Revision and new species of the African genus Mischogyne (Annonaceae)

Mischogyne (Annonaceae, tribe Monodoreae) is a genus of small- to medium-sized tropical trees and shrubs. It is characterised by a combination of: (1) stamens and carpels on a more or less extended torus; (2) carpels divergent from each other at the apex of the torus above the anthers; (3) anthers linear and anther connectives not expanded above the thecae; (4) inflorescences extra-axillary (or sometimes terminal in M. michelioides) with usually solitary flowers; (5) petals reflexed at anthesis (except M. michelioides), and (6) prominent reticulate tertiary veins. The genus is found in the lowland tropical rainforests of Africa with annual precipitation of 1000 – 4000 mm and in the dry coastal foothills of Angola. Five species and one variety of Mischogyne are recognised. One newly discovered species from the Eastern Arc Mountains, M. iddii, is described. The variety M. elliotiana var. glabra is reduced into synonymy with M. elliotiana var. elliotiana. Mischogyne elliotiana var. gabonensis is raised to species level as M. gabonensis. Specimens from the Congo are recognised as a new species, M. congensis. Preliminary conservation status assessments are provided for each species, as well as an identification key and detailed species descriptions. An unusual distribution pattern for the genus is discussed: dry coastal Angola as well as West, Central and East African wet forest.

Mischogyne is a genus of five species characterised by the combination of: (1) stamens and carpels on a more or less extended torus; (2) carpels divergent from each other at the apex of the torus above the anthers; (3) anthers linear and anther connectives not expanded above the thecae; (4) inflorescences extraaxillary (or sometimes terminal in M. michelioides) with usually solitary flowers; (5) petals reflexed at anthesis (except M michelioides), and (6) prominent reticulate tertiary veins.

Taxonomic History
The genus Mischogyne was erected by Exell (1932) to accommodate Mischogyne michelioides Exell, first collected by Gossweiler in 1930. Exell (1932 noted in h i s d e s c r i p t i o n t h e e l o n g a t e a t t e n u a t e "androgynophore" that sets this species apart from any other genus of Annonaceae, and "gives to the flower a distinctly magnoliaceous aspect". He specu-lated on the relationship to the Magnoliaceae, which turned out to be, as he also said, "no more than an extraordinary parallelism." The genus was considered monotypic until 1952 when R. E. Fries circulated a preprint (the formal publication was Fries 1955) in which he proposed that the species Uvariastrum elliotianum (Engl. & Diels) Sprague & Hutch. was better placed in the genus Mischogyne (Fries 1955), now confirmed by molecular markers. Mischogyne elliotiana (Engl. & Diels) Le Thomas has a cylindrical torus, but not nearly as prominent as the long-stalked one of M. michelioides. Fries (1955) noted the similar anthers lacking an extended connective. The two species share the strongly reticulate tertiary venation characteristic of Mischogyne although this was not mentioned by Fries. Turner (2013) noted that Fries' new combination failed to fully reference the place of publication of the basionym as required by the International Code of Nomenclature (Lanjouw 1952) which came into effect in 1953. Turner therefore credited Le Thomas (1969) with the first valid publication of the combination M. elliotianum and we incorporate this attribution. The basionym for the species was Uvaria elliotiana Engl. & Diels (Engler 1901) and the epithet was correctly changed to the masculine by Sprague & Hutchinson (1916) when they transferred the species to Uvariastrum. Unfortunately, Fries (1955) did not change the epithet to the feminine in his transfer to Mischogyne. Evrard (1967) corrected Keay's M. elliotianum var. glabrum to M. elliotianum var. glabra, but without correcting the species epithet. (Note that this change does not change the author according to article 32.2 of the ICN Turland et al. 2018) and that the citation Mischogyne elliotianum var. glabra (Keay) Evrard is incorrect). We make the gender correction here to Mischogyne elliotiana, and to the varietal epithets as well.
At about the same time, Keay (1952) published new taxa in preparation for the publication of the second edition of the Flora of West Tropical Africa. In examining the material of Uvariastrum elliotianum, he described two new varieties in addition to the typical species. He stated that they "have been noted in the MS [for the flora] as new species, but, I consider, are best treated as varieties." These are: (1)  Finally, Le Thomas (1969) published a fourth variety based on a nomen nudum of Pellegrin (1949): Mischogyne elliotianum (Engl. & Diels) R.E.Fries var. gabonensis Pellegr. ex Le Thomas.

