Taxonomic revision of the African genus Greenwayodendron (Annonaceae)

Abstract Greenwayodendron (Annonaceae) is a tropical African genus of trees occurring mainly in rain forests. Until recently, Greenwayodendron contained only two species: Greenwayodendronoliveri from West Africa and Greenwayodendronsuaveolens from Central and East Africa. Genetic data, using chloroplast haplotypes and nuclear microsatellites as well as morphometric analyses, provided important information on the delineation of species. Greenwayodendron now contains six species, including two new species (Greenwayodendronglabrum Lissambou, Hardy & Couvreur, sp. nov. and Greenwayodendronlittorale Lissambou, Dauby & Couvreur, sp. nov.). Greenwayodendronsuaveolensvar.gabonica and Greenwayodendronsuaveolenssubsp.usambaricum are recognised as distinct species: Greenwayodendrongabonicumcomb. nov. and Greenwayodendronusambaricumcomb. nov., respectively. A key, detailed descriptions of morphology and geographic distributions, as well as notes on their ecology and uses are presented for all species. Preliminary conservation assessments following IUCN criteria are also provided. Two species are preliminarily identified as threatened, one as Endangered and one as Vulnerable.

Greenwayodendron contains two species (Table 1), namely G. oliveri (Engl) Verdc. in West Africa and G. suaveolens (Engl. & Diels) Verdc. in Central Africa (Le Thomas 1969;Verdcourt 1969). Greenwayodendron suaveolens was further divided into two subspecies (subsp. usambaricum Verdc. and subsp. suaveolens Verdc.), with the latter subspecies containing two varieties (var. gabonica and var. suaveolens). However, population genetic analyses of short plastid DNA sequences revealed that individuals of var. gabonica represent a genetically distinct lineage from var. suaveolens (Dauby et al. 2010). This genetic differentiation between the two varieties was further confirmed based on the analyses of nuclear microsatellites (Piñeiro et al. 2017;Lissambou et al. in prep.). A species delimitation study within Greenwayodendron, based on eight nuclear microsatellites (Piñeiro et al. 2016) coupled with a detailed morphometric dataset of 33 vegetative, floral and fruit traits, confirmed the existence of at least four potentially different species within the G. suaveolens complex (Lissambou et al. in prep.). Indeed, besides confirming the strong genetic differentiation between the gabonica and suaveolens varieties, the latter study identified two new taxa. The subspecies usambaricum, although geographically isolated from Central African species, is differentiated from suaveolens by vegetative characters, essentially determined by the number of lateral veins (≤ 12 veins in suaveolens and ≥ 14 in usambaricum. In addition, a molecular phylogeny of the genus based on numerous nuclear markers (unpublished data), confirmed that G. suaveolens subsp. usambaricum is phylogenetically distinct from G. suaveolens subsp. suaveolens. Most of these discriminant traits had already been recognised by several authors (Le Thomas 1969;Verdcourt 1969;Dauby et al. 2010;Piñeiro et al. 2016) and argue that the taxon G. suaveolens subsp. suaveolens var. gabonica and G. suaveolens subsp. suaveolens var. suaveolens can be elevated to the rank of species, as already proposed by Dauby et al. (2010).
These new insights into the genetic and morphological diversity of Greenwayodendron spp. warrant the need for a taxonomic revision of the genus. Here, we formally describe two new species and undertake two new combinations.
phylogenetic studies (Chatrou et al. 2012) validated Greenwayodendron as a phylogenetically and morphologically distinct genus from Polyalthia s.l. Finally, Polyalthia s.l. is a mainly Southeast Asian genus (but see these articles for recent changes: Mols et al. 2004;Chatrou et al. 2012;Xue et al. 2014).

Material and methods
Around 500 specimens of Greenwayodendron present in the following herbaria BRLU, BM, BR, COI, K, LBV, P, WAG and YA (abbreviations follows Holmgren et al. (1990)) were examined for this study. The online resources JSTOR Global Plants (http://plants.jstor.org), IPNI (http://www.ipni.org), Tropicos (http://www.tropicos. org), the Museum of Natural History, Paris (http://www.mnhn.fr) and the Herbarium at the University of Coimbra (http://coicatalogue.uc.pt) were consulted for the study of type specimens. Species descriptions are based on living and herbarium specimens. The open source software QGis was used to generate the different taxon distribution maps. The Latin name Greenwayodendron is neutral (derived from the Greek word "dendron" meaning tree). New epithets names and combinations proposed here are thus concordant with the neutral gender.
