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(American Journal of Botany. 2009;96:519-530.) doi: 10.3732/ajb.0800195 © 2009 Botanical Society of America, Inc. |
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Systematics and Phytogeography |
2 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, China 3 Department of Biological Sciences, Boise State University, 1910 University Drive, Boise, Idaho 83725 USA
Received for publication 13 June 2008. Accepted for publication 10 October 2008.
ABSTRACT
Accurate classification systems based on evolution are imperative for biological investigations. The recent explosion of molecular phylogenetics has resulted in a much improved classification of angiosperms. More than five phylogenetic lineages have been recognized from Scrophulariaceae sensu lato since the family was determined to be polyphyletic; however, questions remain about the genera that have not been assigned to one of the segregate families of Scrophulariaceae s.l. Rehmannia Liboschitz and Triaenophora Solereder are such genera with uncertain familial placement. There also is debate whether Triaenophora should be segregated from Rehmannia. To evaluate the phylogenetic relations between Rehmannia and Triaenophora, to find their closest relatives, and to verify their familial placement, we conducted phylogenetic analyses of the sequences of one nuclear DNA (ITS) region and four chloroplast DNA gene regions (trnL-F, rps16, rbcL, and rps2) individually and combined. The analyses showed that Rehmannia and Triaenophora are each strongly supported as monophyletic and together are sister to Orobanchaceae. This relation was corroborated by phytochemical and morphological data. Based on these data, we suggest that Rehmannia and Triaenophora represent the second nonparasitic branch sister to the remainder of Orobanchaceae (including Lindenbergia).
Key Words: DNA sequence • familial placement • Orobanchaceae • phylogenetic relations • Rehmannia • Scrophulariaceae sensu lato • Triaenophora
Nothing in biology makes sense except in the light of evolution. —Theodosius Dobzhansky (1964)
As a corollary to Theodosius Dobzhansky’s famous quote, understanding the evolutionary history of organisms can improve our understanding of biology. Recent molecular phylogenetic analyses have resulted in a major rearrangement of angiosperm classification that now better reflects the evolutionary history of these plants. However, many species remain unsampled and thus unplaced in the new classification scheme. A series of molecular systematic studies of Scrophulariaceae s.l. have revealed that the traditionally circumscribed Scrophulariaceae is polyphyletic (Olmstead and Reeves, 1995; dePamphilis et al., 1997; Olmstead et al., 2001; Oxelman et al., 2005). These studies have resulted in recircumscriptions and new descriptions of families to encompass the monophyletic lineages that were recovered. However, questions remain about the genera that have not been assigned to one of the segregate families of Scrophulariaceae s.l., such as Rehmannia and Triaenophora.
The genus Rehmannia Liboschitz consists of six species endemic to China (Chin, 1979), in which R. glutinosa is widely distributed in central China and cultivated in Japan and Korea (Rix, 1987) and is an important species in traditional Chinese medicine. Rehmannia has longstanding controversies surrounding its systematic placement at both the generic and familial levels. It was originally included within Digitalis (Gaertner, 1770) and was established by Liboschitz in 1835 because of its corolla shape and fruit dehiscence (Fischer and Meyer, 1835). Since then, Rehmannia has usually been placed in the tribe Digitaleae in Scrophulariaceae s.l. (Bentham, 1876; Solereder, 1909; Li, 1948; Chin, 1979). Others have suggested Rehmannia to be part of subfamily Cyrtandroideae in Gesneriaceae based on reports of its unilocular ovary (de Candolle, 1845; Hemsley, 1895; Solereder, 1909; Li, 1948; Burtt, 1954).
Some species initially described as Rehmannia have been segregated as monotypic, i.e., Triaenophora and Titanotrichum (Solereder, 1909). Titanotrichum was transferred to Gesneriaceae (Solereder, 1909), a move that is supported by both morphological and molecular data (Burtt, 1954; Wang et al., 1990, 1992, 2002, 2004; Smith et al., 1997a, b; Pan et al., 2002). On the contrary, Triaenophora, which now contains three species (Solereder, 1909; Chin, 1979; Li et al., 2005), has received almost no attention besides Li (1948) who returned it to Rehmannia, and Chin (1979) who later segregated it.
As for the phylogenetic relations of Rehmannia and Triaenophora, one issue is whether there is any phylogenetic affinity with Digitalis (Gaertner, 1770). Digitalis, and Rehmannia/Triaenophora are remarkably different from each other in their corolla shapes and a series of morphological characters such as inflorescence morphology and fruit dehiscence, as well as geographic distribution (Chin, 1979; Wang and Wang, 2005). All phylogenetic analyses of Scrophulariaceae s.l. have placed Digitalis in Plantaginaceae (Olmstead et al., 2001; APG II, 2003; Albach et al., 2005; Oxelman et al., 2005; Tank et al., 2006). A second, equally likely probability is that Rehmannia and Triaenophora are members of Gesneriaceae because Titanotrichum, a segregate from Rehmannia, has been convincingly placed there (Burtt, 1954; Wang et al., 1990, 1992, 2002, 2004; Smith et al., 1997a, b; Pan et al., 2002).
Recently, Rehmannia was included in a cladistic analysis of DNA sequence data for the further disintegration of Scrophulariaceae, in which a single species of Rehmannia was sister to Lancea and Mazus (subfamily Mazoideae of Phyrmaceae sensu Beardsley and Olmstead, 2002) and in a clade with Paulownia, Orobanchaceae, and Phrymaceae (Oxelman et al., 2005). Sampling all six species of Rehmannia, Albach et al. (2007), using nuclear ITS and chloroplast trnL-F and rps16 sequences, placed Lindenbergia as sister to Rehmannia. However, the low sampling of species other than Rehmannia weakens their result (only four species including outgroups are sampled outside Rehmannia).
