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Systematics and Phytogeography |
University of Missouri–St. Louis, 1 University Boulevard, St. Louis, Missouri 63121 USA; Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299 USA
Received for publication December 4, 2006. Accepted for publication June 14, 2007.
ABSTRACT
We used the chloroplast gene ndhF to reconstruct the phylogeny of the moonseed family (Menispermaceae), a morphologically diverse and poorly known cosmopolitan family of dioecious, primarily climbing plants. This study includes a worldwide sample of DNA sequences for 88 species representing 49 of the 70 genera of all eight traditionally recognized tribes. Phylogenetic relationships were estimated, and the Shimodaira–Hasegawa test was used to compare the likelihood of alternative phylogenetic hypotheses and to evaluate the monophyly of tribes currently in use. The monospecific Indo-Malesian Tinomiscium is sister to the remaining members of the family, within which are two major clades. Within these two clades, well-supported clades correspond to four of the eight traditionally recognized tribes, while others, such as Menispermeae, are polyphyletic. Mapping of major morphological characters on the phylogeny indicates that the crescent-shaped seed is derived from a straight seed, the tree habit has arisen multiple times, endosperm has been lost many times, but unicarpellate flowers evolved only once. Morphological synapomorphies for Menispermaceae include the presence of a condyle, a large embryo, and druplets. The phylogeny provides for the first time a detailed molecular-based assessment of relationships in Menispermaceae and clarifies our understanding of morphological diversification within the family.
Key Words: Menispermaceae • moonseed • ndhF • phylogeny • Ranunculales • synandrium
Menispermaceae are a cosmopolitan family of mostly dioecious climbing plants, rarely trees, shrubs, or herbs, consisting of about 70 genera and approximately 500 species. Most members are tropical, but a few are found in temperate regions (i.e., all Menispermum L. and Cocculus DC. species). Menispermaceae are included in Ranunculales (in the early diverging eudicots [APG, 1998
, 2003
]), and molecular data have confirmed its monophyly and its sister relationship to the Ranunculaceae + Berberidaceae clade (Hoot et al., 1999
; Kim et al., 2004
). Most of the woody members of Menispermaceae are characterized by the presence of successive cambia, which form rings or partial rings of vascular bundles that are radially separated by multiseriate rays. Tangentially, the rings are separated by a connective tissue of mixed stone cells and parenchyma cells (Mennega, 1982
; Carlquist, 1988
, 1996
; Ortiz, 2000
). The flowers are unisexual, inconspicuous, and although usually trimerous and actinomorphic, a few genera such as Cissampelos L., Stephania Lour., and Cyclea Arn. ex Wight have zygomorphic pistillate flowers (Wang et al., 2006
). Carpels are free and usually 3–6 in number, although the three zygomorphic genera have flowers with only a single carpel. Moreover, up to 30 carpels have been reported in some African species [e.g., Tiliacora dinklagei Engl., T. latifolia Troupin, Triclisia sacleuxii (Pierre) Diels, and T. lanceolata Troupin], and up to 60 carpels in Triclisia dictyophylla Diels (Forman, 1986
). The stamens are mainly free, although in several genera they form a synandrium. The leaves are typically simple, but compound leaves are observed in the Malagasy Burasaia Thouars and in the South American Disciphania cubijensis (R. Knuth) Sandwith. Leaf venation is usually palmatinerved, but a few genera have pinnate venation. Leaves are typically more or less subpeltate, with the insertion of the petiole being scarcely in from the margin of the lamina, but a few taxa are strongly peltate. The petiole is pulvinate at both ends. Despite the extensive variation displayed by most morphological traits, the main feature historically used to define the family is the curved seed found in many of the genera, hence the common name "moonseed" family. Concomitant with the curving of the seed is the curving of the embryo. Moreover, the endocarp is variously ornamented and provides important taxonomic characters for distinguishing between and within genera.
Several members of the family are known for their medicinal and toxic components. Among these, the best known is curare, the South American dart and arrow poison, traditionally used by the rainforest people for hunting. Knowledge of that use led to the synthesis of D-tubocurarine, a muscle relaxant utilized in surgical procedures in Western medicine until the early 1980s and for which the sole source was Chondrodendron tomentosum Ruiz and Pav. (Sneader, 1985
; Phillips, 1991
). Menispermaceae are also economically important as hosts of the larvae of fruit-piercing moths, which as adults are pests for a range of commercial fruits and cause considerable loss of crops in Australia and in most of the Old World tropics (Fay, 1996
). Several other uses are reported for a number of Menispermaceae species (see Phillips, 1991
and citations therein for reports of those uses in Asia and in Africa).
Furthermore, Menispermaceae, with its predominantly climbing habit, also plays a large role in many aspects of tropical forests (Schnizer and Bongers, 2002
), and its importance may be increasing with global climate change (Phillips et al., 2002
; Wright et al., 2004
). The family also contributes to overall species diversity, most significantly in lowland tropical forests (Gentry, 1991
; Burnham, 2002
).
Previous classifications have stressed primarily not only characters of the fruit (endocarp), but also of the seeds (endosperm and embryos). However, different weights attached by different authors to these characters have resulted in the recognition not only of heterogeneous assemblages, but also contentious suprageneric classifications.
Miers (1851)
outlined the first suprageneric taxonomic system of the family and recognized six tribes (Table 1). Those were reduced to five by Hooker and Thomson (1855)
. Miers's original tribes were either split or lumped into their newly named tribes. Later, Bentham and Hooker (1862)
recognized only four tribes. These were essentially Hooker and Thomson's tribes, but their Coscinieae were included in Bentham and Hooker's Tinosporeae. Miers (1864)
later expanded his original six tribes to seven (Table 1). In 1888, Prantl (1888)
recognized four tribes. Prantl followed mainly Bentham and Hooker's system but included their Cissampelideae in Cocculeae and created Limacieae (Table 1).
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, who recognized eight tribes, the tribe Menispermeae being further divided into three subtribes (Table 2). Like earlier workers, Diels relied mainly on characters of the seeds in combination with those of floral parts and of endocarps. Characters such as endosperm presence or absence, type of endosperm when present, carpel number, and endocarp ornamentation were most important. Features of secondary importance in Diels's classification include a combination of other characters such as endocarp position, shape of the condyle (intrusion of the endocarp into the seed), number and arrangement of floral parts, and cotyledon texture. Kessler (1993)
circumscribed the tribes more broadly than Diels and recognized only five tribes. His tribal classification depended heavily on a small number of seemingly morphologically homogeneous characters; these characters have since been shown to be phylogenetically unreliable, thus leading to the recognition of paraphyletic assemblages. Moreover, Kessler did not mention several genera (including Pachygone Miers of his Pachygoneae, although this omission has recently been corrected [Kubitzki, 2007
]).