Preliminary conservation assessments of each taxon were assessed using the IUCN Red List Categories and Criteria (IUCN 2012) using criterion B based on the distribution of each species inferred from georeferenced herbarium specimens (Schatz 2002). The extent of occurrence (EOO) and the area of occupancy (AOO) were estimated using the ConR R package ). The minimum AOO was estimated based on a user defined grid cell of 4 km 2 , as recommended by IUCN (2017). Each collecting locality was regarded as a separate subpopulation. The number of 'locations' (as defined by IUCN 2017) was calculated with regard to the type of threats, such that a single 'location' may encompass more than one adjacent subpopulation. Vernacular names are mainly taken from herbarium specimen label information and from Raponda-Walker and Sillans (1961) and Aubréville (1936) for species found in Gabon.

Morphology of Greenwayodendron
Habit. The different species of Greenwayodendron vary from large trees up to 45 metres tall (G. suaveolens) to small trees, no higher than 5 m (G. littorale). The trunks of Greenwayodendron are generally straight and cylindrical with a grey bark covered in large white/grey spots. The slash is bright orange with a distinct black ring under the bark. This black ring is also found in some other African species of Annonaceae such as Anonidium mannii Engl. & Diels. In terms of phylotaxis of the main axis, species in Greenwayodendron follow the spiral pattern, which is common to all other members of the tribe Piptostigmateae (Johnson 2003).
Branches. In terms of pubescence, young branches vary from tomentose (i.e. dense brown pubescence) in G. gabonicum to sparsely pubescent to glabrous in the other four species. The pubescence tends to disappear with age and branches are generally sparsely pubescent to glabrous when older. However, in G. gabonicum, the pubescence persists even in old branches.
Leaves. The leaves of Greenwayodendron follow the typical Annonaceae characters: they are estipulate, simple, entire, distichous and alternate. The petiole of all species can be tomentose (G. gabonicum) to glabrous (G. glabrum). The leaf lamina varies from elliptic to narrowly elliptic in shape, while the base is rounded or cuneate. The apex of the leaves can be very variable even within species. For example, in G. glabrum, the apex varies from acuminate to emarginated (see for example Fig. 3D-G). Leaf size varies from 4.2-7.8 cm in G. littorale up to 10.0-26.3 cm long in G. gabonicum. In most species (G. glabrum, G. littorale and G. oliveri), the leaf lamina is glabrous on the upper side and sparsely pubescent on the lower side. Only G. gabonicum is characterised by a densely pubescent leaf upper side.
Midrib. As for most African genera Le Thomas 1969), the midrib is impressed on the upper side. The midrib varies from densely pubescent on both sides (G. gabonicum) to glabrous on both sides (G. glabrum). Secondary veins are brochidodromous or loop-forming (in contrast to other common Annonaceae venation type eucamptodromous) with prominent intermarginal veins and secondary veins that are straight then arcuate towards the margin (Klucking 1986;Scharaschkin and Doyle 2006). Tertiary veins are reticulate (forming a network) in all species, in contrast to other genera of the tribe (Couvreur et al. 2015) that are either percurrent (Brieya, Piptostigma, Polycertocarpus) (parallel) or intermediate between reticulate and percurrent (Annickia and Mwasumbia).
Inflorescence. The inflorescences in Annonaceae are characterised by a terminal flower with more lateral branched partial inflorescences (Weberling and Hoppe 1996). The inflorescence of the genus Greenwayodendron is terminal (leaf opposed) and generally extra-axillary on old branches. The majority of genera in Piptostigmateae, except Annickia Setten & Maas, have axillary inflorescences Couvreur et al. 2015). Inflorescences are reduced to a compact rhipidium which contains 1-4 flowers. Very often, there is only one flower that develops. In general, the pedicels of all Greenwayodendron species are bibracteate, meaning they have two bracts. This conforms to the type 2 of Fries (1955) and is common to most Annonaceae species. The bract, just under the calyx, is referred to as the upper bract and the lower bract corresponds to the bract located in the lower half of the pedicel.
