In the context of producing a revised phylogenetic Linnean taxonomy of angraecoid orchids, the monotypic and narrow-endemic genus Ossiculum is synonymised with Calyptrochilum. Accordingly, a new combination in Calyptrochilum is proposed for Ossiculum aurantiacum. The morphological and DNA-based evidence for this transfer is discussed. Moreover, Calyptrochilum aurantiacum is here firstly reported outside Cameroon, with a record from the Republic of the Congo. The Red List conservation status of this species is reassessed and it is to be downgraded from “Critically Endangered” (CR) to “Endangered” (EN), following the recent discovery of additional subpopulations in Cameroon.

En vue de produire une classification taxonomique linnéenne des orchidées angraecoïdes, le genre monotypique Ossiculum, longtemps considéré comme endémique du sud-ouest Cameroun, est placé en synonymie de Calyptrochilum. En conséquence, une nouvelle combinaison dans Calyptrochilum est proposée pour Ossiculum aurantiacum. Les données morphologiques et moléculaires qui justifient cette combinaison sont discutées. L’espèce est signalée pour la première fois hors du Cameroun, en République du Congo. Suite à la découverte récente de sous-populations supplémentaires au Cameroun, le statut de conservation de cette espèce est réévalué selon la liste rouge de l'UICN. Bien que toujours menacée, l’espèce Calyptrochilum aurantiacum est transférée de la catégorie “En danger critique” (CR) vers la catégorie “En danger” (EN).

Angraecoid orchids, Calyptrochilum , ex situ conservation, IUCN Red List Categories and Criteria, Mungo River Forest Reserve, Odzala National Park, Ossiculum aurantiacum

While describing the monotypic genus Ossiculum P.J.Cribb & Laan, van der Laan and Cribb (1986) were the first to point out its morphological resemblance to Calyptrochilum Kraenzl, an angraecoid genus comprising two species widespread in tropical Africa. In fact, both genera possess a sigmoid clavate spur and their column bears a somewhat elongate bifid rostellum. Moreover, Ossiculum aurantiacum P.J.Cribb & Laan and Calyptrochilum christyanum (Rchb.f.) Summerh. have a yellow lip and a spur of similar shape. Later, Gasson and Cribb (1986), when describing the leaf anatomy of O. aurantiacum, found that Ossiculum and Calyptrochilum share an array of unique features which sets them apart from other angraecoid orchids: the same epidermal cell arrangements, spiral thickenings on some mesophyll cells, the presence of water storage cells, along with the absence of hypodermal and mesophyll sclereids and that of a palisade layer. By pooling the anatomical evidence with morphological and cytological data, these authors concluded that Ossiculum is most closely related to Calyptrochilum. The study of Arends and van der Laan (1986) on the cytology and morphology of angraecoid orchids recognised four groups based on rostellum length and basic chromosome number. One of these groups (the third one) comprised only Calyptrochilum and Ossiculum (rostellum elongated, x = 17 or 18). Twenty years later, the study of Carlsward et al. (2006) on comparative vegetative anatomy and systematics of 142 angraecoid orchid species also showed that Ossiculum aurantiacum and the two Calyptrochilum species have a very similar leaf anatomy. However, with its brightly yellow-orange flowers, its rostellum lobes resembling pincers and with a bipartite basal callus on the lip, Ossiculum is readily distinguished from the two species of Calyptrochilum, which bear predominantly white and green flowers, straight rostellum lobes and a three-lobed lip lacking a basal callus. In addition, the latter two species have a horizontal to pendent habit (vs an erect habit in Ossiculum) with the leaves more or less flat and arranged in a single plane (vs leaves V-shaped in cross-section).

Ossiculum aurantiacum was first discovered in the Mungo River Forest Reserve, Cameroon in 1980. At that time, the only specimens known were the type and a young seedling, both collected by the Dutch botanist H. J. Beentje. The young seedling flowered in cultivation in November 1983 at the University of Wageningen (The Netherlands) from which a voucher was collected (van der Laan 718). During the next 20 years, O. aurantiacum was not seen again despite the several thousand botanical collections that were made within 50 miles of the type locality (Pollard 2011). Based on that unique collection, the species was assessed as “Critically Endangered” (CR) on the IUCN Red List by Pollard and Darbyshire (2004).

