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

  • Hugo J de Boer1Email author,

    Affiliated with

    • Hanno Schaefer2,

      Affiliated with

      • Mats Thulin3 and

        Affiliated with

        • Susanne S Renner4

          Affiliated with

          BMC Evolutionary Biology201212:108

          DOI: 10.1186/1471-2148-12-108

          Received: 10 February 2012

          Accepted: 21 June 2012

          Published: 3 July 2012

          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.

          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].
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_2127_Fig1_HTML.jpg
          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].
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_2127_Fig2_HTML.jpg
          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].
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_2127_Fig3_HTML.jpg
          Figure 3

          Chronogram for Trichosanthes and outgroups obtained from the same sequence data as used for Figure 1 , 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).

          http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_2127_Fig4_HTML.jpg
          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
          (2)
          Department of Organismic and Evolutionary Biology, Harvard University
          (3)
          Department of Systematic Biology, Uppsala University
          (4)
          Systematic Botany and Mycology, University of Munich (LMU)

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          © de Boer et al.; licensee BioMed Central Ltd. 2012

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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