Acta Botanica Gallica (2002) 149 : 49-66 The genus Olea: molecular approaches of its structure and relationships to other Oleaceae Guillaume Besnard (1), Peter S. Green (2), and André Bervillé (3) (1) INRA, UR-GAP, UMR-DGPC, 2 P. Viala, Bât. 33, F-34060 Montpellier cedex 1. (2) Royal Botanic Gardens, Kew, Richmond, UK Author for correspondence : A Bervillé, INRA, UR-GAP, 2 P. Viala, Bât 33, F34060 Montpellier cedex 1. Tel: 33 (0)4 99 61 22 33, Fax: 00 33 4 99 04 54 15, E-mail: berville@ensam.inra.fr. ABSTRACT: Ribosomal DNA RFLP and RAPD polymorphisms were examined in taxa belonging to Oleaceae genera. The objective of this study was to determine the taxonomic position of the genus Olea and of its components: subg. Olea section Olea, subg. Olea section Ligustroides, subg. Paniculatae and subg. Tetrapilus. Ribosomal DNA polymorphisms supported that O. subg. Tetrapilus was separated from other Olea species and appeared close to other Oleaceae genera as Nestegis, Chionanthus and Phillyrea. Moreover, RAPDs enabled us to distinguish the four groups of the genus Olea. Despite of limited number of informative RAPDs, O. subg. Tetrapilus was also shown closer to Nestegis and Chionanthus than to the other Olea species. Thus, Tetrapilus should be considered as a genus. Our results sustained the common origin of the sections Ligustroides and Olea. Key words: Olea - Oleaceae - Olive - taxonomy - Tetrapilus - ribosomal DNA – RAPD RÉSUMÉ : Nous avons révélé des RFLP de l'ADN ribosomique et des marqueurs RAPD dans différents genres appartenant à la famille des Oléacées. L'objectif de cette étude était de déterminer la position taxonomique du genre Olea et de ses composantes : sous-genre Olea section Olea, sous-genre Olea section Ligustroides, sous-genre Paniculateae et sous-genre Tetrapilus. Les polymorphismes de l'ADN ribosomique soutiennent que le sous-genre Tetrapilus est séparé des autres espèces du genre Olea, et apparaît plus proche des genres Nestegis, Chionanthus et Phillyrea. De plus, les marqueurs RAPD nous permettent de distinguer les quatre groupes du genre Olea. Bien que le nombre de marqueurs RAPD informatifs soit limité, il apparaît que le sous-genre Tetrapilus est plus apparenté à Nestegis et Chionanthus que des autres espèces du genre Olea. Ainsi, Tetrapilus devrait être considéré comme un genre. Par ailleurs, nos résultats soutiennent l'origine commune des sections Ligustroides et Olea. Mots-clés : ADN ribosomique - Olea - Oleaceae - Olivier - taxonomie - Tetrapilus – RAPD I. INTRODUCTION The family Oleaceae contains 25 genera and around 500 to 600 species which are separated in two sub-families: Jasminoideae and Oleideae (review of the classification in Rohwer, 1996). Olea is classified in the sub-family Oleideae and in the tribe Oleeae. The genus Olea includes around 30 to 40 species that are distributed in Oceania, in Asia, in Africa, and in the Mediterranean Basin. Olive tree (Olea europaea L. subsp. europaea) is an important crop in the Mediterranean basin. At the present time, many interests are focused on its genetic resources and on the elucidation of domesticated olive origins (Besnard et al., 2001a), but little is known about the relationships between the different species of the genus Olea. The species closely related to the Mediterranean olive have been particularly studied by botanists and are grouped in the subgenus Olea section Olea which is currently named “O. europaea complex” (Green & Wickens, 1989). The other Olea species are less known and some (notably O. capensis) are supposed to be incompatible with O. europaea (Dyer, 1991). Apart from the olive tree, several Olea species could prove of economic importance. In Africa, several taxa such as 1 O. capensis subsp. macrocarpa and O. perrieri produce fruits rich in oil, and can be used in human alimentation (Palmer & Pitman, 1972; Perrier de la Bathie, 1952). Lastly, O. paniculata wood is used in fine carving or for flooring blocks in Australia (Kiew, 1979). Several Olea classifications have been proposed, notably by De Candolle (1844), Bentham & Hooker (1876) and Knoblauch (1895). Confusions between the different genera have sometime occurred (Altamura et al., 1987). Within the genus Olea, a group of species from South Eastern Asia has been distinguished and classified in the genus Tetrapilus by Johnson (1957) who has drawn it nearer to Linociera (presently considered a synonym of Chionanthus) based on the length of the corolla tube. Furthermore, Tetrapilus species were distinguished from the other Olea using pollen morphology (Nilsson, 1988) and flavonoid composition (Harborne & Green, 1980). Nevertheless, Tetrapilus as a genus has not been generally retained by botanists but considered as being part of the genus Olea (Kiew, 