Morphology
Measurements were made on dried herbarium specimens and on calibrated specimen images; characters such as shape and colour are based on field notes and photos. Plane shape description terminology is based on Systematics Association Committee for Descriptive Biological Terminology (1962); other terminology, including indumentum, is based on Beentje (2015). All known specimen records of Mischogyne were identified, georeferenced and examined and are listed in the supplement S1 of the paper. Specimens examined are listed in the relevant section of the species accounts ("!" indicates a specimen seen and "*" that a high-resolution image of the specimen was viewed). In addition to herbarium searches at K and BM, specimen records from GBIF and Chatelain et al. (2011) were evaluated and georeferenced. Specimen images were viewed using the portals for BR, L (WAG), LISC and P. Measurements of digital images were made using ImageJ (Schneider et al. 2012). In the case of the Ghana and Ivory Coast herbaria, label information is not available and the identifications and georeferencing are assumed to be correct in plotting species distributions. In all other cases georeferencing based on label data was done using GoogleEarth (https://www.google.com/intl/en_uk/ earth/desktop/) and online searches.
Preliminary conservation assessments for each species follow IUCN criteria (IUCN 2012) and are based on the distribution of herbarium specimens (Schatz 2002). We used the online tool GeoCAT (Bachman et al. 2011) to calculate Extent of Occurrence (EOO) and Area of Occupancy (AOO). AOO is calculated using a cell-size of 4 km 2 . The map was generated with ArcMap 10.1 (ESRI 2011).

Molecular methods
New sequences were generated for three chloroplast regions (rbcL gene, trnH-psbA intergenic spacer (IGS), trnL intron and trnL-trnF IGS) from Mischogyne herbarium specimens representing three accepted species and two putative new species to science. This data was supplemented with previously published chloroplast sequence data (Couvreur et al. 2008a;Parmentier et al. 2013) from 16 species representing 10 related genera. Voucher information is provided in Table 1.
After tissue homogenisation (Mixer Mill, Retsch GmbH & co., Haan, Germany), total DNA was extracted from 5 -20 mg of dried material using a modified cetyl trimethyl ammonium bromide (CTAB) protocol (Doyle & Doyle 1990). The rbcL gene was amplified using the primers 1F and 724R or using the primers 636F (Fay et al. 1997) and 1460R (Fay et al. 1998 the trnH-psbA IGS was amplified using primers trnH (GUG) and psbA (Hamilton 1999) and the universal primers C/D and E/F (Taberlet et al. 1991) were used to amplify the trnL intron and trnL-F spacer.
Initial denaturation was set to 2 min at 94°C [rbcL 1F-724R, trnL-F], to 3 min at 94°C [rbcL 636F-1460R], and to 5 min at 96°C [trnH-psbA]. After this, polymerase chain reaction (PCR) was performed for 30 cycles (28 for rbcL 636F-1460R, 35 for trnH-psbA) of denaturation (1 min  The PCR products were electrophoresed on 1% agarose gels in 1× Tris-borate-EDTA (TBE) buffer (pH 8.0) and stained with ethidium bromide to confirm a single product. Afterwards, the cleaned PCR products were sequenced on a 48-capillary ABI 3730 DNA Analyzer using the same primers as in the PCR reactions. A total of 12 new sequences were generated in this study.
The alignments of the three chloroplast regions were concatenated for the downstream analyses. PartitionFinder 2.1.1 (Lanfear et al. 2012) was used to determine an appropriate data-partitioning scheme from potential partitions that were defined a priori (in this case, each marker was treated as a separate partition plus each codon for the coding marker rbcL), as well as the best-fitting model of molecular evolution for each partition, using the Bayesian Information Criterion. PartitionFinder suggested the following data-partitioning scheme (rbcL codon1, rbcL codon 2 + codon3, trnH-psbA, trnL-trnF).
Bayesian Inference (BI) analyses were conducted in MrBayes 3.2.6 (Ronquist et al. 2012). For the BI analysis, the F81 model of sequence evolution was determined to be the best-fitting model available in MrBayes for the rbcL codon1partition, the HKY+G model for the rbcL codon 2 + codon3 and the trnH-psbA partitions, and the HKY model for the trnL-trnF partition in the concatenated data set. 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 values and burn-in values of the different runs were verified with Tracer 1.5 (Rambaut 2010). The first 25% of the trees from all runs were excluded as burn-in before making a majority-rule consensus of the 7500 posterior distribution trees using the "sumt" function. All phylogenetic analyses were run using the CIPRES portal (http:// www.phylo.org/; Miller et al. 2010). Trees were drawn using FigTree 1.4.3 (Rambaut 2016) and adapted in Adobe Photoshop CS5.

