Summary
Afrothismia is a genus of non-photosynthetic mycoheterotrophs from the forests of continental tropical Africa. Multiple phylogenetic inferences using molecular data recover the genus as sister to a clade comprising mycoheterotrophic Thismiaceae and the photosynthetic family Taccaceae, contrary to earlier placements of Afrothismia and Thismiaceae within Burmanniaceae. Morphological support for separating Afrothismia from the rest of Thismiaceae has depended on the zygomorphic flowers of Afrothismia (although some South American species of Thismia are also zygomorphic) and their clusters of root tubers, each with a terminal rootlet. The number of described species of Afrothismia has recently increased substantially, from four to 16, which has provided additional morphological characters that support its distinction from Thismiaceae. Most notably, the ovary in Afrothismia has a single stalked placenta, and circumscissile fruits from which seeds are exserted by placental elevation (in Thismiaceae, in contrast, there are three placentas, a deliquescing fruit lid, and the seeds are not exserted). Afrothismia stamens are inserted in the lower perianth tube where they are attached to the stigma, and individual flowers are subtended by a single large dorsal bract (in Thismiaceae, stamens are inserted at the mouth of the tube, free of and distant from the stigma, and each flower is subtended by a loose whorl of (2-)3(−4) bracts). Here we formally characterise Afrothismiaceae and review what is known of its development, seed germination, interactions with mycorrhizal Glomeromycota, biogeography, phylogeny and pollination biology. All but one (Afrothismia insignis; Vulnerable) of the 13 species assessed on the IUCN Red List of Threatened Species are either Endangered or Critically Endangered; one species (A. pachyantha Schltr.) is considered to be extinct.
Introduction
The non-photosynthetic genus Afrothismia Schltr. (Schlechter 1906) is currently placed in the achlorophyllous tribe Thismieae (Burmanniaceae), together with Thismia Griff. (Griffith 1845), Haplothismia Airy Shaw (Airy Shaw 1952), Oxygyne Schltr. (Schlechter 1906), and the more recently described Tiputinia P.E. Berry & C.L. Woodw. (Woodward et al. 2007). Based on early molecular phylogenetic analyses that included plastid rbcL, atpB and nuclear 18S rDNA genes (Caddick et al., 2000a; 2000b), APGII (2003) and subsequent classifications combined Burmanniaceae sensu stricto with former Thismiaceae into a single family, Burmanniaceae sensu lato. However, the molecular-based analyses in these papers included contaminant plastid sequences (Lam et al., 2016), a common issue when using polymerase chain reaction (PCR) amplification to recover plastid DNA sequence data from mycoheterotrophic taxa. By contrast, morphological analysis (Caddick et al. 2002a) indicated the paraphyly of this group with respect to other Dioscoreales, based partly on the absence of septal nectaries in Thismiaceae (Caddick et al. 2000b; 2002a). In addition, more recent molecular phylogenetic data based in nuclear and mitochondrial sequences and whole plastid genomes (Merckx et al. 2009; 2010; Merckx & Smets 2014; Lam et al. 2016; 2018; Shepeleva et al. 2020; Lin et al. 2022) endorse earlier treatments (e.g., Jonker 1938; Maas van der Kamer 1998; APG I 1998) that support the separation of Thismiaceae from Burmanniaceae in different subclades of Dioscoreales, with Thismiaceae instead associated with Taccacaeae (e.g., Merckx et al. 2009; Merckx & Smets 2014). Moreover, studies based on mitochondrial and nuclear gene sets (Merckx et al. 2009; Merckx & Smets 2014; Merckx et al. 2017) have shown that Thismiaceae is itself paraphyletic, with Afrothismia recovered as the group of a clade comprising photosynthetic Taccaceae plus the rest of Thismiaceae. Strong support for this arrangement was recently recovered in phylogenomic analyses based on mitochondrial genomes (Lin et al. 2022). A general consensus has therefore emerged that Afrothismia is a phylogenetic lineage that is distinct from Burmanniaceae, Taccaceae and Thismiaceae (Fig. 1).