1979). A complete taxonomy of the described Olea species is in preparation (Green, in preparation). The characters retained as the most informative are displayed in Table I. This enabled us to distinguish three subgenera and two sections. The subgenus Tetrapilus is the most distinct group and presents a greater variability. Subgenus Olea is separated into two sections: Olea (comprising cultivated olive trees and its wild relatives) and Ligustroides. The main characters used to distinguish these two sections are the indumentum and the panicle position. Lastly, the subgenus Paniculatae is distinguished from the subgenus Olea notably by the presence of domatia on the undersides of the leaves and of both axillary and terminal panicles. The relationships between these different groups of Olea species remain unclear. Currently, molecular approaches are routinely used in taxonomy and phylogeny studies. Comparison of restriction maps of genes (Olmstead & Palmer, 1994) - as ribosomal genes (rDNA), cytoplasmic DNA - and gene sequences (APG, 1998) have been used in plant phylogeny studies. Within the family Oleaceae, a phylogeography of Fraxinus has been proposed based upon sequences of ITS (Jeandroz et al., 1997), a phylogeny of Syringa has been proposed on chloroplast polymorphisms (Kim & Jansen, 1998) and a phylogeny of most of the genera of the family has been proposed based upon chloroplast DNA sequences (Wallander & Albert, 2000). Use of RAPDs or RFLPs presents the advantage of covering a greater number of genome regions. Thus, these tools make it possible to study polymorphisms in different genomic regions. In Oleaceae, RAPDs have been used to detect interspecific hybridization in Fraxinus (Jeandroz et al., 1996a), or to study the relationships between related taxa belonging to Olea europaea (Besnard et al., 2001b). Nevertheless, these markers could be too variable when distant taxa are compared, and this should lead to a low level of informative characters. The study of the genetic relationships between the species of Olea is required to determine the relationships of Olea with other Oleaceae genera and to understand the phylogeny of Olea species leading to the present structure of its diversity. Moreover, this will show the species complexes and will make it possible to define the history of cultivated olive. We analyzed different taxa of the genus Olea and related genera of Oleaceae with restriction maps of ribosomal RNA genes and with RAPDs to check the position of Olea within the family and to propose a molecular taxonomy of the genus in comparison with the morphologic classification. II. MATERIAL AND METHODS A. Plant material The plant material was collected in the wild, in the collections of botanical gardens, and in those of the Instituto di Ricerche sulla Olivicoltura (C.N.R., Perugia) and of the Institut Nationale de Recherche Agronomique (INRA, Montpellier). Forty-eight samples belonging to the different Olea subgroups (Table II), and fifteen other Oleaceae (Table III) were studied. B. Ribosomal DNA study The DNA extraction protocol has been described by Besnard et al., (2000). Three µg of total DNA were restricted (8 U/µg, 37 ° C, 4-5 h) separately by BamHI, EcoRI, EcoRV and SacI plus their pairwise combinations BamHI-EcoRI, EcoRI-EcoRV, and SacI-EcoRI. The restriction fragments were electrophoresed onto 0.8 % agarose gel at 1.8 V/cm for 16 h. The Southern transfers were successively 2 Table I. Geographical distribution and discriminant morphological characters of the subgenus and sections of Olea. Subgenus Tetrapilus Geographical distribution South Eastern Asia Groups of Olea Subgenus Olea sect. Olea Subgenus Olea sect. (O. europaea complex) Ligustroides Benth. & Hook. From China at East Southern Africa, Saharan mountains, Mediterranean basin, Canaria. Central and Southern Africa Olea paniculata Subgenus Paniculatae From India at Australia Flower characters Corolla tube longer than the corolla lobe Stigma shortly bilobed or capitate capitate capitate capitate Dioecy +/- - - - +/- membranous +/- coriace axillary axillary (or subterminal) terminal (and sometime axillary) axillary and terminal Leaf blade margins entire or serrate entire entire entire Peltate scales - + + + Domatia in axils of veins - - - + Calyx tube Panicles position equal or shorter than the corolla equal or shorter than the corolla equal or shorter than the corolla lobe lobe lobe Leaf characters 3 Table II. Origin and code of the studied Olea accessions. VS = Voucher samples deposited at the herbarium of the Botanical Institute from the University Montpellier II (MPU); NA = Number of studied accessions for a taxon; Y = characterized for rDNA restriction sites; N = not characterized for rDNA restriction sites; * means