et al., 1990) or N-nc18S10 and C26A (Wen & Zimmer, 1996). Nuclear external transcribed spacer (ETS) was amplified and sequenced using primers ETS1F and 18S-IGS (Pell et al., 2008). The nuclear NIA-i3 intron was sequenced using primers NIA-i3F and NIA-i3R (Howarth & Baum, 2002). The chloroplast ndhF gene (Olmstead & Sweere, 1994) and the trnL-F region (Taberlet et al., 1991) were also sequenced in this study. Amplification reactions were carried out in a 25 μL volume containing 1.5 mmol/L MgCl₂, 0.2 mmol/L each dNTP, 0.2 μmol/L each primer, 1 U Taq polymerase, and approximately 25 ng DNA template. The sequencing reaction was carried out in a 10 μL final volume using the BigDye Terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions and viewed with an ABI 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were edited using Sequencher (version 4.1.4; Gene Codes Corporation, Ann Arbor, MI, USA) and aligned with ClustalX version 1.83 PC version (Thompson et al., 1997), followed by manual adjustment in BioEdit (Hall, 1999). All sequences have been deposited in GenBank with accession numbers (Table 1).

1.3 Phylogenetic analyses

Phylogenetic analyses were carried out using maximum parsimony (MP) and Bayesian inference. MP analysis was done using PAUP* version 4.0b10 (Swofford, 2003) with heuristic search, random taxon addition, tree bisection and reconnection branch-swapping, and the Mulpars and Steepest descent options. Parsimony bootstrap (PB) support was obtained using 1000 replicates, with the random taxon addition limited to 10.

Bayesian inference was carried out using MrBayes version 3.12 (Huelsenbeck & Ronquist, 2001). Nucleotide substitution model parameters were determined for the datasets using the Akaike information criterion in Modeltest version 3.7 (Posada & Crandall, 1998; Posada & Buckley, 2004). Bayesian analyses started from random trees and used four Markov chain Monte Carlo (MCMC) runs, monitoring over two million generations and re-sampling trees every 100 generations. Runs were repeated twice to confirm results. After discarding the burn-in trees, the remaining trees were imported into PAUP* and a 50% majority-rule consensus tree was produced to obtain posterior probabilities (PP) of the clades. Internodes with PP ≥95% in the consensus trees were considered statistically significant.

To evaluate the congruence of the chloroplast and nuclear datasets, we used an incongruence length difference (ILD) test (Farris et al., 1994) as a conservative initial test of data partition congruence (Hipp et al., 2004). The ILD test was carried out with 100 replicates of heuristic searches using tree bisection and reconnection branch-swapping with 10 random sequence additions.

1.4 Dating of divergence times

All nuclear and chloroplast sequences were used for dating divergence times because higher accuracy and more confidence can be inferred based on multiple sequences (Renner, 2005). A likelihood ratio test (Felsenstein, 1988) ruled out a global molecular clock (P < 0.05) for our dataset. Bayesian dating method under a relaxed clock model relying on uncorrelated rates was used in this study (Drummond et al., 2006).

The Bayesian approach to estimate divergent times and their credibility intervals was implemented in Bayesian evolutionary analysis by sampling trees (BEAST) version 1.4.8, which uses an MCMC to co-estimate topology, substitution rates, and node ages (Drummond & Rambaut, 2007). The BEAUti interface (distributed with BEAST) was used to create the input file to run in BEAST. All analyses were done using the HKY model of nucleotide substitution (Hasegawa et al., 1985). Different sequence regions were partitioned and model parameters were unlinked across partitions, with rate variation among sites modeled using a gamma distribution and four rate categories. Our analyses were performed based on a relaxed molecular clock model with uncorrelated rates drawn from a log-normal distribution and the birth-death tree prior (Drummond et al., 2006). Posterior distributions of parameters were approximated using two independent MCMC analyses of 10 million steps each, with the first 10% being discarded. The log files were combined to check for convergence on the same distribution and to ensure adequate sample sizes using Tracer version 1.4 (Rambaut & Drummond, 2007). The samples from the posterior were summarized on the maximum clade credibility tree using TreeAnnotator version 1.4.8 and visualized using FigTree version 1.2. Means and 95% higher posterior densities (HPD) of age estimates were obtained from the combined outputs using Tracer version 1.4.