As we have outlined, the familial placement of Rehmannia and Triaenophora has not been well resolved either from a morphological or a molecular perspective. The debate regarding the familial placement of Rehmannia and Triaenophora in morphology is mainly based on the selection of characters used for classification (Solereder, 1909; Burtt, 1954), many of which have been revealed to be convergent (Olmstead et al., 2001) and thus provide little insight regarding the evolutionary relations for the classification system. Meanwhile, the controversy in molecular data lies in the lack of sufficient sampling among the putative relatives of Rehmannia and Triaenophora and the relation between these two genera.
Greater sampling of taxa putatively close to Rehmannia and Triaenophora and the analysis of additional DNA regions are necessary to determine the familial placement of these two genera and their closest relatives in Lamiales s.l. This study is thus conducted with a comprehensive sampling of putative relatives of Rehmannia and Triaenophora in Lamiales s.l. and the use of five DNA regions (ITS, trnL-F, rps16, rbcL, and rps2) that have been shown to be particularly informative in the Lamiales s.l. (dePamphilis et al., 1997; Smith et al., 1997a, b; Nickrent et al., 1998; Young et al., 1999; Olmstead et al., 2001; Beardsley and Olmstead, 2002; Albach et al., 2005; Oxelman et al., 2005; Wolfe et al., 2005; Tank et al., 2006). The goal of this study was to (1) evaluate the phylogenetic relation between Rehmannia and Triaenophora, (2) find their closest relatives, and thereby (3) verify their familial placement.
MATERIALS AND METHODS
Taxon sampling
We sampled all six species of Rehmannia and two of three species of Triaenophora. To fully examine the putative relatives of Rehmannia and Triaenophora, we selected one species of Paulownia, two genera of Gesneriaceae, six genera of Plantaginaceae (including Digitalis), seven genera of Scrophulariaceae sensu stricto, six genera of Phyrmaceae (four in Phrymoideae and two in Mazoideae including four species of Mazus in addition to the one species sampled in previous studies), 12 representative genera of four major clades in Orobanchaceae sensu lato (including the nonparasitic genus Lindenbergia), three genera of Acanthaceae, two genera of Bignoniaceae, one genus of Lamiaceae, and one genus of Pedaliaceae. Taxon sampling was based on recent molecular systematic studies (dePamphilis et al., 1997; Smith et al., 1997a, b; Nickrent et al., 1998; Young et al., 1999; Olmstead et al., 2001; Beardsley and Olmstead, 2002; Albach et al., 2005; Oxelman et al., 2005; Wolfe et al., 2005; Bennett and Mathews, 2006; Tank et al., 2006). Voucher specimens are deposited in the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE). The materials studied and details of voucher specimens are shown in Table 1.
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and used as the template in the polymerase chain reaction (PCR). The entire ITS region, comprising ITS1, 5.8S rDNA, and ITS2, was amplified with primers ITS1 and ITS4 (Wendel et al., 1995). The trnL-F region was amplified with primers c and f of Taberlet et al. (1991). The rps16 intron was amplified with primers rps16-2F and rps16-R3 (Bremer et al., 2002). The rbcL and rps2 gene regions were amplified with primers RH1 and Z1352R (Olmstead and Reeves, 1995; Wolfe and dePamphilis, 1997; Olmstead et al., 2001), and rps2-18F and rps2-661R (dePamphilis et al., 1997), respectively. PCR products were purified with the UNIQ-10 PCR purification kit (Sangon, Shanghai, China). Sequencing primers were the same as amplification primers. Automated sequencing was performed on a MegaBACE 1000 automatic sequencer (Amersham Biosciences, Sunnyvale, California, USA) using manufacturer’s protocols. The DNA sequences reported in the paper have been deposited in GenBank with accession numbers shown in Table 1. For some genera, sequences of the five regions sampled here were available only for different species. Rather than limit our sampling of either genera or sequences, we combined sequences from different species into a single genus in our analyses provided that there was evidence that the genus was monophyletic and the genus was not the primary focus of our study.
Sequence alignment and phylogenetic analysis
Sequence alignments were made with the program CLUSTAL_X (Thompson et al., 1997) and refined manually for the maximization of sequence homology using the program BioEdit 5.0.9 (Hall, 1999).
Parsimony analysis for each matrix was carried out using maximum parsimony (MP) methods in the program PAUP* version 4.0b10 (Swofford, 2002). All characters and character-state changes were specified as unordered and weighted equally, and gaps were coded as missing data. Heuristic searches were performed with 1000 replicates of random addition, one tree held at each step during stepwise addition, tree-bisection-reconnection (TBR) branch swapping, Multrees in effect, and steepest descent off. To examine the robustness of various clades, we ran a bootstrap analysis (Felsenstein, 1985) with 500 replicates using a heuristic search with 1000 replicates of random sequence addition and TBR branch swapping.
Bayesian inference (BI) was conducted using the program MrBayes version 3.0b4 (Ronquist and Huelsenbeck, 2003). The program Modeltest 3.06 (Posada and Crandall, 1998) was employed to determine the appropriate model of sequence evolution for each DNA data set. Four chains of Markov chain Monte Carlo (MCMC) were each run for 10000000 generations and were sampled every 100000 generations, starting with a random tree. For each run, the first 50 samples before the chains reached stationarity were discarded as burn-in. Posterior probability (PP) was used to estimate robustness.
For combined sequence data, the incongruence-length-difference (ILD) test (Farris et al., 1994) was conducted, using the partition homogeneity test in PAUP* 4.0b10 (Swofford, 2002), to examine the congruence between nuclear ITS and chloroplast data sets. Test settings were 100 random stepwise additions and 1000 replicates of heuristic search with TBR branch swapping (Farris et al., 1994). The resulting P value was used to determine whether the two data sets had significant incongruence (P < 0.05). We excluded species from the combined data set if they only had ITS or cpDNA sequences available.