that only one individual was characterized for rDNA analyses. B. G. = Botanical Gardens. Taxa Origin country Locality Botanical Gardens, collections, herbarium N Subgenus Tetrapilus (Lour.) O. tsoongii (Merr.) Green O. tsoongii (Merr.) Green O. brachiata (Lour.) Merr. China China Indonesia Yunnan Bangka, Sumatra Edinburgh B. G. accession: 19 931 835 Edinburgh B. G. accession: 19 697 316 Bogor Botanical Garden 1 1 1 Y Y Y T.TSO1 T.TSO2 T.BRA China India Iran Iran Yemen Kenya South Africa Reunion Guéno, Bandar Abas Marzondar, Ahmady Almhiwit Timau, Kenya Mount Kirstenbosch, Cape Town Sentier de la Providence, Reunion CNR Perugia collection CNR Perugia collection VS. H Hosseinpour F2 INRA Montpellier collection INRA Montpellier collection INRA Montpellier collection Madrid Botanical Garden INRA Montpellier collection 1 1 1 1 2 2 2 2 Y Y Y N Y* Y* Y* Y* E.CC E.CI E.CR1 E.CR2 E.CH1-2 E.AK1-2 E.AS1-2 E.AR1-2 Syria France Härim, Oronte Valley Ostricone, Corsica INRA Montpellier collection 2 2 Y* Y* E.ES1-2 E.EC1-2 Algeria Morocco Morocco La Source, Hoggar Immouzzer, Atlas Mentaga, Atlas INRA Montpellier collection INRA Montpellier collection VS. A Bervillé 1 1 1 1 Y Y N E.LA E.MA1 E.MA2 Canary Islands Santa Rosalia, La Palma 2 Y* E.CE1-2 Subgenus Olea section Ligustroides Benth. & Hook. O. lancea Lam. O. lancea Lam. O. lancea Lam. O. exasperata Jacq. O. woodiana Knobl. O. woodiana Knobl. O. welwitschii (Knobl.) Gilg & Schellenb. O. capensis L. subsp. capensis O. capensis subsp. macrocarpa (Wright) Verd. O. capensis subsp. macrocarpa (Wright) Verd. O. capensis subsp. madagascariensis (Boiv.) O. capensis subsp. madagascariensis (Boiv.) O. capensis subsp. madagascariensis (Boiv.) O. perrieri Chev. O. perrieri Chev. Mauritius Reunion Madagascar South Africa South Africa Kenya Kenya South Africa South Africa Zimbabwe Madagascar Madagascar Madagascar Madagascar Madagascar Cap Noir, Reunion Tsinjoarivo Betty's Bay, Western Cape Umzimkulu River, Natal Kilifi, Indian Ocean coast Kakamega Forest, Mt Elgon Kirstenbosch, Cape Town Bloukranspas, Tsitsikama, Southern Cape Pungwe River, Inyangani Montagne d'Ambre Ambatovy Ambohitantely Andasibe Marojejy VS. L Forget 1 & 2 INRA Montpellier collection VS. RNF 016 & RNF 017 VS. A Costa 01 VS. A Costa 02 VS. H Sommerlate 1 Montpellier collection VS. A Costa 03 VS. A Costa 04 Harare Botanic Garden accession: 6041 VS. RNF 008, 009 & O11 VS. RNF 030 & 031 VS. ROR 193 VS. RNF 045 VS. RNF 006 2 2 2 1 1 1 2 1 1 1 3 2 1 1 1 Y* Y* N N Y N N Y Y Y N N N Y N L.LM1-2 L.LR1-2 L.LA1-2 L.EX L.WOS L.WOK L.CW1-2 L.CC L.CMS L.CMZ L.MA1-3 L.MA4-5 L.MA6 L.PE1 L.PE2 Subgenus Paniculatae O. paniculata R.Br. O. paniculata R.Br. Australia Australia Brisbane, Queensland Kew B. G. accession: 19 66 67 111 VS. C Lambrides 1 1 1 Y N P.KEW P.BRI Subgenus Olea section Olea (= O. europaea complex) [- subspecies cuspidata (Wall. ex DC) Ciferri] O. cuspidata Wall. O. cuspidata Wall. O. cuspidata Wall. O. cuspidata Wall. O. chrysophylla Lam. O. africana Mill. O. africana Mill. O. africana Mill. [- subspecies europaea] var. sylvestris (Miller) Lehr. var. sylvestris (Miller) Lehr. [- subspecies laperrinei (Batt. & Trab.) Ciferri] O. laperrinei Batt. & Trab. O. maroccana Greut. & Burd. O. maroccana Greut. & Burd. [- subspecies cerasiformis (Webb & Berth.) Kunk. & Sund.] O. cerasiformis Webb & Berth. rDNA analyses Code 4 Table III. List and origin of the studied Oleaceae accessions. VS = Voucher samples deposited at the herbarium of the Botanical Institute from the University Montpellier II (MPU); B. G. = Botanical Gardens. ENSAM: Ecole Nationale Supérieure Agronomique from Montpellier. Species Origin country Locality (for wild prospection) Nestegis sandwichensis (A.Gray) Deg. Hawaii (USA) Kokee State Park, Hawaii – VS. T Flynn 6329 Osmanthus fragrans Lour. India Madrid Botanical Garden accession – VS. P Villemur 03 Phillyrea latifolia L. Morocco Immouzzer, Atlas Ligustrum vulgare L. France Collias, Gard Noronhia emarginata (Lam.) Thouars Madagascar Olu Pua Gardens, Hawaii – VS. T Flynn 6331 Chionanthus ramiflorus Roxb. [sect. Linociera (Sw.)] USA Kauai National Tropical Botanical Garden accession: 75 094 70 01 - VS Chionanthus virginicus L. USA Kew B. G. accession: 19 76 292 Syringa vulgaris L. France ENSAM park, Ornamental plant Forestiera neomexicana A.Gray USA Madrid Botanical Garden accession – VS. P Villemur 04 Fraxinus angustifolia Vahl. subsp. oxycarpa (M.Bieb. ex Willd.) Franco & Rocha France Fontanesia phillyreoides Labill. Turkey Montpellier Botanical Garden accession Jasminum officinale L. - Montpellier Botanical Garden accession Jasminum fruticans L. France Forsythia x intermedia Zabel - ENSAM park, Ornamental plant Schrebera alata (Hochst.) Welw. - Kew B. G. accession: 19 69 188 26 