ИСТИНА

ANATOMY OF EXTRAFLORAL NECTARIES IN LEUCADENDRON MUIRII PHILLIPS AND MIMETES CUCULLATUS R. BR. (PROTEACEAE) Maxim I. Antipin, Vladimir V. Choob Lomonosov Moscow State University, Moscow, Russia sagefool@rambler.ru Leaf margin characters are taxa specific and may serve as an important tool in systematics, including identification of fossil records (Hickey, Wolfe, 1975; Hickey, 1979). Shapes of lamina and leaf margin in Proteaceae in general are quite diverse, but Proteaceae of Cape Floristic region are more uniform regarding leaf margin characters, having either an entire leaf edge or just a few teeth at the lamina apex. Both entire and dentate leaves often terminate with special structure, generally referred to as mucro (Rebelo, 2001). Its shape may range from needle- 14 or awn-shaped to rounded/hemispherical. It differs from the rest of the lamina by its indumentum features (it is usually glabrous but in some cases it has trichomes that are arranged in a specific way), by shape, size and colour of epidermal cells and underlying mesophyll cells (often brightly colored with anthocyanins) and by smoother cuticle. The rounded ‘mucrones’ inherent in several genera of Cape Proteaceae of so called Cape Clade (Weston, Barker, 2002) may be classified among salicoid teeth (“a dark, but not opaque, nondeciduous spherical callosity fused to the tooth apex”) according to the classification of Hickey &Wolfe (1975). Teeth of this type in Salicaceae (including some genera of former Flacourtiaceae) are often secretory (see Wilkinson, 2007), appearing to be resin glands or foliar nectaries. Rebelo (2001) prefers to call such mucrones in Mimetes and Leucospermum the ‘glandular teeth’, but he refers to them simply as ‘glands’ in Leucadendron, the genus whose leaf margin is entire in all species and only some species have leaf apices terminating with structures of this type. Brightly coloured rounded leaf glands, or salicoid teeth, of many species of Mimetes, Leucospermum and Leucadendron are documented to attract insects and referred to as extrafloral nectaries (Midgley, 1987; Rebelo, 1995; Zachariades, Midgley, 1999). Although ant-attracting nectaries are usually considered an adaptation that reduces herbivory due to attracted ants preying on herbivorous insects, experiments had shown that excluding ants from nectary-bearing plants of extant Cape Proteaceae does not increase herbivory levels (Zachariades, Midgley, 1999). Lawton & Heads (1984) suppose that nectaries in such cases may be ‘ghosts of predation past’, meaning that insect phytophages avoid visiting plants with nectaries (and ants), thus the exclusion of ants has no noticeable effect on herbivory. Midgley (1987) suggests that nectaries in extant Cape Proteaceae represent an atavistic defence mechanism that eventually became less and less needed with leaves were becoming smaller, more terete and sclerophyllous in responce to recent climate aridification in the Cape. Existing works on leaf morphology of Cape Proteaceae did not attribute any functions to these organs, just mentioning them as ‘teeth’ (Rourke, 1984) or ‘apical callus’ (Williams, 1972). No works on anatomy of these structures or on leaf dentition and distribution of substances they produce have been published yet. This work aimed to describe the anatomy features of a typical ‘rounded mucro’ (salicoid tooth, apical foliar nectary) in species of Mimetes and Leucadendron, to test the structures for mono- and disaccharide occurrence and spatial distribution using in vivo methods of hystochemical testing, and to compare them with morphologically similar secretory structures of Salicaceae/Flacourtiaceae. Live shoots of Mimetes cucullatus R.Br. were collected near Fernkloof, South Africa, in June 2017. Those of Leucadendron muirii Phillips were taken from plants grown in Botanical garden of Lomonosov Moscow State University from seeds collected in the wild in the vicinity of Struisbaai, South Africa. 