Kranz distinctive cells in the culm of ArundineUa (Arundinelleae; Panicoideae; Poaceae)

The transectional anatomy of photosynthetic flowering culms of Arundinella berteroniana (Schult.) Hitchc. & Chase and A. hispida (Willd.) Kuntze from South America and A. nepalensis Trin. from Africa is described and illustrated. The vascular bundles are arranged in three distinct rings, the outermost being external to a continuous sclerenchymatous band. Each of these peripheral bundles is surrounded by two bundle sheaths, a complete mestome sheath and an incomplete, outer, parenchymatous Kranz sheath, the cells of which contain large, specialized chloroplasts. Kranz bundle sheath extensions are also present. The chlorenchyma tissue is also located in this narrow peripheral zone and is interrupted by the vascular bundles and their associated sclerenchyma. Dispersed throughout the chlorenchyma are small groups of Kranz distinctive cells, identical in structure to the outer bundle sheath cells. No chlorenchyma cell is. therefore, more than two cells distant from a Kranz cell. The structure of the chlorenchyma and bundle sheaths indicates that the C4 photosynthetic pathway is operative in these culms. This study clearly demonstrates the presence of the peculiar distinctive cells in the culms as well as in the leaves of Arundinella. Also of interest is the presence of an inner bundle sheath in the vascular bundles of the culm whereas the bundles of the leaves possess only a single sheath. It has already been shown that Arundinella is a NADP-me C4 type and the anatomical predictor of a single Kranz sheath for NADP-me species, therefore, either does not hold in the culms of this genus or the culms are not NADP-me. This is only the second reported breakdown of this association between MS anatomy and the NADP-me biochemical C4 type.


INTRODUCTION
Some species of Arundinella Radii are characterized by the presence of Kranz distinctive cells in the mesophyll of the leaf blades. Arundinella is a C4 genus which possesses the Kranz syndrome (Brown 1977) and these distinctive cells are very similar to those of the Kranz mestome sheath which surrounds the vascular bundles (Brown 1975). The distinctive cells have thicker walls than those of the radially arranged chlorenchyma cells between which they are embedded (Carolin el al. 1973) and these walls also stain heavily. They contain abun dant specialized chloroplasts which store starch (Brown 1975;Renvoize 1982a singly between the vascular bundles or are found in groups of two to six cells without accompanying vascu lar tissue. Some authors are of the opinion that the distinctive cell files connect with the parenchyma sheath cells (Carolin et al. 1973;Ellis 1977). However, in paradermal view it is evident that they are not continuous with the vascular tissue but are contiguous isolated Kranz cell strands that lie parallel to the vascular bundles and are not in contact with them (Crookston & Moss 1973). They are presumably functionally linked to the vascular bundles at intervals by cross veins (Crookston & Moss 1973;Crookston 1980;Renvoize 1982a). Where the cross veins traverse strands of distinctive cells some of these Kranz cells become appressed to the cross vein, so forming a functional link with the vascular tissue.
These cells were first reported by Vickery (1935) in the leaf blade transection of A. nepalensis Trin. Tateoka (1956b) was the first to designate these cells calling them distinctive cells. Subsequently various authors have used differing terminology and a historical review of the study and terminology of these cells is given in Table 1.
This tabulated summary (Table 1) shows that the term distinctive cells appears to be the most widely accepted for these structures and it will be used in this paper. However, the term distinctive cell does not convey the structure or function of these cells and the proposal of the term auxiliary bundle cells (Renvoize 1982a;Clayton & Renvoize 1986) has some merit since these cells undoubt edly are part of the photosynthetic system, being auxil iary photosynthetic strands. Hattersley et al. (1977) have demonstrated that these cells are isolated photosynthetic carbon reduction (PCR, Kranz) strands embedded in the primary carbon assimilation (PCA) chlorenchyma tissue; and they exhibit NADP-me activity (Reger & Yates 1979). Ultrastructurally they are also seen to be similar to the Kranz mestome sheath cells. Both have large agranal chloroplasts containing numerous starch grains (Crookston & Moss 1973;Crookston 1980) and a suberized lamella is located in the cell walls (Hattersley & Browning 1981). The chloroplasts of the chlorenchyma cells, on the other hand, are free of starch and have well developed grana and the cell walls lack a suberized lamella. The distinctive cells are, therefore, undoubtedly Kranz cells and will be designated as such.
