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ISSN OOel-6799 Acta Phytotax. Geobot. 48 (2): 151-158 (1997)

- Anatomy and Systematics in the ofer C. Iucanusianus Complex (Costaceae)

H. O. EDEOGAi" and B. E. OKOLI2

i*Department ofBotany, Edo State Uitiversity, P. M, B. 14, Ekpoma, Edo State, Mgeria; 2Department ofBotany, [J}iiversity ofPort-Harcourt, P. M, B. 5323, Port-Harcourt, IVigeria

Abstract. The anatomy of larnina, and root of three taxa of Costus was investigated with a view of establishing interrelationships among the previously confuseci of the . Differences in features of the vegetative anatomy suggest that a separate specific status for C. cijler and C, lucanusianus is justified as opposed to the conspecific treatment given to them by previous researchers. A hybrid collection of these Costus species does not merit a separate specifie status, although it possessed distinct anaternical features, The systernatic irnportance of this investigation is clear since a 3-4

layered spongy mcsophyll of C, cijlar is different from a 4-5 layered spongy mesophyll of C. Iueanasianus and the 2-3 layers of the putative hybrid, Similarly the hexarch, nonarch and polyarch vascular bundle of the root of C, wfler, C. I"canusianus and the Costus hybrid respectively, are arl distingrLishable attributes of these taxa that are important in systematies.

Key words: anatomy, Costus, Costaceae, lamilla, rhizome, root,

Received June 28, 19PZ accepted Scptember 1, 1997

The genus Costus L. is a member of the lily family, Costaceae (K. Schum.) Nakai, a former subfamily of , treated by Hutchinson (1959) merely as a tribe but which merits separate family status (Tomlinson, 1969), The Costaceae were first raised to the rank of a family by Nakai (1941). The family is one of the most distinctive and isolated members of the order (Dahlgren et al., 1985). Before the elevation to family status, Engler and Prantl (193e) recognised Costoideae as a subfamily under Zingiberaceae. Several anatomical and morphoiogicai features support this isolated position including often well developed aerial shoots with distinct, rigid and commonly branched stems. The are inserted in a low spiral with divergences unlike any other flowering . The sheats are always tubular, and the plants are arornatic, rhizomatous except in Costus englerianus. The production of new plants by asexual reproduction (apomixis) has recently been reported in Costus lucanusianus (Tomlinson, 1969; Edeoga and Okoli, 1991, 1996). The family Costaceae consists of four genera and approximately 200 species (Airy Shaw, 1973). The genus Costus is the largest in the family with about 150 species that are mainly tropical in distribution (Humphries, 1985; Hickey and King, 1981). In addition to its small size and distinctive morphology, Costus has a pantropical distribution especially in the forest

'Author for correspondence.

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and savanna regions. In West , there are about 11 species of Costus with nine species recorded in Nigeria. There are three in the northern part of Nigeria and six, including C. tijkr Ker Gawl and C. Iucanusianus J. Braun & K. Schum, in the southern part of Nigeria (Hutchinson and Dalziel, 1968). Some specie of Costus from West Africa, namely, C. cij}7r, C. detytelli and C lucanttsianus, are considered to constitute a species complex, the Costus cefler complex by some workers including Oteng-Yeboah (1981). This is as a result of the reported occurrence of continuous variations in the morphological and cytological features of these plants. This systematic problem demands a thorough investigation into the lamina, rhizome and root anatomy in relation to systematics in this cornplex. Tomlinson (1956, 1962, 1969) presented some reasons for treating the Costaceae as a separate family based on a number of anatomical characters of the vegetative organs. Some of these diagnostic characters were summarized by Tomlinson (1969). They include the uniseriate filamentous hairs, isodiametric, irregular, polygonal or transversely extended thin-walled epidermal cells, tetracytic stomata (stomata with subsidiary cells), slightly asymmetrical nature of guard cells, colourless and continuous hypodermis, clearly differentiated cortex, well-developed vascular-plexus at each node, restriction of si]ica to internal tissues close to vascular bundles, solitary bodies stellately-spherical or druse-like, vessels restricted to roots, etc, Edeoga (1991) compared the epidermal morphology of the Costus cofizr- C. Iucanusianus complex and assessed its relevance in systematics, noting that multicellular and unicellular trichomes differentiate the two taxa. Other available information on the morphology of CostL{s species include those of Morton (1968) and Edeoga and Okoli (1991, 1996). Apart from the preliminary investjgation of Tomlinson (1969) on the anatomy of some members of Cos.taceae no other information on the lamina, rhizome and root anatomy of these Costus species have been documented to the best of our knowledge. The use of anatomical features in the systematic consideration of different taxa is no more a rare event by different authors. The work of Ayensu (1972) in Dioscoreaceae, Gibson (1981) in Anacardiaceae, Decadas and Beck (1972) in Rosaceae and Leguminosae, Heo (1996) in Monimiaceae,,Metcaife and Chalk (1950) in,Dicotyledons as a whole, Tomlinson (1969) in Commelinales and Zingiberales are classical examples. Emphasis is on these Costus species due to the problems of identification by field collectors despite the medicinal and ornamental values of these plants elaborated by Oliver (1959), Williarnson (1970) and Burkill (1985).

