The systematic value of the leaf indumentum in Lobostemon ( Bora ginaceae )

The character states pertinent to the indumentum of Lobostemon Lehm. leaves are tabulated and discussed. Lobostemon possesses similar trichome and indumentum types as described for Echium L.. with the exception of the ‘Alpine’ indumen­ tum type. Due to the environmentally induced variation, the leaf indumentum characters of Lobostemon are viewed to be of limited phylogenetic value, although they do diagnose a number of taxa.


INTRODUCTION
In terms of Lobostemon Lehm.systematics, the most recent revision by Levyns is noteworthy because she delimits five sections based on floral characters and pre sents a branching diagram (Levyns 1934: 412) to eluci date relationships within the genus.The aforementioned constitute falsifiable hypotheses.
One of the most striking features of the herbaceous forms of the Boraginaceae is the covering of thickwalled, harsh, unicellular trichomes (Metcalfe & Chalk 1950: 945), so much so that Klotz (1959) was of the opinion that one cannot ignore these characters when studying the Boraginaceae.Numerous studies of the leaf indumentum in the Boraginaceae have been undertaken e.g.Revedin (1902), Jonova (1926), Bider (1935), Klotz (1959), Lems & Holzapfel (1968).Bramwell (1972) and Selvi & Bigazzi (2001).Yet.opinions differ with regard to the taxonomic value of these characters.Because the largely European Echium L. is considered to be a sister taxon of the South African endemic Lobostemon (Bohle et al. 2001), the focus henceforth will for the moment fall on Echium.Klotz (1959), during his revision of the genus, used indumentum and trichome characters to key out species, series and sections.Lems & Holzapfel's (1968) in depth study of the genus on the Canary Islands, leads to an optimistic view about their use, claiming that there are at least 14 criteria which can be applied in com paring the Canary Island Echium species with one anoth er, including not only the types of trichomes present, but their relative abundance, their total coverage of the leaf surface, the size of the pustules, and the orientation of the trichomes.Gibbs (1971: 31, 32), confining himself to the Spanish echiums, formed three groups based on indu mentum characters and used a number of characters to key out species.Bramwell (1972), working in Macaronesia, chose to differ and suggested that the indumen tum: 'is more or less useless and certainly of secondary importance ... in the consideration of the evolutionary and ... the phenetic relationships'.Levyns (1934), for the first time in Lobostemon, mentioned that indumentum and trichome characters are particularly influenced by environmental and tem poral factors.Failure to realise this earlier led especial ly De Candolle (1846) and Wright (1904) to describe a myriad of names, most of which were placed in syn onymy by Levyns (1934: 403).Levyns (1934), in cer tain instances, still made use of vegetative characters in her key, e.g. in the section Trichotomi Levyns.Dif ficulty in identifying species using this key invariably occurs when using vegetative characters like indumen tum or trichome type.
While it is true that like all taxonomic criteria, epider mal characters must be interpreted with great circumspec tion, Barthlott (1981) voiced the opinion that the major problem in their systematic application is that we do not yet have enough data [compare also Cole & Behnke (1975)].Despite the vast amounts of SEM micrographs published, many of the data are not comparable because of a lack of standardized terminology and often no structural interpre tation of the characters illustrated.
In the light of the aforementioned, this paper aims to contribute to epidermal related data in the Boraginaceae.Existing terminology is followed as far as possible, and for this reason is expanded in Material and methods below.Two issues are investigated, namely: 1, can SEM analyses of indumentum characters reveal states that can diagnose Lobostemon species, and 2, does the grouping of taxa based on these characters correlate with existing hypotheses of infrageneric relationships within the genus?MATERIAL AND METHODS Voucher specimens were collected for all taxa exam ined and are listed in Table 1.
Material collected from plants in their natural habitat in the field was fixed in FAA.The indumenta of the fixed leaves were studied with a JOEL scanning electron micro scope (SEM).using secondary-electron detection and an acceleration voltage of 4-5 kV.All fresh material used in the SEM study was collected during the peak flowering season between August and October.Additional observa tions were made with a light microscope on herbarium specimens as well as on potted plants.In the latter case, both young to mature leaves were analysed.Lems & Holzapfel (1968) and Bramwell (1972) iden tified three basic trichome types in Macaronesian species of Echium: Glandular trichomes (Gl) occur mainly on the abaxial surface of the midrib or more rarely over wide areas of the abaxial surface of the leaf in most of the Macaro nesian species of Echium.Selvi & Bigazzi (2001) distin guished two types of glandular trichomes in the genera of the tribe Boragineae: Type 6 trichomes consisting of a single stalk cell, and Type 7 trichomes consisting of three or more stalk cells.