From Table 1 it can also be seen that distinctive cells have only been reliably reported in four genera belong ing to four small tribes of the Panicoideae: Arundinella of the Arundinelleae (Tateoka 1956a(Tateoka , 1958; Garnotia of the Gamotieae (Tateoka 1956b(Tateoka , 1958; Arthropogon of the Arthropogoneae (Tateoka 1963) and Anthephora of the Anthephoreae (Johnson & Brown 1973). Watson et al. (1986) record 'circular cells' in nine genera of the Panicoideae. The reports of distinctive cells in Trichopteryx and Loudetia of the Arundinelleae (Brown 1977) appear to be misleading and probably refer to the very reduced vascular bundles surrounded by only three or four Kranz sheath cells which are known from these genera which also lack cross veins (Renvoize 1982a). Many authors have confirmed the occurrence of distinc tive cells in Arundinella and Garnotia (Table 1) and Arthropogon xerachne (Sánchez & Arriaga 1988) but verification of their reported presence in Anthephora is required. We have examined leaves of 25 specimens of four species of Anthephora, all of which have very small minor vascular bundles consisting of only three or four bundle sheath cells surrounding a minute vascular strand. Consequently we query the reported presence of distinctive cells in this genus. We have also examined leaves of Tristachya lejostachya and Loudetia flammida (Sánchez & Arriaga 1988), L. pedicellata and L. simplex (Ellis 1977) without detecting the presence of distinctive cells. In all these cases a few xylem vessels were de tected in association with the Kranz cells but these are not considered to be distinctive cells.
Nevertheless, as presently known, distinctive cells are characteristic of and unique to these four small tribes of the Panicoideae and may indicate phylogenetic relationships between them. Johnson & Brown (1973) consider the possession of distinctive cells to be suffi cient grounds for considering these four tribes as consti tuting one tribe or even a supertribe. Garnotia and Arun dinella. in particular, are very closely related (Renvoize 1982b) and appear to constitute a distinct and related group of genera sharing this interesting anatomical fea ture, as well as spikelet characteristics. Distinctive cells are not a characteristic of the tribe Arundinelleae but are only a feature of some species of Arundinella (Renvoize 1982a).
The culms (aerial stems) of grasses display consider able anatomical variation but, in contrast to the leaf blade, have been poorly documented (Sabnis 1921;Can field 1933;De Wet 1960;Metcalfe 1960;Auquier & Somers 1967). Some of these studies include members of the Arundinelleae. De Wet (1960) describes the peri pheral vascular bundles of the culm of Arundinella as being surrounded by a parenchymatous bundle sheath composed of small cells. Auquier & Somers (1967) con sider the anatomical structure of the culm of Arundinella as belonging to the 'panicoid type' with the peripheral bundles surrounded by a well developed parenchymatous sheath. None of these authors refer to the presence of Kranz anatomy in the cortical zone of the culm. Sánchez (1979, 1981a, 1981b, 1983a, 1983b, 1984) is the first worker to report the presence and development of Kranz anatomy in flowering and stoloniferous culms. Kranz anatomy is only developed in the upper exposed parts of flowering culms and not the basal parts which are covered by the leaf sheath (Sánchez 1981a); it is there fore essential to examine comparative material.
The objective of this study is to determine whether Arundinella exhibits Kranz structure in the flowering culm. If this is so then it will also be of interest to see whether distinctive Kranz cells are also present. This paper describes the structure and arrangement of these cells in the culms of three species of Arundinella: A. berteroniana (Schult.) Hitchc. & Chase and A. hispida (Willd.) Kuntze from Argentina and A. nepalensis from South Africa. A. hispida from the New World and A. nepalensis from the Old World appear to be closely related and Phipps (1967) included them both in the Nepalenses series which he considered to be central to the genus.

MATERIALS AND METHODS
Transverse and longitudinal sections of flowering culms were made from segments taken from the centre of the first intemode below the inflorescence. Both herba rium and field collected material fixed in FAA was used. Sections were either free-hand or the material was desilicified, embedded in wax and sectioned on a rotary microtome. These sections were stained with Alcian Blue and Safranin (Cutler 1978) or Fast Green and Safranin (Johansen 1940). Uncleared sections were soaked in 5% NaOH for 5 -1 0 minutes to restore turgidity and were then used to observe chloroplast position in the Kranz cells. found to be ± 2 mm in A. berteroniana and A. hispida and ± 1 ,5 mm in A. nepalensis.
The epidermis is simple. Stomata were observed adja cent to the chlorenchyma zones and the subsidiary cells are at the same level as the epidermal cells ( Figures 2D;  4C). No prickle hairs or hooks were observed.
A discontinuous ring of chlorenchyma is present be low the epidermis. This ring consists of 1 -6 layers of rachymorph cells none of which are more than two cells distant from a Kranz cell. This tissue is interrupted at regular intervals by the sclerenchyma girders of the peri pheral vascular bundles ( Figures IB; 2B (Figures 1C; 4C). Some of the larger bundles may exhibit a partial or complete periphloematic sheath (Caro 1961). The smaller third order peripheral bundles without metaxylem vessels do not have scleren chyma girders attaching them to the epidermis ( Figures  1C; 2F; 4C). These bundles are surrounded by only a single incomplete Kranz parenchyma sheath, interrupted where it adjoins the sclerenchymatous ring. These smal ler bundles may also possess bundle sheath extensions.