In 2004, a new locality for this species was discovered in the Banyang-Mbo Wildlife Sanctuary, Cameroon. This wildlife sanctuary is widely held to be one of the most biologically important forest complexes in West Central Africa, harbouring 325 documented bird species, 71 amphibians, 63 reptiles and 33 large mammals (Riley and Riley 2005) and several narrow endemic plants (e.g. Cheek 2002; Sonké et al. 2002). Several large-scale anthropogenic disturbances (e.g. oil palm plantations in the west, timber exploitation in the north) have taken place around the sanctuary, menacing its biodiversity, notably the narrow range species which occur within the protected area and its surroundings (Asaha and Deakin 2016). The discovery of this new specimen in an additional location in Cameroon cast away the fears that O. aurantiacum might already be extinct and has fostered new specific surveys and a conservation programme dedicated to this species.

Thanks to intensive fieldwork in Cameroon in 2011 and 2017, we discovered new localities of O. aurantiacum. Newly collected specimens of the species were included into a phylogenetic study of African angraecoid orchids (Simo-Droissart et al. 2018a). In the meantime, examination of dried material deposited in BRLU (herbarium acronym according to Thiers 2018) led one of us (V. Droissart) to discover that the species has been collected in the Republic of the Congo in 1996, but had been misidentified as Cyrtorchis sp. This finding, along with new collections made at the type location in Cameroon in 2017, prompted us to reassess the IUCN conservation status of the species.

Based on these new data, this paper aims to (i) reappraise the taxonomy of Ossiculum aurantiacum and its generic status, (ii) update its distribution in light of the new collections in Cameroon and the first record of this species for the Republic of the Congo and (iii) define its ecology and threats and reassess its IUCN conservation status.

On-site ecology and conservation status of Ossiculum aurantiacum were investigated during three main field campaigns in the Southwest Region of Cameroon (2004, 2011 and 2017). To look for additional collections, herbarium specimens of BR, BRLU, K, WAG and YA (herbaria acronyms according to Thiers 2018) were examined. The geographical distribution of the species was determined from data given on the herbarium sheets. New country records were identified by comparing the species distribution with the information provided by Govaerts et al. (2017). The species distribution map was prepared using ArcMap 10.4.1 (ESRI 2016).

Phylogenetic relationships between African angraecoid orchids, including Ossiculum and Calyptrochilum, were inferred on the basis of DNA sequence data from three regions (ITS, matK, and the trnL-trnF intergenic spacer) and 555 accessions representing 316 species from 43 genera (see Simo-Droissart et al. 2018a). We performed the parsimony and the Bayesian analyses on the combined matrix using, respectively, the computer programme PAUP* v.4.0 a 146 (Swofford 2002) and MrBayes 3.2.5 (Ronquist and Huelsenbeck 2003; Ronquist et al. 2012).

Using the IUCN Red List Categories and Criteria (IUCN 2012), we made a preliminary risk of extinction assessment for the species. We imported georeferenced specimen data into the ConR package (Dauby et al. 2017) to calculate the area of occupancy (AOO) and extent of occurrence (EOO). The cell size for AOO was set 2 × 2 km as recommended by IUCN (2017). We calculated the number of ‘locations’ (as defined by IUCN 2017) with regard to the kind of threats, such that a single ‘location’ may encompass more than one adjacent population.

The integration of molecular evidence into current taxonomical practice and the adoption of a strictly phylogenetic Linnean framework in orchid classification have been altering many generic boundaries (Chase et al. 2015). For instance, two other African angraecoid genera, Podangis Schltr. and Sphyrarhynchus Mansf. considered monotypic, were recently recircumscribed and now comprise, respectively, two (Cribb and Carlsward 2012) and three species (Martos et al. 2017). Our recent molecular phylogeny on African angraecoid orchids (Simo-Droissart et al. 2018a) substantially improved the understanding of taxonomic relationships among the angraecoid orchids, leading us to describe a new genus to accomodate continental African species of Angraecum Bory sect. Pectinaria (Benth.) Schltr. (Simo-Droissart et al. 2018b) and to transfer Ossiculum aurantiacum to Calyptrochilum.

Our recent observations on the rapid degradation of the natural habitat of Calyptrochilum aurantiacum within the Mungo River Forest Reserve and around the Banyang-Mbo Wildlife Sanctuary both in Cameroon, stress the need for urgent actions to protect the last remaining patch of intact lowland forests from human activities. The survival of several other plant species endemic to this area, such as Afrothismia winkleri (Engl.) Schltr. (Burmanniaceae, IUCN status = CR, IUCN SSC East African Plants Red List Authority 2013), Cola metallica Cheek (Malvaceae, IUCN status = CR, Cheek 2003), Tricalysia lejolyana Sonké & Cheek (Rubiaceae, IUCN status = EN, Sonké et al. 2004), also relies on the conservation of these forests occurring below 600 m elevation.