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, 1972
; Forman, 1975
, 1986
; Kessler, 1993
). Although several recent regional taxonomic and/or floristic treatments have been published (e.g., Troupin, 1962
; Barneby, 1970
, 1972
; Barneby and Krukoff, 1971
; Forman, 1986
; Ortiz, 1997
, 2001
, 2005
), phylogenetic studies in Menispermaceae have been largely neglected (but see Hong et al., 2001
), most likely because of its cosmopolitan and primarily tropical distribution, predominantly scandent habit, and inconspicuous flowers. Hence, while most other families in Ranunculales have been the foci of phylogenetic studies, and even model systems for gene expression patterns (Kramer et al., 2003
), in Menispermaceae the most comprehensive taxonomy and classification date back to the early 1900s.
Earlier workers of the family (Diels, 1910
; Forman, 1986
; Thanikaimoni, 1986
) suggested that characters such as the arborescent habit, simple leaves with entire lamina, and pinnate leaf venation were ancestral. Similarly, ancestral fruit characters were suggested to be a sessile drupe with an apical stylar scar, thick and smooth endocarps lacking a condyle, and a seed with fleshy cotyledons lacking endosperm. Conversely, the reduction and fusion of the stamens, modification of the anther position, and the reduction in the number of carpels (Diels, 1910
; Thanikaimoni, 1986
; Forman, 1986
) were suggested as derived conditions.
Our understanding of the affinities and therefore character diversification has been hindered by the lack of a comprehensive phylogenetic hypothesis for the family, as was widely acknowledged in a symposium on the Ranunculiflorae (Endress, 1995
; Hoot and Crane, 1995
; Loconte et al., 1995
).
Therefore, the general objective of this study is to develop a phylogenetic hypothesis for Menispermaceae based on sequences of the chloroplast gene ndhF. Specific objectives are (1) to reevaluate previous classifications by testing the monophyly of the tribes, and (2) to explore the evolution of several morphological characters with emphasis on those that have been traditionally used in infrafamilial classifications.
The results presented here provide an important contribution to our understanding of the phylogenetic affinities in Menispermaceae and also provide a background within which to examine the diversification of morphological traits. Additionally, our results lay the foundations for developing a new classification of the family that more closely reflects phylogenetic relationships and to study the biogeographic history of this geographically widespread plant family.
MATERIALS AND METHODS
Taxon sampling
This study includes a broad sampling across the eight tribes and the three subtribes of Menispermeae recognized by Diels in 1910 (Table 2), thus representing most of the morphological variation encountered in the family. We sampled 49 of the 70 genera and 88 of the 500 species in the family Menispermaceae (see Appendix). We also sampled four other taxa from the closely related family Lardizabalaceae. Sequences for five additional taxa—one Lardizabalaceae, two each of Berberidaceae and Ranunculaceae—were downloaded from GenBank. The compiled matrix includes sequences of 97 taxa for the ndhF chloroplast gene.
DNA isolation, amplification, and sequence alignment
Total DNA was extracted from silica gel-dried and herbarium material using modified CTAB protocols of Doyle and Doyle (1987)
, Porebski et al. (1997)
, S. Stefanovi
(University of Washington, unpublished data), as well as the DNeasy plant mini Kit (Qiagen, Valencia, California, USA). The chloroplast ndhF gene for six species (Appendix) was initially amplified using primers described by Olmstead and Sweere (1994)
. PCR fragments were purified, cloned, and sequenced as described in Lohmann (2006)
. Aligned sequences for the six cloned species were subsequently submitted to the primer prediction program (Primaclade) (Gadberry et al., 2005
) to obtain suitable primers for Menispermaceae. Thus, all primers used in amplification were specifically designed for this study, with exception of the 5F region, which was amplified using the primer developed by Beilstein et al. (2006)
. We amplified the plastid ndhF gene usually in two overlapping fragments, 5F/1204R and 995F/2097R (see Appendix S1, Supplemental Data accompanying the online version of this article). However, for difficult taxa, the gene was amplified in smaller fragments, namely 5F/995R and 807F/1204R, 995F/1534R, and 1204F/2097R (Table 3). PCR reactions contained a total volume of 30 µL for the first fragment (5F/1204R) and 40 µL for the second fragment (995F/2097R). The different volumes were designed to accommodate the number of ambiguous nucleotides or degenerate sites present in the 2097R primer. In addition to 1 µL of DNA template, the mix contained 5 µL 10x reaction buffer, 5 µL 2.5 mM MgCl2, 3 µL dNTP 2.5 mM, and 4 µL or 10 µL of each primer (10 µM solution) for the first and second fragment respectively. A hot-start PCR was used; PCR cycles included 4 min at 94°C at the end of which 0.5 µL Taq polymerase (5 units/ µL) (Promega, Madison, Wisconsin, USA) was added to the reactions. Thermocycling conditions were as follows: 40 cycles of 1 min at 94°C, 1 min at 52°C, 3 min at 68°C, followed by a 10 min extension at 72°C. PCR products were purified either by using a QIAquick PCR purification kit (Qiagen) or by running the total volume in a 4% agarose gel. The product was recovered by using a QIAquick gel extraction kit (Qiagen).
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), and only sequences with PHRED scores 
20 were kept for further analysis. Sequences ranged from 80–98% double-stranded with the exception of Stephania rotunda Lour. and Tinomiscium petiolare, which were only 77% and 78% double-stranded, respectively. Sequences were translated to check for stop codons in MacClade 4.06 for OS X (Maddison and Maddison, 2003
). Alignment was made both by eye at the amino acid level in MacClade 4.06 for OS X (Maddison and Maddison, 2003
) and in ClustalX version 1.81, then manually modified in MacClade. Only the data matrix from the MacClade edited ClustalX alignment was used in subsequent analyses. Although protein sequences were used as an aid to alignment, all analyses were done using nucleotides.
Phylogenetic analysis
Phylogeny was estimated using maximum parsimony (MP), maximum likelihood (ML), and Bayesian approaches, with characters equally weighted and indels coded as missing data. All analyses were run on the Beowulf Cluster at the University of Missouri-St. Louis.
Parsimony analysis
The parsimony ratchet was conducted using the program PAUPMacRat (Sikes and Lewis, 2001
) implemented in PAUP* version 4.0b 10 for UNIX (Swofford, 2002
). Twenty replicates of 200 iterations each were performed, with 15% of the characters reweighted for each iteration. Resulting trees were retrieved and a strict consensus tree was computed in PAUP* version 4.0b 10 (Swofford, 2002
). Support for individual branches or clades was estimated using bootstrap values (Felsenstein, 1985
). Bootstrap values were from 1000 full heuristic bootstrap replicates, one random sequence addition, tree-bisection-reconnection (TBR) swapping, and MULTREES=yes.
Maximum likelihood analysis
The TVM + I + G model was chosen as the best-fit model of nucleotide substitution using both the AIC and LRT criteria in Modeltest (version 3.6; Posada and Crandall, 1998
). Maximum likelihood searches were implemented in PAUP* using 10 random sequence additions, TBR branch swapping, and MULTREES=yes. To assess support for individual clades, a likelihood bootstrap analysis of 100 replicates with one random sequence addition, TBR swapping, and MULTREES=yes was carried out.
