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  • Research article
  • Open Access
  • Evolution and loss of long-fringed petals: a case study using a dated phylogeny of the snake gourds, Trichosanthes(Cucurbitaceae)

    • 1Email author,
    • 2,
    • 3 and
    • 4
    BMC Evolutionary Biology201212:108

    https://doi.org/10.1186/1471-2148-12-108

    • Received: 10 February 2012
    • Accepted: 21 June 2012
    • Published:

    Abstract

    Background

    The Cucurbitaceae genus Trichosanthes comprises 90–100 species that occur from India to Japan and southeast to Australia and Fiji. Most species have large white or pale yellow petals with conspicuously fringed margins, the fringes sometimes several cm long. Pollination is usually by hawkmoths. Previous molecular data for a small number of species suggested that a monophyletic Trichosanthes might include the Asian genera Gymnopetalum (four species, lacking long petal fringes) and Hodgsonia (two species with petals fringed). Here we test these groups’ relationships using a species sampling of c. 60% and 4759 nucleotides of nuclear and plastid DNA. To infer the time and direction of the geographic expansion of the Trichosanthes clade we employ molecular clock dating and statistical biogeographic reconstruction, and we also address the gain or loss of petal fringes.

    Results

    Trichosanthes is monophyletic as long as it includes Gymnopetalum, which itself is polyphyletic. The closest relative of Trichosanthes appears to be the sponge gourds, Luffa, while Hodgsonia is more distantly related. Of six morphology-based sections in Trichosanthes with more than one species, three are supported by the molecular results; two new sections appear warranted. Molecular dating and biogeographic analyses suggest an Oligocene origin of Trichosanthes in Eurasia or East Asia, followed by diversification and spread throughout the Malesian biogeographic region and into the Australian continent.

    Conclusions

    Long-fringed corollas evolved independently in Hodgsonia and Trichosanthes, followed by two losses in the latter coincident with shifts to other pollinators but not with long-distance dispersal events. Together with the Caribbean Linnaeosicyos, the Madagascan Ampelosicyos and the tropical African Telfairia, these cucurbit lineages represent an ideal system for more detailed studies of the evolution and function of petal fringes in plant-pollinator mutualisms.

    Keywords

    • Markov Chain Monte Carlo
    • rbcL
    • Last Glacial Maximum
    • Sunda Shelf
    • Pollinator Attraction

    Background

    Deeply divided or fringed petal lobes are known from a range of angiosperm families, including Caryophyllaceae, Celastraceae, Cucurbitaceae, Myrtaceae, Orchidaceae, Saxifragaceae, and Tropaeolaceae [1]. While the origin and function of subdivided petals vary between groups, division of perianth edges is especially common among nocturnal hawkmoth-pollinated species (such as Trichosanthes[2], Figure 1), where the fringes, in combination with a light petal color, may enhance visibility and thus increase pollination success [3, 4]. Experiments have shown that diurnal and nocturnal hawkmoths are attracted by floral scent but also rely on visual clues to find and recognize flowers even at extremely low light intensity [5, 6]. A preference for high contrasts might help them find their nectar sources, and it seems plausible that fringed petals enhance the sharp contrast between the petal margin and a dark background [4].
    Figure 1
    Figure 1

    Fully expanded flower of Trichosanthes pilosa Lour. showing the characteristic feather-like fringes along the petal margins. Picture courtesy of Ken Ishikawa.

    In Cucurbitaceae, long-fringed petals are known in five genera that occur in Madagascar, tropical Africa, the Caribbean, and East and Southeast Asia [7, 8]. The largest of them is Trichosanthes with currently 90–100 species of mainly perennial, 3 to 30 m long climbers that are usually dioecious and have medium-sized fleshy fruits. Referring to the petal fringes, Linnaeus formed the genus name from the Greek words for 'hair' (genitive τριχός) and 'flower' (Άνθoς). Trichosanthes has its center of diversity in Southeast Asia, but ranges from India throughout tropical and subtropical Asia east to Japan, and southeast to New Guinea, Australia, and Fiji [9]. One species, the snake gourd, T. cucumerina L., is a widely cultivated vegetable in tropical and subtropical regions around the globe, and another 15 species are commonly used in Asian traditional medicine [10]. While floristic treatments are available for most of its range [9, 1116], a comprehensive revision of the nearly 300 names published in Trichosanthes is lacking (but see [17] for a synopsis).

    Trichosanthes belongs in the tribe Sicyoeae, a group of 12 genera and c. 270 species that is supported by morphological and molecular data [18]. Based on a limited number of Trichosanthes species sequenced, it appeared that the genus might be paraphyletic, with the genera Gymnopetalum Arn. (four species; [19]) and Hodgsonia Hook.f. & Thomson (two species; [9]) possibly nested inside it [20]. Both share with Trichosanthes the white flowers, elongated receptacle-tubes, and free filaments. Hodgsonia also has long-fringed petals (Figure 2J), but differs from Trichosanthes and Gymnopetalum in its much larger fruits (up to 25 cm across) and unusual seeds. The petal margins in Gymnopetalum are entire (Figure 2A, 2E) or in one species shortly fimbriate [9]. Geographically, Gymnopetalum and Hodgsonia largely overlap with the distribution area of Trichosanthes except for their absence from New Guinea and Australia, and from much of the northeastern range of Trichosanthes (temperate China, Taiwan, Japan) [9].
    Figure 2
    Figure 2

    Bayesian consensus tree with posterior probabilities (>0.80) and maximum likelihood bootstrap values (>60%) shown at the nodes. Photos on the right illustrate the floral morphology of the different sections and belong to the following species: A) Gymnopetalum chinense ; B) Trichosanthes odontosperma ; C) Trichosanthes montana ssp. crassipes ; D) Trichosanthes pubera ssp. rubriflos ; E) Gymnopetalum tubiflorum ; F) Trichosanthes beccariana ; G) Trichosanthes subvelutina ; H) Trichosanthes postarii ; I) Trichosanthes villosa . Pictures courtesy of W. J. de Wilde and B. Duyfjes (A, C, D, F, H, I), W. E. Cooper (B), N. Filipowicz (E), H. Nicholson (G), and P. Brownless (J). Inferred losses of petal fringes are marked by an asterisk.

    Based on mainly fruit and seed characters, the 43 species of Trichosanthes occurring in the Flora Malesiana region have been grouped into six sections, the typical sect. Trichosanthes and sections Cucumeroides (Gaertn.) Kitam., Edulis Rugayah, Foliobracteola C.Y.Cheng & Yueh, Involucraria (Ser.) Wight, and Asterosperma W.J.de Wilde & Duyfjes [21, 22]. The mainland Asian species, T. truncata C.B.Clarke, is in its own section, Truncata C.Y.Cheng & C.H.Yueh [23]. The four species of Gymnopetalum have been allocated to two sections that differ in flower morphology, the typical sect. Gymnopetalum with just one species from southern India and Sri Lanka and sect. Tripodanthera (M.Roem.) Cogn. with three southeast Asian and Malesian species [24].

