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International audience; For decades coffees were associated with the genus Coffea. In 2011, the closely related genus Psilanthus was subsumed into Coffea. However, results obtained in 2017-based on 28,800 nuclear SNPs-indicated that there is not substantial phylogenetic support for this incorporation. In addition, a recent study of 16 plastid fullgenome sequences highlighted an incongruous placement of Coffea canephora (Robusta coffee) between maternal and nuclear trees. In this study, similar global features of the plastid genomes of Psilanthus and Coffea are observed. In agreement with morphological and physiological traits, the nuclear phylogenetic tree clearly separates Psilanthus from Coffea (with exception to C. rhamnifolia, closer to Psilanthus than to Coffea). In contrast, the maternal molecular tree was incongruent with both morphological and nuclear differentiation, with four main clades observed, two of which include both Psilanthus and Coffea species, and two with either Psilanthus or Coffea species. Interestingly, Coffea and Psilanthus taxa sampled in West and Central Africa are members of the same group. Several mechanisms such as the retention of ancestral polymorphisms due to incomplete lineage sorting, hybridization leading to homoploidy (without chromosome doubling) and alloploidy (for C. arabica) are involved in the evolutionary history of the coffee species. While sharing similar morphological characteristics, the genetic relationships within C. canephora have shown that some populations are well differentiated and genetically isolated. Given the position of its closely-related species, we may also consider C. canephora to be undergoing a long process of speciation with an intermediate step of (sub-)speciation.

Coffee is a beverage enjoyed by millions of people worldwide and an important commodity for millions of people. Beside the two cultivated species (Coffea arabica and Coffea canephora), the 139 wild coffee species/taxa belonging to the Coffea genus are largely unknown to coffee scientists and breeders although these species may be crucial for future coffee crop development to face climate changes. Here we present the Wild Coffee Species database (WCSdb) hosted by Pl@ntNet platform (http://publish.plantnet-project.org/project/wildcofdb_en), providing information for 141 coffee species/taxa, for which 84 contain a photo gallery and 82 contain sequencing data (genotyping-by-sequencing, chloroplast or whole genome sequences). The objective of this database is to better understand and characterize the species (identification, morphology, biochemical compounds, genetic diversity and sequence data) in order to better protect and promote them. DATABASE URL: http://publish.plantnet-project.org/project/wildcofdb_en

Background and AimsLike other clades, the Coffea genus is highly diversified on the island of Madagascar. The 66 endemic species have colonized various environments and consequently exhibit a wide diversity of morphological, functional and phenological features and reproductive strategies. The trends of interspecific trait variation, which stems from interactions between genetically defined species and their environment, still needed to be addressed for Malagasy coffee trees.MethodsData acquisition was done in the most comprehensive ex situ collection of Madagascan wild Coffea. The structure of endemic wild coffees maintained in an ex situ collection was explored in terms of morphological, phenological and functional traits. The environmental (natural habitat) effect was assessed on traits in species from distinct natural habitats. Phylogenetic signal (Pagel’s λ, Blomberg’s K) was used to quantify trait proximities among species according to their phylogenetic relatedness.Key ResultsDespite the lack of environmental difference in the ex situ collection, widely diverging phenotypes were observed. Phylogenetic signal was found to vary greatly across and even within trait categories. The highest values were exhibited by the ratio of internode mass to leaf mass, the length of the maturation phase and leaf dry matter content (ratio of dry leaf mass to fresh leaf mass). By contrast, traits weakly linked to phylogeny were either constrained by the original natural environment (leaf size) or under selective pressures (phenological traits).ConclusionsThis study gives insight into complex patterns of trait variability found in an ex situ collection, and underlines the opportunities offered by living ex situ collections for research characterizing phenotypic variation.

International audience; A comprehensive and meaningful phylogenetic hypothesis for the commercially important coffee genus(Coffea) has long been a key objective for coffee researchers. For molecular studies, progress has been lim-ited by low levels of sequence divergence, leading to insufficient topological resolution and statisticalsupport in phylogenetic trees, particularly for the major lineages and for the numerous species occurringin Madagascar. We report here the first almost fully resolved, broadly sampled phylogenetic hypothesisfor coffee, the result of combining genotyping-by-sequencing (GBS) technology with a newly developed,lab-based workflow to integrate short read next-generation sequencing for low numbers of additionalsamples. Biogeographic patterns indicate either Africa or Asia (or possibly the Arabian Peninsula) asthe most likely ancestral locality for the origin of the coffee genus, with independent radiations acrossAfrica, Asia, and the Western Indian Ocean Islands (including Madagascar and Mauritius). The evolutionof caffeine, an important trait for commerce and society, was evaluated in light of our phylogeny. Highand consistent caffeine content is found only in species from the equatorial, fully humid environmentsof West and Central Africa, possibly as an adaptive response to increased levels of pest predation.Moderate caffeine production, however, evolved at least one additional time recently (between 2 and4 Mya) in a Madagascan lineage, which suggests that either the biosynthetic pathway was already inplace during the early evolutionary history of coffee, or that caffeine synthesis within the genus is subjectto convergent evolution, as is also the case for caffeine synthesis in coffee versus tea and chocolate

