Studies in the Hypoxidaceae. I. Vegetative morphology and anatomyt

The leaf and corm morphology and anatomy of representatives of the genera  Spiloxene , Pauridia and Empodium were studied. The corms are annual and tunicated, except in the group Aquaticae of Spiloxene . They are swollen stems consisting of a number of internodes. In Spiloxene and Pauridia the roots grow from the base of the corm, while in Empodium they develop from the sides. The epidermis of the older corms is replaced by several layers of thin-walled cork. Characters of the corm coverings are used to divide  Spiloxene into six groups. Four leaf forms are recognised namely carinate, terete, canaliculate and plicate. Multicellular processes and unicellular hairs occur occasionally. The leaf stomata are paracytic. Most species have mucilage canals containing pectic compounds of mucopoly­saccharides. The vascular bundles have complete or incomplete bundle sheaths and larger bundles have sclerenchyma caps.


INTRODUCTION
The Hypoxidaceae is represented in South Africa by five genera, namely: Hypoxis L., Rhodohypoxis Nel, Spiloxene Salisb., Empodium Salisb. and Pauridia Harv. Empodium is sometimes included in the tropical genus Curculigo Gaertn. and Spiloxene in Hypoxis. Hypoxis and Rhodohypoxis occur mainly in the sum mer rainfall areas. Spiloxene and Pauridia are confined to the winter rainfall region while Empodium is widespread from the south-west Cape to the Transvaal Drakensberg.
The genera are in need of taxonomic revision and the aim o f this study is to compare anatom ical and morphological features o f the genera Spiloxene, Empodium and Pauridia with special reference to characters that could be of taxonomic value. This paper covers the corm and leaf, while a second will deal with the floral anatomy.

MATERIAL A ND METHODS
Fresh material was collected in the field and voucher specimens are housed in the Governm ent Herbarium, Stellenbosch (STE).
Fresh The leaves of the following species were studied: Spiloxene aquatica, S. capensis, S. flaccida (Nel) Garside, S. minuta (L.) Fourc., S. ovata (L.f.) Garside, S. schlechteri (Bol.) Garside, S. serrata (Thunb.) Garside, Empodium plicatum, Pauridia longituba M. F. Thompson and P. minuta. The Spiloxene species were chosen to represent the groups defined by Nel (1914a). The material used for the cross-sections of the leaves was taken from the middle of the first two or three foliage leaves (i.e. excluding the completely sheathing leaves). K arpechenko's modification of Navashin's fluid was found to be the best fixative. The fixed material was embedded in paraffin wax, sectioned at 15-20 fim, stained with safranin and fast green and mounted in Depex.

THE CORM
(1) Morphology (i) General Two main types of underground storage organs are found in the Hypoxidaceae of Southern Africa: an erect, tuberous perennial rhizome in Hypoxis and Rhodophypoxis and an annual corm in Spiloxene, Empodium and Pauridia.
The annual corm is a swollen stem consisting o f a number of internodes formed by the apical meristem of the shoot. There is no normal elongated stem and the cataphylls, foliage leaves and axillary inflore scences arise directly from the upper half o f the corm. The branching is, therefore, monopodial and the growth is usually orthotropic. In S. aquatica the growth is usually somewhat plagiotropic i.e. horizontal ( Fig. 1 A), a condition brought about by the horizontal growth of the apical bud. This differs from the plagiotropic growth o f the Iridaceae where the branching is sympodial and a new corm is formed from the uppermost axillary bud, the terminal bud having produced a flowering shoot in the previous season (Troll, 1937). E together below by a ridge (Fig. 1 Da) and remaining attached to the basal disc, branched or evenly latticed at the base of the corm or reticulate, ending in a point above ( Fig. ID & IDa), e.g. S.flaccida.
(6) Pectinatae N e': Corm subovate, with basal discs, fibres pectinate, not branched, apiculate, spreading at the neck of the corm, normally adhering to the discs, if separated from the disc then tending to remain joined together at the base (Fig. 1E), e.g. S. schlechteri.

