The taxonomic value of epidermal characters in the leaf of Heteromorpha and some related genera (Apiaceae)

All 17 species of Heteromorpha Cham. & Schltdl. (sensu Humbert 1956), all three species of Polemannia Eckl. & Zeyh. and the monotypic Polemanniopsis B.L. Burtt were investigated for leaf epidermal characters. Stomatal type was anomocvtic. with an exception in only one Madagascar species, H. hetsileensis. The distribution and density of stomata (on both leaf surfaces) are diagnostic for some species. The number, size and outline of normal epidermal cells are different in juvenile and adult leaves and these differences vary between species. Seven trichome types are recognized which, when combined with dispersion pattern, also serve to characterise the various species and forms. UITTREKSEL Al 17 spesies van Heteromorpha Cham. & Schltdl. (sensu Humbert 1956), al drie spesies van Polemannia Eckl. & Zeyh. en die monotipiese Polemanniopsis B.L. Burtt se blaar-epidermale kenmerke is ondersoek. Die stomata-tipe was anomosities, met 'n enkele uitsondering by een Madagassiese spesie. H. hetsileensis. Die verspreiding en digtheid van stomata (op beide oppervlakke) is diagnostics vir sommige spesies. Die aantal. grootte en buitelyn van gewone epidermisselle verskil by jeugen volwasse blare en hierdie verskille varieer tussen spesies. Sewe trigoomtipes word onderskei wat, saam met verspreidingspatroon. kenmerkend is vir die verskillende spesies en vorme.


INTRODUCTION
Heteromorpha Cham. & Schltdl. (sensu stricto) is a genus of trees, shrubs or suffrutices occurring throughout most of temperate and subtropical Africa. Seven species are restricted to the African continent and Yemen. Hum bert (1956) broadened the generic concept to include eight Madagascar species. The African contingent shares the woody habit with the related Polemannia Eckl. & Zeyh. from the Drakensberg region, and fruit characters with the Western and Northern Cape genus Polemanniopsis B.L. Burtt (Burtt 1988), which have been included here as potential outgroups.
Most species are either glabrous or have trichomes which are not readily visible to the naked eye. Phenotypic variation as well as the broad species concepts employed in past surveys (mostly Flora treatments and therefore of a limited, regional nature), have tended to obscure the taxonomic relevance of vestiture. However, trichome type and trichome distribution have been used to distinguish //. trifoliata from species with basally tuberculate trichomes. and to characterise //. papillosa and H. gossweileri (Townsend 1985(Townsend , 1989. Epidermal characters are therefore clearly of taxonomic value in Heteromorpha, especially when the paucity of information on other char acters is considered. Stomatal type has been recorded for only one of the woody African Apiaceae. namely H. arborescens. which was found to be anomocytic (Guyot 1971).  Mean stomatal densities were determined under the light microscope, by a minimum of six counts per specimen over an area along the eyepiece scale bar. Counts were not taken directly adjacent to the margin or midvein, only in the secondary vein interstices. Dispersion was scored according to patterns of local presence or con centration.
After germinating seeds of five African species \H. ar borescens. H. trifoliata, H. involucrata, H. pubescens and H. transvaalensis], juvenile leaves were available to in vestigate ontogenetic changes during leaf development. Trichome types were studied by SEM on one specimen of each taxon. To investigate the structure of each trichome type, mature leaves were embedded in glycol methacry late (GMA) according to a modification (Tilney 1986) of the method of Feder & O'Brien (1968). Sections were obtained with a Porter Blum MT-1 ultramicrotome and stained according to the so-called periodic acid-Schift' /Toluidine Blue (PAS/TB) staining method. Draw ings were done with the aid of a camera lucida attachment on a Zeiss com pound light m icroscope. Vestiture (trichome distribution) was studied on several specimens with the aid of a dissecting stereomicroscope. The inves tigation was limited to the laminar region of the leaf.

