ARTICLE IN PRESS MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx www.elsevier.com/locate/ympev

Phylogenetic relationships among Passiflora species based on the glutamine synthetase nuclear gene expressed in chloroplast (ncpGS) Roxana Yockteng* and Sophie Nadot Laboratoire E
cologie, Syst
ematique et E
volution, Universit
e Paris-XI, CNRS UMR 8079, Orsay, France Received 21 April 2003; revised 10 July 2003

Abstract This paper presents the first molecular phylogeny of the genus Passiflora encompassing almost all sections of this large genus. The nuclear-encoded chloroplast-expressed glutamine synthetase gene (ncpGS) was used to examine the relationships among Passiflora species (passionflowers), which was then compared with the new classification proposed by Feuillet and MacDougal. The resulting Bayesian, likelihood, and parsimony trees are congruent and well supported. The 90 Passiflora species examined apparently split into eight main subgenera: Plectostemma, Granadilla, Astrophea, Deidamioides, Polyanthea, Dysosmia, Tetrapathea, and Tryphostemmatoides. These results are in overall agreement with the Feuillet and MacDougalÕs classification but here we propose that three additional subgenera, Polyanthea, Dysosmia, and Tetrapathea, should be maintained. We observe a striking overall correlation between the phylogenetic position of the different species and their chromosome number. The first clade contains the arborescent species of the subgenus Astrophea, with n ¼ 12. The second clade, subgenus Plectostemma, includes species from four subgenera of KillipÕs classification with n ¼ 6 chromosomes. The last clade, subgenus Granadilla, includes species of seven old subgenera with n ¼ 9. Subgenus Dysosmia, with a variable chromosome number of n ¼ 9–11, is considered here as a separate subgenus closely related to the subgenus Granadilla. Ó 2003 Published by Elsevier Science Inc. Keywords: Passiflora; Phylogeny; ncpGS

1. Introduction The genus Passiflora L. is well known for its commercial uses. Many species are widely cultivated for fruit production such as P. edulis Sims. (passion fruit), P. tripartita var. mollissima (Kunth) Holm-Niel and Jørg (banana passion fruit) or P. ligularis Juss. (sweet granadilla). The sedative properties of P. incarnata L. are exploited in the pharmaceutical industry. Several other species are used as ornamental plants for their intricate and richly coloured flowers. Furthermore, the specific relationships with their herbivores, butterflies of the Heliconiine group, have been widely studied (Benson, 1978; Benson et al., 1975; Gilbert, 1980, 1982, 1983; Spencer, 1986). * Corresponding author. Fax: +33-1-69-15-73-53. E-mail address: [email protected] (R. Yockteng).

1055-7903/$ - see front matter Ó 2003 Published by Elsevier Science Inc. doi:10.1016/S1055-7903(03)00277-X

The Passifloraceae are distributed throughout the tropics and many warm temperate areas, from humid rain forests to deserts. It is classified in the order Malphigiales and appear closely related to the Violaceae and Flacourtiaceae (Chase et al., 2002; Soltis et al., 1999). Passifloraceae are divided into two tribes, the African tribe Pariopsae with 6 genera and the worldwide tribe Passiflorae with 11 genera. Passiflora L. (tribe Passiflorae) is the largest genus of the family with over 450 herbaceous, woody, and climbing plant species. The genus Passiflora is mainly distributed in tropical America with a few species in Asia and Oceania. Passionflowers are usually vines, with alternate and simple leaves, extrafloral nectaries on the petioles or the leaf surfaces. The large hermaphroditic flowers are generally colourful and composed of five stamens, five sepals, generally five petals, and three styles. The genus is assumed to be monophyletic on the basis of three

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R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx

diagnostic characters: the series of the corona filaments, axillary tendrils, and specialized flowers (Judd et al., 1999). The genus Passiflora was established in 1735 by Linnaeus who described 22 species. There are now between 475 and 485 recognized species of Passiflora (Vanderplanck, 2000). The first infrageneric classification encompassing 355 species of the genus recognized 22 subgenera (Killip, 1938) (Table 1) based on variation in the following characters: shape and size of operculum, number of series of filaments in the corona, size and shape of calyx tube, and finally position, size, and shape of the glands in petioles. A new subgenus and several sections were subsequently added to the genus (Escobar, 1988, 1989). The old world Passiflora species were studied by Cusset (1967) and deWilde (1972). More recently, Feuillet and MacDougal (1999) proposed a new infrageneric classification with only four subgenera: Astrophea, Deidamioides, Passiflora, and Plectostemma. They argued that many species are linked by strong affinities and hence must be grouped. At present, the number and nature of the subdivisions of this genus, as well as the position of the monospecific Tetrapathea group of New Zealand classified either as an independent genus or as a subgenus of Passiflora, remain to be resolved. Although studies of molecular genetic variation based on RAPD and AFLP markers have been carried out on the cultivated species of subgenera Tacsonia and Granadilla (Fajardo et al., 1998; S
anchez et al., 1999), no molecular phylogenetic analysis encompassing all subgenera of Passiflora has been yet published. Therefore, in order to clarify the evolutionary history of Passiflora, we sequenced the nuclear-encoded glutamine synthetase gene expressed in the chloroplast (ncpGS EC 6.3.1.2) and we constructed a molecular phylogeny of this gene using parsimony, likelihood, and Bayesian methods. Though variation of chloroplast genes and the widely used nuclear ITS region (Internal Transcribed Spacers) is often insufficient for resolving relationships among closely related species, ncpGS is comparable and has been proven useful for phylogenetic reconstruction and identification of processes of reticulate evolution (Emshwiller and Doyle, 1999, 2002). This gene belongs to a multigene family responsible for the nitrogen metabolism. It diverged 250–300 Myr ago from the cytosolicexpressed glutamine synthetase genes (Kumada et al., 1993; Pesole et al., 1991) and plays an independent role in the assimilation of the ammonia produced during photorespiration. Although the coding region is extremely conserved among angiosperms (1365 bp), the total length of the gene ncpGS is highly variable due to the presence of 11 introns distributed along the gene. Our main objectives are: (a) to examine the subgeneric boundaries in Passiflora; (b) to test the monophyly of the subgenera proposed by Killip (1938) and Feuillet

and MacDougal (1999); and finally (c) to propose a preliminary scenario of the evolutionary history of the genus.

