phylogenetic relationships among passiﬂora species based...

MOLECULARPHYLOGENETICSAND

Molecular Phylogenetics and Evolution 31 (2004) 379–396

EVOLUTION

www.elsevier.com/locate/ympev

Phylogenetic relationships among Passiflora species basedon the glutamine synthetase nuclear gene expressed

in chloroplast (ncpGS)

Roxana Yockteng* and Sophie Nadot

Laboratoire �Ecologie, Syst�ematique et �Evolution, 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 Trypho-

stemmatoides. These results are in overall agreement with the Feuillet and MacDougal�s 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

Killip�s 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.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Passiflora; Phylogeny; ncpGS

1. Introduction

The genus Passiflora L. is well known for its com-

mercial uses. Many species are widely cultivated for fruit

production such as P. edulis Sims. (passion fruit),

P. tripartita var. mollissima (Kunth) Holm-Niel andJørg (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 � 2003 Elsevier Inc. All rights reserved.

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 Mal-

phigiales and appear closely related to the Violaceae and

Flacourtiaceae (Chase et al., 2002; Soltis et al., 1999).

Passifloraceae are divided into two tribes, the Africantribe Pariopsae with 6 genera and the worldwide tribe

Passiflorae with 11 genera. Passiflora L. (tribe Passiflo-

rae) 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 theleaf 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

mail to: [email protected]

380 R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution 31 (2004) 379–396

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 be-

tween 475 and 485 recognized species of Passiflora

(Vanderplanck, 2000). The first infrageneric classifica-

tion encompassing 355 species of the genus recognized

22 subgenera (Killip, 1938) (Table 1) based on variationin 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 re-

cently, Feuillet and MacDougal (1999) proposed a newinfrageneric 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 indepen-

dent genus or as a subgenus of Passiflora, remain to beresolved.

Although studies of molecular genetic variation based

on RAPD and AFLP markers have been carried out on

the cultivated species of subgenera Tacsonia and Gra-

nadilla (Fajardo et al., 1998; S�anchez et al., 1999), no

molecular phylogenetic analysis encompassing all sub-

genera 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 hasbeen proven useful for phylogenetic reconstruction and

identification of processes of reticulate evolution (Ems-

hwiller and Doyle, 1999, 2002). This gene belongs to a

multigene family responsible for the nitrogen metabo-

lism. It diverged 250–300Myr ago from the cytosolic-

expressed glutamine synthetase genes (Kumada et al.,

1993; Pesole et al., 1991) and plays an independent role

in the assimilation of the ammonia produced duringphotorespiration. Although the coding region is ex-

tremely 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 subgen-

eric 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 apreliminary 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 recog-

nized 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 obtainedfrom the wild, from botanical gardens, and from several

national collections (Table 2). Sampled leaves were

preserved in silica gel. Herbarium specimens were de-

posited at the Herbarium of the Museum National

d�Histoire 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 calledPassiflora subgenus and Incarnatae for the Passiflora

series (Table 2) (Killip, 1938). Since, the new classifica-

tion of Feuillet and MacDougal (1999) is not yet pub-

lished in full, the discussion is based on Killip�sclassification.

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 manufacturer�s in-

structions.

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 687and 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 TGCTCT 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), 0.2U of Taq

polymerase (Q-Biogene), 1.5mM of MgCl2, 250 lMof dNTPs (Promega), and 0.2 lM of each primer.

Table 1

Infrageneric classification in alphabetic order of Passiflora proposed by Killip (1938) and modified by Escobar (1988, 1989)