Materials and Methods

Mature and fresh lamina, rhizornes and roots of Costus cijlar, C lucanusianus and a putative Costus hybrid (Edeoga and Okoli, 1996) obtained from living specimens of each plant were fixed in FAA (1:1:18 40% formaldehyde: glacial acetic acid: 70% ethanol (vlv)) for 48-72 hours. These were then rinsed in several changes of distilled water and

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passed through alcohol series (30, 50, 95, 100%). The dehydrated materials were infiltrated with wax by passing through different proportions of alcohol and chloroform (vfv 3:1, 1:1, I:3). As the chloroform gradually replaced the alcohol, pure chloroform and wax were added in the bottles. The idea was to gradually infiltrate the tissues with wax which would be hard enough for microtoming. The bottles were then left on a hot plate (37-40C) for 24 hours before being transferred to the oven (58-60C). This step was designed to evaporate the chloroform. The wax having reached its melting point completely infiltrated the tissue in it. After a period of 2-3 days with constant addition of wax by the use of metal,moulds and melted wax. The moulds were later removed and the specimens with the wax-cube were trimmed and sectioned using a Reichert rotary microtome at 20-24ym. The ribbons were placed on clean slides, smeared with a thin film of Haupt's albumen, and allowed to dry and drops of water added prior to mounting. The slides were placed on a hot plate at 40C for a few minutes to allow the ribbons to expand and were stored overnight. The slides were immersed in pure xylene for 2-5 minutes in a solution of xylene and absolute alcohol with 1:1 ratio (v!v) for a few minutes. The slides were then transferred to another solution of xylene and alcohol in the ratio 1: 3 (vlv) for a few minutes, to 95%, 90%, 70% and 50% alcohol. Drops of alcian blue were put on the specimens for five minutes, washed off with water and counter-stained with safranin for two minutes, then dehydrated in a series of alochol 50%, 70%, 80%, 90%, xylenelabsolute alcohol solution (i.e. 1:3 and 1:1 vlv), and pure xylene at intervals of a few seconds and mounted in Canada balsam. Coloured photomicrographs were taken using a WILD-MPS camera fitted with Leitz LaborluX-1.2 mlcroscope.

Results

The anatomical features of the lamina, rhizome and root of the Costus species and the putative hybrid are summarized in Table 1 and illustrated in Figs. 1 and 2. In outline, the leaf is isolaterally flattened with the midrib and major veins being prominent abaxially. The spongy mesophyll which is confined to the centre of the lamina is composed of 4-5 layers of cells which are irregular in shape in C. Iucanusianus (Fig. Ib). The Iamina in C. cijbr is characterized by well-developed sclerenchymatous cells (Fig. Ia) and the presence of mesophyll that is 3-4 cells thick outside the midrib and up to 8 cells thick within the midrib. The mesophyll cells in the hybrid are larger than in the putative parents and are 'Both2-3 cells thick outside the midrib but up to 6 cells thick near the midrib. the uppex and lower epidermis are made up of a single tier of cells in the three collections studied. The vascular bundles are bicollateral in the three taxa. The smallest bundles are composed of 1-3 tracheids and few phloem elements. In each bundle, the xylem pole consists of tracheary elements. The midrib is very conspicuous. The abaxial surface in C. Iucanusianus is concave. In C. a,fer, the abaxial surface is also convex

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TABLE1. Anatomical features of the lamina, rhizome and root in the Costus species investigated.