Distinctive Kranz cells are present in the peripheral zone ( Figures 1C; 2B; 4B & C). These cells are similar in structure to the Kranz parenchyma sheath cells of all the vascular bundles of this zone. They have thicker walls and larger, predominately centrifugally located chloroplasts than do the chlorenchyma cells and are found singly or in groups of 1 -3(-4) without associated xylem or phloem cells.
In paradermal view the Kranz distinctive cells form long rows, 1 or 2 cells wide, that lie parallel to the vascular bundles but are not accompanied by vascular tissue ( Figure ID). They are connected by lateral cross veins which traverse from one vascular bundle to an other. These interconnections consist only of xylem ele ments and they are not accompanied by bundle sheath cells. No phloem cells were seen. Interconnections are relatively common in the chlorenchymatous zone in the culms of Arundinella.
A second and third (seldom a fourth or fifth) circle of collateral vascular bundles is situated on the inner side of the sclerenchymatous ring. The second circle is partially embedded in these fibres but the other circles are located in the parenchymatous ground tissue of the pith. These inner two circles of bundles consist only of larger, first order vascular bundles surrounded by a single mestome sheath ( Figures 1B;2B, D & F ; 3B ,C ;4B ).

DISCUSSION AND CONCLUSIONS
The presence of these rare and specialized Kranz dis tinctive cells has previously been confirmed in only two genera, Arundinella and Garnotia. However, there has been much confusion in the literature regarding the ter minology for these cells (Table 1) and, although Watson et al. (1986) record circular cells in nine genera, it is not clear whether these are all homologous with the particu lar cells described here. This situation is confusing and unsatisfactory and it is proposed that the term Kranz distinctive cells should in future be employed only for isolated groups of, or single, Kranz cells in the mesophyll which are not associated with contiguous vascular tissue. The term distinctive cells enjoys historical prece dent (Tateoka 1956b) and Kranz distinctive cells also gives an indication of their function. Furthermore this designation is explicit even when translated into other  This study has clearly shown that the unique Kranz distinctive cells of the leaf blades of Arundinella ( Figure  5A, B & C) also occur in the photosynthetic culms. In culm transections they are seen to be rounded cells with thickened walls which contain specialized chloroplasts. They are distinctly larger than the chlorenchyma cells in which they are embedded. These distinctive cells occur as isolated groups or strands in the chlorenchyma com prising one to three contiguous Kranz cells without ac companying vascular tissue. In paradermal view it is clear that they do not form part of the vascular tissue and, therefore cannot be considered as degenerate inter calary vascular bundles (Brown 1977). Instead they are seen to be long, isolated Kranz cell columns not physi cally connected to the vascular bundles but presumably functionally linked at regular intervals by vascular strands. This structure is virtually identical to that described for the distinctive cells of the leaf blades of several Arundinella species. There can be no doubt that these cells which are reported here in the culms of Arun dinella for the first time, represent homologous struc tures in leaf blades and photosynthetic culms. The occur rence of Kranz anatomy in both culms and leaves is noteworthy, because grass species with Kranz leaf ana tomy do not necessarily exhibit Kranz structure in the culm as well (Sánchez unpublished).
The fact that the peripheral first order vascular bundles are surrounded by two bundle sheaths, a complete mes tome sheath and an interrupted Kranz parenchyma sheath, is of considerable interest because this configura tion ( Figure 5) differs from the condition in the leaf blade as reported in the literature. Many authors record a single bundle sheath in the leaf of Arundinella species, which is referred to as the XyMS condition by Hattersley & Wat son (1976) or as the MS type by Brown (1977). Exam ples are Vickery (1935), Brown (1958Brown ( , 1977, Metcalfe (1960), Jacques-Félix (1962), Crookston & Moss (1973), Hattersley & Watson (1975, 1976, Ellis (1977) and Renvoize (1982a). Other workers have reported dou ble bundle sheaths in the leaf blades of Arundinella. Tateoka (1956a) illustrates this PS condition for A. hirta, Conert (1957) for A. decempedalis, Tateoka (1958) for A. leptochloa and A. villosa and Li & Phipps (1973) for A. bengalensis. Some of these latter workers (Tateoka 1958;Li & Phipps 1973) studied A. nepalensis and A. berteroniana, which were also examined in the present study, and found them to have only a single bundle sheath. Eight specimens of A. nepalensis and one of A. berteroniana examined in this study were all observed to have only a single Kranz sheath in the leaf blade. It was, therefore, most unexpected to observe a definite, inner fibrous sheath in the first order bundles of the culm of all three species studied. This may reflect a general condi tion present in all NADP-me grasses or may be an excep tional condition limited to Arundinella and other taxa with distinctive cells. This interesting observation re quires further study as it represents a rare exception to the XyMS character for predicting the NADP-me bio chemical C4 type (Hattersley 1987). This anatomical pre dictor for the NADP-me type may, therefore, apply to leaf blades only or leaves and culms of a given grass may have different photosynthetic pathways.