To date, Afrothismia has been distinguished from Thismia by two morphological characters: zygomorphic flowers (but some South American species of Thismia are zygomorphic), and clusters of root tubers, each with a terminal rootlet (Maas van der Kamer 1998). However, in the last two decades, additional morphological data potentially support separate family status for Afrothismia, including new data associated with the description of numerous new species of Afrothismia (Cheek 2004a; 2007; 2009; Cheek et al. 2019; Cheek & Jannerup 2006; Dauby et al. 2008; Franke 2004; Franke et al. 2004; Maas-van de Kamer 2003; Sainge & Franke 2005; Sainge et al. 2005; 2013) and studies on ontogeny, floral morphology and mycorrhizal organization (Imhof & Sainge 2008; Imhof et al. 2020; Shepeleva et al. 2020). Here we review these recent studies, present evidence in support of family status for the genus, and formally describe and diagnose Afrothismiaceae as a new, fully mycoheterotrophic and monogeneric family in Dioscoreales.
Taccaceae Dumort., which is sometimes placed in or near Dioscoreaceae R.Br., is a pantropical family of 10–20 species, all placed in Tacca J.R. Forst. & G. Forst. Superficially these large, pantropical photosynthetic, terrestrial herbs seem unconnected with the non-photosynthetic Thismiaceae and Afrothismiaceae. However, as Rübsamen (1986) stated (translated from the German): “…Remarkable parallels exist in the structure of the anthers between Taccaceae and Burmanniaceae: the six stamens (of Tacca) look confusingly similar to those of Afrothismia winkleri or Haplothismia; the plate- or umbrella-shaped stigma (or the cap-like appearance of the style) is also reminiscent of the more bowl-shaped stigma of Afrothismia.”
Methods
Nomenclature follows the Code (Turland et al. 2018). Authorship of names follows IPNI (continuously updated). The format of the description follows e.g. Cheek et al. (2019). Morphological terms follow Beentje & Cheek (2003). Herbarium codes follow Index Herbariorum (Thiers, continuously updated). All specimens cited have been seen. The conservation assessments cited follow the IUCN (2012) categories and criteria.
Taxonomic Results
Diagnostic characters for Afrothismia are presented in Table 1 and Figs 1–4, including a comparison with Thismiaceae (Haplothismia, Oxygyne, Thismia, Tiputinia) and Taccaceae (Tacca).
Taxonomic Treatment
Afrothismiaceae Cheek & Soto Gomez fam. nov.
Type genus: Afrothismia Schltr. (1906)
A single genus, Afrothismia, restricted to continental tropical Africa.
Description as for the genus (see below).
Afrothismia Schltr. (Schlechter 1906; Jonker 1938; Cowley 1988; Maas-van de Kamer 1998; Cheek 2009)
Type of genus: Afrothismia winkleri (Engl.) Schltr. Jonker (1938: 223)
Perennial non-photosynthetic mycoheterotrophic herbs entirely lacking green tissue, with only the flower or fruit emerging above the leaf-litter. Stem (rhizome), colourless, opaque, succulent, concealed in substrate, spreading more or less horizontally, becoming vertical when flowering, sparsely branched or unbranched. Scale-leaves sparse, alternate, ovate-triangular, minute, axillary buds globose, minute. Bulbil clusters subglobose in outline, bulbils 15–40, each globose, with an apical rootlet (Fig. 4B).
Inflorescence 1–few-flowered, terminal or sympodial where more than 1 flower (Fig. 2B). Flowers strongly or weakly zygomorphic, subtended by a large dorsal colourless ovate bract (Fig. 2A, 3A, 3B, 4D). Perianth tube usually colour-patterned, translucent, white, red and/or purple, erect to horizontal, straight, S-shaped or angled, globose or subcylindrical, constricted or not, separated internally usually by an annulus, into two parts, upper and lower, outer surface smooth, ribbed or papillate; mouth of tube projecting beyond insertion of the lobes, ribbed or not, partly covered with an operculum (corona) or not, aperture orbicular, hemi-orbicular or elliptic; post-anthesis deliquescing. Perianth lobes six, yellow, white, red or purple, patent, forward-directed or curved, equal or unequal, triangular or filiform, entire or lacerate.
Stamens 6, inserted on distal part of lower perianth tube (epipetalous) below the annulus, staminal filaments dorsiventrally slightly flattened or clavate, the apex and basal part of the connective swollen and sometimes geniculate (Fig. 4H), arching inward and downward to stigma, glabrous, papillate or hairy; anther thecae two, elliptic, introrse, separated by and often embedded in the connective, distal connective appendage papillate (Fig. 4G), firmly adnate to stigmatic surface (Fig. 4E), pollen monoporate, surface reticulate.
