Leaf anatomy of the genus Ehrharta ( Poaceae ) in southern Africa : the Villosa group

The leaf blade anatomy of Ehrharta villosa Schult. f. var. villosa, var. maxima Stapf and E. thunbergii Gibbs Russell is described and illustrated. These three taxa, constituting the Villosa species group, share a diagnostic leaf anatomy distinguished by the absence of a distinct midrib, adaxial semi-radiate mesophyll with the abaxial chlorenchyma palisade-like in arrangement, rectangular long cells and the stomatal apertures which are overlapped by four cuticular flanges projecting from the two adjacent interstomatal cells. These combined attributes characterize this species group, and the stomatal flanges are unique to this group in the genus Ehrharta Thunb. Microhairs are absent in E. villosa but are present in E. thunbergii which also possesses abaxial prickles and plentiful, rounded silica bodies not associated with cork cells as in E. villosa. These two taxa can. therefore, be separated anatom­ ically. Nevertheless, they share many features and are undoubtedly closely related and their classification in the same species group is substantiated by the anatomical evidence presented in this paper.

identified as E. villosa and 10 as E. thunbergii ( = E. gigantea) but considered as a single species.
This paper describes and illustrates the leaf blade anatom y of the taxa of the Villosa group and dis cusses the affinities of these taxa and of the species group by reference to this anatomical evidence.By im plication the anatom ical data is com pared and contrasted with the morphological data as it is reflec ted in the classification of the group (Gibbs Russell 1987).The herbarium voucher specimens used in this anatom ical study were included in the sample on which the above taxonom ic conclusions were based.The m ethodology is described in Gibbs Russell & Ellis (1987) and the form at of the paper follows that of the first paper in the series (Ellis 1987a).

INTRODUCTION
The species of the Villosa group of the genus Ehr harta Thunb.are distinguished morphologically by their large spikelets with profusely hairy, conspicu ously bearded and m ucronate sterile lemmas (Gibbs Russell & Ellis 1987).The leaf blades are reduced and rolled and the culms are suffrutescent, som e times with swollen or tuberous bases.Creeping, underground rhizom es occur in all taxa.
Taxa included in this group are Ehrharta villosa Schult.f. var.villosa and var.maxima Stapf, and E. thunbergii Gibbs Russell (= E. gigantea Thunb.).Chippindall (1955) considered E. villosa var.villosa and E. thunbergii to be conspecific, whereas Smook & Gibbs Russell (1985) synonomize E. villosa var.maxima and E. thunbergii.In the present treatm ent E. thunbergii is considered as a separate species fol lowing Gibbs Russell (1987) and consequently, three taxa are assigned to the Villosa species group.
The leaf blade anatom y of taxa belonging to this species group has received very little attention from previous workers.M etcalfe (1960) gives a full de scription of E. villosa var.maxima and Engelbrecht (1956) also describes the leaf anatom y of E. villosa based on a representative sample of 18 specimens, 8 R ounded adaxial ribs are associated with all the vascular bundles (Figures 1.2,2.2,2.4,3.2,3.4),those of the first order bundles being slightly larger.Shallow, but rather narrow, furrows are present be tween all the ribs.The mesophyll tissue is unusual in that it is semiradiate in arrangem ent, particularly the adaxially situated cells located in the ribs (Figures 1.2,2.2,2.4), but the arrangem ent of the abaxial layers of chlorenchym a cells is palisade-like (Figure 2.2, 2.4) and may be conspicuous due to denser chloroplast concentrations (Figure 3.4).The chlorenchyma cells are relatively large, somewhat variable in shape but tightly packed so that no large intercellular air spaces are visible in transection (Figures 2.2,2.4).The chloroplasts are evenly but densely distributed throughout all the chlorenchyma cells.

