A TAXONOMIC STUDY OF THE BLEPHARIS EDULIS COMPLEX (ACANTHACEAE) IN EASTERN AFRICAItambo MalombeEast African Herbarium, National Museums of KenyaP.O. Box 45166 Nairobi, Kenyamalombe@avu.orgEnsermu KelbessaNational Herbarium, Addis Ababa UniversityP.O. Box 3434, Addis Ababa, Ethiopianat. heb@telecom.net.etMuthama MuasyaEast African Herbarium, National Museums of KenyaP.O. Box 45166 Nairobi, Kenyaplants@africaonline.co.keABSTRACTThe variable species Blepharis edulis, as well as the recently described B. boranensis, is investigated in Eastern Africa using phenetic analysis of gross morphological data supplemented with field observations and pollen morphology studies. Two species, B. edulis and B. boranensis, occuring in the drylands of Eastern Africa are confirmed, with the latter occuring in Borana region (Kenya, Ethiopia and Somalia) and Sodere, Ethiopia, while the former remains variable and widespread. The two species are separated by number and distribution of spines on leaf margin: fewer, sparse or restricted towards leaf base in B. edulis whereas more and evenly distributed in B. boranensis. An identification key and species descriptions are provided.INTRODUCTIONBlepharis Juss. (Acanthaceae) is composed of 129 species, distributed predominantly in tropical Africa, about half occuring in Southern Africa, and extending through Arabia and India to S. E. Asia (Vollesen, 2000). In Eastern Africa, Blepharis edulis (Forssk.) Pers. is commonly found in the arid regions, in habitats that are fragile and undergoing the processes of desertification, which necessitates systematic studies to establish species biodiversity status (Claridge et al., 1997; Bates, 2000).The genus has been divided into three subgenera, namely Blepharis, Ebracteata Vollesen and Acanthodium (Del.) Oberm. Subgenera Blepharis and Acanthodium are further subdivided into three (Blepharis, Inopinata Vollesen and Scorpioidea Oberm. Ex Vollesen) and two sections (Acanthodium (Del.) T.Anderson and Biflora Vollesen) respectively. Blepharis edulis is placed in the Sect. Acanthodium (Furness, 1996; Vollesen, 2000). Taxonomic studies of Blepharis in tropical Africa are few, and include that of Clarke (1899-1900), Napper (1970), Agnew & Agnew (1994) and Vollesen (2000). Some specimens of B. edulis in various herbaria (e.g., EA and ETH) have been identified as B. ciliaris (L.) B.L.Burrt. or B. linariifolia Pers. Recently, Vollesen (2000) also separated B. boranensis from B. edulis, based on position and number of spines or flower sizes. B. edulis has a wide variation in gross morphology, at times blurring the species boundaries with related taxa. For example, in Ethiopia near Bale-Harerge massif where some unique B. boranensis forms are found, collections of B. edulis are characterized by large flowers.According to Vollesen (2000), B. edulis and allied taxa are more or less geographically separated in Eastern Africa. B. edulis grows in northern Tanzania, Kenya, Ethiopia and into Saudi Arabia. B. boranensis grows in the Borana region of Kenya/Ethiopia and overlaps with B. edulis. B. linariifolia is distributed from southern Sudan westwards, whereas B. ciliaris is found only in Asia (Oman, Iran and Pakistan).This study re-examines the infraspecific taxonomy of B. edulis complex, based on phenetic analysis of gross morphological data.MATERIALS AND METHODSMorphological studies were based on herbarium and field observations. Specimens from EA, ETH, MAL, NU and WAG were investigated for 21 morphological characters (table 1). The vegetative and reproductive parts were measured using a ruler and a WILD M3 dissecting microscope. Between five and ten measurements were taken per specimen for every part observed at standardized positions and an average calculated. A total of 171 specimens (Operational Taxonomic Units, OTUs) were analysed for phenetic relationships using STATISTICA (version 4.1), prior to character standardization. In