Terminology
Simple trichomes (Si) correspond to the Type 3 tri chomes of Selvi & Bigazzi (2001).They are mostly short, stiff, hollow trichomes without a large pustular base, and are present in the cotyledons of all the Echium species.However, they do not persist into the adult stage in all species.Simple trichomes occurring in mature leaves are usually short, often curved and closely appressed to the leaf surface [the appressed trichomes correspond to the Type 2 trichomes of Selvi & Bigazzi (2001)].Though the base of the trichome is usually swollen, there is generally little or no cell differentiation of the surrounding epidermal cells.Lems & Holzapfel (1968) record simple trichomes longer than 400 mm always possessing one or more rows of dif ferentiated cells around the base.It is well established that the number of rows of epidermal cells involved in the pus tule formation in the Boraginaceae is a product of environ mental and temporal variables.
Pustular trichomes (Pu), a term coined by Lems & Holzapfel (1968), correspond to the Hiigelhorsten of Bider (1935), nodular bristles of Metcalfe & Chalk (1950), Hockerborsten of Klotz (1959) and Type 1 trichomes of Selvi & Bigazzi (2001).One or more concentric rings of strongly differentiated epidermal cells which contain cystolith-like structures and whose walls are strongly im pregnated with calcareous material surround the bases of the hollow, pustular trichomes.In very large trichomes, some of the cells of the upper palisade layer of the mesophyll are also involved in the pustule structure and may also be calcified.Uphof (1962) referred to these subepidermal areas as pedestals.Lems & Holzapfel (1968) furthermore discerned four main indumentum types in the Boraginaceae: Spinous indumentum (Sp) consists of stiff spines and is found on the leaf surface or is often confined to the margins and the midrib of the leaf; Appressed to ascending silky indumentum (Ap).Here the leaf surface is covered with a dense layer of appressed trichomes that are either simple or with small basal cells; Umbonate indumentum (Um), spinous, consisting of relatively sparsely distributed pustular trichomes with a large, round, basal region on the otherwise glabrous leaf surface.I designate the term umbonate to describe this type of indumentum.Klotz (1959) referred to these as  diskusartig bases.Selvi & Bigazzi (2001) referred to the trichomes forming this indumentum type as Type 4 tri chomes; Alpine indumentum (Al), dense, ascending to erect, with long trichomes with small bases, found only in subalpine zone species.
Trichome and indumentum types were identified according to those recognized by Lems & Holzapfel (1968).All observations of indumentum presence, abun dance and type were confined to the surface of the lamina (excluding margins).Leaves were considered to be glabrous when no sign of any trichomes could be seen (including on the midrib) with a light microscope or SEM.
Transverse sections of paraffin wax-embedded lami nae were cut with a rotary microtome and stained with a mixture of Safranine O and Alcian green (Joel 1983).Sections were taken through the middle of the laminae.
The cluster analysis was done using the Statistica 6.1 package with the following settings: tree clustering; Ward's (1963) method of minimum-variance clustering under the amalgamation rule and percentage disagree ment as a measure of distance.Characters were coded as qualitative presence/absence data.Data for the spinous indumentum type and glandular trichome type were ex cluded from the analysis due to the presence of unknown/ uncertain states in one or more taxa.

RESULTS
Table 1 presents a summary of the various leaf indumen tum and trichome characters codified for Lobostemon.

Trichomes
In Lobostemon, the tendency for epidermal cells at the base of the trichomes to become organized into pustules extends sometimes to several concentric rows, the num ber of which seem to vary with the climatic conditions under which the plant grew and the stage of ontogenetic development.Trichomes typically longer than 400 mm tend to develop pustular bases.In trichomes with a large base, the underlying parenchyma also becomes involved in pustule formation (Figure 1).The pustular trichome type occurs in all Lobostemon species (Figure 2).Most of the species in Lobostemon possess glandular tri- chomes on their leaf surfaces.These trichomes have a tendency to fall flat from an early age (Figure 3).

Length
With regards to the leaves, only four species of Lobo stemon, namely L argenteus (PJ.Bergius) H.Buek, L fru ticosus (L.) H.Buek, L paniculatus (Thunb.)H.Buek and L stachydeus A.DC. clearly possess a dimorphic indumen tum in the adult stage (Figure 4A).The shorter trichome is usually of the simple trichome type.Some forms of L echioides Lehm., L gracilis Levyns, L trichotomus (Thunb.)A.DC. and L paniculiformis A.DC. possess a heteromorphous indumentum.Here the variation in trichome length is discerned to be continuous (Figure 4B).

Distribution
Species with trichomes on both the abaxial and adaxial leaf surfaces are the most common.L regulareflorus (Ker Gawl.)M.H.Buys is unique in only its adaxial leaf surface being hairy.L collinus C.H.Wright is a good example of how climate or age can influence trichome distribution.Plants collected in spring generally have both sides of the leaf hairy.Those collected in late summer (January-April) have glabrous adaxial surfaces.In L. capitatus (L.) H.Buek, however, the opposite holds true.Young leaves collected in spring appear to be hairy only on the abaxial surface.Older leaves from the previous year's growth are hairy on both the adaxial and abaxial surfaces.

Micropapillae
Only a limited number of species seem to have tri chomes without micropapillae (Figure 5A).In the major ity of taxa, trichomes examined in young leaves as well as flower buds display micropapillae.Micropapillae may either be smooth (Figure 5B) or undulated (Figure 5C).In terms of shape, both round and elongate micropapillae have been observed on single trichomes (Figure 5D).The round micropapillae in the aforementioned figure are confined to the base of the trichome, whereas the elongate micropapillae occupy the distal parts.L. decorus Levyns and L. marlothii Levyns have been observed to possess trichomes with or without micropapillae.