A baxial epidermis
Costal and intercostal zones are clearly differen tiated (Figure 1.3, 2.5) due to differential staining, although the epiderm al cells of these two zones do not necessarily differ greatly in structure (Figure 2.6).The costal zones lack stom ata and consist of narrow er cells (Figure 1.4).The intercostal long cells are rather short, and rectangular with slightly undu lating walls.The cells of the central files of each zone may tend to be longer and wider than the lateral cells (Figure 2.5).These larger cells are sometimes also evident in the leaf sections (Figure 1.2, 2.4).
Stom ata are common in 3-5 files in each intercos tal zone (Figure 1.3, 2.5).They are clearly sunken well below the level of the rest of the epidermis with the guard and subsidiary cells being overlapped by four distinct cuticular flanges extending over the stomatal aperture from the adjacent interstom atal long cells (Figures 1.2,2.2,2.4).In surface view a distinct cross-shaped aperture is formed by these flanges, below which the stomatal apparatus is located (Fig ure 1.4, 2.6).SEM studies reveal that the flanges are papilla-like .
Costal silica bodies are not well differentiated and are usually small, rounded and intimately associated with an enfolding cork cell (Figure 1.4, 2.6).In less typical specimens, however, the silica bodies may be much m ore evident and numerous (Figures 6.1,6.3).Prickles are absent but prickles are present on the adaxial costal zones which are equivalent to the ribs as seen in transverse section (Figures 1.2,2.4,3.2).No microhairs were seen either with the light or the scanning electron microscope (Figures 1.4,2.6,.

Comments
E. villosa possesses the characteristic leaf anatomy of the Villosa group being distinguished by the ab sence of a keel or m idrib, the palisade-like abaxial m esophyll, the flanged stom ata, and the rectangular long cells.The anatom y of var.villosa and var.m ax ima is very similar and these two taxa appear to show close affinities, being indistinguishable on leaf blade anatom y, a fact which appears to corroborate their separation at only the varietal level.
A lthough the var.maxima anatom ical sample used in this study is inadequate, the specimen exam ined (Ellis 601) conform s in all respects to the de scription given by M etcalfe (1960) for m aterial from W estern A ustralia even though his m icrotechnique procedures did not allow a detailed exam ination of the mesophyll.These two specimens reveal that the leaf anatom y of var.maxim a conforms very closely with that of var.villosa, with some specimens of the latter being virtually indistinguishable from var. m axim a in leaf anatom y ).
E. villosa var.villosa is a rather variable taxon anatom ically.Some specimens of var.villosa corre spond very closely in leaf size and thickness to the relatively large leaves of var.maxima, as a com pari   E. thunbergii and the interface between these two taxa is not very distinct.
The interm ediate nature of var.villosa is also evi dent in its spikelet size and habitat requirem ents and several specimens have proved difficult to assign to either var.villosa or E. thunbergii on morphological criteria.This is particularly the case if the rhizome characters are not evident.But E. villosa is a species of deep, loose sand of the lowland fynbos and only occurs at higher altitudes where drift sand occurs as a result of wind or water deposition.
The clinal variation in anatomical structure in var.villosa appears to be a reflection of these habitat gradients.Those specimens most resembling var.maxima are all from coastal dune habitats (Figures 2.1-2.6) to which var. maxima appears to be con fined.With increasing altitude and distance from the sea the var.villosa specimens 6.1,6.3)tend to merge with E. thunbergii, which is a species of higher altitudes, heavier soils and the m ountain fynbos.