S. minula.
With the exception o f the Serratae, I regard these groups as well-defined. The group Serratae, which Nel separates from the Ovatae mainly because o f its serrate leaves, is not considered worthwhile upholding as this character is variable in several o f the groups. In the Stellatae, for example, S. capensis has serrated leaves while the leaves of all the other representatives are entire. The Serratae should thus be placed under the Ovatae.
The description by Nel (1914a) of the structure of the corm contains certain anomalies. He uses the term Zwiebelknolle or bulbous corm and states that this organ consists o f leaf sheaths (p. 244 " besteht aus Blattscheiden"). The corm is, in fact, a swollen stem usually covered with the vascular remains of old leaf sheaths. Nel also makes the erroneous statement that the bases of the leaves may remain as discs (" Scheibe" ) A new corm is formed annually on top o f the old one, the old shrunken storage organs of previous seasons remaining at the base as discs (Fig. 2B). The corm is either naked (in which case it is referred to as a tuber) or it has a fibrous covering o f tunics formed from the remains of the sheathing foliage leaf bases and cataphylls.

(ii) Spiloxene
As Nel (1914a, b) has shown, Spiloxene (=Ianthe) can be divided into groups on the basis o f the fibrous tunics of the corms. He recognized seven groups of which six have tunicated corms and one a naked fleshy tuber.
(4) Stellatae Nel: Corms with basal discs, fibres easily separable from the old corm, much branched below the middle or in the lower third and ending in numerous pungent ribs pointing upwards ( Fig. 1C & ICa), e.g. S. capensis.
(5) Flaccidae Nel: Corm subovate with basal discs, the upper (inner) fibres adnate to the corm, the lower (outer) fibres easily separable but adpressed to the body of the corm, spreading above, joined Autumn Spring but this could possibly be a misprint for " Scheide"sheath. In his key and description of the groups Nel describes all the corms except those of the Aquaticae, as " discoideus" . This presumably refers to the old corms which remain as discs at the base o f the func tional corm. His description of the discs as two lamellae with a nutrient rich space in between fits this concept. In his description of the groups (1914b) he does use the terms subdiscoid and subovate in addition to discoid. In autum n the new roots grow from the top of the swollen corm, run under the tunics (where present) to the widest part of the corm and pass through the reticulations ( Fig. 2A). After further development during the winter growing season, it becomes clear that these roots arise from the base o f the present season's new corm. By spring the young corm has become the main storage corm with the roots growing from its base (Fig. 2B). The old corm remains as a disc. In most Iridaceae, by contrast, the roots arise from the base of a swollen storage corm and remain functional the year after this corm has acted as a storage corm (Troll, 1937;De Vos, 1970).
The position of the roots is the same in the autum n/ winter-flowering 5. schlechteri and the springflowering S. aquatica and 5. capensis.
The old storage corm provides the nutrients for leaf formation and, in the autumn flowering species, for flowering.
An observation by M arkotter (1936) that 5. schlechteri frequently reproduces vegetatively was confirmed. In this species new corms appear to develop from axillary buds on the base of corms, a phenomenon seldom observed in other members of the genus.
(iii) Empodium E. plicatum has a corm with coverings resembling those o f the Stellatae group in Spiloxene, i.e. the reticulate vascular fibres of the tunic are free at the base. The dried-up intercostal tissue o f the leaf bases tends to remain longer than in Spiloxene, resulting in a more scale-like covering, the reticulations being less obvious.
The roots do not arise at the top of the summer storage corm, but on its sides in the lower half and pass immediately through the meshes o f the tunics (Fig. 2E). The roots develop after the first good autum n rains and then the flowers appear before the leaves.
The new corm is formed by the apical meristem above the old one in a monopodial orthotropic fashion. Cormlets may be formed in axillary posi tions at the base o f the developing corm towards the end of the growing season (Fig. 2F).
Pauridia has a corm similar to that of the Flaccidae group in Spiloxene, i.e. it is covered with reticulating fibres which remain attached to the basal discs formed by the previous seasons' corms. As in Spiloxene the roots arise at the base of the developing corm (Fig. 2C).
(2) Anatomy Anatomically the corms o f the three genera, including all the groups o f Spiloxene, are very similar.
In the young corm the epidermis consists o f a single layer of thin-walled cells. Immediately interior to this is a layer of parenchyma three to four cells deep which lacks starch. The epidermis of the mature corm is replaced by several layers of thin-walled cork cells arising from a phellogen situated in the sub-epidermal parenchyma (Fig. 3).
The main body o f the corm is made up of a densely packed ground tissue of parenchyma cells rich in starch. Throughout the ground tissue there are mucilage canals, which are not associated with the vascular bundles. Canals are more numerous in the m ature corm than in the younger one. As in the leaf, the canals are surrounded by radially flattened cells. Scharf (1892) found that in Hypoxis the canals arise schizogenously near the growing point and that they have no membrane but are embedded in a ring of smaller cells.
Numerous vascular bundles run irregularly through the ground tissue and supply the roots and leaves. The bundles normally have three to four tracheal elements with ring or spiral thickenings.