    Here we test the monophyly and phylogenetic placement of Trichosanthes using a broad sampling of some 60% of its species, including the type species of each section name, plus representatives of Gymnopetalum, Hodgsonia, and other Sicyoeae as well as more distant outgroups. The well-resolved phylogeny, combined with field observations on flower shape and color, allows us to test whether petal fringes in Old World Sicyoeae evolved just once as would be the case if Gymnopetalum and Hodgsonia were nested inside it [20] or multiple times as would be implied by these genera having separate evolutionary histories. A combination of molecular-dating and ancestral area reconstruction permits reconstructing the biogeographical history of the Trichosanthes clade.

    Results and discussion

    Phylogenetic analyses and taxonomy

    Phylogenies obtained under Bayesian or Maximum Likelihood (ML) optimization revealed no statistically supported incongruences, defined as nodes with Bayesian posterior probabilities (PP) >0.95 or ML bootstrap support >75. A Bayesian consensus tree is shown in Figure 2. It reveals that the genus Trichosanthes is paraphyletic because Gymnopetalum is embedded in it, while Gymnopetalum is polyphyletic because its four species do not group together. Instead, G. tubiflorum (Wight & Arn.) Cogn. groups with species from sections Trichosanthes and Cucumeroides (1.00 PP/84 ML support), while G. orientale W.J.de Wilde & Duyfjes, G. chinense (Lour.) Merr., and G. scabrum (Lour.) W.J.de Wilde & Duyfjes are sister to section Edulis (1.00 PP/86 ML). The Trichosanthes/Gymnopetalum clade (56 species sampled; 0.99 PP/62 ML support) is sister to Luffa, a genus of seven or eight species of which we included five. This sister group relationship, however, is only weakly supported (Figure 2). The genus Hodgsonia (two species with long-fringed flowers, one sampled here) is only distantly related to the Trichosanthes/Gymnopetalum clade.

    Of the seven sections previously proposed in Trichosanthes (see Background), three are supported by the molecular results, namely sections Asterosperma (1.00 PP/100 ML; three species, two of them sampled here), Cucumeroides (1.00 PP/93 ML; seven species, five sampled), and Edulis (1.00 PP/75 ML; nine species, five sampled). Three other sections with more than one species (Involucraria, Foliobracteola, Trichosanthes) are not monophyletic in their current circumscriptions. To achieve a more natural classification, a revised infrageneric classification has been proposed including two new sections [17].

    The biogeographic history of the Trichosanthes clade

    Based on a fossil-calibrated Bayesian relaxed molecular clock model, Trichosanthes originated during the Oligocene (Figure 3), an estimate influenced by our prior constraint of the crown node of the Trichosanthes/Gymnopetalum clade to 34 Ma. This constraint is based on Trichosanthes-like seeds from the Upper Eocene of Bulgaria [25] dating to c. 34 Ma and seeds from the Oligocene of West Siberia [26] dating to c. 23.8 Ma [27]. Seeds assigned to Trichosanthes have also been reported from Miocene and Pliocene sites in France, Germany, Italy, and Poland [2830], and Pliocene Trichosanthes-like leaves are known from France [31]. The biogeographic analysis (Figure 4) inferred an East Asian origin of the genus (region C in Figure 4), but this inference is based only on the living species, while the just-discussed fossils indicate a more northern (Eurasian) range of Trichosanthes before the global climate cooling at the end of the Oligocene. Many other extinct elements of the European Oligocene, Miocene, and Pliocene floras, such as Taxodium, Craigia, Fagus kraeuselii, Ilex, and tropical Araceae, such as Caladiosoma, also have nearest living relatives in tropical Southeast Asia [31, 32].
    Figure 3
    Figure 3

    Chronogram for Trichosanthes and outgroups obtained from the same sequence data as used for Figure1, but modeled under a relaxed molecular clock. Node heights represent mean ages and bars the 95% highest posterior density intervals for nodes that have a posterior probability of ≥ 0.95. Fossil constraints used were: (A) Cucurbitaceae seeds from the London Clay (see Material and Methods ), (B) Trichosanthes seeds from Eocene sediments in Bulgaria [25] and Oligocene sediments in West Siberia [26], and (C) Miocene leaves assigned to Marah. Inset B shows the Bulgarian seeds ([25], Figure thirteen) to the left and Middle Pliocene seeds from Poland ([29], Figures sixteen to seventeen) to the right: Inset C shows the Marah leaf (photos provided by M. Guilliams and D.M. Erwin, University of California, Berkeley).

    Figure 4
    Figure 4

    Ancestral range reconstruction for Trichosanthes and outgroups inferred on 8000 output trees resulting from the Bayesian dating analysis and distribution ranges for all species. Letters in the legend correspond to the colored distribution ranges in the map (inset), and letters adjacent to taxon names correspond to the geographic origin of the sampled plant. Wallace’s Line is shown as a broken line between Borneo and Sulawesi, Lydekker’s Line is shown as a broken line between New Guinea and the Moluccas. The three numbered clades and inferred transoceanic disjunctions are discussed in the text.

    Collision between the Eurasian and Australian tectonic plates started in the Late Oligocene, about 25 Ma ago, and the Sahul Shelf (carrying New Guinea) and Sunda Shelf (Sumatra, Java, and Borneo) reached their present proximity only by the Late Miocene, some 10 Ma [33, 34]. Mid-Miocene pollen records indicate a warm, moist climate and rainforest expansion on these newly forming islands [35], allowing groups adapted to humid forest conditions, such as the liana clade Trichosanthes, to spread and diversify. Such plant groups would have benefited from land bridges that during times of sea level changes repeatedly connected New Guinea and Australia on the one hand, and Indochina, Sumatra, Java, and Borneo on the other. The lowest sea levels, during the last glacial maximum (LGM), were approximately 120 m below those of today, resulting in the complete exposure of the Sunda Shelf; even sea level reduction by just 40 m already connected Indochina, Sumatra, Java, and Borneo [35, 36]. No land bridges, however, ever connected the islands on the Sunda Shelf with those in “Wallacea,” that is, Sulawesi, the Moluccas, and the Lesser Sunda Islands, or the latter with New Guinea and Australia on the Sahul Shelf. In zoogeography, these two boundaries are known as Wallace’s Line and Lydekker’s line, but their significance as floristic boundaries is doubtful [37, 38].