2. Materials and methods 2.1. Plant samples For this study, 91 Passiflora species (138 individuals) were selected, representing 17 of 23 subgenera recognized by Killip (1938) and Escobar (1989) (Table 1). All non-monospecific subgenera were represented by at least two species with the exception of subgenera Psilanthus and Deidamioides. We used two Adenia species and one Dilkea species of the tribe Passiflorae (Passifloraceae) as outgroups to root the trees. Specimens were obtained from the wild, from botanical gardens, and from several national collections (Table 2). Sampled leaves were preserved in silica gel. Herbarium specimens were deposited at the Herbarium of the Museum National dÕHistoire Naturelle (Paris, France). To avoid confusion between the section and subgenus Decaloba, we used the name Plectostemma (Killip, 1938) for the subgenus. In the same way, we used Granadilla for the also called Passiflora subgenus and Incarnatae for the Passiflora series (Table 2) (Killip, 1938). Since, the new classification of Feuillet and MacDougal (1999) is not yet published in full, the discussion is based on KillipÕs classification. 2.2. DNA extraction, amplification, and sequencing For each sample, DNA was isolated from 0.2 g of dry leaves. Extractions were completed using the Dneasy Plant Mini Kit (Qiagen) following manufacturerÕs instructions. The primers designed by Emshwiller and Doyle (1999) were tested on a selected subset of Passiflora DNAs. These primers (GScp687f and GScp994r) were designed to amplify the 50 region between positions 687 and 994 bp of the coding region, which includes four introns of variable lengths. We found that these primers were not specific for the region ncpGS in Passiflora since in some species the cytosolic glutamine synthetase was amplified instead. The sequences of ncpGS obtained were used to design a new specific pair of primers, named GScp839f (50 CAC CAA TGG GGA GGT TAT GC 30 ) and GScp1056r (50 CAT CTT CCC TCA TGC TCT TTG T 30 ). Fig. 1 represents the sequenced region of the ncpGS gene, localized between positions 836 and 1061 of the coding region in Juglans nigra. PCR amplifications were performed in a 40 ll volume reaction using 1
buffer (Q-Biogene), 0.2 U of Taq polymerase (Q-Biogene), 1.5 mM of MgCl2 , 250 lM of dNTPs (Promega), and 0.2 lM of each primer.

ARTICLE IN PRESS R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx

3

Table 1 Infrageneric classification in alphabetic order of Passiflora proposed by Killip (1938) and modified by Escobar (1988, 1989) Subgenus Adenosepala, Killip, 1938 Apodogyne, Killip, 1938 Astephia, Killip, 1938 Astrophea (DC.) Masters, 1871

Section

Series

Botryastrophea Cirrhipes Dolichostemma Euastrophea Leptopoda Pseudoastrophea

Calopathanthus (Harms) Killip, 1938 Chloropathanthus (Harms) Killip, 1938 Deidamioides (Harms) Killip, 1938 Distephana (Juss.) Killip, 1938 Dysosmia (DC.) Killip, 1938 Dysosmioides, Killip, 1938 Granadilla, (Medic.) Mast., 1871

Quadrangulares Digitatae Tiliafoliae Marginatae Laurifoliae Serratifoliae Setaceae Pedatae Incarnatae Palmatisectae Kermesinae Imbricatae Simplicifoliae Lobatae Menispermifoliae

Manicata (Harms) Escobar, 1988 Murucuja (Medic.) Mast, 1871 Plectostemma, Masters, 1871

Auriculatae Heterophyllae Sexflorae Ap
etalae Luteae Organenses Miserae Punctatae Eudecaloba * Polyanthea* Hahniopathanthus Hollrungiella* Mayapathanthus Pseudodysosmia Pseudogranadilla Xerogona

Ampullacea Boliviana Bracteogama Colombiana Colombiana Fimbriatistipula Parritana

1 1 1 11 1 2 15 1 16 1 2 2 11 11 4 2 1 10 1 13 4 1 1 6 1 8 2 14 29 4 5 4 25

Cieca Decaloba

Polyanthea (DC.) Killip, 1938 Porphyropathantus Escobar, 1989 Pseudomurucuja (Harms) Killip, 1938 Psilanthus, Killip, 1938 Rathea (Karst.) Killip, 1938 Tacsonia (Juss.) Tr. & Planch, 1873

Number of species

Leptomischae Colombianae

2 2 4 2 3 7 5 47 19 16 3 1 1 18 6 8 1 1 4 4 2 1 1 12 7 8 2 1

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R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx

Table 1 (continued) Subgenus

Section

Series

Number of species

Poggendorffia Tacsonia Tacsoniopsis Trifoliata

5 3 2 1 5 1 1 2

Tacsonioides (DC.) Killip, 1938 Tacsoniopsis (Tr. & Planch) Killip, 1938 Tetrapathea * Banks ex DC., 1828 Tryphostemmatoides (Harms) Killip, 1938

Asiatic and Oceania taxa are indicated by an asterisk (deWilde, 1972; Harms, 1925). The last column shows the estimated number of species for each taxonomic group (Vanderplanck, 2000). The taxa represented in this study are in bold cases. Table 2 List of species of Passiflora and outgroups used in this study according to classifications from Killip (1938); deWilde (1972), Escobar (1988, 1989), and MacDougal (1994) Species

Infrageneric classification

Collection data

Voucher

Accession No.

Adenia gummifera (Harv.) Harms Adenia sp. Forssk.

1

Outgroup

Blois-France-Greenhouse

Yockteng-146 (P)

AY261533

1

Outgroup

Gilbert-9135

AY261534

Dilkea sp. Mast.

1

Outgroup

USA, Austin, Texas University Greenhouse USA, Austin, Texas University Greenhouse

Gilbert-9246

AY261535

Passiflora species adenopoda D.C.

1

Plectostemma, Pseudodysosmia GranadillaDistephana Granadilla, Quadrangulares Plectostemma, Decaloba, Sexflorae Plectostemma, Decaloba, Punctatae Granadilla, Laurifoliae Granadilla, Laurifoliae Granadilla, Lobatae Granadilla, Lobatae Astrophea, Botryastrophea Astrophea, Botryastrophea Tacsonia, Colombiana, Leptomischae Tacsonia, Colombiana, Leptomischae Astrophea

aimae Annonay & Feuillet alata Curtis

1 1

Allantophylla Mast.

1

alnifolia Kunth

1

ambigua Hemsl.

2

ambigua Hemsl.

3

amethystina Mikan

1

amethystina Mikan

2

amoena Escobar

1

amoena Escobar

2

antioquiensis Karst.

1

antioquiensis Karst.

2

arborea Spreng.

1

aurantia G. Forst.

1

auriculata Kunth

1

auriculata Kunth

3

biflora Lam.

1

biflora Lam.

2

Plectostemma, Decaloba, Eudecaloba Plectostemma, Decaloba, Auriculatae Plectostemma, Decaloba, Auriculatae Plectostemma, Decaloba, Punctatae Plectostemma, Decaloba, Punctatae

Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse French Guiana, Piste St. Elie

Vecchia-1

AY261536

Yockteng-4 (P)

AY261537

Blois-France-Greenhouse

Yockteng-5 (P)

AY261538

Blois-France-Greenhouse

Houel-34 154 (P)

AY261539

Orsay, France, Greenhouse University Paris XI, Seeds from Belize Italy, Ripalta Cremasca, Collection Nationale Italie, Greenhouse Panama, Pipeline Road, Panama Canal Zone, Nal.Park Soberania Blois-France-Greenhouse

Yockteng-6 (P)

AY261540

Vecchia-2 8 (P)

AY261541

Yockteng-7 (P)

AY261542

Yockteng-9 (P)

AY261543

Blois-France-Greenhouse

Yockteng-9 (P)

AY261544

Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse French Guiana

Vecchia-3

AY261545

Yockteng-10 (P)

AY261546

Vecchia-4

AY261547

Yockteng-147 (P)

AY261548

Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Blois-France-Greenhouse Panama, Quebrada Aleman, Fortuna Reservoir, Chiriqu
ı Blois-France-Greenhouse

Yockteng-148 (P)

AY261549

Yockteng-13 (P)

AY261550

French Guiana, Piste St. Elie

Yockteng-15 (P)

AY261551

Colombia, Municipio San Miguel, Antioquia

Yockteng-17 (P)

AY261552

Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse

Morse-199800107

AY261553

Yockteng-19 (P)

AY261554

ARTICLE IN PRESS R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx

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Table 2 (continued) Species

Infrageneric classification

Collection data

Voucher

Accession No.

Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse, Brazil French Guiana

Vecchia-8 152 (P)

AY261555

Yockteng-22 (P)

AY261556

Morse-199500133

AY261557

Yockteng-24 (P)

AY261558

Yockteng-25 (P)

AY261559

Vecchia-10 26 (P)

AY261560

ANBG-66949

AY261561

Yockteng-28 (P) Vecchia-12

AY261562 AY261563

Yockteng-29 (P)

AY261564

Morse-200000042 Vecchia-14 32 (P)

AY261565 AY261566

Morse-199200361

AY261567

Yockteng-34 (P)

AY261568

Yockteng-37 (P)

AY261569

JBAVH-3319 Yockteng-40 (P)

AY261570 AY261571

Vecchia-18

AY261572

Yockteng-41 (P)

AY261573

Morse

AY261574

Yockteng-46 (P)

AY261575

Yockteng-43 (P)

AY261576

Yockteng-44 (P)

AY261577

Yockteng-48 (P)

AY261578

Yockteng-50 (P)

AY261579

Yockteng-49 (P)

AY261580

Yockteng-51 (P) Yockteng-52 (P) Yockteng-55 (P)

AY261581 AY261582 AY261583

Morse-200000041

AY261584

Houel-Guy13

AY261585

caerulea L.

1

Granadilla, Lobatae

candida (Poep & Endl.) Mast. capsularis L.

1

cerasina Annonay & Feuillet cincinnata Mast.

1

Astrophea, Pseudoastrophea Plectostemma, Xerogona Granadilla, Laurifoliae

1

Granadilla, Incarnatae

cincinnata Mast.

2

Granadilla, Incarnatae

Cinnabarina Lindl.

1

cirrhiflora Juss. citrifolia (Juss.) Mast.

1 1

citrifolia (Juss.) Mast.

2

coccinea Aubl. coccinea Aubl.

1 3

Plectostemma, Decaloba, Eudecaloba Polyanthea Astrophea, Pseudoastrophea Astrophea, Pseudoastrophea Distephana Distephana

coriacea Juss.

1

Plectostemma, Cieca

coriacea Juss.

2

Plectostemma, Cieca

coriacea Juss.

5

Plectostemma, Cieca

coriacea Juss. crassifolia Killip

8 1

crenata Feuillet & Cremers crenata Feuillet & Cremers edulis Sims

1

Plectostemma, Cieca Granadilla, Menispermifoliae Granadilla

2

Granadilla

1

Granadilla, Incarnatae

edulis Sims

2

Granadilla, Incarnatae

edulis Sims

3

Granadilla, Incarnatae

edulis Sims

4

Granadilla, Incarnatae

edulis fo.flavicarpa Degener edulis fo.flavicarpa Degener edulis fo.flavicarpa Degener elegans Mast. exura Feuillet foetida L.

1

Granadilla, Incarnatae

3

Granadilla, Incarnatae

4

Granadilla, Incarnatae

1 1 4

Granadilla, Lobatae Granadilla, Lobatae Dysosmia

1

Dysosmia

1

Dysosmia

Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Orsay, France, Greenhouse University Paris XI Orsay, France, Greenhouse University Paris XI Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Orsay, France, Greenhouse University Paris XI Orsay, France, Greenhouse University Paris XI, Seeds from Colombia Blois-France-Greenhouse French Guiana Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Ecology & Evolutionary Biology Conservatory, Univ. Connecticut French Guiana

1

Dysosmia

Blois-France-Greenhouse

Yockteng-58 (P)

AY261586

3

Granadilla

French Guiana, Comte

Houel-Guy15

AY261587

1 2

Granadilla, Lobatae Granadilla, Lobatae

French Guiana, Belizon French Guiana, Belizon

Yockteng-63 (P) Yockteng-63 (P)

AY261588 AY261589

foetida var. arizonica Killip foetida var. hastata (Bertol.) Mast. foetida var. hirsutissima Killip gabriellana Vanderplanck garckei Mast. garckei Mast.

1

Ecology & Evolutionary Biology Conservatory, Univ. Connecticut French Guiana, Route de Kaw, Km.6 Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Orsay, France, Greenhouse University Paris XI Australia, Booroomba Rocks, along Apollo Road, via Naas. French Guiana, Belizon Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse French Guiana, Pont de la Riviere, Comt
e, Route de lÕEst Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Panama, Pipeline Road, Panama Canal Zone, Parque Nal. Soberania Colombia, Ibague, Tolima Botanical Garden Blois-France-Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse French Guiana

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Table 2 (continued) Species

Infrageneric classification

Collection data

Voucher

Accession No.

Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Blois-France-Greenhouse

Yockteng-65 (P)

AY261590

Vecchia-25

AY261591

Yockteng-66 (P)

AY261592

Blois-France-Greenhouse

Houel-7

AY261593

Blois-France-Greenhouse

Yockteng-67 (P)

AY261594

Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Blois-France-Greenhouse

Yockteng-70 (P)

AY261595

Yockteng-71 (P)

AY261596

Blois-France-Greenhouse

Yockteng-72 (P)

AY261597

Blois-France-Greenhouse

Yockteng-73 (P)

AY261598

French Guiana

Yockteng-74 (P)

AY261599

French Guiana, Route de Kaw

Yockteng-75 (P)

AY261600

Blois-France-Greenhouse

Yockteng-76 (P)

AY261601

Morse-199900009 Yockteng-77 (P)

AY261602 AY261603

Yockteng-84 (P)

AY261604

Vecchia-67

AY261605

Yockteng-83 (P)

AY261606

JBAVH-3318 Vecchia-33

AY261607 AY261608

JBMedellin 85 (P) Yockteng-86 (P) Yockteng-87 (P) Yockteng-88 (P)

AY261609 AY261610 AY261611 AY261612

Vecchia-38

AY261613

Yockteng-90 (P)

AY261614

Houel-14 158 (P) JBAVH-3317 Yockteng-92 (P)

AY261615 AY261616 AY261617

Yockteng-91 (P)

AY261618

Yockteng-93 (P)

AY261619

Yockteng-159 (P) Yockteng-94 (P) Morse-199900602

AY261642 AY261620 AY261621

Vecchia-45 96 (P) Yockteng-98 (P)

AY261622 AY261623

Yockteng-99 (P)

AY261624

hahnii (Fourn.) Mast.

1

hahnii (Fourn.) Mast.

2

helleri Peyr.

1

Herbertiana Ker Gawl.

1

hirtiflora Jørg. & Holm-Niels. Indecora Kunth.

1 2

jorullensis Kunth

1

kalbreyeri Mast.

1

karwinskii Mast.

1

kawensis Feuillet

1

kawensis Feuillet

1

lancetillensis MacDougal & Meerman laurifolia L. laurifolia L.

1

Plectostemma, Hahniopathanthus Plectostemma, Hahniopathanthus Plectostemma, Decaloba, Organenses Plectostemma, Decaloba, Eudecaloba Plectostemma, Pseudogranadilla Plectostemma, Pseudogranadilla Plectostemma, Organenses Plectostemma, Pseudogranadilla Plectostemma, Pseudodysosmia Astrophea, Pseudoastrophea Astrophea, Pseudoastrophea Deidamioides

1 7

Granadilla, Laurifoliae Granadilla, Laurifoliae

ligularis Juss.

1

Granadilla, Tiliafoliae

ligularis Juss.

2

Granadilla, Tiliafoliae

ligularis Juss.