Montpellier, Hérault Botanical Gardens, Parks and Herbarium VS. A Moukhli 01 ENSAM park Montpellier, Hérault 5 hybridized in a 7 % SDS, 0.25 M Na2HPO4 and 1 mM EDTA solution at 65 ° C for 18 h with the 18S rRNA gene from sunflower (Choumane & Heizmann, 1988), the 25S rRNA gene and the entire unit from flax (Goldsbrough & Cullis, 1981) as a probe. The membranes were rinsed three times at 65 °C in 2X SSC, 0.1 % SDS and in 0.2X SSC, 0.1 % SDS for 25 min and were exposed to Hyperfilm MP (Amersham) for 6-18 h. The entire Oleaceae sample was studied with this approach (Table III), whereas, only a sub-sample of plants of the genus Olea was analyzed (Table II). C. RAPD The RAPD amplification and electrophoresis procedures were described by Quillet et al., (1995). Eight primers (Bioprobe, France), previously selected (Besnard et al., 2001b) were used on the DNA’s from all individuals: A1, A2, A9, A10, C9, C15, E15, O8. After electrophoresis, gels were placed in a 0.25 N HCl solution for 30 min. Then, the DNA was transferred by a 0.4 N NaOH solution onto a Nylon membrane Hybond N+ (Amersham) with a transblotter (Life Technologies) under a depression of 60 Pa for 1 h. The membranes were rinsed in 2X SSC solution (300 mM NaCl, 30 mM sodium citrate) and then baked to 80 ° C for 2 h. Some RAPD fragments were used as a probe. These were picked up on agarose gels and then purified with Wizard plus Minipreps (Promega). Recovered DNA was labeled using 74 mBq of [32P]dCTP (111 Tbq/mmol). The hybridization conditions were those previously described. The well separated and intense fragments were noted. Hybridization profiles enabled us to verify specificity of some fragments or the homology of sequence of fragments present in different species. In addition, hybridization enabled us to read some fragments without ambiguity. All individuals were characterized with this method except Jasminum fruticans. D. Data analysis - Ribosomal DNA data Ribosomal DNA restriction maps were constructed. A matrix of presence/absence of each polymorphic site was established. Wagner parsimony phylogenetic trees (Farris, 1970) were constructed with these data using the phylogenetic inference package (PHYLIP, version 3.4) written by Felsenstein (1989). - RAPD data A matrix of presence/absence of fragments was established for Olea species. We computed Jaccard similarity (Jaccard, 1908) indexes: Sij = a / (a + b + c) where a is the number of common bands between i and j, and b and c the bands present in one individual (i or j respectively). We used both the Neighbor Joining Method (Nei, 1987) and the UPGMA algorithm (Benzécri, 1973) to construct phenetic trees. III. RESULTS A. Ribosomal DNA restriction maps Seventeen rDNA restriction maps of Oleaceae taxa were obtained. Five were exhibited by Olea species (Fig. 1). All the polymorphisms were found located in the internal gene spacer (IGS) and in the internal transcribed spacers (ITS). Firstly, O. tsoongii and O. brachiata (subgenus Tetrapilus) were distinguished from the other Olea species by the presence of an additional SacI site (S5), the absence of the BamHI site (B1), and the 4 kb EcoRV fragment (V4-V7) instead of the 4.5 kb fragment (V3V7) in O. africana, O. woodiana and O. capensis subsp. capensis. Furthermore, O. capensis subsp. macrocarpa, O. lancea and O. perrieri were distinguished from O. africana, O. woodiana and O. capensis subsp. capensis by a 5 kb EcoRV fragment (V2-V7) instead of the 4.5 kb fragment. An additional EcoRI fragment of 1 kb, only hybridized with the entire unit, was present in the section Olea, except in O. africana. The corresponding additional EcoRI site (E5) cannot be placed on the map because it was not revealed with the 18S and 25S probes. Lastly, O. paniculata also exhibited a similar additional fragment of 1.2 kb. A diagnostic fragment (B1-B2) of 0.6 kb enabled us to recognize the genus Noronhia and most of the Olea species, but it was absent in the subgenus Tetrapilus. 6 IGS 18S 5.8S B2 B3 S4 E3 V7 V3 E5? B1 E2 S6 V3 E2 S6 V3 E6? B1 E2 S6 B1 E2 S6 V4 E2 S5 S6 V4 E2 S6 V4 S1B1 E2 S6 B1 V2 25S S7 IGS B4 S8 E4 Conserved sites E5? Olea europaea complex without O. africana O. africana, O. woodiana & O. capensis ssp capensis E6? O. paniculata O. capensis ssp macrocarpa, O. lancea & O. perrieri O. brachiata & O. tsoongii (Tetrapilus) E2 deletion 50 bp S6 Nestegis sandwichensis & Phillyrea latifolia Noronhia emarginata Chionanthus virginicus S6 Chionanthus ramiflorus E2 V4 Fraxinus angsutifolia Syringa vulgaris & Ligustrum vulgare E2 S2 E2 V5 E2 V4 S6 Osmanthus fragrans Forsythia intermedia & Jasminum officinale Jasminum fruticans V6 E2 Fontanesia phillyreoides E1 Forestiera neomexicana V1 S3 Schrebera alata 1 Kb Fig. 1. Ribosomal DNA restriction maps of the different