Ovary inferior, campanulate, unilocular, placentation axile, placenta globose sometimes 3-lobed, massive, attached at base and apex by cylindrical stalks; ovules numerous, anatropous often on long funicles (Fig. 3A). Style cylindrical, short, hollow (Fig. 3B, C); stigma obconical thick, usually slightly 6-lobed or not, surface densely papillate or hairy (Fig. 4E).
Fruit campanulate, dehiscence circumscissile, the lid of the fruit (perianth floor) detaching completely, exposing the seed-covered placenta which is projected more or less completely above or far above the fruit wall by the elongation of the placental base or placentophore. Seeds numerous, narrowly obovoid or ellipsoid, reticulate, lacking appendages or with a swollen structure (possible elaiosomes) at each end. (Fig. 2).
RECOGNITION (diagnosis)
Afrothismiaceae fam. nov. differing from Thismiaceae Aghardh in that the ovary has a single stalked, globose placenta, fruits circumscissile (pyxidium), seeds exserted from fruit by placental elevation (vs. parietal, fruit lid deliquescing, seeds included), rootstocks with clusters of globose root tubers, each with a terminal root (vs. a single tuber or roots fleshy), stamens inserted in mid or lower part of perianth tube, below the annulus; anther appendages connected to stigma at anthesis (vs. inserted near mouth, distant and free from stigma).
DISTRIBUTION & BIOGEOGRAPHY
Continental tropical Africa, from Nigeria east to Kenya, south to Malawi. Of the 16 described species, 12 occur in Cameroon, one of these extending west to Nigeria, another East to Gabon. Gabon has one described endemic species, Uganda another (currently treated at varietal rank), Kenya and Malawi both have one, and Tanzania two. However, several undescribed species are known to have been collected in Cameroon (Sainge et al. 2017), Gabon (MC, pers. obs) and one in Tanzania (Afrothismia “arachnites”, Rübsamen (1986)). In Cameroon, the species are concentrated in the Cross-Sanaga Interval (Cheek et al 2001), where nine of the 12 described Cameroon species occur, seven of which are endemic. This area contains the highest vascular plant species and generic diversity per degree square in tropical Africa, with endemic genera such as Medusandra Brenan (Peridiscaceae, Barthlott et al. 1996; Dagallier et al. 2020; Soltis et al. 2007; Breteler et al. 2015). The species of Afrothismia in this area show the full range of floral and root morphology documented in the genus. The genus is unrecorded from the Congo basin and there is a c. 2200 km disjunction between the species of Lower Guinea (West-Central Africa) and the westernmost East African record in Uganda.
The single most species-diverse area for Afrothismia is Mt Kupe in S.W. Region, Cameroon, within the Cross-Sanaga Interval. Here five species have been recorded: A. saingei (Franke 2004), A. fungiformis (Sainge et al. 2013), A. winkleri (Cheek et al. 2004), A. hydra (Onana & Cheek 2011) and A. kupensis (Cheek et al. 2019). The first and last species are thought to be endemic to Mt Kupe. The Mt Kupe area has been the source of numerous other new species to science e.g. (Stoffelen et al. 1997; Cheek & Csiba 2002; Cheek 2003) and even new genera, including Kupeantha Cheek (Rubiaceae, Cheek et al. 2018b) and (another non-photosynthetic mycoheterotroph) Kupea Cheek & S.A. Williams (Triuridaceae, Cheek et al. 2003) subsequently found to extend to East Africa (Cheek 2004b).
At Mt Kupe, Afrothismia species occur with other achlorophyllous mycoheterophic plant species in the families Gentianaceae, Burmanniaceae and Triuridaceae, including one observed site of c. 20 m x 20 m with six mycoheterotrophic species, including Afrothismia kupensis (Cheek & Williams 1999; Cheek 2006), equalling the record in the other most species-diverse site ever recorded in Africa, at Moliwe, Mt Cameroon (Cheek & Ndam 1996, analysing collection records from Schlechter), which is now converted into agricultural plantations.
The first records of several of the mycoheterophs at Mt Kupe were made in the course of intensive botanical surveys conducted over several seasons, including non-photosynthetic mycoheterophic plant specialists, to support conservation management. The surveys usually resulted in a botanical conservation checklist (in the case of Mt Kupe, Cheek et al. 2004). However similar surveys at several other locations in Cameroon with lowland or submontane forest that might be expected to host Afrothismia failed to uncover any plants, even in the appropriate late wet season (Cheek et al. 2000; 2010; 2011; Harvey et al. 2004; 2010). These records suggest that Afrothismia species are not ubiquitous in any apparently suitable habitat, but genuinely localised and rare, even in Cameroon, where the majority of the known species are recorded.