Molecular Study
The relationships between the taxa studied are shown in the majority consensus multiple-locus BI tree with the associated posterior probability (PP) values are shown in Fig. 1. As the data for the outgroup taxa are largely based on previous studies (Couvreur et al. 2008a;Parmentier et al. 2013), the relationships in Monodoreae recovered here largely match those already published (Couvreur et al. 2008a;Chatrou et al. 2012;Guo et al. 2017)). Within Monodoreae, a well-supported clade of Orphypetalum and Sanrafaelia (PP=1) is sister to the rest of the tribe which consists of two main clades. One of these main clades includes two subclades, one (PP=0.82) representing Asteranthe as sister to Hexalobus + Uvariastrum (PP=0.9); and the other (PP=0.99) including Isolona and Monodora. The other main clade (PP=1) also includes two subclades, one (PP=1) representing Uvariodendron as sister to Monocyclanthus and Uvariopsis; and the other consisting of the genus Mischogyne. Mischogyne is well-supported as a monophyletic genus (PP=1). Within the genus, two well-supported clades are recognisable. The first clade (PP=1) includes the taxon Mischogyne elliotiana var. gabonensis (further accepted as Mischogyne gabonensis) as sister to two other varieties of Mischogyne elliotiana (PP=1). The second clade includes Mischogyne michelioides and two species new to science (described below).

Morphological Study
Study of herbarium specimens supports the recognition of five species and one variety described below. We were unable to discover any discontinuous characters discriminating Mischogyne elliotiana var. glabra from specimens of M elliotiana var. elliotiana.