    The most striking transoceanic disjunctions in Trichosanthes are numbered in Figure 4. They are (i) the disjunction between the Australian species T. subvelutina F.Muell. ex Cogn. and its sister clade on the Asian mainland and areas of the Sunda Shelf, dated to 23.8 (29.4-18.4) Ma; (ii) the disjunction between T. edulis Rugayah, T. dentifera Rugayah, T. laeoica C.Y.Cheng & L.Q.Huang, T. schlechteri Harms from New Guinea, and T. odontosperma W.E.Cooper & A.J.Ford from Australia on the one hand, and Gymnopetalum chinense, widespread in Asia as far East as Flores, and G. orientale in Sulawesi, the Lesser Sunda Islands, and the Moluccas on the other (this is dated to 16.7 (22.1-11.2) Ma, but the position of G. scabrum relative to G. chinense and G. orientale remains unclear; compare Figures 2, 3, and 4); and (iii) the disjunction between T. wawrae Cogn. from Thailand, peninsular Malaysia, Sumatra, and Borneo, and its sister clade T. papuana F.M.Bailey/T. pentaphylla F. Muell. ex Benth. from New Guinea and Australia, which dates to 7.1 (11.2-3.3) Ma.

    Trichosanthes range expansion between New Guinea and Australia occurred during the Pliocene/Pleistocene, when these two regions were repeatedly connected due to the above-mentioned sea level changes [36]. Thus, the estimated divergence time of the Australian species T. odontosperma (a member of clade ii in Figure 4) from its New Guinean sister species, T. edulis, is 3.9 (6.4-1.6) Ma, while that of the sister species pair T. papuana from the Aru Islands and New Guinea, and T. pentaphylla from Australia (clade iii in Figure 4) is 4.0 (7.1-1.4) Ma; considering their error ranges, these ages fall in the Pliocene/Pleistocene.

    The geographic history of T. pilosa Lour. (including the synonyms T. baviensis Gagnep. and T. holtzei F.Muell. [16]), a widespread species here represented by seven samples from Queensland (Australia), Thailand, Vietnam, and Japan, cannot be inferred because the within-species relationships lack statistical support (Figure 2). Inferring the origin of the snake gourd, T. cucumerina, a vegetable cultivated in tropical and subtropical regions around the globe (represented by a single sample from Sri Lanka) also would require population-level sampling. Both species have fleshy red fruits and small seeds, probably dispersed by birds.

    Evolution and loss of petal fringes

    The phylogeny obtained here implies that long-fringed corollas evolved independently in the Asian genera Hodgsonia and Trichosanthes and were lost in three of the four species formerly placed in the genus Gymnopetalum (petals still bear c. 5 mm-long fringes in G. orientale). The two inferred losses (marked with an asterisk in Figure 2) coincide with shifts from nocturnal to diurnal flowering times (HS personal observation of G. scabrum and G. chinense in Cambodia, Jan. 2010, and China, Sept. 2005; N. Filipowicz, Medical University of Gdansk, personal observation of G. tubiflorum in India, Nov. 2010), and it therefore seems likely that there is a shift from predominantly nocturnal sphingid pollinators to diurnal bee or butterfly pollinators. The loss of fringes does not coincide with long-distance dispersal events to insular habitats (where hawkmoths might be absent), and the trigger for the pollinator shifts so far is unknown.

    The adaptive function of the corolla fringes in pollinator attraction requires experimental study. An innate preference for radial patterns [39] and high contrasts might help hawkmoths find their nectar sources [5, 6], and one possible explanation for the evolution of fringed petals is that they help create such a radial pattern and sharper contrasts between the petals and a dark background [4]. In a diurnal, hawkmoth-pollinated Viola species, more complex corolla outlines correlate with higher fruit set [40] but it remains to be tested if this is also the case in the nocturnal Trichosanthes-hawkmoth system. Another untested possibility is that the fringes with their highly increased surface area and exposed position might be involved in scent production (B. Schlumpberger, Herrenhaeuser Gardens, Hannover, pers. comm., Feb. 2012) or produce a waving motion, which has been shown to increase pollinator attraction in other systems [41]. Anatomical studies of the petal tissue of Trichosanthes, wind tunnel experiments with naive hawkmoths, and detailed field observations are required to test these possibilities.

    Conclusions

    Molecular evidence supports the inclusion of Gymnopetalum into a then monophyletic Trichosanthes[17]. Our molecular phylogenies reveal that long-fringed petals evolved independently in Hodgsonia and Trichosanthes/Gymnopetalum, followed by two losses of corolla fringes in the latter clade, most likely associated with pollinator shifts. Molecular dating and a biogeographic analysis indicate an Oligocene initial diversification of Trichosanthes in mainland Asia. The lineage then diversified and spread in Malaysia (the Malesian biogeographic region) during the late Miocene and Pliocene, reaching the Australian continent several times.

    Methods

    Morphology

    Herbarium specimens from A, BRI, CNS, E, GH, K, KUN, KYO, L, LE, M, MO, P, S, UC, UPS and US were obtained on loan or studied during herbarium visits. Determination of herbarium material was verified using identification keys [9, 1116, 19, 42]. All species in Trichosanthes have corolla fringes, and these are absent in three of the four Gymnopetalum species, except G. orientale, which can have short-fimbriate petal margins (fringes up to 5 mm length).

    Sampling, DNA extraction and amplification

    We included six DNA regions, namely the nuclear ribosomal ITS region (ITS1-5.8S-ITS2), the chloroplast genes rbcL and matK, the trnL and trnL trnF intron and spacer, and rpl20-rps12 spacer. Data for rbcL and the trnL region were taken from previous studies [7, 18, 20, 43, 44]. Only plant samples for which two or more markers were successfully sequenced were included in the analyses, and the combined dataset included one of the two species of Hodgsonia, all four of Gymnopetalum, and 52 of Trichosanthes, representing approximately 60% of the accepted species in the latter genus. Type species of all sections were included: Gymnopetalum tubiflorum (Wight & Arn.) Cogn. (G. sect. Gymnopetalum), Gymnopetalum chinense (Lour.) Merr. (G. sect. Tripodanthera), Trichosanthes postarii W.J.de Wilde & Duyfjes (T. sect. Asterosperma), Trichosanthes pilosa Lour. (T. sect. Cucumeroides), Trichosanthes edulis Rugayah (T. sect. Edulis), Trichosanthes kirilowii Maxim. (T. sect. Foliobracteola), Trichosanthes wallichiana (Ser.) Wight (T. sect. Involucraria), Trichosanthes villosa Blume (T. sect. Pseudovariifera), Trichosanthes cucumerina L. (T. sect. Trichosanthes), Trichosanthes truncata C.B.Clarke (T. sect. Truncata), Trichosanthes subvelutina F.Muell. ex Cogn. (T. sect. Villosae). Species names and their authors, specimen voucher information, and GenBank accession numbers for all sequenced markers (including 262 new sequences) are summarized in Table 1.
    Table 1

    Voucher information and GenBank accession numbers

    Species

    No.

    Voucher (Herbarium)

    Origin of the sequenced material

    ITS

    rpl20-rps12 IS

    matK

    rbcL

    trnL-trnF IS

    trnL intron

    Austrobryonia micrantha (F.Muell.) I.Telford

     

    I. R. Telford 8173 (CANB)

    Australia, New South Wales

    EF487546

    EF487567

    EF487559

    EF487552

    EF487575

    EF487575

    Bryonia dioica Jacq.