Characters C. dEfer C. I"can"sianus Costus hybrid

Lamina: Spongy mesophyll 3-4 layers 4-5 layers 2-3 layers Vascular bundle Bicolateral Bicolateral Bicoiateral Rhizome: Sclerenchyma Almost single layer 2-3 layers 2L3 layers Xylem vessel SmallVaried LargeMostly LargeMostly Starch grains in shape slipper and oval slipper and oval Root:

Vascular bundle Hexarch and small Nonarch and Iarge Polyarch and 1arge Casparian strip Poor]y defined Clearly defined Clearly defined Endodermis Poerly differentiated Wel]-developed Well-developed

Passage cel]s Absent Present PTesent

FIGs. 1. Anatomical sections of lamina, rhizome and root in Costtts. a. Transverse section (TS) of lamina of C, tifkr, showing well-develeped sclerenchyma and bieollateral vascular bundle, × 130, b. TS ef Iamina of C. tucanusianus'. Note the absence of palisade mesephyll. × 130, c. TS of root of C, tucanusianus showing nonarch vascular bundle. × 50, d, Section of root of Costus hybrid showing well-developed and cielimited endoderrnis. Note presence of passage ceLls. × 130.

while the adaxial is concave. The same shape was also observed in the

Costus hybrid. The vascular bundles in the of the three taxa are different from one another (Figs. 2a and 2b). The xylem vessels are smaller in

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FIGs. 2. Anatomical sections of rhizome and root in Costus, a. C, cijiir with a single vascu],ar bundle in rhizome, Note that sclerenchyma is single layered and completely surrounded by the vascular bundle, × 130, b. C. I"canusiantts with,asingle yascular bundle in rhizome, Sclerenchyma is 2m3 layered and compietely surrounded by the vascular bundle. × 200. c. Section of root of Costus hybrid., showing that the Casparian suip and endodennis are well-defined, and passage cell conspicuous. × 200. d. Section of root of C. tucanusinnus, showing the Casparian strip and we!1 definedendodermis. × 200,

Costus cder and a sclerenchymatous layer surrounds the phloem and xylem. The vessels in the rhizomes of C. Iucanusianus and the Costus hybrid are surrounded by a two-three layered sclerenchyma (Fig. 2b). The phloem in each taxon occupies a relatively small region of the vascular bundle. Interesting differences are also found in the shape of starch grains present in the rhizomes of these Costus species. In C. coflar, simple, oval, slipper, circular and irregular shaped starch grains are present (Fig. 2a), In C. I"canusianus, slipper and oval shaped starch grains were predominant while the Costus hybrid is characterized by the presence of all the types of starch grains in the rhizome of C. tijler and C. Iucanusianus. The root in Costus cofizr has six xylem strands alterna･ting with six phloem strands (hexarch). The endodermis is not clearly differentiated, The vascular bundles in the root of C. Iucanusianus are larger than those of C cofler. In C. Iucanusianus the vascular bundles are nonarch, and the endodermis is well-developed with the Casparian strip clearly defined (Fig, 2c). In the Costus hybrid, there are 22 xylem strands alternating with 22 phloem (polyarch). The endodermis is well defined and clearly delimited from the rest of the root cells. The Casparian strip with passage cells are

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conspicuously defined (Fig. 2d).

