A new species of Chlorophytum (Asparagaceae) from the succulent Karoo biome, Namibia – with an updated key for Chlorophytum of Namibia

Until recently (Angiosperm Phylogeny Group 2009), the genus Chlorophytum Ker-Gawl. was referred to family Anthericaceae J.G.Agardh, a segregate of Liliaceae s. lat. (see Dahlgren et al. 1985). The genus, together with several others from related families, was recently placed in family Asparagaceae (subfam Agavoideae, Angiosperm Phylogeny Group 2009), and is now one of the largest genera of that family. With more than 200 species, and with a wide tropical and subtropical Old World distribution, Chlorophytum has its center of variation in tropical and subtropical Africa (Kativu & Nordal 1993, Bjorå 2008). The generic circumscription of the group was reviewed for tropical African taxa by Obermeyer (1962), Marais & Reilly (1978), Nordal et al. (1990) and Kativu & Nordal (1993). The latter transferred most of the tropical African species previously referred to the genus Anthericum L. to Chlorophytum. In tropical and subtropical southern Africa, Kativu et al. (2008) enumerated 56 species of Chlophytum for Flora Zambesiaca. Obermeyer (1962) had previously recorded 37 species (seventeen from Anthericum sensu Marais & Reilly (1978), and twenty from Chlorophytum s. str.) from South Africa. Recently, Kativu et al. (2012) presented twelve species for Flora of Namibia. Details on the taxonomic history of Chlorophytum are provided in Marais & Reilly (1978), Kativu & Nordal (1993) and Bjorå et al. (2008). Since its generic re-circumscription (Kativu & Nordal 1993), studies on Chlorophytum provided descriptions for several new taxa from across Africa: nine new species from the Horn of Africa in Nordal & Thulin (1993), four new species from central and East Africa in Kativu (1993a), one new species from south-eastern Africa in Nordal & Poulsen (1998), two new species from Ethiopia by Sebsebe & Nordal (in Sebsebe et al. 2005), four new species from Zambia and Malawi in Bjorå et al. (2008), and one new species from Burundi and Tanzania (Meerts 2011). Information on phylogenetic relationships in genus Chlo­ rophytum is limited. Kativu (1993b) presented preliminary results of a cladistic analysis on 93 species of the genus based on morphological, anatomical and cytological characters. The study poorly supported some of the traditional generic groups (Dasystachys, Baker 1878, Anthericum sensu Marais & Reilly 1978 and Chlorophytum sensu Marais & Reilly 1978), though with extensive homoplasy. Bjorå et al. (2008) carried out a DNA-based phylogenetic analysis of Chlorophytum from representative subgroups of the genus in which they evaluated the taxonomic affinities of some four newly described species. Meerts & Bjorå (2012) used DNA