Flowers. Flowers in Greenwayodendron are actinomorphic, cyclic and trimerous, with 9 tepals. The external whorl of three tepals is typically referred to as sepals, whereas the two inner whorls of three petals each are termed outer and inner petals. Greenwayodendron species are androdioecious, with male and hermaphrodite individuals. Within Piptostigmateae, only two other genera, Sirdavidia Couvreur & Sauquet and Polycerato-carpus Engl. & Diels are also androdiecious, the rest of the genera being bisexual Couvreur et al. 2015). For G. glabrum and G. littorale, we did not observe staminate flowers whereas for G. gabonicum only male flowers have been seen to date. It is not excluded that the former two species are in fact truly bisexual, but more data needs to be gathered before we can conclude on their sexual systems. Indeed, some African genera of Annonaceae are generally androdioecious but also have functionally bisexual or even monoecious species (Verdcourt 1986;Couvreur and Luke 2010). In contrast to some other African genera such as Uvariopsis Engl. (Le Thomas 1969), there is no apparent sexual dimorphism between staminate and hermaphrodite flowers, both being similar in their general aspect such as pedicel length or tepal shape and size. The sepals are small, never longer than 5 mm. The two whorls of petals, are sub-equal in length and similar in shape. We thus did not differentiate them in the descriptions. In vivo, the petals spread out horizontally and recurve slightly downwards towards the apex. Petals range in size from 22-24 mm long in G. gabonicum to 11.5-12.5 mm long in G. littorale.
In staminate flowers, the stamens range from 16-33 and are packed into several whorls whereas in the bisexual flowers the stamens are fewer in number (4-15) and form a single whorl around the carpels. The connective shield, i.e. the apical extension of the connective between both thecae, is generally tongue shaped and also termed umbonate (Maas et al. 2003) but is flattened and discoid or lobulated in G. oliveri. Connective shape has also been shown to be an important taxonomic character in other genera such as Duguetia A.St.-Hil. (Maas et al. 2003) or Uvariastrum Engl. & Diels (Couvreur 2014). The carpels are free (apocarpic) as for most Annonaceae genera (van Heusden 1992) and vary from 8-20 in number.
Fruits. Fruits in Greenwayodendron are composed of several shortly stipitate monocarps. The stipes and the fruit pedicels are sparsely pubescent to glabrous and generally the same colour as the monocarp. The monocarps are broadly ellipsoid to globular, sparsely pubescent to glabrous and green to dark purple at maturity. ellipsoid to globular, sparsely pubescent to glabrous and green to dark purple at maturity.
Seeds. The seeds of Greenwayodendron range from 1-4 per monocarp. They are ellipsoid to globular, flattened when there is more than one seed per monocarp and surrounded by a furrow. The surface of the seed is covered with a rough white integument and the raphe is always impressed.

Greenwayodendron gabonicum (Le Thomas) Lissambou
Distribution. Mainly occurring in Gabon and one collection from the Republic of Congo; 10-500 m (Fig 2).
Habitat and ecology. In primary and secondary forests, also occurring in forestsavannah mosaics (Lope and Wonga Wongué).
Phenology. In Gabon, G. gabonicum flowers from January to March, immature fruits May to October and mature fruits November to December (Amman Bush; Personal communication), also based on herbaria.
Preliminary conservation status of IUCN. Least Concern [LC]. The extent of occurrence (EOO) of Greenwayodendron gabonicum is estimated to be over 106,375.19 km 2 , whereas its area of occupancy (AOO) is estimated to be 128 km 2 (which falls within the limits for Vulnerable status under criterion B2). The species, recorded from Gabon and the Republic of Congo, is now known from at least 35 specimens representing 22 subpopulations. These 22 subpopulations represent 20 different locations (sensu IUCN 2012), many more than 10 locations, which are the upper limit for Vulnerable status under the subcriterion 'a'. Greenwayodendron gabonicum has been collected in 5 protected areas in Gabon (National Park: Moukalaba-Doudou, Lopé, Ivindo, Waka) and Wonga Wongue Reserve and from unprotected areas. This taxon is relatively low in abundance except in two localities (Wonga Wongue Reserve and Lopé Park) where the relative abundance is high. The main threat to G. gabonicum is its habitat destruction resulting from logging activities, especially in the coastal part of Gabon. Notwithstanding these human activities, with varying levels of impact, the species appears not as threatened as it is an abundant and quite widespread species. The available information suggests that the number of subpopulations and mature individuals of G. gabonicum, as well as its EOO and AOO, will not decrease noticeably in the near future.