Subgenus Section Series Number of species

Adenosepala, Killip, 1938 1

Apodogyne, Killip, 1938 1

Astephia, Killip, 1938 1

Astrophea (DC.) Masters, 1871 Botryastrophea 11

Cirrhipes 1

Dolichostemma 2

Euastrophea 15

Leptopoda 1

Pseudoastrophea 16

Calopathanthus (Harms) Killip, 1938 1

Chloropathanthus (Harms) Killip, 1938 2

Deidamioides (Harms) Killip, 1938 2

Distephana (Juss.) Killip, 1938 11

Dysosmia (DC.) Killip, 1938 11

Dysosmioides, Killip, 1938 4

Granadilla, (Medic.) Mast., 1871 Quadrangulares 2

Digitatae 1

Tiliafoliae 10

Marginatae 1

Laurifoliae 13

Serratifoliae 4

Setaceae 1

Pedatae 1

Incarnatae 6

Palmatisectae 1

Kermesinae 8

Imbricatae 2

Simplicifoliae 14

Lobatae 29

Menispermifoliae 4

Manicata (Harms) Escobar, 1988 5

Murucuja (Medic.) Mast, 1871 4

Plectostemma, Masters, 1871

Cieca 25

Decaloba

Auriculatae 2

Heterophyllae 2

Sexflorae 4

Ap�etalae 2

Luteae 3

Organenses 7

Miserae 5

Punctatae 47

Eudecaloba* 19

Polyanthea* 16

Hahniopathanthus 3

Hollrungiella* 1

Mayapathanthus 1

Pseudodysosmia 18

Pseudogranadilla 6

Xerogona 8

Polyanthea (DC.) Killip, 1938 1

Porphyropathantus Escobar, 1989 1

Pseudomurucuja (Harms) Killip, 1938 4

Psilanthus, Killip, 1938 4

Rathea (Karst.) Killip, 1938 2

Tacsonia (Juss.) Tr. & Planch, 1873 Ampullacea 1

Boliviana 1

Bracteogama 12

Colombiana Leptomischae 7

Colombiana Colombianae 8

Fimbriatistipula 2

Parritana 1

R. Yockteng, S. Nadot / Molecular Phylogenetics and Evolution 31 (2004) 379–396 381

Table 1 (continued)

Subgenus Section Series Number of species

Poggendorffia 5

Tacsonia 3

Tacsoniopsis 2

Trifoliata 1

Tacsonioides (DC.) Killip, 1938 5

Tacsoniopsis (Tr. & Planch) Killip, 1938 1

Tetrapathea * Banks ex DC., 1828 1

Tryphostemmatoides (Harms) Killip, 1938 2

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

1 Outgroup Blois-France-Greenhouse Yockteng-146 (P) AY261533

Adenia sp. Forssk. 1 Outgroup USA, Austin, Texas University

Greenhouse

Gilbert-9135 AY261534

Dilkea sp. Mast. 1 Outgroup USA, Austin, Texas University

Greenhouse

Gilbert-9246 AY261535

Passiflora species

adenopoda D.C. 1 Plectostemma,

Pseudodysosmia

Italy, Ripalta Cremasca, Collection

Nationale Italie Greenhouse

Vecchia-1 AY261536

aimae Annonay &

Feuillet

1 Granadilla-

Distephana

French Guiana, Piste St. Elie Yockteng-4 (P) AY261537

alata Curtis 1 Granadilla,

Quadrangulares

Blois-France-Greenhouse Yockteng-5 (P) AY261538

Allantophylla Mast. 1 Plectostemma,

Decaloba, Sexflorae

Blois-France-Greenhouse Houel-34 154 (P) AY261539

alnifolia Kunth 1 Plectostemma,

Decaloba, Punctatae

Orsay, France, Greenhouse University Paris

XI, Seeds from Belize

Yockteng-6 (P) AY261540

ambigua Hemsl. 2 Granadilla,

Laurifoliae


Nationale Italie, Greenhouse

Vecchia-2 8 (P) AY261541

ambigua Hemsl. 3 Granadilla,

Laurifoliae

Panama, Pipeline Road, Panama Canal

Zone, Nal.Park Soberania


amethystina Mikan 1 Granadilla,

Lobatae


amethystina Mikan 2 Granadilla,

Lobatae


amoena Escobar 1 Astrophea,

Botryastrophea



Vecchia-3 AY261545

amoena Escobar 2 Astrophea,

Botryastrophea

French Guiana Yockteng-10 (P) AY261546

antioquiensis Karst. 1 Tacsonia, Colombiana,

Leptomischae



Vecchia-4 AY261547

antioquiensis Karst. 2 Tacsonia, Colombiana,

Leptomischae


arborea Spreng. 1 Astrophea Panama, Quebrada Aleman, Fortuna

Reservoir, Chiriqu�ıYockteng-148 (P) AY261549

aurantia G. Forst. 1 Plectostemma,

Decaloba, Eudecaloba


auriculata Kunth 1 Plectostemma,

Decaloba, Auriculatae

French Guiana, Piste St. Elie Yockteng-15 (P) AY261551

auriculata Kunth 3 Plectostemma,

Decaloba, Auriculatae

Colombia, Municipio San Miguel,

Antioquia


biflora Lam. 1 Plectostemma,

Decaloba, Punctatae

Ecology & Evolutionary Biology

Conservatory, Univ. Connecticut

Morse-199800107 AY261553

biflora Lam. 2 Plectostemma,

Decaloba, Punctatae

Veerweg, Holland, Nationale Collectie

Passiflora�s Greenhouse



Table 2 (continued)


classification


caerulea L. 1 Granadilla, Lobatae Italy, Ripalta Cremasca, Collection

Nationale Italie Greenhouse, Brazil

Vecchia-8 152 (P) AY261555

candida (Poep & Endl.)