Bayesian analysis
A Bayesian analysis was conducted using MrBayes version 3.1 (Huelsenbeck and Ronquist, 2003) and the GTR +I + G model obtained with the aid of MrModeltest 2.2 (Nylander, 2004
). Two independent analyses were each run for 5 000 000 generations, with four chains, and sampling every 1000 trees. After termination of the independent runs, we first identified and discarded the sample points that were collected before convergence of the chains. To confirm that the chains had achieved stationarity, we plotted log-likelihood scores, tree length, and the shape parameter of the gamma distributed rate variation against generation time for each individual run. This was estimated to be at ca. 143 000 and ca. 137 000 generations, respectively. To be conservative, we discarded the first 200 000 generations corresponding to 2000 trees of each analysis as burn-in. The remaining trees (3001 from each run) were loaded into PAUP* and summarized in a 50% majority rule consensus tree.
Shimodaira–Hasegawa test
We performed the Shimodaira–Hasegawa test (S-H) (Shimodaira and Hasegawa, 1999
; Goldman et al., 2000
) to statistically compare the most likely tree with alternative phylogenetic hypotheses obtained by parsimony and Bayesian approaches. Additionally, we used the S-H test to evaluate support for the monophyly of narrowly circumscribed tribes of Diels (1910)
and broadly circumscribed tribes of Kessler (1993)
; 19 constraint trees were constructed in MacClade. These trees corresponded to all eight tribes of Diels (1910)
and all five of those of Kessler (1993)
as monophyletic both simultaneously and individually, and also all three of Diels's subtribes of Menispermeae as monophyletic both simultaneously and individually. The different constraint tree topologies were then evaluated in PAUP* for likelihood estimates by using the model of evolution and parameters from the original likelihood analysis. Lastly, the topologies recovered under likelihood, Bayesian, and parsimony analyses, as well as the most likely topologies inferred under the constraints were input into PAUP*, and a Shimodaira-Hasegawa test was performed using 1000 bootstrap replicates and the RELL (resampling estimated log likelihoods) option.
Evaluation of major morphological transitions
We explored the diversification of selected morphological characters such as (1) growth habit—climber, tree/shrub, herb; (2) gynoecium merosity—1, 3, 6, more than 6; (3) endosperm condition—present, absent; (4) embryo type—straight, curved, coiled; (5) fruit type—druplet, berry, follicle; (6) condyle condition—absent, present; (7) embryo size—small, large; (8) leaf type—simple, compound; and (9) leaf venation type—pinnatinerved, palmatinerved. We focused on these traits not only because they have traditionally been considered important in infrafamilial classifications, but also to hypothesize the evolution of those characters in the family. We scored the traits for taxa represented in the phylogeny—for ingroup and outgroup taxa—using information either from the literature (mainly Diels, 1910
; Troupin, 1962
; Forman, 1986
) or from personal observation. We used MacClade 4.06 (Maddison and Maddison, 2003
) to trace the selected characters by overlaying them onto the parsimony ratchet strict consensus tree. Additionally, because the accelerated transformation (ACCTRAN) and delayed transformation (DELTRAN) options can not be applied when polytomies are present, we used one of the 326 most parsimonious trees on which the polytomies were manually resolved in MacClade 4.06 to explore the impact of alternative topologies in character-state reconstruction. On this tree we explored all most-parsimonious optimizations, including ACCTRAN and DELTRAN options.
RESULTS
Sequence variation and divergence in ndhF
The data alignment was 2091 nucleotides (nt) long. The longest sequences were obtained for Abuta grandifolia (Mart.) Sandwith and Telitoxicum peruvianum Moldenke, with 2148 and 2147 nucleotides, respectively, while the shortest sequence was observed in Triclisia subcordata Oliv., with 1977 nucleotides. However, sequences for most taxa ranged between 2002 and 2098 nucleotides. Menispermaceae ndhF sequences are A-T rich, as expected, and have the following mean base frequencies: A = 0.28, C = 0.16, G = 0.17, and T = 0.37. A 9-nt deletion (positions 631–639) appeared only in Calycocarpum lyonii (Pursh) A. Gray, while a 9-nt insertion (positions 1930–1938) was shared by the five Cissampelos species and Cyclea fansipanensis Gagnep. On the other hand, a 12-nt deletion (positions 1591–1602) is shared by Tinospora smilacina Benth. and Limacia esiangkara F.M. Bailey, whereas a different 12-nt deletion (positions 1720–1731) sets apart Stephania japonica (Thunb.) Miers. Of the 2091 characters used, 911 (44%) were variable and 573 (27%) were potentially parsimony informative. Uncorrected pairwise sequence divergence within the ingroup varied from 0% between Menispermum dauricum DC. and M. canadense L., to 9.4% between Odontocarya tripetala Diels and Stephania rotunda Lour.; including outgroups, the greatest divergence is 16%, between S. rotunda and Berberis higginsiae Munz.
Phylogenetic analyses
The 20 parsimony ratchet replicates of 200 iterations yielded a total of 4020 trees; of those, 326 were equally parsimonious with a length of 2121 steps, a consistency index excluding uninformative characters = 0.50, and a retention index = 0.84. The maximum likelihood searches produced a tree with a –ln L = 15394.15718. The 6002 trees retained after the burn-in from the two runs of the Bayesian analysis were summarized into a majority rule consensus tree.
Phylogenetic hypotheses resulting from parsimony ratchet, likelihood, and Bayesian approaches are largely congruent as shown by the number of shared nodes and estimated measures of support (likelihood and parsimony bootstrap vs. posterior probabilities) (Fig. 1).
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(Fig. 1). Coscinieae are strongly supported (100/100/100) and include Anamirta cocculus (L.) Wight & Arn. and Arcangelisia flava (L.) Merr.
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) Anomospermeae (100/96/100), Tiliacoreae (100/100/100), and Hyperbaeneae (100/98/100) (Fig. 1). Anomospermeae, plus the former Menispermeae Legnephora moorei (F. Muell.) Miers, Pericampylus glaucus (Lam.) Merr., Sarcopetalum harveyanum F. Muell., and Hypserpa decumbens (Benth.) Diels, form a strongly supported clade (100/100/100), here termed clade A. Relationships between these former Menispermeae and Anomospermeae are unresolved (Fig. 1).
Sister to clade A is a clade with moderate support (88/72/100). Within this clade, the early branch consists of Limacia blumei (Boerl.) Diels, a southeast Asian species, which is in turn sister of a larger, well-supported clade (100/100/100) that includes clade B as sister to clade C + Tiliacoreae. Clade B has moderate support (94/71/100) and contains many former Menispermeae taxa; among those, the Malagasy Rhaptonema sp. and Strychnopsis thouarsii are sister to a clade strongly supported as monophyletic (100/100/100) that includes species of Cissampelos, Cyclea, and Stephania. Clade B is in turn sister to a weakly supported clade (78/60/—), consisting of Tiliacoreae and clade C; both clades are individually strongly supported (100/100/100) (Fig. 1).
