Open Access
Research article

BMC Evolutionary Biology

, 12:108

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

Authors

  • Hugo J de Boer
    • Department of Systematic BiologyUppsala University
  • Hanno Schaefer
    • Department of Organismic and Evolutionary BiologyHarvard University
  • Mats Thulin
    • Department of Systematic BiologyUppsala University
  • Susanne S Renner
    • University of Munich (LMU)Systematic Botany and Mycology

DOI: 10.1186/1471-2148-12-108

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].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_Article_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].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_Article_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].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_Article_2127_Fig3_HTML.jpg
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).

https://static-content.springer.com/image/art%3A10.1186%2F1471-2148-12-108/MediaObjects/12862_2012_Article_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.

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.

Supplementary material

12862_2012_2127_MOESM1_ESM.pdf (2 mb)
Authors’ original file for figure 1
12862_2012_2127_MOESM2_ESM.pdf (642 kb)
Authors’ original file for figure 2
12862_2012_2127_MOESM3_ESM.tiff (3 mb)
Authors’ original file for figure 3
12862_2012_2127_MOESM4_ESM.pdf (1.1 mb)
Authors’ original file for figure 4

Copyright information

© 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.