Coffee genetic resources are being lost at a rapid pace, leading to loss of genetic diversity. Various threats contribute to the erosion of coffee genetic diversity such as human population pressures leading to conversion of land to agriculture, deforestation and land degradation; low coffee prices leading to abandoning of coffee trees in forests and gardens and shifting cultivation to other more remunerative crops; and climate change. Additionally, the cultivated species of coffee (Coffea arabica L.) has a very narrow genetic base. Increased incidence of pests and diseases associated with climate change is leading to significant crop losses, threatening livelihoods in many coffee growing countries. A comprehensive conservation strategy for coffee should take into account complementary methods of in situ and ex situ conservation. The development of molecular techniques has expanded the possibilities and tools for genetic analysis for efficient conservation and use of coffee genetic resources. Before it is too late, a thorough evaluation of existing germplasm should be performed based upon which a comprehensive conservation strategy could be developed. A case study using four different Coffea species in Madagascar is discussed.

The Coffea genus, 124 described species, has a natural distribution spreading from inter-tropical Africa, to Western Indian Ocean Islands, India, Asia and up to Australasia. Two cultivated species, C. arabica and C. canephora, are intensively studied while, the breeding potential and the genome composition of all the wild species remained poorly uncharacterized. Here, we report the characterization and comparison of the highly repeated transposable elements content of 11 Coffea species representatives of the natural biogeographic distribution. A total of 994 Mb from 454 reads were produced with a genome coverage ranging between 3.2 and 15.7 %. The analyses showed that highly repeated transposable elements, mainly LTR retrotransposons (LTR-RT), represent between 32 and 53 % of Coffea genomes depending on their biogeographic location and genome size. Species from West and Central Africa (Eucoffea) contained the highest LTR-RT content but with no strong variation relative to their genome size. At the opposite, for the insular species (Mascarocoffea), a strong variation of LTR-RT was observed suggesting differential dynamics of these elements in this group. Two LTR-RT lineages, SIRE and Del were clearly differentially accumulated between African and insular species, suggesting these lineages were associated to the genome divergence of Coffea species in Africa. Altogether, the information obtained in this study improves our knowledge and brings new data on the composition, the evolution and the divergence of wild Coffea genomes.

A comprehensive and meaningful phylogenetic hypothesis for the commercially important coffee genus (Coffea) has long been a key objective for coffee researchers. For molecular studies, progress has been limited by low levels of sequence divergence, leading to insufficient topological resolution and statistical support in phylogenetic trees, particularly for the major lineages and for the numerous species occurring in Madagascar. We report here the first almost fully resolved, broadly sampled phylogenetic hypothesis for coffee, the result of combining genotyping-by-sequencing (GBS) technology with a newly developed, lab-based workflow to integrate short read next-generation sequencing for low numbers of additional samples. Biogeographic patterns indicate either Africa or Asia (or possibly the Arabian Peninsula) as the most likely ancestral locality for the origin of the coffee genus, with independent radiations across Africa, Asia, and the Western Indian Ocean Islands (including Madagascar and Mauritius). The evolution of caffeine, an important trait for commerce and society, was evaluated in light of our phylogeny. High and consistent caffeine content is found only in species from the equatorial, fully humid environments of West and Central Africa, possibly as an adaptive response to increased levels of pest predation. Moderate caffeine production, however, evolved at least one additional time recently (between 2 and 4Mya) in a Madagascan lineage, which suggests that either the biosynthetic pathway was already in place during the early evolutionary history of coffee, or that caffeine synthesis within the genus is subject to convergent evolution, as is also the case for caffeine synthesis in coffee versus tea and chocolate.