species of Olea and of Oleaceae. B. Phylogenetic reconstruction based on rDNA restriction sites Thus, 17 characters were considered of which 5 were informative (E2, V3, V4, S6, B1). Wagner parsimony based on these data enabled us to detect two homoplasic sites: E2 and V4. For these sites, we can suppose that at least two mutational events have led to their appearance and disappearance. E2 has likely appeared in Jasminoideae, for which it is polymorphic, and it has likely disappeared in Chionanthus ramiflorus (but it is present in Chionanthus virginicus). In addition, Forestiera, which did not display E2, displayed another EcoRI site (E1) in the IGS which could derive from E2 by a rearrangement (insertion) of the IGS region. Secondly, the EcoRV sites (V1-6) have likely been moved by insertion-deletion in the IGS. These sites should have disappeared several times and this might explain the homoplasy observed for the V4 site. Using all the information, 94 most 7 parsimonious trees were obtained. All these trees supported the separation of Tetrapilus from the other species of Olea. The rDNA consensus phylogenetic tree supported a separation of Oleeae from Jasminoideae, Fraxinae and Forestiera neomexicana (data not shown). Nevertheless, the nodes were not well supported due to a high frequency of homoplasic characters. Thus, we constructed and presented a tree only on the tribe Oleeae based on 12 characters of which 3 were informative (B1, V3 and V4) (Fig. 1 and 2). One homoplasy (V4) was detected in this data sample. O. brachiata and O. tsoongii (Tetrapilus) were not grouped with the other Olea species (this is supported by B1, V2 and V3 sites), but appeared related to Nestegis and Phillyrea. Chionanthus ramiflorus O. brachiata O. tsoongii = Tetrapilus E2 Chionanthus virginicus Syringa vulgaris Ligustrum vulgare O. woodiana O. c. ssp capensis O. africana V4 S6 O. paniculata B1 S5 Phillyrea latifolia Nestegis sandwichensis V4 V2 indel Autapomorph trait E6 S1 S2 Osmanthus fragrans V3 Noronhia emarginata Synapomorph trait E5 O. perrieri O. europaea complex O. lancea (except O. africana) O. c. ssp macrocarpa Homoplasy Fig. 2. Most parsimonious tree of the tribe Oleeae based on rDNA restriction sites. C. RAPD analysis The number of common bands according to their size between the different genera was very low, and we can consider that these were not informative without sequence homology verification. Consequently, we noted only the bands in the genus Olea. These were coded: primer-size in bp. Fiftyone Olea fragments were used as a probe (Table IV). A1-975 probe hybridized another fragment of approximately 925 bp (A1-925) and A10-1400 probe hybridized another fragment of approximately 1200 bp (A10-1200). Thus, 53 markers were verified for their sequence homology. The verification of the sequence homology of two fragments did not confirm the lecture based on the size homology. Thus, we considered such bands separately: A1-200a/A1-200b, A2-425a/A2-425b. In addition, 39 other intense and well-separated fragments were noted without sequence homology verification leading to a total of 92 markers. In the genus Olea, most of the markers (79/92 = 86 %) were unique to a subgenus or a section: fourteen markers for the section Olea, twenty-nine for the section Ligustroides, sixteen for the species O. paniculata and twenty for the subgenus Tetrapilus. Three non-polymorphic markers were found in the genus Olea and these were found again in other genera of Oleaceae (Table V). Several genera displayed common bands with the different groups of the genus Olea, in particular Nestegis and Chionanthus (Table V). Based on these data, the subgenus Tetrapilus appeared more closely related to Chionanthus and Nestegis than to the other species of Olea (Table V). 8 Table IV. List of the picked fragments, which were used as a probe. Each fragment was coded: primer - size in bp - accession code of origin (see Table II). Fragment A1-200a A1-675 A2-425a A2-675 A9-200 A9-225 A9-465 A9-525 A9-700 A9-725 C9-800 C15-450 C15-775 E15-800 A1-200b A1-400 A1-510 A1-975 C9-475 C9-900 C15-800 C15-1350 E15-475 E15-600 O8-350 O8-1100 Individual code T.TSO1 T.TSO1 T.TSO1 T.TSO2 T.TSO2 T.GRA T.TSO1 T.GRA T.GRA T.GRA T.TSO2 T.TSO1 T.TSO1 T.TSO2 E.LA E.MA1 E.LA E.MA1 E.CR2 E.MA1 E.ES1 E.MA1 E.ES1 E.ES1 E.ES1 E.ES1 Fragment A1-450 A1-480 A1-970 A2-480 A2-1050 A10-525 A10-750 A10-775 A10-1400 C9-975 C15-1000 E15-350 E15-650 O8-450 O8-600 A1-750 A1-950 A2-200 A9-475 C9-450 C9-525 C9-850 C15-500 C15-1100 O8-1200 Individual code L.LR1 L.WOS L.LR1 L.WOS L.WOS L.WOS L.WOK L.WOS L.LR1 L.PE1 L.WOS L.PE1 L.MAS L.MAZ L.LM1 P.KEW P.KEW P.KEW P.KEW P.KEW P.KEW P.KEW P.KEW P.KEW P.KEW The phenetic trees constructed on these data (Fig. 3) show a clear separation