Within Tiliacoreae, the three neotropical members sampled (Chondrodendron Ruiz & Pav., Curarea Barneby & Krukoff, and Sciadotenia Miers) form a well-supported clade (100/99/100). Another strongly supported (100/100/100) monophyletic group is formed by Pycnarrhena longifolia (Decne. ex Miq.) Becc. + P. novoguineensis Miq., whereas the clade including Albertisia porcata Breteler + Anisocycla linearis Pierre ex Diels, Tiliacora gabonensis Troupin + T. funifera (Miers) Oliv., and Triclisia dictyophylla Diels + T. subcordata Oliv., has only moderate support (100/96/62). Relationships among those three small clades are unresolved, as is the placement of Beirnaertia cabindensis (Exell & Miers) Troupin (Beirnaertia is monospecific) and Carronia protensa (F. Muell.) Diels (Fig. 1).
Clade C includes the well-supported Hyperbaeneae, considered by Diels to be monogeneric and confirmed here as containing solely the neotropical genus Hyperbaena Miers ex Benth. Sister to the Hyperbaeneae, although with low support (97/69/—), is Cocculus orbiculatus (L.) DC. Other groupings within clade C include the sister relationships between Cocculus carolinus (L.) DC. and C. diversifolius DC. (100/100/100), and a moderately supported (100/92/—) sister relationship between Cocculus laurifolius DC., Pachygone ovata (Poir.) Diels + P. loyaltiensis Diels. The last relationship is strongly supported (100/100/100) (Fig. 1).
S-H test and tribal classification
The S-H test rejects the enforced monophyly of Diels's tribal classification (when monophyly was enforced simultaneously for all eight tribes). Menispermeae and Tinosporeae were rejected when monophyly was enforced for each tribe individually, but Fibraureae and Peniantheae were not rejected. On the other hand, the S-H test rejected the enforced monophyly not only of Kessler's system as a whole (topology of all five tribes enforced as monophyletic simultaneously), but also the enforced monophyly of each individual tribe (Table 4).
DISCUSSION
Phylogenetic affinities and morphological characterizations
The phylogenetic hypothesis presented here is based on a broad taxonomic sampling that also reflects the range of morphological variation found within Menispermaceae. The monophyly of the family and several relationships within are strongly supported in our parsimony, likelihood, and Bayesian analyses of sequences of the ndhF chloroplast gene. Additionally, several clades are also distinguished morphologically and congruent taxonomically with the infrafamilial classification of Diels (1910)
, while others are not. Our results revealed that Menispermaceae are characterized morphologically by the presence of druplets, the presence of a condyle, and large embryos. The condyle—the ventral intrusion of the endocarp into the seed, or the formation of a ventral cavity (Forman, 1986
)—was characterized by Miers (1864)
as resulting from an enlargement of the placenta that shaped the developing ovule. However, later anatomical observations suggest that the curvature in the seed, and consequently the condyle, results from the development of the funicle (Joshi, 1939
; Sastri, 1954
).
All analyses consistently recovered two major clades within Menispermaceae, with Tinomiscium petiolare, an Indo-Malesian centered taxon, as sister to the remainder of the family (Figs. 1, 2). Tinomiscium petiolare is characterized by a straight endocarp and by a straight embryo that has foliaceous and imbricate cotyledons (Fig. 2). Additionally, Tinomiscium has tricolporate pollen with longitudinally elongate endoapertures (Harley, 1985
). Although this type of pollen is rare in the family, it also occurs in Fibraurea Lour., Chasmanthera Hochst., Odontocarya Miers, and Tinospora Miers (Harley, 1985
), all of which are included in our expanded Tinosporeae.
Further, the two major clades are morphologically distinguished mainly by the position of the fruit and embryo with respect to the peduncle and by the shape of the embryo and position of the cotyledons. Thus, within clade I, the larger clade that corresponds to our "expanded Tinosporeae" includes taxa that share a straight fruit, with the style-scar at the apex and a straight embryo with the hypocotyl and the radicle at the apical end of the fruit (Fig. 2). Nested within this clade are former members of Peniantheae (Penianthus Miers and the monospecific Sphenocentrum Pierre) and some former representatives of Fibraureae (Fibraurea and Burasaia). Our examination of fruits and seeds shows that those taxa indeed share straight fruits and straight embryos with the other members of our expanded Tinosporeae. However, while the cotyledons of Fibraurea and Burasaia are foliaceous and divaricate, cotyledons in Penianthus and Sphenocentrum are fleshy and collateral.
The smaller, sister clade of the expanded Tinosporeae, Coscinieae, has a subglobose fruit with a sublateral style-scar. Although no mature fruits of the taxa included in this clade were available in this study, the embryo of Anamirta Colebr., as illustrated by Miers (1864)
, is rather straight, and the embryo of Arcangelisia flava is said to be divaricate and "much folded" (Forman, 1986
, p. 210). Although, the third member of Coscinieae, Coscinium Colebr., was not sampled in this study, its inclusion here is likely, because it shares the subglobose fruit shape with a sublateral style and divaricate cotyledons that are said to be "folded and divided" (Forman, 1986
, p. 216).
Clade II contains 31 of the 49 sampled genera. Members in this clade share a variously curved fruit with the style being sublateral to basal and near the peduncle. There is comparable variation in the embryo—crescent-shaped (C-shaped), hippocrepiform (
-shaped), or uncinate (J-shaped). Within clade II, a small clade that contains the two Menispermum species is sister to the two remaining major clades. The Menispermum clade is distinguished by a laterally compressed, crescent-shaped seed (Fig. 2), the character that has given its signature to the family, i.e., "moonseed family." Embryos in this clade are relatively small with subcylindrical cotyledons that are somewhat larger than the radicle.
One of the two major clades found within the Menispermum clade is clade A, which contains the monophyletic, entirely neotropical Anomospermeae plus four taxa formerly in Menispermeae. Clade A has no obvious morphological synapomorphy, but taxa in Anomospermeae are all characterized by a deeply ruminate endosperm (Fig. 2). Most members in this clade have fruits with the remnant of the style near the base; thus the fruits and embryos are hippocrepiform in species of Abuta Aubl., Telitoxicum Moldenke, Caryomene Barneby & Krukoff, and in the monospecific Elephantomene Barneby & Krukoff. However, Anomospermum Miers has subglobose fruits with a sublateral to lateral style and uncinate embryos, and Orthomene Barneby & Krukoff has fruits with an apical or subapical style and only weakly uncinate embryos. Moreover, Anomospermum as currently recognized is not monophyletic; one species is sister to Abuta, two other species are nested within Orthomene, and two more are unplaced within Anomospermeae. Cotyledons in the Anomospermeae clade are semicylindric and much longer than the radicle.