International audience; The completion of the genome assembly for the economically important coffee plant Coffea canephora (Rubiaceae) has allowed the use of bioinformatic tools to identify and characterize a diverse array of transposable elements (TEs), which can be used in evolutionary studies of the genus. An overview of the copy number and location within the C. canephora genome of four TEs is presented. These are tested for their use as molecular markers to unravel the evolutionary history of the Millotii Complex, a group of six wild coffee (Coffea) species native to Madagascar. Two TEs from the Gypsy superfamily successfully recovered some species boundaries and geographic structure among samples, whereas a TE from the Copia superfamily did not. Notably, species occurring in evergreen moist forests of eastern and southeastern Madagascawere divergent with respect to species in other habitats and regions. Our results suggest that the peak of transpositional activity of the Gypsy and Copia TEs occurred, respectively, before and after the speciation events of the tested Madagascan species. We conclude that the utilization of active TEs has considerable potential to unravel the evolutionary history and delimitation of closely related Coffea species. However, the selection of TE needs to be experimentally tested, since each element has its own evolutionary history. Different TEs with similar copy number in a given species can render different dendrograms; thus copy number is not a good selection criterion to attain phylogenetic resolution.

† Background and Aims The coffee genus (Coffea) comprises 124 species, and is indigenous to the Old World Tropics. Due to its immense economic importance, Coffea has been the focus of numerous genetic diversity studies, but despite this effort it remains insufficiently studied. In this study the genetic diversity and genetic structure of Coffea across Africa and the Indian Ocean islands is investigated. † Methods Genetic data were produced using 13 polymorphic nuclear microsatellite markers (simple sequence repeats, SSRs), including seven expressed sequence tag-SSRs, and the data were analysed using model- and non-model-based methods. The study includes a total of 728 individuals from 60 species. † Key Results Across Africa and the Indian Ocean islands Coffea comprises a closely related group of species with an overall pattern of genotypes running from west to east. Genetic structure was identified in accordance with pre-determined geographical regions and phylogenetic groups. There is a good relationship between morpho-taxonomic species delimitations and genetic units. Genetic diversity in African and Indian Ocean Coffea is high in terms of number of alleles detected, and Madagascar appears to represent a place of significant diversification in terms of allelic richness and species diversity. † Conclusions Cross-species SSR transferability in African and Indian Ocean islands Coffea was very efficient. On the basis of the number of private alleles, diversification in East Africa and the Indian Ocean islands appears to be more recent than in West and West-Central Africa, although this general trend is complicated in Africa by the position of species belonging to lineages connecting the main geographical regions. The general pattern of phylogeography is not in agreement with an overall east to west (Mascarene, Madagascar, East Africa, West Africa) increase in genome size, the high proportion of shared alleles between the four regions or the high numbers of exclusive shared alleles between pairs or triplets of regions.

Madagascar has 59 species of Coffea, of which 42 are listed as Critically Endangered, Endangered, or Vulnerable by criteria of the Red List Category system of the World Conservation Union. In an attempt to assess the conservation value of ex situ collections of Malagasy coffee species, a study was undertaken using the field genebank collections maintained at the Kianjavato Coffee Research Station. Three species were selected for this purpose, C. kianjavatensis, C. montis-sacri, and C. vatovavyensis, and for comparative purposes extant, in situ populations of the same species were studied. Parentage analyses of ex situ propagated offspring of C. kianjavatensis and C. montis-sacri were performed to assess if crossing with other Coffea species maintained in the field genebank is compromising the genetic integrity of the collection. For these three species, higher genetic diversity was observed in the ex situ populations compared to the in situ populations, highlighting the importance of preserving the plants currently in ex situ collections. Parentage analyses of seed-propagated offspring of C. kianjavatensis and C. montis-sacri revealed that cross contamination with pollen from other Coffea species in the ex situ field genebank is occurring. These results have significant implications for the conservation management of wild Coffea species and for the management of ex situ genebanks.

Madagascar has 59 described species of Coffea, of which 42 are listed as critically endangered, endangered, or vulnerable by the criteria of the Red List Category system of the World Conservation Union (IUCN). The littoral forest of Madagascar is a distinctive type of humid evergreen forest restricted to unconsolidated sand located within a few kilometers of the Indian Ocean, now persisting only as small fragments with ca. 10 % of its original range remaining. In an attempt to understand the genetic diversity of Madagascan coffee species, we studied ex situ and in situ populations of Coffea commersoniana, an endemic species of the littoral forests of southeastern Madagascar and soon to be impacted by mining activities in that region. The in situ populations studied showed higher genetic diversity than the ex situ population. The genetic partitioning among the two in situ populations of C. commersoniana was high enough to necessitate keeping the two populations separate for restoration purposes. Based on these findings, recommendations for conservation management (in situ and ex situ) are made.