Transverse section
Blade loosely to rather tightly inrolled (Figures 5.3,5.5).A slight keel may sometimes be devel oped, as evidenced by the presence of additional colourless parenchym a associated with the median vascular bundle (Figures 5.3,5.5).This developm ent is not equally evident in all specimens and several have the m edian bundle structurally identical to the lateral first order bundles, without additional paren chyma (Figure 5.1).One or two third order bundles occur between consecutive first order bundles.
Adaxial ribs are slight but rounded (Figure 5.2) or may be more conspicuous but then abaxial intercos tal ribs alternate with the adaxial costal ribs (Figures 5.4,5.5).Adaxial furrows are shallow and wider than in E. villosa.
The mesophyll is rather variable but all specimens conform to the general pattern so characteristic of this group.Examples with semi-radiate chloren chyma with an abaxial palisade-like layer are illus trated in Figures 5.2

Abaxial epidermis
Costal and intercostal zones are always distin guishable 6.2,6.4,7.2,).Cell size and shape differ m arkedly between these two zones on all the specimens examined.The inter costal long cells are often much m ore elongated than in E. villosa but this character is variable with Fig ures 5.6, 5.7 and 6.4 representing the two extremes encountered in this species.The long cell shape is usually rectangular but may be diam ond-shaped (Figure 7.4).The m arkedly elongated long cells may also stain with safranin (Figures 5.7,5.8).
Stom ata occur in 2-3 files on either side of each costal zone but are absent from the central files of the intercostal zones.These stom ata are always sunken and overlapped by cuticular flanges although these are not always easily visible with the light m icroscope (Figures 5.6,5.7).The specimens with thinner leaves and elongated long cells have less con spicuous flanges associated with m ore superficial stom ata.Those specimens tending toward E. villosa in leaf anatom y have this characteristic well devel oped (Figures 6.2, 6.4), as do the specimens showing similarities with the Calycina group (Figure 7.4) or the R am osa group (Figures 7.5,7.6).Although vari able, this attribute is evident on all the specimens studied and is confirm ed by the SEM (Figures 4.5-4.7).
Costal silica bodies are generally well differen tiated, being conspicuous and rounded and alternat ing along the costal files (Figures 5.8,6.4,7.2).C res cent-shaped, enfolding cork cells do not appear to occur in this species.Abaxial costal prickles are com m on and were observed on all specimens with two specimens (Ellis 1152 and 5102) even possessing large intercostal prickles associated with the stomatal bands (Figure 5.8).M icrohairs, although very small, were detected on all specimens, even those resem bling E. villosa in other anatomical character istics.U ltrastructurally these hairs are seen to have a tapering distal cell (Figures 4.6,4.8).