The second major clade, sister to clade A, contains the poorly known southeast Asian Limacia blumei plus clade B as early diverging lineages while clade B is in turn sister to clade C + Tiliacoreae. Our clade B includes some of the taxa formerly placed in Menispermeae; of those, the clade consisting of Rhaptonema sp. and Strychnopsis thouarsii is characterized by a coiled embryo (Fig. 2). Furthermore, a close relationship between both genera plus Spirospermum penduliflorum Thouars—not sampled here—has already been suggested based on shared tricolpate pollen with a narrow rugulate operculum (Harley and Ferguson, 1982
). The clade of Stephania, Cyclea, and Cissampelos has female flowers with a single carpel as a synapomorphy (Fig. 2) and is further characterized by having relatively small hippocrepiform seed and embryo. The genus Antizoma Miers, confined to the arid regions of South Africa and not represented in this study, is the only other genus in the family that has flowers with a single carpel; it may well belong in this clade. Moreover, Cyclea, Cissampelos, and Antizoma also share tricolporate pollen with a smooth operculum, while the pollen in Stephania is triporate (Harley and Ferguson, 1982
). Triporate pollen has also been reported in Coscinium of Diels's Coscinieae (Ferguson, 1978
; Thanikaimoni et al., 1984
) and in Tiliacora Colebr. (Thanikaimoni, 1986
).
Clade B is sister to the clade consisting of the traditionally recognized Tiliacoreae + clade C. Most of the genera included in Tiliacoreae share hippocrepiform seeds lacking endosperm, and the cotyledons are therefore large (Fig. 2). Tiliacora is the only genus in this clade to have seeds with endosperm; this motivated Kessler (1993)
to assign this genus to his Anomospermeae, a placement, however, not supported in this study. Although most members of Tiliacoreae and of the whole family share a tricolpate or tricolporate type of pollen, Tiliacora is unique in having inaperturate pollen (Ferguson, 1975
; but see Thanikaimoni, 1986
).
While relationships within Tiliacoreae are not fully resolved, our analyses recovered a strongly supported clade containing three of the four neotropical members (e.g., Chondrodendron, Curarea, and Sciadotenia); the fourth and monospecific genus Ungulipetalum Moldenke, known only from a few collections from southeast Brazil, was not sampled.
Our clade C contains Diels's Hyperbaeneae plus the genera Cocculus and Pachygone. Within clade C, Hyperbaena Miers ex Benth. and Pachygone have large and fleshy embryos that lack endosperm, while Cocculus DC. has subfoliaceous embryos that are embedded in a nonruminate endosperm. Relationships are well resolved within Hyperbaeneae, and also the sampled members of Pachygone form a monophyletic clade; however, Cocculus is polyphyletic. Furthermore, relationships within clade C are still uncertain, and taxonomic decisions about the suggested congeneric status of Pachygone and Cocculus (Hong et al., 2001
) require an extensive sampling, particularly in the poorly known genus Pachygone.
Implications for infrafamilial classifications
To date, infrafamilial classifications in Menispermaceae remain unsettled, largely due to the complex morphological variation across the family that made interpretation of important evolutionary traits difficult. The tribal classification of Diels (1910)
(Table 2) has been followed by many authors, but subsequent authors have noted that the characters used to circumscribe the tribes were weakly correlated. Thus, a reassessment of the classification has often been suggested (Barneby, 1972
; Forman, 1986
). Kessler (1993)
reduced the number of tribes to five by combining most of Diels's Tiliacoreae and all his Peniantheae and Hyperbaeneae into Pachygoneae, Kessler's newly resurrected tribe. He also transferred Arcangelisia (Coscinieae) and Burasaia (Fibraureae) into Tinosporeae, included Anamirta and Coscinium in Fibraureae, and placed Tiliacora in Anomospermeae.
In this study, we find that all five of Kessler's newly reorganized tribes correspond to either para- or polyphyletic assemblages. However, our results also reveal strongly supported monophyletic clades that correspond to Diels's circumscription of Coscinieae, Hyperbaeneae, Tiliacoreae, and Anomospermeae. Similarly, the constraint trees forcing monophyly of the individual tribes are not significantly different from the most likely tree (Table 4).
On the other hand, most of Diels's Fibraureae and all his Peniantheae are here found to be nested within Tinosporeae. A simple classificatory solution is to include Fibraureae (except for Tinomiscium petiolare) and Peniantheae in Tinosporeae, making this large clade (our "expanded Tinosporeae") monophyletic. While the inclusion of Fibraureae (Burasaia, Fibraurea, and Tinomiscium) in Tinosporeae was previously suggested (Miers, 1851
, 1864
; Barneby, 1970
, 1972
; Forman, 1986
), the inclusion of Peniantheae in Tinosporeae has never been suggested. It is important to note, however, that despite strong support in this study for the inclusion of most members of Fibraureae and all Peniantheae in our "expanded Tinosporeae," it is unclear why the enforced monophyly of these tribes was not rejected in the S-H test. Additionally, should the placement of Burasaia in our "expanded Tinosporeae" be confirmed, the long-unused tribal name "Burasaieae" has nomenclatural priority.
On the other hand, our results do not support earlier suggestions that Odontocarya, a neotropical genus, be combined with the Old World Chasmanthera and Tinospora (Barneby, 1970
, 1972
; Forman, 1985
, 1986
). However, the monospecific neotropical Borismene Barneby is nested within Odontocarya, and thus, Odontocarya as currently conceived is paraphyletic.
Another significant finding in this study is that the tribe Menispermeae, as conceived by Diels, is polyphyletic, thus contradicting previous results based on a reduced sampling (Hong et al., 2001
). While most of the 12 genera of the traditional Menispermeae in our analysis are placed in other well-supported clades within the large clade II, five genera do form a monophyletic group. Within this group, Rhaptonema sp. and Strychnopsis thouarsii are sister to a clade consisting of Stephania, Cyclea, and Cissampelos; a morphological synapomorphy for this clade is flowers with a single carpel. This clade corresponds to Diels's subtribes Cissampelinae (Cissampelos and Cyclea) and Stephaniinae (Stephania). Moreover, results of the S-H test show that the enforced monophyly of the individual subtribes are not statistically different from the most likely tree (Table 4).