Morphology
Mischogyne are shrubs or small trees 3 -7 m tall or medium-sized trees to 25 m (M. gabonensis, M. orientalis). Trunks are cylindrical or branching from the base (M. michelioides). Phyllotaxis is distichous. As is typical for Annonaceae, the very young leaf buds, twigs and leaves are covered with a soft brown appressed indumentum which is quickly lost. Leaves are simple, entire, and exstipulate. Venation is brochidodromous. The tertiary venation is strongly and prominently reticulate and a good character for recognising the genus. Specimens of M. congensis were originally identified as M. elliotiana var. glabra; despite being in different clades, the morphological differences in the taxa (see recognition below) are in the leaf venation, acumen, and leaf texture (and of course geographical range). Species can be recognised from the leaves: M. congensis and M. gabonensis have at least some leaves with a linear acumen, with M. congensis having much less ascendant nerves. M. elliotiana and M. iddii have triangular acumens or are attenuate, with M. iddii having much larger leaves. M. michelioides is deciduous with broad, usually attenuate leaves and grows in a semi-arid environment.
Inflorescences are extra-axillary and one-flowered with the exception of Mischogyne elliotianum which frequently has terminal flowers, and M. gabonensis where the type collection has some inflorescences of two flowers. The fundamental inflorescence type for Annonaceae is determinate with a terminal flower. "Apart from (few) exceptions, inflorescences (sometimes reduced to a single flower) appear as lateral structures to the observer, rather than terminal. In the majority of genera this is due to overtopping by a lateral shoot originating from the axil of a leaf immediately below the floral region…" (Maas et al. 2007;Weberling & Hoppe 1996). This analysis is confirmed for Mischogyne by the often terminal flowers in M. michelioides and examination of young shoots in specimens of M. elliotianum where a young bud can be seen as terminal with a lateral shoot starting to overtop it. Bracts are generally reduced to a tuft or ring of hairs at the base of the pedicel. Small (c. 2 mm diam.) caducous bracts are sometimes visible on M. michelioides.
Flowers in Mischogyne species are bisexual, trimerous with one whorl of three free sepals (except M. gabonensis) and two subequal alternate whorls of three free petals each. The sepals are generally reduplicate-valvate (van Heusden 1992) in bud, mean- Table 1. List of the samples used in the molecular analysis with species names, voucher information, distribution and GenBank accession numbers for the DNA sequences of the three chloroplast regions (*indicates new accessions); a dash (-) indicates missing data.
ing the margins are curved outwards. Reduplicatevalvate sepals are noted as common in genera with middle-sized to large flower buds (van Heusden 1992).
Within the tribe Monodoreae this character is also found in the genus Uvariastrum Engl. and in Asteranthe Engl. & Diels (Couvreur 2014). Mischogyne gabonensis has a membranous connate apiculate calyx which ruptures into two or more fragments as the petals expand and soon falls (Fig. 2D). Van Heusden (1992) records a rupturing calyx in Fusaea, Letestudoxa, some Uvaria, and one Duguetia and Guatteria. The petals are in two whorls with inner and outer petals subequal, white, and covered with a dense indumentum that is finer on the inside. They are reflexed at anthesis, except in M. michelioides, curving backwards towards the pedicel and forming a cup-shape at the base enclosing the anthers (Figs 4A, 5A and 7A). David Johnson (review of this paper) points out that reflexed petals are unusual in the Annonaceae and the strong resemblance of the flowers to Hornschuchia Nees. Greenwayodendron Verdc. also has slightly reflexed oblong petals. The receptacle is more or less extended to form a torus (sometimes referred to as an androgynophore) with stamens spirally arranged on the lower portion and the ovaries in a cluster at the apex. Since the stamens are borne from the base of the receptacle, this structure does not conform to the traditional definition of "androgynophore" which implies a stalk below the stamens (Beentje 2015). It is densely white pubescent. Mischogyne michelioides has a flower with an elongate flexuous torus up to 3 cm long. The genus Toussaintia Boutique also has an extended cylindrical receptacle (Deroin & Luke 2005), but is in a different tribe (Uvarieae). Deroin (2000) demonstrates that the gynoecium of Toussaintia is anatomically typical of Annonaceae. To our knowledge, within the Annonaceae only Toussaintia, Mischogyne, and Balonga buchholzii Le Thomas (now Uvaria buchholzii Engl. & Diels; Zhou et al. 2010) have been described as having an androgynophore. However, many genera have an extended receptacle (torus) (van Heusden 1992), often ovoid with a constricted base; these include Monocyclanthus, Uvariastrum, Uvariopsis and Uvariodendron within the Monodoreae. The receptacle of Mischogyne thus appears to be exceptional only in the degree of elongation of the torus which is made more apparent by the falling of the anthers following anthesis.
The androecium consists of numerous latrorse linear stamens with a connective that lacks the broadened apical prolongation expanded over the anthers typical in the Annonaceae (van Heusden 1992). The connective extension is either absent (Mishogyne michelioides) or slender and rounded, but never expanded over the anthers and glabrous (M. michelioides) or with a tuft of hairs. In most other species of the tribe the connective extensions are compacted together into a pavement-like surface. The stamens are appressed against the torus The gynoecium consists of 3 -12 (to 40 in Mischogyne michelioides) separate carpels. The carpels are cylindrical to ovoid covered with a white indumentum as the torus. They are divergent from each other except in M. michellioides and extend beyond the petal reflexure. The stigmas are bilobed and sessile except in M. elliotiana where a distinct constriction forms a short style.
Fruits consist of a 1 -3 monocarps (field obs.); in herbarium specimens only a single monocarp has been observed. These are ellipsoid or ellipsoid-ovoid, sometimes constricted at one end, and yellow to green with cream or white spots; 3 -12 seeds per monocarp (Setten et al. 1992).
As with many African groups, nothing is recorded regarding pollination, dispersal, or phytochemistry.