Mast.

1 Astrophea,

Pseudoastrophea


capsularis L. 1 Plectostemma,

Xerogona



Morse-199500133 AY261557

cerasina Annonay &

Feuillet

1 Granadilla, Laurifoliae French Guiana, Route de Kaw, Km.6 Yockteng-24 (P) AY261558

cincinnata Mast. 1 Granadilla, Incarnatae Veerweg, Holland, Nationale Collectie



cincinnata Mast. 2 Granadilla, Incarnatae Orsay, France, Greenhouse University

Paris XI

Vecchia-10 26 (P) AY261560

Cinnabarina Lindl. 1 Plectostemma,


Australia, Booroomba Rocks, along

Apollo Road, via Naas.

ANBG-66949 AY261561

cirrhiflora Juss. 1 Polyanthea French Guiana, Belizon Yockteng-28 (P) AY261562

citrifolia (Juss.) Mast. 1 Astrophea,

Pseudoastrophea



Vecchia-12 AY261563

citrifolia (Juss.) Mast. 2 Astrophea,

Pseudoastrophea

French Guiana, Pont de la Rivi�ere,

Comt�e, Route de l�EstYockteng-29 (P) AY261564

coccinea Aubl. 1 Distephana Morse-200000042 AY261565

coccinea Aubl. 3 Distephana Italy, Ripalta Cremasca, Collection


Vecchia-14 32 (P) AY261566

coriacea Juss. 1 Plectostemma, Cieca Ecology & Evolutionary Biology


Morse-199200361 AY261567

coriacea Juss. 2 Plectostemma, Cieca Veerweg, Holland, Nationale Collectie



coriacea Juss. 5 Plectostemma, Cieca Panama, Pipeline Road, Panama Canal

Zone, Parque Nal. Soberania


coriacea Juss. 8 Plectostemma, Cieca Colombia, Ibague, Tolima Botanical Garden JBAVH-3319 AY261570

crassifolia Killip 1 Granadilla,

Menispermifoliae


crenata Feuillet &

Cremers

1 Granadilla Italy, Ripalta Cremasca, Collection


Vecchia-18 AY261572

crenata Feuillet &

Cremers

2 Granadilla French Guiana Yockteng-41 (P) AY261573

edulis Sims 1 Granadilla, Incarnatae Ecology & Evolutionary Biology


Morse AY261574

edulis Sims 2 Granadilla, Incarnatae Veerweg, Holland, Nationale Collectie



edulis Sims 3 Granadilla, Incarnatae Orsay, France, Greenhouse

University Paris XI


edulis Sims 4 Granadilla, Incarnatae Orsay, France, Greenhouse

University Paris XI


edulis fo.flavicarpa

Degener

1 Granadilla, Incarnatae Veerweg, Holland, Nationale Collectie




Degener

3 Granadilla, Incarnatae Orsay, France, Greenhouse

University Paris XI



Degener

4 Granadilla, Incarnatae Orsay, France, Greenhouse

University Paris XI, Seeds from Colombia


elegans Mast. 1 Granadilla, Lobatae Blois-France-Greenhouse Yockteng-51 (P) AY261581

exura Feuillet 1 Granadilla, Lobatae French Guiana Yockteng-52 (P) AY261582

foetida L. 4 Dysosmia Veerweg, Holland, Nationale Collectie



foetida var. arizonica

Killip

1 Dysosmia Ecology & Evolutionary Biology


Morse-200000041 AY261584

foetida var. hastata

(Bertol.) Mast.

1 Dysosmia French Guiana Houel-Guy13 AY261585

foetida var. hirsutissima

Killip

1 Dysosmia Blois-France-Greenhouse Yockteng-58 (P) AY261586

gabriellana

Vanderplanck

3 Granadilla French Guiana, Comte Houel-Guy15 AY261587

garckei Mast. 1 Granadilla, Lobatae French Guiana, Belizon Yockteng-63 (P) AY261588

garckei Mast. 2 Granadilla, Lobatae French Guiana, Belizon Yockteng-63 (P) AY261589


Table 2 (continued)


classification


hahnii (Fourn.) Mast. 1 Plectostemma,

Hahniopathanthus




hahnii (Fourn.) Mast. 2 Plectostemma,

Hahniopathanthus



Vecchia-25 AY261591

helleri Peyr. 1 Plectostemma,

Decaloba, Organenses


Herbertiana Ker Gawl. 1 Plectostemma,


Blois-France-Greenhouse Houel-7 AY261593

hirtiflora Jørg. &

Holm-Niels.