Discussion

The Costus species investigated exhibit a wide variety of 1amina, rhizome and root anatomical features. C. coflar, C. Iucanusianus and the hybrid collection each have a characteristic vegetative anatomy that could be used to distinguish it from other members of the Costaceae. The Costus species studied, however, appear to be a homogenous entity united by a series of lamina, rhizome and root anatomical characters, including isolaterally flattened lamina, bicollateral vascular bundle, presence of starch grains, and presence of tracheary elements. These features of the lamina, rhizome and root anatomy endorse the opinion of Oteng-Yeboah (1981) that some species of Costus from West Africa constitute a species complex, the Costus tefLrr complex. Differences in vegetative anatomy among such members of Costaceae as Costus and Ttipeinochilus (Tomlinson, 1956) have received some emphasis in phylogenetic discussion. As regards epidermal and vegetative morphology, the Costus species investigated are heterogenous. Variation in the epidermal and floral morphology of C. qfer, C. Iucanusianus and the putative hybrid (Edeoga, 1991; Edeoga and Oko]i, 1991, 1996) has also received too much attention from systematic point of view. Most probably, these differences are related to differences in habitat, C. cijbr preferring extrernely to shady environment than C. Iucanusian"s and the putative hybrid. The anatomical structures of the lamina, rhizome and root of the Costus hybrid strongly resemble that of C lucanusiantas. Moreover, in 'are both taxa the shapes of the starch grains almost similar. Thus vegetative anatomy of the Costns hybrid suggests that C. Iucanusianus and the hybrid should be accommodated in the same supraspecific taxon. Further studies are needed to show whether this will be at a subspecies level within C. Iucanusianus or within a separate species of Costtts. Edeoga and Okoli (1996) have suggested that apomictic event observed in the C. ceflar-C. tucanusianus complex has led to intermediacy in fioral features among the Costvts collections they investigated. The opinions of different authors based on differences in morphological and anatomical structures also point at the fact that such taxa in which this type of intermediary exists should be treated as either sister taxa or even contaxa, i.e. conspecific, congeneric, etc. (Oteng-Yeboah, t981; van Heel, 1992). However, in as much as a separate specific status of C. Iucanusianus and C. cofler is justified, it is not justified for the Costus hybrid. Apart from macromorphological similarities, the three taxa of the Costus vary in thejr basic lamina, rhizome and root anatomy especially in the nature of spongy mesophyll, number of vascular bundles in root and the morphology of starch grains (Table 1). Temlinson (1957, 1962, 1969) presented some reasons for treating Costaeea.e as a separate family from Zingiberaceae, based on vegetative anatomy of some members of the Scitamineae. The lamina, rhizome and root anatomy･ of these Costus species investigated seems to agree with the

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Tomlinson's (1956, 1962, 1969) reasons for supporting the raising of the genus to the rank of family by Nakai (1941). However, more studies are still required in such areas as cytology, palynology and phytochemistry before generalizing and upholding all the observations and suggestions 'Ilie arising from our present investigation. systematic importance of this study could be seen since the 3-4 layered spongy mesophyll of C. ofer is different from the 4-5 layered spongy mesophyll of C lucanusianus and the 2-3 layered spongy mesophyll of the putative hybrid. Similarly, the hexarch vascular bundle of the root in C. afer is different from the nOnarch and polyarch of C. Iucanusianus and the Costus hybrid respectively. Hence each taxon could be distinguished from one another on the basis of these anatomical characters. Tomlinson (1969) outlined some diagnostic features of the leaf, stem, rhizome and root anatomy in the Zingiberales as a whole but did not specify how these could be used in resolving critical systematic problems as in the presently investigated C afar-C lucanusianus complex. The need to incorporate information on the lamina, rhizome and root anatomy in systematic consideration of Costus species is therefore incontestable.

References

Airy Shaw, H. K, 1973. J. C. Willis' a Dictionary of the Flowering Plants and Ferns. 8th ed. Cambridge University Press, Cambridge, Ayensu, E, S, 1972. Anatomy.of the VI. Dioscoreales. Clarendon Press, Oxford, Burkill, I. H. 1984. The Useful Plants ef West Tropical Afriea Vol, 1, Families A-D. Royal Botanic Garden, Kew, Dahlgren, R, M. T., H. T. Clifford and P. F. Yeo. 1985. The Families of the Monocotyledons. Structure, EvQlution and Taxonomy. Springer Verlag, Berlin. Devada, C, and C, B, Beck. 1972. Comparative morphology of the primary vascular systems in some species of Rosaceae and Leguminosae. Amer, J, Bot, 59: 557-567. Edeoga, H. O, 1991. Comparative morphology of the leaf epidermis in Costus cijler-C tucanusianus (Costaceae) complex and its systematic importance. Nig. Ann, Nat. Sci. 1: 25-30. and B. E. Okoli. 1991. Comparative morphology of the vegetative and floral characteristics of Costus qfer- C, tucanasianus (Costaceae) complex, J. Exp. Appl. BioL 3: 139-153. and . 1996. Apomictic behaviour in Costus toflar-C. tucanusianus (Costaceae) complex in Nigeria. Feddes Repert. 107: 75-82. Engler, A, and K. Prantl. 1930. Die NatUrlichen Pfianzenfamilien. Reihe: Scietamineae. 2nded, 15a:547-640. Gibson, A. C. 1981. Vegetative anatomy of Pachycorrnus (Anacardiaceae), Bot. J. Linn. Soe, 83: 273-284, Heel, W. A. van. 1992, Roral morphelogy of Stemonaceae and Pelltastemonaceae. Blumea 36: 481-499. Heo, K, 1996. Woed and bark anatomy of Hortonia (Monimiaceae). Acta Phytotax. Geobot. 47: 53-59. Hickey, M. and C. J. King. 1981, 100 Families of Flewering Plants. Cambridge University Press, Cambridge, Humphries, C. J, 1985. Zingiberaceae. Ginger, Cardamon and Turmeric. Iit V, H. Heywood (ed.), Flowering Plants ef the World. Equinox, Oxford, pp, 296-298.