     

    (1) S. Renner 2187 (M)

    (1) Switzerland, cult. BG Zürich

    (2) EU102709

    (1) DQ648157

    (1) DQ536641

    (1) DQ536791

    (1) DQ536791

    (1) DQ536791

      

    (2) A. Faure 66/76 (M)

    (2) Algeria, Lamoriciere

          

    Cyclanthera pedata (L.) Schrad.

     

    S. Renner et al. 2767 (M)

    Germany, cult. BG Mainz

    HE661293

    DQ648172

    DQ536667

    DQ535749

    DQ536767

    DQ536767

    Ecballium elaterium (L.)A.Rich. ssp. elaterium

     

    (1) M. Chase 922 (K)

    (1) UK, cult. RBG-K

    (2) EU102746

    (1) AY968541

    (1) AY973019

    (1) AY973023

    (1) AY973006

    (1) AY973006

      

    (2) S. Renner et al. 2768 (M)

    (2) Germany, cult. BG Mainz

          

    Echinocystis lobata (Michx.) Torr. & A.Gray

     

    S. Renner et al. 2829 (M)

    Germany, cult. BG Mainz

    -

    DQ648174

    DQ536673

    DQ535809

    DQ536814

    DQ536814

    Gymnopetalum chinense (Lour.) Merr.

     

    H. Schaefer 2005/661 (M)

    China, Guangdong

    HE661294

    EU155612

    EU155606

    EU155601

    EU155621

    EU155630

    Gymnopetalum orientale W.J. de Wilde & Duyfjes

     

    M. van Balgooy 7553 (L)

    Indonesia, Bali

    HE661301

    HE661468

    HE661397

    -

    -

    -

    Gymnopetalum scabrum (Lour.) W.J. de Wilde & Duyfjes

    1

    W. de Wilde & B. Duyfjes 22269 (L)

    Thailand, Central

    HE661295

    DQ536556

    DQ536683

    DQ535754

    DQ536824

    DQ536824

    Gymnopetalum scabrum (Lour.) W.J. de Wilde & Duyfjes

    2

    J. Maxwell 16-11-2002 (CMU)

    Thailand

    HE661296

    HE661469

    HE661398

    -

    -

    -

    Gymnopetalum scabrum (Lour.) W.J. de Wilde & Duyfjes

    3

    C.H. Wong, J. Helm & J. Schultze-Motel 2071 (LE)

    China, Hainan

    HE661297

    HE661470

    HE661399

    -

    -

    -

    Gymnopetalum tubiflorum (Wight & Arn.) Cogn.

    1

    N. Filipowicz & Z. Van Herwijnen NF25a (M)

    India, Kerala

    HE661298

    HE661471

    HE661400

    -

    -

    -

    Gymnopetalum tubiflorum (Wight & Arn.) Cogn.

    2

    A. Alston 1670 (UC)

    Sri Lanka, Veragantota

    HE661299

    HE661472

    HE661401

    -

    -

    -

    Gymnopetalum tubiflorum (Wight & Arn.) Cogn.

    3

    G.H.K. Thwaites CP1625 (K)

    Sri Lanka

    HE661300

    HE661473

    HE661402

    -

    -

    -

    Hodgsonia heteroclita Hook.f. & Thomson

     

    (1) P. Phonsena 4705 (L)

    (1) Thailand, Nan

    (1) HE661302

    (1) HE661474

    (1) HE661403

    -

    (2) EU155631

    -

      

    (2) L. Loeffler s.n. (M)

    (2) Bangladesh

          

    Lagenaria siceraria (Molina) Standl.

     

    M. Merello 1331 (MO)

    Ghana

    HE661303

    HE661475

    HE661404

    AY935747

    AY935788

    AY968570

    Linnaeosicyos amara (L.) H.Schaef. & Kocyan

     

    M. Mejia, J. Pimentel & R. Garcia 1877 (NY)

    Dominican Republic

    HE661304

    DQ536602

    DQ536741

    DQ535774

    DQ536873

    DQ536873

    Luffa acutangula (L.) Roxb.

     

    (1) S. Renner et al. 2757 (M), seeds from D. S. Decker-Walters & A. Wagner TCN 1130 (FTG)

    (1) Germany, cult. BG Munich, seeds from India, Ahmadnagar, Maharasthra

    (1) HE661305

    (1) HE661476

    (2) DQ536695

    (2) DQ535826

    (2) DQ536835

    (2) DQ536835

      

    (2) L.X. Zhou s.n., no voucher

    (2) China, cult. BG Guangzhou

          

    Luffa aegyptiaca Mill. (incl. L. cylindrica L.)

     

    D.Z. Zhang 15 April 2003, no voucher

    China, cult. BG Guangzhou

    HE661306

    HE661477

    HE661405

    DQ535827

    DQ536836

    DQ536836

    Luffa echinata Roxb.

     

    G. Schweinfurth 555 (M)

    Egypt

    HE661307

    HE661478

    HE661406

    -

    EU436357

    EU436357

    Luffa graveolens Roxb.

     

    S. Renner & A. Kocyan 2758 (M), seeds from D. Decker-Walters 1543 (FTG 121855)

    Germany, cult. BG Munich, seeds from India, USDA PI540921

    HE661308

    EU436334

    EU436409

    EU436385

    EU436358

    EU436358

    Luffa quinquefida (Hook. & Arn.) Seemann

     

    (1) R. Berhaut 7308 (M)

    (1) Senegal

    (2) HQ201986

    (1) EU436335

    (2) DQ536697

    -

    (1) EU436359

    -

      

    (2) S. Renner & A. Kocyan 2754 (M), seeds from D. S. Decker-Walters TCN 1440 (FTG 118010)

    (2) Germany, cult. BG Munich, seeds originally from Louisiana, USA

          

    Marah macrocarpa (Greene) Greene

     

    (1) M. Olson s.n. (MO)

    (1) USA, Sonoran Desert

    (2) AF11906-7

    (1) DQ536566

    (2) AY968453

    (2) AY968524

    (1) AY968387

    (1) AY968571

      

    (2) D. Arisa & S. Swensen 1009 (RSA)

    (2) USA, Sonoran Desert

          

    Momordica charantia L.

     

    S. Renner et al. 2775 (M)

    Germany, cult. BG Munich

    HE661309

    DQ491013

    DQ491019

    DQ535760

    DQ501269

    DQ501269

    Nothoalsomitra suberosa (F.M.Bailey) I.Telford

     

    I. Telford 12487 (NE)

    Australia, SE Queensland

    HE661310

    DQ536575

    DQ536709

    DQ535762

    DQ536844

    DQ536844

    Sicyos angulatus L.