THE LEAF
(1) General The leaves in the Hypoxidaceae are radical with a more or less sheathing base. The outerm ost leaves of each year are reduced to bifacial leaves or cataphylls. The foliage leaves are generally bifacial, but may be unifacial and terete. In the bifacial forms the lip is usually unifacial and represents the " Vorlauferspitze" o f Troll (1939).
The following description relates to the species listed under M aterial and M ethods and the main features are summarized in Table 1.
(2) Anatomy (i) Epidermis The leaf epidermis of all the species studied consists of a single layer of largely thin-walled cells, covered by a smooth thin cuticle. In surface view the cells are rectangular, elongated in the direction o f the long axis of the leaf, and have straight walls. The walls of the epidermal cells along the leaf margin and keel are sometimes thickened as in S. capensis (Fig. 4). Some epidermal cells may be enlarged to form bulliform cells as in the angle of the V o f many o f the carinate leaf forms e.g. in S. serrata and P. minuta and in the folds in Empodium. In most o f the bifacial leaves the cells on the adaxial surface are larger than those on the abaxial side, but are not enlarged to form bulli form cells (e.g. S. ovata).
In Spiloxene and Pauridia the stom ata are scattered on both surfaces, while in Empodium they are confined to the intercostal zone. In all three genera they are paracytic, i.e. with a single subsidiary cell parallel to and flanking each of the guard cells (Fig. 5E). This agrees with the observations of Stebbins & Kush (1961), but Shah & Gopal (1970) found that in Curculigo orchioides Gaertn. sixty per cent o f the stomata were tetracytic (i.e. with four to six subsidiary cells). Shah & Gopal report further that in this species the epidermal cells are not rectangular, as in the hypoxid leaves which I have studied, but have an irregular shape and arrangement.
The structure o f the guard cells varies only slightly in the species studied. The anterior cavity is welldeveloped due to an outer ledge formed by the cell wall and cuticle. The posterior cavity is poorly   On the ridges o f the folds of Empodium plicatum are unicellular hairs surrounded at their bases by slightly raised epidermal cells (Fig. 5B) which do not form part of the hair. These hairs resemble the cushion macro-hairs o f the Gramineae (Metcalfe, 1960). The cuticle, thin throughout, continues over the trichomes.