Implications for morphological diversification
Tracing character state distributions on the parsimony strict consensus tree indicates clearly that the climbing habit, three carpels, presence of endosperm, and a straight embryo are plesiomorphic conditions in the moonseed family. Within the family, a few characters have unambiguous changes, while others are clearly ambiguous, and the inferred ancestral state changes depending on the topology and the optimization used (e.g., parsimony strict consensus tree or one of the most parsimonious trees, and all most parsimonious states at each node, ACCTRAN or DELTRAN). The tree habit shows at least five and possibly seven independent origins (Fig. 3A). Because the position of Penianthus in relation to the other taxa with tree habit (i.e., Burasaia madagascariensis DC. + Orthogynium sp. and Sphenocentrum jollyanum Pierre) is not resolved on the parsimony strict consensus tree, it is unclear whether the tree habit has evolved once or three times on this part of the tree. In any event, our results do not support previous assumptions of a plesiomorphic tree habit.
|
Character state transitions for embryo type show no ambiguity in their reconstruction. This character was used in earlier tribal classifications of the family (Miers, 1864
), but because of the difficulty in obtaining a good sample from herbarium specimens, its use was later discontinued (Forman, 1984
). In this study we found that, while a straight embryo is present in the early diverging Tinomiscium petiolare and in taxa contained in clade I, and thus is the plesiomorphic condition for the family (Figs. 1, 2), a curved embryo is a synapomorphy for clade II (Figs. 1, 2), while a coiled embryo is a synapomorphy for the Malagasy Rhaptonema sp. + S. thouarsii clade (Fig. 2). Similarly, the presence of druplets, the presence of a condyle, and a large embryo (>6 mm long) are likely synapomorphies for Menispermaceae (Fig. 1).
Further, examination of several other morphological transitions shows that a simple leaf with palmate venation is the plesiomorphic condition in the family, whereas pinnate venation seems to have evolved multiple times (Fig. 3D). Compound leaves arose independently at least twice (not shown here): once in Burasaia and possibly once in Disciphania cubijensis (R. Knuth) Sandwith (because the latter taxon was not sampled here, this interpretation should be taken with caution until its placement on the phylogeny is confirmed).
Another highly homoplasious character (not shown here) is pollen type. While Tinomiscium petiolare, sister taxon to all other Menispermaceae, has tricolporate pollen with longitudinally elongate endoapertures, most Menispermaceae have tricolporate pollen with circular endoapertures. Thus, the statement that tricolpate pollen is ancestral in Menispermaceae (Thanikaimoni, 1986
) is not supported in this study.
Still, a few taxa have tricolpate, triporate, or triporate-operculate pollen types. Some other pollen types, such as the inaperturate pollen of Tiliacora (Ferguson, 1975
; but see Thanikaimoni, 1986
) and the cryptoporate pollen in Leptoterantha Louis ex Troupin, are apomorphic, while tricolpate-operculate pollen is synapomorphic for the Rhaptonema + S. thouarsii clade.
Future prospects
This study provides the first comprehensive molecular phylogeny resulting from a wide-ranging taxonomic, morphological, and geographical sampling of the moonseed family. Phylogenetic analyses using different approaches consistently recovered similar evolutionary histories. The Indo-Malesian species Tinomiscium petiolare is sister to two major clades; while no morphological synapomorphy is evident for clade I, clade II is characterized by curved embryos. Moreover, the phylogenetic hypothesis for Menispermaceae presented here provides evidence for the monophyly of four of the eight tribes circumscribed by Diels and shows that the four remaining tribes are polyphyletic. In addition, the molecular phylogeny presented here provides a framework to evaluate character transformation within the family, thus allowing us to identify traits that have potential predictive phylogenetic value. We show that the tree habit has evolved multiple times from the climbing habit, whereas endosperm had multiple losses. On the other hand, single carpels and curved embryos show single origins within Menispermaceae, and the straight embryo is the plesiomorphic condition for the moonseed family.
Furthermore, given that the genera and species sampled in this study cover the broadest morphological and taxonomic diversity displayed in the family, in addition to the confirmed utility of the ndhF chloroplast gene for reconstructing relationships at the family level, we are confident that the general evolutionary framework presented here will be preserved with increased taxonomic sampling. Therefore, the phylogenetic structure recovered in this study is the basis for a revised infrafamilial classification, a project currently underway (R. Ortiz, unpublished manuscript).
Moreover, the current framework will also allow future investigations into comparative development of key morphological characteristics such as straight vs. curved embryos. Similarly, the phylogeny also lays the foundations to investigate patterns of ecological diversification of a primarily lowland tropical family of which only a few members are dwellers of temperate or subtropical deciduous forest. Further, the uneven distribution of species diversity across clades gives the opportunity to investigate correlations between particular morphological traits and species-rich clades as compared with their typically species-poor sister clades. Additionally, the robust phylogeny presented here also provides the means to examine and date patterns of geographical distribution, which will allow us to gain insights into how this cosmopolitan family has attained its present day distribution. Hence, the results presented in this study constitute an important contribution toward the understanding of the affinities and morphological diversification within Menispermaceae and as such provide the framework on which to address questions in the fields of comparative development, ecology, and biogeography.
APPENDIX
Greenhouse-grown specimens are noted before the voucher information. Voucher specimens are deposited at the following herbaria: Herbario Amazonense (AMAZ), Jardin Botanique National de Belgique (BR), Research Center for Biodiversity, Academia Sinica (HAST), Instituto Nacional de Biodiversidad (INB), Missouri Botanical Garden (MO), Muséum National d'Histoire Naturelle (P), Herbario Museo Ecuatoriano de Ciencias Naturales (QCNE), Australian National Herbarium–CSIRO (QRS), Parc de Tsimbazaza (TAN), Centre National de la Recherche Appliquée au Development Rural (TEF), and Wageningen University (WAG). Species sequenced for designing primers are marked with an asterisk (*).
Taxon; ndhF GenBank accession no., Voucher specimen, Source, Collection locale, Herbarium.