HABITAT
Lowland and submontane evergreen forest; c. 200-1150 m altitude.
ETYMOLOGY
Taken to signify “African Thismia”.
CONSERVATION STATUS
Of the 17 published taxa (16 species) of Afrothismia, 14 have been assessed for their IUCN extinction risk status (Table 2). One has been assessed as Vulnerable, two Endangered and 11 of the 14 are assessed as Critically Endangered (CR), the highest category of threatened status before extinction. The level of CR species in the Afrothismia (c.78%) is exceptionally high and exceeds that of extremely threatened genera of comparable size e.g. Inversodicraea (Podostemaceae) with 14/30 (c.46%) CR species (Cheek et al. 2017). Afrothismia pachyantha has subsequently been declared extinct (Cheek et al. 2019), based on multiple attempts to relocate non-photosynthetic mycoheterotrophic plants at its sole locality by teams of mycotroph specialists since 1991. Its habitat has been cleared for plantation and small-holder agriculture, including rubber (Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg.).
Other locations lost due to agricultural clearance include the type location of Afrothismia winkleri at Muea (Onana & Cheek 2011). This represents the main threat faced by Afrothismia species; they are acutely susceptible because most occur in either a single or only two to three locations, and at each location, their population may occupy only 2–10 m2. It is expected that additional species will shortly become extinct, if they are not already so. The forest at Mt Kala, type and sole locality for A. amietii and A. pusilla, is being cleared for housing development. Afrothismia baerae has not been seen for 20 years since it was first collected in 2002, despite annual monitoring (Quentin Luke pers. comm. to MC 2022). However, other species not seen for decades, such as A. zambesiaca, unseen since the type gathering in 1955, cannot be assumed extinct because there have been no targeted efforts to refind them. In order to support their protection, in 2022, almost all Cameroon Afrothismia species were included within a network of Important Plant Areas (IPAs or TIPAs, Darbyshire et al. 2017; https://www.kew.org/science/our-science/projects/tropical-important-plant-areas-cameroon), however, these species lack legal protection. Eight Afrothismia species are included in the Cameroon Red Data Book (Onana & Cheek 2011: 353-356).
Non-photosynthetic mycoheterotrophic plants such as Afrothismia have never been recorded as being successfully cultivated. We do not yet know which autotrophic plant species their soil fungal symbionts depend upon. Presumably, to succeed in growing Afrothismia from seed, one would need to have both the required species of fungal symbiont and suitable autotrophic plant partner(s) already established. There is no record that this goal has either been achieved or attempted. It is likely that Afrothismia might have orthodox seeds since they are small and dry, but no species are known to be seed-banked, and seed banking is of little value if the seeds cannot be grown to produce viable plants.
The IUCN convention is that species are not considered for inclusion on their website (iucnredlist.org) until they are formally published. This makes it more urgent to publish species so that they can be formally Red Listed and afforded a higher level of protection than they would otherwise obtain (Cheek et al. 2020). Therefore, publication of the seven known but still undescribed species of Afrothismia remains a priority.
Typification
In the generic protologue, two species were published (Schechter 1906). Jonker effectively chose one of these as the lectotype of the genus by giving Afrothismia winkleri as “Type species” (Jonker 1938: 223).
Species discovery
Afrothismia winkleri was first published as Thismia winkleri (Engler 1905) before becoming the foundation of Schlechter’s genus Afrothismia Schltr., joined by A. pachyantha Schltr. (Schlechter 1906). Both species were first collected on the slopes of Mt Cameroon. The third species was collected in the Usambara Mts, now in Tanzania in East Africa, by Peter (s.n., B), who labelled it as Afrothismia arachnites n. sp., but never formally published it. Until today, this species seems to have been overlooked, except by Rübsamen (1986). Two new taxa were added to the genus more than 80 years later by Cowley (1988), Afrothismia winkleri var. budongensis Cowley (Uganda) and A. insignis Cowley (Tanzania). By the end of the 20th Century, Afrothismia was known to have only three species, two of which occurred at Mt Cameroon, one endemic (Cheek & Ndam 1996). A large increase in species discovery and publication, arising mainly from Cameroon, began 15 years after Cowley (1988). Afrothismia gesnerioides H.Maas (Maas-van der Kamer 2003) of Cameroon, was soon followed by Afrothismia baerae Cheek (2004) of Kenya, and four species from S.W. Region, Cameroon: A. saingei T.Franke (2004), A. foertheriana T. Franke et al. (Franke et al. 2004), A. hydra Sainge & T.Franke and A. korupensis Sainge & T.Franke (Sainge et al. 2005; Sainge & Franke 2005). Afrothismia mhoroana Cheek of Tanzania (Cheek & Jannerup 2006), A. amietii Cheek (2007) of Cameroon, A. gabonensis Dauby & Stévart (Dauby et al. 2008) of Gabon, and A. zambesiaca Cheek (2009) of Malawi. The most recently published taxa are three species from Cameroon: Afrothismia pusilla Sainge & Kenfack, A. fungiformis Sainge & Kenfack (both Sainge et al. 2013), A. kupensis Cheek & S.A. Williams (Cheek et al. 2019).