Wood and pollen fossils of Anacardiaceae have been dated to the Paleocene (Hsü, 1983; Muller, 1984). Thus, a normally distributed calibration prior with the mean 60 mya and standard deviation 3.0 mya (roughly matching the Paleocene epoch 55–65 mya) was constrained for the root age of the family. Reliable macrofossils of Rhus were found from western North America in the middle Eocene (Manchester, 1994). They share many characters with the extant Rhus including lateral compression, longitudinal vascular bundles forming ribs in the mescocarp, and two or more layers of columnar cells in the endocarp (Manchester, 1994). The minimum stem age of Rhus was thus calibrated to be 44 with standard deviation of 1.0 mya. Other earlier fossils are leaf impressions dated back to the late Palaeocene–early Eocene (MacGinitie, 1969; Wilf, 2000) and not used for calibrations.

2 Results

2.1 Molecular analysis

The aligned trnL-F sequences generated a data matrix of 1094 bp with 82 variable sites, 45 of which were parsimony-informative. The ndhF dataset had 2021 aligned positions, with 180 variable sites, 80 of which were parsimony-informative. Because there is no recombination in the chloroplast DNA (cpDNA), we combined the ndhF and trnL-F data directly. The aligned matrix of the combined cpDNA data had 3115 characters including 125 parsimony informative sites. The MP analysis yielded more than 10,000 most parsimonious trees (MPTs) with a length of 350 steps, a consistency index (CI) of 0.82, CI excluding uninformative characters = 0.69, a retention index (RI) of 0.91, and a rescaled consistency index (RC) of 0.74. Phylogenetic trees derived from both MP and Bayesian inference were topologically almost identical and their strict consensus tree with PB and PP values is shown in Fig. 2.

Figure 2. Strict consensus tree of over 100,000 of the most-parsimonious trees of Toxicodendron based on the concatenated chloroplast sequences of trnL-F and ndhF (tree length = 350 steps, consistency index = 0.82, retention index = 0.91, and rescaled consistency index = 0.74). The bootstrap values for 1000 replicates are shown above the branches and the Bayesian posterior probabilities higher than 95% are indicated below.

The ITS dataset comprised 770 aligned positions including 151 parsimony-informative sites. The ETS dataset contained 395 aligned positions, 76 of which were parsimony-informative. The ILD test did not detect any conflict between ITS and ETS, thus we combined the two datasets. The ITS-ETS matrix contained 1165 characters with 227 parsimony informative sites. Over 100,000 MPTs were generated with a length of 934 steps (CI= 0.58, CI excluding uninformative characters = 0.47, RI= 0.77, and RC= 0.45). The MP and the Bayesian trees were topologically similar and only the strict consensus tree with PB and PP values is shown in Fig. 3.

Figure 3. Strict consensus tree of over 100,000 most-parsimonious trees of Toxicodendron based on nuclear internal transcribed spacer and external transcribed spacer sequences (tree length = 934 steps, consistency index = 0.58, retention index = 0.77, and rescaled consistency index = 0.45). The bootstrap values for 1000 replicates are shown above the branches and the Bayesian posterior probabilities higher than 95% are shown below.

The NIA-i3 dataset included 1388 aligned positions including 194 parsimony-informative characters. The MP analysis generated more than 10,000 MPTs with a length of 519 steps, CI= 0.83, CI excluding uninformative characters = 0.75, RI= 0.92, and RC= 0.76. The NIA-i3 strict consensus tree is similar to the Bayesian topology and only the former with PB and PP values is shown in Fig. 4.

Figure 4. Strict consensus tree of over 100,000 most-parsimonious trees of Toxicodendron based on sequences of the nuclear NIA-i3 intron (tree length = 519 steps, consistency index = 0.83, retention index = 0.92, and rescaled consistency index = 0.76). The bootstrap values for 1000 replicates are shown above the branches and the Bayesian posterior probabilities higher than 95% are below.

Results of the ILD test showed significant incongruence among cpDNA, ITS-ETS, and NIA-i3 datasets (P= 0.001), so we did not combine the three datasets in the phylogenetic analyses.

Toxicodendron sections Toxicodendron and Griffithii were well supported to be monophyletic by all analyses (Figs. 2–4), but taxon relationships within section Toxicodendron were not well resolved. For example, samples from North America and eastern Asia were not clearly separated (Figs. 2–4). The Asian Toxicodendron trichocarpum and the neotropical Toxicodendron striatum appear as two highly isolated species in both the ITS-ETS and the cpDNA phylogenies (Figs. 2, 3), but the latter grouped with section Griffithii in the NIA-i3 tree (Fig. 4).