Uses. Unknown.
Notes. Greenwayodendron gabonicum is easily differentiated from all other species of the genus by its dense tomentose pubescence mainly along the petiole and midrib. In addition, G. gabonicum is the only species to have a densely pubescent or sparsely pubescent upper leaf lamina and has the longest leaves, petals and monocarps of the genus. This species was initially described as a variety of G. suaveolens. However, phylogeographics (Dauby et al. 2010), genetic data (Piñeiro et al. 2017;Lissambou et al. in prep.) and morphological characters (leaf size, pubescent, flower and fruit size) clearly support the hypothesis that this entity represents a distinct species altogether leading us to make this new combination.
Since there were two specimens of Le Testu 7936 in Paris, we chose the specimen barcoded P00363322 as the holotype of G. gabonicum. Specimen P0036331 is thus an isotype.
Habitat and ecology. A sub-canopy tree, in dense moist evergreen mature and secondary forests.
Phenology. Flowering and fruiting are not well known. A flowering sample was collected in February and several fruiting samples were collected in November in Gabon and in February in Cameroon.
Vernacular names. Unknown. Uses. Unknown. Preliminary conservation status of IUCN. Least Concern [LC]. The extent of occurrence (EOO) of Greenwayodendron glabrum is estimated to be over 78,284.28 km 2 , whereas its area of occupancy (AOO) is estimated to be 108 km 2 (which falls within the limits for Vulnerable status under criterion B2. The species, recorded from Gabon and Cameroon, is now known from at least 31 specimens representing 22 subpopulations. These 20 subpopulations represent 20 different locations (sensu IUCN 2012), many more than 10 locations, which are the upper limit for Vulnerable status under subcriterion 'a'. Greenwayodendron glabrum has been collected in 3 protected areas in Cameroon (Campo-Ma'an National Park), Equatorial Guinea (Monte Alen Nation), Gabon (National Park of Crystal Mountains) and from unprotected areas. This taxon has a low relative abundance except in localities from the region going from the northwest of Gabon to the south of Cameroon where the relative abundance is high. The main threat to G. glabrum is its habitat destruction resulting from logging activities and intensive agriculture in Cameroon. Notwithstanding these human activities, with varying levels of impact, the species appears not as threatened as it is abundant. The available information suggests that the number of subpopulations and mature individuals of G. glabrum, as well as its EOO and AOO, will not decrease noticeably in the next ten years.
Phenology. Flowering and fruiting times are not well known. However, a flowering specimen was collected in March and a fruiting one in September.
Distribution. Restricted to the southern coastal part of Gabon and northern Republic of Congo, 5-50 m (Fig. 6).
Habitat and ecology. Growing on coastal and periodically inundated forests, on sandy soils. Vernacular names. Unknown. Use. Unknown.

Preliminary conservation status of IUCN.
Endangered [EN]. The extent of occurrence (EOO) of Greenwayodendron littorale is estimated to be over 4,506 km 2 and its minimal area of occupancy (AOO) is estimated to be 24 km 2 (within the limits for Endangered status under criterion B2). Greenwayodendron littorale is endemic to western Gabon and the Republic of Congo and develops in the lowland coastal forest where it is a dominant species in the undergrowth. The species is found in protected areas (Loango National Park). It is also known from several unprotected forests subjected to logging and habitat destruction due to human activities. Greenwayodendron littorale is known from eight specimens representing five subpopulations. These 5 subpopulations represent a total of 5 "locations" (sensu IUCN 2012), falling within the limit for Endangered status. We project that the ongoing loss of its habitat will induce a continuous decline in the number of mature individuals. Greenwayodendron littorale is therefore assigned a preliminary status of EN B1 ab(iii,v)+2ab(iii,v).
Habitat and ecology. In moist and semi-deciduous forests. Phenology. In Ivory Coast and Ghana, flowering from February to April. Fruits are immature from May to September and fruits are mature from October to December. Vernacular names. Ivory Coast: Mpahouéfon (Abé), Baouéfou, Sierra Leone. Gatema (Mendé).