Sainge et al. (2017) mentioned three further still undescribed species (referred to as sp. a, b, d, respectively) that he had collected in Cameroon and a fourth that he had seen from Gabon, which he termed sp. c (Boupouya et al. 674). The first author has seen photos of two additional undescribed species from the Cristal Mts of Gabon, Bidault 5044 and Bidault 5478. Additionally, there is Peter s.n. (Afrothismia “arachnites”) of Tanzania (Rübsamen 1986). Thus, seven undescribed species remain to be published, which would take the total number of species in the genus and family to 23, exceeding the 20 species currently accepted in Taccaceae (Plants of the World online, continuously updated).
Taking existing point data records for Afrothismia and using Maxent and an ecological niche modelling approach to map the potential range, Sainge et al. (2017) predicted highly suitable areas where additional species might be found in Sierra Leone, Liberia, Ivory Coast, Nigeria, Cameroon, Equatorial Guinea, Gabon, Republic of Congo, and Democratic Republic of Congo. However, regarding the first three countries, which equate to Upper Guinea, no Afrothismia species have been found to date, although other recent discoveries of achlorophyllous mycoheterophic plant species have been made there by specialists (e.g. Cheek & van der Burgt 2010).
Mycorrhizal relationships in Afrothismia
Combined molecular results from 18S rDNA, ITS and atpA were used to study the phylogeny of several species of Afrothisma and their fungal partners from three locations in SW Region Cameroon (Merckx & Bidartondo 2008). The species of Afrothismia included were A. winkleri, A. hydra, A. foertheriana, A. kupensis (as A. gesnerioides) and A. korupensis. All the fungal symbionts were placed in the Glomus sp. A lineage of the Glomeromycota, and there was no fungal lineage overlap among the different species of Afrothismia. No other fungi or fungal-like organisms were identified, apart from a stramenopile (non-mycorrhizal, assumed pathogen) in A. foetheriana. Franke et al. (2006), investigating the symbionts of seven taxa of Afrothismia also from S W Region, Cameroon, had also found them all to be exclusively Glomus sp A. lineage. However, Imhof (2006) reported an unidentified second fungal species in material of A. gesnerioides. A delayed co-speciation pattern between the plant species and the Glomus lineages was revealed (Merckx 2008; Merckx & Bidartondo 2008): the divergence time estimates for the Glomus nodes were older than for their corresponding nodes in Afrothismia.
Glomeromycota form vascular arbuscular mycorrhiza with about 90% of all land plants (Cheek et al. 2020). Glomus group A are also symbionts with Taccacaeae (Merckx 2008). There is evidence for the origin of Glomeromycota before 400 Mya (Strullu-Derrien et al. 2018).
Many other non-photosynthetic mycoheterophic plants also depend on Glomeromycota as symbionts, including Thismiaceae s.s., Burmanniaceae s.s., and Triuridaceae s.s. However non-photosynthetic mycoheterophic Ericaceae and some Orchidaceae are mainly ectomycorrhizal and depend on Ascomycota and Basidiomycota as symbionts.
Dates of origin and diversification of Afrothismia
Using two different relaxed molecular clock models on the same study set as used above, the origin of Afrothismia was estimated as 91±11 Mya and 120± 11 Mya, with diversification of the genus starting around 50± 13 Mya and 78± 9 Mya (Merckx 2008; Merckx & Bidartondo 2008; Merckx et al. 2010; Merckx & Smets 2014; Merckx et al. 2017). Afrothismia kupensis diverged 50 Mya (its fungal symbiont 219 Mya), A. korupensis 34 Mya (its symbiont and that of A. foetheriana 122 Mya), while A. hydra and A. winkleri diverged from each other 0.8 Mya (their symbionts 66 Mya).