All analyses suggested that section Venenata was not monophyletic (Figs. 2–4). Three clades were recognized within section Venenata. Clade A comprised Toxicodendron succedaneum and Toxicodendron sylvestre with strong support in the ITS-ETS tree (PB = 82%, PP = 100%, Fig. 3), but collapsed in the NIA-i3 strict consensus tree (Fig. 4). Clade B contained only Toxicodendron grandiflorum with robust support in the cpDNA tree (PB = 99%, PP = 100%, Fig. 2). Clades A and B grouped together with strong support in the cpDNA and the NIA-i3 trees, but this clade was not well supported in the ITS-ETS data. All data supported clade C as comprising the eastern Asian Toxicodendron vernicifluum and the eastern North American Toxicodendron vernix (Figs. 2–4).

Relationships among deeper clades of the genus were not well resolved by the cpDNA or ITS-ETS data. However, the NIA-i3 trees revealed that section Griffithii together with T. striatum was sister to a clade including all other Toxicodendron members (Fig. 4).

2.2 Bayesian dating

Results of the age estimates for major clades are presented in Fig. 5. Two clades with disjunct sister pairs between temperate eastern Asia and North America were detected in our phylogenetic analysis. One was from section Toxicodendron with an estimated divergence time of 13.46 mya (7.95–19.42 mya). The other was between the eastern North American T. vernix and the eastern Asian T. vernicifluum, which was estimated to have diverged approximately 7.53 mya (2.76–12.86 mya). A tropical–subtropical disjunct pair between the neotropical T. striatum and the Asian section Griffithii was also recognizable in the results from combined data, and estimated to have diverged 20.84 mya in the early Miocene (11.16–30.52 mya).

Figure 5. Chronogram of Toxicodendron and relatives from within Anacardiaceae based on the combined chloroplast and nuclear sequences inferred using Bayesian evolutionary analysis by sampling trees (BEAST) software. New World taxa are shown in bold; figures in parentheses indicate 95% higher posterior densities. Black stars, fossil constraints; black circles, intercontinental disjunct lineages with age estimates.

3 Discussion

3.1 Phylogeny of Toxicodendron and its position in the Rhus complex

Phylogenetic analyses of both chloroplast and nuclear datasets robustly supported the monophyly of Toxicodendron (Figs. 2–5). Toxicodendron and Rhus s. str. are two major groups nested within the paraphyletic Rhus complex (Heimsch, 1940; Gillis, 1971; Young, 1979; Miller et al., 2001). Rhus s. str. was supported as a distinct genus based on its fruits with red hairs and terminal inflorescences (Barkley, 1937a) and it was strongly supported to be monophyletic by both chloroplast and nuclear datasets (Yi et al., 2004, 2007). Our results also showed Toxicodendron as a clade distinct from other Rhus taxa. Toxicodendron seems to be characterized by a combination of characters including axillary inflorescences, whitish to dune-colored exocarps, which lack glandular pubescence, and the presence of toxic catechols (Barkley, 1937a, 1963; Brizicky, 1963; Gillis, 1971). Nevertheless, these characters also co-occur in other genera in the Rhus complex. For example, based on fruit and flower morphology, Toxicodendron is most closely related to Actinocheita with axillary inflorescences, but chemically it is more similar to Smodingium, Metopium, and Pseudosmodingium, which have toxic resins (Barkley, 1937b; Gillis, 1971; Aguilar-Ortigoza et al., 2004; Aguilar-Ortigoza & Sosa, 2004).

Although most genera in the Rhus complex are well supported to be monophyletic, phylogenetic relationships among these genera have been difficult to resolve (Miller et al., 2001; Yi et al., 2004), possibly due to their early rapid divergence or reticulate evolution. Our chloroplast and ITS data could not convincingly resolve the sister group of Toxicodendron (Figs. 2, 3). However, based on analysis of the NIA-i3 data, Actinocheita was well supported as the sister group of Rhus (PB = 86%, PP = 97%; Fig. 4). More taxon sampling, especially from Pseudosmodingium, Comocladia, and Metopium, and more molecular characters need to be used to resolve the relationships within the Rhus complex.

3.2 Phylogenetic relationships within Toxicodendron

Based on analysis of all three datasets, section Griffithii was well supported as monophyletic (Figs. 2–4). Morphologically, the thick coriaceous leaves and dehiscent exocarps are defining characters of this section. The neotropical T. striatum was initially found to be closely related to this section by the NIA-i3 data (Fig. 4), but this relationship is not supported by the ITS-ETS and chloroplast data (Figs. 2, 3). Morphologically, T. striatum shares more characters with section Venenata and was traditionally placed in that section based on their shared characters including thin and pinnately compound leaves, pendulous infructescences, and glabrous fruits with non-dehiscent exocarps (Barkley, 1937a; Min, 1980). The sometimes erect inflorescence of T. striatum (Woodson et al., 1967) is similar to that of taxa of section Griffithii.