cluster analysis, the degree of similarity between pairs of OTUs was measured using the Euclidean distance coefficient. Pairs of OTUs were then clustered by Unweighted Paired-Group Method using arithmetic Averages (UPGMA). Principal Component Analysis (PCA) was performed on the data to elucidate patterns of relationship and test separation of groups obtained in cluster analysis.Pollen grains were obtained from mature flowers of herbarium specimens. The acetolysis method described by Erdtman (1969), using a mixture of glacial acetic anhydride and concentrated sulphuric acid (3:1), was followed in preparation of grains for Light Microscopy (LM) and Scanning Electron Microscopy (SEM) studies. Measurements of the LM pollen were taken using an ocular micrometer at magnification x400 and micrographs made at xl000, respectively. In SEM dry pollen was placed on clean bronze (stub) and coated with gold. The pollen were scanned and investigated using a Jeol JSM 840 Scanning Electron Microscope.RESULTS AND DISCUSSIONGross morphologyIntuitive interpretation of gross morphology is the basis of the previous species concepts on the Blepharis edulis complex. Phenetic relationships of specimens fitting broadly into B. edulis complex below are analysed and presented here, without a priori allocation of the specimens into taxonomic groups.(i) Cluster analysisThe cluster analysis yielded two major clusters above 17.5 Euclidean linkage distance, delineated as B1 and B2 in figure 1. The clusters, B1 and B2, differ in some gross morphological characters and can be separated by vegetative and floral characters. There is discrete variation in number of spines on leaf margins and length of terminal spine on bracts, numbering 2-18 versus 20-36, and length below 4 mm versus 5-9 mm, respectively. In addition, there is geographical separation between the clusters, with B2 having a wide distribution from northern Tanzania through Kenya and Ethiopia into Eritrea whereas B1 is predominantly restricted to Borana region of Kenya/Ethiopia. Cluster B1 has peculiar inclusion of two specimens from Southern Kenya, which have overall gross morphological similarity to B2.Specimens (OTUs) studied in the cluster analysis above were assigned into the corresponding clusters (B1 and B2) and phenetic relationships further evaluated using Principal Component Analysis (PCA).(ii) Principal Component Analysis (PCA)The variance attributed to each of the four principal components (PC) was found to be 42.07%, 9.14%, 7.66% and 6.15% for PC 1, 2, 3 and 4, respectively (table 1). Thus, PC 1 and 2 contributed up to 51.21% of the total variance. A scatter plot of PC1 against PC2 resulted to segregation of the clusters (figure 2). When the data was plotted in three dimensions, based on PC 1 and 2, the two clusters were observed to be unambiguously separated (figure 3). The partial overlap observed in figure 2 is interpreted to be a product of diagrammatic presentation of pattern in two dimensions.Table 1. Factor loading of the gross morphological characters included in cluster and principal components (PC) analysisPC1PC2PC3PC4Leaf margin spine number-.708620.242020-.214988.148265Bract length-.658309.170862.130438-.479591Bract length / width.141566.378410-.065338-.755718Bract spine length-.711161.174444-.487341-.088250Bract length / spine number-.228695-.743574-.007986-.225794Bractiole length-.757717-.422272-.232721-.087505Bractiole length / width-.320957.518671.182924-.239506Anticous calyx length-.876954.061251-.101589.017006Anticous calyx length / width-.617951-.234957.315540-.145810Posticous calyx length-.889260-.088653-.164651.073983Posticous calyx length / width-.687175-.217953.233313.104824Ventral calyx length-.742064.034804-.296624.031452Corolla length-.897245.119383.154178.008338Corolla length / width.147617.083368-.745357-.144175Filament length-.656531.267216-.023920.137086Process length-.500841-.173179-.149015.245945Anticous