INTRODUCTION
Until recently (Angiosperm Phylogeny Group 2009), the genus Chlorophytum Ker-Gawl.was referred to family Anthericaceae J.G.Agardh, a segregate of Liliaceae s. lat.(see Dahlgren et al. 1985).The genus, together with several others from related families, was recently placed in family Asparagaceae (subfam Agavoideae, Angiosperm Phylogeny Group 2009), and is now one of the largest genera of that family.With more than 200 species, and with a wide tropical and subtropical Old World distribution, Chlorophytum has its center of variation in tropical and subtropical Africa (Kativu & Nordal 1993, Bjorå 2008).
Since its generic re-circumscription (Kativu & Nordal 1993), studies on Chlorophytum provided descriptions for several new taxa from across Africa: nine new species from the Horn of Africa in Nordal & Thulin (1993), four new species from central and East Africa in Kativu (1993a), one new species from south-eastern Africa in Nordal & Poulsen (1998), two new species from Ethiopia by Sebsebe & Nordal (in Sebsebe et al. 2005), four new species from Zambia and Malawi in Bjorå et al. (2008), and one new species from Burundi and Tanzania (Meerts 2011).
Information on phylogenetic relationships in genus Chlo rophytum is limited.Kativu (1993b) presented preliminary results of a cladistic analysis on 93 species of the genus based on morphological, anatomical and cytological characters.The study poorly supported some of the traditional generic groups (Dasystachys, Baker 1878, Anthericum sensu Marais & Reilly 1978 and Chlorophytum sensu Marais & Reilly 1978), though with extensive homoplasy.Bjorå et al. (2008) carried out a DNA-based phylogenetic analysis of Chlorophytum from representative subgroups of the genus in which they evaluated the taxonomic affinities of some four newly described species.Meerts & Bjorå (2012) used DNA Pl. Ecol. Evol. 149 (3), 2016 markers to clarify the taxonomic position of some central African species.
A recent revision of family Anthericaceae for Flora of Namibia (Kativu et al. 2012)  The neighbouring South African province of Northern Cape has similar climatic conditions to those of southern Namibia.Ai-Ais Hotsprings Game Park is part of the Succulent Karoo biome, an arid ecoregion of high botanical diversity.This ecoregion is home to more than 5,000 higher plant species, nearly 40% of which are endemic, and 18% are threatened (Hilton-Taylor 1996).It has the richest succulent flora in the world, harboring about one-third of the world's approximately 10,000 succulent species.The ecoregion includes high diversity of miniature succulents (435 species) and geophytes (630 species), many with very limited distribution (Hilton-Taylor 1996).Many of them are threatened or endangered, largely because they occupy extremely small ranges We aim to revise the new collections of Chlorophytum and update the key to species of Chlorophytum for Flora of Namibia.

MATERIAL AND METHODS
An unusual specimen of Chlorophytum was collected by L. Nyanyeni and A. Burke in September 2012 during a botanical survey of the Orange River Mountain (Ai-Ais Hotsprings Game Park) area in the extreme south of Namibia.Ai-Ais Hotsprings Game Park is a remote, inhospitable and under-collected area.Its climate is extremely arid, with rainfall averaging 0-50 mm per annum along the Orange River and southeastern portion of the park, increasing to 50-100 mm per annum towards the northeast (Burke 2011).The park lies within a transitional zone between summer and winter rainfall areas.It is characterised by cold and frosty winters, and high summer temperatures.Temperature maxima range between 34 and 36°C (Burke 2011).Annual average temperatures tend to mask these extremes, ranging between 16 and 18°C.
The specimen considered here was morphologically compared to all known Chlorophytum species from Namibia and neighbouring Northern Cape Province from literature descriptions and herbarium material from B, BOL, CT, K, SRGH, WAG and WIND.To elucidate the taxonomic affinity of the specimen, a molecular analysis was carried out on representative taxa of all Chlorophytum subgroups of central and southern Africa.
Total genomic DNA was extracted from herbarium specimens or silica dried leaf samples using the DNeasy Plant mini kit (Qiagen, Hilden, Germany).This was PCR amplified and sequenced from one nuclear (nrITS1) and two plastid (trnLF, rps16) DNA regions.For the amplification of nrITS1, modified versions of primers ITS5 and ITS2 (White et al. 1990) were used.The e and f primers (Taberlet et al. 1991) and rpsF and rpsR2R (Oxelman et al. 1997) were used to PCR amplify the trnLF and rps16, respectively.All amplifications were performed on an Eppendorf Mastercycler EP gradient S. Prepared amplicons contained 1 μL purified PCR product, 1 μL of 10 μM primer (the same primer as used for the PCR), and 8 μL milliQ H2O.Sequences were processed on an ABI 3730 DNA analyser (Applied Biosystems).Sequences were assembled and edited using the ContiqExpress module in Vector NTI Advance ™ 11.0 (Invitrogen Corporation, CA, USA).GenBank accession numbers of sequences included in the present study are presented in table 1.Sequences from the 38 accessions were manually aligned using BioEdit 7.0.9.0 (Hall 1999).Maximum parsimony analyses were performed using NONA (Goloboff 1999) in combination with WinClada v. 1.0 (Nixon 2002).Parsimony jack-knifing was undertaken with 1000 replicates and otherwise default setting.The nuclear (ITS) and the chloroplast (trnLF and rps16) regions were analyzed separately.The jack-knife support values of the nuclear analysis were placed above the branches and chloroplast values below.