(ii) Mesophyll
The cells o f the mesophyll are mostly irregular, rounded to oval in shape, rich in chloroplasts and with large inter-cellular spaces. Scattered cells contain raphides and lack chlorophyll. The bifacial leaf forms of Spiloxene and Pauridia show a tendency to form pallisade tissue towards the adaxial side. There is no pallisade in the plicate leaf of Empodium. The cells on either side o f the midrib in the carinate leaves may be enlarged and without chlorophyll as in S. minuta and S. serrata. In the terete leaf of S', aquatica the meso phyll has an aerenchymatous central part.
The mucilage in Spiloxene and Pauridia is confined to mucilage canals which lie adaxially to, or, in terete leaves centripetally to, the larger vascular bundles (Figs. 4 & 6). The canals are bounded by radially flattened cells. In Empodium the canals are scattered in the mesophyll independently of the vascular bundles. Mucilage canals are lacking in the leaves o f S. aquatica, although they are present in the corm.
In sections of fresh leaves the mucilage showed no reaction with Schultze's solution and gave a reddish colour with ruthenium red, indicating that the mucilage is a pectic and not a cellulosic substance. Tests with Alcian blue indicated the presence of mucopolysaccharides. Scharf (1892) records that the " colourless slime turns hard and brown when treated with alcohol" . I found that the mucilage was dispersed by fixatives containing alcohol and that it is soluble in alcohol and not precipitated by it. Karpechenko's fluid (without alcohol) did not effect the position of the mucilage.

(iii) Vascular bundles and sheaths
Cheadle & Uhl (1948) described five different types o f vascular bundles found in monocotyledons. The larger vascular bundles of the species studied fall into their Type IV which possesses one or two large central metaxylem elements. These elements are tracheids with ring or spiral thickenings. Cheadle (1968) recorded primitive vessels with scalariform perfora tion plates in the roots of Hypoxidaceae but not in the rest o f the plant. The phloem consists of sieve tubes, com panion cells and phloem parenchyma, irregularly arranged. The bundles often have sclerenchyma caps at one or both poles (Fig. 4).
The smaller bundles generally lack sclerenchyma and resemble Type I of Cheadle & Uhl (1948) which has no larger metaxylem elements.
The vascular bundles are surrounded by a single layered bundle sheath of parenchymatous cells with few or no chloroplasts. The sheaths may be (i) complete, i.e. continuous round the bundle, e.g. S. capensis (Fig. 4), or (ii) incomplete, i.e. interrupted by sclerenchyma caps at one or both poles, e.g. in S. flaccida and Empodium plicatum.
When the bundle sheath continues round the mucilage canal to the adaxial epidermis, as in S. capensis (Fig. 4), it may be regarded as a bundle sheath extension.
(3) Leaf forms On the basis of morphology and general anatomy the leaves of the species studied may be divided into four groups, namely, carinate, canaliculate, terete and plicate (See Table 1).
The greater part o f the length of the leaf is V-shaped in cross-section with a large vascular bundle in the angle o f the V, forming a definite midrib and some times a marked keel as in S. capensis and S. flaccida. S. serrata and S. minuta do not show as definite a keel but the central bundle is still larger than the rest of the bundles. In S. serrata and sometimes in S. minuta the cells o f the central adaxial mesophyll are enlarged (up to 50 /zm) and lack chlorophyll. The mucilage canals lie adaxially to the larger vascular bundles, except in S. minuta where they are lacking. The bundles may have sclerenchyma caps as in S. capensis. The sheathing part of the leaf does not have a marked midrib. The tip o f the leaf is a unifacial " Vorlauferspitze" (Troll, 1939). This is the most common type o f leaf found in Spiloxene. Geerinck (1968), in comparing the leaves o f Haemodoraceae and Hypoxidaceae, regards this as the typical hypoxidaceous leaf. (ii) The canaliculate leaf Example: S. ovata. (Fig. 6D).
The leaf is bifacial, U-shaped in cross-section and without a midrib.
The adaxial epidermal cells are larger (about 50 /xm) than the abaxial (about 30 /xm). The mesophyll consists o f more or less even-sized irregular cells without the formation of a palisade. Mucilage canals bounded by radially flattened cells lie adaxially to the vascular bundles. The 15-30 bundles are more or less equal in size throughout the width of the leaf or the alternating ones are somewhat larger. There is little or no sclerenchyma associated with the vascular bundles.
This type of leaf is less common than the carinate leaf and, among the species studied, only occurs in S. ovata. Garside (1942) describes this type in S. canaliculata Garside and considers the leaf shape im portant in separating S. canaliculata from S. capensis.
The leaf is unifacial with a ring o f ectophloic bundles. The peripheral mesophyll is denser than the central part which may be aerenchymatous (e.g. S. aquatica). Mucilage canals, when present, lie centripetally to the vascular bundles.
There is a gradual transition from the bifacial, canaliculate sheath to the unifacial terete part (Fig.  7a-d). The adaxial surface decreases gradually until the entire leaf surface consists o f the abaxial epidermis. In the species investigated the terete condition appears to develop differently from that in the Haemodoraceae as described by Geerinck (1968) where the leaves are " folded-welded" . Geerinck described the Haemodoraceae as having a folded (V-shaped in cross-section) basal part which fuses to form a vertically flattened part with vascular bundles alternating in direction, or a terete portion with bundles in a ring with a peripheral phloem.
The leaf is bifacial and is folded longitudinally with two or three folds on either side of the midrib.
Within the folds (ad-and abaxial) the epidermal cells are enlarged to form bulliform cells. On the ridges the the cell walls, especially the outer, are thickened. Trichomes occur along the ridges and margins (Fig.  5B). Stom ata are frequent in the inter-costal zones of both surfaces. The mesophyll is uniform without differentiation o f a palisade. Mucilage canals occur irregularly in the mesophyll and are not associated with the vascular bundles as in Spiloxene.
Unlike the palm leaf, which has a prom inent rib at each fold (Tomlinson, 1961), not every fold has a vascular bundle at the ridge. The midrib is the only large bundle in an abaxial ridge. The adaxial ridges all have large bundles. Arber (1922) said that the folds in Curculigo recurvata develop by alternating dorsal (i.e. abaxial) and ventral (i.e. adaxial) invaginations of the petiole or leaf sheath. The ventral sinuses occur between the lateral bundles and opposite the midrib while the dorsal sinuses occur opposite the main lateral bundles (cf Fig. 6F).

