14. Phylogenetic analysis of the tribe Oryzeae: Total chloroplast DNA restriction fragment analysis (A preliminary report)

Institut Francois de Recherche Scientifique pour le Developement en Cooporation (ORSTOM), Centre de Montpellier, BP 5045, 34032 Montpellier Cedex, France. 1) Present address: Department of Biology, Zhongshan University, Guangzhou 510275, China 2) Present address: Department of Plant Breeding and Biometry, Cornell University, Ithaca, N.Y. 14853, U.S.A.

The tribe Oryzae is highly heterogeneous and has a worldwide distribution. It is estimated to have diverged early in the phylogenesis of grasses, as compared with most other tribes of the Poaceae. Interest for a better understanding of the genetic structure of this tribe comes from two points of view: 1) genetic resources for rice 2) potential interest from the standpoint of evolutionary studies (Second 1985). Along with an ongoing study of the phylogenesis of the genus 0ryza, a study of the phylogenesis of the tribe Oryzeae was undertaken.

A live collection comprised of at least one representative of all recognized genera in the tribe is being assembled. It is available on request. So far, it lacks two genera only. Maltebrunia Kunth from the rain forest of Africa and Madagascar and Prosphytochloa Schweick from South Africa. Accessions of these may become available in the near future, at least from Africa.

The material studied in the present work is presented in Table 1. It comprises the breadth of the collection of the tribe and one accession each of wheat,oat and maize chosen as outgroups.

Chloroplast DNA was purified according to the Method of Dally and Second (1989) and restriction patterns of the following enzymes were observed from their ethidium bromide fluorescence after electrophoresis in agarose: SalI, Psti, KpnI and BamHI.

No intra-specific polymorphism was found between the four accessions of Oryza brachyantha originating from different African countries nor the two accessions of both Leersia hexandra and L. oryzoides originated from different continents or subcontinents, as noted in Table 1.

A matrix of distances between accessions was computed according to the method of Nei and Li (in Nei 1987, plO6-107) based on the proportion of restriction bands in common. An average-linkage dendrogram was then drawn from this matrix as shown in Fig. 1, and relationships among accessions of different clusters (of the Oryzeae tribe only, for better resolution) was further studied through Principal Coordinate Analysis; 75% of the variation was extracted by the first 4 axis. The first two axis are illustrated in Fig. 2.

Fig. 1. An average linkage dendrogram based on genetic distances for cpDNA RFLPsamong individual representatives of various species and genera of the Oryzeae tribe and three other genera of grasses (see Table 1 for signification of symbols). The scale shows the actual genetic distance values.

Fig. 2. A plot of individual representatives of various species and genera of the Oryzaee tribe in the first plan of a Principal Coordinate Analysis of their genetic distance matrix for cpDNA RFLPs (see Table 1 for signification of symbols). The percentage of total variation explained by axis 1 and 2 is indicated.

three main groups each with more or less the same level of diversity: 1) Oryza- Porteresia, 2) Leersia, and 3) one group composed of the other seven genera with Zizaniopsis and Luziola standing somewhat apart.

In other words, two genera appear to be particularly diverse while seven others cluster together. This contrasts with the relationship among genera of this tribe as recorded at morphological level (see Clayton and Renvoize 1986, p6O) and also as reflected by this classification of this tribe in genera.

Leersia tisserantii and L. perrieri, morphologically the most closely related Leersia species, were in the past classified in the genus Oryza. The present data support their placement in the genus Leersia but show how divergent they are from each other at chloroplast DNA level. Leersia perrieri, a rare endemic in Madagascar, is on the other hand surprisingly closely related to the weedy worldwide species L. hexandra. Leersia tisserantii, a rare endemic in Africa is more closely related to the American L. virginica than to its above mentioned counterpart from Madagascar. Leersia virginica is in turn closely related to L. oryzoides, a weedy species found in the relatively high latitudes of the northern hemisphere.

Porteresia and Rhynchoryza were also classified in the past in the genus Oryza. In the present data, Porteresia as well as O. meyeriana appear as equidistance to Oryza and Zizanial/Chikusichloa. This is not apparent in Fig. 1 but appears in the distance matrix and on axis 1 of its principal coordinate analysis as shown in Fig. 2.

O. longiglumis was analyzed by digesting the DNA with the enzymes Sal1, KpnI and PstI only. It appeared most closely related to O. meyeriana and O. brechyantha. The data thus supported the current treatment of the genus Oryza as a natural group. When the chloroplast DNA RFLP data previously obtained in the Sativa section (Dally 1988) is considered, the present data support the recognition of the Oryza section (O. sativa, O. latifolia and O. collina in the present material) as a natural group within the genus. Orya collina was not included in the study of Dally (1988). It appeared that O. collina presents a plastotype not previously observed, with a closest affinity in the O. officinalis complex.

The seven genera Zizania, Chikusichloa, Rhynchoryza, Potamophila, Hygroryza, Zizaniopsis and Luziola, although generally endemic in different continents, clustered together. The case of Potamophila, endemic in Australia, does not favor the hypothesis of Cretaceaous breakup of the Gondwanaland being involved in the origin of the pattern of distribution of the tribe because the closest relative (as read in the distance matrix) of Potainophila is Chikusichloa which is endemic in Asia. A common Asian ancestor niore likely migrated from South-East Asia to Australia. Similarly, various Oryzeae would have entered America by land from Eurasia, the latest being Zizania, the North American "wild rice".

The same material is being studied at the level of nuclear and mitochondrial DNA RFLPs and should allow further phylogenetic relationships to be drawn.

Clayton, W. D. and S. A. Renvoize, 1986. Genera Graminum. Kew Bulletin additional series XIII. 389 p.

Dally, A., 1988. Analyse cladistique de mutations de l'ADN chloroplastique et phylogenie des riz (section Eu-Oryza du genre Oryza). Coll. Etudes et Theses. ORSTOM, Paris. 153 p.

Nei, M., 1987. Molecular Evolutionary Genetics. Columbia Univ. Press, N. Y. 512 p.

Second, G., 1985. Geographic origins, genetic diversity and the molecular clock hypothesis in the Oryzeae. In Genetic Differentiation and Dispersal in Plants. P. Jacquard et al. (eds.) NATO ASI Series, Vol. G5, Springer-Verlag, Berlin Heidelberg.