Justicia gigantophylla (Acanthaceae), an endangered litter-gathering species from southern Cameroon

Background and aims – The taxonomic status of Duvernoia gigantophylla Lindau from southern Cameroon is considered in relation to the wider Justicia extensa-laxa complex of tropical Africa. Its unusual litter-gathering habit (currently unique in Acanthaceae) is also discussed. Methods – This study was based on normal practices of herbarium taxonomy and morphological analysis. All relevant protocols were followed in the production of the conservation assessment and selection of the lectotype. Key results – A new combination Justicia gigantophylla (Lindau) H.J.Sm. & C.Moran is proposed for this narrowly endemic and endangered species, which is re-instated, with a new description and conservation assessment. A basic key to the wider J. extensa-laxa complex is presented. Justicia gigantophylla is assessed as Endangered (EN B1ab(iii)+2ab(iii)) according to the 2012 criteria of IUCN and a new lectotype is also designated.


INTRODUCTION
With 500 species (almost certainly an underestimate), Justicia L. s. lat. is the most species-rich genus within the Acanthaceae family (Vollesen in Darbyshire et al. 2010). Recognized as one of world's 'megagenera' (Frodin 2004), the sheer scale and morphological diversity of Justicia continues to pose significant challenges to taxonomists, with comprehensive treatments being unavoidably complex and difficult to produce. Over the last two centuries, hundreds of species and several whole genera have been moved in and out of Justicia; most accounts treating only particular geographic regions and taxonomists disagreeing on whether to take a broad or narrow approach to the genus overall (Graham 1988). The taxonomic intricacy of Justicia extends to the Justicieae tribe, which has been described as 'the most taxonomically difficult group in Acanthaceae' (Kiel et al. 2017) and which continues to resist clarity even with contemporary molecular methods.
This study focuses on a small group of African species of Justicia, the J. extensa T.Anderson-J. laxa T.Anderson complex (Anderson 1863). The species within this complex are rainforest understorey perennial herbs and shrubs occurring across the "Guineo-Congolian" forest belt (White 1983), extending from Guinea in the west to Kenya in the east and to northern Angola in the south. The complex is currently divided into two widespread species: J. extensa and J. laxa (Heine 1963(Heine , 1966; Vollesen in Darbyshire et al. 2010). Each of these species has several nomenclatural synonyms, a result of formerly segregated taxa having been subsumed within the two more broadly circumscribed species. Justicia laxa is listed as of Least Concern (LC) on the IUCN Red List of Threatened Species, and although not on the formal Red List, J. extensa has also been assessed as of Least Concern following the IUCN (2012) criteria (Cheek et al. 2004).
However, preliminary studies of the large volume of herbarium material available for this group (c. 900 specimens at K, P, BR and WAG alone) have revealed a large range of morphological variation within each species, with at least some of this variation separable into discrete taxa. Some of these entities also appear to have a restricted range and so may be of high conservation significance yet, as with many cryptic species, they are currently hidden within widespread species of low perceived conservation value. Besides Champluvier's unpublished efforts to unravel the complex within Central Africa (annotated specimens seen at BR and K), the exact relationships between and within these species are yet to be fully resolved. This study aims to begin that process by providing a basic key to the J. extensa-laxa complex and by resurrecting Duvernoia gigantophylla Lindau with a new combination in Justicia; a particularly distinct and highly threatened member of the group hitherto obscured by an incomplete taxonomy.

MATERIAL AND METHODS
This study is based on detailed morphological studies of herbarium material, using specimens held at BR, BRLU, K, P, and WAG (herbarium abbreviations following Thiers continuously updated) as well as digitised type material seen on JSTOR. Data on distribution, ecology and nomenclature were taken from specimen labels and from field observations by Martin Cheek, one of the most recent collectors of the species. The geographical coordinates for each specimen were databased and error-corrected where necessary. Specimen data was imported into SimpleMappr (Shorthouse 2010) and used to produce distribution maps. The online tool Geo-CAT (http://geocat.kew.org/, Bachman et al. 2011) was used to calculate the area of occupancy (AOO) and extent of occurrence (EOO). Both figures were then used alongside the IUCN red list categories (IUCN 2012) to produce a full conservation assessment for J. gigantophylla. Due to the limited material available, floral measurements were based on a total of three flowers (taken from Cheek 17553 and Letouzey 4095). These were two whole, dried flowers and one dissected flower, which was first rehydrated using Aerosol OT 5% solution. Nomenclature adheres to the protocols of Turland et al. (2018) and binomial authorities to IPNI (continuously updated). All cited specimens have been seen by the authors.