Comments
The diagnostic anatomical attributes of the Villosa group are all present in E. thunbergii although they may be som ewhat modified on some specimens.Thus only a median vascular bundle is normally present but in a few specimens additional colourless parenchym a is associated with the median bundle, which, by definition, constitutes a slight keel.The sem i-radiate adaxial, and palisade-like abaxial meso phyll, so characteristic of this group, is evident in most specimens.However, in a few, particularly those with thinner leaves and with abaxial intercostal ribs, this pattern may be modified slightly.In all specimens the stom ata are sunken and overlapped by four papillate epiderm al flanges.However, in those specimens with elongate intercostal long cells the stom ata may be almost flush with the level of the epiderm is and the flanges are tiny.These diagnostic features are common to all taxa of the Villosa group and serve to unite E. villosa and E. thunbergii in a group separated from all the other species of Ehr harta.
In addition, several characters serve to separate E. thunbergii from E. villosa, although this distinc tion is not very clear-cut.Examples are the presence of microhairs and abaxial prickles, both of which are lacking in E. villosa.The costal silica bodies of E. thunbergii are also well differentiated and plentiful and alternate with costal short cells.They are not associated with cork cells as in E. villosa.These two taxa can, therefore, be distinguished anatomically.
Yet other attributes intergrade between the taxa of this species group, and the leaf anatomy of the E. thunbergii specimens studied shows a certain degree of variation.A distinct gradation is evident from those specim ens closely resembling E. villosa (Fig ures 6.2, 6.4, 7.1, 7.3) to the extrem e type with thin ner leaves and elongated long cells ).The interface with E. villosa is indistinct.A continuum is discernible from those specimens re sembling E. villosa to the extrem e specimens which may display characteristics of some of the other Ehr harta species groups, the Calycina group in particu lar.Calycina type features observed are the fusiform intercostal long cells as in Figure 7.4, the tendency to stain with safranin ) and the inter costal abaxial ribs (Figure 5.4) or the inflated central cells of the intercostal zones as illustrated for E. vil losa (Figures 2.4,2.5).A single specimen, Ellis 4642, although not illustrated, resem bles E. calycina particularly closely, even having straight-walled fusi form long cells and intercostal m acrohairs which were not observed on any other E. thunbergii speci men.H ow ever, flanged stom ata indicate the true identity of this specimen.
One o ther interesting and deviant specimen is Ellis 4693 (Figures 7.5,7.6)which shows similarities with the R am osa group of species.The sinuous, rect angular long cells, all separated by conspicuous cork/silica cell pairs and the irregular, dum bbell shaped silica bodies, are reminiscent of the Ramosa group and were not seen in any other E. thunbergii specimens.H ow ever, this specimen also has dis tinctly flanged stom ata.
The anatom ical sample examined in this study is heavily biased tow ard the north-w estern parts of the distribution range of E. thunbergii.Those specimens from high altitudes in the extrem e north at Van R hyn's Pass (Ellis 1145, Figures 5.5, 5.6;Ellis 4626, Figures 7.3, 7.4) show anatom ical similarities with E. calycina.A specimen (Ellis 4642) from lower alti tude in the strandveld at Langvlei resem bles E. caly cina very closely indeed.On the other hand, few specimens from the east have been classified as E. thunbergii (these being mainly identified as E. vil losa) and Ellis 4693 from C loete's Pass in the eastern Langeberge resem bles the Ram osa group in certain respects.These observations may reflect transitions to these oth er Ehrharta species groups but a much m ore representative sample must be studied before this can be confirmed.N evertheless, this does serve to dem onstrate that the Villosa group is not discrete, and that characteristics of some other groups are evident, as is the case throughout the genus.
These observations are largely in agreem ent with the findings of E ngelbrecht (1956) and the few ex ceptions noted will be briefly discussed.For the m a jority of specimens the epiderm is is described as be ing hom ogenous with costal and intercostal zones not being distinguishable (Engelbrecht 1956).In the present study the condition is described where these zones are structurally identical, as in E. villosa var.maxima for exam ple, but are distinguishable on ac count of their differential staining.Different staining procedures, therefore, may account for this appar ently superficial difference between the findings of these two studies.Engelbrecht (1956) does record the absence of m icrohairs and prickles associated with the hom ogenous type of epiderm is (which ap pears to be hom ologous with E. villosa) whereas the epiderm is with distinct epiderm al zonation was asso ciated with the presence of these hairs.This correla tion was observed in the present study and is consid ered to be a specific difference between E. villosa and E. thunbergii but Engelbrecht (1956) did not attribute any taxonom ic significance to it.He also records cuticular stom atal flanges for all the speci mens he examined and notes the uniqueness of this feature in the genus.

