Kupeantha (Coffeeae, Rubiaceae), a new genus from Cameroon and Equatorial Guinea

Two new coffee relatives (tribe Coffeeae, Rubiaceae), discovered during botanical expeditions to Cameroon, are examined for generic placement, and the placement of three previously known species (Argocoffeopsis fosimondi, A. spathulata and Calycosiphonia pentamera) is reinvestigated using plastid sequence (accD-psa1, rpl16, trnL-F) and morphological data. Seed biochemistry of the new species and pollen micromorphology (only one of the two species) are also studied. Based on the plastid sequence data, the new taxa are nested in a well-supported monophyletic group that includes Argocoffeopsis and Calycosiphonia. Within this clade, three well-supported subclades are recovered that are morphologically easy to diagnose: (1) Calycosiphonia (excluding C. pentamera), (2) Argocoffeopsis (excluding A. fosimondi and A. spathulata), and (3) a clade including the above excluded species, in addition to the new species. Based on the results, Kupeantha, a new genus of five species, is described, including two new Critically Endangered taxa from the Highlands of Cameroon: Kupeantha ebo and K. kupensis. Phytochemical analysis of Kupeantha seeds reveals compounds assigned as hydroxycinnamic acid derivatives, amino acids and ent-kaurane diterpenoids; caffeine was not detected. Kupeantha is the first new genus described in tribe Coffeeae in 40 years.


Introduction
Distribution data, although important to ensure effective conservation management and sustainable utilisation of natural resources [1], is lacking for many organisms in tropical Africa, including vascular plants necessitating renewed efforts for botanical exploration of the region [2]. Based on the RAINBIO dataset containing 600,000+ georeferenced records representing 22,000+ plant species, priority target areas for future sampling efforts were proposed in Tanzania, Atlantic Central Africa and West Africa [2]. One of the areas, which remained underexplored until recently, is Cameroon in West Central Africa. During the 1990s, the Royal Botanic Gardens, Kew and partners in Cameroon began a series of botanical expeditions to PLOS  them formally in accordance with the International Code of Nomenclature (ICN) [25], we hope to draw attention to their conservation. Furthermore, high-resolution LC-UV-MS/MS seed analyses of the new species was carried out in order to contribute knowledge of the seed chemistry of the genera in the coffee tribe, the chemotaxonomic significance of the chemical constituents, and their distribution across the group. While the chemistry of the genus Coffea L. has been extensively investigated [26][27][28], there is a lack of knowledge about the chemistry of other genera, with the exception of species currently placed in Diplospora DC. (previously assigned to Tricalysia A.Rich ex DC) [27,[29][30][31][32].

Ethical statement
This study is based mainly on herbarium specimens and field observations made in Cameroon during a series of botanical surveys beginning in 1991. These surveys were mainly led by the first author. So far, they have resulted in 52,450 specimens being studied at K and YA, of which 37,850 were newly collected. Data are stored in the Kew-Cameroon specimen Access database (Gosline, p. 11 in Cheek et al. 2004). The top set of specimens was initially deposited at SCA, and later at YA, and duplicates sent to K. Robbr., Tricalysia and Xantonnea Pierre ex Pit. was consulted at BR, BRLU, K, MO, P, SCA, US, WAG and YA [33]. All specimens cited have been seen, unless otherwise indicated. Total sampling for the molecular phylogenetic study comprises 38 accessions, including 2/3 of Calycosiphonia species (DNA could not be extracted from material of C. pentamera and, therefore, its placement was inferred from morphological characters only), 6/10 of Argocoffeopsis species, and two species new to science. The samples with species names, voucher information, origin, and GenBank accession numbers, are given in S1 Appendix.
The matrices of the three chloroplast regions were concatenated for the downstream analyses. PartitionFinder 2.1.1 [39] was used to determine an appropriate data-partitioning scheme from potential partitions that were defined a priori (each marker treated as a separate partition), as well as the best-fitting model of molecular evolution for each partition, using the Bayesian Information Criterion. The optimal data-partitioning scheme was accD-psa1+rpl16 and trnL-F, and the GTR+I+Γ (invgamma) model of sequence evolution was determined to be the best-fitting model for the accD-psa1+rpl16 partition, while the GTR+Γ (gamma) model of sequence evolution was determined to be the best-fitting model for the trnL-F partition in the concatenated data set.
Maximum likelihood (ML) analyses of the optimally partitioned data were performed using RAxML 8.2.10 [40]. The search for an optimal ML tree was combined with a rapid bootstrap analysis of 1000 replicates. Partitioned analyses were conducted using Bayesian Inference (BI) in MrBayes 3.2.6 [41]. The parameters of each of the partitions were the same as in the ML analysis. Rate heterogeneity, base frequencies, and substitution rates across partitions were unlinked. The analysis was allowed to run for 100 million generations across four independent runs with four chains each, sampling every 10000 generations. Convergence, associated likelihood values, effective sample size (ESS) values and burn-in values of the different runs were verified with Tracer 1.5 [42]. The first 25% of the trees from all runs were excluded as burn-in before making a majority-rule consensus of the 30000 posterior distribution trees using the "sumt" function. All phylogenetic analyses were run using the CIPRES portal (http://www. phylo.org/) [43]. Trees were drawn using TreeGraph2 [44].