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」 Hutchinson ， ． 1959， Families of Flowering Plants． Vo1 ．2． Monocotyledons ． 2nd ed ． Oxford and M ．」． Dalzie1， 1968， Flora Qf West Tropical Africa． Vo1 ．3． Crown Agents for Overseas Govem 皿 ent and Administration， London． Metcalfe C ． R ． and L ． Chalk． 1950． Anatomy of Dicotyledons ， ．Vol ．1and 2． Clarendon Press， Oxford ． − Nakai ，T ． 1941． Notulae ad plantas Asiae orientalis （XVI ）． J． Jap． Bot ．17： 189 210， ’ ． − Oliver， B ． 1959． Nigerias U seful Plants II、（1）． Njg． Field 24： 13 34， − Oteng Yeboah， A ． A ． 198ユ． Morphelogical variation and karyotype studies in Costus afer complex （family Zingiberaceae）． Bu11． Sc． Assoc ． Nig．7： 1 Tomlinson ， P ． B ． 1956． Studies ｛n the systematic anatomy of the Zingiberaceae ， 」． − Linn． Soc．，Bot，55： 547 592． ． 1962． Phylogeny of tllc Scitamineae−morphological and anatomical − considerations ． Evolution 16： 】．92 213． Williamson， K ． 1970． Some food plants names in the Niger Delta． InteL J． Amer ， − Ling．36： 156 167，

摘 要

H E ・ ・ ． ．Edeogai B 、E ． Okoli2： Co5傭 ψ r C ． lucanusianus complex （オ オホ ザ キ ア ヤ メ 科 ）の 解 剖 と分類

こ れ ま で 分類 が 混乱 し て い た オ オ ホ ザ キ ア ヤ メ 属 の 3 分 類 群 の 関係 を 明 ら か に す る た め

い に，葉身，根茎 ，根 の 比 較解 剖 を お こ な っ た 。 解剖学的 な違 に よ っ て ， こ れ ま で 同種 と し

て きた 考 え に 反 し て ，Co 鋤 5 ψ 厂 と C ． lucanusianusは 別 種 と す る こ と の 妥 当性 が 示 さ れ

た 。 しか し，こ れ ら の 種 の 交雑 個体 は，解剖 学 的 な差異 は あ る も の の ，別 種 とす る必 要 は な

い こ の に よ れ ば の は afer で − 。 研 究 ， 葉 肉 厚 さ Costus は 3 4 細 胞 層 で ，C ．lucanusuanus で は 4 〜5 で で は 2 〜3 細 胞層 ， 推 定交 雑 個体 細 胞層 で あ っ た 。 根 の 維 管束 系 は C ．afer で は 6

原型 ，C ，1”casianus で は 9 原 型 ，交 雑個 体 で は 多原 型 で ， こ の 形 質 は 種 を区別 す る 重 要 な

特 徴 を示 す 。 1 2 エ ー ・ ー ー （ナ イ ジ ェ リ ア ， ド州 立 大 学植 物 学教 室 ； ナ イ ジ ェ リ ア ， ポ ト ハ コ ト大 学植 物学 教 室）

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