     

    M. Chase 979 (K)

    North America

    HE661311

    DQ648189

    DQ536732

    DQ535847

    DQ536777

    DQ536777

    Trichosanthes adhaerens W.J. de Wilde & Duyfjes

     

    S. Lim, J. J. Postar & G. Markus SAN 143273 (L)

    Malaysia, Borneo, Sabah

    HE661312

    HE661479

    -

    -

    -

    -

    Trichosanthes auriculata Rugayah

     

    A. Kalat, I. Abdullah, & J. Clayton BRUN 17016 (L)

    Borneo, Brunei

    HE661313

    HE661480

    HE661407

    -

    -

    -

    Trichosanthes baviensis Gagnep.

     

    N.M. Cuong 1248 (P)

    Vietnam

    HE661314

    HE661481

    -

    -

    -

    -

    Trichosanthes beccariana Cogn. ssp. beccariana

     

    W. de Wilde et al. SAN 142229 (L)

    Malaysia, Borneo, Sabah

    HE661315

    HE661482

    HE661408

    -

    -

    -

    Trichosanthes borneensis Cogn.

     

    C. Argent et al. 93127 (E)

    Indonesia, Borneo, Kalimantan Timur

    HE661316

    HE661483

    -

    -

    -

    -

    Trichosanthes bracteata (Lam.) Voigt

     

    T. Haegele 20 (M)

    India, Kochin

    HE661317

    HE661484

    EU155608

    EU155602

    EU155622

    EU155632

    Trichosanthes celebica Cogn.

     

    W. de Wilde & B. Duyfjes 21903 (L)

    Indonesia, Sulawesi

    HE661318

    HE661485

    HE661409

    -

    -

    -

    Trichosanthes cucumerina L.

    1

    H. Schaefer 2007/327 (M)

    Germany, cult. BG Munich

    HE661319

    EU155614

    EU155609

    EU155603

    EU155623

    EU155633

    Trichosanthes cucumerina L.

    2

    N. Lundqvist 11380 (UPS)

    Sri Lanka

    HE661320

    HE661486

    HE661410

    -

    -

    -

    Trichosanthes dentifera Rugayah

     

    J.H.L. Waterhouse 445-B (L)

    Papua New Guinea, Bougainville Is.

    HE661321

    HE661487

    -

    -

    -

    -

    Trichosanthes dioica Roxb.

     

    O. Polunin, W. Sykes & J. Williams 5925 (E)

    Nepal

    HE661322

    HE661488

    HE661411

    -

    -

    -

    Trichosanthes edulis Rugayah

     

    W. Avé 4076 (L)

    Indonesia, Irian Jaya

    HE661323

    HE661489

    HE661412

    -

    -

    -

    Trichosanthes elmeri Merr.

     

    E.F.J. Campbell 43 (E)

    Malaysia, Borneo, Sabah

    HE661324

    HE661490

    -

    -

    -

    -

    Trichosanthes globosa Blume

     

    W. de Wilde et al. SAN 144003 (L)

    Malaysia, Borneo, Sabah

    HE661325

    HE661491

    HE661413

    -

    -

    -

    Trichosanthes holtzei F.Muell.

     

    B. Gray 7482 (CNS)

    Australia, N Queensland

    HE661326

    HE661492

    HE661414

    -

    -

    -

    Trichosanthes homophylla Hayata

     

    Y.-C. Kao 499 (GH)

    Taiwan

    HE661327

    HE661493

    HE661415

    -

    -

    -

    Trichosanthes hylonoma Hand.-Mazz.

     

    Wuling Mt Exp 1646 (KUN)

    China

    HE661328

    HE661494

    HE661416

    -

    -

    -

    Trichosanthes intermedia W.J. de Wilde & Duyfjes

     

    V. Julaihi et al. S 76602 (L)

    Malaysia, Borneo, Sarawak

    HE661329

    HE661495

    -

    -

    -

    -

    Trichosanthes inthanonensis Duyfjes & Pruesapan

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 3930 (L)

    Thailand, Chiang Mai

    HE661330

    HE661496

    HE661417

    -

    -

    -

    Trichosanthes inthanonensis Duyfjes & Pruesapan

    2

    K. Pruesapan et al. 67 (L)

    Thailand, Kanchanaburi

    HE661331

    HE661497

    HE661418

    -

    -

    -

    Trichosanthes kerrii Craib

     

    P. Phonsena, W. de Wilde & B. Duyfjes 3959 (L)

    Thailand, Nan

    HE661333

    HE661498

    -

    -

    -

    -

    Trichosanthes kinabaluensis Rugayah

     

    J. Postar et al. SAN 144260 (L)

    Malaysia, Borneo, Sabah

    HE661334

    EU155615

    HE661419

    -

    EU155624

    EU155634

    Trichosanthes kirilowii Maxim. var. japonica (Miq.) Kitam.

    3

    H. Takahashi 20711 (GIFU)

    Japan

    HE661335

    DQ536603

    DQ536742

    DQ535855

    DQ536874

    DQ536874

    Trichosanthes kirilowii Maxim. var. japonica (Miq.) Kitam.

    1

    K. Kondo 05090401e (KYO)

    Japan

    HE661332

    HE661499

    HE661420

    -

    -

    -

    Trichosanthes kirilowii Maxim. var. japonica (Miq.) Kitam.

    2

    K. Deguchi, K. Uchida, K. Shiino & H. Hideshima s.n. (KYO)

    Japan

    -

    HE661500

    HE661421

    -

    -

    -

    Trichosanthes laceribractea Hayata

    1

    S. Fujii 9623 (KYO)

    Japan

    HE661336

    HE661501

    HE661422

    -

    -

    -

    Trichosanthes laceribractea Hayata

    2

    S. Fujii 9978 (KYO)

    Japan

    HE661337

    HE661502

    HE661423

    -

    -

    -

    Trichosanthes laceribractea Hayata

    3

    Liang Deng 7090 (KUN)

    China

    HE661338

    HE661503

    -

    -

    -

    -

    Trichosanthes laeoica C.Y.Cheng & L.Q.Huang

    1

    M. Coode et al. NGF 32585 (E)

    Papua New Guinea, Eastern Highlands

    HE661339

    HE661504

    -

    -

    -

    -

    Trichosanthes laeoica C.Y.Cheng & L.Q.Huang

    2

    P. Katik LAE 77807a (BRI)

    Papua New Guinea

    HE661340

    HE661505

    -

    -

    -

    -

    Trichosanthes lepiniana (Naud.) Cogn.

    1

    J.D.A. Stainton 8522 (E)

    Nepal

    HE661341

    HE661506

    HE661424

    -

    -

    -

    Trichosanthes lepiniana (Naud.) Cogn.

    2

    Shanzu Wen 85 (KUN)

    China

    HE661342

    HE661507

    HE661425

    -

    -

    -

    Trichosanthes lepiniana (Naud.) Cogn.