1 Plectostemma,

Pseudogranadilla


Indecora Kunth. 2 Plectostemma,

Pseudogranadilla




jorullensis Kunth 1 Plectostemma,

Organenses


kalbreyeri Mast. 1 Plectostemma,

Pseudogranadilla


karwinskii Mast. 1 Plectostemma,

Pseudodysosmia


kawensis Feuillet 1 Astrophea,

Pseudoastrophea


kawensis Feuillet 1 Astrophea,

Pseudoastrophea

French Guiana, Route de Kaw Yockteng-75 (P) AY261600

lancetillensis MacDougal

& Meerman

1 Deidamioides Blois-France-Greenhouse Yockteng-76 (P) AY261601

laurifolia L. 1 Granadilla, Laurifoliae Morse-199900009 AY261602

laurifolia L. 7 Granadilla, Laurifoliae Veerweg, Holland, Nationale Collectie



ligularis Juss. 1 Granadilla, Tiliafoliae Veerweg, Holland, Nationale Collectie



ligularis Juss. 2 Granadilla, Tiliafoliae Italy, Ripalta Cremasca, Collection


Vecchia-67 AY261605

ligularis Juss. 3 Granadilla, Tiliafoliae Orsay, France, Greenhouse University

Paris XI


ligularis Juss. 4 Granadilla, Tiliafoliae Colombia, Ibague, Tolima Botanical Garden JBAVH-3318 AY261607

Macrophylla Mast. 1 Astrophea Italy, Ripalta Cremasca, Collection


Vecchia-33 AY261608

maliformis L. 2 Granadilla, Tiliafoliae Colombia, Medellin, Botanical Garden JBMedellin 85 (P) AY261609

manicata (Juss.) Pers. 1 Manicata Blois-France-Greenhouse Yockteng-86 (P) AY261610

mathewsii (Mast.) Killip 1 Tacsonia, Tacsonia Blois-France-Greenhouse Yockteng-87 (P) AY261611

Membranacea Benth. 1 Plectostemma,

Hahniopathanthus


menispermifolia Kunth 1 Granadilla,

Menispermifoliae



Vecchia-38 AY261613

menispermifolia Kunth 2 Granadilla,

Menispermifoliae

Panama, Pipeline Road, Panama Canal

Zone, Parque Nal. Soberania


mixta L. f. 1 Tacsonia, Tacsonia Blois-France-Greenhouse Houel-14 158 (P) AY261615

mixta L. f. 2 Tacsonia, Tacsonia Colombia, Ibague, Tolima Botanical Garden JBAVH-3317 AY261616

mooreana Hook. 1 Granadilla, Lobatae Veerweg, Holland, Nationale Collectie



mooreana Hook. 2 Granadilla, Lobatae Italy, Ripalta Cremasca, Collection



morifolia Mast. 1 Plectostemma,

Pseudodysosmia

Orsay, France, Greenhouse University

Paris XI


multiflora L. 1 Apodogyne Blois-France-Greenhouse Yockteng-159 (P) AY261642

murucuja L. 1 Murucuja Blois-France-Greenhouse Yockteng-94 (P) AY261620

nephrodes Mast. 1 Granadilla,

Menispermifoliae



Morse-199900602 AY261621

nitida Kunth 1 Granadilla, Laurifoliae French Guiana Vecchia-45 96 (P) AY261622

oerstedii var. choconiana

(Watson) Killip

1 Granadilla,

Simplicifoliae


ornithoura Mast. 1 Plectostemma,

Decaloba, Organenses



Table 2 (continued)


classification


penduliflora Bertero

ex DC.