Morphological trends and evolution: flowers
The species of Afrothismia included in the molecular phylogenetic analyses of Merckx & Bidartondo (2008) and Merckx et al. (2009) are representative of the range of variation in floral morphology currently known in Afrothismia. Here, we briefly describe these patterns, referring to other species that share similar morphology, accepting that this similarity might result in part from convergence rather than recent common ancestry. It is likely that these apparent morphological trends are associated with attracting pollinators (see below; pollination biology).
To date, no molecular phylogenetic analyses have included all 16 described species of Afrothismia. Merckx & Bidartondo (2008) and Merckx et al. (2009) included five and six species respectively, and Shepelova et al. (2020) included six species. In these analyses, A. kupensis (as gesnerioides) was sister to the remaining Afrothismia species. Both A. gesnerioides and A. kupensis share similar morphology and are probably closely related, both species possessing tepals that are triangular and non-filamentous and a horizontal perianth tube that is only slightly sinusoidal in the upper part. Among other species, A. korupensis shows the more-or-less filamentous perianth lobes that are present in all other species of the genus except A. gesnerioides and A. kupensis. In A. korupensis, the perianth tube is vertical and the proximal and the majority of the distal part aligned on the same axis. Only the mouth and uppermost part of the distal tube is orientated horizontally, with a projecting corona partly occluding the mouth. This pattern, of a vertical perianth tube with horizontal mouth, is also seen in A. pusilla, A. fungiformis (both also in Cameroon), A. baerae (Kenya) and A. “arachnites” (Tanzania). Another species, A. foertheriana, has a highly reduced distal perianth tube, the campanulate proximal tube comprising the majority and being about as wide as long, and the lobes and corona bear numerous short projections that are also seen in A. pusilla. Afrothismia amietii of Cameroon also shares this pattern, differing in the distal tube being entirely absent. The remaining species examined, A. hydra and A. winkleri, are notable for the proximal perianth tube being ± horizontal, the distal tube being angled vertically, forming an L-shape. This pattern is seen, with variations, throughout the range of the genus, occurring also in A. gabonensis (Gabon), A. insignis, A. mhoroana (both Tanzania) and A. zambeziaca (Malawi). It is also among these species that yellow is included among the flower colours. All other species are coloured in a combination of purple or dark dull red, usually with white (though white is absent from the perianth of A. foertheriana). The flowers of Afrothismia mhoroana are yellow and white in colour, lacking purple or dark red colouring entirely.
Mycorrhizal trends and evolution
A series of studies of the mycorrhizal structures of Afrothismia (Imhof 1999; 2006; Imhof et al. 2013; 2020) also found signs of substantial ongoing evolutionary diversification. The studies involved Afrothismia winkleri (identification to be confirmed, based on Wilks 1179 of Gabon, Imhof 1999), Afrothismia gesnerioides (based on de Winter 91(L), S Region Cameroon, Imhof 2006), Afrothismia kupensis (as A. gesnerioides) and likely Afrothismia winkleri (as Afrothismia saingei) (Imhof et al. 2013) and Afrothismia hydra, A. korupensis, A. gesnerioides and likely Afrothismia winkleri (as Afrothismia saingei) (Imhof et al. 2020).
According to Imhof et al. (2020), the root-shoot combination of Afrothismia winkleri exhibits one of the most complex mycorrhizal colonization patterns described to date. It shows four different hyphal shapes (straight, looped, inflated coils, degenerating coils) in six separate tissue compartments (filiform root, root epidermis, third root layer, root cortex parenchyma, shoot cortex at root clusters, shoot cortex apart from root clusters). Interconnections between all hyphal shapes demonstrated that they belong to the same fungus. In addition, the long filiform roots of this species were interpreted as being especially efficient in facilitating penetration by fungi. In contrast, the mycorrhizal pattern in A. gesnerioides is comparatively simple, with three hyphal forms in five tissue compartments, and the short blunt roots interpreted as being less efficient. Afrothismia hydra and A. korupensis were found to be intermediate in complexity between the foregoing species.