    3

    H. de Boer HB49, coll. 1865 (P)

    France, cult BG Paris

    HE661343

    HE661508

    -

    -

    -

    -

    Trichosanthes miyagii Hayata

     

    T. Yamazaki 310 (KYO)

    Japan

    HE661344

    HE661509

    HE661426

    -

    -

    -

    Trichosanthes montana Rugayah ssp. crassipes W.J. de Wilde & Duyfjes

     

    J. Postar et al. SAN 144259 (L)

    Malaysia, Borneo, Sabah

    HE661346

    EU155616

    HE661427

    -

    EU155625

    EU155635

    Trichosanthes montana Rugayah ssp. montana

     

    W. de Wilde et al. 22279 (L)

    Indonesia, Java

    HE661345

    HE661510

    -

    -

    -

    -

    Trichosanthes mucronata Rugayah

     

    W. de Wilde & B. Duyfjes SAN 139459 (L)

    Malaysia, Borneo, Sabah

    HE661347

    HE661511

    HE661428

    -

    -

    -

    Trichosanthes multiloba Miq.

    1

    S. Tsugaru, G. Murata & T. Sawada s.n. (KYO)

    Japan

    HE661348

    HE661512

    HE661429

    -

    -

    -

    Trichosanthes multiloba Miq.

    2

    S. Fujii 9957 (KYO)

    Japan

    HE661349

    HE661513

    HE661430

    -

    -

    -

    Trichosanthes nervifolia L.

     

    B. Jonsell 3828 (UPS)

    Sri Lanka

    HE661350

    HE661514

    HE661431

    -

    -

    -

    Trichosanthes obscura Rugayah

     

    K.M. Wang 1581 (L)

    Borneo, Brunei

    HE661351

    HE661515

    -

    -

    -

    -

    Trichosanthes odontosperma W.E.Cooper & A.J.Ford

    1

    H. Schaefer 2007/09 (M)

    Australia, Queensland

    HE661352

    EU037013

    HE661432

    -

    EU037011

    EU037010

    Trichosanthes odontosperma W.E.Cooper & A.J.Ford

    2

    B. Gray 9147 (UPS)

    Australia, Queensland

    HE661353

    HE661516

    HE661433

    -

    -

    -

    Trichosanthes odontosperma W.E.Cooper & A.J.Ford

    3

    I. Telford 11285 (CNS)

    Australia, Queensland

    HE661354

    HE661517

    HE661434

    -

    -

    -

    Trichosanthes pallida Duyfjes & Pruesapan

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 4658 (L)

    Thailand, Phetchaburi

    HE661355

    HE661518

    HE661435

    -

    -

    -

    Trichosanthes pallida Duyfjes & Pruesapan

    2

    P. Phonsena, W. de Wilde & B. Duyfjes 3981 (L)

    Thailand, Phetchaburi

    HE661356

    HE661519

    HE661436

    -

    -

    -

    Trichosanthes papuana F.M.Bailey

     

    W. Takeuchi & D. Ama 17069 (L)

    Papua New Guinea

    HE661357

    HE661520

    HE661437

    -

    -

    -

    Trichosanthes pedata Merr. & Chun

     

    Jiangiang Li 239 (KUN)

    China

    HE661358

    HE661521

    HE661438

    -

    -

    -

    Trichosanthes pendula Rugayah

     

    J. Postar et al. 144100 (L)

    Malaysia, Borneo, Sabah

    HE661359

    EU155617

    HE661439

    -

    EU155626

    EU155636

    Trichosanthes pentaphylla F.Muell. ex Benth.

    1

    W. Cooper 2094 (CNS)

    Australia, Queensland

    HE661360

    HE661522

    HE661440

    -

    -

    -

    Trichosanthes pentaphylla F.Muell. ex Benth.

    2

    W. Cooper 2061 (CNS)

    Australia, Queensland

    HE661361

    HE661523

    HE661441

    -

    -

    -

    Trichosanthes phonsenae Duyfjes & Pruesapan

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 4419 (L)

    Thailand, Phetchaburi

    HE661362

    HE661524

    HE661442

    -

    -

    -

    Trichosanthes phonsenae Duyfjes & Pruesapan

    2

    P. Phonsena, W. de Wilde & B. Duyfjes 3980 (L)

    Thailand, Phetchaburi

    HE661363

    HE661525

    HE661443

    -

    -

    -

    Trichosanthes pilosa Lour.

    1

    H. Schaefer 2007/17 (M)

    Australia, Queensland

    HE661364

    EU155620

    EU155611

    -

    EU155629

    EU155639

    Trichosanthes pilosa Lour.

    2

    P. Phonsena, W. de Wilde & B. Duyfjes 3913 (L)

    Thailand, Chiang Mai

    HE661365

    HE661526

    HE661444

    -

    -

    -

    Trichosanthes pilosa Lour.

    3

    H. Takahashi 20755 (GIFU)

    Japan

    -

    DQ536604

    DQ536743

    DQ535856

    DQ536875

    DQ536875

    Trichosanthes pilosa Lour.

    4

    H. Schaefer 2007/09 (M)

    Australia, Queensland

    HE661366

    HE661528

    HE661445

    -

    -

    -

    Trichosanthes pilosa var. roseipulpa W.J. de Wilde & Duyfjes

     

    P. Phonsena, W. de Wilde & B. Duyfjes 4694 (L, holotype)

    Thailand, Nan

    HE661367

    HE661529

    HE661446

    -

    -

    -

    Trichosanthes postarii W.J. de Wilde & Duyfjes

    1

    J. Postar et al. SAN 144066 (L, isotype)

    Malaysia, Borneo, Sabah

    HE661368

    EU155618

    HE661447

    -

    EU155627

    EU155637

    Trichosanthes postarii W.J. de Wilde & Duyfjes

    2

    J. Postar et al. SAN 144098 (L)

    Malaysia, Borneo, Sabah

    HE661369

    HE661530

    HE661448

    -

    -

    -

    Trichosanthes pubera Blume ssp. rubriflos (Cayla) Duyfjes & Pruesapan var. fissisepala Duyfjes & Pruesapan

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 4451 (L)

    Thailand, Chiang Mai

    HE661370

    HE661531

    HE661449

    -

    -

    -

    Trichosanthes pubera Blume ssp. rubriflos (Cayla) Duyfjes & Pruesapan var. fissisepala Duyfjes & Pruesapan

    2

    K. Pruesapan et al. 56 (L)

    Thailand, Kanchanaburi

    HE661371

    HE661532

    HE661450

    -

    -

    -

    Trichosanthes pubera Blume ssp. rubriflos (Cayla) Duyfjes & Pruesapan var. rubriflos

    1

    R. Zhang 1 (M)

    China, cult. South China BG, Guangzhou

    HE661372

    DQ536560

    DQ536688

    DQ535819

    DQ536828

    -

    Trichosanthes pubera Blume ssp. rubriflos (Cayla) Duyfjes & Pruesapan var. rubriflos