Topological congruence between the trees constraining Phrymaceae as monophyletic and no constraint was evaluated with the Templeton (1983) test using PAUP* 4.0b10 (Swofford, 2002). The phylogenetic analysis herein was divided into two steps as follows. We first conducted cladistic analyses of combined cpDNA (trnL-F, rps16, rbcL, and rps2) from all sampled taxa. The four chloroplast regions (cpDNA) included in this study formed a single linkage group as part of the chloroplast genome, so conflicts that might arise between data partitions from different sources subject to different evolutionary histories should not exist (Olmstead et al., 2001). The genus Calceolaria was selected as the outgroup based on Olmstead et al. (2001). Attempts to use ITS sequences at this level resulted in numerous ambiguities, and analyses of these sequences resulted in spurious relations among some taxa. Second, based on the described analyses, together with results of previous molecular systematic studies (Beardsley and Olmstead, 2002; Oxelman et al., 2005), we selected different taxa for separate and combined analyses of nrDNA ITS and combined cpDNA (e.g., excluding Scrophulariaceae s.s., Plantaginaceae, Gesneriaceae), focusing on clades presumably closest to Rehmannia/Triaenophora and Orobanchaceae. The representative species of Acanthaceae, Bignoniaceae, Lamiaceae, and Pedaliaceae were selected as the outgroups.
RESULTS
A comparison of the sequences we generated with those published from Albach et al. (2007) indicates that the ITS sequences are identical to each other, the rps16 sequences were 99.76–100% similar, and the trnL-F sequences were 99.29–100% similar. These comparisons confirm the accuracy of both the sequences generated herein and those of Albach et al. (2007) for Rehmannia.
Analyses with all sampled taxa
Data for one or two of the four chloroplast regions are missing for 15 genera, but no genus was missing more than two sequences of the four genes. The entire cpDNA data set consists of 4125 bp, of which 2554 (61.9%) were constant, 721 (17.5%) were variable but uninformative, and 850 (20.6%) were parsimony informative. Parsimony analyses resulted in nine trees of 3511 steps each (consistency index [CI] = 0.623; retention index [RI] = 0.608). One most parsimonious (MP) tree of cpDNA data (Fig. 1) was congruent with the Bayesian tree (the best-fit model GTR + I + G) in topology. The MP tree comprised six main clades labeled A–G. Titanotrichum oldhamii was sister to Streptocarpus in Gesneriaceae (clade A) with high support (bootstrap support [BS] = 94%; posterior probability [PP] = 100%). Clades B and C contained the species of Plantaginaceae (BS = 91%; PP = 100%) and Scrophulariaceae sensu stricto (BS = 87%; PP = 100%), respectively. Clade D was Acanthaceae, Pedaliaceae, Lamiaceae, and Bignoniaceae (BS = 73%, PP = 100%) and clade E was Mazoideae (BS = PP = 100%). Clade F comprised Phrymoideae with BS = PP = 100%. Clade G included Rehmannia, Triaenophora, and Orobanchaceae with BS = 62% and PP = 99%. Rehmannia and Triaenophora formed one strongly supported lineage (BS = 100%; PP = 100%) and was sister to Orobanchaceae, that was likewise strongly supported as monophyletic (BS = 88%; PP = 100%). Paulownia was sister to clade G with low support.
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Phylogenetic relation between Rehmannia and Triaenophora
Triaenophora has been considered closely related to Rehmannia in traditional systematics (Forbes and Hemsley, 1890; Solereder, 1909; Li, 1948; Chin, 1979). Our molecular data show that Rehmannia and Triaenophora form a strongly supported clade, in which Triaenophora is sister to Rehmannia. The sister relation between Rehmannia and Triaenophora is also corroborated by allozymic variability (Li et al., 2007) and numerical analysis of morphological data (Li et al., 2008). This sister relation is not surprising because Rehmannia and Triaenophora share a series of uniform synapomorphies, such as two lateral bracteoles at the base of the pedicel just above the subtending bract (they are aborted early in development in R. chingii, R. solanifolia, and R. glutinosa), four stamens with a gap at the expected site of the adaxial staminode, and the unidirectional initiation of corolla lobes and stamens from the abaxial to the adaxial side (Wang and Wang, 2005). Triaenophora has a series of unique traits distinctive from those of Rehmannia, i.e., five trifid calyx lobes; dense, white, spreading, lanose-villous hairs on the stems, leaves, and pedicels; and a bilocular ovary (Chin, 1979; Wang and Wang, 2005). Rehmannia, as a strongly supported monophyletic group distinct from Triaenophora, is characterized by five revolute and undivided calyx lobes; brown or white glandular hairs on stems, leaves, and pedicels; and one ovarian locule (Chin, 1979; Wang and Wang, 2005).
Our results regarding the relations among species within Rehmannia are in agreement with Albach et al. (2007) and Li et al. (2007, 2008). Albach (D. C. Albach, Johannes Gutenberg-Universität Mainz, Germany, unpublished data) conducted a study similar to ours with the exception that a single species of Triaenophora was included (T. rupestris) and in place of rps2, analyzed ndhF sequences. The results of these two independently conducted studies provide mutual confirmation that Triaenophora and Rehmannia are sister to each other and together are sister to Orobanchaceae. Likewise, both studies find evidence against the monophyly of Phyrmaceae (discussed later). Our results further show that the monotypic genus Titanotrichum initially described as a species of Rehmannia, is not closely related, but better included in Gesneriaceae (Burtt, 1954; Wang et al., 1990, 1992, 2002, 2004; Smith et al., 1997a, 1997b; Pan et al., 2002).