Imhof et al. (2020) suggested that the differences between four Afrothismia species reflected a transitional change towards increasing functional complexity and strict partitioning of conveyance (straight hyphae) and storage purposes (inflated hyphal coils). They concluded that since investigations on the mycorrhizal structures of Thismia spp. describe a disparate and much less sophisticated colonization pattern than in Afrothismia, this difference supports taxonomic separation from Thismiaceae.
Ontogeny
Caddick et al. (2000) described comparative floral ontogeny in several Dioscoreales, including two species of Thismia and four species of Tacca. Nuraliev et al. (2021) provided a detailed description of both flower and inflorescence ontogeny in several species of Thismia, noting variation in placentation in different species, including some in which placentation is parietal at the base and columnar above. Flower buds of Afrothismia hydra, Haplothismia exannulata and two species of Thismia are illustrated here (Fig. 3). Imhof & Sainge (2008) documented development in Afrothismia hydra from seed to seed-dispersal.
In A. hydra, seeds germinate with root tissue only, disrupting the seed coat and developing a primary ovoid root tubercle. The hypocotyl, cotyledons and shoot are not visible during germination. A second tubercle arises at the proximal end of the first one and subsequent root tubercles with filiform extensions develop sequentially, resulting in a small root aggregate. The root aggregate enlarges, forming a central axis to which all roots are connected. This axis has a growth pole where new root tubercles arise; it later develops into a stem with scale leaves, finally terminating in a flower. After anthesis, the corolla tube disintegrates, leaving a pyxidium which opens by means of a peculiar elongating placenta, which Imhof & Sainge (2008) termed a ‘placentophore’, also shown in our material of A. hydra (Fig. 3A). The placentophore later elevates the placenta with attached seeds above the flowering level and is interpreted as an adaptation to ombrohydrochory (rain-operated seed dispersal). The placentophore can extend to 4–5 times the length of the fruits and seems to be the result of meristem growth rather than cell elongation (Imhof & Sainge 2008).
Pollen structure
In her excellent systematic exploration of the embryology, seeds, and pollen of Burmanniaceae s.l. and Corsiaceae, Rübsamen (1986) made an SEM study of the pollen and seeds of two species of Afrothismia, A.winkleri (based on Zenker 3613, B) and A. “arachnites” (based on Peter s.n., B), using dried herbarium material. Caddick et al. (1998) described microsporogenesis and pollen morphology in most genera of Dioscoreales, including Thismia and Tacca. In Afrothismia, pollen grains are released as single units, as in most Dioscoreales (rarely as tetrads in some Burmanniaceae and Thismia species); they are plano-convex, monoporate (ulcerate), with pollen lengths of the two species 24 μm and 14–16 μm, respectively (Rübsamen 1986). The surface sculpture is coarsely reticulate, the muri “caterpillar like”, occasionally with tiny pores, compared with more finely perforate (rarely smooth) in Thismia (Rübsamen 1986). Rübsamen (1986) stated that “…Because of the exine sculpture, the genus Afrothismia (with a reticulate exine surface) is also clearly separated from the genus Thismia.“
Pollination biology
Within Afrothismia, pollination has been recorded only in A. kupensis (Cheek et al. 2019). Observations over a seven-day period of floral visitors to a plot with six flowering plants recorded ten visitors entering the flowers, all representing a species of a mosquito-like insect.
Two specimens were caught on departure from the flowers, preserved and found to be carrying pollen consistent with Thismiaceae. The insects were identified as scuttle flies (Phorideae), probably the genus Megaselia, which in other plant groups has a mutualism in which pollination is affected in exchange for the larvae feeding on the decomposing flowers (Sakai 2002; Hall & Brown 1993). It might be expected that other species will have different pollinators since they have pollination structures not seen in A. kupensis, such as brightly coloured yellow flowers (vs dull purple and white) and long filiform antennae-like perianth lobes (vs flat, triangular) postulated to disperse pollinator attractant volatiles (Merckx 2008).
Within Thismia, “trap” flowers have been suggested, as pollinators have been observed temporarily restrained inside the hypanthium chamber (Guo et al. 2019; Nuraliev et al. 2021). Self-fertilization could also occur widely in Dioscoreales, including both Thismia and Afrothismia. The unusual flower structure of Afrothismia, with anther appendages extending to the stigma surface at anthesis, indicates this possibility. In contrast, self-fertilzation is strongly indicated by our anatomical sections of Haplothismia exannulata (Fig. 3 K, L) showing a mucilagenous mass of germinating pollen tubes growing directly into the style between the carpel margins.
