Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids (;125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

DNA sequences from the chloroplast trnL-F region of 154 Rubiaceae and 11 outgroup taxa were analyzed cladistically. An emphasis was placed on the tribes Rondeletieae, Sipaneeae, and Condamineeae. Sipaneeae are not close to Rondeletieae and belong in the Ixoroideae. There is no support for a widely distributed Rondeletieae in a broad sense. Instead, Rondeletieae sensu stricto form an almost entirely Antillean clade. Support was found for the separation of Arachnothryx, Rogiera, Roigella, and Suberanthus from Rondeletia. The Guettardeae as well as Gonzalagunia are found close to a complex formed by Arachnothryx, Javorkaea, and Rogiera. Condamineeae, in a strict sense, belongs in the Ixoroideae. A number of Rondeletieae genera should be transferred to Condamineeae or other parts of Ixoroideae. Support is found for an emended tribe Naucleeae, comprising several genera with spherical pseudanthia. For the first time, tribal or subfamilial affiliation based on molecular sequence data is suggested for Allenanthus, Blepharidium, Chione, Coutaportla, Dolichodelphys, Mazaea, Neobertiera, Neoblakea, Phialanthus, Phyllacanthus, Phyllomelia, Schmidtottia, and Suberanthus.

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The classification of the Catesbaeeae and Chiococceae tribes, along with that of the entire Rubiaceae, has long been debated. The Catesbaeeae-Chiococceae complex (CCC) includes approximately 28 genera and 190 species primarily concentrated in the Greater Antilles (nearly 70% of the species), Central and South America, and in the western Pacific (three genera). Previous molecular studies, with broad sampling of the Rubiaceae, have shown the CCC to be a monophyletic group. The present study is a more detailed examination of the generic relationships within the CCC using two data sets, the nuclear ribosomal ITS regions and the trnL-F chloroplast intron and spacer. Maximum parsimony analyses lend further support to the previous hypotheses that the CCC is monophyletic and sister to Strumpfia maritima. However, within the complex several genera do not form monophyletic groups. Previous studies of the Rubiaceae suggest that the ancestral fruit type in the CCC is a multiseeded capsule. Indehiscent, fleshy fruits appear to have evolved three to four times within this lineage. Changes in floral morphologies within the complex tend to correspond to cladogenesis among and within genera. Finally, molecular analyses suggest one or possibly two long-distance dispersals from the Americas to the western Pacific.Key words: biogeography; Caribbean; Catesbaeeae; Chiococceae; flower evolution; fruit evolution; islands; ITS; Neotropics; Pacific; Rubiaceae; systematics; trnL-F.The Rubiaceae is one of the largest and most diverse families of flowering plants, with approximately 650 genera and 13 000 species (Delprete, 2004), mostly of pantropical distribution. Historically fruit and seed characters have been used to infer the classification of the family and in several cases to define subfamilies (de Candolle, 1830; Hooker, 1873;Schumann, 1891). The subfamilial and tribal classification went through several minor modifications until Bremekamp (1966), using many additional characters, divided the family into eight The authors thank Lynn Raulerson and Agnus Rinehart (University of Guam), and Ritidian National Wildlife Refuge, Guam, David Lorence (National Tropical Botanical Garden, Lawai, Hawaii), George Proctor (Institute of Jamaica, Natural History Division, Kingston, Jamaica), Andreas Oberli (Hope Gardens, Kingston, Jamaica), Luke Lee and Kevin Boswell (Forestry Department of Jamaica), Teodoro Clase (Jardín Botánico Nacional, Santo Domingo, Dominican Republic), Tanguay Jaffré and Jean-Christphe Pintaud (IRD, Noumea, New Caledonia), David Orr (Waimea Arboretum, Hawaii), and Barry Hammel (Missouri Botanical Garden) for field assistance and advice; Kobinah Abdul-Salim (NYBG) for statistical advice; Claes Persson (Gö-teborg University) and anonymous reviewers for their helpful suggestions, the curators of NOU and NY herbaria, and the National Tropical Botanical Garden, Waimea Arboretum and the Hope Gardens for permission to sample herbarium and/or living collections.

The Rubiaceae is the fourth Angiosperm family in number of species in the World and in the Neotropics. Its overwhelming diversity and presence in most biomes, and at most vegetation layers, makes this family one of the most important components of tropical vegetation. During the last two decades, family classification went through several reorganizations, mostly influenced by the advent of molecular phylogenetic studies, and many taxonomic revisions and floristic studies on Brazilian Rubiaceae have become available. In view of the considerable amount of literature that has recently been produced on Neotropical Rubiaceae, the present work has two main objectives: the first is to offer an overall view of the most recent family classification with emphasis on the genera of Rubiaceae occurring in Brazil, and to indicate particular taxa that are still in need of phylogenetic and taxonomic studies; the second objective is to present a short discussion on the state of floristic and taxonomic knowledge with respect to the various regions of Brazil, indicating the taxa and the geographic areas that need to be studied. Key words: Rubiaceae, Brazil, classification, floristics, systematics, taxonomy. ResumoRubiaceae é a quarta família em número de espécies entre as Angiospermas no Mundo e no Neotrópico. A grande diversidade de espécies com representantes na maioria dos bioma, ocupando os diferentes estratos vegetacionais, fazem desta família um dos mais importantes componente da vegetação tropical. Durante as duas últimas décadas a classificação da família sofreu várias reorganizações, principalmente pela influência de estudos filogenéticos moleculares, e muitos estudos florísticos e revisões taxonômicas foram recentemente produzidos sobre Rubiaceae Neotropicais. Considerando a grande quantidade de literatura que foi recentemente produzida sobre estes assuntos, o presente trabalho tem dois objetivos principais: o primeiro é fornecer uma visão geral sobre a mais recente classificação da família com ênfase nos gêneros de Rubiaceae que ocorrem no Brasil, indicando os táxons que necessitam de estudos filogenéticos e taxonômicos; o segundo, é apresentar uma breve discussão sobre o estado de conhecimento florístico e taxonômico nas várias regiões do Brasil, indicando os táxons e as áreas geográficas que precisam de mais estudos.

The genera of tribe Sipaneeae have been either included within tribe Rondeletieae or treated as its sister tribe, and historically placed in subfamily Cinchonoideae. The herbaceous habit was the only character used to separate Sipaneeae from Rondeletieae. Recent molecular phylogenies included Maguireothamnus, Sipanea, Sipaneopsis, and Neobertiera in Sipaneeae, and positioned the tribe in subfamily Ixoroideae. These results stimulated a detailed analysis of this tribe and an evaluation of the position and delimitation of several genera believed to be related because of their morphological similarity. Our study indicates that tribe Sipaneeae is monophyletic, and confirms its placement within Ixoroideae. The Sipaneeae clade included Chalepophyllum, Dendrosipanea, Limnosipanea, Maguireothamnus, Neobertiera, Sipanea, and Sipaneopsis. Several genera of this group are small shrubs, and the woody habit is shown to be basal in Sipaneeae. Sipanea and Limnosipanea are shown to be monophyletic and not sister taxa, indicating that the herbaceous habit evolved two times in the tribe. Sequences of Steyermarkia, Pteridocalyx and Neblinathamnus were not available in this study, but because of overall morphological similarity with the genera of Sipaneeae, they are tentatively included in the tribe.

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