    2

    P. Phonsena, W. de Wilde & B. Duyfjes 3907 (L)

    Thailand, Saraburi

    HE661373

    HE661533

    HE661451

    -

    -

    -

    Trichosanthes quinquangulata A.Gray

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 4416 (L)

    Thailand, Phetchaburi

    HE661374

    HE661534

    HE661452

    -

    -

    -

    Trichosanthes quinquangulata A.Gray

    2

    N. Koonthudthod et al. 326 (L)

    Thailand, Phetchaburi

    HE661375

    HE661535

    HE661453

    -

    -

    -

    Trichosanthes quinquefolia C.Y.Wu

     

    K. Nanthavong et al. BT 705 (L)

    Laos, Khammouan

    HE661376

    HE661536

    HE661454

    -

    -

    -

    Trichosanthes reticulinervis C.Y.Wu ex S.K.Chen

     

    X.F. Deng 131 (IBSC)

    China, Guangdong

    HE661377

    DQ536605

    DQ536744

    DQ535857

    DQ536876

    DQ536876

    Trichosanthes rosthornii Harms

    1

    Jingliang Chuan 5654 (KUN)

    China

    HE661378

    HE661537

    HE661455

    -

    -

    -

    Trichosanthes rosthornii Harms

    2

    A. Henry 1626 (LE)

    China, Hubei

    HE661379

    HE661538

    -

    -

    -

    -

    Trichosanthes schlechteri Harms

     

    W. Takeuchi & D. Ama 15663 (LAE)

    Papua New Guinea

    HE661380

    EU155619

    EU155610

    EU155605

    EU155628

    EU155638

    Trichosanthes sepilokensis Rugayah

     

    J. Postar et al. SAN 151201 (L)

    Malaysia, Borneo, Sabah

    HE661381

    HE661539

    -

    -

    -

    -

    Trichosanthes smilacifolia C.Y.Wu

     

    Qiwu Wang 85620 (KUN)

    China

    HE661382

    HE661540

    -

    -

    -

    -

    Trichosanthes subvelutina F.Muell. ex Cogn.

    1

    I. Telford 9778 (CANB)

    Australia, Queensland

    HE661383

    HE661541

    HE661456

    -

    -

    -

    Trichosanthes subvelutina F.Muell. ex Cogn.

    2

    F. Davies 1541 (CANB)

    Australia, Queensland

    HE661384

    HE661542

    HE661457

    -

    -

    -

    Trichosanthes subvelutina F.Muell. ex Cogn.

    3

    N. Nicholson 3110 (BRI)

    Australia, New South Wales

    HE661385

    HE661543

    HE661458

    -

    -

    -

    Trichosanthes tricuspidata Lour spp. javanica Pruesapan & Duyfjes

     

    P. Phonsena, W. de Wilde & B. Duyfjes 4414 (L)

    Thailand, Phetchaburi

    -

    HE661592

    HE661591

    -

    -

    -

    Trichosanthes tricuspidata Lour. ssp. tricuspidata

     

    P. Phonsena, W. de Wilde & B. Duyfjes 4007 (L)

    Thailand, Nakhon Sawan

    HE661386

    HE661544

    HE661459

    -

    -

    -

    Trichosanthes truncata C.B.Clarke

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 3917 (L)

    Thailand, Chiang Mai

    HE661387

    HE661545

    HE661460

    -

    -

    -

    Trichosanthes truncata C.B.Clarke

    2

    P. Phonsena, W. de Wilde & B. Duyfjes 4490 (L)

    Thailand, Chiang Mai

    HE661388

    HE661546

    HE661461

    -

    -

    -

    Trichosanthes truncata C.B.Clarke

    3

    P. Phonsena, W. de Wilde & B. Duyfjes 6329 (L)

    Thailand, Chiang Mai

    HE661389

    HE661547

    HE661462

    -

    -

    -

    Trichosanthes villosa Blume

    1

    P. Phonsena, W. de Wilde & B. Duyfjes 4669 (L)

    Thailand, Chiang Mai

    -

    EU037006

    EU037007

    EU037005

    EU037009

    EU037008

    Trichosanthes villosa Blume

    2

    P. Phonsena, W. de Wilde & B. Duyfjes 6331 (L)

    Thailand, Chiang Mai

    HE661390

    : HE661548

    HE661463

    -

    -

    -

    Trichosanthes villosa Blume

    3

    P. Phonsena, W. de Wilde & B. Duyfjes 4449 (L)

    Thailand, Chiang Mai

    HE661391

    HE661549

    HE661464

    -

    -

    -

    Trichosanthes villosa Blume

    4

    P. Phonsena, W. de Wilde & B. Duyfjes 4000 (L)

    Thailand, Phetchaburi

    HE661392

    HE661550

    -

    -

    -

    -

    Trichosanthes villosa Blume

    5

    K. Pruesapan et al. 60 (L)

    Thailand, Kanchanaburi

    HE661393

    HE661551

    HE661465

    -

    -

    -

    Trichosanthes fissibracteata C.Y.Wu ex C.Y.Cheng & Yueh

     

    Shaowen Yu 974 (KUN)

    China, Yunnan

    HE661394

    HE661552

    HE661466

    -

    -

    -

    Trichosanthes wallichiana (Ser.) Wight

     

    A. Henry 9432 (LE)

    China, Yunnan

    HE661395

    HE661553

    -

    -

    -

    -

    Trichosanthes wawrae Cogn.

     

    B. Gravendeel et al. 631 (L)

    Indonesia, Java

    HE661396

    HE661554

    HE661467

    -

    -

    -

    Total DNA was extracted using the Carlson/Yoon DNA isolation procedure [45] and a Mini-Beadbeater (BioSpec Products) to pulverize the plant material. Extracts were purified using the GE Illustra GFX™ PCR DNA and Gel Band Purification Kit following the standard protocol.

    Polymerase chain reaction (PCR) amplification of purified total DNA was performed in 200 μl reaction tubes with a total volume of 50 μl. Each tube contained a mixture of 5 μl reaction buffer (ABgene, 10x), 3 μl MgCl2 (25 mM), 1 μl dNTP’s (10 μM), 0.25 μl Taq-polymerase (ABgene; 5U/μl), 0.25 μl BSA (Roche Diagnostics), 12.5 μl of each primer (2 mM), 14.5 μl Milli-Q water and 1 μl template DNA. The ITS region was amplified using the primer pair ITS-P17 and ITS-26 S-82R [46] with the following PCR protocol 97°C 5 min., (97°C 30 s., 55°C 1 min., 72°C 1 min.) x 35, 72°C 10 min., 4°C ∞; matK with primers matK-2.1a [47] and matK-1440R [48], 95° 5 min., (95° 30 s., 50° 1 min., 72° 1 min.) x 35, 72° 10 min., 4° ∞; and rpl20 rps12 using the primers rpl20 and rps12[49], 95° 5 min., (95° 30 s., 53° 1 min., 72° 1 min.) x 35, 72° 10 min., 4° ∞. Sequencing was performed by Macrogen Inc. (Seoul, South Korea) on an ABI3730XL automated sequencer (Applied Biosystems). The same primers as used in the PCR were used for the sequencing reactions.