3

Granadilla, Tiliafoliae

ligularis Juss. Macrophylla Mast.

4 1

Granadilla, Tiliafoliae Astrophea

maliformis L. manicata (Juss.) Pers. mathewsii (Mast.) Killip Membranacea Benth.

2 1 1 1

menispermifolia Kunth

1

menispermifolia Kunth

2

mixta L. f. mixta L. f. mooreana Hook.

1 2 1

Granadilla, Tiliafoliae Manicata Tacsonia, Tacsonia Plectostemma, Hahniopathanthus Granadilla, Menispermifoliae Granadilla, Menispermifoliae Tacsonia, Tacsonia Tacsonia, Tacsonia Granadilla, Lobatae

mooreana Hook.

2

Granadilla, Lobatae

morifolia Mast.

1

multiflora L. murucuja L. nephrodes Mast.

1 1 1

nitida Kunth oerstedii var. choconiana (Watson) Killip ornithoura Mast.

1 1

Plectostemma, Pseudodysosmia Apodogyne Murucuja Granadilla, Menispermifoliae Granadilla, Laurifoliae Granadilla, Simplicifoliae Plectostemma, Decaloba, Organenses

1

Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Orsay, France, Greenhouse University Paris XI Colombia, Ibague, Tolima Botanical Garden Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Colombia, Medellin, Botanical Garden Blois-France-Greenhouse Blois-France-Greenhouse Blois-France-Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Panama, Pipeline Road, Panama Canal Zone, Parque Nal. Soberania Blois-France-Greenhouse Colombia, Ibague, Tolima Botanical Garden Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Orsay, France, Greenhouse University Paris XI Blois-France-Greenhouse Blois-France-Greenhouse Ecology & Evolutionary Biology Conservatory, Univ. Connecticut French Guiana Blois-France-Greenhouse Blois-France-Greenhouse

ARTICLE IN PRESS R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution xxx (2003) xxx–xxx

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Table 2 (continued) Species

Infrageneric classification

Collection data

Voucher

Accession No.

penduliflora Bertero ex DC. perfoliata L. pittieri Mast.

1

Astephia

Blois-France-Greenhouse

Yockteng-100 (P)

AY261625

1 1

AY261626 AY261627

2

Blois-France-Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Colombia, Medellin, Botanical Garden

Yockteng-101 (P) Vecchia-49

pittieri Mast.

JBMedellin

AY261628

platyloba Killip

1

Pseudomurucuja Astrophea, Dolichostemma Astrophea, Dolichostemma Granadilla, Tiliafoliae

Vecchia-51

AY261629

platyloba Killip

2

Granadilla, Tiliafoliae

Vecchia-50

AY261630

punctata L.

1

Yockteng-104 (P)

AY261635

Quadrangularis L.

1

Morse-19990006

AY261631

Quadrangularis L.

2

Yockteng-105 (P)

AY261632

Quadrangularis L.

3

Vecchia-54

AY261633

racemosa Brot. reflexiflora Cav. riparia Mart. Ex Mast. rubra L.

1 1 1 1

rufostipulata Feuillet sandrae MacDougal

1 1

Plectostemma, Decaloba, Punctatae Granadilla, Quadrangulares Granadilla, Quadrangulares Granadilla, Quadrangulares Calopathanthus Tacsonioides Granadilla, Laurifoliae Plectostemma, Xerogona Granadilla, Laurifoliae

sanguinolenta Mast. & Linden seemanii Griseb

1

Psilanthus

1

Granadilla, Tiliafoliae

serrato-digitata L.

1

Granadilla, Digitatae

serrato-digitata L.

1b

Granadilla, Digitatae

serrato-digitata L.

2b

Granadilla, Digitatae

serrulata Jacq.

1

Granadilla, Tiliafoliae

sexflora Juss.

2

sprucei Mast.

1

Plectostemma, Decaloba, Sexflorae Granadilla, Lobatae

standleyi Killip

1

Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Blois-France-Greenhouse Blois-France-Greenhouse Blois-France-Greenhouse Orsay, France, Greenhouse University Paris XI French Guiana Panama, Pipeline Road 8Kms, Panama Canal Zone, Parque Nal. Soberania Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Orsay, France, Greenhouse University Paris XI Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Veerweg, Holland, Nationale Collectie PassifloraÕs Greenhouse Italy, Ripalta Cremasca, Collection Nationale Italie Greenhouse Blois-France-Greenhouse

tacsonioides Killip talamancensis Killip

1 3

telesiphe Knapp & Mallet tetrandra Banks ex DC. trialata Feuillet & MacDougal trifasciata Lem.

1

tripartita var mollissima (Kunth) Holm-Niel & Jørg tripartita var mollissima (Kunth) Holm-Niel & Jørg

1 1

Plectostemma, Decaloba, Punctatae Pseudomurucuja Plectostemma, Decaloba, Punctatae Plectostemma

1b

Tetrapathea Granadilla, Quadrangulares Plectostemma, Decaloba, Miserae Tacsonia, Bracteogama

2

Tacsonia, Bracteogama

1

Yockteng-106 Yockteng-107 Yockteng-108 Yockteng-109

(P) (P) (P) (P)

AY261634 AY261636 AY261637 AY261638

Yockteng-111 (P) Yockteng-112 (P)

AY261639 AY261640

Morse-199800014

AY261641

Yockteng-116 (P)

AY261643

Morse-199900027

AY261644

Yockteng-148 (P)

AY261645

Vecchia-58

AY261646

Yockteng-119 (P)

AY261647

Yockteng-120 (P)

AY261648

Vecchia-60

AY261649

Yockteng-121 (P)

AY261650

Blois-France-Greenhouse Blois-France-Greenhouse

Yockteng-124 (P) Yockteng-125 (P)

AY261651 AY261652

Blois-France-Greenhouse

Yockteng-127 (P)

AY261653

Blois-France-Greenhouse French Guiana

Yockteng-128 (P) Yockteng-129 (P)

AY261654 AY261655

Blois-France-Greenhouse

Yockteng-130 (P)

AY261656

Blois-France-Greenhouse

Yockteng-132 (P)

AY261657

Orsay, France, Greenhouse University Paris XI, Seeds from Colombia, Cajica

Yockteng-153 (P)

AY261658

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Table 2 (continued) Species

Infrageneric classification

Collection data

Voucher

Accession No.

3

Tacsonia, Bracteogama

Colombia, La Calera

Yockteng-131 (P)

AY261659

4

Tacsonia, Bracteogama

Colombia, La Calera

Yockteng-131 (P)

AY261660

1

Tacsonia, Bracteogama

Ecology & Evolutionary Biology Conservatory, Univ. Connecticut

Morse-Student

AY261661

1 2

Manicata Tryphostemmatoides

Blois-France-Greenhouse Colombia-Valle del Cauca Yumbo Monta~ nitas KM6 Via Pavitas San Jose Blois-France-Greenhouse Blois-France-Greenhouse

Yockteng-133 (P) Yockteng-134 (P)

AY261662 AY261663

Yockteng-136 (P) Houel-27 157 (P)

AY261664 AY261665

Blois-France-Greenhouse French Guiana, Crique Plomb

Houel-30 Yockteng-137 (P)

AY261666 AY261667

Ecology & Evolutionary Biology Conservatory, Univ. Connecticut Colombia, Ibague, Tolima Botanical Garden

Morse-198700236

AY261668

JBAVH-3320

AY261669

tripartita var mollissima (Kunth) Holm-Niel & Jørg tripartita var mollissima (Kunth) Holm-Niel & Jørg tripartita var mollissima (Kunth) Holm-Niel & Jørg trisecta Mast. tryphostemmatoides Harms tulae Urb umbilicata (Griseb.) Harms variolata Poep & Endl. vespertilio L.