RESULTS AND DISCUSSION
Five characters were chosen for analysis:

Stomatal apparatus
The stomatal apparatus proved difficult to interpret be cause cell boundaries were indistinct in the stomal region and because cells adjacent to the stomata differed super ficially from the surrounding epidermal cells. The cuticula appears to be much thicker in this region, making the radial cell walls and guard cell boundary almost indiscer nible. This at first suggested a tetracytic or actinocytic arrangement, but the number of surrounding cells was too inconsistent for this to be the case. Stomata are juxtaposed in some instances, or separated by one cell only ( Figure  IB), confirming the perigenous anomocytic origin. Cells surrounding the stomata sometimes differ from other epidermal cells in their reaction to staining, but there is no evidence of any structural difference, hence our inter pretation that these are not true subsidiary cells.
As found by Guyot (1971) for H. arborescens, the rest of the genus-with one exception-as well as the related genera, all possess the anomocytic type stomatal apparatus ( Figure IB). The stomata of the Apiaceae are generally considered to be more or less intermediate between anomocytic and anisocytic (Guyot 1971;Ostroumova 1987 in Baranova 1992).
The single exception, the Madagascan species H. betsileensis, has an anisocytic type stomatal apparatus (Fig  ure 1A). The radial cell walls of subsidiary cells are shorter than the tangential walls, giving them a thinner shape than the normal epidermal cells. The characteristic clustering of the stoma with three surrounding cells sug gests a common origin from one stomatal initial cell (mesogenous type), although it must be emphasized that the ontogeny was not studied (see Baranova 1992 for a detailed discussion of morphological vs. ontogenetic clas sification of stomata).

Stomatal dispersion
Stomata sometimes occur in crypts formed by the prominently raised veins on the abaxial surface, as in H. pubescens.
Four main patterns of stomatal dispersion over the adaxial surface were identified. Stomata are either absent, occur along margin and/or midrib only, occur mostly along margin and veins, or occur throughout the entire surface. These data are listed in Table 1. In the Madagas can species and the other genera stomata are either absent or dispersed randomly across the entire surface. Heteromorpha sensu stricto by contrast, shows great variation. //. stenophylla and //. gossweileri differ from the other African species in their stomatal dispersion pattern which is shared with all Polemanniopsis and Polemannia spe cies, but with only two Madagascan species (H. andohahelensis and //. bojeriana). In H. papillosa this state co-occurs with states 2 and 3 of Table 1.

Stomatal distribution and density
The results of the stomatal density study are presented in Figure 2. The data show that the variation in this char acter is independent of habit and also not logically corre lated with mesophytic or xerophytic habitats. Compared to Heteromorpha. the other two genera are relatively in variant in terms of abaxial density (Figure 2A

Epidermal ontogeny
As the leaves develop from the juvenile to the mature stage, the proportion of stomata to normal epidermal cells ( Figure 3) remains constant in H. involucrata and H. pubescens, whereas it decreases in both the typical and the frutescent forms of H. trifoliata. This is not due to fewer stomata being formed, but to a reduction in size of the normal epidermal cells and thus a higher epidermal cell density.

Trichome type
The v ariation in mature trichome types as well as their distribution is summarized in Table 1. Various types com prising seven character states were recognised and are shown in Figure 4 and Figure 5. The papillate type ( Figures 4A; 5C2), which is also the juvenile type, has a wide occurrence (present in all three genera), and is also the only type for Polemanniopsis and Polemannia. Com pared to the Madagascan group where only the filamen tous, multicellular type is found in H. betsileensis alone, the African species show a high diversity of form.
Papillate trichomes, similar to those shown in Figure  4A and 5C. were observed on the juvenile leaf margins of Heteromorpha. These were present on the juvenile leaves in all forms of the five African species investigated. This feature appears to be variously lost or modified by means of apical and sometimes basal cell differentiation in the ontogeny of the mature types.
Tuberculate trichomes occur either as simple tubercles ( Figure 5C) or with one ( Figures 4C: 5A. B & E) or many ( Figure 4E) trichomes of another type affixed apically. This condition was described by Townsend (1985) as papillose, and is characteristic of H. pubescens and most forms of //. involucrata. The term 'verruculose' used by Townsend (1985) to describe the vestiture in H. goss weileri suggests the simple tuberculate type of trichome ( Figure 5C1). Although the SEM survey showed only the papillate type ( Figure 4A). which does bear a superficial resemblance to the tuberculate type, light microscopy con firmed the presence of both types ( Figure 5C).
The filamentous/filiform type ( Figures 4E: 5E) is found only in //. pubescens and H. betsileensis. and in addition to distribution and density of hairs, characterizes these species. Short cylindrical hairs (Figures 4B: 51)> are char-   acteristic of //. arborescens and H. trifoliata, but arc also found in //. papillosa, the West African form of H. tri foliata, and occasionally (with other types) in some forms of //. involucrata.
Other types that can be identified are 1, a unicellular conical hair ( Figures 4F; 5A), usually present in all forms of H. involucrata; and 2. a unicellular or multicellular long cylindrical hair ( Figures 4D; 5B), present in the central African forms of //. involucrata.