The GCAs are new tetraploid interspecific hybrids developed in Madagascar from Coffea eugenioides, C. canephora and C. arabica. Selected GCA having genotype UF1023 contained 0.37% DW caffeine and no detectable theobromine in green beans. Low caffeine accumulation in GCA plants is due mainly to the low biosynthetic activity of purine alkaloids, possibly the extremely weak N-methyltransferase reactions in caffeine biosynthesis. No significant catabolic activity of caffeine was found in GCA-UF1023, in common with almost all coffee plants including C. arabica.

A recently proposed delimitation of the genus Coffea has raised new questions of geographical distribution, its evolutionary history, and mechanisms of its speciation. Global chromosome morphology and uniform C-banding patterns argue for a unique and common Coffea genome. However, evolution of the Coffea genus was associated with genome size variation and fine chromosomal changes (number of secondary constrictions, number and physical localization of rDNA loci and repetitive elements). The Coffea genome is small to medium-sized. Following the paleo-ancestral hexaploidization common to all dicotyledons, it did not undergo a whole-genome duplication. Although genus size variation did not provoke strong variations of the genome structure, it has played a role in its genus speciation. Indeed, the higher the difference in genome size of two species, the less likely they could be intercrossed. On a molecular scale, transposable element-insertion patterns produced complementary chronological information related to the events that shaped the evolutionary history of genus Coffea .

Caffeine, a purine alkaloid, was not detected in leaves of two Mascarocoffea species, Coffea millotii and Coffea perrier. Trigonelline, a pyridine alkaloid, occurred in these species, but the levels (3−4 μmol/g fresh weight) were much lower than that of Robusta coffee (Coffea canephora) (36 μmol/g fresh weight). Feeding experiments with [8-14C]adenine indicated that purine alkaloid biosynthesis was terminated at 7-methylxanthine formation and as a consequence theobromine and caffeine were not produced in Coffea millotii and Coffea perrier. The adenine salvage activity was lower but its degradation activity was higher in leaves of these Mascarocoffea species than those in Coffea canephora. The metabolic fate of the purine nucleosides, [8-14C]inosine, [8-14C]guanosine and [8-14C]xanthosine was investigated in leaves of Coffea millotii. The biosynthesis of 7-methylxanthine, but not theobromine or caffeine, from these precursors was detected. Large amounts of these purine nucleosides were catabolized via allantoin. Limited amounts of [8-14C]inosine and [8-14C]guanosine were salvaged and utilized for RNA synthesis, however, no [8-14C]xanthosine salvage was observed. Little or no 14C-metabolites were observed when [8-14C]theobromine and [8-14C]caffeine were applied to leaf disks of Coffea millotii. From the results obtained in this study, possible metabolic pathways of purines in Mascarocoffea species are discussed.

The genetic diversity of endemic Madagascan Coffea species has not received any considerable attention, despite the high extinction threat facing most of the species on the island. In order to address this shortfall, we assessed and compared the genetic diversity and allelic richness of ex situ and in situ populations for four selected species, and 18 ex situ populations maintained at the Kianjavato Coffee Research Station. We also investigated the possibility of in situ and ex situ hybridization events for Madagascan coffee, both within and between species. For these purposes, we used 18 nuclear microsatellite markers, on a total of 37 Coffea populations (4 in situ and 33 ex situ) representing a total of 398 genotypes (96 in situ and 302 ex situ) for 23 species. Our study showed (1) strong differentiation be- tween Madagascan Coffea species; (2) good differentiation between all the populations studied, including those from the same locality (even when the area of the locality was restricted); (3) good evidence for both in situ and ex situ hybridization, although in situ hybridization appeared to be limited; (4) much higher genetic diversity within in situ collections compared to ex situ collections; and (5) that, despite limitations, the Kianjavato Coffee Research Station collection is an extremely valuable resource of Madagascan coffee germplasm. Recommendations for in situ and ex situ germplasm conservation are discussed.