Uses. Unknown. Preliminary conservation status of IUCN. Least Concern [LC]. The extent of occurrence (EOO) of Greenwayodendron oliveri is estimated to be over 260,482,084 km 2 , whereas its area of occupancy (AOO) is estimated to be 136 km 2 (which falls within the limits for Endangered status under criterion B2). The species, recorded from five countries (Ivory Coast, Guinea Conakry, Ghana, Liberia and Sierra Leone), is now known from at least 34 specimens representing 26 subpopulations. These 26 subpopulations represent 22 different locations (sensu IUCN 2012), many more than 10 locations, which are the upper limit for Vulnerable status under subcriterion 'a'.
Greenwayodendron oliveri has been collected in 10 protected areas in the following countries (Ivory Coast, Guinea Conakry, Ghana, Liberia and Sierra Leone). The main threat to G. oliveri is its habitat destruction resulting from logging activities and intensive agriculture in West Africa. Notwithstanding these human activities, with varying levels of impact, the species appears not as threatened as it is abundant. The available information suggests that the number of subpopulations and mature individuals of G. oliveri, as well as its EOO and AOO, will not decrease noticeably in the next ten years.
Habitat and ecology. Moist evergreen and semi-deciduous lowland and mid-altitude 30-1600 m in forests.
Phenology. In Gabon and southwest Cameroon, flowering from January to April. Fruits are mature from November to December. Uses. The wood of Greenwayodendron suaveolens is used for carpentry and construction of habitats, but also for the manufacture of hunting and fishing spears (Le Thomas 1969). Bark decoctions are used as a laxative to facilitate childbirth and as a stimulant for women's fertility in the Republic of Congo (Boutique 1951). In Cameroon and Gabon, bark ash is rubbed in scarifications, on the forehead to treat psychosis and bark paste is applied externally to treat rheumatism, headache, epilepsy, toothache and malaria (Le Thomas 1965, Raponda-Walker andSilians 1961). In the Democratic Republic of Congo, decoctions of the bark are used to calm colic. In Nigeria, the leaf has been recorded as being taken internally for menorrhagia.
Preliminary IUCN conservation status. Least Concern [LC]. The extent of occurrence (EOO) of Greenwayodendron suaveolens is estimated to be over 2,316,419 km 2 , whereas its area of occupancy (AOO) is estimated to be 476 km 2 (which falls within the limits for Vulnerable status under criterion B2). The species, recorded from 10 countries (Nigeria, Cameroon, Republic of Congo, Gabon, Equatorial Guinea (Rio Muni), Central African Republic, Uganda, Sao Tome and Principe, Democratic Republic of Congo and Angola (Cabinda)), is now known from at least 126 specimens representing 88 subpopulations. These 88 subpopulations represent 93 different locations (sensu IUCN 2012), many more than 10 locations, which are the upper limit for Vulnerable status under subcriterion 'a'. Greenwayodendron suaveolens has been collected in 13 protected areas in the following countries: Nigeria, Cameroon, Republic of Congo, Gabon, Equatorial Guinea (Rio Muni), Central African Republic, Uganda, São Tomé-and-Prìncipe, Democratic Republic of the Congo, Angola (Cabinda). The main threat to G. suaveolens is its habitat destruction resulting from urbanisation in Central Africa as well as intensive agriculture and mining in the Democratic Republic of Congo. Notwithstanding these human activities, with varying levels of impact, the species appears not as threatened as it is abundant. The available information suggests that the number of subpopulations and mature individuals of G. suaveolens, as well as its EOO and AOO, will not decrease noticeably in the next ten years.
Notes. In Gabon, G. suaveolens and G. gabonicum occupy the same habitat (mature tierra firme forest) and can occur in sympatry. However, G. gabonicum has a clearly tomentose pubescence of the lower face of the lamina which is sparsely pubescent in G. suaveolens.
In the original description of P.? acumianata, Oliver cited two syntypes (Mann 841 and Thomson 109). However, both represent different species (Verdcourt 1969). The former is the type species of G. oliveri and the latter a specimen of G. suaveolens.