Morphology, palynology, distribution and conservation
Measurements, colours and other details given in the descriptions of the new species Kupeantha ebo and K. kupensis are based on living material, spirit, and herbarium specimens, and data and photographs derived from field notes. Pollen samples were collected only from Kupeantha kupensis (Cheek 7882 K). Whole, unacetolysed anthers were placed on a stub using double-sided tape and sputter-coated with platinum in a Quorom Q150T coater for 30 seconds and examined in a Hitatchi 54700 scanning electron microscope at an acceleration voltage of 4kV.
The conservation status was assessed using the IUCN Red List Category criteria [45]. The distribution of the species was mapped using SimpleMappr [46].

Nomenclature
The new taxon names generated as part of this study satisfy the requirements of the International Code of Nomenclature for algae, fungi, and plants, and are hereby effectively published. In addition, they have been submitted to The International Plant Names Index (IPNI), from where they will be made available to the Global Names Index (http://gni.globalnames.org/). The IPNI LSIDs will resolve and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS, and Ghent University Academic Bibliography.

Phytochemical analysis
The extracts were prepared by extracting ground seed material from the newly described species Kupeantha kupensis (one accession) and Kupeantha ebo (two accessions). There was insufficient or no material available for the other species. Extracts were made in 80% aqueous methanol (100 mg/ml) for 24 h, prior to centrifugation. The supernatants were then subjected to LC-UV-MS/MS analysis. Analyses were performed on a Thermo Scientific system consisting of an 'Accela' U-HPLC unit with a photodiode array detector and an 'LTQ Orbitrap XL' mass spectrometer fitted with an electrospray source (Thermo Scientific, Waltham, MA, USA). Chromatography was performed on 5 μl sample injections onto a 150 mm x 3 mm, 3 μm Luna C-18 column (Phenomenex, Torrance, CA, USA) using the following 400μl/min mobile phase gradient of H 2 O/CH 3 OH/CH 3 CN +1% HCOOH: 90:0:10 (0 min), 90:0:10 (5 min), 0:90:10 (60 min), 0:90:10 (65 min), 90:0:10 (67 min), 90:0:10 (70 min) followed by return to start conditions and equilibration in start conditions for 5 min before the next injection. The ESI source was operated with polarity switching and the mass spectrometer was set to record high resolution (30 k resolution) MS1 spectra (m/z 125-2000) in positive mode using the orbitrap and low resolution MS1 spectra (m/z 125-2000) in negative mode and data dependent MS2 and MS3 spectra in both modes using the linear ion trap. Detected compounds were assigned by comparison of accurate mass data (based on ppm), and by available MS/MS data, with reference to the published compound assignment system [47] and with supportive UV spectra.