The genus Coffea, mainly native to Africa and to the Indian Ocean islands (Mascarocoffea), accounts for 124 species. Genome size data are available for 23 African species. The aim of this study was to assess the genome size of 44 Mascarocoffea species and to investigate possible association with species geographic distribution, stomata traits, and species relationships. 2 C values were measured using flow cytometry. A lyophilization procedure for leaves was tested. The 2 C nuclear DNA content of Mascarocoffea species ranged from 0.96 to 1.41 pg. Coffea mauritiana and Coffea humblotiana have the smallest genomes and Coffea millotii has the largest. Mean 2 C DNA for Mascarocoffea and Africa is 1.19 and 1.43 pg, respectively. The overall DNA values corresponded to two partially overlapped normal distributions: one harboring species from east Africa Mascarocoffea, the other harboring species from west/central Africa. Plotted on a geographical map according to the native origin of species, these values showed a gradient in Madagascar and Africa. Genome sizes increased following a north to southeast gradient in Madagascar and an east to west gradient in Africa. None, or only weak correlations were noted between genome size and stomata parameters. Genetically close species could be highly distinctive in their genome size while divergent species could be similarly sized. The non-random geographic distribution and habitat of species, and the absence of correlation between genome size and genetic relationships, suggest that during Coffea genome evolution, both DNA content increase and/or decrease occurred independently in Africa and in the Indian Ocean Islands.

BACKGROUND AND AIMS The phenolic composition of Coffea leaves has barely been studied, and therefore this study conducts the first detailed survey, focusing on mangiferin and hydroxycinnamic acid esters (HCEs). METHODS Using HPLC, including a new technique allowing quantification of feruloylquinic acid together with mangiferin, and histochemical methods, mangiferin content and tissue localization were compared in leaves and fruits of C. pseudozanguebariae, C. arabica and C. canephora. The HCE and mangiferin content of leaves was evaluated for 23 species native to Africa or Madagascar. Using various statistical methods, data were assessed in relation to distribution, ecology, phylogeny and use. KEY RESULTS Seven of the 23 species accumulated mangiferin in their leaves. Mangiferin leaf-accumulating species also contain mangiferin in the fruits, but only in the outer (sporophytic) parts. In both leaves and fruit, mangiferin accumulation decreases with ageing. A relationship between mangiferin accumulation and UV levels is posited, owing to localization with photosynthetic tissues, and systematic distribution in high altitude clades and species with high altitude representatives. Analyses of mangiferin and HCE content showed that there are significant differences between species, and that samples can be grouped into species, with few exceptions. These data also provide independent support for various Coffea lineages, as proposed by molecular phylogenetic analyses. Sampling of the hybrids C. arabica and C. heterocalyx cf. indicates that mangiferin and HCE accumulation may be under independent parental influence. CONCLUSIONS This survey of the phenolic composition in Coffea leaves shows that mangiferin and HCE accumulation corresponds to lineage recognition and species delimitation, respectively. Knowledge of the spectrum of phenolic accumulation within species and populations could be of considerable significance for adaptation to specific environments. The potential health benefits of coffee-leaf tea, and beverages and masticatory products made from the fleshy parts of Coffea fruits, are supported by our phenolic quantification.

Biosynthesis of caffeoylquinic acids occurs via the phenylpropanoid pathway in which the phenylalanine ammonia-lyase (PAL) acts as a key-control enzyme. A full-length cDNA (pF6), corresponding to a PAL gene (CcPAL1), was isolated by screening a Coffea canephora fruit cDNA library and its corresponding genomic sequence was characterized. Amplification of total DNA from seven Coffea species revealed differences in intronic length. This interspecific polymorphism was used to locate the gene on a genetic map established for a backcross progeny between Coffea pseudozanguebariae and C. dewevrei. The CcPAL1 gene was found on the same linkage group, but genetically independent, as a caffeoyl-coenzyme A-O-methyltransferase gene, another gene intervening in the phenylpropanoid pathway. In the same backcross, a lower caffeoylquinic acid content was observed in seeds harvested from plants harbouring the C. pseudozanguebariae CcPAL1 allele. Involvement of the CcPAL1 allelic form in the differential accumulation of caffeoylquinic acids in coffee green beans is then discussed.

The presence of theobromine (up to 0.14%) and of caffeine (up to 0.76%) has been established for the first time in beans from two populations of Coffea lancifolia (A320 and A405) and in one population of C. kianjavatensis (A213). This indicates that the Mascarocoffea , once thought to be purine-free, includes some members storing methyl xanthines in seeds, but the virtual absence of caffeine from another C. kianjavatensis (A602), and of both caffeine and theobromine from three populations of C. homollei (A574, A743 and SZ108) indicates the biochemical diversity of these taxa.

Unequivocal evidence is presented for the presence of 0.55–0.81% caffeine in the beans of population A213 of Coffea kianjavatensis , one taxon of the Mascarocoffea , which traditionally have been viewed as caffeine-free.