Familial placement of Rehmannia and Triaenophora
Rehmannia and Triaenophora were traditionally placed in the Digitaleae (Scrophulariaceae s.l.) with close affinity to Digitalis (Bentham, 1846, 1876; Forbes and Hemsley, 1890; Wettstein, 1891; Li, 1948; Chin, 1979). However, the inclusion of Rehmannia within Digitaleae was questioned when Oxelman et al. (2005) placed one species of Rehmannia as sister to Mazus and Lancea (Scrophulariaceae s.l. or Mazoideae sensu Beardsley and Olmstead, 2002). In all trees herein, Rehmannia and Triaenophora are shown to not have a close affinity with any Digitaleae. Our results also show no close relation between Rehmannia and Gesneriaceae including Titanotrichum, a relation that has been traditionally suggested (de Candolle, 1845; Hemsley, 1895; Solereder, 1909; Li, 1948; Burtt, 1954).
Rehmannia and Triaenophora, as a monophyletic group, are shown herein to be sister to Orobanchaceae (including Lindenbergia) with moderate to high support BS = 92 for ITS, 59–62 for cpDNA, 94 for combined and high to maximum PP (99–100) from all analyses. Lindenbergia is sister to other parasitic genera of Orobanchaceae with high support in trees of cpDNA and combined ITS and cpDNA data as seen in previous molecular phylogenies (Young et al., 1999; Olmstead et al., 2001; Wolfe et al., 2005; Bennett and Mathews, 2006; Tank et al., 2006) rather than sister to Rehmannia and Triaenophora (Albach et al., 2007). Our results further confirm that Orobanchaceae is a well-supported lineage that includes the holoparasitic members traditionally treated in Orobanchaceae, the hemiparasitic taxa previously treated under Scrophulariaceae s.l. and the nonparasitic genus Lindenbergia (dePamphilis et al., 1997; Nickrent et al., 1998; Wolfe and dePamphilis, 1998; Young et al., 1999; Olmstead et al., 2001; Wolfe et al., 2005; Bennett and Mathews, 2006; Tank et al., 2006). The sister relationship between Rehmannia and Mazoideae recognized in Oxelman et al. (2005) is not supported by our molecular data with greater sampling both in Rehmannia and Mazoideae. Rehmannia and Triaenophora together with Orobanchaceae are sister to Paulownia and Phrymoideae.
On the basis of observations of morphological characters, together with previous reported data (Chin, 1979; Zhang, 1990a), we conclude that Rehmannia, Triaenophora, and Orobanchaceae often have capsules that are half or partly exserted from the persistent calyx tubes, whereas in Phrymaceae, the capsules are completely included in the persistent calyx tube (Fig. 4). Seed coat characters are seldom used for systematic analyses at higher taxonomic levels. However, seed coat characters may provide synapomorphies for some of the relations recovered in our molecular based trees. Rehmannia and Triaenophora are characterized by numerous minute seeds with alveolate and reticulate testa similar to seeds of Orobanchaceae with alveolate, pitted and reticulate testa (except for seeds of Melampyrum with smooth testa; Fig. 4) (Musselman and Mann, 1976; Zhang, 1990b; An and Hong, 2003; Plaza et al., 2004). Meanwhile, Phrymaceae possess minute seeds with smooth testa, and Paulownia is characterized by seeds with membranous wings (Tsoong, 1979; Yang, 1979; Fig. 4). Along with Lindenbergia, Rehmannia, and Triaenophora have tricolporate pollen with reticulate sculpting of the exine (Ying et al., 1993; Hjertson, 1995; Wang et al., 1997), which differs from the characteristic tricolpate pollen (and its variants) with retipilate sculpting of the exine in other parasitic genera in Orobanchaceae (Tsoong and Chang, 1965; Minkin and Eshbaugh, 1989; Bolliger and Wick, 1990; Abu Sbaih et al., 1994; Zhang, 1990b; Lu et al., 2007). The evolutionary trends of pollen exine ornamentation from reticulate to retipilate sculpting have been seen in Dichapetalaceae and in many different groups (Punt, 1976; Minkin and Eshbaugh, 1989), as well as from tricolporate to tricolpate pollen in some taxa (Ferguson and Skvarla, 1981; Hong, 1984; Minkin and Eshbaugh, 1989; Martínez-Ortega et al., 2000). The tricolpate pollen with retipilate sculpting of the exine in parasitic genera of Orobanchaceae may be derived from tricolporate pollen with reticulate sculpting in the nonparasitic genera Lindenbergia, Rehmannia, and Triaenophora.
In addition, phytochemical characters are often used to complement or improve molecular trees (Grayer et al., 1999). Iridoids have been found as natural constituents in most taxa of Lamiales s.l. (Jensen, 1992). Comparison of iridoid glycosides distributed among Rehmannia/Triaenophora, Orobanchaceae, Scrophulariaceae s.s and Plantaginaceae shows that catalpol and aucubin are widely present in these taxa (Kitagawa et al., 1971; Oshio and Inouye, 1982; Grayer et al., 1999; Albach et al., 2007). However, Rehmannia/Triaenophora and most Orobanchaceae (including Lindenbergia) lack harpagide and 6-rhamnopyranosyl-catalpol and their esters, which are characteristic for many Scrophulariaceae s.s. (Jensen et al., 2008). This distribution pattern supports the sister relationship between Rehmannia/Triaenophora and Orobanchaceae. Meanwhile, the lack of sorbitol as the reserve carbohydrate in Rehmannia and its presence among members of Digitaleae (Kitagawa et al., 1971; Taskova et al., 2005; Albach et al., 2007) is further evidence against a close affinity between Rehmannia and Digitaleae.