between four groups in accordance with the morphological classification of the genus (P.S. Green, in preparation). However, O. woodiana from Kenya is related to the section Olea although it should be related to the section Ligustroides. This individual displayed no specific markers of Ligustroides. The identification of this species (performed by H. Sommerlatte C/O GTS Nairobi, Kenya) should be incorrect. The phenetic tree based on the Neighbor joining method allows revealing that the sections Ligustroides and Olea have proximal positions in comparison to O. paniculata and the subgenus Tetrapilus. This is due to a sample too limited for the latter species and to their high genetic divergence in comparison to the subgenus Olea. The genetic proximity of the two sections of the subgenus Olea are supported by the phenetic tree constructed using the algorithm UPGMA. In each section, the relationships between species deduced from morphologic data were not confirmed here. In the section Ligustroides, the different taxa were well recognized except for two species from Madagascar, O. c. subsp. madagascariensis and O. perrieri, which are mixed. The O. capensis complex (subsp. macrocarpa, subsp. capensis, subsp. madagascariensis) is not supported by RAPD and ribosomal data. The four subspecies of the section Olea are not found. Three groups only can be distinguished based on RAPD data: O. africana-O. chrysophylla, O. cuspidataO. laperrinei and O. europaea-O. cerasiformis-O. maroccana. IV. DISCUSSION A. Position of subgenera and sections of genus Olea within the Oleaceae Our rDNA data are based mainly on IGS polymorphisms, but a high level of reorganization exists in this sequence, as already shown in Fraxinus (Jeandroz et al., 1996b). Consequently, we have to be prudent in the interpretation of these data. Nevertheless, our results support the evidence that species 9 Table V. Olea markers revealed by hybridization in Oleaceae taxa. 1 = presence of the fragment; - = absence of the fragment; (-) means that the fragment is polymorphic in the group of considered taxa; * marker with a lower intensity. In brackets, the size of the revealed fragments is indicated when it is different of the used probe. O. subg. Olea sect. Olea O. subg. Olea sect. Ligustroides O. subg. Paniculatea O. subg. Tetrapilus Nestegis sandwichensis Chionanthus virginicus Chionanthus ramiflorus Noronhia emarginata Osmanthus fragrans Phillyrea latifolia Ligustrum vulgare Syringa vulgaris Fraxinus angustifolia Forestiera neomexicana Schrebera alata Jasminum officinale Forsythia x intermedia Fontanesia phillyreoides A1-400 A10-525 E15-475 C15-1000 08-1100 C15-800 A1-200a A2-675 1 1 1 1 1* 1 (450 bps) 1 1* 1 (450 bps) 1* - 1 1 1 1 1 1 1 1 1 1 1 1 - 1 1 1 1* 1* (500 bps) 1* 1* 1* - 1 1 1 1 1 1* 1 1 1 1 1 - 1 (-) 1 1 1 1 1* 1* 1* (1050 bps) 1* - 1 1 (-) 1* 1 1 1* 1 1 (-) 1 1 - 1 (-) 1 (525 bps) 1 (500 bps) - A9-700 C15-775 1 1 1 (500 bps) 1* 1* 1* 1* 1* 1* (750 bps) 1* 1* 1* 1* - 10 A. B. Olea section Ligustroides section Paniculatae subg enus Tetrapilus subg enus 0.1 O. madaga scariensis - L. MA5 O. madaga scariensis - L. MA6 O. madaga scariensis - L. MA4 O. pe rrieri - L. PE1 O. pe rrieri - L. PE2 O. madaga scariensis - L. MA3 O. madaga scariensis - L. MA1 O. madaga scariensis - L. MA2 O. lanc ea - L.LM1 O. lanc ea - L.LA2 O. lanc ea - L.LR2 O. lanc ea - L.LR1 O. lanc ea - L.LM2 O. lanc ea - L.LA1 O. c. ssp cap ensis - L.CC O. exa sperata - L. EX O. c. ssp we lwit schii - L.C W1 O. c. ssp we lwit schii - L.C W2 O. woodiana - L.C WOS O. c. ssp macroca rpa - L.C MS O. c. ssp macroca rpa - L.C MZ O. t soongii -T.TSO1 O. t soongii -T.TSO2 O. bra chiata - T.BR A O. panicu lata - P.KEW O. panicu lata - P.BR I O. af ricana - E. AS2 O. af ricana - E. AS1 O. af ricana - E. AK1 O. woodiana - L. WOK O. af ricana - E. AR1 O. af ricana - E. AR2 O. ch rysophyl la - E.C H1 O. ch rysophyl la - E.C H2 O. af ricana - E. AK2 O. e. ssp cerasiformis - E.C E1 O. e. ssp cerasiformis - E.C E2 O. e. ssp europaea - E. ES2 O. e. ssp europaea - E. EC1 O. e. ssp europaea - E. EC2 O. ma roccana - E. MA1 O. ma roccana - E. MA2 O. e. ssp europaea - E. ES1 O. cu spidata - E.CR2 O. cu spidata - E.CR1 O. cu spidata - E.C I O. cu spidata - E.CC O. lape rrine i - E. LA O. madaga scariensis - L. MA4 O. madaga scariensis - L. MA6 O. pe rrieri - L. PE1 O. madaga scariensis - L. MA5 O. pe rrieri - L. PE2 O. madaga scariensis - L. MA1 O. madaga scariensis - L. MA3 O. madaga scariensis - L. MA2 O. exa sperata - L. EX O. lanc ea - L. LM1 O. lanc ea - L. LA2 O. lanc ea - L. LR2 O. lanc ea - L. LA1 O. lanc ea - L. LR1 O. lanc ea - L. LM2 O. woodiana - L. WOS O. c. ssp cap ensis - L.CC O. c. ssp macroca rpa - L.C MS O. c. ssp macroca rpa - L.C MZ O. c. ssp welwit schii - L.C W1 O. c. ssp