Indumentum
Three main indumentum types can be recognized in the genus.The spinous indumentum type (Figure 2A) is the most prevalent.The appressed indumentum type (Figure 2B) is commonly found on those taxa exhibiting silvery leaves.Forms of L argenteus, L curvifolius H.Buek, L echioides, L fruticosus and L trigonus (Thunb.)H.Buek that grow in less arid and more sheltered conditions tend to have appressed trichomes.Generally appressed indumenta also become more spinous as the season progresses, i.e. as it becomes drier and warmer.L trichotomus and L. gracilis appear to be the only species with an appressed indumen tum without an accompanying complement of spinous indumentum (Figure 2C).The umbonate indumentum type is the most difficult to identify due to the general absence of calcified cells around the trichome bases (Figure 2D).This indumentum type appears to be absent from sections Argentei Levyns and Fruticosi Levyns, but most prevalent in section Trichotomi Levyns.Only L regulareflorus and L sanguineus Schltr.appear to possess an umbonate indu mentum type to the exclusion of a spinous indumentum on the leaf surfaces.The remainder of species generally have a spinous indumentum on the margins in addition to the umbonate indumentum of both leaf surfaces.Although the leaves of L glaucophyllus (Jacq.)H.Buek appear to be glabrous to the naked eye, this study has shown the preva lence of minute umbonate indumentum on especially the abaxial surface (Figure 6).
A cluster analysis of the data in Table 1 (excluding char acters with unknown/uncertain states) created a number of clusters with taxa possessing identical character states viz.:   I alluded above to Barthlott's (1981) reasons as to why indumentum and trichome characters have not been applied to systematics with great success.Following this study, and those by Levyns (1934) and Bramwell (1972), it seems clear that in the absence of data to the contrary, a lack of knowledge concerning the influence of habitat and ontogenetic development probably more than any thing else places a damper on the use of indumentum and trichome data for systematic studies in the Boraginaceae.However, even though there is uncertainty about their systematic use, and grouping of plants based on epider mal features does not agree with those based on repro ductive features, the need remains to provisionally describe and organize into a system, all data whereby relationships between the various patterns can be mean ingfully approached in the future (Klucking 1995).
This study has revealed that trichomes in Lobostemon should not be defined as purely epidermal but rather as emergences.In Lobostemon, young plants possess straight, simple, unicellular trichomes (Table 1: Si) whose swollen bases are part of the epidermal layer.From this simple type, present on juvenile leaves, differ ent developments may occur both in the ontogeny and in the transition from juvenile to adult foliage.
Most of the species in Lobostemon possess glandular trichomes on their leaf surfaces.By contrast, Klotz (1959) in his revision of the genus Echium, found the chief occurrence of glandular trichomes to be on the stems of E. humile Desf.and E. trygorrhizum Pomel.although E. gaditanum Boiss.was observed with glandu lar trichomes on their leaf surfaces.tends to undergo a second growing period later in the season that results in the indumentum becoming sparser as the leaf size increases.Moreover, this study has shown that trichomes tend to fall or break off in some species of Lobostemon, leaving behind their hardened bases and manifesting a sparser coverage.
Some taxa can be diagnosed on a single or a combi nation of indumentum characters.The possession of a dimorphic indumentum in Lobostemon is confined to L. argenteus, L. stachydeus, L. paniculatus and L. frutico sus.L. curvifolius and L. fruticosus have often been mis taken for each other: the two taxa are distinguishablethe dimorphic indumentum being absent in L. curvi folius.L. regulareflorus and L. belliformis M.H.Buys, although morphologically similar, differ in that L. belli formis possesses trichomes on both leaf surfaces where as L. regulareflorus possesses trichomes confined to the adaxial surface.
Keeping the shortcomings of phenetics in mind, the aforementioned cluster analysis created groups that could not be correlated to current sectional divisions sensu Levyns (1934: 412).Some clusters, however, can be found as subgroups within Levyns' sections e.g. the L. decorus, L. marlothii, L. muirii and L. oederiaefolius cluster and to an extent the L. glaucophyllus, L. hotten toticus Levyns, L. laevigatus and L. paniculiformis clus ter.The L. echioides, L. gracilis and L. trichotomus clus ter, although not all members of the same section, repre sent the basal taxa in Levyns' branching diagram.

FIGURE 5 .
FIGURE 5.-Trichome micropapillae in Lobostemon.A, no micropapillae in L muirii: B, round and smooth in L paniculatus; C, round and undu late in L belliformis; D, round and elongate smooth in L curvifolius.Scale bars: 100 pm.

DISCUSSION
FIGURE 6.-Minute umbonate indumentum on abaxial leaf surface of

FIGURE 7 .
FIGURE 7.-A phenogram of Lobostemon based on indumentum characters employing Ward's method of minimum-variance clustering and per centage disagreement as a measure of distance.

TABLE 1 .
-Distribution of indumentum types and trichome characteristics in Lobostemon (with voucher specimens housed in NBG)