Lastly, Rehmannia and Triaenophora are characterized by two lateral bracteoles borne at the flower pedicel just above the leaf-like subtending bract (Wang and Wang, 2005). In some species of Rehmannia, they are aborted early in development and cannot be detected at anthesis (Wang and Wang, 2005). Similarly, two lateral bracteoles frequently occur in Orobanchaceae, where they are borne at the pedicel above the leaf-like subtending bract or just below the flower due to a much shortened pedicel (Zhang, 1990a). Rehmannia/Triaenophora and Orobanchaceae (s.s.) are characterized by simple racemes, while Lindenbergia has compound racemes with each branch composed of flowers and several pairs of bracts, all of which are subtended by a large leaf-like bract (Yang, 1979; Zhang, 1990a). The reduction from inflorescence branch to a single or double flower frequently occurs in several major clades of angiosperms, such as Silene in Caryophyllaceae, Salvia in Lamiaceae, and Rhynchoglossum in Gesneriaceae (Weber, 1978; Weberling, 1989; Wang and Li, 2002). The two lateral bracteoles in Rehmannia/Triaenophora and Orobanchaceae (s.s.) might be the result of a transformation from compound racemes to simple racemes; however, further developmental analyses will be necessary to resolve this.
In comparison to other groups in Lamiales s.l. with respect to the distribution of related phytochemical and morphological characters, the combination of the aforementioned features are synapomorphies for Rehmannia/Triaenophora and Orobanchaceae. Based on the molecular results, corroborated by phytochemical and morphological data, we suggest that Rehmannia and Triaenophora represent the second nonparasitic branch sister to the remainder of Orobanchaceae s.l. (including Lindenbergia) or a clade at the rank of family sister to Orobanchaceae. Our results recognizing this sister relationship represent the first step toward better understanding the relations of Rehmannia and Triaenophora with other segregate families of Scrophulariaceae s.l. Further detailed studies are needed to better understand morphological and anatomical synapomorphies among these species.
The familial status of Paulownia and Mazus/Lancea (Mazoideae) remain uncertain in the results presented here. Paulownia has been placed alternately in the Scrophulariaceae s.l., Bignoniaceae, or assigned to a family of its own (Nakai, 1949; Beardsley and Olmstead, 2002). It is distinctively different from Orobanchaceae, Scrophulariaceae s.s. and Phrymaceae in its woody habit, capsule with a persistent woody calyx tube, and seeds with membranous wings (Fig. 4). The phylogenetic analyses herein, with increased sampling of Mazus, indicate that Mazus and Lancea (Mazoideae) may not be included in Phrymaceae as previously suggested by Oxelman et al. (2005). However, Templeton’s tests (see Results, Analyses with selected taxa) do not reject the inclusion of Mazoideae in Phrymaceae. The systematic position of Paulownia and Mazoideae deserves further detailed studies with greater taxon sampling among their putative relatives and new DNA regions together with genetic or evolutionary developmental methods to gain a comprehensive understanding about their phylogenetic history.
FOOTNOTES
1 The authors thank Professor De-Yuan Hong for helpful comments on the manuscript. This study was supported by the National Natural Science Foundation of China Grant 30570105, and CAS Grant KSCX2-YW-R-135. 
4 Author for correspondence (e-mail: wangyz{at}ibcas.ac.cn)
LITERATURE CITED
Abu Sbaih, H. A., D. M. Keith-Lucas, AND S. L. Jury. 1994. Pollen morphology of the genus Orobanche L. (Orobanchaceae). Botanical Journal of the Linnean Society 116: 305–313.[Web of Science]
Albach, D. C., AND M. W. Chase. 2004. Incongruence in Veroniceae (Plantaginaceae): Evidence from two plastid and a nuclear ribosomal DNA region. Molecular Phylogenetics and Evolution 32: 183–197.[CrossRef][Web of Science][Medline]
Albach, D. C., H. Q. Li, N. Zhao, AND S. R. Jensen. 2007. Molecular systematics and phytochemistry of Rehmannia (Scrophulariaceae). Biochemical Systematics and Ecology 35: 293–300.[CrossRef][Web of Science]
Albach, D. C., M. M. Martínez-Ortega, AND M. W. Chase. 2004. Veronica: Parallel morphological evolution and phylogeography in the Mediterranean. Plant Systematics and Evolution 246: 177–194.[Web of Science]
Albach, D. C., H. M. Meudt, AND B. Oxelman. 2005. Piecing together the "new" Plantaginaceae. American Journal of Botany 92: 297–315.
An, B. C., AND S. P. Hong. 2003. Systematic application of seed morphology in Korean Orobanchaceae. Korean Journal of Plant Taxonomy 33: 411–420.
APG II [Angiosperm Phylogeny Group]. 2003. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141: 399–436.[CrossRef][Web of Science]
Beardsley, P. M., AND R. G. Olmstead. 2002. Redefining Phrymaceae: The placement of Mimulus, tribe Mimuleae, and Phryma. American Journal of Botany 89: 1093–1102.
Beardsley, P. M., S. E. Schoenig, J. B. Whittall, AND R. G. Olmstead. 2004. Patterns of evolution in western North American Mimulus (Phrymaceae). American Journal of Botany 91: 474–489.
Bennett, J., AND S. Mathews. 2006. Phylogeny of the parasitic plant family Orobanchaceae inferred from phytochrome A. American Journal of Botany 93: 1039–1051.
Bentham, G. 1846. Scrophulariaceae. In A. de Candolle [ed.], Prodromus systematis naturalis regni vegetabilis, vol. 10, 180–586. Victoris Masson, Paris, France.
Bentham, G. 1876. Scrophulariaceae. In G. Bentham, and J. D. Hooker [eds.], Genera plantarum, vol. 2, 913–980. Reeve and Co., London, UK.
Bolliger, M., AND L. Wick. 1990. The pollen morphology of Odontites (Scrophulariaceae) and its taxonomic significance. Plant Systematics and Evolution 173: 159–178.[CrossRef][Web of Science]
Bremer, B., K. Bremer, N. Heidari, P. Erixon, R. G. Olmstead, A. A. Anderberg, M. Källersjö, AND E. Barkhordarian. 2002. Phylogenetics of asterids based on 3 coding and 3 non-coding chloroplast DNA markers and the utility of non-coding DNA at higher taxonomic levels. Molecular Phylogenetics and Evolution 24: 274–301.[CrossRef][Web of Science][Medline]
Burtt, B. L. 1954. Studies in the Gesneriaceae of the Old Word I: General introduction. Notes from the Royal Botanic Garden Edinburgh 21: 185–218.