We lectotypified the type specimen of G. suaveolens, as the specimen H. Soyaux 218 from Berlin is thought to have been destroyed. In describing Polyalthia mortehani, De Wildeman (De Wildeman, 1914) did not designate a type specimen. The observation of the two specimens present in BR (Mortehan 362; De Giorgi 1576), led us to select the sample De Giorgio 1576 as the lectotype because it contains the best flowers' information.
In their description of Polyalthia oliveri, Engler and Diels (1901) identified the Zenker G.A. specimens from Cameroon as P. oliveri. In the Flore Forestière de la Côte d'Ivoire (1936), Aubréville indicates that these Zenker specimens are not P. oliveri and introduces the name P. aubrevillei (now a synonym of G. suaveolens). He refers to the illustration of Engler and Diels (1901) of P. oliveri as being Polyalthia aubrevillei. He provided a key to separate both species (P. oliveri and P. aubrevillei) with "Etamines à connectif très allongé aigu" [Stamens with very long and acute connective] for P. aubrévillei. Following the code (Art. 38.13, McNeill et al. 2012), this description is valid because it contains a diagnose (the key) and was published prior to 1953 validating the direct reference of a previous description for the specimens belonging to this new name. Based on the specimens cited by Engler and Diels (1901) from Cameroon, we selected Zenker 1306 as the lectotype because it contains numerous opened flowers.
Finally, it must be noted that analyses of morphology and genetic diversity (Lissambou et al. in press) identified a group of specimens collected in São Tomé and Prìncipe as possibly distinct. However, to date, the status of this group of specimens is inconclusive, partly related to the lack of fertile material.
Habitat and ecology. Mature forest in mid-altitude mountain. Phenology. Flowering and fruiting are not well known. Nevertheless, floral buds samples were collected in November and December. Fruit specimens were collected from August to November. Vernacular names. Unknown. Uses. Unknown. Preliminary IUCN conservation status. Vulnerable [VU]. The extent of occurrence (EOO) of Greenwayodendron usambaricum is estimated to be over 2 km 2 (within the 20,000 km 2 upper limit for Endangered status under criterion B1) whereas its minimal area of occupancy (AOO) is estimated to be 36 km 2 which falls within the limits for Endangered status under criterion B2. Greenwayodendron usambaricum is endemic to the Usambara Mountains in Tanzania where it is dominated under canopy. It has been collected in one protected area (Mount Usambara). This species is known from 9 specimens representing 7 subpopulations. These 7 subpopulations represent a total of 7 "locations" (sensu IUCN 2012), exceeding the upper limit for Endangered status, but falling within the limit for Vulnerable status. The main G. usambaricum threat is habitat destruction resulting from logging and intensive agriculture in Tanzania. We project that the ongoing loss of its habitat will induce a strong continuous decline in the number of subpopulations and mature individuals in the next ten years as well as an important decline of its EOO and AOO. Greenwayodendron usambaricum is therefore assigned a preliminary status of VU B1ab(i,ii,iii,iv,v) + B2ab (i,ii,iii,iv,v).
Notes. Initially, individuals from the Usambara Mountains in Tanzania were considered part of G. oliveri. Indeed, Engler and Diels (1901) cited the specimen Scheffler 74 (Usambaras) under Polylathia oliveri. Verdcourt (1969) then described the Usambara specimens as a subspecies of G. suaveolens, recognising little morphological differentiation with G. suaveolens. Here, we consider these individuals as representing a separate species from G. suaveolens. Indeed, besides its disjunct and isolated distribution, G. usambaricum can be distinguished from G. suaveolens by the higher number of secondary veins (≥ 14 versus ≤ 12 for G. suaveolens). Nevertheless, more flowering material is needed to better understand the morphological differences between these two species. These observations are corroborated to a certain extent by population genetic data such as microsatellites (Lissambou et al. in prep.) and especially nuclear phylogenetic analyses of the genus (Couvreur et al. in prep.). In the latter case, G. usambaricum was reconstructed as sister with maximum support to a clade containing G. gabonicum, G. glabrum, G. litorale and G. suaveolens. Specimens examined. Tanzania. Tanga: Bomole, Amani, hill trail ca. halfway to summit in area of natural forest, 5°02'S, 39°10'E, 4 Jun 1996, Johnson, D.M. 1943