anther length-.853582.135202-.031912.020108Anticous anther length / width-.249498.125429.318528-.355310Posticous anther length-.819098.088809.081081-.008983Posticous anther length / width-.156811.513822.474052.238117Style length-.854121.166648.009147.145332% Variance42.072159.138677.657486.14562Figure 1. Dendrogram showing cluster pattern of the Blepharis specimens studied. ? indicates unexpected position of the two collection from southern (K4 in Tsavo area) Kenya.Figure 2: Scatter plot of the two clusters B1 (?) and B2 (0) along PC1 and PC2 ordination spaceFigure 3: Three dimensional scatterplot showing differences of B1 and B2 along PCI axis.Figure 4. SEM micrographs of pollen grains of B. edulis complex. A: hexagonal reticulum and smooth muri, B: constriction along colpi in b1 pollen and C: circular to triangular polar view and elliptic equatorial view, respectivelyPollen morphologyThe pollen grains were of medium size, based on terminology by Erdtman (1969), with polar axis length (P) ranging between 36.4-48.4 µm, equatorial axis (E) 15.9-24.1 µm, and P:E ratio 1.9-2.5. The pollen is isopolar, tri-colpate and circular to triangular in polar view, and elliptic in equatorial view (figure 4). The pollen exine has hexagonal reticulum with smooth muri and columellae appearing in the perforations of lumen (figure 4). Results presented here are in agreement with previous observations on pollen morphology in the genus Blepharis (Furness, 1996, 1997).Pollen size does not distinguish the clusters observed in phenetic analysis of gross morphology, as there is size continuum with cluster B1 pollen falling within the range of cluster B2. Cluster B1 can be distinguished from cluster B2 by presence of a constriction along colpi, a feature observed in few grains. However, this feature might have arisen due to abnormal pollen development. Furness (1996, 1997) notes that abnormal pollen occur in Blepharis sect. Acanthodium where grains are few and the sculpturing patterns are disrupted near the apertures. Examples of taxa with abnormal pollen include B. mitrata C.B. Clarke , B. capensis (L.f.) Pers., B. diversispina (Nees) C.B.Clarke, B. grossa (Nees) T.Anderson, B. obmitrata C.B.Clarke and B. subvolubilis C.B.ClarkeTAXONOMIC TREATMENTTwo species are recognized based on phenetic analysis of gross morphological data and pollen morphological studies. Cluster B1 is treated as B. boranensis Vollesen and B2 circumscribed as B. edulis (Forssk.) Pers.Taxonomic keyAnnual herb; bract spines under 4.4 mm long; leaf marginal spines absent or 2-18, rarely more, stem glabrous to puberulous, sericeous 1. B. edulisPerennial herb; bract spines 4.4-9.2 mm long; leaf marginal spines 20-36; stem puberulent to densely pubescent, ascending2. B. boranensis1. Blepharis edulis (Forssk.) Pers., Syn. P1. 2: 180 (1806); Spreng., Syst. Veg. (ed. 16) 2: 820 (1825); T. Anderson in J. Linn. Soc, Bot. 7: 36 (1863); Lindau in Engler & Prantl, Nat. Pflanzenfam. IV, 3b: 318 (1895) & in Engler, Planzenw. Ost-Afr. C: 369 (1895) & C.B. Clarke in Thiselton-Dyer, Fl. Trop. Afr. 5: 102 (1899). Type: Yemen, Forsskal 905 (C, holotype; K, phototype!).Acanthus edulis Forssk., Fl. Aegypt-Arab.: 114 (1775); Vahl, Symb. Bot. 1: 48 (1790); Wild., Sp. (ed. 4) 3: 400 (1801).Blepharis linariifolia sensu Lindau, Ann. R. Ist. Bot. Roma 6: 76 (1896); C. B. Clarke in Thiselton-Dyer, Fl. Trop. Afr. 5: 100 (1899), p.p.; Agnew, Upl. Kenya Wild Fl.: 579 (1974); Kokwaro, Med. Pl. East Afr. (ed. 2): 22 (1993); Furness, Rev. Palaeobot. & Palyn. 92: 256 (1996).Blepharis ciliaris (L.) B.L.Burtt, sensu Collenette, Fl. Saudi Arabia: 29 (1985); Furness, Rev. Palaeobot. & Palyn. 