Preliminary key to the Justicia extensa-laxa complex
Based on the preliminary results of this study, J. laxa is provisionally separated into three taxa of which J. gigantophylla is the most distinctive and discrete. J. gigantophylla is separated ahead of a full revision due to its highly threatened position and consequent need for formal recognition. The closely related J. extensa complex will likely also warrant subdivision following further investigation.
Pollen prolate, 4-colporate, 8-pseudocolpate; P = 39-46 µm, E = 25-34 µm, P/E = 1.15-1.5; colpi and pseudocolpi approximately equal in length or pseudocolpi somewhat longer, extending to near the poles but not meeting; pori prominent, ± 6.5 µm wide; colpi and pseudocolpi microverrucate, interapertural and polar exine reticulate with microverrucate lumina. -Fig. 3. Habitat, ecology and phenology -Litter-gathering understory herb found mostly at roadsides and within forest clearings, sometimes near streams. Atlantic Equatorial Coastal Forests (White 1983). Mostly at elevations between c.300-900 m. Flowering occurs during rainy season between May/ June and September/October. Conservation assessment -A full IUCN conservation assessment is in press (Smith in press), which gives this species the current status of Endangered -EN B1ab(iii)+2ab(iii). This is based on the limited EOO of < 5,000 km 2 (3,427 km 2 ) and AOO of < 500 km 2 (44 km 2 ), the small and fragmented number of locations, and the imminent/currents threats of open-cast mining, logging and population growth in the immediate vicinity of the known locations. Uses -Thus far, there are no described uses for this taxon, though its close relatives J. laxa and J. extensa are known for their economic and medicinal value. Both species are used as fish poisons throughout their native range in West Africa, and J. extensa is reportedly used as a treatment for a wide range of ailments; from headaches to epilepsy (Burkill 1985). Notes -Justicia gigantophylla is based on Lindau's (1913) Duvernoia gigantophylla (see Discussion). It is clearly related to J. laxa T.Anderson, which it closely resembles in characters of the inflorescence, corolla, and anthers, but can be immediately separated by its oblanceolate, sessile or very shortly petiolate leaves that clasp the stem to form litter-gathering rosettes. The leaves are also larger than in J. laxa and usually bear more lateral veins, and the calyx lobes are much wider, with the margins adpressed to one another, forming a star shape in cross-section. J. laxa has more elliptic, regularly spaced leaves with a longer petiole, and narrowly lanceolate calyx lobes that are held flat.

Distribution
Due to limited suitable material available for study, the pollen description for J. gigantophylla presented here is based on a single accession, Cheek 12553. The 4-colporate, 8-pseudocolpate reticulate pollen of that specimen is very similar to that reported for J. extensa, except that it is 4-aperturate whereas J. extensa is 3-aperturate (Graham 1988;Kiel et al. 2017). 3-colporate, 6-pseudocolpate pollen (as in J. extensa) is hypothesized to be synapomorphic for Justicieae as a whole (Kiel et al. 2017). However, aperture number is variable within the justicioids and it is clear that aperture number has evolved homoplastically across this clade (Kiel et al. 2017). Whilst pollen grains with 2 or 3 apertures are the most commonly encountered, some New World justicioids have up to 6 apertures, and 4-aperturate grains have been reported in several justicioid taxa, such as Anisotes guineensis Lindau (Baden 1981). Given this variation and homoplasy, pollen characters appear to be of limited taxonomic within the justi-

