New taxonomic and conservation status of Ossiculum (Vandeae, Orchidaceae), a highly threatened and narrow-endemic angraecoid orchid from Central Africa

Abstract 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.


Introduction
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.

Material and methods
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 represent-ing 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. -Droissart et al. (2018a) included Ossiculum aurantiacum in their molecular study of African angraecoid orchids and showed that, in both parsimony and Bayesian analyses, O. aurantiacum was sister to the two species of Calyptrochilum. This relationship is supported with strong bootstrap (BS 100) and posterior probability (PP 1), while the sister relationship of the two Calyptrochilum species is supported moderately and only by the Bayesian analysis (PP 0.77). Given the morphological and cytological similarities between Ossiculum and Calyptrochilum (see above) and the molecular data, we consider the monotypic genus Ossiculum as a junior synonym of Calyptrochilum and propose the following amended generic diagnosis.  Distribution. Cameroon and the Republic of the Congo (Figure 1). This species is reported here for the first time in the Republic of the Congo. Its presence in a forest with Gilbertiodendron dewevrei suggests that Calyptrochilum aurantiacum might be more widely distributed, as this type of monodominant forest extends from Nigeria to the Democratic Republic of the Congo and northern Angola. Ecology and habitat. Calyptrochilum aurantiacum occurs between 240 to 600 m elevation in mature dense lowland forest (sometimes degraded), in plantations and in Gilbertiodendron dewevrei monodominant forest. It was initially collected growing as an epiphyte 35 Figure 2. Habitat degradation and forest clearance at the type locality of Calyptrochilum aurantiacum (Mungo River Forest Reserve). In less than 10 years, hundreds of hectares of mature lowland rainforest have been converted to cocoa plantations along the Mungo River (pers. comm. by local communities). A Freshly cleared forest where plantain and bananas will be cultivated during two years prior to being replaced by cocoa trees. B A ten-year old cocoa plantation on the banks of the Mungo River; only a few shade trees remain from the original forest. C Plantain and banana bunches that are harvested every day along the Mungo River. Photographs by V. Droissart (June 2017). m above the ground on the lower branches of a primary forest tree (van der Laan and Cribb 1986). In 2017, we collected the species near the type locality on Desbordesia glaucescens (Irvingiaceae) and Scottellia sp. (Achariaceae) that had recently been felled to establish a new cocoa and banana plantation (Figure 2). In the same locality, the species was also collected on kola and cocoa trees in a 7-year old cocoa plantation. Within and around the Banyang-Mbo Wildlife Sanctuary, it was collected in the canopy of large trees (i.e. Duguetia staudtii (Engl. & Diels) Chatrou (Annonaceae) and Klainedoxa gabonensis (Pierre ex Engl.) (Irvingiaceae)).

Simo
Calyptrochilum aurantiacum is a heliophile epiphyte growing on branches of 5 to 20 cm in diameter under the canopy of large trees (from 50 to more than 100 cm diameter at breast high). In most observed trees (ten observations), Calyptrochilum aurantiacum was found growing together with the angraecoid orchid Diaphananthe plehniana (Schltr.) Schltr. and with the fern Microgramma owariensis (Desv.) Alston (Polypodiaceae) (Figure 3). On Duguetia staudtii and Desbordesia glaucescens, we observed more than 100 individuals per tree. The flowering and fruiting of C. aurantiacum occur during the large rainy season, from May to November.

Conservation
The extent of occurrence (EOO) of Calyptrochilum aurantiacum is estimated to be 19948.1 km 2 , which falls within the limits for "Vulnerable" (VU) category under criterion B1, whereas its area of occupancy (AOO) is estimated to be 28 km 2 , which falls within the limits for "Endangered" (EN) category under criterion B2. Calyptrochilum aurantiacum is an epiphyte of lowland evergreen rainforest. The species occurs within two officially protected areas: the Banyang-Mbo Wildlife Sanctuary in Cameroon and the Odzala National Park in Republic of the Congo. These two protected areas appear well managed. In other sites where C. aurantiacum occurs in Cameroon (within the Mungo River Forest Reserve and outside of the Banyang-Mbo Wildlife Sanctuary), its habitat is currently experiencing a great deal of human pressure (i.e. lowland forest is currently under threat from logging, farming practices, establishment of crop plantations, population growth and human migration, leading to urban expansion). From our recent observations made in June 2017 (Figure 2), the Mungo River Forest Reserve in Cameroon can no longer be considered as an effective protected area. Indeed, the recent construction of a road along the border of the Mungo River Forest Reserve provides greater accessibility, resulting in intensification of forest clearance inside and outside of the reserve's boundary. The main threats to the species are timber exploitation in the north of the Banyang-Mbo Wildlife Sanctuary, cocoa, plantain and banana plantations in the Mungo River Forest Reserve and shifting agriculture and oil palm plantation on the eastern part of the Banyang-Mbo Wildlife Sanctuary. These activities are gradually transforming the species habitat and we think that this degradation will continue in the future.
There are 20 herbarium specimens of Calyptrochilum aurantiacum collected in six localities that represent three subpopulations: two in Cameroon (the Mungo River Forest Reserve and the Banyang-Mbo Wildlife Sanctuary) and the third in the Republic of the Congo. These three subpopulations represent five locations (sensu IUCN 2017), which fall within the limits for the IUCN category "Endangered" (EN) under criterion B2. The projected ongoing loss of its habitat leads us to predict a continuous decline in mature individuals of the species. Thus, C. aurantiacum is assigned an IUCN conservation status of EN B2ab(iii,v).

Discussion
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 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. 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.

Conclusion
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.