92: 256 & 263 (1996); Agnew & Agnew, Upl. Kenya Wild Fl: 274 (1994).Creeping to ascending annual herbs growing up to 45 cm high. Stem puberulent to densely puberulent on grey young branches and becoming glabrate, sericeous to puberulous, glandular, with age. Leaves green; midrib glaucous; abaxial surface puberulent to densely puberulent; lamina lanceolate to elliptic; 1.9-10.4 ×0.3-1.8 cm; acute to acuminate; apiculate; sessile to subsessile; margin lacerate, spines usually 2-18 or absent, scattered from base up to two-thirds towards apex, occasionally to tip of leaf; terminal spine 0.2-4.5 mm long, straight or on young plants falcate. Spikes erect, strobilate, each comprising of at least 2 mature flowers; 1.8-17.7 ×1.4-7.0 cm. Bracts green to glaucous, 1.4-3.5 × 0.4-1.6 cm; apex recurved, 0.4-2 cm long; marginal spines usually 3-8, terminal one less than 4.0 mm long; bracteole 6.5-20.0 × 0.2-1.5 mm, linear to lanceolate. Sepals densely pubescent; dorsal 1-2.5 × 0.2-1.0 cm, broadly ovate; ventral 0.8-1.9 × 0.2-0.8 cm, oblong; lateral pair 0.4-1.0 cm long, broadly ovate. Corolla blue to purplish; 1.2-3.1 × 0.5-1.8 cm; puberulent with dense strip of indumentum from base towards apex; veins blue, conspicuous; 5 lobed, two lower most lateral lobes vestigial, terminal lobe 0.1-0.6 mm long. Filament purple striped, 5.3-12 mm long with basal tuft of hair, dorsal pair flattened with apical appendages and broader anthers. Style 3.5-11.0 mm long. Capsule 4.3-10.0 × 2.5-5.8 mm, lanceolate to elliptic. Seed 2 per capsule; yellow; discoid; 4.4-6.8 × 3.0-4.5 mm; covered with white stellate hygroscopic hairs 0.3-5.3 mm long.Distribution and habitatIn eastern Africa B. edulis is distributed from central Tanzania through Kenya to Eritrea and Somalia; altitude 20-1,900 m altitude. It grows in sandy or rocky, granite lava on open grounds in semi desert vegetation dominated by Acacia spp. or Commiphora woodland and annual grasses.Selected specimens.Ethiopia. Arsi Region: Sodere, Chilalo Awraja, 3 May 1971, Thulin 1309; Harerge Region: Dire Dawa, Isa, 18 Sept. 1985, Ensermu 1349; Dire Dawa, Melka Jelda, 29 June 1982, Mesfin 2908; Gursum Awr, 20 Jan. 1987, Ensermu & Petros 1869; Batie, 9 July 1966, de Wilde 9722; Shewa Region: Walenchiti, 26 Sept. 1980, Ensermu & Tamra 401; Koka Dam, 14 Mar. 1971, Ash 749.Eritrea. Ocule, 15 Apr. 1902, Pappi 1272; Gahatli Ailet, 19 Mar. 1989, Ryding 1816; Zula, 6 Mar. 69, Robertson 1089; Zula, 6 Mar. 1969, Bally B6909.Kenya. Rift Valley Province: Baringo, Marigat, 23 Oct. 1964, Leippert 5187; L. Baringo, Ol Kakwa Is, 2 June 1977, Gilbert 4711; Kajiado, Nairobi-Magadi Rd., Bally B9767; Turkana, Kainuk, 30 Dec. 1981, Coppock 24; Muasya & Malombe 1587; L. Turkana, Central Is., Modha 11. North Eastern, Marsabit, Milgis, 21 May 1970, Magogo 1442; Garissa, 29 May 1977, Gillett 21201. Eastern Province: Machakos, Kiboko, 22 Dec. 1960, Ossent 533; Mwingi, 4 Mar. 1973, Sanagi 934. Coast Province: Manyani, 15 Sep. 1967, Ivens 2236; Voi, 22 Feb. 1955, Assent 32.Tanzania. Lushoto, Mkomazi, 1955, Evens 572; Evens 572; Njooro, 25 Mar. 1977, Peterson 433; Lushoto, 1 May 1953, Drummond & Hemsley 2324.NotesAlthough this study reconfirms separation of B. edulis complex as described, the taxon remains to represent variable forms, which cannot be separated phenetically. As Vollesen (2002) observed, the species can be very small in arid conditions to semi-perennial multi branched ascending semi-woody stems upto 45 cm in a favourable climate. Three major forms were observed in figure 1 but lacked discontinuous characters for separation. One form was characterized by a few widely spaced leaf spines and large bracts (up to 3.5 cm long). Its distribution includes Ethiopia and Eritrean Rift Valley from Zula through Dire Dawa, Kombolcha sites to Northern Kenya in Lake Turkana area such as Koobi Fora and near the Ferguson Gulf. The second form has the smallest flower sizes (up to 1.3 cm wide) and lacks spine or are very few restricted to first half of the leaf. This group is widely distributed in the region mainly in Kenya and Gamo Gofa Region of Ethiopia. The third form is closely related to B. boranensis on the basis of leaf spines but differ on the number of spines