Seed structure and seed dispersal
Rübsamen (1986) described seeds of two species of Afrothismia, A.winkleri (based on Zenker 3613, B) and A. “arachnites” (based on Peter s.n., B). The seeds of the two Afrothismia species are 0.7–0.91 x 0.17–0.23 mm, twice the length of the range of the six Thismia species described by Rübsamen (1986). She characterised the seeds as elongated, 3–5 cells along the longitudinal axis, and having rows of epidermal cells that are twisted either clockwise or counter-clockwise. The epidermal cells are elongate with raised anticlinal walls showing a suture. The outer periclinal wall is smooth to verrucose, usually collapsed, showing the parallel-bar-like thickenings on the inner periclinal wall (Rübsamen 1986: tafel 82 f–h). Rübsamen (1986) neither discussed nor depicted terminal seed appendages (Fig. 2C).
Burmanniaceae and Thismiaceae are usually classified as having “dust seeds”, which have the possibility of being dispersed by the wind, although in forest floor habitats, even breezes are usually absent, so Maas et al. (1986) considered that they are more likely to be dispersed in runnels of water. However, Afrothismia seeds were reported by Rübsamen (1986) to be about twice as large as those of a sample of six species of Thismia, reaching 0.7–0.91 mm long in A. winkleri and A. “arachnites”. Measurements of Afrothismia hydra at 0.7–0.8 mm long also fit this range (Sainge & Franke 2005). However, seeds of A. zambesiaca were recorded at 0.5 mm long Cheek 2009), and those of A. foertheriana at 0.5–0.6 mm long (Franke et al. 2004), yet those of A. kupensis are c. 1.5 mm long (Cheek et al. 2019). For other species, seed dimensions are not recorded. In both A. zambesiaca (Cheek 2009) and A. “arachnites” (Cheek, pers. obs. 2022) the base and apex of the seed have attached white bodies, possibly elaiosomes (Fig. 2C), which suggests that ant dispersal might be possible, rather than the “splash-cup” mechanism believed to occur in Thismia. Since the seeds are elevated above the fruit on a “placentophore” in all known Afrothismia species where fruits have been observed, the splash-cup mechanism suggested for Thismia (Stone 1980) cannot occur in Afrothismia.
Discussion & Conclusions
Setting aside the seven undescribed species listed above, Afrothismia (Afrothismiaceae) with 16 accepted species, are the most species-diverse fully mycotrophic genus and family in Africa. In recent years many facets of the biology of this fascinating group have been uncovered, yet important aspects remain poorly known, such as pollination biology, or entirely unreported, such as microsporogenesis, cytology, and seed dispersal, and, we do not yet know which autotrophic plant species their soil fungal symbionts depend upon.
However, the highest priority for the family must be to protect from extinction the known species, to conserve them in their natural habitats e.g. by including them in Important Plant Areas (Darbyshire et al. 2017) and developing species conservation action plans to improve the likelihood of their survival (e.g. Couch et al. 2022). This is crucial since ex situ conservation is currently not possible. For its size, the genus must be amongst the most highly threatened on the planet, because 11 of the 14 assessed species are globally Critically Endangered, the highest level of threat. Although only one of these species is considered extinct, several others have not been seen alive in decades. Searching for these long-lost species is urgent. Afrothismia accepted species numbers have increased in the last 20 years by 300%, from 4 to 16. It can be projected that more await discovery so long as unsurveyed suitable habitat survives, and it is vital to find and protect these also.
Until species are documented, described and known to science, it is difficult to assess them for their IUCN conservation status, and therefore the possibility of conserving them is reduced (Cheek et al. 2020). Documented extinctions of plant species continue. In the Cross-Sanaga Interval of Cameroon, the centre of diversity for Afrothismia with half the accepted species, the best documented global species extinction is another fully mycoheterotrophic species, Oxygyne trianda Schltr. (Thismiaceae, Cheek & Onana 2011; Onana & Cheek 2011; Cheek et al. 2018a). Examples of species becoming extinct before they are known to science appear to be on the increase. In Cameroon, inside the Cross-Sanaga Interval, examples are Vepris bali Cheek and Monanthotaxis bali Cheek (Cheek et al. 2018c; Cheek et al. 2022). In all cases, anthropogenic habitat clearance for agriculture has been the cause of these extinctions.
Acknowledgements
The first author thanks R. Vogt (B) for access to material of Afrothismia ‘arachnites’, Janis Shillito for typing and Anne Marshall for retrieval of literature.