PHYLOGENY OF THERUBIACEAE-RUBIOIDEAE, INPARTICULAR THE TRIBERUBIEAE: EVIDENCE FROM ANON-CODING CHLOROPLASTDNA SEQUENCE'Alessandro Natali2, Jean-Fran�ois Manen,2and Friedrich Ehrendorfer3ABSTRACT A phylogenetic analysis of 39 species of the tribe Rubieae and of 15 taxa belonging to 12 other tribes of Rubiaceaehas been performed using the DNA sequence of the chloroplast atpB-rbcL intergene region. The subfamily Rubioideaemay be characterized as a monophylum, i.e., by a characteristic 204-bp deletion, shared by the representative tribesCoccocypseleae, Psychotrieae, Hedyotideae (paraphyletically linked to Spermacoceae), Anthospermeae, Theligoneae,and Paederieae, which, in this order, step-wise approach the advanced Rubieae. This tribe is clearly monophyleticand characterized by an additional 50-bp deletion. Five clades can be recognized within Rubieae, which mostlycorroborate, but also partly contradict, traditional groupings (i.e., Galium and Asperula appear to be of polyphyleticorigin); some of these results may have taxonomic implications. By comparing the nucleotide sequences of a well-defined chloroplast intergene region among differ-ent genera and species, we hope to contribute tothe reconstruction of the phylogeny of the tribeRubieae. This tribe belongs to the huge, mostlywoody, tropical and subtropical family Rubiaceae,one of the largest of ail angiosperm families, withabout 637 genera and more than 10,000 species(Mabberley, 1987). In contrast, the Rubieae, con-taining predominantly perennial to annual herbswith pseudowhorls of leaves and leaflike stipules,and composed of about 13 genera (Ehrendorfer,unpublished), is centered in temperate and tropical-mountain regions. The evolutionary radiation ofthe Rubieae has resulted in a worldwide distribu-tion, but apparently is relatively recent. A surveyof the literature (Muller, 1981) indicates that thefirst fossil pollen records of the Rubieae are fromthe Upper Miocene for the genus Rubia (VanCampo, 1976) and the Pliocene for the genusGalium (Menke, 1976). Because the tribe Rubieaeis supposedly of relatively recent origin, instead ofthe widely used rbcL sequence, a non-coding se-quence of the chloroplast DNA, the atpB-rbcLspacer, was chosen. We thought that this sequence,being under lower selective constraints, would ex-hibit higher variability between the different studiedgenera and species. This assumption has been pro-posed by Gielly & Taberlet (1994) for a study ofthe genus Gentiana, using other non-coding cpDNAsequences. By comparison of this non-coding sequence (Ma-nen et al., 1994), we had presented a phylogeneticanalysis of 25 species of the tribe Rubieae, usingsix tropical genera from other tribes of Rubiaceaeas outgroups. In a separate paper (Ehrendorfer etal., 1994) we briefly discussed the relationshipsamong these outgroup tribes and their affinitieswith the tribe Rubieae. In this paper we extendthese data by adding the sequences of 23 moreRubiaceae taxa. This allows a precise and usefuldelimitation of the subfamily Rubioideae by a verycharacteristic deletion, as well as, in spite of theaddition of many taxa, the confirmation of thepreviously suggested general traits of the phylog-eny of the tribe Rubieae and of the polyphyly ofthe genera Galium and Asperula.MATERIALS AND METHODS The list of the Rubiaceae taxa studied so far isshown in Table 1. It represents 8 genera, 39 spe-' We thank R�gine Straessle for the amplification and sequencing of many of these DNA fragments. We alsothank Michael Kiehn, Institute of Botany, University of Vienna, for kindly sending several samples of tropical speciesas well as for various critical suggestions and valuable additions to our manuscript. This work was partially supportedby the Fonds National Suisse de la Recherche Scientifique (contracts 31-28757.90 and 22.214.171.124). 2 Conservatoire et Jardin Botaniques de Gen�ve, 1, Ch. de l'Imp�ratrice, 1292 Chamb�sy/Gen�ve, Switzerland. Institut fir Botanik und Botanischer Garten der Universitit Wien, Rennweg 14, 1030 Wien, Austria.ANN. MISSOURI BOT. GARD. 82: 428-439. 1995.