DISCUSSION A N D CONCLUSIONS
The three taxa of the Villosa group, share a dis tinctive leaf anatom y characterized by a unique com bination of attributes as well as similar vegetative m orphology and a specific habitat.These distin guishing features correlate with the diagnostic large, hairy spikelets, and their assignment to the same small species group appears to be fully justified by the anatom ical as well as the morphological evidence (Gibbs Russell 1987).This group also appears to represent a natural grouping.
The leaf anatom y is characterized by the absence of a keel, palisade-like mesophyll abaxially located, rectangular long cells and stomatal apertures which are overlapped by four cuticular flanges projecting from the two adjacent interstom atal long cells.This latter feature is unique to this species group in the genus Ehrharta.
A lthough Engelbrecht (1956) studied only uni fixed leaf blade m aterial he noted that the form of the cells of the abaxial chlorenchyma layer differed from the rem ainder, an observation confirm ed in this study.How ever, he also reports cell wall invagi nations as being present and characteristic of E. vil losa.These invaginations were not observed on all chlorenchym a cells, however, but appeared to be confined to those cells adjacent to the vascular bun dles or adjoining the adaxial epiderm is.This obser vation was not confirmed in the present study, in which field-fixed m aterial was examined, and ap pears to be an artefact probably resulting from im perfect rehydration of the mesophyll tissue.Engelbrecht (1956) recognized two basic groups of species in Ehrharta -one with invaginated chloren chyma and one without.E. villosa is placed in the group with invaginations together with taxa of the Setacea and Ram osa species groups as here consti tuted (Gibbs Russell & Ellis 1987).The present find ings are in disagreem ent with Engelbrecht's (1956) grouping, as the Setacea group is the only group in which arm cells were observed (Ellis 1987) and the Setacea and Villosa groups are not considered to be closely related.
A lthough he examined a large sam ple, E ngel brecht (1956) was unable to distinguish E. villosa and E. thunbergii either anatomically or m orpholo gically and concluded that they do not represent two separate species.The present study is not in full agreem ent with this conclusion as E. villosa and E. thunbergii were found to differ in several respects such as the presence of microhairs and prickles as well as differences in silica bodies.A lthough these differences appear to be consistent and diagnostic, it must be rem em bered that the interface betw een these two species is not distinct as far as most other characters are concerned and a continuum is evident between them without clear character disjunctions.E. villosa and E. thunbergii, therefore, intergrade to a certain extent and, although their extremes are anatomically quite distinct, a small proportion of specimens are som ewhat interm ediate.The decision to consider these two taxa as being conspecific (Chippindall 1955;Engelbrecht 1956), therefore, has some m erit.How ever, the placing of E. thunber gii in synonomy under E. villosa results in a very variable, polym orphic entity with a wide ecological tolerance.The recognition of three taxa seems to be a more practical solution which probably reflects the natural situation more accurately.However, a cline undoubtedly exists from E. villosa var.maxima through var.villosa to E. thunbergii with each of these taxa occupying slightly different habitats and differing in morphology and leaf anatomy.
The relationships of the Villosa group to the rest of the genus are not very clear*from anatomical evi dence alone.The group does not occupy such an isolated position within the genus as does the Seta cea group (Ellis 1987) which possesses such taxonomically significant diagnostic features as arm cells and distinct microhairs and silica bodies.There are also no anatom ical interm ediates between the Seta cea group and any of the other species groups.A l though the Villosa group is readily diagnosed by its flanged stom ata, this feature cannot be accorded the high taxonomic value that arm cells and microhair and silica body shape have in the classification of the Poaceae, because it is encountered independently in different subfamilies.
In addition, several E. thunbergii specimens dis play strong Calycina group attributes in their leaf anatom y, and both these groups have very similar microhairs.The Villosa and Calycina groups also share very similar hairy spikelets, which differ mainly in size and profuseness of vesture, but occur in no other Ehrharta species group.The indications are, therefore, that the Villosa group is more closely related to the Calycina group than to any of the other groups.However, as is common in this genus, a reticulate pattern of relationships can be expected and Ram osa group characteristics were also ob served on a few specimens.
The Villosa group, although distinct in m orpho logy, anatom y and ecology, does show certain affini ties with the Calycina group and undoubtedly be longs to the genus Ehrharta.This group, therefore, appears to be a specialized perennial line with strong underground rhizomes and suffrutescent culms which has become adapted to a sandy habitat.
, 7.1 and 7.3 and correspond closely to the E. villosa specimens illustrated in Fig ures 3.2 and 3.4.O ther E. thunbergii specimens, with thinner leaves and fewer chlorenchyma cell lay ers differ slightly from this pattern (Figure 5.4).The chlorenchym a cells themselves remain rather large, somewhat angular and tightly packed with very small intercellular air spaces (Figures 5.2, 5.4).