Abuta grandifolia (Mart.) Sandwith*; EF624258; Ortiz et al. 221, Estación Experimental de Allpahuayo, Loreto, Peru, AMAZ, MO. Abuta rufescens Aubl.; EF624259; Ortiz et al. 226, Carretera Iquitos-Nauta, Km 26, Loreto, Peru, AMAZ, MO.Abuta sandwithianana Krukoff & Barneby; EF624260; Ortiz et al. 305, Carretera Iquitos-Nauta, Km 58, Loreto, Peru, AMAZ, MO. Albertisia porcata Breteler.; EF624261; McPherson 16678, East of Lastourville, Ogooué-Lolo, Gabon, MO. Anamirta cocculus (L.) Wight & Arn. EF624262; Cultivated BR 19514617; BR.; Anisocycla linearis Pierre ex Diels EF624263; Hong-Wa et al. 466, Madagascar, MO. Anomospermum bolivianum Krukoff & Moldenke; EF624264; Ortiz et al. 294, Oxapampa, Pasco, Peru, MO.Anomospermum chloranthum Diels s. l.; EF624265; Ortiz & Aguilar 324, Península de Osa, Puntarenas, Costa Rica, INB, MO. Anomospermum grandifolium Eichler; EF624266; Ortiz et al. 243, Requena, Loreto, Peru, AMAZ, MO. Anomospermum reticulatum (Mart.) Eichl. ssp. idroboi (Krukoff & Barneby); EF624267; Kriebel et al. 3501, Sarapiquí, Heredia, Costa Rica, INB, MO. Anomospermum solimoesanum (Moldenke) Krukoff & Barneby; EF624268; Ortiz & Vargas 198, Jatún Sacha, Napo, Ecuador, MO, QCNE. Arcangelisia flava (L.) Merr.; EF624269; Arifiani 452, North Celebes, Indonesia, MO. Beirnaertia cabindensis (Exell & Mendoca) Troupin; EF624270; Walters & Niangadouma 1267, Hau-Ogooué, Gabon, MO. Borismene japurensis (Mart.) Barneby; EF624271; Ortiz et al. 307, Carretera Iquitos-Nauta, Km 26.5, Loreto, Peru, MO. Burasaia madagascariensis DC.; EF624272; Rabenantoandro et al. 1262, Vohibola forest, Toamasina, Madagascar, MO. Calycocarpum lyonii (Pursh) A. Gray; EF624273; Ortiz et al. 335, Bollinger, Missouri, USA, MO. Carronia protensa (F. Muell.) Diels; EF624274; van der Werff & Gray 17049, Atherton Tableland, Timber Reserve 1230, Boonjee, Queensland, Australia, MO. Caryomene grandifolia Barneby & Krukoff; EF624275; Zárate 2136, Río Yavarí, Lago Preto, Mariscal Ramón Castilla, Loreto, Peru, AMAZ, MO. Chasmanthera dependens Hochst.; EF624276; Cultivated BR 19780026; BR. Chasmanthera welwitschii Troupin; EF624277; Ewango 3028, Réserve de Faune à Okapis, Province Orientale, Republique Democratique du Congo, MO. Chondrodendron tomentosum Ruiz & Pav.*; EF624278; Ortiz & Vásquez 217, Estación Experimental de Allpahuayo, Loreto, Peru, AMAZ, MO. Cissampelos andromorpha DC.; EF624279; Ortiz et al. 302, Carretera Iquitos-Nauta, Km 58, Loreto, Peru, AMAZ, MO. Cissampelos grandifolia Triana & Planch.; EF624280; Ortiz et al. 301, Carretera Chatarra-Cacazú, Oxapampa, Pasco, Peru, MO. Cissampelos ovalifolia DC.; EF624281; Zardini & Chaparro 60300, Mbaracayú Biosphere Reserve, Canindeyú, Paraguay, MO. Cissampelos owariensis Beauvais ex DC.; EF624282; Cultivated BR 19871151; BR. Cissampelos tropaeolifolia DC.; EF624283; Ortiz et al. 229, Carretera Iquitos-Nauta, Km 30, Loreto, Peru, MO. Cocculus carolinus (L.) DC.; EF624284; Ortiz & Pruski 349, Algiers section of New Orleans, Orleans, Louisiana, USA, MO. Cocculus diversifolius DC.; EF624285; Cruz-Espinoza 405, San Juan Bautista Cuicatlán, Oaxaca, Mexico, MO. Cocculus laurifolius DC.; EF624286; Cultivated BR 19084083; BR. Cocculus orbiculatus (L.) DC.; EF624287; Chung 1645, Tao-Yan, Taiwan, MO. Curarea candicans (Rich. ex DC.) Barneby & Krukoff; EF624288; Torke 310, Guyana, MO. Curarea cuatrecasasii Barneby & Krukoff; EF624289; Ortiz & Aguilar 323, Península de Osa, Puntarena, Costa Rica, INB, MO. Curarea tecunarum Barneby & Krukoff; EF624290; Ortiz & Vásquez 214, Estación Experimental de Allpahuayo, Loreto, Peru, MO. Curarea toxicofera (Wedd.) Barneby & Krukoff; EF624291; Ortiz 184, Estación Experimental de Allpahuayo, Loreto, Peru, AMAZ, MO. Curarea sp.nov.*; EF624292; Ortiz & Vargas 194, Jatún Sacha, Napo, Ecuador, MO, QCNE. Cyclea fansipanensis Gagnep.; EF624293; van der Werff et al. 17424 Tra Linh, Quoc Toan Community, Cao Bang, Vietnam, MO. Dioscoreophyllum cumminsii (Stapf) Diels; EF624294; Cultivated BR 19640448; BR. Disciphania killipii Diels; EF624295; Ortiz & Zárate 310, Carretera Iquitos-Nauta, Loreto, Peru, AMAZ, MO. Disciphania lobata Eichler; EF624296; Ortiz 266, Carretera Santo Tomás, Loreto, Peru, MO. Elephantomene eburnea Barneby & Krukoff*; EF624297; Ortiz et al. 237, Centro de Investigación Jenaro Herrera, Requena, Loreto, Peru, MO. Fibraurea tinctoria Lour.; EF624298; Larsen et al. 42660, Koh Hong, Prov. Songkla, Thailand, MO. Hyperbaena domingensis (DC.) Benth.; EF624299; van der Werff et al. 19586, Reserva San Francisco, Zamora-Chinchipe, Ecuador, MO. Hyperbaena eladioana Q. Jiménez; EF624300; Ortiz et al. 327, Valle del Candelaria, San José, Costa Rica, MO. Hyperbaena leptobotryosa (Donn. Sm.) Standl.; EF624301; Ortiz et al. 320, Península de Osa, Puntarenas, Costa Rica, MO. Hyperbaena smilacina Standl.; EF4302; Ortiz et al. 330, Parque Nacional Rincón de la Vieja, Guanacaste, Costa Rica, MO. Hyperbaena tonduzii Diels; EF624303; Ortiz et al. 326, Valle del Candelaria, San José, Costa Rica, MO. Hypserpa decumbens (Benth.) Diels; EF624304; van der Werff 17057, Longland's Gap, Heberton Range, Queensland, Australia, MO. Jateorhiza macrantha (Hook. f.) Exell & Mendonca; EF624305; Kenfack & Zapfack 2039. Southwest Province, Fako, Bimbia-Bonadikombo, Cameroon, MO. Kolobopetalum leonense Hutch. & Dalziel; EF624306; Schmidt et al. 3435, Ankasa Forest Resource Reserve, Western, Ghana, MO. Legnephora moorei (F. Muell.) Miers; EF624307; van der Werff & Gray 17053, Atherton Tableland, Timber Reserva 1230, Queensland, Australia, MO. Leptoterantha mayumbensis (Exell) Troupin; EF624308; Ewango 3005, Réserve de Faune à Okapis, Province Orientale, Republique Democratique du Congo, MO. Limacia blumei (Boerl.) Diels; EF624309; Arifiani 513, Bogor