1 1

Murucuja Tacsonioides

1 3

vitifolia (Harv.) Harms

1

Distephana Plectostemma, Decaloba, Punctatae Distephana

vitifolia (Harv.) Harms

5

Distephana

Collection data, voucher, and GenBank accessions numbers are given. Specimens are deposited at the Herbarium of the Museum National dÕHistoire Naturelle (Paris, France (P)). JBMedellin, Botanical Garden Medellin, Colombia; JBAVH, Botanical Garden Alejandro Von Humboldt, Tolima, Colombia; Morse, University of Connecticut, USA; Houel, Passiflora National Collection, Blois, France; Vecchia, Passiflora National Collection, Italy; ANBG, Australian National Botanical Gardens; Gilbert, Passiflora collection, Greenhouse University Texas, Austin.

Fig. 1. Location of the amplified and sequenced region of the chloroplast-expressed glutamine synthetase (ncpGS) for Passiflora. The intron-exon distribution is with respect to the ncpGS sequence of Juglans nigra (GenBank Accession No. AF169795).

The thermal cycler (PTC-100 MJ-Research) was programmed for an initial step of 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 45 s at 55 °C, 1.5 min at 72 °C, and a final step of 7 min at 72 °C. PCR products were visualized on a 2% agarose gel, purified using the PEG precipitation protocol (Rosenthal et al., 1993), and both strands were sequenced using the same primer combination as for PCR amplifications. Cycle sequencing products were run on ABI capillary sequencers (MWG Biotech). Sequences were deposited in GenBank. 2.3. Phylogenetic analyses Clustal X (Thompson et al., 1997) was used to produce a first alignment, which was corrected manually using the Bioedit program (Hall, 1999). The indels of the non-coding regions were coded using GapCoder, a program developed by Neil Young and based on the

Simmons and OchoterenaÕs method that codes indels as separate characters in a data matrix (Simmons and Ochoterena, 2000). We examined nucleotide and translated sequences to search for patterns characteristic of specific taxonomic groups. The significance of the taxonomic group effect on intron length was tested by one-way ANOVA (Excel, Microsoft). Phylogenetic reconstruction was carried out using either the whole alignment or two subsets of this alignment, one consisting of the coding regions, the other consisting of the non-coding regions (introns). 2.4. Parsimony Maximum parsimony (MP) analyses were conducted using PAUP 4.0b10a* (Swofford, 2001). Heuristic searches were conducted with tree-bisection reconnection

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(TBR) branch swapping, multiple trees ON, and 1000 random taxon addition replicates limiting the rearrangements to 100,000 per replicate and saving all most parsimonious trees. Support for internal nodes was assessed by bootstrapping (100 replicates). Decay indexes were calculated using the Autodecay 4.0 program (Eriksson, 1998). 2.5. Maximum likelihood Due to the large number of taxa included in our study we conducted the maximum likelihood (ML) analyses using the Quartet Puzzling method (Strimmer and vonHaeseler, 1996) implemented in PAUP 4.0b10a* (Swofford, 2001), which takes a reasonable calculation time. We used 1000 steps and the model of evolution GTR + I + G with a gamma shape parameter equal to 1.9758, estimated using Modeltest version 3.06 (Posada, 2001). This model was used in the analyses based on the whole alignment and on the intron subset. For the exon subset, we specified that the sequence corresponded to a coding region. 2.6. Bayesian approach Additionally, a Bayesian analysis was conducted with MrBayes 2.01 program (Huelsenbeck and Ronquist, 2001). We used uniform, prior probabilities, the general time reversible + I + G model of molecular evolution, and a random starting tree. Four chains of the Markov Chain Monte Carlo were run simultaneously, sampled every 100 generations for a total of 150,000 generations. Stationarity conditions were reached around generation 82,000; thus the first 820 trees were burnt in of the chain (eliminated). The majority rule consensus tree was calculated with PAUP 4.0b10a* (Swofford, 2001). Phylogenetic inferences were based on this consensus. 2.7. Character evolution The patterns of phenotypic evolution based on 19 morphological characters and the divergence in the chromosome numbers were assessed by overlaying the different states on the subgenera and outgroups branches. The character-state evolution was reconstructed using parsimony method in MacClade 3.08 program (Maddison and Maddison, 1992). The morphological characters selected consist principally of the characters used by Killip (1938) to establish the different subgenera. The character states of different subgenera were established based on Killip (1938), deWilde (1974), MacDougal (1994), Escobar (1988, 1994), Cervi (1997), Vanderplanck (2000), and several floras. We used the outgroup states as the ancestral states (deWilde, 1974; Killip, 1938).

9

3. Results 3.1. Characteristics of ncpGS sequences in Passiflora The primers used by Emshwiller and Doyle (1999) resulted in the amplification, in several Passiflora species, of the cytosolic-expressed glutamine synthetase (cytGS) instead of the expected chloroplast-expressed glutamine synthetase (ncpGS). These preliminary results revealed a first division within the genus Passiflora. The cytosolic glutamine synthetase was amplified for species belonging to two subgenera: Murucuja and Plectostemma. The chloroplast-expressed glutamine synthetase (ncpGS) was amplified for all other species belonging to the subgenera Distephana, Granadilla, Tacsonia, and Tacsonoides. The newly designed pair of primers allowed specific amplification of the ncpGS gene for all Passiflora species. The sequences length ranges between 401 and 659 bp. The final alignment comprises 814 positions. The coding subset includes 226 characters, corresponding to 75 amino acids and the intron subset comprises 587 positions. The exons of the ncpGS region analyzed are quite conserved but nonetheless they present motifs containing taxonomic information. As an example, the 46th amino acid splits the species into two groups. The first group, holding all species of the subgenera Deidamioides, Distephana, Granadilla, Polyanthea, Tacsonia, and Tetrapathea, excepting the species representing the series Lobatae and Menispermifoliae (subgenus Granadilla), present a Serine (S). At this position, all the other species except P. rubra and P. sexflora, and the 13 species of Lobatae and Menispermifoliae share a Threonine (T). A similar pattern is observed with positions 22, 27, and 44. The introns, with their numerous indels, display a reasonable variability, suitable for phylogenetic inference. The mean pairwise intron divergence of 0.147 is three times higher than the exon divergence (0.043). Only a few indels were due to the outgroup. The majority were produced by the ingroup alignment. ANOVA tests show that the differences in total intron length among subgenera are significant (Table 3). The noncoding region was significantly longer in subgenera Astrophea, Distephana, Dysosmia, Granadilla, Manicata, Tacsonia, and Tacsonoides, than in Murucuja, Plectostemma, and Pseudomurucuja. Moreover, the length of the first intron of Astrophea was significantly different from that of the other subgenera. The analyses did not include the individuals of subgenera Calopathanthus, Psilanthus, Polyanthea, Deidamioides, Tetrapathea, Apodogyne, and Astephia because the number of individuals available in these subgenera was less than two. However, the intron lengths of Calopathanthus, Psilanthus, Polyanthea, Deidamioides, Tetrapathea, Tryphostemmatoides, and the outgroup were