15 Live leaves of both species were longitudinally sectioned with hand razor and mounted on slides. Molisch test was immediately performed on some of the sections (alpha-naphthol alcohol solution and concentrated sulphuric acid). Both stained and control leaf sections were observed and photographed using light microscope with digital camera (Olympus CX41). Fully developed leaves of Mimetes cucullatus vegetative shoots usually terminate with three rounded teeth (some smaller leaves only have a single tooth). The teeth are somewhat swollen structures thicker than the leaf blade, they are brightly coloured with anthocyanins, covered with a thick and smooth cuticle, and glabrous. The rest of the blade is covered with abundant trichomes and its cuticle is rough. The leaf blade of Leucadendron muirii acropetally narrows and terminates with single brightly coloured salicoid tooth with distinctly more smooth cuticle. Its apical area is somewhat flattened or concave. Molisch test revealed sugars in vascular bundles reaching the tooth in Mimetes cucullatus. The tooth part of the section is wider than the blade per se; its epidermal and subepidermal cells are red of anthocyanin. Sugar is also noted in large polygonal mesophyll cells situating abaxially in relation to the vascular bundle and almost lacking the chlorophyll. It is detected in loose round parenchyma cells between these polygonal cells and the abaxial epidermis. Cells of tooth epidermis are square or elongated (palisade-like sensu Wilkinson, 2007), noticeably longer radially than flatter epidermal cells of the blade; they are longest on the abaxial side of the tooth. Mesophyll cells and intercellular spaces underlying the epidermis are most stained in the proximal part of the tooth at its abaxial side. This seems to be an area where exudates reach the gland surface through epidermis. In Leucadendron muirii leaf sections, the borderline between the gland and the rest of the lamina is clearly seen in the most apical narrowing part of the leaf. Epidermal cells of the gland are only slightly elongated compared to the blade epidermal cells, but mesophyll cells underlying them are quite distinct as they are bright purple-red and contain no chloroplasts. The most apical, flattened part of the gland is characterized by the longest, square epidermal cells. Parenchyma cells in the flattened apical part of the gland are large and polygonal and lack both chlorophyll and anthocyanins. There is a small space between these large parenchyma cells and the epidermal layer of the flattened apex of the gland, which stains orange with Molisch reagent. In genera of Salicaceae s. str. and former Flacourtiaceae, the secretory epidermis of the glandular salicoid teeth consists of elongated, palisade-like cells perpendicular to the leaf surface (Wilkinson, 2007). Subepidermal mesophyll consists of rounded or polygonal cells lacking chloroplasts. It is supplied by one to several veins and more or less modified for nectar or resin production; some authors refer to it as ‘nectariferous parenchyma’ (Thadeo et al., 2008). Nectar or other excretes 16 may concentrate either in a cavity between the outer epidermal walls and the cuticle (Prockia, Thadeo et al., 2008), or between the loose spongy cells of partly disintegrated mesophyll under palisade-like epidermis (Polyothyrsis, Idesia, Wilkinson, 2007). Secretory teeth of Idesia and Populus are glabrous, while the rest of the leaf is covered with trichomes (Wilkinson, 2007). The same pattern of indumentum distribution is observed in apical leaf glands of Proteaceae. Teeth of Leucadendron muirii and Mimetes cucullatus strickingly liken Idesia foliar nectaries in their morphology and anatomy. In spite of that, the epidermal cells on Proteaceae salicoid teeth are less elongated radially than the typical palisade-like epidermal cells of secretory glands of Salicaceae/Flacourtiaceae, these cells seem to have changed their shape due to their secretory function. As the secretory teeth usually actually produce nectar only at certain stages of leaf developmetn, it is worth to perform a detailed study of the changes these structures experience in the course of leaf development. The genus Leucadendron is characterized by a wide adaptive radiation. Therefore, studies to cover the whole range of mucro forms within the genus could shed some light on evolutionary history of foliar nectaries as a highly specialized structure of great ecological importance. References Hickey L.J. 1979. A revised classification of the architecture of dicotyledonous leaves. In: Metcalfe C.R., Chalk L. (eds.): Anatomy of the dicotyledons. 2nd ed. Oxford: Clarendon Press. V. 1. P. 25–39. 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