1 Astephia Blois-France-Greenhouse Yockteng-100 (P) AY261625

perfoliata L. 1 Pseudomurucuja Blois-France-Greenhouse Yockteng-101 (P) AY261626

pittieri Mast. 1 Astrophea,

Dolichostemma



Vecchia-49 AY261627

pittieri Mast. 2 Astrophea,

Dolichostemma

Colombia, Medellin, Botanical Garden JBMedellin AY261628

platyloba Killip 1 Granadilla, Tiliafoliae Italy, Ripalta Cremasca, Collection


Vecchia-51 AY261629

platyloba Killip 2 Granadilla, Tiliafoliae Italy, Ripalta Cremasca, Collection


Vecchia-50 AY261630

punctata L. 1 Plectostemma,

Decaloba, Punctatae




Quadrangularis L. 1 Granadilla,

Quadrangulares



Morse-19990006 AY261631


Quadrangulares





Quadrangulares



Vecchia-54 AY261633

racemosa Brot. 1 Calopathanthus Blois-France-Greenhouse Yockteng-106 (P) AY261634

reflexiflora Cav. 1 Tacsonioides Blois-France-Greenhouse Yockteng-107 (P) AY261636

riparia Mart. Ex Mast. 1 Granadilla, Laurifoliae Blois-France-Greenhouse Yockteng-108 (P) AY261637

rubra L. 1 Plectostemma,

Xerogona

Orsay, France, Greenhouse

University Paris XI


rufostipulata Feuillet 1 Granadilla,

Laurifoliae


sandrae MacDougal 1 Panama, Pipeline Road 8Kms, Panama

Canal Zone, Parque Nal. Soberania


sanguinolenta Mast. &

Linden

1 Psilanthus Ecology & Evolutionary Biology


Morse-199800014 AY261641

seemanii Griseb 1 Granadilla, Tiliafoliae Orsay, France, Greenhouse

University Paris XI


serrato-digitata L. 1 Granadilla, Digitatae Ecology & Evolutionary Biology


Morse-199900027 AY261644

serrato-digitata L. 1b Granadilla, Digitatae Veerweg, Holland, Nationale Collectie



serrato-digitata L. 2b Granadilla, Digitatae Italy, Ripalta Cremasca, Collection


Vecchia-58 AY261646

serrulata Jacq. 1 Granadilla, Tiliafoliae Veerweg, Holland, Nationale Collectie



sexflora Juss. 2 Plectostemma,

Decaloba, Sexflorae




sprucei Mast. 1 Granadilla, Lobatae Italy, Ripalta Cremasca, Collection


Vecchia-60 AY261649

standleyi Killip 1 Plectostemma,

Decaloba, Punctatae


tacsonioides Killip 1 Pseudomurucuja Blois-France-Greenhouse Yockteng-124 (P) AY261651

talamancensis Killip 3 Plectostemma,

Decaloba, Punctatae


telesiphe Knapp &

Mallet

1 Plectostemma Blois-France-Greenhouse Yockteng-127 (P) AY261653

tetrandra Banks ex DC. 1 Tetrapathea Blois-France-Greenhouse Yockteng-128 (P) AY261654

trialata Feuillet &

MacDougal

1 Granadilla,

Quadrangulares


trifasciata Lem. 1 Plectostemma,

Decaloba, Miserae


tripartita var mollissima

(Kunth) Holm-Niel

& Jørg

1b Tacsonia, Bracteogama Blois-France-Greenhouse Yockteng-132 (P) AY261657


(Kunth) Holm-Niel

& Jørg

2 Tacsonia, Bracteogama Orsay, France, Greenhouse

University Paris XI,

Seeds from Colombia, Cajica



Table 2 (continued)


classification



(Kunth) Holm-Niel

& Jørg

3 Tacsonia, Bracteogama Colombia, La Calera Yockteng-131 (P) AY261659


(Kunth) Holm-Niel

& Jørg

4 Tacsonia, Bracteogama Colombia, La Calera Yockteng-131 (P) AY261660


(Kunth) Holm-Niel

& Jørg

1 Tacsonia, Bracteogama Ecology & Evolutionary Biology


Morse-Student AY261661

trisecta Mast. 1 Manicata Blois-France-Greenhouse Yockteng-133 (P) AY261662

tryphostemmatoides

Harms

2 Tryphostemmatoides Colombia-Valle del Cauca Yumbo

Monta~nitas KM6 Via Pavitas San Jose


tulae Urb 1 Murucuja Blois-France-Greenhouse Yockteng-136 (P) AY261664

umbilicata (Griseb.)