welwit schii - L.C W2 O. panicu lata - P.KEW O. panicu lata - P.BR I O. t soongii - T. TSO1 O. t soongii - T. TSO2 O. bra chiata - T.BR A O. cu spidata - E.C I O. cu spidata - E.CC O. lape rrine i - E. LA O. cu spidata - E.CR1 O. cu spidata - E.CR2- E.CR2 O. cu spidata O. ma roccana - E. MA1 O. ma roccana - E. MA2 O. e. ssp europaea - E. EC1 O. e. ssp europaea - E. EC2 O. e. ssp cerasiformis - E.C E1 O. e. ssp cerasiformis - E.C E2 O. e. ssp europaea - E. ES2 O. e. ssp europaea - E. ES1 O. af ricana - E. AS1 O. af ricana - E. AS2 O. af ricana - E. AK1 O. woodiana - L. WOK O. af ricana - E. AR1 O. af ricana - E. AR2 O. ch rysophyl la - E.C H1 O. ch rysophyl la - E.C H2 O. af ricana - E. AK2 0.1 Fig 3. Phenetic trees of the species of Olea based on RAPD data: A. Phenetic tree based on Jaccard similarity (1908) and constructed with UPGMA algorithm. B. Phenetic tree based on Jaccard similarity (1908) and constructed with Neighbor Joining algorithm. 11 of the subgenus Tetrapilus species are more closely related to Nestegis, Chionanthus and Phillyrea than to the other Olea species. Such result was also supported by cpDNA information (Wallander & Albert, 2000). Furthermore, RAPD technology has led to a low level of informative characters (14 % of markers) when different genera, subgenera or sections were compared. Nevertheless, RAPD data also support that Tetrapilus species are related to Chionanthus and Nestegis. Combining rDNA and RAPD data, the genera Nestegis and Chionanthus appeared in an intermediary position between Tetrapilus and the other Olea species. Thus, Tetrapilus should be considered as a genus as proposed by Johnson (1957): Tetrapilus Lour. Nevertheless, an exhaustive study of the subgenus Tetrapilus is necessary to accumulate evidences. Moreover, we suggest that sequencing of ribosomal DNA ITS should lead to more informative characters and should avoid homoplasy. B. Structure of the genus Olea revealed by molecular markers In a recent study, AFLPs have led to the distinction of O. lancea and O. paniculata from the section Olea (Angiolillo et al., 1999), but this work was limited to a few accessions of Olea. In our work, we analyzed a larger number of accessions and these belong to the different sections and subgenera of Olea. The distinction of four groups of Olea obtained with RAPD data is in accordance with the taxonomy based on morphological characters, except possibly for O. woodiana subsp. disjuncta from Kenya, if the material was correctly named. We suspect that the molecular proximity is correlated to the cross-ability between the species as already shown for Syringa (Kim & Jansen, 1998). Thus, the four Olea groups could correspond to four species complexes. Within section Ligustroides, the three subspecies of O. capensis studied were as clearly separated as all the other taxa, except O. c. subsp. madagascariensis and O. perrieri. The taxa of this section have a discontinuous distribution but some are probably sympatric in some areas (notably in Central Africa or in South Africa). From the great genetic proximity between the different taxa of this section and the sympatric distribution, we can suspect that gene flow occurred or could occur between some taxa. Within the section Olea, the relationships deduced from rDNA RFLP and RAPD data were not in accordance with the morphological classification (Green & Wickens, 1989). Firstly, the O. laperrinei individual did not appear related to O. maroccana and O. cerasiformis, two other northern African taxa considered as relic forms of an ancient northern African population. This led us to suppose that the Northwestern African taxa and Saharan populations could derive from different ancestral populations. This hypothesis is also supported by cytoplasmic markers, which enabled to clearly separate O. maroccanaO. cerasiformis from O. laperrinei (Besnard & Bervillé, 2000). Otherwise, O. chrysophylla from Yemen and O. africana from Eastern and Southern Africa were grouped together with RAPDs but displayed a different rDNA unit. These two taxa displayed also different chlorotypes indicating their distinct origins (Besnard & Bervillé, 2000). These observations lead us to suspect gene flows between the different taxa during favorable periods. Thus, gene flow could have contributed to the evolution of the Mediterranean olive as suggested by Green & Wickens (1989) and Besnard et al. (2001b). C. Origin of the genus Olea We cannot deduce the geographical origin of the genus Olea with our molecular data. Nevertheless, O. paniculata is well separated from the subgenus Olea and this supports an ancient separation of the genus Olea between Asia-Oceania (subgenus Paniculatae) and Africa (subgenus Olea). The distribution of the subgenus Olea is mainly