Taxonomy
As previously circumscribed, the genus Mischogyne consisted of one variable species in West and Central Africa and another quite different species in Angola. Resulting from our morphological and molecular phylogenetic study, we add a new East African species (M. iddii, described below), a species in the coastal rainforest of Cameroon and Gabon (M. gabonensis, previously accepted as M. elliotiana var. gabonensis), and a species from the Congo river basin (M. congensis, described below). Specimens of the latter had previously been identified as M. elliotiana var. glabra.
We recognise two varieties of Mischogyne elliotiana (Engl. & Diels) Le Thomas. Mischogyne elliotiana var. glabra Keay was previously recognised on the basis of a lack of indumentum on the branches, but we find the degree of hairiness within the species to vary continuously and reject the variety. On the other hand, the specimens of M. elliotiana var. sericea Keay are so distinct in indumentum and leaf size, shape and venation that we maintain the taxon.

Distribution
The species occur in evergreen or semideciduous equatorial or gallery forest with 1000 -4000 mm precipitation per annum (ppa), apart from the Angolan Mischogyne michelioides Exell which occurs in rocky terrain beneath the coastal escarpment with 650 -1000 mm ppa (Map 1).
Apart from Mischogyne michelioides in Angola, the species distribution of the genus now falls into a familiar pattern for tropical African genera: one rather variable species west of the Cross River ("Upper Guinea", White 1979); several (or one in our case) species in Lower Guinea; an isolated mid-Congo riverine species; and one or more species in East Africa, often in the Eastern Arc mountains. Mischogyne is unusual in also having a species, M. michelioides, in semi-arid coastal Angola. Most Annonaceae species in Angola occur in Cabinda or Lunda provinces in moist Congolian forest. While the other species of Mischogyne are fairly similar to each other, M michelioides has an impressively long receptacle (to 3 cm) and very long stamens quite unlike any other Annonaceae (van Heusden 1992). (Sadly we have been unable to locate a photograph of a live flower). It is described as a shrub branched from the base growing in cracks between boulders. The origin and evolution of Mischogyne michelioides is unclear.
Research on tropical African species distribution and speciation has often emphasised Pleistocene refugia as a driving force (e.g. Duminil et al. 2015).  Couvreur et al. (2008b) demonstrate that the West-East vicariance in other Monodorioid genera occurred in the late Miocene 8 -6 million years BCE (Myr). This separation reflects a world-wide cooling and drying of ecosystems (Herbert et al. 2016). A presumed pan-African rainforest existed in the mid-Miocene Climatic Optimum (17 -15 Myr) and explains the common Guinean -Congolean -East African distribution. Estimated Miocene rainforest extent does not show it extending into Angola (Morley 2007). There are small fragments of wet forest in Angola today which Gossweiler (Gossweiler & Mendonça 1939) refers to as "laurasilva", but these occur to the north of the Mischogyne michelioides distribution. One hypothesis is that wet forest extended along the coast of Angola in the Miocene, and that the population there adapted to drier conditions and possibly a specific pollinator, producing its unusual morphology.