Phylogenetic analyses
The concatenated ML and BI analyses did not generate significantly different topologies, therefore, we present the relationships shown in the 50% majority consensus multiple-locus BI tree, with the associated PP values and the bootstrap values of the multiple-locus ML tree (Fig 1). Only BS values above 70% and posterior probabilities (PP) above 0.75 are shown. The best scoring ML tree is available for comparison in S1 Fig. Tribe Coffeeae is recovered as monophyletic, with the monophyletic genus Coffea strongly supported as sister to the rest of the tribe (PP = 1, BS = 100). The rest of the Coffeeae is represented by two main clades. The first of these, the Argocoffeopsis clade (PP = 1, BS = 99) indicated in grey (Fig 1), includes the genus Argocoffeopsis (PP = 1, BS = 86), which becomes monophyletic after exclusion of Argocoffeopsis fosimondi and A. spathulata, the monophyletic genus Calycosiphonia (PP = 1, BS = 87), and a monophyletic group (PP = 0.99, BS = 92) here named Kupeantha comprising Argocoffeopsis fosimondi and A. spathulata in addition to two new species described in this study. The placement of Calycosiphonia pentamera in Kupeantha was inferred based only on its morphology because DNA could not be extracted.
The other main clade includes the rest of the generic diversity of the tribe. Included in this study are members of Sericanthe, Diplospora, Empogona, Discospermum, Xantonnea, Belonophora, and Tricalysia.

Morphology and palynology
Characters separating Argocoffeopsis, Calycosiphonia and Kupeantha gen. nov. are provided in Table 1. Characters separating Argocoffeopsis fosimondi, A. spathulata, Calycosiphonia pentamera, and the new species are documented in Table 2. The descriptions in the taxonomic treatment largely follow [48].

Phytochemical analysis
LC-UV-MS/MS seed analysis (Table 3) revealed the detection of a range of hydroxycinnamic acid derivatives, including caffeoyl-, coumaroyl-and feruloyl-quinic acids, in all three extracts, one of Kupeantha kupensis (Cheek 7882), and two of Kupeantha ebo (Alvarez 11, Fenton 134), which were assigned from their observed [M+H] + ions, with supportive UV spectra and MS/ MS interpretation ( Table 3). The main hydoxycinnamic acid detected in the positive ionisation mode in the three extracts with m/z 339.1070 eluted at the retention time (Rt) of 11.0 min was assigned as 5-O-coumaroylquinic acid. Amino acids were also detected in all three extracts and were assigned from their observed [M+H] + ions and supportive MS/MS interpretation as asparagine, phenylalanine and tryptophan, or their isomers (Table 3).
In the present study, a compound eluting at Rt 15.5 min with m/z 497.2737 was detected in both new species and was assigned with the molecular formula C 26 H 40 O 9 , determined from the [M+H] + ion, which is that of the diterpenoid glucoside, villanovane I. Also, detected in all of the extracts analysed were compounds eluting at Rt 24.4 min and 38.8 min, with m/z 495.2575 and 516.3161, respectively. These compounds were assigned with the molecular formulae from their observed [M+H] + ions as C 26 H 38 O 9 , which is that of tricalysioside B or C, and as C 26 H 42 O 9 , which is that of tricalysioside P. Other compounds assigned as ent-kaurane diterpenoids or their glycosides were only detected in the seed extract of Cheek 7882. These included a compound eluting at Rt 19.6 min with m/z 333.2059 that was assigned with the

Relationships in tribe Coffeeae
The relationships recovered in tribe Coffeeae (Fig 1) largely match those obtained in previous studies [21,34], with Coffea sister to the rest of the tribe, but with little support for the relationships among most genera. [34] removed the species of Empogona from Tricalysia since they are more closely related to the genus Diplospora. This relationship is confirmed here (Fig 1). As in [21], we find a possible sister relationship between Discospermum and Xantonnea, although only a single taxon of each was sampled and therefore, this relationship needs further investigation. According to our results (Fig 1), species previously included in Argocoffeopsis and Calycosiphonia, in addition to two new taxa, form a well-supported monophyletic group. This clade is here indicated as the Argocoffeopsis clade, which is the generic name with priority.