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Table 3 ANOVA test of the significance of total intron length differences among the different subgenera N ¼ 108

df

Mean square

F

P >F

Subgenera Subgenera
total intron length Error

9 98 107

14400.59 230.75 152219.07

62.4

1.27E ) 36

similar to the third group. In contrast, the intron length of Apodogyne and Astephia species was closer to that of the second group. 3.2. Phylogenetic analysis The total alignment displayed 329 parsimony-informative sites and 356 invariable sites. Most informative sites were located in the intron regions (272 sites). The number of informative sites rose to 412 when the indels were coded. MP analyses of the whole alignment (coding and noncoding regions) yielded 32,573 most parsimonious trees of 1579 steps. The strict consensus tree (Fig. 2) is highly resolved and most of the nodes are well supported, as shown both by the bootstrap values and decay indexes. The topology did not change using coded or not coded indels. However, the indels were important to identify the main groups, and for this reason the analyses were performed with coded indels. Both maximum likelihood and Bayesian analyses resulted in trees well supported and highly congruent with the MP consensus tree. Separate analyses of the coding and non-coding regions produced less resolved trees. For each dataset the different methods used yielded similar topologies. The non-coding region produced topologies very similar to the trees obtained using the entire alignment, although some nodes were less supported. When using the coding region, only the internal nodes defining the main taxonomic groups were well supported. The individuals of the same species are branched together or they are at least in the same clade in the tree. The overall topology revealed three well-supported clades (Fig. 2), each including one of the three largest subgenera: Plectostemma, Granadilla and Astrophea. Clade 1 is composed of the subgenera Astrophea and Tryphostemmatoides; clade 2 contains the species of the subgenera Astephia, Deidamioides, Murucuja, Plectostemma, Pseudomurucuja, and Tetrapathea; and clade

3 consists of the subgenera Calopathanthus, Distephana, Dysosmia, Granadilla, Manicata, Psilanthus, Tacsonia, and Tacsonoides. P. cirrhiflora Juss., from the monospecific Polyanthea subgenus, is sister group to all other Passiflora species. Figs. 3a–c present the evolution of 19 morphological characters and the chromosome numbers in different subgenera. These reconstructions helped us to support or reject the different clades.

4. Discussion The chloroplast-expressed glutamine synthetase gene proved its usefulness for resolving the relationships among species of the genus Passiflora. In spite of the relatively short length of the ncpGS alignment, the level of variation was satisfactory with 329 parsimony-informative sites (40%) compared to other DNA regions. For example, the alignment of ITS sequences for 36 Passiflora species (GenBank Accession Nos. AY1023461– AY1023831, AY0328231–AY0328431, AY0327811– AY0328011) presents 21% of informative sites. The alignment of 38 sequences of the chloroplast trnL–trnF region from Passiflora species (GenBank Accession Nos. AY0327601–AY0327801, AY1023861–AY1024031) displays 11% of informative sites. The comparison of ncpGS, cytosol-expressed glutamine synthetase (cytGS), and the chloroplastic gene matK shows that the ncpGS has a higher level of divergence than matK and gives an acceptable phylogenetic signal (Yockteng and Nadot, 2003). As expected at this taxonomic level, the noncoding region provides most of the phylogenetic information. Interestingly, the length of the introns proved particularly informative, creating indels in the alignment that had a great impact on the resolution of the trees. Although the coding region was highly conserved, the information it contained allowed identification of clear, well supported major taxonomic divisions within the genus.

c Fig. 2. Strict consensus tree of 32,573 most parsimonious trees based on the alignment of chloroplast-expressed glutamine synthetase (ncpGS) sequences. Bootstrap support values (BS) greater than 70% and decay indices greater than 4 are indicated above branches. Thick lines indicate BS values of 100%, dots indicate nodes supported by 100% in the Bayesian tree. The subdivisions according to Killip (1938) are indicated for each taxon. The sections and series names are abbreviated as follows: Cie, Cieca; Dec, Decaloba; Auri, Auriculatae; Sexf, Sexflorae; Orga, Organenses; Mise, Miserae; Punc, Punctatae; Eud, Eudecaloba; Xero, Xerogona; Psedy, Pseudodysosmia; Psegr, Pseudogranadilla; Hah, Hahniopathanthus; Lept, Leptomischae; Col, Colombianae; Bract, Bracteogama; Tac, Tacsonia; Quad, Quadrangulares; Digi, Digitatae; Tili, Tiliafoliae; Laur, Laurifoliae; Inca, Incarnatae; Simp, Simplicifoliae; Loba, Lobatae; Meni, Menispermifoliae; Doli, Dolichostemma; Euas, Euastrophea; Pseas, Pseudoastrophea. The three clades emerging from the analysis are indicated on the right.

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Fig. 3. Character evolution of 19 morphological characters and chromosome numbers for different subgenera. The different states are represented by black tick marks, densely dotted tick marks, dispersed dotted tick marks, and double tick marks. Double pattern tick mark indicates a polymorphic character state (two different states). The character states indicated on the basal branch are the outgroup states. (a) Vegetative characters: 1. tendrils; 2. tendrils terminating the peduncle; 3. leaves; 4. polymorphic leaves (two different leaf shapes in the same individual); 5. hooked trichomes or uncinate trichomes in the leaf blade; 6. petiolar glands; 7. bracteal glands with three different states absent, present at margin or present at base; 8. bracts; (b) anatomical characters and chromosome numbers: 9. tree species: yes or no; 10. dioecious plants; 11. seed ridges; 12. pollen type: type 1 (reticulum with less than 5 lm wide and exine with 2 12–4 lm wide) or type 2 (reticulum with more than 7 lm wide and exine with more than 6 lm wide) (Presting, 1965); 13. chromosome number (n); and (c). floral characters: 14. inflorescences; 13. colour flower: light (white, green, pinkish, yellowish) or hot (red, rose, violet, yellow carmine); 14. calyx tube: not tubular (bowl-shaped, funnel-shaped, patelliform), short tubular (less than 2 cm long) or long tubular (more than 2 cm long); 15. petals; 16. corona filaments: linear/filiform, verrucose, forming a tubular membrane or tubercles-shaped; 17. operculum, and 18. ovary shape.

All the phylogenetic analyses conducted resulted in congruent trees that have well resolved and well supported topologies. The strongest support values were obtained with the Bayesian analysis, which has recently become popular in phylogenetics, and accessible through the MrBayes program (Huelsenbeck and Ronquist, 2001). Although Suzuki et al. (2002) pointed out that this method creates an overestimation of the probabilities of the internal nodes with respect to other methods, the strong support values obtained with our Bayesian analysis were corroborated by the bootstrapping assessment and the decay indexes conducted in the MP analysis (Fig. 2).