Taxonomic Treatment
Mischogyne Exell (1932); R. E. Fries (1959); Paiva (1966); Le Thomas (1969). Type species: Mischogyne michelioides Exell Trees or shrubs. Trunk straight, sometimes fluted, cylindrical, or shrubs branching from the base (M. michelioides). Very young leaf buds, leaves, and twigs with a soft brown indumentum of appressed hairs, quickly glabrous or glabrescent. Twigs striate, light grey to dark grey to brown. Petioles terete, deeply grooved adaxially, glabrous to densely pubescent. Leaves simple, entire, alternate, petiolate and exstipulate, lamina narrowly elliptic to obovate, chartaceous to coriaceous, often slightly bullate, glabrous to pubescent; dark glossy green above, lighter green below, drying olive green concolorous or paler below; midrib impressed on the upper side, raised on the lower side, glabrescent; secondary venation brochidodromous, prominent above and below; tertiary venation markedly reticulate on both surfaces of the lamina with reticulation net cells 0.6 -1.8 mm wide. Inflorescence extra-axillary or terminal (M. michelioides) on young branches, single flowered (occasionally two flowers in M. gabonensis). Bracts generally reduced to a tuft of hairs or early caducous. Flowers bisexual. Sepals 3, reduplicate-valvate, free, enclosing the receptacle until anthesis ( Fig. 2A -C), or united in a thin membranous capsule rupturing at anthesis (M. gabonensis, Fig. 2D). Petals 6, in two alternating whorls of three each, white, free, subequal, elliptic to ovate, pubescent, reflexing at anthesis except M. michelioides where the petals spread from the base. Receptacle extended as a torus, conical, cylindrical, or elongate in a thin flexible column, with stamens attached to the lower portion and carpels attached at the apex. Stamens numerous, spirally arranged, linear, latrorse, basifixed, appressed at least in lower part against the torus; filament minimal or absent; connective and thecae nearly the length of the anther; connective pubescent or glabrous, without expanded terminal extension, connective apex glabrous or terminated by a tuft of hairs. Sepals, petals and stamens dropping at end of anthesis, leaving the receptacle and carpels exposed. Carpels 3 -12 (to 40 in M. michelioides), cylindrical to ovoid, densely pubescent; style short or absent, stigma bilobed, ovules numerous, lateral, biseriate, placentation parietal. Fruits monocarps 1 -3, ellipsoid, oblong to globose, pubescent to glabrous, smooth or with slight longitudinal ribs, constricted or not around the seeds, shortly stipitate, apiculate. Seeds 3 -12 per monocarp, flattened to transversely ellipsoid, raphe raised to flat, ruminate with 15 -20 membranous semicircular invaginations.
RECOGNITION. Similar to Mischogyne elliotiana in being a small rainforest shrub or tree with leaves less than 20 cm long, but differing in: many leaves having a linear acumen arising from the rounded apex of the leaf and 5 times or more as long as wide rather than apex attenuate or acuminate with a triangular acumen 1 -3 times long as wide; secondary nerves ascendant 10 -25% of the leaf length before anastomosing at a distinct angle to a marginal nerve vs ascendant 25 -30% of the leaf length before anastomosing almost parallel with the margin; the leaves drying dark glossy green above and below rather than matte below; the older petioles glabrous rather than having at least some indumentum (Fig. 3). DISTRIBUTION (Sprague & Hutchinson 1916: 159), (Keay 1952), (Keay et al. 1952); (Hutchinson et al. 1958); (Aubréville 1959); (Keay et al. 1964). Uvaria elliotiana Engl. & Diels in Engler (1901: 28 Shrub or small tree up to 4 -7 (-10) m tall with slender arching habit, trunk cylindrical, up to 12 cm in diam.; old branches glabrous; young branches glabrous to sparsely pubescent rapidly glabrescent, hairs 0.1 -0.3 mm long, appressed, brown. Bark blackish-green, finely rugose, pustulate; slash yellowish-white, fibrous, strongly scented, "smells like green tomatoes" (Nimba Botanic Team 974).

Mischogyne gabonensis (Le
RECOGNITION. Similar to Mischogyne elliotiana in its flowers and fruits. Distinct in being a medium-sized tree to 25 m tall rather than a small tree or shrub 3 -10 m tall; in having 3 -7 carpels rather than 7 -12; and in having sepals united in a capsular membranous calyx rather than three free reduplicate-valvate sepals; in the carpel indumentum velvety appressed with hairs 0.05 -2 mm long, rather than with longer bristly semi-erect hairs 0.8 -1 mm long.  2) The specimen from the south of Cameroon is not in the Campo-Ma'an National Park, but in an area designated as a production forest.