Generic delimitation of Argocoffeopsis and Calycosiphonia
Within the Argocoffeopsis clade, three well-supported subclades are recovered: (1) Calycosiphonia (excluding C. pentamera due to unavailability of DNA, but transferred to Kupeantha based on morphology), (2) Argocoffeopsis (excluding A. fosimondi and A. spathulata), and (3) Kupeantha, a clade including the above excluded species, in addition to the newly described K. kupensis and K. ebo (Fig 1). These three subclades are supported by numerous morphological characters (Table 1).

Generic delimitation of Kupeantha and its species
The morphological affinities of the new species, K. kupensis and K. ebo, shown in Table 2, appear to lie with Calycosiphonia pentamera, Argocoffeopsis fosimondi and A. spathulata. In all five species, distinctive supra-axillary buds are developed, and the distal internodes of the stem dry dull black, becoming abruptly white and spongy in one of the more proximal internodes. The lobes of the calyculi are minute, and never foliose, except in deviant cases [19]. The calyx tube is reduced to a minute rim shorter than the disc, and lobes are absent. These characters set this group apart from Argocoffeopsis and Calycosiphonia. Although these five species, now included in the new genus Kupeantha, resemble Calycosiphonia in being evergreen small trees or shrubs, with glabrous leaves and axillary, calyculate, 1-flowered inflorescences, they lack the locellate anthers and 7-8-merous flowers found in that genus. Instead, flowers are (5-)6-merous, with non-locellate anthers. Superficially, they seem unrelated to Argocoffeopsis since they appear not to bear flowers on short leafy spur shoots.
Kupeantha kupensis differs from all other species of Kupeantha in having ellipsoid-rostrate fruits (the other species lack rostrate fruits). The long (12-17 mm) corolla lobes are only exceeded by those of Kupeantha fosimondi (16-20 mm) and these two species share characters not seen in Kupeantha pentamera and Kupeantha spathulata, such as large anthers 9-10 mm long (instead of anthers c. 2 mm long), and a submontane habitat (instead of a lowland habitat). The affinities of Kupeantha ebo are more difficult to ascribe since flowers were lacking. In shape and size of leaves and fruits it resembles most closely K. pentamera, but differs by lacking the finely reticulate nervation of that species (the quaternary nerves are inconspicuous), and by having 6-9 secondary nerves (not 10-13) on each side of the midrib. Keys to the genera, species descriptions, and nomenclatural changes are provided in the taxonomic treatment below.

Phytochemical analysis
Hydroxycinnamic acid derivatives are known constituents of the seeds of Coffea species, which are used to prepare the beverage coffee [49,50], and were also detected in all of the extracts of Kupeantha kupensis and Kupeantha ebo analysed in the present study. However, whilst the purine alkaloid caffeine is widely documented to occur in Coffea seeds, it was not detected in any of the seeds analysed in this study, nor in other previously studied genera in the Coffeeae [32]. A compound assigned as villanovane I (or isomer) was detected in both Kupeantha ebo and K. kupensis, and is a diterpenoid glucoside previously reported to occur in Coffea species [27]. Additionally, compounds assigned as tricalysioside B or C, and as tricalysioside P (or their isomers) were detected. These ent-kaurane glycosides have been reported to occur in species previously placed in the genus Tricalysia and now included in Diplospora [27]. Other compounds assigned as ent-kaurane diterpenoids or their glycosides were only detected in Kupeantha kupensis. These included compounds assigned as tricalysiolide A, tricalysiolide B or G (or tricalysin D), and tricalysioside E (or their isomers). These ent-kaurane compounds have also been reported to occur in species now placed in Diplospora [27], as indicated in Table 3. In summary, the detection of hydroxycinnamic acid derivatives, amino acids and ent-kaurane diterpenoids, or their isomers, which also occur in Diplospora species, are reported here for the first time in seeds of species in the Argocoffeopsis clade.