Chen, S., K. Guan, Z. K. Zhou, R. Olmstead, AND Q. C. B. Cronk. 2005. Molecular phylogeny of Incarvillea (Bignoniaceae) based on ITS and trnL-F sequences. American Journal of Botany 92: 625–633.
Chin, T. L. 1979. Rehmannia and Triaenophora. In P. C. Tsoong, and H. P. Yang [eds.], Flora reipublicae popularis sinicae, vol. 67, part 2, Scrophulariaceae, 212–222. Science Press, Beijing, China.
de Candolle, A. 1845. Cyrtandraceae. In A. de Candolle [ed], Prodromus systematis naturalis regni vegetabilis, vol. 9, 258–286. Victoris Masson, Paris, France.
de Pamphilis, C. W., N. D. Young, AND A. D. Wolfe. 1997. Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation. Proceedings of the National Academy of Sciences, USA 94: 7367–7372.
Dobzhansky, T. 1964. Biology, molecular and organismic. American Zoologist 4: 443–452.[Web of Science][Medline]
Farris, J. S., M. Källersjö, A. G. Kluge, AND C. Bult. 1994. Testing significance of incongruence. Cladistics 10: 315–319.[CrossRef][Web of Science]
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791.[CrossRef][Web of Science]
Ferguson, I. K., AND J. J. Skvarla. 1981. The pollen morphology of the subfamily Papilionoideae (Leguminosae). In R. M. Polhill, and P. H. Raven [eds.], Advances in legume systematics, part 2, 859–896. Royal Botanic Gardens, Kew, UK.
Fischer, F. E. L., AND C. A. Meyer. 1835. Rehmannia. Index Seminum Hortus Botanicus Imperialis Petropolitanus Promutua Commutione Offert 1: 36.
Forbes, F. B., AND W. B. Hemsley. 1890. Enumeration of all the plants known from China Proper, Formosa, Hainan, the Corea, the Luchu Archipelago and the Island of Hongkong, together with their distribution and synonymy. Journal of the Linnean Society of London. Botany 26: 193–195.
Gaertner, J. 1770. Observationes et descriptions botanicae. Novi Commentarii Academiae Scientiarum Imperialis Petropolitanae 14: 531–547.
Grayer, R. J., M. W. Chase, AND M. S. J. Simmonds. 1999. A comparison between chemical and molecular characters for the determination of phylogenetic relationships among plant families: An appreciation of Hegnauer’s "Chemotaxonomie der Pflanzen." Biochemical Systematics and Ecology 27: 369–393.[CrossRef][Web of Science]
Hall, T. A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
Hemsley, W. B. 1895. Description of some new plants from eastern Asia, chiefly from the island of Formosa, presented by Dr. Augustine Henry, F.L.S., to the Herbarium, Royal Gardens, Kew. Annals of Botany 9: 154–155.
Hjertson, M. L. 1995. Taxonomy, phylogeny and biogeography of Lindenbergia (Scrophulariaceae). Botanical Journal of the Linnean Society 119: 265–321.[Web of Science]
Hong, D. Y. 1984. Taxonomy and evolution of the Veroniceae (Scrophulariaceae) with special reference to palynology. Opera Botanica 75: 1–60.
Jensen, S. R. 1992. Systematic implications of the distribution of iridoids and other chemical compounds in the Loganiaceae and other families of the Asteridae. Annals of the Missouri Botanical Garden 79: 284–302.[CrossRef][Web of Science]
Jensen, S. R., H.-Q. Li, D. C. Albach, AND C. H. Gotfredsen. 2008. Phytochemistry and molecular systematics of Triaenophora rupestris and Oreosolen wattii (Scrophulariaceae). Phytochemistry 69: 2162–2166.[CrossRef][Web of Science][Medline]
Jobson, R. W., AND V. A. Albert. 2002. Molecular rates parallel diversification contrasts between carnivorous plant sister lineages. Cladistics 18: 127–136.[CrossRef][Web of Science]
Kitagawa, I., T. Nishimura, A. Furubayashi, AND I. Yosioka. 1971. Constituents of rhizome of Rehmannia glutinosa f. hueichingensis. Yakugaku Zasshi 91: 593–596.[Web of Science][Medline]
Li, H. L. 1948. A revision of the genus Rehmannia. Taiwania 1: 71–82.
Li, J. H. 2008. Phylogeny of Catalpa (Bignoniaceae) inferred from sequences of chloroplast ndhF and nuclear ribosomal DNA. Journal of Systematics and Evolution 46: 341–348.[Web of Science]
Li, X. D., J. Q. Li, AND Y. Y. Zan. 2005. A new species of Triaenophora (Scrophulariaceae) from China. Novon 15: 559–561.[Web of Science]
Li, X. D., J. Q. Li, AND Y. Y. Zan. 2007. Allozymic variability in Rehmannia and Triaenophora (Scrophulariaceae). Acta Phytotaxonomica Sinica 45: 561–569.[CrossRef][Web of Science]
Li, X. D., Y. Y. Zan, J. Q. Li, AND S. Z. Yang. 2008. A numerical taxonomy of the genera Rehmannia and Triaenophora (Scrophulariaceae). Journal of Systematics and Evolution 46: 730–737.[Web of Science]
Lohan, A. J., AND K. H. Wolfe. 1998. A subset of conserved tRNA genes in plastid DNA of nongreen plants. Genetics 150: 425–433.