    Sequence alignment

    Sequence trace files were compiled into contigs with the program Gap4 and edited using Pregap4 [50], both part of the Staden package [51]. Sequences were aligned manually in Se-Al [52]. The final matrix included rpl20-rps12 (100% of taxa), ITS (96%), matK (84%), trnL-F spacer (31%), trnL intron (28%), and rbcL (20%). The three latter regions increased statistical support values at early-branching clades. Sequences were concatenated, and gap-coded using the Simmons and Ochoterena simple method [53] implemented in SeqState [54].

    Phylogenetic analyses

    Selection of best-fit models of nucleotide substitution for the nuclear and plastid data partitions relied on the Akaike Information Criterion (AIC and AICc) as implemented in JModelTest version 0.1.1 [55, 56]. Likelihood calculations were carried out for 88 substitution models on an ML-optimized tree. The best-fitting model for the combined data was the general time-reversible (GTR) model, with a proportion of invariable sites (I) and rate variation among sites (G) with four rate categories. Maximum likelihood tree searches and bootstrapping of the combined data (using 1000 replicates) relied on RAxML version 7.2.6 [57] on the CIPRES cluster [58].

    Bayesian tree searching used MrBayes [59] on the CIPRES cluster [58]. The combined data were analyzed using three partitions (nuclear, plastid, gap data), allowing partition models to vary by unlinking gamma shapes, transition matrices, and proportions of invariable sites. Markov chain Monte Carlo (MCMC) runs started from independent random trees, were repeated twice, and extended for 10 million generations, with trees sampled every 1000th generation. We used the default priors in MrBayes, namely a flat Dirichlet prior for the relative nucleotide frequencies and rate parameters, a discrete uniform prior for topologies, and an exponential distribution (mean 1.0) for the gamma-shape parameter and branch lengths. Convergence was assessed by checking that the standard deviations of split frequencies were <0.01; that the log probabilities of the data given the parameter values fluctuated within narrow limits; that the convergence diagnostic (the potential scale reduction factor given by MrBayes) approached one; and by examining the plot provided by MrBayes of the generation number versus the log probability of the data. Trees saved prior to convergence were discarded as burn-in (10 000 trees) and a consensus tree was constructed from the remaining trees.

    The data matrix and trees have been deposited in TreeBASE (http://www.treebase.org; study number 12339).

    Divergence time estimation

    Divergence times were estimated using the Bayesian relaxed clock approach implemented in BEAST version 1.6.2 [60]. Searches used a Yule tree prior, the GTR + G substitution model, and 50 million MCMC generations, sampling every 1000th generation. Six monophyletic groups were defined based on the results of our phylogenetic analyses and previously published phylogenies [18, 20, 44]. Tracer version 1.5 [61] was used to check that effective sampling sizes had all reached >200, suggesting convergence of the chains. TreeAnnotator, part of the BEAST package, was then used to create a maximum clade credibility tree, with the mean divergence ages shown for all nodes with >95% highest posterior density.

    Calibration relied on Cucurbitaceae fossils assigned to particular nodes (labeled A--C in Figure 3), using a gamma prior distribution with the fossil age as the offset and shape and scale parameter chosen to add a 95% CI of c. 10 Ma older than the fossil. (A) The root node, that is, the most recent common ancestor of Momordica and Trichosanthes, was constrained to 55.8 Ma with a shape parameter of 1.0 and a scale of 1.0, based on seeds from the Paleocene/Eocene Felpham flora representing the oldest Cucurbitaceae and dated to c. 55.8 Ma [62]. (B) The crown node of the Trichosanthes/Gymnopetalum clade was constrained to 34 Ma with a shape parameter of 1.0 and a scale of 3.4, based on Trichosanthes seeds from the Upper Eocene of Bulgaria [25] dated to c. 34 Ma and seeds from the Oligocene of West Siberia [26] dated to c. 23.8 Ma [27]. (C) The divergence of Marah and Echinocystis was set to 16 Ma with a shape parameter of 1.0 and a scale of 3.35, based on leaves and a fruit representing Marah from the Miocene of Stewart Valley, Nevada (M. Guilliams and D. M. Erwin, University of California, Berkeley, in preparation; the fruit comes from the Fingerrock Wash site, dated to c. 16 Ma, the leaf from the Savage Canyon Formation, dated to c. 14.5 Ma). Absolute ages were taken from the geologic time scale of Walker and Geissman [63]. We also tested lognormal and exponential prior distributions, which gave very similar age estimates (results not shown).

    Biogeographical analysis

    Biogeographic reconstruction relied on statistical dispersal-vicariance analysis using S-DIVA version 2.0 [64] as implemented in RASP, which carries out parsimony inference on the chain of trees obtained from an MCMC search [65, 66], in our case the 8000 post burn-in Bayesian trees resulting from the BEAST dating analysis. S-DIVA averages the frequencies of an ancestral range at a node in ancestral reconstructions over all trees, with alternative ancestral ranges at a node weighted by the frequency of the node [64]. Range information for all species was compiled from taxonomic treatments [9, 11, 1316], and the coded distribution areas were: A) Australia and New Guinea, B) Wallacea, C) Insular Sunda Malesia, D) Mainland Southeast Asia, E) India and adjacent countries, F) Africa, Europe and the New World.

    Declarations

    Acknowledgments

    We thank W.J. de Wilde and B. Duyfjes for leaf samples, advice on species sampling and taxonomy, and comments on preliminary results; W.E. Cooper, N. Filipowicz, C. Jeffrey, and I. Telford for leaf samples; L. Nauheimer for Figure 3, B. Schlumpberger and A. Kelber for advice on function of petal fringes, M. Guilliams and D.M. Erwin for information on Marah fossils, and curators of the herbaria A, BRI, CNS, E, GH, K, KUN, KYO, L, LE, M, MO, P, S, UC, UPS and US for samples, loans, or help during visits to their institutions. This research was supported by SIDA-SAREC grant SWE-2005-338, Anna Maria Lundins stipendiefond, Helge Ax:son Johnsons stiftelse, Regnells botaniska resestipendium, SYNTHESYS grant GB-TAF-4255, and Knut och Alice Wallenbergs medel till rektors förfogande.

    Authors’ Affiliations

    (1)
    Department of Systematic Biology, Uppsala University, Norbyvägen 18 D, Uppsala, SE-75236, Sweden
    (2)
    Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue, Cambridge, MA, 02138, U.S.A
    (3)
    Department of Systematic Biology, Uppsala University, Norbyvägen 18 D, Uppsala, SE-75236, Sweden
    (4)
    University of Munich (LMU), Systematic Botany and Mycology, Menzinger Str. 67, Munich, 80638, Germany

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