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Background and Aims: Like 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., Methods: Data 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 Results: Despite 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)., Conclusions: This 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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chloroplast sequences are widely used for phylogenetic analysis due to their high degree of conservation in plants. Whole chloroplast genomes can now be readily obtained for plant species using new sequencing methods, giving invaluable data for plant evolution However new annotation methods are required for the efficient analysis of this data to deliver high quality phylogenetic analyses. In this study, the two main tools for chloroplast genome annotation were compared. More consistent detection and annotation of genes were produced with GeSeq when compared to the currently used Dogma. This suggests that the annotation of most of the previously annotated chloroplast genomes should now be updated. GeSeq was applied to species related to coffee, including 16 species of the Coffea and Psilanthus genera to reconstruct the ancestral chloroplast genomes and to evaluate their phylogenetic relationships. Eight genes in the plant chloroplast pan genome (consisting of 92 genes) were always absent in the coffee species analyzed. Notably, the two main cultivated coffee species (i.e. Arabica and Robusta) did not group into the same clade and differ in their pattern of gene evolution. While Arabica coffee (Coffea arabica) belongs to the Coffea genus, Robusta coffee (Coffea canephora) is associated with the Psilanthus genus. A more extensive survey of related species is required to determine if this is a unique attribute of Robusta coffee or a more widespread feature of coffee tree species., Competing Interests: Dominique Crouzillat is employed by Nestlé Centre Tours. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

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 Madagascar were 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.

Caffeine is the most consumed alkaloid stimulant in the world. It is synthesized through the activity of three known N-methyltransferase proteins. Here we are reporting on the 422-Mb chromosome-level assembly of the Coffea humblotiana genome, a wild and endangered, naturally caffeine-free, species from the Comoro archipelago. We predicted 32,874 genes and anchored 88.7% of the sequence onto the 11 chromosomes. Comparative analyses with the African Robusta coffee genome (C. canephora) revealed an extensive genome conservation, despite an estimated 11 million years of divergence and a broad diversity of genome sizes within the Coffea genus. In this genome, the absence of caffeine is likely due to the absence of the caffeine synthase gene which converts theobromine into caffeine through an illegitimate recombination mechanism. These findings pave the way for further characterization of caffeine-free species in the Coffea genus and will guide research towards naturally-decaffeinated coffee drinks for consumers. [ABSTRACT FROM AUTHOR]

Coffea arabica (the Arabica coffee) is an allotetraploid species originating from a recent hybridization between two diploid species: C. canephora and C. eugenioides. Transposable elements can drive structural and functional variation during the process of hybridization and allopolyploid formation in plants. To learn more about the evolution of the C. arabica genome, we characterized and studied a new Copia LTR-Retrotransposon (LTR-RT) family in diploid and allotetraploid Coffea genomes called Divo. It is a complete and relatively compact LTR-RT element (~5 kb), carrying typical Gag and Pol Copia type domains. Reverse Trancriptase (RT) domain-based phylogeny demonstrated that Divo is a new and well-supported family in the Bianca lineage, but strictly restricted to dicotyledonous species. In C. canephora, Divo is expressed and showed a genomic distribution along gene rich and gene poor regions. The copy number, the molecular estimation of insertion time and the analysis at orthologous locations of insertions in diploid and allotetraploid coffee genomes suggest that Divo underwent a different and recent transposition activity in C. arabica and C. canephora when compared to C. eugenioides. The analysis of this novel LTR-RT family represents an important step toward uncovering the genome structure and evolution of C. arabica allotetraploid genome. [ABSTRACT FROM AUTHOR]

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 4 Mya) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 between 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1- copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution. [ABSTRACT FROM AUTHOR]

Miniature Inverted-repeat Transposable Elements (MITEs) are small nonautonomous class-II transposable elements distributed throughout eukaryotic genomes. We identified a novel family of MITEs (named Alex) in the Coffea canephora genome often associated with expressed sequences. The Alex-1 element is inserted in an intron of a gene at the CcEIN4 locus. Its mobility was demonstrated by sequencing the insertion site in C. canephora accessions and Coffea species. Analysis of the insertion polymorphism of Alex-1 at this locus in Coffea species and in C. canephora showed that there was no relationship between the geographical distribution of the species, their phylogenetic relationships, and insertion polymorphism. The intraspecific distribution of C. canephora revealed an original situation within the E diversity group. These results suggest possibly greater gene flow between species than previously thought. This MITE family will enable the study of the C. canephora genome evolution, phylogenetic relationships, and possible gene flows within the Coffea genus. [ABSTRACT FROM AUTHOR]

Of the 103 accepted Coffea species, 70% are threatened with extinction but only a few of them have been studied. A set of 40 polymorphic microsatellite markers was developed using a GA/GT-enriched Coffea canephora genomic library. Amplification of these markers was tested in accessions of C. heterocalyx (a Critically Endangered species) and C. pseudozanguebariae (a Vulnerable species) belonging to different African geographical clades. All microsatellites were polymorphic in C. canephora, with a mean allele number per polymorphic locus of more than 3 (at least 9 genotypes were tested). Observed and expected heterozygosities calculated for C. canephora and C. pseudozanguebariae varied from 0.10 to 0.91 and from 0.20 to 0.77, respectively. In total, 38 primer pairs (95%) were amplified in C. heterocalyx and C. pseudozanguebariae, indicating their high level of transferability across the genus Coffea. This large marker set will be useful for more extensive genetic studies of threatened Coffea species. Soixante-dix pour cent des 103 espèces de Coffea actuellement identifiées sont menacées d’extinction, mais peu d’entre elles sont étudiées. Un jeu de 40 marqueurs microsatellites polymorphes a été développé en utilisant une banque génomique de Coffea canephora enrichie en GA/GT. L’amplification de ces marqueurs a été testée sur des accessions de C. heterocalyx (une espèce déclarée « Gravement Menacée ») et C. pseudozanguebariae (une espèce déclarée « Vulnérable »), appartenant à différents clades géographiques Africains. Tous les microsatellites sont polymorphes chez C. canephora, avec un nombre moyen d’allèles par locus de plus de 3 (sur un minimum de 9 génotypes testés). Les hétérozygoties observées et attendues, calculées pour les espèces C. canephora et C. pseudozanguebariae varient respectivement entre 0,10 et 0,91; et entre 0,20 et 0,77. Au total, 95 % des paires d’amorces ont permis une amplification chez C. heterocalyx et C. pseudozanguebariae, indiquant un fort taux de transfert au sein du genre Coffea. Ce large jeu de marqueurs sera utile pour des études génétiques approfondies des espèces menacées de Coffea. [ABSTRACT FROM AUTHOR]

• The nuclear and chloroplast phylogenetic trees are incongruent for most of Coffea species. • Most of Coffea species are closely related to African Psilanthus than to Coffea. • Incongruences may be explained by retention of ancestral polymorphism and by hybridization. • Coffea rhamnifolia may be replaced into Psilanthus. • C. canephora might be a complex of species or currently in a state of ongoing speciation. 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 full-genome 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. [ABSTRACT FROM AUTHOR]