Section Toxicodendron was supported to be monophyletic by all molecular data (Figs. 2–4). Taxa of this section are woody lianas with three leaflets. Bayesian analysis based on the combined data revealed that the eastern Asian T. radicans subsp. hispidum is sister to all trifoliate members from the New World, rather than T. radicans subsp. radicans (Fig. 5), although T. radicans from eastern North America and eastern Asia are morphologically similar to each other based on the presence of entire leaves (Gillis, 1971). Other taxa in this section are morphologically distinct from T. radicans, such as Toxicodendron diversilobum from western North America, in producing leaves sometimes with more than three leaflets and leaflet margins always irregularly lobed.

With the exclusion of section Griffithii, the newly delimited section Venenata seems to be paraphyletic (Figs. 2–4). Nevertheless, three clades (A, B, and C) were consistently recognized within section Venenata, with the exception of clades A and B not supported by the NIA-i3 dataset (Fig. 4). The clades A and B form a group robustly supported by NIA-i3 and chloroplast datasets (Figs. 2, 4). This clade is characterized morphologically by highly asymmetrical and compressed fruits. Clade C comprises a disjunct lineage of the Asian T. vernicifluum and the North American T. vernix. Species in this clade usually have pinnately compound leaves with 7–13 leaflets, greenish flowers, and reniform drupes that are symmetrical. The Chinese T. trichocarpum appeared to be isolated phylogenetically (Figs. 2–4). This species is similar to taxa of section Venenata in that they are shrubs or trees with thin and pinnately compound leaves. However, T. trichocarpum is unique in possessing young branches and fruits covered with bristle-like hairs, unlike any other Toxicodendron taxa, which bear glabrous fruits.

Combined with morphological evidence, our analysis of both plastid and nuclear sequences of Toxicodendron yields several important insights into the phylogeny of the group. Sections Toxiodendron and Griffithii seem to be monophyletic based on the current sampling. Section Venenata, however, is polyphyletic even with the exclusion of section Griffithii. Two additional sections might be segregated from this section, one including T. trichocarpum and the other composed of T.vernix and T. vernicifluum. In addition, the neotropical T. stratium should perhaps be included in the newly recognized section Griffithii (Min, 1980). The infrageneric classification of Toxicodendron will be revised in a separate paper, when we obtain samples of the monotypic sect. Simplicifolia from northern Borneo.

3.3 Intercontinental biogeographic disjunctions

It may seem inappropriate to calibrate the Rhus–Pistacia clade as the stem clade of Rhus because the phylogenetic position of Rhus is not well resolved. But the Rhus complex seems to have radiated. Our estimates under this calibrating scheme are minimum ages for the clades of biogeographic interests in Toxicodendron.

The two distinct north temperate disjunctions recognized by the molecular phylogenetic evidence are not consistent with those traditionally observed based on morphology. For the disjunction in section Venenata, only T. vernicifluum and T. vernix were supported to constitute a disjunct species pair and their divergence time was estimated to be in the middle Miocene to the Pliocene (7.53 mya with 95% HPD of 2.76–12.86 mya; Fig. 5). This disjunct pair is mostly restricted to the north temperate regions, with T. vernicifluum common in eastern Asia ranging from Japan and Korea to India, and T. vernix scattered in swampy areas of eastern North America. Another disjunction is in section Toxicodendron between T. radicans subsp. radicans from eastern North America and subsp. hispidum from central and eastern China. Although each dataset did not well resolve this disjunct lineage within section Toxicodendron, the chronogram generated from BEAST recognized that the eastern Asian subspecies is sister to all the trifoliate taxa from North America rather than its eastern North American counterpart (Fig. 5). This disjunction was estimated to have occurred at 13.46 mya (95% HPD: 7.95–19.42 mya) during the middle to late Miocene.

Studies of various disjunct taxa among eastern North America, western North America, and eastern Asia have shown a closer relationship between eastern and western North America than between eastern North America and eastern Asia, even though taxa from eastern Asia and eastern North America may be morphologically more similar to each other, for example, Aralia sect. Aralia (Wen et al., 1996, 1998), Cornus (Xiang et al., 1998a, b), and Calycanthus (Wen et al., 1996; Zhou et al., 2006a). These results suggested that the close floristic relationship between eastern and western North America and that the morphological similarity of disjunct taxa between eastern North America and eastern Asia could be attributed to similar habitats as a result of morphological convergence (Wen, 1999, 2001; Ickert-Bond et al., 2007).