Lu, L., H. Wang, S. Blackmore, D. Z. Li, AND L. N. Dong. 2007. Pollen morphology of the tribe Rhinantheae (Orobanchaceae) and its systematic significance. Plant Systematics and Evolution 268: 177–198.[CrossRef][Web of Science]
Martínez-Ortega, M. W., J. Sánchez Sánchez, AND E. Rico. 2000. Palynological study of Veronica Sect. Veronica and Sect. Veronicastrum (Scrophulariaceae) and its taxonomic significance. Grana 39: 21–31.[CrossRef][Web of Science]
Mayer, V., M. Möller, M. Perret, AND A. Weber. 2003. Phylogenetic position and generic differentiation of Epithemateae (Gesneriaceae) inferred from plastid DNA sequence data. American Journal of Botany 90: 321–329.
McDade, L. A., S. E. Masta, M. L. Moody, AND E. Waters. 2000. Phylogenetic relationships among Acanthaceae: Evidence from two genomes. Systematic Botany 25: 106–121.[CrossRef][Web of Science]
Minkin, J. P., AND W. H. Eshbaugh. 1989. Pollen morphology of the Orobanchaceae and rhinanthoid Scrophulariaceae. Grana 28: 1–18.[Web of Science]
Musselman, L. J., AND W. F. Mann. 1976. A survey of surface characteristics of seeds of Scrophulariaceae and Orobanchaceae using scanning electron microscopy. Phytomorphology 26: 370–378.[Web of Science]
Nakai, T. 1949. Classes, ordinae, familiae, subfamilieae, tribus, genera nova quae attinent ad plantas Koreanas. Journal of Japanese Botany 24: 8–14.
Nickrent, D. L., R. J. Duff, A. E. Colwell, A. D. Wolfe, N. D. Young, K. E. Steiner, AND C. W. de Pamphilis. 1998. Molecular phylogenetic and evolutionary studies of parasitic plants. In P. S. Soltis, D. E. Soltis, and J. J. Doyle [eds.], Molecular systematics of plants II: DNA sequencing, 211–241. Kluwer, Boston, Massachusetts, USA.
Olmstead, R. G., C. W. de Pamphilis, A. D. Wolfe, N. D. Young, W. J. Elisons, AND P. A. Reeves. 2001. Disintegration of the Scrophulariaceae. American Journal of Botany 88: 348–361.
Olmstead, R. G., AND P. A. Reeves. 1995. Evidence for the polyphyly of the Scrophulariaceae based on chloroplast rbcL and ndhF sequences. Annals of the Missouri Botanical Garden 82: 176–193.[CrossRef][Web of Science]
Oshio, H., AND H. Inouye. 1982. Iridoid glycosides of Rehmannia glutinosa. Phytochemistry 21: 133–138.[CrossRef][Web of Science]
Oxelman, B., P. Kornhall, R. G. Olmstead, AND B. Bremer. 2005. Further disintegration of Scrophulariaceae. Taxon 54: 411–425.[Web of Science]
Pan, K. Y., Z. Y. Li, AND Y. Z. Wang. 2002. Floral organogenesis of Titanotrichum oldhamii (Gesneriaceae). Acta Botanica Sinica 44: 895–902.
Plaza, L., I. Fernández, R. Juan, J. Pastor, AND A. Pujadas. 2004. Micromorphological studies on seeds of Orobanche species from the Iberian Peninsula and the Balearic Islands, and their systematic significance. Annals of Botany 94: 167–178.
Posada, D., AND K. A. Crandall. 1998. Modeltest: Testing the model of DNA substitution. Bioinformatics 14: 817–818.
Punt, W. 1976. Evolutionary trends in the pollen grains of Dichapetalaceae. In I. K. Ferguson, and J. Muller [eds.], The evolutionary significance of the exine, 139–146. Academic Press, London, UK.
Ree, R. H. 2005. Phylogeny and the evolution of floral diversity in Pedicularis (Orobanchaceae). International Journal of Plant Sciences 166: 595–613.[CrossRef][Web of Science]
Rix, M. 1987. The genus Rehmannia. Plantsman 8: 193–195.
Rogers, S. O., AND A. J. Bendich. 1988. Extraction of DNA from plant tissues. Plant Molecular Biology A6: 1–10 [manual].[CrossRef]
Ronquist, F., AND J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
Rønsted, N., M. W. Chase, D. C. Albach, AND M. A. Bello. 2002. Phylogenetic relationships within Plantago (Plantaginaceae): Evidence from nuclear ribosomal ITS and plastid trnL-F sequence data. Botanical Journal of the Linnean Society 139: 323–338.[CrossRef][Web of Science]
Smith, J. F., K. D. Brown, C. L. Carroll, AND D. S. Denton. 1997a. Familial placement of Cyrtandromoea, Titanotrichum and Sanango, three problematic genera of the Lamiales. Taxon 46: 65–74.[CrossRef][Web of Science]
Smith, J. F., J. C. Wolfram, K. D. Brown, C. L. Carroll, AND D. S. Denton. 1997b. Tribal relationships in the Gesneriaceae: Evidence from DNA sequences of the chloroplast gene ndhF. Annals of the Missouri Botanical Garden 84: 50–66.[CrossRef][Web of Science]
Solereder, H. 1909. Uber die Gattung Rehmannia. Berichte der Deutschen Botanischen Gesellschaft 27: 390–404.
Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.0 b10. Sinauer, Sunderland, Massachusetts, USA.
Taberlet, P., L. Gielly, G. Pautou, AND J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109.[CrossRef][Web of Science][Medline]
Tank, D. C., P. M. Beardsley, S. A. Kelchner, AND R. G. Olmstead. 2006. Review of the systematics of Scrophulariaceae s.l. and their current disposition. Australian Systematic Botany 19: 289–307.[CrossRef][Web of Science]
Tank, D. C., AND R. G. Olmstead. 2008. From annuals to perennials: Phylogeny of subtribe Castillejinae (Orobanchaceae). American Journal of Botany 95: 608–625.