Taxonomic history and position of the species
Justicia gigantophylla was first described in 1913 by Gustav Lindau under the name Duvernoia gigantophylla and noted by the author for its resemblance to J. extensa, which Lindau also transferred to Duvernoia Nees. Duvernoia was first described from South Africa (D. adhatodoides E.Mey. ex Nees) but subsequently much expanded by Lindau. Despite Lindau's confidence in Duvernoia, its validity as a genus was challenged by Burkill & Clarke (1900) in the Flora of Tropical Africa (FTA) who noted that Lindau appeared to have circumscribed Duvernoia based on one character; that of the lack of tubercles on the smooth bands of the pollen. Burkill & Clarke (1900) wrote "we find the group contrary to nature itself; it appears not a genus, but a handful of species taken at random from every part of the genus Justicia (in a very wide sense)". Consequently, Burkill & Clarke moved all known species of Duvernoia into Justicia. Most recent treatments have followed this decision (e.g. Lebrun & Stork 1997;Scotland & Vollesen 2000;Vollesen in Darbyshire et al. 2010Manning & Goldblatt 2014).
However, Lindau continued to uphold Duvernoia post-FTA and although Duvernoia gigantophylla was first collected in 1898 it was not described until 1911, nearly a decade after Clarke's FTA treatment. Duvernoia gigantophylla has not been covered in any subsequent floristic treatment. It is not mentioned in the Flora of West Tropical Africa (Heine 1963), which only extends as far east as the former 'British Cameroons' (modern-day Northwest and Southwest Cameroon Regions), i.e. outside the range of D. gigantophylla, which is only known from the South Cameroon Region. Similarly, Heine (1966) in Flore du Gabon did not discuss D. gigantophylla in relation to J. extensa or J. laxa. The name is entirely omitted from modern checklists of the vascular plants of Cameroon (Onana 2011(Onana , 2013 or of continental Africa (Lebrun & Stork 1997;Klopper et al. 2006;African Plants Database 2018). Duvernoia gigantophylla has therefore been left untreated, neither synonymized nor transferred, and essentially lost to science for over 100 years.
Since the first collection of this species in 1898 and the subsequent type specimen of 1911, no more specimens were collected until the 1960s (all of which were then mis-identified as either J. extensa or J. laxa). In most herbaria, this species has effectively been synonymised under J. laxa s. lat., due mainly to it having glabrous ovaries/capsules, the primary characteristic used by Burkill & Clarke (1900) and Heine (1963Heine ( , 1966 to differentiate J. laxa from J. extensa (the latter having pubescent ovaries/capsules). Some of the earliest specimens of D. gigantophylla (e.g. Zenker 1840) were labelled as "Justicia andersonii Lindau", a name that was subsequently applied in 2004 to other specimens of this species by D. Champluvier (then of BR) who agreed that this species is distinct from J. laxa. However, J. andersonii is an unpublished manuscript name and would in any case be an illegitimate later synonym of D. gigantophylla (the name J. andersonii has already been published for two other species: Justicia andersonii Ramamoorthy from India and Justicia andersonii Wassh. from Brazil). The rediscovery of this species in 2014 and again in 2015, and observations on its unusual litter-gathering habit, prompted the re-examination of its taxonomic status which has led to its resurrection and transfer to Justicia here.
Recent molecular phylogenetic studies (Kiel et al. 2017(Kiel et al. , 2018 have demonstrated that Justicia s. lat. is highly paraphyletic and have outlined the need for serious re-evaluation and revision of the generic delimitation within the "justicioid" lineage. However, as the sampling in those studies was insufficient to reach firm conclusions on revised generic circumscriptions, a broadly circumscribed Justicia will remain for the immediate future, but it also seems likely that it is only a matter of time before major updates are implemented. What is clear from the results of Kiel et al. (2017) is that Duvernoia sensu Lindau is polyphyletic. Included in their sample are both J. extensa and J. aconitiflora (A.Meeuse) Cubey, the latter taxon being one of the two species previously treated in Duvernoia s.s. (see Manning & Getliffe-Norris 1995) and closely allied to the type species D. adhatodoides E.Mey. ex Nees (= J. adhatodoides (E.Mey ex Nees) V.A.W. Graham). Justicia extensa and J. aconitiflora are resolved as not closely related in this phylogeny. Hence, even if Justicia s. lat. is eventually broken up into segregate genera and Duvernoia is resurrected, the name D. gigantophylla will not stand. Therefore, it is appropriate that this species is transferred to Justicia s. lat. pending future generic recircumscription.
Besides the progress made with J. gigantophylla, it is clear that further work is necessary to resolve the J. extensa-laxa complex with any satisfaction. Molecular analysis would be a helpful addition here and may offer a logical and informative next step in the study of this group.

Litter-gathering habit
The distinctive litter-gathering habit of J. gigantophylla immediately separates it from the non-litter gathering species with clearly petiolate leaves seen across the rest of the J. extensa-laxa complex. The large leaves are tightly clasped around the stem and, due to the short internodes towards the stem apices, the opposite-decussate pairs form together a pseudo-rosette which creates a basket-like effect that collects humus and leaf litter from the forest. This habit appears to be unique in Acanthaceae though it has evolved in many different families (Zona & Christenhusz 2015;Lachenaud & Jongkind 2013) and may represent a remarkable case of convergence. The precise role of this adaptation remains unclear for J. gigantophylla, though in other litter-gathering plants commensal organisms are believed to utilise the caught debris as food and/or housing, whilst the host plant benefits from nutrients as this debris is broken down (Zona & Christenhusz 2015). Litter-gathering plants such as this are surprisingly understudied, especially as far as Africa is concerned. It appears that they are often gregarious, with a strong tendency for several litter-gathering species to grow together (Lachenaud & Jongkind 2013). The reasons for this are unclear, but the litter-gathering habit may represent an adaptation to nutrient-poor soils. Nutrients might also be absorbed by the stem epidermis, or by underground roots as debris is washed away to the base of the plant (Hawthorne 2013). Cheek et al. (2008) described Psychotria kupensis (Rubiaceae), another litter-gathering shrub from Central Africa. Much like J. gigantophylla, this species was noted by the author for gathering forest litter in "funnel-like" rosettes, which indeed remains preserved in some herbarium specimens. Cheek et al. suggest that this litter-gathering form may benefit the plant through the uptake of "nutrient-enriched rainwater that percolates down from the funnel" (Cheek et al. 2008: 244); it is likely that a similar process occurs in J. gigantophylla.