Botanic Garden, West Java, Indonesia, MO. Limacia esiangkara F. M. Bailey; EF624310; Gray 8927, Portland Roads Road, Australia, MO. Menispermum canadense L.; EF624311; Ortiz et al. 235, Jefferson County, Missouri, USA, MO. Menispermum dauricum DC.; EF624312; Lee & Won 0404084, Mt. Chonghyun, Korea, MO. Odontocarya amazonum Barneby; EF624313; Ortiz & Zarate 314, Carretera Iquitos -Nauta, km 22, Loreto, Peru, AMAZ, MO. Odontocarya diplobotrya Diels; EF624314; Ortiz & Vílchez 269, Jenaro Herrera, Loreto, Peru, AMAZ, MO. Odontocary klugii (A. C. Smith) Barneby; EF624315; Ortiz & Cahuamari 272, Mazan, Quebrada Sharara, Loreto, Peru, AMAZ, MO. Odontocarya tripetala Diels*; EF634316; Ruiz 5601, Mariscal Ramón Castilla, Loreto, Peru, MO. Odontocarya truncata Standl.; EF634317; Hammel & Perez 22567, Quebrada Coobó, Buenos Aires, Puntarenas, Costa Rica, MO. Orthogynium sp.; EF624318; Birkinshaw & Rabenantoandro 549, Betampona, Toamasina, Madagascar, MO, P, TAN. Orthomene hirsuta (Krukoff & Moldenke) Barneby & Krukoff; EF624319; Ortiz et al. 308, Carretera Iquitos-Nauta, Km 26.5, Loreto, Peru, AMAZ, MO. Orthomene schomburgkii (Miers) Barneby & Krukoff; EF624320; Ortiz & Vargas 201, Estación Biológica Jatun Sacha, Napo, Ecuador, MO, QCNE. Pachygone loyaltiensis Diels; EF624321; Lowry et al. 5614, Upper Amoa River Valley, Province du Nord, New Caledonia, MO. Pachygone ovata (Poir.) Diels; EF624322; Gray 8851, White Cliff Point, Australia, MO, QRS. Penianthus longifolius Miers; EF624323; Sweeney et al. 1436, Mt. Cameroon area, Southwest, Cameroon, MO. Pericampylus glaucus (Lam.) Merr; EF624324; Chung 1644, Yangming-Shan National Par, Tai-Pei, Taiwan, MO. Pychnarrhena longifolia (Decne. ex Miq.) Becc.; EF624325; Arifiani 519, Bogor Botanic Garden, West Java, Indonesia, MO. Pychnarrhena novoguineensis Miq.; EF624326; Gray 8794, Goldsborough Valley Road, Australia, MO. Rhaptonema sp.; EF624327; McPherson 18854, Sahaka, Antsiranana, Madagascar, MO. Rhigiocarya racemifera Miers; EF624328; Kenfack 1655, Fako, Limbe, Southwest, Cameroon, MO. Sarcopetalum harveyanum F. Muell.; EF624329; van der Werff 17058, CSIRO Arboretum, Atherton, Queensland, Australia, MO. Sciadotenia amazonica Eichler; EF624330; Ortiz & Zárate 264, Carretera Nina Rumi, Loreto, Peru, AMAZ, MO. Sciadotenia brachypoda Diels; EF624331; Ortiz & Farroñay 222, Puerto Almendras, Loreto, Peru, AMAZ, MO. Sciadotenia mathiasiana Krukoff & Barneby; EF624332; Ortiz et al. 259, Carretera Iquitos-Nauta, Km 1.5, Loreto, Peru, AMAZ, MO. Sciadotenia toxifera Krukoff & A. C. Sm.; EF624333; Ortiz et al. 231, Carretera Iquitos-Nauta, Km 30, Loreto, Peru, AMAZ, MO. Sphenocentrum jollyanum Pierre; EF624334; Daramota 30, Onigambari forest reserve, Oluyole, Nigeria, MO. Stephania japonica (Thunb.) Miers; EF624335; Huang 2054, Nantou Hsien, Yuchin Hsiang, Taiwan, HAST. Stephania rotunda Lour.; EF624336; Cultivated BR 19680593; BR. Strychnopsis thouarsii Baill.; EF624337; Schatz et al. 3728, Reserve Speciale Perinet-Analamazaotra, Toamasina, Madagascar, MO, P, TEF, WAG. Telitoxicum krukovii Moldenke; EF624338; Ortiz et al. 261, Carretera Iquitos-Nauta, Km 1.5, Loreto, Peru, MO. Telitoxicum peruvianum Moldenke*; EF624339; Ortiz et al. 218, Estación Experimental de Allpahuayo, Loreto, Peru, AMAZ, MO. Tiliacora funifera (Miers) Oliv.; EF624340; D. Kenfack 2100, Greater Accra, Legon campus, Ghana, MO. Tiliacora gabonensis Troupin; EF624341; Walters & Niangadouma 1159, Plateaux Bateke National Park, Haut-Ogooué, Gabon, MO. Tinomiscium petiolare Hook. f. & Thomson; EF624342; Maxwell 97–223, Doi Luang National Park, SW side, Chiang Mai, Thailand, MO. Tinospora smilacina Benth.; EF624343; Gray 8798, Copperload Dam Road, Australia, MO. Triclisia dictyophylla Diels.; EF624344; Kenfack & Zapfack 2038, Soutwest Province, Fako, Bimbia-Bonadikombo, Cameroon, MO. Triclisia subcordata Oliv.; EF624345; Kenfack 2101, Greater Accra, Legon campus, Ghana, MO.
Outgroup taxa
Akebia longeracemosa Matsum.; EF624346; Huang 2045, Nantou Hsien, Chichi Town, Taiwan, HAST. Boquila trifoliolata (DC.) Decne.; EF624347; Zapata 108, Camino entre Pucón y Currehue, Araucania, Chile, MO. Stauntonia obovatifoliola Hayata; EF624348; Huang 2041, Hsinchu Hsien, Wufeng Hsiang, Taiwan, HAST. Stauntonia hexaphylla Decne.; EF624349; Cultivated BR 19791807; BR.
FOOTNOTES
1 The authors thank P. Stevens, A. Doust, J. Pruski, F. Zapata, M. Richardson, and members of the Kellogg laboratory at the University of Missouri–St. Louis (UMSL) for helpful discussions and comments on versions of the manuscript. R.O. thanks E. Kay and L. Lohmann for training in techniques; M. Beilstein and A. Doust for help with data analysis; P. Sweeney for help with editing the trees; D. Arifiani, D. Kenfack, P. Sweeney, F. Zapata, C. Ewango, B. Gray, B. Hammel, R. Zárate, K.-F. Chung, C.-I. Peng, C. Hong-Wa, B. Torke, K. Kriebel, and staff of The National Botanical Garden of Belgium (BR) and the Missouri Botanical Garden DNA bank for providing leaf material; R. Vásquez, R. Zárate, and C. Amasifuén (in Peru) and N. Zamora, F. Morales, A. Soto, O. Vargas, and R. Aguilar (in Costa Rica) for help with logistics and assistance in the field; and the curators of A, BR, COL, F, GH, HUA, K, MEXU, MO, NY, P, and US for loans. B. Gunn kindly made the line drawings. This work is based on part of the Ph.D. dissertation of R.O. Funding was provided in part by the E. Desmond Lee and Family Laboratory of Plant Systematics at the University of Missouri–St. Louis (UMSL), the International Center for Tropical Ecology (UMSL), the Missouri Botanical Garden Alumni Fund, and the Missouri Botanical Garden–Peru program. 
4 Author for correspondence (rosa.ortiz-gentry{at}mobot.org
)
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