throughout the African continent. The common origin of the sections Ligustroides and Olea is supported by our molecular study. Verdoorn (1956) supposed that the axial and terminal flowering exhibited in O. woodiana could be an ancestral character. Thus, this author considered that O. woodiana was in an intermediary position between O. capensis and O. europaea. This is partially supported by rDNA restriction maps: O. africana and O. woodiana from Africa were not distinguished with rDNA restriction maps. The absence of the E5 restriction site may be an ancestral state. Thus, we can suppose that the subgenus Olea originated in Africa rather in Asia where it is also present. Quézel (1978) and Maley (1980) have already suggested an African origin for O. europaea species in the Rand-Flora (indigenous African flora adapted to Mediterranean climate). Consequently, the section Olea and the section Ligustroides probably originated from the Rand-Flora about 10 to 20 millions years ago. At the present time, we cannot determine whether the differentiation of the subgenus Olea occurred before, during or after the Rand-Flora formation. 12 Dyer (1991) has reported an incompatibility between O. capensis subsp. capensis (section Ligustroides) and O. africana (section Olea). In contrast, cross-ability between O. africana and O. europaea was evidenced (Besnard et al., 2001b), and this leads us to consider the O. europaea complex as a primary genetic resource of the cultivated olive tree. A study of the evolution of the wild and domesticated olive has to be performed on the whole O. europaea complex. ACKNOWLEDGMENTS: This work is supported by the FAIR program from CT 95-0689. We thank the persons who provided us the vegetal material: Dr G.Abdullah, Dr A. Alhakimi, Dr L. Amsellem, Dr L. Baldoni, Dr C. Costa, Dr T. Flynn, Dr L. Forget, Dr H. Hosseinpour, Dr L.Humeau, Dr C. Lambrides, Dr A. Mapaura, Dr A. Ouksili, Dr F. Rakotonasolo, Dr M. Sanchez, Dr A. Santos, Dr C.W. Smith, Dr H. Sommerlate, Pr I. Umboh, Dr K.S. Walter, We thank Dr P.A. Schäfer, Dr J. Mathez, Dr P. Lashermes and Dr B. Khadari for their help in the data analysis and for their comments. Thanks are also due to Dr A. Tagmount and D. Chevalier for their technical support. 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Iran, Pakistan, India, China Arabia, Eastern Africa Eastern and SouthernAfrica O. laperrinei Batt. & Trab. O. maroccana Greut. & Burd. Saharan Mountains Southern Morocco O. cerasiformis Webb & Berth. O. maderensis Lowe Canary Islands Madeira Olea O. europaea L. subsp. europaea subsp. cuspidata (Wall. ex G. Don) Ciferri subsp. laperrinei (Batt. & Trab.) Ciferri subsp. cerasiformis Kunk. & Sund. Ligustroides Benth. & Hook. O. capensis L. subsp. capensis subsp. enervis (Harv.) Verd. subsp. macrocarpa (Wright) Verd. subsp. madagascariensis (Boiv. ex Perr.) ined. Southern Africa Southern Africa Southern Africa Madagascar Central Africa Southern Africa Tanzania (Tanganyika) Madagascar Southern Africa Kenya, Tanzania Madagascar Mascareignes, Madagascar South Africa Zimbabwe, Mozambique O. hochstetteri Bak. O. welwitschii (Knobl.) Gilg & Schellenb O. schliebenii Knobl. O. perrieri Chev. ex Perr. O. woodiana Knobl. subsp. woodiana subsp. disjuncta ined. O. ambrensis Perr. O. lancea Lam. O. exasperata Jacq. O. chimanimani Kupicha Paniculatae ined. O. paniculata R Br. O. bournei Fyson, O. glandulifera Desf., O. thozetii Panch. & Seb. India, Nepal, Northern Oceania, Australia, New Caledonia Appendix 1, continued Subgenus Sections Tetrapilus (Lour.) ined. Species O. borneensis Boer. O. brachiata (Lour.) Merr. O. caudatilimba Chia O. cordatula Li O. decussata (Heine) Kiew O. dentata DC. O. dioica Roxb. O. gamblei Clarke O. hainanensis Li O. javanica (Blume) Knobl. O. laxiflora Li O. neriifolia Li O. obovata (Merr.) ined. O. palawanensis Kiew O. parvilimba (Merr. & Chun) Miao O. polygama Wight O. rosea Craib. O. salicifolia Wall. O. tetragonoclada Chia O. tsoongii (Merr.) Green O. wightiana Wall. ex G. Don Subspecies Main synonyms O. maritima Wall. ex G. Don O. graciliflora Koor. & Val. O. gagnepainiana Knobl O. guangxiensis Miao O. penengiana Ridl. O. rubrovenia (Elm.) ined. O. heyneana Wall. ex DC. O. gardneri Thw. O. densiflora Li O. oblanceolata Craib. O. yuennanensis Hand.-Mazz Geographical distribution Indonesia, Malaysia (Borneo) China, Cambodia, Thailand, Malaysia, Indonesia (Java, Sumatra) China (Yunan) Vietnam Indonesia, Malaysia (Borneo) Burma, Malaysia (Penang), Vietnam, Philippines, China India India China (Hainan) Indonesia, Malaysia China (Yunan) China (Hainan) Philippines Philippines China (Hainan) India Cambodia, Laos, Vietnam, China, Thailand China (Xizang), India, Burma China, India, Burma China (Guangdong, Sichuan,Yunan) India