Taskova, R. M., C. H. Gotfredsen, AND S. R. Jensen. 2005. Chemotaxonomy markers in Digitalideae (Plantaginaceae). Phytochemistry 66: 1440–1447.[CrossRef][Web of Science][Medline]
Templeton, A. R. 1983. Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and the apes. Evolution 37: 221–244.[CrossRef][Web of Science]
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, AND D. G. Higgins. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882.
Tsoong, P. C. 1979. Paulownia. In P. C. Tsoong, and H. P. Yang [eds.], Flora reipublicae popularis sinicae, vol. 67, part 2, Scrophulariaceae, 28–44. Science Press, Beijing, China.
Tsoong, P. C., AND K. T. Chang. 1965. Palynological study of Pedicularis and its relation with the taxonomic systems of the genus. Acta Phytotaxonomica Sinica 10: 257–281, 357–374.
Wang, C. N., M. Möller, AND Q. C. B. Cronk. 2004. Phylogenetic position of Titanotrichum oldhamii (Gesneriaceae) inferred from four different gene regions. Systematic Botany 29: 407–418.[CrossRef][Web of Science]
Wang, F. H., N. F. Chien, Y. L. Zhang, AND H. Q. Yang. 1997. Rehmannia. In F. H. Wang [ed.], Pollen flora of China, 390. Science Press, Beijing, China.
Wang, L., AND Y. Z. Wang. 2005. Floral development of Triaenophora (Veronicaceae) and phylogenetic implication. Plant Systematics and Evolution 250: 69–79.[CrossRef][Web of Science]
Wang, W. T., K. Y. Pan, AND Z. Y. Li. 1990. Gesneriaceae. In W. T. Wang [ed.], Flora reipublicae popularis sinicae, vol. 69, 125–581. Science Press, Beijing, China.
Wang, W. T., K. Y. Pan, AND Z. Y. Li. 1992. Key to the Gesneriaceae of China. Edinburgh Journal of Botany 49: 5–74.
Wang, Y. Z., AND Z. Y. Li. 2002. Inflorescence development of Whytockia (Epithemateae, Gesneriaceae) and phylogenetic implications within Gesneriaceae. Plant Systematics and Evolution 236: 45–54.[CrossRef][Web of Science]
Wang, Y. Z., Z. Y. Li, K. Y. Pan, AND X. H. Zou. 2002. Pattern and significance of seedling development in Titanotrichum oldhamii (Gesneriaceae). Acta Botanica Sinica 44: 903–907.
Weber, A. 1978. Beiträge zur Morphologie und Systematik der Klugieae und Loxonieae (Gesneriaceae). VII. Spross-Infloreszenz-und Blütenbau von Rhynchoglossum. Botanische Jahrbücher für Systematik 99: 1–47.
Weberling, F. 1989. Morphology of flowers and inflorescences. Cambridge University Press, Cambridge, London, UK.
Wendel, J. F., A. S. Schnabel, AND T. Seelanan. 1995. Bidirectional inter locus concerted evolution following allopolyploid speciation in cotton (Gossypium). Proceedings of the National Academy of Sciences, USA 92: 280–284.
Wettstein, R. 1891. Scrophulariaceae. In A. Engler, and K. Prantl [eds.], Die Natürlichen Pflanzenfamilien, 39–107. Wilhelm Engelmann, Leipzig, Germany.
Wolfe, A. D., AND C. W. dePamphilis. 1997. Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche. Plant Molecular Biology 33: 965–977.[CrossRef][Web of Science][Medline]
Wolfe, A. D., AND C. W. dePamphilis. 1998. The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants. Molecular Biology and Evolution 15: 1243–1258.[Abstract]
Wolfe, A. D., C. P. Randle, S. L. Datwyler, J. J. Morawetz, N. Arguedas, AND J. Diaz. 2006. Phylogeny, taxonomic affinities, and biogeography of Penstemon (Plantaginaceae) based on ITS and cpDNA sequence data. American Journal of Botany 93: 1699–1713.
Wolfe, A. D., C. P. Randle, L. Liu, AND K. E. Steiner. 2005. Phylogeny and biogeography of Orobanchaceae. Folia Geobotanica 40: 115–134.[CrossRef]
Yang, H. B. 1979. Lindenbergia, Lancea, Mimulus and Mazus. In P. C. Tsoong, and H. P. Yang [eds.], Flora reipublicae popularis sinicae, vol. 67, part 2, Scrophulariaceae, 94–196. Science Press, Beijing, China.
Ying, T. S., Y. L. Zhang, AND D. E. Boufford. 1993. The endemic genera of seed plants of China. Science Press, Beijing, China.
Young, N. D., K. E. Steiner, AND C. W. dePamphilis. 1999. The evolution of parasitism in Scrophulariaceae/Orobanchaceae: Plastid gene sequences refute an evolutionary transition series. Annals of the Missouri Botanical Garden 86: 876–893.[CrossRef][Web of Science]
Zhang, Z. Y. 1990a. Orobanchaceae. In W. T. Wang [ed.], Flora reipublicae popularis sinicae, vol. 69, 69–124. Science Press, Beijing, China.
Zhang, Z. Y. 1990b. Studies on the pollen morphology and seed coat of the genus Cistanche (Orobanchaceae) in China. Acta Phytotaxonomica Sinica 28: 294–298.
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50%) are above the branches; Bayesian posterior probabilities (PP) (
part of capsule exserted from the persistent calyx tube; 
capsule included in persistent calyx tube; 
capsule almost completely exserted from the persistent calyx tube; 
seeds with alveolate and pitted testa; 
seeds with smooth or anomalous reticulate surfaces; 
seeds with membranous wings; 
calyx of connate sepals with spinescent apices; 
calyx with long triangular and lanceolate calyx lobes; 
corolla with two highly reduced and triangular upper lobes; 
corolla with two oblong and orbicular upper lobes.