Harms

1 Tacsonioides Blois-France-Greenhouse Houel-27 157 (P) AY261665

variolata Poep & Endl. 1 Distephana Blois-France-Greenhouse Houel-30 AY261666

vespertilio L. 3 Plectostemma,

Decaloba, Punctatae

French Guiana, Crique Plomb Yockteng-137 (P) AY261667

vitifolia (Harv.) Harms 1 Distephana Ecology & Evolutionary Biology


Morse-198700236 AY261668

vitifolia (Harv.) Harms 5 Distephana Colombia, Ibague, Tolima Botanical Garden JBAVH-3320 AY261669

Collection data, voucher, and GenBank accessions numbers are given. Specimens are deposited at the Herbarium of the Museum National

d�Histoire 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 pro-

grammed for an initial step of 5min at 94 �C, followedby 35 cycles of 1min at 94 �C, 45 s at 55 �C, 1.5min at

72 �C, and a final step of 7min at 72 �C. PCR productswere 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 se-

quencers (MWG Biotech). Sequences were deposited in

GenBank.

2.3. Phylogenetic analyses

Clustal X (Thompson et al., 1997) was used to pro-

duce 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 Ochoterena�s method that codes indels as

separate characters in a data matrix (Simmons and

Ochoterena, 2000).

We examined nucleotide and translated sequences tosearch 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 align-

ment, one consisting of the coding regions, the otherconsisting of the non-coding regions (introns).

2.4. Parsimony

Maximum parsimony (MP) analyses were conducted

using PAUP 4.0b10a* (Swofford, 2001). Heuristic sear-

ches were conducted with tree-bisection reconnection


(TBR) branch swapping, multiple trees ON, and 1000random taxon addition replicates limiting the rear-

rangements to 100,000 per replicate and saving all most

parsimonious trees. Support for internal nodes was as-

sessed by bootstrapping (100 replicates). Decay indexes

were calculated using the Autodecay 4.0 program (Eri-

ksson, 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 vo-

nHaeseler, 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 to1.9758, 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 Ron-

quist, 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 simulta-

neously, sampled every 100 generations for a total of

150,000 generations. Stationarity conditions werereached 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 bran-

ches. 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 charactersused by Killip (1938) to establish the different subgen-

era. 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).

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 spe-

cies, of the cytosolic-expressed glutamine synthetase

(cytGS) instead of the expected chloroplast-expressed

glutamine synthetase (ncpGS). These preliminary resultsrevealed a first division within the genus Passiflora. The

cytosolic glutamine synthetase was amplified for species

belonging to two subgenera: Murucuja and Plec-

tostemma. The chloroplast-expressed glutamine synthe-

tase (ncpGS) was amplified for all other species

belonging to the subgenera Distephana, Granadilla,

Tacsonia, and Tacsonoides.

The newly designed pair of primers allowed specificamplification of the ncpGS gene for all Passiflora spe-

cies. 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 contain-ing taxonomic information. As an example, the 46th

amino acid splits the species into two groups. The first

group, holding all species of the subgenera Deidamio-

ides, 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 ofLobatae 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 infer-

ence. 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 ma-

jority were produced by the ingroup alignment. ANO-VA tests show that the differences in total intron length

among subgenera are significant (Table 3). The non-

coding region was significantly longer in subgenera

Astrophea, Distephana, Dysosmia, Granadilla, Manicata,

Tacsonia, and Tacsonoides, than in Murucuja, Plec-

tostemma, 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 sub-

genera Calopathanthus, Psilanthus, Polyanthea, Deida-

mioides, Tetrapathea, Apodogyne, and Astephia because

the number of individuals available in these subgenera

was less than two. However, the intron lengths of Cal-

opathanthus, Psilanthus, Polyanthea, Deidamioides, Tet-

rapathea, Tryphostemmatoides, and the outgroup were

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 9 14400.59

Subgenera� total intron length 98 230.75 62.4 1.27E) 36

Error 107 152219.07


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-infor-

mative sites and 356 invariable sites. Most informativesites 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 non-

coding 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 datasetthe different methods used yielded similar topologies.

The non-coding region produced topologies very

similar to the trees obtained using the entire align-

ment, although some nodes were less supported. When

using the coding region, only the internal nodes de-

fining the main taxonomic groups were well sup-

ported.

The individuals of the same species are branchedtogether 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, Plec-

tostemma, Pseudomurucuja, and Tetrapathea; and clade

Fig. 2. Strict consensus tree of 32,573 most parsimonious trees based on the

quences. Bootstrap support values (BS) greater than 70% and decay indices

values of 100%, dots indicate nodes supported by 100% in the Bayesian tree. T

The sections and series names are abbreviated as follows: Cie, Cieca; Dec, D

Miserae; Punc, Punctatae; Eud, Eudecaloba; Xero, Xerogona; Psedy, Pseu

Leptomischae; Col, Colombianae; Bract, Bracteogama; Tac, Tacsonia; Quad

Inca, Incarnatae; Simp, Simplicifoliae; Loba, Lobatae; Meni, Menispermifoli

The three clades emerging from the analysis are indicated on the right.