Taxonomic treatment
A key to the genera in the Argocoffeopsis clade is provided in below.  Differs from Calycosiphonia Robbr. and Argocoffeopsis Lebrun in the conspicuous, spikelike, supra-axillary buds, which are raised 1-4 mm above the stipule sheaths (not axillary, concealed within the stipule sheaths); epidermis of leafy stems drying black in the distal (1-)2(-4) internodes, becoming white and spongy in proximal internodes (not pale brown or grey, or, if black, only for a few mm from the most distal internode); calyx tube a minute indistinct rim above the hypanthium, shorter than the disc; calyx lobes absent (not with a distinct tube longer than disc with minute lobes or with a well-developed tube, half as long or more than ovary); the calyculi with foliar lobes minute, c. 0.2 mm long (not with foliar lobes 3.5 mm or more long, or with calyculi not formed).

Key to the species of Kupeantha
Distribution and habitat: Kupeantha ebo is endemic to the Ebo Proposed National Park in the Littoral Region in Cameroon. The species is found in submontane, evergreen forest, on well-drained sandy to loamy and rocky soil from 770 to 832 m in elevation.  [51,52]. The forest, where this new species was found, was a logging and farming area and it currently seems to be slowly regenerating, since these activities stopped some years ago, although there are still gaps of secondary vegetation among the primary forest. Considering the Ebo Forest does not have a protected status, the survival of this species will depend on the collaboration between locals and the scientific community. There are threats from oil palm plantations and iron ore mining, in addition to the resumption of slash and burn small-holder agriculture. Given that several botanists have spent cumulatively many months over several years collecting specimens at Ebo, and that only two specimens of this species have resulted, it is clear that K. ebo is genuinely infrequent and rare. Accordingly, K. ebo is here assessed as Critically Endangered CR B2ab(iii) [45].

Kupeantha kupensis
Distribution and habitat: Kupeantha kupensis is restricted to the SW Region in Cameroon (Fig 4). This new species is known only from Mt Kupe. The area from which K. kupensis is known supports a submontane (cloud) forest with Santiria trimera (Oliv.) Aubrév. . This evergreen forest has a closed-canopy with many epiphytes and a rich herb layer, classified as Biafran evergreen forests [53], rich in Fabaceae-Caesalpinoideae. The Kupe area is now well known as an area of high endemism in Cameroon [6]. Elevation 1100-1600(-2000?) m.
Phenology: Flowering in February, November; fruiting in January, June, July, October and November.
Etymology: Named for Mt Kupe, the only known location for the species. The survival of this species is in the hands of the Bakossi people and others who live around the mountain.
Conservation status: This assessment maintains that of Cheek [5: 260]. Kupeantha kupensis is only known from two sites at a single location. Many thousands of specimens have been collected at Mt Kupe [6] so the area cannot be considered undercollected. Although the Mt Kupe Integrated Ecological reserve has been created in the last 10 years, both sites for Kupeantha kupensis fall outside its boundary. One site is under threat of forest clearance (the type collection was made where a coffee plantation had been created inside the forest). Kupeantha kupensis is here assessed as Critically Endangered CR B2ab(iii) [45]. B2 indicates that the total area of occupancy is less than 10 km 2 (in fact the area of occupancy is 8 km 2 using the IUCN preferred 4 km 2 cells for each of the two sites known at this location), while b(iii) indicates "continuing decline inferred in area, extent and quality of habitat". Further exploration is needed to find more locations for this tree, if they exist, however, the western slopes of Mt Kupe have been relatively intensively surveyed for plants. We hope that the people of Mt Kupe will seek to protect this beautiful species from extinction. Note: Kupeantha kupensis is the latest of many narrowly endemic species recently described from Mt Kupe. Other examples are Diospyros kupensis Gosline [54], Memecylon kupeanum R. D.Stone, Ghogue & Cheek [55], Dracaena kupensis Mwachala, Cheek, Eb.Fisch. & Muasya [56] and Psychotria ngollengollei Cheek [57].