CONCLUSIONS
The similarity in the vegetative anatom y and morphology of Pauridia and Spiloxene suggests that these genera are closely related. Pauridia has a corm like the group Flaccidae of Spiloxene and there are no m ajor differences in the leaf structure of the two genera.
The differences between Empodium and the other two genera are greater. The corm, although super ficially similar, differs in the position o f the roots. The plicate leaf form is only found in Empodium. The mucilage canals of the leaves are scattered in the mesophyll in Empodium, while in Spiloxene and Pauridia, where present, they are associated with the vascular bundles (Table 1). The stom ata are scattered on both leaf surfaces in Spiloxene and Pauridia while in Empodium they are confined to the intercostal zones. Empodium has unicellular hairs while the epidermal processes observed in Spiloxene, were all multicellular.
Within the genus Spiloxene the nature o f the corm coverings can be used to define groups (Nel, 1914a, b). With the exception of the Serratae, which should be combined with the Ovatae, the groups of Nel are accepted.
The leaf form does not appear to be a good basis for grouping species in any of the three genera, as other wise dissimilar species can have the same leaf form. On the other hand, in the group Ovatae o f Spiloxene carinate [S. cuspidata (Nel) Garside, S. gracilipes (Schltr.) Garside], canaliculate [5. ovata, S. linearis (Andr.) Garside] and terete [S. dielsiana (Nel) Garside] forms occur. However, the type o f leaf can be used to distinguish between some closely related species such as S. capensis and S. canaliculata (Garside 1942).
A part from the fact that the internal structure reflects the outward morphology, the leaf anatomy was not found to provide characters o f taxonomic value. The presence of aerenchyma in 5. aquatica is probably a habitat response. Mucilage canals are absent from the leaves in species as different as S. minuta and S. aquatica. In Empodium however, their arrangement is characteristic and different from the other species investigated. The bundle sheaths are generally indistinct and the presence o f sclerenchyma is not constant.
The morphology o f the corm was found to provide the vegetative characters most useful to the taxono mist.