3 consists of the subgenera Calopathanthus, Distephana,

Dysosmia, Granadilla, Manicata, Psilanthus, Tacsonia,

and Tacsonoides. P. cirrhiflora Juss., from the mono-

specific 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 supportor 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 therelatively short length of the ncpGS alignment, the level

of variation was satisfactory with 329 parsimony-infor-

mative 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–trnFregion 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 non-coding region provides most of the phylogenetic infor-

mation. 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.

alignment of chloroplast-expressed glutamine synthetase (ncpGS) se-

greater than 4 are indicated above branches. Thick lines indicate BS

he subdivisions according to Killip (1938) are indicated for each taxon.

ecaloba; Auri, Auriculatae; Sexf, Sexflorae; Orga, Organenses; Mise,

dodysosmia; Psegr, Pseudogranadilla; Hah, Hahniopathanthus; Lept,

, Quadrangulares; Digi, Digitatae; Tili, Tiliafoliae; Laur, Laurifoliae;

ae; Doli, Dolichostemma; Euas, Euastrophea; Pseas, Pseudoastrophea.

c

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 12–4lm wide) or type 2 (reticulum with more than 7lm wide and exine with more than 6lm

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 tu-

bercles-shaped; 17. operculum, and 18. ovary shape.


All the phylogenetic analyses conducted resulted in

congruent trees that have well resolved and well sup-

ported topologies. The strongest support values wereobtained with the Bayesian analysis, which has recently

become popular in phylogenetics, and accessible

through the MrBayes program (Huelsenbeck and Ron-

quist, 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 bootstrap-ping assessment and the decay indexes conducted in the

MP analysis (Fig. 2).

The general topology presents three main well sup-

ported monophyletic groups (Fig. 2). The first clade,

which consists of the species representing subgeneraAstrophea 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), Killipcreated a separate subgenus for this species in spite

noting its apparent relationship to the subgenus

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) inwhich 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 whichAstrophea 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 Ben-

son et al. (1975). Its pollen type shared with Plec-

tostemma, Polyanthea, and the outgroups (Presting,

1965) (Fig. 3b) could then considered as a pleisiomor-phy. The position of P. candida (Poep & Endl.) Mast.,

separated from the Astrophea clade in our trees, is un-

explained. 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 Trypho-

stemmatoides on the basis of the presence of a non-pli-cate 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 inPlectostemma. However, the relationship between P.

tryphostemmatoides and Astrophea subgenus is well

supported in our phylogeny (Bootstrap Support¼ 88).

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 (BS¼ 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, (repre-

sented 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 ofFeuillet and MacDougal (1999), which includes the

subgenera Apodogyne, Astephia, Murucuja, Plec-

tostemma, 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 Plec-

tostemma group. The monophyly of this subgenus is

supported by a rare combination of features like the

plicate operculum, trifoliate leaves and peduncles ter-

minating in a 2-flowered tendril (Figs. 3a and c). The

position of P. tetrandra, intermediate between P.

lancetellensis and the Plectostemma group, indicatesthat Tetrapathea is not an independent monotypic ge-

nus. This result suggests that the particular reproduc-

tive 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 inde-

pendent origin from Passiflora.

Likewise, this clade includes five subgenera initially

considered by Killip (1938) as independent. Our resultsshow that these subgenera are embedded within Plec-

tostemma, 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 re-

garded in the past as belonging to Plectostemma until

they were placed in three different subgenera for pos-

sessing many segregating characters (Killip, 1938). Forexample, P. multiflora was excluded from Plectostemma


and moved to a new monotypic subgenus Apodogyne onthe 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. Simi-larly, 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 Plec-

tostemma (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 forMurucuja (Fig. 3c). Passiflora pendulifloraBertero ex DC. was also considered as a separate sub-

genus by Killip (1938) because of its lack of an oper-

culum (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 Plec-

tostemma, it would be necessary, in a further study of

the genus Passiflora, to include the Asian and NewGuianan species regrouped in the three sections Deca-

loba subsect. Polyanthea, Holrungiella, and Octandran-

thus (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 differ-

ent sections. One section would be composed of all theseries 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, namelyPuntactae but appearing derived from Pseudodysosmia

in our trees. This last result is in agreement with Mac-

Dougal (1994) who described this section as monophy-

letic 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

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 (BS¼ 100) includes all

the species representing the subgenera Calopathanthus,

Distephana, Dysosmia, Granadilla, Manicata, Psilanthus,

Tacsonia, and Tacsonoides. The subgenus Dysosmia isthe sister clade to the rest of the species. The monophyly

of the species P. foetida L. is well supported (BS¼ 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 spe-

cies in a separate subgenus.