Trichome distribution
however, only rarely the case in H. involucrata, where the abaxial midrib is usually more pilose than the adaxial midrib. The presence of trichomes along the leaf margin is the rule in H. involucrata (Figure 6 B & C) and the exception in H. trifoliata. The West African form of H. trifoliata can be distinguished from the typical form by the presence of small tubercles, albeit sparse in some specimens, along the leaf margin. The leaf margin in typi cal H. trifoliata is usually glabrous, or if pubescent (com monly in young growth), then with short cylindrical hairs. Both forms have cy lindrical trichomes along the adaxial midrib.
Patterns of laminar distribution of trichomes are sum-H. involucrata is the only species with trichomes on marized in Table 1. Hairs, when present, are usually lo-the areas between the veins (and not on the veins and cated at least on the adaxial midrib (Figure 6 A). This is. margin only, as in all other species). H. pubescens has morphological features in common with H. involucrata, and also shares the hairy abaxial midrib and presence of hairs on the adaxial secondary venation (Townsend 1985) in addition to those found elsewhere ( Figure 6D).
H. gossweileri has a vestiture pattern, which for its particular trichome type, is characteristic. The leaf margin, midrib and major adaxial veins all have a variable number of trichomes, often concentrated apically or basally (Townsend 1985).

TAXONOMIC IMPLICATIONS
The anomocytic type of stomatal apparatus seems con servative, occurring throughout, with the exception of H. betsileensis, suggesting a different position for this species. This is also the only Madagascan species with any degree of vestiture on the laminar region. The occur rence of a second stomatal type could indicate a polyphyletic grouping of the Madagascan species, as this feature appears to be rather conservative, and is a potential character for tribal delimitation. A re-investigation of generic limits is suggested. Abaxial stomatal density is a potential grouping character within the Madagascan con tingent, and could support the division of the group into two or more genera when these species are investigated further.
The typical and frutescent forms of H. trifoliata, for which seedlings were investigated, show an ontogenetic feature not present in H. pubescens and H. involucrata, namely a decrease in size of normal epidermal cells.
Three of the African species of Heteromorpha can be distinguished from the others on the basis of the uniformly dispersed adaxial stomata, a character which is shared with Polemannia, Polemanniopsis and two Madagascan species. This character may be logically correlated with the high adaxial stomatal density in these species. H. stenophylla, previously considered to be merely a form of H. trifoliata (Schreiber 1967;Townsend 1985), is charac terized by having less than 100 abaxial stomata per mm2 and a substantially higher adaxial density than other African species.
Papillate trichomes seem to be the primitive state from which other states were derived. If the cell differentiation in the development of the other trichome types were in vestigated, this could lead to a better understanding of the phylogeny of Heteromorpha. The presence of small tubercles (although sparse in some specimens) along the leaf margin of the West African form of H. trifoliata raises some doubt as to its present taxonomic position, and sug gests a closer affinity to the suffrutescent species H. gossweileri, H. involucrata and H. pubescens. The abaxial stomatal density distinguishes it from other suffrutescent types with which it may be confused, namely some central African forms of H. involucrata. The formal description of this taxon has been indicated by other evidence, and will be published elsewhere.
variation than the H. arborescens complex, and formal recognition of infraspecific taxa may be useful.

CONCLUSIONS
Anomocytic stomata and papillate trichomes are con servative characters at the generic level. The density and distribution of stomata are of limited value at this level, but show some pattern at the species level. Heteromorpha sensu stricto differs in the diversity of trichome types, and this character is useful to distin guish some of the species.
Epidermal characters provide supporting evidence for a less conservative treatment of the African species than those of Cannon (1978) and Townsend (1985Townsend ( , 1989. The degree of woodiness in Heteromorpha species is an im portant character and would seem to be correlated with epidermal features. Without attention to habit, other char acters tend to be difficult to interpret.

H.
pubescens and most forms of H. involucrata share, along with other morphological characters, the distribution of trichomes along abaxial midrib and venation, but are distinguished on the basis of trichome type. Some forms of H. involucrata exhibit deviation from the typical con dition o f a hairy abaxial midrib. As currently cir cumscribed, this species shows as much if not more