RESULTS AND DISCUSSION
Material of the putative new taxon did not morphologically match any of the taxa so far recorded in central and southern Africa.A close examination of the specimen against four of the Namibian species not included in a DNA analysis, namely Chlorophytum viscosum, C. calyptrocarpum, C. krause anum and C. rangei led to the following observations.Chlo rophytum calyptrocarpum is characterised by wiry roots that bear elongated tubers or occasionally has its roots reduced to elongated tubers.Its inflorescence is a wiry, glandular panicle.These characters are in contrast to the fusiform roots and non-glandular, condensed racemes of the putative new taxon.Chlorophytum viscosum is glandular on all parts with a laxly branched panicle.Chlorophytum krauseanum and C. rangei are tufted, grass-like species with wiry leaves and paniculate inflorescences, and are thus significantly different from the putative new taxon.
The nuclear DNA analysis placed the Orange River taxon in an isolated position, separate from taxa that superficially resemble it (fig.1).The analysis resolved the putative new taxon as sister to the Dasystachys sensu Baker and Euchlorophytum sensu Bjorå (2008) clades, however, with low support (JK = 65).In the chloroplast analysis, the taxon also resolved as sister to the Dasystachys sensu Baker clade (JK = 78).The taxon thus does not appear to have any close Cult.

KU880781 KU880880 KU880826
A. angustifolium Hochst.ex A.Rich.connection with any of the species included in the phylogenetic analyses.
In the genus Chlorophytum, subterranean organs usually represent a useful character for assigning species to informal subgroups.The roots of the specimen (fig.2D) resemble those of C. subpetiolatum in being swollen at base and tapering at the tips, not commonly found in the genus.The similarity in the root system apparently does not reflect any phylogenetic relationship as seen in the molecular analyses and deviating morphological characters.In addition, C. subpetiolatum is a semi-humid to humid tropical species that has its most southerly extent in neighbouring Angola and Zambia, more than a thousand kilometres to the north, and separated from the putative taxon by the dry Namib Desert.The phylogenetic analysis places the species in a position separate from all other analysed central and southern African species, hence supporting its recognition as a distinct taxon.Kativu, sp. nov.Type: Namibia, Luderitz Distr., Ai-Ais Hotsprings Game Park, Orange River Mountain (Boom River), on a granite mountain, in mid-slope, on well drained soils that overly a stony/rocky substrate, 12 Sept. 2012, fl., fr., L. Nanyeni 380 (holo-: WIND; iso-: WIND, SRGH).Species is only known from the type locality and is listed as occasionally occurring.Efforts should be made to collect more material from the area.