The two temperate disjunctions are estimated to have occurred during the late Miocene (to Pliocene), which likely favored the migration route by way of the BLB rather than the North Atlantic land bridge (NALB). The BLB was open to terrestrial organisms from at least the early Paleocene until the late Miocene, whereas the NALB was no longer available by the early Miocene (Hopkins, 1967; Axelrod, 1975; Tiffney & Manchester, 2001).

It is difficult to differentiate fossils of Toxicodendron, Rhus, and other closely related taxa, except for the trifoliate section Toxicodendron. Fossils that were assigned to the genus mostly belong to leaves of section Toxicodendron (MacGinitie, 1937; Gillis, 1971, 1975; Ramírez & Cevallos-Ferriz, 2002). Fossil leaves similar to T. radicans before the early Oligocene in western North America and Europe (Gillis, 1975) appeared to be unreliable, because they were much older than our estimated age of section Toxicodendron (Fig. 5). Leaf fossils after the Oligocene were also common in western North America and Mexico (MacGinitie, 1937; Ramírez & Cevallos-Ferriz, 2002). These fossils are similar to the poison ivy of eastern Asia or eastern North America, rather than the western poison oak (T. diversilobum) from the same area (Gillis, 1971). The fossil data seem to suggest that species from section Toxicodendron were once more widely distributed in the two continents. Subsequent climatic changes beginning in the middle Miocene and geologic events including the uplift of the Rocky Mountains in western North America might have caused the extinction of T. radicans and development of T. diversilobum in this area. Fossils of the western poison oak have been found in the Pleistocene of western North America and they were very similar to those of the present-day T. diversilobum (Gillis, 1975). Fossil data from Toxicodendron indicated that western North America may have served as a conduit between eastern Asia and eastern North America in the eastward migration by way of the BLB.

A subtropical to tropical intercontinental disjunct lineage was supported by the NIA-i3 (Fig. 4) and the combined nuclear and plastid sequence data between section Griffithii and T. striatum (Fig. 5), with the former mainly restricted to subtropical to tropical forests in southwestern China to northeastern India, and Nepal (with only Toxicodendron griffithii extending to warm temperate area), and the latter scattered in tropical Americas (from Mexico to Brazil and Peru).

A Laurasian migration route connecting the neotropics and tropical Eurasia through North America and subsequently the NALB has been used to explain the pantropical disjunctions between the Old and the New World (Davis et al., 2002a; Zhou et al., 2006b). The tropical–subtropical Toxicodendron disjunction was estimated to have occurred in the early Miocene (20.84 mya; Fig. 5), matching the last opening period of the NALB at 20–25 mya (Wolfe, 1975; Tiffney, 1985b). Floristic similarities between North America and Europe in the Oligocene and Miocene suggest that some exchange of temperate or deciduous taxa may have continued after the Eocene (Hably et al., 2000). Islands might have served as stepping stones for the post-Eocene exchanges (Tiffney, 1985a, 1985b, 2000; Tiffney & Manchester, 2001). The estimated divergence of the tropical Toxicodendron disjunction coincides with the availability of an NALB migration route after the Eocene, as proposed for Cercis (Davis et al., 2002b), Cornus (Xiang et al., 2005), and Liquidambar (Ickert-Bond & Wen, 2006).

In addition to the NALB route, the BLB has been proposed as an alternative route for the spread of the Northern Hemisphere biota throughout much of the Tertiary (Hopkins, 1967; Tiffney, 1985a). In the case of the subtropical–tropical disjunct lineage of Toxicodendron exchange by way of the BLB seems less plausible, because the high latitude of BLB almost certainly prohibited the migration of thermophilic taxa for most of the time in the Tertiary (McKenna, 1983; Tiffney, 1985a; Wen, 1999; Davis et al., 2002a). However, due to the evolutionary complexity of the Toxicodendron and Rhus groups, additional work needs to be conducted to better resolve the phylogeny and biogeography of the complex.

Acknowledgments

Acknowledgements  This study was supported by grants from the National Basic Research Program of China (973 Program, grant no. 2007CB411601), the National Natural Science Foundation of China (grant nos. 30625004 and 40771073 to H. Sun), the Yunnan Natural Science Foundation (grant no. 2008CC013), and the John D. and Catherine T. MacArthur Foundation (to J. Wen). Laboratory work was carried out in and partially supported by the Laboratory of Analytical Biology of the National Museum of Natural History, Smithsonian Institution, Washington DC, USA. We thank Quentin LUKE for his kind assistance in collecting samples in Kenya.