on the bracts. It is distributed in Kenya (Voi and Taita-Taveta districts), Tanzania and Ethiopia from Jijiga eastwards towards Somali mainly Harerge Region. Therefore, the specimen Beentje 1860, from Tsavo in southern Kenya was considered to be more closely related to B. edulis even though clustered with B. boranensis specimens in figure 1. More material from Tsavo should be examined to further ascertain their affinity.B. edulis is found in degraded areas and can be a useful indicator species for ecological change. According to Itani 78/78 (EA) it provides forage for all livestock and flowers are used for staining fat. Leaves are grinded and burned to treat mouth diseases in children (Mus 181, EA). Water from boiled seeds is mixed with milk and taken to cure inflammation of the liver (Newbould 5936, EA). Dry inflorescence or root concoction is used to treat mouth wounds in children, kidney ailments, hasten childbirth, and against scorpion stings among the Kalenjin, Maasai, Somali and Turkana communities, respectively.2. Blepharis boranensis Vollesen. Vollesen, Blepharis, 110-112 (2000). Type, Ethiopia, Burger 3560 (K, holotype!, EA, ETH, isotype! )Creeping to ascending perennial herb, emanating from a woody rootstock, up to 35 cm high. Stem densely puberulent on the young branches and becoming glabrate, sericeous to puberulous, glandular, with age. Leaves green sometimes with purplish patches, midrib glaucous; abaxial surface glabrous, puberulent to densely puberulent; lamina lanceolate to elliptic; 5.8-11.6 × 0.7-1.6 cm; acute, acuminate; mucronate; sessile to subsessile; dentulate, rarely lacerate; spines 20-36, evenly distributed from base to apex, terminal spine 0.1-3.7 mm long, straight. Spike erect, strobilate, each comprising of at least 2 mature flowers per spike; 3.8-13.2 × 1.7-4.8 cm. Bracts glaucous, 1.3-3.3 × 0.6-1.6 cm; apex slightly recurved, 0.8-1.7 cm long; marginal spine 5-11, terminal spine up to 9.2 mm long; bracteole 9.3-19.0 × 0.2-1.8 mm, linear to lanceolate. Sepals densely pubescent; dorsal 1.5-2.4 × 0.6-7.8 cm, elliptic; ventral 1.1-1.8 × 0.3-0.8 cm, oblong; lateral pair, 0.7-1.0 cm long, ovate. Corolla blue with yellowish base; 2.1-3.3 × 0.8-1.9 cm; puberulent with a brown dense strip of indumentum from base towards apex; vein dark bluish, conspicuous; 5- lobed, two lower side lobes vestigial, terminal lobe 0.3-0.8 cm long. Filament purple striped, 6.1-11.6 mm long with basal tuft of hair, dorsal pair flattened with apical appendages and broader anthers. Style 9-12.2 mm long. Capsule 7.5-10.4 × 3.0-5.8 mm, elliptic. Seeds 2 per capsule; yellow; ovoid, 4.5-6 × 3.0-4.3 mm; covered with white hygroscopic stellate hairs 1.8-8.7 mm long.Distribution and habitatBlepharis boranensis is found in Borana region (Kenya, Ethiopia and Somalia) and also around Sodere, Ethiopia between 1,350-1,850 m in limestone soil of Acacia-Commiphora woodland.Selected specimensEthiopia. Sidamo Region: 33 km on Neghelle-Filtu road, 20 May 1982, Friis, Mesfin & Vollesen 3138; Negelle, Borana Awraja, 8 May 1987, Puff et al. 870508; Negelle, Borana Awraja, 20 Dec. 1998, Ensermu & Aschalew 4012; Malka Guba, near Dawa Parma River, 30 May 1996, Ensermu & Dessalegn 3798; Arsi Region: Sodere, 27 Jan. 1980, Mesfin 925.Kenya. Eastern Province: Marsabit District, Koloba Hill, Jan. 1990, Powys 927.ACKNOWLEDGMENTSWe acknowledge discussions and comments from Dr K. Vollesen and S. Simiyu at various stages in this study. The curators of EA, ETH, MAL, NU and WAG herbaria are acknowledged for allowing the use of their specimens for this study. The study was undertaken as part of M.Sc. funded by Swedish Agency for Research Cooperation (Research Programme on Sustainable Use of the Dryland Biodiversity, RPSUD).REFERENCESAgnew, A. D. Q. & S. Agnew (1994). Upland Kenya Wild Flowers. East Africa Natural History Society, Nairobi.Bates, P. (2000). 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