Chlorophytum boomense
Plant erect, up to 32 cm high.Rhizome short, bearing fibrous remains of old leaf bases.Roots many, swollen at base, without tubers.Leaves (sub-)distichous, 2-several, lowermost reduced to cataphylls, linear to linear-lanceolate, clasping, soft, flat, glabrous, 5-12.5 × 0.8-1.2cm.Peduncle slightly arcuate at base, terete, glabrous, 2-3 bracteate, with lowermost bract 1.9 cm long, linear, acuminate, upper ones smaller.Inflorescence a loose, simple raceme, exerted above the leaf rosette.Floral bracts broad and clasping at base, acute, 4-5 brownish veined, 2 mm long.Pedicels 2-several per node, articulated below the middle, c. 9 mm long in fruit.Perianth white, star-shaped, tepals free, 3-veined, c. 10 × 1.5 mm.Filaments terete, sparsely papillate, longer than the anthers.Style exerted, slightly declinate.Young capsule longer than broad, sharply trigonal, smooth, c. 6.5 mm long.Seeds unknown.Figure 2. Etymology -Chlorophytum boomense borrows its name from Boom River, where it grows in the vicinity.Habitat and phenology -The species occurs on a granite mountain, in mid-slope, at altitude 257 m, on well drained soils that overly a stony/rocky substrate.It flowers and fruits in September.Distribution -Chlorophytum boomense is endemic to southern Namibia and appears to be of limited distribution.The type locality lies in a protected national park area.Preliminary IUCN Conservation Status -The species is given a Red List status of Vulnerable [VU D2].The extent of occurrence (EOO) of Chlorophytum boomense was not estimated since it is only known from one subpopulation, whereas its area of occupancy (AOO) is estimated to be 4 km 2 (estimated using a cell size of 2 km × 2 km as recommended  by IUCN 2014), which falls within the limits for Critically Endangered status under the subcriterion B2.The species is endemic to south Namibia, and is known from one specimen, representing one subpopulation, which thus represents a total of one "location" (sensu IUCN 2014).This number falls within the limits for Critically Endangered status under the condition'a' of B2.The species was collected in 2012 in the Ai-Ais Hotsprings Game Park.Despite extensive herbarium and field studies made for Flora of Namibia in 2012, and additional herbarium studies for this publication, this species was not observed elsewhere, so it apparently occurs only in this protected area.No previous decline of population has been observed, and in absence of threats, we cannot anticipate a degradation of the quality of its habitat or a decline in the number of subpopulations, mature individuals, EOO, AOO.Since the conditions for applying condition b under subcriterion B2 are not met, Chlorophytum boomense cannot be regarded as threatened following Criteria B.
However, the species is only known from the type locality, the Orange River Mountain (Boom River), where it was collected on a granite mountain, in mid-slope, on well drained soils that overly a stony/rocky substrate.It was also listed as occasionally occurring.Due to this the very limited distribution in a rather harsh environment, the species could be vulnerable to rapid stochastic events.On the basis of what is known, an assessment of Vulnerable under D2 reflects the fact that it is only known from a single locality with a small population.Chlorophytum boomense is thus assigned a preliminary status of VU D2.The species should be reassessed if additional survey reveals further records outside the protected area.Recognition -The species is morphologically similar to C. subpetiolatum (Baker) Kativu (Kativu & Nordal 1993), particularly in its spongy, fusiform roots that are characteristically swollen at base and tapered towards the tips.It differs, however, through its filaments that are longer than the anthers (anthers longer than filaments in C. subpetiolatum) and leaves that clasp at base.The morphologically similar C. subpetiolatum is a tropical species that is confined to the north of the Tropic of Capricorn and so far has never been recorded in Namibia.

Figure 1 -
Figure 1 -Strict consensus tree based on ITS1 data from representative groups of Anthericaceae illustrating the position of Chlorophytum boomense from among central and southern African Chlorophytum taxa.Parsimony jackknife values above 50% are shown above the branches.Below the branches, jackknife support values for the concatenated chloroplast trnLF and RPS16 regions are included.

Figure 2 -
Figure 2 -Photographic illustrations of Chlorophytum boomense from type locality (specimen L. Nanyeni 380): A, plant in bud; B, flowering plant in habitat; C, part of inflorescence and flower detail; D, rhizome and root system.All photographs by A. Burke.
recognised twelve species of Chlorophytum.Nine of the species have a wide distribution in southern Africa, while C. rangei Engl.& Krause and C. viscosum Kunth are confined to Namibia and South Africa and C. krauseanum (Dinter) Kativu to Namibia, South Africa and Botswana.So far, there are no endemic species of Chlo rophytum recorded for Namibia.The majority of Namibian species are distributed in the north and central districts of the country, e.g. C. calyptrocarpum (Baker) Kativu, a species used in our morphological comparson below.Only five are found south of the 26° latitude, namely Chlorophytum came ronii (Baker) Kativu, C. krauseanum, C. longifolia Schweinf.ex Baker, and the mentioned C. rangei and C. viscosum.