The rest of the species are distributed in three

well-supported groups of unresolved relationship. Thepresence 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 clade2 (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 Calopa-

thanthus, 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 besubdivided into three different sections. The first section

(BS¼ 92) would correspond to the subgenera Calopa-

thanthus, Manicata, and Tacsonia. Both latter subgenera

are mainly distributed in the Andes Mountains and

share a long calyx tube (Fig. 3c). In contrast, P. race-

mosa Brot. (Calopathanthus) is mainly distributed in

Brazil and has an operculum that forms a cylindrical

tube. However, its relationship with Manicata andTacsonia is strongly supported, suggesting that these

characters are derived. The second section would cor-

respond to the series Quadrangulares, Incarnatae, and

Laurifoliae (subgenus Granadilla) and the subgenus Di-

stephana. 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. cincinnataMast. (subgenus Granadilla) within Distephana is unex-

pected, since this species does not display these specific

morphological features. As this subclade consists of a

large polytomy, an explanation could be that P. cincin-

nata is actually the sister group to Distephana. The po-

sition of the recently described French Guiana species

P. aimae confirms its relationship with Distephana, as


suggested by Annonay and Feuillet (1998) P. gabriell-

ana, 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 budswould represent a synapomorphy of this sub-clade. The

other subsection contains the series Lobatae, Menispe-

rmifoliae, and Simplicifoliae (subgenus Granadilla),

which share foliaceous and verticillate bracts. The sub-

genus 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 tubefavours their inclusion in Tacsonia (Harms, 1925)

whereas the 2–4 seriate corona supports their inclusion

in the disappeared section Tacsonoides (subgenus Gra-

nadilla) (De Candolle, 1828). Our results validate this

latter suggestion. Since Tacsonoides appears paraphy-

letic 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 tohummingbird 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 cladesbased on DNA sequence data from the glutamine syn-

thetase 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 chromo-

somes for clade 1 (Astrophea s.l.), x ¼ 6 for clade 2

(Plectostemma s.l.), and x ¼ 9 for clade 3 (Granadillas.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 agreementwith Killip (1938). The similarity of chromosome num-

ber between P. sanguinolenta and Plectostemma could

result from convergence.

Furthermore, the unique chromosome count avail-

able for the Deidamioides subgenus (Gilbert and Mac-

Dougal, 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) wouldsupport 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 var-

iability 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 aconstant hybridization process (Killip, 1938; Vanderp-

lanck, 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 an-cestral chromosome number of Passiflora. The presence

of n ¼ 12 chromosomes in the outgroup Adenia (Passi-

floraceae) supports this possibility. To confirm this hy-

pothesis, chromosome counts for P. cirrhiflora, sister

group to all other Passiflora species, and for P. trypho-

stemmatoides (basal in clade 1) are necessary.

5. Conclusion

The study presented here is the first comprehensive

molecular phylogenetic analysis of the large genus Pas-

siflora. 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 infra-

generic classification proposed by Feuillet and Mac-

Dougal (1999), in which four subgenera are recognized

(Plectostemma, Astrophea, Deidamioides, and Grana-

dilla), but our results give evidence for the existence of

three additional subgenera. In spite of the good resolu-

tion provided by the ncpGS gene, further studies usingadditional molecular markers, such as chloroplast re-

gions, must be undertaken in order to confirm our re-

sults. It would also be necessary to increase the number

of species and in particular to include the Asian mem-

bers of Passiflora as well as species of subgenera not

represented in this analysis.

Acknowledgments

We are extremely grateful to Christian Houel (Pas-

siflora 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),


Cor Laurens (Passiflora National Collection, Holland),Maurizio Vecchia (Passiflora National Collection, It-

aly), 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 An-

des, Bogota, Colombia), H�ector Eduardo Esquivel

(Botanical Garden Alejandro Von Humboldt, Tolima,

Colombia), Alexandra Hiller (Giessen University, Ger-many), 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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