Due to the ongoing destruction of its natural habitat, Calyptrochilum aurantiacum has been the focus of an ex situ conservation project. The species has been cultivated since 2004 in our ex situ collection in Yaoundé, Cameroon. Living specimens, collected from fallen branches and trees in the field, have been successfully grown and they have flowered for several consecutive years. Presently, about 150 living individuals are being cultivated in the shadehouse. Since November 2015, we have performed eight hand pollination experiments on C. aurantiacum in the shadehouse and the species has been introduced in our seed bank in Yaoundé (Figure 4). However, due to the small size of its flowers, manual pollination of C. aurantiacum is still challenging as the fruit set (resulting from hand self-pollination) remains low (~25%). On-site studies on reproductive biology of the species are thus needed to identify pollination mechanisms involved and to check if the pollinator(s) is/are also threatened by forest clearance.

The patchy distribution of Calyptrochilum aurantiacum seems surprising and suggests that additional surveys should be made to search for new populations. The discovery of the satellite population in the Republic of the Congo also raised the question whether the Cameroonian and Congolese populations of C. aurantiacum, separated by more than 650 km, have different species of pollinators. Seemingly, the intriguing pollinator may be absent in Yaoundé since we have never observed “natural” pollination on the living plants in cultivation.

Figure 4. 

Seed banking of Calyptrochilum aurantiacum in Yaoundé (Cameroon). In 2017, an ex situ conservation programme was initiated to support the long term preservation of C. aurantiacum. A Due to the small size of the flowers, manual pollination has been performed under a stereomicroscope. B Fruit development and maturation takes place in a shadehouse established in Yaoundé. C Finally, viable seeds have been harvested and conserved in the freezer (-20°C). Before being preserved at low temperature, seed viability is assessed using the tetrazolium test (red colouration of living embryo). Photographs A and C by V. Droissart, B by Gyslène Kamdem.

Notwithstanding the success of our ex situ conservation programme, strategies to ensure the long-term survival of C. aurantiacum must focus primarily on in situ conservation efforts. During the 2017 survey, we found one individual growing on a cocoa tree within a plantation at Ebonji, Cameroon. Since clearance of forest for conversion to agricultural plantations will continue to increase, we performed in September 2017 the transplantation of 45 individuals on three cocoa trees (15 on each) in Ebonji village, in order to see if the species could maintain viable populations in those plantations. If the transplantation tests prove successful, the next step will be to ensure that the pollinator will also survive in this human-transformed habitat.

Morphological and DNA-based evidence led to the transfer of the narrow-endemic Ossiculum aurantiacum to the widespread genus Calyptrochilum and, thus, recognises the monotypic genus Ossiculum as a junior synonym of Calyptrochilum. Thanks to recent intensive field trips and laboratory work on central African orchids, new localities of Calyptrochilum aurantiacum have been discovered, a situation that contributed to downgrade the IUCN conservation status of the species. Calyptrochilum aurantiacum is currently known from five locations in two countries (Cameroon and Republic of the Congo) and is assessed as Endangered [EN B2ab(iii,v)] according to the IUCN Red List Categories and Criteria. Fieldwork within and around the Odzala National Park is required to evaluate the species habitat and the state of the subpopulation there. In situ studies on reproductive biology would be necessary for greater efficiency in conservation measures.

We express our gratitude to the Mohamed bin Zayed Species Conservation Fund (Grant n°16255698, V. Droissart as PI) and the National Geographic Society (Grant C303-15, V. Droissart as PI) who supported fieldwork and monitoring of the orchid living collection in Cameroon. The U.S. National Science Foundation (Grant 1051547, T. Stévart as PI, G. M. Plunkett as Co-PI) also supported fieldwork and herbarium visits. We are grateful to the curators of the following herbaria BR, BRLU, K, WAG and YA for making their collections available and for facilities kindly provided. The American Orchid Society provided financial support to the work of the first and the last author in Cameroon. We are grateful to the authorities of Higher Teachers’ Training College, University of Yaoundé I, to host the Yaoundé shadehouse and for allowing authors to access their facilities. We would like to thank Marie-Noël Djuikouo, Gyslène Kamdem, Charlemagne Nguembou and Lise Zemagho for collecting vouchers and taking photographs of the species in the Yaoundé shadehouse in Cameroon. Finally, we thank João N. M. Farminhão for commenting and improving the English of the manuscript and the two reviewers for their constructive comments on an earlier version of the manuscript.