The general topology presents three main well supported monophyletic groups (Fig. 2). The first clade, which consists of the species representing subgenera Astrophea and Tryphostemmatoides, is sister group to two derived clades. These two clades regroup 15 of the 17 subgenera represented in this study. The most basal group appears to be the monotypic subgenus Polyanthea, with its one species P. cirrhiflora from French Guiana. On the basis of morphological characteristics such as the presence of compound leaves and glands at the margin of bracts (Fig. 3a), Killip created a separate subgenus for this species in spite noting its apparent relationship to the subgenus

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13

Fig. 3. (continued)

Astrophea because of the shared presence of verrucose outer corona filaments and a truncate and 3-angled ovary (Fig. 3c). Our results show that P. cirrhiflora and Astrophea are sister groups to the rest of the genus, and paraphyletic, indicating that the characters shared by these two taxa could be considered to be plesiomorphic. Their paraphyly is strongly supported in all trees: 14 changes separate the two groups. We were unable to include SE Asian Passiflora species, but it would have been interesting to examine the relationship between species from this geographical region and P. cirrhiflora, since both groups have been considered as subsections of the section Decaloba by Harms (1925). So far, our results support the classification of Killip (1938) in which P. cirrhiflora forms an independent group. 4.1. Clade 1 The subgenus Astrophea forms a very well supported clade with bootstrap support of 100. This result is in agreement with the new classification of Passiflora proposed by Feuillet and MacDougal (1999), in which Astrophea is considered as one of the four subgenera. Morphological features such as their arboreal habit and scar-like petiolar glands (Killip, 1938) (Figs. 3a and c)

constitute therefore synapomorphies of this group. The basal position of Astrophea suggests that the arborescent habit could be ancestral in the genus, as assumed Benson et al. (1975). Its pollen type shared with Plectostemma, Polyanthea, and the outgroups (Presting, 1965) (Fig. 3b) could then considered as a pleisiomorphy. The position of P. candida (Poep & Endl.) Mast., separated from the Astrophea clade in our trees, is unexplained. This position is possibly the result of a wrong identification of this species. Passiflora tryphostemmatoides Harms appears closely related to the Astrophea clade. This species has been classified in an independent subgenus called Tryphostemmatoides on the basis of the presence of a non-plicate operculum, of a tendril terminating the peduncle and finally the presence of specialized branching-tendril (Killip, 1938; Vanderplanck, 2000) (Fig. 3a and c). Several species from the subgenus Plectostemma were described as presenting also a non-plicate operculum (Holm-Nielsen and Jørgensen, 1986; Killip, 1938), leading the authors to argue that all members of the subgenus Tryphostemmatoides should be included in Plectostemma. However, the relationship between P. tryphostemmatoides and Astrophea subgenus is well supported in our phylogeny (Bootstrap Support ¼ 88).

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Fig. 3. (continued)

Therefore, we consider subgenus Tryphostemmatoides as the sister group of subgenus Astrophea. 4.2. Clade 2 Clade 2 consists of a well supported group (BS ¼ 87) of 36 species belonging to six different subgenera identified by Killip (1938). Two small subgenera are found at the base of this clade: Deidamioides, (represented by P. lancetellensis MacDougal & Meerman), and the monospecific Tetrapathea from New Zealand (P. tetrandra Banks ex DC). Most of this clade consists of species representing the subgenus Plectostemma of Feuillet and MacDougal (1999), which includes the subgenera Apodogyne, Astephia, Murucuja, Plectostemma, and Pseudomurucuja recognized by Killip (1938). The status of Deidamioides as an independent subgenus, suggested by Harms (1923) and accepted by Killip (1938) and Feuillet and MacDougal (1999), is supported by our results, as shown by the presence of 21 changes between P. lancetellensis and the Plectostemma group. The monophyly of this subgenus is

supported by a rare combination of features like the plicate operculum, trifoliate leaves and peduncles terminating in a 2-flowered tendril (Figs. 3a and c). The position of P. tetrandra, intermediate between P. lancetellensis and the Plectostemma group, indicates that Tetrapathea is not an independent monotypic genus. This result suggests that the particular reproductive features of this species such as dioecy (Fig. 3b), never observed elsewhere in the genus Passiflora, are derived character states and do not reflect an independent origin from Passiflora. Likewise, this clade includes five subgenera initially considered by Killip (1938) as independent. Our results show that these subgenera are embedded within Plectostemma, making this group non-monophyletic. We suggest therefore that only one subgenus should be maintained: Plectostemma. In fact, P. multiflora, P. penduliflora, P. perfoliata, and P. tacsonoides were regarded in the past as belonging to Plectostemma until they were placed in three different subgenera for possessing many segregating characters (Killip, 1938). For example, P. multiflora was excluded from Plectostemma

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and moved to a new monotypic subgenus Apodogyne on the basis of distinctive characters such as glandular petioles and inflorescences in fascicles (Figs. 3a and c). Our results shed a new light on the interpretation of morphological features. For example, P. multiflora and Plectostemma species closely related in our trees share a plicate operculum and also transversely sulcate seeds (Figs. 3b and c). These arguments support the inclusion of the subgenus Apodogyne into Plectostemma. Similarly, characters like the absence of glands in petioles and transversely sulcate seeds argue for the inclusion of P. perfoliata L., P. tacsonoides Killip (Pseudomurucuja), P. tulae Urb., and P. murucuja L. (Murucuja) in Plectostemma (Figs. 3a and b). These latter species were classified in a separate subgenus on the basis on their coloured flowers and the corona forming a tubular membrane for Murucuja (Fig. 3c). Passiflora penduliflora Bertero ex DC. was also considered as a separate subgenus by Killip (1938) because of its lack of an operculum (Fig. 3c). The position of P. murucuja, P. tulae, P. penduliflora, P. tacsonoides, and P. perfoliata in a same sub-clade is not unexpected since all these species are mainly distributed in the West Indies. Furthermore, the trees confirmed the position of the three Australian species, P. herbertiana Ker Gawl., P. aurantia G. Forst., and P. cinnabarina Lindl. described before as belonging to Plectostemma. These were grouped in a subsection called Eudecaloba within section Decaloba of Plectostemma (deWilde, 1972; Harms, 1925) (Table 1). To confirm the hypothesis of deWilde (1972) that old world indigenous species belong to Plectostemma, it would be necessary, in a further study of the genus Passiflora, to include the Asian and New Guianan species regrouped in the three sections Decaloba subsect. Polyanthea, Holrungiella, and Octandranthus (deWilde, 1974). On the basis of these results, we suggest retention of the three following subgenera in clade 2: Deidamioides, Tetrapathea, and Plectostemma. We also suggest a subdivision of subgenus Plectostemma into three different sections. One section would be composed of all the series of Decaloba, sections Xerogona, Cieca and Pseudogranadilla and also the species P. murucuja, P. tulae, P. penduliflora, P. tacsonoides, and P. perfoliata. The newly described species from Panama, P. sandrae (ined.) should be included in this section, according to our results. The second section would be composed of the section Pseudodysosmia and also include P. alnifolia Kunth., initially classified in a different series, namely Puntactae but appearing derived from Pseudodysosmia in our trees. This last result is in agreement with MacDougal (1994) who described this section as monophyletic because of the presence uncinate trichomes in the shoot apex: consequently he named the section the ‘‘hooked trichomes group’’ (Fig. 3a). Finally, the species from the series Hahniopathanthus with their reticulate

15

sulcate seeds (Fig. 3b) would form the last section, which is sister to the rest of Plectostemma species. 4.3. Clade 3 This strongly supported clade (BS ¼ 100) includes all the species representing the subgenera Calopathanthus, Distephana, Dysosmia, Granadilla, Manicata, Psilanthus, Tacsonia, and Tacsonoides. The subgenus Dysosmia is the sister clade to the rest of the species. The monophyly of the species P. foetida L. is well supported (BS ¼ 100). The presence of unique characters such as pinnatisect bracts, petiolar glands modified in hairs, and absence of tendrils (Fig. 3a) supports the maintenance of this species in a separate subgenus. The rest of the species are distributed in three wellsupported groups of unresolved relationship. The presence of P. sanguinolenta Mast. & Linden from the subgenus Psilanthus is unexpected in clade 3. In fact, several morphological characters such as the pollen type and the absence of bracts (Figs. 3a and b) suggest an affinity of P. sanguinolenta with the species of clade 2 (Killip, 1938; Snow and MacDougal, 1993). However, the presence of a red tubular calyx and a non-plicate operculum of P. sanguinolenta distinguish it from clade 2 (Fig. 3c). Further analyses including more species of the subgenus Psilanthus are necessary to clarify its relationship with the species of the Granadilla group. Since the species representing subgenera Calopathanthus, Distephana, Manicata, Psilanthus, Tacsonia, and Tacsonoides are nested within Granadilla in the trees, we suggest that this group should be united in one subgenus. We also propose that Granadilla should be subdivided into three different sections. The first section (BS ¼ 92) would correspond to the subgenera Calopathanthus, Manicata, and Tacsonia. Both latter subgenera are mainly distributed in the Andes Mountains and share a long calyx tube (Fig. 3c). In contrast, P. racemosa Brot. (Calopathanthus) is mainly distributed in Brazil and has an operculum that forms a cylindrical tube. However, its relationship with Manicata and Tacsonia is strongly supported, suggesting that these characters are derived. The second section would correspond to the series Quadrangulares, Incarnatae, and Laurifoliae (subgenus Granadilla) and the subgenus Distephana. This latter subgenus consists of species with scarlet flowers and corona filaments forming a tubular membrane suggesting a monophyletic origin for this group of species (Fig. 3c). The position of P. cincinnata Mast. (subgenus Granadilla) within Distephana is unexpected, since this species does not display these specific morphological features. As this subclade consists of a large polytomy, an explanation could be that P. cincinnata is actually the sister group to Distephana. The position of the recently described French Guiana species P. aimae confirms its relationship with Distephana, as

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suggested by Annonay and Feuillet (1998) P. gabriellana, also a French Guiana species recently described as being part of Granadilla subgenus (Vanderplanck, 2000), appears to be related to P. laurifolia inside this group confirming its classification. The last section that we propose can be split into two subsections. The first subsection is formed by the series Tiliafoliae and Digitatae (subgenus Granadilla). The presence of united bracts at the base enveloping the buds would represent a synapomorphy of this sub-clade. The other subsection contains the series Lobatae, Menispermifoliae, and Simplicifoliae (subgenus Granadilla), which share foliaceous and verticillate bracts. The subgenus Tacsonoides, represented by P. umbilicata and P. reflexiflora, is paraphyletic, in a basal position within this subsection. The classification of P. reflexiflora and P. umbilicata has varied over time. Their long calyx tube favours their inclusion in Tacsonia (Harms, 1925) whereas the 2–4 seriate corona supports their inclusion in the disappeared section Tacsonoides (subgenus Granadilla) (De Candolle, 1828). Our results validate this latter suggestion. Since Tacsonoides appears paraphyletic in the trees, the similarity between the two species could be due to the retention of plesiomorphies inherited from a common ancestor or a convergent adaptation to hummingbird pollination (MacDougal, 1994). To summarize, we suggest that two subgenera should be recognized in clade 3: Dysosmia and Granadilla, and we propose to subdivide Granadilla into three sections. 4.4. Chromosome number The division of Passiflora into three main clades based on DNA sequence data from the glutamine synthetase nuclear gene expressed in the chloroplast, and our subsequent suggestion to group 10 subgenera in three subgenera, complies with variation in chromosome numbers. Each of our three clades is characterized by a different base chromosome number: x ¼ 12 chromosomes for clade 1 (Astrophea s.l.), x ¼ 6 for clade 2 (Plectostemma s.l.), and x ¼ 9 for clade 3 (Granadilla s.l.) (De Melo et al., 2001; Snow and MacDougal, 1993) (Fig. 3b). The only incongruence between the classification and the chromosome number concerns P. sanguinolenta. The single chromosome count available for this species gave n ¼ 6 chromosomes, leading Snow and MacDougal (1993) to place it with Plectostemma, whereas our results support its inclusion within Granadilla, in agreement with Killip (1938). The similarity of chromosome number between P. sanguinolenta and Plectostemma could result from convergence. Furthermore, the unique chromosome count available for the Deidamioides subgenus (Gilbert and MacDougal, 2000) gave n ¼ 9 chromosomes (Fig. 3b), relating it to clade 3. The difference in the chromosome

number of Deidamioides and Plectostemma (s.l) would support the independent origin and the monophyly of Deidamioides. Dysosmia, considered here as a separate subgenus within clade 3, has variable chromosome numbers from n ¼ 9 to n ¼ 11 (Fig. 3b). The high morphological variability displayed by the species belonging to Dysosmia and reflected by their subdivision in several subspecies and varieties has been suggested to be the product of a constant hybridization process (Killip, 1938; Vanderplanck, 2000), a likely explanation for the chromosomal variability of this group. Despite this variability, the chromosome numbers observed support the sister group relationship between Granadilla and Dysosmia. Raven (1975) affirmed that the base number for Passiflora is x ¼ 9. The position of Astrophea in the phylogeny suggests in contrast that n ¼ 12 is the ancestral chromosome number of Passiflora. The presence of n ¼ 12 chromosomes in the outgroup Adenia (Passifloraceae) supports this possibility. To confirm this hypothesis, chromosome counts for P. cirrhiflora, sister group to all other Passiflora species, and for P. tryphostemmatoides (basal in clade 1) are necessary.

5. Conclusion The study presented here is the first comprehensive molecular phylogenetic analysis of the large genus Passiflora. It allowed the identification of eight clades within Passiflora, and we suggest that each of them should be considered as a separate subgenus, namely Astrophea, Deidamioides, Dysosmia, Granadilla, Plectostemma, Polyanthea, Tetrapathea, and Tryphostemmatoides. Our phylogeny is in overall agreement with the new infrageneric classification proposed by Feuillet and MacDougal (1999), in which four subgenera are recognized (Plectostemma, Astrophea, Deidamioides, and Granadilla), but our results give evidence for the existence of three additional subgenera. In spite of the good resolution provided by the ncpGS gene, further studies using additional molecular markers, such as chloroplast regions, must be undertaken in order to confirm our results. It would also be necessary to increase the number of species and in particular to include the Asian members of Passiflora as well as species of subgenera not represented in this analysis.

Acknowledgments We are extremely grateful to Christian Houel (Passiflora National Collection, Blois, France) who provided a great part of the plant material. We deeply thank his continuous help and availability. We are also indebted to Catalina Estrada (University of Texas, Austin, USA),

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Cor Laurens (Passiflora National Collection, Holland), Maurizio Vecchia (Passiflora National Collection, Italy), Clinton Morse (University of Connecticut, USA), Chris Jiggins (Smithsonian Tropical Institute, Panama), Margarita Beltr
an (Smithsonian Tropical Institute, Panama), Alejandro Merch
an (Universidad de los Andes, Bogota, Colombia), H
ector Eduardo Esquivel (Botanical Garden Alejandro Von Humboldt, Tolima, Colombia), Alexandra Hiller (Giessen University, Germany), Lawrence Gilbert (University of Texas, Austin, USA), and the Australian National Botanical Gardens for generously providing leaf and seed material for DNA extractions. We thank C
eline Devaux, Bernard Lejeune, Jacqui Shykoff, and two anonymous reviewers for their helpful comments on the manuscript.

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